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

Abstract

In contrast to the large number of sidechain protecting groups available for cysteine derivatives in solid phase peptide synthesis, there is a striking paucity of analogous selenocysteine Se-protecting groups in the literature. However, the growing interest in selenocysteine-containing peptides and proteins requires a corresponding increase in availability of synthetic routes into these target molecules. It therefore becomes important to design new sidechain protection strategies for selenocysteine as well as multiple and novel deprotection chemistry for their removal. In this paper, we outline the synthesis of two new Fmoc selenocysteine derivatives [Fmoc-Sec(Meb) and Fmoc-Sec(Bzl)] to accompany the commercially available Fmoc-Sec(Mob) derivative and incorporate them into two model peptides. Sec-deprotection assays were carried out on these peptides using 2,2'-dithiobis(5-nitropyridine) (DTNP) conditions previously described by our group. The deprotective methodology was further evaluated as to its suitability towards mediating concurrent diselenide formation in oxytocin-templated target peptides. Sec(Mob) and Sec(Meb) were found to be extremely labile to the DTNP conditions whether in the presence or absence of thioanisole, whereas Sec(Bzl) was robust to DTNP in the absence of thioanisole but quite labile in its presence. In multiple Sec-containing model peptides, it was shown that bis-Sec(Mob)-containing systems spontaneously cyclize to the diselenide using 1 eq DTNP, whereas bis-Sec(Meb) and Sec(Bzl) models required additional manipulation to induce cyclization.

Figure 1

Listing of known Se-protected Sec SPPS derivatives.

Figure 2

Prior results on protected Cys- and Sec-containing peptides. a) DTNP methodology allows facile deprotection of a number of common Cys S-protectants in either the presence or absence of thioanisole. b) Sec(Mob) deprotection required only catalytic amounts of DTNP in the absence of thioanisole.

Figure 3

Synthetic routes into Sec derivatives 3a-c.

Figure 4

Results of DTNP assay on protected Sec-containing test peptides 7-9.

Figure 5

Deprotection/Cyclization sequence for oxytocin peptides 10-12, illustrating graphically the different requirements between the Sec(Mob)-bearing peptides and the Sec(Meb) or Sec(Bzl)-bearing peptides.