One-pot chemical synthesis of small ubiquitin-like modifier protein–peptide conjugates using bis(2-sulfanylethyl)amido peptide latent thioester surrogates

Abstract

Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO-peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of peptide segments derivatized by thioester or bis(2-sulfanylethyl)amido (SEA) latent thioester functionalities and the one-pot assembly of the SUMO-peptide conjugate by a sequential native chemical ligation (NCL)/SEA native peptide ligation reaction sequence. This protocol also enables the isolation of a SUMO SEA latent thioester, which can be attached to a target peptide or protein in a subsequent step. It is compatible with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, and it gives access to homogeneous, reversible and functional SUMO conjugates that are not easily produced using living systems. The synthesis of SUMO-peptide conjugates on a milligram scale takes 20 working days.

Accelerating chemoselective peptide bond formation using bis(2-selenylethyl)amido peptide selenoester surrogates

“…Chemical synthesis is especially useful when proteins having unusual architectures1–6 or well-defined modifications at specific sites are desirable. An application of protein chemical synthesis which is of utmost importance in biological sciences is the study of some complex post-translational modifications such as glycosylation,7 ubiquitination8–10 or sumoylation 11,12…”

“…The reaction of a SEA peptide with an N-terminal cysteinyl peptide yields a native peptide bond to cysteine and proceeds efficiently in the presence of 4-mercaptophenylacetic acid (MPAA40). The usefulness of the SEA ligation has been demonstrated by the total synthesis of several functional proteins 11,12,41–45…”

“…SEA peptides are easily accessible by the Fmoc SPPS using SEA polystyrene32,48 or PEG-based resins 11,12. We therefore sought to convert SEA peptides 1 into SeEA derivatives 5 by using the exchange reaction shown in Scheme 2, which takes advantage of the capacity of SEA amide form 1 to equilibrate in water with the SEA transient thioester form 2 29,32,49.…”

Total Chemical Synthesis of SUMO and SUMO‐Based Probes for Profiling the Activity of SUMO‐Specific Proteases

“…SUMO conjugates have been prepared before by semi‐synthesis using intein chemistry15 or by synthesis and ligation of peptide fragments 16, 17, 18, 19. In this report, we present a fully synthetic linear solid‐phase peptide synthesis (SPPS) approach towards full‐length SUMO conjugates, which enables facile parallel synthesis procedures.…”

Combination of Thiol‐Additive‐Free Native Chemical Ligation/Desulfurization and Intentional Replacement of Alanine with Cysteine

“…We synthesized stresscopin-related peptidea nd neuroendocrine regulatory peptide-2 (NERP-2) by this method. [4] In order to overcome this problem,s everal groups have developedt echniques for the removal of MPAA from reaction mixtures (liquid-liquide xtraction, [5] functional solid supports, [6] etc.). [1] Various synthetic methods have been reported, but native chemical ligation (NCL) is one of the most frequently used techniques.…”