Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Protein Structure
Protein Structure
Chemical Fragmentation - Mechanisms of Chemical Fragmentation Reactions

Cyanogen bromide Cleavage of Methionine is based on the reaction of cyanogen bromide with the thioether group of methionine, yielding a cyanosulfonium salt. The pathway of the subsequent transformations of this salt is illustrated in the scheme below.

Class="center">

Cyanogen bromide was first employed in studies of Ribonuclease. One of the longest protein chains successfully cleaved using cyanogen bromide is ß-galactosidase from E. coli (1021 amino acid residues).

Reaction conditions: Room Temperature, 15–30 hours, medium: 70% HCOOH, 100-fold molar excess of cyanogen bromide. Met-X bonds are cleaved by 90–100%. This reaction is uniquely selective. For the cleavage of highly resistant Met-Ser and Met-Tyr fragments, it is preferable to carry out the reaction in aqueous trifluoroacetic acid. In all resulting fragments, the C-terminal residues are homoserine lactone.

Side Reactions: In the presence of cyanogen bromide, Cysteine is oxidized to cystine and further to cysteic acid. Excess cyanogen bromide brominates Tyrosine residues and cleaves peptide chains at Tryptophan residues.

Replacing 70% formic acid with the stronger hexafluorobutyric acid and increasing the excess of cyanogen bromide (up to 10,000 mol/mol of protein) increases the yield of products resulting from the Hydrolysis of peptide chains at tryptophan.

Cleavage at tryptophan. Methods for the Selective Cleavage of peptide bonds at tryptophan residues rely on the high reactivity of the indole ring. Several such methods are available.

1. Cleavage at tryptophan using N-Bromosuccinimide.

Conditions: Room temperature, 2 hours, pH = 4.0, 2–3-fold excess of NBS. Peptides are cleaved by 50–90%, and Proteins by 10–60%.

2. Recently, 2-(2-nitrophenylsulfenyl)-3-bromo-3-methylindolenine (BNPS-skatole) has been predominantly used for the cleavage of Peptides and Proteins at tryptophan residues. This reagent is prepared by brominating 2-(2-nitrophenylsulfenyl)-3-methylindole (I) with N-bromosuccinimide in glacial acetic acid:

Compared to N-bromosuccinimide, BNPS-skatole acts more selectively. Numerous studies have been conducted on the application of BNPS-skatole in Protein Chemistry. The yield of selective cleavage products at tryptophan ranges from 40 to 80%. It is possible that the yield depends on the quality of the reagent.

BNPS-skatole decomposes at room temperature upon exposure to light, releasing bromine. However, well-purified crystalline preparations (pale yellow star-shaped crystals from ligroin) can be stored for a sufficiently long time in airtight packaging at 20°C.

3. Cleavage at tryptophan in the DMSO-hydrogen halide system

Cleavage at tryptophan using the DMSO-HBr mixture is carried out in acetic acid. In terms of product yield, this method is comparable to cleavage with BNPS-skatole and is preferable due to the greater availability of Reagents and simplicity of execution.

The reaction mechanism involves the Oxidative Halogenation of the tryptophan indole ring. Halogenation of the ring is effected by reaction intermediates formed between DMSO and HGal (sulfide dihalide (I), halosulfonium halide (II), free halogen).

Direct interaction of tryptophan-containing peptides with DMSO-HBr leads to The formation of an intermediate product, 2-dimethylsulfoniumtryptophan:

4. Cleavage at Trp using o-iodosobenzoic acid is performed in 80% acetic acid supplemented with 4 M guanidine hydrochloride. According to recent data, this method is not highly specific, as side Cleavage at tyrosine residues also occurs. If halogens are removed from the reaction mixture, peptide bond cleavage does not take place. It is suggested that The Mechanism of this process is analogous to other reactions employing halogenating agents (including N-bromosuccinimide), and that oxidative halogenation is a property of the o-iodosobenzoic acid : 4 M guanidine hydrochloride system. The likelihood of cleavage at tyrosine can be reduced by adding p-cresol. Under these conditions, the method becomes suitable for selective cleavage at tryptophan.

5. Other methods for cleavage at tryptophan are also known. Among these is cleavage using active iodine, which is generated in the system: hydrogen peroxide - iodide salts - lactoperoxidase (or horseradish peroxidase). The yield is 30–40%. Similar yields are obtained using the iodinating agents J₂, J₃^-, JCl, or the chloramine T / KI mixture.

Cleavage at the carboxyl group of tryptophan in an acidic medium can be achieved using N-chlorosuccinimide. Other bonds remain unaffected, including those at tyrosine or Histidine. Reaction yields and rates are lower than those obtained with N-bromosuccinimide.

Peptide bonds of tryptophan in PROTEINS AND PEPTIDES can be selectively cleaved using 2,4,6-tribromo-4-methylcyclohexa-2,5-dienone (tribromocresol):

The peptide bonds of tyrosine and histidine are not affected. However, tyrosine is converted into a 3,5-dibromo derivative, cysteine is oxidized to cysteic acid, methionine is converted to methionine-S-oxide, and histidine undergoes modification.

Cleavage at tyrosine residues. If the protein molecule lacks tryptophan residues, selective cleavage at tyrosine can be achieved using N-bromosuccinimide.

Dienospirolactone is a strong chromophore with λ_mах = 260 nm (s = 10000-11000).

This method is frequently used to study Histones, which do not contain tryptophan residues. The yield is typically non-quantitative, but increases with a higher excess of the reagent.

Reaction conditions: 20∘С, 6 hours, 50% СН₃СООН, reaction yield 30-65%. In the presence of tryptophan, tyrosine cleavage occurs only with a large excess of NBS.

Cleavage at the cysteine residue. The method for selective peptide bond cleavage at the amino group of cysteine is based on converting SH groups into thiocyanate groups. Cleavage at Cysteine using cyanides was first performed in 1964–65. The reaction mechanism is shown in the scheme below.

The reaction initially yields thiocyanoalanine residues, which cyclize into acyliminothiazolidine. This is followed by rapid hydrolysis of the peptide bond. Disulfide bond cleavage by cyanides leads to partial and random formation of thiocyanate groups.

A method has been proposed for the direct and quantitative conversion of SH groups into their corresponding thiocyanate derivatives using 2-nitro-5-thiocyanobenzoic acid:

It has also been demonstrated that 1-cyano-4-dimethylaminopyridinium salts act as active cyanating agents for SH groups in proteins:

Other chemical Methods for Peptide bond cleavage

Cleavage at the Asn-Gly bond by hydroxylamine relies on the ability of the asparagine side chain to cyclize, forming a substituted succinimide. The latter undergoes nucleophilic attack by hydroxylamine, resulting in peptide bond cleavage. The enhanced lability of the Asn-Gly bond in this reaction is apparently due to the lack of steric hindrance to cyclization.

A brief outline of the reaction mechanism is as follows:

The Gly-Gly bond is not cleaved. Conditions: 20∘С, 4 hours, pH = 9, 6 M guanidine •HCl + 2% NH_2OH•HCl

Cleavage at the Asp-Pro bond. This bond is the most labile under acidic hydrolysis conditions and is readily cleaved even under mild conditions: 10% CH₃COOH + pyridine, pH 2.5, 7 M guanidine hydrochloride, 40 C, 4 days.

A method is known for cleaving the peptide bonds of Serine and Threonine, based on the preliminary oxidation of the hydroxyl group by dicyclohexylcarbodiimide (DCC) in the presence of phosphoric acid in a DMSO medium, followed by reaction with phenylhydrazine. The intermediate phenylhydrazone product cyclizes into a pyrazole with the simultaneous elimination of a fragment containing a free N-terminal amino group.

Another possible approach involves the formation of an intermediate hydrazone under the action of hydroxylamine. It cyclizes into an isoxazoline, yielding a peptide chain fragment with a free amino group. The reaction scheme is shown below: