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One-pot thiol-amine bioconjugation to maleimides: simultaneous stabilisation and dual functionalisation.

ABSTRACT: Maleimide chemistry is widely used in the site-selective modification of proteins. However, hydrolysis of the resultant thiosuccinimides is required to provide robust stability to the bioconjugates. Herein, we present an alternative approach that affords simultaneous stabilisation and dual functionalisation in a one pot fashion. By consecutive conjugation of a thiol and an amine to dibromomaleimides, we show that aminothiomaleimides can be generated extremely efficiently. Furthermore, the amine serves to deactivate the electrophilicity of the maleimide, precluding further reactivity and hence generating stable conjugates. We have applied this conjugation strategy to peptides and proteins to generate stabilised trifunctional conjugates. We propose that this stabilisation-dual modification strategy could have widespread use in the generation of diverse conjugates.

SUBMITTER: Wall A

PROVIDER: S-EPMC8162801 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Orthogonal dual thiol-chloroacetyl and thiol-ene couplings for the sequential one-pot assembly of heteroglycoclusters.

Project description:We describe the first one-pot orthogonal strategy to prepare well-defined cyclopeptide-based heteroglycoclusters (hGCs) from glycosyl thiols. Both thiol-chloroactetyl coupling (TCC) and thiol-ene coupling (TEC) have been used to decorate cyclopeptides regioselectively with diverse combination of sugars. We demonstrate that the reaction sequence starting with TCC can be performed one-pot whereas the reverse sequence requires a purification step after the TEC reaction. The versatility of this orthogonal strategy has been demonstrated through the synthesis of diverse hGCs displaying alternating binary combinations of α-D-Man or β-D-GlcNAc, thus providing rapid access to attractive heteroglycosylated platforms for diverse biological applications.

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A Facile One-Pot Construction of Succinimide-Fused Spiro[Pyrrolidine-2,3'-Oxindoles] via 1,3-Dipolar Cycloaddition Involving 3-Amino Oxindoles and Maleimides.

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Pot economy and one-pot synthesis.

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One-Pot

Project description:Data files and supplementary data associated with the one-pot method. These files are the results of one-pot enrichment of both acetylated and succinylated peptides.

2017-12-08 | MSV000081794 | MassIVE

A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format

Project description:Glycosylation, including N-glycosylation and O-glycosylation is generally characterized and controlled as a critical quality attribute for therapeutic glycoproteins because glycans can impact protein-based drug product efficacy, half-life, stability, and safety. Analytical procedures to characterize N-glycans are relatively well-established, but the characterization of O-glycans is challenging due to the complex workflows and lack of enzymatic tools. Here we present a simplified chemoenzymatic method to simultaneously profile N- and O-glycans from the same sample using a one-pot format by mass spectrometry (MS). N-glycans were first released by PNGase F, followed by O-glycopeptide generation by Proteinase K, selective N-glycan reduction, and O-glycan release by β-elimination during permethylation of both N- and O- glycans. Glycan structural assignments, and determination of N- to O-glycan ratio was obtained from the one-pot mass spectra. The streamlined, one-pot method is an accurate and reproducible approach that will facilitate advanced characterizations for quality assessments of therapeutic glycoproteins.

2024-08-01 | PXD052172 | Pride

Simultaneous Quantification of the Acetylome and Succinylome by One-Pot Affinity Enrichment

Project description:Protein post-translational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi-PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here we describe a streamlined, quantitative label-free proteomic workflow – “one-pot” PTM enrichment – which enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein. Coupled with a label-free, data-independent acquisition (DIA), we identified and quantified 2235 acetylated and 2173 succinylated peptides with the one-pot method and show that peak areas are highly correlated between the one-pot and traditional single-PTM enrichments. The ‘one-pot’ method makes possible detection of multiple PTMs occurring on the same peptide, and we show that it can be used to make unique biological insights into PTM crosstalk. Compared to single-PTM enrichments, the one-pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue.

2018-01-09 | MSV000081906 | MassIVE

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