Biotransformation of quinoxaline by Streptomyces badius

J.B. SUTHERLAND, F.E. EVANS, J.P. FREEMAN AND A.J. WILLIAMS. 1996. Quinoxaline, a mutagenic azaarene produced in foods during cooking, was added to cultures of Streptomyces badius ATCC 39117. After 24 h, the cultures were extracted with extracted with ethyl acetate. Two major metabolites were purified by liquid chromatography and identified by mass spectrometry and nuclear magnetic resonance spectroscopy as 3,4–dihydro‐2(1 H)‐quinoxalinone and 2(1 H)‐quinoxalinone.

Little is known about the metabolism of quinoxaline except that Pseudomonas putida metabolizes it to quinoxaline cis-5,6dihydrodiol, 5-hydroxyquinoxaline, and 2( 1H)-quinoxah o n e (Boyd et al. 1987(Boyd et al. , 1993. We have found that Streptomyces badius has the ability to cometabolize the heterocyclic ring of quinoxaline.
After incubation for another 24 h, the cultures and controls were extracted with equal volumes of ethyl acetate. The solvent was dried over anhydrous sodium sulphate and evaporated in vacuo. MetaChem Technologies, Torrance, CA, USA) was used with an isocratic mobile phase (65% ammonium acetate buffer [SO mmol 1-I, pH 5.51 and 35% methanol; 1 ml min-'). The U.V. detector was operated at 254 nm. Peaks were collected as they eluted from the column, concentrated in uacuo, and redissolved in deionized water.
The collected metabolites were applied to a preconditioned Sep-pak Vac C,, column (Waters Associates, Milford, MA, USA), washed with deionized water, and eluted from the column with methanol. Visible/u.v. absorption spectra were obtained in methanol with a Shimadzu UV-2lOlPC spectrophotometer.
Mass spectra were obtained by electron ionization (Sutherland et al. 1990) on a Finnigan M A T (San Jose, CA, USA) series 4000 quadrupole mass spectrometer that had been upgraded to model 4500.
Proton nuclear magnetic resonance (NMR) spectra were recorded at 500.13 MHz on a Bruker AM500 NMR spectrometer (Billerica, MA, USA) ; the samples were dissolved in acetone-d, (99.96 atom Yo 'H). Chemical shifts are reported on the 6 scale by assigning the residual proton signal of acetone to 2.05 ppm. Data acquisition and processing conditions were similar to those used previously for one-dimensional NMR spectra (Evans et al. 1994).

RESULTS
The HPLC chromatogram of the ethyl acetate extract from cultures of S. badius grown with quinoxaline ( Fig. 1) shows three peaks at 7.2 min (metabolite I), 9.6 min (metabolite 11), and 14.4 min (residual quinoxaline). No metabolites were found in any of the controls.

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The NMR spectrum of metabolite I in acetone-d, (Table  1) showed eight major resonances, two of which were equivalent and located in the aliphatic region (H3ab, 3.80 pprn). Two resonances exhibited broadening, which is characteristic of exchangeable protons (NH4, 5.23 ppm and NH1, 9.15 pprn). Homonuclear decoupling experiments established that the H3ab resonances were coupled to NH4. Saturation of the NH4 resonance resulted in a nuclear Overhauser effect (NOE) to the H3ab resonances and to a doublet in the aromatic region (H5, 6.73 ppm). Saturation of the NH1 resonance resulted in an NOE to another doublet (H8, 6.84 ppm). These experiments and additional homonuclear decoupling experiments enabled the assignment of all of the resonances in 3,4-dihydro-2( 1H)-quinoxalinone.
The NMR spectrum of metabolite I1 (Table 1) was also the same as that of authentic 2( lw-quinoxalinone. The NMR spectral assignments are based in part on the observation of an NOE to the doublet at 7.39 ppm (H8) resulting from saturation of the N H l resonance.

DISCUSSION
Metabolite I was shown to be 3,4-dihydro-2( lH)-quinoxalinone, a lactam form that predominated over 3,4-dihydro-2-hydroxyquinoxaline, the lactim form. The mechanism of formation by S. badius is unknown but may involve a hydratase-catalysed addition of water.