The title compound was obtained by McElroy et al. as a crude product without definite characterization due to its instability and the lack of a suitable analytical method other than bioluminescence. In this research, reliable methods for analyzing and isolating the unstable luciferyladenylates were established by means of HPLC. Firefly L-luciferin was condensed with adenylic acid, the chiral center of L-luciferin being epimerized when adenylated. After evaporating the solvent, the residue was washed with EtOAc and CH2C12, and extracted with 6 M urea. The extract was chromatographed on a reversed-phase C18-HPLC column. The D-luciferyl-D-adenylate contained less than 8% luciferin and its diastereomeric purity was more than 98%. The luciferyladenylates are extremely unstable; they are hydrolyzed to luciferin and adenylic acid, and epimerized quite easily in neutral to basic buffers. L-Luciferyl-D-adenylate emits light with firefly luciferase of about 1/3 the light yield compared to that of the D, D-isomer, possibly because of partial epimerization of the L, D-isomer to the D, D-isomer during the reaction.
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Imai et al. (1988) studied this question.