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Maleimide-mediated methodologies are among the most used in bioconjugation. [5] [6] Due to fast reactions and high selectivity towards cysteine residues in proteins, a large variety of maleimide heterobifunctional reagents are used for the preparation of targeted therapeutics, assemblies for studying proteins in their biological context, protein-based microarrays, or proteins immobilisation. [7]
Reaction with thiols occur in the pH range 6.5–7.5, NEM may react with amines or undergo hydrolysis at a more alkaline pH. NEM has been widely used to probe the functional role of thiol groups in enzymology. NEM is an irreversible inhibitor of all cysteine peptidases, with alkylation occurring at the active site thiol group (see schematic ...
TCEP is particularly useful when labeling cysteine residues with maleimides. TCEP can keep the cysteines from forming di-sulfide bonds and, unlike dithiothreitol and β-mercaptoethanol, it will not react as readily with the maleimide. [2] However, TCEP has been reported to react with maleimide under certain conditions. [3] [4]
The nucleophilic lysine residue is commonly targeted site in protein bioconjugation, typically through amine-reactive N-hydroxysuccinimidyl (NHS) esters. [3] To obtain optimal number of deprotonated lysine residues, the pH of the aqueous solution must be below the pKa of the lysine ammonium group, which is around 10.5, so the typical pH of the reaction is about 8 and 9.
The specificity of this reaction is ideal for situations where the cysteine is located away from the desired epitope (e.g. in peptides where a terminal cysteine can be added to either end of the peptide). Maleimide activated KLH, where the first part of this two step procedure has been completed, is commercially available.
Fig. 8 FSLs can be easily created from cysteine-containing peptides, proteins or any other thiols of biological interest. The mixing of a cysteine containing peptide with FSL-RFG(Maleimide), in 4MMF buffer, allows the peptide to react with the maleimide residue of the FSL (through the well-known Michael nucleophilic addition reaction ...
Spin label is attached to Cysteine 102 residue. Site-directed spin labeling (SDSL) was pioneered in the laboratory of Dr. W.L. Hubbell. [2] [3] In SDSL, sites for attachment of spin labels are introduced into recombinantly expressed proteins by site-directed mutagenesis. Functional groups contained within the spin label determine their specificity.
In general, the conjugation methodology is based on three main reactions; a reaction between activated carboxyl groups and amino groups which yields an amide bond, a reaction between pyridyl dithiols and thiols which yields disulfide bonds, and a reaction between maleimide derivatives and thiols, which yields thioether bonds.” [6]