Oxidative Deselenization of Selenocysteine: Applications for Programmed Ligation at Serine

Abstract

Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under-utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high-yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)-free protein eglin C.

One‐Pot Ligation–Oxidative Deselenization at Selenocysteine and Selenocystine

“…[6a] This methodology can also be extended to ligation-deselenization at selenol-derived amino acid residues. [7] Independently, our laboratory [8] and Metanis and co-workers, [9] recently demonstrated that, in the presence of an oxidant to outcompete hydrogen atom abstraction, Sec can be transformed into serine (Ser) at the ligation junction which provides a further expansion of Sec-based ligations. While this oxidative deselenization can be performed using O2 as an oxidant, [8][9] peptide cleavage products were observed on larger peptide targets.…”

“…[7] Independently, our laboratory [8] and Metanis and co-workers, [9] recently demonstrated that, in the presence of an oxidant to outcompete hydrogen atom abstraction, Sec can be transformed into serine (Ser) at the ligation junction which provides a further expansion of Sec-based ligations. While this oxidative deselenization can be performed using O2 as an oxidant, [8][9] peptide cleavage products were observed on larger peptide targets. [8] For this reason, following optimization, we arrived at oxone® as an oxidant that could facilitate clean conversion of Sec to Ser in minutes in the presence of TCEP (Scheme 1B).…”

Accessing human selenoproteins through chemical protein synthesis

“…Our SELM synthesis is based on four segments with three sequential Sec-NCL reactions, and utilizes a protected form of Sec, selenazolidine (Sez), 43 as well as a deselenization reaction (Scheme 1) that we and others have recently developed. 34,44,45 The shortest member of the selenoprotein family, SELW, was prepared from two peptide segments with a single Cys-NCL reaction (Scheme 2). …”

Utilizing Selenocysteine for Expressed Protein Ligation and Bioconjugations

“…10,11 Therefore, it is possible to convert Sec into an abundant amino acid, leaving no trace of the original ligation method. The ligation site then can be selected to increase the probability of the fragments reacting, such as loops and other surface accessible regions.…”

“…Sec-mediated ligation also offers much greater flexibility in the choice of ligation site because, following ligation, the Sec can be selectively converted into an alanine or serine under mild conditions. 8–11 Due to the abundance of these amino acids it is straightforward to generate the protein of interest with the desired sequence with no trace of the ligation. The distinct advantage of Sec deselenization to Ala is that it is compatible with the presence of cysteines in the target protein.…”