Cyanogen bromide

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Cyanogen bromide

Cyanogen bromide is a chemical primarily used to 'activate' biopolymers, for example, polysaccharides (such as Sepharose, for use in chromatography columns), or protein materials (for immunoassays), for easy subsequent covalent coupling to other reagents under relatively mild conditions. CNBr reacts electrophilically with the pendant hydroxyl groups of proteins and polysaccharides, forming imidocarbonate and carbamate groups. When using this 'pre-activated' protein or polysaccharide, the imidocarbonate groups react with amino groups belonging to subsequents reagents (such as proteins being passed through the column) with the formation of stable covalent linkages, which are generally broken in a controlled fashion by changing pH during elution of the column.

Cyanogen bromide is also used to fragment proteins and peptides into smaller pieces. This is useful in the production of methionine-free peptides and for peptide mapping in proteomics, since the process occurs exclusively at methionine residues. It is disfavored due to the toxicity of cyanogen bromide, both in neat (solid) and solution forms. However, very little cyanogen bromide is required to quantitatively cleave proteins and peptides; unlike most proteases which are usually used to cleave peptides, cyanogen bromide cleaves a peptide cleanly at the C-side of Methionine residues. The leading potease which does this is Factor Xa and cyanogen bromide has a great cost advantage over this, especially in industrial processes.

The mechanism is as follows:

Cyanogen bromide is strongly electrophilic and is attacked by the nucleophilic sulfur on the methionine. The resulting adduct is now most electrophilic at the gamma-carbon position, and since a five-membered ring is more kinetically favorable than the six-membered ring, the C-terminal carbonyl of the methionine attacks the electrophilic position with assistance from its amide nitrogen.

Cysteine, while also nucleophilic, fails to be cleaved because the cyanide adduct becomes rapidly deprotonated, the sulfur is uncharged, and the beta carbon of the cysteine is therefore not electrophilic. Instead, the strongest electrophile is the cyanide nitrogen, which is most likely attacked by water, liberating hydrogen cyanide and the original cysteine.

Cyanylation techniques (under different conditions) have also been reported which cleave proteins and peptides with a clean C-terminus at cysteine, selectively in the presence of methionine.