Thiol groups are encountered in biological systems in cysteine residues and in cofactors such as lipoamide and lipoic acid. They are also called sulfhydryl and mercapto groups. When Zeise discovered C2H5SH in 1834, he called it "mercaptan" (corpus mercurium captans) because the formation of mercury derivatives was a striking characteristic. The thiol group is the most chemically reactive group that is normally encountered in biological systems. It is a powerful nucleophile that undergoes a wide variety of chemical reactions, many of which are exploited in its biological functions. A most important property is its tendency to ionize,
to the thiolate anion, which is usually the reactive species. The nonionized thiol group is usually unreactive. Most alkyl thiol groups, such as those of cysteine residues, have p^a values close to 9.
Therefore, they are reactive only at alkaline pH values, where significant amounts of thiolate anion are present. The thiolate anion is known as a soft nucleophile, poorly solvated, highly polarizable, with vacant d-orbitals and nucleophilic power much greater than would be predicted from its basicity. Thiol groups have only very weak hydrogen-bonding capabilities.
Thiol groups under too many chemical reactions to catalogue completely. Only the most important are described here.
Thiol groups are readily oxidized by oxygen, especially in the presence of trace amounts of metal ions, such as; it is likely that the complex of metal and thiol (see below) is the actual reactant with oxygen. Thiol groups may be oxidized to various oxidation states, but some of them are intrinsically unstable. In addition to the thiol form, only two oxidation states are generally encountered, the disulfide and the sulfonic acid. The disulfide is usually the end product of air oxidation:
Disulfide bonds between cysteine residues are often found in proteins, especially those secreted from cells. These disulfide bonds are not produced by air oxidation, however, but are incorporated by the catalyst protein disulfide isomerase .
The sulfonic acids are produced by more potent oxidizing agents. For example, performic acid oxidizes both thiol and disulfide forms of Cys residues of proteins to cysteic acid, with the CH^-SO-
3 side chain. The intermediate sulfenate (-SO-) and sulfinate (-SO^) oxidation states are generally unstable and not normally present. They have been identified, however, when specifically stabilized in a protein structure, as in the case of the cysteine sulfenic acid that is involved in the catalytic mechanism of NADH peroxidase (1).
2. Thiol-disulfide exchange
Thiolate anions react rapidly with disulfide bonds, displacing one sulfur atom of the disulfide bond and taking its place:
Such thiol-disulfide exchange reactions will continue among all the thiol and disulfide species present until an equilibrium mixture is generated. Thiol-disulfide exchange reactions are extremely specific, and that with 5,5′-dithio-bisnitrobenzoic acid (DTNB), or Ellman’s reagent, is the most convenient and accurate method of assaying thiol groups quantitatively.
If one of the sulfur atoms of the disulfide is oxidized to the sulfonate, it does not take part in thiol-disulfide exchange, and only a single reaction will take place if the reagent is present in great excess over the thiol group:
Such reactions are very useful for making specific mixed disulfide species in stoichiometric quantities. The same can be accomplished with thiocyanate derivatives in which the thiol displaces the cyanide ion:
Thiolate anions react rapidly with alkyl halides, such as iodoacetamide, iodoacetate, and methyl iodide:
Such reactions are irreversible and the adduct generated is very stable.
4. Addition across double bonds
Thiolate anions are sufficiently nucleophilic to add across C=C double bonds, as in maleic anhydride. #-Ethylmaleimide is the classic reagent that is used most frequently to modify thiol groups:
With cysteine residues in proteins, the resulting side-chain is now positively charged and is analogous to a lysine or arginine side-chain. Consequently, proteolytic enzymes such as trypsin will cleave the peptide bond after such a residue, so this modification is used frequently in peptide mapping studies of proteins.
5. Metal ions
Thiol groups form complexes of varying stabilities with a variety of metal ions. The most stable are those with divalent mercury, Hg , but its divalency means that complexes with a variety of stoichiometries are formed. Consequently, univalent organic mercurials of the type R-Hg+ tend to be used instead, because they more reproducibly form one-to-one complexes with thiol groups. Such reactions with mercurials are the most obvious and one of the more useful ways to make heavy-atom derivatives for X-ray crystallography determination of protein structure (see Isomorphous Replacement).
Thiol complexes with silver are less stable than those of mercury, but univalent Ag+ reacts stoichiometrically and can be used to titrate thiol groups. Copper, iron, zinc, cobalt, molybdenum, manganese, and cadmium ions all form various complexes with thiol groups.