Disulfides oxidative cleavage

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

Figure 4-3. Oxidative cleavage of adjacent polypeptide chains linked by disulfide bonds (shaded) by per-formic acid (left) or reductive cleavage by 3-mercap-toethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively.

Witkiewicz, P.L. and Shaw, C.F. Ill (1981) Oxidative cleavage of peptide and protein disulfide bonds by gold(III) amechanism for gold toxicity. Journal of the Chemical Society, Chemical Communications, (21), 1111-1114. 

The proposed mechanism of the oxidative cleavage of S-protecting groups by the chlorosilane/sulfoxide procedure is outlined in Scheme 8. 95 The first reaction is considered to be formation of the sulfonium cation 9 from diphenyl sulfoxide (7) and the oxygenophilic silyl compound 8. The formation of a sulfonium ion of this type is known and has been utilized for the reduction of sulfoxides. 97 Subsequent electrophilic attack of 9 on the sulfur atom of the S-protected cysteine residue leads to the formation of intermediate 10, whereby the nature of the silyl chloride employed should be the main factor that influences the electrophilicity of 9. The postulated intermediate 10 may then act as the electrophile and react with another S-protected cysteine residue to generate the disulfide 11 and the inert byproduct diphenyl sulfide (12). This final step is analogous to the reaction of a sulfenyl iodide as discussed in Section 6.1.1.2.1. 

The simplest approach is offered by parallel homodimers which require only two orthogonal protecting groups. After deprotection of one thiol group mild oxidation by one of the methods discussed in Section 6.1.1.1 or following direct selective oxidative cleavage of one S-protected cysteine residue by the methods described in Section 6.1.1.2, the second disulfide bond has to be produced by selective procedures compatible with the presence of a disulfide as described in Section 6.1.2. 

Thus, oxidation reactions are possible under very mild conditions and with high selectivities. Therefore, this method is especially useful for oxidative deprotections. For example, the oxidative cleavage of the carbon-sulfur bond is easily possible according to Eq. (103). In this way, disulfides and follow-up products of carbenium... 

The first step, protein purification, is discussed in chapter 5. Once the protein is pure, sequence analysis can begin, with cleavage of the disulfide bonds. Cleavage is achieved by oxidizing the disulfide linkages with performic acid (fig. 3.17). Sometimes this step results in the production of two or more polypeptide chains, in which case the individual chains must be separated. 

A final problem is to determine which cysteines are connected by disulfide links. Usually, the disulfide bonds are broken by either oxidative cleavage with performic acid or reductive cleavage with 2-mercaptoethanol before initial sequencing is undertaken ... 

The relative ease of oxidation of a model disulfide, di-DNP-cystine, is illustrated in Table XIXe, where the amount of oxidant used was just in 50% excess (Ramacbandran, 1961). The yield of 73% DNP-cysteic acid is not far from values for yields of cysteic acid (80-90 %) from performic acid oxidation of cystine (Schram et ah, 1954). Proteins with reactive —SH, —S—S—, or free —NH2 groups therefore pose problems when side reactions in the oxidative cleavage of peptide bonds is to be avoided (cf. Section V, B). 

Oxidative cleavage of disulfides can be made by chemical and electrochemical means thus anodic oxidation at platinum or carbon electrodes of different kinds of disulfides in the presence of alkenes in acetonitrile affords products of acetamidosulfenylation with cyclic alkenes a high selectivity for trans addition is observed. The reaction may be formulated ... 

The actual oxidizing species present depends on pH and is either chlorine or hypochlorous acid (HOCl). Apparently, the hypochlorous acid is the more active species on hair given that degradation is greater at lower pH. Although the chemistry of these interactions was not examined, one would expect disulfide bond cleavage and peptide bond fission similar to the effects shown for the reaction of chlorine and wool fiber [42]. 

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