Disulphides formation

Other mild oxidising agents which abstract the terminal hydrogen atoms and thus facilitate disulphide formation may be used as vulcanising agents. They include benzoyl peroxide, p-nitrosobenzene and p-quinone dioxime. 

Nitrosothiols can also be reduced with sodium borohydride, leading (with SNAP14) to the disulphide formation (equation 18). 

Substrate analogues containing the mercaptan functionaUty have been extensively investigated as collagenase inhibitors, and some other sulphur-based functionalities have also been explored [1,161,172-185]. The mercap-tans tend to be very potent inhibitors of all of the MMP, presumably due to the strong interaction between the active site Zn(II) and the mercaptide anion. Unfortunately, these compounds tend to undergo inactivation by oxidative disulphide formation. However, the rate at which this occurs varies widely and depends on the structure of the inhibitor. The most common synthetic route to these derivatives again leads to a diastereomeric mixture. 

Among the out-of-register mercaptans, derivatives (115) and (116) are equivalent in potency to their in-register counterparts (90) and (91), but with a reversal in stereoselectivity as indicated by the modest preference for the L-Leu analogue stereoisomer in subsite Pj. The thiophenol derivative (118) is very potent, at least in the presence of y9-mercaptoethanol which presumably suppresses disulphide formation. 

The photostimulated reactions of thiolate anions with 2-halo-2 -nitropropane derivatives yield both oc-nitrosulphides via an S l pathway and disulphides (equation 71a)282 284. In contrast with the case of the oxidative dimerisation products of the mono-enolates, the disulphides are formed via an ionic mechanism nucleophilic attack by the thiolate anion on the a-halogen and subsequent reaction of a second thiolate with the sulphenyl halide. As expected for such a process, disulphide formation is favoured (and thus a-nitrosulphide formation is disfavoured) the more nucleophilic the thiolate (i.e. derived from a less acidic thiol) and the easier the abstraction of the halo-substituent (i.e. I > Br > Cl). Use of the protic solvent methanol instead of the usual dipolar aprotic solvents for the reaction of equation 71a is detrimental to the yield of the S l substitution products exclusively disulphides are formed285 (equation 71b). Methanol solvation probably retards the dissociation of the radical anion intermediate in the SRN reaction, into radical and anion, and hence retards the chain reaction relative to the ionic reaction. The non-nucleophilic methylsulphinate ion gives only an S l reaction product with 2-bromo-2-nitropropane286. 

Zahn and coworkers effected the interchain oxidation of resin-bound cystein peptides to cystin peptides by intersite disulphide formation in good yields 149>150>. 

Ethane thiol reacts more readily than the methyl compound, but the main features of the reaction (e.g. the effect of reactant concentration) are similar. The products include some acid (assumed to be peracetic or acetic) as well as sulphur dioxide and acetaldehyde the large sulphur deficit is again ascribed to disulphide formation. In oxygen-rich mixtures all the sulphur is converted to sulphur dioxide. 

NO-induced protein S-glutathionylation was proposed for the first time in 1988 by J.W. Park as a possible mechanism for the inactivation of yeast alcohol dehydrogenase by NO [32]. However, it took almost 10 years until the possibility that NO might be able to direct the incorporation of GSH to protein sulfhydryls was reconsidered. In 1997. it could be demonstrated that micromolar concentrations of GSNO inhibit aldose reductase through site-specific mixed disulphide formation at a conserved cysteine residue in its catalytic site... 

Diphasic kinetic behaviour in the reduction of cyt c(iii) by glutathione, which has the potential to recognize different protein forms, suggests - the existence of two cyt c(iii) conformers which may be interconverted by protonation (pl ra 7) followed by a slow conformational change. Transient glutathione-cyt c(iii) complexes were detected at the 339 nm isosbestic and the retention of the 695 nm absorption indicates that the site of complexation is not close to the haem iron. Decay of the transient at 339 nm is consistent with slow disulphide formation. 

Like disulphide formation, the coupling of thiols and primary or secondary amines is of great industrial importance in the manufacture of rubber vulcanisation accelerators, the products being sulphenamides. 

Oxidation is carried out with 30% H2O2 at pH 10-10.5, 50°C, in the presence of excess amine to minimise disulphide formation, the sulphena-mide separating as a precipitate or melt [239]. Higher pH tends to overoxidise the thiol to sulphinate etc. Hypochlorite can also be used for this oxidation, the factors affecting oxidant choice being described in section 9.5.1.8.2. Chief commercial products include iV-cyclohexyl-, AT-r-butyl- and A/ -morpholino-2-thiobenzothiazole sulphenamides, and total production is of the same order as the disulphides. 

However, a major disulphide contribution to structures within the cell is made unlikely by the observation that disulphide bonds are relatively rare in intercellular proteins. In fact we have already discussed the possible role of glutathione in maintaining protein thiols in the reduced state. It is really with proteins that operate outside the cell that one finds the great importance of disulphide-stabilized structures. One can reasonably rationalize this fact in two ways. Since the protein must operate without the protective environment of the cell, random disulphide formation would eventually occur. By initially fixing most thiols as disulphides in an active configuration the chances for deleterious random disulphide formation... 

The oxidation of thiols with sulphoxides presents several attractive features like the simplicity of the reaction, the high yield and the selectivity of disulphide formation. It has to be noticed, however, that tertiary thiols do not react with sulphoxides or they give very little disulphide even in the presence of amine catalysts. Reaction temperatures higher than 100°C give rise to extensive decomposition ... 

This puts some doubt on the hypothesis that disulphide formation stems from dimerization of free thiol radicals as indicated in the simplified mechanism (Scheme 7) reported below. 

Sometimes, the reduced peptide is prone to rapid re-oxidation, not necessarily to the desired products. In these cases, it is recommended to carry out a rapid (< 2 min) desalting by passing the reduced peptide through a PD-10 column (Sephadex G-2SM, Pharmacia), eluted with the buffer chosen for the disulphide formation/folding process. 

Figures. Intramoecular disulphide formation mediated by solid-phase Ellman s reagents.

A. Disulphide formation through activation with DTNP (121) and... 

Collectively, these results support the use of basic pH to inhibit hydrolysis of the peptidyl thioester, and the strong reducing environment of a smaU thiol and trialkylphosphine to prevent A ,5-diacyl by-products and to reduce disulphide formation. Using these optimized conditions, peptides ranging from 9 to 88 amino adds with 60-88% yield were prepared. The methods for preparation of the thioester resin and ligation are described below. 

Incidental reference has been made to disulphide formation elsewhere in this Chapter, and a number of alternative methods have been explored /3-dimethylaminoethyl and y-dimethylaminopropyl diselenides have been prepared from the corresponding alkyl chloride and NagSeg cyanolysis... 

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