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1030 G. SCHÖLLNHAMMER AND P. HEMMERICH

Photo-addition Reactions in the Benzenoid Subnucleus of Flavoquinones

G . SCHÖLLNHAMMER a n d P . HEMMERICH

Department of Biology, University of Konstanz, Germany

(Z. Naturforsch. 27 b, 1030—1031 [1972] ; received May 10, 1972)

Photo-addition, Flavoquinones

By photochemical attack on flavin cations, compounds substituted in the benzenoid subnucleus have been prepared. Water and alcohols can be added in this way and under certain conditions the alcoholic groups of the flavin side chain are involved. Structures and properties of photo-products are described.

It has been shown by TE NIJENHUIS and BERENDS2 that the benzenoid (or better: o-quinoid) subnucleus of alloxazines can be attacked photo-chemically. We found that with flavins the reaction products bear a hydroxyl group either in the 6 or in the 9 position of the benzenoid nucleus. The reactive species is the protonated flavin, the rate of the reaction has an optimum at pH 0 — 1. At still lower pH dynamic proton quenching decreases the rate. The fastest reaction is given by formylmethyl-flavin (1) which is fully protonated at pH 1 (pK = 3 .5 2 ) and by 1,10-substituted flavinium salts (Schemel, No. 1 and 2 ) .

R = —CHjj-CO-R"1

R*= H , C H 3

R"; OH

0 CH2 R' CHJ-^JV^NU+N^O CH -U^X^NR»

0 Scheme I. Hydroxylation of protonated flavins.

Requests for reprints should be sent to Prof. Dr. P. HEM-MERICH, Fachbereich Biologie der Universität, D-7750 Kon-stanz, Germany.

CHO i i CH2 '

N Y )

0

The reaction involves water addition either at the 6,7- or at the 8,9-position and subsequent oxi-dation of the hydrate as Scheme I shows. Experi-ments done in D 2 0 with formylmethylflavin showed that the remaining aromatic proton in the reaction products was not exchanged against deuterium, which proved the absence of 6,9-addition.

The very stable cyclic hemiacetals (2) are formed during the photolysis of N(10)-2'-carbonyl flavins via the addition of the 9-OH group to the carbonyl function, thus forming cyclic acetals. All these com-pounds, i. e. alcohols or cyclic acetals show UV-spectra with a characteristic absorption centered at 405 nm: the hydroxylated compounds are comple-tely non-fluorescent, the cyclic acetals show only a weak brownish-red fluorescence.

In methanolic sulfuric acid, methanol adds to the benzenoid subnucleus yielding 9-methoxy com-pounds exclusively. The hydroxyl groups (irrespec-tive of being in the 6 or in the 9-position) show a relatively low pK around 7 owing to the electron deficiency of the flavoquinone nucleus. UV-spectra of 9-hydroxy-3-methyl lumiflavin are given in Fig. 1. An unequivocal assignment of the posi-tion of the hydroxyl groups can be made by chelate formation -with copper (II) perchlorate in acetate buffer at pH 6 or in acetone. In the case of 6-substitution metal chelates of the structure 3 can be formed, which is not possible in the case of 9-substituted compounds. As an example the proof of structure for hydroxylated

i) R, R',R" = CH3

2) R - R1 _ C H , - C H , ~ 1 1

R" = H

3) R R1

R"

= CHJ = H = CH3 or H

I) R

R1 R"

= variable side chain

= H

PHOTOHYDRATION REACTIONS OF FLAVINS 1031

200 nm —

Fig. 1. UV-spectra of 9-hydroxy-3-methyl lumiflavin. 6 N HCl, 0,1 N HCl, pH 7, • • • pH 9.

1,10-ethano-flavinium Perchlorates is given in Fig. 2. Only the spectrum of the 6-hydroxylated compound changes, due to chelate formation, whereas the spectra of the unsubstituted and of the 9-hydroxylated compound remain unchanged. UV-spectra of flavin metal complexes have been de-s c r i b e d b y HEMMERICH a n d LAUTERWEIN 3 .

20

15

0.5

403

415 6-0H/Cu(E)-chelate

cto;

a) R=H ,R'=H b) R=H ,R'=0H (6-OH) c)R=0H, R'=H (9-OH)

-unsubstituted

650 6-0H/Cu(fl)-chelate \/

300 400 500 700 800

Fig. 2. Chelate formation of hydroxylated 1,10-ethano-flavi-nium Perchlorate with copper (II) Perchlorate in acetone. Fla-vin concentration 5 x 1 0 - 5 M, copper/flavin ratio 100/1.

Flavins with a 2'-hydroxy group in the side chain (4) undergo photoreactions as shown in Scheme II.

H , • k y Lumichrome

NH " 10

hv fast R = H slow R = CHj

0 * H+(pk=0,5>

CH,C0R I Y r W ,

CH.

CH2CH0HR

tvyV. CH.

H+(pk 3,5 R = H 1.0 R = CH3)

R I

H,C — C -OH I nrYY,

hv slow hv

fast

H . R 0 " CH,

CH.

H(K R

0 CH, iYVYT0 t̂yyy cyclic ether cyclic acetal

Scheme II. Photoreactions of flavins with a 2'-OH group in the side chain.

The 2'-OH group adds to the protonated flavin nucleus in the way alcohols do, to give cyclic ethers (6). The reaction is slow, because protonation (4, 5 pK 0.5) occurs at a pH where dynamic proton quenching becomes predominant. The cyclic ethers (6) are also found as photolysis products of neutral flavins (6) in the presence of divalent anions 4 .

The neutral flavin (4) undergoes fast photolysis to lumichrome (10) or oxidation of the 2 -CHOH group to the corresponding carbonyl function (4, 7) by intramolecular hydrogen abstraction. The thus formed carbonyl compounds are protonated in a pH region (pK 1 — 3.5) where proton quenching occurs to a lesser extent and undergo fast hydroxy-lation and acetal formation. The carbonyl com-pound 7 (R = CH3) is quite stable to further lumi-chrome decay, which shows that oxidation of the 2'-CHOH group is not the first step in photolytic lumichrome formation.

We want to thank Miss W I L K E for technical as-sistance.

1 B. TE NIJENHUIS and W. BERENDS, Photochem. Photobiol. 6 ,491 [1967].

2 C. H. SUELTER and D. E. METZLER, Biochim. biophysica Acta [Amsterdam] 44 ,23 [ I960] .

3 P. HEMMERICH and J. LAUTERWEIN, "Inorganic Biochem-istry", Ed. G. L. EICHHORN, Elsevier Publishing Company, Amsterdam 1972, in press.

4 M . SCHUMANN JÖRNS a n d P . HEMMERICH, Z . N a t u r f o r s c h . 27 b, 1040 [1972].