Light‐Induced Alkylation and Dealkylation of the Flavin Nucleus

Abstract

The spectral course of irreversible photoreduction of the flavin nucleus by arylacetates and related compounds has been investigated in detail. The resulting benzylated dihydroflavins and their reoxidation products have been characterized spectrophotometrically, among them 5-benzylflavosemiquinone radical and 5-benzylflavoquinonium cation. Reversible and irreversible flavin reduction corresponding to hydride-or group-transfer from substrate to flavin are discussed with respect to their scope and limitations and possible biological implications in the action mechanism of flavin dependent dehydrogenases.Flavin sensitized photodecarboxylation of phenylacetate has been reported previously by our group [ 1,2] and was found to result in a new and irreversible type of flavin photoreduction, i . e. benzyl group transfer (rather than hydride or electron transfer) towards the acceptor positions 5 or 4a of the flavin nucleus (I). While "H-transfer" (the term "H-transfer" is meant formally regardless of the mechanism, i . e. hydride, H-atom or whatsoever), obtained for example with mandelic acid, is reversible under all conditions tested, the group-transfer reaction is I complicated by the fact that either product, 4a-benzyl-4a,5-dihydroflavin (11, "4a-~CH2FlredH") and 5-benz yl-1,Ei-dihydroflavin (111, "5-cpCH2F1,,dH"), can be isolated and has its own way of reoxidation with 0,, which is different from that of the unsubstituted Flr,aH, ("leucoflavin" or 1 ,5-dihydroflavin), the only dihydroflavin known before [3]. These pathways are illustrated in Scheme 1.This shows that also group transfer is reversible under certain conditions. The conditions imply action Trivial Names and Unusual Abbreviations. Flavin, 10-substituted 7,8-dimethyl-isoalloxazine ; lumiflavin, 7,8,10-trimethyl-isoalloxazine; Elox, flavin in the neutra!, oxidized state; Fl,,aH,, reduced flavin (dihydroflavin) ; HFI, flavosesemiquinone radical; pCH,, benzyl. of light for 4a-yCH,Flr,dH and of mild acid forIt is evident that, in order to interpret the structure and function of intermediates in flavin dependent biological oxidations, the physical properties of group transfer as well as H-transfer products and all possible types of reduced flavin should be characterized thoroughly. This alone might help to understand the reasons, originating from the substrate structure or environmental conditions, which switch the course of flavin dependent "substrate" oxidation from group towards H-transfer and vice versa. and polar (R, = CH,COO-) environments or a t least sufficient solubility stretching out over a wide polarity range from water to toluene (R, = CH,). 5-@3HzFlredH. MATERIALS AND METHODS AllAll other reagents were best grade from Fluka A. G. (Buchs, Switzerland), with the exception of tetraacetylisoriboflavin, obtained by reductive alloxane condensation (and subsequent acetylation in pyridine) of Z-phenylaz0-3,4-dimethyl-N-ribitylaniline, which in turn was a gift from HoffmannLa Roche.Optical spectra were run on Cary 14 instruments.The photochemical light sou...

Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors

“…This spectrum is similar to the corresponding adducts obtained from S. shibatae and T. thermophilus flavin-vIPP isolated under aerobic conditions (15). Thus, the flavin-vIPP adducts are not sensitive to oxygen, unlike the behavior anticipated for reduced flavins alkylated at N5 (21). In addition, N5-alkylated flavins are reported to have absorbance maxima between 320 and 355 nm at pH 5-8, whereas C4a adducts have maxima between 360 and 385 nm with a shoulder at approximately 300 nm (19,(22)(23)(24).…”

“…The bathochromic shift in strongly acidic solutions is characteristic of C4a-flavin adducts, in contrast to the hypsochromic shift observed for N5 adducts. In addition, the adducts were stable in the presence of oxygen at pH 6 or, in 6N HCl, under conditions where N5 adducts typically oxidize to give long-lived semiquinones (21). Although the possibility of an N5 to C4a rearrangement cannot be ignored, it is typically slow (21) and is unlikely to account for our observations.…”

“…Oxidation of N5 and C4a flavin adducts has also been well studied (21,25). The N5 adducts are unstable in the presence of oxygen, whereas C4a adducts require light to initiate oxidation.…”

Structure of the flavin adduct formed in the suicide reaction of .alpha.-hydroxybutynoate with D-lactate dehydrogenase

“…Scheme IV). In contrast to the intermediate IVa, which has a 1,5-dihydroflavin structure, the cyclic adduct V should not react with oxygen, as N(5)-substitution of enzyme-bound or free flavins reduces their oxygen sensitivity drastically (Ghisla et al, 1973;Walker et al, 1970;Massey & Ghisla, 1975).…”

Oxidation Kinetics of 1, 5-Dihydroflavin by Oxygen in Non-aqueous Solvent

“…The preequilibrium formation of the flavoperoxide complex was measured by changing stepwise the initial concentration of oxygen, [ .for reuctions (7) und (18), respectively, calculatedas described in text solvent 1171 appears unlikely in view of the very low amount of OH-ions present in buffered dimethylformamide, and non-alkylated flavin has never been reported to yield a metastable carbinolamine adduct. Moreover, such an exchange could not end in the expulsion of the peroxy substituent as molecular oxygen in order to explain a selective effect over the rapid preequilibrium step.…”