Deazaflavins have been found to act as potent catalysts in the photoreduction of flavoproteins in the presence of EDTA and other "photosubstrates". In distinction to the catalysis brought about by normal flavins which involves dark reaction of the photoreduced flavin catalyst, the mechanism of the catalysis by deazaflavins has been shown to involve unstable, strongly reducing radicals which are generated by photolysis of a preformed covalent dimer. By this new method it is possible to reduce not only flavoproteins but a variety of other redox proteins, including heme proteins and iron-sulfur proteins. By virtue of its great catalytic efficiency, it is possible to employ concentrations of deazaflavin sufficiently low as not to interfere with the spectral evaluation of the reduced proteins obtained.

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Evidence is given for the fact that the excited flavin triplet (3F10*x) exhibits competitive le-and 2e-transfer chemistry, depending on the nature of the photosubstrate. As an 'external' photoreductant, the 2e-donor borohydride has been investigated. Borohydride is found to compete effectively with the 'internal' 1 e-donors, namely excess starting flavin in the ground state (Flax) and, as primary product, (alky1)dihydroflavin (RFl,,dH). It will be shown that some of the flavin radicals, observed by earlier authors in the reaction of 3Fl& with CH or C -COO-substrates or in the autophotolytic side-chain cleavage of riboflavin, are due to the dye-dye reaction (I) :(1) H i In contrast flavin photoreduction by borohydride or hydrocarbon substrates need not involve radicals, but may in fact be a hydride or 'carbanion-plus-proton' addition towards the highly polar and considerably basic (pK = 4.4) acceptor triplet (cf. reaction 11) RCOO-The products are much more photoreactive than the starting substrates, which leads to the secondary photocomproportionation (111) : 3Fl,*, + R-Fl,,dH + HFl + RFl, (R = alkyl or H). (111) This latter reaction is the second source of radicals in the system. This (cf. 11) 'photohydrogenation' of flavin is mechanistically related to the biological reduction of flavin by CH substrates.The photochemistry of flavins is in fact older than the corresponding ground-state chemistry and even biochemistry, since the side-chain photolysis of vitamin Bz was the first flavin reaction to be discovered [l]. The very complex course of this reaction, yielding lumiflavin at alkaline pH and lumichrome at neutrality, is still not completely resolved [2] (and references therein). This is largely due to a common mistake of insisting that such reactions must involve radical Dedicated to Professor Hellmut Bredereck, Universitat Stuttgart, on his 75th birthday.Abbreviations. Fl,,, flavoquinone, flavin; 3Fl,*,, excited flavin triplet; HFI Fl-, acidic and basic flavosemiquinone; Fl', oxidized flavin radical; HZFlred, flavohydroquinone, 1,5-dihydroflavin.

Ausgehend von der vielfachen Ahnlichkeit im chemischen Verhalten von lichtangeregtem Flavin und Protein-gebundenem Flavin haben wir die Flavin-abhangige Photodehydrierung systematisch untersucht: 3-Benzyllumiflavin (1) reagiert photochernisch mit Carbonsauren wie 3-Indolessigsaure, Phenoxyessigsaure oder (tert-Buty1thio)essigsaure unter Decarboxylierung und Bildung der 4a-alkylierten 4a,S-Dihydroflavinderivate 3a-c. In Gegenwart von Thiodiessigsaure oder Dithiodiessigsaure entsteht das 5-Carboxyrnethyl-1,5-dihydroflavinderivat 4 a bzw. das 4a-(Carboxymethy1thio)derivat 3d. - ReductivePhotoalkylation of theFlavin Nucleus; Structure andReactivity of the Photoproductsl.2)O n the basis of the manifold similarities between flavin photochemistry and flavin biocatalysis, we have made an effort toward systematic investigation of flavin-dependent photodehydrogenation: 3-benzyllumiflavin (1) reacts photochemically with carboxylic acids, e. g. 3-indoleacetic acid, phenoxyacetic acid and (tert-buty1thio)acetic acid, to give 4a-alkylated 4a,5-dihydroflavin derivatives 3 a -c and COz. In the presence of thiodiacetic acid and dithiodiacetic acid the 5-carboxymethyl-l,5-dihydroflavin derivative 4 a and the 4a-carboxymethylthio derivative 3d, respectively, are formed. -Alkyl sulfides are attacked in the cr-position yielding 5-substituted 1,5-dihydroflavin derivatives, which undergo trans-elimination if P-CHz-groups are present; in the case of the thiolane the adduct 4d is found to be stable because of sterical hindrance. With benzaldehyde the 5-benzoyl-l,5-dihydro derivative 4 e is formed. -Alkenes are attacked in the allylic position yielding 4a-alkylated 4a,5-dihydro derivatives, 3e -g. I n the presence of 1,4-cyclohexadiene the 1,5-dihydroflavin 4 c is formed, as also in the case of *) Korrespondenz bitte an diesen Autor richten.

The catalytic action of 5-deazaflavin in the photochemical reduction of flavin and iron proteins [Massey, V. and Hemmerich, P. (1978) Biochemistry, 17, 9-17] is shown to be due to the highly reactive 5-deazaflavosemiquinone. This radical is generated in a complex sequence of reactions, which involves (a) covalent photoaddition of the substrate residue to the deazaflavin, (b) fast secondary photoreaction of this adduct with starting deazaflavin to yield a covalent radical dimer, accompanied by the liberation of the oxidized substrate, and (c) deazaflavin-sensitized cleavage of the radical dimer to the monomers. The structure and properties of this radical (redimerisation or dismutation) and the precursor intermediates as well as the mechanism of the photoreaction are described. Deazaflavins and their natural parent compounds are compared with respect to their different redox behavior and radical stability. The syntheses of 5-deuterated deazaflavins are described and their redox reactions are compared with those of normal deazaflavins.Among the three principal types of flavoprotein activities, i.e. (de)hydrogenation, 0 2 activation and e-transfer, only the first is retained in deazaflavoproteins, and even this to rather limited extent in most cases [l -71. The initial overestimation of deazaflavins as flavin analogs must be ascribed to the lack of chemical knowledge about this heterocyclic system. For example, Briistlein and Bruice [8] were among the first to carry out chemical studies and they concluded, from the retention of H label in the course of reduction, that deazaflavin was transferring hydride and that, by analogy, flavin was a hydride-transferring entity also. Edmondson et al. [2] have been studying the system more extensively, but they have been deceived by the fact that there is a physical analogy between the oxidized species Fl,, and dF1, which, however, does not extend to the radical HdFl nor to the fully reduced state HZdFl,,d. These authors, however, were the first to state the lack of radical stability in deazaflavoproteins as compared to natural flavoproteins. This was corroborated by the inactivity of modified D-amino acid oxidase radical produced photochemically, as reported by Jorns and Hersh [6]. Based upon theoretical studies, Sun and Song [9] claimed that 'the reactivity of position 5 is approximately independent of the identity of atom (N or C)'.On the contrary, we want to show that the analogy between flavin and deazaflavin is rather limited, deazaflavin being a 'flavin-shaped nicotinamide model ' [ 11. Spencer et al. [lo] were the first to see 'carbanion adducts' and 'two-electron disproportionation', a phenomenon which should rather be termed 'interdeazaflavin hydride transfer'. This study does not yield much information on the half-reduced deazaflavin. Radicals do not occur as essential intermediates in deazaflavin-linked oxidoreduction, unless enforced le--transfer methods are applied, as we will show below.Based on our new and simple synthesis of the deazaflavin skeleton [ll], ...

Eingegdngen am 26. Oktober 1973 10-Phenylisoalloxazin (2a) reagiert in1 ersten angeregten Triplett-Zustand bei der Umsetzung mit verschiedenen Substraten analog dem Lumiflavin (-7,8,IO-Trimcthylisoalloxazin) unter Bildung des I ,S-Dihydroisoalloxazins bzw. 5-odcr 4a-alkylierter Dihydro-Derivate. -. Der erste angcregte Singulett-Zustand reagiert hingegen unter Photocyclisieruag in cincr protonenkatalysierten Reaktion, die als intramolckulare, elcktrophile Photosubstitution des N( I)-Atoms a m Phenyl-Substitucntcn beschrieben werden kann. Dabei entsteht das neue heterocyclischc System des SH-BenzimidazoI 1,2,3-ii]~enzo[g]pteridin-6,8(7H)-dions (1 1). das auch durch Photocyclisierung des 1.3-Diphenylalloxazins (6) zuglnglich ist. Der oxidative Abbau von 11 wird untersucht. The Photochemistry of 10-Phenylisoalloxazine: Intramolecular Singlet and lntermolrcular Triplet Reactions 1)F r o m the first excited triplet state of 10-phcnylisoalloxazine ( 2 4 photoreductiox or photoalkylation occurs in a way analogous to lumiflavin (= 7.8.10-trimethylisoall~xazine) to yield I ,5-dihydroisoalloxazine o r 5-and 4a-alkylated dihydro-derivatives thereof. .-The first excited singlet state, however, undergoes photocyclization in a proton-catalyzed reaction. which can be described as an intramolecular. electrophilic photosubstitution of the N(1)-atom on the phenyl-sutstituent, yielding the new hctcrocyclic system of 5H-benzimidazo[l,2,3-ij]bcnzo[g]pteridine-6,8(7H)-dione (11). which is also formed on photocyclization OF I ,3-diphenylalloxazinc (6). The oxidative degradation of 11 has been studied.Die photochemische Aktivitlt des Isoalloxazin-Systems erstreckt sic5 uber einen bemerkcnswert wciten Bereich innerhalb des Chromophors: Eine scheinbar so einfache Umsetzung wie die Photodecarboxylierung von Phenylessiisaure kann beispielsweise die 8-, die N(5)und die 4a-Position betreffen2 5 ) . Daruter hinaus treten bei der I'hotolysc im saureil pH-Hcreich nuclcophile Additionen am Benzolkern in 6-und 9-Position ein6.7). Auch fur Dunkelredktionen ist die Anzahl der moglichen Rcaktionsorte bctrachtlich: Beispielsweise wiesen Bruice und Mitarbb.8.9) kurzlich fur die Rcaktion von Isoalloxazin mit Sulfit die Beteiligung 1 ) Teilweise vorgetragen auf dem ,,Mikrosymposium uber Photochemie der Heterocyclen" in Muhlheim/Ruhr a m 24. I I. 1972. 1615 der Positionen 4a, 5 , 6 und 8 nach. Am 1,3,1O-Trimethylisoalloxain-Kation greifen Hydroxyl-Ionen reversibel die Positionen 6 und 810) und irrcversibel die Position IOa an, wobei einc Verengung des Pyrimidindion-Ringes zum Spirohydantoin eintritt 11). Die Redktion des I ,5-Dihydroisorlloxazins mit molekularem Saucrstoff ist umstritten: Mager und Berendsl2) postulieren fur die Redktion des 1,3,1O-Trimethyl-l,5-dihydroisoalloxazins mit Sauerstoff, Spector und Massey 13) fur die Redktion des reduzierten Fkdvoproteins p-Hydroxybenzoat-Hydroxylase ebenfalls mit Sauerstoff ein IOd-Hydroperoxid als Inteimedilrprodukt. Hemmerich, Muller und Mitarbb. 10914.15) hingegen argumentieren an Hand von Elcktronenspektre...

The photoreactions of the flavin triplet with unsaturated hydrocarbons, aldehydes, sulphur compounds, and carbonic acids are reported. These reactions proceed by attack at the allylic C-H-, the RCO-H-, or the α-C-H-bond, respectively; in the case of carbonic acids decarboxylation occurs. In all the reported reactions covalent adducts are formed, whose structure and mode of decay is characterized.