“…34 Initially, ratiometric labelling suggested the loss of the C-4 proton of D-glucose in the course of 2-DOS biosynthesis. 35,36 The in vitro conversion of D-glucose-6-phosphate into 2-DOS was observed in cell-free extracts of S. fradiae and was found to be NAD + -dependent, as expected for the proposed mechanism. 37, 38 Whereas the aforementioned in vivo labelling studies indicated loss of the C-4 hydrogen in the course of the cyclisation, in vitro conversion of [4-2 H]-D-glucose to 2-DOI was found to proceed with retention of the deuterium label; 39,40 the discrepancy may be explained by the kinetic isotope effect, which could cause untritiated substrate to be preferentially converted to product over the labelled compound, leading to depletion of the label in the product.…”