Coenzyme models. Part 29. On the unusual spectroscopic behaviour of 10-dodecyl-3-methylisoalloxazine (amphiphilic flavin analogue) in aqueous solution

Abstract

At room temperature in aqueous solution, the visible absorption peak (S1) of the title isoalloxazine(3) gave a well resolved, three-band fine structure which is believed to appear only in apolar solvents or in enzymatic hydrophobic pockets where isoalloxazine is free of hydrogen-bonding effects. On the other hand, 10-ethyl-3-methylisoalloxazine (1) and another amphiphilic isoalloxazine, 10-butyl-3-hexadecylisoalloxazine (2) did not show this fine structure. We have observed that (i) the fine structure disppears upon addition of organic solvents (pyridine or ethanol) or surfactants above the critical micelle concentration, (ii) the blue shift of another absorption peak in the u.v. region (S2) does not occur, and (iii) the fluorescence intensity of aqueous (3) is much smaller than that of aqueous (1) but increases upon addition of organic solvents or surfactant micelles. These results are explicable on the basis of an aggregation-deaggregation equilibrium of (3) in aqueous solution, and the fine structure of aqueous (3) is attributed to the ‘stacking’ association of the isoalloxazine molecules. The reactivity of (3) in aqueous solution has been estimated through an investigation of the hydrolytic decomposition and oxidation of N-benzyl-1,4-dihydronicotinamide (BzIMH) and several thiols. In most cases, the rate constants for (3) were smaller by a few orders of magnitude than those for (1) but were significantly enhanced by the addition of organic solvents or cetyltriethylammonium bromide micelle. The results indicate that the reactions mediated by isoalloxazine, which mostly involve polarised transition states, are extremely unfavourable in the hydrophobic region of aggregated (3). These novel findings have significant implications for the chemistry of flavoproteins, because the surroundings of the binding sites of flavoprotelns have frequently been discussed in terms of the change in the absorption spectra of bound flavin coenzymes.