The Removal of S‐Cysteine Protection by Means of 2‐Pyridine Sulfenyl Chloride and the Subsequent Formation of Disulfide Bonds. Preliminary communication

Abstract

Summary2-Pyridine sulfenyl chloride (PS-Cl) is a useful reagent for simultaneously deprotecting and activating the mercapto-group of cysteine and of cysteine-peptides preliminary to disulfide-bond formation. The S-protecting groups that are amenable to this reaction include trityl, diphenylmethyl, acetamidomethyl, t-butyl, and t-butylsulfenyl.In order to produce a peptide cysteine bridge, two S-protected cysteine residues are usually introduced during the synthesis. They are then deprotected and finally oxidized to the required disulfide bond. A wide variety of S-protecting groups have been described in the literature. The conditions for their removal are sometimes quite strong, For example, the cleavage of S-trityl cysteine with HBr or trifluoroacetic acid (TFA) affords only 60% of free cysteine because the removal of the S-trityl group in acid is in equilibrium with its attachment [I]; S-Diphenylmethyl (S-Dpm) requires 15-30 min of boiling in TFA and phenol [l]; HBr affords only a 50% yield at 50". However, S-trityl and S-Dpm-cysteine can be deblocked with Ag+-or Hg2+-ions [2]. S-t-butyl-cysteine can be cleaved either by sulfitolysis [3] or with o-nitrophenyl-sulfenyl chloride [4]. The S-acetamidomethyl group [5] has also been widely used, the conditions for deprotection with Ag+ or Hg2+ are very mild, The oxidation step is also critical. While many procedures offer high yields for cysteine or cysteine derivatives [6] they sometimes fail in more complicated situations.We wish to report here a rather general and mild method for deprotecting S-protected cysteine-residues which affords, after reaction, a reactive mixed disulfide which can react further with free SH-groups to provide an other disulfide.Scheme1 shows an outline of this S-deprotection by means of 2-pyridine sulfenyl chloride (PS-Cl, compound I).PS-C1 is extremely reactive and decomposes immediately with moisture. However it is a solid and can be easily handled in water-free glacial acetic acid. The i)Abbreviations are according to the IUPAC-IUB Commission on Biochemical Nomenclature.

Insulin—From its Discovery to the Industrial Synthesis of Modern Insulin Analogues

“…[46] The addition of slight excesses of strong acids,such as hydrochloric acid to possibly protonate the indole group,orofAc-Trp-OH, as ascavenger, were found to be equally as effective. [48] Although the rate of thiolysis increases as the pH increases,the rate of disproportionation is concomitantly enhanced, thereby leading to accumulation of symmetric disulfides.N evertheless,t he rates are sufficiently high even at pH 5-7;however, the authors used pH 8.5, which led to al arge accumulation of the B-chain homodimer and consequently to the poor yield of 15 %ofthe HPLC-purified heterodimer.This reaction has been significantly improved by the use of 3-nitro-2-pyridylsulfanyl- [49] and more recently of 5-nitro-2-pyridylsulfanylcysteine [50] derivatives,which allow for high rates of thiolysis even at pH 3.5-6, where such side reactions are completely suppressed. This allowed Kiso and co-workers to use established cysteinepairing procedures for the sequential, unambiguous formation of the human insulin heterodimer,a so utlined in Scheme 6.…”

Of Thiols and Disulfides: Methods for Chemoselective Formation of Asymmetric Disulfides in Synthetic Peptides and Polymers

“…Further electron‐withdrawing activating group such as 2‐ and 4‐nitrophenyl ( S ‐Nps) and 2‐ and 4‐pyridyl groups ( S ‐Pyr) have been used because the disulfide formation is promoted by the low p K a of the aromatic thiol leaving group (Scheme C). The introduction of thiol reactive moieties by 2‐pyridinesulfenyl chloride is, however, susceptible to hydrolysis . An alternative strategy includes N‐terminal‐thiol deprotection followed by activation with excess 2,2′‐dipyridyldisulfide and coupling with a free thiol in the presence of DTT at pH 7 .…”

Some peculiarities of synthesis of cysteine-containing peptides

“…PyS has been proposed for deprotection of cysteine protected with Trt, Dpm, Acm, Bu t , and SBu t groups and subsequent preparation of asymmetrical disulfides. 108 Reactions with this reagent are carried out in glacial acetic acid. The reactive mixed disulfide formed reacts with a free SH group to give the target disulfide.…”

“…Free SH groups may be liberated by the action of an excess of thiols (e.g., b-mercaptoethanol). 108 Nps-Cl was used to remove Trt, Dpm, Ddm, and Acm protections 109 and intermediate mixed disulfides can be isolated. Free SH-derivatives can be obtained under the action of an excess of a thiol (e.g., b-mercaptoethanol or dithiothreitol).…”

“…Deprotection and formation of disulfide bonds using Nps-Cl occur much more slowly than with PyS-Cl. 108 The use of aromatic sulfenyl halides is limited because of difficulties associated with their synthesis and isolation (these compounds are easily hydrolysed even in humid air) and due to the fact that tryptophan can undergo side reactions under the action of these compounds.…”