Acid-promoted tautomeric lactonization and oxidation-reduction of pyrroloquinoline quinone (PQQ)

Abstract

Acid-treatment facilitates PQQ detection by electron ionization mass spectroscopy with a molecular ion atM/e 330 and a base ion formed by triple decar☐ylation atM/e 198. Other ions found probably arise through acid-catalyzed tautomeric lactonization of PQQ to PQQ-lactone (PQQL) with subsequent oxidation of PQQL and reduction of PQQ. We propose that a car☐yl group, presumably the 9-car☐yl, attacks a double bond in PQQ, reversibly converting the 4,5-orthoquinone into an 4,5-enediol and forming an isomeric lactone, PQQL, of 330 daltons. The masking of carbonyls may explain the low reactivity of PQQ with carbonyl reagents in acid. Acid-promoted tautomeric lactonization with carbonyl-masking is known to occur with fluoresceins, phenolphthalein and other compounds, but has not been recognized before with PQQ. Acid-treated PQQ demonstrates molecular and other ions derived from reduced PQQ (PQQ(2H)) or its lactone atM/e 332 with a base ion atM/e 200. There is compelling evidence for a dehydrogenated lactone, PQQ(−2H)L), atM/e 328 with a base ion atM/e 196. We suggest that PQQ, in tautomeric equilibrium with PQQL, oxidizes PQQL to PQQ(−2H)L (328 daltons), with its concurrent reduction to PQQ(2H) (332 daltons). With acidified D 2O, PQQ shows deuterated products with ions atM/e values consistent with lactonization and oxidation-reduction. An analytically useful quinoxaline adduct, formed from PQQ and 2,3-diaminonaphthalene (PQQ-DAN) of 452 daltons, also undergoes acid-tautomerization-lactonization and oxidation-reduction similar to PQQ showing molecular ions atM/e 450, 452 and 454 and decar☐ylation-derived strong (base) ions atM/e 318, 320 and 322. Esterification of PQQ-DAN acid-products with triethyloxonium tetrafluoroborate gives triethyl esters derived from the 452 and 454 dalton compounds with very strong molecular ions atM/e 536 and 538, respectively. Ions derived from diethyl esterification of the 450 dalton compound atM/e 506 identify the 450 compound as the dehydrogenated monolactone of PQQ-DAN. Acid-hydrolyzed PQQ placed in neutral buffers yields PQQ(2H) as the main product with some PQQ also present. Both catalyze redox cycling and amplified formazan formation with glycinate and nitroblue tetrazolium. Therefore, acid hydrolysis of quinoproteins releases PQQ-products, detected well by redox-cycling. These products are not detected efficiently with carbonyl reagents in acid since acid-treatment of PQQ leads to 1) carbonyl group reduction and PQQ(2H) formation and 2) carbonyl masking of remaining PQQ by tautomeric lactonization.