[New fluorogenic dienophile: synthesis, reaction with vitamin D, vitamin A and microcystins, and application to...

We designed and synthesized a new sensitive and highly reactive fluorogenic reagent (1a, DMEQ-TAD) targeting a conjugated diene. DMEQ-TAD reacted quantitatively with major vitamin D metabolites and synthetic analogs under a variety of conditions to yield the corresponding 6,19-cycloadducts as a mixture of the C(6) epimers. The stereochemistry of the adducts was determined by their CD spectra on the basis of the exciton chirality method. The fluorescent products can be quantified linearly down to 10 fmol by HPLC. The new fluorometric method was successfully used in the assays of 25-OHD3, 24,25-(OH)2D3 and 25,26-(OH)2D3 in the human plasma. The method was proved to be reliable and precise compared with the HPLC-UV assay method. Reactions of DMEQ-TAD with retinoic acid (21a) and its geometrical isomers (21b-d) having a conjugated pentaene system afforded 7,10-adducts (24a-d) as major products together with 5,8-adducts (26a-d) in about 9:1 ratio (70-95% yield) except for the 9Z-isomer (21e) which gave only a 5,8,11,14-bis-adduct (27). DMEQ-TAD reacted with microcystins LR, YR and RR, at the conjugated diene part to yield 4,7-cycloadducts as a pair of epimers in good yield.