Synthesis of a Selenocysteine-Containing Peptide by Native Chemical Ligation

Gieselman, Matt D.; Xie, Lili; Donk, Wilfred A. van der

doi:10.1021/ol015712o

Cited by 223 publications

(210 citation statements)

References 46 publications

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“…Analytical HPLC analyses of these crude product mixtures resulted in extremely complex chromatograms. Some of the peaks were identified as iodinated adducts or as a dehydroalanine-containing peptide (data not shown), consistent with previous reports [15,23]. We then attempted to improve this procedure by protecting the neighboring group cysteine as a disulfide using a StBu group.…”

Section: Model Cyclization Reaction Involving Sec(mob) and Cys(mob)supporting

confidence: 84%

“…It had been previously demonstrated by van der Donk and coworkers. that the oxidative deprotection of Mob groups with I 2 could be improved if the peptide also contained a disulfide bond [23]. We noticed that when performing a control reaction in which we cleaved the target Sec(Mob) containing peptide with a vicinal disulfide bond (Cys(StBu)-Sec(Mob)-Gly-PAL-Resin) using a cleavage cocktail containing TFA/water (95/5) that more peaks than expected were present in the analytical HPLC chromatogram, especially a large peak that eluted very early in the profile (Figure 1(A)).…”

Section: Model Cyclization Reaction Involving Sec(mob) and Cys(mob)mentioning

confidence: 99%

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Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

Harris¹,

Flemer²,

Hondal³

2006

Journal of Peptide Science

102

113

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Abstract:We present here a simple method for deprotecting p-methoxybenzyl groups and acetamidomethyl groups from the sidechains of cysteine and selenocysteine. This method uses the highly elecrophilic, aromatic disulfides 2,2 -dithiobis(5-nitropyridine) (DTNP) and 2,2 -dithiodipyridine (DTP) dissolved in TFA to effect removal of these heretofore difficult-to-remove protecting groups. The dissolution of these reagents in TFA, in fact, serves to 'activate' them for the deprotection reaction because protonation of the nitrogen atom of the pyridine ring makes the disulfide bond more electrophilic. Thus, these reagents can be added to any standard cleavage cocktail used in peptide synthesis.The p-methoxybenzyl group of selenocysteine is easily removed by DTNP. Only sub-stoichiometric amounts of DTNP are required to cause full removal of the p-methoxybenzyl group, with as little as 0.2 equivalents necessary to effect 70% removal of the protecting group. In order to remove the p-methoxybenzyl group from cysteine, 2 equivalents of DTNP and the addition of thioanisole was required to effect removal. Thioanisole was absolutely required for the reaction in the case of the sulfur-containing amino acids, while it was not required for selenocysteine. The results were consistent with thioanisole acting as a catalyst. The acetamidomethyl group of cysteine could also be removed using DTNP, but required the addition of >15 equivalents to be effective. DTP was less robust as a deprotection reagent. We also demonstrate that this chemistry can be used in a simultaneous cyclization/deprotection reaction between selenocysteine and cysteine residues protected by p-methoxybenzyl groups to form a selenylsulfide bond, demonstrating future high utility of the deprotection method.

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Section: Model Cyclization Reaction Involving Sec(mob) and Cys(mob)supporting

confidence: 84%

Section: Model Cyclization Reaction Involving Sec(mob) and Cys(mob)mentioning

confidence: 99%

Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

Harris¹,

Flemer²,

Hondal³

2006

Journal of Peptide Science

102

113

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“…12,13 Long selenopeptides that possess a couple of Sec residues along the chain have also been synthesized by application of selenocysteine-based native chemical ligation (Sec-NCL). [14][15][16] To synthesize selenopeptides in a flask, particular Sec derivatives have to be utilized. (Figure 1), which have Boc protection on the amino group and MBn or MPM protection on the selenium atom, are useful for Boc-based selenopeptide synthesis as the MBn and MPM groups in these targets can be deprotected under satisfactory conditions in the final step of selenopeptide synthesis.…”

Section: Introductionmentioning

confidence: 99%

“…[20][21][22] Later, more reactive bromoalanines were utilized for the synthesis of Sec derivatives. 15,[23][24][25][26] 12 the amino group of L-serine (3) was protected with Boc, and the obtained carbamate 4 was converted into methyl ester 5 (Scheme 1). The Ser derivative thus protected was tosylated to 6 and then selenated to 7 by reaction with a selenolate, which was generated from di(p-methylbenzyl) diselenide (MBnSeSeMBn) and NaBH 4 in MeOH, according to a similar procedure reported by Braga.…”

Section: Introductionmentioning

confidence: 99%

Improved synthetic routes to the selenocysteine derivatives useful for Boc-based peptide synthesis with benzylic protection on the selenium atom

Shimodaira¹,

Iwaoka²

2016

Arkivoc

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Selenocysteine (Sec) derivatives, i.e., Boc-Sec(MBn)-OH (1) and Boc-Sec(MPM)-OH (2), which are useful for chemical synthesis of selenopeptides, were obtained from L-serine in five steps with total yields of 73% and 74%, respectively. The enantiomeric excesses were confirmed to be >99% e.e. by optical resolution using a chiral column on HPLC. On the other hand, for the case of a Fmoc-protected Sec derivative, i.e., FmocSec(MPM)-OH, similar reactions resulted in low yields and partial racemization taking place.

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“…A variety of ligation chemistries has been developed for this purpose (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Application of the chemical ligation principle (1) has led to practical chemical syntheses of a wide variety of different classes of proteins (16).…”

mentioning

confidence: 99%

His₆tag-assisted chemical protein synthesis

Bang

Kent

2005

Proc. Natl. Acad. Sci. U.S.A.

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To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses. (16). Synthetic access to protein molecules has been used to elucidate the molecular basis of protein folding and stability (17), to elucidate the molecular basis of protein function (18), to design and build proteins of novel structure (19), and to determine the molecular structure of proteins by both NMR (20) and x-ray crystallography (21). Chemical protein synthesis has also been used to develop candidate protein therapeutic molecules with improved properties (21,22).Thus far, most research on chemically synthesized protein molecules (16) has been focused on relatively small proteins in the size range of 50 to Ϸ150 aa (i.e., made from two or three peptide segments). This size limitation is the result of two phenomena: (i) practical constraints on the size of the unprotected peptide segment building blocks (23) and (ii) technical challenges in the chemical ligation of more than two or three peptide segments. Even highly optimized stepwise solid-phase peptide synthetic procedures are limited to Ϸ50 amino acid residues for the practical preparation of high-purity unprotected peptides (24). Thus, the chemical synthesis of a protein of typical size (Ϸ300 aa) (25) would require the use of at least six synthetic peptide segments as building blocks.The most effective way of covalently joining unprotected peptide segments to form a protein molecule is native chemical ligation (3,26). Native chemical ligation involves the reaction of a peptide-␣ thioester with a Cys-peptide; reversible thioesterthiol exchange with the N-terminal Cys residue gives a thioesterlinked intermediate that undergoes an irreversible intramolecular rearrangement to give a near-quantitative yield of a single product linked by a native peptide bond at the ligation site. The native chemical ligation reaction is both practical and highly effective. ¶ Each ligation product can be purified by reverse-phase HPLC and characterized with great precision by electrospray M...

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Synthesis of a Selenocysteine-Containing Peptide by Native Chemical Ligation

Cited by 223 publications

References 46 publications

Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

Improved synthetic routes to the selenocysteine derivatives useful for Boc-based peptide synthesis with benzylic protection on the selenium atom

His₆tag-assisted chemical protein synthesis

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Synthesis of a Selenocysteine-Containing Peptide by Native Chemical Ligation

Cited by 223 publications

References 46 publications

Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

Improved synthetic routes to the selenocysteine derivatives useful for Boc-based peptide synthesis with benzylic protection on the selenium atom

His6tag-assisted chemical protein synthesis

Contact Info

Product

Resources

About

His₆tag-assisted chemical protein synthesis