Nucleophilic Addition at the Photoexcited Flavin Cation: Synthesis and Properties of 6- and 9-Hydroxy-Flavocoenzyme Chromophores

Abstract

1. The method of "photohydroxylation" of azaheteroaromatics, known in the phenazine and alloxazine series, has been applied to flavin ( = isoalloxazine) derivatives FbxH. The products habe been identified as 6-and 9-hydroxyflavins (FlOx-6/9-OH). On this basis it was possible to assign a 6-hydroxyflavin structure to the new flavocoenzyme chromophores described by Mayhew et al. in the accompanying paper.2. I n agreement with the data obtained by Dekker et al. in a recent laser study on alloxazine, we find that the reaction proceeds via addition of water, alcohols or carboxylic acids by the lowest excited singlet state of flavin with synchronous intramolecular hydrogen shift from the site of RO-addition [= C(6) or C(9)l towards N(5) and subsequent oxidation of the 6/9-hydroxy-1 $-dihydroflavin formed.3. The photoaddition reaction occurs in competition with photodehydrogenation of available CH-bonds by the lowest excited flavin triplet, in which case the CH-substrate may be provided by the solvent ROH or, when R = H, by methyl groups of a second flavin molecule (= flavin autophotolysis). This photodehydrogenation may be suppressed by 0,-dependent triplet quenching and/or by applying a positive charge to the flavin (RFl,,H+, R = H or alkyl) in order to inhibit the reaction with itself.4. Differentiation of 6-and 9-hydroxyflavin isomers was possible by double resonance evaluation of the proton coupling between the residual Ar-H and the adjacent methyl group, by evaluation of the electron paramagnetic resonance hyperhe coupling for the residual Ar-H in the radical cations 1 ,5-RHlk6/9-OH, and by formation of cupric chelates from 6-hydroxy-flavin as compared to the lack of metal affinity in the 9-isomer. lH-Nuclear magnetic resonance, electron paramagnetic resonance and visible spectra and their dependence on the state of ionisation of hydroxyflavins are discussed. Hydroxyflavin anions as well as their N( 1)-alkylated meso-ionic derivatives are shown to exhibit characteristic long-wavelength absorption bands ranging from 50 to 800 nm with maxima around 600 nm and low molar absorption coefficients between 500 and 3000 M-bm-l (as compared to =lo4 for the first n, n* transition a t %400 nm), which might be readily mistaken for "flavin-substrate charge transfer'' bands. 5. 9-Methoxy-flavins could be spectroscopically related to 9 : 10-bridged cyclic hemiacetals obtained upon ring closure of 9-hydroxy groups with lob-carbonyl functions. Such cyclic hemiacetals can be trapped in the acid photolysis of 10-(hydroxyalky1)-flavins, e.g. riboflavin or flavocoenzymes, in a sequence of 10-side-chain dehydrogenation and N ( 1)-cycloaminal cation formation of the lob-carbonyl formed, which results in a quenching of the further photolysis in favor of 9-photoaddition.Since the discovery of covalent 8a-dimerization of riboflavin and its derivatives [3], evidence has been accumulating with respect to the reactivity of the benzenoid subnucleus of flavoquinone (Fl0,H) Abbreviations. EPR, electron paramagnetic resonance ; NMR, nuclear magnet...

Chemical synthesis and some properties of 6-substituted flavins

“…In this compound, conjugation of the nitrogen with the aromatic ring system is blocked. Similar effects have been obtained upon oxidation of the sulfur moieties in 6-thio flavins (Steenkamp et aI., 1978a) and in 8-thio flavins (Moore et aI., 1979), as well as acetylation of 6-hydroxy and 8-hydroxy flavins (Schollnhammer & Hemmerich, 1974;Ghisla & Mayhew, 1976).…”

“…The 6-amino flavin chromophore is characterized by relatively small absorbance changes with pH at wavelengths greater than 500 nm and by a single, relatively narrow band in the 390-430-nm region, the position of which is pH dependent (Figure 3). Their spectral properties are similar to those of 6-hydroxy flavin anions (Mayhew et al, 1974;Schollnhammer & Hemmerich, 1974) and to 6-keto flavins, in which an "orthoquinoid structure" is obtained by alkylation at the N(l) position of the flavin (Schollnhammer & Hemmerich, 1974). By analogy, it appears that the neutral and the monoanionic forms of 6-amino flavins, which have very similar spectra, should have the orthoquinoid structure shown in Figure 3.…”

New flavins for old: artificial flavins as active site probes of flavoproteins

“…3). The neutral molecules are protonated at position 6a in free solution, while the anions are characterized by longwavelength absorption, which is typical of N(l)-blocked 6-hydroxyflavins [35]. This suggests that in 6-substituted flavins the paraquinoid form shown in Fig.…”

Preparation and some properties of 6-substituted flavins as active site probes for flavin enzymes

“…In addition, a differentiation of structures Sa and 8b was attempted by complexation with transition metals. It has been shown (Schollnhammer & Hemmerich, 1974) that 6-hydroxyflavins form chelate complexes with Cuz+, and this feature was utilized for obtaining information on the nature of the 6-substitution in these compounds. Addition of Cu2+ to a freshly prepared solution of the compound indeed caused a major spectral change (Figure 6), which is ascribed to formation of the complex 10.…”