Removal of the 5-nitro-2-pyridine-sulfenyl protecting group from selenocysteine and cysteine by ascorbolysis

Abstract

We previously reported on a method for the facile removal of 4-methoxybenzyl and acetamidomethyl protecting groups from cysteine (Cys) and selenocysteine (Sec) using 2,2'-dithiobis-5-nitropyridine dissolved in trifluoroacetic acid, with or without thioanisole. The use of this reaction mixture removes the protecting group and replaces it with a 2-thio(5-nitropyridyl) (5-Npys) group. This results in either a mixed selenosulfide bond or disulfide bond (depending on the use of Sec or Cys), which can subsequently be reduced by thiolysis. A major disadvantage of thiolysis is that excess thiol must be used to drive the reaction to completion and then removed before using the Cys-containing or Sec-containing peptide in further applications. Here, we report a further advancement of this method as we have found that ascorbate at pH 4.5 and 25 °C will reduce the selenosulfide to the selenol. Ascorbolysis of the mixed disulfide between Cys and 5-Npys is much less efficient but can be accomplished at higher concentrations of ascorbate at pH 7 and 37 °C with extended reaction times. We envision that our improved method will allow for in situ reactions with alkylating agents and electrophiles without the need for further purification, as well as a number of other applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Keywords: ascorbate; cysteine; deprotection; disulfide; orthogonal; protecting group; selenocysteine.

Figure 1

Deprotection of Sec(Mob) using TFA/DTNP followed by thiolysis. Deprotection of Cys(Mob) works similarly, but requires the use of thioanisole as an additive.

Figure 2

Deprotection of Sec(Mob) using TFA/DTNP followed by ascorbolysis.

Figure 3

HPLC chromatograms and mass spectrometry of the Sec-containing test peptide treated with ascorbate under various conditions for 24 hrs with a 5:1 ascorbate: peptide ratio. (A) HPLC chromatogram of the Sec-containing peptide without ascorbate treatment (control). (B) HPLC chromatogram of the Sec-containing peptide treated with ascorbate at 25 °C and pH = 4.5. (C) Mass spectrum of the Sec-containing peptide without ascorbate treatment (control). (D) Mass spectrum of the Sec-containing peptide treated with ascorbate at 25 °C and pH = 4.5.

Figure 4

HPLC and mass spectroscopic analysis of the Cys-containing test peptide treated with 100-fold excess ascorbate. (A) HPLC chromatogram of the Cys(5-Npys)-containing peptide treated with ascorbate at 37 °C, pH = 7 for 24 hrs. (B) Mass spectrum of the Cys-containing peptide treated with ascorbate at 37 °C and pH = 7.0 for 24 hrs.

Figure 5

Strategy for creating an interstrand selenosulfide bond between two peptide chains. Directed disulfide bond formation could occur (1), followed by conversion of Sec(Mob) to Sec(5-Npys) (2). Treatment with Asc would liberate the selenol, which could then be coupled to a Cys residue on a second peptide strand.