Cyclization with multiple S-S bonds

Disulfide-rich peptides (DSRs) are a rapidly expanding class of bioactive peptides that is widely found in nature, e.g. in bacteria, fungi, plants, and animals (venom toxins). DSRs are attracting increasing interest from drug developers as a result of their remarkably high chemical stabilities in comparison to linear peptides. The various disulfide bonds in these peptides play a crucial role in enforcing their native conformation. Thereby, the correct connectivity between the cysteines is absolutely crucial for expressing the correct biological function. As a consequence, their chemical synthesis needs to be performed with absolute precision in order to ensure the correct SS-bond topology.

In practice, SS-bond formation in cysteine-rich peptides is often performed by subjecting the peptide to freely oxidative folding conditions using a mixture of either cysteine (SH)/ cystine (SS) or glutathione (SH/SS). For selected cases (e.g. cyclotides), this procedure is believed to produce only the (single) thermodynamic isomer, but even if so the obtained product needs to be verified as to whether it possess the right SS-bond topology. However, there are numerous examples for which the SS-oxidized product is formed as a complex mixture of different topological isomers. Therefore, it is of utmost importance to apply the pairwise protection/deprotection of cysteines using orthogonal protective group strategies in order to be able to ensure product formation as a unique and single topological isomer.

Pepscan has a long-standing experience in the synthesis of DSRs. We applied and optimized the use of a series of different cysteine-protective groups (i.e. Mmt, Trt, Mob, Acm, Dpm, Nbz, tBu, StBu, Phacm, Msbh, etc.) that allow for a simple and straightforward synthesis of DSRs including up to 6 different SS-bonds, all with fully defined topologies as the result of the stepwise protection/deprotection strategies mentioned above.

Disulfide-rich Peptides

(A) Structure of omega-atracotoxin-Hv2a (37 aa) containing three disulfide bonds (cysteines at 4-18 (1), 11-22 (2), and 17-36 (3)); (B) Formation of single topological isomer via stepwise formation of SS-bonds using an orthogonal protection/deprotection strategy; (C) Formation of a mixture of at least three topological isomers resulting from a native folding strategy.

In our experience, we learnt that many of the protective groups strategies as reported in literature do work for that particular example, but practically are not applicable to other DSRs (ref. 1). We have therefore carefully reviewed all existing procedures and protective group strategies one-by-one, optimized their conditions for applications to other peptides, and combined these in various different ways in order to obtain a general set of conditions that basically works for all peptides. With this experience at hand, Pepscan is the partner to contact in case you want your DSRs to be synthesized at the highest possible quality and purity, obviously as a single and unique topological isomer.


Dekan Z. et al.: Total Synthesis of human hepcidin through regioselective disulfide bond formation by using the safety-catch cysteine protecting group 4,4-dimethylsulfinylbenzhydryl. Angew. Chem. Int. Ed. Engl. 2014, 53, 2931-2934.