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

Abstract

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.