Reactions of aliphatic thiyl radicals in the solid state: photoisomerization of trans-4,5-dihydroxy-1,2-dithiacyclohexane and oxidation of dithiothreitol.

A description of free-radical reactions in the solid state is important for some processes causing long-term stability problems of natural and synthetic products. Recent studies revealed that, in the solid state, mercaptooctadecanethiyl radicals, C(18)H(37)S., do not abstract a hydrogen atom from mercaptooctadecane, C(18)H(37)SH, but yield perthiyl radicals, C(18)H(37)SS., via a net sulfur transfer (Faucitano et al. ChemPhysChem 2005, 6, 1100-1107). Here, we demonstrate that such a sulfur transfer is not a general phenomenon of thiyl-radical reactions in the solid state, providing experimental evidence for a solid-state hydrogen-transfer reaction between a dithiyl radical, generated through the photolysis of trans-4,5-dihydroxy-1,2-dithiacyclohexane (DTT(ox)), and dithiothreitol. The photolysis of crystalline solid deposits of DTT(ox) yields two isomers of 2,3-dihydroxy-1-mercaptotetrahydrothiophene with a combined quantum yield of Phi(F) = 0.39 +/- 0.02. This quantum yield was increased to Phi(F) = 0.87 +/- 0.13 when the solid deposits contained an additional dithiol, dl-1,4-dimercapto-2,3-butanediol (DTT), at a ratio of DTT/DTT(ox) = 10:1. This increase in quantum yield depended, in part, on the presence of oxygen but was independent of residual moisture in the solid samples. Mechanistically, the formation of 2,3-dihydroxy-1-mercaptotetrahydrothiophene can be rationalized by the H transfer from DTT to a photochemically formed dithiyl radical from DTT(ox), yielding 2 equiv of monothiyl radicals from DTT, followed by a series of radical transformations.

Solid-state photodegradation of bovine somatotropin (bovine growth hormone): evidence for tryptophan-mediated photooxidation of disulfide bonds.

Lyophilized recombinant bovine somatotropin (rbST; bovine growth hormone) is sensitive to photoinduced degradation. The underlying mechanisms of these processes are identified and presented. Lyophilized rbST was photolyzed with near-ultraviolet (UV) light between 305 and 410 nm, and the protein content was analyzed by various bioanalytical techniques, including tryptic mapping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino acid analysis, and fluorescence, UV, Raman and Fourier transform infrared (FTIR) spectroscopy. The solid-state photodegradation of rbST by near-UV light exclusively targets the protein disulfide bonds. The reaction is initiated by photoionization of tryptophan (Trp) and one-electron reduction of the disulfide. However, in contrast to the behavior of other proteins in solution, rbST appears to undergo back electron transfer to restore Trp and yield a pair of cysteine (Cys) thiyL radicals, which add molecular oxygen and ultimately recombine to yield alpha-disulfoxide, thiosulfinate, and/or thiosulfonate. Photodegradation is strictly dependent on the presence of molecular oxygen, but does not involve singlet oxygen. Between 0.4 and 10%, residual moisture levels do not affect the rate of photodegradation. Our results show a novel mechanism for Trp-mediated photodegradation of protein disulfide bonds via formation of a pair of thiyL radicals followed by addition of molecular oxygen.