Cyanogen bromide

Template:Chembox new

Cyanogen bromide is the chemical compound with the formula CNBr. This colorless solid is widely used to modify biopolymers and fragment proteins and peptides. It is also used in organic synthesis.

Synthesis, basic properties, structure

Although the formula is written CNBr, cyanogen bromide is a molecule with the connectivity BrCN. Carbon and bromine are linked by a single bond and carbon and nitrogen by a triple bond. It is a linear species, as is the related cyanogen chloride (CNCl). Cyanogen bromide can be prepared by oxidation of sodium cyanide with bromine. This reaction proceeds via the intermediacy of cyanogen ((CN)₂).

NaCN + Br₂ → BrCN + NaBr

The compound is molecular, although polar. As such it dissolves in polar organic solvents and has a significant vapor pressure.

Biochemical applications

The three main uses of cyanogen bromide are to activate proteins, fragment proteins by cleaving peptide bonds, and synthesize cyanamides and other molecules. Side reactions can occur during cleavage.

Activating proteins

Activating proteins allows for covalent coupling to other reagents under mild conditions. Cyanogen bromide activation is the most common method for preparing affinity gels because it entails a simple procedure, cyanogen bromide reacts with the hydroxyl groups that are in many matrices, and the pH conditions of this method are mild enough for many sensitive biomolecules. CNBr reacts with hydroxyl groups on agarose to form cyanate esters or imidocarbonates, which are uncharged. These groups react with primary amines under mild conditions, resulting in the covalent coupling of a ligand to the agarose matrix. These reactions are shown in the figure below.

Although this method is widely used for immobilization of proteins onto agarose resin, there are disadvantages to this technique: CNBr is highly toxic and sensitive to oxidation. Also, the ligand is attached to the agarose resin by an isourea bond, which is unstable because it is positively charged at neutral pH. Consequently, isourea derivatives may act as weak anion exchangers.[1] Reductive amination is often used as an alternative method to covalently immobilize proteins onto agarose supports.

Cleaving proteins

Cyanogen bromide is also used to fragment proteins and peptides because of its ability to hydrolyze peptide bonds exclusively at the C-terminus of methionine residues. This cleavage is useful for characterizing complex proteins by separating and identifying peptides.

As shown in the figure on the right, CNBr is strongly electrophilic and is attacked by the nucleophilic sulfur on the methionine. This is followed by the formation of a five-membered ring. If a six-membered ring was formed, it would have a resonance structure with a double bond in the ring between the alpha carbon and adjacent nitrogen. Because a double bond forces the ring to assume a rigid conformation, the five-membered ring is formed instead so that the double bond is outside the ring. Although cysteine also has a nucleophilic sulfur atom, the peptide bond following this amino acid is not cleaved. This is because the cyanide adduct becomes rapidly deprotonated, leaving the sulfur uncharged and the beta carbon of the cysteine not electrophilic. Instead, the strongest electrophile is the cyanide nitrogen, which is most likely attacked by water, liberating toxic hydrogen cyanide gas and the original cysteine.

Common buffers for peptide bond cleavage include 0.1N HCl (hydrochloric acid) and 70% HOF (formic acid). There are advantages and disadvantages to both. Using HCl is often presented as an alternative to HOF because peptides containing hydroxyl groups will react with HOF to form formyl esters. However, HOF is still often used because it dissolves most proteins and can be easily removed by evaporation. Also, the oxidation of methionine to form methionine sulfoxide, which prevents CNBr cleavage, occurs more readily in HCl than in HOF, presumably because HOF is a reducing acid. Alternative buffers for cleavage include guanidine in HCl and urea in HCl.

Side reactions

When methionine is followed by serine or threonine, side reactions can occur that destroy the methionine without peptide bond cleavage. Normally, once the iminolactone is formed, water and acid can react with the imine to cleave the peptide bond and form a homoserine lactone and new N-terminal peptide. However, if the adjacent amino acid to methionine has a hydroxyl or sulfhydryl group, this group can react with the imine to form a homoserine without peptide bond cleavage. These two cases are shown in the figure.

Note that water is required for normal peptide bond cleavage of the iminolactone intermediate. In a formic acid matrix, cleavage of Met-Ser and Met-Thr bonds can be enhanced by increasing the water concentration, thereby driving the kinetics toward addition of water across the imine rather than reaction of the hydroxyl from the serine with the imine. It is also believed that lowering the pH of the matrix results in faster cleavage rates by lowering levels of methionine oxidation.

Organic synthesis

Cyanogen bromide is also widely used in synthesis since the electrophilic cyanide ion is attacked by nucleophiles such as amines, alcohols, and thiols. In the synthesis of cyanamides and dicyanamides, primary and secondary amines react with CNBr to yield mono- and dialkylcyanamides. This can further react with amines and hydroxylamine to yield guanidines and hydroxyguanidines. In the von Braun reaction, tertiary amines react with CNBr to yield disubstituted cyanamides and an alkyl bromide. CNBr can be used to prepare aryl nitriles, nitriles, anhydrides, and cyanates. It can also serve as a cleaving agent.

Toxicity, storage, and deactivation

Cyanogen bromide is extremely moisture-sensitive. As long as it is kept in very dry conditions at 2-8 °C, it can be stored for at least eighteen months. CNBr is also acutely toxic and can readily absorbed by body tissues through the skin. To deactivate CNBr in a solution not exceeding 60g CNBr/liter (dilute if necessary), the recommended method is to add 1_MNaOH and NaOCl in volumes of ratio 1:1:2 (CNBr solution:NaOH:NaOCl). The aqueous alkali hydroxide instantly hydrolyzes CNBr to alkali cyanide and bromide. The cyanide can then be oxidized by sodium or calcium hypochlorite to the less toxic cyanate ion. Note that deactivation is extremely exothermic.

References

Kaiser, Raymond; Metzka, Lorraine (1999), "Enhancement of Cyanogen Bromide Cleavage Yields for Methionyl-Serine and Methionyl-Threonine Peptide Bonds.", Analytical Biochemistry, 266 (1): 1–8

Lunn, George; Sansone, Eric B. (1985), "Destruction of Cyanogen Bromide and Inorganic Cyanides.", Analytical Biochemistry, 147 (1): 245–50

Schroeder, W.A.; Shelton, Joan Balog; Shelton, J. Roger (1969), "An Examination of Conditions for the Cleavage of Polypeptide Chains with Cyanogen Bromide: Application to Catalase.", Archives of Biochemistry and Biophysics., 130 (1): 551–6

Gross, Erhard; Witkop, Bernhard (1962), "Nonenzymatic Cleavage of Peptide Bonds: The Methionine Residues in Bovine Pancreatic Ribonuclease.", The Journal of Biological Chemistry, 237 (6): 1856–60

Kumar, Vinod (2005), "Cyanogen Bromide (CNBr)." (PDF), Thieme, 10: 1638–9

"Cyanogen Bromide Activated Matrices." (PDF), Sigma Product Information

"Cyanogen bromide." (PDF), NIH Division of Occupational Health and Safety

External links