Cysteine, Npys-protected

Besides being presently the most efficient method for site-directed interchain disulfide bridging of two different cysteine peptides, this procedure is also recommended for a controlled peptide-protein conjugation by reacting Npys-protected cysteine peptides with properly thiol-functionalized protein carriers. 1761 In this conjugation procedure the amount of peptide grafted to the protein is quantitatively determined by measuring spectro-photometrically at 430 nm the amount of 3-nitropyridine-2(l//)-thione liberated in the reaction. An example of this approach is outlined in Scheme 18. 

These sulfides are prepared from other sulfur-protected cysteine derivatives by reaction with the sulfenyl chloride, The Npys group can also be introduced directly by treatment of the thiol with NpysCl, ... 

FIGURE 6.20 Synthesis of cystine-containing peptides from cysteine-containing peptides by removal of other protectors followed by (A) deprotection of the sulfhydryls and their oxidation to the disulfide, and (B) formation of the disulfide bond by reaction of a liberated sulfhydryl with a sulfhydryl that is protected and activated by 3-nitro-2-pyridylsulfanyl (Npys).89... 

Similarly to the methoxycarbonylsulfanyl-protected cysteine derivatives, the S-Npys compounds are stable toward strong acid treatment1 64 165 172 and thus, they can be exploited in the dual purpose of S-protection and S-activation. The Npys group has been used with considerable success in combination with Boc chemistry for the preparation of peptides. 173-175 This S-protecting/activating group, however, is labile to piperidine treatment and can, therefore, not be used in Fmoc-based peptide synthesis. 

Alternatively, both peptide chains could be protected at one cysteine residue as a 5-Acm derivative and at the second cysteine residue by an acid-labile [Trt, Mob, Xan, or Bzl(4-Me)], base-labile (Fm), or reduction-labile (5-tBu) group. Both peptide chains may then be separately converted into the free thiol/Acm-protected form for selective activation of one chain as S-SPy or. S -Npys derivatives by reaction with di(2-pyridyl)disulfide or di[5-nitro(2-pyridyl)]disulfide, or as a sulfenohydrazide derivative by reaction with azodicarbocylic acid derivatives for formation of the first interchain disulfide bridge. 

Scheme 8 Formation of Two Interchain Disulfide Bonds with Different Connectivities Using Two Kinds of Protecting Groups for the Cysteine Residues and Activation via the Npys Derivative. Synthetic Schemes for the Two Isomers of the ro-Conotoxin MVIIC Fragment Obtained by Digestion with Lysyl Endopeptidase and Thermolysin19 ...

In order to obtain a heterodimeric disulfide bridge the cysteine residue of one component, either PNA or peptide, must be derivatized. 3-Nitro-2-pyridine-sulphenyl (NPys)-derivatized Cys is specifically reactive toward free thiols. Npys-labeled Cys is commercially available and with special cautions (see Note 3) can be assembled into a peptide chain like a commonly protected amino acid. 

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