Synthesis of cyclodextrin composites incorporating targeting and drug carrying capabilities : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry, Massey University, Turitea Campus, Palmerston North, New Zealand
A selective, versatile, and robust methodology for the bi-functionalisation of -
cyclodextrin has been developed which allows for the attachment of peptides and/or
sulfonamides in varying C- and N-terminal combinations on resin using NFluorenylmethoxycarbonyl
(Fmoc) Solid Phase Peptide Synthesis (SPPS). Mono-6Afluorenylmethyloxycarbonylamino-
mono-6X-succinyl-b-cyclodextrin, an amino acid
based bi-functionalised derivative of b-cyclodextrin, has been functionalised with the
bioactive peptide, bradykinin, and/or sulfonamides using Fmoc SPPS on Rink resin.
The all-in-one molecule contains a carrier (cyclodextrin), targeting agent (bradykinin)
and/or drug (sulfonamide). Varying combinations of these bradykinin-focused
molecules have been synthesised in an attempt to determine the structure-function
relationship against cancer cell lines using cell-based screening in vitro.
This study commenced with the synthesis of two linkers on to cyclodextrin. This
enabled selective binding directly on to the resin, or a peptide attached to the resin.
Peptide growth and/or cleavage from the resin followed allowing for the synthesis of
peptide-cyclodextrin species in various combinations. Fmoc SPPS techniques have been
employed to allow for the addition and synthetic extension of peptides on to
cyclodextrin. Peptide purification was achieved by reverse phase high pressure liquid
chromatography, and nuclear magnetic resonance spectroscopy and mass spectrometry
were used to determine the success of the coupling reactions and identification of
cyclodextrin regio isomers. Sulfonamide additions to the cyclodextrin and/or peptide compounds were obtained after numerous studies investigating the optimal reaction
conditions. 4-Fluorenylmethyloxycarbonylaminobenzenesulfonyl chloride was found to
give the highest yields for the synthesis of C-terminal peptide sulfonamides with 4-
carboxybenzenesulfonamide giving the highest optimal yields for N-terminal peptide
sulfonamides. Peptide coupling efficiency of cyclodextrin and sulfonamides were
investigated and optimised by comparing different SPPS resins and solvents. The
incorporation of spacers between the peptide/cyclodextrin and/or resin have also been
investigated in an attempt to improve overall reaction yields.
Preliminary bioassay testing against tumour cell lines HT-29 Human Duodenum,
Hs700T Human pancreatic adenocarcinoma, and MA-104 Human pancreatic
adenocarcinoma were performed. The MTT assay and the flow cytometry assay were
used to show the effect of varying combinations of these cyclodextrin-peptidesulfonamide
molecules against the three cell lines and compared to a known anticancer
drug, 5-Fluorouracil. Despite employment of simple entities in the construction of these
compounds, an increase in cell proliferation (ca 10-20%) was seen for some
cyclodextrin-bradykinin complexes. In addition, an exposed C-terminus on the
bradykinin-sulfonamide moiety and an exposed N-terminus on the cyclodextrinbradykinin
sulfonamide moiety both gave positive results. Mixed results were obtained
with the addition of a linker between the cyclodextrin and the bradykinin molecules
(less then 5% increase or decrease) compared to their non-linker counterparts.