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N-Hydroxysuccinimide (NHS) is an organic compound with the formula (CH 2 CO) 2 NOH. It is a white solid that is used as a reagent for preparing active esters in peptide synthesis. It can be synthesized by heating succinic anhydride with hydroxylamine or hydroxylamine hydrochloride. [2]
The ester linkage is formed between the N-hydroxyphthalimide and a carboxylic acid by elimination of water, the coupling achieved with N,N′-dicyclohexylcarbodiimide (DCC). For peptide synthesis, the N-terminus of the growing peptide is protected with tert-butyloxycarbonyl while its C-terminus (Z–NH–CH(R)–COOH) is coupled to N ...
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.
Amine reactive: BS3 is amine-reactive in that its N-hydroxysulfosuccinimide (NHS) esters at each end react specifically with primary amines to form stable amide bonds in a nucleophilic acyl substitution-type reaction in which the N-hydroxysulfosuccinimide acts as the leaving group. [9]
Because the target for the ester is a deprotonated primary amine, the reaction is favored under basic conditions (above pH 7). Hydrolysis of the NHS ester is a major competing reaction, and the rate of hydrolysis increases with increasing pH. NHS- and sulfo-NHS-esters have a half-life of several hours at pH 7 but only a few minutes at pH 9.
In protein synthesis (such as Fmoc solid-state synthesizers), the N-terminus is often used as the attachment site on which the amino acid monomers are added. To enhance the electrophilicity of carboxylate group, the negatively charged oxygen must first be "activated" into a better leaving group. DCC is used for this purpose.
Active esters are often used in peptide synthesis, e.g., N-hydroxysuccinimide, hydroxybenzotriazole. [1] Active esters of acrylic acid are precursors to polymers with reactive side chains. [4] The concept of active esters extends to esters of phosphoric and sulfuric acids. One such case is dimethylsulfate, a strong methylating agent.
Common uses for this carbodiimide include peptide synthesis, protein crosslinking to nucleic acids, but also in the preparation of immunoconjugates. EDC is often used in combination with N-hydroxysuccinimide (NHS) for the immobilisation of large biomolecules. Recent work has also used EDC to assess the structure state of uracil nucleobases in RNA.