Deprotection of the p-Methoxybenzyl Group of Selenocysteine by Neighboring Group Participation

Abstract

Introduction Selenocysteine (Sec) is now recognized as the 21 amino acid in the universal genetic code and the discovery of 25 human proteins containing selenocysteine has generated renewed interest in synthetic peptides containing this rare amino acid [1]. The weakness of the carbon-selenium bond renders trityl-type protecting groups ineffective during peptide synthesis, thus benzyl (Bzl) and p-methoxybenzyl (Mob) groups have been used to protect the selenol side-chain [2,3]. While the Mob group is more acid labile than a benzyl group, very strong Lewis acids such as TMSBr and TMSOTf are still required for its removal. This is problematic because these reagents are not very soluble in ether, which makes work up of the peptide difficult. Thus, an alternative method of removal of the Mob group is needed. Our group has been working on methods for making the biologically important peptide Cys-Sec-Gly, which corresponds to the C-terminus of mammalian thioredoxin reductase. A major difficulty in making this peptide is in the deprotection of the Mob group from the Sec residue. In order to address this problem, we started to explore other deprotection methods that did not include silyl reagents. One of our early attempts at removing the Mob group involved oxidative deprotection with I2. As van der Donk had noted, deprotection of the Mob group from Sec using I2 in the presence of a peptide disulfide bond resulted in oxidative deprotection of Sec(Mob) with concomitant formation of a selenylsulfide bond [4]. We found the use of I2 as a reagent for the removal of Mob groups challenging, as many side products resulted that were unidentifiable by us. During the course of this investigation, we noticed spontaneous deprotection of the Mob group of Sec when the neighboring Cys residue was protected with a S-t-butyl group. However, very little deprotection was observed when the adjacent Cys residue was protected with a trityl group. When we further activated the neighboring Cys residue with a 5-Npys group [5], 100% deprotection of the Mob group of Sec was achieved in the presence of TFA, thioanisole, and phenol. This led us to use 2,2 dithiobis (5-nitropyridine) (DTNP) as a reagent for deprotecting Mob groups from Sec residues in peptides where no other Cys residue was present in the sequence.