Specific Cleavage of Peptides at Cysteinyl Residues

Degani, Yair; Patchornik, A.; Maclaren, J. A.

doi:10.1021/ja00966a069

Cited by 12 publications

(17 citation statements)

References 2 publications

(2 reference statements)

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“…Decomposition of the SCN-labeled peptide is likely via one of the pathways proposed previously in the literature, possibly accompanied by scission of the peptide backbone. 21,23,48 No further experiments were performed to identify decomposition products, and any sample with noticeably reduced C≡N band intensity was discarded. All spectra reported here are for intact cyanylated peptide samples.…”

Section: Resultsmentioning

confidence: 99%

The Effects of α-Helical Structure and Cyanylated Cysteine on Each Other

et al. 2010

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β-Thiocyanatoalanine, or cyanylated cysteine, is an artificial amino acid that can be introduced at solvent-exposed cysteine residues in proteins via chemical modification. Its facile post-translational synthesis means that it may find broad use in large protein systems as a probe of site-specific structure and dynamics. The C≡N stretching vibration of this artificial side chain provides an isolated infrared chromophore. To test both the perturbative effect of this side chain on local secondary structure and its sensitivity to structural changes, three variants of a model water-soluble alanine-repeat helix were synthesized containing cyanylated cysteine at different sites. The cyanylated cysteine side chain is shown to destabilize, but not completely disrupt, the helical structure of the folded peptide when substituted for alanine. In addition, the C≡N stretching bandwidth of the artificial side chain is sensitive to the helix−coil structural transition. These model system results indicate that cyanylated cysteine can be placed into protein sequences with a native helical propensity without destroying the helix, and further that the CN probe may be able to report local helix formation events even when it is water-exposed in both the ordered and disordered conformational states. These results indicate that cyanylated cysteine could be a widely useful probe of structure-forming events in proteins with large in vitro structural distributions.

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Section: Resultsmentioning

confidence: 99%

The Effects of α-Helical Structure and Cyanylated Cysteine on Each Other

et al. 2010

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“…In fact, selective peptide-cleavage methods at the Cys residue employing an N-acylation strategy have been reported for protein sequencing (Scheme 1 a). [7] In these methods, N-acylation is induced by an electrophilic auxiliary group introduced on a Cys side chain (e.g. S-carbonyl group), and this auxiliary group provides the activation of the peptide bond and subsequent hydrolysis.…”

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confidence: 99%

A Synthetic Approach to a Peptide α‐Thioester from an Unprotected Peptide through Cleavage and Activation of a Specific Peptide Bond by N‐Acetylguanidine

2011

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In modern procedures for total chemical protein synthesis, the concept of chemical ligation plays an essential role in the assembly of target protein polypeptide chains.[1] The peptide a-thioester is the key component for chemical ligation such as native chemical ligation (NCL), direct segment coupling methods, or traceless Staudinger ligation.[2] Thus, substantial effort has been expended on the establishment of peptide-athioester synthesis based on conventional Boc or Fmoc solidphase peptide synthesis (SPPS, Boc = tert-butoxycarbonyl, Fmoc = 9-fluorenylmethyloxycarbonyl).[3] However, because of the inherent limitations of SPPS, synthesis of peptide athioesters with more than 50 amino acids (aa) is still challenging. For the preparation of such a polypeptide athioester, an expression method using the intein system has come to be recognized as a robust technology with the capacity to provide polypeptide a-thioesters of more than 50 aa.[4] Inspired by the biological system, we explored an inteinlike chemical methodology for preparing a long peptide athioester by using a native (unprotected) peptide as the starting material. Some groups recently reported thioesterification of E. coli-expressed peptides using acid treatment, but these intriguing methods lead to epimerization of the Cterminal amino acid residue and still have sequence limitations.[5] Thus we set out to find a widely usable new methodology.The key point was the manipulation of an unprotected peptide to install a C-terminal a-thioester. This task appears to require the selective activation of a native amide bond and subsequent thiolysis. Recently, several groups reported elegant methods of activating the peptide backbone to install an a-thioester at the peptide C terminus.[6] In these methods, the activation of the peptide bond was performed by a selective acylation strategy of an Na-amide nitrogen atom at a specific amino acid residue. To examine the thioesterification of unprotected peptides, we focused on the cysteine (Cys) residue, because Cys possesses a thiol group, which might be more easily modified than the other amino acid side chains and thus selectively induce N-acylation. In fact, selective peptide-cleavage methods at the Cys residue employing an Nacylation strategy have been reported for protein sequencing (Scheme 1 a). [7] In these methods, N-acylation is induced by an electrophilic auxiliary group introduced on a Cys side chain (e.g. S-carbonyl group), and this auxiliary group provides the activation of the peptide bond and subsequent hydrolysis. These reports encouraged us to hypothesize the feasibility of the selective activation of a native amide bond at the Cys residue and subsequent thiolysis, instead of hydrolysis, to obtain a peptide a-thioester. However, our preliminary examination of thiolysis for the activated Cys residue did not proceed and regenerated unprotected peptide substrate or hydrolyzed product instead of the desired peptide athioester (Scheme 1 a). Therefore, we have explored a new nucleophile as an alternative to a...

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“…In 1950, Edman reported a stepwise peptide cleavage from the N-terminal using phenyl isothiocyanate [107]. Thereafter, selective peptide bond cleavage methods at specific residues [108,109] or a Cys [110,111] residue and S-acylation of a Cys [112,113] residue (Fig. 17a).…”

Section: Site-specific Peptide Cleavage Through Side Chain Modificationsmentioning

confidence: 99%