What are Cyclotides?
Uses of Cyclotides
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Discovery of Cyclotides
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For a primer on peptide synthesis try this site from the Michigan State University

Naturally occurring cyclotides may be extracted in significant quantity (up to 1g cyclotide/kg wet tissue weight) from plants but the ability to synthesize modified cyclotides is an important consideration in determining their utility in drug design applications. Although cyclotides possess a rather complex structure they have been shown to be amenable to solid phase peptide synthesis (Daly, et al., 1999b, Tam and Lu, 1997). In addition to chemical methods, it may also be possible to apply recombinant technologies to the production of cyclotides as the gene sequence has recently been discovered (Jennings, et al., 2001).

Chemical synthesis offers the most direct and rapid route to individual compounds and the greatest amount of control over subsequent formation of tertiary structure. Figure One summarizes two general strategies that have been applied in the synthesis of the cyclotides, i.e., either oxidizing the disulfide bonds in a linear precursor peptide prior to cyclization, or cyclizing prior to oxidation. An advantage of the former is that by correctly forming the disulfide bonds the termini are proximate, thereby potentially facilitating cyclization over competing polymerisation reactions. There are in principle as many possible linear precursors as there are amino acids in the cyclic peptide (29 in the case of kalata B1). In planning a synthesis the breakpoint in the sequence should preferentially be chosen so that the terminal amino acids are small to avoid steric hindrance in the subsequent cyclization reaction, and in a region not involved in crucial elements of secondary structure. In the case of kalata B1 a ligation point involving a Gly-Gly pair in a turn region had the further advantage of eliminating potential problems from racemization on ligation of the termini (Daly, et al., 1999b). While this strategy successfully produced correctly folded kalata B1, it appears that oxidation to form the correct disulfide bond isomer occurs more efficiently if the cyclic backbone is formed first (Daly, et al., 1999b). Furthermore, cyclization following oxidation of the cysteine residues can be complicated by side reactions, so cyclization prior to oxidation is in general the preferred strategy.

Figure One:Two pathways of cyclotide synthesis - oxidation followed by cyclisation or cyclisation followed by oxidation. Cyclisation prior to oxidation is the preferred strategy.

The most robust method of cyclization of the cyclotides involves a thiazip mechanism (Daly, et al., 1999b, Tam and Lu, 1997, Tam, et al., 1999a, Tam, et al., 1999b). This overcomes a disadvantage of direct macrocyclization, which frequently results in peptide polymerization due to the low probability of the termini meeting. The key to this mechanism is the reaction of an electrophilic C-terminal thioester (added during chain assembly) and nucleophilic N-terminal amine to produce backbone cyclization under basic conditions. If the intervening sequence contains cysteine side chains these can act as intermediate nucleophiles, effectively 'zipping' the activated C-terminus along the peptide toward the N-terminus. Efficient backbone cyclisation takes place via an irreversible S,N-acyl migration at an N-terminal cysteine. The requirement for an N-terminal cysteine is easily accommodated in the cysteine rich cyclotides; a small C-terminal residue also improves the reaction rate.

After cyclization the propensity for a particular cyclotide to form the correct three-dimensional fold on oxidation appears to vary with the sequence. For example, kalata B1 has been reported to efficiently fold to the correct disulfide isomer once cyclized (Daly, et al., 1999b) but selective formation of individual disulfide bonds was required for the folding of circulin A (Tam, et al., 1999b).

The ability to synthesise cyclotide molecules has opened the possibility of grafting other bioactive sequences onto this framework to take advantage of its exceptional stability as a template in drug design (Craik, 2001).


Craik DJ: Plant cyclotides: circular, knotted peptide toxins. Toxicon (2001) 39:1809-1813.

Daly NL, Love S, Alewood PF and Craik DJ: Chemical synthesis and folding of large cyclic polypeptides: Studies of the cystine knot polypeptide kalata B1. Biochemistry (1999b) 38:10606-10614.

Jennings C, West J, Waine C, Craik D and Anderson M: Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis. Proc. Natl. Acad. Sci. U. S. A. (2001) 98:10614-10619.

Tam JP and Lu Y-A: Synthesis of large cyclic cystine-knot peptide by orthogonal coupling strategy using unprotected peptide precursors. Tetrahedron Lett. (1997) 38:5599-5602.

Tam JP, Lu Y-A and Yu Q: Thia zip reaction for synthesis of large cyclic peptides: Mechanisms and applications. J. Am. Chem. Soc. (1999a) 121:4316-4324.

Tam JP, Lu YA, Yang JL and Chiu KW: An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides.Proc. Natl. Acad. Sci. U S A (1999b) 96:8913-8918.