Cysteine Thioaldehydes: Photolytic Generation, Reactivity and Biological Implications

Cysteine thioaldehydes are putative intermediates in formylglycine-generating enzyme (FGE) catalysis and in the biosynthesis of penicillin, coenzyme A and some ribosomally synthesized and posttranslationally modified peptides (RiPPs). Norrish type-II photolysis of phenacylsulfides has been used to generate cysteine thioaldehydes, but this process can be inefficient owing to competing -scission, which leads to unproductive formation of cysteine disulfides and oxidising enol radicals. We show that irradiation of phenacylsulfides bearing non-conjugating electron-withdrawing substituents (e.g. 4-trimethylammonium) leads to efficient Norrish type-II fragmentation. In aqueous buffer, photolysis of cysteine phenacylsulfides leads to high conversion into cysteine enethiol (pKa 4.3–4.8) and isothiazolone, which can be interconverted through the addition of an oxidant (Ellman’s reagent) or reductant (TCEP). Cysteine enethiolates are relatively stable in oxygen-free aqueous buffer at neutral-to-high pH, but undergo cyclocondensation to cysteine thiazole at low pH. Cysteine thioaldehydes within short peptides in aqueous buffer do not undergo appreciable levels of hydrolysis to give formylglycine; instead, tautomerization/deprotonation to the enethiol/enethiolate is dominant. However, such hydrolysis is observed in the absence of buffer and for longer peptides with high -helical character, being most prevalent for peptides displaying the FGE consensus sequence. Hydrolytically released dihydrogen sulfide efficiently traps cysteine thioaldehyde, ultimately leading to dithioacetal and 1,2,4-trithiolane derivatives. Depending on peptide sequence and secondary structure, nucleophilic participation of the proximal peptide backbone carbonyl group on cysteine thioaldehydes can lead to peptide oxazoles. These results reveal that the hydrolysis of cysteine thioaldehydes into formylglycine, a transformation required for the activation of sulfatases, is not trivial and highlights the essential role of the enzymatic pocket of sulfatase maturing enzymes, like FGE, for high efficiency and a rationale for the identity and location of certain amino-acid–substrate interactions. Furthermore, the facility by which cysteine thioaldehydes undergo transformations into peptide thiazoles and oxazoles suggests that alternative oxidation-before-cyclocondensation biosynthetic pathways deserves strong consideration for some niche families of these natural products