Abstract
A new sensitive and highly reactive fluorescence-labeling reagent (DMEQ-TAD) targeting conjugated dienes was developed. The fluorescent dienophile (DMEQ-TAD), in which fluorescent dimethoxyquinoxalinone (DMEQ) group is substituted via ethylene spacer on a dienophile, 1,2,4-triazoline-3,5-dione (TAD), was synthesized in 8 steps in 24% overall yield from dinitroveratrole. The reactions of DMEQ-TAD with six major vitamin D metabolites and some synthetic analogs were examined under various conditions. The reaction produced the corresponding 6,19-cycloadduct as a pair of the C (6) epimers (8 and 9) in quantitative yield. The structures of the adducts (8 and 9) including the stereochemistry at C (6) were unambiguously determined. The fluorescence-labeled vitamins were analyzed by HPLC with fluorescence detector. The new fluorometric method was used in the assay of plasma 25-OH-D_3, 24,25 (OH)_2D_3, and 25,26 (OH)_2D_3. The method was proved to be precise and reliable by comparing with the HPLC-UV method. The reaction of DMEQ-TAD with vitamin A metabolites and fluorometric assay of plasma retinoic acid was also examined.
