Occurrence and Minimization of Cysteine Racemization during Stepwise Solid-Phase Peptide Synthesis1,2

Han, Yongxin; Alberício, Fernando; Bárány, George

doi:10.1021/jo9622744

Cited by 217 publications

(234 citation statements)

References 19 publications

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“…347 It is used for the Fmoc/ t Bu strategy although Fmoc-Cys(Trt) can undergo racemization in basic carboxyl activation conditions. 332 It can also be removed by oxidation with iodine, thereby leading to a dilulfide bridge by reaction with a free Cys side chain. Other oxidative removals are listed in the table.…”

Section: Protecting Groups Removed By Acidmentioning

confidence: 99%

Amino Acid-Protecting Groups

2009

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Section: Protecting Groups Removed By Acidmentioning

confidence: 99%

Amino Acid-Protecting Groups

2009

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“…Success of all deblocking and amino acid couplings were monitored qualitatively using a ninhydrin test [20]. Elongation of the peptides used standard Fmoc SPPS chemistry with 4 equivalents (equivalents relative to peptide synthesis scale in mmoles) 2-(1H-benzotriazol-1-YL)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (0.05 M final concentration), 4 equivalents HOBt, 4 equivalents Fmoc-AA-OH, and 4 equivalents 2,4,6-trimethylpyridine (TMP) (collidine) in a 50% DCM/50% DMF solution with no preactivation time [21]. For the synthesis of peptides with a C-terminal carboxamide, Fmoc-PAL-Resin (0.35 mmol/g loading) was used, and each synthesis was done on a 0.15 mmole scale.…”

Section: Peptide Synthesesmentioning

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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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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“…22| Add the N-terminal Cys residue in its pentafluorophenol-activated and t Buthio-protected form. Coupling of this residue requires vortexing with 6 equivalents of Fmoc-Cys( t Buthio)-OPfp and 6 equivalents of HOBt relative to peptide at final concentrations of 0.5 M each for 1.5 h. Wash the resin 2-3 times with DMF as described in Step 19 and deblock the Fmoc-protecting group as described in Steps 18 and 19. m CRITICAL STEP Cys residues have a propensity to racemize when coupled using DIPEA 38 . To avoid Cys racemization, the N-terminal Cys residue is coupled as an activated OPfp ester obviating the need for a base.…”

mentioning

confidence: 99%

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

2007

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Expressed protein ligation (EPL) allows semisynthesis of a target protein with site-specific incorporation of probes or unnatural amino acids at its N or C termini. Here, we describe the protocol that our lab has developed for incorporating fluorotyrosines (F n Ys) at residue 356 of the small subunit of Escherichia coli ribonucleotide reductase using EPL. In this procedure, the majority of the protein (residues 1-353 out of 375) is fused to an intein domain and prepared by recombinant expression, yielding the protein in a thioester-activated, truncated form. The remainder of the protein, a 22-mer peptide, is prepared by solid-phase peptide synthesis and contains the F n Y at the desired position. Ligation of the 22-mer peptide to the thioester-activated R2 and subsequent purification yield full-length R2 with the F n Y at residue 356. The procedure to generate 100 mg quantities of Y 356 F n Y-R2 takes 3-4 months. INTRODUCTION Problem under investigationThe Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of all four nucleotides to their corresponding 2¢-deoxynucleotides and consists of two homodimeric subunits: R1 and R2 (refs. 1-3). R1 is the homodimeric subunit where nucleotide reduction occurs. It contains binding sites for nucleoside diphosphate substrates as well as for deoxynucleoside triphosphate and ATP allosteric effectors, which govern substrate specificity and turnover rates. R2 is the homodimeric subunit, which houses the diiron-tyrosyl radical cofactor (Y 122 ) essential for catalysis. Each turnover requires radical migration from the Y 122 site on R2 to the active site of R1, over a distance of 35 Å . The mechanism of this long-range radical propagation event is an active area of research 4,5 .Structures of active RNR, a 1:1 complex of R1 and R2, are not available. However, Uhlin and Eklund 6 have generated a docking model of this complex from the individual structures of R1 and R2 (refs. 7,8

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Occurrence and Minimization of Cysteine Racemization during Stepwise Solid-Phase Peptide Synthesis¹^,²

Cited by 217 publications

References 19 publications

Amino Acid-Protecting Groups

Amino Acid-Protecting Groups

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

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

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