Cyclotides are a unique class of head-to-tail cyclic cysteine-knot microproteins that exhibit a wide range of biological activities ranging from insecticidal to anti-HIV. They have a unique structure in which two disulfide bridges form a ring through which a third disulfide bridge is threaded, making cyclotides exceptionally stable against thermal and proteolytic degradation. This characteristic makes them of great interest in drug discovery. MCoTI-I and MCoTI-II, which originally were extracted from Momordica cochenchinesis, are examples of macrocyclic cysteine knot microproteins that display extremely potent trypsin inhibitory activity. In this thesis, two distinct complementary approaches have been developed for the first total synthesis of MCoTI-I and MCoTI-II. In the first, termed thia zip native chemical ligation (NCL), the cyclic backbone and three disulfide bonds are formed in a one pot reaction. In the second, backbone ligation is achieved via a novel immobilised protease-mediated ligation reaction.
These methodologies have been further applied to the synthesis of novel analogues of the natural MCoTI-II structure modified at a key residue in the active loop (Lys10) that have altered protease inhibition specificity together with minimizing of MCoTI-II size. These wild-type and re-engineered MCoTI cyclotides were found to be active against not only trypsin-like serine proteases; trypsin, chymotrypsin, leukocyte elastase, HCV NS3/4A, β-tryptase and matriptase, but they are also active against cysteine protease; foot-and-mouth disease virus (FMDV 3Cpro).