Abstract
Cells employ post-translational modifications (PTMs) as key mechanisms to expand proteome diversity beyond the inherent limitations of a concise genome. The ability to incorporate post-translationally modified amino acids into protein targets via chemical ligation of peptide fragments has enabled the access to homogeneous proteins bearing discrete PTM patterns and empowered functional elucidation of individual modification sites. Native chemical ligation (NCL) represents a powerful and robust means for convergent assembly of two homogeneous, unprotected peptides bearing an N-terminal cysteine residue and a C-terminal thioester, respectively. The subsequent discovery that protein cysteine residues can be chemoselectively desulfurized to alanine has ignited tremendous interest in preparing unnatural thiol-derived variants of proteogenic amino acids for chemical protein synthesis following the ligation-desulfurization logic. Recently, the 21st amino acid selenocysteine, together with other selenyl derivatives of amino acids, have been shown to facilitate ultrafast ligation with peptidyl selenoesters, while the advancement in deselenization chemistry has provided reliable bio-orthogonality to PTMs and other amino acids. The combination of these ligation techniques and desulfurization/deselenization chemistries has led to streamlined synthesis of multiple structurally-complex, post-translationally modified proteins. In this review, we aim to summarize the latest chemical synthesis of thiolated and selenylated amino-acid building blocks and exemplify their important roles in conquering challenging protein targets with distinct PTM patterns.
Highlights
In the past two decades, native chemical ligation (NCL) technology has been broadly utilized for the convergent building of peptide and protein targets
Kent demonstrated that sequential Native chemical ligation (NCL) reactions can be achieved by harnessing the differential reactivity of peptidyl thioesters (Bang et al, 2006): a bifunctional peptide fragment possessing an N-terminal Cys and a dormant alkyl thioester on the C terminus was ligated with a peptide phenyl thioester without the concern of head-to-tail peptide cyclization; aryl thiol additives [i.e., thiophenol and 4-mercaptophenylacetic acid (MPAA)] were added subsequently to activate the inert thioester which allowed the NCL reaction to proceed in situ (Scheme 1B)
The acyl hydrazide moiety is unresponsive to NCL conditions and acts as a protecting group for the C-terminal carboxylate, which can be converted to acyl azide by NaNO2 followed by thiolysis to unleash the capacity for NCL (Fang et al, 2011; Qi et al, 2019) (Scheme 1C)
Summary
In the past two decades, native chemical ligation (NCL) technology has been broadly utilized for the convergent building of peptide and protein targets. Kent demonstrated that sequential NCL reactions can be achieved by harnessing the differential reactivity of peptidyl thioesters (Bang et al, 2006): a bifunctional peptide fragment possessing an N-terminal Cys and a dormant alkyl thioester on the C terminus was ligated with a peptide phenyl thioester without the concern of head-to-tail peptide cyclization; aryl thiol additives [i.e., thiophenol and 4-mercaptophenylacetic acid (MPAA)] were added subsequently to activate the inert thioester which allowed the NCL reaction to proceed in situ (Scheme 1B) This sequential ligation concept has been furthered by the use of 2,2,2-trifluoroethanethiol (TFET) to enhance the rate of peptide ligation through in situ generation of a productive. Flood et al demonstrated that peptide hydrazides can be activated by a stoichiometric amount of acetyl acetone to produce peptide acyl-pyrazoles that will readily undergo transesterification reactions with aryl thiols to afford activated peptide thioesters for peptide ligation (Flood et al, 2018)
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