Photolumazines, new naturally occurring inhibitors of riboflavin synthetase

B{ochimica et Biophysica Acta, 313 (1973) 229-234 © ElsevierScientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA R e p o r t BBA 21370 P h o t o l u m a z i n e s , n e w n a t u r a l l y occurring inhibitors o f riboflavin s y n t h e t a s e

A. SUZUKIand M. GO.TO Department of Chemistry, Gakushuin University, Tokyo (Japan) (Received June 1st, 1973)

SUMMARY Photolumazines A, B, C, and 7-hydroxy-6-methyl-8-(1-D-ribityl)lumazine (Masuda's Compound V) were isolated from Photobacteriurn phosphoreum. Photolumazine C was identical with an authentic sample of 7-hydroxy-8-(1-D-ribityl)lumazine, previously isolated from Pseudomonas ovalis. Photolumazine A and B are new compounds and they were characterized as 7-hydroxy-6-(L-1,2-dihydroxyethyl)-8-(1-D-ribityl)lumazineand 7-hydroxy-6-hydroxymethyl-8-(1-D-ribityl)lumazine,respectively. The lumazine compounds isolated were proved to be inhibitors of riboflavin synthetase.

It was reported that the chemical change catalyzed by riboflavin synthetase involves 6,7-dimethyl-8-(1-D-ribityl)lumazineI . The reactivity of riboflavin synthetase was tested with a number of lumazine compounds, and 7-hydroxy-6-methyl-8-(1-D-ribityl)lumazine, the only compound of natural origin tested, was proved to be a potent inhibitor of riboflavin synthetase 2. We have previously isolated the following 8-(1-D-ribityl)lumazines from microorganisms: 7-hydroxy-6-(3-indolyl)-8-(1-D-ribityl)lumazine3'4, 7-hydroxy-6-(p-hydroxyphenyl)-8-( 1-D-ribityl)lumazine3'4 , 6-(2-carboxyethyl)-7-hydroxy-8-(1-D-ribityl)lumazine 4 (Putidolumazine), and 7-hydroxy-8-(1-D-ribityl)lumazines . In this paper we describe the isolation and characterization of three lumazine compounds (designated as Photolumazines A, B, and C in this paper) from Photobacterium phosphoreum Photolumazine C was identified as 7-hydroxy-8-(1-D-ribityl)lumazine. Photolumazines A and B are new compounds and they were characterized as 7-hydroxy-6-(L-1,2dihydroxyethyl)-8-(1-D-ribityl-)lumazine and 7-hydroxy-6-hydroxymethyl-8-(1-D-ribityl)lumazine, respectively, on the basis of chemical degradations and syntheses. All

230

BBA REPORT H

I Photolumazine A ,R: --C~CH20H ' OH

HN O~N//L H

N

R

/~O N I

Photolumazine B,R: --CId2OH Phetolumazine C,R:--H Compound V, R: --CH 3

CH2(CHOH)3CH2OH

7-hydroxy-8-(1-D-ribityl)lumazine compounds hitherto isolated from microorganisms were proved to be inhibitors of riboflavin synthetase. Isolation of the compounds: The strain dPhotobacterium phosphoreum was obtained from the Fermentation Research Institute of the Takeda Chemical Industries, Osaka. P. phosphoreum was cultivated for 48 h in a fermentation jar (capacity: 5 1) at 25 °C with aeration (see ref. 6). The culture was acidified with acetic acid to pH 4.0 and treated with charcoal (30 g). The charcoal was filtered and washed with water (2 1). The adsorbed fluorescent compounds were eluted from this with a mixture of 4% aqueous ammonia and 50% ethanol (1:1, v/v)(21). The eluate was evaporated to dryness in vacuo below 40 °C, and the residue (one blue- and four purple-fluorescent compounds were chromatographically detected) was purified by chromatography, as shown in Fig. 1. Structures: By comparison with synthetic pteridines, two fluorescent compounds were identified as 7-hydroxy-6-methyl-8-(1-D-ribityl)lumazine (Masuda's Compound V) 7 and 7-hydroxy-8-(1-D-ribityl)lumazines (Photolumazine C), respectively. The other two compounds (Photolumazines A and B) were supposed to be new pteridines. The ultraviolet spectra of these compounds are similar to those of Compound V and this suggests that these are 7-hydroxylumazines with alkyl side chains at the N-8 and C-6 positions. On reduction with aluminum amalgam these compounds gave 7-hydroxy-8-(1-D-ribityl)lumazine by losing the side chain at the C-6 position 8 . On reduction with zinc powder in O. 1 M HCI, Photolumazine B gave Compound V and a little amount of Photolumazine C. From these results the structure of 7-hydroxy-6hydroxymethyl-8-(1-D-ribityl)lumazine was postulated for Photolumazine B. This compound was synthesized as described below, and its identity with the natural product was confirmed (Table I). On reduction with zinc powder in 0.1 M HC1, Photolumazine A gave Photolumazine C, but not Compound V. On oxidation with periodate followed by reduction with sodium borohydride, Photolumazine A yielded 7-hydroxy-8-(2-hydroxyethyl)-6-hydroxymethyllumazine prepared from Photolumazine B by the same manner. This evidence suggests that Photolumazine A has a - C H O H - C H O H - R group at position 6.7-Hydroxy-6-(D-1,2-di_hydroxyethyl)-8-(1-D-ribityl)lumazine and 7-hydroxy-6-(L-1,2-dihydroxyethyl)-8-(1-Dribityl)lumazine were synthesized as described below. Photolumazine A was proved to be 7-hydroxy-6-(L-1,2-dihydroxyethyl)-8-(1-D-ribityl)lumazineby comparison with the synthetic materials (Table I). S. Matsuura et al. have recently isolated the same compound as

BBA REPORT

231

Concentrated eluate

I

Separation by paper chromatography Solvent: Ill(see Table I)

I

I

RF 0.28 (blue)

RF 0.40 (purple)* /

Separation by a DEAE-clellulosecolumn (4 cm X 24 cm; the column was washed with water (1 1) and the band developed with 0.003 M HC1)

Fr 1 (purple) Fr 2 (purple) Fr 3 (p rple) Compound V Photo(approx. 1.6 nag) lumazine C (approx. 0.4 mg)

l

Paper chromatography Solvent:III,I,IV

I

Neopterin-cyclic phosphate (approx. 0.33 mg) (see ref. 6)

Separation by paper chromatography Solvent: III

I RF 0.73 (purple)

I Paper chromatography

RF 0.65 (purple)

Solvent: IV, II

Paper chromatography Solvent: IV, II

Photolumazine A

Photolumazine B

(approx. 0.1 m8)

(approx. 0.08 mg)

* The color of fluorescence in parenthesis Photolumazine A from the same microorganisms; they claim, however, that the isolated material is a mixture of stereoisomers (personal communication). Synthesis of 7-hydroxy.6-hydroxymethyl-8-(1-D-ribityl)lumazine: 2,6-Dihydroxy5-nitro-4-D-ribitylaminopyrimidine (400 nag) in water (30 ml) was hydrogenated over a platinum oxide catalyst (150 mg). After the theoretical amount of hydrogen was taken up, the catalyst was removed by f'dtration. The solution was acidified with acetic acid (1 ml). Hydroxypyruvic acid 9, prepared from bromopyruvic acid (600 mg), was added, and the

232

BBA REPORT

TABLE I PAPER CHROMATOGRAPHY OF PTERIDINES Solvents: I, 2-propanol- 2% ammonium acetate (1 : 1, v/v); II, 2-propanol- 1% ammonia (2:1, v/v); III, 3% aqueous NH4C1; IV, 1-butanol-acetic acid-water (4:1:1, v/v); V, 4% sodium citrate. Substance

Photolumazine A Photolumazine B Photolumazine C Compound V R-6-CHOHCH2 OH* (L-isomer) R-6-CHOHCH2 OH (D-isomer) R-6-CH2OH RH

R F values I

H

III

IV

V

0.42 0.40 0.45 0.48 0.42

0.18 0.18 0.23 0.26 0.18

0.63 0.57 0.60 0.61 0.63

0.05 0.07 0.10 0.14 0.05

0.55 0.48 0.52 0.51 0.55

0.38

0.16

0.60

0.05

0.52

0.40 0.45

0.18 0.23

0.57 0.60

0.07 0.10

0.48 0.52

*R, 7-hydroxy-8-(1-D-ribityl)lumazine.

mixture was heated for 1 h on a water bath. The products were absorbed on charcoal (5 g), which was then washed well with water. The compounds on charcoal were eluted with a mixture of 50% ethanol and 4% ammonia (1:1, v/v) (300 ml), and the eluate was evaporated to dryness in vacuo. The residue was dissolved in water (50 ml) and the products were purified by chromatography using a DEAE-cellulose column (3.5 cm × 20 cm; elution: 0.01 M HC1) and a cellulose column (5 cm × 22 cm; elution: 2-propanol-1% ammonia (2:1, v/v)). On the last column, the products were separated into two fractions; each eluate was concentrated to dryness in vacuo. The products were identified as 7-hydroxy8-(1- D-ribityl)lumazine s (15 rag) and the desired product (30 rag), which on crystallization from aqueous ethanol gave 23 mg of pure material, m.p. > 265 °C (decomposition). Found: C, 39.36; H, 4.80; N, 15.41%. Calculated for C12H16NaOa "H20: C, 39.78; H, 5.01;N, 15.47%. _2sonm _2atom Ultraviolet absorption: in 0.1 M HC1, emi n 3280, ema x 11900, e_30o minnm 6680, _24snm A~(~O _259 nm 5080, _267nm 47Rfl _289nm e_aaonm max 1 1 100; in water, e rain _ / _ v , ernax emi n . . . . . ema x 10200, eaoTnm q7~13 _asonm 12800. mill . . . . , e max

Synthesis of 7-hydroxy-6-(D- 1,2-dihydroxyethyl)-8-(1-D-ribityl)lumazine: 2,6-Dihydroxy-5-nitro-4-D-ribitylaminopyrimidine (300 mg) in water (20 ml) was hydrogenated over a platinum oxide catalyst (100 mg). After the theoretical amount of hydrogen was taken up, the catalyst was removed by filtration. The solution was acidified with acetic acid (0.5 ml). a-Keto-D-erythronate, prepared from the osone 1° of D-erythrose (500 mg) by oxidation with bromine 11 , was added, and the mixture was heated for 1 h on a water bath. The products were adsorbed on charcoal (5 g), which was then washed well with water. The compounds on charcoal were eluted with a mixture of 50% ethanol and 4% ammonia

BBAREPORT

233

(1:1, v/v) (300 ml), and the eluate was evaporated to dryness/n vacuo. The residue was dissolved in water (50 ml) and the products were purified by chromatography using a DEAE cellulose column (4 cm X 20 cm; elution: 0.005 M HCI). Further purification was achieved by paper chromatography using the following solvents: A, 3% NH4 C1; B, 1-butanol-acetic acid-water (4:1:1, v/v). The products obtained were 7-hydroxy-8-(1-D-ribityl)lumazine (yield, 8 mg), 7-hydroxy-6-hydroxymethyl-8-(1-D-ribityl)lumazine(5 nag) and the desired product (5 mg); characterizations were achieved by paper chromatography. M.p. > 265 °C (decomposition). Found: C, 39.56;H, 5.03; N, 14.20%. Calculated for CIaHIaN409 "H2 O: C, 39.80; H, 5.14; N, 14.28%. On reduction with A1-Hg or Zn/HC1, the product gave 7-hydroxy-8-(1-D-ribityl)lumazine. It consumed 4 moles of periodate per mole. The ultraviolet spectra of the compound were essentially identical with those of 7.hydroxy-6-hydroxymethyl-8-(l-D-ribityl)lumazine. 7-Hydroxy-6-(L-1,2-dihydroxyethyl)-8-(1-D-ribityl)lumazinewas similarly prepared by condensation of 2,6.dihydroxy-5-amino-4-D-ribitylaminopyrimidine and a-ketoL-erythronate. Found: N, 14.20%. Calculated for CIaHaaN409 "H20: N, 14.28%. On reduction with AI-Hg or Zn/HCI, the product gave 7-hydroxy-8-(1-D-ribityl)lumazine. It consumed 4 moles of periodate per mole. The ultraviolet spectra of the compound were practically identical with those of 7-hydroxy.6-hydroxymethyl-8-(1-D-ribityl)lumazine. Inhibition of riboflavin synthetase by ribityllumazines: Enzyme preparation, conditions of incubation and determination of riboflavin were carried out as described by Plant and co-workers2. Purified enzyme from Eremothesium ashbyii (activity: 50 to 90 nmoles of riboflavin formed per ml of the enzyme solution per hour at 37 °C) was used TABLE II INHIBITION OF RIBOFLAVINSYNTHETASEBY SUBSTITUTEDLUMAZINEDERIVATIVES Riboflavin was determined by the direct spectrophotometric method. 6-Substituted 7-hydroxy-8(1-l~ribitylJlumazines

Inhibitor concentration* (raM)

Competitive inhibition (Ki)** (M)

6-Methyl 6-Hydroxymethyl 6-H 6-(L-l,2-Dihydroxyethyl) 6-(D-1,2-Dihydroxyethyl) 6-(2-Carhoxyethyl) 6-(p-hydroxyphenyl)

0.010, 0.0033 0.017, 0.0058 0.015, 0.0049 0.016, 0.0054 0.014, 0.0048 0.031, 0.010 0.024, 0.0080

2.9.10 -~ 3.0"10-~ 1.1.10 -s 1.7.10 -s 1.7.10 -s 2.7.10 -5 2.8.10 -s

* Values in 3 ml o f the e n z y m e preparation. * * Estimated b y the use o f the D i x o n plot t2 .

234

BBA REPURT

throughout these studies. The reaction was initiated by addition of the enzyme preparation, and the mixture was incubated for 1 5 - 2 0 rain at 37 °C. At the end of the incubation period, 0.5 ml of 30% trichloroacetic acid was added to the reaction mixture (3 ml). Coagulated protein was removed by centrifugation. The supernatant was utilized directly for the spectrophotometric estimation of riboflavin. The following concentrations of 6,7-dimethyl-8-(1D-ribityl)lumazine were used: 0.202 mM, 0.101 mM and 0.051 mM. The inhibitor constants determined for a series of naturally occurring 6-substituted 7-hydroxy-8-(1-D-ribityl)lumazine compounds are summarized in Table II. These compounds were proved to be fairly potent inhibitors of riboflavin synthetase tested. Thus, 7-hydroxy-8-(1-D-ribityl)lumazines (Putidolumazine, Photolumazines A, B and C and others), frequently found in microorganisms, might have a function as regulators for the biosynthesis of riboflavins. The origin of these lumazine compounds is still unknown; they may be derived from Compound G (6,7-dimethyl-8-(1-D-ribityl)lumazine) or its unknown pyrimidine precursors. Further work along this line is in progress. This paper was dedicated to the 60th anniversary of Professor Dr. Max Viscontini, University of Zurich. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12

Plant, G.W.E. (1963)J. Biol. Chem. 238, 2225-2243 Winestock, C.H., Aegaichi, T. and Plaut, G.W.E. (1963)J. Biol. Chem. 238, 2866-2874 Takeda, I. and Hayakawa, S. (1968) Agric. Biol. Chem. 32, 873-878 Suzuki, A. and Goto, M. (1971)Bull. Chem. Soc. Japan 44, 1869-1872 Suzuki, A., Miyagawa, T. and Goto, M. (1972)Bull. Chem. Soc. Japan 45, 2198-2199 Suzuki, A. and Goto, M. (1973)Biochim. Biophys. Acta 304, 222-224 Masuda, T., Kishi, T. and Asai, M. (1958) Chem. Pharm. Bull. 6, 291-299 Matsuura, S., Nawa, S., Goto, M. and Hirata, Y. (1955) 42,413-416 Sprinson, D.B. and Chargaff, E. (1946)J. Biol. Chem. 164, 417-432 Hamilton, J.K. and Smith, F. (1952) J. Am. Chem. Soc. 74, 5162-5163 Overend, W.G., Stacey, M. and Wiggins, L.F. (1949) J. Chem. Soc. 1358-1363 Dixon, M. and Webb, E.C. (1958) in Enzymes pp. 22-27, Academic Press, New York