Side reactions during photochemical cleavage of an alpha-methyl-6-nitroveratryl-based photolabile linker

The mechanisms of reactions causing irreversible inhibition of the activity of enzymes when irradiated in the presence of the recently developed alpha-methyl-6-nitroveratryl-based photolinker [Holmes CP. J. Org. Chem. 1997; 62: 2370-2380] have been investigated. Several experiments based on the interaction of the photolinker with model peptides or n-butylamine have been accomplished. A complexity of products, resulting from the side reactions competing with the 'normal' photocleavage of the linker, have been found. The amino and thiol groups of the molecules present in the solvents upon irradiation were recognized as having a major influence on the course of photolysis. Some of these side products resulting from the interaction with amines were identified and the mechanisms by which they can be generated are discussed. The mechanism of the interaction of the thiol groups present in peptides or proteins with the photolinker is unclear and it remains to be further elucidated. It was found that the undesirable effects are favored by a basic pH and are largely reduced by a slightly acidic pH, together with the presence of dithiothreitol. Significant positive effects of dithiothreitol have been observed on the rate as well as the yield of the photocleavage. These results demonstrate that the use of photolabile linkers in biological media can be accompanied by undesired effects, which can be largely reduced by choosing appropriate conditions and additives.