2,2′‐Dithiobis(5‐nitropyridine) (DTNP) as an effective and gentle deprotectant for common cysteine protecting groups

Abstract

Of all the commercially-available amino acid derivatives for solid phase peptide synthesis, none has a greater abundance of sidechain protection diversity than cysteine. The high reactivity of the cysteine thiol necessitates its attenuation during peptide construction. Moreover, the propensity of cysteine residues within a peptide or protein sequence to form disulfide connectivity allows the opportunity for the peptide chemist to install these disulfides iteratively as a post-synthetic manipulation through the judicious placement of orthogonal pairs of cysteine S-protection within the peptide's architecture. It is important to continuously discover new vectors of deprotection for these different blocking protocol in order to achieve the highest degree of orthogonality between the removal of one species in the presence of another. We report here a complete investigation of the scope and limitations of the deprotective potential of 2,2′-dithiobis(5-nitropyridine) (DTNP) on a selection of commercially-available Cys S-protecting groups. The gentle conditions of DTNP in a TFA solvent system show a remarkable ability to deprotect some cysteine blocking functionality traditionally removable only by more harsh or forcing conditions. Beyond illustrating the deprotective ability of this reagent cocktail within a cysteine-containing peptide sequence, the utility of this method was further demonstrated through iterative disulfide formation in oxytocin and apamin test peptides. It is shown that this methodology has high potential as a stand-alone cysteine deprotection technique or in further manipulation of disulfide architecture within a more complex cysteine-containing peptide template.

The use of 2,2′‐dithiobis(5‐nitropyridine) (DTNP) for deprotection and diselenide formation in protected selenocysteine‐containing peptides

“…Following quenching of the NaBH 4 , the free selenols quickly dimerized and were quantified as their corresponding diselenides via HPLC assay. As shown in Figure , the results of these general assays illustrate some interesting differences and parallels when compared with their corresponding Cys‐protected analogs .…”

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

“…The side chain protectedA -chain, by tert-butyl-based groups (P) includingB oc protection of the depsidipeptide unit (A8-A9), was selectivelyd eprotected at Cys(StBu) A6 with 25 % b-mercaptoethanol/ DMF, RT,2 1.5 h, and then treated with 10 equiv 2,2'-dithiobis(5nitropyridine, DTNP) [50] to produce Cys(Npys) A6 .C ys(Mmt) A11 was deprotected with 1.1 %TFA, 5% TIS (TIS = triisopropylsilane) in dichloromethane (6 2min) to form the intrachain Cys A6 -Cys A11 disulfide by thiolysis. [58] Although Cys(tBu) A7 is deprotected by 10 %t rifluoromethanesulfonic acid (TFMSA) in TFAa nd concomitantly activated by an excess of dipyridyl disulfide to yield Cys(Pyr) A7 , [59] the photolysis of Cys(oNv) B7 occurs quantitatively in 45 min to generate insulin in 36 % yield over these last two steps. Oxidation with I 2 in AcOH/H 2 O, purificationb yHPLC, and lyophilizationf rom NH 4 HCO 3 produced human insulin in 82 %y ield.…”

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

“…Introduction of the Npys group is performed by reaction of either the protected cysteine or conversion of the free thiol with Npys halides . An alternative pathway includes conversion with 2,2′‐dithiobis(5‐nitropyridine) (DTNP) into Npys and quantitative disulfide formation upon addition of DTT (with and without addition of thioanisole) . Limiting factors are the high stability, which complicates removal and requires enhanced temperatures and an access of DTNP.…”

“…[140] An alternative pathway includes conversion with 2,2'-dithiobis(5-nitropyridine) (DTNP) into Npys and quantitative disulfide formation upon addition of DTT (with and without addition of thioanisole). [141] Limiting factorsa re the high stability,w hich complicates removal and requires enhanced temperatures and an access of DTNP.F urthers tudies include Npys disulfidec oupling assisted by microwavei rradiation [142] and immobilizedN pys on resin enabling disulfide formationa nd facilitatedw ork-up. [143] The chemical stability of the Npys group allows for Boc/benzyl synthesis conditions, but cannotb ea pplied to "low-high" cleavage protocols or to bases employedi nt he Fmoc-strategy.…”