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. Synthesis and Analysis of Libraries of Potential Flavour Compounds A thesis presented in partial fulfillment of the requirements of the degree of Doctor of Philosophy m Chemistry at Massey University Palmerston North New Zealand by Kyong-A (Karen) Bang July 2006 Science is organised knowledge. Wisdom is organised life. Immanuel Kant (1724 - 1804) Abstract The goal of this project was to synthesise potential flavour compounds combinatorially and identifY key components for further investigation as flavourants in dairy products. This thesis describes the design and synthesis of libraries of ketones and y-Iactones that will be evaluated for flavour potential. Gas chromatography-mass spectrometry (GC­ MS), the Fox, and gas chromatography-olfactometry (GC-O) were used throughout this study. Ketones were synthesised individually via a two-step sequence: a Grignard reaction fol lowed by the oxidation of the resulting alcohol in Chapter 2. Some compounds selected from the Fox analysis were assessed by GC-O. The analysis gave promising results for aromatic and cyclopropyl ketones and a library of cyclopropyl ketones was prepared. Individual racemic lactones were synthesised via a two-step sequence: the L instead modification of the Knoevenagel reaction and subsequent lactonisation in Chapter 3 . Libraries of racemic y-Iactones (Cs-Cn), including a-substituted y-Iactones, were produced combinatorial ly. Further, synthesis of a library of y-thionolactones was achieved by treatment of a library of y-Iactones with Lawesson's reagent . The libraries were analysed by GC-O. A (R)-dodecalactone was synthesised from L-glutamic acid and the (S)-enantiomer was synthesised by the same sequence from D-glutamic acid in Chapter 4. Asymmetric syntheses of both enantiomeric series of y-Iactones utilizing the Sharp less asymmetric dihydroxylation reaction was employed to give the libraries in Chapter 5 . Libraries of a-substituted and � -substituted y-Iactones were synthesised combinatorially and analysed by GC-O. Acknowledgements I would like to thank Associate Professor Carol M . Taylor for the opportunity to carry this project for my PhD degree. I appreciate her valuable advise and help during the work. I am so grateful to have co-supervisors Associate Professor David R. K. Harding in Massey University and Or. Sharon Bisley at Fonterra Research Centre for their support and consu lting. I would like to give special thanks to the team at Fonterra Research Centre ; Or. Ross Holland as the leader of this project, Or. Gary Depree, Or. Owen Mills and Or. Frank Visser for their analytical advise in dairy and flavour industry; Mr. Andrew Broome for his technical help with GC-MS and GC-O. I am so pleased to get my PhD scholarship from the Foundation for Research, Science and Technology via the Fonterra Research Centre (Project No. A 1 347 1 . 1 ). I could not complete the work without Or. Krishanthi Jayasundera for her endless knowledge and advice in chemistry and life . I enjoyed the time sharing a laboratory and an office with her. I also want to thank to my co-worker, Mr. David Lun for his contribution to th is project. There are so many friends, lecturers and Massey staff I would l ike to thank for their support and warm smiles . Finally, I thank to my husband Dr. Peter Shin for his support and endless love and I am so lucky to have him in my life . 11 Table of contents Abstract Acknowledgements Table of contents Tables Figures Schemes Abbreviations Chapter 1. Background and introduction: food flavour and combinatorial chemistry 1.1 Background 1 .2 Cheese flavour 1.3 Biosynthesis of cheese flavour compounds lA Flavour compounds in other foods 1.5 Instrumental analysis of food flavours 1.6 Combinatorial chemistry 1.7 Aims and objectives of this project Chapter 2. Synthesis of a library of ketones as potential flavour compounds 2.1 Introduction 2.2 Strategy for the chemical synthesis of the library of ketones 2.3 Synthesis III 11 iii VI VlI lX XIV 7 17 20 22 29 30 34 35 2.4 Screening of first ketone library 2.S Combinatorial chemistry 2.6 Summary 2.7 Experimental procedures Chapter 3: Synthesis of a library of racemic lactones as potential flavour compounds 3.1 Introduction 3.2 Generation of y-lactones in nature 3.3 Previous syntheses ofracemic y-lactones 3.4 Racemic lactones 3.S Thionolactones 3.6 Summary 3.7 Experimental procedures Cha pter 4: Syntheses of chiral lactones from amino acids 4.1 Introduction 4.2 Previous synthesis of chiral y-lactones 4.3 Syntheses of chiral lactones 4.4 Attempted synthesis of chiral 8-1actones 4.S Summary 4.6 Experimental procedures IV 42 46 SO S I 66 68 72 7S 98 106 107 118 120 129 140 141 142 Chapter 5: Asymmetric synthesis of chiral y-lactones utilizing the Sharpless asymmetric dihydroxylation reaction 5.1 Introduction 5.2 Strategy for the chemical synthesis of chiral lactones 5.3 Synthesis of stereoisomerically pure y-Iactones 5.4 Synthesis of optically active y-Iactones with unsaturated y-side chains 5.5 Summary 5.6 Experimental procedures Chapter 6: Summary and future work References v 150 155 156 168 172 173 192 197 Tables Table 1.1 Table 1.2 Table 2.1 Table 2.2 Table 2.3 Table 2.4 Table 2.5 Table 2.6 Table 2.7 Table 2.8 Table 3.1 Table 3.2 Table 3.3 Table 3.4 Table 3.5 Table 3.6 Table 3.7 Table 4.1 Table 4.2 Table 4.3 Table 5.1 Important flavour compounds in selected cheeses. Odour descriptions of compounds found in dairy products. Flavour notes of straight chain methyl ketones in cheese. Flavour notes of other ketones in cheese. The fIrst library of 20 ketones as potential flavour compounds. Ketones prepared via Swem oxidation of secondary alcohols. A library of the ketones by the DDQ oxidation. FOX results which quantify the relationship between each ketone and four cheeses. The properties of secondary alcohols from the Grignard reaction. The amount of ketone added in a beaker for the sample preparation. Structure, name and odourous properties of lactones. Fonning p,y-unsaturated carboxylic acids under the two sets of reaction conditions. Condensation of a-alkyl malonic acids in the presence of amine bases. The odour description of y-Iactones. Odour description for thionolactones. The thionation of lactones. The structure and odour description of thionolactones. y- and b-Lactones (Cg-C IS) from various cheeses. Enzymatic resolution of racemic lactones. Cuprate additions reported by Monache. Odour descriptions for enantiomerically optical y-Iactones VI 2 21 30 31 33 38 42 45 52 63 67 83 91 97 102 104 106 119 122 138 159 Figures Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure I.S Figure 1.6 Figure 1.7 Figure 1.8 Figure 1.9 Figure 1.10 Figure 1.11 Figure 1.12 Figure 1.13 The major amino acids for aroma formation in cheese. Fatty acids in cheese flavour. Ketones in cheese flavour. Alcohols in cheese flavour. Lactones in cheese flavour. Esters in cheese flavour. Aldehydes in cheese flavour. Sulfur compounds in cheese flavour. Nitrogen-containing compounds in cheese. Chemical structures of hop bitter acids. Chemical structure of the bell pepper pyrazine. Flavour compounds in chocolate. Mercaptoakohols, mercaptoketones and mercaptoaldehydes with desirable properties. S 7 8 9 12 12 13 IS 16 18 19 19 28 Figure 1.14 Potential flavour compounds. 29 Figure 2.1 The general structure of ketone targets. 32 Figure 2.2 Commercially available aldehydes and solutions of Grignard reagents being used in the synthesis of ketones. 36 Figure 2.3 The ketones that were not sufficiently stable for analysis. 42 Figure 2.4 Figure 2.S Figure 2.6 Figure 2.7 Figure 2.8 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.S PCA map for the blank, 4 cheeses and 16 ketones, with illustration of angle determination for compound 2.29. 44 The structure and odour description of lead ketones from the first library. 46 Oligocyclopropane-containing compounds. 47 PCA map for the blank, camembert and the library of four ketones. 49 GC trace for the library of cyc\opropyl ketones and their odour descriptions. 49 General structures of lactones. 66 Long-chain hydroxy fatty acid precursors. 70 IH NMR spectra (400 MHz, COCl}) of the crude reaction products. 77 Mass spectrum of dihydro-S-octyl-2(3H)-furanone. 78 (a) NMR data and (b) GC trace of the mixture 00.33 and 3.34. 80 Vll Figure 3.6 I H NMR spectrum (400 MHz, CDCb) of the crude reaction mixture containing 3.34 and a trace of 3.33. 82 Figure 3.7 (a) Four diastereomers of compound 3.66 and (b) the NMR trace of the mixture. 94 Figure 3.8 GC traces of (a) five carboxylic acids and (b) five racemic lactones. 95 Figure 3.9 GC traces of (a) five carboxylic acids and (b) five dihydro-3-methyl-5- alkyl-2(3H)-furanones. 96 Figure 3.10 The structures of natural flavour compounds in fruits and cheeses. 98 Figure 3.11 Sulfur-containing compounds in roasted coffee. 98 Figure 3.12 Examples of thiolactone rings in natural products. 99 Figure 3.13 2 ,4-his( 4-methoxyp henyl)-1 ,3 -dithia -2 ,4-dip hosphetane-2 ,4-disulfide. 101 Figure 3.14 GC trace for library of five thionolactones. 105 Figure 4.1 Chiral lactones isolated from nature. 118 Figure 5.1 (DHQhPHAL and (DHQD)2PHAL. 151 Figure 5.2 Synthesis of libraries ofy-substituted lactones with GC trace at each step. 158 Figure 5.3 Mass spectrum for the mixture of 3S,5R-3.66 and 3R,5R-3.66. 161 Figure 5.4 NOESY spectrum of lactone 3S,5R-3.66 (CDCb, 500 MHz). 162 Figure 5.5 A library of four 3-substituted (5R)-dihydro-5-pentyl-2(3H)-furanones with the GC trace and the odour description. 163 Figure 5.6 NOESY spectrum of lactone (+)-trans-531 (CDCh, 500 MHz). 166 Figure 5.7 A library of three 4-substituted (5R)-dihydro-5-pentyl-2(3H)-furanones with the GC trace and the odour description. 167 Figure 5.8 Insect pheromones. 168 Figure 6.1 The structures of lead ketones from Chapter 2. 192 Figure 6.2 Some of the ketones produced in libraries. 193 Figure 6.3 Structures of libraries of racemic y-Iactones. 194 Vlll Schemes Scheme 1.1 Scheme 1.2 Scheme 1.3 Scheme 1.4 Scheme 1.5 Scheme 1.6 Scheme 1.7 Scheme 1.8 Scheme 1.9 Scheme 1.10 Scheme 1.11 Scheme 1.12 Scheme l .l 3 Flavour development in cheese. Conversion of methionine to volatile sui fur compounds. Enzymatic hydrolysis of triglyceride. The biosynthesis of undecan-2-one from dodecanoic acid. Enantioselective hydrolysis of (d,l)-menthyl acetate. Ehrlich pathway for 2-phenylethanol synthesis. Lipoxygenase-mediated generation of 3S,5Z-oct-5-en-3-01 from a­ linolenic acid. The alcoholysis of a milk fat monoglyderide and ethanol by LAB. Lipoxygenase-mediated generation of 3Z-hexenal from a-linolenic acid. The formation of methanethiol. Decarboxylation of amino acids. Solid-phase peptide synthesis according to Merrifield. Synthesis of a combinatorial library from cubane tetraacid chloride and amines. Scheme 1.14 Synthesis of S-methyl thioesters from carboxylic acids and methyl 3 6 8 9 10 10 11 13 14 15 16 23 25 chlorothiolformate. 26 Scheme 1.15 Synthetic pathways for production of mercaptoalcohols from a,�- Scheme 1.16 Scheme 2.1 Scheme 2.2 Scheme 2.3 Scheme 2.4 Scheme 2.5 Scheme 2.6 Scheme 2.7 Scheme 2.8 Scheme 2.9 Scheme 2.10 Scheme 2.11 unsaturated ketones. Synthesis of mer cap to esters. The metabolic pathway for the formation of methyl ketone. Retrosynthetic analysis for ketone library. The general mechanism for the Grignard reaction. The overall transformation of the Swem oxidation. Mechanism of the Swem oxidation. The oxidation of(lOE,12Z)-10,12-octadecadiene-1,-9-diol with POC. The attempted oxidation of allylic alcohol 2.47 with POe. The mechanistic possibility ofE2. The mechanistic possibility of El . The oxidation of 1 ,3-diphenyl-3 -hydroxy-I-propene by OOQ. Mechanism for cyclopropane formation in nature. IX 27 28 32 34 35 37 37 39 39 40 40 41 48 Scheme 2.12 Combinatorial synthesis of a library of four cyclopropyl ketones. 48 Scheme 3.1 Possible pathway for dihydro-5-octyl-2(3H)-furanone formation by Penicillium roqueforti. 69 Scheme 3.2 Lactones from ricinolenyl-CoA. 71 Scheme 3.3 Synthesis of (Z)-5-octen-4-0Iide. 72 Scheme 3.4 Synthesis of (Z)-6-nonen-4-0Iide. 72 Scheme 3.5 Synthesis of dihydro-5-heptyl-2(3H)-furanone. 73 Scheme 3.6 Undergraduate laboratory experiment of making dihydro-5 -pentyl- 2(3H)-furanone. 73 Scheme 3.7 Mechanism of the classical Knoevenagel condensation. 74 Scheme 3.8 Dehydration and decarboxylation of a P-hydroxy-a-dicarboxylic acid. 74 Scheme 3.9 Retrosynthetic analysis for racemjc lactones. 75,88 Scheme 3.10 Mechanism for lactone formation. 76 Scheme 3.11 Reaction applied to an unsaturated aldehyde 3.32 (Method A). 79 Scheme 3.12 Reaction applied to an unsaturated aldehyde 3.32 (Method B). 81 Scheme 3.13 Lactonisation of p,y-unsaturated acids. 84 Scheme 3.14 Proposed mechanism of lactonisation. 85 Scheme 3.15 Proposed mechanism for the lactonisation of a p,y-unsaturated carboxylic acid with Amberlyst-15 resin. 86 Scheme 3.16 A mechanism for the formation ofy-Iactones from unsaturated fatty acids proposed by Ansell and Palm er. 87 Scheme 3.17 The perchloric acid-catalysed isomerisation of oleic acid into y- stearolactone. 87 Scheme 3.18 Unsuccessful pathway for lactonisation of a more highly unsaturated acid 3.34. 88 Scheme 3.19 Condensation of a-substituted malonic acid 3.45 and aldehyde 1.61. 89 Scheme 3.20 The metal-catalysed decarboxylative aldol reaction. 89 Scheme 3.21 The Knoevenagel reaction of p-nitrobenzaldehyde with methyl malonic acid. 90 Scheme 3.22 The condensation reaction followed by dehydration and decarboxylation. 92 Scheme 3.23 The Knoevenagel reaction of he pt anal with methyl malonic acid in the presence of diethylamine. 93 Scheme 3.24 Synthesis of a-substituted lactones, 3.66 and 3.67. 93 x Scheme 3.2S Synthesis of a library of y-lactones. 9S Scheme 3.26 Kharasch and Langford's synthetic route to y-thiolactone. 100 Scheme 3.27 Kaloustian's approach to oxygen-sulfur exchange. 100 Scheme 3.28 A proposal for the mechanism of thionation of a y-lactone with Lawesson's reagent. 101 Scheme 3.29 Individual syntheses of y- and b-thiono1actones. 102 Scheme 3.30 Synthesis of thiono-derivative of whiskey lactone 3.114. 103 Scheme 3.31 Enzymatic resolution of racemic y-lactones and thionation of chira1 1actones. 103 Scheme 3.32 Combinatorial synthesis of a library of five thionolactones. lOS Scheme 4.1 Chemical resolution of cyano alcohols followed by hydrolysis and 1actonisation. 121 Scheme 4.2 Synthesis of a racemic lactone 3.91 from ethyIS-oxo-hexanoate. 122 Scheme 4.3 Stereoselective reduction of a ketone in the generation of a chiral lactone. 123 Scheme 4.4 Synthesis of (SR)-dihydro-S-octyl-2(3H)-furanone. 124 Scheme 4.S Synthesis of a chrial y-Iactone 4.26 starting from ally1ic alcohol 4.21. 12S Scheme 4.6 Retrosynthetic analysis of (+)-cryptophycin. 12S Scheme 4.7 Synthesis of ehiral y-laetone 4.29 by utilising an enantioselective Corey- Bakshi-Shibata (CBS) reduction. 126 Scheme 4.8 Syntheses of chiral y-butyrolactones via reduction with fermenting bakers' yeast. 127 Scheme 4.9 Synthesis of ehiral y-Iactone 4.43, a precursor to a Phe-Arg hydroxyethylene isostere. 128 Scheme 4.10 Retrosynthetie analysis of chiral y-lactones. 129 Scheme 4.11 Diazotisation of L-glutamic acid. 130 Scheme 4.12 Reduction of (SR)-2-oxotetrahydrofuran-S-carboxylic acid. 130 Scheme 4.13 Two routes to (SS)-S-(bromomethyl)-2(3H)-furanone. 131 Scheme 4.14 Two routes to (SS)-S-(iodomethyl)-2(3H)-furanone. 131 Scheme 4.1S Retrosynthetic analysis of the y-1actone via an olefinic handle. 132 Scheme 4.16 Reisner's extension of the y-side chain via olefm metathesis. 132 Scheme 4.17 The condensation of halides and allyltributyl tin in the presence of AlBN. 133 Xl Scheme 4.18 Retrosynthetic approach to a y-lactone via a Stille coupling. 133 Scheme 4.19 Reduction of S-4.45. 134 Scheme 4.20 The zinc-mediated one-pot Wurtz-type reductive coupling reaction. 134 Scheme 4.21 Wurtz-type reductive coupling reaction of S-4.44. 134 Scheme 4.22 Retrosynthetic approach to a y-lactone via a Wittig reaction. 135 Scheme 4.23 Wittig reaction of S-4.44. 135 Scheme 4.24 Synthesis and proposed side reactions of phosphonium salt 4.66. 136 Scheme 4.25 Maurer and Hauser's synthetic route to y-Iactones. 137 Scheme 4.26 Retrosynthetic approach to a y-Iactone via a cup rate displacement. 137 Scheme 4.27 Silverstein and Ravid's cuprate displacements of tosylate S-4.72. 138 Scheme 4.28 Formation of (5R)-dihydro-5-octyl-2(3H)-furanone. 139 Scheme 4.29 Synthesis of (5S)-dihydro-5-octyl-2(3H)-furanone from D-glutamic acid. 139 Scheme 4.30 Proposed synthetic route for the formation of chiral b-Iactones. 140 Scheme 4.31 Syntheses of both enantiomers of dihydro-5-octyl-2(3H)-furanone. 141 Scheme 5.1 Two proposed mechanisms for the osmium-catalysed dihydroxylation. 150 Scheme 5.2 Sharp!ess' asymmetric dihydroxylation of olefms. 151 Scheme 5.3 Enantiofacial selectivity of AD reaction. 152 Scheme 5.4 The asymmetric dihydroxylation of p,y- and y,b-unsaturated esters. 153 Scheme 5.5 Synthesis of a chiral building block 4R,5R-5.9. 153 Scheme 5.6 Synthesis of enantiopure lactone R-1.52. 154 Scheme 5.7 Cuprate addition of the butenolide R-5.13. 154 Scheme 5.8 The a-alkylation of �-hydroxy-y-lactone 5.14. 155 Scheme 5.9 Retrosynthetic analysis of substituted lactones. 155 Scheme 5.10 Synthesis of (5S)-dihydro-5 -octyl-2(3H)-furanone. 156 Scheme 5.11 Synthesis of (5R)-dihydro-5-octyl-2(3H)-furanone. 157 Scheme 5.12 Synthesis of dihydro-3-methyl-5-pentyl-(3H)-furanone. 160 Scheme 5.13 Introducing a butyl group into the lactone R-3.]. 162 Scheme 5.14 Cuprate addition of the butenolide R-5.22. 164 Scheme 5.15 Formation ofy-lactone 4S,5R-5.27. 164 Scheme 5.16 Cuprate addition of bute noli de. 165 Scheme 5.17 Cuprate addition for the formation of y-lactone 4S,5R-5.31. 165 Scheme 5.18 Cuprate addition for the formation of y-Iactone 4S,5R-5.27. 166 Scheme 5.19 Formation of 3,4,5-tri-substitueted dihydro-5-pentyl-2(3H)-furanone. 168 XII Scheme 5.20 Regioselective dihydroxyation. 169 Scheme 5.21 Steric effects of dihydroxyation. 170 Scheme 5.22 Possible synthetic pathway for the production of compound R-3.43. 170 Scheme 5.23 Hydrogenation of the Ca-Cp double bond in the lactone ring. 172 Scheme 6.1 Synthetic route for making ketones in Chapter 2 . 192 Scheme 6.2 Combinatorial synthesis of a library of four cyclopropyl ketones. 193 Scheme 6.3 Synthetic route for racemic y-Iactone in Chapter 3. 194 Scheme 6.4 Synthetic route for both enantiomers of 1.52 in Chapter 4. 195 Scheme 6.5 Synthesis of libraries of enantiomerically pure y-Iactones. 195 Scheme 6.6 Retrosynthetic analysis of substituted lactones in Chapter 5. 196 Xlll Abbreviations AD AEDA AIBN aq. b.p. Boc CBS CoA DDC DDQ (DHQ)2PHAL (DHQD)2PHAL DMAP DMF DMSO E l E2 EN EtOAc FD FLD FMDV g GC-MS GC-O h hex HLE HRMS IR L asynunetric dihydroxylation aroma extract dilution analysis Azobisisobutyronitrile aqueous boiling point butyloxycarbonyl Corey-Bakshi-Shibata coenzyme A N,N' -dicyclohexyldicarbodiimide 2,3 -dichloro-5 ,6-dicyanobenzoquinone dihydroquinidine derivative of the phthalazine class of ligands dihydroquinine derivative of the phthalazine class of ligands 4-( dimethyl amino )pyridine N,N-dimethylformamide dimethyl sulfoxide elimination, first order elimination, second order electronic nose ethyl acetate factor of dilution flame ionisation detector foot and mouth disease virus gram gas chromatography-mass spectrometry gas chromatography-olfactometry hour hexane horse liver esterase high resolution mass spectrometry infra-red litre XIV m.p. M.S. mg min mL MsCI ilL N NMR NOESY PC PCA PDC pKa PLE ppm RI RT RT s sat'd TBDPS TFAA THF TLC TMS lactic acid bacteria molecular ion melting point molecular sieve milligram minute milliliter methanesulfonyl chloride micro litre normal nuclear magnetic resonance nuclear Overhauser effect spectroscopy principal component principal component analysis pyridinium dichromate acid dissociation constant pig liver esterase parts per million retention factor room temperature retention time second saturated tert-butyldiphenylsilyl trifluoroperacetic acid tetrahydrofuran thin layer chromatography tetramethylsilane xv Chapter 1 Chapter 1: Background and introduction: food flavour and combinatorial chemistry 1.1 Background The flavour industry has grown dramatically in recent years because of the high demand of consumers vis-a-vis taste and flavour. There is a huge diversity of compounds that comprise flavours in nature . While many of these compounds in isolation do not have flavour or aroma attributes s imilar to the food from which they were isolated, it is a goal of flavour chemists to identifY a group of flavour compounds, often referred to as character impact compounds, which have the distinct character of the natural food from which they were derived. Important flavour compounds have been isolated from many food products including fruits, vegetables, breads, meats, beverages and dairy products . This project has been focussed on the flavour of dairy products, due to the experience and interests of our collaborators at Fonterra, and the potential economic importance of this industry to New Zealand. 1.2 Cheese flavour Dairy products originate from milk, their flavour components tend to be similar and the secret of their varied and unique characters is in the balance of those components. In particular, cheese flavour is one of the most complicated and interesting subjects in the flavour industry because of the variation of cheese flavours among varied types of cheese. There is not a single compound or class of compounds which is responsible for the full flavour of cheese. The correct balance of a mixture of volatile components contributes to the flavour of cheese and this is known as the "component balance theory". I The flavour of cheese is derived from degradation of milk protein (e.g., casein), fat, and carbohydrate (i.e., lactose) generating the complex, balanced flavour of aged cheese. The compounds identified in the flavour of cheeses are fatty acids, methyl ketones, alcohols, phenolic compounds, lactones, esters, aldehydes, sulfur compounds, pyrazines and amines. Some cheese flavour compounds, identified in selected cheeses, are l isted in Table 1 . 1 . Table 1 . 1 : Important flavour compounds in selected cheeses .2 Cheese Compounds Cheddar Methanethiol, dimethyldisulfide, diacetyl, 3-methylbutan-1 -01, acetic acid, butyric acid Nonan-2-one, oct-I-ene-3 -01, N-isobutylacetamide, 2 - Camembert phenylethanol, 2 -phenylethylacetate, heptan-2-01, non an-2-01, ammonia, isovaleric acid, isobutyric acid, hydroxybenzoic acid, hydroxyphenylacetic acid Romano Butanoic acid, hexanoic acid, octanoic acid Butanoic acid, hexanoic acid, octanoic acid, ethyl Parmesan butyrate, ethyl hexanoate, ethyl acetate, ethyl octanoate, ethyl decanoate, methyl hexanoate Provolone Butanoic acid, hexanoic acid, octanoic acid Surface ripened cheese 4-Methyloctanoic acid, 4-ethyloctanoic acid, p-cresol, m-cresol, 3 ,4-dimethylphenol Muenster Dimethyl disulfide, isobutyric acid, 3 -methylvaleric acid, isovaleric acid, benzoic acid, phenylacetic acid Blue cheeses Heptan-2-one, nonan-2-one, methyl esters of C4,6,8,IO,12 acids, ethyl esters of C \,2,4,6,8, 10 acids Brie Isobutyric acid, isovaleric acid, methyl ketones, sulfur compounds, oct-I -en-3 -ol Mozzarella Ethyl isobutanoate, ethyl 3 -methylbutanoate 2 Many of the flavour molecules in cheese arise from the transformation of compounds in milk by microorganisms, enzymes and chemical reactions . F lavour generation in cheese involves a complex series of reactions and interactions of lactose, fatty acids and amino acids. These reactions are e ither biochemical or chemical in nature. The numerous compounds involved in cheese aroma are mainly derived from three major metabolic pathways: glycolysis and fermentation, l ipolysis and proteolysis (Scheme 1 . 1 ) .3 Each of these will be discussed in more detail in the following sections. Glycolysis and Fermentation Acetic acid Diacetyl Acetaldehyde Ethanol Propionic acid Lactic acid Lipolysis Methyl ketones Aldehydes Lactones A1cohols Interaction products e.g., esters, thioesters Scheme 1 . 1 : Flavour development in cheese.3 3 Amines Sulfur compounds Acids Aldehydes A1cohols Pyrazines 1.2.1 Glycolysis (Lactose into lactic acids) Most of the lactose in milk is removed in the whey as lactose or lactic acid, and that retained in the cheese curd is metabolised to lactate, partly during curd manufacture and partly during the early stages of ripening, normally by the starter culture . The pathway for lactose metabolism is driven by starter bacteria (i.e. , Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris). 4 The conversion of lactose into lactic acid makes a major contribution to the flavour of acid-coagulated cheeses and contributes significantly to the flavour of young, rennet-coagulated cheeses. 1.2.2 Lipolysis (Milk fat into fatty acids) The fat fraction of cheese has a major effect on cheese texture and is important for the perception and development of cheese flavour. The fat in cheese is converted into fatty acids by hydrolysis in the presence of lipases . Lipases in cheese originate from milk, starter, adjunct starter or non-starter bacteria and mammalian enzymes (e.g., calf, kid and lamb pregastric esterases ).5 1.2.3 Proteolysis (Milk protein into amino acids) Proteolysis is the most complex, and the most extensive, series of biochemical reactions during the maturation of most cheese varieties. Proteolysis is mainly responsible for softening of the texture of cheese during the early stages of ripening and helps the development of cheese flavour via the formation of amino acids and peptides . Proteolytic enzymes involved in the various stages of protein and peptide degradation include the milk-clotting enzyme (from calf rennet), natural milk proteinases and proteolytic enzymes from lactic acid bacteria (i. e. , Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris) used as starter organisms .6 Proteolysis is the pathway by which milk proteins 4 are hydrolysed to produce free amino acids and peptides in cheese. Free amino acids, the final products of proteolysis, have been used as indices of ripening in cheese for many years. 7 1 .2.4 Catabolism of amino acids Amino acid catabolism is a major process for flavour formation in cheese. The conversion of amino acids to aroma compounds by microorganisms, especially by lactic acid bacteria and Brevibacterium linens, is the main pathway in many cheeses .8 The identification of the key aroma compounds of various cheeses shows that amino acid degradation is a major process for aroma formation in cheese and that aromatic amino acids (1 . 1 - 1 .3), branched-chain amino acids (1 .4-1 .6) and methionine (1 .7) are the major precursors of these aroma compounds (Figure 1 . 1 ). OH ~ o COOH COOH gyOH HN � NH2 NH2 NH2 phenylalanine tyrosine tryptophan 1 .1 1 .2 1 .3 �OH COOH COOH COOH �NH2 yNH2 ........ S�NH2 NH2 leucine isoleucine valine methionine 1.4 1 . 5 1 . 6 1 . 7 Figure 1 . 1 : The major amino acids for aroma formation in cheese. The conversion of amino acids to aroma compounds proceeds by two different pathways . The first is initiated by elimination reactions catalysed by amino acid lyases that cleave the side chain of amino acids, especially for aromatic amino acids and methionine (1 .7). 5 The second pathway goes through a-keto acid intermediate 1 .8 and is mainly initiated by a transamination reaction catalysed by amino acid aminotransferases or by a-deaminase (liberating ammonia in the latter case) and has been observed for aromatic amino acids, branched-chain amino acids and methionine ( 1 .7). a-Keto acid 1 .8 is further degraded to various compounds either by enzymatic reactions or by chemical reactions (Scheme 1 .2) .9 o HO�S" meth ionine 1 . 7 Transaminase Lyases or methionine a-deaminase HOJy o 2-oxo-butyric acid 1 . 1 2 OX;;;/ "S .... S" d i m ethy ldisu lfide 1 . 1 3 "S .... S .... s.......­ dimethyltrisu lfide 1 . 1 4 o HO�S" o a-ketomethiobutyric acid meth aneth iol 1 . 1 1 Fatty a cids 1 .8 H�S" o 3-methy lthiopropanal 1 .9 Esterases HiT" o 1 . 1 0 o �SH n = 1 -4 thioesters ( 1 . 1 5 - 1 . 1 8 ) Scheme ] .2 : Conversion of methionine (1 .7) to volatile sulfur compounds . Controlling amino acid catabolism by cheese microorganisms is carried out by selecting strain s of microorganisms with interesting catabolic activities . 6 1.3 Biosynthesis of cheese flavour compounds 1.3.1 Fatty acids Fatty acids are important as flavour compounds in the aroma of cheeses e.g., mold-ripened cheese, Italian style cheeses and hard cheese. They are also precursors of methyl ketones, alcohols, lactones, aldehydes and esters. The hydrolysis of fat in cheese has been widely described and is important in various cheeses. The even-numbered carbon free fatty acids, ranging from acetic (1.19) to octadecanoic acid (1.20), isobutyric (1.21) and isovaleric acid ( 1 .22) are present (Figure 1 .2) . IO o �OH 1 .1 9 �COOH 1 .20 Figure 1 .2: Fatty acids in cheese flavour. iCOOH 1 .21 �COOH 1 .22 Milk fat contains neutral l ip ids, from which the hydrolysi s by lipases produces free fatty acids in cheeses. These lipases hydrolyse triglycerides to form diglycerides, monoglycerides, glycerol (1.23), and free fatty acids (Scheme 1 .3 ) . 1 1 A controlled enzymatic hydrolysis using lipases gives a fatty acid composition dependent upon the specificity of the lipases used. Certain lipases e.g., mamalian pregastric esterases, contribute a high degree of specificity towards the short chain fatty acids. 1 2 Other l ipases preferentially liberate long chain fatty acids and some do not display any particular preference.13 Lipolysed milk fat products have found wide appl ications , for example for the enhancement of butter-like flavours 1 4 and flavour development in milk chocolate . IS 7 E OH OH E OGOR1 OH 1 .23 OCOR2 + OCOR3 E OGOR1 E OH E OGOR1 enzymatic hydrolysis OCOR2 OCOR2 OH + OH OCOR3 OCOR3 3H2O + 0 0 0 R1, R2, R3 = alky l groups R1JlOH R2JlOH R3JlOH Scheme 1 .3 : Enzymatic hydrolysis of triglyceride . 13.2 Ketones The homologous series of odd-chain methyl ketones, from C3 to Cl?, are some of the most important compounds in the aroma of blue cheese and surface mold-ripened cheese. 16 The two major odour impact methyl ketones are heptan-2-one (1 .24) and nonan-2-one ( 1 .26). E leven other methyl ketones (i.e . , all alkan-2-ones from C4 to C13 as well as octan-3-one), have been isolated from camembert cheeses by vacuum distillation. Each ketone presents a characteristic odour and can be described as a note: octan-2 -one (1 .25, floral), nonan-2- one (1 .26, fruity), decan-2-one ( 1 .27, fruity), undecan-2-one ( 1 .28, floral) and tridecan-2- one ( 1 .29, musty) . Oct- l -en-3-one ( 1 .30) has a mushroom note in an aqueous layer and a metallic note in a lip id layer. Acetophenone ( 1 .3 1 ) has an orange blossom note . Diacetyl (1 .32) and acetoin ( 1 .33) are well known for their buttery notes . The mushroom, musty and buttery notes are extremely important in camembert cheese.loa, 1 6b Ketones are also present in cheddar, gruyere, and French cantal cheese (Figure 1 .3 ) . 17 0 0 0 0 OH � � if + 0 0 0 1 . 24 - 1 .29: n = 4 - 8, 1 0 1 . 30 1 .3 1 1 .32 1 .33 Figure 1 .3 : Ketones in cheese flavour. 8 Methyl ketones are produced by the oxidation of free fatty acids and glyceride esters, fol lowed by decarboxylation of the resulting �-keto acids (e.g., 1 .35, Scheme 1 .4). Another possible mechanism is that �-keto acids in milkfat as glyceride esters are directly decarboxylated. This pathway is important because 60% of the carbonyl compounds produced by enzymes are methyl ketones. 1 8 Other ketones, especially diacetyl ( 1 .32) and acetoin ( 133) are obtained from pyruvate, stemming from lactose and citrate metabolism by the activity of lactic acid bacteria. 1 9 eOOH 1 . 34 J Il-0xidatlon (0 H,U e�o 1 .35 I d eCarbOXY�tion � 1 .28 Scheme 1 .4 : The biosynthesis ofundecan-2-one ( 1 .28) from dodecanoic acid ( 134). 1 .3.3 Alcohols Primary and secondary alcohols, along with ketones, are important aroma compounds in cheese. Oct- l -en-3-01 (1 .36) has a mushroom note and 3-methylbutan- l -ol (1 .37) gives an alcoholic, floral note . 2-Phenylethanol (1 .38) has a rose floral note and octa-l ,5E-dien-3- 01 ( 1 .39) has been found to have a celluloid taste (Figure l .4)?O �OH V OH � OH � �OH 1.36 1.37 1.38 1.39 Figure 1 .4 : Alcohols in cheese flavour. 9 Lipases have been used to perfonn enantiospecific hydrolyses to yield one enantiomer of aliphatic and terpene alcohols. An example is I-menthol (/- 1 .4 1 ), which is one of the most important terpene alcohols in the fragrance and flavour industry. It is the main component of peppermint oil . Many microbial l ipases can resolve racemic menthyl esters (e. g. , d,l- 1 .40) via the selective hydrolysis of the I-ester (Scheme 1 .S) .2 1 0 QOH 2'o� O� .. + - � d,/-1 .40 /-1 .41 d-1 .40 Scheme 1 .S : Enantioselective hydrolysis of (d,l)-menthyl acetate. Some alcohols are derived from the Ehrlich pathwal2 of amino acid metabolism (Scheme 1 .6) or from aldehyde degradation . 8 eOOH coo 6N H 2 transaminase 6 0 decarboxylase I � 7" ""\- � \ - o 2-keto-glutarate L-glutamate I 0 t02 L-P henylalanine Pheny l pyruvate ( 1 . 1 ) ( 1 .42) dehy drogenase 7�· NADH+ W NAD+ OH 6 1 .38 Phenylacetaldehyde ( 1 .43 ) Scheme 1 .6 : Ehrlich pathway for 2 -phenylethanol ( 1 .38) synthesis . Linoleic acid (1 .44) and linolenic acid (1 .45) are precursors of some alcohols, particularly 3S,SZ-oct-S -en-3-01 (1 .46),23 oct-2-en- 1 -01 ( 1 .47), SE-octa- 1 ,S -dien-3-01 ( 1 .39) and 5Z- 1 0 octa-1 ,5 -dien-3-o1 (1.48). The principal enzymes implicated in this alcohol synthesis are l ipoxygenase and a hydroperoxide lyase found in moIds (Scheme 1.7). HOO 1.45 l 1 D-LiPoxyg enase o OH � 1 .49 1 Hydrop eroxide lyase ~ OH 1 .39 1l Dehyd rog enase ~ OH 1 .46 o OH Scheme 1 .7 : Lipoxygenase-mediated generation of 3S,5Z-oct-5 -en-3 -o1 (1.46) from u- linolenic acid (1.45). 1 .3.4 Lactones Lactones which occur In cheese include dihydro-5-hexyl-2(3H)-furanone (1.50), tetrahydro-6-pentyl-(2H)-pyran-2-one (1.51), dihydro-5 -octyl-2(3H)-furanone (1.52) and tetrahydro-6-heptyI-(2H)-pyran-2-one (1.53) (Figure 1 .5) . The nature and levels of these 1 1 compounds are dependent on the diet of the animal producing the milk. Lactones are generally characterised by fruity notes (i. e. , peach, apricot and coconut). 1 6b �o 1 .50 �OyO 1 .51 V 1.52 o �o 1 .53 Figure 1 .5 : Lactones in cheese flavour. Hydroxylated fatty acids are the precursors of lactones. The closing of the ring occurs by the action of pH, microorganisms, or both. Hydroxyacids can be present as triglycerides in milk and lipases can liberate them. The fatty acids are then cyclised. Dihydro-5-octyl- 2(3H)-furanone (1 .52) can be formed, from long-chain saturated fatty acids (linoleic acid, 1 .44 and l inolenic acid, 1 .45).24 More detai ls are described in Chapter 3 . 1 .3.5 Esters Esters are common volatile components of cheeses. Ethyl esters of the straight-chain fatty acids [e.g., ethyl butanoate (1 .54) and ethyl hexanoate (1 .55) and 2-phenylethyl acetate (1 .56) are frequently found in cheese (Figure 1.6)). Most esters encountered in cheeses are described as having fruity, floral notes.25 o /'--o� 1 .54 o /'--o� 1 .55 Figure 1.6 : Esters in cheese flavour. 12 �Oy V 0 1 .56 A wide range of enzymes and mechanisms are involved in ester-forming reations?6 An example involves the alcoholysis of milk fat glycerides by lactic acid bacteria (LAB) in a transferase reaction (Scheme 1 .8) .27 o 11 E OC-R OH OH LAB EtOH .. E OH R-COO-Et + OH OH Scheme 1 .8 : The alcoholysis of a milk fat mono glyceride and ethanol by LAB. 1 .3.6 Aldehydes The main aldehydes encountered in camembert cheese and brie are hexanal (1 .57), heptanal (1 .58), nonanal (1 .60), 2-methylbutanal (1 .63), 3-methylbutanal (1 .64) and benzaldehyde ( 1 .65) . Hexanal (1 .57) and 2E-hexenal (E-1 .66) give the green note of immature fruit. Octanal (1 .59), nonanal (1 .60), decanal ( 1 .61) , and dodecanal (1 .62) are described as having an orange note and benzaldehyde (1 .65) is described as having an aromatic note of bitter almond (Figure 1 .7)?8 o �H o �H o �H D H 1 .57-1 .62: n = 4 - 8, 1 0 1 .63 1 .64 1 .65 E-1.66 Figure 1 .7 : Aldehydes in cheese flavour. 3Z-Hexenal (Z-1 .68) arises through the action of l ipoxygenase on the unsaturated fatty acid, linolenic acid (1 .45) fol lowed by the action of hydroperoxide lyase. Further enzymatic transformation gives rise to 2E-hexenal «E)_1 .66).29 Aldehyde Z-1 .68 can be reduced to the corresponding alcohol (1 .70, Scheme 1 .9) . 1 3 0 OH 1 .45 I Lipoxyg enase 0 OH OOH 1 .67 I Ald ehyde-lyase ( ) 0 0 � H + H OH 0 (Z)-1 .68 1 .69 Reductase I somerase o �OH � H (Z)-1 .70 (E)-1 .66 Scheme 1 .9 : Lipoxygenase-mediated generation of 3Z-hexenal (Z-1 .68) from a-linolenic acid ( 1 .45). 1 .3.7 Sulfur compounds Four sulfur compounds that form a garlic note fraction have been isolated from camembert and brie cheeses: 2 ,4-dithiopentane ( 1 .71 ), diethyldisulfide ( 1 .72), 2 ,3 ,5-trithiohexane ( 1 .73), and 2 ,4-dithio-3-methylthiopentane (1 .74) (Figure 1 .8) . Other sui fur compounds are also encountered in cheeses and they are described as having a strong garlic, very ripe cheese odour. lOa 1 4 SH SH � S, SH SH AA SH SH � SH 1 .71 1 .72 1 . 73 1 . 74 Figure 1.8 : Sulfur compounds in cheese flavour. Many microorganisms are able to produce sulfur compounds in cheeses . Methionine is an amino acid which is a precursor to methanethiol. Methionine demethiolase cleaves a C-S bond (Scheme 1. 10). 30 o HO�S' demethio/ase CH3SH metha n ethiol 1 . 1 1 methionine 1 .7 2-oxo-butyric acid 1 .1 2 Scheme 1 . 1 0 : The formation ofmethanethiol (1 .1 1 ) . 1 .3.8 Nitrogen-containing compounds Amino acid decarboxylation leads to carbon dioxide and free amine (Scheme 1 . 1 1 ) . This reaction needs pyridoxal-phosphate as a coenzyme. Phenylalanine (1 . 1) thus gIVes phenylethylamine (1 .75), tyrosine (1.2) gIves tyramine (1 .76), and leucine ( 1 .4) gIves i sobutylamine ( 1 .77).31 Numerous volatile amines have been identified in the headspace of h b" 32 a c eese; some cause Ittemess. 1 5 Amino acids, e.g. , o YCOOH NH2 phenylalanine 1 . 1 I phenylethyla m i n e 1 .75 OH COOH NH2 tyrosine 1 .2 leucine 1 . 4 I decarboxylalion I Amines OH N H 2 tyra mine 1 .76 � N H 2 isobutylamine 1 .77 Scheme 1 . 1 1: Decarboxylation of amino acids. 2-Acetyl- I -pyrrol ine (1 .78, in Figure 1.9) has been identified in a variety of processed and cooked food, including dry and fresh milk, camembert and Swiss gruyere cheese, rennet casein, and liquid cheddar whey.33 Compound 1 .78 and pyrazines (e.g., 2-sec-butyl-3 - methoxy pyrazine, 1 .79, in Figure 1.9) are formed by the Maillard reaction between carbohydrate fragments and amino acid residues . Indole ( 1 .80) and 3-methyl indole ( 1 .8 1 , skatole) are considered to be important flavour compounds i n water buffalo mozzarella and emmental, respectively. � (J(:: Q:) oi � N � N 0 H H 1 .78 1 . 79 1 .80 1 .81 Figure 1 .9 : Nitrogen-containing compounds in cheese. 16 1.4 Flavour compounds in other foods Many of the flavour compounds in dairy food occur in other types of food as well . The ultimate objective of this programme is to utilise lead flavour compounds in foods of all types (i. e . , not restricted to dairy products). A brief survey of flavour compounds in other types of food is therefore included. 1 .4.1 Cerea) flavours34 Flavours in raw cereals originate from the grain . Cereal products obtain their flavour enzymatically, mainly in treatments l ike dough-kneading and femlentation, and non­ enzymatically, in processing for instance by different types of heat treatment. Special attention has been paid to the formation of alkyl pyrazines that are specially produced from the yeast metabolites in extrusion and roasting processes .35 Off-flavour in cereal and cereal products is caused by advanced lipid oxidation and growth of microorganisms on the grain . 1 .4.2 Meat flavour34 There are over 1 000 volatile compounds found in cooked meats . Meat flavour develops during cooking from the complex interaction of precursors derived from both the fat and lean components of meat. Heterocycl ic compounds, such as pyrazines and thiazoles contribute to roast and gri l led aromas, while certain aliphatic and heterocyclic sulfur compounds provide some of the characters of boiled meat. Sulfur compounds are extremely important in meat flavour and certain furanthiols and furan disulfides possess the meaty aroma. 1 7 1 .4.3 Beer fla vour34 Beer contains numerous taste and aroma compounds . Some of them are derived from the raw materials, malt and hops, but by far the majority are formed during the brewing process. Hops contribute s ignificantly to beer bittemess and beer flavour. The main compounds in hops are bitter resins called a-acids or humulones ( 1 .82-1 .84) and �-acids or lupulones ( 1 .85-1 .87) (Figure 1 . 10). After bottling, the beer flavour is unstable and changes considerably with time. OH 0 OH 0 R R a-acids ( h u m u l ones) 1 .82-1 .84 �-acids (Iupulones ) 1 . 85-1.87 R = \1 n-hum ulone ( 1 .82) I n-Iupulone ( 1 .85) �-< coh u m ul one ( 1 .83) / co l u p u lone ( 1 .86) \� a d h u m ulone ( 1 .84) / a d l u p u lone ( 1 .87) Figure 1. 10: Chemical structures of hop bitter acids . 1 .4.4 Wine flavours34 Wine flavour i s affected by an enormous number of variables . The grape variety and factors affecting wine development and berry composition exert major influences on distinctive flavours.36 There are two classes of compounds, terpenes and pyrazines (e.g. , the bell pepper pyrazine, 2-sec-butyl-3-methoxy pyrazine, 1 .79, Figure 1 . 1 1) , identified in grapes that are flavour impact compounds. Terpenes contribute to the distinctive floral 18 aromas and the bell pepper pyrazine 1 .79 has an aroma described as h erbaceous or vegetative . 1 .79 Figure 1 . 1 1 : Chemical structure of the bell pepper pyrazine ( 1 .79). 1 .4 .5 Cocoa flavour34 Cocoa makes a unique contribution to the flavour of chocolate . The following 1 3 flavour compounds contribute to the overall chocolate flavour (Figure 1 . 1 2) . D H .)(1 0 �OH DOH 3-methylbutanal 2-ethyl-3,5-dimethylpyrazine 2-methylbutanoic acid 3-methylbutanoic acid sweet malty potato chip-like sweet (1.64) (1.88) (1.89) (1.90) 0 0 � � .)(1- Et)[N1 Et N H 5-methyl-2E-hepten-4-one hazelnut-like 1-octen-3-one mushroom-like (1.30) 2-ethyl-3,6-dimethylpyrazine 2 ,3-d iethyl-5-methylpyrazine nutty, earthy potato chip-like (1.91) (1.92) (1.93) o o o �H �H �H 2Z-nonenal green, tallowy (1.94) 2E,4E-decadienal fatty, waxy (1.95) �o (6R)-tetrahydro-6-pentyl-(2H)-pyran-2-one sweet, peach-like (R-1.51) Figure 1 . l 2 : Flavour compounds in chocolate . 1 9 2E,4E-nonadienal fatty (1.96) /s_s�o 2-methyl-3-( methyldith io )furan (1.97) 1 .5 Instrumental analysis of food flavours There are indications that only a small fraction of the complex mixture of volatiles occurrmg m foods causes the overall odour. The determination of odour-active compounds from volatiles having l ittle or no odour is the task to be solved in flavour analysis . The odour detection threshold is the lowest concentration of a certain odour compound that is perceivable by the human sense ofsmel 1 .37 1 .5.1 GC-O, GC-MS and EN The chemical analysis of flavour in dairy products is complicated since significant levels of l ip ids, proteins and carbohydrates in milk make it difficult to separate flavour-active chemicals . GC-MS is an excellent tool to separate the volatile compounds by gas chromatography (GC) and then identify and analyse the components by mass spectrometry (MS). However, GC-MS is an indirect method to identify the odour-active compounds in dairy products, since the unknown volati les separated by gas chromatography are identified by mass spectrometry rather than odour impact.38 GC-O is the combination of techniques that combine o lfactometry, or the use of human detectors, to assess odour activity in defined air streams with the gas chromatographic separation of volatiles. GC-O i s used to determine odour thresholds (e.g., from Aroma extract dilution analysis (AEDA)39 and Charm analysis40) in various food including meat, breads, vegetables, beverages and dairy productS .4 1 The appl ication of GC-O to dairy products has included the analysis of fresh and heated milk, cheeses , yogurt, and milk chocolate .42 Some of the aroma compounds identified in dairy products using GC-O are listed in Table 1 .2 . 20 Table 1 .2 : Odour descriptions of compounds found in dairy products. Compound Odour description ethyl butanoate banana, pineapple , sweet ethyl hexanoate fruity, pineapple heptanal green, sweet l -octen-3 -0 I mushroom-like dimethyl sulfide intense, boiled cabbage, sulphurous 2-heptanone fruity, spicy, c innamon 2-undecanone floral, rose-like tetrahydro-6-heptyl-(2H)-pyran-2-one coconut diacetyl buttery In addition to classical studies on correlation models based on physico-chemical properties there are an increasing number of studies using an electronic nose (EN) system.43 The electronic nose (EN) is an analytical instrument that compares the odours of samples with those of standards. It has been applied extensively in the chemical, cosmetic and food and beverage industries. There are a number of different technologies available and EN analyses the complete headspace above a product by passing it over a series of sensors . These sensors are semi-conductors and they react with volatile chemicals i n the sample headspace . The FOX 4000 is a commercial EN and it has been used to characterise odourants in food, particulary in the wine industry.44 2 1 1.6 Combinatorial chemistry 1 .6 .1 Background Combinatorial chemistry IS a relatively new way of generating a large number of compounds rapidly in a fonnat that is described as a library. Combinatorial chemistry was first reported by Mario Geyson and his colleagues in 1984.45 He was interested in VP 1 , an immunologic ally important, 2 13 residue, coat protein of the foot and mouth disease virus (FMDV). A l ibrary of hexapeptides corresponding to the 208 possible overlapping hexapeptides of the viral protein was screened for binding to the antibodies generated in response to the intact FMDV. This proved which regions of the FMDV protein (epitopes) were important in tenns of recognition of the antibodies . Residues 146 to 1 52 were identified as being crucial for binding. Combinatorial chemistry can be divided into two challenges : making the library and finding the active compound. The big advantage of combinatorial chemistry is that a library is generated exponentially and then screened for its biological activity. The chemical library is then scrutinised to find the active compound. Robotic instruments and computational tools have enabled the identification of biologically active compounds by screen ing a mixture of thousands of compounds in libraries. 22 1 .6.2 Generating a chemical library 1.6. 2. 1 Solid-phase organic synthesis Since the first report of solid-phase organic synthesis by Merrifie ld46 in 1963 , the general field of solid-phase organic synthesis has grown enonnously. The fundamental technique is based on polymeric resin beads to which a reactive functional group is bonded covalently. This immobilized group serves as the starting unit for the assembly of additional covalently bonded units. When the desired array of molecular building b locks i s assembled, the original covalent bond to the resin is cleaved, to generate the new molecule as a separate entity. The Merrifield approach to solid-phase peptide synthesis is shown in Scheme 1 . 12 . R1 R1 ,{' Boc, N tCOOH � yl NHBOC �ylNH2 Y ___ H______ Y 0 CF3COOH. Y 0 P p P H '-':::: 0 R2 CF3COOH. '-':::: 0 H R2 DCC • I 0 I 0 DCC P R1 0 R3 P Q oyl� � NHBOC '-':::: 0 R2 0 • l o p = p:lymer P DCC - <==>-N=C=N-<==) N, N' -dicyclohexylcarbodiimide Scheme 1 . 1 2 : Solid-phase peptide synthesis according to Merrifield.48 23 The advantage of solid-phase organic synthesis is that the growing product is always chemically bound to the resin bead. Thus reactions can be driven to completion by using excess reagents, and waste products can be separated readily and repeatedly from reactants by simple filtration and extensive washing. The rapid production of l ibraries of molecules by solid phase synthesis has led to modifications related to miniaturisation, automation, and robotics. 1 .6. 2.2 Solution-phase organic synthesis Although solid-phase organic synthesis has been widely developed, particularly in the peptide industry, solution-phase organic synthesis is also used for producing small, drug­ related molecules in the field of combinatorial chemistry. The concept of generating libraries of small organic molecules with a drug-related structure is that a rigid core molecule supporting mUltiple reactive s ites is combined with a mixture of building blocks to produce a random mixture of polyfunctionalised structures. For example, cubane tetra acid chloride (1 .98) (Scheme 1 . 1 3 ) could be combined with four molar equivalents of an equimolar mixture of amines A-Z to produce tetra-substituted cubane compounds A, A, A, A, through to Z, Z, Z, Z. This method of library generation has several advantages . First it is a powerful method of generating molecular diversity in a single combinatorial step. Reactions are pushed to completion by the use of excess quantities of the reactive reagent, and products are isolated by solvent-solvent extraction without further purification.47 24 �CI O Cl o Cl 0 Cl 1 .98 + I : etc. 1, O�NHA AH:�O AHN etc. ZHN Scheme 1 . 1 3 : Synthesis of a combinatorial l ibrary from cubane tetraacid chloride (1 .98) and amines. The advantage of solution synthesis relative to solid phase synthesis is that the compounds are in a free form to be assessed for activity. Solution-phase organic synthesis is a reliable way to apply traditional organic reactions (e.g., Diels-Alder, aldol, Mitsunobu, Stille, Suzuki, Heck, Grignard, Wittig) .4R 1 .6.3 Combinatorial chemistry in food flavour The application of combinatorial chemistry to food flavour is an emerging area that has the potential to dramatically reduce the time and cost associated with the synthesis and evaluation of potential flavour compounds. The introduction of combinatorial chemistry to food flavou r has been pioneered by two research groups. 2 5 Volfson 's group prepared a l ibrary of S-methyl thioesters and screened it for sensory unique components.49 Thioesters have been identified as one of the known classes of compounds which are formed in maturing cheeses and contribute to their characteristic aroma with very low thresholds . Synthesis of a combinatorial library of eleven S-methyl thioesters was effected by reaction between methyl chlorothiolformate (1 .99) and eleven carboxylic acids with vanous carbon chain lengths in the presence of 4- (dimethylamino)pyridine (DMAP, 1 .100) (Scheme 1 . 1 4) .50 The l ibrary of synthetic S- methyl thioesters was screened using GC-O and identified S-methyl thiopropionate (1 .102, n = 1 ) as having a possible characteristic aroma of camembert cheese.5 1 [�hCH3 l - o • � I ,11 + �n'SCH3 1 . 1 0 1 - 1 . 1 1 1 n = 0 - 1 0 Y H�Et3�1 N(CH3h 1 . 1 00 o 0 CIASCH3 + H3CS�SCH3 1 .99 1 . 1 1 2 by-product Scheme l . 1 4 : Synthesis of S-methyl thioesters from carboxylic acids and methyl chlorothiolformate (1 .99). CoBins ' group studied sensorial properties and syntheses of mercaptoketones,52 mercaptoaldehydes,53 and mercaptoesters.54 Libraries of p-mercaptoketones were synthesised via two synthetic pathways by conjugate addition of (a) hydrogen sulfide 26 ( 1 . 1 13) or (b) thioacetic acid (1 . 1 14) to a,/3-unsaturated ketones with various substituents. Subsequent reduction of the mixture of ketones led to the formation of mercaptoalcohols (Scheme 1 . 1 5 ) .55 Each intermediate step was monitored by thin layer chromatography (TLC) and the composition of the l ibraries was identified by GC-MS. The sensorial analysis of l ibraries was completed by GC-O to provide the odour descriptions and threshold values . (a) (b) R2 0 R)�.)lR4 R3 + 0 �SH 1 . 1 14 6 THF H 0 Mercaptoketone SH OH Rll.. 1- R4 -�;y- , R3 Mercaptoai cohoi Scheme 1 . 1 5 : Synthetic pathways for production of mercaptoalcohols from a,/3- unsaturated ketones. 5-Methyl 4-mercaptohexan-2-one (1 .1 1 5) and one diastereoisomer of 5 -methyl 4- mercaptohexan-2-01 (1 .1 16) were the most pleasant odours perceived at the sniffing port. 4-Mercapto-4-methylpentan-2-one (1 .1 17) and 4-mercapto-3 -methylpentan-2-o1 ( 1 . 1 18) were identified with very low detection limits of 0 .004 and 0.0 1 2 ng L- 1 , respectively. 27 Combinatorial synthesis of mercaptoaldehydes was achieved via a similar approach starting with a,p-unsaturated aldehydes. Three 3-mercapto-2-methylaldehydes ( 1 . 1 1 9- 1 . 1 2 1 ) were identified to possess meat aroma (Figure 1. 13) . SH 0 SH OH SH 0 SH OH � r +A y exotic fruit rhubarb, lemon 1 . 1 1 7 1 . 1 1 8 1 . 1 1 5 1 . 1 1 6 r\rH �H �H SH 0 SH 0 SH 0 meat aroma meat aroma meat aroma 1 . 1 19 1.120 1 .1 21 Figure 1 . 1 3 : Mercaptoalcohols, mercaptoketones and mercaptoaldehydes with desirable properties . Mercaptoesters were synthesised combinatorially from thioacetic acid and the mercaptoalcohols described above (Scheme 1. 16). Mercaptoesters are known flavour compounds in foods and beverages . For example, 3-mercapto-3 ,3 -methylbutylformate ( 1 .1 22 , R I = Me, R2 = H, R3 = H, R4 = H) is characterised by an exceptionally low odour threshold (2-5 ppt) and has been found in coffee and beer. ,>= 0 Cl Mercaptoketone or Mercaptoaldehyde Mercaptoalcohol Scheme 1 .16 : Synthesis of mer cap to esters . 28 Mercaptoester R1 , R2, R3, R4 = H or a lkyl groups SH 0 �O� 1 . 1 22 1 . 7 Aims and objectives of this project The area of flavour and fragrance is huge and is still an area of development vis-a-vis contributions to the food industry. Specifically, the flavour of dairy products arises from complex mixtures of flavour compounds . This project was initiated to synthesise potential flavour compounds combinatorially and identifY key components for further investigation as flavourants in dairy products. Solution-phase combinatorial synthesis is a good way to generate potential flavour compounds rapidly to investigate further structure-activity relationships in food flavour. Previous work in this area by researchers at Fonterra Research Centre Limited illustrated the potential of ketones and lactones in food products (Figure 1 . 1 4). Further investigation of these classes of compounds, particularly with regard to unsaturated and stereochemically pure derivatives, was considered worthwhile. o � blue cheese l ike 1.24 �o fruity, peach 1. 50 Figure 1 . 1 4 : Potential flavour compounds . The generation and screening of a library ofketones is discussed in Chapter 2 . Libraries of racemic y-Iactones were investigated in Chapter 3 and approaches to enantiomerically enriched y-lactones are described in Chapters 4 and 5 . 29 Chapter 2 Chapter 2 : Synthesis of a library of ketones as potential flavour compounds 2.1 Introduction Ketones, and most notably methyl ketones, are key compounds in the flavour of mold- ripened cheeses.56 In camembert and blue veined cheeses, h igh concentrations of methyl ketones (Table 2 . 1 ) and 2-hydroxyalkanes (i.e . , CH3CHOHR) are found, including some unsaturated methyl ketones such as octa- l ,5 -dien-3-one (2.8), and oct - l -en-3-one ( 1 .30) and non-3-en-2-one (2.9) (Table 2 .2) .57 Table 2 . 1 . F lavour notes of straight chain methyl ketones in cheese. I I Name Structure Flavour note 0 � Propan-2-one (2.1 ) n = O Acetone Butan-2-one (2.2) n = l Acetone Pentan-2-one (2.3) n = 2 Fruity, acetone Hexan-2-one (2.4) n = 3 Floral , fruity Heptan-2-one ( 1 .24) n = 4 B lue cheese, Roquefort cheese Octan-2-one (1 .25) n = 5 Fruity, musty Nonan-2-one (1 .26) n = 6 Fruity, musty Decan-2-one (1 .27) n = 7 Fruity, musty Undecan-2-one (1 .28) n = 8 Floral, herbaceous Tridecan-2-one (1 .29) n = 1 0 Fruity, green mushroom, fruity 30 Table 2 .2 : F lavour notes of other ketones in cheese . Name Structure Flavour note 0 Octan-3-one (2.5) � Fruity, green mushroom, fruity 4-Methyl-pentan-2-one (2.6) � Fruity, green mushroom, fruity 4-Methyl-hexane-2-one (2.7) JJl Geranium leaf, soil 0 Octa- l ,5-dien-3 -one (2 .8) � Geranium leaf, soil 0 Oct- I -en-3 -one (1 .30) � Mushroom 0 Non-3 -en-2-one (2.9) � Mushroom 0 Acetophenone (1 .3 1 ) if Orange blossom 0 DiacetyI (1 .32) 0 Buttery 0 OH Acetoin ( 1 .33 ) + Buttery 0 0 Damascenone (2.10) ~ Woody Methylfurylacetone (2 . 1 1 ) o� Woody 0 Propiophenone (2.12) ~ Woody 3 1 Methyl ketones are formed by enzymatic oxidative decarboxylation of fatty acids58 as described briefly in Chapter 1 , and by the spores of Penicillium roqueforti, mycelium, and P . '11 ' b ' 1 8b 1 6c 59 Th b I ' h .c. h · .c. " h . emcl lurn carnern ertl. " e meta 0 lC pat way lor t elr lormatlOn IS s own In Scheme 2 . 1 . The free fatty acids are oxidised to �-ketoacids, followed by decarboxylation to give methyl ketones. The methyl ketones can be metabolised further to secondary alcohols by Penicillium roqueforti. milk triglycerides Upases o 13-keto-acyl-CoA (2. 13) S .... CoA I thiohydrolase C�O o decarboxylase .. � reductase 1 .28 1 . 13-oxidation COOH 2. f3-keto-thiolase 13-keto-acid (1 .35) OH � 2.14 Scheme 2 . 1 : The metabolic pathway for the formation of methyl ketone . The chemical synthesis of ketones was the first step in our efforts to produce compounds that may have application to cheese and fresh dairy flavours. The l ibrary design was based on the generic structure shown in Figure 2 . 1 with two points of diversity, RI and R2. R I represents long alkyl chains with unsaturation in some cases and variation in the position and orientation of double bonds. R2 is a relatively small alkyl group. The set of target molecules is outl ined in Table 2 . 3 . Figure 2 . 1 : The general structure of ketone targets . 32 VJ VJ Table 2 .3 : The first l ibrary 0[20 ketones as potential flavour compounds 0 R)lR2 �- ,� R I 0 0 �, � � 1 .28 2.19 0 0 �, � � 2.15 2.20 �, � \ 0 2.1 6 2.21 0 0 V' � � I � � 2.17 ,;:; 2.22 0 0 �, � 2.18 2.23 --- R2 '\.� �---MgBr 2 .37 o � H 2.38 o � H 1 .95 o H 2.39 Figure 2 .2 : Commercially available aldehydes and solutions of Grignard reagents being used in the synthesis ofketones . The Grignard reactions were performed in THF at 0 qc. In each case, after an hour, TLC indicated the formation of a new, more polar compound. After standard workup and purification by flash chromatography, the 20 alcohols were obtained. One compound from the library of a1cohols will be discussed to illustrate the salient features of the NMR spectra. The lH NMR spectrum ofundecan-2-o 1 (2.14) exhibited a three-proton doublet at () 1 .23 that was assigned to the methyl protons in the RCHOHCH3 group. The 1 3C NMR spectrum of undecan-2-01 (2.14) included signals at () 25 .7 (RCHOHCH3) and () 68 . 1 (RCHOHCH3), confirming the presence of the secondary alcohol. 2.3.2 Swern oxidation The Swem oxidation is a mild method for the oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, respectively (Scheme 2 .4).62 36 1 . DM80, (COClh OH CH2CI2, -78 QC 0 R1�R2 2. Amine base • R1JlR2 Scheme 2 .4 : The overall transformation of the Swem oxidation. The first step of the Swem oxidation involves the interaction between oxalyl chloride (2.4 1 ) and dimethyl sulfoxide (DMSO) (2.42) at -78 QC to afford chlorosulfonium chloride (11) (Scheme 2.5). Reaction of this species with the alcohol affords an alkoxysulfonuim salt (Ill) and forms the desired carbonyl species and dimethyl sulfide (2.43) (Scheme . 2.5).63 �,�)� CI-C-C-CI � 2.41 e) o I .... 8, Me (B Me 2 .42 --- o \ 0 " 0 (BS-O-C-C-CI C / �o e I Cl --- + Scheme 2.5 : Mechanism of the Swem oxidation. + CO2 + CO + CI- Both aldehydes and ketones may be made by the Swern oxidation. Double bonds and activated C-H bonds are unreactive under the mild and specific conditions of the Swern oxidation. A disadvantage of the Sw ern oxidation is that strictly anhydrous conditions are required due to the moisture-sensitivity of oxalyl chloride. Moreover, DMSO (2.42) and oxalyl chloride (2.41 ) explode when combined at room temperature and therefore low temperatures are necessary. 37 The oxidation of secondary aIcoho l s with saturated R I and R2 groups occurred smoothly via the Swem protocoL Those compounds with doub le bonds remote to the alcohol func tional group were also successfully ox idised (Table 2 .4). A three-proton singlet was obse rved at S 2 . 1 3 in the IH NMR spectrum of undecan-2-one ( 1 .28) that was assigned to the methyl group located next to the carbonyl group of the ketone. The resonance at S 209 . 1 in the I3C NMR spectrum was attributed to the newly formed carbonyl group . Table 2.4: Ketones prepared via Swem Oxidation of Secondary Alcohols. OH 0 R1,(R2 - R)lR2 � Rl CH3- CH3CHr CH2=CH- [:>- " 1 .2 8 2 . 1 9 2.24 2 .29 7 1 % 47 % 60 % 53 % �" 2 . 1 5 2 .2 0 2.25 2 .30 66 % 96 % 6 % 30 % - �, 2 . 1 6 2.2 1 2.26 2 .3 1 47 % 94 % 38 % 73 % Unfortunately, allylic and/or benzylic secondary aIcohols gave, at best, l ow yields of ketones via the Swern protocol. Alternative oxidation methods were therefore i n ve stigated to get good yields of these ketones. 2 .3 .3 Oxidation with PDC In 1 979 , Corey and Schmidt64 introduced pyridinium d ichromate, (C5HsNH+)2Cr20l- (PDC, 2.44), for the oxidation of secondary and allyl i c alcohols. Compound 2.44 i s 38 reported to be a more useful oxidant for allylic aIcohols than for their saturated analogs. The oxidation of (1 OE, 1 2Z)- 1 O, 1 2-octadecadiene- l ,-9-diol (2.45) with PDC (2 .44) i n N.N-dimethylformamide (DMF) gave rise t o the keto acid 2 .46 (Scheme 2 .6). 65 OH C5H1 1 OH 2.45 ! PDC (2 .44) DMF ?"" 0 OH CSH1 1 0 2.46 0 Scheme 2 .6 : The oxidation of ( 1 0E,1 2Z)- 1 0, 1 2-octadecadiene- l ,9-diol (2.45) with PDC (2 .44) . 66 When this procedure was app lied to the oxidation of 4-phenyl-but-3-en-2-ol (2.47), the compound isolated was analysed by NMR . The IH NMR spectrum exhibited two new resonances in the 0 6. 1 -6.6 ppm region that were assigned to o lefini c protons of the conjugated d iene 2 .4 8 . There was no singlet in the 0 2 .0-2 .5 ppm region as would be expected for the methyl group of the desired methyl ketone (2 .1 7) (Scheme 2 .7). OH � l) 2.47 (0 \ Cr,O,'- + � )2 2.44 - - - - - - - - - - - - - - - - - - -� ~ Target Compound (2.1 7) � V Product (2.48) Scheme 2 .7 : The attempted oxidation of allylic alcohol 2 .47 with PDC (2 .44). 39 Both the Swem and PDC oxidations fai led to fonn the des ired ketone from allylic alcohols. Our mechanistic explanation for this is based on the observed product from the PDC oxidation . Once the alkoxysulfonium salt (via the Swem oxidation), or the alkoxychromium salt (via the PDC oxidation) is fonned, p -elimination would give rise to a diene (2.48) via an E l or E2 mechanism (Scheme 2 .8). An E l mechanism would procede via a carbocation that is stabil ised by the adjacent 1t-bond system (Scheme 2 .9) . 8 o via PDC � V _ _ _ 2.48 Scheme 2 . 8 : The mechanistic possib ility ofE2. X ��O' H I � � H ,C-H // H x = + SMe2 ( via Swern) X = Cr03- ( via PDC) <±> H �� ? �� ""=::: I " - I // H H 0 2.48 Scheme 2 .9 : The mechanistic possibil ity of E l . 40 2.3.4 Oxidation with DDQ (2 .49) 2,3-Dichloro-5 ,6-dicyanobenzoquinone (DDQ, 2.49) is a potential qumone that se lectively oxidises allylic alcohols.66 Compound 2.49 is generally used under anhydrous conditions and often in large excess. The oxidation of allylic alcohols us ing a catalytic amount of DDQ in a biphasic, sl ightly acidic benzene/water system, in the presence of periodic acid was carried out at room temperature overnight. The oxidation of 1 ,3- diphenyl-3 -hydroxy- l -propene (2.50) was reported as a typical example (Scheme 2 . 1 0).67 OH � U 2 .50 U o NCx)cCI I I NC Cl o DDQ (2 .49) (80 %) o • � U 2.51 U Scheme 2 . 1 0 : The oxidation of 1 ,3 -diphenyl-3 -hydroxy- I -propene (2 .50) by DDQ (2.49).70 The example in Scheme 2 . 1 0 provided good precedent for the oxidation of alcohols that had undergone e limination rather, than oxidation, under other conditions . Indeed, after workup and purification by flash chromatography, the allylic ketones were obtained with reasonably high yields (Table 2 .5) . 4 1 Table 2 . 5 : A l ibrary of the ketones produced by DDQ Oxidation. OH 0 � - Jl R1 R2 R1 R2 � R1 CH3- CH3CHT CH2=CH- [>- �" 2.17 2.22 2.27 2.32 1 -& 59 % 56 % 60% 8 1 % �, 2.18 2.23 2.28 2 .33 68 % 40 % 72 % 45 % 2.4 Screening of the first ketone library For the first library of twenty ketones, each compound was synthesised individually and characterised by IH and 1 3C NMR spectroscopy. S ixteen ketones were suitable for screening by the Fox 400068 and GC-O. Four ketones (2 .25, 2 .28 , 2 .30, and 2.33, Figure 2 .3) appeared to be unstable; it seems oxidative decomposition occurred while the samples were in storage. These ketones were therefore not available for analysis. The respective odour characteristics of the 1 6 remaining ketones were analysed by the Fox 4000, an instrument that compares the odour of compounds and that of standards (e.g., cheeses such as cheddar, gouda, parmesan and camembert). 2.25 � � 2.28 o o o 2 .30 o 2.33 Figure 2 .3 : The ketones that were not sufficiently stable for the Fox analysis . 42 2.4. 1 The FOX 4000 The e lectronic nose (EN) has emerged as an exciting new analytical tool having broad application. In flavour chemistry, the electronic nose has the potential to reproducibly monitor flavours, thus maintaining product consistency and quality. In a headspace analysis, volatile molecules are injected into the EN and they react with the sensor array. The chemical nature and concentration of the analyte affect the response characteristics. Once the gas-phase molecules are at the sensors, they need to be able to react with the sensors to produce a response. In thi s study, an Alpha M .O.S . Fox 4000 EN was equipped with 1 8 metal-oxide sensors to analyse pure chemical samples . The sensors react to the presence of organic compounds by changes in their electrical resistance. The raw data of a single analysis run consists of 1 8 numbers, representing the maximum relative change in resistance experienced by the sensors. The visual result from principal components analysis (PCA) was performed by the computer software and the clusters of data points represented the aroma of samples. The distance between two clusters represented the difference between the aromas of the two respective samples. The individual ketones were evaluated at low and high concentrations by comparison with each cheese and a blank reference. For the data analys is, the overall PCA was used to show the relationships between each ketone and the four cheeses (Figure 2 .4). 2 1 Two principal components, PC I and PC2 gave a measure of how close the aroma of each ketone was to those of each cheese. The relationship of each ketone with the four cheeses was quantified as a vector. The calculated angles between zero-cheese (vector 1 ) and zero-ketone (vector 2 ) are summarised in Table 2 .6 . 43 � � PC2 I ci· : 25. 87% 0 .<400 - 0 . 3 00 - 0 . 200 - 0 . 1 00 - 0 . 000 - -0 . 1 00 - - 0 . 2 0 0 - -0 . 3 0 0 - -0. <4 0 0 - -0 .500 - -0 .600 - -0 .70 0 - 1 -0 . 5 -0 . 2 vector 1 (zero-camembert) �·��",\,n --7 0 . 0 0 . 2 C H�AR 0 . 5 0 . 8 1 . 0 PC1 C1 : 66 . 99% G�A PA� N 1 . 2 1 . 5 1 . 8 Figure 2 .3 : peA map for the blank, 4 cheeses and 1 6 ketones, with i l lustration of angle determination for compound 2.29. 2 . ( Table 2 .6 : FOX results which quantify the re lationship between each ketone and four cheeses (C l = camembert; G = gouda; C2 = cheddar and P = parmesan). Angles are those determined in accordance with Figure 2 . 3 . 0 0 0 0 � � � � 1 .28 2.19 2.24 C l G C2 P C l G C 2 P C l G C 2 P C l G I C2 I P 3 5 ° 5 7 ° 59° 62° 3 9° 5 2° 54° 57° 5 0° 5 2° 54° 5 7° 1 5° 23 ° I 2 2° I 2 7° 0 0 0 0 � � � � 2.15 2.20 2.25 C l G C2 P C l G C 2 P C l G C 2 P C l G I C 2 I p 60° 6 1 ° 63 ° 66° 4 1 ° 42° 43 ° 48° unstable unstable ~ \ 0 \ 0 \ 0 .,?- � 2.16 2.21 2.26 C l G C2 P C l G C 2 P C l G C2 P C l G 1 C 2 I p 54° 65 ° 67° 69° 45° 6 1 ° 64° 66° 69° 70 ° 7 3° 74° 83 ° not deternined 0 0 0 0 � � � � I ,,:; 2.27 ,,:; 2.17 ,,:; 2.22 ,,:; 2.32 C l G C2 P C l G C 2 P C l G C2 P C l G 1 C2 I p 1 1 ° 2 2° 1 7° 2 6° 23° 2 7° 2 6° 33 ° 1 2° not deternined 1 3° not deternined 0 0 0 0 � � � � 2.18 2.23 2.28 2.33 C l G C2 P C l G C 2 P C l G C2 P C l G I C2 I p 3 6° 64° 67° 69° 2 2° not deternined unstable unstable These results revealed six compounds that gave relatively small angles and thus were promising vis-a-vis the aroma of cheeses, especially camembert cheese. The cells for these compounds have been shaded in Table 2 .6 . 45 2.4.2 GC-O and GC-MS Six ketones identified from Fox 4000 analysis were further analysed by GC-O for the sensory aspect and this GC-O was conducted by the author only. The odour description of each ketone is shown in Figure 2 .3 and compound 2.23 gave no odour. The purity of each individual ketone was good by GC-MS. o 2 .29 m ushroom-like, metallic o ~ 2 . 1 7 toast, cooked coconut, fruity, spicy o � V 2 .27 m etall ic, iro n , spicy o ~ 2.22 musty, sil icon, g l ue-like, sweet aroma o ~ 2.32 fruity, grassy, mango-like Figure 2 .3 : The structure and odour description of lead ketones from the first library. 2.5 Combinatorial chemistry In the previous work, the synthesis of each ketone was performed individually via a 2- step sequence. The synthesis of this initial batch of compounds was essential for identifying and solving the problems associated with the oxidation of secondary a1cohols in highly conjugated systems . Sixteen ketones were screened by the FOX 4000 and selected compounds by GC-O for sensory aspects of each compound. The ketones with 46 cyclopropyl and phenyl groups showed potential. Therefore, it was desirable to make more of these compounds in a combinatorial way to reduce time . 2.5.1 A Library of ketones containing a cyclopropane Cyclopropane-containing compounds (Figure 2 .6) have been isolated from the fermentation broth of Streptoverticillium /ervens.69 The biosynthetic pathway and metabolism of cyclopropane-containing natural products has been studied.70 The most likely mechanism for cyclopropane ring formation involves electrophilic attack of a carbenium ion on a homoconjugated double bond of an unsaturated fatty acid. This mechanism has been derived from an observed peroxide fragmentation in the female gametes of Ectocarpus siliculosus (Scheme 2 . 1 1 ).7 1 o y � H U-1 06305 (2.52) FR-900848 (2.53) Figure 2 .6 : Oligocyclopropane-containing compounds.72 47 hyd roperox i de-Iyase COOH I Female gametes of E. si/ieu/osus H feOOH O� 2.55 2.56 Scheme 2 . 1 1 : Mechanism for cyclopropane formation in nature. A library of four cyclopropyl ketones was synthesised in a combinatorial manner, involving the Grignard reaction of four commercially available aldehydes with cyclopropyl magnesium bromide, fol lowed by Swem oxidation (Scheme 2 . 1 2) . We focussed our attention on smaller molecules than those in the original library, since they were deemed to be more volatile and odiferous . o �H [>- MgBr OH . � Swern oxidation o � n = 4-7 (2.57-2.60) Scheme 2 . 1 2 : Combinatorial synthesis ofa library of four cyclopropyl ketones . This library of cyclopropyl ketones was tested against camembert cheese only in the FOX 4000 analysis, since cyclopropyl ketones were shown earl ier to be most compatible with camembert cheese (Table 2 .6). The PCA map is shown in Figure 2 .7 . The relationship between the odour from our library of ketones and camembert cheese was still good, since the angle was calculated to be 29° . 48 0.600 0.500 0.400 o.:m CAM!IoII!E R T 0.200 ZBiilO N 0.100 U 0... 0.000 {lloo -0.200 � -0.300 .0.400 {l500 �� {l600 , , 0- , .0.'40 , , , I I 0.90 ·1 .20 -1.00 {lOO {l60 {l20 0.00 0.20 0.40 0.60 P C 1 Figure 2.7 : PCA map for the blank, camembert and the library of four ketones (high and low concentrations) (2.57-2.60). The library of cyclopropyl ketones was also analysed by GC and the compounds eluted in the order of increasing mass. The l ibrary was also assessed by GC-O (Figure 2 .8). � 2.65: Sweet green f10ral� 2.64: Fresh, green fruity Retention Time (min) o / � 2.66: Fatty. oily with sharp note 1 \ 0 : � 2.67: Buttery oily with sharp note Figure 2 . 8 : GC trace for the l ibrary of cyc lopropyl ketones and their odour descriptions. 49 2.6 Summary Synthesis and analysis of ketones were the focus of this chapter. Ketones were synthesized individually via a two-step sequence. The Grignard reaction was the first step to produce an alcohol from a commerc ially available aldehyde, using 1 -3 carbon Grignard reagents which were also commercially available . The oxidation of the secondary alcohol ensued, to produce a ketone . Different reaction conditions were required for the oxidation: Swem oxidation was effective for the oxidation of saturated, long-chain alcohols, while oxidation with DDQ was superior for alcoho ls in highly conjugated systems. Sixteen ketones were synthesised individually and each ketone was screened by the Fox 4000 . Some compounds selected from the Fox analysis were assessed by GC-O. The analysis gave promising results for aromatic and cyclopropyl ketones . The first l ibrary of cyclopropyl ketones was synthes ised and screened. 50 2.7 Experimental procedure 2.7. 1 Genera l procedure General methods. I H NMR spectra were recorded at 270 MHz, and I 3C NMR spectra were recorded at 67.5 MHz. All chemical shifts are reported relative to residual TMS as an internal reference . Mass spectra were recorded on a Shimadzu GCMS-QP5000 mass spectrometer. The MS was operated in electron impact mode with an ionization potential of 70 eV and a scanning rate at 0.5 sec/scan over a mass range of mlz 29-350 . Silica gel flash column chromatography was performed with Scharlau Sil ica gel 60, 0 .4�0 .6 mm, 230-400 mesh. Experiments requiring anhydrous conditions were perfonned under a nitrogen atmosphere. Analytical thin layer chromatography (TLC) was performed with Merck pre-coated TLC plates, silica gel 60 F254, thickness 0.25 mm. Tetrahydrofuran (THF) was disti l led under a nitrogen atmosphere from sodiumlbenzophenone. Dichloromethane (CH2Cb) was distil led under a nitrogen atmosphere from calcium hydride. Dimethyl sulfoxide (DMSO) and triethylamine (Et3N) were disti l led under a nitrogen atmosphere from calcium hydride and were stored over potassium hydroxide. Grignard reaction. A solution of the Grignard reagent (commercially availab le solution in THF, 1 .2 equiv.) was added dropwise to a solution of the aldehyde (250 mg, 1 .0 equiv.) in THF (5 mL) at 0 QC. After stirring for 1 h at 0 QC, water ( 1 0 mL) was added and the mixture was extracted with Et20 (3 x 20 mL). The combined extracts were washed with brine ( l 0 mL), dried (MgS04), and concentrated in vacuo. The crude oil was purified by chromatography (hex-EtOAc, 5 : 1 ) and the secondary alcohol (Table 2 .7) was used directly in the next step . 5 1 Table 2 . 7 : The properties of secondary alcohols from the Grignard reaction. Grignard Aldehyde Scale Rf (5 : 1 Physical (mmol) hex/EtOAc) Property MeMgBr Decanal 1 .6 0.2 1 Colourless (2.34) oi l 2-trans Decenal 1 .6 0 .45 Colourless oi l 4-cis Decenal 1 .6 0.52 Colourless oi l Cinnamaldehyde 1 .8 0.22 Yellow oil 2 ,4-trans, trans 1 .2 0 .47 Yellow oil Decadienal EtMgBr Decanal 1 .6 0 .6 1 Colourless (2 .35) oil 2-trans Decenal 1 .6 0.59 Colourless oi l 4-cis Decenal 1 .6 0 .58 Colourless oil Cinnamaldehyde 1 .8 0.50 Yellow oil 2 ,4-trans, trans 1 .2 0 .45 Yellow oil Decadienal (CH2=CH)MgBr Decanal 1 .6 0.30 Colourless (2 .36) oi l 2-trans Decenal 1 .6 0 .4 1 Colourless oil 4-cis Decenal 1 .6 0 .40 Colourless oi l Cinnamaldehyde 1 .8 0.33 Yellow oil 2 ,4-trans, trans 1 .2 0 .40 Yellow oil Decadienal [>-MgBr Decanal 1 .6 0.52 Colourless (2.37) oil 2 -trans Decenal 1 .6 0.45 Colourless oi l 4-cis Decenal 1 .6 0 .40 Colourless oi l Cinnamaldehyde 1 .8 0.29 Yellow oil 2 ,4-trans, trans 1 .2 0.40 Yellow oil Decadienal General procedure fo r Swern oxidation. To a cooled (-73 °C), stirred solution of oxalyl chloride ( 1 .5 equiv.) in CH2Ch (7 mL) was added DMSO (2 equiv.) dropwise. The 52 mixture was stirred at -73 °C for 30 min, and a solution of the alcohol (0.25 g, 1 equiv.) in CH2Ch ( 1 mL) was added dropwise over 5 min. After 30 min, Et3N (5 equiv.) was added, and the mixture was then allowed to warm to room temperature. Water ( 1 0 mL) was added, and the layers were separated. The aqueous layer was extracted with CHCb (3 x 30 mL). The combined organic layers were washed with water (3 x 1 0 mL) and brine (20 mL), dried (MgS04), and concentrated in vacuo. The resulting oil was redissolved in Et20 (40 mL) and washed with water (3 x 1 0 mL) and brine (20 mL) . After drying (MgS04), the ethereal solution was concentrated in vacuo to provide the ketone. General procedure for oxidation with DDQ. To a stirred solution of allylic alcohol (0.25 g, 1 equiv.) and DDQ (OJ equiv.) in benzene (7 mL) was added periodic acid (0 .9 equiv.) in O . l N hydrochloric acid (30 mL). The resultant mixture was stirred at room temperature for 16 h, then poured into saturated aqueous sodium carbonate solution (50 mL), and extracted with dichloromethane. The extracts were washed with water, dried with magnesium sulfate, and concentrated in vacuo to provide the ketone. 2.7.2 Experimental data The chemical structure of each compound i s given in Tab le 2 .2 . Undecan-2-one (1 .28) prepared via Swem oxidation. The residue was purified by chromatography ( 1 0 : 1 hex-EtOAc) to give 1 .28 ( 1 90.4 mg, 86 %) as a yel low oi l : Rr = 0 .40 ( 1 0 : 1 hex-EtOAc); IH NMR (270 MHz, CDCb) 8 0.88 (t, J= 6. 1 Hz, 3H, CH3CHr ), 1 .22-1 .27 (m, 1 2H, 6 x -CH2-) , l .57 (p, J = 7.3 Hz, 2H, -CH2CH2CO-), 2 . 1 3 (s, 3H, 53 CH3CO-), 2 .42 (t, J = 7.3 Hz, 2H, -CH2CO-) ; I JC NMR (67 .5 MHz, CDCh) 8 1 4 . 1 , 22.7, 23 .9 , 29.2, 29 .3 , 29 .4, 29 .8, 3 1 .9, 43 .8, 209. 1 ; MS obsd. for C l IH220 (M +) : 1 70 . 3E-Undecen-2-one (2.15) prepared via Swem oxidation. The residue was purified by chromatography ( 1 0 : 1 hex-EtOAc) to give 2.1 5 ( 1 75 .8 mg, 66%) as a yellow oil: Rf = 0.55 ( 1 0 : 1 hex-EtOAc) ; IH NMR (270 MHz, CDCh) 8 0.95 (t, J = 7 .0 Hz, 3H, CH3CH2- ), 1 .30-1 .35 (m, 6H, 3 x -CHr), 1 .49- 1 .53 (m, 6H, 2 x -CHr), 2 . 3 1 (s, 3H, CH3CO-), 6. 1 3 (d, J = 1 5 .9 Hz, I H, -CH=CHCO-), 6 .88 (dt, J = 1 5 .9 Hz, 6 .8 Hz, I H, -CHr 1 3 CH=CH-); C NMR (67.5 M Hz, CDCh) 8 1 4 . 1 , 22 .6, 26.8, 28 . 1 , 29 .0, 29 . 1 , 3 1 .7, 32 .5 , 1 3 1 . 1 , 1 48 .4, 1 98 .4 ; MS obsd. for C l I H200 (M+) : 1 68 . 5Z-Undecen-2-one (2.16) prepared via Swem oxidation . The residue was purified by chromatography ( 1 0 : 1 hex-EtOAc) to give 2.16 ( 1 1 7 .5 mg, 47%) as a yel low oi l : Rf = 0.43 ( 1 0 : 1 hex-EtOAc); IH NMR (270 MHz, CDCh) 8 0 .89 (t, J = 7.2 Hz, 3H, CH3CHr ), 1 .28- 1 .38 (m, 6H, 3 x -CH2-), 2 .03 (q, J = 6 .6 Hz, 2H, -CH2-), 2 . 1 5 (s, 3H, CH3CO-), 2 .32 (q, J = 6.8 Hz, 2H, -CH2-), 2 .48 (t, J = 7 . 1 Hz, 2H, -CHr), 5 .28-5 .42 (m, 2H, - CH=CH-); I 3C NMR (67.5 MHz, CDCh) 8 1 4 . 1 , 2 1 .7 , 22 .6, 27 . 1 , 29 .3 , 29.9, 3 1 .5 , 43 .6, 1 27.4, 1 3 1 . 1 , 208 .2 ; MS obsd. for C I I H200 (M+): 1 68 . 4-Phenyl-3E-buten-2-one (2.17) prepared via DDQ oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2 .1 5 ( 1 47.5 mg, 59%) as a dark yellow oil : Rr= 0.53 (5 : 1 hex-EtOAc); IH NMR (270 MHz, CDCh) 8 2 .39 (s , 3H, CH3CO-), 6.70 (d, J = 1 6 .2 Hz, I H, =CH-Ph), 7 .38-7 .45 (m, 3H, ArH), 7 .48-7 .50 (m, I H, =CHCO-), 7 .48- 54 1 3 7 .53 (m, 2H, ArH); C NMR (67 .5 M Hz, CDCb) 8 27 .5, 1 27 .0 , 1 28 . 1 , 1 28 .8 , 1 30 .4, 1 34.2 , 1 43 .3 , 1 98 .2 ; MS obsd. for C IOH I OO (M+): 1 46 . 3E,5E-Undecadien-2-one (2.18) prepared via DDQ oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2 .18 ( 1 29 .7 mg, 68%) as a dark yellow oil : Rr = 0.45 (5 : 1 hex-EtOAc); I H NMR (270 MHz, CDCI3) 8 0 .79 (t, J = 6 .8 Hz, 3H, CH3CO-), 6 .04 (d, J = 5.6 Hz, 1 H, -CH=CH-CO-), 6 . 1 7-6.20 (m, 2 H, -CHrCH=CH-), 7.06-7. 1 5 (m, 1 H, -CH=CH-CH=CH-CO-); I 3C NMR (67.5 MHz, CDCb) 8 1 3 .8 , 22 .3 , 26.9, 28 .2 , 3 1 .2 , 32 .9 , 1 28.4, 1 28 .5 , 1 43 .7, 145 .4, 1 98 . 1 ; MS obsd. for C 1 1H I 80 (M+) : 1 66 Dodecan-3-one (2.19) prepared via Swem oxidation . The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.19 (322.7 mg, 47 %) as a yellow oil : RI = 0 .36 (5 : 1 hex-EtOAc); IH NMR (270 MHz, CDCb) 8 0.88 (t, J = 6.6 Hz, 3H, CH3CHr), 1 .04 (t, J = 7.4 Hz, 3H, CH3CH2CO-), 1 .25- 1 . 29 (m, 1 2H, 6 x -CH2-), 1 .56 (q , J = 6 .8 Hz, 2H, -CH2CH2CO-), 2 .35-2.44 (m, 4H,-CH2COCH2-); I 3C NMR (67 .5 MHz, CDCh) 8 7 .5 , 1 3 .8 , 22 .5 , 23 .7 , 29. 1 , 29.3, 3 1 .7 , 3 5 .5 , 42 . 1 , 2 1 0 .7 ; MS obsd. for C 1 2H240 (M+) : 1 84. 4E-Dodecen-3-one (2.20) prepared via Sw em oxidation . The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.20 (239 .8 mg, 96%) as a yellow oil: Rl= 0 .75 (5 : 1 hex-EtOAc); I H NMR (270 MHz, CDCh) 8 0.88 (t, J = 7 . l Hz, 3H, CH3CH2-), 1 . 1 0 (t, J = 7 .2 Hz, 3H, CH3CH2CO-), 1 .23 - 1 .30 (m, 8H, 4 x -CH2-)' 1 .46 (p , J= 7 . 1 Hz, 2H, - 55 CH2CH2CH=CH-), 2 .2 0 (q, J = 6 .8 Hz, 2H, -CH2CH=CH-), 2 .56 (q, J = 7 .2 Hz, 2H, - COCH�CH3), 6 . 1 0 (dt, J = 5 .8 Hz, 1 .3 Hz, 1 H, -CH=CHCO-), 6. 89-6.78 (dt, J = 5 .8 Hz, 6.8 Hz, 1 H, -CH=CHCO-); l 3C NMR (67.5 MHz, CDCh) 0 8. 1 , 1 4 . 1 , 22 .6, 28 . 1 , 29 .0, 29 . 1 , 3 1 .7 , 32 .4 , 33 . 1 , 1 29 .8 , 1 47.0, 200 .9; MS obsd. for C ' 2H220 (M +) : 1 82 . 6Z-Dodecen-3-one (2.21 ) prepared via Swem oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.21 (235 . 1 mg, 94%) as a yellow oil: Rr= 0 .76 (5 : 1 hex-EtOAc) 'H NMR (270 MHz, CDCh) 0 0.89 (t, J = 7 . 1 Hz, 3H, CH3CHr), 1 .05 (t, J = 7.2 Hz, 3H, CH3CH2CO-), 1 .22- 1 .39 (m, 6H, 3 x -CH2-), 2 .02 (q, J = 7.0 Hz, 2H, CH2CH=CH-), 2 .22-2 .35 (m, 2H, -CH=CHCH2-), 2 .3 9-2 .47 (m, 4H, -CH2COCH2-), 5 .28-5 .44 (m, 2H, -CH=CH-); I 3C NMR (67.5 MHz, CDCh) 0 7 .9, 1 4 . 1 , 2 1 .8, 22 .6, 27 .2 , 29.4, 3 1 .5 , 36 .0 , 42 .3 , 1 27.6, 1 3 1 . 1 , 2 1 0 .9 ; MS obsd. for C 1 2H220 (M+) : 1 82 . 1 -Phenyl-2E-penten-3-one (2.22) prepared via DDQ oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2 .22 ( 1 4 1 .2 mg, 56%) as a dark yellow solid : Rj = 0.83 (5 : 1 hex-EtOAc); 'H NMR (270 MHz, CDCh) 0 1 . 1 6 (t, J = 7.5 Hz, 3H, CH3CH2CO-), 2 .67 (q , J = 7 .2 Hz, 2H, -COCH2CH3), 6.73 (d, J = 1 6.3 Hz, 1 H, - CH=CHCO-), 7 .36-7 .38 (m, 5 H, ArH), 7 . 52-7 .58 (m, 1 H, PhCH=CH-); I 3C NMR (67 .5 MHz, CDCh) 0 8 . 1 , 33 .9, 1 25 .8 , 1 28 .0, 1 28 .7, 1 30 . 1 , 1 34 .3 , 1 4 1 .9, 200 . 5 ; MS obsd. for C l lH ' 20 (M+) : 1 60 . 4E,6E-Dodecadien-3-one: (2.23) prepared via DDQ oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.23 ( 1 0 1 .5 mg, 40%) as a yellow oil : Rr = 56 0.67 (5 : 1 hex-EtOAc); IH NMR (270 MHz, CDCb) () 0.89 (t, J = 7.0 Hz, 3H, CH3CH2-), 1 . 1 1 (t, J = 7.4 Hz, 3 H, CH3CH2CO-), 1 .25-- 1 .38 (m, 4H, CH3CH2CH2CH2CH2CH=), 1 .44 (p, J = 6 .4 Hz, 2H, CH3CH2CfuCH2CH2CH=), 2 . 1 S�2.2 1 (m, 2H, CH3CH2CH2CH2CH£H=), 2 .56 (q, J 7 .2 Hz, 2H, CH3CfuCO-), 6.08 (d , I S .4 Hz, I H, -CH=CH-CO-), 6 . 1 S--6 . 1 9 (m, 2H, -CHz-CH=CH-), 7 . 1�7.30 (m, I H, -CH=CH­ CH=CH-CO-); I 3C NMR (67 .5 MHz, CDCh) () 8 .3 , 1 4 .0, 22 .4 , 28 .4, 3 1 .3 , 33 .0 , 33 . S , 1 27 .4 , 1 28.7, 1 42 .6 , 1 45 .3 , 20 1 .0; MS obsd. for C 1 2H200 (M) : 1 80. Dodec-l -en-2-one (2.24) prepared via Swem oxidation. The residue was puri fied by chromatography ( 1 0 : 1 hex-EtOAc) to give 2.24 ( 1 50 mg, 60%) as a ye l low oil : Rf= 0 .66 ( 1 0 : 1 hex-EtOAc); I H NMR (270 MHz, CDCh) () 0.88 (t, J 7 .0 Hz, 3H, CH]CH2-), 1 .24- 1 .29 (m, 1 2H, 6 x -CH2-), 1 .62 (p, J = 7 .0 Hz, 2H, -CH2CH2CO-), 2 .58 (t, J = 7.4 Hz, 2H, -CfuCO-), 5 . 8 1 (dd, J 1 0 .4 Hz, 1 .5 Hz, I H, -COCH=Cfu), 6 .20 (dd, J = 1 7 .7 Hz, 1 .4 Hz, 1 H, -COCH=Cfu), 6 .36 (dd, J 1 7 .7 Hz, 1 0 .2 Hz, 1 H, -COCH=CH2); l 3C NMR (67 .5 MHz, CDCb) () 1 4. 1 , 22.7, 24. 1 , 29.3, 29.5, 3 1 .9 , 39.7, 1 27 .7 , 1 36 .S , 200.8; MS obsd. for C I2HnO (M'!} 1 82 . 1 ,4E-Dodecdien-2-o ne (2 .25) prepared via Swem oxidation . The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2 .2 5 ( 1 1 . 5 mg, 6%) as an orange oi l : Rr 0 .49 (5 : 1 hex-EtOAc) ; I H NMR (270 MHz, CDCh) () 0.88 (t, J = 6 .8 Hz, 3H, CH3CH2-), 1 .26- 2 .3 1 (m, lOH, 5 x �CHr), 1 .48 (p , J 7.4 Hz, 2H, -CH2CH2CH=CH-), 2 .25 (qd, J = 7 . 1 Hz, 1 .5 Hz, 2H , -CfuCH=CH-), 5 .80 (dd, J 1 0. 5 Hz, 1 .3 Hz, 1 H, -CH=CH�), 6 .28 (dt, J = 7.3 Hz, 1 .3 Hz, I H, -CH=CHCO-), 6.36 (dt, J = 1 5 .6 Hz, 1 .6 Hz, I H, -CH=CH,), 6 .67 57 (dd, J = 7.6 Hz, 0.6 Hz, I H, -COCH=CH2), 6.95 (dt, J = 1 5 .9 Hz, 7.2 Hz, I H, - CH2CH=CHCO-); I 3C NMR (67.5 MHz, CDCh) 8 1 4 . 1 , 22.7, 28 . 1 , 29. 1 , 29.2 , 3 1 . 8, 32 .8 , 1 28 .0, 1 28. 1 , 1 34 .8 , 1 49. 1 , 1 89.6. 1 ,6Z-Dodecdien-2-one (2 .26) prepared via Swem oxidation . The residue was purified by chromatography ( 1 0 : 1 hex-EtOAc) to give 2.26 (56.7 mg, 38%) as a yellow oil: Rf= 0.52 (5 : 1 hex-EtOAc); I H NMR (270 MHz, CDCI 3) 8 0.89 (t, J= 6.8 Hz, 3H, CH3CH2-) , 1 .25- 1 .37 (m, 6H, 3 x -CH2-), 2 .03 (q, J = 6.8 Hz, 2H, -CfuCH=CH-), 2 .36 (q, J= 6.5 Hz, 2H, -CH=CHCfu-), 2 .64 (t, J = 7.8 Hz, 2H, -CH2CO-), 5 .3 1 -5 .44 (m, 2H, -CH2CH=CHCH2- ), 5 .83 (dd, J = 1 0 . l Hz, 1 .6 Hz, 1 H, Cfu=CHCO-), 6.2 1 (dd, J = 7 .8 Hz, 1 .6 Hz, I H, Cfu=CHCO-), 6 .35 (dd, J = 7 .7 Hz, 1 0 .2 Hz, I H, CH2=CHCO-); I 3C NMR (67.5 MHz, CDCh) 8 1 4 . l , 2 1 . 8 , 22 .6, 27 .2 , 29 .3 , 3 l .5 , 39 .6 , 1 27.5 , 1 27 .9, 1 3 l .2 , 1 36 .4, 200.0; MS obsd. for C I 2H200 (M): 1 80 . I -Phenyl- l ,3E-pentadien-3-one (2 .27) prepared via DDQ oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.27 ( 1 50 mg, 60%) as an orange oil : Rf= 0.34 (5 : 1 hex-EtOAc); I H NMR (2 70 MHz, CDCh) 8 5 .90 (dd, J = 1 0 .5 Hz, 1 H, -COCH=Cfu), 6.3 7 (d, J = 1 6 . 1 Hz, I H, -COCH=Cfu), 6 .70 (t, J = 1 0 .6 Hz, I H , - COCH=CH2), 7.03 (d, J = 1 6 . 1 Hz, 1 H, PhCH=CHCO-), 7 .38-7 .44 (m, 5H, ArH), 7 .68 (d, J = 1 6 Hz, I H, PhCH=CHCO-); 1 3C NMR (67.5 MHz, CDCh) 8 1 23 .9, 1 28 .2 , 1 28 .5 , 1 2 8.8, 1 30 .5 , 1 34 .5 , 1 35 .3 , 1 43 .8, 1 89.3 ; MS obsd. for C 1 1H I OO (M+): 1 58 . 58 1 ,4E,6E-Undecatrien-3-one : (2.28) prepared via DDQ oxidation . The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.28 ( 1 80.2 mg, 72%) as a yellow oi l : Rf = 0 .65 (5 : 1 hex-EtOAc); l H NMR (270 MHz, CDCb) (5 0.89 (t, J = 7 .0 Hz, 3H, CH3CH2-), 1 .26- 1 .36 (m, 4H, CH3CfuCH2CfuCH2CH=), 1 .44 (p, J = 7 .5 Hz, 2H, CH3CH2CfuCH2CH2CH=), 2 . 1 5-2 .23 (m, 2H, CH3CH2CH2CH2CfuCH=), 5 .79 (dd, J = 1 0 .6 Hz, 0.6 Hz, 2H, -COCH=Cfu), 6 .20-6 .25 (m, 1 H, -CH2-CH=CH-), 6.3 1 -6.38 (m, 1H , -CH2-CH=CH-CH=CH-CO-), 6 .60 (dd, J = 1 7 .4 Hz, 1 0 .5 Hz, 2H, =CHCOCH=), 7 .22-7 .32 (m, 1 H, =CH-CH=CHCO-); l 3C NMR (67.5 MHz, CDCh) (5 1 4 .0, 22.5, 28 .3 , 3 1 .4 , 33 . 1 , 1 2 5 .6, 1 27 .7, 128 .8 , 1 35 .2, 1 44.4, 1 46 .4 , 1 89.6. l -Cyclopropyl decan-l -one (2.29) prepared via Swem oxidation . The residue was purified by chromatography ( 1 0 : 1 hex-EtOAc) to give 2.29 ( 1 29 .7 mg, 53%) as a yellow oil : Rf = 0.77 (5 : 1 hex-EtOAc); lH NMR (270 MHz, CDCb) (5 0 .8 1 -0.99 (m, 4H, cycloH), 1 .00 (t, J = 7 .4 Hz, 3H, CH3-), 1 .25 - 1 .29 (m, 1 2H, -CH2-), 1 .55 - 1 .63 (m, 1 H, cycloH), 1 .84- 1 .97 (m, 2H, -CH2CH2CO-) , 2 .53 (t, J = 7 .3 Hz, 2H, -Cfu-CO-); l 3C NMR (67.5 MHz, CDCb) (5 6.6, 1 0 .5 , 1 9 .3, 20 .3 , 22 .7, 29.2, 29 .3 , 29.4, 29.5 , 39 .0, 43 .5 , 77.4; MS obsd. for C 1 3H240 (M +): 1 96. l -Cyclopropyl-2E-decen-l -one: (230) prepared via Swem oxidation. The residue was purified by chromatography ( 1 0 : 1 hex-EtOAc) to give 2.30 (75 mg, 30 %) as a yellow oil : Rf = 0 .67 (5 : 1 hex-EtOAc); l H NMR (270 MHz, CDCb) (5 0 .83-0 .93 (m, 4H, cyc1oH), 1 .08 (t, J = 4 .2 Hz, 3H, CH3-), 1 .2 7- 1 .3 1 (m, l OH, -Cfu-), 1 .43- 1 .5 1 (m, 1 H, cycloH), 2 .0 1 -2 .32 (m, 2H, -Cfu-CH=CH-), 6.22 (d, J=1 5 .0 Hz, I H, -CH=CHCO-), 6 .9 1 59 (dt, J = 1 5 .8 , 6 .9 Hz, I H, -CH=CHCO-); l 3C NMR (67 .5 MHz, CDCb) 8 1 1 .0 , 1 4 . 1 , 1 8 .6 , 22 .6, 28 .2 , 29 . 1 , 29.2, 3 1 .7 , 32 .5 , 1 30 .3 , 1 46.9, 200 . 1 . 1 -Cyclopropyl-4Z-decen-1-one: (2.3 1 ) prepared via Swem oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.31 ( 1 83 . 1 mg, 73%) as a yellow oi l : Rr = 0 .74 (5 : 1 hex-EtOAc); IH NMR (270 MHz, CDCb) 8 0 .8 1 -0 . 9 1 (m, 2H, cycloH), 1 .0 1 (p , J = 4 .2 Hz, 2H, cycloH), 1 .23- 1 .39 (m, 5H . CH3CHr) 1 .88- 1 .97 (m, I H, cycloH), 2 .02 (q, J = 6 .6 Hz, 2H, -ClliCH=CH-), 2 .33 (q, J = 6 .9 Hz, 2H, - CH2CH2CO-), 2 .60 (t, J=7 . 1 Hz, 2H, -ClliCO-), 5 .27-5 .44 (m, 2H, -CH=CH-) ; I 3C NMR (67.5 MHz, CDCh) (5 1 0 .4, 1 3 .9, 20.2, 2 1 .7 , 22 .5 , 27 .0, 29 .2, 3 1 .4, 43.2, 1 27 .6, 1 30 .7, 209.8; MS obsd. for C 1 3H220 (M+) : 1 94 . 1 -Cyclopropyl-3-phenyl-2E-propen-1 -one: (2.32) prepared via DDQ oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.32 (202 .4 mg, 8 1 %) as a yellow oil : Rr= 0.57 (5 : 1 hex-EtOAc); IH NMR (270 MHz, CDCh) 8 0 .9 1 - 1 . 00 (m, 2H, cycloH), 1 . 1 2 - 1 . 1 7 (m, 2H, cycloH), 2 . 1 8-2.27 (m, I H, cycloH), 6.85 (dd, J = 1 6 . 1 Hz, 2 .6 Hz, I H, -CH=CH-CO-), 7.32-7 .36 (m, 3H, ArH), 7 .5 1 -7 .54 (m, 2 H, ArH), 7 .59 (d, J = 1 6 .2 Hz, -CH=CH-CO-); 1 3C NMR (67.5 MHz, CDCb) 8 1 1 .2 , 1 9 .4 , 1 26 . 1 , 1 27 .9, 1 28 .6, 1 30.0, 1 34 .3 , 1 4 l .6, 1 99 .5 ; MS obsd. for C I 2H I20 (M) : 1 72 . l -Cyclopropyl-2E,4E-decadien-l -one: (2.33) prepared via DDQ oxidation. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give 2.33 ( l 1 2 .5 mg, 45%) as a yellow oi l : Rr = 0.37 (5 : 1 hex-EtOAc); IH NMR (270 MHz, CDCI3) (5 0.87-0 .93 (m, 4H, 60 cycloH), 1 .08 (t, J=4.4 Hz, 3H, CH3-), 1 .23 - 1 .39 (m, 6H, -CH2-), 1 .4 1 - 1 .49 (m, 1 H, cycloH), 2 .07-2.22 (m, 2H, -CH2-CH=CH-), 4 . l 1 (q, J = 7 . 1 Hz, 1 H, -CH=CH­ CH=CHCO-), 6 . 1 8 -6 .26 (m, 2H, -CH=CH-CH=CHCO-), 7 . 1 6-7 .28 (m, I H, -CH=CH­ CH=CHCO-); 1 3C NMR (67 .5 MHz, CDCb) 8 1 0.9, 1 4 .0, 1 9 .2, 22.5, 28 .4 , 3 1 .4, 33 . 1 , 1 27 .8 , 1 28 .7 , 1 42 .4 , 145 .5, 1 99 .6. Simultaneous synthesis of l-cyclopropyl ketones (2.64 , 2.65, 2.66, and 2.67) Formation of l-cyclopropyl alcohols: A solution of the cyclopropyl magnesium bromide (0 .5 M in THF, 9.6 mL, 4 .8 equiv.) was added dropwise to a solution of hex anal ( l OO mg, 1 22 ilL, 1 .0 mmol , 1 .0 equiv.), heptanal ( 1 1 4 mg, 1 40 ilL, 1 .0 mmol, 1 .0 equiv.), octanal ( 1 28 mg, 1 56 ilL, 1 .0 mmol, 1 .0 equiv.) and nonanal ( 1 42 mg, 1 72 ilL , 1 .0 mmol , 1 .0 equiv.) in THF (20 mL) at 0 qc . After stirring for 1 h at 0 QC, water (20 mL) was added and the mixture was extracted with Et20 (3 x 20 mL). The combined extracts were washed with brine ( 1 0 mL), dried (MgS04), and concentrated in vacuo . The crude oil was purified by chromatography (hex-EtOAc, 5 : 1 ) to give a colourless oil (four alcohols : 462 mg, 7 1 %) which was used directly in the next step. Swern oxidation: To a cooled (-73 °C), stirred solution of oxalyl chloride (533 mg, 360 mL, 4 .2 mmol, 6 .0 equiv.) in CH2Ch ( 1 5 mL) was added DMSO (437 mg, 397 mL, 5 .6 mmol, 8 equiv.) dropwise. The mixture was stirred at -73 °C for 30 min, and a so lution of the four alcohols obtained above (462 mg, �0 .7 mmol each of the four alcohols) in CH2Cb (5 mL) was added dropwise over 5 min. After 30 min, Et3N (5 equiv.) was added, and the mixture was then allowed to warm to room temperature . Water ( 1 0 mL) was added, and the layers were separated. The aqueous layer was extracted with CHCh (3 x 6 1 30 mL). The combined organic layers were washed with water (3 x 20 mL) and brine (20 mL), dried (MgS04), and concentrated in vacuo. The resulting oil was redissolved in Et20 (40 mL) and washed with water (3 x 20 mL) and brine (20 mL). After drying (MgS04), the ethereal solution was concentrated in vacuo to provide the ketones. The residue was purified by flash chromatography eluting with 5 : 1 HexlEtOAc to give a mixture of four ketones (2.64, 2 .65, 2.66, and 2.67) as a yellow oil (28 1 mg; 62 %); MS obsd. for C9H 1 90 (M+) : 142; MS obsd. for C l OH200 (M+) : 1 56 ; MS obsd. for C l lH220 (M+) : 1 70 ; MS obsd. for C1 2H240 (M +) : 1 84 . 2 .7.3 Experimental procedure for screening with the FOX 4000 The ketone samples were prepared in lower and higher concentrations (Table 2 .8) to get a Fox response to compare with four cheeses (e.g., cheddar, gouda, pannesan and camembert cheeses). The amount of each ketone was placed in a suitable beaker (e.g., 2 L and 5 L) along with three FOX vials that could capture the odour. The sealed beaker with the ketone and the vials was heated at 60 QC for 1 5 min. The vials with vacuum­ sealed lids were ready for the FOX analysis . The cheese samples were prepared by coring and wire-cutting disks to give the same size ( 1 cm length x 0.5 cm diameter) of each cheese. The cheese sample was placed in a FOX vial with a sealed lid on . The blank was prepared by adding one drop of water with air present in a FOX vial. Five blanks and three duplicates of each ketone were p laced in two trays at room temperature, and three duplicates of each cheese were placed in a cooled sample tray (2-4 QC). Then 2 ,5 mL of heads pace was injected into the FOX for analysis , 62 Table 2 . 8 : The amount of ketone added in a beaker for the sample preparation. High Compound* amount (ilL) vol. of beaker (L) amount (ilL) 1 .28 1 2 1 2 . 19 1 .5 2 0.5 2.24 3 2 1 2.29 10 2 5 2.1 5 0.5 2 0 .7 2.20 2 2 0 .7 2 . 16 1 5 0 .4 2.2 1 0.8 2 OJ 2.26 0.8 2 OJ 23 1 0.8 2 OJ 2.1 7 1 .5 2 0 .5 2.22 33 .6 mg 2 14 .8 mg 2.27 0.8 mg 2 1 2 .0 mg 232 48 .4 mg 2 1 6 .3 mg 2.18 1 2 1 2.23 5 2 2 *The structure of each compound IS gIven In Table 2 .2 . The FOX Low vol. of beaker (L) 5 2 2 2 5 2 5 2 2 2 2 2 2 2 5 2 The FOX 4000 (AlphaMOS, Toulouse, France) electronic nose system equipped with the standard set of 1 8 metal-oxide sensors, a CTC HS 1 000 head space autosampler, and an 63 ACU 5000 air conditioning unit providing humidified air (20% relative humidity) at 36 qc . The carrier gas was ultra-grade compressed air (79% nitrogen and 2 1 % oxygen). The headspace (2 .5 mL) was taken from the vial ( 1 0 mL) and was injected into a stream of synthetic air passing at 1 50 mL/min over metal oxide sensors at 60°C . Acquisition time for each sample vial was 3 min. 2.6.4 Experimental procedure of screening with GC-MS and GC-O The sample ( 1 ).tL) was dissolved in EhO ( 1 mL) and was analysed by GC-MS and GC­ O. GC-MS GC-MS analyses were carried out using a Shimadzu 6C- 1 7 A gas chromatograph coupled to a Shimadzu GCMS-QP5000 mass spectrometer. Samples ( 1 ).tL) were injected by split / splitless injection at 250 DC (splitless time: 3 0 sec). The mass detector was maintained at 250 DC. For separation, an Alltech Econo-CapTM EC- l OOO™ (30 m x 0.25 mm i.d. with 0.25 ).tm film) was employed. Helium was used as the carrier gas at a constant flow-rate of 1 .8 mLlmin. The column temperature was programmed from 35 °C to 230 QC at a rate of 5 °C/min and then held for 2 1 min at 230 DC . The MS was operated in electron impact mode with an ionization potential of 70 eV and a scanning rate at 0 .5 sec/scan over a mass range ofmlz 29-350. GC-O GC-O analysis was carried out using a Shimadzu GC9A series (Shimadzu corporation, Kyoto , Japan). The chromatograph was equipped with an Alltech Econo-CapTM EC- 1 000TM (30 m x 0.25 mm with 0 .25 ).tm film). The oven temperature was programmed 64 from 35 cC to 230 cC at a rate of 5 cC/min and then held for 2 1 min. Samples (2 ilL) were injected by split I splitless injection at 250 QC. Helium was used as the carrier gas at a flow rate of 1 mLlmin. The end of the column was equipped with an effluent splitter connected to two identical lengths of fused silica detectivated capil lary tubing, one going to the flame ionisation detector (FID, 250 QC) and the other to the custom built smelling port via a heated tube. A stream of humidified air (75 mL/min) was used to sweep the effluent through a glass cone to the sniffer. Time and odour descriptions were recorded when an odour was detected. 65 Chapter 3 Chapter 3 : Synthesis of a library of racemic lactones as poten tial flavour compounds 3 . 1 I ntroduction A lactone is a cyclic ester (Figure 3 . 1 ). lt is the intramolecular condensation product of an alcohol and a carboxylic acid. Lactone nomenclature contains a prefix that indicates the nng SIZe: �-lactone (4-membered), y-Iactone (5 -membered), 8-lactone (6-membered ring). 51: R P R8IOyO � a P p-Iactone y-Iactone 8-lactone Figure 3 . 1 : General structures of lactones. Lactones are important flavour components in many food products including fiuits, vegetables, breads, meats, beverages and dairy products. Lactones are wide ly used by the flavour industry because of their characteristic organoleptic properties, including fru ity, coconut-l ike, buttery, sweet, or nut-like.73 In particu lar, Cg to C I4 y- and 8-lactones have been identified in low concentrations in various types of cheese, and identified as the principal contributors to flavour and aroma because of their low thresholds.74 The flavour of lactones is influenced by the ring size (4, 5 or 6), the length of the lateral carbon chain and the presence of un saturation (Table 3 . 1 ) . The nature and intensity of 66 odour has been reported to vary with the configuration at Cy and C8 of the y- and 8- I . I 75 actones respectIve y. Table 3 . 1 : Structure, name and odourous properties of lactones.76 Structure Name Odorant notes �o dihydro-5 -pentyl-2(3H)- coconut, fatty fruity, 3.1 furanone aniseed �o dihydro-5-hexyl-2(3H)- peach, fatty, fruity 1 .50 furanone 0 0 dihydro-5 -octyl-2(3H)- peach, butter, fatty 1 . 52 furanone �o tetrah ydro-6-bu ty 1-(2H)- fatty, oily, sweet, nutty pyran-2-one 3.2 �o tetrahydro-6-pentyl-(2H)- peach, oily, creamy pyran-2-one 1 .51 �o tetrahydro-6-heptyl-(2H)- peach, buttery, coconut pyran-2-one 1 .53 �o 5-hexyl-2 (5H)-furanone mushroom 3.3 �o 5-hexyl-2 (3H)-furanone fruity, oi ly, fatty 3 .4 67 3.2 Generation of y-Iactones in nature Lactones in cheeses arise biosynthetically from the hydrolysis of l ipids to hydroxy acids that undergo �-oxidation cycles, followed by cyc lisation to form lactones, during heat treatment of the milk. Another possible mechanism of formation of lactones in cheese supposes a lactonisation of hydroxy fatty acids present in milkfat as glyceride esters . Lactones are also formed by the enzymatic reduction of milk oxy fatty acids known to exist in milk fat to produce the hydroxy acids which then lactonise.77 Spores from Penicillium roqueforti, isolated from Roquefort cheese, have been used to synthesise dihydro-5-octyl-2 (3H)-furanone ( 1 .52, Scheme 3 . 1 ) . The precursor fatty acids, released by the action of Candida cylindracea lipase on soybean oil, are predominantly the C ' 8 fatty acids (e.g., oleic acid and linoleic acid). Oleic acid (3.5) was regioselectively hydroxylated at C , o. It is poss ib le to obtain either 1 0-(R)-hydroxystearic acid (3.6), or a mixture of the two enantiomers, depend ing on the organism employed. The acid is then activated as i ts coenzyme A ester (3.7) and its C ' 8 chain degraded by three �-oxidation cycles to fonn the resulting 4-hydroxy-dodecanoyl-CoA (3 . 1 0) . (5R)-Dihydro-5 -octyl- 2 (3H)-furanone (R-1 .52) was formed, following hydrolysis and lactonisation (Scheme 3 . 1 ) . 78 68 HO COOH 3.5 j 1 0-( R)-hydratase 3 . 6 1 C o A ester activatio n I �-oxidation 3.7 CoA o 's I c�o j HSCoA (retro-Claisen reaction) o 11 C' SCoA + 3.8 o .)l scOA 3.9 j j 2 X p-ox;dal;on I relm-Cla;sen reacl;on o SCoA OH 3 . 1 0 j hydrolysis lactonisation o R-1 .52 Scheme 3 . 1 : Possible pathway for dihydro-5-octyl-2(3H)-furanone formation by Penicillium roqueforti. 69 It is possible to produce lactones through b iotechnology. Some fungi79 and yeast species80 were identified for their abi lity to produce smal l amounts of lactones as aroma compounds . Yarrowia /ipolytica has been employed to produce y-lactones. The series of biotransformation begins with a long-chain hydroxy fatty acid precursor like ricinoleic acid (3.1 1 ), a major component of castor oil, which is activated as its coenzyrne A ester 3.12 (Figure 3 .2) . OH 3 . 1 1 : R = OH 3 .12 : R = SCoA o R Figure 3 .2 : Long-chain hydroxy fatty acid precursors . The accepted pathway from ricinolenyl-CoA (3. 12) to dihydro-5 -hexyl-2(3H)-furanone (1 .50) involves three �-oxidation cycles, followed by reduction . The last �-oxidation produces 4-hydroxy-decanoyl-CoA (3. 13 ), which then cyclises to give dihydro-5-hexyl- 2(3H)-furanone ( 1 .50) (Scheme 3 .2) . 76 Scheme 3 .2 ilIustrates how Yarrowia lipolytica produces dihydro-5 -hexyl-2(3H)-furanone (1 .50), 5-hexyl-2(5H)-furanone (33), 5-hexyl- 2 (3H)-furanone (3.4), dihydro-3-hydroxy-5-hexyl-(3H)-furanone (3.16) and dihydro-3- keto-5-hexyl-(3H)-furanone (3 .18) under various conditions. 70 OH o 3.12 SCoA j 1 . 3 X �-oxidation and retro-Claisen reaction 2. reduction 3. p-oxidation o �SCOA-�O OH 3.13 aCYI-coA j oxidase o �SCOA-OH 3.14 2-enOYI-coAj hydratase OH 0 1 .50 �O 3.3 1 �SCOA_ �Oyo __ OH 3.15 > ___ 1 HO 3. 1 6 3-hYdrOXyaCYI-coAj 1 dehydrogenase o 0 �scoA- �Oyo OH 3.17 .);-1. o 3 . 1 8 Scheme 3 .2 : Lactones from ricinolenyl-CoA (3. 12). �O 3.4 The synthesis of racemic lactones is discussed in this chapter, and the study of stereoisomerically pure lactones is the subject of subsequent chapters of this thesis. 7 1 3.3 Previous racemic syntheses ofy-lactones There are a large number of syntheses of y-lactones in the literature. The fol lowing is a selection of relatively short racemic syntheses that might be applied in a combinatorial approach. In 1 982, Maurer and Hauser8 1 reacted acetylenic Grignard reagents with ethyl 4- oxobutanoate (3. 1 9). The acetylenic y-lactone 3.20 was hydrogenated over Lindlar's catalyst to give the corresponding lactone (3.2 1 ) (Scheme 3 .3) . H 2 BrMgC=C-Et �O o L i ndlar's catalyst �O 0 � C H O • -.;;::::: . . . EtOOC T H F -5 cc q U i no l i ne 3 . 19 (20 %) 3.20 (85 %) 3.21 Scheme 3 .3 : Synthesis of (Z)-5 -octen-4-0Iide (3.2 1 ). Another approach by Maurer and Hauser8 1 involved addition of an allyl zinc species to ethyl 4-oxobutanoate (3. 19). A Wittig reaction was used to form the lactone (3 .24) with a Z-double bond in its side chain (Scheme 3 .4). EtOOC � C H O 3 . 19 � ZnCI • � OVO /"y °� O U 0 3/EtOAC . OHC U 3.22 3.23 Scheme 3 .4 : Synthesis of (Z)-6-nonen-4-0Iide (3.24). 72 In 1 998, Krie-r2 reported the synthesis of y-lactones in a one-pot transformation from an alkylidenecyclopropane 3 .25. Trifiuoroperacetic acid, generated in situ from trifluoroacetic anhydride and hydrogen peroxide, was the reagent of choice for the reaction leading to the intermediate epoxide 3 .26, which subsequently underwent expansion to form dihydro-5 -heptyl-2(3H)-furanone (3 .27) (Scheme 3 .5 ). � TFAAlH202 • nC7H1 5 CH2CI2, 20 °C, 1 2 h 3.25 Scheme 3 .5 : Synthesis of dihydro-5-heptyl-2(3H)-furanone (3.27). In 1 990, Bunce and Reeves modified a long-standing undergraduate laboratory experimentS3 to make dihydro-5-pentyl-2(3H)-furanone (3.1 ) . The synthesis begins with the Linstead modification of the Knoevenagel condensation between malonic acid (3.28) and heptanal ( 1 .58), in the presence of triethylamine . The unsaturated acid 3 .29 i s the major product with >95 % selectivity for the � ,y-double bond isomer. The unsaturated acid 3 .29 is then lactonised using an acidic resin in heptane at reflux to form dihydro-5 - pentyl-2(3H)-furanone (3. 1 ) (Scheme 3 .6).84 o � H 1 .58 + HOOC �COOH 3.28 � 0 �OH 3.29 Amberlyst-1 5 . heptane � (76 %) �O 3.1 Scheme 3 .6 : Undergraduate laboratory experiment of making dihydro-5-pentyl-(3H)- furanone (3. 1 ). 73 Ragoussis et al. have investigated the Linstead modification of the Knoevenagel condensation in detai l and have optimized reaction conditions to provide >99 .9 % of the �,y double-bond i somer.85 The ratio of malonic acid, aldehyde and piperidinium acetate was 2 : 1 :0.2 . The classical Knoevenagel reaction is i l lustrated in Scheme 3 .7 . The decarboxylative el imination of water from the �-hydroxymalonic acid normally leads to the a,�-unsaturated acid. H eOOH R� + < o eOOH .. �H Ry\y'eOOH H e=o W R�eOOH a,l3-unsaturated acid Scheme 3 .7 : Mechanism of the classical Knoevenagel condensation. In the presence of piperidinium acetate (i.e . , a dehydration catalyst rather than a base) the intermediate �-hydroxymalonic acid is dehydrated to give the isomeric unsaturated malonic acids (Scheme 3 .8). Only the � ,y-unsaturated decarboxylic acid is isolated since there is reversible hydration-dehydration, and the a,�-unsaturated dicarboxylic acid cannot decarboxylate under the reaction conditions (Scheme 3 .8). �H J � HtN� eOOH - j R eOOH � � _ '---../' _ R�eOOH H eOOH R � eOOH R � eOOH p,y-unsaturated acid AcO ) e R�COOH _ eOOH eOOH . \ -- n o decarboxylation R�eOOH Scheme 3 .8 : Dehydration and decarboxylation of a �-hydroxy-a-dicarboxylic acid. 74 3.4 Racemic Lactones 3 .4 . 1 Stra tegy for the chemical synthesis of a library of y-Iactones Libraries of racemic lactones can be envisaged to arise via the lactonisation of p ,y- unsaturated carboxylic acids (Scheme 3 .9). The unsaturated carboxylic acids can in turn be obtained via the Linstead modification of the Knoevenagel condensation of alkyl malonic acids with a carbonyl compound (Scheme 3 .9) . � + R3 HOOC-( COOH Scheme 3 .9 : Retrosynthetic analysis for racemic lactones. 75 3.4.2 Synthesis of racemic dihydro-5-octyl-2(3H)-furanone The Linstead modification of the Knoevenagel condensation was first applied to the synthesis of dihydro-5-octyl-2(3H)-furanone ( 1 .52) utilising malonic acid (3 .28) and decanal (1.6 1 ) utilising the base as the solvent (i. e., triethylamine, Scheme 3 . 1 0). eOOH o � H + 1 .61 < 3.30 j decarboxylation l 3.31 eOOH 3.28 O H a e O O H � �-hydroxy dica rboxylic acid e O O H eOOH o O H Amberlyst- 1 5 heptane t, .. Scheme 3 . 1 0 : Mechanism for lactone formation. 1 .52 o A broad singlet was observed at 8 1 1 .30 ppm in the lH NMR spectrum of 3 -dodecenoic acid (3.3 1 ), which was assigned to the carboxylic acid proton (Figure 3 .3a) . A multiplet at 8 5 .56 ppm corresponding to the two olefinic hydrogens was also observed. Following lactonisation, these s ignals disappeared and a new signal at 8 4.49 ppm appeared 76 corresponding to Hy in the newly formed lactone functionality of compound 1 .52 (Figure 3 .3b) . (a) 8 1 1 .3 0 ppm (b) I 10 I 10 3.31 H 1 .52 H I 8 COOH o 85.56 ppm I 6 I 4 84.49 ppm I • I 4 I 2 I 2 I [ppm] (pp m] Figure 3 .3 : IH NMR spectra (400 MHz, CDCb) of the crude reaction products: (a) the � ,y-unsaturated carboxylic acid (331 ) and (b) the y-lactone (1.52) . 77 The purity and identity of dihydro-5-octyl-2(3H)-furanone (1 .52) were demonstrated by GC-MS. The mass spectrum showed a molecular ion at mlz 1 98 . The base peak at mlz 85 is a diagnostic feature of the mass spectra ofy-lactones. This results from loss of the side chain as i l lustrated in Figure 3 .4 .86 mlz 1 98 Line#: I R. Time:3 5.250(Scan# :3991) MassPeaks: 128 RawModc:Singlc 35.250(399 1 ) BasePeak:85 . 00( 14758635) BO Mode:None 8S 1 0000000 29 I I 41 43 + �. I I I I 100 �����n4j.,l+i=�4t.,...�,.jb-r=+!��f'�':� I "li8, ,. �41 I I ,162, 1 I I.�O", "I ' ,,,19.�, ,,� 20 30 40 50 60 70 80 90 1 00 1 1 0 120 1 30 140 1 50 1 60 1 70 1 80 1 90 200 mlz Figure 3 .4 : Mass spectrum ofdihydro-5 -octyl-2(3H)-furanone (1 .52). While the Linstead modification of the Knoevenagel condensation gave >95 % selectivity for the �,y-double bond isomer when saturated aldehydes were employed, a problem arose when there was unsaturation in the aldehyde. cis-4-Decenal (332) and malonic acid (3.28) were heated at reflux in triethylamine (these reaction conditions will be referred to 78 as Method A) to give the a,p- (3.33) and p ,y-double bond (3.34) isomers as a mixture (Scheme 3 . 1 1 ). 5 1 COOH Et3N 7 6 4 �CHO 3.33 COOH + 3.32 < 5 3 1 COOH -::?' COOH 3.28 7 6 4 2 (32 % ) 3.34 Scheme 3 . 1 1 : Reaction applied to an unsaturated aldehyde 3.32 (Method A). In the I H NMR of the product mixture (Figure 3 .5a), there is a multiplet at 8 5 .34-5 .45 ppm corresponding to the two olefinic hydrogens at the 6- and 7- positions, as in the spectrum of cis-4-decenal. A multiplet at 8 5 .56-5 .59 ppm was assigned to H-3 and HA of the i somer 3.34. A doublet at 8 5 .84 ppm with J2•3 = 1 5 .6 Hz was attributed to H-2 of compound 3.33 and a doublet of triplets at 8 7 .09 ppm with J2•3 = 1 5 .6 Hz and J3,4 = 6.7 Hz represented the other olefinic hydrogen, i. e. , H-3 , for the isomer 3.33 . The ratio of isomers was calculated by integration of the peaks in the GC trace and by integration of NMR signals (Figure 3 .5 ). 79 (a) // /� -,------, r - 3� If-3 7 6 3.33 3.34 , 10 -JW� __ -' L ___ _ (b) , 7.0 10.0 H3 COOH H2 5 COOH H4 2 r j r----r··-----' .----, S 6 { I I ( ( 4 ; I I I r ! J f J J Lt �----,-,---, 2 ,/r � / 334 33,G� H-3 {H-4 r� W-2 Ij! 3.33 and 334 _�_�=Jj���A�j��N��H-7_ 20.0 , 6.5 , 6.0 3.33 (57 %) 30.0 40.0 3.34 (43 %) 50.0 , 5.5 60.0 miJ> Figure 3 . 5 : (a) NMR data and (b) GC trace of the mixture of3.33 and 3.34 . 80 , (ppm) , [ppm) Therefore, cis-4-decenal (3.32) and malonic acid (3 .28) were reacted, by what we shall refer to as Method B (Scheme 3 . 1 2), as originally described by Ragoussis et al.8s It gave the isomeric ally pure p,y-unsaturated carboxylic acid 3 .34. �CHO + 3.32 COOH < COOH 3.28 DMSO 100°C (91 %) 3.34 Scheme 3 . 1 2 : Reaction appl ied to an unsaturated aldehyde 3 .32 (Method B). COOH The IH NMR spectrum of this product (Figure 3 .6) included a multiplet at 8 5 .32-5 .49 ppm corresponding to the olefinic hydrogens on the 6 ,7-cis double bond and a multiplet at 8 5 . 53 -5 .59 ppm corresponding to the p,y-olefinic hydrogens . There was no sign of the a,p-double bond isomer 3.33 . These reaction conditions (Method B) were applied to both saturated and unsaturated aldehydes to compare with the original reaction conditions in Method A. The yields and purity of the products were improved. Table 3 .2 summarises the chemical yields and product distribution under the two sets of reaction cond itions. 8 1 I 7.0 7 6 3.34 10 5 COOH 2 8 6.5 6 I 6.0 4 I 5.5 2 r I [ppm] I [pp m) Figure 3 .6 : lH NMR spectrum (400 MHz, CDCl3) of the crude reaction mixture containing 3.34 and a trace of 3.33 . 82 Table 3 .2 : Fonning �,y-unsaturated carboxylic acids under the two sets of reaction conditions. �,y-unsaturated carboxylic acid Yield (%) Method A Method B �eooH 3.29 50 % 77 % -;?' eOOH 84 % 83 % 3.31 - -;?' eOOH 32 %* 9 1 % 3.34 -;?' -;?' eOOH 1 9 %* 58 % 3.35 0 �N Nil 68 % o � eOOH 3.36 * As a mixture of a,�- and �,y-unsaturated carboxylic acids Method A: malonic acid ( l .0 equiv.), aldehyde ( l .0 equiv.), EbN (solvent), reflux, 1 h; Method B: malonic acid (2 .0 equiv.), aldehyde ( l .0 equiv.), piperidinium acetate (0 .2 equiv.), DMSO (so lvent), 1 00 °C, 5 h . 83 3.4.3 Lactonisation of B,y-unsaturated acids There are a number of ways in which B,y-unsaturated acids might be converted to y- lactones, including halolactonisation,87 selenolactonisation88 and via the Sharpless asymmetric dihydroxylation.89 However, one-step, acid-catalysed lactonisation held obvious appeal (Scheme 3 . 1 3) . Various acid catalysts have been employed: sulfuric acid,90 p-toluenesulfonic acid,9 1 or acidic ion exchange resins (e.g., Amberlyst- 1 5 and Dowex Hl .92 Amberlyst- 1 5 resin was chosen as the heterogeneous catalyst for the lactonisation of unsaturated carboxylic acids, since it is a porous sulfonated polystyrene resin that serves as an excellent source of strong acid in nonaqueous media. The advantages of using resins are that they require less rigour in handling, react faster, and possess higher loading capacities than the other l iquid acidic methods. An additional advantage is that the catalyst can be regenerated and used several times. Amberlyst- 1 5 .. �o 0 �COOH n Heptane n = 4 (3.29) n = 7 (3 .31 ) n = 4 (3 .1 ) ; 84 % n = 7 (1 .52); 88 % Scheme 3 . l 3 : Lactonisation ofp ,y-unsaturated acids (3 .29 and 3 .3 1 ). Although lactonisation of simple p,y-unsaturated carboxylic acids was achieved in good yield (Scheme 3 . 1 3 ), we had difficulty forming lactones in substrates with double bonds elsewhere in the carbon skeleton . Unfortunately, only trace amounts of product were obtained when more highly unsaturated acids [e.g., 3(E),6(Z)-dodeca-3 ,6-dienoic acid (3.34)] were subjected to these conditions . To try to understand this, we needed to 84 consider the mechanism of the reaction. At first we considered a concerted mechanism for cyclisation in which protonation of the double bond is accompanied by lactonisation (Scheme 3 . 1 4, Route a) but this is disfavored according to Baldwin 's rules.93 An alternative, involving regioselective addition of the acid across the double bond, followed by intramolecular substitution seems much more l ikely. This was also suggested by Sakai et al. in 1 987 who reported the formation of dihydro-5 -ethyl-2(3H)-furanone (3 .39) from hex-3-enoic acid (3.37), presumably via the addition of hydrogen iodide across the olefin, fol lowed by intramolecular SN2 displacement of the iodide (Scheme 3 . 1 4) .94 H I R-C�"vO R0'i � �dO-tri9 R COOH R '(O�O � LJ Route� R1CYf. � % t t H� b � -exo- e ;J Me3SiCI �COOH _N_a_, ,_ H_20 ____ [ �, COOHj _ �o hexane, RT 3.37 (660/ ) 3.38 3.39 /0 Scheme 3 . 1 4 : Proposed mechanism of lactonisation. If the mechanism proposed (Scheme 3 . 14 , Route b) is in operation, then it is possible that either doub le bond of a diene might interact with the acidic resin (Scheme 3 . 1 5) . An intermediate y-sulfate 3.42 might undergo lactonisation via intramolecular substitution as outlined above. Clearly this is not a competitive pathway. Attachment of the acid to the 85 resin via a more remote double bond (e.g., formation of 3 .40) cannot lead to y-lactone 3.43 and indeed formation of a macro lactone would be unlikely for entropic reasons . Interestingly, the formation of a methyl ester (3.41 ) can be performed in the presence of Amberlyst- 1 5 by heating in methanol . Intennolecular Fischer esterification is therefore faster than the i ntramolecular processes . COOH Am berlyst - 15 heptane, � 3.34 COOH Amberlyst- 1 5 MeOH, � Amberlyst- 1 5 heptane, � O� I O=S=O 3.42 0 - - - - - - - - - - .... COOMe 3.41 o 3.43 Scheme 3 . 1 5 : Proposed mechanism for the lactonisation of a /3,y-unsaturated carboxylic acid with Amberlyst- 1 5 resin. Ansell and Palmer proposed a mechanism for the formation of y-lactones from unsaturated fatty acids .95 The lactonisation of short-chain fatty acids was achieved in the presence of large excesses of sulfuric or trifluoroacetic acids at 1 50 °C. The cyclisation of fatty acids via a carbocation derived from a double bond in the 4- or 5- position of the 86 backbone leads to a y-lactone. Since only the y-Iactone is produced arising from both 3 - and 4- positions o f the double bond, carbocation migration is possible (Scheme 3 . 1 6) . + R�COOH 3 / R�COOH � R�COOH • R�O + R�COOH , 4 R�COOH Scheme 3 . 1 6 : A mechanism for the formation of y-lactones from unsaturated fatty acids proposed by AnseIl and Palmer.95 It is possible that the rate-determining step in lactonisation of an olefinic acid is faster when the positive charge is localised at the 4-position that leads directly to the 5- membered ring. This explanation was discussed by Showell and Swern.96 They developed a method for the perchloric acid-catalysed i somerisation of oleic acid (3.5) into y-stearolactone (3.44) (Scheme 3 . 1 7) . This result provided evidence that the carbocation must migrate along the carbon chain to the 4-position where y-lactone formation is favoured. 9 0 OH 3 .5 I HCIO, 1 00°C 0 Scheme 3 . 1 7 : The perchloric acid-catalysed isomerisation of oleic acid (3.5) into y- stearolactone (3.44). 87 The possib ility of double bond migration may explain the difficulty in isolating homogeneous products from attempts to lactonise more h ighly unsaturated acids. The possibility of migration of carbonium ions makes many structures possible . Even though 5 -membered ring formation is preferred to give the y-lactone, the positional integrity of double bonds in the s ide chain cannot be preserved (Scheme 3 . 1 8) . COOH o 3 .34 3.43 Scheme 3 . 1 8 : Unsuccessful pathway for lactonisation of a more highly unsaturated acid 3 .34. 3.4 .4 Substituted y-lactones If the generation of y-lactones by condensation and cyclisation were possible, according to the retrosynthetic analysis in Scheme 3 .9 (reiterated be low from p.75), then we would have an expedient route to variously substituted y-Iactones, albeit as mixtures of stereoisomers . Thus, the use of substituted malonic acids (R3 =I- H) would lead to the incorporation of a substituent at the a-position . Incorporation of a ketone (instead of an aldehyde; R 2 =I- H) would introduce a �-substituent. Ketones were unreactive, as observed by Ragoussis,85 thus making it impossible to introduce a �-substituent via this approach. HOOC Scheme 3 .9 : Retrosynthetic analysis for racemic lactones. 88 We investigated the scope of the reaction us ing et-substituted malonic acids, i.e., methyl malonic acid (3.45) and ethyl malonic acid (3.46) in the presence of triethylamine (Method A). et-Substituents could be incorporated, but the yields were poor and formation of saturated carooxylic acids as by-products resulted. Condensation in the presence of piperidinium acetate (Method B) of et-substituted malonic acids and aldehydes gave no reaction at all . This provided an important clue : for once, Method A gave superior results to Method B . Perhaps basicity, as well as dehydrating capacity, is important in the case of a-substituted malonic acids (Scheme 3 . 1 9) . �C H O 1 .61 COOH + -< COOH 3.45 �COOH 3.47 M ethod A: 1 1 % Method B : 0 % Scheme 3 . 1 9 : Condensation of a-substituted malonic acid 3.45 and aldehyde 1 .61 . There are examples of condensations between a-substituted malonic acids and aldehydes in the literature . Lalic et al. reported the metal-catalysed decarboxylative aldol reaction depicted in Scheme 3 .20.97 o 0 � + Ph� C H O BnS O H 3 .48 3.49 Cu( ethy l hexanoate)2 N V OMe (! I N � H 3.50 THF (82 %) o OH B n S � Ph 3.51 Scheme 3 .20: The metal-catalysed decarboxylative aldol reaction. 89 Tanaka et al. studied the Knoevenagel reaction of p-nitrobenzaldehyde (3.52 ) with methyl malonic acid (3.45) (Scheme 3 .2 1 ). The reaction was monitored by IH NMR, which demonstrated reversibility between the starting materials and the j3-hydroxy intermedi ate 3.53 . The Knoevenagel reaction in the presence of piperid in e proceeded via bis-piperidide intem1ediate 3 .54 . The bis-piperidide intermediate 3 .54 is a stronger electrophile and reacts with the malonate an ion as shown by NMR experiments. Decarboxylation and elimination ensued to give the unsaturated carboxyl ic acid 3.57 as the major product.98 + -< COOH COOH 3.45 /� 02N� "- #COOH 02NI() O COOH � OH 3.53 0 j - co, J 3.54 02N� I 02N� ' '" �COOH �COOH COOH 3.55 OH ON j 3.5S 02N� I COOH 3.57 Scheme 3 .2 1 : The Knoevenagel reaction of p-nitrobenzaldehyde (3 .52) with methyl malonic a c id (3.45). 90 In l ight of this alternative mechanistic pathway, which was productive for a-substituted malonic acids, we investigated the Knoevenagel reaction with methyl malonic acid (3.45) and heptanal ( 1 .58) in the presence of different bases (Table 3 .3) . Tertiary amines, (i.e . , triethylamine and pyridine), gave low yields and saturated carboxylic acids were formed as by-products . Secondary amines, (i.e. , piperidine and diethylamine) gave greatly improved yields of the desired products . The condensation with ethyl malonic acid (3.46) and heptanal ( 1 .58) was also investigated and gave similar results (Table 3 .3) . Table 3 .3 : Condensation of a-alkyl malonic acids in the presence of amine bases. � H 1 .58 0 + COOH R-\ CO OH R Base -CH3 NEt3 -CH3 NHEt2 N -CH3 0 H -CH3 0 -CH2CH3 NEt3 H -CH2CH3 0 -CH2CH3 NHEt2 R base .. �COOH R = M e , 3.47 R = Et, 3.58 pKa of Base Yield 1 1 .0 1 1 % 1 0 .5 60 % 5.3 1 1 % 1 1 .2 26 % 1 1 .0 0 % 1 1 .2 1 2 % 1 0 .5 60 % 9 1 Dehydration and decarboxylation were key steps in the condensation reaction following attack of the malonate anion on a bis-diimide derivative. In an attempt to investigate the order of these events, we set up a s imple experiment with a protected malonic acid, t.e . , diethyl malonate (3.59), which cannot undergo decarboxylation. An unsaturated diester (3.60) was isolated and the resonances at 0 1 28.6 and 0 150 .0 ppm in the 1 3C NMR spectrum were attributed to the carbons at the newly fonned double bond in 3.60, indicating that dehydration could occur before decarboxylation . The unsaturated diester was hydrolysed and then decarboxylation was carried out to give the desired product 3 .29 (Scheme 3 .22). � H + COOEt < 1 .58 o COOEt 3.59 KOH COOH · �COOH 3.61 ( 1 0 %) COOEt �COOEt 3.60 Amberlyst 1 5 II (92 % ) .. �COOH 3.29 Scheme 3 .22 : The condensation reaction fol lowed by dehydration and decarboxylation. Therefore, the choice of base in the reaction is based on steric bulk and basicity. The final choice of base was a secondary amine, diethylamine, which gave a better result than other basic conditions (Scheme 3 .23). 92 �H + o G:N , Et Et H I 1 2 N _ �H Et _�N'Et Er' ' Et (11, I 3.62 Et'�' Et Et,N 'Et 3.63 1 .58 H Ef'·�'Et + eOOH -< 3.45 eOOH ( H eOOH �eOOH eOOH I - H HQ ' Et -�eOOH -�eOOH 'N 3.64 3.65 3.47 Et'Ef)' Et Scheme 3 .23 : The Knoevenagel reaction of heptanal (3.52) with methyl malonic acid (3.45) in the presence of diethylamine. Once the a-substituted f3,y-unsaturated carboxylic acids were in hand, lactonisation in the presence of acid catalyst was straightforward (Scheme 3 .24). �H + R-< eOOH 1 .58 0 eOOH R = Me (3 .45) Et (3.46) R Et2NH I " �eOOH R = Me (3 .47, 60 %) Et (3.58, 60 %) Amberlyst- 15 .. � O yo Heptane y � R R = Me (3.66, 72 % ) Et (3.67, 56 % ) Scheme 3 .24: Synthesis o f a-substituted lactones (3.66 and 3.67). There were four isomers expected and the ratio of the two pairs of enantiomers present was calculated by integration of the NMR signals (Figure 3 .7) . We presume that the major products have the trans-orientation of the two substituents since this would be thermodynamically more stable. 93 Cb) 3S,5S-3.66 3R,5R-3.66 , I H5 from 3R,SS- and 3S,5R (87 %) 3S,5R-3.66 3R,5S-3.66 H3 from 3R,5S- and 3S,5R I (87 %) I '� I�il; H3 fro " m 3R,5R­ I ; and 3S,5S '� ( 1 3 %) �� _ _ �_---- 111 t_Ji��� 4.5 t 4.0 t 3.5 t 3.0 2.5 [ppm] Figure 3 .7 : Ca) Four diastereomers of compound 3.66 and (b) the NMR trace of the mixture . 3.4.5 Synthesis of a library of racemic y-lactones The Linstead modification of the Knoevenagel condensation, under the vanous conditions described, enabled us to prepare � ,y-unsaturated carboxylic acids, which can lactonise to give y-Iactones. We sought to prepare a library of racemic lactones in a combinatorial fashion. Mixtures of aldehydes and malonic acids could be used to produce l ibraries of y-Iactones. The first l ibrary involved five aldehydes in combination with malonic acid (3.28) to fonn �,y-unsaturated acids and cyclisation of p,y-unsaturated acids 94 were fol lowed (Scheme 3 .25). The GC traces of l ibraries of five acids and lactones showed peaks corresponding to five compounds in each library (Figure 3 .8). The peaks could be attributed to each compound and identified by the MS. eo OH < eOOH Base �eOOH_�O n = 3; 1 .57 n = 4; 1 .58 n = 5; 1 .59 n = 6; 1 .60 n 7; 1 .61 3.29 n ::: 3; 3.68 n :::: 4; 3.29 n ::: 5; 1 .69 n = 6 ; 3.70 n = 7 ; 1 .31 Scheme 3 .25 : Synthesis of a library of y-lactones (n 3-7) . (a) �COOH n (b) �O n = 3 4 5 6 7 n = 3 4 5 6 7 n 3; 3.71 n = 4; 3.1 n :;;: 5 ; 1 .50 n :::: 6; 3.27 n = 7; 1 .52 Figure 3 .8 : GC traces of (a) five carboxyl i c acids and (b) five racemic l actones, n = 3 �7 . 95 A mixture of the same five aldehydes was reacted with five equivalents of methyl malonic acid (3.45), leading to a library of racemic diastereomers of lactones containing a methyl group at the 3 -position. The l ibrary was analysed as for the previous library by GC-MS (Figure 3 .9) to establ ish their identities and purities. The experiment with ethyl malonic acid (3.46) was also carried out to synthesise five racemic dihydro-3-ethyl-5- alkyl-(3H)-furanones and the l ibrary was again analysed by GC-MS to establish their identities and purities . (a) (b) �COOH n �o n = 3 96 4 5 6 7 , N ote: Each peak represents a pair of enantiomers . 40.0 min n = 3 4 5 6 7 Note: Each peak represents major diastereomers . The intensities of five lactones were not equal since some o f l ac tones were lost during purification. , , .J i I I) 11)" k�," , 1 0.0 20.0 30.0 40.0 min Figure 3 .9 : GC traces of (a) five carboxylic acids and (b) five dihydro-3 -methyl-5-alkyl- (3H)-furanones . 96 3.4.6 Screening of the lactone libraries The odour descriptions for y-lactones were obtained from screening by GC-O and their details are shown in Table 3 .4. Table 3 .4 : The odour description ofy-Iactones. �o R RT Compounds Odour Description Lit. Odour" (min) Library 1 (R = H) 25 .9 dihydro-5 -butyl-2(3H)-furanone coconut coconut (3.7 1 , Cs) 28 .4 dihydro-5 -pentyl-2(3H)-furanone apricot, coconut coconut, fatty (3. 1 , C9) fruity, aniseed 30 .8 dihydro-5-hexyl-2(3H)-furanone fruity juice, coconut peach, fatty, ( 1 .50, CIO) fruity 3 3 . 1 dihydro-5 -heptyl-2 (3H)-furanone peach, strawberry musty, fruity, (3 .27, Cl l) peach 3 5 .3 dihydro-5-octyl-2(3H)-furanone sweet peach peach, butter, ( 1 .52, C1 2) fatty Library 2 (R = Me) 24.2 dihydro-3-methyl-5 -butyl-(3H)- coconut, sweet, green , coconut furanone (3.72, C9) herbaceous 26.8 dihydro-3 -methyl-5 -pentyl-(3H)- sweet, coconut. furanone (3.66, C IO) milky, fruity 29 .2 dihydro-3 -methyl-5-hexyl-(3H)- peach, fruity, sweet furanone (3.73, C I I ) 3 1 .4 dihydro-3-methyl-5-heptyl-(3H)- burning sweet furanone (3.74, C 12 ) peach 33 .7 dihydro-3 -methyl-5-octyl-(3H)- fatty, sl ightly peach, furanone (3.75, C 13) sweet Library 3 (R = Et) 25 .8 dihydro-3 -ethyl-5-butyl-(3H)- fatty, coconut furanone (3.76, CIO) 27.9 dihydro-3-ethyl-5 -pentyl-(3H)- fatty, fruity furanone (3.67, Cl I ) 30 .3 dihydro-3 -ethyl-5 -hexyl-(3H)- sweet peach furanone (3.77, Cn) 32 .4 d ihydro-3 -ethyl-5 -heptyl-(3H)- sweet, fatty, slightly furanone (3.78, C13) peach 34.5 dihydro-3 -ethyl-5 -octyl-(3H)- fatty, waxy, sweet furanone (3.79, C 14) 97 3.5 Thionolactones 3.5.1 Introduction Sulfur-containing compounds play prominent roles in the flavour of many kinds of fruits, such as passion fruit, blackcurrant and grapefruit. lOo For example, 3 (S)-sulfanylhexanol (3.80)101 is found in yellow passion fruit. Guava contains 3(S)-methylthiohexanol (3.81 ),102 2(R)-methyl-4(S)-propyl- l ,3 -oxathiane (3.82) and 3 (S)-propyl-)'-sultone (3.83) (Figure 3 . 1 0) . 1 03 Some of them are identified as the principal contributors to cheese flavour (e.g., methanethiol l . l l and dimethyldisulfide 1 .13 in cheddar). S H 0$/ ~ - -�OH �OH 0 '� 3.80 3.81 O::: S ' S .... S , - S H 3.82 3.83 1 . 1 1 1 .1 3 Figure 3 . 1 0 : The structures of natural flavour compounds in fruits and cheeses. Thiol-substituted furans, such as 2-methyl-3-furanthiol (3.84), 2-furanmethanethiol (3.85), and the corresponding disulfides , bis(2-methyl-3 -furyl)disulfide (3.86) and bis(2- furylmethyl)disulfide (3.87) are found in roasted coffee at low concentrations (Figure 3 . l 1 ). 1 04 SH fi-o S - S {.( )O � S - S J) o 0 � S H o 3.84 3.85 3.86 3.87 Figure 3 . l 1 : Sulfur-containing compounds in roasted coffee. 98 The importance of sulfur compounds in meat flavour was reported by Brennan and Bemhard in 1 964 . They identified hydrogen sulfide and methyl , ethyl, propyl and butyl mercaptans in the headspace of canned cooked beef. 105 Liebich identified dimethyl disulfide and dimethyl sulfone in boiled beef 106 Since sui fur-containing compounds are identified as key compounds in a variety of food, the conversion of lactones into thionolactones has the potential to produce interesting flavour compounds. Thio- (1), thiono- (ll) and dithio-derivatives (Ill) of lactones have been studied with respect to their unique odours. A number of natural products containing a thiolactone ring have been isolated [e.g. , thiolactomycin (3.88), thiote tromycin (3.89), thiocoumarin (3.90)) (Figure 3 . 1 2) . 1 07 o 3.88 3.89 3.90 Figure 3 . 1 2 : Examples of thiolactone rings in natural products. 3.5.2 Previous synthetic work The chemistry of thionoesters has been an area of interest in both organic and flavour chemistry because of their unique behaviour and odour. In 1 963 , Kharasch and Langford reported the conversion of dihydro-5-methyl-(3H)-furanone (3.9 1 ) into the corresponding thiolactone (3 .92) via isothiouronuim bromides (Scheme 3 .26). 1 08 99 "(y0 H Br 2NaOH ° '- /'- Jl e <±) '-( ......, ' O Na 3.91 SH H CI "Ci0 3.92 Scheme 3 .26: Kharasch and Langford 's synthetic route to y-thiolactone. In the late 1 970's , Kaloustian synthesised thiono lactones via a two-step procedure of sulfhydrolysis / acetylation of N,N-dimethyliminolactonium salts at low temperature (Scheme 3 .27a). I09 In 1 98 1 , the same group reported a shorter and more convenient route (Scheme 3 .27b),"o although a by-product (3.95) was obtained, reducing the yield of the desired product. (a) "(y0 (b) 3.91 1 . N a S H / Me2CO 2 . AcCI / C5H5N -78 QC "(y0 3.91 "Ci0 3.92 (93 % ) (78 % ) 8 B F4 <±) yYN M e2 3.94 H Oy � � O E t 1 0 % 3.95 Scheme 3 .27 : Kaloustian 's approach to oxygen-sulfur exchange. 100 In 1 979, Lawesson introduced 2 ,4-bis(4-methoxyphenyl)- 1 ,3-dithia-2,4-diphosphetane- 2,4-disulfide (3.96, Lawesson 's reagent, Figure 3 . 1 3) to synthesise thiono and dithiolactones . Lawesson ' s group found that thiono lactones were generally pale yellow oils or colourless crystals that are stable, whereas the orange-red oily dithiolactones gave a most unpleasant odour and decomposed upon s torage . 1 1 1 Figure 3 . 1 3 : 2 ,4-bis( 4-methoxyphenyl)- 1 ,3-dithia-2,4-diphosphetane-2,4-disulfide (Lawesson ' s reagent, 3 .96). A mechanistic proposal for the replacement of the oxygen atom of the carbonyl group of the y-Iactone with sulfur is i l lustrated in Scheme 3 .28 . .. Scheme 3 .28 : A proposal for the mechanism ofthionation ofa y-Iactone with Lawesson' s reagent. 1 0 1 Since 2 ,4-bis(4-methoxyphenyl)-1 ,3 -dithia-2,4-diphosphetane-2,4-disulfide (3.96) seems to be a convenient th ionation reagent, it has been widely adopted. Hayashi 's group made y- and 8-thionolactones individually (Scheme 3 .29) and reported physical and chemical . 1 1 2 propertIes . �o�o _3_.9_6_..­nu toluene n = 0; 3.91 n = 1 ; 3.97 n = 2; 3.98 n = 3; 3.71 n = 4 ; 3.1 n = 5; 1 .50 n = 6; 3.27 n = 7; 1 .52 11, 3 h �s n = 0; 3.92 n = 1 ; 3.99 n = 2; 3.1 00 n = 3; 3. 101 n = 4; 3.1 02 n = 5; 3. 1 03 n = 6; 3. 104 n = 7; 3.1 05 n = 1 ; 3.1 06 n = 2; 3. 107 n = 3; 3.2 n = 4 ; 1 .51 3 .96 toluene 11, 5 h Scheme 3 .29 : Individual syntheses ofy- and 8-thionolactones . n = 1 ; 3.108 n = 2 ; 3. 109 n = 3; 3. 1 1 0 n = 4 ; 3. 1 1 1 Also, in 1 998 , Beck and Mosandl prepared y- and 8-thionolactones by Lawesson ' s method and analysed their odour by GC-O utilising a chiral column (Table 3 .5) . 1 1 3 Table 3 . 5 : Odour description for thionolactones . 1 1 3 Compound Absolute Odour description configuration y-C6 R green, grassy 3.99 S green, grassy, mint, burnt y-Cs R mushroom-like odour, hay-like odour 3.101 S mushroom-like odour, hay-like odour, pungent y-C IO R fruity 3.103 S sweet, fruity y-C 12 R fruity 3.105 S fruity 8-C6 R maggi-like odour, spicy, burnt 3.1 12 S maggi-like odour, sulfurous, burnt 8-Cs R mushroom-like odour, grassy, green 3.109 S mushroom-like odour, grassy, hay like odour, sulfurous 8-C IO R green, sl ightly sweet, s lightly fruity 3.1 1 1 S sweet, fruity 8-C 1 2 R sweet, fruity 3. 1 13 S sweet, fruity 1 02 In 200 1 , Schmarr, Eiseneich and Engel synthesised thio-, thiono- and dithio-derivatives of whiskey l actone (3.66) by Lawesson 's method and described their odour (Scheme 3 .30). 1 14 �o 3.66 coconut-like Lawesson's Reagent toluene, reflux, 3 h . �s 3.1 14 mushroom-like Scheme 3 .30: Synthesis of thiono-derivative of whiskey lactone 3.1 14 . Final ly, in 2002 , Lizzani-Cuvelier's group resolved racemic y-lactones enzymatically and then applied Lawesson 's method to exchange oxygen and sulfur (Scheme 3 .3 1 ). 1 1 5 �o n = 0; 3.91 n = 1 ; 3.97 n = 2; 3.98 n = 3; 3.71 n = 4; 3.1 n = 5; 1 .50 lipase . )o) n " ('Oyo 60 % "---1 n = 0; S-3.91 n = 1 ; S-3.97 n = 2; 5-3.98 n = 3; 5-3 .71 n = 4 ; S-3.1 n = 5; S-1 .50 Lawesson's Reage�t X -)(j ('Oys toluene, reflux, 4 h U n = 0; S-3.92 n = 1 ; S-3.99 n = 2; 5-3.1 00 n = 3; S-3.1 01 n = 4; S-3. 102 n = 5; 5-3.1 03 Scheme 3 .3 1 : Enzymatic resolution of racemic y-Iactones and thionation of chiral lactones. 3.5.3 Synthesis of a library of racemic thionolactones We first synthesised the thionolactone derived from commercial ly available dihydro-5- methyl-(3H)-furanone (3 .91) . We investigated the use of Lawesson 's reagent in different stoichiometric ratios to optimise the yield (Tab le 3 .6). The main problem faced with this dihydro-5 -methyl furan-2-thione (3.92) was its volatility; care must be taken not to leave it under vacuum too long. Dihydro-5-octyl furan-2-thione (3.105), the h ighest molecular 1 03 weight compound of the proposed l ibrary, was also synthesised by the oxygen-sulfur exchange of dihydro-5-octyl-(3H)-furanone ( 1 .52) (Table 3 .6). Both compounds were characterised by NMR, IR and MS and they were in agreement with l i terature values. I 1 6 Significant spectroscopic evidence confirmed the conversion of the C=O functionality in the lactones to the C=S moiety in the thionolactones . Dihydro-5-octyl-2(3H)-furanone has a peak at cS 1 77 .4 ppm in its I 3C NMR spectrum attributable to the lactone C=O, the corresponding thionolactone C=S signal appears at cS 222.6 ppm. Changes are also observed in the IR spectrum, with u(C=O) at 1 769 cm-l for dihydro-5-octyl-2(3H)- furanone and u(C=S) at 1 273 cm- I for dihydro-5-octyl furan-2-thione. The mass spectrum showed that the molecular ion peak had increased by 1 6 amu fol lowing conversion to the thionolactone. Thus good evidence was provided for formation of the thionolactone. Table 3 .6 : The thionation of lactones. .. �s �o Lawesson's reagent toluene Compounds Lawesson's reagent, equiv. Yield (%) n = O 0.5 37 1 56 3.92 2 70 n = 7 2 8 1 3 . 105 The individual syntheses of dihydro-5 -methyl furan-2-thione (3.92) and dihydro-5-octyl furan-2-thione (3. 105) gave us confidence to prepare a l ibrary of thionolactones using this methodology. A l ibrary of five thionolactones was prepared according to Scheme 3 .32 and analysed by GC-MS (Figure 3 . 1 4). Dihydro-5 -methyl furan-2-thione (3.92), 1 04 dihydro-5 -ethyl furan-2-thione (3.99) and dihydro-5-propyl furan-2-thione (3.100) were synthesised individually and assessed for their odour properties (Table 3 .7) . �o 3.71 : n :::: 3 3 . 1 : n = 4 1 .50: n :::: 5 3.27: n == 6 1 .52: n :::: 7 Lawesson's reagent toluene .. �s 3 .1 01 : n = 3 3 . 102 : n = 4 3 .103 : n = 5 3.104: n = 6 3.105: n :::: 7 Scheme 3 .32 : Combinatorial synthesis of a library of five thionolactones. �s n = 3 4 5 6 7 20.0 40.0 Figure 3 . 1 4 : GC trace for library of five thionolactones (n = 3-7). 50.0 min The resulting library of eight thionolactones was checked for purity and composition by GC-MS and then screened by GC-O. The odour descriptions of the individual compounds are given in Table 3 .7 , along with descriptions from the literature where compounds have been reported previously. 1 05 Table 3 .7 : The structure and odour description ofthionolactones. �s n Odour Description Lit. Odour Descriptionl l3 0 3.92 sulfury, sl ightly fruity 1 3.99 green, burnt, mint sweet, sulfury, burnt 2 3 . 100 sweet, metal l ic , burnt 3 3. 1 0 1 metal lic , blue cheese like, chives mushroom homogenate, coconut, sweet, sulfury 4 3.1 02 mushroom, creamy, spring onion 5 3. 1 03 softer note, butter, mushroom fruity, fatty, rancid 6 3.1 04 pasta, mushroom 7 3 . 1 05 sweeter note, peach peel, mushroom slightly fruity, soapy 3.6 Summary Syntheses and analyses of racemic lactones were the focus of th is chapter. Individual racemic lactones were synthesised via a two-step reaction . The Linstead modification of the Knoevenagel condensation to produce a �,y-unsaturated acid followed by lacton isation were applied to the synthesis of dihydro-5 -octyl-2(3H)-furanone . Different reaction conditions were required for the Knoevenagel condensation of di fferent substrates. Once the reaction chemistry was optimised, libraries of dihydro-5 -alkyl-(3H)- furanones (C8-C I 2), and 3-substituted dihydro-5-alkyl-(3H)-furanones were produced. Further, synthesis of a library of eight y-thionolactones was achieved from a library ofy- lactones with Lawesson ' s reagent. The l ibraries were analysed by GC-MS and GC-O. 1 06 3.7 Experimental procedure 3 .7 . 1 General procedure for racemic lactones and thionolactones General methods. As described in Chapter 2, with the fol lowing modifications . IH NMR spectra were recorded at 400 MHz, and I3C NMR spectra at 1 00 MHz on a Broker Avance 400 spectrometer. High resolution mass spectra were recorded on a VG 70SE mass spectrometer, operating at a nominal accelerating voltage of 70 eV. Pyridine, piperidine and diethylamine (Et2NH) were distilled from calcium hydride and stored over KOH pellets . Piperidinium acetate was freshly prepared for each occasion, by combin ing piperidine (99 !lL, 85 mg, 1 mmol) and acetic acid (57 !lL, 60 mg, 1 mmol) in DMSO ( 1 mL). General procedure for the Linstead modification of the Knoevenagel reaction. Method A. A mixture of malonic acid ( 1 0 mmol, 1 .0 equiv.) and aldehyde ( 1 0 mmol, 1 .0 equiv.) in Et3N (2 mL) was heated at reflux for 1 h . The mixture was cooled to RT and transferred to a separatory funnel using Et20 (20 mL). Method B. Piperidinium acetate (as prepared above, 0.02 mmol , 0 .02 equiv .) in DMSO (20 ilL) was treated wth malonic acid (20 mmol, 2 .0 equ iv.) and aldehyde ( 1 0 mmol, 1 .0 equiv.) in DMSO (20 mL), and stirred under nitrogen at RT for 20 min . The mixture was then heated at 1 00 QC for 5 h, cooled to RT and transferred to a separatory funnel u sing Et20 (20 mL). Method C. A mixture of aldehyde ( l 0 mmol, 1 .0 equiv.) and Et2NH (50 mmol , 5 .0 equiv.) in CH2Cb (20 mL) was heated at reflux for 1 h . An a-substituted malonic acid 1 07 (20 mmol , 2 .0 equiv.) was added into the mixture and heated at reflux overnight. The mixture was cooled to RT and transferred to a separatory funnel using EhO (20 mL). Work-up (same for methods A, B and C). The mixture was washed once with i ce cold 1 0% HCI ( l 0 mL) followed by 5% NaOH ( 1 0 mL). The alkaline aqueous extract was washed once with Et20 ( l 0 mL), acidified by the addition of 1 0% HCI ( l 0 mL) and extracted with Et20 ( 1 0 mL). The organic layer was washed with brine (20 roL), dried over MgS04, filtered, and concentrated. The carboxylic acids thus obtained were employed directly in the next step, without purification . (3E)-Non-3-enoic acid (3.29): prepared from heptanal and malonic acid, giving a colourless solid; 0 .87 g (50 %, Method A), 1 .33 g (77 %, Method B). Rr = 0.48 (5 : 1 hex­ EtOAc). IH NMR (400 MHz, CDCh) 0 0 .88 (t, J = 6 .9 Hz, 3H, CH3-), l .24- 1 .3 1 (m, 4H, CH3CH2CHr), l .3 7 (p, J = 7.2 Hz, 2H, -CH2CH2CH=CH-), 2 .04 (q, J = 7 .0 Hz, 2H, - CH2CH=CH-), 3 .06 (dd, J = 6.6, 0.9 Hz, 2H, -CH=CH-CH2COOH), 5 .49-5 .53 (rn, 1 H , - CH=CHCH2COOH), 5 .53-5 .59 (m, I H, -CH=CHCH2COOH), 1 1 .60 (s, I H, -COOH); l 3C NMR ( 1 00 MHz, CDCb) 0 14 .0, 22 .5 , 28 .7 , 3 1 .3 , 32 .4, 37 .8, 1 20 .6, 1 35 . 5 , 1 78 .9 ; HRMS calcd for C9H 1 602 (M +) : 1 56. 1 1 503 ; obsd : 1 56. 1 1 505 . (3E)-Dodec-3-enoic acid (3.31 ): prepared from decanal and malonic acid, gIvmg a colourless solid; l .66 g (84 %, Method A), 1 .64 g (83 %, Method B). Rr = 0.50 (5 : 1 h ex­ EtOAc). IH NMR (400 MHz, CDCb) 0 0.88 (t, J = 6 .8 Hz, 3H, CH3-), l .23 - 1 .3 1 (m, I OH, 5 x -CHz-), 1 .3 8 (p, J = 6 .9 Hz, 2H, -CH2CH2CH=CH-), 2 .03 (q, J = 6 .8 Hz, 2H, - CH2CH=CH-), 3 .06 (d, J = 6 .4 Hz, 2H, -CH=CH-CH2COOH), 5 .47-5 .56 (m, I H, - CH=CHCH2COOH), 5 .56-5 .63 (m, I H, -CH=CHCH2COOH), 1 1 .30 (s, I H, COOH); I 3C 1 08 NMR ( 1 00 MHz, CDCh) 8 1 4 . 1 , 22 .6, 29. 1 , 29. 1 , 29.2, 29.4, 3 1 .9 , 32 .5 , 37 .8 , 1 20 .6, 1 3 5 .5 , 1 80 .7 ; HRMS calcd for C 1 2H2202 (M): 1 98. 1 6 1 98 ; obsd: 1 98 . 1 6 1 70 . (3E,6Z)-Dodeca-3,6-dienoic acid (3 .34): prepared from (4Z)-dec-4-enal and malonic acid, giving a yel low oil; 0 .62 g (32 %, Method A), l . 78 g (9 1 %, Method B). Rr = 0 . 1 7 (5 : 1 hex-EtOAc). l H NMR (400 MHz, CDCh) 8 0.89 (t, J = 7 .0 Hz, 3H, CH3-), 1 .26- 1 .3 1 (m, 4H, 2 x -CH2-), 1 .34 (p, J = 7 .3 Hz, 2H, -ClliCH2CH=CH-), 2 .02 (q, J = 7.0 Hz, 2H, -CH2ClliCH=CH-), 2 .79 (app . t, J = 6.7 Hz, 2H, -CH=CH-Cfu-CH=CH-), 3 .08 (d, J = 5 .0 Hz, 2H, -CH=CH-CfuCOOH), 5 . 35 -5 .46 (m, 2H, cis-CH=CH-), 5 . 54-5 .58 (m, 2H, trans-CH=CH-), 1 1 .28 (s, 1 H, COOH); l 3C NMR ( l OO MHz, CDCb) 8 1 4 .0, 22 .5 , 27 . 1 , 29 .2 , 30 .2 , 3 1 .4 , 37 .8 , 1 2 1 .2 , 1 26.4, 1 3 1 .3 , 1 33 .5 , 1 78 . 8 ; HRMS calcd for C \zH1oOz (M+): 1 96. 1 4633 ; obsd: 1 96. 1 4633 . (3E)-Dodeca-3,1 1 -dienoic acid (335): prepared from dec-9-enal and malonic acid, giving a yel low oil ; 0.37 g ( 1 9 %, Method A), 1 . 1 3 g (58 %, Method B). Rj= 0.25 (5 : 1 hex-EtOAc). IH NMR (400 MHz, CDCb) 8 1 .27- 1 .37 (m, 8H, 4 x -CHd, 2 .04 (q, J = 6.7 Hz, 4H, =CHCfu(CHz)4CfuCH=), 3 .06 (dd, J = 6.6 Hz, 0 .8 Hz 2H, -CH=CH­ CHzCOOH), 4 .94 (dq, J = 1 7 . 1 , 2 .0 Hz, 1 H, trans-Cfu=CH-), 4 .94 (m, 1 H, cis-Cfu=CH- ), 5 .45 -5 . 5 5 (m, 1 H, -CH2CH=CHCH2COOH), 5 .55-5 .63 (m, I H, CH2CH=CHCH2COOH), 5 . 8 1 (ddt, J = 1 7 .0, 1 0 .3, 6 .7 Hz, 1 H, CH2=CH-), 1 1 . 1 7 (s, 1 H, COOH); l 3C NMR ( 1 00 MHz, CDCh) 8 28 .8 , 28.9, 28 .9, 29.0, 32 .4, 3 3 .7 , 3 7 .8, 1 1 4.2, 1 20.7, 1 3 5 .4 , 1 39 . 1 , 1 78 .9; HRMS calcd for C l2H2002 (M+): 1 96 . 1 4633; obsd: 1 96 . 1 4624 . 1 09 COOH + < COOH o I N� c< 7 5 3 COOH � 6 4 2 o 7-Phthalimido-3-heptenoic acid (3 .36): prepared from 5-phthalimido pentanal and malonic acid, giving a colourless solid; 1 . 1 3 g (68 %, Method B). Rf = 0.25 (5 : 1 hex- EtOAc). ' H NMR (400 MHz, MeOH) 8 1 .72 (p, J = 7.3 Hz, 2H , H-6), 2 . 1 6 (q, J = 7 .5 Hz, 2H, H-5) , 3 .03 (d , J = 5 .2 Hz, 2H, H-2 ), 3 .35 (t, J = 7 .0 Hz, 2H, H-7), 5 .6 1 -5 .63 (m, 2H, H-3 and H-4), 7 .42-7.95 (m, 4H, Ph), 1 1 . 1 7 (s, I H, COOH); l 3C NMR ( l OO MHz, MeOH) 8 29 .4, 30.9, 38 .7 , 40.4 , 1 24.0, 1 28 .7 , 1 30 .5 , 1 30 .8 , 1 3 1 .2 , 1 33 .0, 1 34.6, 1 39 .6 , 1 69 .5 , 1 72 .8 ; HRMS calcd. for C , sH , sN04 (M+) : 273 . l 00 1 1 ; obsd : 273 . 1 0025 . Diethyl (E)-hept-l -enylmalonate (3.60): prepared from heptanal and diethyl malonate, giving a colourless oil ; 640 mg (25 %, Method C). Rf= 0 .56 (3 : 1 hex-EtOAc). 'H NMR (400 MHz, CDCh) 8 0.80 (t, J = 6.8 Hz, 3H, CH3-), 1 . 1 5- 1 .45 (m, 1 2H, 3 x -CH2-, 2 x - OCH2CH3), 2 .2 5 (q, J = 7 .6 Hz, 2H, -CfuCH=CH-), 4 .09-4 . 1 8 (m, l H, -CH(COOEt)2), 4 .23 (q, J = 7 . 1 Hz, 4H, 2 X -OCH2CH3), 5 .58-5 .62 (m, 2H, -CH=CH-); l 3C NMR ( l OO MHz, CDCh) 8 1 4 . 1 , 22 .5 , 2 8 .2, 28 .9 , 29.7, 3 1 .5 , 6 1 . 1 , 6 1 .2 , 1 28.6, 1 50 .0 , 1 65 .6 . (3E)-Non-3-en-oic acid (3.29): Diethyl ester 3.60 ( 1 28 mg, 0.5 mol) was added into a solution of potassium hydroxide ( 1 28 mg) in water ( 1 0 mL). The mixture was heated under reflux for 3 h until hydrolysis was complete. The reaction mixture was diluted with water (20 mL) and then the ethanol produced in the reaction, along with water (total volume 20 mL) was removed by disti llation. Amberlyst 1 5 ( \ 28 mg) was added into the cooled residue and the mixture was heated under reflux for 2 h. The reaction mixture was extracted with Et20 (3 x 20 mL), washed with brine (20 mL), dried over MgS04 and 1 1 0 concentrated to give a colourless solid, 3.29 (80 mg, 92 %). Physical data was in agreement with that described in detai l above. (3E)-2-Methyl-non-3-enoic acid (3.47): prepared from heptanal and methyl malonic acid, giving a colourless oil ; 1 . 1 2 g (66 %, Method C). R( = 0 .4 1 (3 : 1 hex-EtOAc) . 'H NMR (400 MHz, CD Cb) 8 0 .89 (t, J = 6.8 Hz, 3H, CH3-) , 1 .2 8- 1 .36 (m, 6H, 3 x -CH2-), 1 .44 (p, J = 7.2 Hz, 2H, -ClliCH2CH=CH-), 1 .84 (d, J = 1 .0 Hz, 3H, CH3CHCOOH), 2 .20 (p, J = 7.0 Hz, 1 H, -CHCOOH), 6 .90-6 .94 (m, 2H, -CH=CH-), 1 1 .30 (s, I H, - COOH); l 3C NMR ( l OO MHz, CDCh) 8 1 1 .9 , 14 .0, 22 .6, 28 .4, 28 .9 , 29.0, 3 1 .6, 1 26 .9, 1 45 . 5 , 1 73 . 8 ; HRMS calcd. for C I OH ' 901 (MH +) : 1 7 1 . 1 3850; obsd: 1 7 1 . 1 3863 . (3E)-2-Ethyl-non-3-enoic acid (3.58): prepared from heptanal and ethyl malonic acid, giving a colourless oi l ; 1 . 1 2 g (6 1 %, Method C). Rr = 0 .48 (3 : 1 hex-EtOAc). 'H NMR (400 MHz, CDCh) 8 0 .75 - 0.85 (m, 6H, 2 x CH3-), 1 . 1 5-1 .76 (m, 8H, 4 x -CH2-), 1 .97 (q, J = 7.2 Hz, 2H, -CH1CH=CH-), 2 . 80 (q, J = 7 .7 Hz, I H, -CH(CH2CH3)COOH), 5 .30- 5 .55 (m, 2H, -CH=CH-), 1 0 .09 (s, I H, -COOH); I 3C NMR ( l OO MHz, CDCb) 8 1 1 .6, 1 4 .0 , 22 .5 , 25 .6, 28 .8 , 3 1 .3 , 32 .4, 50.8 , 1 26.7, 1 34.3, 1 8 1 . 1 ; HRMS calcd. for C l lH2 10z (MH+) : 1 84 . 1 4633 ; obsd: 1 84 . 14582 . eOOH Amberlyst-1 5 MeOH 1 2 10 8 5 1 1 9 7 6 4 COOMe 2 Methyl (3E,6Z)-Dodeca-3,6-dienoate (3.41 ): A solution of (3E,6Z)-dodeca-3 ,6-dienoic acid ( 1 .96 g, 1 0 .0 mmol) in methanol (50 mL) was heated at reflux in the presence of Amberlyst- 1 5 (2 .0 g) for 1 h, then cooled. The Amberlyst- 1 5 was removed by filtration and rinsed with EtzO (50 mL). The filtrate and washings were concentrated and the residue purified by chromatography (3 : 1 hex-EtOAc) to give 3.41 as a light yel low oil 1 1 1 ( l .9 1 g, 9 1 %). Rf= 0.57 (3 : 1 hex-EtOAc); lH NMR (400 MHz, CDCb) 8 0.87 (t, J = 6.9 Hz, 3H, H- 1 2 ), 1 .22- 1 .38 (m, 6H, H-9, H-1 0, H- 1 1 ), 2 .02 (q, J = 6.9 Hz, 2H, H-8), 2 .78 (t, J = 7 . l Hz, 2H , H-5), 3 .04 (d, J = 4 .4 Hz, 2H, H-2), 3 .68 ( s , 3H, OCH3), 5 .33 -5 .58 (m, 4H, H-3 , H-4, H-6, H-7); l 3C NMR ( l OO MHz, CDCb) 8 1 4 . l , 22 .5 , 27 . l , 29 .3 , 30.3 , 3 l .5 , 37 .9, 5 1 .8 , 1 2 1 .8 , 1 26 .6 , 1 3 1 .2 , 1 3 3 .0 , 1 72 .5 ; HRMS calcd. for C 1 3H2302 (MH+): 2 1 l . 1 6980; obsd: 2 1 1 . 1 7002 . General procedure for lactonisation: A suspension of the carboxylic acid ( 1 .0 g) and Amberlyst- 1 5 ( 1 .0 g) in heptane (2 mL) was heated at reflux for 1 h, then cooled. The Amberlyst- 1 5 was removed by filtration and rinsed with EhO (5 mL). The filtrate and washings were concentrated, and the residue purified as specified. Dihydro-5-pentyl-2(3H)-furanone (3.1) : on a scale of 8 .5 mmol, to give the title compound, purified by distil lation , as a pale yellow oil ( 1 . 1 2 g, 84%). Rl= 0.70 (5 : 1 hex­ EtOAc), b.p. 1 35 - 1 45°C/30 mm Hg. lH NMR (400 MHz, CDCh) 8 0.90 (t, J = 6.3 Hz, 3 H, CH3-), 1 .27 - l .89 (m, 8H, 4 x -CH2-), 2 .33 (app. sextet, J = 6.6 Hz, 2H, Hp), 2 .54 (dd, J = 9 .4, 2 .5 Hz, 2H, Ha), 4.49 (p , J = 6 .5 Hz, 1 H, Hy); l 3C NMR ( 1 00 MHz, CDCb) 8 1 3 .7 , 22 .3 , 24. 5 , 27 .6, 28 .6, 3 1 .6, 3 5 .3 , 80 .9 , 1 77 .4 ; HRMS calcd. for C9H 1 702 (MH+) : 1 57 . l 2285 ; obsd: 1 57 . 1 229 1 . Dihydro-5-octyl-2(3H)-furanone (1 .52): on a scale of 8 .3 mmol, to give the title compound, purified by distillation, as a pale yellow oil; 1 .44 g, 88%, R{ = 0.73 (5 : 1 hex­ EtOAc), b.p. 1 85 - 1 95°CI30 mm Hg. lH NMR (400 MHz, CDCh) 8 0.88 (t, J = 6.8 Hz, 3H, CH3-), 1 .27- 1 .88 (m, 1 4H, 7 x -CH2-), 2 .33 (app. sextet, J = 7 . 1 Hz, 2H, Hp), 2 .54 1 1 2 (dd, J = 6 .9 , 2 .5 Hz, 2H, Ha), 4 .49 (p, J=6.9 Hz, I H, Hy) ; I 3C NMR ( 1 00 MHz, CDCh) 8 1 3 .9 , 22.4, 25 . 1 , 27 .9, 28 .8 , 29 . 1 , 29.2, 3 1 .7 , 33 .9 , 3 5 .4, 8 1 .0, 1 77 .4 ; HRMS calcd. for C'2H2302 (MH+) : 1 99 . 1 698 1 ; obsd : 1 99 . 1 702 1 . Dihydro-3-methyl-5-pentyl-(3H)-furanone (3.66): on a scale of 1 .0 mmol, to give the title compound, purifi ed by chromatography (5 : 1 hex-EcOAc) to give a colourless oil ( 1 34 mg, 72 %). Rj = 0.36 (5 : 1 hex-EtOAc). ' H NMR (400 MHz, CDCh) 8 0.90 (t, J = 6.8 Hz, 3H , CH3-), 1 .28 (d, J = 7.3 Hz, 3H, -CH3 at a), 1 .30- 1 .75 (m, 8H, 4 x -CH2-), 2 .0 1 (dt, J = 1 2 .8, 7 . 5 Hz, I H, Hp), 2 . 1 2 (ddd, J = 1 2 .8 , 9 .0, 5 .0 Hz, I H, Hp')' 2 .69 (m, I H, Ha), 4 .5 1 (tt, J = 7 .9, 5 .3 Hz, I H, Hy) ; I 3C NMR ( 1 00 MHz, CDCh) 8 1 3 .8 , 1 5 .8 , 22 .4 , 24.9, 3 1 .4, 3 3 .9 , 3 5 .3 , 3 5 .4, 78 .4, 1 80 .0 ; HRMS calcd. for C IOH ' 902 (MH +) : 1 7 1 . 1 3850; obsd : 1 7 1 . 1 39 1 0 . Dihydro-3-ethyl-5-pentyl-(3H)-furanone (3.67): on a scale of 1 .0 mmol, to give the title compound, purifi ed by chromatography (5 : 1 hex-EcOAc) to give a colourless oil ( 1 03 mg, 56 %). Rj = 0 .62 (5 : 1 hex-EtOAc). ' H NMR (400 MHz, CDCh) 8 0.90 (t, J = 6.8 Hz, 6H, 2 x CH3-), 1 .22- 1 .77 (m, I OH, 5 x -CH2-), 2 .00 (dt, J = 1 2 .9, 7 .5 Hz, I H, Hp), 2 . 1 3 (ddd, J = 1 3 . 1 , 8 . 8 , 4.5 Hz, I H, Hp'), 2 .64-2 .75 (m, I H, Ha), 4 .46-4 .56 (m, I H, Hy); ' 3C NMR ( 1 00 MHz, CDCh) 8 1 4 .0, 1 5 .9, 22 .6, 25 .3 , 29 . 1 , 29.4 , 3 1 .8 , 34 .0, 35 .4, 78 .5 , 1 80. 1 ; HRMS calcd. for Cl lH2 ,02 (MH) : 1 85 . 1 3850; obsd : 1 85 . 1 39 1 0 . General procedure for thionolactone formation: A mixture of the y-lactone ( 1 .0 mmol, 1 .0 equiv.) and Lawesson 's reagent (2.0 mmol, 2.0 equiv. ) in dry toluene ( 1 0 mL) was 1 1 3 heated at reflux for 4 h and, cooled, filtered and concentrated. The thionolactone was isolated by chromatography (5 : 1 hex-EtOAc). Dihydro-5-methyl furan-2-thione (3.92): from dihydro-5-methyl-2(3H)-furanone, to give a yellow oil (8 1 mg, 70 %). Rj= 0.37 (5 : 1 hex-EtOAc). IR (u max) 1 238 (C=S) cm-1 (for dihydro-5 -methyl-(3H)-furanone : IR (u max) 1 764 (C=O) cm-1 ) ; I H NMR (400 MHz, CDCh) 8 1 .5 2 (d, J = 6.3 Hz, 3H, CH3-), 1 .88 - 1 .92 (m, I H, Hp), 2 .4 1 -2 .44 (m, I H, Hp')' 3 .06 (dt, J = 1 8 .8 , 9.5 Hz, I H, Ha), 3 . 1 9 (ddd, J = 1 8 .9, 8 .9 , 4 .0 Hz, I H, Hq')' 5 .04 (sextet, J = 6 .7 Hz, I H, Hy); l 3C NMR ( l OO MHz, CDCh) 8 20.3, 3 1 . 1 , 44.9, 87 . 1 , 222 .5 ; HRMS calcd for CsHsOS (M+) : 1 1 6 .02959; obsd: 1 1 6.02962 . Dihydro-5-ethyl furan-2-thione (3.99): from dihydro-5-ethyl-2(3H)-furanone, to give a yellow oil (72 mg, 55 %). Rl= 0 .38 (5 : 1 hex-EtOAc). lH NMR (400 MHz, CDCh) 8 0.89 (t, J = 6 .9 Hz, 3H, CH3-), 1 .27- l .30 (m, 2 H, -CHr), 1 .87 - 1 .92 (m, I H, Hp), 2 .36-2 .40 (m, 1 H, Hp')' 3 .03 (dt, J = 1 8 .8, 9 .2 Hz, I H, Ha), 3 . 1 7 (ddd, J = 1 8 .9 , 8 .9, 3 .9 Hz, I H, Hq:), 4 .88 (p, J = 7 .2 Hz, I H, Hy); 1 3C NMR ( 1 00 MHz, CDCb) 8 14 .0, 22 .3 , 3 1 . 1 , 44 .9, 87 . 1 , 222 .5 ; HRMS calcd for C6H l OOS (M+) : 1 30 .04524; obsd : 1 30 .04539. Dihydro-5-propyl furan-2-thione (3. 100): from dihydro-5 -propyl-2(3H)-furanone, to give a yellow oil (67 mg, 47 %). Rl= 0.42 (5 : 1 hex-EtOAc) . lH NMR (400 MHz, CDCb) 8 0.88 (t, J = 6.9 Hz, 3H , CHr), 1 .26-1 .34 (m, 4H, -CH2-), 1 . 87-1 .92 (m, I H, Hp) , 2 .38- 2 .42 (m, I H, HW)' 3 .03 (dt, J = 1 8 .8 , 9 .2 Hz , I H, Ha), 3 . 1 7 (ddd, J = 1 8 .9 , 8 .9 , 3 .9 Hz, I H, Hq'), 4.88 (p, J = 7 .2 Hz, I H, Hr); 1 3C NMR ( 1 00 MHz, CDCh) 8 1 4 .0 , 22 .3 , 25 .6, 3 1 .0 , 44 .8 , 87 .0, 222 .5 ; HRMS calcd for C7H 1 20S (M +) : 1 44 .06089; obsd: 1 44.06099. 1 1 4 Dihydro-5-octyl-furan-2-thione (3. 105): from dihydro-5 -octyl-2(3H)-furanone, to give a yellow o i l ( 1 73 mg, 8 1 %). Rf= 0 .58 (5 : 1 hex-EtOAc). IR (u max) 1 273 (C=S) cm-l (for dihydro-5-octyl-2(3H)-furanone: IR (u max) 1 769 (C=O) cm-l) ; lH NMR (400 MHz, CDC13) 0 0 .88 (t, J = 6.9 Hz, 3 H, CH3-), 1 .27- 1 .35 (m, 1 4H, -CHr), 1 .87- 1 .92 (m, I H, Hp), 2 .34-2 .37 (m, I H, Hp')' 3 .0 1 (dt, J= 1 8 .9 , 9 . 1 Hz, 1 H, Ha), 3 . 1 6 (ddd, J = 1 8 .9 , 9 .0, 3 .8 Hz, I H, Hq'), 4.86 (p, J = 7 .3 Hz, 1 H, Hy) ; l 3C NMR ( l OO MHz, CDCh) 0 1 4. 1 , 22 .6, 25 .3 , 29.2, 29 .3 , 29 .4, 29.6, 3 1 .8 , 34.9, 44.9, 9 1 . 1 , 222.6; HRMS calcd for C1 2H220S (M+): 2 14 . 1 3 9 1 4; obsd: 2 1 4 . 1 3925 . 3.7.2 Simultaneous synthesis ofy-Iactones and y-thionolactones Formation of f3,y-unsaturated acids: Malonic acid ( 1 . 1 8 g, 1 0 mmol, 1 0 equiv.) was added to a solution of hexanal ( 1 20 JlL, 1 00 mg, 1 mmol, 1 equiv.), heptanal ( 1 40 JlL, 1 1 5 mg, 1 mmol, 1 equiv.), octanal ( 1 60 /-1L 1 3 1 mg, 1 mmol, 1 equiv.), nonanal ( 1 70 /-1L, 1 4 1 mg, 1 mmol, 1 equiv.), and decanal ( 1 90 /-1L, 1 58 mg, 1 mmol, 1 equiv.) in DMSO (20 mL). Piperidinium acetate ( l OO /-1L of the solution prepared as described above, -0. 1 mmol) was added and the mixture was stirred at room temperature for 20 min, then heated at 1 00 QC for 5 h. The mixture was cooled, diluted with Et20 (20 mL) and washed with ice cold 1 0 % HCI (20 mL). The organic layer was extracted with 5 % NaOH (20 mL), washed with Et20 (20 mL) and then acidified by the addition of 1 0 % HCl (20 mL). The mixture of acids was then extracted with Et20 (2 x 20 mL), dried over MgS04, filtered and concentrated to give a colourless oi l (five p ,y-unsaturated acids : 730 mg, 86 %) that was used without purification in the next step . 1 1 5 Lactonisation: A mixture of the five l3,y-unsaturated carboxylic acids obtained above (6 1 0 mg, �0.7 mmol each of the five p ,y-unsaturated carboxylic acids) was dissolved in heptane ( 1 0 mL) and Amberlyst- 1 5 (6 1 0 mg) was added. The mixture was heated at reflux for 1 h, cooled, filtered (washing the resin well with Et20), and concentrated. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give a mixture of five y­ lactones as a yel low oil (480 mg; 79 %); MS obsd. for CgH1 S02 (M+) : 1 42 ; MS obsd. for C9H 1702 (M+) : 1 56 ; MS obsd. for C IOH 1 902 (M+) : 1 70 ; MS obsd. for C ) ) H2 102 (M+): 1 84 ; MS obsd. for C 1 2H2302 (M+) : 1 98 . Thionation: A mixture of the five y-Iactones (339 mg, -0.36 mmol each, 1 equiv. each) and Lawesson's reagent ( 1 .62 g, 4 mmol, 1 0 equiv.) in dry toluene (60 mL) was heated at reflux for 4 h. After cooling, filtration and evaporation the residue was purified by chromatography (5 : 1 hex-EtOAc) to give a mixture of five y-thionolactones (278 mg, 73 %) as a yellow oi l : Rr = 0.68 (5 : 1 hex-EtOAc); MS obsd. for CsH ) 40S (M) : 1 5 8 ; MS obsd. for C9H ) 60S (M+) : 1 72 ; MS obsd. for C I OH ) sOS (M+): 1 86; MS obsd. for C ) )H200S (M+) : 200 ; MS obsd. for C l 2HnOS (M+) : 2 1 4 . Formation of a-substituted p,y-unsaturated acids : EhNH (25 mmol , 2 5 equiv.) was added to a solution of hexanal ( 1 20 ilL, 1 00 mg, 1 mmoI, 1 equiv.), heptanaI ( 1 40 ilL, 1 1 5 mg, 1 mmoI, 1 equiv.), octanal ( 1 60 ilL 1 3 1 mg, 1 mmoI, 1 equiv.), nonanaI ( 1 70 ilL, 1 4 1 mg, 1 mmol, 1 equiv.), and decanal ( 1 90 ilL, 1 58 mg, 1 mmol, 1 equiv.) in CH2Ch (20 mL). The mixture was heated at reflux for 1 h, and then an a-substituted malonic acid ( l a mmol , 1 0 equiv., methylmalonic acid or ethylmalonic acid) was added to the mixture. The reaction mixture was heated at reflux overnight. The mixture was cooled, diluted with Et20 (20 mL) and washed with ice cold 1 0 % HCI (20 mL). The organic layer was 1 1 6 extracted with 5 % NaOH (20 mL), washed with Et20 (20 mL) and then acidified by the addition of 1 0 % HCI (20 mL). The mixture of acids was then extracted with Et20 (2 x 20 mL), dried over MgS04, filtered and concentrated to give a colourless oil (five a­ methyl �;y-unsaturated acids : 343 mg, 37 %; five a-ethyl �,y-unsaturated acids : 370 mg, 37 %) that was used without purification in the next step. Lactonisation: The mixture of five �,y-unsaturated catboxylic acids obtained above (343 mg, -0.4 mmol each of the five a-methyl � ,y-unsaturated carboxylic acids; 370 mg, -0.4 mmol each of the five a-ethyl �,y-unsaturated carboxylic acids) was dissolved in heptane ( 1 0 mL) and Amberlyst- 1 5 (the same amount by weight as the acid mixture) was added. The mixture was heated at reflux for 1 h, cooled, filtered (washing the resin well with Et20), and concentrated. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give a mixture of five y-lactones as a yellow oil (the five dihydro-3-methyl-5 -alkyl­ (3H)-furanones : 1 3 8 mg, 40 %, MS obsd. for C9H l 602 (M +) : 1 56 ; MS obsd. for C l OH 1 S02 (M+) : 1 70; MS obsd. for C 1 1H2002 (M +) : 1 84 ; MS obsd. for C I 2H2202 (M+) : 1 98 ; C 1 3H2402 (M+) : 2 1 2 ; the five dihydro-3-ethyl-5 -alkyl-(3H)-furanones : 1 30 mg, 3 5 %; MS obsd. for C l OH 1 s02 (M): 1 70 ; MS obsd. for C l IH2002 (M+) : 1 84; MS obsd. for C 1 zH2202 (M+) : 1 98 ; C 1 3HZ402 (M+) : 2 1 2 ; MS obsd. for C I 4H240Z (M+) : 226). 3 .7.3 GC-MS and GC-O analyses GC-MS: As described in Chapter 2 . GC-O: As described in Chapter 2 , with the following modifications . The chromatograph was equipped with an Alltech Econo-CapTM EC-I000TM (30 m x 0.53 mm with l .20 !lm film). 1 1 7 Chapter 4 Chapter 4 : Syntheses of chiral lactones from amino acids 4. 1 I ntroduction Optical ly active y-substituted butano l ides [y-substituted-2(5H)-furanones] and butenol ides have emerged as important compounds in their own right, and as synthons for the synthesis of complex natural products . Examples include antibiotics, pheromones, antifungal I 1 7 and flavour compounds of fruits , dairy products and fermented foods . l I S (4S,5S)-Dihydro-4-methyl-5-butyl-2(3H)-furanone, (4S,5S-4. 1 , widely known as (-)-cis- Quercus lactone)"9 is isolated together with a isomer (4S,5R-4. 1 ) from oak wood and aged spirits or wines stored in casks made from oak wood . 5 -(3E,6-Heptadienyl)dihydro- 2(3H)-furanone (4.2) is a melon-fly pheromone l20 and (-)-methylenolactocin (4.3) 1 2 1 and (-)-protol ichesterinic acid (4.4) 1 22 are antitumour antibiotic lactones (Figure 4 . 1 ) . /'/""(y0 �o �o , , 4S,5S-4.1 4S,5R-4.1 4.4 Figure 4 . 1 : Chiral lactones isolated from nature. 1 1 8 4.2 The structures and stereochemistry of lactones in various cheeses have been analysed. Enantiomeric ratios for some y- and 8-lactones (CS-C I S) are l isted in Table 4. 1 . 1 23 The enantiomeric distributions show an excess of the (R)-enantiomer for most lactones. Further investigation is warranted to determine the s ignificance of each enantiomer toward the overall flavour of cheese. The synthesis of chiral y-Iactones is therefore the aim of this chapter. Table 4. 1 : y- and 8-Lactones (Cs-C 1 s) from various cheeses. 1 23 (R):(S) (%) of Lac tones Cheese y-C IO y-C1 2 8-C8 8-C 1o 8-C 12 Type �o �o �o Bs(yo �o 1 .50 1 .52 3 . 107 1 .5 1 1 .53 Pam1esan N/A N/A R>82 R>82 R>87 Cheddar Trace R>75 1 7 :83 8 1 : 1 9 90 : 1 0 Lill1burger Trace 85 : 1 5 7 8 :22 78 :22 R>87 Ell1ll1ental Trace 85 : 1 5 8 5 : 1 5 76 :24 90 : 1 0 Blue Trace 83 : 1 7 Trace 80 :20 89: 1 1 1 1 9 8-C14 8-C 16 �o �o 10 4.5 4.6 86 : 1 4 Trace 8 5 : 1 5 Trace 83 : 1 7 Trace 83 : 1 7 N/A 8 7 : 1 3 Trace 4.2 Previous syntheses of chiral y-Iactones y-Butyrolactones have been used as intennediates in the production of comp lex natural products. There have been many approaches to the synthesis of chiral y-Iactones. These syntheses can be classified into three categories, as follows : • Resolution of racemic compounds; • Asymmetric synthesis; and • Synthesis where the stereochemistry is derived form the chiral pool. 4.2.1 Resolution The classical approach to generating enantiomerically pure compounds is the resolution of racemic compounds by chemical or enzymatic methods. Several resolutions of lactones have been reported. Pirkle and Adams1 24 reported the isolation of enantiomerially pure cyano alcohols 4.7 . This was achieved by reaction of racemic cyano alcohol (±)-4.7 and (R)-(-)- l -( l ­ naphthyJ)ethyl isocyanate (4.8) 1 25 to afford diastereomeric cyano carbamates (4.9a and 4.9b) . The diastereomers were separated by acidic alumina chromatography and then individually silanol ised to generate the enantiomeric cyano alcohols S-4.7 and R-4.7 . The cyanofunctionality of 4.7 was hydrolysed and lactonisation afforded the enantiomerically pure 8-lactones 4.10 (Scheme 4. 1 ). 1 20 OH �C=N I� ":(CO CU 4.8 4.9a OH �C=N 5-4.7 j 1 . KOH, H20, t,. 2. HCI, PhH, t,. (97 % ) 5-4.1 0 {±)-4.7 1 . (R)-(-)- 1 -( 1 -naphthyl )ethyl isocyanate (4.8) PhH , t,. 2. chromatography (81 % ) <;H3 0 _ : Jl . C=N a-naph"""" N O� H 4.9b I HSiCI3, Et3N PhH, t,. (92 % ) OH �C=N R-4.7 1 . KOH, H20, t,. 2. HCI, PhH , t,. (97 % ) R-4.10 Scheme 4. 1 : Chemical resolution of cyano alcohols followed by hydrolysis and lacton isation. Racemic y-Iactones can be resolved by enzyme-catalysed enantioselective hydrolysis. 1 26 For example, Fougue and Rousseau prepared a racemic lactone 3.91 from ethyl 5-oxo- hexanoate (4.1 1 ), by reduction of the carbonyl group and acid-catalysed lactonisation 1 2 1 (Scheme 4.2). 1 27 Enzymatic resolution of the racemic lactone using pig liver esterase (PLE) and horse l iver esterase (HLE) at room temperature in a buffered solution of sodium dihydrogenphosphate at pH 7.2 is summarised in Table 4.2 . o 0 �O� _1_. _N_aB_H_4_' _E_tO_H_ ..... � -er0 2. TsO H , benzene 4 . 1 1 � 3.91 Scheme 4 .2 : Synthesis of a racemic lactone 3.91 from ethyl 5-oxo-hexanoate (4.1 1 ). Table 4 .2 : Enzymatic resolution of racemic lactones . -er 0 o 1, • • . (-yO ------- U enzyme, pH 7.2 RT (±) -3 .91 S-3.91 O H + � COOH R-4.1 2 conversion time %ee of 8-3.91 %ee of R-4.12 enzyme (%) (h) PLE 63 1 70 40 HLE 60 0.7 95 64 4.2.2 Asymmetric synthesis Many syntheses ofy-lactones have used asymmetric reagents to introduce the chirality. 1 28 For example, Midland and Tramontanol 29 synthesised an optically active y-Iactone via propargylic alcohol 4.15 (88 % ee), prepared by reduction of acetylenic ketone 4.13 in the presence of a chiral reagent, B-3 -pinanyl-9-BBN (4. 14). 1 30 The propargylic alcohol 4.15 was then hydrogenated and lactonised to give (5S)-dihydro-5 -ethyl-2(3H)-furanone (S-3 .97) in 4 1 % overall yield (Scheme 4.3) . 1 22 0 @ fJJ OH �O� . ,,8 THF, RT �O� + � .. (58 %) 4.1 3 0 4.14 4.1 5 0 88 % ee 1 . H2, Pd / C, MeOH /" "(y0 .. 2. H+ (70 %) $-3.97 87 % ee Scheme 4 .3 : Stereoselective reduction of a ketone in the generation of a chiral l actone. In 1 98 1 , Solladie and Matloubi-Moghadam reported the synthesis of (5R)-dihydro-5- octyl-2(3H)-furanone (R-1 .52, Scheme 4 .4) from chiral sulfoxides . 1 3 1 The synthesis of the y-Iactone began with the condensation of the anion of (R)-(+ )-tert-butyl (p- tolylsulfinyl)acetate (4.16) and pelargonaldehyde ( 1 .60). The crude adduct 4.17 was desulfurised with aluminium amalgam. Protection of the hydroxyl group with dihydropyran and reduction of the ester function gave a primary alcohol 4.18 . This was converted to the mesylate, and then the mesylate displaced with potassium cyanide to give 4.19 . Finally, after removal of the protecting group, the nitrile 4 .20 was hydrolysed to the carboxyl group and the product was cyclised to give (5R)-dihydro-5-octyl-2(3H)- furanone (R-1 .52 , Scheme 4.4) 1 23 .. o � H (Bu M g B r 1 .60 OTHP rOH 1 . AIIHg, T H F- 1 0 % H20 1 . MsCI , T H F , Et3N OTH P • H ' , .� ./"'-- C N • 4.18 2. KCN, THF-Me2SO �l? � .. '" 70DC 4.19 2. PPTS , D H P , CH2CG 3. LiAIH4' Et20 O H P PTS, EtO H , 55°C � 1 . NaO H , H20-EtOH _______ H " · _______ ... .. C N l? 2 . p-TsOH, Benzene 4.20 �O R-1 .52 Scheme 4 .4 : Synthesis of(5R)-dihydro-5-octyl-2(3H)-furanone (R-1 .52). In 2000, Martin synthesised both enantiomers of y-lactone 4.26 starting from 2 ,3 -epoxy alcohol 4.22. 1 32 The chirality was introduced during the Sharpless asymmetric epoxidation of allylic alcohol 4.2 1 . This reaction typically leads to compounds with >95 % ee. m The epoxy alcohol 4.22 was transformed into the allyl ic alcohol 4.23 by reductive opening of the corresponding iodide obtained from the epoxy tosylate . The ozono lysis of 4.23 and homologation of the resulting aldehyde with the sodium salt of (trimethylphosphono )acetate yielded the y-hydroxy a,�-unsaturated ester 4.24 that was benzoylated and hydrogenated to yield the saturated diester 4.25. Basic hydrolysis, fol lowed by acidification, afforded the desired lactone 4.26 in 40 % overall yield (Scheme 4 .5) . 1 34 1 24 OH OH 1 . benzoylation .. n-C1 3 H27�C02Me .. 2. NaH, benzene o 1 1 ( MeOhP ............... COOMe (70 % ) OBz n-C 1 3H27�C02Me 4.25 1 . NaH , M e O H 2. W (90 % ) 4.24 2. H2 , Pd/C �/).-(O,?O 12"---1 4.26 ( 9 9 % ) Scheme 4 .5 : Synthesis ofa chiral y-lactone 4.26 starting from allylic alcoho l 4.2 1 . In 2003 , Gosh reported an enantioselective synthesis o f (+)-cryptophytic 5 2 (4.27), a potent antibiotic, antismog agent. 1 35 One of the fragments in this synthesis (shown in red in Scheme 4 .6) was obtained from (3S,5R)-dihydro-3 -methyl-5-phenyl-(3H)-furanone (4.29) . Me OH Me ;. � H V � 4.28 4.27 II Glao 4.29 Scheme 4.6: Retrosynthetic analysis of (+)-cryptophytic (4.27); the key fragment in red. 125 The optically active y-Iactone 4.29 was prepared on a milligram scale uti l ising an enantioselective Corey-Bash-Shibata (CBS) reduction. Enantioselective CBS reduction of 4.30 with 8 mol % oxazaborolidine 4.3 1 , to produce the corresponding (S)-alcohol , was performed as described by Corey. 1 36 The resulting 4-hydroxyester 4.32 was lactonised by heating under reflux with catalytic acetic acid in toluene to give the l actone 4.33. The 3 - methyl group was introduced by alkylation of the lactone enolate (Scheme 4.7). Ph D O OMe __ (R_)_-0_x_a_z_ab_o_r_OI..;;..id_in_e_4_.3_1_(8_m_OI_%_o_)_ BH3 ·THF • 4.30 r '\;H P h L N/ - (- Ph 'B-O I Me 4.31 k Ph y °'):::: O AcOH, toluene, reflux • U LiHMDS, Mel , -78 QC. Ph'-(y0 (84 % ) 4.33 >97 % ee (75 %) , 4.29 Scheme 4 .7 : Synthesis of chiral y-Iactone 4.29 by utilising an enantioselective Corey- Bakshi-Shibata (CBS) reduction. Access to chiral y-butyrolactones via reduction with fermenting bakers ' yeast l 37 has also been reported . The asymmetric reduction of y- and 8-keto acids using bakers ' yeast gave y- and 8-lactones respectively with >98% ee (Scheme 4.8) . 126 o � COOH 4.34 Baker's yeast .. [ O H l -.tt:.. / '" (O � O �COOHJ (37 % ) . U 4.35 S-3.97 >98 % ee o [ 9H 1 H+ / /" 'crO 0 � COOH Baker's yeast � COOH -.. ( 3 5 % ) 4.36 4.37 S-3.1 06 >98 % ee Scheme 4 .8 : Syntheses of chiral y-butyrolactones via reduction with fermenting bakers ' yeast. 4.2.3 The chiral pool as a source of starting materials for lactone synthesis Chiral y-Iactones have also been synthesised using starting materials from the chiral pool, . . h I 1 I I 1 38 'b 1 1 39 1 1 40 I 1 4 1 mcludmg suc mo ecu es as evog ucosenone, n ono actone, g ucose, xy ose, . 'd 142 d 1 . 'd 1 43 tartanc aCl an g utamlC aCl . In 200 1 , Brewer and Rich 1 1 7c synthesised a fully functionalized Phe-Arg hydroxyethylene isostere as a tripeptide derivative for inhibition of the botulinium family of neurotoxins. Enantiomerically pure y-lactone 4.43 was synthesised as a precursor to the tripeptide derivative (Scheme 4 .9). 1 44 Aldol condensation of a-lithioethyl acetate with N-Boc-L- leuc inal (4.38) provided (3R,4S)-N-Boc-statine (48 %) and (3R,4S)-N-Boc-statine (32 %) (4.39) as a pair of diastereomers which could be separated by silica gel chromatography. 1 45 Protection of the hydroxyl and Boc-NH functional groups followed by reduction with diisobutylaluminum hydride, gave alcohol 4.41 . Cyanide displacement 1 27 of the mesylate derivative afforded nitrile 4.42 in 61 % yield. Hydrolysis and lactonisation followed, to give lactone 4.43 (Scheme 4.9). BOCHN� o 4.38 , L�e�OEI � g OEt )-OCH3 BoeN � BocHN ----.... - "-L 2. chromatography OH 0 POCI3 I - 0 (32 %) 0 4.39 ( 1 00 % ) 4.40 D I BAL 1 . MsCI, Et3N � toluene BOCN � 2 . NaCN BOCN� o QC ro OH D MSO ro CN (81 %) 4.41 1 . NaOH-H202 l' EtOH o QC -----C:...........c:............. __ .... BocH N '-. H20 2. HOAc 'Q (57 % ) 4.43 0 o (61 % ) 4.42 OEt Scheme 4 .9 : Synthesis of chiral y-Iactone 4.43, a precursor to a Phe-Arg hydroxyethylene isostere. 4.2.4 Aim of this chapter The literature examples surveyed above give variable stereoselectivity in their generation of the y-carbon stereo genic centre . Many examples are specific and the y-substituent is introduced early in the synthetic sequence . For our purposes, flexibility in the introduction of the y-substituent was desired, with unambiguous control of stereochemistry. Therefore, the synthesis of optically pure y-butyrolactones, in a combinatorial fash ion, presented a s ignificant chal lenge. 1 2 8 4.3 Synthesis of chiral y-Iactones The chemical synthesis of enantiomerically pure y-lactones was inevitably going to require several more steps than the racemic approach presented in Chapter 3. According to the philosophy of Section 4 .2 .3 , L- and D-glutamic acids were viewed as a promising source ofthe required stereogenicity (Scheme 4 . 1 0). + R'-G N H X �0"VO ==? :- 2 U HOOC�COOH X = Br (S-4.44) L·4.46 or I (S-4.45) Scheme 4 . l 0: Retrosynthetic analysis of chiral y-Iactones. For the synthesis of a library of y-lactones, with variation III the y-side chain, we envisioned disconnection of the Co-Cc bond (Scheme 4. 1 0) . The CB-cation synthon is real ised by an alkyl halide . Both the bromide (S-4.44) and the iodide (S-4.45) are known compounds, available by well-established routes from glutamic acid. S ince both enantiomers of glutamic acid are available at reasonable cost, it was possible to access both R- and S-lactones . A number of approaches were considered for the attachment of the s ide chain . These will be discussed in the following sections. 4.3.1 Syntheses of alkyl halides (5S)-Dihydro-5 -(hydroxymethyl)-2(3H)-furanone (S-4.47) was prepared via appropriate modifications of a literature procedure from I-glutamic acid (L-4.46). 1 46 Diazotisation of I-glutamic acid (L-4.46) led to (5S)-2-oxotetrahydrofuran-5-carboxylic acid (S-4.47) (Scheme 4. 1 1 ). 147 1 29 tj H2 H O O C� COOH L-4.46 N a N02 HCI I H 20 (66 % ) .. HOO C'(yo 5-4.47 Scheme 4 . 1 1 : Diazotisation of L-glutamic acid (L-4.46). The carboxylic acid in S-4.47 was reduced with borane-dimethyl sulfide complex to give (5S)-dihydro-5-(hydroxymethyl)-2(3H)-furanone (S-4.48, Scheme 4 . 1 2). 1 48 The lactone acid is very hygroscopic. It was found that azeotropic disti l lation with toluene was required, to remove water, in order to get a good yield of the primary alcohol S-4.48 . The I H NMR spectrum of S-4.48 exhib ited two doublet of doublets at 8 3 .67 and 3 .89 ppm that were assigned to the diastereotopic protons at the 8-carbon . The I 3C NMR spectrum of S-4.4 7 has a resonance at 8 173 .4 that is characteristic of the carbonyl group at CE. This signal is gone in the I 3C NMR spectrum of S-4.48 and there is a new resonance at 8 63 .7 ppm which is consistent with the -CH20H functionality. o )1, 0 0 HO E " a 5-4.47 BH 3· M e2S THF RT (83 %) o HO�O 5-4.48 [a)D20 = +3 1 . 3 l it . 148 [a)D20 = +3 3 . 0 Scheme 4 . 1 2 : Reduction of(5R)-2-oxotetrahydrofuran-5 -carboxyl ic acid (S-4.47). Conversion of the primary alcohol S-4.48 to the corresponding alkyl bromide S-4.44 was approached in two ways , Displacement of the mesylate S-4.49 with l ithium bromide l49 gave (5S)-5-(bromomethyl )-2(3H)-furanone (S-4.44) in 58 % overall yield. The transformation could be achieved in better yield in a single step, using carbon tetrabromide in combination with triphenyJphosphine (Scheme 4. 1 3) . 1 50 1 30 HO � O� O � MS O�O� O Li Br, TH� U (63 %) U 60 °C o Br�O S-4.48 S-4.49 (92 %) I CBr4 , PPh3, MeCN (93 % ) S-4.44 t Scheme 4 . 1 3 : Two routes to (5S)-5-(bromomethyl)-2(3H)-furanone (S-4.44). Formation of the analogous primary alkyl iodide S-4.45 was accomplished with iodine, imidazole and triphenylphosphine. 1 5 1 Alternative conditions, using methyl iodide and carbonyl diimidazole, 1 52 gave a higher yield and a product that could be used without further purification (Scheme 4. 1 4) . I � O� O . 12 , PPh3 U i m idazole S-4.45 (71 %) o H O � v � O ____ M __ el____ _ U carbonyl S-4.48 d i i m idazole (88 %) I�O S-4.45 [a]o20 = +2.2 l it . 1 5 1 [a]o20 = +2.3 Scheme 4 . 1 4 : Two routes to (5S)-5-(iodomethyl)-2(3H)-furanone (S-4.45). 4.3.2 Elaboration of the y-side chain Several approaches were considered for attachment of the side chain . 4.3.2. 1 Via an olefinic handle This retrosynthetic analysis of the y-lactone begins with the o lefin metathesis of the double bond to give compound 4.50 which has been reported previously (Scheme 4. 1 5) . Compound 4.50 can be prepared by the radical condensation of an alkyl halide and allyltributyl tin (4.52) in the presence of a catalytic amount of azobisisobutyronitrile (AIBN, 4.5 1 ) . 149 The alkyl halide was avai lable from the previous section (Scheme 4 . 1 3 and 4 . 1 4 in Section 4 .3 . 1 ). 1 3 1 olefin metathesis radical reaction =======» �SnBu3 Me Me Me�N=NkMe 4.52 NC CN 4.51 + > R� + X�O X = Br (5-4.44) X = I (5-4.45) �o 4.50 Scheme 4 . 1 5 : Retrosynthetic analysis of the y-lactone via an olefinic handle. Precedent for the metathesis step was provided by the work of Reisner's group (Scheme 4 . 1 6). 1 53 .. 4.53 4.55 Scheme 4 . 1 6 : Reisner's extension of the y-side chain via olefin metathesis . In 1 982 , Keckl 54 reported the condensation of bromide 4.56 and allyltributyl tin in the presence of AIBN (4.51 ) to yield corresponding lactone 4.57 (Scheme 4 . l 7a). Keck's method was applied in some other cases (Scheme 4. 1 7b and 4. 1 7c ) 1 55 providing good precedent for our proposal. Keck's method was attempted with lactone S-4.45 and was unsuccessful, recovering starting materials. 1 3 2 (a) � S n Bu 3 + 4.52 (b) � S n B u3 + 4.52 (c) � SnBu3 4.52 BC/"" 'q.0 P h + P h 4.56 0 }-" H :et:> I ' N , H \ f Cbz 4.58 I/I, . ,(y-0 5-4.45 A I B N (4.51 } �" q.0 • to luene Ph 80 QC P h (88 %) 4.57 0 }-" H � AI B N (4.51 } • toluene 80 QC ' N (77 % ) , H \ f Cbz 4.59 A I B N (4.51) �"'(y-0 .. toluene 80 QC (9 1 %) 4.50 Scheme 4 . 1 7 : The condensation ofhal ides and allyltributyl tin in the presence of AIBN. 4.3.2.2 A Stille coupling A retrosynthetic approach to a y-Iactone via a Stille coupling has two possible routes . Route (a) is a Stille coupling between a vinyltrialkyl tin reagent and alkyl haIide 4.44. This approach held little promise s ince the cross-coupling of Csp3-X electrophiles is not desirable due to s low transmetallation. 1 56 o R�O (a) > Stille reaction o + BU3sn� O 5-4.60 o B r�O 5-4.44 ==:::::::» Br�O 5-4.44 Scheme 4 . 1 8 : Retrosynthetic approach to a y-Iactone via a Stille coupling. 1 3 3 Route (b) reverses the roles, with various vinyl halides and the lactone-based tin reagent S-4.60 (Scheme 4 . 1 8). If we could make the lactone-based tin reagent, we could control the extension of the alkyl chain at the y-position with a variety of commercially available vinyl bromides. SciFinder Scholar revealed one previous report of S-4.60. However, inspection of this reference revealed that Kosikowski et al. did not isolate the trialkyl tin species. It is a presumed intermediate in the reduction of S-4.45 (Scheme 4. 1 9) . 1 57 BU3SnH .. A I B N toluene Scheme 4 . 1 9 : Reduction of S-4.45. Whi le allyltributyltinhydride (4.52) is commercially available, the zinc-mediated, one-pot Wurtz-type reductive coupling reaction of alkyl halides (e.g., 4.61 ) with tributyltin chloride (4.62) could be expected to be a useful approach to the synthesis of more complex allylstannanes than 4.52 (Scheme 4.20). 158 � B r + B U3SnCI 4.61 4.62 Scheme 4 .20 : The zinc-mediated one-pot Wurtz-type reductive coupling reaction . The one-pot synthesis Wurtz-type reductive coupling reaction of alkyl bromide S-4.44 was tried and it was not successful (Scheme 4 .2 1 ). o B r�O 5-4.44 BU3SnCI - - - - - - - - -)( - - - - - - - - - - - - .. Zn I H20 ( N H 4CI) I T H F o B U3sn�O 5-4.60 Scheme 4 .2 1 : Wurtz-type reductive coupling reaction of S-4.44. 1 34 4.3.2.3 A Wittig reaction We proposed that y-Iactones could be prepared by a Wittig reaction between various aldehydes and the phosphonium salt which could be formed from the corresponding lactone halide (Scheme 4 .22). R�O � RCHO + e Br (£) 0 Ph 3P �O S-4.63 o Br�O S-4.44 Scheme 4 .22: Retrosynthetic approach to a y-Iactone via a Wittig reaction . In our synthesis, the reaction of bromide S-4.44 with triphenylphosphine yielded the phosphonium salt S-4.63 . A multiplet was observed at D 7.25-7 .73 ppm in the IH NMR spectrum of the phosphonium salt S-4.63 , which was assigned to the I S protons of the triphenylphosphonium group. The phosphonium salt S-4.63 was treated with dimsyl sodium, this choice was made on the basis of bases used previously in Wittig reaction s . 1 59 Heptanal was then introduced to the reaction mixture (Scheme 4.23 ) . The desired compound S-4.64 was not produced. The phosphonium salt S-4.63 was recovered from the aqueous layer. PPh3 Br � O¥ O toluene U 89 % S-4.44 1 . Na H . D M SO 2. heptanal X � S-4.63 S-4.64 Scheme 4 .23 : Wittig reaction of S-4 .44. 1 35 o Some other attempts at using Wittig chemistry in related systems gives insight into the fai lure of this reaction. In 2003 , Grayson synthesised the phosphonium salt 4 .66 from the lactone iodide 4 .65 with triphenylphosphine (Scheme 4.24). 160 Their Wittig reagent thus had two additional - CH2- units relative to compound 4.63 in our synthesis . The phosphonium salt 4 .66 was then deprotonated and exposed to an aldehyde. This led to recovery of the phosphonium salt 4.66 from the aqueous phase fol lowing work-up of the reaction. They speculated that competitive deprotonation a to the lactone carbonyl function, or the intervention of intramolecular condensation reactions (formation of 4 .67), might account for the failure f h W· . . 1 6 1 o t e Ittlg reactlOn . I�O PPh3 • 4.65 H H LOA �o RCHO �O�o Ph3PG) H - - - -� - - -... R U e H �a se 4.67 4.66 wor� Scheme 4 .24: Synthesis and proposed side reactions of phosphonium salt 4 .66. We considered the poss ibility of swapping the roles of the lactone and s ide chain in the Wittig reaction . However, the work of Maurer and Hauser revealed other problems (Scheme 4.25) .8 1 Aldehyde 4.70 was prepared in 50 % overall yield in three steps from 1 3 6 glutamic acid L-4.46. They had difficulty in characterising the lactone-aldehyde 4 .70 because of its instability. The key step in this synthesis was the reaction of aldehyde 4.70 with a Wittig reagent to produce 4.71 . They obtained the final compound 4.71 , as a racemic mixture of 4 : 1 cis-trans isomers. The optical activity was lost via enolisation of the unstable aldehyde 4.70. �H2 HOOC�COOH L-4.46 o 0 H N 02/HCI .. �O� O SOCI2 .. � O� O HO' 'Lr Cl' U H 2/Pd .. [ �O�O J BaS04 H " U S-4.70 (50 % over 3 steps) S-4.47 S-4.69 cis-4.71 trans-4.71 Scheme 4 .2 5 : Maurer and Hauser' s synthetic route to y-Iactones . 4.3.2.4 A cuprate displacement The retrosynthetic approach which ultimately gave us a useful result, was a cup rate displacement1 62 oflactone halide S-4.45 (Scheme 4 .26) X�O X = I (S-4.45) Scheme 4 .26: Retrosynthetic approach to a y-Iactone via a cuprate displacement. Silverstein and Ravid 1 63 reported the cup rate displacement of tosylate S-4.72 to afford y- lactones (R-3.97, R-3 .7 1 , and R-4.43 , Scheme 4.27). The R-isomers were also produced. 1 37 o TSO�O S-4.72 R2CuLi , -70 to -50 DC R = Me, R-3.97 (66 %) R = nBu , R-3.71 (4 1 % ) R = �" R-3.43 ( 1 2 % ) Scheme 4 .27 : Silverstein and Ravid's cuprate displacements of tosylate S-4.72 . The cuprate displacement was widely appl ied and recently Monache elongated the y-side chain of lactone iodide 4.73 using organocopper reagents, generated in s itu from Grignard reagents, according to Table 4 .3 . 164 Table 4 .3 : Cuprate additions reported by Monache. 164 I/" ·(y-O C bz H N' 4.73 Compound 4.74 4.75 4.76 4.77 4.78 RMgBr, C u i , -50DC R Ethyl Dodecyl Isopropyl Cyclohexyl Phenyl Yield 69 % 72 % 6 1 % 75 % < 1 5 % Following this precedent, (5R)-dihydro-5-octyl-2(3H)-furanone (R- 1 .52) was synthesised according to Scheme 4.28. The cuprate was generated in situ from heptylmagnesium bromide. The purification of the copper iod ide was essential to the success of this reaction. l 38 nC7H 1 5MgBr, Cu i , -50°C (71 %) R-1 .S2 [a]o20 = +38.3 lit. 1 65 [a]o20 = +36.8 o Scheme 4 .2 8 : Fonnation of (5R)-dihydro-5-octyl-2(3H)-furanone (R-1 .52). The I H NMR spectrum of lactone iodide R-4.45 had a multiplet at 8 3 .34 ppm which was assigned to protons of the diastereotopic -CH21 group. This signal had gone in the spectrum of compound R-1 .52 and new upfield resonances at 8 0 .88 and 1 .27-1 .88 ppm were consistent with the rep lacement of the electron-withdrawing halide by extension of the alkyl chain . H igh resolution mass spectrometry also supported the formation of compound R-1 .52. The route from L-glutamic acid was thus successfully completed in an overall yield of 33 %. The analogous conversion of D-glutamic acid (D-4.46) to furanone S-1 .52 was successful in an overall yield of 29 %. Each compound was analysed by NMR and its optical rotation measured (Scheme 4 .29). D-4.46 Mel carbonyl d i i m i dazole (87 % ) [a]020 = -2. 1 7 l it . 1 5 1 [a]o20 = -2.3 HO/1, . . (y0 R-4.48 [a]o20 = - 3 1 . 3 3 1 it . 1 48 [a]o20 = -33 . 0 [a]020 = -35 . 7 1 it . 165 [a]o20 = -36 . 8 Scheme 4.29 : Synthesis of (5S)-dihydro-5-octyl-2(3H)-furanone (S-1 .52) from D- glutamic acid (D-4.46). 1 39 4.4 Attempted synthesis of chiral 8-lactones We hoped we might extend thi s synthetic route to the synthesis of o-lactones starting with L-a-aminoadipic acid (L-4.79) which has an additional -CH2- unit relative to glutamic acid. L-a-Aminoadipic acid is commercial ly available, although expensive. D-a- Aminoadipic acid (D-4.79), required for (R)-o-Iactones, is not commercially available. (6S)-( + )-Oxo-tetrahydro-2H-pyran-2-carboxylic acid (S-4.80) was synthesised by diazotisation of L-a-aminoadipic acid. The reduction of the carboxylic acid S-4.80 with borane-dimethyl sulfide complex did not give (6S)-(+)-6-hydroxymethyl-tetrahydro-2H- pyran S-4.81 (Scheme 4.30). This synthetic route needs further investigation if we are to pursue the synthesis of chiral o-lactones. t:;J H2 NaN02/HCI HOOC�COOH L-4.79 Mel . _ - - - - - - - - - .... carbonyl d i i m idazole (59 % ) RMgBr, C u i , -50°C - - - - - - - - - - - - - - - - - - � . �_��.���� .. HO � OyO T H F V S-4.81 Scheme 4.30: Proposed synthetic route for the formation of chiral o-lactones . 1 40 4.5 Summary (5R)-Dihydro-5-octyl-2(3H)-furanone (R-1 .S2) was synthesised from L-glutamic acid (L- 4.46) in four steps in 33 % overall yield (Scheme 4 .3 1 ). The formation of the alkyl hal ide followed literature procedures, with each step being modified to optimise the yield. The last step, to attach the y-side chain, was a big challenge as many reaction conditions were unsuccessful. The final choice was a cup rate displacement. The (S)-enantiomer (S-1 .S2) was also synthesised by following the same sequence from D-glutamic acid (D-4.46) in 29 % overall yield. Both series of compounds were characterised by lH and 1 3C NMR spectroscopy, and optical rotation. �H2 HOOC�COOH L-4.46 � HOOC'Lr0 S-4.47 ° HO\:y° S-4.48 t I�O S-4.45 (overall yield 33 %) o NH2 HOOC � COOH D-4.46 + HOOC/ .(yo R-4.47 + HO/" '(yO R-4.48 • I/ " "(y° R-4.45 t �/"'(r0 S·1 .52 (overall yield 29 %) Scheme 4 .3 1 : Syntheses of both enantiomers of dihydro-5 -octyl-2(3H)-furanone (R-1 .52 and S-1 .52). 1 4 1 4.6 Experimental procedures 4.6.1 Genera l procedure General methods as described earlier with the following exceptions. Optical rotations were measured on a Perkin-Elmer 34 1 polarimeter. Acetonitrile (CH3CN) was disti lled from calcium hydride. Toluene was dried and distilled from sodium. Methanesulfonyl chloride (MsC I) was dried and distilled from phosphorous pentoxide. Copper iodide (CuI) was freshly purified by dissolving CuI in boiling saturated aqueous NaI over a period of 3 0 min. The mixture was cooled, diluted with water, filtered and washed sequentially with water, EtOH, EtOAc, Et20, and hexane and dried in vacuo for 24 h . 4.6.2 Experimental procedures and data t::J H2 HOOC�COOH L-4.46 N a N 02 • HOOC -( O)::::=- o H C I \--.f S-4.47 (5S)-2-0xotetrahydrofuran-5-carboxylic acid (S-4.47): Hydrochloric acid (2N, 1 20 mL) was added to a stirred solution of L-glutamic acid (L-4.46) (29 .4 g, 0 .2 mol , 1 .0 equiv.) in water (200 mL). The resultant clear solution was cooled to 0 °C and a solution of sodium nitrite ( 1 6.6 g, 0.24 mol, 1 .2 equiv.) in water ( 1 20 mL) was added dropwise with stirring over 1 5 min while carefully maintaining the solution at 0 qc. The pale yellow solution was stirred overnight at room temperature. Water was removed by distillation at 45 °C under reduced pressure. The residue was dissolved in ethyl acetate (250 mL) and anhydrous magnesium sulfate (30 g) added. The mixture was stirred for 2 h, filtered and concentrated to yield a clear yellow o il which solidified overnight. The solid was dissolved in ether ( 1 20 mL) and stirred at room temperature for 30 min, cooled 1 42 to -20 °C and stirred for 5 h . A yellow crystall ine solid S-4.47 ( 1 6.7 g, 64 %) was isolated by filtration : m.p. 70-72 °C (Lit. 1 47 m.p . 7 1-73 °C); Rr = 0 .40 ( 1 0 : 1 CH2Ch­ MeOH); [a]o20 = + 1 5 .8° (c 2 .00, EtOH), Lit. 1 47 [a]o20 = + 1 5 .6° (c 2.00, EtOH); 1H NMR (400 MHz, CD30D) 8 2 .33 (m, 2H, H-3), 2 .61 (m, 2H, H-4), 5 .04 (t, J = 6. 1 Hz, I H, H- 5) ; 1 3C NMR ( 1 00 MHz, CD30D) 8 26 .8 , 27.7, 77 .4, 1 73 .4 , 1 79. 1 . (5R)-2-0xotetrahydrofuran-5-carboxylic acid (R-4.47) was prepared in an analogous fash ion on a scale of 0 .2 mol to give D-4.46 as a yellow crystalline solid ( 1 7 .2 g, 66 %): m.p. 7 1 -73 °C (Lit. 1 47, m.p. 7 1-73 °C); Rr = 0.40 ( 1 0 : 1 CH2Ch-MeOH); [a]o20 = - 1 5 .7° (c 2 .00, EtOH), Lit . 147 [a]o20 = - 1 5 .6° (c 2 .00, EtOH). H OOC�O S-4.47 �O� O HO L-J S-4.48 (5S)-Dihydro-5-(hydroxymethyl)-2(3H)-furanone (S-4.48): Borane-dimethyl sulfide complex (6 mL, 2.0 M in THF, 1 .2 mol , l .2 equiv.) was added dropwise, over a period of 30 min, to a stirred solution of S-4.47 (dried by azeotropic distillation with toluene, 1 .30 g , 1 0 mmol, 1 .0 equiv.) in dry THF (50 mL) at room temperature. After stirring for 3 h, the reaction mixture was quenched by the cautious addition of anhydrous methanol (50 mL). The mixture was concentrated to give the crude product which was purified by chromatography ( 1 : 1 hex-EtOAc) to give S-4.48 as a colourless o i l (960 mg, 83 %) : Rf= 0.27 ( 1 : 1 hex-EtOAc); [a]o20 = +3 1 .3° (c 3 .00, EtOH), Lit. 1 48 [a]o20 = +33 .0° (c 3 .00, EtOH); 1H NMR (400 MHz, CDCh) 8 2 . 1 1 -2 .20 (m, I H, H-3 ), 2 .24-2 .34 (m, I H, H-3 ' ), 2 .50-2 .68 (m, 2H, H-4), 3 .67 (dd, J = 1 2 .5 , 4 .7 Hz, 1 H, H-6), 3 .89 (dd, J = 1 2 .5 , 2 .9 Hz, 143 1 H, H-6 '), 4.63 -4 .68 (m, 1H, H-5); l 3C NMR ( 1 00 MHz, CDCh) 0 23 .0, 28 .5 , 63 .7 , 80.9, 1 78 .2 . (5R)-Dihydro-5-(hydroxymethyl)-2(3H)-furanone (R-4.48) was prepared in an analogous fashion on a scale of 1 0 mmol to give R-4.48 as a colourless oil (94 1 mg, 8 1 %). Rj= 0.27 (3 : 1 hex-EtOAc); [a]D20 = -3 1 .3° ( c 3 .00, EtOH), Lit. 1 48 [a]D20 = -3 3 . 0° (c 3 .00, EtOH). (5S)-5-(Bromomethyl)dihydro-2(3H)-furanone (S-4.44): �Oyo H O � MsCI -- S-4.48 Method A (via mesylate). o 0 M SO� S-4.49 LiBr �Oy o Br � S-4.44 (5S)-5-(Mesyloxymethy l)dihydro-2 (3H)-furanone (S-4.49): Methanesul fonyl ch loride ( 1 55 ilL, 229 mg, 2 mmol, 2 equiv.) was added to a stirred solution of S-4.48 ( 1 1 6 mg, 1 mmol, 1 equiv.) and E!3N (55 8 ilL, 405 mg, 4 mmol , 4 equiv.) in CH2Ch ( 1 0 mL) at - 30°C. The mixture was stirred at this temperature for 30 min, quenched with water ( 1 0 mL), and extracted with CH2Ch (3 x 20 mL). The organic layer was washed with brine (20 mL), dried over MgS04, filtered, and concentrated. The residue was purified by chromatography ( 1 :3 hex-EtOAc) to give S-4.49 as a co lourless oil ( 1 22 mg, 63 %). Rj= 0 .40 ( 1 :3 hex-EtOAc). [a]o20 = +32 .5° (c 1 .00, CHCh), Lit. I SO [a]o20 = +33 .3° (c 1 .00, CHCh); IH NMR (400 MHz, CDCh) 8 2 . l 0-2 . 1 5 (m, I H, H-3), 2 . 1 8-2 .25 (m, I H, H -3 '), 2 .49-2 .54 (m, 2H, H-4), 3 .0 1 (s, 3H, CH 3S02-) , 4 .23 (dd, J = 1 2 .4, 4.5 Hz, I H, H-6), 4 .37 144 (dd, J = 1 2 . 5 , 2 . 8 Hz, I H, H-6 '), 4 .70-4 .77 (rn, I H, H-5) ; 1 3C NMR ( l OO MHz, CDCb) 8 1 4 . 1 , 22 .7 , 23 .9, 29.2 , 29.3, 29 .4, 29 .8, 3 1 .9 , 43.8, 209. 1 . (5S)-5-(Bromomethyl)dihydro-2(3H)-furanone (S-4.44): Lithium bromide ( 1 09 mg, 1 .2 mmol , 1 .5 equiv.) was added to a stirred solution of S-4.49 ( 1 22 mg, 0.6 mmol, 1 equiv.) in THF (3 mL). The solution was stirred at 60 °C overnight. The reaction was quenched by the addition of water ( 1 0 mL) and extracted with EtOAc (3 x 20 mL). The organic layer was washed with brine (20 mL), dried over MgS04, filtered and concentrated to give a residue which was purified by chromatography ( 1 :3 hex-EtOAc) to give e S-4.44 as a colourless oil ( 1 04 mg, 92 %). Rr = 0 .40 ( 1 :3 hex-EtOAc); [a]o20 = + 1 .7° (c 2 .70, CHCb), Lit. 1 50 [a]o20 = +2.0° (c 2.70, CHCb) lH NMR (400 MHz, CDCh) 8 2 . 1 5 (m, I H, H-3), 2 .45 (rn, I H, H-3 ') , 2 .62 (rn, 2H, HA), 3 .52-3 .6 1 (m, 2H, H-6), 4 .73-4 .80 (rn, I H, H-5 ) ; 1 3C NMR ( 1 00 MHz, CDCh) 8 26 . 1 , 28 .3 , 34 . l , 77 .8 , 1 76 .2 . �°'y::- O H O 'LJ S-4.48 �°'y::- O Br 'LJ S-4.44 Method B. Triphenylphosphine (525 mg, 2 mmol, 2 equiv.) was added in portions to a stirred solution of S-4.48 ( 1 1 6 mg, 1 mmol, 1 equiv.), K2C03 (690 mg, 5 mmol, 5 equiv.) and CBr4 (662 mg, 2 mmol, 2 equiv.) in CH3CN (5 mL) at ° °C. The mixture was stirred overnight at room temperature and concentrated. The residue was taken up in ether ( 1 0 mL) and filtered. The filtrate was evaporated, and the residue was purified by chromatography ( 1 :3 hex-EtOAc) to give S-4.44 as a yellow oil ( 1 67 mg, 93 %). Data as above. 1 45 �Oyo Br L-J S-4.44 S-4.63 [(5S)-5-(Methyl)dihydro-2(3H)-furyl] triphenylphosphonium bromide (S-4.63): The bromo lactone S-4.44 ( 1 80 mg, 1 mmol, 1 equiv.) was heated at reflux in toluene (5 mL) with PPh3 (288 mg, l . 1 mmol, l . 1 equiv.) for 5 h. The solvent was decanted from the solid product that was then washed with warm toluene (2 x 1 0 mL). The solid was dried to give the crude phosphonium salt S-4.63 as a pale yellow solid (393 mg, 89 %). A sample recrystallised from ethyl acetate had m.p. 1 38 - 142 °C: Rr = 0.40 ( 1 0 : 1 hex- EtOAc); [a]D20 = +3 l .3° (c 1 .00, EtOH); IH NMR (400 MHz, CDCh) 8 2 .05-2 . 1 5 (m, I H, H-4), 2 .37-2 .47 (m, I H, H-4') , 2 .50-2 .70 (m, 2H, H-3), 3 .48-3 .58 (m, 2H, H-6), 4 .69- 4.76 (m, I H, H-5), 7 .25-7 .73 (m, 1 5H, ArH) ; I 3C NMR ( 1 00 MHz, CDCh) 8 26. 1 , 28 .3 , 34 .0 , 77 .8 , 1 28 .4, 1 28 .6, 1 33 .5 , 1 33 .7 , 1 76. l ; HRMS ca1cd. for C23HnBr02P (MH+) : 44 l .06 1 36 ; obsd: 44 1 .07475 . �Oyo HO L-J S-4.48 � or M e l I�O S-4.45 (5S)-5-(lodomethyl)dihydro-2(3H)-furanone (S-4.45): Method A. Triphenylphosphine (524 mg, 2 mmol, 2 equiv.) was added to a stirred solution of S-4.48 ( 1 1 6 mg, 1 mmol , 1 equiv.), iodine (508 mg, 2 mmol , 2 equiv.) and imidazole ( 1 36 mg, 2 mmol, 2 equiv.) in CH3CN ( 1 0 mL) at 0 0c. The mixture was heated at reflux overnight. The reaction was cooled and the mixture was extracted with EhO (3 x 20 mL) . The combined extracts were 146 washed with water (20 mL) and brine (20 mL), dried over MgS04, filtered and concentrated to give the crude product which was purified by chromatography (3 : 1 hex­ EtOAc) to give S-4.45 as a yel low oil ( 1 60 mg, 7 1 %) : Rj= 0 .33 (3 : 1 hex-EtOAc); [0,]020 = +2 . 1 ° (c 2 .4 , CH2Ch), Lit. I S I [0,]020 = +2.3° (c 2 .4, CH2Ch); IH NMR (400 MHz, CDCh) 8 1 .89- 1 .99 (m, I H, H-3), 2 .38-2 .52 (m, I H, H-3 '), 2 .53-2 .65 (m, 2 H, H-4), 3 .28- 3 .38 (m, 2H, H-6), 4 .47-4 .54 (m, I H, H-5 ); DC NMR ( l OO MHz, CDCh) 8 7 .9 , 27.7, 28 .6 , 78 . 1 , 1 76 .0 . Method B. A stirred solution of S-4.48 ( 1 1 6 mg, 1 mmol, 1 equiv .) in dry CH3CN (5 mL) was treated with carbonyl diimidazole (324 mg, 2 mmol, 2 equiv.). After a clear solution was obtained, methyl iodide (0.3 1 mL, 5 mmol, 5 equiv.) was added. The mixture was stirred at room temperature for 30 min followed by heating under reflux for 2 h. The reaction was cooled and water ( 1 0 ml) was added. The mixture was extracted with EtOAc (3 x 20 mL). The combined extractions were washed with saturated aq. Na2S203 (2 x 1 0 mL) and dried over MgS04. The mixture was filtered and concentrated in vacuo to give the crude product that was purified by chromatography (5 : 1 hex-EtOAc) to give R-4.45 as a yellow oil ( 1 99 mg, 88 %). Data as above. (5R)-5-(Iodomethyl)dihydro-2(3H)-furanone (R-4.45) was prepared in an analogous fashion on a scale of 1 mmol to give R-4.45 as a yel low oil ( 1 97 mg, 87 %); Rf = 0 .33 (3 : 1 hex-EtOAc); [0,]020 = -2.2° (c 2 .4, CH2Ch), Lit. 1 5 1 [0,]020 = _2 .3° (c 2 .4, CH2Ch). 147 I�O nC7H 15Mg Br. o Cui S-4.45 R-1 .52 (SR)-Dihydro-S-octyl-2(3H)-furanone (R-l .S2): Heptylmagnesium bromide (3 mL, I M i n Et20, 3 mmol, 3 equiv.) was added over 1 0 min to a suspension of CuI (286 mg, 1 .5 mmol, 1 .5 equiv.) in THF ( 1 0 mL) at -50 °C. The mixture was stirred at -50 °C for 1 h, then iodolactone R-4.4S (226 mg, 1 mmol, 1 equiv.) in THF (5 mL) was added dropwise over 5 min. The mixture was stirred at -50 °C for 1 .5 h. The reaction was quenched by the addition of saturated aq. NH4CI (20 mL), stirred for an additional 10 min and extracted with EtOAc (3 x 20 mL). The extracts were washed with brine (20 mL), filtered, dried over MgS04, and concentrated. The residue was purified by chromatography (3 : 1 hex-EtOAc) to give R-1 .S2 as a yel low oil ( 1 4 1 mg, 7 1 %): Rr = 20 · 1 35 ] 20 36 80 0.59 (3 : 1 hex-EtOAc); [a]o = +38 .3° (c 0 .30, MeOH), LIt. [a 0 = + . (c 0.30, MeOH); IH NMR (400 MHz, CDCb) 8 0.88 (t, J = 6.8 Hz, 3H, CH3-), 1 .27- 1 .88 (m, 1 4H, -CH2-), 2 .33 (app . sextet, J = 7 . 1 Hz, 2H, H-4), 2 .54 (dd, J = 6.9, 2 .5 Hz, 2 H, H-3 ), 4.49 (p, J= 6.9 Hz, I H, H-5); I 3C NMR ( 1 00 MHz, CDCb) 8 1 3 .9, 22.4, 25 . 1 , 27 .9, 28 .8 , 29 . 1 , 29 .2 , 3 1 .7 , 33 .9 , 35 .4, 8 1 .0, 1 77 .4; HRMS calcd. for C 1 2H2302 (MH +) : 1 9 9 . 1 698 1 ; obsd: 1 99 . 1 702 1 . (SS)-Dihydro-S-octyl-2(3H)-furanone (S-1 .S2) was prepared in an analogous fashion on a scale of 1 mmol to give S-1 .S2 as a yellow oil ( 1 23 mg, 62 %) : Rr = 0.59 (3 : 1 hex- EtOAc). [a]o20 = -3 5 .7° (c 0.30, MeOH), Lit. 1 65 [a]o20 = -36.8° (c 0 .30, MeOH). 1 48 L-4.79 S-4.80 (6S)-6-0xo-tetrahydro-.2H-pyran-2-carboxylic acid (S-4.80) : Hydrochloric acid (2N, 2 .5 mL) was added to a stirred solution of L-a-aminoadipic acid (S-4.79, 1 6 1 mg, 1 mmol, 1 .0 equiv.) in water ( 1 6 mL). The resultant clear solution was cooled to 0 DC and a solution of sodium nitrite ( 1 1 0 mg, 1 .6 mmol, 1 .6 equiv.) in water ( 1 2 mL) was added dropwise with stirring over 1 5 min while carefully maintaining the solution at 0 DC. The pale yellow solution was stirred overnight at room temperature. Water was removed by dist i llation at 45 DC under reduced pressure. The residue was dissolved in ethyl acetate (20 mL) and anhydrous magnesium sulfate (3 g) added. The mixture was stirred for 2 h , filtered and concentrated to yield a clear yellow oil which solidified overnight. The solid was dissolved in ether (20 mL) and stirred at room temperature for 30 min, cooled to -20 DC and stirred for 5 h. A yellow crystalline solid S-4.80 (85 mg, 59 %) was isolated by filtration: m.p. 1 02-1 04 DC; Rr= 0 .2 1 (9 : 1 EtOAe-MeOH) ; [a]o20 = +12 .3D (c 1 .00, H20); IH NMR (400 MHz, CD30D) 8 0 .94-1 . 1 0 (m, 2H, H-4), 1 . 1 8- 1 .32 (m, 2H, H-5), 1 .45- 1 .76 (m, 2H, H-3), 2 .88 (t, J = 7 . 1 Hz, 1 H, H-6), 7.68 (s, 1 H, -COOH) ; 1 3C NMR ( l OO MHz, CD30D) 8 2 1 .6, 34.2, 34 .4 , 7 1 .3 , 1 75 .6, 1 76 .2 ; HRMS caled. for C6H904 (MH+) : 1 45 .05008 ; obsd: 1 45 .04959 . 149 Chapter 5 Chapter 5 : Asymmetric synthesis of chiral y-Iactones utilizing the Sharpless asymmetric dihydroxylation reaction 5.1 Introduction 5.1.1 Background A number of asymmetric reactions have emerged in order to tackle the syntheses of b iologically active chiral compounds. Some applications to the synthesis of y-Iactones were summarised in Chapter 4. The osmium-catalysed dihydroxyiation reaction has been investigated and two different mechanisms have been suggested. Boseken and Criegee l66 proposed a concerted [3+2] pathway (Scheme 5 . 1 , path A) while Sharpless' group suggested a stepwise reaction (Scheme 5 . 1 , path B). 1 67 Path B is initiated by a [2+2]-like addition of the olefin across an Os=O bond followed by rearrangement of the resulting osmaoxetane intermediate. OS04 + L + = Scheme 5 . 1 : Two proposed mechanisms for the osmium-catalysed dihydroxylation. 1 5 0 The mild and stereoselective Sharp less' asymmetric dihydroxylation (AD) has been widely adopted in organic synthesis and a typical example is in Scheme 5 .2 . 1 68 5.1 AD-mix-� methyl sulfonamide OH H20ltB u OH .. /'0... /'0... .-l /'0... ,/ (97 %) ,/ ........., Y ........., ........., O H 5.2 97 % ee Scheme 5 .2 : Sharpless' asymmetric dihydroxylation of olefins . The AD-mix formulation was optimised by triaI l ing a number of reaction conditions. The key elements are the osmium salt and trace amounts of the phthalazine class of l igands (5.3a, 5 .3(3) (Figure 5 . 1 ) . M eO Et N = N Et� 01f/; 0&" . H I � /; :Y" I '-":: � � N OMe OMe ( D HOh-P HAL (5 .3a) Ligand used i n AD-m ix-u ( D H O D )rPHAL (5.3� ) Ligand used in AD-mix-� Figure 5 . 1 : (DHQ)2PHAL (5.3a) and (DHQD)2PHAL (5.3(3). The osmium-ligand ensemble can be regarded as a chiral oxygen-donating group. The prochiral double bond can be attacked from either of two faces, with discrimination being provided by the bulky asymmetric l igands. The dihydroquinine derivative (DHQ)2PHAL 1 5 1 (5.3a), attacks from the bottom face (i. e., the a-face), and the dihydroquinidine complex containing (DHQD)2PHAL (5.3P), attacks an olefinic group from the top face (i.e., the p- face) (Scheme 5 .3) . I q, ,,0 ,Os, O· J ·O L Bottom (a)- attack L = l igand AD-mix-p AD-mix-a Rs = small al kyl g roup RM = med i u m alkyl group RL = large a l kyl group Scheme 5 .3 : Enantiofacial selectivity of AD reaction. The AD-mix formulation is prepared with K3Fe(CN)6 (3 .0 equiv.), K2C03 (3 .0 equ iv.), K20s02(OH)4 (0.2 mol %) and (DHQ)2PHAL ( 1 mol %) for AD-mix-a or (DHQD)2PHAL ( 1 mol %) is for AD-mix-� . These reagents are ground together to give a fine powder and kept dry. These two AD-mix formulations are now available from Aldrich. 1 52 5.1 .2 Previous applications of Sharpless' asymmetric dihydroxylation to the synthesis of y-Iactones In 1 992 , Sharpless' group reported the asymmetric dihydroxylation of � ,y- and y,o- unsaturated esters gave 4-hydroxyl y-Iactones and muricatacins, respectively (Scheme 5 .4). 1 69 '- \ AD-mix-a /\ '1- ( O� O methyl sulfona mide 5 � H20/tBuO H . . Ho" (85 %) � COOMe 5 AD-mix-� � m _ e _ t _ hY _ I _ SU _ lf _ on _ a _ m _ id _ e _ 5 0 0 H20/tB uOH • (84 %) HO 4S,SS-S.S >99 % ee S.4 4R,SR-S.S 99 % ee OH AD-m ix-a AD-mix-� . OH :: 0 methyl sulfonamide methyl su lfonamlde � W\J" 0 H20/B uOH �COOMe H20/tBuOH 1 1 0 1 1 . � • (82 %) 11 (84 %) SS,6S-S.7 (-) m uricatacin 95 % ee S.6 SR,6R-S.7 (+) mu ricatacin 96 % ee Scheme 5 .4 : The asymmetric dihydroxylation of� ,y- and y,o-unsaturated esters . Sato 's group reported the synthesis of a chiral bui lding block, (4R,5R)-4 ,5 -dihydro-4- hydroxy-5-trimethylsilyl-2(3H)-furanone (4R,5R-5.9), with AD-mix-� (Scheme 5 .5) . 1 70 M e 3 S i �CO OEt S.8 AD-mix-13 meth yl su lfo n a mide H20/S uO H (75 %) .. Me3Si VO H O 4R,SR-S.9 99 % ee Scheme 5 .5 : Synthesis ofa chiral building block 4R,5R-5.9 . For the purposes of the current investigation, the most relevant example of the synthesis of enantiopure lactones was reported by Harcken and Bruckner. 17 1 Their synthesis began with a trans-�,y-unsaturated acid (1 .3 1 ) that was transformed into its methyl ester (5.10). Under the conditions of the dihydroxylation, a �-hydroxy-y-lactone 4R,5R-5.11 was 1 53 obtained. This was dehydrated with mesyl chloride and triethylamine. The saturated y- lactone R-152 was obtained by palladium-catalysed hydrogenation of the double bond (Scheme 5 .6). � COOH 7 1 .31 MeO H , CSA C H C I3 (90 %) • AD-mix-j3 methyl su lfona mide � COOM e H20/tS u O H 7 :?" S.10 (81 %) yo MsCI, Et3N �O H2, Pd/C �O C H2CI2 EtOAc (91 %) (94 %) H O 4R,SR-S.1 1 R-S.12 R-1 .S2 92 % ee Scheme 5 .6 : Synthesis of enantiopure lactone R-1.S2. Harcken and Brucknerl47 reported the elaboration of intermediates in Scheme 5 .4 to give chiral disubstituted 4S,SR-4.1 . Lithium dimethyl cuprate added to the butenolide (R-S.1 3) to give the desired trans-configured 1 ,4-addition product 4S,SR-4.1 and none of its cis isomer (Scheme 5 .7). �O (77 %) �O R-S.1 3 4S,SR-4.1 Scheme 5 .7 : Cuprate addition of the butenolide R-S.13 . Furthermore, their report described the synthesis of trisubstituted y-Iactones . Compound S.14 was accessed by analogy to reactions depicted in Scheme 5 .4. The a-substituted lactone S.1S was synthesised by the a-alkylation of dilithiated �-hydroxy-y-Iactone S.14. The a-substituent was oriented trans to the �-OH group (Scheme 5 .8). 1 54 po HO 5 . 1 4 78 % ee LOA, Bul THF ( 5 3 % ) '( O yo HOr.t'--h 5.1 5 3 Scheme 5 .8 : The a-alkylation of p-hydroxy-y-Iactone 5.14 . 5.2 Strategy for the chemical synthesis of variously substituted chiral lactones This chapter is focussed on the util ization of Sharpless ' asymmetric dihydroxylation chemistry. The introduction of substituents by Harcken and Bruckner gave us reason to believe that this could be done combinatorially. Our approach is il lustrated retrosynthetically in Scheme 5 .9 . Scheme 5 .9 : Retrosynthetic analysis o f substituted lactones. The a-substituted y-Iactone I can be accessed via alkylation of the enolate of lactone 11 . An a,p ,y-tri-substituted y-lactone I I I might be synthesised via alkylation of the enolate of lactone IV that already contains a p-substituent. I t is difficult to predict the stereochemical outcome of such a reaction. A p-substituted y-Iactone IV can be obtained by conjugate addition of a dialkylcuprate to butenolide V, an intermediate in the enantioselective synthesis of y-Iactones (Scheme 5 .9 ). The saturated lactone 11 can be formed by catalytic 1 55 hydrogenation of butenolide V, which is obtained by dehydration of the lac tone VI. The enantioenriched l3-hydroxy-y-substituted lactone VI is obtained by cyclisation of a l3,y- unsaturated methyl ester employing Sharpless' asymmetric dihydroxylation. This takes advantage of the l3,y-unsaturated acids avai lable from our racemic synthesis via the Linstead modification of the Knoevenagel reaction in Chapter 3 . 5.3 Synthesis o f stereoisomerically pure y-Iactones 5.3 .1 Chiral dihydro-5-octyl-2(3H)-furanone Before attempting to generate a library of optically pure y-Iactones in a combinatorial way, we prepared (5S)-dihydro-5-octyl-2(3H)-furanone (as synthesised in racemic form in Chapter 3) using Sharpless ' dihydroxylation methodology (Scheme 5 . l 0). Amberlyst- 1 5 �COOH Methanol , � 7 � 85 % 1 .31 S-5 .12 [a ]020 = + 70.9 AD-m ix-a methyl sulfonamide (, ) 0 U - COOMe H 20fSuOH A I " UO ��� • 7 7 89 % " 5.1 0 H O' H2 , Pd/C EtOAc 1 00 % .. S-1 .52 [a]020 = -39 , 3 4S,5S-5 . 1 1 [a]020 = -42 . 9 Lit. 128b [a]020 = +69.2 Lit. 1 73a [a]020 = -36.8 Scheme 5 . l 0 : Synthesis of (5S)-dihydro-5 -octyl-2(3H)-furanone. l3 ,y-Unsaturated acid 1 .3 1 , available from our racemic synthesis, was esterified. The lH NMR spectrum of ester 5.10 had a singlet at (5 3 .68 ppm that was assigned to protons of the -COOCH3 group. After lactonisation of e ster 5.1 0 in the presence of AD-mix-a, there was no corresponding signal in the spectrum of compound 4S,5S-5.1 1 . There was, 1 56 however, a mUltiplet at 8 4.45-4.53 ppm which was assigned to Hy of the y-Iactone. The hydroxy lactone was converted to butenolide S-5.12 via p-el imination of a mesylate . The I 3C NMR spectrum of hydroxy lactone 4S,5S-5.1 1 had a resonance at 8 69. 1 ppm that was assigned to Cp, which bore the -OH group. This signal had gone in the spectrum of compound S-5.12 and there were new resonances at 8 12 1 .3 and 1 56.4 ppm that were assigned to the carbons of the double bond. Palladium-catalysed hydrogenation afforded the desired product, (5S)-dihydro-5-octyl-2(3H)-furanone (S-1 .52, Scheme 5 . 1 0) . There were no J 3C resonances in the 1 20- 1 60 ppm region of the spectrum, supporting reduction of the double bond. Mass spectrometry supported the formation of compound S-1 .52 by exhibiting a molecular ion at mlz 1 98 in the mass spectrum, consistent with the desired molecular formula C 12H2202 . The R-enantiomer was synthesised by the same sequence of reactions utilizing AD-mix-p in the stereoselective lactonisation step (Scheme 5 . 1 1 ) . ==.�� .... �o 0 �COOMe= 7 5.10 R-1 .52 [a]D20 = +40.0 Lit. 1 73a [a]D20 = +36 .8 Scheme 5 . 1 1 : Synthesis of (5R)-dihydro-5-octyl-2(3H)-furanone. 1 5 7 5.3.2 Generating libraries of chiral y-Iactones A library of five (S)-y-Iactones was synthesised in a combinatorial fashion via the reaction of a mixture of five esters with AD-mix-a and each step was analysed by GC-MS . All compounds in this series were of the 5S-configuration. The 4-hydroxy intennediates were of the 4S-configuration (Figure 5 .2) . n = 3 ; 3.68 n = 4 ; 3.29 n = 5; 1 .69 �COOH n = 6; 3.70 n n = 7; 1 .31 Amberlyst- 1 5 M ethanol, !'1 (81 % ) n = 3; 5.16 n = 4 ; 5.17 n = 5; 5.4 n = 6 ; 5.18 n = 7; 5.10 �COOMe n I AD mix a H20fB u O H (89 % ) n = 3 ; 5.19 n = 4 ; 5.20 n = 5; 5.5 n = 6; 5.21 n = 7; 5. 1 1 n = 3 ; 5-5 . 1 3 n = 4; 5-5.22 n = 5; 5-5.23 n = 6; 5-5.24 n = 7; 5-5 . 1 2 MsCI Et3N CH2C12 (73 %) H2 Pd/C EtOAc 2 . 5 bar ( 1 00 %) n = 3 ; 5-3.71 0 n = 4 ; 5-3.1 )- It - aO n = 5; 5-1.50 n n = 6; 5-3.27 n = 7; 5-1.52 n = 3-7 �.o 20.0 3�.O 30.0 I I \ ! [ I I ' I ull , 30.0 400 J ..J.o 50.0 1 "'.0 600 ruin �� . 600 mm Figure 5 .2 : Synthesis oflibraries ofy-substituted lactones with GC trace at each step. 1 58 A library of R-enantiomers was synthesised by the same sequence of reactions utilising AD-mix-� in the stereoselective lactonisation step. The odour descriptions of each compound were assessed by GC-O and detail s are shown in Tab le 5 . 1 . Table 5 . 1 : Odour descriptions for enantiomerically optical y-Iactones. Structure Odour Description -k'�nao n = 1 ; S-3.71 coconut, faint n = 2 ; S-3.1 milky, white chocolate n = 3 ; S-1 .50 milky, apricot n = 4; S-3.27 buttery, apricot n = 5 ; S-1.52 soapy, apricot �o n = 1 ; R-3.71 coconut, mandarin peel, coconut n = 2 ; R-3.1 apricot with skin on, coconut n = 3 ; R-1 .50 fermented apple, fruity juice, coconut n = 4 ; R-3.27 burnt peach, strawberry jam n = 5 ; R-1 .52 sweet peach 1 59 L· Od D . f 1 72 It. our escnp Ion creamy coconut fatty fatty milky soft fatty fruity, sweet, fatty sweet coconut sweet, spicy, coconut fatty, weak coconut strong fatty fruity peach 5.3.3 3-Substituted (5R)-dihydro-5-pentyl-2(3H)-furanones To investigate the influence of a-substitution in y-Iactones, we chose to focus on compounds derived from (SR)-dihydro-S-pentyl-2(3H)-furanone. The size of lactones has an impact on flavour and compounds b igger than dihydro-S-octyl-2(3H)-furanone produced l ittle interest from the flavour aspect. 1 72 As described in Chapter 4, the (R)- enantiomer is the major isomer in nature and the more potent. We therefore elected to pursue the SR series of dihydro-S-pentyl-2(3H)-furanone . (SR)-Dihydro-S-pentyl-2(3H)-furanone R-3.1 was synthesised by the same sequence of reactions described in section S .3 . 1 starting from p,y-unsaturated acid 3.29. An a-methyl group was then introduced via alkylation of the lactone enolate R-3.1 to give (3S,SR)- dihydro-3 -methyl-S-pentyl-(3H)-furanone 3S,SR-3.66 as the major product (trans :cis= 1 7 :3) in Scheme S . 1 2 . The trans:cis ratio was calculated by integration of the NMR signals . The alkylation of the lactone enolate followed a typical procedure from the l iterature . 1 73 Previous reports indicated that the stereoselectivity of the alkylation led to a trans:cis ratio of9 : l . 1 72c•d Amberlyst- 1 5 (� /'0. COOH Methanol , � '" � '-./ 85 % 3.29 75 % �O R-5.22 1 . LDA, THF -78°C AD m ix P � COOM e H20/tB u O H 4 5.1 7 8 9 % �o H O 4R,5R-5.20 H 2 , Pd/C EtOAc 1 00 % �O R-3.1 2. Mel 82 % �O + �Oyo 3R,5R-3 .6� 3S, 5R-3.66 trans:cis = 1 7 : 3 Scheme S . 1 2 : Synthesis of dihydro-3-methyl-S-pentyl-(3H)-furanone. 1 60 MS analysis supported the incorporation of a methyl group with a weak molecular ion at mlz 1 70 and a base peak at mlz 99. The base peak arises from loss of the side chain as i llustrated in Figure 5 .3 . �+ �Oyo _____ �o'[o _ +(0,[0 � m/z 99 � m/z 1 70 � 3.66 LineN:l RTime:26.750(Scan# 2971) Ma�sPeaIcs: 122 RawMode:Single 26.750(297 1 ) BasePeak 98.95(20713383) BG Modc:None + r .. �. - --- -'- - _ .. o.· _ . ___ __ �·_ - -0 - --- - , 20000000 1 0000000 Figure 5 .3 : Mass spectrum for the mixture of 3S,SR-3.66 and 3R,SR-3.66. mlz The newly incorporated methyl group gave rise to a doublet at 81 .25 ppm in the IH NMR spectrum. Nuclear Overhauser effect spectroscopy (NOESY) provided evidence for the trans relative stereochemistry of the major product 3S,SR-3.66. In the lH NOESY spectrum, there was no correlation between Ha and Hy since they were on opposite faces of the ring. There was a correlation between Hy and the methyl group at Ca, providing evidence that they were on the same face of the lactone ring (Figure 5 .4). 1 6 1 H� - �-- Hw e 0 Ha .�L_ j Hy 0 _�� l� o �" " " " " 'cr" " " " " " " " " " " " " " '" ... ......... .................... ............... 1 •.••.••.....•...•..... u ····�dll··········, : ,� ' I MF 4 ! Jt ........ .... , .................. . ! t .... � . ........... * . ................................... ······································ ··············;··f ··········11 ··· 11 ·························· t '" ··tg·� ··· ··· ··· · · · ·· ·· ········· ·· ······ ····· ·· ············ ····· ··· ·· i· ............................. ., ... ... ............ ,.............. '''!' ......................................... . 3 2 F2 [ppm) Figure 5 .4 : NOESY spectrum oflactone 3S,SR-3.66 (CDCb, 500 MHz). To test the viability of introducing larger substituents, a butyl group was added via alkylation of the lactone enolate of R-3.1 to give a trans:cis mixture of 3S,SR-S.25 and 3R,SR-S.25 (Scheme 5 . l 3). GC-MS analysis supported the incorporation of a butyl group with a base peak at mlz 1 4 1 , arising from loss of the y-side chain. �o �o + 1 . LOA, THF -78°e 2. n-Bul 74 % 3S,5R-5.25 \ 3R,5R-5.25 R-3.1 trans:cis = 7 : 3 Scheme 5 . 1 3 : Introducing a butyl group into the lactone R-3.1 . 1 62 5.3.4 Generating a library of 3-substituted dihydro-5-pentyl-2(3H)-furanones A l ibrary of four 3 -substituted dihydro-5 -pentyl-2(3H)-furanones was synthesised ill a combinatorial fashion via alkylation of the lactone enolate with four alkyl iodides. The library was analysed by GC-MS with the compounds e luting in order of increasing size . The mass spectrum corresponding to each peak in the GC trace indicated a base peak at mlz 99, 1 1 3 , 1 27 and 14 1 , respectively, reflecting an additional -CH2- unit for each successive member in the series (R = Me, Et, n-Pr and n-Bu). The intensities of the five lactones were not equal due to partial losses of the smaller lactones, more volatile during work-up and purification. The odour descriptions of each compound were assessed by GC-O (Figure 5 .5) . 1 . 4.8 eq .LDA, THF -78 QC �o�o 2. 1 eq. each Mel , Ell , n-Prl , n-Bul • U 34 % I 20.0 R-3.1 R = n-Bu R = n-Pr b uttery milky R = E t milky, fru ity R = Me coconut I 30.0 I 40.0 min �o +�o R R R = Me, Et, n-Pr, n-Bu Figure 5 .5 : A library of four 3-substituted (5R)-dihydro-5-pentyl-2(3H)-furanones with the GC trace and the odour description. 1 63 5.3.5 4-Substituted (5R)-dihydro-5-pentyl-2(3H)-furanones A 4-substituent can be introduced onto a butenolide via conjugate addition of an alkyl cup rate reagent, i.e., dialkyl copper lithium and dialkyl copper magnesium bromide. For example, whisky lactone 4S,5R-4.1 was obtained by a stereospecific 1 ,4-addition of lithium dimethylcuprate to butenolide R-5.22 (Scheme 5 . 1 4) . 1 74 �o R-S.22 Me2CuLi Et20 , -78DC (80 %) �o 4S,SR-4.1 Scheme 5 . 1 4 : Cuprate addition of the butenolide R-5.22 . In an analogous fashion, we investigated the conjugate addition of "Bu2CuLi (generated in situ) in our synthesis. The butyl group attacked the butenolide exclusively from the less hindered face, and only the trans-configured 1 ,4-addition product was isolated (Scheme 5 . l 5 ). Mass spectroscopy analysis supported the incorporation ofa butyl group with a base peak at mlz 1 4 1 that arises from loss ofthe y-side chain. �o R-S.22 nBU2CuLi TH F , -78DC (3 1 %) ., Scheme 5 . 1 5 : Formation ofy-Iactone 4S,5R-5.27. Grignard reagents can also be employed as precursors to cuprate reagents. Some examples by Hanessian ' s group are given in Scheme 5 . l 6 that shows excellent yields and diastereoselectivity. 1 75 They looked at both enantiomeric series with a range of Grignard reagents. 1 64 TBDPSO�O 5-S.28 T B D PSO / '· · ·a-° R-S.28 iBuMgBr, Cu i Me2S, T H F , -78 QC (98 % ) CH2= C H M g B r, C u i o TBDPSO�O � S.29 Me2S, T H F , -78 QC // 0 0 • TBDPSO .... . �. (80 % ) � S.30 Scheme 5 . 1 6 : Cuprate addition of bute nol i de. A cuprate addition with methyl magnesium bromide as the precursor gave the trans- con figured 1 ,4-addition product 4S,5R-5.31 (Scheme 5 . 1 7). �o 5-S.22 [a] D20 = -9 3 . 3 MeMgBr, C u i Me2S, T H F - 7 8 QC (71 % ) �O 45,5R-5.31 [a]D20 = +50. 1 Lit. 1 50 [alo20 = +48 . 3 Scheme 5 . 1 7 : Cuprate addition for the formation ofy-lactone 4S,5R-5.3 1 . Analysis by GC-MS supported the incorporation of a methyl group with a base peak at mlz 99. The newly incorporated methyl group gave rise to a doublet at 8 1 .07 ppm in the IH NMR spectrum. Nuclear Overhauser effect spectroscopy (NOESY) supported the trans- orientation of the p-substituent relative to the pre-existing y-substituent. In the IH NOESY spectrum, there are no correlations between HP and Hy s ince trans-configured protons give no space connectivity while there is a correlation between the CH3 group at the p- position and Hy, since they are on the same face of the lactone ring (Figure 5 .6). 1 65 HI3 � CH3 Ha 0 Ha' Hy 0 j H'Y !,-._ - . Pent __ �l ----------.:.-- -.... 5�----..�:i I······················· .......................... (9 .......................... ................................................................. :� ...................................................... . --=�-l.. � = , i�· .... · .... .. · .. .......... ¥ ........... ........................... . .. ........... ...... .. .. . . .......................... .................... . " j < . ...... ............ .......................... .. ... .e... .. o . ........................ ! ... . . ........ . � ... . . D 3 2 F2 [ppm] Figure 5 .6 : NOESY spectrum oflactone (+)-trans-S.31 (CDCh, 500 MHz). E c-a u: A cuprate addition with butyl magnesium bromide was attempted and the trans-configured 1 ,4-addition product 4S,SR-S.27 was synthesised with a better yield than the conjugate addition of l1Bu2CuLi (Scheme 5 . 1 8) . �o 5-5.22 [a]o20 = -93. 3 nBuMgBr, Cui T H F , - 7 8 QC .. (68 % ) �o ,,' J 45,5R-5.27 r [a]o20 = +25.4 Scheme 5 . 1 8 : Cuprate addition for the formation of y-Iactone 4S,SR-S.27. 1 66 5.3.6 Generating a library of 4-substituted (5R)-dihydro-5-pentyl-2(3H)-furanones A library of three 4-substituted (5R)-dihydro-5-pentyl-2(3H)-furanones was synthesised in a combinatorial fashion with three Grignard reagents via a cup rate addition to the butenolide. The mass spectrum corresponding to each peak in the GC trace indicated a base peak at mlz 99, 1 1 3 and 1 4 1 , respectively, reflecting an additional -CH2- unit for each successive member in the series (R = Me, Et and nBu). The odour descriptions of each compound were assessed by GC-O (Figure 5 .7). 2 eq . MeMgBr, EtMgBr, nBuMg Br, �o 3 eq . C u i , 1 . 5 eq . M e2 S , T H F -78 DC. �0"Vo 8 1 % U S-5.22 R = Et milky white chocolate 20.0 R = Me coconut 30.0 R = n-Bu b u ttery 40.0 min R = Me, (+)-trans-5.31 Et, (+)-trans-5.32 nBu, (+)-trans-5.27 Figure 5 . 7 : A l ibrary of three 4-substituted (5R)-dihydro-5 -pentyl-2 (3H)-furanones with the GC trace and the odour description. 1 67 5.3.7 An 3,4,5-tri-alkyl y-Iactone An 3 ,4,5-tri-substituted dihydro-5 -pentyl-2(3H)-furanone 5.33 can be synthesised via alkylation ofthe enolate oflactone 4S,5R-53 1 that already contains a 4-substituent to give (3R,4S,5R)-dihydro-3-methyl-4-methyl-5-pentyl-(3H)-furanone 3R,4S,5R-5.33 as a major product (major:minor = 4: 1 , Scheme 5 . 1 9) . The ratio was calculated by integration of the NMR signals (see experimental section). The odour of compound 533 was assessed by GC-O as described in Scheme 5 . 1 9 . �o 1 . LDA, TH F -78°C 2. Mel 50 % 4S,5R-5.31 • ,/,�OyO " ... � 3R,4S,5R-5.33 maj or (4: 1 ) 3S,4S,5R-5.33 m i n o r spicy, fruity, coconut Scheme 5 . 1 9 : Formation of3 ,4,5 -tri-substituted d ihydro-5 -pentyl-2(3H)-furanone 5.33 . 5.4 Syntheses of optically active y-lactones with unsaturated y-side chains 5.4.1 Introduction (5R, 1Z)-5 -( 1 -Decenyl)dihydro-2(3H)-furanone (5.34) was isolated from a female Japanese beetle (Popilliajaponica); only this isomer attracts males of the species in field bioassays . The unsaturation of compound 5.34 is important for bioactivity since male insects showed no response to a saturated analogue 5.35 in Figure 5 .8 . o o 5.34 o o 5.36 R-3.43 Figure 5 . 8 : Insect pheromones . 1 68 Insect pheromones are often recovered from flower fragrances and they play a role in the fragrance industry, e.g., (5R, 1 Z)-5-( 1 -decenyl)dihydro-2(3H)-furanone (5.34), (5R,2Z)-5- (2-decenyl)dihydro-2(3H)-furanone (5.36) and (5R,2Z)-5-(2-octenyl)dihydro-2(3H)- furanone (R-3.43) in Figure 5 . 8 . 1 76 Pheromones isolated from nature are optical active, 1 77 and early syntheses were only racemic. 1 78 Optically active lactones have been obtained via resolution of an intermediate or final product. 1 79 As discussed in Chapter 4, chemical and . . h h b d 1 80 enzymatIc asymmetnc synt eses aye een reporte . To introduce the unsaturation into an optically active y-lactone is the focus of this section since chirality and unsaturation of a target compound are important in a b ioactive form and its aroma. 5.4.2 The regioselectivity of Sharpless' asymmetric dihydroxylation Regioselectivity in the dihydroxylation of a polyene is detennined by both e lectronic and steric effects. Recently, it was shown that rate constants for the dihydroxylation of isolated double bonds are much larger with trans- l ,2-disubstituted and trisubstituted olefins than with cis- l ,2-disubstituted and terminal alkenes. Electronic factors influence the regioselectivity, and the osmylation of un symmetrical polyenes preferentially occurs at the most electron-rich double bond (Scheme 5 .20). 1 8 1 o �OEt 5.37 AD-mix-p H20fBuOH (93 %) OH 0 �OEt OH 5.38 95 %ee Scheme 5 .20 : Regioselective dihydroxyation. 1 69 Steric effects may play a decisive role in systems with electronically very s imilar double bonds . Generally, the sterically most accessib le site is osmylated preferentially. The cis- double bond of a cis, trans-polyene will not be attacked to an appreciable extent during asymmetric dihydroxylation of the trans-double bond with these l igands (Scheme 5 .2 1 ) . 1 82 �AC 5.39 AD m ix j3 H20fSuOH (82 %) Ho' S::2r 5.40 93 %ee Scheme 5 .2 1 : Steric effects of dihydroxyation. 5.4.3 Synthesis of a y-Iactone with an unsaturated y-substituent We attempted the synthesis of (5R,2Z)-5 -(2-octenyl)dihydro-2(3H)-furanone (R-3 .43) by the same sequence of reactions described in section 5 .3 . l . The � ,y-unsaturated dodecadienoic acid 3.34 (available from Chapter 3 ) was esterified and lactonised with AD-mix-� (Scheme 5 .22). Amberlyst - 1 5 COOH Methanol, ,A, COOMe .. � 9 1 % 3E,7Z-3.41 3.34 AD mix j3 MsCl, Et3N H20fSuOH .. 0 CH2CI2 0 69 % .. 5 1 % 3R,4R-S.41 R-S.42 - - - - - - - - - - - - - - - - - -.. o R-3.43 Scheme 5 .22 : Possible synthetic pathway for the production of compound R-3.43 . 1 70 The 1H NMR spectrum of ester 3E,7Z-3.41 displayed a s inglet at 83 .68 ppm which was assigned to protons of the -COOCH3 group . There was no corresponding signal in the spectrum of compound 3R,4R-S.41 , following lactonisation. There was, however, a triplet at 8 4.46 ppm that was assigned to Hy of the lactone. The I 3C NMR spectrum of hydroxyl lactone 3R,4R-S.41 has a resonance at 8 68.6 ppm that was assigned to Cp, which bore the -OH group. The hydroxy lactone 3R,4R-S.41 was converted to the corresponding mesylate and in situ p-elimination gave butenolide R-S.42 . The CHOH s ignal had gone in the l 3C NMR spectrum of compound R-S.42 and there were new resonances at 8 1 2 1 . 1 and 1 5 5 .8 ppm that were assigned to the carbons o f the double bond in the lactone ring. The final step to complete the synthesis of (R,Z)-5-(2-octenyl)dihydro-2(3H)-furanone (R- 3.43) was the regioselective reduction of the Cu-Cp double bond in the lactone ring (Scheme 5 .22) . The regioselective hydrogenation of the Cu-Cp double bond in the lactone ring was reported previously by Midland1 29 with copper hydride 183 (Scheme 5 .23a). Koseki ' s group more recently reported hydrogenation with tributyltin hydride and trimethylsilyl chloride in the presence of copper iodide and lithium chloride in 82 % yield (Scheme 5 .23b). 1 84 Koseki ' s reaction conditions were attempted in butenolide R-S.42 to give R-3.43 and the starting material was recovered. In the future, this step needs more attention in order to complete this synthesis. 1 7 1 (a) LiCuH2 �o Et20, -78 QC �O • (88 %) R-S.43 R-3.43 (b) BU3Sn H , Me3SiCI �O LiCI, C u i , T H F , -78 QC �O • (82 %) R-S.43 R-3.43 Scheme 5 .2 3 : Hydrogenation of the C:cCp double bond in the lactone ring. 5.5 Summary Syntheses and analyses for l ibraries of chiral lactones were the focus of this chapter. Both enantiomers of y-substituted lactones (CS-C 12 ) were synthesised via a four-step reaction sequence including the Sharp less asymmetric dihydroxylation. Libraries of a-substituted y-Iactones and • -substituted y-lactones were produced and analysed by GC-MS and GC- O. Further, synthesis ofa y-Iactone with an unsaturated y-substituent was attempted. 1 72 5.6 Experimental procedures General method: as described earlier. eO OH .. 1 .31 1 2 1 0 8 6 4 1 1 9 7 5 5.10 2 1 eOOM e (3E)-Methyl 3-dodecanoate (5.10): A solution of 3E-dodec-3-enoic acid ( 1 .0 g, 5 . 0 mmol) in methanol ( 50 mL) was heated at reflux in the presence of Amberlyst- 1 5 ( 1 .0 g) for 1 h . The mixture was cooled, the Amberlyst- 1 5 removed by filtration and rinsed with Et20 (20 mL). The filtrate and washings were concentrated and the residue purified by chromatography (5 : 1 = hex-EtOAc) to give 5.1 0 as a l ight yellow oil (900 mg, 85 %). Rr= 0.83 (3 : 1 hex-EtOAc); IH NMR (400 MHz, CDCh) <3 0.88 (t, J = 6 .8 Hz, 3H, H-1 2), 1 .32 (m, 1 2H, H-6 , H-7, H-8, H-9, H- I 0, H- l 1 ), 2 .02 (q, J = 6.6 Hz, 2H, H-5), 3 . 03 (d, J = 5 .6 1 3 Hz, 2H, H-2), 3 .68 (s, 3H, OCH3), 5 .47-5 .60 (m, 2H, H-3 , H-4); C NMR ( l OO MHz, CDCh) <3 1 4 .0, 22.6, 29 . l , 29 .2 , 29.4, 3 1 .8 , 32 .4, 37 .9 , 5 1 .7, 1 2 1 .3 , 1 34.9, 1 72 .6. 5.1 0 e OOMe AD-Mix�a. 7" 5" 3" 1 " 1 � /'. ?( ° 'y:? O 8" 6" 4 " 2" 4"---1 2 Ho" 3 4S,5S-5.1 1 (4S,5S)-4,5-Dihydro-4-hydroxy-5-octyl-2(3H)-furanone, 4S,5S-5.1 1 : AD-mix-a ( 1 .4 g) and the ester 5.10 (2 1 2 mg, l .0 mmol , 1 .0 equiv.) were added to a mixture of lBuOH (20 mL) and H20 (20 mL) at 0 QC . The mixture was stirred for 48 h, then quenched by the addition of sat 'd aq. Na2S03 (20 mL). The mixture was extracted with CH2Ch (3 x 20 mL), dried over MgS04, filtered and concentrated. The residue was purified by 1 73 chromatography (5 : 1 = hex-EtOAc � 1 :5 = hex-EtOAc) to give 4S,5S-5.1 1 as a colourless oil ( 1 9 1 mg, 89 %). Rr= 0 . 1 4 (5 : 1 hex-EtOAc); [a]D20 = -42 .9° (c 1 .0 , CHCh); I H NMR (400 MHz, CDCh) 8 0.85 (t, J = 6.9 Hz, 3H, H-8"), 1 .2 1 -2 . 0 1 (m, 1 4H, H- l ", H- 2", H-3", H-4", H-5", H-6", H-7" ) , 2 .50 (dd, J = 1 7 .7, 0 .8 Hz, I H, H-3) , 2.77 (dd, J = 1 7 .7 , 5 .5 Hz, I H, H-3 '), 4.30-4 .37 (m, I H, H-4), 4 .44-4.48 (m, I H, H-5), 5 .30 (s, I H, - OH); J 3C NMR ( l OO MHz, CDCb) 8 1 4 . 1 , 22 .6, 2 5 .5 , 28.2, 29.2, 29 .4, 29 .5 , 3 1 .8 , 39.4, 69 . 1 , 84.7, 1 75 .5 . 5.10 � C OOMe AD-Mix-u .. o 4R,5R-5. 1 1 (4R,5R)-4,5-Dihydro-4-hydroxyl-5-octyl-2(3H)-furanone, 4R,5R-S.1 1 was prepared with AD-mix-� ( 1 .4 g, 1 .4 g/ 1 mmol of ester) and the ester S.10 (2 1 2 mg, 1 .0 mmol, 1 .0 eguiv.) as above to give a colourless oil ( 1 9 1 mg, 89 %); [a]D20 = +43 .5° (c 1 .0, CHCb). � /" ' aO 45,55-5.1 1 1" 5" 3" 1 " 1 �/'.?(0"Y.:-0 8" 6" 4" 2" \d 2 4 3 5-5.1 2 (5S)-5-0ctyl-2(SH)-furenone (S-S.12): Triethylamine ( 1 46 /lL, 1 06 mg, 1 .05 mmol, 2 . 1 eguiv.) and methanesulfonyl chloride (43 /lL, 6 3 mg, 0.55 mmol, 1 . 1 eguiv.) were added to a solution of lactone 4S,SS-S.1 1 ( 1 07 mg, 0 .5 mmol, 1 .0 equiv.) in CH2Cb ( l 0 mL) at 0 °C. After 1 h the reaction was quenched by adding sat'd ag . NH4C I solution ( 1 0 mL) and water (20 mL). The mixture was extracted with CH2Cb (3 x 20 mL), dried over MgS04, filtered and concentrated. The residue was purified by chromatography (3 : 1 = hex-EtOAc) to give S-S.12 as a colourless oil (74 mg, 75 %). RI = 0.47 (3 : 1 hex-EtOAc); [a]D20 = 1 74 +70.9° (c 2 .0, dioxane), l it. 1 28b [a]o20 = +69.2° (c 2 . 1 0, dioxane) ; IH NMR (400 MHz, CDCb) 8 0 .88 (t, J = 6.9 Hz, 3H, H-8"), 1 .2 1 - 1 .47 (m, 12H , H-2", H-3", H-4", H-5" , H- 6", H-7" ), 1 .63-1 .82 (m, 2H, H - l "), 5 .03-5 .07 (m, I H, H-5), 6 . 1 0 (dd, J = 5 .7, 2.0 Hz, I H, H-4), 7.48 (dd, J = 5 .7, 1 .5 Hz, I H, H-3); 1 3C NMR ( l OO MHz, CDCb) 8 1 4 .0, 22 .5, 24.8, 29 .0, 29 . 1 , 29 .2, 3 1 .7 , 33 .0, 83 .4, 1 2 1 .3 , 1 56 .4, 1 73 . 1 . o o 4R,SR-S.1 1 R-S.1 2 (5R)-5-0ctyl-2(5H)-furenone (R-5.12) was prepared in an analogous fash ion on a scale of 1 mmol to give R-5.1 1 as a colourless oil (74 mg, 75 %); [a]o20 = -73 .8° (c 2 . 1 0, dioxane), lit . 1 2Sb [a]o20 = -69 .2° (c 2 . 1 0, dioxane) . �/" 'ao 5-5 . 12 7" 5" 3" 1 " 1 � /, , 5( 0'r:: 0 8" 6" 4" 2 " U 2 4 3 5-1 .52 (5S)-Dihydro-5-octyl-2(3H)-furanone (S-1 .52): Pd/C ( 1 8 mg, 1 0 % Pd on charcoal, 0 .02 mmol, 0.05 equiv.) was added to a solution of lactone S-5.12 (74 mg, 0 .4 mmol , 1 .0 equiv.) in EtOAc (2 mL). The mixture was shaken at RT overnight under H2 (2.5 bar). The mixture was filtered through Celite , which was then washed well with EtOAc (20 mL). The filtrate was concentrated to give S-1 .52 as a colourless oil (74 mg, 1 00 %). Rl= 0 .46 (3 : 1 hex-EtOAc); [a]o20 = -39 .3° (c 0 .30, Me OH), l it . 1 73a [a]o20 = -36.8° (c 0.30, MeOH); lH NMR (400 MHz, CDCh) 8 0.88 (t, J = 6.9 Hz, 3H, H-8"), 1 .26-1 .34 (m, 1 2H, H-2", H- 3", H-4", H-5", H-6", H-7"), 1 .60-1 .74 (m, 2H, H- l "), 1 .82- 1 .89 (m, I H, H-4), 2.33 (sext, J = 6.6 Hz, 1 H, H-4 '), 2 .53 (dd, J = 9.7, 6 .9 Hz, 2H, H-3), 4 .45-4.53 (m, I H, H-5); 1 3C 1 75 NMR ( l OO MHz, CDCb) 0 1 4.0, 22.6, 2 5 . 1 , 2 7 . 9 , 2 8 . 8 , 2 9 . 1 , 2 9 .2 , 2 9 . 3 , 3 1 . 7 , 3 5 . 5 , 8 1 . 0 , 1 77 . 3 . o o R-5.12 R-1 .52 (5R)-Dihydro-5-octyl-2(3H)-furanone (R-1 .52) was prepared in an analogous fashion on a scale of 1 mmol to give R-1 .52 as a yellow oil ( 1 1 2 mg, 1 00 %); [a]o20 = +40.0° (c 0 .3 0 , MeOH), lit . 1 73a [a]o20 = +3 6 .8 ° (c 0 . 3 0 , MeOH). � COOH .. �CO OM e n = 3 , 4 , 5 , 6 , 7 Esterification. A mixture of the five �,)'-unsaturated carboxylic acids obtained from Chapter 3 (42 6 mg, �0.5 mmol each) were dissolved in methanol ( I O mL) and Amberlyst- 1 5 (426 mg, the same amount by weight as the acid mixture) was added. The mixture was heated at reflux for 1 h, cooled, filtered (washing the resin well with Et20), and concentrated. The residue was purified by chromatography (5 : 1 hex-EtOAc) to give a mixture of five esters as a light yellow oil (374 mg; 8 1 %); GC-MS: RT 1 4 .2 min (n = 3 , min (n = 5 , C ) ) H2002, M+ obsd mlz 1 84); RT 2 l .6 min (n = 6 , C 1 2H220Z, M+ obsd mlz 1 98 ) ; �CO OMe AD-Mix-!) ... yo HO n = 3 , 4 , 5 , 6 , 7 1 76 Asymmetric lactonisation: AD-mix-� ( 1 .4 g) and the five esters ( 1 87 mg, �0 .2 mmol each of the five esters) were added to a mixture of lBuOH (20 mL) and H20 (20 mL) at 0 QC. The mixture was stirred for 4 8 h, then quenched by the addition of sat 'd aq. Na2S03 (20 mL). The mixture was extracted with CH2Cb (3 x 20 mL), dried over MgS04, filtered and concentrated. The residue was purified by chromatography (5 : 1 = hex-EtOAc � 1 5 : 1 = CH2Cb-MeOH) to give a colourless oil ( 1 65 mg, 8 9 %); GC-MS: R T 42 .4 min (n = 3 , (n = 5 , C I OH 1 803, M+ obsd mlz 1 86); RT 5 3 .6 min (n = 6 , C 1 1H2003, M+ obsd mlz 200); Rr 5 9 .7 min (n = 7 , C I 2H2203, M+ obsd mlz 2 1 4) . �COO Me AO-M ix-a • );J'�Cr° Ho" n = 3 , 4 , 5 , 6 , 7 (4S,5S)-series were prepared in an analogous fashion on a scale of esters ( 1 87 mg, �0 .2 mmol each of the five esters) as a colourless oil ( 1 63 mg, 88 %); GC-MS : RT 42 . 1 min (n RT 5 9 . 5 min (n = 7 , C I 2H2203, M+ obsd mlz 2 1 4) . MsCI C HCI2 �O n = 3 , 4 , 5 ,6 , 7 Elimination: Triethylamine (209 ilL , 1 5 2 mg, 1 . 5 mmol, 1 0 .5 equiv.) and methanesulfonyl chloride (60 ilL, 89 mg, 0 .7 8 mmol, 5 . 5 equiv.) were added to a solution of the five lactones ( 1 3 3 mg, 0 . 1 4 mmol each, 1 equiv.) in CH2Cb ( 1 0 mL) at 0 qc. After 1 77 1 h the reaction was quenched by the addition of sat 'd aq. NH4Cl solution ( 1 0 rnL) and water (20 mL). The mixture was extracted with CH2Cb (3 x 20 mL), dried over MgS04, filtered and concentrated. The residue was purified by chromatography (3 : 1 = hex-EtOAc) to give a colourless oil (50 mg, 42 %); GC-MS : RT 2 6 .2 min (n = 3, CgH1202, M+ obsd mlz obsd mlz 1 68 ); RT 3 2 .9 min (n = 6, C I lH 1 g02, M+ obsd mlz 1 82 ) ; RT 3 5 .0 min (n = 7 , MsCI CHCI2 n = 3 ,4 ,5 , 6 , 7 (5S)-series were prepared in an analogous fashion as a colourless o il (63 mg, 53 %); GC- obsd mlz 1 54); RT 3 0 . 7 min (n = 5, C lOH 1602, M+ obsd mlz 1 68); RT 3 2 .7 min (n = 6, �o _____ � o n = 3 ,4 , 5 , 6 , 7 Hydrogenation: Pd/C ( 1 6 mg, 10 % Pd on charcoal, S mol%) was added to a solution of lactones (50 mg, 0.06 mmol each) in EtOAc (2 mL). The mixture was shaken at RT overnight under H2 (2 .5 bar). The mixture was filtered through Celite, which was then washed well with EtOAc (2 0 mL). The filtrate was concentrated to give a co lourless oil ( 5 0 mg, 1 00 %); GC-MS : RT 2 5 .2 min (n = 3 , CgH 1402, M+ obsd mlz 1 42 ) ; RT 2 7 . 6 min (n 1 78 1 98) . n = 3 ,4 , 5 , 6,7 Five (5R)-series were prepared in an analogous fashion to give lactones as a yellow oil (63 mg, 1 00 %); GC-MS: RT 2 5 .4 min (n = 3, CSH I402 , M + obsd mlz 142); Rr 27 .8 min (n 32 .6 min (n = 6, C I IH2002, M + obsd mlz 1 84); Rr 34 .6 min (n = 7 , C 1 2H2202 , M+ obsd mlz 1 98) . �COOH 3.29 (3E)-Methyl 3-nonenoate (5.17) : A solution of 3-nonenoic acid (3 .46 g, 5 .0 mmol) in methanol (50 mL) was heated at reflux in the presence of Amberlyst- 1 5 (3 .46 g) for 1 h. The mixture was cooled, the Amberlyst- 1 5 was removed by filtration and rinsed with Et20 (20 mL). The filtrate and washings were concentrated and the residue purified by chromatography (5 : 1 = hex-EtOAc) to give 5.17 as a l ight yellow oi l (2 .69 g, 89 %). Rl= 0.78 (3 : 1 hex-EtOAc); IH NMR (400 MHz, CDCh) 8 0 . 8 8 (t, J = 7 . 0 Hz, 3H, H-9), 1 .32 (m, 6H, H-6, H-7, H-8), 2 .02 (q, J = 6.6 Hz, 2 H, H-5), 3 .03 (d, J = 5 . 5 Hz, 2 H, H-2), 3 .68 (s, 3 H, -OCH3), 5 .47-5 .6 1 (m, 2H, H-3 , H-4); DC NMR ( l OO MHz, CDCh) 8 1 3 .9, 22 .4, 28 .7 , 3 1 .2 , 32 .3 , 37 .8 , 5 1 .5 , 1 2 1 .3 , 1 34 .8 , 1 72 .4 . 1 79 �COOMe 5.1 7 AD-Mix-p • 5" 3" 1 " 1 � °'f;; O 4" 2': . _� 2 H O 3 4R,5R-S.20 (4R,SR)-4,S-Dihydro-4-hydroxy-S-pentyl-2(3H)-furanone (4R,SR-S.20): AD-mix-p ( l .4 g) and the ester S.17 ( 1 87 mg, 1 .0 mmol , 1 .0 equ iv.) were added to a mixture of 'BuOH (20 mL) and H20 (20 mL) at 0 dc. The mixture was stirred for 48 h, then quenched by the addition of sat'd aq. Na2S03 (20 mL). The mixture was extracted with CH2Cb (3 x 20 mL), dried over MgS04' filtered and concentrated. The residue was purified by chromatography (5 : 1 = hex-EtOAc � 1 5 : 1 = CH2Ch-MeOH) to give 4R,SR- 5.20 as a colourless oil ( 1 85 mg, 98 %). Rr= 0 .80 ( 1 5 : 1 CH2Ch-MeOH); [a]D20 = +57 .80 (c 1 .0 , CHCI3), lit. 1 1 7a [a]D 20 = +62 .9° (c 1 .0, CHCh); IH NMR (400 MHz, CDC13) 8 0 .85 (t, J = 6 .9 Hz, 3H, H-5 " ), 1 .67- 1 .89 (m, 8H, H- l ", H-2", H-3", HA"), 2 .54 (dd, J = 1 7 .8 , 0 .8 Hz, 1 H, H-3), 2 .80 (dd, J = 1 7 .8 , 5 .5 Hz, I H, H-3 '), 4 .35 -4.42 (m, I H, H-4), 4.44-4,48 (m, I H, H-5 ), 5 .38 (s, I H, -OH),; l 3C NMR ( l OO MHz, CDCh) 8 1 3 .8, 22 .4, 2 5 . 1 , 2 8 . 1 , 3 1 .5 , 39 .4 , 68 .6, 85 .5 , 1 76.9. �o HO 4R,5R-S.20 M sCI 5" 3" 1" 1 �O� O 4" 2" \=.1 2 4 3 R-S.22 (SR)-5-Pentyl-2(5H)-furanone (R-5.22): Triethylamine (293 ilL, 2 1 3 mg, 2 . 1 mmol, 2 . l equiv.) and methanesulfonyl chloride (85 ilL, 1 26 mg, 1 . 1 mmol, 1 . 1 equiv.) were added to a solution of lactone 4R,SR-5.20 ( 1 85 mg, 1 .0 mmol, 1 .0 equiv.) in CH2Ch ( l 0 mL) at o qc. After 1 h the reaction was quenched by the addition of sat'd aq. NH4Cl solution ( 1 0 1 80 mL). The mixture was extracted with CH2Ch (3 x 20 mL), dried over MgS04, filtered and concentrated. The residue was purified by chromatography (3 : 1 = hex-EtOAc) to give R- 5.22 as a colourless oil ( 129 mg, 75 %). Rj= 0.4 1 (3 : 1 hex-EtOAc); [a]o20 = -93.3° (c 1 .0, CHCb); IH NMR (400 MHz, CDCh) 8 0 .89 (t, J = 7 . 1 Hz, 3H, H-5"), 1 .27- 1 .52 (m, 6H, H-2", H-3", H-4"), 1 .63- 1 .82 (m, 2H, H- l "), 5 . 02 -5 .08 (m, I H, H-5), 6 . 1 0 (dd, J = 5 .7, 2 .0 Hz, I H, H-4), 7 .48 (dd, J = 5 .7 , 1 .5 Hz, I H, H-3); 13C NMR ( l OO MHz, CDCb) 8 1 3 .8, 22 .3 , 24.5 , 3 1 .3 , 33 .0, 83 .4, 1 2 1 .3 , 1 56.4, 1 73 .2 . �o R-5.22 5" 3" 1 " 1 �OyO ------ 4" 2 " U 2 4 3 R-3 .1 (5R)-Dihydro-5-pentyl-2(3H)-furanone (R-3.1) : Pd/C ( 1 8 mg, 10 % Pd on charcoal, 0 .02 mmol, 0 .05 equiv.) was added to a solution of lactone R-5.22 (74 mg, 0.4 mmol) in EtOAc (2 mL). The mixture was shaken at RT overnight under H2 (2 .5 bar). The mixture was filtered through Celite, washing well with EtOAc (20 mL). The filtrate was concentrated to give R-3.1 as a colourless oil (74 mg, 1 00 %). Rf = 0 .4 1 (3 : 1 hex-EtOAc); 20 1· 1 48 20 I [a]o = +50 . 1 ° (c 3 .0, MeOH), It. [a]o = +50.4° (c 3 .0, MeOH); H NMR (400 MHz, CDCh) 8 0.90 (t, J = 6.5 Hz, 3H, H-5" ), 1 .24- 1 . 9 1 (m, 8H, H- l ", H-2", H-3", H- 4"), 2 .28-2 .37 (m, 2 H, H-4), 2 .54 (dd, J = 9.4, 7 .0 Hz, 2H, H-3), 4 .45-4 . 54 (m, I H, H-5); DC NMR ( l OO MHz, CDCh) 8 1 3 .8 , 22 .4, 24 .8 , 27 .9, 28 .8 , 3 1 .4, 35 .4, 8 1 .0, 1 77 .3 . 1 8 1 �o R-3.1 ______ • �o 3S,SR-3.66 �o 3R,5R-3.66 trans:cis = 1 7 : 3 (3S,5R)-Dihydro-3-methyl-5-pentyl-(3H)-furanone (3S,SR-3 .66) and (3R,5R)- Dihydro-3-methyl-5-pentyl-(3H)-furanone (3R,5R-3.66): nBuLi (2 .5 M in hexane, 480 �L, l .2 mmol, l .2 equiv.) was added to a solution of ipr2NH ( 1 2 1 mg, 1 69 ).lL, l .2 mmol, l .2 equiv . ) in THF ( 1 0 mL) at 0 QC. The mixture was cooled to -78 QC and stirred for 30 min. A solution of lactone R-3.1 ( 1 73 mg, l .0 mmol, 1 .0 equiv.) in THF ( l 0 mL) was added to the mixture dropwise and stirring continued for 2 h at this temperature. Methyl iodide ( 142 mg, 62 ).lL, 1 .0 mmol , l .0 equiv.) was added and stirred for 3 h at -78 QC. The reaction was quenched by adding MeOH ( 1 0 mL) and sat 'd NH4Cl (20 mL). The aqueous layer was extracted with Et20 (3 x 20 mL) and the combined organic l ayers were washed with brine (20 mL), dried over MgS04 fi ltered and concentrated . The residue was purified by chromatography (3 : 1 = hex-EtOAc) to give a mixture of3S,5R-3 .66 and 3R,5R-3 .66 as a colourless oil ( 1 53 mg, 82 %). Rr = 0.60 (3 : 1 hex-EtOAc) ; 'H NMR (400 MHz, CDCh) 8 0 .87 (t, J = 6.6 Hz, 3H, CH3CHr), 1 .2 5 (d, J = 7.3 Hz, 3H, -CH3 at C-3), 1 .27- 1 .72 (m, 8H, -CH2-), 1 .97 (dt, J = 1 2 .8 , 7 . 5 Hz, I H, H-4), 2 .09 (ddd, J = 1 2 .8 , 9 .0 , 5 .0 Hz, 1 H, H- 4 '), 2 .43-2 . 5 1 (m, 0 . 1 5 H, cis H-3), 2 .60-2 .72 (m, 0 .85H, trans H-3), 4 .24-4.35 (m, 0. 1 5H , cis H-5), 4 . 5 1 (tt, J = 7 .7 , 5 .3 Hz, 0 . 85H, trans H-5); I 3C NMR ( 1 00 MHz, CDCb) 8 1 3 .8 , 1 5 .8 , 22 .4 , 24.9, 3 1 .4, 33 .9, 3 5 .3 , 35 .4 , 78 .6, 1 80 .0; HRMS ca1cd. for C I OH ' 902 (MH +) : 1 7 l . 1 3850; obsd: 1 7 l . 1 3 825 . 1 82 �o R-3.1 ___ � o 3S.5R-S.25 \ trans:cis = 7:3 3R,5R-5.25 (3S,5R)-Dihydro-3-butyl-S-pentyl-(3H)-furanone (3S,SR-5.2S) and (3R,SR)-Dihydro- 3-butyl-5-pentyl-(3H)-furanone (3R,5R-5.2S): nBuLi (2.5 M in hexane, 480 ilL, 1 .2 mmol , 1 .2 equiv.) was added to a solution of ipr2NH ( 1 2 1 mg, 1 69 ilL, 1 .2 mmol, 1 .2 equiv.) in THF (20 mL) at 0 qc. The mixture was cooled to -78 QC and stirred for 30 min. A solution of l actone R-3.1 ( 1 73 mg, 1 .0 mmol, 1 .0 equiv.) in THF (5 mL) was added to the mixture dropwise and stirring continued for 2 h . Butyl iodide ( 1 84 mg, 1 1 4 ilL, 1 .0 mmol, 1 .0 equiv.) was added, and stirred for 3 h at -78 QC. The reaction was quenched by the addition of MeOH ( l 0 mL) and sat 'd aq. NH4CI (20 mL). The aqueous layer was extracted with Et20 (3 x 20 mL) and the combined organic layers were washed with brine (20 mL), dried over MgS04 filtered and concentrated. The residue was purified by chromatography (3 : 1 = hex-EtOAc) to give a mixture of 3S,5R-S.2S and 3R,SR-S.2S as a colourless oil ( 1 56 mg, 74 %). Rr = 0.62 (5 : 1 hex-EtOAc) ; lH NMR (400 MHz, CDCh) () 0 .84 (t, J = 7 .2 Hz, 6H, CH3-), 1 . 1 9-1 .82 (m, 1 4H, -CH2-), 1 .97 (dd, J = 8 . 1 , 6.4 Hz, 2H, H-4) , 2 .36-2 .42 (m, 0 .3H, cis H-3), 2 .46-2 .5 8 (m, 0 .7H, trans H-3), 4 .2 1 -4.3 1 (m, 0 .3H, cis H-5), 4.40-4.45 (m, 0 .7H, trans H-5); 1 3C NMR ( l OO MHz, CDCh) () 1 3 .9 , 1 3 .9 , 22 .4, 22 .5 , 25 .0, 29 .5 , 30.6, 3 1 .5 , 33 .5 , 35 .6, 39 .3 , 78.8, ] 79.6; HRMS calcd for C 13H2S02 (MH+) : 2 l 3 . 1 8546; obsd: 2 1 3 . 1 8543 . 1 83 �o _�. �o R R-3.1 R == Me, Et, npr, nSu Four (3,SR)-Dihydro-3-alkyl-S-pentyl-(3H)-furanones: nBuLi (2 . 5 M in hexane, 960 ilL, 2.4 mmol, 4.8 equiv.) was added to a solution of ipr2NH (243 mg, 337 ilL, 2 .4 mmol, 4.8 equiv.) in THF (20 mL) at 0 QC. The mixture was cooled to -78 QC and stirred for 30 min. A solution of lactone R-3 .1 (346 mg, 2.0 mmol, 4.0 equiv.) in THF ( 1 0 mL) was added to the mixture drop wise and stirring continued for 2 h at this temperature . Methyl iodide ( 142 mg, 62 ilL, l .0 mmol, l .0 equ iv.), ethyl iodide ( 1 56 mg, 8 1 ilL, 1 .0 mmol, l .0 equiv.), n-propyl iodide ( 1 70 mg, 98 ilL, 1 .0 mmol, 1 .0 equiv.), and n-butyl iodide ( 1 84 mg, 1 1 4 ilL, 1 .0 mmol, 1 .0 equ iv.) were dissolved in THF (5 mL) and the solution was added dropwise to the reaction mixture at -78 QC, and stirred for 3 h at this temperature . The reaction was quenched by addition of Me OH ( 1 0 mL) and sat'd NH4CI (20 mL). The aqueous layer was extracted with Et20 (3 x 20 mL) and the combined organic layers were washed with brine (20 mL), dried over MgS04, filtered and concentrated . The residue was purified by chromatography (3 : 1 = hex-EtOAc) to give a colourless oil ( 1 45 mg, 34 %); GC-MS: RT 26.8 min (R = Me, C I OH 1 s02, M+ obsd. mlz 1 70); RT 29.2 min (R = Et, min (R = nBu, C 1 3H2402, M+ obsd. mlz 2 1 2) . 1 84 �o R-S.22 ___ � o 4S,SR-S.31 (4S,SR)-Dihydro-4-methyl-S-pentyl-(3H)-furanone (4S,SR-S31) : Methyl magnesIUm bromide (2 .7 mL, 3 M in EhO, 8 .0 mmol, 8 .0 equ iv.) was added over 1 0 min to a suspension of CuI (762 mg, 4.0 mmol, 4 .0 equiv.) in THF (20 mL) at -78°C. After 1 0 min, dimethylsulfide ( 1 24 mg, 1 47 ilL, 2.0 mmol, 2 .0 equiv.) was added and the mixture was stirred at the same temperature for 2 h. Butenolide R-S.22 ( 1 7 1 mg, 1 .0 mmol , 1 .0 equiv.) in THF (5 mL) was added dropwise over 5 min at -78 DC. The mixture was warmed to 0 DC and stirred for 3 h . The reaction was quenched by the addition of sat'd aq. NH4CI (20 mL), stirred for an additional 1 0 min, concentrated to remove to bulk of the THF and extracted with Et20 (3 x 20 mL). The extracts were washed with brine (20 mL), dried over MgS04, fi ltered and concentrated. The residue was purified by chromatography (3 : 1 hex- EtOAc) to give 4S,5R-5.31 as a colourless oil ( 1 20 mg, 64 %). Rr= 0.56 (3 : 1 hex-EtOAc); MHz, CDCh) 8 0.83 (t, J = 6.6 Hz, 3H, CH3-) , 1 .07 (d, J = 6 .5 Hz, 3H, -CH3 at C-4), 1 .22- 1 .66 (m, 8H, -CH2-), 2 .08-2 .20 (m, 2H, H-3), 2 .54-2.65 (m, I H, H-4), 3 .90-3 .97 (m, I H, H-5); I 3C NMR ( l OO MHz, CDCh) 8 1 4 .0, 1 7 .5 , 22 .5 , 25 .4, 3 1 .6, 34.0, 36 . 1 , 37 . 1 , 87 .5 , 1 76 .6 ; HRMS calcd for C IOH 1 902 (MH+) : 1 7 1 . 1 3850; obsd : 1 7 1 . 1 383 1 . 1 85 �o R-5.22 _______ �O (' 4S,5R-5,27 (4S,SR)-Dihydro-4-buty I-S-pentyl-(3 H)-furanone (4S,5R-5.27): n-Butyl magnesIUm bromide (4 .68 mL, 1 .7 1 M in 1 : 1 THF/toluene, 8 .0 mmol, 8 .0 equiv.) was added over 1 0 min to a suspension o f CuI (762 mg, 4.0 mmot, 4 .0 equ iv.) i n THF (20 mL) at -78°C. After 1 0 min, d imethylsulfide ( 1 24 mg, 1 47 �L, 2 .0 mmol, 2.0 equiv.) was added and the mixture was stirred at the same temperature for 2 h and then butenolide R-5.22 ( 1 7 1 mg, 1 .0 mmol, 1 .0 equiv.) in THF (5 mL) was added dropwise over 5 min at -78 °C. The m ixture was stirred at 0 °C for 3 h . The reaction was quenched by the addition of sat 'd aq. NH4CI (20 mL), stirred for an additional l a min, concentrated to remove the bulk of the THF and extracted with Et20 (3 x 20 mL). The extracts were washed with brine (20 mL), dried over MgS04, filtered, concentrated and the residue purified by chromatography (3 : 1 hex-EtOAc) to give 4S,5R-5.27 as a colourless oil (74 mg, 4 1 %). Rf = 0.56 (5 : 1 hex- EtOAc); [a]o20 = +24.5° (c 1 .0, CH2Ch) ; lH NMR (400 MHz, CDCh) (3 0 .93 (t, J = 6.5 H2, 6H, CH3-), 1 .25 - 1 .38 (m, 1 2H, -CHr), l .50-1 .69 (m, 2H, OCHCfu-), 2 .05-2 .24 (m, 2H, H-3 , HA), 2 .66 (dd, J = 1 7 .3 , 8 .3 Hz, I H, H-3 '), 4 .03-4 . 1 2 (m, I H, H-5) ; 1 3C NMR ( l OO MHz, CDCh) (3 1 3 .9, 1 4 .0 , 22 .5 , 22 .6 , 25 .4, 29.7, 3 1 .5 , 32 .8 , 34.6, 35 .3 , 4 1 .2, 86 .2, 1 76 .8 ; HRMS caIcd for C 1 3H2S02 (MH+) : 2 1 3 . 1 8546; obsd: 2 1 3 . 1 8548 . 1 86 �o ______ • �o � R-5.1 5 R = Me, Et, nBu Three (4S,SR)-Dihydro-4-alkyl-S-pentyl-(3H)-furanones: Methyl magnesium bromide (667 I-tL, 3 M in Et20, 2 .0 mmol, 2 .0 equiv.), ethyl magnesium bromide (667 I-tL, 3 M in EhO, 2 .0 mmol , 2 .0 equiv.) , and n-butyl magnesium bromide ( 1 . 1 7 mL, 1 .7 1 M in 1 : 1 THF/toluene, 2 .0 mmol, 2 .0 equiv.) were added over 1 0 min to a suspension of CuI (57 1 mg, 3 .0 mmot, 3 .0 equiv.) in THF (20 mL) at -78°C . After 1 0 min, dimethylsulfide (93 mg, 1 1 0 I-tL, 1 .5 mmot, 1 .5 equiv.) was added and the mixture was stirred at the same temperature for 2 h. Butenolide R-S.22 ( 1 7 1 mg, 1 .0 mmol , 1 .0 equiv.) in THF (5 mL) was added dropwise over 5 min. The mixture was stirred at -78 °C for 3 h. The reaction was quenched by the addition of sat 'd aq. NH4Cl (20 mL), stirred for an additional 1 0 min, concentrated to remove the bulk of the THF and extracted with Et20 (3 x 20 mL). The extracts were washed with brine (20 mL), dried over MgS04, filtered, and concentrated . The residue was purified by chromatography (3 : 1 hex-EtOAc) to give a colourless o i l ( 1 54 mg, 8 1 %); GC-MS: RT 26.0 min (R = Me, C IOH 1 S02, M + obsd mlz 1 70); RT 2 8 . 1 min (R = 1 87 4S,5R-5.31 1 . LOA. THF -78°e 2. Mel • �Oyo + �o " ,.� " 3R,4S,5R-5.33 major 4 : 1 3S,4S,5R-5.33 minor (3R,4S,5R)-Dihydro-3,4-dimethyJ-5-pentyJ-2 (3/1)-fura none (3R,4S,SR-5.33) and (3S,4S,SR)-dihydro-3,4-dimethyl-5-pentyl-2(3H)-furanone (3R,4S,SR-5.33): nBuLi (2 .5 M in hexane, 480 ilL, 1 .2 mmol , 1 .2 equiv.) was added to a solution of ipr2NH ( 1 2 1 mg, 1 69 ilL, 1 .2 mmol, 1 .2 equiv.) in THF ( 1 0 mL) at 0 qc. The mixture was cooled to -78 QC and stirred for 30 mm. A solution of 4S,5R-5.32 ( 1 70 mg, 1 .0 mmol , 1 .0 equiv.) m THF ( 1 0 mL) was added to the mixture dropwise and stirring continued for 2 h at this temperature. Methyl iodide ( 1 42 mg, 62 ilL, 1 .0 mmol, 1 .0 equiv.) was added and stirred for 3 h at -78 °C . The reaction was quenched by the addition of Me OH ( 1 0 mL) and sat'd NH4CI (20 mL). The aqueous layer was extracted with Et20 (3 x 20 mL) and the combined organic l ayers were washed with brine (20 mL), dried over MgS04, fil tered and concentrated. The residue was purified by chromatography (3 : 1 = hex-EtOAc) to give a mixture of 3R,4S,5R-5.33 and 3S,4S,SR-S.33 as a yellow oil (93 mg, 50 %). Rr= 0 .66 (3 : 1 hex-EtOAc); I H NMR (400 MHz, CDCh) 8 0 .86 (t, J 6.9 Hz, 3H, CH3-), 0 .99 (d, J 7 . 1 Hz, 3H, -Clh at C-4), 1 . 1 1 (d, J = 6 . 5 Hz, 3H , -Cl-h a t C-3 ), 1 .25 - 1 .73 (m, 8H , -CH2-), 2 . 1 4-2 .2 1 (m, 1 H, H-4), 2 .58-2 .73 (m, 1 H, H-3 ), 3 .82-3 .94 (m, 0 .2H, H-5 as minor), 3 .94- 4.04 (m, 0.8H, H-5 as major); l 3C NMR ( 1 00 MHz, CDCb) 8 1 3 .8, 1 4 .0, 1 7 . 1 , 22.4, 25 .3 , 3 1 .5 , 33 .9, 36.0, 3 7 .0, 87 .4, 1 76 .5 ; HRMS calcd for C 1 1H2 102 (MH+) : 1 85 . 1 54 15 ; obsd: 1 85 . 1 5428. 1 88 COOH 3.34 1 1 9 7 6 4 2 1 2 1 0 8 3.41 � COOMe 5 3 1 Methyl 3E,6Z-undecadienoate (3.41) : A solution of 3E,6Z-undecadienoic acid (3 .46 g, 5 .0 mmol) in methanol (50 mL) was heated at reflux in the presence of Amberlyst- 1 5 (3.46 g) for 1 h . The mixture was cooled, the Amberlyst- 1 5 was removed by filtration and rinsed with EtzO (20 mL). The filtrate and washings were concentrated and the residue purified by chromatography (5 : 1 = hex-EtOAc) to give 3.41 as a l ight yellow oil (2 .69 g, 9 1 %). Rf= 0 .78 (3 : 1 hex-EtOAc); lH NMR (400 MHz, CDCb) 0 0 .89 (t, J= 6.9 Hz, 3H, H- 1 2), 1 .23 - 1 .3 8 (m, 6H, H-9, H- l O, H- 1 1 ), 2 .03 (q, J = 6.9 Hz, 2H, H-8), 2 .76-2 . 8 1 (m, 2H, H-5), 3 .05 (d, J = 4.4 Hz, 2H, H-2), 3 .68 (s, 3H, OCH3), 5 .33-5 .57 (m, 4H, H-3 , H-4, H-6, H-7) ; l 3C NMR ( l OO MHz, CDCh) 0 1 4 . 1 , 22 .5 , 27 . 1 , 29 .3 , 30.3, 3 1 .5 , 37 .9, 5 1 .8 , 1 2 1 .8, 1 26 .6, 1 3 1 .2 , 1 33 .0, 1 72 .5 ; HRMS calcd for C 1 3H2302 (MHl : 2 1 1 . 1 6980; obsd: 2 1 1 . 1 7002 . COOMe AD-mix-� • 3.41 8' 6' 4 ' 7' 5' 3' 2' 5.41 o (4R,SR)-4,5-Dihydro-4-hydroxy-5-(2 ' Z)-oct-2-enyl-2(3H)-furanone (5.41 ): AD-mix-f3 ( l .4 g) and the ester 3.41 (2 1 0 mg, 1 . 0 mmol, 1 .0 equiv.) were added to a mixture of 'BuOH (20 mL) and H20 (20 mL) at 0 qc. The mixture was stirred for 48 h, then quenched by the addition of sat 'd aq . Na2S03 (20 mL). The mixture was extracted with CH2Ch (3 x 20 mL), dried over MgS04, filtered and concentrated. The residue was purified by chromatography (5 : 1 = hex-EtOAc -)- 1 5 : 1 = CHzCh-MeOH) to give 5.41 as a 1 89 colourless oil ( 146 mg, 69 %). Rj = 0 .63 ( 1 5 : 1 CH2Ch-MeOH); [a]D20 = +46 . 1 ° (c 0.65, CHCb), l it. l 83 [a]o20 = +44.5° (c 0.65, acetone); lH NMR (400 MHz, CDCh) 8 0 .86 (t, J = 6 .9 Hz, 3H, H-8 ' ), 1 .23-1 .36 (m, 6H, H-5 ' , H-6 ' , H7 ') , 2 .06 (g, J = 7. 1 Hz, 2H, H-4 ') , 2 .5 1 -2 .65 (m, 2H, H-1 '), 2 .77 (dd, J = 17 .8 , 5 . 5 Hz, 2H, H-3) , 4 .32-4.39 (m, 1 H, H-4), 4 .45-4 .49 (m, I H, H-5), 5 .36-5 .42 (m, I H, H-3 '), 5 .52-5 .63 (m, I H, H-2') ; l 3C NMR ( l OO MHz, CDCh) 8 14 .0, 22.5, 26.5 , 27.4, 29. 1 , 3 l .4, 39 .2 , 68.6, 84.3, 1 22 .4 , 134 . l . 1 76.0; HRMS calcd for C l 2H2003 (M+): 2 1 2 . 1 4 124; obsd: 2 1 2 . 1 4 1 1 6 . Et3N 8' 6' 4' l ' 0 0 • MsCI CH2CI2 4 3 5.35 5 .3 6 (5R)-S-Oct-2 'Z-enyl-2(5H)-furenone (5.42): Triethylamine ( 1 46 fJ.L, 1 06 mg, 1 .05 mmol, 2 . 1 equiv.) and methanesulfonyl chloride (43 fJ.L, 63 mg, 0.55 mmol, I . l eguiv.) were added to a solution of lactone 5.41 ( 1 05 mg, 0.5 mmol, 1 .0 equiv.) in CH2C h ( 1 0 mL) at 0 0c. After 1 h the reaction was quenched by the addition of sat'd ag. NH4CI solution ( 1 0 mL). The mixture was extracted with CH2Ch (3 x 20 mL), dried over MgS04, filtered and concentrated . The residue was purified by chromatography (3 : 1 = hex-EtOAc) to give 5.42 as a colourless oil (49 mg, 5 1 %). R/= 0 .38 (3 : 1 hex-EtOAc); [a]D20 = - 1 34.7° (c I .O, CHCb), lit. l S} [aJo20 = - 1 34. 1 ° Cc 0.9, CHCh); lH NMR (400 MHz, CDCh) 8 0.86 (t, J = 6 .9 Hz, 3H, H-8 ' ), l .22- l .35 (m, 6H, H-5 ' , H-6' , H-7 ' ) , 1 .99 (q, J = 7 .3 Hz, 2H, H4 '), 2 .50 (p, J = 7.9 Hz, 2H, H-l '), 4 . 98-5 .04 (m, 1 H, H5), 526-5 .35 (m, I H, H-3 '), 5 .53-5 .62 (m, I H, H-2 '), 6. 1 0 (dd, J = 5.7, 2 .0 Hz, I H, H4), 7 .42 (dd, J = 5 .7 , 1 .5 Hz, H- 1 90 3); 1 3C NMR ( l OO MHz, CDCh) 8 1 4 .0, 22 .5 , 27 .3 , 29.0, 3 1 .0, 3 1 .4, 82.7, 1 2 1 . 1 , 1 2 1 .9 , 1 34.9, 1 55 . 8 , 1 72 .9 ; HRMS calcd. for C1 2H 1 902 (MH): 1 95 . 1 3850; obsd: 1 95 . 1 3925 . 1 9 1 C h ap t e r 6 Chapter 6: Summary and future work The goal of this project was to synthesise potential flavour compounds combinatorially and identifY key components for further investigation as flavourants in dairy products. This chapter aims to summarise our accomplishments and put them in context. The synthesis and analysi s of ketones were the foc i of Chapter 2 . Ketones were synthesised individually via a two-step sequence. The Grignard reaction was the first step to produce an alcohol and the oxidation of the secondary alcohol ensued, to produce a ketone (Scheme 6. 1 ). [01 .. .. Scheme 6 . 1 : Synthetic route for making ketones in Chapter 2 . Twenty ketones were synthesised individually and sixteen were sufficiently stable to b e screened by the Fox 4000. Some compounds selected from the Fox analysis were assessed by GC-O. Results from the preliminary screenings indicated aromatic and cyclopropyl ketones as compounds of interest (Figure 6. 1 ). The first library of cyclopropy\ ketones was synthesised and screened (Scheme 6 .2 ). o o � U 2.1 7 o � U 2.22 2 .29 o o � lJ 2.27 ~ 2.32 Figure 6. 1 : The structures of lead ketones from Chapter 2 . ] 92 o �H [>-MgBr OH · Uv Swern oxidation .. o � n = 4-7 (2 .57 -2 .60) S c heme 6 .2 : Combinatorial synthesis of a l ibrary of four cyclopropyl ketones. Further ketone l ib raries were produced by a co-worker, David Lun. He synthesised l ib raries o f ketones including ethyl ketones and cyclopropyI ketones. Selected compounds are i llustrated in Figure 6.2 and their odour and potency was assessed by the l aboratory technician fro m Fonterra. o � o o � � Figure 6 . 2 : Some o f the ketones produced in l ibraries. The synthesis and analysis of racemic l actones were the foc i of Chapter 3. Individual racemic lactones were synthesised via a two-step p ro cess. Conden sation of aldehydes with malon ic acids, in the Linstead modification of the Knoevenagel reaction, gave rise to (3E)- al k-3-enoic acids. For unsubstituted malonic acid, catalytic p iperidinium acetate was the m os t effective reagent. For 2-alkylmalonic acids, di ethylamine was the base of choice . Ketones (R2 *' H ) were unreactive in this condensation. Acid-catalysed cyclisation of these unsaturated acids gave rise to y-lactones which were converted to y-thionolactones using Lawesson 's reagent. (3E)-A lk-3-enoic acids containing additional unsaturation gave 1 9 3 complex product mixtures on attempted cyclisation, due to rearrangements of the intermediate carnocations (Scheme 6.3) . + eOOH R 3 -< eOOH base • Scheme 6 .3 : Synthetic route for racemic y-lactone in Chapter 3 . Libraries of racemic y-lactones (C8-e l 2) , and a-substituted y-lactones were produced combinatorially according to this method. Further, synthesis of a library o f y- thionolactones was achieved from a library of y-l actones with Lawesson's reagent. The l ibraries were analysed by GC-O (Figure 6.3) . The odour of the lactones was generally characterised by fruity notes (i.e. , peach, apricot and coconut). �O �o �o �s Figure 6 .3 : Structures ofl ibraries of racemic y-Iactones (n = 3 , 4, 5 , 6, 7). (5R)-Dihydro-5-octyl-2 (3H)-furanone (R-1 .52) was synthesised from L-glutamic acid (L- 4.44) and the (S)-enantiomer (8-1 .52) was synthesised by analogy from D-glutamic acid (D-4.44) in Chapter 4 (Scheme 6 .4). 1 94 !'::1Hz HOOC�COOH L-4.46 4 steps ° NHz HOOC�COOH D-4.46 4 steps . � /' · · ao 5-1 .52 Scheme 6 .4 : Synthetic route for both enantiomers of 1 .52 in Chapter 4 . This route was for the synthesis of chiral y-Iactones individually and more effic ient methods are needed for variety of divers ities with good overall yields in order to ach ieve l ibraries of compounds combinatorially. Asymmetric syntheses of both enantiomeric series of y-lactones utilizing the Sharp less asymmetric dihydroxylation reaction were employed to give libraries (Scheme 6 .5) . � CoOM e ___ )-'/�.'('O'):::::O )-'/), ,(,0,):::::0 ___ )-'/) . . (,0,):::::0 n n ,,---! - n\d nU Scheme 6 .5 : Synthesis ofl ibraries of enantiomerically pure y-lactones (n = 3 , 4, 5 , 6, 7) . According to the retrosynthetic route for optically active y-Iactones in Chapter 5, there are three points of diversity (Scheme 6.6) . a-Alkylation of a p ,y-disubstituted y-lactone has been used to introduce R3. Cup rate addition to the intennediate butenolide is highly stereoselective, delivering R2 trans relative to RJ . The configuration at Cy, bearing Rl , is controlled by the Sharpless asymmetric dihydroxylation . We have investigated the introduction of substituents in each position. It was our ultimate goal to produce libraries 1 95 in thi s manner, whereby five aldehydes (R1CH2CH=O), five alkylmetal species (R2M), and five alkyl hal ides (R3X) would give a l ibrary of 1 25 compounds . R�O = R"(Y0 = R'-(:r0 = R2 R3 R2 + HOOC /'--. COO H Scheme 6 .6 : Retrosynthetic analysis of substituted lactones in Chapter 5 . I t was a fundamental objective ofthis project t o generate flavour compound l ibraries using combinatorial chemistry. Understanding the synthetic chemistry required for applying it to the combinatorial approach was the biggest challenge. Introducing modem analytical techniques to the evaluation of our synthetic compounds gave an opportunity to identifY new potential flavour compounds. Further assessment of potential flavour compounds is required for rigorous screening to identity their potency. 1 96 References References Chapter 1 1 . Mtilder, H . Neth. Milk Dairy 1. 1952, 6, 1 5 7- 1 67 . 2 . Urbach, G . Int. 1. Dairy Technol. 1997, 50, 79-89. 3. 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