Abstract: Photocages have been successfully applied in cellular signaling studies for the controlled release of metabolites with high spatio-temporal resolution. Commonly, coumarin photocages are activated by UV light and the quantum yields of uncaging are relatively low, which can limit their applications in vivo. Here, syntheses, the determination of the photophysical properties, and quantum chemical calculations of 7-diethylamino-4-hydroxymethyl-thiocoumarin (thio-DEACM) and caged adenine nucleotides are reported and compared to the widely used 7-diethylamino-4-hydroxymethyl-coumarin (DEACM) caging group. In this comparison, thio-DEACM stands out as a phosphate cage with improved photophysical properties, such as red-shifted absorption and significantly faster photolysis kinetics

Abstract: Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. However, in anaerobic cholesterol-degrading bacteria such as Sterolibacterium denitrificans the primary carbon of the isoprenoid side chain is oxidised to a carboxylate in the absence of oxygen. Here, we identify an enzymatic reaction sequence comprising two molybdenum-dependent hydroxylases and one ATP-dependent dehydratase that accomplish the hydroxylation of unactivated primary C26 methyl group of cholesterol with water: (i) hydroxylation of C25 to a tertiary alcohol, (ii) ATP-dependent dehydration to an alkene via a phosphorylated intermediate, (iii) hydroxylation of C26 to an allylic alcohol that is subsequently oxidised to the carboxylate. The three-step enzymatic reaction cascade divides the high activation energy barrier of primary C-H bond cleavage into three biologically feasible steps. This finding expands our knowledge of biological C-H activations beyond canonical oxygenase-dependent reactions