The main uses of cyanogen bromide are to immobilize proteins, fragment proteins by cleaving
peptide bonds, and synthesize
cyanamides and other molecules.
Protein immobilization Cyanogen bromide is often used to immobilize proteins by coupling them to
reagents such as
agarose for
affinity chromatography. Because of its simplicity and mild
pH conditions, cyanogen bromide activation is the most common method for preparing affinity gels. Cyanogen bromide is also often used because it reacts with the
hydroxyl groups on agarose to form
cyanate esters and
imidocarbonates. These groups are reacted with
primary amines in order to couple the protein onto the agarose matrix, as shown in the figure. Because cyanate esters are more reactive than are cyclic imidocarbonates, the amine will react mostly with the ester, yielding
isourea derivatives, and partially with the less reactive imidocarbonate, yielding substituted imidocarbonates. The disadvantages of this approach include the toxicity of cyanogen bromide and its sensitivity to oxidation. Also, cyanogen bromide activation involves the attachment of a
ligand to agarose by an isourea bond, which is positively charged at neutral pH and thus unstable. Consequently, isourea derivatives may act as weak
anion exchangers. These are the most common buffers for cleavage. An advantage to HCl is that formic acid causes the formation of formyl esters, which complicates protein characterization. However, formic is still often used because it dissolves most proteins. Also, the oxidation of methionine to
methionine sulfoxide, which is inert to BrCN attack, occurs more readily in HCl than in formic acid, possibly because formic acid is a reducing acid. Alternative buffers for cleavage include
guanidine or
urea in HCl because of their ability to
unfold proteins, thereby making methionine more accessible to BrCN. Water is required for normal peptide bond cleavage of the
iminolactone intermediate. In formic acid, cleavage of Met-
Ser and Met-
Thr bonds is enhanced with increased water concentration because these conditions favor the addition of water across the
imine rather than reaction of the side chain hydroxyl with the imine. Lowered pH tends to increase cleavage rates by inhibiting methionine side chain oxidation.
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 (refer to figure), water and acid can react with the imine to cleave the peptide bond, forming a
homoserine lactone and new C-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. == Organic synthesis ==