Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Supramolecular Helical Arrangement of Porphyrins Along DNA A thesis submitted in the partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry Massey University, Palmerston North, New Zealand Adam Wayne Ian Stephenson 2010 ii i Abstract Porphyrins are useful chromophores and have been used in numerous biological applications including light harvesting, oxygen transport and energy transfer. DNA is a perfect template for the controlled assembly of organic chromophores. By combining DNA and porphyrins in a controlled manner we have developed a novel range of porphyrin-DNA supramolecular constructs for future applications in nanobiotechnology. A number of β-pyrrolic functionalised porphyrin precursors were synthesised to be used as building blocks in the construction of both covalently and non-covalently modified DNAs. Using these porphyrins we have created several lipophilic porphyrin-DNA complexes through non-covalent attachment methods. Using a CuI catalysed azide alkyne cycloaddition (CuAAC) reaction of azido functionalised porphyrins we have developed a versatile approach for the covalent, site specific internal porphyrin insertion into oligonucleotides in a post-synthetic manner. We have investigated a number of duplex structures where porphyrins were located in the major or minor grooves of the duplex. Additionally, porphyrins were studied as intercalating moieties when they were inserted as a bulge in the middle of the duplexes or parallel triplexes. Additionally, when porphyrins were placed in both strands of the duplex they formed a zipper type structure in the minor groove. This resulted in a significant increase in the duplex thermal stability due to the formation of porphyrin H-aggregates. UV-Vis and CD spectroscopy as well as molecular modelling were used to help understand the interactions between porphyrins in the duplex. These findings lay the foundation for the future design of artificial DNA-chromophore supramolecular architectures and for their applications in material science and nanotechnology. ii Acknowledgements I would like to firstly thank my supervisors Associate Professor Ashton Partridge and Dr Vyacheslav Filichev for their support and assistance throughout the duration of this project. Especially Vyacheslav for the enthusiasm he showed towards the project and the time taken to show me various techniques used in DNA chemistry. Thanks also to Niels Bomholt for his assistance with the DNA modelling of porphyrin modified oligonucleotides. Thanks to all former and present colleagues in the NRC and MacDiarmid Centre of Advanced Materials and Nanotechnology for their assistance over the years. Especially Dr Wayne Campbell for his help with DSSCs and Dr Pawel Wagner for solving X-ray crystal structures. I am also grateful for the assistance of departmental staff over the years I have been at Massey University. I would like to acknowledge important financial support from the Marsden grant administrated by the Royal Society of New Zealand (grants MAU0407, MAU0704). Finally, a big thanks to family and friends for their support over the past few years and putting up with the mood swings associated with research and writing. iii Table of Contents Abstract ..............................................................................................................................i Acknowledgements ...........................................................................................................ii Table of Contents .............................................................................................................iii List of Figures .................................................................................................................vii List of Tables..................................................................................................................xiii List of Abbreviations......................................................................................................xiv Chapter 1: Introduction 1.1 Porphyrins ...................................................................................................................1 1.1.1 Porphyrins and DNA................................................................................3 1.2 Deoxyribose Nucleic Acids - DNA.............................................................................3 1.3 Methods for Studying DNA Secondary Structures.....................................................6 1.3.1 Gel Electrophoresis ..................................................................................6 1.3.2 UV-Vis Spectroscopy...............................................................................7 1.3.2 Exciton Coupling .....................................................................................8 1.3.4 Circular Dichroism (CD) Spectroscopy.................................................10 1.4 Automated DNA Synthesis .......................................................................................12 1.5 Incorporation of Porphyrins into DNA .....................................................................13 1.6 Non-covalent Attachment of Porphyrins to DNA.....................................................14 1.7 Covalent Attachment of Porphyrins to DNA............................................................16 1.7.1 External Modification ............................................................................18 1.7.2 Internal Modification .............................................................................20 1.8 Thesis Objectives ......................................................................................................22 1.9 Thesis Structure.........................................................................................................22 Chapter 2: Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 2.1 Introduction ...............................................................................................................24 2.2 Chapter Summary......................................................................................................26 iv 2.3 Synthesis of Porphyrins for Development of Non-Covalently Attached Lipophilic Porphyrin-DNA Complexes............................................................................................28 2.3.1 Route A – Wittig Reaction.....................................................................29 2.3.1.1 The Wittig Reaction 2.3.1.2 Isomerisation 2.3.1.3 Pyridinium Salt Formation 2.3.1.4 Alkene to Alkane Reduction and Pyridinium Salt Formation 2.3.2 Route B – Schiff Base Formation ..........................................................34 2.3.3 Route C – Amide Bond Formation ........................................................37 2.3.3.1 Synthesis of Amino Porphyrins 2.3.3.2 Amide Bond and Pyridinium Salt Formation 2.4 Synthesis of Porphyrins for the Covalent Attachment to Nucleosides and DNA..... 41 2.4.1 Synthesis of Porphyrin for Use in Pre- and Post-synthetic Sonogashira Chemistry...............................................................................................42 2.4.2 Synthesis of Porphyrin for Use in Pre- and Post-synthetic CuAAC Reactions................................................................................................45 2.4.2.1 Synthesis of Aromatic Azides 2.42.2 Synthesis of Aliphatic Azides 2.5 Conclusion ................................................................................................................49 Chapter 3: Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives via the Modified Horner-Emmons Reaction 3.1 Introduction...............................................................................................................50 3.2 Chapter Summary .....................................................................................................53 3.3 Synthesis of β-Pyrrolic Ethynyl Porphyrins..............................................................54 3.3.1 Phosphonate Synthesis...........................................................................54 3.3.2 Modified Horner-Emmons Reaction......................................................55 3.3.3 Synthesis of β-Pyrrolic Ethynyl Dyes for Dye Sensitised Solar Cells...58 3.4 UV-Vis Spectroscopy and DSSC Testing.................................................................60 3.4.1 UV-Vis Absorption Spectroscopy .........................................................61 3.4.2 X-ray Crystallography............................................................................61 3.4.3 Dye Sensitised Solar Cells (DSSCs)......................................................63 3.5 Conclusion ................................................................................................................65 v Chapter 4: Construction of Lipophilic Porphyrin-DNA Complexes 4.1 Introduction ...............................................................................................................67 4.2 Chapter Summary......................................................................................................68 4.3 Porphyrin Solubility..................................................................................................69 4.4 Complex Formation ..................................................................................................70 4.4.1 Singe Stranded and Duplex DNA Porphyrin Loading Ratios................71 4.4.2 G-Quadruplex (GQ) Complex Formation..............................................73 4.5 Conclusion ................................................................................................................75 Chapter 5: Covalent Attachment of Porphyrins to DNA 5.1 Introduction ...............................................................................................................76 5.2 Chapter Summary......................................................................................................77 5.3 Pre-synthetic Sonogashira and CuAAC Reactions ...................................................78 5.3.1 Pre-synthetic Sonogashira Reaction.......................................................78 5.3.2 Pre-synthetic CuAAC Chemistry...........................................................80 5.4 Post-synthetic Sonogashira and CuAAC Reactions..................................................83 5.4.1 Post-synthetic Sonogashira Reaction .....................................................83 5.4.2 Post-synthetic CuAAC Reaction............................................................84 5.5 UV-Vis and CD Studies of Porphyrin-DNA Conjugates..........................................90 5.5.1 Single Stranded Oligonucleotides ..........................................................90 5.5.2 Triplexes and Duplexes Containing Internal Porphyrin Modifications .94 5.5.2.1 Triplexes 5.5.2.2 Duplexes 5.6 Conclusion ..............................................................................................................105 Chapter 6: Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 6.1 Introduction .............................................................................................................107 6.2 Chapter Summary....................................................................................................108 6.3 Post-synthetic CuAAC Chemistry and Oligonucleotide Purification.....................109 6.3.1 Microwave Accelerated Post-synthetic CuAAC Reaction ..................109 6.3.2 Purification and Characterisation of Oligonucleotides Possessing Two Porphyrin Modifications ......................................................................111 6.3.3 Application of the CuAAC Reaction to ZnII and FeIII Porphyrins .......112 vi 6.4 UV-Vis and CD Spectroscopic Studies of DNA-Porphyrin Conjugates Containing Multiple Porphyrins ......................................................................................................115 6.5 Conclusion ..............................................................................................................127 Chapter 7: Summary and Future Directions..............................................129 Chapter 8: Experimental Methods 8.1 Reagents an Equipment used for the Synthesis of Porphyrin Derivatives..............133 8.2 Reagents and Equipment used for the Synthesis of Porphyrin Possessing Oligonucleotides ...........................................................................................................137 8.3 Experimental Procedures for Chapter 2 - Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification ...............................................................................140 8.4 Experimental Procedures for Chapter 3 - Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives via the Modified Horner-Emmons Reaction .............................................161 8.5 Experimental Procedures for Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes.....................................................................................................................183 8.6 Experimental Procedure for Chapter 5 and Chapter 6 ............................................185 Appendix A...................................................................................................................194 References .....................................................................................................................196 vii List of Figures Number Page Figure 1.1 Structure of the porphyrin showing labelling (A), IUPAC numbering (B) and the orthogonal meso rings (C) ...........................................................................................1 Figure 1.2 Aromaticity and tautomeric forms in the free-base (A and B), dianionic (C) and metallated (D) forms of porphyrin .............................................................................2 Figure 1.3 UV-Vis absorption spectrum of ZnTPP in dichloromethane showing the Soret and Q bands. ............................................................................................................2 Figure 1.4 Nucleotides in DNA and RNA .......................................................................4 Figure 1.5 Watson-Crick hydrogen bonding in DNA......................................................4 Figure 1.6 A, B and Z DNA.............................................................................................5 Figure 1.7 Structure of triplex DNA showing the TFO strand in blue (A), and the Hoogsteen base pairing in the TFO strand (B)..................................................................6 Figure 1.8 Hoogsteen type hydrogen bonding in a G-quartet (left) and different topologies of G-quadruplexes ...........................................................................................6 Figure 1.9 UV melting of duplex DNA............................................................................7 Figure 1.10 A) Determination of the Tm value using either the maximum of the first derivative (triangles) or the median method. B) Typical melting curve of a parallel triplex.. ..............................................................................................................................8 Figure 1.11 Proposed direction for the transition dipole moment (µ) vector in a β- pyrrolic modified porphyrin..............................................................................................8 Figure 1.12 Schematic representation of the relationship between chromophore arrangement and spectral shift based on the molecular exciton theory ............................9 Figure 1.13 A) Schematic representation of H-aggregates showing face-to-face stacking of molecules with the transition dipoles located parallel to the lines joining their centres. B) Schematic representation of J-aggregates showing head-to-tail stacking of molecules with the transition dipoles located parallel to each other but perpendicular to the lines joining their centres.........................................................................................................10 Figure 1.14 A) CD spectra of duplex B DNA exhibiting a Cotton effect signal in the porphyrin Soret band region. B) A positive CD bisignate curve resulting from exciton coupling of dipoles that are in a clockwise orientation. C) A negative CD bisignate viii curve resulting from exciton coupling of dipoles that are in an anticlockwise orientation .......................................................................................................................11 Figure 1.15 Automated DNA synthesis .........................................................................13 Figure 1.16 The cationic porphyrin meso-tetrakis[4-(N-methylpyridiumyl)]porphyrin, TMPyP ............................................................................................................................14 Figure 1.17 An example of a porphyrin that binds selectively to the top and bottom of the G-quadruplex preventing telomere extension ...........................................................15 Figure 1.18 Illustration of one of the first lipophilic DNA complexes ..........................16 Figure 1.19 Pre- and post-synthetic DNA modifications...............................................17 Figure 1.20 Examples of porphyrins incorporated into DNA........................................17 Figure 1.21 The CuI catalysed azide–alkyne cycloaddition (CuAAC) reaction ............18 Figure 1.22 A porphyrin molecular cap that stabilises noncanonical DNA ..................19 Figure 1.23 Stacking of 5ʹ porphyrin capped DNA by Berova et al..............................19 Figure 1.24 Example of porphyrin modified dinucleotides ...........................................20 Figure 1.25 Molecular modelling of a meso linked porphyrin-uridine complex by Stulz et al. showing porphyrin stacking in singled stranded (A) and duplex (B) DNA...........21 Figure 1.26 A porphyrin zipper in the major groove that when formed allowed for energy transfer between porphyrins ................................................................................21 Figure 2.1 5,10,15,20-Tetraphenylporphyrin (TPP) showing the β-pyrrolic and meso positions ..........................................................................................................................24 Figure 2.2 The synthesis of 2-bromo-5,10,15,20-tetraphenylporphyrin 1. ....................25 Figure 2.3 The synthesis of TPPps.................................................................................26 Figure 2.4 Synthetic plan for the development of DNA-porphyrin supramolecular structures. ........................................................................................................................27 Figure 2.5 Routes to lipophilic porphyrins ....................................................................28 Figure 2.6 Synthesis of lipophilic porphyrin 6 via the Wittig reaction and pyridinium salt formation ..................................................................................................................29 Figure 2.7 Synthesis of compound 5 via the Wittig reaction. ........................................30 Figure 2.8 Synthesis of compound 6..............................................................................31 Figure 2.9 1H NMR spectrum of the pyridinium salt 6 in CDCl3 (B) and d6-DMSO (A). Note the two sets of signals in CDCl3.............................................................................32 Figure 2.10 Reduction of the alkene (5) to an alkane (8)...............................................33 Figure 2.11 Synthesis of pyridinium salt 9 ....................................................................33 Figure 2.12 Route B .......................................................................................................34 ix Figure 2.13 Attempted synthesis of Schiff base 12........................................................35 Figure 2.14 Failed Schiff base reaction..........................................................................36 Figure 2.15 Attempted synthesis of imine 15 ................................................................36 Figure 2.16 Lipophilic porphyrin from route C .............................................................37 Figure 2.17 Synthesis of 2-amino-5,10,15,20-tetraphenylporphyrin from Zhu et al.....38 Figure 2.18 Synthesis of amino functionalised porphyrins............................................39 Figure 2.19 Synthesis of lipophilic porphyrin 27...........................................................40 Figure 2.20 Summary of lipophilic porphyrins synthesised ..........................................40 Figure 2.21 Synthetic outline for the development of DNA-porphyrin supramolecular structures using covalent attachment methods................................................................42 Figure 2.22 Target compounds for use in Sonogashira chemistry.................................42 Figure 2.23 Synthesis of precursors for the Sonogashira reaction.................................43 Figure 2.24 Synthesis of precursor 40 for the Sonogashira reaction .............................44 Figure 2.25 Target compounds for use in CuAAC reactions (M = H2, NiII, CuII, ZnII, FeIII) ................................................................................................................................45 Figure 2.26 Synthesis of aromatic azides 41 and 42 for use in CuAAC reactions ........46 Figure 2.27 Synthesis of aromatic azide 45 for use in CuAAC reactions......................47 Figure 2.28 Synthesis of aliphatic azides 48-51 for use in CuAAC reactions ...............49 Figure 3.1 The mechanism of the Horner-Emmons reaction for the synthesis of alkenes.............................................................................................................................51 Figure 3.2 The mechanism of the modified Horner-Emmons reaction for the synthesis of alkynes ........................................................................................................................51 Figure 3.3 Schematic representation of a Grӓtzel cell ...................................................52 Figure 3.4 β-Pyrrolic substituted TPP showing various linkers and binding groups (BG) used for DSSCs ...............................................................................................................53 Figure 3.5 Synthesis of chloro and bromophosphonates from the appropriate aldehydes.........................................................................................................................55 Figure 3.6 The modified Horner-Emmons reaction showing the formation of the ethynyl linkage and the undesired halovinyl intermediate (X = Br or Cl)......................56 Figure 3.7 The molecular structure of the halovinyl intermediate 39a as defined by defined by X-ray crystallography....................................................................................56 Figure 3.8 Synthesis of porphyrins 73 and 74................................................................57 Figure 3.9 Synthesis of benzoic acid 82 for use in DSSCs............................................59 x Figure 3.10 Proposed sequence for the conversion of the aldehyde 80 into the methyl ester 81 ............................................................................................................................59 Figure 3.11 Synthesis of the cyanoacetic acid 83 ..........................................................59 Figure 3.12 Synthesis of malonic acid 84 showing the production of the decarboxylated product 85................. ......................................................................................................60 Figure 3.13 UV-Vis spectra of the ethynyl, alkene and alkane porphyrinic acids in DMF at 25 ˚C. .................................................................................................................61 Figure 3.14 Crystal structure of 2-(4ʹ-formyl)phenylethynyl-5,10,15,20- tetraphenylporphyrinato zinc (II) 80 with methanol coordinated to the zinc .................62 Figure 3.15 Crystal structure of 4-{trans-2ʹ-[2ʹʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ- tetraphenylporphyrinato copper (II) yl) ethen-1ʹ-yl])-1-benzaldehyde 88 taken from Bonfantini et al................................................................................................................62 Figure 3.16 Malonic acid and cyanoacetic acid dyes tested in DSSCs..........................63 Figure 3.17 DSSC cell holder containing a TiO2 cell with bound porphyrin. ...............64 Figure 4.1 A) An illustration of one of the first lipophilic-DNA complexes B) A TEMPO lipophilic salt used in the construction of DNA batteries.................................67 Figure 4.2 Lipophilic compounds used to create DNA-porphyrin complexes ..............68 Figure 4.3 Synthesis of compound 90............................................................................70 Figure 4.4 Example of a DNA-porphyrin complex of compound 27 ............................71 Figure 4.5 Possible stacking arrangement of lipophilic porphyrins on DNA ................73 Figure 4.6 UV-Vis spectra of GQ-porphyrin complex (thicker line) and unreacted porphyrin 27 (thinner line) in CHCl3 ..............................................................................74 Figure 5.1 Synthetic outline for the development of DNA-porphyrin conjugates.........76 Figure 5.2 Porphyrin precursors used in Sonogashira chemistry...................................78 Figure 5.3 Porphyrin precursors used in CuAAC chemistry .........................................78 Figure 5.4 Attempted pre-synthetic Sonogashira coupling involving compounds 35 and 92 .........................................................................................................................79 Figure 5.5 Pre-synthetic Sonogashira coupling reactions. .............................................80 Figure 5.6 Synthesis of model triazole linked porphyrin nucleosides ...........................82 Figure 5.7 Post-synthetic Sonogashira reactions ...........................................................83 Figure 5.8 Acetylene containing nucleotides incorporated into DNA for use in CuAAC chemistry.........................................................................................................................84 Figure 5.9 Porphyrin-DNA monomers obtained via the post-synthetic CuAAC chemistry.........................................................................................................................86 xi Figure 5.10 CuAAC reaction between azido porphyrins 41 or 50 and oligonucleotides containing monomers V, Y or Z .....................................................................................87 Figure 5.11 HPLC profiles of the reaction between azide 50 and ON5 ........................88 Figure 5.12 Denaturing 20% PAGE (7 M urea) of porphyrin modified oligonucleotides. .............................................................................................................89 Figure 5.13 CD spectra of single stranded oligonucleotides ON10-15 at pH 6.0..........90 Figure 5.14 CD spectra of single stranded oligopyrimidine ON15 at pH 5.0, 6.0 and 7.2 (A), cytosine+-cytosine base pair (B) and possible i-tetraplex of ON13 (C) .................92 Figure 5.15 CD spectra of single stranded oligopyrimidine ONwt at pH 5.0, 6.0 and 7.2 .......................................................................................................................92 Figure 5.16 CD spectra of single stranded purine-pyrimidine oligonucleotide ON17 at pH 5.0 and 7.2 .................................................................................................................93 Figure 5.17 CD spectra showing the thermal melting of single stranded oligopyrimidine ON15 from 30-60 °C at pH 5.0.......................................................................................93 Figure 5.18 A representation of AMBER* force field lowest energy minimised structures of the porphyrin possessing triplex ON14/D1 showing two possible porphyrin orientations......................................................................................................................96 Figure 5.19 TINA structures incorporated into TFO strands used as bulged insertions in to stabilise triplexes.........................................................................................................96 Figure 5.20 1,2,3-Triazole linked benzyl moiety incorporated into TFO strands..........97 Figure 5.21 CD spectra of triplex ONwt and ON15 with D1, and duplex D1 alone at pH 5.0 (20 °C).................................................................................................................99 Figure 5.22 CD spectra of triplex ONwt and ON15 with D1 and duplex D1 with the triplex strand excluded at pH 6.0 (20 °C) .......................................................................99 Figure 5.23 A representation of the lowest energy AMBER* force field minimised structures of duplexes involving ON10 (A), ON11 (B), ON12 (C) and ON13 (D).....101 Figure 5.24 A representation of the lowest energy structures duplexes involving ON14 (A) and ON15 (B) .........................................................................................................102 Figure 5.25 CD spectra of duplexes ON10-15 with ON22 at pH 6.0 (20 °C).............104 Figure 5.26 CD spectra of duplexes involving ONwtm and ON16-21 at pH 6.0 (20 °C).....................................................................................................................104 Figure 5.27 CD spectra of duplex ON13/ON22 at 20 °C, 70 °C and after cooling and incubation at 20 °C for 30 minutes ...............................................................................105 Figure 6.1 β-Pyrrolic functionalised azido porphyrin 50 .............................................109 xii Figure 6.2 Synthesised oligodeoxynucleotides ............................................................110 Figure 6.3 Representative PAGE (20% with 7 M urea) of unmodified oligonucleotide ON25 and porphyrin modified oligonucleotides ON31 and ON35 .............................112 Figure 6.4 FeIII and ZnII porphyrin azides....................................................................113 Figure 6.5 Possible modes for the coordination of 50 (A) and 46 (B).........................114 Figure 6.6 Representative UV melting profiles (260 nm) of unmodified and modified duplexes. .......................................................................................................................115 Figure 6.7 A representation of the lowest energy AMBER* force field minimised structures of duplex 2 (A) and duplex 10 (B) ...............................................................118 Figure 6.8 Representative UV-Vis melting profiles. ...................................................119 Figure 6.9 AMBER* force-field lowest energy minimised structures of porphyrin modified duplexes... ......................................................................................................120 Figure 6.10 Plausible arrangement of porphyrins in duplex 11 showing the flipped and non-flipped conformations............................................................................................121 Figure 6.11 CD spectra of duplexes 1, 11, 12 and 13 ..................................................121 Figure 6.12 A representation the lowest AMBER* force field minimised structures of duplexes possessing two zipping porphyrins ................................................................122 Figure 6.13 Coronene modified LNA possessing short (A) and long (B) linkers .......123 Figure 6.14 Plausible arrangements of porphyrins in duplexes with three modifications.................................................................................................................124 Figure 6.15 A representation of the lowest energy ABMER* force field minimised structures of duplexes 23 (A) and 24 (B) viewed into the minor groove......................125 Figure 6.16 CD spectra of duplexes 1, 4, 9 and 11-24.................................................126 Figure 6.17 Representative UV melting profiles (260 nm) of duplexes 3 (a), 13 (b), 18 (c) and 24 (d).................................................................................................................127 Figure 7.1 Possible metal free nitrile oxide post-synthetic chemistry .........................131 Figure 7.2 Incorporation of possible anionic (A), cationic (B) 5,10,15,20- tetraphenylporphyrins or 5,15-diphenylporphyrin derivatives .....................................131 Figure 8.1 1H NMR spectroscopic assignments for β-pyrrolic functionalised porphyrins .....................................................................................................................135 xiii List of Tables Number Page Table 2.1 Synthetic attempts for the synthesis of azide 45 ............................................48 Table 3.1 Phosphonates synthesised and their yields.....................................................55 Table 3.2 β-Pyrrolic alkyne porphyrins 31, 39 and 67-72 and their yields ....................57 Table 3.3 Results of DSSC testing .................................................................................64 Table 3.4 Dye loading on TiO2.......................................................................................65 Table 4.1 Solubility at RT of 1 mg of porphyrin 9 or 27 ...............................................69 Table 4.2 Loading ratios for porphyrins 9 and 27 ..........................................................72 Table 5.1 Oligonucleotides before and after CuAAC reactions with azides 41 or 50 ...86 Table 5.2 Melting temperatures of parallel DNA triplexes and antiparallel DNA duplexes containing a single porphyrin modification .....................................................98 Table 5.3 Annealing temperatures of parallel DNA triplexes and antiparallel DNA duplexes containing a single porphyrin modification .....................................................98 Table 5.4 Melting temperatures of antiparallel duplexes containing mixed purine/pyrimidine oligodeoxynucleotides.....................................................................103 Table 5.5 Annealing temperatures of antiparallel duplexes containing mixed purine/pyrimidine oligodeoxynucleotides.....................................................................103 Table 6.1 ONs synthesised and their mass spectroscopic analysis ..............................111 Table 6.2 Arrangement of porphyrins in DNA duplexes and their melting temperatures ...........................................................................................................116-117 xiv Abbreviations A adenosine ACN acetonitrile AcOH acetic acid aq aqueous Ar aromatic ATR Attenuated total reflection BHT 2,6-bis(1,1-dimethylethyl)-4-methylphenol BIAB [bis(acetoxy)iodo]benzene bp base pair br broad C cytosine Calcd calculated CD circular dichroism spectroscopy CDCl3 deuterated chloroform conc concentrated COSY correlation spectroscopy CPG controlled porous glass CTAB cetyl trimethylammonium bromide CuAAC CuI catalysed Huisgen 1,3-dipolar azide alkyne cycloaddition d doublet dA 2ʹ-deoxyadenosine Da daltons DBU 1,8-diazabicyclo[5.4.0]undec-7-ene dC 2ʹ-deoxycytosine DCE 1,2-dichloroethane DCM dichloromethane DDQ 2,3-dichloro-5,6-dicyanobenzoquinone DFT density functional theory dG 2ʹ-deoxyguanosine DMAP 4-dimethylaminopyridine DMEA N,N'-Dimethyl-1,2-ethanediamine xv DMF N,N-dimethylformamide DMT 4,4ʹ-dimethoxytrityl DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DSSC dye sensitised solar cell EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EDTA ethylenediaminetetraacetate acid eq equivalent ESI electrospray ionisation EtOH ethanol FAB fast atom bombardment FF fill factor G guanosine GQ guanosine quadruplex h hour(s) HOMO Highest occupied molecular orbital HPLC high preformance liquid chromatography HRMS high resolution mass spectrometry IR infra-red spectroscopy ITO indium tin oxide Jsc short circuit current L litres LHCP left hand circularly polarised LNA locked nucleic acid LRMS low resolution mass spectrometry LUMO Lowest unoccupied molecular orbital m multiplet M mol/L MALDI matrix assisted laser desorption ionisation MeOH methanol min minute mL millilitres mmol millimole NBS N-bromosuccinimide xvi NMR nuclear magnetic resonance ON oligodeoxynucleotide PAGE polyacrylamide gel electrophoresis PCC pyridinium chlorochromate Ph phenyl ppm parts per million Rf retention factor RHCP right hand circularly polarised RT room temperature s singlet t triplet T thymidine Ta annealing temperature TBAF tetrabutyl ammonium fluoride Td denaturing temperature TFO triplex forming oligonucleotide TEAA triethylammonium acetate TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl THF tetrahydrofuran TINA twisted intercalating nucleic acid TLC thin layer chromatography Tm melting temperature TMPyP tetrakis[4-(N-methylpyridiumyl)]porphyrin TMS tetramethylsilane TOF time of flight TPP 5,10,15,20-tetraphenylporphyrin TPPCH3 2-methyl-5,10,15,20-tetraphenylporphyrin TPPCHO 2-formyl-5,10,15,20-tetraphenylporphyrin TPPps TPP phosphonium salt U uridine UV-Vis ultraviolet-visible spectroscopy Voc open circuit voltage µL microlitres µmol micromole Chapter 1 – Introduction 1 Chapter 1 Introduction 1.1 Porphyrins Porphyrin type compounds are found throughout nature where they are employed in a myriad of biological roles such as oxygen transport, reaction catalysts, electron transfer, energy transfer and light harvesting.1 Porphyrins consist of four pyrrole rings linked via methylene groups to give a fully conjugated ring system. The most simple version of the porphyrin family is the porphin molecule depicted in Figure 1.1 including the standard nomenclature (A) used to locate the substituents attached to the porphyrin structure, and the standard numbering system (B). A commonly used porphyrin is 5,10,15,20- tetraphenylporphyrin (TPP, C), consisting of four phenyl rings in the meso positions, which are orthogonal to the plane of the porphyrin core. N NH N HN α β meso N NH N HN 1 2 3 4 5 6 7 8 9 10 11 12 1314151617 18 19 20 21 22 2324 N NH N HN A B C Figure 1.1 Structure of the porphyrin showing labelling (A) IUPAC numbering (B) the orthogonal meso rings (C). The nitrogens of the porphyrin can either be protonated (more commonly known as the free base, Figure 1.2, A) or deprotonated (Figure 1.2, C) and exist in two tautomeric forms (Figure 1.2, A and B). On deprotonation, a cavity is formed that is of the ideal size to allow the binding of most metals. Porphyrins, which are approximately flat, are highly aromatic, however, only 18 of the pi electrons can exist in an internal aromatic ring, with the peripheral double bonds being isolated. On metallation (Figure 1.2, D), there exists both an internal and an external aromatic ring system. It is the aromaticity of Chapter 1 – Introduction 2 the porphyrin molecule that gives it its high thermodynamic stability, a high molar absortivity coefficient and the ability to have its electronic properties tuned by the binding of different metals. N N N N H H N N N N M N N N N 2- N N N N H H A B C D Figure 1.2 Aromaticity and tautomeric forms in the free-base (A and B), dianionic (C) and metallated (D) forms of the porphyrin. Porphyrins are highly coloured and have characteristic electronic absorption spectra. Figure 1.3 shows the UV-visible spectrum for 5,10,15,20-tetraphenylporphyrin (TPP) and its ZnII analogue, as an example of a typical electronic spectrum for a porphyrin molecule. The components include a strong Soret (or B) band (due pi-pi* transitions) commonly between 400 – 420 nm and weaker Q bands (four in free base porphyrins and two in metalled porphyrins) between 520-600 nm. Modification of the porphyrin at one of the β-pyrrolic positions results in decreased intensity and broadening of the Soret band.2 In general, porphyrins are also strongly fluorescent. This fluorescence (which usually occurs at approximately 650 nm) is dependent on a number of factors including metallation, for example, ZnII and free base porphyrins are highly fluorescent while NiII porphyrins are virtually non-fluorescent. Figure 1.3 UV-Vis absorption spectrum of ZnTPP in dichloromethane showing the Soret and Q bands (reproduced from Campbell).3 Chapter 1 – Introduction 3 1.1.1 Porphyrins and DNA DNA is a perfect template or scaffold for the creation of functional pi systems as it has a number of favourable characteristics such as:4-6 • The spontaneous self assembly of oligonucleotides with complementary sequences. • The duplex has a well known and characteristic structure. In B DNA the base pair distances along the helical array is 3.4 Å providing the ideal basis with for pi stacking. • DNA is relatively easy to synthesise with predictable positioning of organic chromophores in various DNA secondary structures. • There are a number of alternative methods to covalently and non-covalently bind molecules to the DNA structure. Porphyrins make an ideal pi system to be incorporated into DNA due to the large aromatic structure and the ability to tune the electronic properties of the complex by implementing small variations in the porphyrin. Therefore, in this thesis we discuss the construction of novel porphyrin-DNA supramolecular structures for the future development of various nanotechnological applications. 1.2 Deoxyribose Nucleic Acids - DNA Nucleic acids consist of simple units called nucleotides which consist of a phosphate group and nucleosides (Figure 1.4). The latter consists of a pentafuranose ring which is ribofuranose for RNA and 2ʹ-deoxyribofuranose for DNA. The four bases found in DNA are heterocyclic compounds of two types: purines and pyrimidines. Adenine (A) and guanine (G) are purines, and cytosine (C) and thymine (T) are pyrimidines. A fifth base called uracil (U) replaces thymine in RNA. Nucleosides are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms in the sugar ring. It is this asymmetry around the phosphates that gives directionality to the strands.4 Chapter 1 – Introduction 4 1 2 3 4 5 6 1 2 3 67 8 9 5' 3' N NN N NH2 NH NN N O NH2 N N NH2 O HN N O O HN N O O Thymine Uracil Cytosine Adenine Guanine Base O XO OP O- O nucleoside nucleotide X = H: DNA X = OH: RNA Purines Pyrimidines Figure 1.4 Nucleotides in DNA and RNA. As a result of Watson-Crick hydrogen bonds (Figure 1.5), pi-pi stacking and electrostatic repulsion between the negatively charged phosphates in the DNA forms a helical structure. Depending on the sequence and the media, the duplex can exist in various conformations from the most commonly found right handed spiral B form, to the compact A form and the left handed Z form (Figure 1.6). The B form duplex forms two grooves, the major groove (22 Å wide) and the minor groove (12 Å wide). The size of the grooves means that the bases are more accessible in the major groove. N N N N N H H N N O O H NN N N O N H H H N N N O H H A T G C Figure 1.5 Watson-Crick hydrogen bonding in DNA. Chapter 1 – Introduction 5 Figure 1.6 A, B and Z DNA. DNA does not exist exclusively as a duplex but can also form various secondary structures. Watson-Crick hydrogen bonds form the above mentioned antiparallel duplex but alternative Hoogsteen base pairing allows for the formation of triplex and quadruplex structures. It is these structures that might be responsible for the regulation of some genes.7 A triple helix is formed when a third single stranded oligonucleotide binds in the major groove of the duplex (Figure 1.7). This third strand is commonly referred to as the triplex-forming oligonucleotide or TFO strand. As illustrated in Figure 1.7, triplexes are formed when the bases of the TFO strand form Hoogsteen or reverse Hoogsteen hydrogen bonds with the purine bases already involved in Watson–Crick base pairs. The directionality of the TFO strand gives rise to parallel and anti-parallel triplexes.4 A homopyrimidine TFO binds in a parallel fashion (i.e. the same 5ʹ to 3ʹ orientation) to the homopurine strand in a DNA duplex. This is achieved by forming T-A··T and C-G··C+ Hoogsteen interactions (· ·), and requires the protonation of the N-3 atom of cytosine in the TFO. The pKa of the imino group of the cytosine (5.2) is well below that of physiological pH making parallel triplex formation pH dependent. Alternatively, triplexes can be formed from a homopurine (GA) TFO strand in a parallel or antiparallel fashion. Chapter 1 – Introduction 6 Figure 1.7 Structure of triplex DNA showing the TFO strand in blue (A), and the Hoogsteen base pairing in the TFO strand (B). Guanosine quadruplex (GQ) is a unique secondary structure that can form at physiological pH and salt concentrations in sequences containing adjacent guanine residues (Figure 1.8). Hoogsteen interactions allow for the formation of intra or intermolecular quadruplexes that can exist in a variety of topologies based on strand polarity, the concentration of metal ions, length of sequence etc.7 Figure 1.8 Hoogsteen type hydrogen bonding in a G-quartet (left) and different topologies of G- quadruplexes. 1.3 Methods for Studying DNA Secondary Structures There are three main techniques for the investigation and characterisation of oligonucleotides: gel electrophoresis, UV-Vis spectroscopy and circular dichroism (CD) spectroscopy. 1.3.1 Gel Electrophoresis Gel electrophoresis is a technique used for the separation and characterisation of DNA. DNA will migrate through a gel towards the positive electrode in the presence of a current, based on size, charge and topology of the strands. Shorter oligonucleotides Chapter 1 – Introduction 7 migrate faster through the gel than longer ones as it is easier for them to travel through the pores of the gel. Gels can be constructed from various polymers depending on the size of the DNA, the most common being agarose (> 150 nucleotides) and polyacrylamide (< 150 nucleotides) gels. Polyacrylamide gel electrophoresis (PAGE) can be performed under denaturing or non-denaturing conditions depending on the information that is desired. The addition of urea and formamide to a gel and oligonucleotide results in the loss of secondary structure of the DNA (denaturing), whereas non-denaturing gels retain the structure and topology on the DNA strands. As DNA only absorbs in the UV region, visualisation is generally achieved through the staining of the oligonucleotides with a visible dye. 1.3.2 UV-Vis Spectroscopy The main tool for determining the effect of a modification on DNA is to measure the thermal stability of the secondary structure. On heating of a structure, be it a duplex, triplex or other secondary structure, disordering of the DNA occurs resulting in hyperchromicity at 260 nm. From the change in absorbance, we are able to determine the temperature of melting (Tm), or more accurately, the temperature of midtransition. This is the point at which half the oligonucleotides exist as a single strands and half as the secondary structure (Figure 1.9). Figure 1.9 UV melting of duplex DNA (reproduced from Mergny et al.).8 Chapter 1 – Introduction 8 There are a number of methods to determine the Tm of DNA secondary structure and two of these are shown in Figure 1.10A. In this report we employed the most common of the two methods where the Tm is the maximum of the first derivative of the melting curve. This is the more effective method when complex transitions, such as the melting of a triplex, is being determined (Figure 1.10B). An alternative method involves drawing baselines at the top and the bottom of the curve, a median line is then placed between these lines. The point at which the median line intersects the melting curve is the Tm. In general, only small difference in the calculated Tm is produced using either method. Figure 1.10 A) Determination of the Tm value using either the maximum of the first derivative (triangles) or the median method. B) Typical melting curve of a parallel triplex. (Reproduced and adapted from Mergny et al.).8 In some instances the DNA does not always display the same midtransition temperature during the melting and annealing processes, giving rise to hysteresis. Hysteresis is common in some secondary structures more than others, such as in the melting and annealing of triplexes. Generally, hysteresis can be overcome by heating and cooling the duplex at a slower rate. It is possible to gather thermodynamic information on a duplex by measuring Tm values at various DNA concentrations, providing no hysteresis occurs.8 Insertion of organic chromophores into DNA leads to the formation of additional peaks in the UV-Vis spectra at the wavelengths of chromophoric absorption. Attachment of several chromophores onto DNA gives rise to complex interacts between aromatic moieties, such as H- and J-aggregates, resulting from excitonic coupling. Chapter 1 – Introduction 9 1.3.3 Exciton Coupling Exciton coupling is an important tool for understanding the relative conformation of closely located molecules, especially when combined with UV-Vis and circular dichroism spectroscopy. According to exciton coupling theory,12 a molecule can be excited from the ground state (GS) to an excited state (E) by a photon of an appropriate wavelength. As a result, the distribution of electrons in the excited state differs to that of the ground state thus creating an electric transition dipole (µ). This dipole is described by a vector that corresponds to the direction of the electron movement and its strength is dependent on the nature of the transition occurring. Although the direction of the transition dipole has been well studied in meso functionalised porphyrins,9 the direction is less well defined in β-pyrrolic substituted porphyrins. Thus in the absence of DFT calculations we must make assumptions to determine the direction of the dipole. By observing the relative contributions and location of the HOMO and LUMO levels in similar β-pyrrolic modified porphyrins,10, 11 where the HOMO is located on the porphyrin core and the LUMO is located on the β- pyrrolic phenyl moiety, we can assume that the electric dipole vector runs along the axis of the β-pyrrolic modification as shown in Figure 1.11. N N N N M Figure 1.11 Proposed direction for the transition dipole moment (µ) vector in a β-pyrrolic modified porphyrin. If multiple molecules are located in close proximity and are of similar energy levels then the excitation can be delocalised over both molecules, resulting in exciton coupling. As a result of this interaction, a pair of degenerate excited states results in two nondegenerate excited states (Eʹ and Eʹʹ) which are of higher and lower energies compared to the original excited state (Figure 1.12). Chapter 1 – Introduction 10 E'' E' E" E'E GS GS H-aggregate Monomer J-aggregate blue shift red shift Figure 1.12 Schematic representation of the relationship between chromophore arrangement and spectral shift based on the molecular exciton theory. The solid arrow represents allowed transitions and the dashed arrow represents forbidden transitions. These excited states are a combination of the relative directions of the dipoles in the excited molecules, either being in phase or out of phase, with out of phase motions being forbidden on electrostatic grounds. The difference between the excited and the ground states energy levels results in either a hypsochromic shift (H-aggregates, Figure 1.13A) or a bathochromic shift (J-aggregates, Figure 1.13B) in the UV-Vis spectra.12 A Electric transition dipole H-Aggregates J-Aggregates B Figure 1.13 A) Schematic representation of H-aggregates showing face-to-face stacking of molecules with the transition dipoles located parallel to the lines joining their centres. B) Schematic representation of J-aggregates showing head-to-tail stacking of molecules with the transition dipoles located parallel to each other but perpendicular to the lines joining their centres. 1.3.4 Circular Dichroism (CD) Spectroscopy Circular dichroism (CD) spectroscopy is a key technique for obtaining information into the structure of DNA.13 This is achieved by studying the shape and intensity of the UV region (200-350 nm) where the DNA bases absorb the light. Information can be gathered to give an indication about the conformation of the DNA, such as B, Z, or A duplexes, G-quadruplexes, triplexes or other DNA secondary structures.13 Circular Chapter 1 – Introduction 11 dichroism spectroscopy is a system that measures the difference in absorption of left handed (LHCP) and right handed circularly polarised (RHCP) light that originates from the interactions of chiral molecules (or molecules that have induced chirality) with circularly polarised electromagnetic radiation.14 The difference in the absorption of LHCP and RHCP light can give rise to either positive or negative peaks in the CD spectrum (Figure 1.14A, Soret band region) which occur at the wavelength that the light is absorped. These positive and negative CD signals are called Cotton effects. Like those observed in UV-Vis spectroscopy, exciton coupling can occur when two chromophores with similar excited state energy levels are located in close proximity. Both the Eʹ and Eʹʹ excited states absorb LHCP and RHCP light to different extents resulting in two Cotton peaks of opposite signs, centered at λmax of the monomer. The resulting observed signal is the sum of the Cotton effects which gives rise to bisignate curves as shown in Figure 1.14B and C. The sign of which is dependent on the relative orientation of the transition dipoles. Figure 1.14 A) CD spectra of duplex B DNA exhibiting a Cotton effect signal in the porphyrin Soret band region. B) A positive CD bisignate curve resulting from exciton coupling of dipoles that are in a clockwise orientation. C) A negative CD bisignate curve resulting from exciton coupling of dipoles that are in an anticlockwise orientation. Chapter 1 – Introduction 12 1.4 Automated DNA Synthesis Automated DNA synthesis allows for the rapid production of oligonucleotides up to 200 bases in length. Unlike DNA synthesis in nature, automated DNA synthesis occurs in the 3ʹ to 5ʹ direction and is achieved through the stepwise addition of nucleoside phosphoramidites or H-phosphonates to each other in a controlled manner. Construction is based on controlled porous glass (CPG) or polystyrene supports, which allows for removal of excess reagents by filtration and eliminates the need for purification steps between base additions. The synthetic cycle is shown in Figure 1.15 and begins with a 3'-hydroxyl nucleoside attached to the support by a base sensitive linker. This nucleoside contains a 4,4ʹ- dimethoxytrityl (DMT) protecting group which is cleaved with trichloroacetic acid, providing a free 5'-hydroxyl for the next coupling reaction (step 1). An activated trivalent phosphoramidite derivative is then added reacting with the free 5'-hydroxyl on the primary nucleotide to create a phosphite bond (step 2). Activation occurs with the simultaneous addition of 1H-tetrazole, protonation the 3ʹ-O-phosphoramidite and promoting faster coupling with the 5ʹ-OH of the nucleoside on the support. Self condensation of the activated nucleotide is prevented by the protection of the 5ʹ-OH with DMT. Generally the elongation occurs in a quantitative yield, however, any chains that do not undergo coupling are capped with acetic anhydride and 1-methylimidazole (step 3). The phosphite linker is then oxidised to phosphotriester by the addition of iodine and water (step 4), and the cycle is continued until the desired chain length is obtained (step 5). When the desired length is obtained the oligonucleotide is cleaved from the support with a strong base (usually concentrated NH4OH), which also removes the 2ʹ-cyanoethyl groups from the phosphates and any protecting groups that are on the nucleobases (step 6). This desired oligonucleotide can be purified from any short oligonucleotide sequences using techniques such as reverse-phase or ion-exchange HPLC or denaturing gel electrophoresis. Chapter 1 – Introduction 13 O O Base1 DMTO O O Base1 HO O O Base2 DMTO P N OCH2CH2CN O O Base1 O O O O Base1 O O Base2 DMTO P OCH2CH2CN O O O Base1 O O Base2 DMTO P OCH2CH2CN O O Oxidation Acid deprotection for continued synthesis Capping Activation and coupling Deprotection = CPG support O HO Base1 O O Base2 DMTO P O O O Base deprotection and cleavage 1 2 3 4 5 6 START FINISH Figure 1.15 Automated DNA synthesis. 1.5 Incorporation of Porphyrins into DNA Many organic molecules have already been incorporated into DNA using various chemical techniques as covered in the reviews of Filichev,4 Wagenknecht5 and Hӓner.15 In this thesis we focus specifically on the incorporation of porphyrins into DNA. The incorporation of porphyrins into DNA is not a novel concept. Various methods have been used successfully to construct supramolecular DNA-porphyrin complexes using two basic approaches – covalent and non-covalent attachment. Non-covalent Chapter 1 – Introduction 14 attachment of cationic porphyrins to DNA is a reasonably well investigated field, however, covalent attachment has been studied less extensively. Current covalent methods are scarce and focus on the attachment of porphyrins via the meso position. It is thus the goal of this thesis to lay the foundation for the development of novel β- pyrrolic functionalised porphyrins-DNA supramolecular structures for the use in future nanotechnological applications. This will be achieved through the synthesis of porphyrin precursors which can then be incorporated into the DNA structure through either non-covalent or covalent attachments. 1.6 Non-covalent Attachment of Porphyrins to DNA The most commonly used porphyrin for non-covalent interactions with DNA is the water soluble meso-tetrakis[4-(N-methylpyridiumyl)]porphyrin, TMPyP (Figure 1.16). This cationic porphyrin has a natural affinity for DNA.16 The four pyridiumyl salts allow for interaction with the negative phosphates of DNA. Intercalation of the porphyrin between base pairs has also been observed17 along with groove binding and stacking along the exterior of the DNA.18 Variation of the metal ions (such as the incorporation of metals with axial ligands),19, 20 porphyrin loading and DNA base sequence allow for some degree of control over the type of interaction that occurs. N NH N HN N N N N Figure 1.16 The cationic porphyrin meso-tetrakis[4-(N-methylpyridiumyl)]porphyrin, TMPyP. Interaction of cationic porphyrins such as TMPyP have been shown to result in the photo cleavage of the DNA in the presence of light, and has been employed in photodynamic therapy for the treatment of cancer cells.21, 22 Formation of complexes containing three, two23 and one24 charged meso derivatives have also been produced for Chapter 1 – Introduction 15 use in photodyanamic therapy studies. Unfortunately, reducing the number of cationic groups also reduces the solubility of the porphyrins in water. The interaction of cationic porphyrins with DNA is not limited to duplexes. Studies have shown that cationic porphyrins such as TMPyP can bind to G-quadruplexes. Binding can occur in three modes, either binding to the top and bottom of quadruplexes (capping),25 binding between strands of G-quadruplexes26 or intercalation between guanine tetrads within a quadruplex.27 All of these binding modes have been shown to increase the stability of the G-quadruplex structure. Of particular interest is the capping of porphyrins to the end of G-quadruplexes resulting in increased stability of the G- quadruplexes. This is biologically important as telomeric ends, which exist at the ends of chromosomal DNA protecting the DNA from degradation, consist of G rich sequences. Stabilising the telomeric G-quadruplex structure, for example by capping it with a porphyrin (Figure 1.17), inhibits the activity of telomerase, an enzyme responsible for the elongation of the telomeres. Inhibiting the elongation of telomeres can help restrict the growth of cancer cells.28-30 N N N N N H H N H N N H O O OO N NN N Mn 5 Cl Figure 1.17 An example of a porphyrin that binds selectively to the top and bottom of the G-quadruplex preventing telomere extension.25 A slight variation on non-covalent attachment is the production of lipophilic DNA complexes. DNA is normally soluble in aqueous solution, however by exchanging the Na+ or Li+ ions along the phosphate backbone for quaternary ammonium salts possessing a long lipophilic chain (Figure 1.18), it is possible to create a complex that is soluble in organic solutions.31, 32 Slow evaporation of organic solvents can produce lipophilic DNA films with aligned DNA strands that have shown to be important in the Chapter 1 – Introduction 16 production of batteries,33 liquid crystals,34 drug release systems,35 photodynamic devices36 and many other systems. Although many materials have been incorporated into DNA-lipid complexes, the incorporation of lipophilic porphyrins is a novel approach. If successful it may allow for the simple construction of porphyrin based arrays. Base O O O P OO N CH3 H3C CH3 O H3C(CH2)9 4 Figure 1.18 Illustration of one of the first lipophilic DNA complexes.31 1.7 Covalent Attachment of Porphyrins to DNA The covalent attachment of porphyrin moieties to DNA can be realised through the use of either pre- or post-synthetic approaches (Figure 1.19). Currently there are only a few examples of post-synthetic porphyrin modifications37-39 (Figure 1.20A and B) although there are many examples of post-synthetic modification involving other chromophores.4, 15, 40 A pre-synthetic approach involves the attachment of a porphyrin to a nucleoside which is then converted to the appropriate porphyrin phosphoramidites or H- phosphonates. These are then incorporated into the DNA structure during DNA synthesis. The pre-synthetic approach can allow for the incorporation of many functionalised nucleotides on a single DNA strand using automated DNA synthesis only if high yielding coupling reactions occur. Post-synthetic modification means that a special functional group of the porphyrin reacts specifically with a pre-synthesised oligonucleotide carrying a complementary functional group. In order to screen different substituents in nucleic acid structures, a post-synthetic approach is more convenient than the time-consuming preparation of an individual phosphoramidite or an H-phosphonate for each modification. Chapter 1 – Introduction 17 Nucleoside DNA synthesis DNA synthesis Post-synthetic Pre-synthetic + Nucleoside Oligonucleotide+ Oligonucleotide = Chromophore= Functional group = Complementary Functional group Figure 1.19 Pre- and post-synthetic DNA modifications. Covalent attachment of porphyrin moieties to DNA has been achieved using a variety of methodologies including: the modification of nucleobases,38, 41-44 ribofuranose residues,37, 45-49 phosphate backbone39, 50-52 and using acyclic linkers.53, 54 This provided structures having porphyrin residues as 3′- or 5′-molecular caps,47, 54-56 introducing them instead of a nucleobase in the middle of the helix53 or as a label in the minor39, 46 and major38, 43, 44 grooves. These porphyrins have been attached to DNA using numerous linkers (Figure 1.20) including maleimido-thiol,38, 39 amide,46-49 phosphate50-52 and alkyne bonds,41-44 the latter being incorporated using the Sonogashira reaction. All of the current methods for covalent attachment are obtained through the meso position of the porphyrin. N N N N SO3H SO3H SO3H N O O HN(CH2)2S Zn P O O O O O O N NH O O ON HNN NH N N N N N N N Zn O O O N HN O O C6H13C6H13 C6H13 C6H13 N NH2 ON O O O P O -O O N NH N HN N N OH A B C D Figure 1.20 Examples of porphyrins incorporated into DNA. A) maleimido-thiol linkage,39 B) amide bond,37 C) phosphate attachment51 and D) alkyne bond.42 Chapter 1 – Introduction 18 There are many other routes available for the incorporation of organic chromophores into DNA.40 Of particular interest is the use of CuI catalysed Huisgen 1,3-dipolar cycloaddition reaction between azides and alkynes (Figure 1.21).57 This reaction is so efficient and reliable that the copper catalysed azide–alkyne cycloaddition (CuAAC) reaction is classified as a “click reaction”. There has been a tremendous number of applications for use of this reaction since the recent discovery that azide–alkyne cycloadditions can be catalysed by CuI.58, 59 The CuAAC reaction has been widely used for the attachment of organic chromophores and biomolecules to DNA.60, 61 This is due to the relatively easy ways at which azide and alkyne functional groups can be incorporated into compounds, the inexpensive CuI catalyst (usually obtained from the reduction of stable sources of CuII such as CuSO4) and the stability of the resulting 1,4- regioisomeric triazoles. The CuAAC reaction has not yet been applied for the attachment of porphyrins to DNA, and those reports on CuAAC reactions involving porphyrins hve been published on meso functionalised porphyrins only.62-69 N N N N N N + CuI Figure 1.21 The CuI catalysed azide–alkyne cycloaddition (CuAAC) reaction. 1.7.1 External Modification Covalent attachment can be achieved through internal or external attachment of porphyrins to DNA. The external modification of DNA, i.e. modification at the at the 5ʹ and or 3ʹ ends, has been shown to stabilise duplexes45, 50 and even G-quadruplexes70 by forming a molecular cap. This cap provides thermodynamic stability as a result of stacking with the nearest nucleobases,71, 72 protecting their hydrogen bonds from the aqueous environment.73 Depending on the modifications made to the porphyrin and the position of attachment to the DNA, various levels of stabilisation have been observed. It has been shown that even noncanonical GA base pairs can be stabilised over Watson- Crick base pairing.50 For example, when the tetraaryl porphyrin shown in Figure 1.22 was placed at the 5ʹ end of a d(GA)4 sequence, formation of a duplex consisting of GA base pairs was detected. In contrast, similar positioning in a duplex consisting of GC Chapter 1 – Introduction 19 base pairs resulted in thermal destabilisation. This is a result of the better overlap of the porphyrin with GA pairs than GC pairs. Other uses of a cap include extensive work by Berova and others47, 49, 51 in the development of UV and CD detectors of B to Z transitions in DNA, where different CD exciton couplings were observed between porphyrins at either end of the duplex depending on the DNA conformation. Figure 1.22 A porphyrin molecular cap that stabilises noncanonical DNA (reproduced from Berova et al.).50 Recently, it has been observed that porphyrin to porphyrin interactions were responsible for the aggregation of duplexes possessing porphyrins as a 5ʹ cap (Figure 1.23).54, 56 Head to tail interduplex aggregates were observed at high salt concentrations at low temperature. This was shown by the conversion of a bisignate CD curve in the Soret region to a multisignate curve on increased salt concentration, suggesting porphyrin- porphyrin stacking. The strongest signals were observed in DNA containing ZnII porphyrin but were also recorded for CuII and free base porphyrins. Figure 1.23 Stacking of 5ʹ porphyrin capped DNA by Berova et al.56 A) Duplex representation and CD spectroscopy in the absence of NaCl showing no duplex stacking, B) Duplex representation and CD spectroscopy in 450 mM NaCl and the proposed porphyrin DNA stacking (reproduced and adapted from Berova et al.)56 Chapter 1 – Introduction 20 1.7.2 Internal Modification With exception to some early work by Richert53 and Kool45 where porphyrins were used as base replacements in DNA duplexes, the internal modification of nucleobases with porphyrins is a relatively new field. Prior to 2007, only work by Stulz74, 75 and Sessler76, 77 existed which focused on the development of meso porphyrin modified nucleosides and dinucleotides (Figure 1.24). Figure 1.24 Example of porphyrin modified dinucleotides (reproduced and adapted from Stulz et al.)74 However, it was not until 2007/2008 that the synthesis of a DNA containing the internal modification of meso functionalised porphyrins was reported, placing the porphyrins in the major38, 43, 44 and minor39, 46 grooves of duplexes. This included work by Stulz’s group where up to eleven porphyrins were incorporated into a single strand using a pre- synthetic approach.43 This allowed for contiguous stacking of porphyrins in single stranded oligonucleotide (Figure 1.25A) and within the major groove of duplexes (Figure 1.25B). In addition, porphyrins based on the modification of 2′-deoxy-5- ethynyluridine showed significant thermal destabilisation of the resulting duplexes by 5- 7 °C per porphyrin modification. Chapter 1 – Introduction 21 Figure 1.25 Molecular modelling of a meso linked porphyrin DNA complex by Stulz et al. showing porphyrin stacking in singled stranded (A) and duplex (B) DNA. (Reproduced from Stulz et al.)43 Further work showed a stabilising effect of +0.5 °C per porphyrin modification when the meso linked porphyrins were placed adjacently in complementary strands, forming a zipper in the major groove of the duplex. The placement of ZnII and free base porphyrins in complementary strands allowed for effective energy transfer between a zinc porphyrin and a free-base porphyrin as shown in Figure 1.26.44 Figure 1.26 A porphyrin zipper in the major groove that when formed allowed for energy transfer between porphyrins (reproduced from Stulz et al.)44 Chapter 1 – Introduction 22 1.8 Thesis Objectives As porphyrins are interesting chromophores and DNA is a perfect template for chromophore scaffolding it was thus the aim of this thesis to create and study DNA- porphyrin supramolecular architectures based on β-pyrrolic modified porphyrins. Investigations into these complexes will be separated into two categories of modification: covalent and non-covalent attachment. For the non-covalent approach we aim to synthesise lipophilic porphyrins and use them to create lipophilic porphyrin-DNA complexes. This may provide DNA porphyrin complexes or films with interesting spectroscopic properties. For the covalent attachment we aim to design and construct a versatile approach for the site specific internal modification of oligonucleotides with multiple porphyrins. Both pre- and post-synthetic Sonogashira and CuAAC reactions will be investigated to modify oligonucleotides using pre-synthesised β-pyrrolic porphyrin building blocks. This work will differentiate from other recently published approaches by incorporating β-pyrrolic modified porphyrins in a post-synthetic manner using Sonogashira and CuAAC reactions. We have also focused on internal modification rather than modification of the 3ʹ and 5′ ends. Once oligonucleotides have been constructed we can investigate the effect of porphyrin modifications when incorporated in various positions of single stranded, duplex and triplex DNA. This should provide information for the future application of porphyrin modified ONs. 1.9 Thesis Structure This thesis discusses the development of novel porphyrin-DNA complexes using β- pyrrolic modified porphyrins. Chapter 2 describes the development of synthetic methods for the synthesis of porphyrin derivatives to be used in the creation of porphyrin-DNA supramolecular constructs. This includes the synthesis of lipophilic porphyrins for the non-covalent Chapter 1 – Introduction 23 attachment to DNA as well as porphyrin precursors for the covalent attachment to DNA. Chapter 3 discusses the development of a modified Horner-Emmons reaction as a new synthetic method for creation of alkyne bonds in the β-pyrrolic position of porphyrins. This project allowed us to synthesise porphyrins for covalent attachment to DNA as well a series of novel alkyne linked porphyrinic acids for photovoltaic devices. This work has been published in Tetrahedron Letters (Stephenson, A. W. I.; Wagner, P.; Partridge, A. C.; Jolley, K. W.; Filichev, V. V.; Officer, D. L., Tetrahedron Letters 2008, 49, (39), 5632-5635) . Chapter 4 develops a method for the construction of novel supramolecular DNA- porphyrin complexes using the non-covalent attachment of lipophilic porphyrins. A manuscript discussing this work is under preparation for submission to Tetrahedron Letters. Chapter 5 uses the porphyrin precursors developed in Chapter 2 to create covalently modified oligonucleotides containing a single internal modification of a β-pyrrolic porphyrin using CuAAC and Sonogashira reactions. The effect of porphyrin modification on the thermal stability of the resulting duplexes and triplexes is discussed. A manuscript involving this work has been prepared for submission to Chemistry – A European Journal. Chapter 6 investigates the effect of porphyrin aggregate formation on the thermal stability of porphyrin modified duplexes. A range of duplexes were formed containing up to four porphyrins in a zipper fashion in the minor groove of the duplex. Thermal stability of each duplex was determined by UV-Vis spectroscopy. CD spectroscopy and molecular modelling was used to help to understand the high thermal stabilities observed for these complexes. This work has been published in ChemBioChem (Stephenson, A. W. I.; Bomholt, N.; Partridge, A. C.; Filichev, V. V., ChemBioChem, 2010, 11, (13), 1833-1839). Future work and applications are discussed in Chapter 7 and experimental procedures are included in Chapter 8. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 24 Chapter 2 Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 2.1 Introduction The structure of the porphyrin allows for two main locations for the extension of the molecule, the β-pyrrolic position and the meso position (Figure 2.1). In this thesis we focus on the synthesis of β-pyrrolic functionalised porphyrins as they have advantages over meso functionalisation. Derivatives attached to the meso phenyl ring have been shown to result in the disruption of the conjugation between the porphyrin core and the attached group as a result of the phenyl meso ring sitting orthogonal to the plane of the porphyrin core. This is a result of steric interactions between the adjacent β-pyrrolic hydrogens and meso phenyl ring which twists the phenyl ring in relation to the porphyrin core. In contrast, alkene and alkyne modifications at the β-pyrrolic position result in a system that is in the same plane as the porphyrin core. N NH N HN meso β-pyrrolic Figure 2.1 5,10,15,20-Tetraphenylporphyrin (TPP) showing the β-pyrrolic and meso positions. To allow for β-pyrrolic modification, a functional group needs to be added to the β- pyrrolic position. This is usually achieved via the synthesis of β-pyrrolic formyl and bromo derivatives which act as building blocks for the extension of the porphyrin core. Formylation, such as that in 2-formyl-5,10,15,20-tetraphenylprophyrin, provides a starting material for the synthesis of many derivatives including 2-alkenes via the Wittig reaction78, 79 and 2-alkynes via the Horner-Emmons reaction80 (see Chapter 3). Bromination, as in 2-bromo-5,10,15,20-tetraphenylporphyrin, allows for the synthesis of 2-alkynes via Sonogashira and related reactions. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 25 2-Bromo-5,10,15,20-tetraphenylporphyrin (1) is generally synthesised through the reaction of TPP with NBS in an organic solvent, usually chloroform.81-86 Synthesis of 2- bromo-5,10,15,20-tetraphenylporphyrin is difficult and requires significant care to limit the quantity of the di- and tri-brominated species, and yet still maximise the yield of 2- bromo-5,10,15,20-tetraphenylporphyrin (Figure 2.2). There are a number of publications detailing the synthesis of 1,81-86 however, these results were generally difficult to reproduce. Purification of the 2-bromo-5,10,15,20-tetraphenylporphyrin from the di- and tri-species is difficult due to the similar Rf of the brominated species and the smearing of bands that occurs during column purification on silica gel. As a result only a small quantity of 2-bromo-5,10,15,20-tetraphenylporphyrin can be purified at one time and purities of above 95% are difficult to achieve. Scaling up to multi-gram quantities of 2-bromo-5,10,15,20-tetraphenylporphyrin, which is required for the synthesis of large quantities of porphyrins, is practically difficult to achieve. N NH N HN Ph Ph Ph Ph N NH N HN Ph Ph Ph Ph N NH N HN Ph Ph Ph Ph N NH N HN Ph Ph Ph Ph Br Br Br i) + + 1 Figure 2.2 The synthesis of 2-bromo-5,10,15,20-tetraphenylporphyrin 1. Reagents and conditions: i) CHCl3, NBS, RT. As an alternative to bromination, 2-formyl-5,10,15,20-tetraphenylporphyrin (TPPCHO, 2) provides an excellent building block for β-pyrrolic functionalisation as it is easy to synthesise in high yields and multi-gram quantities without the need for difficult silica gel chromatography. Synthesis of 2 is achieved in three steps from 5,10,15,20- tetraphenylporphyrin (TPP) in high yields (Figure 2.3).87 The aldehyde can be used in many different reactions for the synthesis of alkene and alkyne derivatives. Conversion of TPPCHO in three steps to the phosphonium salt (TPPps, 3) provides a building block for the synthesis of alkenes using Wittig chemistry87 (Figure 2.3). Thus, due to the ease of synthesis, 2-formyl-5,10,15,20-tetraphenylporphyrin was used as a building block for the development of porphyrin compounds in this thesis. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 26 N NH N HN Ph Ph Ph Ph CHO N N N N N Ph Ph Ph PhCu N NH N HN Ph Ph Ph Ph N NH N HN Ph Ph Ph Ph N NH N HN Ph Ph Ph Ph N NH N HN Ph Ph Ph Ph CHOOHCl P+Ph3Cl- 2 3 i) ii) iii) iv)v) vi) + Figure 2.3 The synthesis of TPPps. Reagents and conditions: i) Propionic acid, reflux, 15-20% ii) Cu(OAc)2·H2O, CHCl3, MeOH, reflux, 100% iii) POCl3, DMF, 1,2-DCE, reflux then H2SO4, 90-95% iv) NaBH4, THF, H2O, RT, 85-100% v) SOCl2, pyridine, DCM, 0 °C to RT, 95-100% vi) PPh3, CHCl3, reflux, 80-90%. 2.2 Chapter Summary In this chapter, we have synthesised alkane, alkene and alkyne β-pyrrolic derivatives from 2-formyl-5,10,15,20-tetraphenylporphyrin for the development of DNA-porphyrin supramolecular structures (Figure 2.4). Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 27 DNA-Porphyrin Complex Covalent Attachment Non-Covalent Attachment Lipophilic Porphyrins CuAAC Chemistry Sonogashira Reaction N N N N NPh Ph Ph Ph N O BrNi N N N N Ph Ph Ph Ph R M M = NiII, CuII, ZnII, FeIII R = N3 or CH2N3 N N N N Ph Ph Ph Ph Zn R R = Br, I or C CH Figure 2.4 Synthetic plan for the development of DNA-porphyrin supramolecular structures. The DNA-porphyrin complexes are divided into two groups based on the method of attachment - covalent or non-covalent. For the non-covalent attachment (lipophilic porphyrins) we have created several pyridinium salts of TPP, discussed in section 2.3. The covalent attachment involved the synthesis of precursors for pre- and post-synthetic attachment to DNA. This includes azido porphyrins as starting materials for Huisgen 1,3-dipolar cycloaddition reactions (CuAAC reactions), and ethylene, bromo and iodo porphyrin derivatives as starting materials for Sonogashira chemistry. All compounds were derived from 2-formyl-5,10,15,20-tetraphenylporphyrin for the reasons covered above. All compounds were characterised using 1H NMR, HRMS, UV-Vis and IR spectroscopy, where required, as discussed in Chapter 8 - Experimental. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 28 2.3 Synthesis of Porphyrins for Development of Non- Covalently Attached Lipophilic Porphyrin-DNA Complexes This work focused on the creation of a lipophilic porphyrin that could interact with DNA via electrostatic interactions with the negatively charged phosphate backbone. This is a novel approach that could be used to create DNA that is soluble in organic solutions, aligned DNA films for use in light harvesting devices,88, 89 or fluorescent labelling of DNA. To ensure solubility of the lipophilic complex in organic solvents an aliphatic chain of C10 or greater is generally required. This may not be required for porphyrins due to the intrinsic solubility of porphyrins in organic solvents, however, there appeared to be little advantage to deviate from this length. Three different synthetic routes were investigated for the synthesis of the novel lipophilic porphyrins (Figure 2.5). These routes are classified by the key reactions used: Route A – Wittig reaction, Route B – Schiff base formation and Route C – amide bond formation. Route C was found to be the most successful method for the large scale synthesis of lipophilic porphyrins. Route A also provided lipophilic porphyrins, however, in a lower yield. DNA-porphyrin Complex N N N N Ph Ph Ph Ph N H Ni O N Br N NH N HN Ph Ph Ph Ph N Br N NH N HN HN LINKER N H N Ph Ph Ph Ph Route A Route B Route C Br Figure 2.5 Routes to lipophilic porphyrins. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 29 2.3.1 Route A – Wittig Reaction The Wittig reaction was the key synthetic step to link the aliphatic 11-bromoundecanal (4) with TPPps (3) allowing for the overall synthesis of the lipophilic compound 6 as shown in Figure 2.6. 11-Bromoundecanal (4) was chosen as it was easily synthesised from the inexpensive 11-bromoundecanol, in 83% yield using PCC.90 11- Bromoundecanal must be prepared freshly as it is difficult to prevent the oxidation of the aldehyde to the acid, which occurs in hours if the compound is left open to air. Additionally, the terminal bromine allows for the easy synthesis of pyridinium salts (6). The synthesis of 6 was broken down into three steps; the Wittig reaction, isomerisation and the pyridinium salt formation. N NH N HN P(Ph3)Cl Br O H Wittig reaction N N NH N HN Br N NH N HN N Br Isomerisation 4 3 5 6 PhPh PhPh Ph Ph Ph Ph PhPh Ph Ph Figure 2.6 Synthesis of lipophilic porphyrin 6 via the Wittig reaction and pyridinium salt formation. 2.3.1.1 The Wittig Reaction The first step in the synthesis of lipophilic porphyrins using route A was the Wittig reaction. Wittig reactions on porphyrins are generally carried out using conditions developed by Bonfantini et al.78, 87 This involves the combination of the phosphonium salt in dry solvent (usually chloroform or toluene) with approximately three equivalents of an aldehyde and DBU. This reaction is completed very quickly and in general produces a cis/trans mixture of the desired porphyrin that can be isomerised to the trans Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 30 product with iodine. Using minor adjustments on the above method (DCM, 3.75 eq of 1, 9 eq of DBU, RT, 20 min, Figure 2.7), the reaction of TPPps 3 with aldehyde 4 produced a cis/trans mixture of 5 and a common by-product of the Wittig reaction, 2- methyl-5,10,15,20-tetraphenylporphyrin91 (TPPCH3, 7). This was observed in a ratio of 1:2:5 (7:5cis:5trans) as judged by 1H NMR spectroscopy with an overall reaction yield of 67%. This by-product of the Wittig reaction generally occurs as a result of a competitive reaction when the aldehyde is slow to react with the ylid. TPPCH3 can usually be separated from the desired compound using silica gel or alumina chromographic techniques. Unfortunately, the by-product 7 moved at the same Rf to the cis:trans mixture of 5 in various solvent combinations on silica and alumina media, therefore, could not be separated from the cis/trans mixture of 5. N NH N HN P(Ph3)Cl Br O H N NH N HN 4 3 5trans N NH N HN Ph PhPh Ph Ph Ph Ph Ph Ph Ph Ph Ph N NH N HN CH3 Ph Ph Ph Ph 75cis i) (CH2)10Br (CH2)10Br + + Figure 2.7 Synthesis of compound 5 via the Wittig reaction. Reagents and conditions: i) DCM, DBU, RT, 20 min, 67%. Previous results have shown that by carrying out the reaction in dry refluxing toluene, the production of 7 could be limited and the formation of the trans product favoured.92 When these conditions were tried results showed little change in the cis:trans ratio, however, the production of 7 was reduced but not completely eliminated. Unfortunately, the overall yield of the reaction decreased from 67% to 43% as well. Although pure 5 could not be obtained, the synthesis was continued on the hope that purification would be more achievable after isomerisation and/or pyridinium salt formation. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 31 2.3.1.2 Isomerisation Two options are commonly used for the isomerisation of alkene bonds to the thermodynamically stable trans orientation, stirring with iodine in complete darkness or refluxing for long periods in a high boiling, inert solvent such as toluene. Both methods were attempted on the cis:trans mixture of 5 with varying results. Refluxing the diastereomeric mixture in toluene for 72 hours showed no change in the ratio of cis:trans by 1H NMR spectroscopy, suggesting that the energy required to isomerise the mixture is greater than that supplied in refluxing toluene. The treatment of the cis:trans mixture with three equivalents of I2 at RT in chloroform for three hours successfully isomerised the mixture as could be observed in the 1H NMR spectrum. This resulted in 5trans that still contained 7, but now the ratio was 6:1 (5trans:7). Attempts to purify this mixture using various chromatographic techniques were unsuccessful. 2.3.1.3 Pyridinium Salt Formation The pyridinium salt 6 was formed by refluxing 5trans in neat pyridine for two days followed by silica gel column purification to remove TPP-CH3 7. This allowed for the formation of the lipophilic porphyrin 6 in an overall yield of 19% from TPPps (Figure 2.8). N NH N HN 5trans Ph Ph Ph Ph i) Br N NH N HN Ph Ph Ph Ph N Br 6 Figure 2.8 Synthesis of compound 6. Reagents and conditions: i) pyridine, reflux, 2 days, 74%. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 32 1H NMR spectroscopic analysis in CDCl3 of the obtained pyridinium salt showed what appeared to be a multiple porphyrin mixture in equal molar concentrations. (Figure 2.9B). As an impure compound was not suitable for the construction of lipophilic porphyrin-DNA constructs, alternative methods for the synthesis of lipophilic porphyrins were investigated (see reduction, route B and route C). It was not until different routes were investigated that high resolution ESI mass spectrometry confirmed the presence of the desired compound 6. Further NMR spectroscopic analysis showed that what appeared to be multiple porphyrin products was in fact a pure single compound. When the solvent was changed to d6-DMSO, the two signals appear to coalesce into one (Figure 2.9A). It is unlikely this is a result of aggregation in CDCl3 as the characteristic broad NMR signals are not present. Due to the limited amount of compound no further studies were undertaken to explain the unusual NMR spectrum (i.e. variable temperature NMR studies in CDCl3). Figure 2.9 1H NMR spectrum of the pyridinium salt 6 in CDCl3 (B) and d6-DMSO (A). Note the two sets of signals in CDCl3. 2.3.1.4 Alkene to Alkane Reduction and Pyridinium Salt Formation Because of the initial thoughts that the pyridinium salt 6 may not be pure, reduction of the cis/trans mixture of alkene 5 to an alkane bond (8) was carried out using 10% Pd on carbon under and atmosphere of H2 (Figure 2.10)3 This resulted in an intractable mixture of the reduced material 8 and the starting impurity TPPCH3 (7). Due to the low yield of the reduction reaction (ca. 40%), the ratio of the desired product (8) to TPPCH3 (7) decreased from 6:1 to 1:1 as judged by 1H NMR spectroscopy. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 33 N NH N HN Ph Ph Ph Ph (CH2)10Br 5cis/trans 8 7 7 6:1 1:1 N NH N HN Ph Ph Ph Ph (CH2)10Br i) + Figure 2.10 Reduction of the alkene (5) to an alkane (8). Reagents and conditions: i) 10% palladium on carbon, formic acid, H2, 50°C, 3.5 h then NaOH, ca. 40%. The pyridinium salt 9 was formed in 30% yield by refluxing the product of the Pd catalysed reduction reaction in pyridine for 48 hours. This was followed by purification through silica gel to remove TPPCH3 7 (Figure 2.11), eluting the desired salt in MeOH:DCM (1:4). Interestingly, no changes were observed when comparing 1H NMR spectra in DMSO and CDCl3, which contrasts to the alkene derivative (Figure 2.9). N NH N HN N NH N HN 8 Ph Ph Ph Ph PhPh Ph Ph Br N Br 9 i) Figure 2.11 Synthesis of pyridine salt 9. Reagents and conditions: i) pyridine, reflux, 2 days, 30%. Due to the problems associated with the Wittig chemistry (i.e. the formation of significant quantities of TPPCH3) its use for the large scale synthesis of lipophilic Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 34 porphyrins would not be feasible. Although enough material was prepared for the possible construction of a limited range of porphyrin-DNA constructs, alternative methods were investigated for the large scale synthesis of lipophilic porphyrins. 2.3.2 Route B – Schiff Base Formation Route B was investigated as an alternative method for the construction of lipophilic porphyrins. This involved the synthesis of Schiff base or imine linkages between the porphyrin and the aliphatic chain (Figure 2.12). Schiff bases or imine forming reactions at the β-pyrrolic position of porphyrins have been scarcely reported.93-95 Schiff bases have been prepared in refluxing toluene under Dean-Stark conditions with the use of the Lewis acid catalyst lanthanumII triflate.95 Reduction of the imine to the stable tertiary amine was performed with NaBH4. DNA-porphyrin Complex N N N N Ph Ph Ph Ph N H Ni O N Br N NH N HN Ph Ph Ph Ph N Br N NH N HN HN LINKER N H Br Ph Ph Ph Ph Route A Route B Route C Schiff base formation Figure 2.12 Route B. Unfortunately, lanthanumII triflate was not available therefore acetic acid was used as this is a common catalyst for the formation of Schiff bases. Reactions were initially performed using an ethylenediamine (10) linker. This allowed for the formation of an imine bond with TPPCHO, leaving a free amino group to be reacted with an appropriate aldehyde such as 11-bromoundecanal. Reaction of TPPCHO (2) with 10 eq of Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 35 ethylenediamine (10) in chloroform or THF at 50 °C, using catalytic amounts of acetic acid, showed the production of imine 11 by 1H NMR and MALDI-TOF spectroscopic analysis (Figure 2.13). Purification of imine 11 using silica and alumina columns was unsuccessful as the imine was labile, resulting in the isolation of TPPCHO starting material. However, the imine could be isolated as a 1:1 mixture of imine 11 and TPPCHO by direct precipitation of the reaction mixture from MeOH. The insertion of the NiII ion into the porphyrin core is known to stabilise previously reactive compounds, however, the imine formed from NiTPP-CHO (obtained from the reaction of TPP-CHO and Ni(OAc)2·4H2O) and ethylenediamine showed the same labile properties as the free base. N NH N HN Ph Ph Ph Ph CHO H2N NH2 N NH N HN Ph Ph Ph Ph N NH2 x 122 10 11 N NH N HN Ph Ph Ph Ph HN NH2 i) ii) Figure 2.13 Attempted synthesis of Schiff base 12. Reagents and conditions: i) CHCl3, AcOH, 50 °C, 1 h ii) NaBH4 or NaCNBH3, THF, RT. Reduction of imine 11 to the corresponding amine (12) was attempted using NaBH4 or NaCNBH3 in THF. TLC analysis of the resulting reaction mixtures suggested that no reaction had occurred. This was confirmed by MALDI-TOF analysis where only the starting imine was detected. The inability to reduce the imine was unexpected as literature has shown that the reduction of porphyrin Schiff bases could successfully be performed using NaBH4.93, 95 Attempts to react the unreduced imine (12) with 11- bromoundecanal (3) without catalytic amounts of acid showed no reaction. The addition of acetic acid to the reaction resulted in production of TPPCHO (2, Figure 2.14). Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 36 N NH N HN Ph Ph Ph Ph CHO N NH N HN Ph Ph Ph Ph N NH211 2 H Br O 4 i) Figure 2.14 Failed Schiff base reaction. Reagents and conditions: i) CHCl3, AcOH, 50 °C. Due to the problems outlined above, 1,4-phenylenediamine (13) was used instead of ethylenediamine as it has the potential to produce a more stable imine due to electron delocalisation between the benzene ring and the porphyrin core. NiTPPCHO (14) was reacted with 1,4-phenylenediamine (13) in the presence of acetic acid (Figure 2.15). This showed, by TLC, the production of a band with lower Rf than the starting aldehyde, consistent with the production of imine 15. Attempts to purify the material through neutral alumina resulted in the decomposition to NiTPPCHO, though unlike 11 some of the suspected imine remained. A 1H NMR spectrum of the resulting solid confirmed the existence of NiTPPCHO and another porphyrin in a 1:1 ratio. Although it was suspected that the other compound was the imine due to the presence of appropriate aromatic signals, its presence could not be confirmed by MALDI and ESI mass spectrometry. Attempts to reduce the imine 15 to the corresponding amine using NaBH4 or NaCNBH3 failed to produce any sign of the desired product. N N N N Ph Ph Ph Ph H2N NH2 CHO Ni 13 N N N N Ph Ph Ph Ph Ni N NH214 15 i) Figure 2.15 Attempted synthesis of imine 15. Reagents and conditions: i) THF, AcOH, RT, overnight. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 37 Alternatively, the reaction between 11-bromoundecanal (4) and a large excess of 1,4- phenylenediamine (13) or ethylenediamine (10) showed the production of an uncharacterisable black tar. From these results we can conclude that the use of Schiff base reactions to produce lipophilic porphyrins is not possible using the conditions described. 2.3.3 Route C – Amide Bond Formation Route C was the most successful route for the synthesis of lipophilic porphyrins and allowed for the multi-gram production of the desired compound 16 (Figure 2.16). This involved two major steps: the Wittig reaction to create a stable amino functionalised porphyrin and amide bond formation to add the lipophilic chain. N N N N Ph Ph Ph Ph N H Ni O N Br 16 Wittig reaction Amide bond formation Figure 2.16 Lipophilic porphyrin from route C. 2.3.3.1 Synthesis of Amino Porphyrins β-Pyrrolic modification was chosen over meso functionalisation as a meso derivative would require the synthesis of a new core porphyrin. Instead, the β-pyrrolic derivative can be obtained from TPP. Direct amine formation at the β-pyrrolic position (Figure 2.17) was avoided as previous attempts showed low stability of 2-amino-5,10,15,20- tetraphenylporphyrinato copper (II) (17).96, 97 Instead, the Wittig reaction was used to indirectly attach an amino functionality to the porphyrin. This produced a more stable amine, particularly, when the NiII ion was located in the core of the porphyrin. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 38 N N N N N N N N N N N N Ph Ph Ph Ph Ph Ph Ph Ph Ph Ph Ph Ph NO2 NH2 17 i) ii) Cu Cu Cu Figure 2.17 Synthesis of 2-amino-5,10,15,20-tetraphenylporphyrinato copper (II) from Zhu et al.96 Reagents and conditions: i) CHCl3, Cu(NO3)2, AcOH, Ac2O, RT, 2 h, 30% ii) DCM, MeOH, 10% palladium on carbon, NaBH4, RT, 2 h, 12%. The treatment of a solution of TPPps (3) in dry DCM with 4-nitrobenzaldehyde (18) and DBU (Figure 2.18) resulted in the rapid formation of a cis/trans (1:2.3) mixture of 19, which was converted exclusively to the trans isomer with iodine treatment. Reduction of compound 19 with SnCl2·2H2O and HCl69 to 20 was found to be problematic. Occasionally, after the addition of Et3N to the reaction, a highly polar material was produced rather than the desired amine. ESI mass spectrometry showed an ion with m/z of 950.24 which was consistent with the M+H+ of the SnIV porphyrin 21. It was not possible to obtain a crystal of the polar material, however IR spectroscopy showed signals consistent with N=O stretches at 1519 and 1640 cm-1 and no NH2 signals. Furthermore, UV-Vis and 1H NMR spectroscopy was consistent with a metalled species. Instead the NiII metal analogue of the nitro porphyrin 19 was prepared using Ni(OAc)2·4H2O in CHCl3:MeOH (10:1). NiII was chosen as it is a stable metal ion that is able to be characterised using NMR spectroscopy. Compound 22 was easily converted to amine 23 without the production of any unwanted polar material. This was achieved by stirring 22 in SnCl2·2H2O and HCl for 48 hours at RT to give 23 in 78% yield, along with the starting material 22 (15% yield). As an aside, it was observed that NiII or ZnII insertion into 20 resulted in the decomposition of the amine in the presence of the metal acetate. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 39 N NH N HN Ph Ph Ph Ph PPh3Cl 3 19 NO2OHC N NH N HN Ph Ph Ph Ph NO2 18 N NH N HN Ph Ph Ph Ph NH2 21 N N N N Ph Ph Ph Ph NO2 N N N N Ph Ph Ph Ph NH2 Ni Ni N N N N Ph Ph Ph Ph Sn Cl Cl NO2 Exact Mass: 949.103 Molecular Weight: 949.467 21 20 OR 22 23 i) ii) iii) iv) Figure 2.18 Synthesis of amino functionalised porphyrins. Reagents and conditions: i) DCM, DBU, RT, 30 min then I2, CHCl3, RT, 3 h, 85% ii) THF, SnCl2·2H2O, HCl, RT, 24 h, Et3N, 87% iii) DCM, MeOH, Ni(OAc)2·4H2O, overnight, reflux, 100% iv) THF, SnCl2.2H2O, HCl, RT, 24 h, Et3N, 78%. 2.3.3.2 Amide Bond and Pyridinium Salt Formation Amide coupling between 11-bromoundecanoic acid 25 (synthesised from 11- bromoundecanol (24) as shown in Figure 2.19) and amino porphyrin 23, to give amide 26, was performed using adapted methods.33, 98 This reaction was shown to proceed cleanly in 48 hours using 7.5 eq of EDC, DMAP and acid 25 in THF at RT, or slower over 96 hours using 2.5 eq of each reactant. Purification was achieved via silica gel chromatography to give 26 in 76% yield using a reaction that could be easily scaled up to multi-gram quantities. The pyridinium salt (27) was formed by refluxing 26 in neat pyridine overnight. Traces of pyridine were removed from 27 by storing the compound under high vacuum for five days. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 40 Br O HO BrHO N N N N Ph Ph Ph Ph NH2 Ni 26 N N N N Ph Ph Ph Ph N H Ni O N Br 27 + N N N N Ph Ph Ph Ph N H Ni O Br 23 25 i) ii) iii) 24 Figure 2.19 Synthesis of lipophilic porphyrin 27. Reagents and conditions: i) acetone, H2O, CrO3, H2SO4, 0 °C, 2 h then RT, 12h, 43% ii) THF, EDC, DMAP, RT, 48 h, 76% iii) pyridine, reflux, overnight, 100%. In summary, three routes were investigated to synthesise lipophilic porphyrins for the construction of DNA-porphyrin supramolecular assemblies. These provided three lipophilic porphyrins (Figure 2.20). Two of the porphyrins (27 and 9) were synthesised in a scale large enough to be useful for the development of supramolecular DNA- porphyrin structures, but only compound 27 could be produced in multi-gram quantities. N N N N Ph Ph Ph Ph N H Ni O N Br N NH N HN Ph Ph Ph Ph N Br N NH N HN Ph Ph Ph Ph N Br 27 6 9 Figure 2.20 Summary of lipophilic porphyrins synthesised. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 41 2.4 Synthesis of Porphyrins for the Covalent Attachment to Nucleosides and DNA This work studied the covalent attachment of porphyrins to DNA for the development of supramolecular structures around a DNA scaffold. This required the site-specific covalent labelling of DNA. Various chemical coupling methods have been previously implemented to achieve this as discussed in Chapter 1.7. The covalent attachment of porphyrins to DNA can take place using either pre- or post-synthetic approaches (Figure 2.21). The pre-synthetic approach involves the synthesis of porphyrin containing phosphoramidites or H-phosphonates which are then incorporated into the DNA structure during DNA synthesis. The post-synthetic approach involves the synthesis of an oligonucleotide containing a functional group that the porphyrin moieties can be attached to later on. Pre-synthetic modification has advantages over post-synthetic modification, such as ability to incorporate large numbers of modifications into a single strand using automated DNA synthesis. Significant disadvantages also exist such as the time consuming preparation of phosphoramidites, which, in some cases may not be stable for long periods and may not necessary couple into DNA with the required high efficiencies. Post-synthetic modification avoids the synthesis of complex phosphoramidites and allows for the attachment of the porphyrins to functional groups that can be positioned in a number of locations in the oligonucleotide. To achieve pre- or post-synthetic modification a linker is required between the nucleotides and the porphyrin. We decided to use two linkers that have been used substantially in oligonucleotide chemistry. The 1,2,3-triazole linker, which is formed using CuI catalysed azide alkyne Huisgen 1,3-dipolar cycloaddition (CuAAC), and the alkyne linker formed using Pd0 catalysed Sonogashira reaction. The synthesis of the porphyrins used in these reactions is detailed below. The pre- and post-synthetic coupling reactions will be discussed in Chapter 5 and 6. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 42 DNA-Porphyrin Complex Covalent attachment to DNA Triazoles Alkyne Coupling method Pre- synthetic Post- synthetic Pre- synthetic Post- synthetic CuAAC reaction Sonogashira reaction Figure 2.21 Synthetic outline for the development of DNA-porphyrin supramolecular structures using covalent attachment methods. 2.4.1 Synthesis of Porphyrin for Use in Pre- and Post-synthetic Sonogashira Chemistry The Sonogashira reaction is a Pd0 catalysed reaction between a terminal alkyne and a halide, preferably an iodo derivative. Three β-pyrrolic functionalised porphyrin derivatives were chosen as target compounds (Figure 2.22). The iodo 32, the bromo 40 and the terminal alkyne 35 were synthesised using modified Horner-Emmons chemistry. The implementation of modified Horner-Emmons chemistry to create β-pyrrolic ethynyl modifications in porphyrins is a novel approach involving the reaction of a phosphonate and an aldehyde in basic conditions to give an alkyne. Further details of the modified Horner-Emmons reactions are discussed in depth in Chapter 3. Direct halogenation at the β-pyrrolic position of the porphyrin core was not considered for reasons discussed previously. ZnTPP I ZnTPP 32 35 ZnTPP Br 40 Figure 2.22 Target compounds for use in Sonogashira chemistry. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 43 Compounds 32 and 35 were synthesised from bromophosphonate 30 and TPPCHO (2) as shown in Figure 2.23. The bromophosphonate 30 was synthesised from phosphonate 29 using tetrabutylammonium bromide, DDQ and triphenylphosphine in a 84% yield using the method of Firouzabadi et al.99 Phosphonate 29 was synthesised in a 90% yield using diphenylphosphite and 4-iodobenzaldehyde,100 which in turn was synthesised in three steps from 4-iodobenzoic acid (28) in a 35% yield.101, 102 The iodo functionalised porphyrin 31 was synthesised via a modified Horner-Emmons reaction80 between the bromophosphonate 30 and TPP-CHO (2) in a 75% yield. This reaction could easily be scaled up to allow for the multi-gram production of 31. ZnII was inserted into the porphyrin core (32) in a quantitative yield in order to prevent copper insertion during the subsequent Sonogashira reactions, in which CuI is the co-catalyst. TPP-CHO TPP IZnTPP IZnTPP Si ZnTPP ZnTPP ZnTPP 2 313233 35 34 I P PhO OPh O OH I COOH 29 I P PhO OPh O Br 3028 i-iv) v) vi) vii)viii) ix) Figure 2.23 Synthesis of precursors for the Sonogashira reaction. Reagents and conditions: i) SOCl2, reflux, overnight, 47% ii) CH3CN, LiClO4, NaBH4, RT, overnight, 87% iii) DCM, BIAB, TEMPO, RT, 2 h, 85% iv) diphenylphosphite, MgO, RT, overnight, 90% v) DCM, DDQ, PPh3, nBu4NBr, RT, overnight, 84% vi) THF, t-BuOK, RT, 3 h, 75% vii) CHCl3, MeOH, Zn(OAc)2·2H2O, RT, 1 h, 99% viii) Et3N, trimethylsilylacetylene, Pd(PPh3)4, CuI, reflux 3 h, 93% ix) DCM, THF, TBAF, RT, 5 min, 97%. Conversion of 32 to 33 was achieved in a 93% yield by refluxing a solution of 2-(4ʹ- iodophenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc (II) (33) and trimethylsilylacetylene in Et3N in the presence of 0.3 eq. of Pd(PPh3)4 and 0.5 eq. of CuI under argon overnight. The use of DMF as a solvent resulted in the isolation of the starting material. It was critical to remove trace quantities of copper and palladium salts by washing the trimethylsilyl protected porphyrin 33 repeatedly with a 5% aq Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 44 Na2EDTA solution followed by 3M NH4OH. Failure to do so resulted in the exclusive formation of the Glaser homodimer by-product 34 upon cleavage of the silyl protecting group. After washing, deprotection of 33 to 35 using TBAF was achieved with no sign of the unwanted Glaser homodimer 34. This synthesis provided both a terminal alkyne and iodo derivatives for the use in pre- and post-synthetic Sonogashira coupling methods. Alternatively, the bromo derivative of compound 32 was synthesised as shown in Figure 2.24. The synthesis of 40 was achieved using similar chemistry to that of 32. Phosphonate 36 was synthesised from 4-bromobenzaldehyde and diphenylphosphate using MgO. The required halophosphonate for the modified Horner-Emmons chemistry was synthesised as the chlorophosphonate 37 and the bromophosphonate 38. The chlorophosphonate 37 was prepared in a 22% yield using POCl3/N,N-diethylaniline103 and the bromophosphonate 38 in a 73% yield using milder reaction conditions of tetrabutylammonium bromide, DDQ and triphenylphosphine.99 Phosphonates 37 and 38 were then reacted separately with TPPCHO (2) under modified Horner-Emmons conditions to produce alkyne 39. Yields were moderately higher using the bromophosphate 38 than the chlorophosphonate 37. Reactions from either phosphonate produced an inseparable mixture of 39 and the halovinyl intermediate (see Chapter 3). This could only be avoided by performing the Horner-Emmons reaction in refluxing THF but as a consequence the yield dropped to 15%. ZnII was inserted into porphyrin 39 to prevent Cu insertion during the following Sonogashira reactions. TPP CHOBr P HO PhO OPhO TPP BrZnTPP Br BrOHC Br P X PhO OPhO X = Cl, 37 X = Br, 38 3940 36 i) ii) or iii) iv) v) 2 Figure 2.24 Synthesis of precursor 40 for the Sonogashira reaction. Reagents and conditions: i) diphenolphosphite, MgO, RT, overnight, 90% ii) POCl3, N,N-diethylaniline, 90 °C, 1 h, 22% iii) DCM, DDQ, PPh3, nBu4NBr, RT, overnight, 73% iv) THF, t-BuOK, RT, 45 min, 81% from 37, 40% from 38 v) CHCl3, MeOH, Zn(OAc)2·2H2O, RT, 1.5 h, 92%. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 45 2.4.2 Synthesis of Porphyrin for Use in Pre- and Post-synthetic CuAAC Reactions Huisgen 1,3-dipolar cycloaddition between an azide and a terminal alkyne (CuAAC reaction) as a variety of the “click reaction” and is an invaluable tool for the modification of DNA and other biomolecules.60, 61 CuAAC chemistry has been used to couple a significant number of organic molecules to DNA.104 Prior to this work coupling had not been achieved using porphyrins. It is difficult to synthesise an azido functionalised DNA, however, alkyne nucleosides are commercially available. Therefore a number of novel azido functionalised porphyrins were synthesised for the use in CuAAC reactions. Although few examples of azido porphyrins are present in literature, it was possible to tune existing chemistry and apply it for the synthesis of the desired compounds (Figure 2.25). The azido porphyrins were classed in two categories depending on the type of azide synthesised: aliphatic and aromatic azides. After purification the presence of the azides was confirmed by observing the IR azide asymmetric stretches at ~2015 cm-1 for aromatic azides and 2095 cm-1 for aliphatic azides. N3 MTPP MTPP N3N3MTPP Aromatic Aliphatic Figure 2.25 Target compounds for use in CuAAC reactions (M = H2, NiII, CuII, ZnII, FeIII). 2.4.2.1 Synthesis of Aromatic Azides The aromatic NiII azido porphyrin 41 was obtained in four steps from TPPps 3 and nitro benzaldehyde as shown in Figure 2.26. As discussed previously, the treatment of a DCM solution of 3 with 4-nitrobenzaldehyde and DBU, using a method adapted from Bonfantini et al.,78, 87 resulted in the rapid formation of a cis/trans mixture of 19. This was converted exclusively to the trans isomer by iodine treatment. Metallation of the nitro compound 19 with Ni(OAc)2·4H2O provided the NiII analogue 22 which was reduced to the amine 23 using SnCl2·2H2O and HCl. Azide formation (41) was Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 46 achieved via diazotisation with H2SO4/NaNO2 in the dark followed by the addition of NaN3.68, 69 The formation of the β-functionalised free base aromatic azide 42 was achieved using a similar method to that described for the NiII porphyrin. N NH N HN PPh3Cl TPP NO2 OHC NO2 NiTPP NH2 TPP N3 3 i) v) vi) iii) NiTPP NO2 TPP NH2 NiTPP N3 iv) 19 22 23 20 41 42 ii) 18 Figure 2.26 Synthesis of aromatic azides 41 and 42 for use in CuAAC reactions. Reagents and conditions: i) DCM, DBU, RT, 30 min then I2, CHCl3, RT, 3 h, 85% ii) DCM, MeOH, Ni(OAc)2·4H2O, overnight, reflux, 100% iii) THF, SnCl2.2H2O, HCl, RT, 24 h, Et3N, 78% iv) THF, H2O, NaNO2, H2SO4, RT, 2 h then NaN3, RT, 20 min, 98% v) THF, SnCl2.2H2O, HCl, RT, 24 h, Et3N, 78% vi) THF, H2O, NaNO2, H2SO4, RT, 2 h then NaN3, RT, 20 min, 96%. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 47 The alkyne linked aromatic azide 45 (Figure 2.27) was successfully synthesised after numerous unsuccessful reactions. Attempts to form azide 45 from nitro 44 (via the corresponding aniline derivative) failed as the nitro precursor (44) could not be obtained using Horner-Emmons chemistry. Attempts to synthesise the nitro porphyrin 44 from the appropriate bromophosphonate 43 and TPPCHO (2) using modified Horner- Emmons chemistry resulted in the isolation of an intractable mixture of unknown compounds. Alternatively, it was possible to obtain the azide from the corresponding iodo derivative 32. The synthesis of the azide 45 was achieved in a 61% yield via the reaction of the iodo precursor 32 with NaN3, sodium ascorbate, N,N-DMEA and Cu(ACN)4PF6 in dry DMSO. Alternative reaction conditions involving the bromo (40) or iodo (32) functionalised porphyrins, as listed in Table 2.1, resulted in the quantitative isolation of the starting material. It is interesting that although iodo 32 could be converted to azide 45, reaction using the ethene equivalent of porphyrin 32 under identical reaction conditions only resulted in the isolation of the starting material. TPP CHO NO2 P Br PhO OPhO TPP NO2 TPP NH2 ZnTPP N3 + ZnTPP I X 2 43 45 44i) ii) 32 Figure 2.27 Synthesis of aromatic azide 45 for use in CuAAC reactions. Reagents and conditions: i) THF, t-BuOK, RT, 3 h ii) DMSO, NaN3, sodium ascorbate, Cu(ACN)4PF6, N,N-DMEA, 70 °C, 48 h, 61%. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 48 Table 2.1 Synthetic attempts for the synthesis of azide 45. Starting material Reagents Reaction Conditions Result Bromo 40 NaN3, sodium ascorbate, CuI, N,N-DMEA DMSO, RT, overnight No reaction Bromo 40 NaN3, sodium ascorbate, CuI, N,N-DMEA DMSO, 70 °C, overnight No reaction Iodo 32 NaN3, sodium ascorbate, CuI, N,N-DMEA DMSO, 70 °C, overnight No reaction Bromo 40 NaN3, sodium ascorbate, CuI, N,N-DMEA DMSO: H2O (9:1), microwave, 1 h, 100 °C No reaction Iodo 32 NaN3, sodium ascorbate, CuI, N,N-DMEA DMSO: H2O (9:1), microwave, 1 h, 100 °C No reaction Iodo 32 NaN3, sodium ascorbate, CuI, N,N-DMEA DMSO: H2O (9:1), 70 °C, overnight No reaction Iodo 32 NaN3, sodium ascorbate, CuI, N,N-DMEA Toluene, 70 °C, overnight No reaction Iodo 32 NaN3, L-proline, CuI, NaOH DMSO, 70 °C, overnight No reaction Iodo 32 nBuLi, tosyl azide THF, -78 °C to RT Unknown products Iodo 32 NaN3, sodium ascorbate, Cu(ACN)4PF6, N,N-DMEA DMSO, 70 °C, 48 h 61 % 2.4.2.2 Synthesis of Aliphatic Azides The desired aliphatic azides (Figure 2.28) were synthesised from aldehyde 47 and TPPps 3 in two steps via the Wittig reaction followed by metal insertion. Aldehyde 47 was obtained from para-tolunitrile in three steps using methods of Schlenoff et al.105 and Barbe et al.106 The aliphatic azide 48 was synthesised in an overall yield of 60% via a Wittig reaction between phosphonium salt 3 and aldehyde 47 followed by iodine isomerisation. The Wittig reaction must be performed using the azido aldehyde 47 as the bromo aldehyde 46 was found to self polymerise in the presence of DBU. Importantly, the aliphatic azide 48 was stable to the conditions used for the insertion of various metal ions (ZnII (49), NiII (50), and FeIII (51)). PtII insertion, which requires refluxing in benzonitrile, could not be obtained as decomposition of the azide occurred at the elevated temperatures. Chapter 2 – Synthesis of β-Pyrrolic Porphyrin Derivatives for DNA Modification 49 TPP N3 NiTPP N3 OHC N3 ZnTPP N3 N NH N HN PPh3Cl OHC Br NC Br NC i) Polymer FeClTPP N3 46 47 3 48 49 50 51 ii) iii) iv) 46 47 v) vi) vii) viii) Figure 2.28 Synthesis of aliphatic azides 48-51 for use in CuAAC reactions. Reagents and conditions: i) CCl4, NBS, light, reflux, 2 h, 62% ii) toluene, DIBAL, 0 °C, 1 h then CHCl3, HCl, RT, 1 h, 72% iii) Acetone, H2O, NaN3, reflux, overnight, 100% iv) DCM, DBU, RT, 20 min v) DCM, DBU, RT, 20 min then CHCl3, I2, RT, overnight, 60% vi) DCM, MeOH, Zn(OAc)2·2H2O, RT, 1 h, 97% vii) CHCl3, MeOH, Ni(OAc)2·4H2O, reflux, overnight, 99% viii) acetonitrile, CHCl3, FeCl2·4H2O, 70 °C, 5 h, then air, 70 °C, overnight, 90%. 2.5 Conclusion In summary, we have synthesised a number of β-pyrrolic functionalised porphyrin precursors for the development of DNA-porphyrin supramolecular constructs. This includes the synthesis of lipophilic porphyrins for the construction of lipophilic DNAs, as well as building blocks required for pre- and post-synthetic Sonogashira and CuAAC reactions. The construction of these complexes will be discussed in Chapters 4-6. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 50 Chapter 3 Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives via the Modified Horner- Emmons Reaction 3.1 Introduction An ethynyl bond is an important linkage for the development of chromophores for use in light harvesting devices88, 89, 107 and supramolecular DNA-porphyrin systems. Molecular modelling calculations have shown that enhanced communication exists in β- pyrrolic ethynyl porphyrins over similar ethenyl compounds.2 It has also been observed that a triple-bonded β-pyrrolic substituent stabilises the porphyrin ring toward oxidation more effectively than the equivalent double-bond systems, suggesting possible advantages of ethynyl linkages in light harvesting devices.108 The synthesis of porphyrins containing ethynyl substituents at the β-pyrrolic position is typically achieved via a Sonogashira108-110 or other metal catalysed coupling reactions from the corresponding 2-bromoporphyrins.81-86 However, Sonogashira coupling reactions on 2-bromo-5,10,15,20-tetraphenylporphyrin are relatively low yielding and restricted to small scales. 76, 7781, 109, 111-115 Sonogashira couplings also require the use of a copper co-catalyst which has the potential to metalate/transmetalate the porphyrin.116 Alternative copper free coupling has been performed using Pd catalysts81 and ligands such as AsPh3.81, 109, 117 However, the restriction imposed by the expense and toxicity of the reagents, together with the harsh reaction conditions (high reaction temperatures for extended times), limits the application of this approach in possible scaled-up reactions. Due to the importance of the ethynyl linkage we investigated a new method for the creation of β-pyrrolic ethynyl bonds in porphyrins. As an alternative, a modified Horner-Emmons (or Horner-Wadsworth-Emmons) reaction (Figure 3.1) was applied to porphyrins for the construction of β-pyrrolic ethynyl linkages. The Horner-Emmons reaction is used for the synthesis of alkenes from an aldehyde and a phosphonate in the presence of a base. Modifying the phosphonate to introduce a Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 51 leaving group enables the synthesis of ethynyl linkages as shown in Figure 3.2. This modified Horner-Emmons reaction negates the need for a halogenated porphyrin and a metal catalyst. Target compounds could be produced in high yielding, scalable reactions from the readily synthesised 2-formyl-5,10,15,20-tetraphenylporphyrin (TPPCHO, 2). P R1 RO RO O O R2 H H P O R1 RO RO HH Base P R1 RO RO H O O H R2 P RO RO O O HR1 R2H R1 R2 P RO RO O O Figure 3.1 The mechanism of the Horner-Emmons reaction for the synthesis of alkenes. P RO RO O O LG R1 R2 H R1 R2 P RO RO O O P O R1 RO RO H LG P R1 RO RO LG O O H R2 P R1 RO RO O O R2 LG H H LG Base R1 R2 Base Figure 3.2 The mechanism of the modified Horner-Emmons reaction for the synthesis of alkynes. The synthesis of β-pyrrolic substituted ethynyl porphyrins also gave us an opportunity to construct and investigate the performance of novel β-pyrrolic functionalised ethynyl Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 52 acid derivatives as light harvesting molecules in a dye sensitised solar cell (DSSC) device. Dye sensitised solar cells are a relatively new class of low-cost photovoltaic devices that have been the focus of research here at Massey University and elsewhere for more than two decades. Porphyrins have shown promise as dyes in DSSCs.88, 89, 107 DSSCs, most commonly known as Grӓtzel cells118 after its inventor, consists of a layered structure comprising of (Figure 3.3): • A conducting ITO covered glass electrode, • A wide band gap semi-conductor, most commonly nanocrystalline anatase TiO2, • A monolayer of dye (porphyrin), • A redox active electrolyte (usually I-/I3- couple) and • A counter ITO covered glass electrode with catalytic Pt coating. Photo-excitation of the dye on the surface of the TiO2 results in the injection of an electron from the dye into the conduction band of the TiO2, which then perculates to the ITO electrode of the glass. The captured electrons can then be used by a device or stored. The electrons then flow back to the counter electrode where they are picked up via an I-/I3- redox couple in the electrolyte. The redox couple then returns the charged dye back to the neutral state, allowing the whole process to repeat upon further excitation. Figure 3.3 Schematic representation of a Grӓtzel cell (reproduced from Campbell).3 Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 53 Previous research has found that three important aspects of the porphyrin dye affect the performance of the dye in light harvesting Grӓtzel cells: the chromophore, the linker and the binding group (Figure 3.4).88 Previous studies have extensively tested a number of β-pyrrolic porphyrins containing a variety of conjugated and non conjugated linkers.10, 88, 89, 119-121 None of these early studies explored the ethynyl linker in these molecules. As we have a route to the synthesis of ethynyl derivatives and the ability to test them it was decided to synthesise and to explore their efficiencies in a DSSC. Comparisons can then be made to the previously studied ethane and ethene linker derivatives. N N N N Zn Chromphore BG = BG Linker Linker = COOH HOOC COOH NC COOH Figure 3.4 β-Pyrrolic substituted TPP showing various linkers and binding groups (BG) used for DSSCs. 3.2 Chapter Summary This chapter discusses the development and limitations of modified Horner-Emmons chemistry for the synthesis of a number of novel β-pyrrolic substituted ethynyl porphyrins. From these we have synthesised several carboxylic acids dyes for use in light harvesting DSSC devices. Spectroscopic properties and application of these molecules in DSSC were investigated and discussed. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 54 3.3 Synthesis of β-Pyrrolic Ethynyl Porphyrins 3.3.1 Phosphonate Synthesis To synthesise the desired alkynes the modified Horner-Emmons reaction requires an aldehyde and a phosphonate containing an appropriate leaving group. We chose to use 2-formyl-5,10,15,20-tetraphenylporphyrin (TPPCHO, 2) and an aromatic phosphate. There are many synthetic routes for the formation of phosphonates from the corresponding aldehydes. Phosphonates 29, 36 and 52-58 (Figure 3.5 and Table 3.1) were obtained from the corresponding aromatic aldehydes using diphenolphosphite and MgO in high yields.100, 122 Aldehydes required were either purchased or synthesised as in the case of 4-iodobenzaldehyde101, 102 and 4-(5,5-dimethyl-2-phenyl-1,3- dioxane)benzaldehyde. To prevent the reaction stopping at the production of the alkene the phosphonates must contain an appropriate leaving group. Previous work has shown the effective use of leaving group such as halogens,122, 123 benzotriazoles and methylsulfonic acids.100 Bromo and chloro leaving groups were selected as they are easy to prepare (Figure 3.5). Halophosphonates were either synthesised as the chlorophosphonates (37, 59-63 and 66, Table 3.1) with phosphoroxychloride and N,N-diethylaniline103 or as bromophosphonates (30, 38, and 64-65) using tetrabutylammonium bromide, DDQ and triphenylphosphine99 (Table 3.1). Halophosphonates were easily purified via silica gel column chromatography or recrystallisation. Although the halophosphonates were present in an mixture of isomers this did not effect the Horner-Emmons reaction. Chlorophosphonates were obtained in a lower yield than the bromophosphonates due to the harsh acidic conditions involved in chlorination. Similarly, chlorination could not be used where the aromatic substituent was acid sensitive such as in 63 where the 1,3- dioxane ring was hydrolysed in acidic conditions. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 55 Ar X P O PhO PhO OHC Ar Ar HO P O PhO PhO i) ii) 29, 36, 52-58 30, 37-38, 59-66 X = Cl or Br Figure 3.5 Synthesis of chloro and bromophosphonates from the appropriate aldehydes. Reagents and conditions: i) diphenylphosphite, MgO, RT, overnight ii) POCl3, N,N-diethylaniline, 90 °C, 1 h or DCM, DDQ, PPh3, nBu4NBr, RT, overnight. Table 3.1 Phosphonates synthesised and their yields. Ar X (compound) Yield (%) X (compound) Yield (%) 4-Iodophenyl OH (29) 84 Br (30) 84 4-Bromophenyl OH (36) 90 Cl (37) 22 Br (38) 84 Phenyl OH (52) 73 Cl (59) 19 4-Methoxyphenyl OH (53) 90 Cl (60) 25 4-Methoxycarbonylphenyl OH (54) 82 Cl (61) 17 4-Pyridyl OH (55) 40 Cl (62) 45 OH (56) 88 Cl (63) 0 4-(5,5-Dimethyl-2-phenyl- 1,3-dioxane) Br (64) 64 4-Benzonitrile OH (57) 69 Br (65) 69 4-Nitrophenyl OH (58) 84 Cl (66) 35 3.3.2 Modified Horner-Emmons Reaction Typically a Horner-Emmons reaction uses equimolar amounts of reactants and two equivalents of a strong base to convert the aldehyde and phosphonate to an ethene derivative. It was found that under these conditions the synthesis of ethynyl- functionalised porphyrins generally (exclusion of pyridine derivative 70 – refer to Table 3.2) resulted in the isolation of the starting material as well as an inseparable mixture of the halovinyl intermediate (31a, 39a, 67a-72a) and the desired product (31, 39, 67-72) (Figure 3.6). Unfortunately, the halovinyl intermediate could not be separated as it had an identical Rf to the ethynyl product on silica gel or alumina columns in various solvents. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 56 N NH N HN Ph Ph Ph Ph CHO ArPPhO OPhO X N NH N HN Ph Ph Ph Ph ArX N NH N HN Ph Ph Ph Ph Ar Halovinyl intermediate + i) 2 30, 37-38, 59-66 31, 39, 67-72 + 31a, 39a, 67a-72a Figure 3.6 The modified Horner-Emmons reaction showing the formation of the ethynyl linkage and the undesired halovinyl intermediate (X = Br or Cl). Regents and conditions: i) THF, t-BuOK, RT, overnight. Conformation of the structure of the halovinyl intermediate was achieved by X-ray crystallography (Figure 3.7). A single crystal of the halovinyl intermediate 39a was obtained by the slow diffusion of MeOH into a mixture of halovinyl intermediate 39a and ethynyl 39 in DCM. Figure 3.7 The molecular structure of the halovinyl intermediate 39a as defined by defined by X-ray crystallography. The removal of the halovinyl intermediate was overcome in moderate to high yields (Table 3.2) by significantly reducing the reaction time from overnight to 1.5 hours and dramatically increasing the concentration of a base to a 10% solution of t-BuOK (ca. 80 eq). This resulted in the formation of only trace amounts of the halovinyl intermediate as confirmed by the NH signal at -2.6 ppm in 1H NMR spectra. Further treatment of the isolated products containing the halovinyl intermediate with t-BuOK in THF resulted in spectroscopically pure materials for all compounds excluding the bromo porphyrin 39. Pure compound 39 could only be obtained in a very low yield by performing the reaction in refluxing THF. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 57 Table 3.2 β-Pyrrolic alkyne porphyrins 31, 39 and 67-72 and their yields. Porphyrin Ar Phosphonate (X) Yield (%) 31 4-Iodophenyl 30 (Br) 75 39 4-Bromophenyl 37 (Cl) 80 38 (Br) 35 67 Phenyl 59 (Cl) 54 68 4-Methoxyphenyl 60 (Cl) 84 69 4-Methoxycarbonylphenyl 61 (Cl) 0 70 4-Pyridyl 62 (Cl) 70 71 4-(5,5-Dimethyl-2-phenyl- [1,3]dioxane) 64 (Br) 88 72 4-Nitrophenyl 66 (Cl) 0 Although the modified Horner-Emmons reaction provided a means of attaching a range of ethynyl-linked aromatic substituents, it was found to be restricted to aryl substituents that did not react with the t-BuOK. For example, the reaction of TPPCHO with diphenyl chloro(4-methoxycarbonylphenyl)methylphosphonate (61) or diphenyl chloro(4- nitrophenyl)methylphosphonate (66) failed to produce the desired products (69 or 72) but instead gave an intractable mixture of multiple products. Additionally, in the case of the cyano bromophosphonate 65 (Figure 3.8) the reaction afforded a readily separated mixture of the desired product 73 and the unexpected 2-(4′-benzamide)ethynyl- 5,10,15,20-tetraphenylporphyrin 74. Although not a common method, the use of base for the hydrolysis of nitriles to amides has been previously reported.124-126 + +N NH N HN Ph Ph Ph Ph CHO PPhO OPhO Br TPP CN CN TPP O NH2 74 73 2 65 i) Figure 3.8 Synthesis of porphyrins 73 and 74. Reagents and conditions: i) THF, t-BuOK, RT, 3 h, 36% for 73, 26% for 74. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 58 Metallation of ethynyl porphyrins 31, 39, 67-68 and 70-71 was performed using Zn(OAc)2·2H2O to obtain ZnII porphyrins 32, 40, 75-78. These compounds were required for DFT studies in collaboration with Otago University. Although results are not included in this thesis, a manuscript is under preparation11 and results will also be published in the PhD thesis of John Earles.127 3.3.3 Synthesis of β-Pyrrolic Ethynyl Dyes for Dye Sensitised Solar Cells Porphyrins are strong chromophores and much effort has been put into their study as light harvesting complexes. Many different dyes, linkers and binding groups have been investigated for the use in DSSC devices. Currently no work has been published on the use of β-pyrrolic functionalised ethynyl porphyrin acids in DSSC applications. This is mainly due to the inherent problems associated with the synthesis of such compounds. The development of the modified Horner-Emmons chemistry in porphyrins has allowed us to easily synthesise the required acid derivatives and study their properties in light harvesting devices. Three commonly used acids for DSSCs - benzoic acid, malonic acid and cyanoacetic acid - were synthesised from the protected aldehyde 71 for the use in DSSCs. Synthesis of the benzoic acid was achieved in five steps from the protected aldehyde in an overall yield of 51% (Figure 3.9). The protecting group was cleaved using trifluroacetic acid in DCM:H2O at room temperature to give the aldehyde 79. The protected (71) and unprotected (79) aldehydes had the same Rf on silica TLC, however, visualisation of the unprotected aldehyde was possible as it turned brown in the presence of 2,4-dinitrophenylhydrazine. ZnII was inserted using Zn(OAc)2·2H2O in CHCl3/MeOH to give 80 which was further oxidised to the methyl ester 81 using a procedure described by Campbell.3 Porphyrin 80 was dissolved in a mixture of THF:MeOH and stirred in the presence of NaCN followed by oxidation with activated MnO2. This reaction is assumed to proceed through the cyanohydrin which is oxidised to an α-keto nitrile (Figure 3.10). The α-keto nitrile is in turn converted to the methyl ester by MnO2 in methanol. The product of the esterification still contained approximately 5% of the starting aldehyde judged by 1H NMR spectroscopy. This was removed by silica gel column purification after hydrolysis of the methyl ester to the benzoic acid 82 using KOH. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 59 ZnTPP CHO TPP CHO TPP O O ZnTPP COOMe ZnTPP COOH 71 79 80 81 82 i) ii) iii) iv) Figure 3.9 Synthesis of benzoic acid 82 for use in DSSCs. Reagents and conditions: i) DCM, TFA, H2O, RT, 1 h, 81% ii) CHCl3, MeOH, Zn(OAc)2·2H2O, RT, 1 h, 99%, iii) THF, MeOH, NaCN, RT, 30 min then MnO2, reflux 18 h, 88% iv) THF, MeOH, H2O, KOH, reflux, 15 h, 73%. ZnTPP CHO 81 ZnTPP H CN OH ZnTPP NC O ZnTPP MeO O NaCN MeOH MnO2 MeOH cyanohydrin α-keto nitrile80 Figure 3.10 Proposed sequence for the conversion of the aldehyde 80 into the methyl ester 81. The cyanoacetic acid porphyrin derivative 83 was produced in a 97% yield from the aldehyde 80. This was achieved using cyanoacetic acid and ammonium acetate in a mixture of acetic acid and THF (Figure 3.11).128 80 ZnTPP CHO ZnTPP CN COOH 83 i) Figure 3.11 Synthesis of the cyanoacetic acid 83. Reagents and conditions: i) THF, acetic acid, cyanoacetic acid, ammonium acetate, 60 °C, 5 h, 97%. The malonic acid 84 shown in Figure 3.12 was problematic to synthesise. Porphyrin malonic acid derivatives are generally synthesised at 70 °C using 6 eq of malonic acid and ammonium acetate in THF:acetic acid.120 Using these general conditions the desired Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 60 malonic 84 and the decarboxylated acid 85 were produced in a 10:90 ratio as determined by the ratio of the aromatic hydrogens to the adjacent β-pyrrolic hydrogens in the 1H NMR spectrum. It was possible to observe the production of the malonic acid and the decarboxylated material by silica TLC, however due to the high polarity of the malonic acid, separation via silica gel column chromatography was not possible. Decarboxylation, which is undesirable as it provides an impure compound for DSSC testing, usually results from over heating the reaction mixture. It was possible to avoid the production of the decarboxylated product by performing the reaction at 40 °C with 20 eq of malonic acid and ammonium acetate. This reaction would not go to completion resulting in the isolation of the starting aldehyde (80) and the malonic acid (84) in a 40:60 ratio. By increasing the equivalents of the acid and the ammonium acetate to 40 eq we were able to push the reaction to completion but unfortunately both the malonic acid and the decarboxylated acid were obtained in a 70:30 ratio. i) ZnTPP CHO ZnTPP COOH COOH ZnTPP H COOH 80 84 85 + Figure 3.12 Synthesis of malonic acid 84 showing the production of the decarboxylated product 85. Reagents and conditions: THF, acetic acid, malonic acid, ammonium acetate, 40 °C, 4 h, 96%. 3.4 UV-Vis Spectroscopy and DSSC Testing The synthesis of the new ethynyl modified porphyrins provided us the opportunity to investigate their spectroscopic and photovoltaic properties in DSSCs. Comparisons could be made to the previously synthesised and studied alkane and alkene porphyrin derivatives. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 61 3.4.1 UV-Vis Absorption Spectroscopy UV-Vis spectroscopy gave us an insight into the relative energy levels of the new ethynyl compounds. Figure 3.13 showed that both the Soret and the two Q bands of the ethynyl benzoic acid 82 were red shifted 1.5, 2.5 and 5.5 nm, respectively, relative to the equivalent ethene compound 87. Shifts of 13.0, 12.5 and 14.5 nm were observed compared to ethane compound 86.88 This suggested that there was increased conjugation, or more precisely an increased electron withdrawing effect,129 between the aromatic ring and the porphyrin core in the ethynyl derivative. This is consistent with observations in other β-alkyne porphyrins.109, 130 N N N N Ph Ph Ph Ph COOH Zn N N N N Ph Ph Ph PhZn N N N N Ph Ph Ph PhZn COOH COOH 8286 87 0 100 200 300 400 400 450 500 550 600 650 λ/nm ε (c m 2 m m ol -1 )/ 10 00 0 5 10 15 20 500 550 600 650 Figure 3.13 UV-Vis spectra of the ethynyl, alkene and alkane porphyrinic acids in DMF at 25 ˚C. Ethynyl 82 (thinner line), alkene 87 (thicker line) and alkane 86 (dotted line). Inserted is an expansion of the Q-band region. 3.4.2 X-ray Crystallography It was not possible to obtain a single crystal of the acid 82, but a crystal of aldehyde 80 suitable for X-ray diffraction was obtained by slow diffusion of methanol into a solution Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 62 of compound 80 in DCM (Figure 3.14). This could be compared to the crystal structure of the previously published CuII ethene derivative 88 (Figure 3.15).78, 131 Figure 3.14 Crystal structure of 2-(4ʹ-formyl)phenylethynyl-5,10,15,20-tetraphenylporphyrinato zinc (II) 80 with methanol coordinated to the zinc. The angle between the benzene ring and the plane of best fit made by the porphyrin ring is 31.98(16) degrees (phenyl rings omitted for clarity). The thermal ellipsoids were set at 50% probability level. The hydrogen atoms are drawn as spheres of arbitrary radii. Figure 3.15 Crystal structure of 4-{trans-2′-[2′′-(5′′,10′′,15′′,20′′-tetraphenylporphyrinato copper (II) yl) ethen-1′-yl]}-1-benzaldehyde 88 taken from Bonfantini et al.78 It has been shown that the extent of conjugation between the porphyrin and the β- pyrrolic substituent increases as the dihedral angle between the two moieties becomes more planar.132 Thus, it would be expected from the UV-Vis spectroscopy that a more planar structure would exist in the ethynyl linkage compared to the alkene linkage. Against expectations, the dihedral angle between the plane of the benzene ring and the Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 63 plane of best fit made by the porphyrin core was 31.98(16) degrees, and almost twice that of the corresponding angle in 88 of 17(2) degrees. Interestingly, the increase in planarity that is suggested in the UV-Vis data is not supported by the crystal structure of compound 80. Thus, it is difficult to draw conclusions relating to the relevant extent of the electronic communication in ethene and ethynyl derivatives. It is possible that this angular deformity may be a result of crystal packing forces. 3.4.3 Dye Sensitised Solar Cells (DSSCs) The synthesised benzoic (82), cyanoacetic (83), and malonic acids (84/85) were tested under conditions as described by Campbell3 and compared to the previously synthesised alkene and alkane acids 86, 87, and 89 (Figures 3.13 and 3.16). N N N N N N N N HOOC COOH HOOC H ZnZn 70% 30% N N N N HOOC CN Zn N N N N HOOC CN Zn 83 89 84/85 Figure 3.16 Malonic acid and cyanoacetic acid dyes tested in DSSCs. Solutions of these porphyrin acids were prepared in THF (0.2 mM, BHT stabilised). Porphyrins were then absorbed onto sintered TiO2 glass electrodes by soaking overnight Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 64 at RT in darkness. The TiO2 electrodes with bound porphyrins were removed from the dye solutions, rinsed, dried under high vacuum and tested immediately. The cells were assembled and tested in an open unsealed cell holder shown in Figure 3.17. Introduction of an I-/I3- redox electrolyte via capillary action between the working electrode and the counter electrode completed the working cell. Four identical cells for each acid were irradiated under the equal intensity of 1 sun and the average values calculated (Table 3.3). The efficiencies (η) presented using an unsealed cell have been shown to be approximately half of those expected in a sealed optimal cell.121 Figure 3.17 DSSC cell holder containing a TiO2 cell with bound porphyrin. Insert is a photograph of TiO2 cells with bound acids 86, 87 and 82 respectively. Table 3.3 Results of DSSC testing. Compound η (%) FF Voc (V) Jsc (mAcm-2) 86 1.29 (1) 0.55 0.555 4.20 87 2.12 (2) 0.52 0.583 6.97 82 1.86 (1) 0.50 0.577 6.39 89 2.65 (3) 0.52 0.611 8.41 83 2.80 (1) 0.53 0.624 8.58 84/85 2.64 (4) 0.57 0.665 7.09 η = cell efficiency (1 standard deviation), FF = fill factor, Voc = open circuit voltage, Jsc = short circuit current. Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 65 The DSSC results can be separated into porphyrins possessing benzoic acids (86, 87 and 82) and those containing cyanoacetic/malonic acids (89, 83 and 84/85). The alkene linkage in compound 87 was found to be the best performing benzoic acid dye followed by the alkyne 82 and alkane 86 respectively, as shown by the higher cell efficiency (η). As can be seen in Table 3.3, the results show that dyes with cyanoacetic and malonic acid binding groups have higher efficiencies than benzoic acids. This is consistent with previously published porphyrinic acids using these binding groups.89 The ethynyl cyanoacetic porphyrin 83 showed higher efficiencies than the alkene variant 89. The malonic acid showed similar efficiency to that of the double bond cyanoacetic acid but it is difficult to compare these as the malonic acid was not pure. It is plausible that variations in cell efficiencies could be the result of differences in dye loading on the TiO2 surface, rather than due to the real efficiency of the chromophore. To investigate this we determined the dye loading of the three benzoic acids dyes (86, 87 and 82) on the TiO2 surface using UV-Vis spectroscopy (see experimental methods section for details). Results showed that varying the linker did not substantially affect the surface coverage of the dye on TiO2 (Table 3.4). It can therefore be assumed that the trends in DSSC efficiency are not a direct result of different dye loading on the surface. Table 3.4 Dye loading on TiO2 Compound Moles absorbed Mass absorbed (g) Dye loading (mol/g of TiO2) 86 2.2 × 10-7 1.8 × 10-4 5.3 × 10-5 87 2.3 × 10-7 1.9 × 10-4 5.5 × 10-5 82 2.3 × 10-7 1.9 × 10-4 5.6 × 10-5 To provide further insight into the acids used in DSSCs a DFT study is being undertaken in collaboration with Otago University (not discussed in the current thesis).11, 127 3.5 Conclusion The use of modified Horner-Emmons conditions has provided a versatile method for the synthesis of ethyne linked substituents at the β-pyrrolic position of TPP. This high Chapter 3 – Synthesis of β-Pyrrolic Ethynyl Porphyrin Derivatives 66 yielding, scalable methodology negated the need of a metal catalyst and did not require the preparation of 2-bromo-5,10,15,20-tetraphenylporphyrin as the starting material. The X-ray crystallography data showed that the dihedral angle between the plane of the porphyrin core and the plane of the aryl substituent was greater in the ethynyl linked molecule compared to an ethylene analogue. Electronic properties indicated a bathochromic shift of the ethynyl linked porphyrin compared to the corresponding ethylene analogue which is in contrast to the information gained in the crystal structure. A number of porphyrin benzoic, cyanoacetic and malonic acids were synthesised and evaluated in DSSCs. Results showed that the obtained ethynyl dyes were effective in DSSCs, however no significant improvement over similar alkene acids was observed. Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 67 Chapter 4 Construction of Lipophilic Porphyrin-DNA Complexes 4.1 Introduction A variation to general TmPyP cationic porphyrin used for the non-covalent attachment to DNA16 is the production of lipophilic DNA complexes. DNA is normally soluble in aqueous solution, however by exchanging the Na+ or Li+ ions along the phosphate backbone for quaternary ammonium salts possessing a long lipophilic chain (Figure 1.15) it is possible to create an organically soluble complex.31, 32 These complexes can be simply prepared by mixing of salmon testes or calf thymus DNA (ca. 1.3 × 106 Da, 2000 bp) and a lipophilic salt in an aqueous environment. A precipitate is formed which on filtration and drying can be resolubilised in an organic solvent. Slow evaporation of the organic solvent has been shown to produce lipophilic DNA films with aligned DNA strands.133, 134 These have shown promise in the production of batteries,33 liquid crystals,34 drug release systems35 and photodynamic devices.36 Although many materials have been incorporated into DNA-lipid complexes, the incorporation of lipophilic porphyrins is a novel approach. If successful it may allow for the simple construction of porphyrin based arrays for use in nanotechnological applications such as light harvesting, fluorescent labelling or low-power photon upconversion. Base O O O P OO N CH3 H3C CH3 O H3C(CH2)9 4 N NHO O O O N N O O BA Figure 4.1 A) An illustration of one of the first lipophilic-DNA complexes31 B) A TEMPO lipophilic salt used in the construction of DNA batteries.33 Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 68 Lipophilic porphyrins were synthesised as discussed in Chapter 2. Three porphyrins were synthesised for the use in lipophilic arrays, compounds 6, 9 and 27 (Figure 4.2). Due to the problems associated with the synthesis of compound 6, not enough material was available for the construction of DNA-porphyrin complexes. Complexes were instead made from the more abundant porphyrins 27 and 9. Along with porphyrin 9 and 27 we created lipophilic DNA using cetyl trimethylammonium bromide (CTAB). N Br N N N N Ph Ph Ph Ph N H Ni O N Br N NH N HN Ph Ph Ph Ph N BrN NH N HN Ph Ph Ph Ph N Br 27 6 9 CTAB Figure 4.2 Lipophilic compounds used to create DNA-porphyrin complexes. 4.2 Chapter Summary This chapter discusses the synthesis of novel porphyrin-DNA supramolecular assemblies around single and double stranded DNA as well as guanosine quadruplexes (GQ). Loading rates and solubility of such compounds were investigated. Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 69 4.3 Porphyrin Solubility A critical feature of the lipophilic porphyrins is that they must be soluble in a solvent that is miscible with water, therefore allowing the lipophilic porphyrin to be properly mixed with the oligonucleotide. The porphyrin-DNA complexes were constructed by the addition of a solution containing porphyrin 9 or 27 to an oligonucleotide in water. Therefore the porphyrins must remain soluble on dilution with water. Failure to do so will result in the porphyrin rather than the porphyrin-DNA complex precipitating from solution. Solubility of 9 and 27 was investigated in water miscible solvents as shown in Table 4.1. Solubility tests were performed at room temperature on 1 mg of porphyrin and solubility properties were identical for both porphyrins. Table 4.1 Solubility at RT of 1 mg of porphyrin 9 or 27. Solvent Solubility H2O Insoluble THF Soluble in 20 µL, remained soluble on dilution with water to 2 mL. DMSO Soluble in 20 µL, remained soluble on dilution with water to 2 mL. DMF Soluble in 20 µL, remained soluble on dilution with water to 2 mL. EtOH Partially soluble in 20 µL. Completely soluble on addition of 20 µL water. Remained soluble on further dilution with water to 3 mL. Acetonitrile Partially soluble in 20 µL. Completely soluble on addition of 20 µL water. Remained soluble on further dilution with water to 10 mL. As can be observed from Table 4.1, porphyrins 9 and 27 were soluble in a number of solvents that could be implemented in the development of lipophilic complexes. The use of DMSO and DMF was avoided as it would be difficult to remove trace quantities of these solvents after the complex has been formed. Out of the remaining three solvents acetonitrile was chosen for the construction of lipophilic complexes. It is worth mentioning that to dissolve the porphyrins in a mixture of ACN:water, the acetonitrile must be added first to suspend the porphyrin followed by the addition of water. The addition of a solution of ACN:water (1:1) will not solubilise either porphyrin. Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 70 To investigate the possibility of reducing the lipophilic chain length compound 90 was synthesised by the reaction of porphyrin 70 with methyl iodide (Figure 4.3). It was observed that compound 90 was soluble in THF, DMSO or DMF but precipitation of the porphyrin occurred on addition of water. This result means that the formation of a lipophilic complex with a shorter tether is not feasible using the current protocol. N NH N HN Ph Ph Ph Ph N N NH N HN Ph Ph Ph Ph N Ii) 9070 Figure 4.3 Synthesis of compound 90. Reagents and conditions: i) DMF, CH3I, overnight, 40 °C, 92%. 4.4 Complex Formation Complexes were formed by the slow addition of a solution of porphyrin to a solution of oligonucleotide. For the creation of the porphyrin-DNA complexes a solution of oligonucleotide (Li+ or Na+ salt) was prepared in water (generally 100 µL, concentration of 100-500 µM). To the oligonucleotide solution either porphyrin 9 or 27 (2.5 mg in 80 µL ACN and then diluted with 920 µL water) was added in 2-5 µL fractions forming a red precipitate. Addition was continued until no more red precipitate was observed and a red colour developed in the solution. To see if any more precipitate was occurring, it was necessary to centrifuge the sample (5 seconds at 13500 rpm) between additions of the porphyrin. When no more precipitate was observed the sample was centrifuged (15 min at 13500 rpm) to form a red pellet of the porphyrin-DNA complex (Figure 4.4). The supernatant was then removed and the pellet was washed twice with H2O (1 mL) then dried under high vacuum for two days to remove any traces of solvent. Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 71 Figure 4.4 Example of a DNA-porphyrin complex of compound 27. Initial complexes were formed from salmon testes DNA (ca. 1.3 × 106 Da, 2000 bp). This is a cheap DNA sample that can be obtained commercially in large quantities. A majority of lipophilic complexes have been constructed around this material and used to produce organically soluble complexes.31, 33-36 On the addition of porphyrin 27 to the sodium salt of salmon sperm DNA, a precipitate was formed. This was washed with H2O and dried under high vacuum for three days. When attempts were made to dissolve the resulting solid in various solvents (DCM, CHCl3, DMSO, DMF, toluene or EtOH) the complex was found to be insoluble. This is most likely a result of the pi stacking of porphyrins which can prevent the solvation, especially in porphyrins based on TPP. This insolubility was unfortunate as it makes the complexes unusable. Because of the insolubility resulting from large DNA sequences, complex formation was investigated using short oligonucleotides (12-15 mers) in the hope that these would be more soluble in an organic media. Complexes formed from the single stranded 12 mer sequence (5ʹAGCTTGCTTGAG) were found to be completely soluble in DCM or chloroform. This allowed us to investigate some of the properties of these complexes. 4.4.1 Singe Stranded and Duplex DNA Porphyrin Loading Ratios To determine the ratio of porphyrin to DNA we calculated the loading of porphyrin on DNA. In doing so it is assumed that all phosphate groups are charged and available to interact with the porphyrins. Loading rates were calculated on single strand and duplex oligonucleotides using a similar method to construct the complex to that described above. Through careful measurements we were able to calculate the approximate number of moles of the porphyrin used to precipitate the oligonucleotide. Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 72 To achieve this a solution of single stranded or duplex DNA was created at a concentration of approximately 100 µM in water (100 µL). 5 µL of this solution was added to 995 µL of water and the concentration of the oligonucleotide solution was accurately determined by UV-Vis spectroscopy. To the remaining 95 µL, a porphyrin solution of a known concentration (approximately 2.5 µmoles per mL) was added gradually until no more precipitation occurred. The pellet was centrifuged and the supernatant removed. Any remaining oligonucleotide in the supernatant was precipitated from LiClO4 and acetone, dissolved in water and the concentration determined by UV-Vis spectroscopy. From the initial and final concentrations of DNA and the moles of porphyrin added, it is possible to determine the ratio of porphyrin to DNA. The results represented in Table 4.2 showed that slight overloading of the DNA occurred for both porphyrins. This suggests that some porphyrin is not bound to the DNA via ionic interactions. This could be due to either the intercalation of the porphyrin between bases or, what is more likely, the stacking of porphyrins with each other creating a second layer of porphyrin around the DNA (Figure 4.5). The loading ratios of the free base porphyrin 9 were found to be slightly higher than that for the NiII porphyrin 27. This may possibly suggest that porphyrin 9 stacks better with itself than porphyrin 27. Table 4.2 Loading ratios for porphyrins 9 and 27. Sequence Porphyrin Loading ratea 5ʹAGCTTGCTTGAG 27 1.2 5ʹAGCTTGCTTGAG 9 1.3 5ʹAGCTTGCTTGAG 3ʹTCGAACGAACTC 27 1.1 5ʹAGCTTGCTTGAG 3ʹTCGAACGAACTC 9 1.2 a Loading rate is calculated as porphyrins per phosphate and are the average of three titrations. Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 73 N N N N N N D N A = Porphyrin Figure 4.5 Possible stacking arrangement of lipophilic porphyrins on DNA. It should be noted that the point at which precipitation was maximised was much harder to determine for compound 9 than it was for 27. In the case of porphyrin 27 there was a distinct transition where the addition of more porphyrin only resulted in an increase of porphyrin in solution. The addition of many equivalents eventually resulted in the resolvation of the complex. In contrast, the transition for compound 9 is not as defined as there was always trace quantity of porphyrin in solution. The continued addition of porphyrin 9 well past the equivalence point (4-5 eq) did not result in an increase in the porphyrin concentration in the solution, however a significant increase in the amount of precipitate was observed without any sign of resolvation. This suggests different stacking of compound 9 and 27 around DNA. 4.4.2 G-Quadruplex (GQ) Complex Formation To extend our study we investigated the formation of lipophilic 2ʹ-deoxyguanosine quadruplexes of thrombin binding aptamer (5ʹ-dGGTTGGTGTGGTTGG) using porphyrin 27 and CTAB. G-quadruplexes (GQ) are formed in G rich sequences in the presence of buffers containing potassium ions.7 A standard buffer for the formation of GQ is 10 mM sodium phosphate and 100 mM KCl, pH 7.0. Unfortunately, the addition of 27 to a solution of the above buffer containing no DNA resulted in the precipitation of the porphyrin. Further investigation showed that the porphyrin was still semi insoluble in salt concentrations as low as 0.62 mM sodium phosphate and 6.25 mM KCl. Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 74 It was observed that the GQ-porphyrin supramolecular complex could be formed by pre-forming the GQ in 10 mM sodium phosphate, 100 mM KCl then precipitating the oligonucleotide in GQ form from LiClO4 and acetone. It was found that when the DNA precipitate was redissolved in water the GQ structure was still preserved, as shown by a characteristic CD signal with minima at 260 nm and maxima at 292 nm.135 On the addition of porphyrin 27 to the GQ solution a precipitate was formed which was isolated, dried and dissolved in CHCl3. UV-Vis spectroscopy of the resulting solution showed a peak at 260 nm that was not present in porphyrin 27 therefore confirming the existence of both oligonucleotide and porphyrin in the chloroform solution (Figure 4.6). It was not possible to determining if the GQ structure still remained after complex formation as the CD spectrum of the porphyrin-DNA complex could not be measured due to the strong HT voltage rising from the porphyrin in the UV region. Dilution of the solution to give a reliable HT voltage showed no CD induced Cotton effects in the porphyrin region. This solution was also too dilute to observe any GQ CD signal. By repeating the experiment using CTAB, a precipitate was produced that could be dissolved in EtOH. This solution had an identical CD spectrum to the unmodified GQ which suggests that the GQ structure in the porphyrin modified GQ still remains. 0 0.5 1 1.5 2 2.5 240 340 440 540 640 740 λ / nm Ab s Figure 4.6 UV-Vis spectra of GQ-porphyrin complex (thicker line) and unreacted porphyrin 27 (thinner line) in CHCl3. Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 75 4.5 Conclusion Lipophilic porphyrin complexes could be synthesised using short sequences of single stranded, duplex and GQ oligonucleotides. Longer DNA sequences resulted in the formation of an insoluble complex. Loading studies on single stranded and duplex DNA showed slight overloading of porphyrins on DNA. Characterisation of the porphyrin- DNA supramolecular structure by CD spectroscopy was not possible due to the overpowering porphyrin signal. Because the non-covalent complexes were either insoluble or unable to be fully characterised, focus switched to the covalent attachment of porphyrins to DNA as discussed in Chapters 5 and 6. Chapter 5 - Covalent Attachment of Porphyrins to DNA 76 Chapter 5 Covalent Attachment of Porphyrins to DNA 5.1 Introduction Porphyrin-DNA supramolecular assemblies are important for the development of functional pi-systems with tunable optical properties. As discussed previously, we have chosen to focus on two coupling methods, Sonogashira and CuI catalysed azide alkyne Huisgen 1,3-dipolar cycloaddition reactions, also known as CuAAC reactions, as a means of the pre- or post-synthetic site-specific modification of oligonucleotides (Figure 5.1). Contrary to the common functionalisation of the porphyrin through the meso positions38, 41-44, 46, 55 which results in a system orthogonal to the porphyrin core, a β-pyrrolic modified porphyrin was used which provided a planar system between the porphyrin core and the adjacent benzene ring. To achieve site-specific modification β-pyrrolic substituted porphyrins containing halogens (Br, I), alkynes or azides, as described in Chapter 2.4, were used in conjunction with pre- and post-synthetic Sonogashira and CuAAC reactions. N3 Br/I Nucleoside Nucleoside Br/I Nucleoside N Nucleoside Nucleoside N N Nucleoside DNA synthesis DNA synthesis Oligonucleotide Oligonucleotide I/BrOligonucleotide N3 I/Br N Oligonucleotide Oligonucleotide N N Oligonucleotide Post-synthetic Pre-synthetic + + + + + + = Porphyrin Figure 5.1 Synthetic outline for the development of DNA-porphyrin conjugates. Chapter 5 - Covalent Attachment of Porphyrins to DNA 77 A pre-synthetic approach involves the attachment of a porphyrin to nucleosides which are then converted to the appropriate porphyrin phosphoramidites or H-phosphonates. These are then incorporated into the DNA structure during DNA synthesis. Due to their chemical nature, stability of porphyrin phosphoramidites as DNA building blocks is usually limited46, 48 and is dependent on the structure of the molecule. H-Phosphonate porphyrin analogues have also been employed in DNA synthesis,46, 55 however coupling yields are usually not satisfactory for their multiple incorporations. A pre-synthetic approach can allow for the incorporation of many functionalised nucleotides in a single DNA strand using automated DNA synthesis, only if high yielding reactions occur. In this regard, a post-synthetic modification of DNA is a more versatile approach compared to the time-consuming preparation of phosphoramidites.40 Post-synthetic modification, where a special functional group of the porphyrin reacts specifically with a pre-synthesised oligonucleotide carrying a complementary functional group, is particularly important for screening different substituents in nucleic acid structures. 5.2 Chapter Summary This chapter investigates the pre-synthetic modification of oligonucleotides using both Sonogashira and CuAAC chemistry. Due to the problems accounted with pre-synthetic modification, focus switched to the development of a method for internal post-synthetic modification using alkyne containing ONs via CuAAC chemistry. We have screened porphyrin substituents using this method with ONs incorporating 2ʹ-deoxy-5- ethynyluridine, 2′-O-propargyl uridine or 4-ethynylphenylmethylglycerol moieties. The synthesis of ONs possessing the internal porphyrin modifications allowed us to undertake UV-Vis and CD thermal stability studies on the resulting duplexes and triplexes. Chapter 5 - Covalent Attachment of Porphyrins to DNA 78 5.3 Pre-synthetic Sonogashira and CuAAC Reactions As discussed in Chapter 2 a number of precursors for the use in Sonogashira and CuAAC reactions were synthesised. These include compounds 32, 35 and 40 (Figure 5.2) for use in Sonogashira chemistry and compounds 41, 42, 45 and 48-51 (Figure 5.3) for use in CuAAC chemistry. From these compounds we have synthesised a number of porphyrin possessing nucleosides and oligonucleotides using both pre- and post- synthetic approaches. ZnTPP I ZnTPP 32 35 ZnTPP Br 40 Figure 5.2 Porphyrin precursors used in Sonogashira chemistry. TPP N3 NiTPP N3 41 42 ZnTPP N3 45 TPP N3 NiTPP N3 ZnTPP N3 FeIIIClTPP N3 48 49 50 51 Figure 5.3 Porphyrin precursors used in CuAAC chemistry. 5.3.1 Pre-synthetic Sonogashira Reaction Pre-synthetic Sonogashira reactions focused on the modification of the commercially available 2ʹ-deoxy-5ʹ-O-DMT-5-iodouridine (91) and 5′-O-DMT-8- bromodeoxyguanosine (N-isobutyryl) (92) to create porphyrin modified nucleosides. Reaction of 92 with porphyrin 35 using various conditions involving CuI and Pd(PPh3)4 resulted in only the production of homodimer 34 as shown in Figure 5.4. This was not completely unexpected as Pd0 coupling involving purines A and G are notoriously difficult due to the suspected interaction of the Pd with the purines. Chapter 5 - Covalent Attachment of Porphyrins to DNA 79 ZnTPP NH N N O N H N O OH DMTO ZnTPP ZnTPP Br 35 92 34 O + Figure 5.4 Attempted pre-synthetic Sonogashira coupling involving 35 and 92. The synthesis of the uridine porphyrin nucleoside 94 was achieved from 2-(4ʹ- ethynylphenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc (II) (35) and pyrimidine 5ʹ-O-DMT protected 2ʹ-deoxy-5-iodouridine (91) in high yield (Figure 5.5). Crucial to the success of the Sonogashira coupling between 35 and 91 was the complete degassing of all solvents and the use of at least 4 equivalents of 35 to maximise the formation of compound 94. It was also essential to mix both reactants in Et3N before the addition of the Pd and Cu catalysts, failure to do so resulted in the exclusive formation of homodimer 34. Although synthesis was successful, purification via silica gel chromatography was problematic and pure porphyrin nucleoside 94 was obtained in only 12% yield after subsequent methanol precipitation. Attempts to couple iodo porphyrin (32) and 2ʹ-deoxy-5ʹ-O-DMT-5-ethynyluridine (93) using the same reaction conditions showed only trace amounts of the desired product by TLC analysis of the reaction mixture. Similarly the reaction of bromo porphyrin 40 with 93 was unsuccessful. The one step demetallation and DMT deprotection of 94 with trifluoroacetic acid provided compound 95 (Figure 5.5), whose spectroscopic properties were compared with the previously reported meso linked uridine nucleoside 96.43 In chloroform, the Soret band of the meso uridine 96 was observed at 420 nm while that for the β-pyrrolic functionalised 95 was detected at 429.5 nm. This 9.5 nm bathochromic shift in the Soret band is a result of a higher degree of conjugation occurring between the porphyrin core and the uracil in the β-pyrrolic linked 95 compared to the meso linked 96. To the best of our knowledge, this is the first reported synthesis of a β-pyrrolic linked nucleoside. Chapter 5 - Covalent Attachment of Porphyrins to DNA 80 NH O ON O OH DMTOZnTPP R ZnTPP NH O ON O OH DMTO ZnTPP NH O ON O OH DMTO 35 93 91 94 I NH O ON O OH HO 95 x i) i) ii) N N H N H N NH O ON O OH HO 96 N N H N HN 32: R = I 40: R = Br + + Figure 5.5 Pre-synthetic Sonogashira coupling reactions. Reagents and conditions: i) Et3N, Pd(PPh3)4, CuI, 70 °C, overnight, 78% crude, 12% pure ii) DCM, TFA, RT, 2 min, 70%. Due to the unexpectedly low quantities of compound 95 obtained, conversion of 95 to the corresponding phosphoramidite was not attempted and efforts were instead focused on pre- and post-synthetic CuAAC DNA modifications. 5.3.2 Pre-synthetic CuAAC Chemistry Pre-synthetic CuAAC chemistry focused on the modification of 2′-deoxy-5- ethynyluridine with azido functionalised porphyrins discussed in Chapter 2.4.2. To investigate the versatility of the CuAAC reaction, reactions were performed with aromatic and aliphatic variants containing different metal ions. Chapter 5 - Covalent Attachment of Porphyrins to DNA 81 The model triazoles 97-101 (Figure 5.6) containing NiII, ZnII, FeIII and CuII metal ions were synthesised from the corresponding azide and 2ʹ-deoxy-5ʹ-O-DMT-5- ethynyluridine 93 with 2 eq of Cu(ACN)4PF6, in THF. A copper catalyst that is soluble in organic solvents was used as it had previously shown positive results in comparison to aqueous CuSO4·4H2O or CuI in CuAAC reactions.68 Silica TLC analysis of the reaction mixtures after two days showed the formation of a more polar material which either moved as one or two spots in MeOH:DCM (1:9). Further analysis showed that the addition of trifluoroacetic acid vapour to a sample of the crude reaction mixture before running the TLC resulted in only a single spot in MeOH:DCM (1:9). This suggested that the two polar TLC spots can be attributed to the complete or partial cleavage of the acid sensitive DMT protecting group during the CuAAC reaction. Conformation of the DMT cleavage was obtained on 1H NMR spectroscopy (where possible) and ESI-MS spectrometry analysis of the polar material after silica gel column chromographic purification. Although it was possible to separate the desired triazoles from the unreacted azides using silica gel column chromatography, it was not possible to separate the DMT on and DMT off products from each other. As expected the copper catalysed reaction of the free base porphyrin azide 48 resulted in the quantitative isolation of the CuII metalled porphyrin azide 102. Isolation and re-reaction of this CuII porphyrin azide resulted in the production of the desired triazole 101 in 44% yield. The synthesis of 97-99 provided the opportunity to investigate and compare the spectroscopic properties of the porphyrin linked uracil derivatives. In the UV-Vis spectra of the triazole linked conjugate 99, the porphyrin Soret band occurs at 435 nm. When compared to the ethynyl derivative 94 (Figure 5.5), which has a Soret band at 440 nm, we can conclude that the introduction of the triazole results in distruption of conjugation compared to the ethynyl linked 94. Similarly, the comparison of the aromatic triazole 97 with the aliphatic triazole 98, in which Soret bands were observed at 427.5 and 426 nm respectively, confirmed that the additional CH2 group results in the disruption of conjugation between the porphyrin core and the uracil. Although a number of 1,4-regioisomeric 1,2,3-triazoles were synthesised and characterised, which proves the possibility to link porphyrins to DNA using CuAAC chemistry, we focused on post-synthetic DNA modification. Chapter 5 - Covalent Attachment of Porphyrins to DNA 82 ZnTPP N3 NiTPP N3 NiTPP N3 NH O ON O OH HO NNNZnTPP NH O ON O OH HO NNN NiTPP NH O ON O OH HO NNN NiTPP TPP N3 NH O ON O OH HO NNN CuTPP CuTPP N3 93 93 93 93 93 97 98 99 100 101 48 102 41 50 45 51 i) i) i) i) i) i) NH O ON O OH HO NNN FeIIIClTPP FeIIIClTPP N3 + + + + + Figure 5.6 Synthesis of model triazole linked porphyrin nucleosides (shown as the DMT off structures). Reagents and conditions: i) THF, Cu(ACN)4PF6, RT, 2-4 days, 36% for 97, 28% for 98, 39% for 99, 42% for 100 and 44% for 101. Chapter 5 - Covalent Attachment of Porphyrins to DNA 83 5.4 Post-synthetic Sonogashira and CuAAC Reactions Post-synthetic oligonucleotide modification is an attractive alternative to the time- consuming preparation of multiple phosphoramidites. This is particularly important for screening various modifications of porphyrin possessing oligonucleotides. Post- synthetic modification is clearly an option for porphyrin derivatives because of the difficultly in synthesising the pre-synthetic analogues and the possibility of the decreased lifetime of porphyrin containing phosphoramidites.43 We have therefore investigated post-synthetic Sonogashira and CuAAC reactions as a means to create porphyrin modified oligonucleotides. 5.4.1 Post-synthetic Sonogashira Reaction For the post-synthetic Sonogashira reaction we prepared DMT on ONs using automated DNA synthesis containing a single internal insertion of one of the following DNA building blocks: 2′-deoxy-5-ethynyluridine (V), 2′-deoxy-8-bromoguanosine (W) or (R)-1-O-(2-iodobenzyl)glycerol (X). This gave ON1-ON3 respectively (Figure 5.7). The phosphoramidites required for oligonucleotide synthesis were either prepared in the case of 2′-deoxy-5-ethynyluridine136 and (R)-1-O-(2-iodobenzyl)glycerol137 or purchased (2′-deoxy-8-bromoguanosine). ON1 5'-CCCCTTVCTTTTTT ON2 5'- TWCGCA ON3 5'- CCCCTTXTCTTTTTT + 35 34 + 32/40 No Reaction HN O O N O O O O O O I NH N N O N H N OO O Br V W X + 35 34 P P P OO O O O O O NCNCNC Figure 5.7 Post-synthetic Sonogashira reactions. Reagents and conditions: Pd(PPh3)4, CuI, DMF, Et3N, 3 h. Chapter 5 - Covalent Attachment of Porphyrins to DNA 84 Investigation into the Pd0 catalysed Sonogashira reaction between CPG bound oligonucleotides ON2 and ON3 and ethylene functionalised porphyrin 35 was performed. Reactions were carried out using methods described earlier138-142 followed by cleavage from the CPG support using 32% NH4OH. TLC of the reaction mixture after the Sonogashira reaction showed the exclusive formation of the Glaser homodimer species 34 (Figure 2.23). UV-Vis spectroscopy of the oligonucleotides cleaved from the support CPG showed only traces of the porphyrin conjugates. Alternatively, the iodo (32) or the bromo (40) functionalised porphyrins were reacted with ON1, as under these conditions the formation of the homodimer porphyrin is infeasible. Unfortunately, reactions failed to produce any porphyrin functionalised oligonucleotides. Because of the failures of the Sonogashira reaction our focus switched to the CuAAC reaction. 5.4.2 Post-synthetic CuAAC Reaction CuAAC reactions require a CuI catalyst that is usually produced from the in situ reduction of CuII with sodium ascorbate. NiII porphyrins were selected for the development of the post-synthetic procedure as these porphyrins are guaranteed to remain metallated throughout the CuAAC reaction. For the post-synthetic reactions ONs containing a single internal insertion of either 2ʹ-deoxy-5-ethynyluridine (V), 2ʹ-O- propargyl uridine (Y) or (R)-1-O-(4-ethynylbenzyl)glycerol (Z) were prepared using automated DNA synthesis (Figure 5.8). The phosphoramidites required for oligonucleotide synthesis were either prepared according to the published procedures in the case of 5-ethynyl-2ʹ-deoxyuridine136 and (R)-1-O-(4-ethynylbenzyl)glycerol137 or purchased (2ʹ-O-propargyl uridine). After the respective CuAAC reactions, these modifications will result in the positioning of the porphyrins in the major (V) or minor (Y) grooves of duplexes or as a bulged intercalating insertion (Z) in duplexes and triplexes. O O HN O O N OO HN O O N OO O V Y Z O P OOO P OO O P OO NC NCNC Figure 5.8 Acetylene containing nucleotides incorporated into DNA for use in CuAAC chemistry. Chapter 5 - Covalent Attachment of Porphyrins to DNA 85 Numerous conditions have been used for the post-synthetic attachment of organic molecules to DNA using CuAAC chemistry.61, 104, 143 Previous studies have shown that post-synthetic oligonucleotide based CuAAC reactions can be accelerated using microwave irradiation in a triethylammomium acetate buffer solution144, 145 or on CPG support.145, 146 Our attempts to achieve the coupling of nickel azido porphyrins 41 or 50 (Figure 5.3) using unfocused microwave irradiation (MicroSYNTH by Milestone Laboratory Systems) either on CPG or when the ON was in a buffer failed to produce any conjugates. The use of various combinations of CuI and CuII catalysts, solvents, and reducing agents at various temperatures did not improve the yields. Instead by increasing the reaction time and avoiding unfocussed microwave irradiation, porphyrin- DNA conjugates of V, Y and Z were produced in medium to high yields (ON10-21, Table 5.1 and Figure 5.9). These reactions were performed by shaking a reaction mixture containing CPG bound ON10-21, azido porphyrins 41 or 50, CuSO4·4H2O and sodium ascorbate in DMSO:H2O for 72 hours at RT. Alternatively, CuBr could be used, however, CuSO4·4H2O is easier to handle due to its greater solubility in water. Oligonucleotide sequences were selected to position the porphyrin modifications internally, as apposed to a 5ʹ or 3ʹ molecular cap. Pure pyrimidine sequences (ON4-6) were designed to allow for antiparallel duplex or parallel triplex formation when combined with the appropriate complementary sequences. Mixed purine/pyrimidine sequences (ON7-10) were selected to investigate the versatility of the CuAAC reaction towards sequences containing purine nucleotides. These sequences also allowed for antiparallel duplex formation when combined with the appropriate complementary sequence. Chapter 5 - Covalent Attachment of Porphyrins to DNA 86 Table 5.1 Oligonucleotides before and after CuAAC reactions with azides 41 or 50. m/z [Da] No. Oligonucleotidec Calcd. found Retention Times (min)a Conversion (%)b ON4 5' –CCCCTTVCTTTTTT - - 18.8 - ON5 5' –CCCCTTYCTTTTTT - - 18.2 - ON6 5' –CCCCTTZTCTTTTTT - - 17.8 - ON7 5' –AGCTVGCTTGAG - - 20.8 - ON8 5' –AGCTYGCTTGAG - - 20.7 - ON9 5' –CTCAAGZCAAGCT - - 20.8 - ON10 5' –CCCCTT1CTTTTTT 4958.9 4956.0 41.6 45 ON11 5' –CCCCTT2CTTTTTT 4972.9 4961.7 41.7 48 ON12 5' –CCCCTT3CTTTTTT 4988.9 4979.8 42.0 64 ON13 5' –CCCCTT4CTTTTTT 5002.9 4997.4 40.8 71 ON14 5' –CCCCTT5TCTTTTTT 5216.9 5202.8 43.2 60 ON15 5' –CCCCTT6TCTTTTTT 5231.0 5229.9 42.6 58 ON16 5' –AGCT1GCTTGAG 4497.8 4507.0 45.1 31 ON17 5' –AGCT2GCTTGAG 4511.9 4517.2 45.0 55 ON18 5' –AGCT3GCTTGAG 4527.8 4525.9 46.2 60 ON19 5' –AGCT4GCTTGAG 4541.8 4541.4 45.2 67 ON20 5' –CTCAAG5CAAGCT 4693.9 4697.2 47.0 19 ON21 5' –CTCAAG6CAAGCT 4707.9 4707.9 46.7 39 a HPLC retention times are ± 0.5 min, see methods for HPLC gradients. b Conversion % is determined from the HPLC peak integrals at 260 nm. cSee Figure 5.9 for numbering. n = 0 (5) 1 (6) n = 0 (1) 1 (2) n = 0 (3) 1 (4) N NN N Ni n R = O O HN O O N OO HN O O N OO O O P OO O P OO O P OO N N N N NN N NN R R R NC NC NC Derived from V Y Z Location of the modification in duplex DNA Major groove Minor groove Bulged insertion Figure 5.9 Porphyrin-DNA monomers obtained via the post-synthetic CuAAC chemistry. Chapter 5 - Covalent Attachment of Porphyrins to DNA 87 CuAAC reactions (Figure 5.10) were achieved by mixing nickel containing porphyrin- azides 41 or 50 (7.67 µmol) and one of the alkyne CPG bound oligonucleotides ON4- ON9 (0.33 µmol) in a micro-centrifuge vial, followed by the addition of DMSO (150 µL). Freshly prepared CuSO4·4H2O (0.2 µmol in 5 µL H2O) and sodium ascorbate (1.0 µmol in 20 µL H2O) solutions were added resulting in the partial precipitation of the porphyrin azide. The reactions were then sealed under argon, to avoid DNA cleavage caused by CuI ions in the presence of oxygen,147 and shaken at RT for 72 hours. O O HN O O N OO HN O O N OO O V Y Z O P OO O P OO O P OO TTTTTTCBTTCCCC TTTTTTCBTTCCCC 3' 5' N N N N Ph PhPh Ph Ni N3 TTTTTTCBTTCCCC NN N NN N = CPG Support ii) / iii) + N N N N Ph PhPh Ph Ni N3 or B = 41 50 NiTPP NiTPP n n = 0 or 1 n n = 0 or 1 i) NC NC NC Figure 5.10 CuAAC reaction between azido porphyrins 41 or 50 and an oligonucleotide containing V, Y or Z. Reagents and conditions: i) CuSO4, sodium ascorbate, DMSO, H2O, RT, 72 h ii) 32% aq. NH4OH, 55 °C, overnight iii) HPLC purification. Chapter 5 - Covalent Attachment of Porphyrins to DNA 88 After shaking, the CPG supports were repeatedly washed with DCM to remove the unreacted azides. The resulting red coloured CPG support provided an indication of the progress of the reaction. The unreacted azide could be recovered in 80-90% yield for the use in future CuAAC reactions by washing the DCM solution containing the azide with H2O, followed by drying over MgSO4 and precipitation from DCM:MeOH. Oligonucleotides were then cleaved from the support using 32% aq NH4OH, and purified using semi-preparative C18 HPLC monitored at both 260 and 427 nm (Figure 5.11). Porphyrin conjugated oligonucleotides showed appreciably increased retention times compared to unmodified oligonucleotides. As can be observed in Table 5.1, ONs possessing 2ʹ-O-propargyl uridine (Y) had superior conversions in comparison to ONs containing 2ʹ-deoxy-5-ethynyluridine (V) and (R)-1-O-(4-ethynylbenzyl)glycerol (Z). Mixed purine/pyrimidine sequences (ON16-21) were found to have lower conversions than pyrimidine sequences (ON10-15). Figure 5.11. HPLC profiles of the reaction between azide 50 and ON5 showing the significant increase in retention times for the porphyrin modified oligonucleotide (left: λ = 260 nm, right: λ = 427 nm). Collected fractions were lyophilised, redissolved in 100 µL H2O and precipitated from LiClO4 and acetone to give deep red oligonucleotides. ONs were dissolved in 100 µL water to create stock solutions. Not all oligonucleotides could be completely solubilised and heating for several hours at 70 °C was required to increase solubility. Oligonucleotides were desalted using C18 zip-tips and then characterised by MALDI- TOF spectrometry (Table 5.1). It should be noted that the purchase and use of a new focused microwave system provided the conditions required for successful microwave accelerated CuAAC reactions to occur. Although not discussed here, this was used to produce porphyrin functionalised oligonucleotides in Chapter 6. Chapter 5 - Covalent Attachment of Porphyrins to DNA 89 Purity of ONs was checked using 20% denaturing PAGE, showing a single red band with a significant retardation compared to the wild type oligonucleotide (Figure 5.12). This is in contrast to work published by Stulz et al.43 which showed increased mobility of oligonucleotides possessing a single porphyrin moiety (based on the structure of porphyrin 96, Figure 5.5) compared to the unmodified oligonucleotide. Figure 5.12 Denaturing 20% PAGE (7 M urea) of porphyrin modified oligonucleotides stained with Stains-All® dye and destained with H2O. Porphyrin modified oligonucleotides were red before staining. Lane 1 is unmodified oligonucleotide ONwt (top) and a marker (bottom), lane 2 is ON17, lane 3 is ON16, lane 4 is ON19 and lane 5 is ON21. To summarise, Sonogashira and CuAAC chemistry have been shown to be useful methods for the creation of β-pyrrolic porphyrin functionalised nucleosides. More importantly, CuAAC reactions were found to be an effective method for the creation of post-synthetic and site-specific, internally modified porphyrin oligonucleotides. Reactions could be performed on aliphatic or aromatic azido NiII porphyrins and CPG bound ONs containing terminal alkyne modifications (V, Y and Z) in moderate to high yields (19-71% conversion). This has allowed synthesis of a number of porphyrin possessing ONs for use in thermal stability studies on single stranded, duplex and triplex DNA. Chapter 5 - Covalent Attachment of Porphyrins to DNA 90 5.5 UV-Vis and CD Studies of Porphyrin-DNA Conjugates Optical and thermal stability properties of the oligonucleotides possessing NiII porphyrins were investigated using UV-Vis and CD spectroscopy. These methods were used to give an indication of the effect of a porphyrin modification on single stranded, duplex and triplex DNA possessing internal porphyrin modifications. 5.5.1 Single Stranded Oligonucleotides Single stranded oligonucleotides ON10-ON21 were prepared at a concentration of 1.0 µM in cacodylate buffer at various pH (5.0, 6.0 and 7.2). Samples were prepared by taking the appropriate quantity of oligonucleotide from the stock solution and diluting it to 1.0 µM with the appropriate pH buffer to obtain a 1.0 µM solution. CD spectra were then recorded at 20 °C from 220-500 nm. CD spectra, at pH 6.0, of all porphyrin modified oligonucleotides showed DNA signals at 248 (negitive ellipticity) and 280 nm (positive ellipticity, not shown) and strong signals around the location of the porphyrin Soret band (Figure 5.13). Results showed, in general, a bisignate curve suggesting that either the porphyrin interacts with the chiral environment of the oligonucleotide or that dipole-dipole electronic interactions between two porphyrins occur.38, 39, 47, 48, 50, 52, 54, 56 -10 -5 0 5 10 15 20 25 380 400 420 440 460 480 500 λ / nm ∆ε / M - 1 c m - 1 ON10 pH 6.0 ON11 pH 6.0 ON12 pH 6.0 ON13 pH 6.0 ON14 pH 6.0 ON15 pH 6.0 Figure 5.13 CD spectra of single stranded oligonucleotides ON10-15 at pH 6.0. Chapter 5 - Covalent Attachment of Porphyrins to DNA 91 Further analysis showed that CD signals were pH dependent for CT sequences (ON10- 15) but not for mixmer sequences ON16-21. For example, the CT sequence ON15 (Figure 5.14A) showed virtually no porphyrin CD signals at pH 7.2, while a strong bisignate curve was observed at pH 5.0 and 6.0. Along with this, CD intensity in the UV region decreased with increasing pH and was blue shifted by 6 nm. This is a characteristic signature of i-motif formation as a result of cytosine protonation (pKa = 5.2) (Figure 5.14C).148, 149 Increasing the pH resulted in cytosine deprotonation, i-motif unfolding and loss of the porphyrin-porphyrin interactions. It is possible that this structure allowed for the orientation of porphyrins in such a manner that porphyrin- porphyrin exciton coupling occurred through porphyrin stacking as shown in a possible i-tetraplex structure (Figure 5.14C). The unmodified oligonucleotide (ONwt, Table 5.2) did not show the characteristic pH dependent i-motif CD signals (Figure 5.15) suggesting that the porphyrin, rather than the ON sequence, triggers the formation of the i-motifs. Native PAGE (pH 5.0) confirmed the absence of an i-motif structure in ONwt. Porphyrin possessing ONs did not penetrate the native PAGE gel at pH 5.0. This significant retardation compared to that observed in denaturing gels confirmed the formation of a secondary structure. Although the molecularity of ON10-15 could not be determined by native PAGE it may be possible using electrospray ionisation mass spectroscopy in pH 5.0 buffers. The pH independent CD spectra observed in mixmers (Figure 5.16) suggested the formation of aggregates rather than i-motifs in mixmer sequences ON16-21. Chapter 5 - Covalent Attachment of Porphyrins to DNA 92 -40 -20 0 20 40 60 80 100 120 220 270 320 370 420 470 λ / nm ∆ ε / M - 1 c m - 1 ON15 pH 5.0 ON15 pH 6.0 ON15 pH 7.2 C C C C T T U C C C C C T T U C C C C C T T U C C C C C T T U C N N N O N N N O H H H H H R R A B C = Porphyrin Figure 5.14 CD spectra of single stranded oligopyrimidine ON15 at pH 5.0, 6.0 and 7.2 (A), cytosine+- cytosine base pair (B, R = furanose ring) and possible i-tetraplex of ON13 (C). -30 -20 -10 0 10 20 30 40 50 60 220 270 320 370 420 470 λ / nm ∆ε / M - 1 c m - 1 ONwt pH 5.0 ONwt pH 6.0 ONwt pH 7.2 Figure 5.15 CD spectra of single stranded oligopyrimidine ONwt at pH 5.0, 6.0 and 7.2. Chapter 5 - Covalent Attachment of Porphyrins to DNA 93 -40 -20 0 20 40 60 80 100 120 140 220 270 320 370 420 470 λ / nm ∆ε / M - 1 c m - 1 ON17 pH 5.0 ON17 pH 7.2 Figure 5.16 CD spectra of single stranded purine-pyrimidine oligonucleotide ON17 at pH 5.0 and 7.2. Thermal melting of the single stranded ONs resulted in the loss of the CD signal around 430 nm and reduced signal intensity at 280 nm (Figure 5.17), suggesting the melting of i-motifs (ON10-ON15) or the separation of aggregates. After melting, a CD signal for the porphyrin was only observed after incubation at 20 °C overnight and the intensity was significantly lower than for the unheated sequence. This suggested that the formation of aggregates or i-motifs was slow to occur at 1.0 µM concentration and that the i-motif or aggregates only form in the concentrated stock solution (ca. 300-1000 µM). -20 0 20 40 60 80 100 220 270 320 370 420 470 λ / nm ∆ε / M - 1 c m - 1 ON15 30°C ON15 40°C ON15 50°C ON15 60°C Figure 5.17 CD spectra showing the thermal melting of single stranded oligopyrimidine ON15 from 30-60 °C at pH 5.0. Chapter 5 - Covalent Attachment of Porphyrins to DNA 94 5.5.2 Triplexes and Duplexes Containing Internal Porphyrin Modifications The thermal stability of triplexes and duplexes containing the synthesised oligonucleotides ON10-ON21 was assessed by UV-Vis thermal denaturation experiments at 260 and 430 nm from 10-70 °C. The melting temperatures (Tm, °C) were determined as maxima of first derivatives of melting curves and are listed in Tables 5.2 and 5.4. Annealing temperatures are shown in Tables 5.3 and 5.5. The triplex forming oligonucleotide (TFO) sequences (ONwt and ON10-15) possessing different porphyrin modifications were studied in a parallel triplex towards the duplex D1 (Table 5.2) and in antiparallel duplexes towards appropriate oligonucleotides ON22-24 (Table 5.2-5.5). Triplexes were formed by mixing the TFO strand at 1.5 µM with 1.0 µM of each duplex strand (D1). Duplexes were formed from 1.0 µM of each strand in the appropriate buffer. Oligonucleotides were heated to 70 °C (triplexes) or 90 °C (duplexes) for 15 minutes then cooled to 10 °C an incubated for 30 minutes. Buffers were made to 20 mM sodium cacodylate, 100 mM NaCl and 50 mM MgCl2 and adjusted to the required pH using dilute HCl according to the procedure described.150 Modifications 1 and 2 (ON10, ON11, ON16 and ON17) were designed to locate the porphyrin in major groove of the duplex, 3 and 4 (ON12, ON13, ON18 and ON19) in the minor groove and modifications 5 and 6 (ON14, ON15, ON20 and ON21) were inserted as an intercalating bulge in the middle of duplexes and triplexes. 5.5.2.1 Triplexes A triple helix is formed when third single stranded oligonucleotide binds in the major groove of the duplex (Figure 1.7). This is achieved by forming T-A··T and C-G··C+ Hoogsteen interactions (· ·), and requires the protonation of the cytosine in the TFO. The pKa of the imino group of the cytosine is 5.2 thus triplex stability increases at a lower pH. The thermal stability of triplexes was assessed by UV-Vis thermal melting at 260 and 430 nm (Table 5.2). At pH 6.0 two transitions were observed at 260 nm for parallel triplexes which correspond to the melting of the triplex at lower temperature and the duplex at 55 °C. At pH 5.0 triplex transitions occurred at higher temperatures (due to the increased protonation of cytosine) which resulted in the overlaid melting profiles for Chapter 5 - Covalent Attachment of Porphyrins to DNA 95 duplexes and triplexes at 260 nm (ON12-15/D1). Sharp melting transitions for the triplexes were also visible at 430 nm, thus confirming the melting of the porphyrin containing TFO strands from the duplex. As can be seen from the Tm data in Table 5.2, the internal insertion of a porphyrin resulted in increased Tm values of the Hoogsteen–type triplexes compared to the wild- type triplex (ONwt/D1). Stabilisation of all porphyrin modified triplexes was observed at pH 6.0 (∆Tm = 3.0-12.0 °C). The TFO strands ON10 and ON14 containing aromatic porphyrins (modifications 1 and 5, Figure 5.9) resulted in slightly higher stabilisation of the triplex at pH 6.0 than for TFO strands ON11 and ON15 containing the aliphatic linked porphyrins (modifications 2 and 6). In the case of ON14 and ON15, where the porphyrin is incorporated as a bulged insertion into the duplex strands, molecular modelling (AMBER* force field151, 152) suggested that stabilisation was most likely due to the intercalation of the porphyrin (Figure 5.18A-D). However, as shown in Figure 5.18 the porphyrin could either penetrate through the duplex DNA (A and B) or intercalate between bases in the duplex strands such that the meso phenyl rings are located in the grooves of the triplex (C and D). These two orientations were possible due to the long linker between the porphyrin and the phosphate backbone (19 Å). Intercalation, similar to that shown in TINA configurations144, 153 (Figure 5.19), was found to be of higher energy (-20034 KJ/mol) than when the porphyrin penetrated the duplex strands and aligned itself in the minor groove (-20086 KJ/mol). This difference in energy is most likely a result of the destabilising bulge in the phosphate backbone of the TFO strand that is required for the porphyrin to intercalate between the bases of the duplex strands. Further thermal stabilisation might be possible by decreasing the linker length and thus reducing the bulge in the backbone of the TFO strand. Although not modelled, the aliphatic linked porphyrin in ON15 is likely to be positioned in a similar manner to the aromatic porphyrin in ON14. Chapter 5 - Covalent Attachment of Porphyrins to DNA 96 Figure 5.18 A representation of the AMBER* force field lowest energy minimised structures of the porphyrin possessing triplex ON14/D1 showing two possible porphyrin orientations. Figures A (side) and B (top) show the porphyrin penetrating through the duplex D1. Figures C (side) and D (top) show the higher energy structure where the porphyrin intercalates between the bases of duplex D1. Phosphorus atoms in the TFO strand have been coloured green. In B and D, nucleotides not directly above or below the porphyrin modification have been removed for clarity. O O O N NN P OO O O O P OO Figure 5.19 TINA structures incorporated into TFO strands used as bulged insertions to stabilise triplexes.138, 144 Chapter 5 - Covalent Attachment of Porphyrins to DNA 97 As expected, the Tm values of triplexes of ON12-15/D1 increased at pH 5.0 due to the increased cytosine protonation. Surprisingly, the Tm values of ON10-11 were almost unchanged. At pH 7.2 no triplex formation was observed above 10 °C. The lower Tm values for the triplexes at 430 nm suggested that the porphyrin was stabilising the triplex and the movement of the porphyrin resulted in the disruption of the triplex soon afterwards. The significantly lower annealing temperatures at 260 nm alludes to the possibility of different kinetics in the annealing and denaturing processes. The importance of the porphyrin in the stabilisation of the triplex strand is emphasised when we observe the previously reported Tm value for the bulged insertion of 1,2,3-triazole linked benzyl moiety (Figure 5.20) in the TFO strand.144 Under identical conditions and using the sequence equivalent to ON15, destabilisation of the resulting triplex by greater than 23 °C was observed at pH 6.0. O O O N NN P OO Figure 5.20 1,2,3-Triazole linked benzyl moiety incorporated into TFO strands.144 98 Table 5.2 Melting temperatures of parallel DNA triplexes and antiparallel DNA duplexes containing a single porphyrin modification. Parallel Triplexa 3’-CTGCCCCTTTCTTTTTT 5’-GACGGGGAAAGAAAAAA (D1) Antiparallel Duplexb 3’-GGGGAAAGAAAAAA (ON22) pH 5.0 pH 6.0 pH 6.0 pH 7.2 ONwt 5' –CCCCTTTCTTTTTT 54.0c 27.0 48.0 48.0 ON10 5' –CCCCTT1CTTTTTT 39.0 (39.0) 39.0 (37.0) 31.0 (30.0) 30.0 (30.3) ON11 5' –CCCCTT2CTTTTTT 40.0 (39.5) 30.0 (37.7) 32.5 (29.0) 33.2 (33.7) ON12 5' –CCCCTT3CTTTTTT 54.5c (53.5) 34.5 (29.3) 35.0 (28.5) 33.2 (33.7) ON13 5' –CCCCTT4CTTTTTT 55.0c (54.0)c 34.5 (32.7) 41.5 (43.8) 41.7 (43.0) ON14 5' –CCCCTT5TCTTTTTT 55.0c (53.0) 38.9 (37.0) 38.7 (24.0) 34.6 (37.0) ON15 5' –CCCCTT6TCTTTTTT 55.0c (53.5) 36.3 (36.8) 33.3 (31.0) 34.3 (34.6) Table 5.3 Annealing temperatures of parallel DNA triplexes and antiparallel DNA duplexes containing a single porphyrin modification. Parallel Triplexa 3’-CTGCCCCTTTCTTTTTT 5’-GACGGGGAAAGAAAAAA (D1) Antiparallel Duplexb 3’-GGGGAAAGAAAAAA (ON22) pH 5.0 pH 6.0 pH 6.0 pH 7.2 ONwt 5' –CCCCTTTCTTTTTT 54.0c 27.0 48.0 48.0 ON10 5' –CCCCTT1CTTTTTT 35.8 (35.0) 35.7 (37.0) 31.5 (31.8) 28.3 (28.1) ON11 5' –CCCCTT2CTTTTTT 40.0 (38.9) 27.5 (30.8) 32.5 (29.0) 32.0 (33.0) ON12 5' –CCCCTT3CTTTTTT 54.5c (47.3) 25.4 (25.4) 33.7 (28.7) 33.3 (31.6) ON13 5' –CCCCTT4CTTTTTT 54.8 (50.9)c 18.0 (26.5) 42.8 (41.7) 42.5 (43.5) ON14 5' –CCCCTT5TCTTTTTT 55.0c (53.3) 24.7 (36.7) 34.0 (24.0) 34.6 (36.4) ON15 5' –CCCCTT6TCTTTTTT 55.0c (54.5) 32.0 (37.0) 33.5 (31.0) 34.9 (34.6) Tm (°C) data for parallel triplex and antiparallel duplex melting, taking from the UV-Vis melting curves at 260 and 430 nm (shown in brackets). aC = 1.5 µM of ON10-15 and ONwt and 1.0 µM of each strand of dsDNA (D1) in 20 mM sodium cacodylate, 100 mM NaCl and 5 mM MgCl2, pH 5.0 and 6.0. bC = 1.0 µM of each strand in 20 mM sodium cacodylate, 100 mM NaCl and 5 mM MgCl2, pH 6.0 and 7.2. cThird strand and duplex melting overlaid. 98 Chapter 5 - Covalent Attachment of Porphyrins to DNA 99 In order to confirm triplex formation, CD spectra of the triplexes was recorded at pH 5.0 (Figure 5.21) and 6.0 (Figure 5.22). A concentration of 1.0 µM of each oligonucleotide strand was used to avoid interference from any excess of the TFO strand. A negative band around 210 nm is considered to be a sign of parallel DNA triplexes.154 As can be seen, a negative band exists at 209 nm, therefore confirming the formation of a triplex in porphyrin modified sequences. Although a negative band exists at 208 nm in duplex D1 the intensity was significantly lower than for the triplex. -250 -200 -150 -100 -50 0 50 100 150 200 205 255 305 355 405 455 λ / nm ∆ε /M - 1 c m - 1 ONwt/D1 ON15/D1 D1 Figure 5.21 CD spectra of triplex ONwt and ON15 with D1, and duplex D1 alone at pH 5.0 (20 °C). -200 -150 -100 -50 0 50 100 150 200 205 255 305 355 405 455 λ / nm ∆ε /M - 1 c m - 1 ONwt/D1 ON15/D1 D1 Figure 5.22 CD spectra of triplex ONwt and ON15 with D1 and duplex D1 alone at pH 6.0 (20 °C). Chapter 5 - Covalent Attachment of Porphyrins to DNA 100 5.5.2.2 Duplexes It was observed (Table 5.2) that the single internal incorporation of a porphyrin in an antiparallel duplex (ON10-15/ON22) resulted in thermal destabilisation of the duplex compared to the unmodified duplex. Variation in the destabilisation depended on the porphyrin incorporated and its location in the duplex, remembering that modifications 1 and 2 were designed to locate the porphyrin in the major groove, 3 and 4 in the minor groove and 5 and 6 as an intercalating bulge in the middle of the duplex. Generalising the results we found that duplexes involving ON10-11 and ON14-15 showed significant thermal destabilisation between 13.4-18.0 °C. Molecular modelling (AMBER* force field) of the duplexes involving ON10-11 (Figure 5.23A and B) and ON14-15 (Figure 5.24A and B) suggested that the porphyrins had very little interaction with the nucleobases and were positioned in the major groove. Porphyrins 1 and 2 in ON10 and ON11 were located either in the major groove space (ON10) or positioned against the side of the major groove (ON11). Due to the linker length, porphyrins 5 and 6 protruded through the duplex, leaving just the phenyl and triazole moieties located in between bases in the duplex core and the porphyrin positioned in the major groove. This intercalation may account for the slight stabilisation of ON14-15 over ON10-11. As the porphyrin is hydrophobic it could be expected that destabilisation is a result of a different hydration of the duplex compared to the wild type. Significant differences were found in the melting temperatures of duplexes containing ON12 and ON13 where the porphyrin was positioned in the minor groove. Duplexes containing the aromatic porphyrin ON12 showed ∆Tm of -13.0 to -14.8 °C, similar to the duplexes ON10-11 and ON14-15, while duplex ON12/ON22, which contained the aliphatic porphyrin 4, had a ∆Tm of -6.3 to -6.5 °C. Molecular modelling suggested that the aromatic (ON12, Figure 5.23C) and the aliphatic porphyrins (ON13, Figure 5.23D) follow the minor groove in a similar manner, however, the aliphatic porphyrin is less destabilising presumably as a result of rotational flexibility around the extra sp3 carbon. The aromatic porphyrin in ON12 is unable to fit as tightly in the minor grove and as a result lower Tm values were observed. Chapter 5 - Covalent Attachment of Porphyrins to DNA 101 Figure 5.23 A representation of the lowest energy AMBER* force field minimised structures of duplexes involving ON10 (A), ON11 (B), ON12 (C) and ON13 (D). On the left is the side view and on the right is the top view (nucleotides above and below the porphyrin modification have been removed for clarity). Chapter 5 - Covalent Attachment of Porphyrins to DNA 102 Figure 5.24 A representation of the lowest energy AMBER* force field minimised structure of duplexes involving ON14 (A) and ON15 (B). On the right is the side view and on the left is the top view (nucleotides not directly above or below the porphyrin modification have been removed for clarity). Duplexes containing mixed purine/pyrimidine stands (Tables 5.4 and 5.5) showed Tm values consistent with that of the porphyrin modified CT sequences. Significant destabilisation was found in all duplexes especially those containing modifications of (R)-1-O-(4-ethynylbenzyl)glycerol (ON20 and ON21/ON24). These duplexes showed ∆Tm of -16.3 to -18.0 °C for the duplexes containing the aromatic porphyrin 5 and even further destabilisation for the aliphatic porphyrin 6 (∆Tm -21 to -22.1 °C). Duplexes containing a porphyrin in the minor groove (ON18 and ON19/ON23) confirmed the melting trend shown in ON10-15 – that being the additional flexibility of the aliphatic porphyrin 4 allows for that porphyrin to fit more comfortably in the minor groove of the duplex resulting in less destabilisation of the duplex. Additionally, the melting profiles showed virtually no hysteresis at either 260 or 430 nm for all duplexes indicating similar kinetics in both denaturing and annealing processes even with the introduction of a porphyrin. Chapter 5 - Covalent Attachment of Porphyrins to DNA 103 Table 5.4 Melting temperatures of antiparallel duplexes containing mixed purine/pyrimidine oligodeoxynucleotides. Antiparallel Duplex 3'–TCGAACGAACTC (ON23) Antiparallel Duplex 3'-GAGTTCGTTCGA (ON24) pH 6.0 pH 7.2 pH 6.0 pH 7.2 ONwtm 5' –AGCTTGCTTGAG 50.0 50.0 - - ON16 5' –AGCT1GCTTGAG 35.0 (32.0) 35.0 (34.0) - - ON17 5' –AGCT2GCTTGAG 34.0 (33.5) 32.0 (NVT) - - ON18 5' –AGCT3GCTTGAG 36.0 (36.0) 34.0 (35.0) - - ON19 5' –AGCT4GCTTGAG 42.0 (39.0) 43.0 (NVT) - - ON20 5' –CTCAAG5CAAGCT - - 32.0 (33.0) 33.3 (NVT) ON21 5' –CTCAAG6CAAGCT - - 29.0 (28.0) 27.9 (29.1) Table 5.5 Annealing temperatures of antiparallel duplexes containing mixed purine/pyrimidine oligodeoxynucleotides. Antiparallel Duplex 3'–TCGAACGAACTC (ON23) Antiparallel Duplex 3'-GAGTTCGTTCGA (ON24) pH 6.0 pH 7.2 pH 6.0 pH 7.2 ONwtm 5' –AGCTTGCTTGAG 50.0 50.0 - - ON16 5' –AGCT1GCTTGAG 34.5 (31.5) 35.0 (34.0) - - ON17 5' –AGCT2GCTTGAG 34.5 (NVT) 32.0 (NVT) - - ON18 5' –AGCT3GCTTGAG 36.0 (36.5) 34.0 (35.0) - - ON19 5' –AGCT4GCTTGAG 42.0 (38.5) 43.0 (NVT) - - ON20 5' –CTCAAG5CAAGCT - - 33.6 (31.0) 32.2 (NVT) ON21 5' –CTCAAG6CAAGCT - - 26.0 (27.5) 28.9 (27.0) Tm (°C) data for antiparallel duplex melting taking from the UV melting curves at 260 and 430 nm (shown in brackets). C = 1.0 µM of each strand in 20 mM sodium cacodylate, 100 mM NaCl and 5 mM MgCl2, pH 6.0 and 7.2. NVT = No visible transition. The CD spectra of duplexes containing ONs ON10-21 were recorded from 220-500 nm at 20 °C. CD spectra showed a negative band at around 245-250 nm and a positive band at 274-280 nm (Figure 5.25 and 5.26), clearly suggesting that the modified duplexes retained their overall B-form double helix structure. These oligonucleotides also showed, in general, a porphyrin CD signal as a bisignate curve around 430 nm. Similar bisignate curves have been assigned to exciton coupling between porphyrin molecules.54, 56 As our structures possess only a single porphyrin modification these interactions would only be possible through duplex aggregation. Aggregation, which is thought to occur over longer time periods, was found to be unlikely as the porphyrin CD signal of a duplex that had been melted returned to the pre-melted level almost instantaneously (Figure 5.27). It was more likely that the porphyrin bisignate curve was a result of the porphyrin somehow interacting with the DNA grooves. Chapter 5 - Covalent Attachment of Porphyrins to DNA 104 Figure 5.25 CD spectra of duplexes ON10-15 with ON22 at pH 6.0 (20 °C). Figure 5.26 CD spectra of duplexes involving ONwtm and ON16-21 at pH 6.0 (20 °C). -100 -50 0 50 100 150 220 270 320 370 420 470 λ / nm ∆ ε/M - 1 c m - 1 ON10/ON22 ON11/ON22 ON12/ON22 ON13/ON22 ON14/ON22 ON15/ON22 -15 -5 5 350 420 490 -150 -100 -50 0 50 100 150 220 270 320 370 420 470 λ / nm ∆ ε / M - 1 c m - 1 ONw tm/ON23 ON16/ON23 ON17/ON23 ON18/ON23 ON19/ON23 ON20/ON24 ON21/ON24 -30 0 30 390 460 Chapter 5 - Covalent Attachment of Porphyrins to DNA 105 -60 -40 -20 0 20 40 60 80 220 270 320 370 420 470 λ / nm ∆ε / M - 1 c m - 1 20 °C 70 °C Cooled 30 min at 20 °C Figure 5.27 CD spectra of duplex ON13/ON22 at 20 °C, 70 °C and after cooling and incubation at 20 °C for 30 minutes. 5.6 Conclusion In conclusion we have developed both CuAAC and Sonogashira coupling methods for the pre-synthetic attachment of ethynyl and azidoporphyrins to 5-iodo and 5-ethynyl-2ʹ- deoxyuridine respectively. From this we have gained an insight into the comparative levels of conjugation between the porphyrin cores and the nucleosides in ethynyl and 1,2,3-triazole linked porphyrin nucleosides. More importantly, the CuAAC reaction was found to be is superior to Sonogashira chemistry for the post-synthetic construction of covalently linked porphyrin modified ONs. Using CuAAC chemistry, coupling of both aliphatic and aromatic NiII azidoporphyrins to ONs containing various terminal ethylnyl bonds was achieved. The effect of various single porphyrin modifications on the structure and thermal stability of single stranded, duplex and triplex DNA was screened. Single stranded ONs containing internal porphyrin modifications formed porphyrin driven i-motif structures in CT sequences and aggregates when the oligonucleotides did not posses the appropriate Chapter 5 - Covalent Attachment of Porphyrins to DNA 106 sequence for i-motif formation. These aggregates and i-motifs were shown not to inhibit duplex and triplex formation. Thermal stability studies were performed on Hoogsteen- type triplexes containing porphyrin modified TFO strands and it was found that porphyrin modifications generally stabilise the triplex. Overall destabilisation was observed when a porphyrin was incorporated internally into a duplex, however, destabilisation was significantly lowered when the aliphatic porphyrin 50 was positioned in the minor groove. Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 107 Chapter 6 Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 6.1 Introduction The helical self-assembled structure of nucleic acids provides the perfect template for the construction and development of functional pi-systems with tunable optical properties.4, 5, 15 Considerably different physico-chemical properties are often observed between chromophores that are closely packed on the same strand, in contrast to being attached to adjacent strands, of the duplex. Porphyrins have been widely studied as labels in a variety of applications such as light harvesting devices/electron transfer systems,88, 89 low-power photon upconversion,155 and the production of reactive oxygen species.22, 156 Covalent attachment of porphyrin moieties to DNA has been achieved using a variety of methodologies (see Chapter 1.7). Recently, the synthesis of a DNA containing 11 meso- functionalised porphyrins attached to 2′-deoxy-5-ethynyluridines was carried out from the corresponding phosphoramidites (Figure 1.22), however this showed a significant thermal destabilisation of the resulting duplex.43 A stabilising effect of +0.5 °C per modification was observed when the porphyrins were placed adjacently in complementary strands.44 Molecular modelling showed that porphyrins were arranged in a zigzag fashion and stacked in pairs in the major groove (Figure 1.23). Finally, an effective energy transfer between a zinc porphyrin and a free-base porphyrin in the major groove of the DNA duplex was observed.44 In this chapter we used a CuAAC chemistry approach developed in Chapter 5 to combine DNAs containing commercially available 2′-O-propargyl uridine or 2′-O- propargyl adenosine157 with β-pyrrolic azido substituted porphyrins that allows us to place multiple functional entities in the minor groove of the DNA duplex.158 Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 108 6.2 Chapter Summary In this chapter we report the synthesis of oligonucleotides containing one or two internal porphyrin modifications via the post-synthetic microwave accelerated CuI catalysed Huisgen 1,3-dipolar azide alkyne cycloaddition reaction. In contrast to the single or double internal incorporations of porphyrins into one of the DNA strands of the duplex, the introduction of porphyrins into the second strand results in the formation of H- aggregates in the minor groove of the DNA helix. This also led to extraordinary enhancement of thermal stability to such an extent that duplexes with four adjacent porphyrins could only be dissociated at a low salt concentration. Molecular modelling indicated that porphyrins could accommodate a number of conformations including one in which the porphyrin sequence did not match the sequence of the nucleotides. Each stacking conformation resulted in a different duplex thermal stability and corresponding CD spectra. Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 109 6.3 Post-synthetic CuAAC Chemistry and Oligonucleotide Purification As discussed in Chapter 5.5.2, the single incorporation of 1,2,3-triazole linked aliphatic NiII porphyrin in the minor groove of a duplex resulted in a thermal destabilisation of approximately 5.5-7.0 °C. This was significantly less than the equivalent aromatic linked porphyrin which showed a destabilisation of 14.7-16.0 °C. This was most likely due to the extra flexibility resulting from the additional sp3 carbon. Therefore, investigation has continued further by incorporating multiple NiII aliphatic porphyrins (50, Figure 6.1) in a zipper fashion in the minor groove. 6.3.1 Microwave Accelerated Post-synthetic CuAAC Reaction β-Pyrrolic functionalised azido porphyrin 50 (Figure 6.1) was synthesised from 5,10,15,20-tetraphenylporphyrin phosphonium salt79, 87 as described in Chapter 2. The NiII ion was inserted into the porphyrin core to prevent the uptake of copper during the CuI catalysed CuAAC reaction. DMT-off ONs on CPG support were prepared containing single or double internal insertions of commercially available 2′-O-propargyl uridine or 2′-O-propargyl adenosine (Figure 6.2) using automated DNA synthesis. N N N N 50 N3 Ni Figure 6.1 β-Pyrrolic functionalised azido porphyrin 50. As discussed in Chapter 5.4.2, the commonly used method involving the shaking of the reaction mixture containing azide 50, CuSO4, sodium ascorbate and an oligonucleotide bound to a CPG support at room temperature resulted in poor conversion of DNA to the desired product for mixmer sequences. It has been previously reported144, 146 that microwave irradiation can be used to push the CuAAC reaction to completion, Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 110 especially for the ONs possessing several terminal alkynes in the sequence. In our case, focused microwave irradiation of CPG bound ONs possessing one or two propargyl nucleotides (Figure 6.2) for 20 minutes at 70 °C resulted in a full conversion of starting ONs as indicated by denaturing PAGE analysis of the cleaved ONs. This was in contrast to the results obtained when using unfocused microwave irradiation. ON25 3' GTACGTATATATAGAGCG ON26 5' CATGCATATATATCTCGC ON27 3' GTACGTATA4ATAGAGCG ON28 5' CATGCATATA4ATCTCGC ON29 5' CATGCATAT7TATCTCGC ON30 5' CATGCATA4ATATCTCGC ON31 5' CATGCAT7TATATCTCGC ON32 5' CATGCA4ATATATCTCGC ON33 5' CATGC7TATATATCTCGC ON34 3' GTACGTA4A4ATAGAGCG ON35 5' CATGCATA4A4ATCTCGC ON36 5' CATGCAT7T7TATCTCGC NH O ON O OO O N N N N Ph Ph Ph Ph Ni N N N N NN N NH2 O O O O N N N N Ph Ph Ph Ph Ni N N N 7 4 Figure 6.2 Synthesised oligodeoxynucleotides. DMT-off oligonucleotides on CPG (0.33 µmol) containing 2′-O-propargyl uridine or 2′- O-propargyl adenosine were removed from their corresponding columns and placed into a microwave reaction vessel together with compound 50 (7.67 µmol, 23 eq) in degassed DMSO (200 µL). Freshly prepared CuSO4·5H2O (0.32 µmol, 0.96 eq, 8 µL of a 40 µmol/mL solution in degassed H2O) and sodium ascorbate (1.25 µmol, 3.8 eq, 25 µL of a 50 µmol/mL solution in degassed H2O) were added. The reaction mixture was then irradiated in a microwave synthesiser (Discover, CEM Corporation, 70 °C, 100 watts, 20 min). The content of the reaction was transferred to a microcentrifuge tube and the CPG supports were repeatedly washed with DCM (1.5 mL) to remove any unreacted 50 followed by H2O to remove any remaining copper. This resulted in a red coloured CPG, which was an indication of the progression of the reaction. Unreacted 50 was recovered in 80-90% yield by washing the DCM solution containing porphyrin with H2O, drying over MgSO4, and precipitating from DCM:MeOH. The obtained DMT-off oligonucleotides bound to CPG supports were cleaved with 32% aq NH4OH (0.5 mL) at RT for 2 hours and then at 55 °C overnight. Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 111 6.3.2 Purification and Characterisation of Oligonucleotides Possessing Two Porphyrin Modifications Cleaved porphyrin possessing ONs were purified using C18 puri-pak columns eluting single modifications in 20% CH3CN:H2O and double modifications in 30-40% CH3CN:H2O. Oligonucleotides could be purified directly from the aq. NH4OH solution using C18 puri-paks which was advantageous to the previously used HPLC purification method. It was observed that significant quantities of oligonucleotides containing two porphyrin modifications remained on the column during purification. After purification ONs were freeze dried, redissolved in H2O (100 µL), precipitated from LiClO4 and acetone (0.01 M lithium perchlorate in acetone (1.6 mL)) and dissolved in 100 µL of water to give a deep red solution of ON27-ON36 (Table 6.1). Heating to 70 °C for 1 hour was required to dissolve some ONs. Oligonucleotides were characterised by MALDI-TOF MS in the negative mode using either 2ʹ,4ʹ,6ʹ-trihydroxyacetophenone, 3- hydroxypicolinic acid or 6-azathiothymine as a matrix and dibasic ammonium citrate as a co-matrix (Table 6.1). Purity was checked using denaturing 20% PAGE, showing a red single band with a significant retardation compared to the wild type oligonucleotide. ON34-ON36 containing two porphyrins did not penetrate into the gel (Figure 6.3). Table 6.1 ONs synthesised and their mass spectroscopic analysis. Strand Sequence m/z, calcd., Da m/z, found, Da ON25 3’ GTACGTATATATAGAGCG 5562.6 - ON26 5’ CATGCATATATATCTCGC 5433.5 - ON27 3’ GTACGTATA4ATAGAGCG 6431.2 6427.7 ON28 5’ CATGCATATA4ATCTCGC 6302.2 6297.6 ON29 5’ CATGCATAT7TATCTCGC 6316.2 6309.4 ON30 5’ CATGCATA4ATATCTCGC 6302.2 6297.0 ON31 5’ CAT GCAT7TATATCTCGC 6316.2 6316.7 ON32 5’ CATGCA4ATATATCTCGC 6302.2 6296.8 ON33 5’ CATGC7TATATATCT CGC 6316.2 6312.8 ON34 3’ GTACGTA4A4ATAGAGCG 7299.8 7305.1 ON35 5’ CATGCATA4A4ATCTCGC 7170.8 7167.5 ON36 5’ CATGCAT7T7TATCTCGC 7198.8 7199.1 Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 112 ON25 ON31 ON35 Figure 6.3 Representative PAGE (20% with 7 M urea) of unmodified oligonucleotide ON25 and porphyrin modified oligonucleotides ON31 and ON35 captured using an Olympus digital camera after staining with Stains-All®. Porphyrin modified oligonucleotides are circled. Dyes were used for ON25 only. 6.3.3 Application of the CuAAC Reaction to ZnII and FeIII Porphyrins The development of the CuAAC reaction using NiII porphyrins allowed us to expand our investigation into ZnII and FeIII porphyrins (Figure 6.4). ZnII porphyrins have been widely used in numerous applications involving light harvesting,88, 89 electron transfer,44, 159 fluorescence, reactive oxygen species production156 etc. Additionally, they can be easily demetallated in acidic conditions to give the free base porphyrin which can be used to obtain various metalled species. Porphyrin containing FeIII ions have been used in many biological applications such as oxygen transport and reaction catalysts.160 The combination of porphyrins containing different metals in close proximity on a DNA could provided systems with very interesting photodynamic and electronic properties. Dye Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 113 ZnTPP N3 45 ZnTPP N3 FeIIIClTPP N3 4951 Figure 6.4 FeIII and ZnII porphyrin azides. Initial CuAAC reactions on FeIII porphyrins failed to produce any sign of the desired conjugates using a CuII catalyst and sodium ascorbate. Comparing the redox potentials of FeIII to FeII (~0.77 V) and CuII to CuI (~0.15 V)161 led us to conclude that the FeIII was being preferably reduced over the CuII. It should be noted that although these redox potentials are not specific to porphyrins they will be a close approximation to the actual redox values. Repeating the reaction using Cu(ACN)4PF6 in THF at RT for three days, followed by the cleavage of the oligonucleotides using aq NH4OH, provided the desired conjugate as a light green solution. The solution was freeze dried and resulting solid was dissolved in H2O. Purity was checked using 20% or 12% denaturing PAGE, however it was found that the oligonucleotide containing an iron porphyrin did not penetrate into the gel. Attempts to purify the oligonucleotide by C18 HPLC or C18 puri- pack columns failed to elute any of the desired oligonucleotide. Inspection of the puri- pack column showed that the porphyrin-ON complex was bound to the solid support. No mass spectra analysis was performed as the desired oligonucleotide could not be purified. CuAAC reactions using ZnII porphyrins 45 and 49 were problematic. In the primary experiment, in which porphyrin 49 and CPG bound ON with a sequence equivalent to the unmodified ON28 were shaken for three days at RT, a green solution was obtained on cleavage with aq NH4OH. Precipitation of the oligonucleotide using LiClO4 and acetone resulted in a green coloured pellet. Denaturing 20% PAGE showed a single green band which migrated between NiII porphyrin-DNA and unmodified ONs. Unfortunately, attempts to repeat this reaction using a second batch of 49 or using porphyrin 45 failed to produce any sign of the desired conjugates. Likewise, performing the reaction under microwave conditions was unsuccessful. On closer observation of the IR signals of 45 and 49, differences were noticed that could account for the variations in reactivity observed. In the initial batch of compound 49 only a single azide asymmetric stretch was observed at 2097.2 cm-1, however the second batch showed two independent Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 114 signals at 2097.1 and 2122.7 cm-1. A similar trend was observed in the IR spectrum of compound 45. This change in energy of the azide signal is consistent with the coordination of the azide to the fifth coordination site of ZnII that occurs perpendicular to the plane of the porphyrin core (Figure 6.5).162, 163 The addition of a stronger ligand such as pyridine to porphyrins 45 and 49 resulted in only a single IR signal at 2097.1 cm-1 and 2015.2 cm-1 respectively, suggesting azide coordination indeed occurred. Although the addition of pyridine was not shown to inhibit the CuAAC reaction NiII porphyrin azides 50, the addition of pyridine to the CuAAC reaction involving porphyrins 45 and 50 failed to produce any DNA conjugates. Zn N ZnN Zn N N N ZnN N N A B N N N N Figure 6.5 Possible modes for the coordination of 49 (A) and 45 (B). Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 115 6.4 UV-Vis and CD Spectroscopic Studies of DNA-Porphyrin Conjugates Containing Multiple Porphyrins The thermal stability of the porphyrin modified DNA duplexes possessing one to four NiII porphyrins in various arrangements in the minor groove was accessed using UV- Vis thermal melting at 260 and 423 nm (Table 6.2, Figure 6.6). The interaction of the porphyrin moieties and DNA backbone structures were also examined by CD spectroscopy. In addition, molecular modelling calculations of plausible DNA structures were performed using the AMBER* force field.151, 152 Figure 6.6 Representative UV melting profiles (260 nm) of unmodified and modified duplexes: a) duplex 1 and porphyrin modified duplexes 3, 9, 11 and 18, C = 1.0 µM of each strand in 20 mM sodium cacodylate, 100 mM NaCl and 5 mM MgCl2, pH 7.2; b) duplex 1 and porphyrin modified duplexes 23 and 24 at low salt concentrations, C = 1.0 µM of each strand in 20 mM sodium cacodylate, 6.25 mM NaCl, pH 7.2. 10 20 30 40 50 60 70 80 90 0.0 0.5 1.0 A bs o rb a n c e (no rm a liz e d) Temperture / °C Duplex 1 Duplex 3 Duplex 9 Duplex 11 Duplex 18 a) 30 40 50 60 70 80 90 0.0 0.5 1.0 A bs o rb a n c e (no rm a liz e d) Temperture / °C Duplex 1 Duplex 23 Duplex 24 b) 116 Table 6.2 Arrangement of porphyrins in DNA duplexes and their melting temperaturesa Duplex Strands Arrangement Td 260 Ta 260 Td 423 Ta 423 ∆Tm 260 ∆T260/P 1 ON25/ON26 5'3' 56.0 (46.1)b 56.0 (46.1) ---- ---- ---- ---- 2 ON27/ON26 5'3' 51.2 51.0 52.0 50.0 -4.8 -4.8 3 ON25/ON28 5' 3' 51.2 50.8 53.3 52.7 -4.8 -4.8 4 ON25/ON29 5' 3' 50.0 50.0 49.2 48.0 -6.0 -6.0 5 ON25/ON30 5' 3' 53.5 53.5 53.8 53.7 -2.5 -2.5 6 ON25/ON31 5' 3' 50.8 50.8 52.0 50.7 -5.2 -5.2 7 ON25/ON32 5' 3' 52.1 52.0 52.0 51.2 -3.9 -3.9 8 ON25/ON33 5' 3' 51.0 51.0 50.1 50.0 -5.0 -5.0 9 ON25/ON35 5' 3' 27.4 26.1 NVTc NVT -28.6 -14.3 10 ON25/ON36 5' 3' 25.0 25.0 NVT NVT -31.0 -15.5 11 ON27/ON28 5' 3' 71.8 (57.8) 72.6 (58.0) 72.2 (58.9) 74.5 (59.8) +15.8 (+11.7) +7.9 (+5.85) 12 ON27/ON29 5' 3' 71.2 71.0 72.6 72.3 +15.2 +7.6 13 ON27/ON30 5' 3' 71.1 71.7 72.3 73.5 +15.1 +7.55 14 ON27/ON31 5' 3' 69.2 68.0 71.8 71.4 +13.2 +6.6 15 ON27/ON32 5' 3' 66.0 65.9 70.6 70.1 +10.0 +5.0 16 ON27/ON33 5' 3' 65.1 65.4 70.0 70.0 +9.1 +4.55 116 117 Table 6.2 cont. Arrangement of porphyrins in DNA duplexes and their melting temperaturesa Duplex Strands Arrangement Td 260 Ta 260 Td 423 Ta 423 ∆Tm 260 ∆T260/P 17 ON34/ON28 5' 3' 71.2 (63.7) 68.8 (61.2) 73.0 (62.7) 70.8 (61.0) +15.2 (+17.6) +5.07 (+5.86) 18 ON34/ON30 5' 3' 70.8 66.2 73.2 70.0 +14.8 +4.93 19 ON34/ON31 5' 3' 71.6 69.6 71.6 70.8 +15.6 +5.2 20 ON34/ON32 5' 3' 68.0 66.2 68.5 67.6 +12.0 +4.0 21 ON27/ON35 5' 3' 69.0 66.5 70.5 69.5 +13.0 +4.33 22 ON27/ON36 5' 3' 71.1 69.8 72.0 70.8 +15.1 +5.03 23 ON34/ON35 5' 3' >90 (76.2) >90 (74.8) >90 (65.8) >90 (65.3) >34 (+30.1) >8.5 (+7.53) 24 ON34/ON36 5' 3' >90 (75.8) >90 (77.5) >90 (75.0) >90 (76.0) >34 (+29.7) >8.5 (+7.43) a Tm (°C) data for antiparallel duplex melting determined using the maximum of the first derivative of the UV-vis melting curves (λ = 260 and 423 nm, 0.5 °C/min). C = 1.0 µM of each strand in 20 mM sodium cacodylate, 100 mM NaCl and 5 mM MgCl2, pH 7.2. b C = 1.0 µM of each strand in 20 mM sodium cacodylate, 6.25 mM NaCl, pH 7.2. cNVT = No visible transition. 117 Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 118 The destabilisation of DNA duplexes that was observed in the cases of a single porphyrin substituent (Table 2, duplexes 2-8, 2.5 to 6.0 °C) is consistent with that observed in Chapter 5. Molecular modelling indicated that the lowest energy structure of duplex 2 held the porphyrin within the minor groove (Figure 6.7A). This is most likely due to a combination of Van der Waals interactions between the porphyrin and the nucleobases and decreased entropy as the solvent cage around the porphyrin is partially removed (hydrophobic effect). Double modifications on the same strand (duplex 9 and 10, Figure 6.7B) resulted in further destabilisation (∆Tm –28.6-31.0 °C) compared to the unmodified duplex 1. Figure 6.7 A representation of the lowest energy AMBER* force field minimised structures of duplex 2 (A) and duplex 10 (B). To establish the effect on duplex stability of stacked porphyrins in the minor groove, duplexes 11-24 were prepared containing two, three or four alternating porphyrin substituents. Significantly enhanced thermal stability (∆Tm +15.1-15.8 °C) was observed for duplexes in which the porphyrins were either attached to adjacent (e.g. duplexes 11 and 13) or complementary bases (e.g. duplex 12). It is well known that as a result of their highly planar aromatic structure porphyrins commonly form aggregates through pi-pi stacking.164 During UV-Vis thermal meltings, recorded from 230 to 500 nm at 10 °C intervals, a bathochromic shift of 1.5-4.0 nm was observed for the Soret band (~420 nm) for duplexes 11-24 in which porphyrins were placed in both strands. In comparison, singly modified duplexes 2-8 did not exhibit this shift (Figure 6.8A). This Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 119 suggested the formation of H-aggregates between interlocked porphyrins bound to complementary strands of the duplex.39 The observed shift in the Soret band upon the formation of H-aggregates is in a similar range to the previously studied DNA-based porphyrin dimers.39 As the distance between the porphyrins on the complementary stands increased (duplexes 14-16) the Tm decreases. Figure 6.8 Representative UV-Vis annealing profiles: a) duplex 2 with a single modification showing an increase in absorbance at 260 nm and a decrease at 420 nm; b) duplex 11 with two interlocking modifications showing an increase in absorbance 260 nm and a bathochromic shift from 417-421 nm during melting. It is interesting to note that for duplexes 11-16 the Tm values for the porphyrin region (423 nm) were slightly higher than those corresponding to the duplex DNA region (260 nm). The largest difference of 4.9 °C corresponded to duplex 16 that has three base- pairs in between overlapped porphyrins. This indicated that the porphyrins dissociate after the dissociation of the DNA duplex. According to molecular modelling, face-to- face pi-pi interactions between two porphyrins placed on opposite strands were observed for duplexes 11, 12 and 13 and to some extent even in the case of duplex 16 due to the flexibility of the system (Figure 6.9). Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 120 Figure 6.9 An AMBER* force field lowest energy minimised structures of porphyrin modified duplexes. A) Duplex 11 with flipped porphyrins, B) duplex 11 with non-flipped porphyrins, C) duplex 12 with flipped porphyrins, D) duplex 12 with non-flipped porphyrins, E) duplex 13 with non-flipped porphyrins and F) duplex 16. It is important to note that due to the flexible nature of the linker, numerous porphyrin conformations can be adopted relative to the DNA duplex, including a flipped conformation (Figure 6.10) in which porphyrins do not follow the sequence of the nucleotides in the duplex. The observed distances between O2′ atoms in duplexes 11 and 12 were between 6.1 - 7.7 Å and independent of the adopted conformation, i.e. flipped or non-flipped. However, the observed distance between O2′ atoms in duplex 13 Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 121 was significantly larger (11.3 Å), therefore, the non-flipped conformation was the preferred option. The orientation of the porphyrin moieties and the adopted conformations was confirmed by CD spectroscopy (Figure 6.11). Figure 6.10 Plausible arrangement of porphyrins in duplex 11 showing the flipped and non-flipped conformations. 300 400 500 -80 -40 0 40 80 120 ∆ε / ∆ε / ∆ε / ∆ε / M − 1 − 1 − 1 − 1 cm − 1 − 1 − 1 − 1 λ / nm Duplex 1 Duplex 11 Duplex 12 Duplex 13 Figure 6.11 CD spectra of duplexes 1, 11, 12 and 13. In case of duplex 13, strong positive and negative bands originating from the porphyrin’s Soret band appeared at 422 and 448 nm, respectively. This negative CD exciton couplet (–/+ pattern) indicates the anticlockwise orientation of the transition dipoles of the porphyrins (Figure 6.12).38, 49, 50 The flipping behaviour was confirmed by the different CD profiles in the region of the Soret band for duplexes 11 and 12. Molecular modelling shows that in the non-flipped structures there is a clockwise orientation of the porphyrins’ transition dipoles which should provide a positive CD Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 122 exciton couplet (+/– pattern). Contrary to this, an anticlockwise orientation is observed for the flipped structures which should give a rise to a negative CD exciton couplet. In fact the pattern of CD spectra for duplexes 11 and 12 in the Soret region is +/–/+ with maximum intensities at 474/441/413 and 460/436/413, respectively. This was attributed to the more dynamic interactions of the porphyrins and existence of both flipped and non-flipped porphyrins in duplexes 11 and 12. A negative band at 245 nm and a positive band around 274 nm in CD spectra clearly suggested that the modified duplexes retain their overall B-form double helix structure. At 85 °C both the positive and negative bands in the Soret region of the CD spectra vanished which indicated that there is no contact between porphyrins when duplexes 11-13 are melted. Figure 6.12 A representation the lowest AMBER* force field minimised structures of duplexes possessing two zipping porphyrins (nucleotides above and below have been removed for clarity). A and C: flipping porphyrins in duplexes 11 and 12 referring to the anticlockwise orientation of porphyrins’ transition dipoles, respectively. B and D: non-flipping porphyrins with clockwise porphyrin orientation in duplexes 11 and 12, respectively. E: duplex 13 with non-flipping porphyrins referring to the anticlockwise orientation of porphyrins. The considerable difference in distances of O2′ atoms in the minor groove of DNA- RNA duplexes has been used in the construction of several nucleic acid zippers based on locked nucleic acids (LNA),165-168 uridine-2′-carbamates169 and O2′-pyrenylmethyl Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 123 nucleotides.170-172 These systems implement multiple chromophore residues (generally pyrenes) which upon duplex formation results in a characteristic excimer fluorescence due to the interaction between pyrenes in electronically excited and ground states. Such systems have been implemented in signalling of full complimentarity (detection of mis- matches165) with the ultimate goal to create multichromophoric arrays. In these studies the strongest interactions between chromophores were ascribed for duplexes equivalent to structures 11 and 12, however in our case an effective communication between porphyrins was also observed for structure 13. The ability for porphyrins in duplex 13 to interact is most likely due to the long flexible linker which allows for ideal positioning of chromophores even when O2ʹ atoms are spaced further apart. This is supported by recent work by Wengel et al.173 in which strong interactions between coronene moieties was observed in LNA duplexes equivalent to 11 and 13 when the coronene was tethered to 2′-amino-LNA by a long flexible linker (Figure 6.13A). Reducing the linker resulted in strong interactions only in structures equivalent to duplex 11 (Figure 6.13A). NH O ON O NO O O O P O ONH O ON O NO OP O O A B Figure 6.13 Coronene modified LNA possessing short (A) and long (B) linkers. The introduction of a third porphyrin moiety into the duplex did not result in a further increase of the thermal stability (Table 6.2, duplexes 17-22). It was observed that Tm values in the porphyrin region for duplexes 17 and 18 (73.0 and 73.2 °C, respectively) were higher than for duplex 20 (68.5 °C), in which flipping of the porphyrin substituents is unlikely to occur (see the discussion above for duplex 13). This suggested the existence of two possible classes of stacking arrangements (Figure 6.14). The first in which the single porphyrin intercalated between the two porphyrins on the complementary strand (duplexes 17 and 18), and the second in which the single Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 124 porphyrin was stacked either above or below the two porphyrins on the complementary strand (duplex 20). This was made possible only by the flexibility of the porphyrin- linker combination which does not require the porphyrin sequence to follow the sequence of the nucleotides. In the second group we also included duplex 21 based on the the change in melting temperature per porphyrin (∆T260/P) which was very close to that of duplex 20. The remaining duplexes with three porphyrins (19 and 22) were also included in the first group, in which all three porphyrins significantly contributed to the thermal stability of the complex. The lower values of ∆T260/P for duplexes 17-22 might also be explained by the increased center-to-center distances between porphyrins (4.8 - 7.5 Å) in comparison with duplexes having double modifications (duplexes 11-13, 4.4 - 5.0 Å) as observed in the molecular modelling studies. A more accurate conclusion about the flipping of porphyrins might be obtained upon combination of NiII porphyrins, which can serve as fluorescent quenchers,174 with different fluorescent molecules attached in the similar manner to the duplex. Figure 6.14 Plausible arrangements of porphyrins in duplexes with three modifications. More dramatic increases in thermal stability were observed for duplexes containing four porphyrins (duplexes 23 and 24). It should be noted that in order to measure the melting transitions of these very stable complexes the salt concentration had to be reduced from 100 mM to 6.25 mM NaCl and MgCl2 excluded completely. Thus, duplex 23 with adjacent porphyrins had a ∆Tm of +30.1 °C and duplex 24 with overlapping porphyrins had a ∆Tm of +29.7 °C when compared to duplex 1 at low salt concentrations. This is a remarkable difference in thermal stability of ~65 °C between duplexes with four zipping porphyrins and duplexes with a double modification in one of the strands (duplexes 9 and 10). Interestingly, a Tm value recorded at 423 nm for duplex 23 is lower than that for duplex 24. This was thought to be a result of the different dynamic motions in the stack of porphyrins during melting. Indeed, molecular modelling showed that Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 125 porphyrins in duplex 23 could exist as a 2+2 stack while a contiguous stacking of all four porphyrins was observed for duplex 24 (Figure 6.15). Molecular modelling also showed that the introduction of the fourth closely attached chromophore leads to increased center-to-center distances between the inner porphyrins (9.6 Å) in duplex 23. In the CD spectra for both duplexes 23 and 24, broad negative bands were observed in the region of 400-450 nm, which can be ascribed to the complex interaction of four porphyrins in these structures (Figure 6.16). Further explaination may be proposed using calculated CD spectra9, 56 based on the four porphyrin DNA models obtained with AMBER* force field molecular modelling. Figure 6.15 A representation of the lowest energy ABMER* force field minimised structures of duplexes 23 (A) and 24 (B) viewed into the minor groove. This clearly shows the 2+2 stack in duplex 23 and the continuous stacking of porphyrins in duplex 24. Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 126 300 400 500 -100 0 100 200 ∆ε / M - 1 c m - 1 λ / nm Duplex 17 Duplex 19 Duplex 20 Duplex 22 300 400 500 -80 0 80 ∆ε / M - 1 c m - 1 λ / nm Duplex 11 Duplex 12 Duplex 13 300 400 500 -80 0 80 ∆ε / M - 1 c m - 1 λ / nm Duplex 13 Duplex 14 Duplex 15 Duplex 16 300 400 500 -60 0 60 ∆ε / M - 1 c m - 1 λ / nm Duplex 1 Duplex 4 Duplex 9 300 400 500 -70 0 70 ∆ε / M - 1 c m - 1 λ / nm Duplex 23 Duplex 24 ∆ε / M - 1 c m - 1 ∆ε / M - 1 c m - 1 ∆ε / M - 1 c m - 1 ∆ε / M - 1 c m - 1 ∆ε / M - 1 c m - 1 Figure 6.16 CD spectra of duplexes 1, 4, 9 and 11-24. The melting profiles showed virtually no hysteresis at either 260 or 423 nm for most of the duplexes studied including the four porphyrin zippers (Figure 6.17). This indicates that the denaturing-annealing kinetics was determined by the interaction of nucleic bases and not by the porphyrins. Also, the observed broadening of the melting curves for the four porphyrin duplexes 23 and 24 is thought to correspond to the unravelling of strands around the porphyrin aggregates. It is assumed that β-pyrrolic functionalisation of the porphyrins is vital as it prevents unfavourable interactions of the meso substituted Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 127 phenyl rings and allows for the easier accommodation of the porphyrins in the DNA environment. Figure 6.17 Representative UV melting profiles (260 nm) of duplexes 3 (a), 13 (b), 18 (c) and 24 (d) showing both denaturing (solid) and annealing (dashed) profiles. C = 1.0 µM of each strand in 20 mM sodium cacodylate, 100 mM NaCl and 5 mM MgCl2, pH 7.2 for duplexes 3, 13 and 18. C = 1.0 µM of each strand in 20 mM sodium cacodylate, 6.25 mM NaCl, pH 7.2 for duplex 24. 6.5 Conclusion In conclusion, a quick and reliable microwave accelerated method for the post-synthetic attachment of azidoporphyrins to ONs containing the commercially available 2′-O- propargyl uridine and 2′-O-propargyl adenosine has been developed. The covalent attachment of adjacent porphyrin moieties into complementary strands of DNA led to the formation of face-to-face or H-aggregates in the minor groove, and resulted in a significant increase in the duplex thermal stability. The interaction of only two porphyrins led to thermal stabilisation of 7.55-7.9 °C per porphyrin with practically no 30 40 50 60 70 80 90 0.0 0.5 1.0d) Ab so rb an ce (no rm a liz e d) Temperture / °C Duplex 24 30 40 50 60 70 80 90 0.0 0.5 1.0 Ab so rb an ce (no rm al iz ed ) Temperture / °C Duplex 3a) 30 40 50 60 70 80 90 0.0 0.5 1.0b) Ab so rb an ce (no rm al iz ed ) Temperture / °C Duplex 13 30 40 50 60 70 80 90 0.0 0.5 1.0c) Duplex 18 Ab so rb an ce (no rm al iz ed ) Temperture / °C Chapter 6 - Porphyrin H-Aggregate Formation in the Minor Groove of the Duplex 128 hysteresis between denaturing and annealing profiles. This is a significant improvement compared to previously studied DNA-porphyrin constructs.38, 39, 42-44 Duplex thermal stability exceeded 90 °C when four porphyrins were stacked in the zipper motif. Based on molecular modelling and CD spectroscopy studies it was proposed that porphyrins may not necessarily follow the sequence of nucleotides in duplexes. However, duplex 13, in which porphyrins actually followed the sequence of nucleotides, should be considered as a model for the DNA-based chromophoric assemblies possessing flexible linkers. We believe these findings in combination with previously published reports on porphyrin modified DNA duplexes lay a foundation for the future design of artificial DNA-chromophore supramolecular architectures and for their applications in material science and nanotechnology. Chapter 7 – Summary and Future Directions 129 Chapter 7 Summary and Future Directions The aim of this thesis was to create and study novel DNA-porphyrin supramolecular architectures, for use in nanotechnology, based on β-pyrrolic modified porphyrins. This has been achieved through the development of both covalently and non-covalently (lipophilic) linked porphyrin complexes. During the synthesis of porphyrin precursors for DNA modification a modified Horner- Emmons reaction was developed for use in porphyrins. This enabled the creation of a versatile synthetic method for the construction of ethynyl linked β-pyrrolic porphyrin derivatives. This high yielding, scalable methodology improved on existing methodologies by negating the need of a metal catalyst and 2-bromo-5,10,15,20- tetraphenylporphyrin. Using the Horner-Emmons method, synthesis and evaluation of the performance of a number of porphyrin dyes in DSSCs applications was performed. To develop this chemistry further it would be advantageous to create a porphyrin modified phosphonate derivative. Such a modification may allow for the easy production of β-pyrrolic ethynyl linkers without the time consuming synthesis of multiple aromatic phosphonates. Using the appropriate lipophilic building blocks, synthesis of non-covalently bound lipophilic complexes from short sequences of single stranded, duplex and GQ oligodeoxynucleotides was achieved. Loading studies on single stranded and duplex DNA showed slight overloading of porphyrins on DNA. Unfortunately, longer DNA sequences, which could be used for the construction of aligned DNA films, resulted in the formation of insoluble complexes. For future work it may be possible to create soluble complexes of large ONs by adapting the synthetic method to reduce the porphyrin loading ratio on DNA. Using covalent modification techniques, methods for the pre-synthetic attachment of ethynylporphyrin and various azidoporphyrins to 5-iodo and 5-ethynyl-2ʹ-deoxyuridine respectively, was achieved using Sonogashira and CuAAC chemistry. From insight into the comparative levels of conjugation between the porphyrin cores and the nucleosides was gained. More importantly, development of conventional and microwave accelerated Chapter 7 – Summary and Future Directions 130 CuAAC reaction methods for the site specific, post-synthetic, internal attachment of NiII β-pyrrolic functionalised azidoporphyrins to ONs was accomplished. Reactions between azido porphyrins, possessing both aliphatic and aromatic linkages, and ONs containing various terminal alkynes allowed us to screen the effect of various single porphyrin modifications on single stranded, duplex and triplex DNAs. Single stranded porphyrin possessing ONs formed pH sensitive i-motifs or pH independent aggregates depending on the nucleotide sequence. Single internal duplex modification resulted in thermal destabilisation of 6.3-18.0 °C (in pyridimine sequences) depending on the location of the porphyrin in the duplex. Single porphyrin modification of a TFO strand led to thermal stabilisation of the resulting triplexes (∆Tm = 3.0-9.0 °C at pH 6.0). Further stabilisation of a triplex possessing a porphyrinic bulged insertion may be possible by reducing the tether length. Using the information gained from duplexes possessing single internal modifications, post-synthetically modified 2′-O-propargyl possessing duplexes with up to four aliphatic porphyrins were synthesised. This provided systems in which adjacent porphyrin moieties placed in complementary strands led to significantly increased duplex thermal stability as a result of face-to-face or H-aggregates formation in the minor groove of the duplex. The interaction of only two porphyrins resulted in thermal stabilisation of 7.55-7.9 °C per porphyrin and duplex thermal stability exceeded 90 °C when four porphyrins were stacked in the zipper motif. This was a significant improvement compared to previously studied DNA-porphyrin constructs, especially those in which the porphyrins are placed in the wider major groove.44 Based on molecular modelling and CD spectroscopy studies it was proposed that porphyrins may not necessarily follow the sequence of nucleotides in duplexes. Currently the post-synthetic CuAAC reaction is limited by the choice of metals within the porphyrin. It is important for the future development to incorporate porphyrins possessing various metal ions, particularly those ones leading to highly fluorescent porphyrins (e.g. ZnII, PtII, MgII or H2). If this is not achievable using CuAAC chemistry it may be possible to use ethynyl possessing DNA with porphyrins using alternative metal free ‘click reactions’ such as nitrile oxide chemistry (Figure 7.1).175, 176 This chemistry would allow for the post-synthetic incorporation of free base porphyrins followed by the insertion of metal ions. Chapter 7 – Summary and Future Directions 131 N NH N HN BaseO O O O P O- O NHO N NH N HN BaseO O O O P O- O O N+ i) Figure 7.1 Possible metal free nitrile oxide post-synthetic chemistry. Reagents and conditions: Chloramine-T or NCS, NaHCO3, DMSO, H2O, RT. Further development of a stacked arrangement of chromophores by incorporating a variety of moieties in the minor groove of DNA could be possible. Such chromophores may include fluorescent anthracene or pyrene moieties in combination with fluorescent quenching NiII porphyrins, which could confirm the flipped and non-flipped arrangement of chromophores in DNA. Further duplex thermal stabilisation and increased solubility may be possible by reducing the hydrophobicity of the porphyrin by incorporating charged groups in the meso positions of 5,10,15,20- tetraphenylporphyrins. This could be particularly interesting if the alternation of stack anionic (Figure 7.2A) and cationic (Figure 7.2B) porphyrins in the minor groove of the duplex was investigated. Possibilities also exist in 5,15-diphenylporphyrin derivatives (Figure 7.2C) where the porphyrin would be attached to DNA via the meso position but not in an orthogonal manner. N NH N HN N N N N BaseO O O O P O- O N N N N NH N HN OOC COO COO OOC N NH N HN A B C Figure 7.2 Incorporation of possible anionic (A), cationic (B) 5,10,15,20-tetraphenylporphyrins or 5,15- diphenylporphyrin derivatives. Chapter 7 – Summary and Future Directions 132 By carefully choosing chromophores, such as combinations of ZnII and free base porphyrins, creation of a molecular wire along the minor groove of the duplex may be possible. This could lead to interesting photonic properties. Of particular interest may be the development of systems for energy upconverstion.155, 177, 178 Energy upconversion, which can occur via triplet-triplet annihilation where one higher energy singlet excited molecule is generated from two lower energy triplet excited molecules, has been shown to occur in solution involving ZnII/PtII porphyrins and anthracene/pyrene moieties. It may be possible to place these moieties in complementary strands such that stacking in the minor groove occurs on duplex formation. This may allow for controllable energy upconversion on a duplex scaffold. Chapter 8 – Experimental Methods 133 Chapter 8 Experimental Methods 8.1 Reagents and Equipment used for the Synthesis of Porphyrin Derivatives Reagents Solvents and reagents were supplied from many different sources, generally as AR grade. Chromatography solvents were distilled laboratory grade. H2O used in porphyrin synthesis was of reverse osmosis grade. DMSO was dried over 4 Å molecular sieves and distilled at reduced pressure. THF was dried by passing argon degassed solvent through activated alumina columns. Dry DCM and DMF were prepared by distilling AR grade solvent over CaH2 under N2 atmosphere. CHCl3 was dried over K2CO3 and distilled under N2 atmosphere. Reaction Monitoring and Chromatographic Purification Due to the coloured nature of the chromophores, most of the reactions were easily monitored with thin layer chromatography (TLC) on silica TLC plates (60 F254) purchased from Merck and were visualised in UV light (254 nm) when necessary. The exception was the incomplete Horner-Emmons reaction where the halovinyl intermediate and the alkyne derivatives had the same Rf. In this case the reaction had to be worked up and the ratio of halovinyl intermediate to alkyne was determined by 1H NMR spectroscopy. Metallation of porphyrins with ZnII generally resulted in compounds with a slightly lower Rf and metallation with NiII and CuII resulted in a higher Rf. Metallation with FeIII resulted in a very polar compound that could only be moved on TLC using a 2-5% methanol in DCM. Malonic acid formation resulted in the production of very polar material that could only be separated from the decarboxylated acid on TLC in MeOH:acetic acid:DCM (5:1:94). Porphyrin nucleosides were very polar and moved as one or two spots in MeOH:DCM (1:9). Chromatographic purification of compounds, Chapter 8 – Experimental Methods 134 when stated, was performed on the bench top using Silica 60 (230-400 mesh, SDS) or Al2O3 (Basic, 70-200 mesh, Scharlau). Various column diameters were used for the separation of different quantities of porphyrin: <50 mg (22 mmdia), 50-100 mg (30 mmdia), 100-200 mg (40 mmdia) and >200 mg (60 mmdia). Precipitation of Porphyrins The experimental methods in the following sections describe the purification of porphyrin derivatives by precipitation from DCM:MeOH. This involved the porphyrin residue being dissolved in DCM, filtered through clean cotton wool followed by the addition of a significant quantity of MeOH. The volume was then reduced in vacuo to approximately 5-10 mL. Additional MeOH was added and the process was repeated until a precipitate formed. An equivalent process was used to precipitate compounds from DCM:hexane. NMR Spectroscopy The primary characterisation technique for the porphyrin compounds was 1H NMR spectroscopy. Porphyrin solutions of approximately 2 x 10-2 M in CDCl3 or d6-DMSO were prepared and 1H NMR spectra were obtained on 400 or 500 MHz Brüker instruments using Topspin software. Chemical Shifts are given relative to TMS or the residual protium (d6-DMSO, 2.51 ppm, pyridine 7.10 ppm) when TMS was not present. Although porphyrin compounds appear to have complex spectra, they are generally first order in nature and are easily assigned with the aid of short range COSY experiments. A typical example of a β-pyrrolic modified TPP derivative (Figure 8.1) is described here. At high field, this spectrum shows the signals of two highly shielded nitrogen protons at the centre of the aromatic porphyrin core, as a broad singlet. This signal is lost on metallation. Moving up-field are the signals of the vinylic hydrogens (H1ʹ and H2ʹ) as doublets with splitting of approximately 12.0 and 16.0 Hz for the cis and trans isomers respectively. Next come the aromatic hydrogens which are shown as doublets with a splitting of approximately 8.0 Hz. These are easily assigned from COSY spectra or coupling constants. The Hortho of the meso functionalised phenyl rings are shown around 7.8 ppm as multiplets due the lack of symmetry in the porphyrin. Likewise, Hmeta Chapter 8 – Experimental Methods 135 and Hpara hydrogens occur as multiplets around 7.4 ppm. The β-pyrrolic hydrogens are further down-field typically between 9.1 and 8.6 ppm. A typical coupling of 4.5 – 4.9 Hz can be observed for these protons but often they converge into overlapping chemical shifts. The furthest down-field signal is generally that of H3 – the β-pyrrolic proton next to the β-pyrrolic substituent. The porphyrin ring current heavily deshields this proton, with an additive contribution from the β-pyrrolic substituent. N NH N HN R Hpara Hmeta Hortho Hβ−pyrrolic H2' HaromaticH1' Figure 8.1 1H NMR spectroscopic assignments for β-pyrrolic functionalised porphyrins. 1H NMR spectroscopy was the primary method for the determination of the presence of the halovinyl intermediate in the desired alkyne compound. The halovinyl could be observed by the presence of multiple NH signals (generally three) at approximately - 2.70 ppm. Mass Spectrometry Low resolution mass spectrometry was used to confirm the identity of porphyrins. Masses were obtained as M+ ions using a Waters Micromass MALDI in the positive ion mode. Molecular ions were generally obtained using a matrix free system and when a matrix was deemed necessary α-cyano-4-hydroxycinnamic acid, retinoic acid or 2-(4'- hydroxybenzeneazo)benzoic acid were used. Loss of N2 was observed in all azido functionalised porphyrins. Low resolution mass spectra of porphyrin nucleosides could not be obtained. Chapter 8 – Experimental Methods 136 High resolution mass spectrometry (HRMS) data for porphyrin compounds (excluding porphyrin nucleosides) were obtained by Raisa Imatdieva (University of Auckland) using a microTOF-Q ESI mass spectrometer or a VG-70SE FAB mass spectrometer with a p-nitrobenzyl alcohol matrix in the positive mode. Typically M+ or (M+H)+ ions were observed. High resolution mass spectrometry data for metallo porphyrin nucleosides was obtained by John Allen (Australian National University) using a Waters LCT ESI-TOF mass spectrometer in the negative mode and the free base porphyrin nucleoside on a Waters LCT Premier XE ESI-TOF mass spectrometer in the positive mode. UV-Vis Absorption Spectroscopy Solution UV-Vis spectroscopy was carried out for all free base and metallo porphyrin derivatives using a Shimadzu UV-3101PC UV-VIS-NIR-scanning spectrophotometer. The typical adsorption spectra were observed for all species, dominated by the Soret and Q bands as described in Chapter 1. Infrared Spectroscopy IR spectra were recorded when deemed necessary on a Nicolet 5700 FT-IR from Thermo Electron Corporation using an ATR attachment. Chapter 8 – Experimental Methods 137 8.2 Reagents and Equipment used for the Synthesis of Porphyrin Possessing Oligonucleotides Reagents Solvents and reagents were supplied from many different sources, generally as AR grade. H2O was used only after purification through a miliQ system. Unmodified short oligonucleotides were purchased from IDT (USA). Salmon testes DNA was purchased from Aldrich. Phosphoramidites used in automated DNA synthesis were commercially available from ChemGenes. Synthesis of Oligonucleotides DMT-off oligodeoxynucleotides were synthesised in a 1.0 µmol scale on 1000 Å CPG supports using a MerMade 4 Automated DNA Synthesiser from BioAutomation Corporation, using 4,5-dicyanoimidazole as an activator and 0.075 M solutions of the corresponding phosphoramidites in dry ACN. 2′-O-Propargyl uridine phosphoramidite or 2′-O-propargyl adenosine phosphoramidite (8 mg) was hand coupled by dissolving the amidite in the activator solution (1 mL) in a plastic syringe which was added to the support after the detritilation and washing cycles (coupling time of 5 minutes). Purification Porphyrin functionalised oligonucleotides were purified by HPLC or puri-pak columns. HPLC purification was achieved on a Waters 600 HPLC fitted with a Waters 2487 dual λ absorbance detector (260 and 427 nm) using a reverse-phase semipreparative Econosil C18 (10 µm, 10 × 250 mm) column. Puri-pak C18 purification cartridges were obtained from ChemGenes. Mass Spectrometry Molecular weights of the oligonucleotides were obtained using a Bruker Daltonics Autoflex MALDI TOF in the negative mode (University of Waikato) using either Chapter 8 – Experimental Methods 138 2ʹ,4ʹ,6ʹ-trihydroxyacetophenone, 3-hydroxypicolinic acid or 6-azathiothymine as a matrix and dibasic ammonium citrate as a co-matrix. Molecular weights of CT sequences were obtained using a Waters Micromass MALDI-TOF in the positive mode. Oligonucleotides were desalted using C18 zip-tips prior to loading on the MALDI plate. Gel Electrophoresis Purity of ON’s was checked using denaturing 20% PAGE (7 M urea). Porphyrin modified oligonucleotides were observed as red bands for NiII containing porphyrins and as green bands for ZnII and FeIII porphyrins. Gels were stained with Stains-All® dye and destained with H2O. Significant retardation of porphyrin modified oligonucleotides was observed compared to wild type oligonucleotides. UV-Vis Spectroscopy UV-Vis Spectroscopy and melting temperature measurements of oligonucleotides were performed on a CARY 100Bio UV-Vis spectrophotometer using a 2 × 6 multicell block with a Peltier temperature controller. Extinction coefficients for porphyrin modified oligonucleotides were calculated using the extinction coefficients of each nucleoside at 260 nm. Extinction coefficients of unmodified nucleotides (L/(mol.cm)): dA (15400), dG (11700), dT (8800), dC (7300). Extinction coefficients for porphyrin modifications (L(mol.cm)): 1 (14800), 2 (14800), 3 (15900), 4 (15900), 5 (9000) , 6 (9000) and 7 (21400). Circular Dichroism Spectroscopy CD spectra were recorded using an Applied Photophysics Chirascan CD spectrophotometer (150 W Xe arc) with a Quantum Northwest TC125 temperature controller. Identical solutions were used for CD spectroscopy to that used for melting studies with the exception of triplex DNA where 1.0 µM of each oligonucleotide strand was used. An average of ten scans was recorded (1 nm intervals, 240 nm/min, 1 cm pathlength), baselined against the appropriate buffer solution then smoothed. Data was recorded in mdeg and converted to delta epsilon. Chapter 8 – Experimental Methods 139 Molecular Modelling Molecular modelling calculations and the construction of duplexes were performed using MacroModel v9.1 from Schrödinger. All calculations were conducted using AMBER* force field and the GB/SA water model.151, 152 11-Mer duplexes containing unmetallated tetraphenylporphyrin modified nucleotides were generated from a B type DNA-DNA duplex using Maestro v9.1 from Schrödinger. Parallel triplexes were built by the consecutive superimposition of triples CGC and TAT, which were generated with Insight II v9.72 from MSI and transported into MacroModel. Bulged insertions 5 and 6 were constructed in several steps. The unmodified duplex or triplex was constructed and the appropriate strand was disconnected by the deletion of the phosphate group at the location of the bulged insertion. Afterwards, the bulged insertion up to and including the triazole ring was built and placed between bases in the structure. The phosphate backbone was reconnected and minimisation was then performed, creating enough space for the bulged insertion. The appropriate porphyrin was then built off the triazole ring. Constraints ensured the planarity of the porphyrins and were based on the metal complexed porphyrin available in the Maestro software (distances: N1-N3, N2-N4; 4.132 Å, force constant 100 and torsion angels: N1-C2-C3-C4, N2-C6-C7-C8, N3-C10- C11-C12, N4-C14-C15-C16; 0.0º, force constant 100). The stochastic dynamics calculations generating 250 structures were performed using an extended cut off potential with a SHAKE algorithm to constrain bond to hydrogen. Simulation temperature was 300 K, simulation time 500 ps and equilibration time 150 ps. All 250 structures were minimised using the PRCG method with convergence threshold of 0.05 KJ/mol and examined with Xcluster from Schrödinger to find representative low-energy structures. Chapter 8 – Experimental Methods 140 8.3 Experimental Procedures for Chapter 2 - Synthesis of β- Pyrrolic Porphyrin Derivatives for DNA Modification 11-Bromoundecanal (4) H Br O C11H21BrO Exact Mass: 248.078 Mol. Wt.: 249.188 11-Bromoundecanol (500 mg, 2 mmol) was added to a mixture of PCC (644 mg, 3 mmol, 1.5 eq) in dry DCM (10 mL) under Ar and stirred at RT until the reaction was complete by TLC (4 hr). The crude reaction mixture was purified by silica gel column chromatography eluting the product with DCM as a colourless band. The solvent was removed in vacuo to give 11-bromoundecanal as a light yellow oil (416 mg, 83%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.76 (t, 1H, J = 1.9 Hz CHO), 3.41 (t, 2H, J = 6.9 Hz, CHO(CH2)9CH2Br), 2.42 (td, 2H, J = 7.5 and 1.9 Hz, CHOCH2(CH2)9Br), 1.85 (dt, 2H, J = 7.7 and 6.9 Hz CHO(CH2)8CH2CH2Br), 1.62 (q, 2H, J = 7.5 Hz, CHOCH2CH2(CH2)8Br), 1.43-1.40 (m, 2H, CHO(CH2)7CH2(CH2)2Br), 1.30-1.28 (m, 10H, CHO(CH2)2(CH2)5(CH2)3Br). 2-(12ʹ-Bromododec-1ʹ-trans-ene)-5,10,15,20-tetraphenylporphyrin (5) N NH N HN Ph Ph Ph Ph C56H51BrN4 Exact Mass: 858.33 Mol. Wt.: 859.935 Br To a solution of TPPps (3, 400 mg, 0.43 mmol) and 11-bromoundecanal (4, 400 mg, 1.61 mmol, 3.75 eq) in dry DCM (20 mL), DBU (0.58 ml, 3.88 mmol, 9 eq) was added and the reaction was stirred under Ar at RT for 20 min. The resulting solution was passed down a plug of silica gel in DCM and the solvent was reduced in vacuo. The crude mixture was precipitated from DCM:MeOH and collected by filtration to give a purple solid (250 mg, 67%). 1H NMR spectroscopic analysis showed a cis/trans mixture of the desired compound 5 and TPPCH3 7. 1H NMR key signals (500 MHz, CDCl3, TMS): δ 6.20 (d, 1H, J = 11.7 Hz, Hcis), 6.07 (d, 2.5H, J = 15.7 Hz, Htrans), 2.58 (s, 1.5H, TPP-CH3). This gave a ratio of 5cis:5trans:TPP-CH3 (7) of 2:5:1. Chapter 8 – Experimental Methods 141 To the cis/trans mixture (33 mg, 38 µmol) in DCM (10 mL), I2 (29 mg, 3 eq) was added and the mixture was stirred at RT under darkness for 3 hrs. The organic layer was then washed with a saturated solution of Na2S2O3, separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting solid was dissolved in DCM and purified by silica gel column chromatography eluting the product with DCM as a red band. The solvent was removed in vacuo and the trans product was precipitated from DCM:MeOH to give a purple powder (25 mg, 76%) that contained TPPCH3 7 (5trans:7 of 6:1). 1H NMR (500 MHz, CDCl3, TMS): δ 8.84 (s, 2.3H, 5 and 7 Hβ-pyrrolic), 8.80- 8.78 (m, 2.3H, 5 and 7 Hβ-pyrrolic), 8.75-8.73 (m, 2.3H, 5 and 7 Hβ-pyrrolic), 8.69 (m, 1.16H, 5 and 7 Hβ-pyrrolic), 8.22-8.19 (m, 7H, Hortho), 8.08-8.06 (m, 2.3H, Hortho), 7.78- 7.69 (m, 14H, Hmeta, para), 6.42 (dt, 1H, J = 15.6 and 6.7 Hz, H2′), 6.08 (d, 1H, J = 15.5 Hz, H1′), 3.41 (t, 2H, J = 6.9 Hz, (CH2)9CH2Br), 2.58 (s, 0.45H, 7 TPPCH3), 2.05-2.01, (m, 2H, CH2(CH2)9Br), 1.90 - 1.84 (m, 2H, (CH2)8CH2CH2Br), 1.47-1.44 (m, 2H, (CH2)7CH2(CH2)2Br), 1.41-1.32 (m, 12H, CH2(CH2)6(CH2)3Br), -2.71 (br s, 2.33H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 274 (42.3), 420.5 (138), 518.5 (8.57), 552.5 (3.98), 594.5 (2.69), 660 (2.29). ESI-LRMS: m/z (%, assignment) cluster at 859-864, 859 (90, (M+H)+). HRMS: Calcd for (M+H)+ (C56H52BrN4): 859.3370, found: 859.3343. 2-(12ʹ-Pyridiumyldodec-trans-1ʹ-ene)-5,10,15,20-tetraphenylporphyrin bromide (6) N NH N HN Ph Ph Ph Ph N BrC61H56BrN5 Exact Mass: 937.372 Mol. Wt.: 939.035 Compound 5trans (25 mg, 29 µmol) was refluxed in pyridine (2 mL) for two days after which the solvent was removed in vacuo. The crude solution was dissolved in DCM and purified through a plug of silica gel, eluting TPPCH3 (4 mg) in DCM then the pyridinium salt in MeOH:DCM (3:20). The pyridinium salt was precipitated from DCM:hexane and collected by filtration. The resulting crystals were washed with a saturated aq. solution of NaBr to give a red solid (20 mg, 74%). 1H NMR (500 MHz, d6- DMSO): δ 9.06 (dd, 2H, J = 5.7 and 1.3 Hz, HPyr), 8.84-8.63 (m, 7H, Hβ-pyrrolic), 9.56 (tt, Chapter 8 – Experimental Methods 142 2H, J = 7.6 and 1.3 Hz, HPyr), 8.22-8.19 (m, 6H, Hortho), 8.12 (t, 1H, J = 7.6 Hz, HPyr), 8.08-8.06 (m, 2H, Hortho), 7.85-7.77 (m, 12H, Hmeta, para), 6.44-6.41 (m, 1H, H2ʹ), 6.02 (d, 1H, J = 15.4 Hz, H1ʹ), 4.57 (t, 2H, J = 7.4 Hz, (CH2)9CH2PyrBr), 2.00-1.98 (m, 2H, CH2(CH2)9PyrBr), 1.93-1.88 (m, 2H, (CH2)8CH2CH2PyrBr), 1.30-1.23 (m, 14H, CH2(CH2)7(CH2)2PyrBr), -2.69 (br s, 2H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 272 (37.5), 423 (120), 519 (8.01), 556.5 (3.65), 594.5 (3.13), 673.5 (4.73). ESI-LRMS: m/z (%, assignment) cluster at 858-861, 858 (100, (M-Br)+). HRMS: Calcd for (M-Br)+ (C61H56N5): 858.4530, found: 858.4480. 2-(12ʹ-Bromododecane)-5,10,15,20-tetraphenylporphyrin (8) N NH N HN Ph Ph Ph Ph Br C56H53BrN4 Exact Mass: 860.345 Mol. Wt.: 861.951 To a cis/trans mixture of 5 (210 mg, 0.24 mmol) in formic acid (21 mL), 10% palladium on carbon (210 mg) was added and the mixture was heated under H2 atmosphere at 50˚C. After 3.5 hrs the reaction was halted by the addition of an aq. solution of NaOH (21 g in 150 mL H2O) causing the porphyrin to precipitate from the solution. The resulting precipitate was collected by filtration then redissolved in DCM. The resulting solution was dried over MgSO4, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography collecting the major red fraction in DCM:hexane (1:1). The resulting solid was precipitated from DCM:MeOH to give a purple solid (85 mg, ca. 40%) in a 1:1 mixture of 8 and TPPCH3 7. 1H NMR (500 MHz, CDCl3, TMS): δ 8.86 (s, 4H, 8 and 7 Hβ-pyrrolic), 8.79 (d, 1H, J = 4.7 Hz, 7 Hβ-pyrrolic), 8.78 (d, 1H, J = 4.7 Hz, 8 Hβ-pyrrolic), 8.75 (d, 2H, J = 4.8 Hz, 7 Hβ- pyrrolic), 8.73 (d, 2H, J = 4.9 Hz, 8 Hβ-pyrrolic), 8.62 (d, 2H, J = 4.7 Hz, 7 Hβ-pyrrolic), 8.60 (d, 2H, J = 4.7 Hz, 8 Hβ-pyrrolic), 8.22-8.18 (m, 12H, 8 and 7 Hortho), 8.10-8.09 (m, 2H, 8 Hortho), 8.07-8.05 (m, 2H, 7 Hortho), 7.78-7.66 (m, 24H, 8 and 7 Hmeta, para), 3.39 (t, 2H, J = 6.9 Hz, 8 (CH2)11CH2Br), 2.31 (t, 2H, J = 7.7 Hz, 8 CH2(CH2)11Br), 2.58 (s, 3H, 7 TPPCH3), 1.87-1.81 (m, 2H, 8 (CH2)10CH2CH2Br), 1.78-1.75 (m, 2H, 8 CH2CH2(CH2)10Br), 1.43-1.38 (m, 2H, 8 (CH2)9CH2(CH2)2Br), 1.31-1.21 (m, 14H, 8 (CH2)2(CH2)7(CH2)3Br), -2.70 (4H, br s, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) Chapter 8 – Experimental Methods 143 274.5 (55.5), 417.5 (510), 514.5 (22.1), 549 (6.34), 588 (6.39), 643.5 (4.43). ESI- LRMS: m/z (%, assignment) cluster at 861-866, 861 (80, (M+H)+). HRMS: Calcd for (M+H)+ (C56H54BrN4): 861.3526, found: 861.3503. 2-(12ʹ-Pyridiumyldodecane)-5,10,15,20-tetraphenylporphyrin bromide (9) N NH N HN Ph Ph Ph Ph N BrC61H58BrN5 Exact Mass: 939.388 Mol. Wt.: 941.051 Compound 8 (80 mg, ca. 0.11 mmol) was refluxed in pyridine (20 mL) for two days, after which the solvent was removed in vacuo. The crude solid was dissolved in DCM and purified through a plug of silica gel, eluting TPPCH3 (7, 34 mg) in DCM then the pyridinium salt in MeOH:DCM (1:4). The pyridinium salt was precipitated from DCM:hexane and collected by filtration. The resulting crystals were washed with a saturated aq. solution of NaBr to give a red solid (30 mg, ca. 30%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.10 (d, 2H, J = 5.2 Hz, HPyr), 8.86 (s, 1H, Hβ-pyrrolic), 8.86 (s, 1H, Hβ- pyrrolic), 8.78 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.73 (d, 2H, J = 4.8 Hz, Hβ-pyrrolic), 8.64 (s, 1H, Hβ-pyrrolic), 8.61 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.21 (t, 1H, J = 1.3 Hz, HPyr), 8.20- 8.18 (m, 6H, Hortho), 8.09-8.07 (m, 2H, Hortho), 7.81 (t, 2H, J = 6.6 Hz, HPyr), 7.79-7.67 (m, 12H, Hmeta, para), 4.75 (t, 2H, J = 7.2 Hz, CH2(CH2)11PyrBr), 2.81 (t, 2H, J = 7.7 Hz, (CH2)11CH2PyrBr), 1.86 (m, 2H, CH2CH2(CH2)10PyrBr), 1.76-1.80 (m, 2H, (CH2)10CH2CH2PyrBr), 1.25-1.20 (m, 16H, (CH2)2(CH2)8(CH2)2PyrBr), -2.81 (s br, 2H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 418 (450), 515 (21.3), 549.5 (6.86), 587.5 (6.64), 643.5 (3.32). ESI-LRMS: m/z (%, assignment) cluster at 860-863, 860 (100, (M- Br)+). HRMS: Calcd for (M-Br)+ (C61H58N5): 860.4678, found: 860.4687. Chapter 8 – Experimental Methods 144 N-[(5,10,15,20-Tetraphenylporphinato-2-yl nickel II)methylene]ethanyl-1,2- diamine (11) N NH N HN Ph Ph Ph Ph N NH2 C47H36N6 Exact Mass: 684.3 Mol. Wt.: 684.829 Ethylenediamine (10, 0.207 mL, 3.11 mmol, 10 eq) was added to a solution of TPPCHO (2, 200 mg, 0.311 mmol) in CHCl3 (50 mL) and heated under Ar at 50 ˚C for 10 min. Acetic acid (1 drop) was added and heating was continued until TPPCHO was no longer visible by TLC (1 hr). The reaction was then precipitated from CHCl3:MeOH and collected by filtration as a purple powder (202 mg).1H NMR spectroscopy revealed the solid to be a 1:1 mixture of product (11) and TPPCHO (2). 1H NMR (500 MHz, CDCl3, TMS): δ 9.26 (s, 1H, CHO), 8.86-8.82 (m, 12H, 11 and 2 Hβ-pyrrolic), 8.72 (d, 1H, J = 4.7 Hz, 11 or 2 Hβ-pyrrolic), 8.57 (d, 1H, J = 4.7 Hz, 11 or 2 Hβ-pyrrolic), 8.24-8.15 (12H, m, 11 and 2 Hortho), 7.86-7.72 (m, 24H, 11 and 2 Hmeta, para), 7.05 (d, 1H, J = 15.8 Hz, CH), 3.43 (t, 2H, J = 5.7 Hz, CH2), 3.05 (t, 2H, J = 5.7 Hz, CH2), -2.62 (2H, br s, 11 or 2 NH), -2.62 (2H,br s, 11 or 2 NH). ESI-LRMS: m/z (%, assignment) cluster at 685-688, 685 (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C47H37N6): 685.3074, found: 685.3082. 2-Formyl-5,10,15,20-tetraphenylporphyrinato nickel II (14) N N N N Ph Ph Ph Ph CHO Ni C45H28N4NiO Exact Mass: 698.162 Mol. Wt.: 699.423 To a solution of refluxing TPPCHO (2, 1.7 g, 2.64 mmol) in CHCl3 (500 mL), Ni(OAc)2·4H2O (6.5 g, 26.4 mmol, 10 eq) in MeOH (50 mL) was added and refluxing was continued overnight. The solvent was removed in vacuo. The resulting solid was dissolved in hot CHCl3 (50 °C, 100 mL) and purified through a plug of silica gel with hot CHCl3 until colour was no longer eluted. The solvent was removed in vacuo and the Chapter 8 – Experimental Methods 145 resulting solid was precipitated from DCM:hexane to give a red solid (1.8 g, 97%). Spectroscopic data is in agreement with Bonfantini et al.87 N-[(5ʹ,10ʹ,15ʹ,20ʹ-tetraphenylporphinato-2ʹ-yl nickel II)methylene]benzene-1,4- diamine (15) N N N N Ph Ph Ph Ph N NH2 Ni C51H34N6Ni Exact Mass: 788.22 Mol. Wt.: 789.549 p-Phenylenediamine (13, 77 mg, 0.71 mmol, 10 eq) was added to a solution of NiTPP- CHO (14, 50 mg, 71 µmol) in THF (15 mL), to this acetic acid (1 drop) was added and the reaction was stirred at RT overnight. The solvent was removed in vacuo to give a red solid which was purified by silica gel column chromatography eluting first the starting material with DCM (24 mg, 48%) then what was suspected to be the desired product with MeOH:DCM (1:9) (25 mg). 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrin-2ʹʹ-yl)ethen-1ʹ-yl]nitrobenzene (19) N NH N HN Ph Ph Ph Ph NO2 C52H35N5O2 Exact Mass: 761.279 Mol. Wt.: 761.867 DBU (315 µL, 2.10 mmol, 3.2 eq) was added to a solution of TPPps (3, 612 mg, 0.661 mmol) and 4-nitrobenzaldehyde (18, 297 mg, 1.98 mmol, 3 eq) in DCM (50 mL) and the reaction was stirred at RT under Ar for 30 min. Upon completion by TLC the solvent was reduced in vacuo and the resulting purple solid was precipitated from DCM:MeOH and collected by filtration as a cis/trans isomeric mixture (437 mg, 87%). The isomeric mixture was dissolved in CHCl3 (30 mL) and I2 (434 mg, 1.72 mmol, 3.0 eq) was added. After stirring under darkness at RT for 3 hrs a saturated aq. solution of Na2S2O3 (100 mL) was added and stirring was continued for an additional 15 min. The Chapter 8 – Experimental Methods 146 organic layer was separated, dried over MgSO4, filtered and the solvent was reduced in vacuo. The resulting solid was precipitated from DCM:MeOH and collected by filtration to give pure trans product as a purple powder (430 mg, 85% overall). Spectroscopic data is in agreement with Bonfantini et al.78 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrin-2ʹʹ-yl)ethen-1ʹ-yl]aminobenzene (20) N NH N HN Ph Ph Ph Ph NH2 C52H37N5 Exact Mass: 731.305 Mol. Wt.: 731.884 To a solution of compound 19 (120 mg, 0.157 mmol) in THF (100 mL), SnCl2·2H2O (1.42 g, 6.30 mmol, 40 eq) and conc. HCl (0.7 mL) were added and stirred in darkness under Ar for 24 hrs at RT. Afterwards, Et3N (2.8 mL) was added forming a light brown coloured solid which was filtered through a glass sinter and washed with THF (20 mL). The filtrate solvent was removed in vacuo. The resulting solid was dissolved in DCM (37 mL), diluted with hexane (7 mL) and purified by alumina column chromatography (activity II, basic) eluting first the starting material with DCM:hexane (1:1) then the desired product with DCM (100 mg, 87%). 1H NMR (500 MHz, CDCl3, TMS): δ 8.98 (s, 1H, Hβ-pyrrolic), 8.85 (s, 2H, Hβ-pyrrolic), 8.81 (d, 1H, J = 4.6 Hz, Hβ-pyrrolic), 8.77-8.76 (m, 2H, Hβ-pyrrolic), 8.75 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.27-8.19 (m, 8H, Hortho), 7.83- 7.70 (m, 12H, Hmeta, para), 7.25 (d, 1H, J = 16.0 Hz, H1ʹ or 2ʹ), 7.10 (d, 2H, J = 7.8 Hz, Haromatic), 6.80 (d, 1H, J = 16.0 Hz, H1ʹ or 2ʹ), 6.68 (d, 2H, J = 7.8 Hz, Haromatic), 3.80 (br s, 2H, NH2), -2.57 (br s, 2H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 278.5 (36.3), 421 (191), 524 (18.7), 568 (13.0), 600 (8.56), 654.0 (2.12). ESI-LRMS: m/z (%, assignment) cluster at 732-735, 732 (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C52H38N5): 732.3122, found: 732.3118. Chapter 8 – Experimental Methods 147 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato-2ʹʹ-yl nickel II)ethen-1ʹ- yl]nitrobenzene (22) N N N N Ph Ph Ph Ph NO2 Ni C52H33N5NiO2 Exact Mass: 817.199 Mol. Wt.: 818.544 Ni(OAc)2·4H2O (1.93 g, 7.81 mmol) in MeOH (6 mL) was added to a refluxing solution of compound 19 (425 mg, 0.56 mmol) in CHCl3 (60 mL) and refluxing was continued overnight. The reaction mixture was taken to dryness, dissolved in DCM and purified through a plug of silica gel with DCM. The solvent was precipitated from DCM:MeOH and collected by filtration to give a red powder (455 mg, 100%). Spectroscopic data is in agreement with Bonfantini et al.78 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato-2ʹʹ-yl nickel II)ethen-1ʹ- yl]aminobenzene (23) N N N N Ph Ph Ph Ph NH2 Ni C52H35N5Ni Exact Mass: 787.225 Mol. Wt.: 788.561 To a solution of compound 22 (1.25 g, 1.52 mmol) in THF (830 mL), SnCl2·2H2O (14.7 g, 65 mmol, 42.8 eq) and conc. HCl (7.25 mL) were added and the reaction was stirred under Ar for 48 hrs at RT. Afterwards, Et3N (29 mL) was added forming a light brown coloured solid which was filtered through a glass sinter and washed with THF (100 mL). The filtrate was concentrated in vacuo. The resulting red solid was dissolved in DCM (30 mL), diluted with hexane (30 mL) and purified by alumina column chromatography (activity II, basic) eluting first the starting material with DCM:hexane (1:1) (187 mg, 15%) then the desired product with DCM (940 mg, 78%). 1H NMR (400 MHz, CDCl3, TMS): δ 8.83 (s, 1H, Hβ-pyrrolic), 8.71-8.66 (m, 6H, Hβ-pyrrolic), 8.04-7.95 (m, 8H, Hortho), 7.75-7.65 (m, 12H, Hmeta, para), 7.05 (d, 1H, J = 15.8 Hz, H1′ or 2′), 7.02 (d, Chapter 8 – Experimental Methods 148 2H, J = 8.2 Hz, Haromatic), 6.67 (d, 2H, J = 8.1 Hz, Haromatic), 6.65 (d, 1H, J = 16.0 Hz, H1′ or 2′), 4.06 (br s, 2H, NH2). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 275 (109), 425 (175), 540 (21.5), 577 (16.8). ESI-LRMS: m/z (%, assignment) cluster at 788-794, 788 (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C52H36N5Ni): 788.2304, found: 788.2139. 11-Bromoundecanoic acid (25) HO Br O C11H21BrO2 Exact Mass: 264.072 Mol. Wt.: 265.187 Chromium trioxide (1.8 g, 18.0 mmol, 1.5 eq) and H2O (1.65 mL) was cooled to 0 °C and to this conc. H2SO4 (1.34 mL, 24.0 mmol, 2 eq) was added followed by H2O (3 mL). After 5 minutes a solution of 11-bromoundecanol (24, 3.0 g, 12 mmol) in acetone (9 mL) was added dropwise over 1 minute. The reaction mixture was stirred for 2 hrs at 0 °C then stirred at RT for 12 hrs. Et2O (100 mL) and H2O (100 mL) were added and the aqueous layer was extracted with Et2O (3 × 100 mL). The combined organic phases were then washed with brine (100 mL), the organic phase was dried over MgSO4, filtered and reduced in vacuo. The resulting solid was purified by silica gel column chromatography eluting the product as a colourless band with DCM:0.5% AcOH affording the product as a white solid (1.30 g, 43%). 1H NMR (500 MHz, CDCl3, TMS): δ 10.18 (br s, 1H, COOH), 3.40 (t, 2H, J = 7.0 Hz, COOH(CH2)9CH2Br), 2.35 (t, 2H, J = 7.5, COOHCH2(CH2)9Br), 1.88-1.82 (m, 2H, COOH(CH2)8CH2CH2Br), 1.66- 1.60 (m, 2H, COOHCH2CH2(CH2)8Br), 1.43-1.40 (m, 2H, COOH(CH2)7CH2(CH2)2Br), 1.35-1.28 (m, 10H, CHO(CH2)2(CH2)5(CH2)2Br). Chapter 8 – Experimental Methods 149 trans-1-{11-{4-[2-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato nickel II)vinyl]phenylamino}-11-oxoundecyl}bromide (26) N N N N Ph Ph Ph Ph N H Ni O C63H54BrN5NiO Exact Mass: 1033.287 Mol. Wt.: 1035.733 Br 11-Bromoundecanoic acid (25, 2.01 g, 7.5 eq, 7.62 mmol) and DMAP (930 mg, 7.5 eq, 7.62 mmol) were dissolved in THF (600 mL) and cooled to 0 ˚C under Ar. To this EDC (1.18 g, 7.5 eq, 7.62 mmol) was added and the reaction mixture was stirred for 30 min at 0 ˚C followed by the addition of 23 (800 mg, 1.01 mmol). The reaction was stirred for 48 hrs at RT, diluted with DCM (300 mL) and washed with H2O (3 × 300 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting solid was purified by silica gel column chromatography eluting the product as the major red fraction in DCM. The solvent was removed in vacuo to give a purple solid (792 mg, 76%). 1H NMR (400 MHz, d6-DMSO): δ 9.92 (s, 1H, NH), 8.85 (s, 1H, Hβ-pyrrolic), 8.67-8.62 (m, 6H, Hβ-pyrrolic), 8.02-7.94 (m, 8H, Hortho), 7.82-7.71 (m, 12H, Hmeta, para), 7.55 (d, 2H, J = 8.5 Hz, Haromatic), 7.17 (d, 1H, J = 16.0 Hz, H1ʹ), 7.11 (d, 2H, J = 8.5 Hz, Haromatic), 6.98 (d, 1H, J = 16.0 Hz, H2ʹ), 3.51 (t, 2H, J = 6.9 Hz, CO(CH2)9CH2Br), 2.31 (t, 2H, J = 7.4 Hz, COCH2(CH2)9Br), 1.78 (dt, 2H, J = 7.3 and 6.8 Hz, CO(CH2)8CH2CH2Br), 1.64-1.56 (m, 2H, COCH2CH2(CH2)8Br), 1.38-1.27 (m, 12H, CO(CH2)2(CH2)6(CH2)2Br). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 425.5 (167), 539.5 (15.7), 576 (11.2). ESI-LRMS: m/z (%, assignment) cluster at 1033-1040, 1035 (100, M+). HRMS: Calcd for M+ (C63H54BrN5NiO): 1035.2841, found: 1035.2804. Chapter 8 – Experimental Methods 150 trans-1-{11-{4-[2-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato nickel II)vinyl]phenylamino}-11-oxoundecyl}pyridinium bromide (27) N N N N Ph Ph Ph Ph N H Ni O N BrC68H59BrN6NiOExact Mass: 1112.329 Mol. Wt.: 1114.833 Compound 26 (560 mg, 0.542 mmol) was refluxed in pyridine (70 mL) overnight. Following this the solvent was removed in vacuo to give a purple solid which was dried under high vaccum for five days (603 mg, 100%). 1H NMR (500 MHz, d6-DMSO): δ 9.96 (s, 1H, NH), 9.10 (dd, 2H, J = 6.7 and 1.2 Hz, Hpyr), 8.86 (s, 1H, Hβ-pyrrolic), 8.70- 8.65 (m, 6H, Hβ-pyrrolic), 8.61 (dt, 1H, J = 7.8 and 1.2 Hz, Hpyr), 8.17 (t, 2H J = 6.7 Hz, Hpyr), 8.04-7.96 (m, 8H, Hortho), 7.83-7.74 (m, 12H, Hmeta, para), 7.56 (d, 2H, J = 8.6 Hz, Haromatic), 7.20 (d, 1H, J = 16.0 Hz, H1ʹ), 7.13 (d, 2H, J = 8.6 Hz, Haromatic), 6.70 (d, 1H, J = 16.0 Hz, H2ʹ), 4.60 (t, 2H, J = 7.4 Hz, CO(CH2)9CH2PyrBr), 2.33 (t, 2H, J = 7.4 Hz, COCH2(CH2)9PyrBr), 1.95-1.89 (m, 2H, CO(CH2)8CH2CH2PyrBr), 1.63-1.58 (m, 2H, COCH2CH2(CH2)8PyrBr), 1.30-1.28 (m, 12H, CO(CH2)2(CH2)6(CH2)2PyrBr). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 425.5 (163), 538.5 (14.9), 575 (10.8). ESI-LRMS: m/z (%, assignment) cluster at 1033-1040, 1033 (100, (M-Br)+). HRMS: Calcd for (M-Br)+ (C68H59N6NiO): 1033.4098, found: 1033.4062. 4-Iodobenzoyl chloride I Cl O C7H4ClIO Exact Mass: 265.900 Mol. Wt.: 266.464 4-Iodobenzoic acid (28, 5.0 g, 20 mmol) was refluxed overnight in SOCl2 (25 mL). After cooling, the reaction mixture was diluted with H2O (25 mL) and extracted into Et2O (3 × 100 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting solid was dissolved in Et2O (200 mL), absorbed onto silica gel and purified by silica gel column chromatography, first eluting the product with DCM then eluting the starting material with ethylacetate (2.2 g, 44%). The product was taken to dryness to give a white solid (3.6 g, 47%). 1H NMR (500 Chapter 8 – Experimental Methods 151 MHz, CDCl3, TMS): δ 7.92 (d, 2H, J = 8.8 Hz, Haromatic), 7.82 (d, 2H, J = 8.8 Hz, Haromatic). 4-Iodobenzyl alcohol I OH C7H7IO Exact Mass: 233.954 Mol. Wt.: 234.034 4-Iodobenzyl alcohol was prepared using a method adapted from Ziyaei-Halimjani and Saidi.102 4-Iodobenzoyl chloride (3.6 g, 13.5 mmol) was dissolved in acetonitrile (20 mL) and to this LiClO4 (1.43 g, 13.5 mmol) and NaBH4 (1.35 g, 35.7 mmol) were added. The reaction then was stirred under Ar overnight at RT. The reaction was completed according to TLC after 24 hrs. The reaction mixture was diluted with H2O (20 mL) and extracted into Et2O (3 × 100 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting white solid was purified by silica gel column chromatography eluting the product with DCM, which was taken to dryness to give a white solid (2.75g, 87%). Spectroscopic data is in agreement with Vassiliou et al.179 4-Iodobenzaldehyde I H O C7H5IO Exact Mass: 231.939 Mol. Wt.: 232.018 4-Iodobenzaldehyde was prepared using a method adapted from De Mico et al.101 To a solution of dry DCM (11 mL), 4-iodobenzyl alcohol (2.75 g, 11.7 mmol) and [bis(acetoxy)iodo]benzene (BIAB) (4.19 g, 13.0 mmol) were added and the reaction was stirred for 5 minutes at RT. Afterwards, TEMPO (177 mg, 1.17 mmol 0.1 eq) was added and stirring was continued for a further 2 hrs until reaction was completed according to TLC. The resulting mixture was diluted with DCM (20 mL), washed with an saturated aq solution of Na2S2O3 (20 mL), NaHCO3 (20 mL) then brine (20 mL), dried over MgSO4, filtered and the solvent removed in vacuo. The resulting solid was purified by silica gel column chromatography first eluting any remaining alcohol with DCM:hexane (1:2) then the desired aldehyde with DCM. The colourless band was taken to dryness to give a white solid (2.30 g, 85%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.98 (s, 1H, CHO), 7.94 (d, 2H, J = 8.5 Hz, Haromatic), 7.61 (d, 2H, J = 8.5 Hz, Haromatic). Chapter 8 – Experimental Methods 152 Synthesis of compounds 29-32 are described in Chapter 8.4 2-[4ʹ-(Trimethylsilyl)ethynylphenyl]ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (33) N N N N Ph Ph Ph PhZn Si C57H40N4SiZn Exact Mass: 872.231 Mol. Wt.: 874.430 To a dry degassed solution of Et3N (25 mL), 2-(4ʹ-iodophenyl)ethynyl-5,10,15,20- tetraphenylporphyrinato zinc II (32, 500 mg, 0.55 mmol) was added and the reaction was stirred for 5 min at RT under Ar. Trimethylsilylacetylene (390 µL, 2.77 mmol, 5 eq) followed by Pd(PPh3)4 (191 mg, 0.16 mmol, 0.3 eq) and CuI (42 mg, 0.22 mmol, 0.5 eq) were added and the solution was refluxed under Ar for 3 hrs. After cooling to RT the solvent was removed in vacuo, the reaction mixture was dissolved in DCM (100 mL), washed with 5% aq. solution of Na2EDTA (3 × 100 ml), 3 M aq. solution of NH4OH (2 × 100 mL) and H2O (100 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The crude purple solid was purified by silica gel column chromatography eluting the product with DCM:hexane (2:1) as a purple band which was taken to dryness to give a purple solid (446 mg, 93%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.24 (s, 1H, Hβ-pyrrolic), 8.95 (s, 2H, Hβ-pyrrolic), 8.93 (s, 2H, Hβ-pyrrolic), 8.90 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.79 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.25-8.20 (m, 8H, Hortho), 7.81-7.60 (m, 12H, Hmeta, para), 7.46 (d, 2H, J = 7.9 Hz, Haromatic), 7.33 (d, 2H, J = 7.9 Hz, Haromatic), 0.31 (s, 9H, CH3). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 431 (351), 556 (33.8), 593 (19.3). ESI-HRMS: Calcd for M+ (C57H40N4SiZn): 872.2308, found: 872.2305. Chapter 8 – Experimental Methods 153 Glaser porphyrin homodimer (34) N N N N Ph Ph Ph Ph Zn N NN N Ph Ph Ph Ph Zn C108H62N8Zn2 Exact Mass: 1598.368 Mol. Wt.: 1602.481 The unwanted homodimer was produced during the deprotection of 33 using TBAF when compound 33 was not adequately washed to remove Cu salts. 1H NMR (500 MHz, CDCl3, TMS): δ 9.27 (s, 2H, Hβ-pyrrolic), 8.96 (s, 4H, Hβ-pyrrolic), 8.93 (s, 4H, Hβ- pyrrolic), 8.91 (d, 2H, J = 4.7 Hz, Hβ-pyrrolic), 8.81 (d, 2H, J = 4.7 Hz, Hβ-pyrrolic), 8.26-8.22 (m, 16H, Hortho), 7.83-7.68 (m, 24H, Hmeta, para), 7.54 (d, 4H, J = 8.2 Hz, Haromatic), 7.38 (d, 4H, J = 7.9 Hz, Haromatic). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 432 (355), 558 (35.0), 593 (20.7). FAB-LRMS: m/z (%, assignment) cluster at 1599-1601, 1601 (100, M+). HRMS: Calcd. for (M+H)+ (C108H63N864Zn2): 1599.3758, found: 1599.3772. 2-(4ʹ-Ethynylphenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (35) N N N N Ph Ph Ph PhZn H C54H32N4Zn Exact Mass: 800.192 Mol. Wt.: 802.249 To a degassed solution of THF:DCM (2.5:1, 6 mL) 2-[4ʹ- (trimethylsilyl)ethynylphenyl]ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (34, 200 mg, 0.230 mmol) was added and the reaction was stirred under Ar for 5 minutes at RT. Tetrabutylammonium fluoride (170 µL of a 1 M solution in THF, 0.74 eq) was added and stirring was continued for 5 minutes. The reaction mixture was diluted with DCM (50 mL), washed with degassed 20% aq. solution of NaHCO3 (50 mL) then with degassed H2O (50 mL). The organic layer was separated, dried over MgSO4, filtered and taken to dryness to give a purple solid which was dissolved in DCM and purified through a plug of silica gel with DCM. The purple solution was taken to dryness to give a purple solid (178 mg, 97%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.25 (s, 1H, Hβ- pyrrolic), 8.95 (s, 2H, Hβ-pyrrolic), 8.93 (s, 2H, Hβ-pyrrolic), 8.89 (d, 1H, J = 4.6 Hz, Hβ-pyrrolic), Chapter 8 – Experimental Methods 154 8.79 (d, 1H, J = 4.6 Hz, Hβ-pyrrolic), 8.25-8.21 (m, 8H, Hortho), 7.84-7.63 (m, 12H, Hmeta, para), 7.48 (d, 2H, J = 8.1 Hz, Haromatic), 7.35 (d, 2H, J = 8.2 Hz, Haromatic), 3.22 (s, 1H, CH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 286 (32.7), 431 (355), 556 (20.2), 591 (6.00). ESI-LRMS: m/z (%, assignment) cluster at 800-808, 800 (100, M+). ESI-HRMS: Calcd for M+ (C52H32N4Zn): 800.1913 found: 800.1906. Synthesis of compounds 36-40 are described in Chapter 8.4 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato-2ʹʹ-yl nickel II)ethen-1ʹ- yl]azidobenzene (41) N N N N Ph Ph Ph Ph N3 Ni C52H33N7Ni Exact Mass: 813.215 Mol. Wt.: 814.559 To a solution of compound 23 (175 mg, 0.22 mmol) in THF (50 mL), NaNO2 (122 mg, 1.76 mmol, 8 eq) in H2O (400 µL) and H2SO4 (1 drop) were added and the mixture was stirred in darkness under Ar until no starting material was present by TLC (2 hrs). Afterwards NaN3 (162 mg, 2.49 mmol, 11.3 eq) in H2O (400 µL) was added and the reaction mixture was stirred for an additional 20 min. The resulting reaction mixture was diluted with DCM (150 mL), washed with H2O (100 mL) then with a saturated aq. solution of NaHCO3 (100 mL), dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting red solid was purified by silica gel column chromatography eluting the product with DCM:hexane (1:1) as a purple/red band. The resulting red solution was reduced in vacuo and the porphyrin was precipitated from DCM:MeOH giving a purple solid (175 mg, 98%). 1H NMR (500 MHz, CDCl3, TMS): δ 8.85 (s, 1H, Hβ-pyrrolic), 8.70-8.66 (m, 6H, Hβ-pyrrolic), 8.02-7.94 (m, 8H, Hortho), 7.74-7.65 (m, 12H, Hmeta, para), 7.15 (d, 2H, J = 8.5 Hz, Haromatic), 7.06 (d, 1H, J = 16.2 Hz, H1ʹ or 2ʹ), 6.96 (d, 2H, J = 8.5 Hz, Haromatic), 6.78 (d, 1H, J = 16.2 Hz, H1ʹ or 2ʹ). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 426 (188), 538 (16.7), 572 (11.0). ESI-LRMS: m/z (%, assignment) cluster at 813-813, 818 (100, M+). ESI-HRMS: Calcd for M+ (C52H33N7Ni): 813.2145, found: 813.2162. IR-ATR (cm-1): 2116.0 (azide). Chapter 8 – Experimental Methods 155 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrin-2ʹʹ-yl)ethen-1ʹ-yl]azidobenzene (42) N NH N HN Ph Ph Ph Ph N3 C52H35N7 Exact Mass: 757.295 Mol. Wt.: 757.881 To a solution of compound 20 (70 mg, 96 µmol) in THF (100 mL), NaNO2 (22 mg, 0.32 mmol, 33 eq) in H2O (50 µL) and H2SO4 (1 drop) were added and the reaction was stirred in darkness under Ar until no starting material was present by TLC (3 hrs). Afterwards NaN3 (27 mg, 0.41 mmol, 42 eq) in H2O (100 µL) was added and the reaction mixture was stirred for an additional 10 min. The resulting reaction mixture was diluted with DCM (150 mL), washed with H2O (100 mL) then with a saturated aq. solution of NaHCO3 (100 mL), dried over MgSO4, filtered and the solvent was removed in vacuo to give a purple solid (69 mg, 96%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.02 (s, 1H, Hβ-pyrrolic), 8.87 (s, 2H, Hβ-pyrrolic), 8.85 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.81 (d, 2H, J = 4.8 Hz, Hβ-pyrrolic), 8.75 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.29-8.21 (m, 8H, Hortho), 7.86- 7.76 (m, 12H, Hmeta, para), 7.27 (d, 1H, J = 16.0 Hz, H1ʹ or 2ʹ), 7.26 (d, 2H, J = 7.4 Hz, Haromatic), 7.02 (d, 2H, J = 7.4 Hz, Haromatic), 6.95 (d, 1H, J = 16.0 Hz, H1ʹ or 2ʹ), -2.55 (br s, 2H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 424.5 (168), 524.5 (15.2), 563.5 (8.68), 598.5, (5.67), 656.5 (1.94). ESI-LRMS: m/z (%, assignment) cluster at 758-761, 758 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C52H35N7): 758.3027, found: 758.3029. IR-ATR (cm-1): 2115.7 (azide). Chapter 8 – Experimental Methods 156 2-(4ʹ-Azidophenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (45) N N N N Ph Ph Ph Ph N3 Zn C52H31N7Zn Exact Mass: 817.193 Mol. Wt.: 819.239 To a solution of 2-(4ʹ-iodophenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (32, 60 mg, 66 µmol) in dry DMSO (3 mL), NaN3 (8.4 mg, 132 µmol, 2 eq), sodium ascorbate (1.2 mg, 6.6 µmol, 0.1 eq) and tetrakis(acetonitrile)copper(I) hexafluorophosphate (9.9 mg, 13.2 mmol, 0.2 eq) were added at once followed by N,N- DMEA (2.04 µL, 19.2 µmol, 0.3 eq). The reaction mixture was heated at 70 °C until TLC showed no starting material (48 hrs), cooled to RT, diluted with DCM (30 mL), washed with H2O (30 mL) then with 5% aq. solution of Na2EDTA (30 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The crude material was purified by silica gel column chromatography eluting the product in DCM:hexane (1:1) which was taken to dryness to give a purple solid (33 mg, 61%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.24 (s, 1H, Hβ-pyrrolic), 8.94 (s, 2H, Hβ- pyrrolic), 8.93 (s, 2H, Hβ-pyrrolic), 8.89 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.78 (d, 1H, J = 4.6 Hz, Hβ-pyrrolic), 8.25-8.21 (m, 8H, Hortho), 7.81-7.66 (m, 12H, Hmeta, para), 7.38 (d, 2H, J = 8.5 Hz, Haromatic), 7.01 (d, 2H, J = 8.8 Hz, Haromatic). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 281 (20.0), 430 (202), 556 (16.2), 593 (7.4). ESI-LRMS: m/z (%, assignment) cluster at 817-723, 817 (100, M+). ESI-HRMS: Calcd for M+ (C52H31N7Zn): 817.1927, found: 817.1948. IR-ATR (cm-1): 2105.2 (azide). 4-(Bromomethyl)benzonitrile NC Br C8H6BrN Exact Mass: 194.968 Mol. Wt.: 196.044 To a solution of para-tolunitrile (11.75 g, 0.1 mmol) in CCl4 (100 mL), NBS (19.58 g, 0.11 mmol, 1.1 eq) was added and the reaction mixture was refluxed for 2 hrs with halogen lamp light irradiation. The reaction mixture was filtered while hot and the filtrate was taken to dryness to give a solid which was recrystallized twice from EtOH to give pure white needles (12.14g, 62%). 1H NMR (400 MHz, CDCl3, TMS): δ 7.64 (d, 2H, J = 8.4 Hz, Haromatic), 7.50 (d, 2H, J = 8.4 Hz, Haromatic), 4.47 (s, 2H, CH2Br). Chapter 8 – Experimental Methods 157 4-(Bromomethyl)benzaldehyde (46) OHC Br C8H7BrO Exact Mass: 197.968 Mol. Wt.: 199.045 4-(Bromomethyl)benzaldehyde was synthesised from 4-(bromomethyl)benzonitrile according to the procedure described by Schlenoff et al.105 4-(Azidomethyl)benzaldehyde (47) OHC N3 C8H7N3O Exact Mass: 161.059 Mol. Wt.: 161.161 4-(Azidomethyl)benzaldehyde was synthesised from 4-(bromomethyl)benzaldehyde according to the procedure described by Barbe et al.106 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrin-2ʹʹ-yl)ethen-1ʹ-yl] azidomethylbenzene (48) N NH N HN Ph Ph Ph Ph N3 C53H37N7 Exact Mass: 771.311 Mol. Wt.: 771.908 DBU (250 µL, 1.67 mmol, 3.1 eq) was added to a solution of TPPps (3, 500 mg, 0.54 mmol) and 4-(azidomethyl)benzaldehyde (47, 260 mg, 1.13 mmol, 3 eq) in DCM (100 mL) and the reaction was stirred at RT under Ar for 20 minutes. After TLC indicated that phosphonium salt had completely reacted, the solvent was reduced in vacuo and the resulting solid was precipitated from DCM:MeOH. The resulting purple solid was collected by filtration as a cis/trans (35:65) isomeric mixture (340 mg 76%) that also contained a small amount of TPPCH3 (7) according to 1H NMR spectroscopy. The isomeric mixture was dissolved in CHCl3 (100 mL) and I2 (103 mg, 0.41 mmol, 1.0 eq) was added. After stirring in darkness at RT overnight, saturated aq. solution of Na2S2O3 (100 mL) was added and stirring was continued for an additional 15 minutes. The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo to give the trans product. The resulting purple solid was dissolved in DCM (20 mL), diluted with hexane (20 mL) and purified by silica gel column chromatography Chapter 8 – Experimental Methods 158 first eluting traces of TPPCH3 with DCM:hexane (1:1) then the desired product as a purple band with MeOH:DCM (1:19). The solvent was reduced in vacuo and the porphyrin was precipitated with DCM:MeOH. The porphyrin precipitate was collected by filtration to give pure trans product (270 mg, 60% overall) as a purple powder. 1H NMR (400 MHz, d6-DMSO): δ 9.02 (s, 1H, Hβ-pyrrolic), 8.83-8.72 (m, 5H, Hβ-pyrrolic), 8.67 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.26-8.18 (m, 8H, Hortho), 7.89-7.80 (m, 12H, Hmeta, para), 7.42 (d, 1H, J = 16.2 Hz, H1ʹ or 2ʹ), 7.36 (d, 2H, J = 8.1 Hz, Haromatic), 7.30 (d, 2H, J = 8.1 Hz, Haromatic), 6.94 (d, 1H, J = 15.9 Hz, H1ʹ or 2ʹ), 4.49 (s, 2H, CH2N3), -2.72 (br s, 2H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 425 (172), 523 (12.4), 559 (7.60), 597 (4.83), 651 (1.79). ESI-LRMS: m/z (%, assignment) cluster at 772-775, 772 (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C53H38N7): 772.3183, found: 772.3183. IR- ATR (cm-1): 2096.6 (azide). 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato-2ʹʹ-yl zinc II)ethen-1ʹ-yl] azidomethylbenzene (49) N N N N Ph Ph Ph Ph N3 Zn C53H35N7Zn Exact Mass: 833.225 Mol. Wt.: 835.282 Zn(OAc)2·2H2O (31.3 mg, 0.14 mmol, 1.1 eq) in MeOH (2.5 mL) was added to a solution of porphyrin 48 (100 mg, 0.13 mmol) in DCM (25 mL) and the reaction was stirred at RT for 1 hr. The solvent was reduced in vacuo and the resulting solid was dissolved in DCM and purified through a plug of silica gel with DCM. The solvent was removed in vacuo to give a purple solid (105 mg, 97%). 1H NMR (500 MHz, d6- DMSO): δ 8.96 (s, 1H, Hβ-pyrrolic), 8.76-8.71 (m, 5H, Hβ-pyrrolic), 8.63 (d, 1H, J = 4.2 Hz, Hβ-pyrrolic), 8.21-7.91 (m, 8H, Hortho), 7.82-7.78 (m, 12H, Hmeta, para), 7.35 (d, 2H, J = 7.3 Hz, Haromatic), 7.29 (d, 2H, J = 7.4 Hz, Haromatic), 7.27 (d, 1H, J = 16.0 Hz, H1ʹ or 2ʹ), 6.98 (d, 1H, J = 16.0 Hz, H1ʹ or 2ʹ), 4.48 (s, 2H, CH2N3). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 428.5 (229), 556 (20.0), 591.5 (7.39). ESI-LRMS: m/z (%, assignment) cluster Chapter 8 – Experimental Methods 159 at 833-842, 834 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C52H36N7): 834.2318, found: 834.2282. IR-ATR (cm-1): 2097.1 (azide). 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato-2ʹʹ-yl nickel II)ethen-1ʹ-yl] azidomethylbenzene (50) N N N N Ph Ph Ph Ph N3 Ni C53H35N7Ni Exact Mass: 827.231 Mol. Wt.: 828.585 Ni(OAc)2·4H2O (240 mg, 0.96 mmol, 12.5 eq) in MeOH (10 mL) was added to a refluxing solution of porphyrin 48 (60 mg, 77.8 µmol) in CHCl3 (90 mL) and refluxing was continued overnight. After cooling the solvent was reduced in vacuo and the porphyrin was precipitated from DCM:MeOH. The desired product was collected by filtration to give a red solid (60 mg, 99%). 1H NMR (400 MHz, d6-DMSO): δ 8.91 (s, 1H, Hβ-pyrrolic), 8.68-8.63 (m, 6H, Hβ-pyrrolic), 8.03-7.95 (m, 8H, Hortho), 7.85-7.73 (m, 12H, Hmeta, para), 7.32 (d, 2H, J = 8.3 Hz, Haromatic), 7.27 (d, 1H, J = 16.3 Hz, H1′ or 2′), 7.21 (d, 2H, J = 8.4 Hz, Haromatic), 6.81 (d, 1H, J = 16.0 Hz, H1ʹ or 2ʹ), 4.46 (s, 2H, CH2N3). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 425 (176), 538 (16.1), 570 (10.2). ESI- LRMS: m/z (%, assignment) cluster at 827-831, 828 (100, (M+H)+).ESI-HRMS: Calcd for M+ (C53H35N7Ni): 827.2302, found: 827.2312. IR-ATR (cm-1): 2096.1 (azide). Chapter 8 – Experimental Methods 160 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato-2ʹʹ-yl iron III chloride)ethen-1ʹ-yl] azidomethylbenzene (51) N N N N Ph Ph Ph Ph N3 Fe Cl C53H35ClFeN7 Exact Mass: 860.199 Mol. Wt.: 861.190 Spectroscopic grade acetonitrile (120 mL) was refluxed under argon for two hours to remove dissolved oxygen. The temperature was lowered to 70 °C and FeCl2·4H2O (320 mg, 1.61 mmol, 15.6 eq) was added. Porphyrin 48 (80 mg, 0.10 mmol) was dissolved in degassed chloroform (15 mL) and added slowly to the reaction mixture over 5 minutes. The temperature was increased and the solution was refluxed for a further 5 hours under Ar then overnight open to the air. The cooled solution was extracted with DCM (200 mL) and the organic layer was washed with 0.1 M HCl solution (6 × 100 mL) to extract the inorganic salts. The organic layer was separated, dried over CaCl2 and the solvent was removed in vacuo to give a brown solid. The solid was dissolved in DCM and purified by silica gel column chromatography (3% MeOH:DCM) eluting the desired product as a brown band. The solvent was removed in vacuo, the purple solid dissolved in DCM (20 mL) and filtered through a #4 glass sinter. The solvent was removed in vacuo to give a purple solid (80 mg, 90%). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 425 (132), 579 (10.1), 615 (6.80). ESI-LRMS: m/z (%, assignment) cluster at 825-827, 825 (100, M-Cl+) and 860-865 (2, M+). HRMS: Calcd for M+ (C53H35ClFeN7): 860.1986, found: 860.2002. IR-ATR (cm-1): 2095.8 (azide). Chapter 8 – Experimental Methods 161 8.4 Experimental Procedures for Chapter 3 - Synthesis of β- Pyrrolic Ethynyl Porphyrin Derivatives via the Modified Horner- Emmons Reaction General Method for the Synthesis of Diphenyl hydroxyphenylmethylphosphonates R P OH OPhO OPh Diphenyl hydroxylphenylmethylphosphonates were prepared using the method from Kondo et al.122 and Katritzky et al.100 Aldehyde (ca. 40 mmol) was melted in a 250 mL round bottom flask using a heat gun if required. Diphenolphosphite (1 eq) followed by MgO (1 eq) were added and the reaction mixture was stirred overnight at RT to give a white solid. The solid was sonicated in CHCl3 (200 mL) for 1 hr, filtered through fluted filter paper and washed with CHCl3 until the eluent was clear by TLC. The solvent was removed in vacuo and the resulting solid was purified (if required) by silica gel column chromatography first eluting traces of the aldehyde then eluting the product. The solvent was removed in vacuo, the residue was dissolved in hot DCM, filtered through a #4 sinter and the solvent was removed in vacuo to give a white solid. Diphenyl hydroxy(4-iodophenyl)methylphosphonate (29) I P OH OPhO OPh C19H16IO4P Exact Mass: 465.983 Mol. Wt.: 466.206 Compound 29 was isolated in 84% yield. Purified by silica gel column chromatography with MeOH:DCM (1:49). 1H NMR (400 MHz, CDCl3, TMS): δ 7.72 (dd, 2H, J = 8.6 and 0.7 Hz, Haromatic), 7.32 (dd, 2H, J = 8.5 and 2.9 Hz, Haromatic), 7.28-7.25 (m, 4H, Haromatic), 7.18-7.13 (m, 2H, Haromatic), 7.07-7.01 (m, 4H, Haromatic), 5.28 (d, 1H, J = 9.3 Hz, CH), 3.40 (br s, 1H, OH). ESI-LRMS: m/z (%, assignment) cluster at 489-491, 489, (100, (M+Na)+). ESI-HRMS: Calcd for (M+Na)+ (C19H16INaO4P): 488.9723, found: 488.9723. Chapter 8 – Experimental Methods 162 Diphenyl hydroxy(4-bromophenyl)methylphosphonate (36) Br P OH OPhO OPh C19H16BrO4P Exact Mass: 417.997 Mol. Wt.: 419.206 Compound 36 was isolated in 90% yield. No chromatographic purification was required. 1H NMR (500 MHz, CDCl3, TMS): δ 7.49 (dd, 2H, J = 8.8 and 0.7 Hz, Haromatic), 7.43 (dd, 2H, J = 8.8 and 2.9 Hz, Haromatic), 7.29-7.24 (m, 4H, Haromatic), 7.17- 7.13 (m, 2H, Haromatic), 7.06-7.01 (m, 4H, Haromatic), 5.24 (d, 1H, J = 9.3 Hz, CH), 4.16 (br s, 1H, OH). FAB-LRMS: m/z (%, assignment) cluster at 419-422, 419, (100, (M+H)+). HRMS: Calcd. for (M+H)+ (C19H1779BrO4P): 419.0048, found: 419.0043. Diphenyl hydroxyphenylmethylphosphonate (52) P OH OPhO OPh C19H17O4PExact Mass: 340.086 Mol. Wt.: 340.31 Compound 52 was isolated in 73% yield. No chromatographic purification was required. Spectroscopic data is in agreement with Katritzky et al.100 Diphenyl hydroxy(4-methoxyphenyl)methylphosphonate (53) MeO P OH OPhO OPh C20H19O5P Exact Mass: 370.097 Mol. Wt.: 370.336 Compound 53 was isolated in 90% yield. No chromatographic purification was required. Spectroscopic data is in agreement with Kondo et al. 122 Diphenyl hydroxy(4-methoxycarbonylphenyl)methylphosphonate (54) MeOOC P OH OPhO OPh C21H19O6P Exact Mass: 398.092 Mol. Wt.: 398.346 Compound 54 was isolated in 82% yield. No chromatographic purification was required. 1H NMR (500 MHz, CDCl3, TMS): δ 8.08 (d, 2H, J = 8.1 Hz, Haromatic), 7.67 (dd, 2H, J = 8.2, 2.2 Hz, Haromatic), 7.32-7.03 (m, 10H, Haromatic), 5.40 (d, 1H, J = 9.8 Hz, CH), 4.25 (br s, 1H, OH), 3.96 (s, 3H, CH3). FAB-LRMS: m/z (%, assignment) cluster at 398-401, 399 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C21H20O6P): 399.0998, found: 399.0995. Chapter 8 – Experimental Methods 163 Diphenyl hydroxy(4-pyridyl)methylphosphonate (55) N P OH OPhO OPh C18H16NO4P Exact Mass: 341.082 Mol. Wt.: 341.298 Reaction was preformed at 0 °C according to the procedure of Kondo e. al.103 and compound 55 was isolated in 82% yield. No chromatographic purification was required. Spectroscopic data is in agreement with Kondo et al. 122 Diphenyl hydroxy[4-(5ʹ,5ʹ-dimethyl-1ʹ,3ʹ-dioxane-2ʹ-yl)phenyl]methyl phosphonate (56) P OH OPhO OPh O O C25H27O6P Exact Mass: 454.155 Mol. Wt.: 454.452 Compound 56 was isolated in 88% yield. No chromatographic purification was required. 1H NMR (500 MHz, CDCl3, TMS): δ 7.61 (dd, 2H, J = 8.3, 2.2 Hz, Haromatic), 7.56 (d, 2H, J = 8.3 Hz, Haromatic), 7.31-7.13 (m, 6H, Haromatic), 7.10-7.08 (m, 2H, Haromatic), 7.06-7.03 (m, 2H, Haromatic), 5.43 (s, 1H, CH), 5.27 (dd, 1H, J = 9.3, 5.0 Hz, CHOH), 3.81 (d, 2H, J = 11 Hz, CH2), 3.81 (d, 2H, J = 11Hz, CH2), 3.72 (br s, 1H, OH) 1.32 (s, 3H, CH3), 0.84 (s, 3H, CH3). FAB-LRMS: m/z (%, assignment) cluster at 453- 457, 455 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C25H28O6P): 455.1623, found: 455.1621. Diphenyl hydroxy(4-benzonitrile)methylphosphonate (57) NC P OH OPhO OPh C20H16NO4P Exact Mass: 365.082 Mol. Wt.: 365.319 Compound 57 was isolated in 70% yield. Purified by silica gel column chromatography with DCM. 1H NMR (500 MHz, CDCl3, TMS): δ 7.65 (dd, 2H, J = 8.2, 1.7 Hz, Haromatic), 7.56 (d, 2H, J = 8.2 Hz, Haromatic), 7.37-6.99 (m, 10H, Haromatic), 5.20 (d, 1H, J = 13.8 Hz, CH). ESI-LRMS: m/z (%, assignment) cluster at 366-368, 366, (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C20H17NO4P): 366.8090, found: 366.0887. Chapter 8 – Experimental Methods 164 Diphenyl hydroxy(4-nitrophenyl)methylphosphonate (58) O2N P OH OPhO OPh C19H16NO6P Exact Mass: 385.072 Mol. Wt.: 385.307 Compound 58 was isolated in 84% yield. No chromatographic purification was required. Spectroscopic data is in agreement with Kondo et al. 122 General Method for the Synthesis of Diphenyl Chlorophenylmethylphosphonates R P Cl OPhO OPh Diphenyl chlorophenylmethylphosphonates were prepared using the method from Kondo et al.103 To the appropriate hydroxymethylphosphates, POCl3 (2 mL/g of hydroxymethylphosphate) and N,N-diethylaniline (0.2 mL/g of hydroxymethylphosphate) was combined and heated at 90 °C for 1 hr. The resulting solution was cooled to RT then poured slowly into ice. The resulting solid was extracted into DCM (150 mL), the organic layer was separated and washed with saturated aq solution of NaHCO3 solution (3 × 150 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting solid was dissolved in DCM and filtered through a plug of silica gel with DCM. The solvent was removed in vacuo to give a white solid that solidified with time. Filtration of the solid gave the desired product. Diphenyl chloro(4-bromophenyl)methylphosphonate (37) Br P Cl OPhO OPh C19H15BrClO3PExact Mass: 435.963 Mol. Wt.: 437.651 Compound 37 was isolated in 22% yield. 1H NMR (500 MHz, CDCl3, TMS): δ 7.56- 7.50 (m, 4H, Haromatic), 7.46-7.27 (m, 4H, Haromatic), 7.23-7.15 (m, 4H, Haromatic), 7.02 (d, 2H, J = 8.8 Hz, Haromatic), 5.23 (d, 1H, J = 14.0 Hz, CH). FAB-LRMS: m/z (%, assignment) cluster at 436-441, 438 (100, (M+H)+). HRMS: Calcd. for (M+H)+ (C19H1681Br35ClO3P): 438.9688, found: 438.9695. Chapter 8 – Experimental Methods 165 Diphenyl chlorophenylmethylphosphonate (59) P Cl OPhO OPh C19H16ClO3P Exact Mass: 358.053 Mol. Wt.: 358.755 Compound 59 was isolated in 19% yield. Spectroscopic data is in agreement with Katritzky et al.100 Diphenyl chloro(4-methoxyphenyl)methylphosphonate (60) MeO P Cl OPhO OPh C20H18ClO4P Exact Mass: 388.063 Mol. Wt.: 388.781 Compound 60 was isolated in 25% yield. Spectroscopic data is in agreement with Kondo et al. 122 Diphenyl chloro(4-methoxycarbonylphenyl)methylphosphonate (61) MeOOC P Cl OPhO OPh C21H18ClO5P Exact Mass: 416.058 Mol. Wt.: 416.791 Compound 61 was isolated in 17% yield. 1H NMR (500 MHz, CDCl3, TMS): δ 8.05 (d, 2H, J = 8.1 Hz, Haromatic), 7.70 (dd, 2H, J = 8.6, 2.0 Hz, Haromatic), 7.32-7.17 (m, 6H, Haromatic), 7.13-7.10 (m, 2H, Haromatic), 7.00-6.99 (m, 2H, Haromatic), 5.30 (d, 1H, J = 14.0 Hz, CH), 3.93 (s, 3H, CH3). FAB-LRMS: m/z (%, assignment) cluster at 416-420, 417 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C21H19O5P35Cl): 417.0659, found: 417.0661. Diphenyl chloro(4-pyridyl)methylphosphonate (62) N P Cl OPhO OPh C18H15ClNO3P Exact Mass: 359.048 Mol. Wt.: 359.743 Compound 62 was isolated in 45% yield. Spectroscopic data is in agreement with Kondo et al.103 Chapter 8 – Experimental Methods 166 Diphenyl chloro(4-nitrophenyl)methylphosphonate (66) O2N P Cl OPhO OPh C19H15ClNO5P Exact Mass: 403.038 Mol. Wt.: 403.753 Compound 66 was isolated in 35% yield. Spectroscopic data is in agreement with Kondo et al. 122 General Method for the Synthesis of Diphenyl Bromophenylmethylphosphonates R P Br OPhO OPh Diphenyl bromophenylmethylphosphonates were prepared using the method from Firouzabadi et al.99 To a solution of DDQ (2 eq), PPh3 (2 eq) and nBu4NBr (2 eq) in dry DCM (30 mL/g of phosphonate), the appropriate hydroxymethylphosphates (1 eq) were added and the reaction was stirred overnight under Ar at RT. On completion, the reaction mixture was washed with H2O (3 × 100 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting oil was purified by silica gel column chromatography eluting the product as a colourless fraction with DCM unless otherwise stated. The solvent was removed in vacuo to give a slightly yellow oil that solidified on cooling Diphenyl bromo(4-iodophenyl)methylphosphonate (30) I P Br OPhO OPh C19H15BrIO3P Exact Mass: 527.899 Mol. Wt.: 529.103 Compound 30 was isolated in 84% yield. 1H NMR (500 MHz, CDCl3, TMS): δ 7.73 (d, 2H, J = 8.2, Hz, Haromatic), 7.40 (dd, 2H, J = 8.2 and 1.5 Hz, Haromatic), 7.38-7.33 (m, 2H, Haromatic), 7.27-7.15 (m, 6H, Haromatic), 6.98 (d, 2H, J = 8.5 Hz , Haromatic), 5.13 (d, 1H, J = 13.1 Hz, CH). ESI-LRMS: m/z (%, assignment) cluster at 529-533, 531, (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C19H16BrIO3P): 528.9060, found: 528.9047. Chapter 8 – Experimental Methods 167 Diphenyl bromo(4-bromophenyl)methylphosphonate (38) Br P Br OPhO OPh C19H15Br2O3P Exact Mass: 479.913 Mol. Wt.: 482.102 Compound 38 was isolated in 84% yield. 1H NMR (500 MHz, CDCl3, TMS): δ 7.54- 7.53 (m, 4H, Haromatic), 7.37-34 (m, 2H, Haromatic), 7.29-7.15 (m, 6H, Haromatic), 7.00-7.15 (m, 6H, Haromatic), 700-6.98 (m, 2H, Haromatic), 5.16 (d, 1H, J = 13.0 Hz, CH). FAB- LRMS: m/z (%, assignment) cluster at 481-485, 483 (100, (M+H)+). HRMS: Calcd. for (M+H)+ (C19H1679Br81BrO3P): 482.9183, found: 482.9179. Diphenyl bromo[4-(5ʹ,5ʹ-dimethyl-1ʹ,3ʹ-dioxane-2ʹ-yl)phenyl]methylphosphonate (64) P Br OPhO OPh O O C25H26BrO5P Exact Mass: 516.07 Mol. Wt.: 517.349 Compound 64 was isolated in 64% yield. Purified by silica gel column chromatography with MeOH:DCM (1:49). 1H NMR (500 MHz, CDCl3, TMS): δ 7.65 (dd, 2H, J = 8.3, 1.8 Hz, Haromatic), 7.56 (d, 2H, J = 8.3 Hz, Haromatic), 7.37-7.10 (m, 8H, Haromatic), 6.97- 9.95 (m, 2H, Haromatic), 5.39 (s, 1H, CH), 5.18 (d, 1H, J = 12.8 Hz, CHOH), 3.77 (d, 2H, J = 11.1 Hz, CH2), 3.65 (d, 2H, J = 11.1 Hz, CH2), 1.28 (s, 3H, CH3), 0.80 (s, 3H, CH3). FAB-LRMS: m/z (%, assignment) cluster at 515-521, 517 (50, (M+H)+). HRMS: Calcd for (M+H)+ (C25H2779BrO5P): 517.0779, found: 517.0764. Diphenyl bromo(4-cyanophenyl)methylphosphonate (65) NC P Br OPhO OPh C20H15BrNO3P Exact Mass: 426.997 Mol. Wt.: 428.216 Compound 65 was isolated in 70% yield. 1H NMR (500 MHz, CDCl3, TMS): δ 7.65 (dd, 2H, J = 8.2, 1.7 Hz, Haromatic), 7.56 (d, 2H, J = 8.2 Hz, Haromatic), 7.37-6.99 (m, 10H, Haromatic), 5.20 (d, 1H, J = 13.8 Hz, CH). FAB-LRMS: m/z (%, assignment) cluster at 427-433, 428 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C20H1681BrNO3P): 430.0031, found: 430.0029. Chapter 8 – Experimental Methods 168 2-(4′-Iodophenyl)ethynyl-5,10,15,20-tetraphenylporphyrin (31) N NH N HN Ph Ph Ph Ph I C52H33IN4 Exact Mass: 840.175 Mol. Wt.: 840.750 TPP-CHO (2, 600 mg, 0.89 mmol) was dissolved in dry THF (80 mL). To this diphenyl bromo(4-iodophenyl)methylphosphonate (30, 1.41 mg, 2.67 mmol, 3 eq) and t-BuOK (8.0 g) were added and the reaction was stirred under Ar at RT for 3 hrs. Following this, DCM (50 mL) was added and the resulting purple solution was washed with H2O (2 × 100 mL) then neutralised with dilute AcOH. The organic layer was separated, dried over MgSO4 and filtered. The solvent was reduced in vacuo and the porphyrin was precipitated from DCM:MeOH to give a red powder that was collected by filtration (560 mg, 75%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.08 (s, 1H, Hβ-pyrrolic), 8.90 (s, 1H, Hβ-pyrrolic), 8.84 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.79 (s, 1H, Hβ-pyrrolic), 8.78 (s, 2H, Hβ- pyrrolic), 8.75 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.25-8.21 (m, 8H, Hortho), 7.85-7.65 (m, 14H, Hmeta, para and Haromatic), 7.10 (d, 2H, J = 8.4 Hz, Haromatic), -2.67 (br s, 2H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 427 (167), 523 (13.9) 557 (4.46), 603 (3.62), 655 (2.87). ESI-LRMS: m/z (%, assignment) cluster at 841-843, 841 (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C52H34IN4): 841.1823, found: 841.1777. 2-(4′-Bromophenyl)ethynyl-5,10,15,20-tetraphenylporphyrin (39) N NH N HN Ph Ph Ph Ph Br C52H33BrN4 Exact Mass: 792.189 Mol. Wt.: 793.749 TPPCHO (2, 100 mg, 0.15 mmol) was dissolved in dry THF (15 mL). To this diphenyl bromo(4-bromophenyl)methylphosphonate (38, 223 mg, 0.46 mmol, 3 eq) or diphenyl chloro(4-bromophenyl)methylphosphonate (37, 202 mg, 0.46 mmol, 3 eq) and t-BuOK (1.5 g) were added and the reaction was stirred under Ar for 45 min at RT. Following this, DCM (25 mL) was added and the resulting purple solution was washed with H2O Chapter 8 – Experimental Methods 169 (2 × 50 mL) then neutralised with dilute glacial acetic acid. The organic layer was separated, dried over MgSO4 and filtered. The solvent was reduced in vacuo and the porphyrin was precipitated from DCM:MeOH to give a red powder which was collected by filtration (48 mg, 40% from Br derivative 38, 100 mg, 81% from Cl derivative 37). 1H NMR (500 MHz, CDCl3, TMS): δ 9.09 (s, 1H, Hβ-pyrrolic), 8.90 (s, 2H, Hβ-pyrrolic), 8.85 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.79 (s, 1H, Hβ-pyrrolic), 8.78 (s, 1H, Hβ-pyrrolic), 8.76 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.25-8.21 (m, 8H, Hortho), 7.83-7.64 (m, 12H, Hmeta, para and Haromatic), 7.48 (d, 2H, J = 8.4 Hz, Haromatic), 7.24 (d, 2H, J = 8.4 Hz, Haromatic), -2.65 (br s, 2H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 426.5 (345), 522.5 (29.2), 558 (9.06), 598.5 (8.13), 655 (5.87). FAB-LRMS: m/z (%, assignment) cluster at 792-795, 794 (70, M+). HRMS: Calcd. for M+ (C52H3379BrN4): 792.1888, found: 792.1874. 2-Phenylethynyl-5,10,15,20-tetraphenylporphyrin (67) N NH N HN Ph Ph Ph Ph C52H34N4 Exact Mass: 714.278 Mol. Wt.: 714.853 TPPCHO (2, 100 mg, 0.155 mmol) was dissolved in THF (15 mL). Compound 59 (151 mg, 0.47 mmol, 3 eq) and t-BuOK (1.5 g) were added and the reaction mixture was stirred under Ar for 1.5 hrs. The solvent was removed in vacuo and H2O (20 mL) was added to the purple solid, resulting in a suspension. Glacial acetic acid was added slowly with stirring until the solution reached pH 5, and then the reaction mixture was extracted with CHCl3 (50 mL). The organic layer was separated, dried over MgSO4, filtered and the porphyrin was precipitated from DCM:MeOH to give a purple solid (60 mg, 54%). This was a mixture of halovinyl intermediate and ethynyl porphyrin 80 that appeared as one spot on TLC. To obtain a spectroscopically pure compound the solid (60 mg) was dissolved in dry THF (5 mL) and t-BuOK (0.25 g) was added. The solution was stirred under Ar at RT for 1 h. The solvent was removed in vacuo and H2O (20 mL) was added. Glacial acetic acid was added slowly, with stirring, until pH 5 then the reaction mixture was extracted into CHCl3 (50 mL). The organic layer was separated, dried over MgSO4, filtered and the porphyrin was precipitated from DCM:MeOH to give a purple solid (30 mg, 50%). Spectroscopic data is in agreement with Ali et al.130 Chapter 8 – Experimental Methods 170 1H NMR (500 MHz, CDCl3, TMS): δ 9.10 (s, 1H, Hβ-pyrrolic), 8.91 (s, 2H, Hβ-pyrrolic), 8.86 (d, 1H, J = 5.0 Hz, Hβ-pyrrolic), 8.81 (s, 2H, Hβ-pyrrolic), 8.78 (d, 1H, J = 5.0 Hz, Hβ-pyrrolic), 8.26-8.23 (m, 8H, Hortho), 7.83-7.65 (m, 12H, Hmeta, para), 7.40-7.34 (m, 5H, Haromatic), - 2.63 (br s, 2H, NH). 1H NMR selected data for mixture of halovinyl intermediate 67a and 67 (500 MHz, CDCl3, TMS): δ -2.58, -2.64, -2.71. UV-Vis (THF): λmax [nm] (ε ×10−3) 424 (324), 521 (21.8), 556 (5.60), 599 (5.00), 656 (2.54). FAB-LRMS: m/z (%, assignment) cluster at 713-716, 715 (100, (M+H)+). HRMS: Calcd. for (M+H)+ (C52H35N4): 715.2862, found: 715.2857. 2-(4′-Methoxyphenyl)ethynyl-5,10,15,20-tetraphenylporphyrin (68) N NH N HN Ph Ph Ph Ph OMe C53H36N4O Exact Mass: 744.289 Mol. Wt.: 744.879 Compound 68 was prepared from 60 using the same method as used in compound 67 (97 mg, 84%). Spectroscopically pure compound was also obtained using the same method as for compound 67 (54 mg, 56%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.04 (s, 1H, Hβ-pyrrolic), 8.86 (s, 2H, Hβ-pyrrolic), 8.81 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.77 (s, 2H, Hβ-pyrrolic), 8.72 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.22-8.20 (m, 8H, Hortho), 7.79-7.66 (m, 12H, Hmeta, para), 7.29 (d, 2H, J = 8.7 Hz, Haromatic), 6.86 (d, 2H, J = 8.7 Hz, Haromatic), 3.86 (s, 3H, OCH3), -2.69 (br s, 2H, NH). 1H NMR selected data for mixture of halovinyl intermediate 68a and compound 68 (400 MHz, CDCl3, TMS): δ -2.60, -2.66, - 2.72. UV-Vis (THF): λmax [nm] (ε ×10−3) 422 (231), 521 (19.7), 558 (7.16), 599 (5.27), 656 (2.45). FAB-LRMS: m/z (%, assignment) cluster at 743-747, 745 (100, (M+H)+). HRMS: Calcd. for (M+H)+ (C53H37N4O): 745.2967, found: 745.2970. Chapter 8 – Experimental Methods 171 2-(4′-Pyridyl)ethynyl-5,10,15,20-tetraphenylporphyrin (70) N NH N HN Ph Ph Ph Ph N C51H33N5 Exact Mass: 715.274 Mol. Wt.: 715.841 TPPCHO (2, 350 mg, 0.54 mmol) was dissolved in dry THF (20 mL). Compound 62 (215 mg, 0.60 mmol, 1.1 eq) and t-BuOK (112 mg, 0.68 mmol, 1.25 eq) were added and the reaction was stirred under Ar for 3 hrs. Following this, additional 62 (50 mg, 0.14 mmol) and t-BuOK (76 mg, 0.46 mmol) were added. On completion the solvent was removed in vacuo and the residue was redissolved in DCM (50 mL). The organic solution was washed with H2O (50 mL), separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (28% aq. NH4OH:MeOH:DCM 1:10:89) eluting the product as a brown band. The solvent was removed in vacuo, the reside was dissolved in DCM and the porphyrin was precipitated from DCM:MeOH to give a purple solid (275 mg, 70%). 1H NMR (400 MHz, d4-pyridine): δ 9.13 (s, 1H, Hβ-pyrrolic), 8.92 (s, 2H, Hβ-pyrrolic), 8.87 (d, 1H, J = 5.0 Hz, Hβ-pyrrolic), 8.87 (m, 3H, Hβ-pyrrolic), 8.58 (dd, 2H, J = 4.4 Hz, 1.6 Hz, Haromatic), 8.25-8.20 (m, 8H, Hortho), 7.84-7.63 (m, 12H, Hmeta, para), 7.23 (dd, 2H, J = 4.4 Hz, 1.6 Hz, Haromatic), -2.67 (br s, 2H, NH). UV-Vis (THF): λmax [nm] (ε ×10−3) 427 (239), 521 (21.2), 556 (7.96), 599 (7.50), 656 (6.94). FAB-LRMS: m/z (%, assignment) cluster at 714-719, 716 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C51H34N5O): 716.2814, found: 716.2814. Chapter 8 – Experimental Methods 172 2-[4′-(5,5-Dimethyl-1,3-dioxane-2-yl)phenyl]ethynyl-5,10,15,20- tetraphenylporphyrin (71) N NH N HN Ph Ph Ph Ph O O C58H44N4O2 Exact Mass: 828.346 Mol. Wt.: 828.996 TPPCHO (2, 400 mg, 0.623 mmol) was dissolved in dry THF (53 mL). Compound 64 (900 mg, 1.74 mmol, 2.8 eq) and t-BuOK (5.3 g) were added and the reaction was stirred under Ar for 1 hr. Following this, DCM (150 mL) was added and the resulting purple solution was washed with H2O (2 × 100 mL). The organic layer was separated, dried over MgSO4 and filtered. The volume of the solvent was reduced to one third in vacuo and the porphyrin was precipitated from DCM:MeOH. The red powder was collected by filtration (453 mg, 88%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.10 (s, 1H, Hβ-pyrrolic), 8.90 (s, 2H, Hβ-pyrrolic), 8.84 (d, 1H, J = 5.0 Hz, Hβ-pyrrolic), 8.80 (s, 2H, Hβ- pyrrolic), 8.76 (d, 1H, J = 5.0 Hz, Hβ-pyrrolic), 8.25-8.21 (m, 8H, Hortho), 7.82-7.63 (m, 12H, Hmeta, para), 7.50 (d, 2H, J = 8.0 Hz, Haromatic), 7.39 (d, 2H, J = 8.2 Hz, Haromatic), 5.45 (s, 1H, CH), 3.85 (d, 2H, J = 11.0 Hz, CH2), 3.72 (d, 2H, J = 11.0 Hz, CH2), 1.38 (s, 3H, CH3), 0.87 (s, 3H, CH3), -2.65 (s, 2H, NH). UV-Vis (THF): λmax [nm] (ε ×10−3) 424 (290), 521 (32.8), 556 (16.3), 599 (14.1), 656 (10.9). FAB-LRMS: m/z (%, assignment) cluster at 827-831, 829 (100, (M+H)+). HRMS: Calcd. for (M+H)+ (C58H45N4O2): 829.3542, found: 829.3531. 2-(4′-Cyanophenyl)ethynyl-5,10,15,20-tetraphenylporphyrin (73) and 2-(4′- benzamide)ethynyl-5,10,15,20-tetraphenylporphyrin (74) N NH N HN Ph Ph Ph Ph CN N NH N HN Ph Ph Ph Ph O NH2 C53H35N5O Exact Mass: 757.284 Mol. Wt.: 757.878 C53H33N5 Exact Mass: 739.274 Mol. Wt.: 739.863 73 74 TPPCHO (2, 100 mg, 0.156 mmol) was dissolved in dry THF (15 mL). Compound 65 (132 mg, 0.31 mmol 2 eq) and t-BuOK (1.5 g) were added and the reaction mixture was Chapter 8 – Experimental Methods 173 stirred under Ar for 3 hrs. H2O (50 mL) and DCM (50 mL) were added forming an emulsion. To this glacial acetic acid was added slowly until the suspension cleared. The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting solid was redissolved in DCM and purified through a plug of silica gel, first eluting 73 with DCM, then 74 with MeOH:DCM (1:20). Both 73 and 74 were precipitated as purple solids from MeOH (30 mg, 26%) and hexane (42 mg, 36%), respectively. Compound 73 1H NMR (500 MHz, CDCl3, TMS): δ 9.13 (s, 1H, Hβ- pyrrolic), 8.92 (s, 2H, Hβ-pyrrolic), 8.87 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.79-8.78 (m, 3H, Hβ- pyrrolic), 8.26-8.22 (m, 8H, Hortho), 7.83-7.68 (m, 12H, Hmeta, para), 7.63 (d, 2H, J = 8.1 Hz, Haromatic), 7.45 (d, 2H, J = 8.1 Hz, Haromatic), -2.64 (br s, 2H, NH). UV-Vis (THF): λmax [nm] (ε ×10−3) 428 (200), 522 (19.2), 557 (7.08), 599 (6.68), 658 (5.83). FAB-LRMS: m/z (%, assignment) cluster at 738-742, 758 (100, M+). HRMS: Calcd for M+ (C53H33N5): 739.2736, found: 739.2736. Compound 74 1H NMR (500 MHz, CDCl3, TMS): δ 9.09 (s, 1H, Hβ-pyrrolic), 8.88 (s, 2H, Hβ-pyrrolic), 8.83 (d, 1H, J = 4.9 Hz, Hβ- pyrrolic), 8.79 (s, 1H, Hβ-pyrrolic), 8.74 (d, 2H, J = 4.9 Hz, Hβ-pyrrolic), 8.23-8.18 (m, 8H, Hortho), 7.79-7.59 (m, 14, Hmeta, para and Haromatic), 7.41 (d, 2H, J = 8.0 Hz, Haromatic), 6.04 (br s, 1H, NH2), 5.60 (br s, 1H, NH2), -2.67 (br s, 2H, NH). UV-Vis (THF): λmax [nm] (ε ×10−3) 426 (229), 522 (20.9), 557 (7.34), 599 (6.37), 656 (4.68). FAB-LRMS: m/z (%, assignment) cluster at 757-761, 758 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C53H36N5O): 758.2920, found: 758.2911. General Method for the Insertion of Zinc into the Porphyrins A solution of Zn(OAc)2·2H2O (2 eq) in MeOH (10 mL) was added to solution of porphyrin (1 eq) in CHCl3 (100 mL) and the reaction was stirred at RT. After stirring for 1 hr TLC analysis indicated that the reaction was complete. The solvent was removed in vacuo and the remaining solid was dissolved in DCM and purified through a plug of silica gel (DCM) if required. The solvent was removed in vacuo and the porphyrin was precipitated from DCM:MeOH to give a purple solid. Chapter 8 – Experimental Methods 174 2-(4′-Iodophenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (32) N N N N Ph Ph Ph Ph I Zn C52H31IN4Zn Exact Mass: 902.088 Mol. Wt.: 904.124 Compound 32 was isolated in 99% yield. 1H NMR (500 MHz, CDCl3, TMS): δ 9.25 (s, 1H, Hβ-pyrrolic), 8.95 (s, 2H, Hβ-pyrrolic), 8.93 (s, 2H, Hβ-pyrrolic), 8.90 (d, 1H, J = 4.7 Hz, Hβ- pyrrolic), 8.79 (d, 1H, J = 4.6 Hz, Hβ-pyrrolic), 8.25-8.20 (m, 8H, Hortho), 7.83-7.63 (m, 14H, Hmeta, para and Haromatic), 7.12 (d, 2H, J = 7.1 Hz, Haromatic). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 272 (26.3), 430 (307), 556 (20.8), 592 (8.70). ESI-LRMS: m/z (%, assignment) cluster at 925-931, 925 (100, (M+Na)+). ESI-HRMS: Calcd for (M+Na)+ (C52H31N4NaZn): 925.0777, found: 925.0730. 2-(4′-Bromophenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (40) N N N N Ph Ph Ph Ph Br Zn C52H31BrN4Zn Exact Mass: 854.102 Mol. Wt.: 857.123 Compound 40 was isolated in 92% yield. 1H NMR (400 MHz, CDCl3, TMS): δ 9.22 (s, 1H, Hβ-pyrrolic), 8.93 (s, 2H, Hβ-pyrrolic), 8.91 (s, 2H, Hβ-pyrrolic), 8.87 (d, 1H, J = 4.8 Hz, Hβ- pyrrolic), 8.76 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.22-8.18 (m, 8H, Hortho), 7.79-7.60 (m, 12H, Hmeta, para), 7.46 (d, 2H, J = 8.5 Hz, Haromatic), 7.23 (d, 2H, J = 8.5 Hz, Haromatic). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 431 (279), 557 (18.3), 590.5 (7.83). FAB-LRMS: m/z (%, assignment) cluster at 853-860, 856 (100, M+). HRMS: Calcd. for M+ (C52H3181BrN464Zn): 856.1003, found: 856.1016. Chapter 8 – Experimental Methods 175 2-Phenylethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (75) N N N N Ph Ph Ph PhZn C52H32N4Zn Exact Mass: 776.192 Mol. Wt.: 778.227 Compound 75 was isolated in 92% yield. 1H NMR (400 MHz, CDCl3, TMS): δ 9.94 (s, 1H, Hβ-pyrrolic), 8.94 (s, 2H, Hβ-pyrrolic), 8.92 (s, 2H, Hβ-pyrrolic), 8.89 (d, 1H, J = 4.8 Hz, Hβ- pyrrolic), 8.79 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.24-8.20 (m, 8H, Hortho), 7.80-7.63 (m, 12H, Hmeta, para), 7.40-7.32 (m, 5H, Haromatic). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 430.5 (211), 557.5 (13.6), 588.5 (5.10). FAB-LRMS: m/z (%, assignment) cluster at 776-782, 776 (100, M+). HRMS: Calcd. for M+ (C52H32N464Zn): 776.1918, found: 776.1913. 2-(4′-Methoxyphenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (76) N N N N Ph Ph Ph Ph OMe Zn C53H34N4OZn Exact Mass: 806.202 Mol. Wt.: 808.253 Compound 76 was isolated in 86% yield. 1H NMR (500 MHz, CDCl3, TMS): δ 9.23 (s, 1H, Hβ-pyrrolic), 8.96 (s, 2H, Hβ-pyrrolic), 8.94 (s, 2H, Hβ-pyrrolic), 8.90 (d, 1H, J = 4.7 Hz, Hβ- pyrrolic), 8.79 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.26-8.22 (m, 8H, Hortho), 7.83-7.68 (m, 12H, Hmeta, para), 7.34 (d, 2H, J = 8.8 Hz, Haromatic), 6.89 (d, 2H, J = 8.8 Hz, Haromatic), 3.85 (s, 3H, OCH3). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 430 (214), 556 (16.9), 589 (6.61). FAB-LRMS: m/z (%, assignment) cluster at 806-812, 806 (100, M+). HRMS: Calcd. for M+ (C53H34N4O64Zn): 806.2024, found: 806.2033. Chapter 8 – Experimental Methods 176 2-(4′-Pyridyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (77) N N N N Ph Ph Ph Ph N Zn C51H31N5Zn Exact Mass: 777.187 Mol. Wt.: 779.215 Compound 77 was isolated in 94% yield. 1H NMR (500 MHz, d4-pyridine): δ 9.55 (s, 1H, Hβ-pyrrolic), 9.09 (s, 2H, Hβ-pyrrolic), 9.08 (s, 2H, Hβ-pyrrolic), 9.05 (d, 1H, J = 4.7 Hz, Hβ- pyrrolic), 8.98 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.81 (d, 2H, J = 5.8 Hz, Haromatic), 8.40-8.36 (m, 8H, Hortho), 7.77-7.70 (m, 12H, Hmeta, para), 7.42 (d, 2H, J = 5.7 Hz, Haromatic), UV- Vis (CH2Cl2): λmax [nm] (ε ×10−3) 433.5 (262), 560 (15.7), 609 (6.27). FAB-LRMS: m/z (%, assignment) cluster at 777-784, 778 (100, (M+H)+). HRMS: Calcd. for (M+H)+ (C51H32N566Zn): 780.1918, found: 780.1922. 2-[4′-(5,5-Dimethyl-1,3-dioxane-2-yl)pheny]ethynyl-5,10,15,20- tetraphenylporphyrinato zinc II (78) N N N N Ph Ph Ph Ph O O Zn C58H42N4O2Zn Exact Mass: 890.26 Mol. Wt.: 892.37 Compound 78 was isolated in 88% yield. 1H NMR (500 MHz, CDCl3, TMS): δ 9.26 (s, 1H, Hβ-pyrrolic), 8.95 (s, 2H, Hβ-pyrrolic), 8.94 (s, 2H, Hβ-pyrrolic), 8.90 (d, 1H, J = 4.7 Hz, Hβ- pyrrolic), 8.80 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.25-8.21 (m, 8H, Hortho), 7.82-7.62 (m, 12H, Hmeta, para), 7.50 (d, 2H, J = 8.1 Hz, Haromatic), 7.41 (d, 2H, J = 8.2 Hz, Haromatic), 5.45 (s, 1H, CH), 3.85 (d, 2H, J = 10.8 Hz, CH2), 3.71 (d, 2H, J = 10.8 Hz, CH2), 1.38 (s, 3H, CH3), 0.86 (s, 3H, CH3), UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 274 (137), 431 (244), 557.5 (15.7), 590.5 (6.15). ESI-LRMS: m/z (%, assignment) cluster at 891-896, 891 (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C58H43N4N4O2Zn): 891.2672, found: 891.2663. Chapter 8 – Experimental Methods 177 2-(4′-Formylphenyl)ethynyl-5,10,15,20-tetraphenylporphyrin (79) N NH N HN Ph Ph Ph Ph CHO C53H34N4O Exact Mass: 742.273 Mol. Wt.: 742.863 Compound 71 (395 mg, 0.48 mmol) was added to a solution of CH2Cl2:TFA:H2O (3:3:1, 46 mL total) and the reaction was stirred at RT for 1 h. The green solution was diluted with H2O (50 mL) and DCM (50 mL) and neutralised with a saturated aq. solution of NaHCO3. The red solution was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting solid was dissolved in DCM and purified by silica gel chromatography (DCM), eluting the product as a purple band. The solvent was reduced in vacuo and the porphyrin was precipitated from DCM:MeOH to give the aldehyde as a purple solid (286 mg, 81%). Spectroscopic data is in agreement with Guldi et al.6 1H NMR (500 MHz, CDCl3, TMS): δ 10.03 (s, 1H, CHO), 9.10 (s, 1H, Hβ-pyrrolic), 8.88 (s, 2H, Hβ-pyrrolic), 8.83 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.76 (s, 2H, Hβ-pyrrolic), 8.74 (d, 1H, J = 4.8 Hz, Hβ-pyrrolic), 8.23-8.18 (m, 8H, Hortho), 7.83 (d, 2H, J = 8.2 Hz, Haromatic), 7.80-7.61 (m, 12H, Hmeta, para), 7.48 (d, 2H, J = 8.2 Hz, Haromatic), -2.65 (s br, 2H, NH). UV-Vis (THF): λmax [nm] (ε ×10−3) 428 (168), 522 (17.0), 558 (6.68), 600 (5.74), 658 (3.91). FAB-LRMS: m/z (%, assignment) cluster at 742-745, 743 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C53H35N4O): 743.2810, found: 743.2802. 2-(4′-Formylphenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (80) N N N N Ph Ph Ph Ph CHO Zn C53H32N4OZn Exact Mass: 804.187 Mol. Wt.: 806.237 A solution of Zn(OAc)2·2H2O (62 mg, 0.283 mmol, 1.27 eq) in MeOH (2 mL) was added to a solution of aldehyde 79 (165 mg, 0.222 mmol) in CHCl3 (20 mL) and the reaction was stirred at RT. After 1 h, TLC analysis indicated that the reaction was complete. The solvents were removed in vacuo and the remaining residue was Chapter 8 – Experimental Methods 178 precipitated from DCM:MeOH to give a purple powder (176 mg, 99%). Spectroscopic data is in agreement with Guldi et al.6 1H NMR (500 MHz, CDCl3, TMS): δ 10.02 (s, 1H, CHO), 9.27 (s, 1H, Hβ-pyrrolic), 8.93 (s, 2H, Hβ-pyrrolic), 8.915 (s, 2H, Hβ-pyrrolic), 8.87 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.77 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.23-8.18 (m, 8H, Hortho), 7.83 (d, 2H, J = 7.8 Hz, Haromatic), 7.80-7.60 (m, 12H, Hmeta, para), 7.51 (d, 2H, J = 8.2 Hz, Haromatic). UV-Vis (THF): λmax [nm] (ε ×10−3) 437 (185), 564 (10.2), 603 (6.63). FAB-LRMS: m/z (%, assignment) cluster at 804-810, 806 (100, M+). HRMS: Calcd for M+ (C53H32N4O64Zn): 804.1867, found: 804.1850. 2-(4′-Methoxycarbonylphenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (81) N N N N Ph Ph Ph Ph COOMe Zn C54H34N4O2Zn Exact Mass: 834.197 Mol. Wt.: 836.263 To a solution of aldehyde 80 (70 mg, 87µmol) in THF:MeOH (1:5, 21 mL), NaCN (85 mg, 3.0 mmol, 20 eq) was added and the reaction was stirred at RT, under Ar, for 30 min. Activated MnO2 (560 mg, 6.5 mmol, 75 eq) was added and the reaction was refluxed for 18 hrs. On cooling to RT the reaction mixture was filtered through a plug of celite (DCM) and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (DCM) and taken to dryness in vacuo to give a purple solid (62 mg, 88%). 1H NMR (500 MHz, CDCl3, TMS): δ 9.24 (s, 1H, Hβ-pyrrolic), 8.93 (s, 2H, Hβ-pyrrolic), 8.91 (s, 2H, Hβ-pyrrolic), 8.87 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.77 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.23-8.18 (m, 8H, Hortho), 7.95 (d, 2H, J = 8.3 Hz, Haromatic), 7.79-7.58 (m, 12H, Hmeta, para), 7.42 (d, 2H, J = 8.2 Hz, Haromatic), 3.91 (s, 3H, CO2CH3). UV-Vis (THF): λmax [nm] (ε ×10−3) 437 (242), 531 (2.69), 566 (15.5), 603 (6.90). FAB-LRMS: m/z (%, assignment) cluster at 833-841, 836 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C54H35N4O64Zn): 835.2051, found: 835.2049. Chapter 8 – Experimental Methods 179 2-(4′-Carboxyphenyl)ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (82) N N N N Ph Ph Ph Ph COOH Zn C53H32N4O2Zn Exact Mass: 820.182 Mol. Wt.: 822.237 KOH (67 mg, 1.2 mmol, 20 eq) in a mixture of MeOH (16 mL) and H2O (1.6 mL) was added to a solution of compound 81 (50 mg, 60 µmol) in THF (16 mL). The mixture was refluxed for 15 hrs under Ar. After cooling to RT, H2O (10 mL), DCM (10 mL) and aq 2.0 M H3PO4 (0.63 ml, 21 eq) were added and the resulting organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (Et2O:DCM 1:4) and the porphyrin was precipitated from DCM:hexane to give a purple solid (36 mg, 73%). 1H NMR (500 MHz, d6-DMSO): δ 13.09 (br s, 1H, COOH), 9.02 (s, 1H, Hβ-pyrrolic), 8.76 (s, 1H, Hβ-pyrrolic), 8.76 (s, 1H, Hβ-pyrrolic), 8.74 (s, 2H, Hβ-pyrrolic), 8.71 (d, 1H, J = 4.5 Hz, Hβ- pyrrolic), 8.77 (d, 1H, J = 4.5 Hz, Hβ-pyrrolic), 8.32-8.21 (m, 8H, Hortho), 7.94 (d, 2H, J = 8.3 Hz, Haromatic), 7.84-7.72 (m, 12H, Hmeta, para), 7.47 (d, 2H, J = 8.3 Hz, Haromatic). UV-Vis (DMF): λmax [nm] (ε ×10−3) 439 (253), 571 (13.4), 611 (4.45). FAB-LRMS: m/z (%, assignment) cluster at 818-826, 821 (100, (M+H)+). HRMS: Calcd for (M+H)+ (C53H33N4O264Zn): 821.1895, found: 821.1906. 2-[4′-(Ethen-1ʹʹ-yl-2ʹʹ-cyano-2ʹʹcarboxylic acid)phenyl]ethynyl-5,10,15,20- tetraphenylporphyrinato zinc II (83) N N N N Ph Ph Ph PhZn COOH NC C56H33N5O2Zn Exact Mass: 871.193 Mol. Wt.: 873.284 A mixture of porphyrin 80 (70 mg, 87 µmol), cyanoacetic acid (44.4 mg, 0.522 mmol, 6 eq) and ammonium acetate (40.2 mg, 0.52 mmol, 6 eq) in a solution of THF:acetic acid (1:1, 7 mL) was heated at 60˚C for 5 hrs. Afterwards Zn(OAc)2·2H2O (76 mg, 0.35 mmol, 4 eq) was added and the solution was heated for an additional 15 min. On Chapter 8 – Experimental Methods 180 cooling to RT the solution was filtered and sufficient H2O was added to precipitate the product as a purple powder (68 mg, 97%). 1H NMR (500 MHz, d6-DMSO): δ 14.05 (s br, 1H, COOH), 9.05 (s, 1H, Hβ-pyrrolic), 8.76 (s, 2H, Hβ-pyrrolic), 8.74 (s, 2H, Hβ-pyrrolic), 8.71 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.60 (d, 1H, J = 4.6 Hz, Hβ-pyrrolic), 8.35 (s, 1H, Hacrylic), 8.21-8.15 (m, 8H, Hortho), 8.06 (d, 2H, J = 8.4 Hz, Haromatic), 7.86-7.70 (m, 12H, Hmeta, para), 7.53 (d, 2H, J = 8.3 Hz, Haromatic). UV-Vis (DMF): λmax [nm] (ε ×10−3) 440.5 (243), 570 (19.8), 607 (10.3). FAB-LRMS: m/z (%, assignment) cluster at 871-877, 872 (100, M+). HRMS: Calcd for M+ (C56H33N5O264Zn): 871.1925, found: 871.1901. 2-[4′-(Ethen-1ʹʹ-yl-2ʹʹ,2ʹʹ-dicarboxylic acid)phenyl]ethynyl-5,10,15,20- tetraphenylporphyrinato zinc II (84) and 2-[4′-(ethen-1ʹʹ-yl-2ʹʹ-carboxylic acid)phenyl]ethynyl-5,10,15,20-tetraphenylporphyrinato zinc II (85) N N N N Ph Ph Ph PhZn COOH COOH C56H34N4O4Zn Exact Mass: 890.187 Mol. Wt.: 892.284 N N N N Ph Ph Ph PhZn COOH H C55H34N4O2Zn Exact Mass: 846.197 Mol. Wt.: 848.274 84 85 To a solution of aldehyde (80, 50 mg, 62.1 µmol) in THF:acetic acid (1:1, 5 mL total), malonic acid (258 mg, 2.48 mmol, 40 eq) and ammonium acetate (191.5 mg, 2.48 mmol, 40 eq) were added and the resulting solution was stirred at 40 °C for 4 hrs under Ar. TLC analysis of the reaction mixture showed the formation of the malonic acid derivative as well as the decarboxylated product. Afterwards, Zn(OAc)2·2H2O (50 mg, 0.22 mmol, 3.7 eq) was added and the reaction was stirred for an additional 15 minutes, filtered and sufficient H2O was added to cause precipitation. The resulting solid was filtered and dried under reduced pressure to give a purple powder that was found to be 70:30 malonic acid (84):decarboxylated (85) material by 1H NMR spectroscopy (ca. 96%). 1H NMR (500 MHz, d6-DMSO): δ 9.01 (s, 0.3H, 85, Hβ-pyrrolic), 8.99 (s, 0.7H, 84, Hβ-pyrrolic), 8.76 (s, 2H, 84 and 85, Hβ-pyrrolic), 8.74 (s, 2H, 84 and 85, Hβ-pyrrolic), 8.70 (d, 0.7H, J = 4.7 Hz, 84, Hβ-pyrrolic), 8.70 (d, 0.3H, J = 4.7 Hz, 85, Hβ-pyrrolic), 8.59 (d, 0.7H, J = 4.7 Hz, 84, Hβ-pyrrolic), 8.58 (d, 0.3H, J = 4.7 Hz, 85, Hβ-pyrrolic), 8.21-8.14 (m, 8H, 84 and 85, Hortho), 7.84-7.70 (m, 12.6H, 84 and 85, Hmeta, para, 84, Haromatic), 7.46 (d, 1.4H, J = 8.2 Hz, 84 Haromatic), 7.38 (d, 1.4H, J = 8.2 Hz, 85, Haromatic), 7.31 (d, 0.6H, J = 8.3 Hz, 84 Haromatic). Note: Hacrylic and Hacid were not visible. ESI-LRMS 85: m/z (%, Chapter 8 – Experimental Methods 181 assignment) cluster at 847-854, 847 (100, (M+H)+). ESI-HRMS: Calcd for (M+H)+ (C55H35N4O2Zn): 847.2046, found: 847.2049. No HRMS could be obtained for compound 84. UV-Visible spectroscopic data for 2-(4′-carboxyphenyl)ethlyane-5,10,15,20- tetraphenylporphyrinato zinc II (86) and 4-(trans-2′-(2′′-(5′′,10′′,15′′,20′′- tetraphenylporphyrinato zinc II yl)ethen-1′-yl))-1-benzoic acid (87). Compound 86: UV-Vis (DMF): λmax [nm] (ε ×10−3) 426 (426), 558.5 (14.8), 596.5 (4.42). Compound 87: UV-Vis (DMF): λmax [nm] (ε ×10−3) 437.5 (215), 567.5 (20.0), 605.5 (8.47). DSSC Testing DSSC were testing under conditions developed by Dr. Wayne Campbell. Solutions of acids were prepared in SDS AR BHT stabilised THF at 0.2 mM. To this, sintered TiO2 on a ITO on a glass support (heated at 490 °C for 30 min) was added and soaked overnight at RT in darkness. The TiO2 plates with bound porphyrins were removed from the dye solutions, rinsed, dried under high vacuum, placed in the cell holder containing a Pt counter electrode and I-/I3- electrolyte was added. Four identical cells for each acid were irradiated under the intensity of 1 sun and the average values calculated excluding any outlying values. Nanocrystalline screen printed TiO2 plates were obtained form Dye-Sol. Electrolyte: 0.1 M LiI, 0.05 M I2, 0.5 M 4-tert-butylpyridine, 0.6 M BMII, 0.5 M BHT in 1:1 valeronitrile:glutaronitrile Dye loading studies To perform the loading studies porphyrins were dissolved in THF (25 mL) at a concentration of approximately 1 × 10-5 M. 3 mL of this solution was added to a vial containing two TiO2 plates that had previously been sintered at 490 °C for 30 min. The vials were sealed and the dye was left to be absorbed overnight (16 hrs) in the dark. The following day the cells were removed and washed with THF (10 × 1 mL) to remove the non-specifically bound porphyrins. In our case no non-specifically bound porphyrin was observed. The UV-Vis spectra of the initial and the final solutions were recorded from Chapter 8 – Experimental Methods 182 220-800 nm (path length 0.1 cm) and the absorbance at λmax was recorded. From this data the quantity of porphyrin bound to the surface of the TiO2 for each dye was calculated. Chapter 8 – Experimental Methods 183 8.5 Experimental Procedures for Chapter 4 - Construction of Lipophilic Porphyrin-DNA Complexes 2-(4′-N-Methylpyridiumyl)ethynyl-5,10,15,20-tetraphenylporphyrin iodine (90) N NH N HN Ph Ph Ph Ph N I C52H36IN5 Exact Mass: 857.202 Mol. Wt.: 857.78 To a solution of compound 70 (80 mg, 0.11 mmol) in dry DMF (4 mL) methyl iodide (36 µL, 0.56 mmol, 5 eq) was added and the reaction was stirred at 40 °C overnight under Ar. The solvent was removed in vacuo to give a brown solid which was dissolved in DCM and precipitated from DCM:hexane. The solid was collected by filtration and the crystals were washed with an aqueous solution of NaI to give a red solid (96 mg, 92%). 1H NMR (400 MHz, CDCl3, TMS): δ 9.26 (s, 1H, Hβ-pyrrolic), 9.06 (d, 2H, J = 6.4 Hz, Haromatic), 8.95 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.92 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.88 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.79 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.76 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.75 (d, 1H, J = 4.9 Hz, Hβ-pyrrolic), 8.25-8.18 (m, 8H, Hortho), 7.87-7.75 (m, 12H, Hmeta, para), 7.72 (d, 2H, J = 6.6 Hz, Haromatic), 4.46 (s, 3H, CH3), -2.59 (br s, 2H, NH). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 411 (176), 475 (103), 533 (23.1) 581 (14.9), 616 (10.9), 677 (9.52). ESI-LRMS: m/z (%, assignment) cluster at 730-733, 730 (100, (M-I)+). ESI-HRMS: Calcd for (M-I)+ (C52H36N5): 730.2965, found: 730.2955. General Synthetic Method for the Formation of Porphyrin-DNA Complexes For the creation of lipophilic porphyrin DNA complexes we prepared a solution of oligonucleotide (Li+ or Na+ salt) in water (generally 100 µL, concentration of 100-500 µM). Porphyrin 9 or 27 (2.5 mg) was suspended in ACN (40 µL, sonicated for 15 minutes) then dissolved by the addition of water (40 µL). This porphyrin solution was further diluted to 1 mL with water. To the oligonucleotide solution the porphyrin solution was added in 2-5 µL fractions forming a red precipitate. Addition was continued until no more red precipitate is observed and a red colour developed in the Chapter 8 – Experimental Methods 184 solution. To see if any more precipitate formed it was necessary to centrifuge the sample between additions of the porphyrin (5 sec at 13500 rpm). When no more precipitate was observed the sample was centrifuged to form a pellet of the porphyrin- DNA complex (15 min at 13500 rpm). The supernatant was removed and the pellet was washed with H2O (2 × 1 mL) then dried under high vacuum for two days to remove any traces of H2O or remaining solvent. The complex was dissolved in CHCl3 (100 µL) to give a red solution. Chapter 8 – Experimental Methods 185 8.6 Experimental Procedure for Chapter 5 - Covalent Attachment of Porphyrins to DNA and Chapter 6 - Porphyrin H- Aggregate Formation in the Minor Groove of the Duplex 2ʹ-Deoxy-5ʹ-O-dimethoxytrityl-5-[4-(5,10,15,20-tetraphenylporphyrinato-2-yl zinc II)ethynylphenyl]ethynyluridine (94) NH O ON O OH DMTO ZnTPP C84H60N6O7Zn Exact Mass: 1328.381 Mol. Wt.: 1330.801 To a dry degassed solution of 2-(4ʹ-ethynylphenyl)ethynyl-5,10,15,20- tetraphenylporphyrinato zinc II (35, 25 mg, 31.1 µmol) in Et3N (1 mL), DMT protected 5-iodo-2ʹ-deoxyuridine (91, 81 mg, 124 µmol, 4 eq) was added and the reaction mixture was stirred for 5 min at RT under Ar. Pd(PPh3)4 (3.6 mg, 3.1 µmol, 0.1 eq) and CuI (1.2 mg, 6.2 µmol, 0.2 mol %) were added and the reaction mixture was stirred under Ar at 70 °C overnight. TLC indicated that the reaction was almost complete. The reaction was diluted with DCM (25 mL), washed with a 5% aq. solution of Na2EDTA (3 × 25 mL), then with a 3 M aq. solution of NH4OH (2 × 25 mL) and finally with H2O (25 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was removed in vacuo. The resulting solid was dissolved in DCM and purified by silica gel column chromatography first eluting the Glaser homodimer (34) and trace starting material (35) with DCM as a red band followed by the desired product (94) with MeOH:DCM (1:9) as a crude mixture (32 mg, 78%). The crude mixture was precipitated from DCM:MeOH to give pure 94 which was collected by filtration as a purple solid (5 mg, 12%). 1H NMR (500 MHz, d6-DMSO): δ 11.83 (s, 1H, NH), 9.02 (s, 1H, Hβ-pyrrolic), 8.77 (s, 2H, Hβ-pyrrolic), 8.75 (s, 2H, Hβ-pyrrolic), 8.72 (d, 1H, J = 4.6 Hz, Hβ-pyrrolic), 8.60 (d, 1H, J = 4.6 Hz, Hβ-pyrrolic), 8.23-8.16 (m, 8H, Hortho), 7.85-7.75 (m, 14H, Hmeta, para and Haromatic), 7.49 (d, 2H, J = 8.3 Hz, Haromatic), 7.44-7.28 (m, 9H, HDMTO), 6.95 (td, 4H, J = 7.0 and 1.9 Hz, HDMTO), 6.48 (s, 1H, H6), 6.18 (t, 1H, J = 5.4 Hz, H1′), 5.47 (d, 1H, J = 4.7 Hz, OH), 4.46 (m, 1H, H3′), 4.06 (m, 1H, H4′), 3.76 (s, 3H, OCH3), 3.75 (s, 3H, Chapter 8 – Experimental Methods 186 OCH3), 3.46-3.38 (m, 2H, H5′), 2.56-2.52 (m, 1H, H2′), 2.33-2.28 (m, 1H, H2′). UV-Vis (CH2Cl2): λmax [nm] (ε ×10−3) 440 (162), 570 (14.3), 605 (8.00). ESI-LRMS: m/z (%, assignment) cluster at 1363-1370, 1365, (100, (M+Cl)-) ESI-HRMS: Calcd for (M+Cl)- (C84H60N6O7Cl64Zn): 1363.3503, found: 1363.3505. 2ʹ-Deoxy-5-[4-(5,10,15,20-tetraphenylporphyrin-2-yl)ethynylphenyl]ethynyluridine (95) NH O ON O OH HO TPP C63H44N6O5 Exact Mass: 964.337 Mol. Wt.: 965.061 To a solution of crude porphyrin nucleoside 94 (15 mg, 11 µmol) in DCM (2 mL) trifluoroacetic acid (1.7 µL, 22 µmol, 2 eq) was added and the reaction mixture was stirred at RT for 2 minutes. To the resulting brown solution CHCl3 (10 mL) was added and the solution was extracted with H2O (5 × 10 mL) then basified with Et3N (5 µL), dried over MgSO4, filtered and the volume reduced to approximately 1 mL in vacuo. The porphyrin was precipitated from DCM:MeOH to give a brown solid which was collected by vacuum filtration (7.5 mg, 70%). 1H NMR (400 MHz, d6-DMSO): δ 12.02 (s, 1H, NH), 8.98 (s, 1H, Hβ-pyrrolic), 8.92 (s, 2H, Hβ-pyrrolic), 8.91 (s, 2H, Hβ-pyrrolic), 8.86 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.73 (d, 1H, J = 4.7 Hz, Hβ-pyrrolic), 8.27-8.21 (m, 8H, Hortho), 7.87-7.79 (m, 14H, Hmeta, para and Haromatic), 7.47 (d, 2H, J = 8.3 Hz, Haromatic), 7.41 (s, 1H, H6), 6.20 (t, 1H, J = 6.1 Hz, H1′), 5.32 (d, 1H, J = 4.7 Hz, 3′OH), 5.22 (d, 1H, J = 5.6 Hz, 5′OH), 4.46 (m, 1H, H3′), 3.95 (m, 1H, H4′), 3.66 (m, 2H, H5′), 2.20 (m, 2H, H2′), -2.81 (br s, 2H, NH). UV-Vis (CHCl3): λmax [nm] (ε ×10−3) 429.5 (112), 524 (12.4), 560 (5.62), 600 (4.24), 659 (3.21). ESI-LRMS: m/z (%, assignment) cluster at 965-967, 965, (20, (M+H)+) ESI-HRMS: Calcd for (M+H)+ (C63H45N6O5): 965.3451, found: 965.3492. Chapter 8 – Experimental Methods 187 General Synthetic Method for Porphyrin Nucleosides 97-101 Appropriate porphyrin azide (30 mg) was dissolved in dry THF (2.0 mL) and the reaction was degassed under Ar for 30 min. To this 2′-deoxy-5′-O-4ʹʹ,4ʹʹʹ- dimethoxytrityl-5-ethynyluridine (93, 1 eq) was added followed by tetrakis(acetonitrile)copper(I)hexafluorophosphate (2 eq). The reaction was stirred at RT for 2 (99-101) to 4 (97 and 98) days at which point the solvent was removed in vacuo and the crude material was purified by silica gel column chromatography with MeOH:DCM (1:9) first eluting the starting azide followed by the product. 2ʹ-Deoxy-5-{1-{4-[2-[4-(5,10,15,20-tetraphenylporphyrinato nickel II)vinyl]phenyl}-1,2,3-triazol-4-yl}uridine (97) NH O ON O OH HO NNN NiTPP C63H45N9NiO5 Exact Mass: 1065.29 Mol. Wt.: 1066.782 Characterisation and yields: Compound 97, 13 mg, 36%. 1H NMR (500 MHz, d6- DMSO): δ 11.80 (br s, 1H, NH), 8.96 (s, 1H, Hβ-pyrrolic), 8.93 (s, 1H, H6 or Htriazole), 8.70-8.65 (m, 7H, Hβ-pyrrolic + H6 or Htriazole), 8.05-7.99 (m, 8H, Hortho), 7.93 (d, 2H, J = 8.6 Hz, Haromatic), 7.91-7.74 (m, 12H, Hmeta, para), 7.38 (d, 2H, J = 8.6 Hz, Haromatic), 6.84 (d, 1H, J = 15.6 Hz, Hvinylic), 6.84 (d, 1H, J = 15.7 Hz, Hvinylic), 6.28 (t, 1H, J = 6.7 Hz, H1′), 5.33 (d, 1H, J = 4.1 Hz, C3′OH), 5.10 (t, 1H, J = 4.9 Hz, CH2OH), 4.34-4.30 (m, 1H, H3′), 3.89 (m, 1H, H4′), 3.66-3.63 (m, 2H, H5′), 2.25-2.23 (m, 2H, H2′). UV-Vis (CHCl3): λmax [nm] (ε ×10−3) 309 (20.7), 427.5 (119), 538 (9.48), 572 (6.87). ESI- LRMS: m/z (%, assignment) cluster at 1064-1070, 1064, (100, (M-H)-). ESI-HRMS: Calcd for (M-H)- (C63H44N9O558Ni): 1064.2819, found: 1064.2828. Chapter 8 – Experimental Methods 188 2ʹ-Deoxy-5-{1-{4-[2-[4-(5,10,15,20-tetraphenylporphyrinato nickel II)vinyl]benzyl}- -1,2,3-triazol-4-yl}uridine and 2ʹ-deoxy-5ʹ-O-dimethoxytrityl-5-{1-{4-[2-[4- (5,10,15,20-tetraphenylporphyrinato nickel II)vinyl]benzyl}-1,2,3-triazol-4- yl}uridine (98) NH O ON O OH HO NN N NiTPP NH O ON O OH DMTO NN N NiTPP C64H47N9NiO5 Exact Mass: 1079.305 Mol. Wt.: 1080.809 C85H65N9NiO7 Exact Mass: 1381.436 Mol. Wt.: 1383.175 Compound 98, in approximate ratio of 4:1 (DMT off: DMT on) 10 mg, 28%. 1H NMR assigned for DMT off product in mixture (400 MHz, d6-DMSO): δ 11.70 (br s, 1H, NH), 8.90 (s, 1H, Hβ-pyrrolic), 8.67-8.63 (m, 6H, Hβ-pyrrolic), 8.57 (s, 1H, H6 or Htriazole), 8.44 (s, 1H, H6 or Htriazole), 8.03-7.94 (m, 8H, Hortho), 7.82-7.72 (m, 12H, Hmeta, para), 7.27 (d, 2H, J = 8.2 Hz, Haromatic), 7.25 (d, 1H, J = 16.1 Hz, Hvinylic), 7.19 (d, 2H, J = 8.2 Hz, Haromatic), 6.79 (d, 1H, J = 16.1 Hz, Hvinylic), 6.24 (t, 1H, J = 7.0 Hz, H1′), 5.64 (s, 2H, CH2), 5.28 (d, 1H, J = 4.2 Hz, C3′OH), 5.02 (t, 1H, J = 4.9 Hz, C5′OH), 4.28 (m, 1H, H3′), 3.85 (m, 1H, H4′), 3.60 (m, 2H, H5′), 2.18 (m, 2H, H2′). UV-Vis (CHCl3): λmax [nm] (ε ×10−3) 309 (22.3), 426 (180), 540 (14.8), 576 (9.60). ESI-LRMS: m/z (%, assignment) cluster at 1078-1084, 1078, (100, DMT off, (M-H)-) and 1380-1385, 1381, (15, DMT on, M-). ESI-HRMS: DMT off; calcd for (M-H)- (C63H46N9O558Ni): 1078.2975, found: 1078.2991, DMT on; calcd for (M)- (C85H65N9O758Ni): 1381.4360, found: 1381.4333. Chapter 8 – Experimental Methods 189 2ʹ-Deoxy-5-{1-{4-[2-[4-(5,10,15,20-tetraphenylporphyrinato zinc II)ethynyl]benzyl}-1,2,3-triazol-4-yl}uridine and 2ʹ-deoxy-5ʹ-O-dimethoxytrityl-5- {1-{4-[2-[4-(5,10,15,20-tetraphenylporphyrinato zinc II)ethynyl]benzyl}-1,2,3- triazol-4-yl}uridine (99) NH O ON O OH HO NNNZnTPP NH O ON O OH DMTO NNNZnTPP C84H61N9O7Zn Exact Mass: 1371.399 Mol. Wt.: 1373.829 C63H43N9O5Zn Exact Mass: 1069.268 Mol. Wt.: 1071.463 Compound 99, in approximate ratio of 3:1 (DMT off: DMT on) 15 mg, 39%. 1H NMR assigned for DMT off product in mixture (400 MHz, d6-DMSO): δ 11.78 (br s, 1H, NH), 9.03 (s, 1H, Hβ-pyrrolic), 8.89 (s, 1H, H6 or Htriazole), 8.76 (s, 2H, Hβ-pyrrolic), 8.74 (s, 2H, Hβ-pyrrolic), 8.71 (d, 1H, J = 4.5 Hz, Hβ-pyrrolic), 8.71 (s, 1H, H6 or Htriazole), 8.59 (d, 1H, J = 4.5 Hz, Hβ-pyrrolic), 8.22-8.15 (m, 8H, Hortho), 7.85-7.74 (m, 12H, Hmeta, para), 7.28 (d, 2H, J = 8.2 Hz, Haromatic), 7.22 (d, 2H, J = 8.3 Hz, Haromatic), 6.27 (t, 1H, J = 6.9 Hz, H1′), 5.31 (d, 1H, J = 4.0 Hz, C3′OH), 5.09 (t, 1H, J = 5.0 Hz, C5′OH), 4.32 (m, 1H, H3′), 3.89 (m, 1H, H4′), 3.65 (m, 2H, H5′), 2.24 (m, 2H, H2′). UV-Vis (CHCl3): λmax [nm] (ε ×10−3) 435 (250), 563 (16.7) 603 (7.54). ESI-LRMS: m/z (%, assignment) cluster at 1068-1075, 1068, (100, DMT off, (M-H)-) and 1370-1375, 1370, (20, DMT on, (M-H)- ). ESI-HRMS: DMT off; calcd for (M-H)- (C63H42N9O564Zn): 1068.2600, found: 1068.2600, DMT on; calcd for (M-H)- (C84H60N9O764Zn): 1370.3907, found: 1370.3933. Chapter 8 – Experimental Methods 190 2ʹ-Deoxy-5-{1-{4-[2-[4-(5,10,15,20-tetraphenylporphyrinato iron III chloride)vinyl]benzyl}--1,2,3-triazol-4-yl}uridine and 2ʹ-deoxy-5ʹ-O- dimethoxytrityl-5-{1-{4-[2-[4-(5,10,15,20-tetraphenylporphyrinato iron III chloride)vinyl]benzyl}-1,2,3-triazol-4-yl}uridine (100) NH O ON O OH HO NN N FeIIIClTPP NH O ON O OH DMTO NN N FeIIIClTPP C64H47ClFeN9O5 Exact Mass: 1112.274 Mol. Wt.: 1113.413 C85H65ClFeN9O7 Exact Mass: 1414.404 Mol. Wt.: 1415.78 Compound 100, in unknown ratio of DMT off: DMT on, 15 mg, 42%. UV-Vis (CHCl3): λmax [nm] (ε ×10−3) 426 (120), 514 (16.6). ESI-LRMS: m/z (%, assignment) cluster at 1074-1079, 1077, (100, DMT off, (M-Cl)-) ESI-HRMS: DMT off; calcd for (M-HCl)- (C64H46N9O556Fe): 1076.2971, found: 1076.2948, No HRMS could be obtained for the DMT on structure. 2ʹ-Deoxy-5-{1-{4-[2-[4-(5,10,15,20-tetraphenylporphyrinato copper II)vinyl]phenyl}--1,2,3-triazol-4-yl}uridine and 2ʹ-deoxy-5ʹ-O-dimethoxytrityl-5-{1- {4-[2-[4-(5,10,15,20-tetraphenylporphyrinato copper II)vinyl]phenyl}-1,2,3-triazol- 4-yl}uridine (101) NH O ON O OH HO NNN CuTPP NH O ON O OH DMTO NNN CuTPP C84H63CuN9O7 Exact Mass: 1372.415 Mol. Wt.: 1374.001 C63H45CuN9O5 Exact Mass: 1070.284 Mol. Wt.: 1071.635 Compound 101, in unknown ratio of DMT off: DMT on, 15 mg, 44%. UV-Vis (CHCl3): λmax [nm] (ε ×10−3) 425 (182), 548 (13.7) 584 (5.94) ESI-LRMS: m/z (%, assignment) cluster at 1068-1075, 1068, (50, DMT off, (M-H)-) and 1371-1075, 1371, (25, DMT on, (M-H)-). ESI-HRMS: DMT off; calcd for (M-H)- (C63H44N9O563Cu): 1069.2761, found: Chapter 8 – Experimental Methods 191 1069.2775, DMT on; calcd for (M-H)- (C84H62N9O763Cu): 1371.4068, found: 1371.4070. 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-Tetraphenylporphyrinato-2ʹʹ-yl copper II)ethen-1ʹ- yl]azidobenzene (102) N N N N Ph Ph Ph Ph N3 Cu C52H33CuN7 Exact Mass: 818.209 Mol. Wt.: 819.411 Compound 102 was produced as an unwanted product of the CuI catalysed CuAAC reaction between 4-[trans-2ʹ-(5ʹʹ,10ʹʹ,15ʹʹ,20ʹʹ-tetraphenylporphyrin-2ʹʹ-yl)ethene-1- yl]azidobenzene (43) and 2′-deoxy-5′-O-4ʹʹ,4ʹʹʹ-dimethoxytrityl-5-ethynyluridine (93). UV-Vis (CHCl3): λmax [nm] (ε ×10−3) 422 (167), 547 (16.5), 584 (9.06). ESI-LRMS: m/z (%, assignment) cluster at 818-825, 818 (100, M+). ESI-HRMS: Calcd for M+ (C52H33N7Cu): 818.2088, found: 818.2042. IR-ATR (cm-1): 2114.1 (azide). Experimental for the Synthesis of Porphyrin Oligonucleotides Post-synthetic CuAAC Reaction For the shaking reaction DMT-off ON4-ON9 on CPG (0.33 µmol) containing the appropriate ethynyl modification were removed from their corresponding columns and added to a microcentifuge vial along with the appropriate azide (7.67 µmol, 23 eq) and degassed DMSO (150 µL). Freshly prepared CuSO4·5H2O (0.2 µmol, 0.6 eq, 5 µL of a 40 µmol/mL solution in degassed H2O) and sodium ascorbate (1.0 µmol, 3 eq, 20 µL of a 50 µmol/mL solution in degassed H2O) were added. The reaction mixture was shaken under Ar in darkness for three days. For the microwave accelerated reaction DMT-off oligonucleotides on CPG (ON25 or ON26, 0.33 µmol) containing 2′-O-propargyl uridine or 2′-O-propargyl adenosine were removed from their corresponding columns and placed into a microwave reaction vessel Chapter 8 – Experimental Methods 192 together with appropriate azide (7.67 µmol, 23 eq) in degassed DMSO (200 µL). Freshly prepared CuSO4·5H2O (0.32 µmol, 0.96 eq, 8 µL of a 40 µmol/mL solution in degassed H2O) and sodium ascorbate (1.25 µmol, 3.8 eq, 25 µL of a 50 µmol/mL solution in degassed H2O) were added. The reaction mixture was then irradiated in a microwave synthesiser (Discover, CEM Corporation, 70 °C, 100 watts, 20 min). The content of the reactions were transferred to a microcentrifuge tube. To the reaction mixture DCM (1.5 mL) was added and the CPG was centrifuged (14500 rpm for 1 minute). The DCM was removed and washing was repeated until the supernatant no longer showed any colour (see recovery of azides). The red CPG was then washed with H2O (1.5 mL) to remove any remaining inorganic salts. Residual solvent was removed under reduced pressure and the obtained DMT-off oligonucleotides bound to CPG supports were treated with 32% aq NH4OH (1 mL) at RT for 2 h and then at 55 °C overnight. Recovery of Azides To the combined DCM washings containing the appropriate reacted azide, H2O (50 mL) was added and the resulting solution was vigorously stirred for 1 hr to remove DMSO and any remaining inorganic salts. The organic layer was extracted into DCM (2 × 50 mL), dried over MgSO4, filtered and the porphyrin was precipitated from DCM:MeOH. The desired product was collected by filtration to give a red solid (approximately 80- 90% recovery). Purification of Oligonucleotides Purification of porphyrin functionalised DMT off ONs by HPLC was accomplished using the following gradient. Buffer A [0.05 M triethylammonium acetate in H2O (pH = 7.0)] and buffer B (75% ACN in H2O), flow 2.5 mL min-1. Gradients: 2 min 100% A, linear gradient to 70% B in 38 min, linear gradient to 100% B in 7 min, 100% B in 3 min and then 100% A in 10 min. Purification of porphyrin functionalised DMT off ONs by puri-pak C18 cartridges was accomplished using the following method. Although it is recommended that the Chapter 8 – Experimental Methods 193 solvents are allowed to drip through the column an increased flow rate of approximately 30 mL per min was found to be adequate for purification. Cartridges were first washed with ACN (2 × 2 mL) then activated with 1.0 M TEAA solution (3 × 2 mL). Crude porphyrin modified oligonucleotide was loaded on to the column in a 1:1 solution of conc. NH4OH:H2O (2 mL) with the eluent being reloaded and eluted until the eluent showed no colour. Cartridges were then flushed with 3% aq. NH4OH solution (4 × 2 mL) followed by H2O (4 × 2 mL). Oligonucleotides were then eluted with a gradient of ACN:H2O (20% ACN to 100% ACN, 10% intervals) eluting single modifications in 20% ACN:H2O and double modifications in 30-40% ACN:H2O. After purification ONs were lyophylised, dissolved in H2O (100 µL, heating to 70 °C for 1 hr was required for some ONs), 0.01 M lithium perchlorate in acetone (1.6 mL) was added and the ONs were stored at -10 °C for 1 h. The precipitated ON pellet was centrifuged (15000 rpm for 30 min), the supernatant was removed and the pellet was washed with acetone (30 µL). Melting Temperature Measurements The triplexes were formed by first mixing the two strands of the Watson-Crick duplex, each at a concentration of 1.0 µM in the corresponding buffer solution followed by the addition of the third TFO strand at a concentration of 1.5 µM (total volume 1 mL). The solutions were then heated to 70 °C for 15 min, cooled and incubated at 10 °C for 30 min. The duplexes were formed by mixing the two strands each at a concentration of 1.0 µM in the appropriate buffer (total volume 1 mL). The solutions were heated at 90 °C for 15 min, cooled and incubated at 20 °C for 30 min. The melting temperatures were determined as the maxima of the first derivative plots of the melting curves obtained by measuring absorbance at 260 nm and 430/423 nm against increasing temperature (10 to 70 (or 90) °C, 1.0 °C per min for duplexes and 5 to 70 °C, 0.5 °C per min for triplexes). All melting temperatures are an average of two denaturing-annealing cycles. Appendix A 194 Appendix A Crystallographic Data Compound 80 N N N N CHO Zn Empirical Formula C54H35N4O2Zn Formula weight 910.15 Temperature 106 Wavelength 0.71073 Crystal system Monoclinic Space Group P2 (1) /c Unit cell dimensions a = 10.161(2) Å α = 90.00 ° b = 35.026(7) Å β = 105.43(3) ° c = 12.550(3) Å γ = 90.00 ° Volume 4305.5(15) Å3 Z 4 Density (calculated) 1.404 Mg/m3 Absorption Coefficient 0.744 mm-1 F(000) 1876 Index ranges -12<=h<=10, -43<=k<=39, -11<=l<=15 Reflections collected 23230 Independent reflections 8616 Completeness to theta 0.978 Refinement method Shelxl-97 Data/restraints/parameters 18 Goodness-of-fit on F2 1.052 Largest diff. peak and hole -1.167 e. Å-3 R factor (all) 0.0995 R factor (gt) 0.0716 wR factor (all) 0.1864 wR factor (gt) 0.1713 Appendix A 195 Compound 39a N NH N HN Br Br Empirical Formula C52H34Br2N4 Formula weight 890.65 Temperature 106 Wavelength 0.71073 Crystal system Triclinic Space Group P-1 Unit cell dimensions a = 9.4970 (19) Å α = 71.08 (3) ° b = 13.337 (3) Å β = 79.17 (3) ° c = 19.031 (4) Å γ = 74.07 (3) ° Volume 2179.7(8) Å3 Z 2 Density (calculated) 1.357 Mg/m3 Absorption Coefficient 1.902 mm-1 F(000) 904 Index ranges -13<=h<=12, -19<=k<=18, -26<=l<=27 Reflections collected 19407 Independent reflections 12212 Refinement method Shelx-97 Goodness-of-fit on F2 1.098 Largest diff. peak and hole 5.272 e. Å-3 R factor (all) ` 0.1380 R factor (gt) 0.1020 wR factor (all) 0.3325 wR factor (gt) 0.2994 References 196 References 1. Kadish, K. M.; Smith, K. M.; Guilard, R., The Porphyrin Handbook. Academic Press: London, 2000; Vol. 4, p 345. 2. Lin, V. S. Y.; DiMagno, S. G.; Therien, M. 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