Nucleoside intermediates in blasticidin S biosynthesis identified by the in vivo use of enzyme inhibitors STEVENJ. GOULD' Departments of Chemistry and of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-4003, U.S.A. JINCAN GUOAND ANJAGEITMANN Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-4003, U.S.A. KARL DFXESUS Department of Chemistry, Oregon State University, Corvallis, OR 97331-4003, U.S.A. Received March 9, 1993 This paper is dedicated to Professors Ian Sperzser and David B. MacLean STEVENJ. GOULD, JINCAN GUO,ANJA GEITMANN,and KARLDmus. Can. J. Chem. 72,6 (1994). Intermediates in the biosynthesis of blasticidin S and its nucleoside co-metabolites were detected by altering fermentation con- ditions. Inhibitors of specific types of biochemical reactions that were expected to be involved in blasticidin biosynthesis were fed to Streptomyces griseochromogenes, in some cases with the inclusion of large quantities of the primary precursors of blas- ticidin S. The types of reactions and inhibitors used were (1) transaminase (aminooxyacetic acid and 2-methylglutamate), (2) amidotransferase (azaserine and 6-diazo-5-0x0-L-norleucine), (3) arginine biosynthesis (arginine hydroxamate), and (4) meth- yltransferase (ethionine). These manipulations apparently distorted the pools of precursors and (or) intermediates, and led to sub- stantial accumulations of three known, previously minor, metabolites of S. griseochromogenes, cytosylglucuronic acid, pentopyranine C, and demethylblasticidin S, and of two new ones, pentopyranone and isoblasticidin S. New cytosyl metabolites were detected by HPLC with photodiode array detection. Fermentations to which arginine hydroxamate and cytosine had been added also produced three aberrant metabolites that were derived from pentopyranone and arginine hydroxamate. STEVENJ. Gorno, JINCAN GUO,ANJA GEITMANNet KARL Dmus. Can. J. Chem. 72.6 (1994). En modifiant les conditions de fermentation, on a pu dttecter les intermediaires dans la biosynthkse de la blasticidine S et de ses comCtabolites nuclCosidCs. Des inhibiteurs de reactions biochimiques spkcifiques attendues dans la biosynthkse de la blasti- cidine ont CtC donnCes au Streptomyces griseochrornogenes, dans certains cas avec inclusion de grandes quantitks de prkcurseurs primaires de la blasticidine S. Les types de reactions et d'inhibiteurs utilisCs sont : (I) la transaminase (acide aminooxyacCtique et le 2-mCthylglutamate); (2) amidotransfLrase (azaserine et 6-diazo-5-0x0-L-norleucine); (3) biosynthkse de I'arginine (hydroxamate d'arginine) et (4) mCthyltransfCrase (Cthionine). Ces manipulations ont apparemment modifiC la repartition des prCcurseurs et (ou) des intermediaires et ont conduit B des accumulations importantes de trois mktabolites connus, mais anterieurement mineurs, du S. griseochromogenes, l'acide cytosylglucoronique, la pentopyranine C et la demethylblasticidine S, ainsi que deux nouveaux metabolites, la pentopyranone et l'isoblasticidine S. Grgce B la CLHP avec une photodiode comme ditecteur, on a pu mettre en evidence la presence de nouveaux metabolites cytosylCs. Les fermentations rCalisCes avec des sub- strats dans lesquels de l'hydroxamate d'arginine et de la cytosine avaient CtC ajoutCs ont aussi conduit B la formation de trois metabolites aberrants dCrivLs de la pentopyranone et de l'hydroxamate d'arginine. [Traduit par la redaction] Blasticidin S, an antifungal antibiotic produced by Stretorny- Blasticidin S is composed of N-methyl-L-P-arginine, 2 (blas- ces griseochrornogenes first isolated by Takeuchi et al. in 1958, tidic acid), and an unusual nucleoside (cytosinine), 3. Seto et al. is used commercially for the control of Piricularia oryzae (rice (13) had established that cytosine, 4, D-glucose, 5, L-a-arginine, blast) (1). Chemical degradation (2-5) and X-ray diffraction 6, and methionine, 7, are the primary precursors (Scheme 1). studies (6, 7) yielded the nucleoside structure 1. Since then, a We first became interested in the biosynthesis of 1 because of number of structurally related nucleoside antibiotics have been the potential relevance of our studies (14) on the L-P-lysine unit characterized, including gougerotin (8), mildiomycin (9), of another nucleoside antibiotic, streptothricin F, to the origin of bagougeramines A and B (lo), arginomycin (1 1), and antibiotic the L-P-arginine moiety embedded in 1. Indeed, both p-amino Sch 36605 (12). acids proved to be derived by 2,3-aminomutase reactions in which an intramolecular migration of nitrogen from C-2 to C-3 and a companion migration of the 3-proR hydrogen to the 2- proR position takes place (15, 16). Feedings with [I-~HI-and [2,3,4,6,6-2~5j-~-g1ucoses,used to probe the biosynthesis of the cytosinine moiety, revealed that the carbinol hydrogens H-1, -2, and -3 were retained in 1, but that H-4 was lost. Retention of H-2 and H-3 indicated that the deoxygenations at these positions could not occur by simple dehydrations (17). Efforts to incorporate more advanced putative intermediates proved inconclusive. We now report these results, as well as '~uthorto whom correspondence may be addressed. those of a subsequent program of feeding inhibitors of specific COULD ET AL. 7 mL) and fed sterilely in two equal portions to a 200 mL production cul- ture (synthetic medium) 48 and 72 h.after inoculation with a seed cul- H2N$+'oH HOAoH ture. After an additional 72 h, the fermentation was bioassayed (122 HO mg of 1 had been produced) and worked up as previously described to NH2 0H 6 yield 29.4 mg of pure 1. Liquid scintillation counting revealed that this \,AS material was not radioactive. ~-N-Ace@lcytosyl-2',3',4',6'-fetraber1zoylglucose,10 N-Acetylcytosine (19) (215 mg, 1.40 mmol) and Hg(CN)2 (783 mg, 3.10 mmol) were suspended in 6 mL of nitromethane in a dry 50-mL round-bottom flask. A distillation head was attached, the mixture heated, and ca. 1 mL of solvent was distilled. At this point, the heat was removed and a-1 -bromotetrabenzylglucose (20) (923 mg, 1.40 mmol) in CH2C12 (1 mL) was added dropwise from the top of the distillation head. An additional aliquot of nitromethane (1 mL) was added, and the mixture once again heated at reflux. After 4.5 h, the yellow suspension was cooled to 0°C and filtered. 'The solid was washed with cold types of biochemical reactions that were expected to be involved nitromethane, 30% aqueous KI, water, and EtOH. After standing over- in the biosynthesis of 1. The latter approach successfully led to night, a second crop could be recovered from the filtrate, and this was the identification of the roles of a number of intermediates in the washed as described above. The combined solids were recrystallized metabolic matrix that includes the biosynthesis of 1. from DMF-water to give 567 mg (77%): mp 250°C (dec.); IR: 1778, 1739, 1717 cm-I; 'H NMR (TFA-dl) 6: 8.72 (d, lH, J = 7.8 Hz), 8.02 Experimental (d,2H,J=7.6Hz).7.93(d,2H,J=7.4Hz),7.85(d,2H,J=7.6Hz), General 7.81 (d, 2H, J=7.6 Hz), 7.69-7.61 (m, 3H), 7.55 (t, lH, J=7.6 Hz), [l-2~]-~-~lucosewas obtained from Cambridge Isotope Laborato- 7.49-7.32(m, 9H), 6.74(bm, lH), 5.56(d, lH, J=9.0Hz), 6.41 (t, lH, ries. Other organic chemicals were obtained from either Aldrich or J=9.5 Hz),6.15(t, lH, J=9.6Hz),5.93(t, lH, J=9.2Hz),4.87(dd, Sigma Chemical Company. IH, J = 12.4, 1.9 Hz), 4.814.73 (m, 2H), 2.43 (s, 3H); 13c NMR (TFA-dl) 6: 178.54, 178.36, 172.63, 171.76, 171.59, 162.24, 161.54, Preparation of [1 -14~]-blastidicacid, 2a 155.83, 154.05, 149,58, 148.34, 138.25, 138.03, 137.82, 137.61, A sample of [l "-14~]-1hydrochloride (485 mg, 1.06 mmol, 5.97 x 132.58, 132.45, 131.61, 131.53, 131.36, 130.39, 129.53, 129.01, lo6 dpdmmol) was dissolved in 6 N HCl(10 mL), and stirred at room 99.57,85.96,78.29,76.60,75.24,71.96,65.64,25.93. Anal. calcd. for temperature for 6 h (these conditions caused significant decomposition C40H13N301,: C 67.52; H 1.84, N 5.9 1; found: C 67.60, H 1.70, N of 3). The mixture was neutralized to pH 4.0 and applied to an anion 5.84. exchange column (Amberlite, IRA-410, 100 mesh, OH-, 2.5 cm x 30 cm), and the initial effluent immediately loaded onto a cation exchange ~-~-~ce@lc~tos~l-2',3',4',6'-tetrabenzo~l[l-~~]~lucose,1Oa column (Amberlite, IRC-50, 50 mesh, H+, 2.5 cm x 20 cm). This was Using the same procedure, a-1 -bromotetrabenzoyl[l -2~]glucose washed first with H70 to neutrality and then with 0.5 N HC1 until the (618 mg, 0.94 mmol), N-acetylcytosine (148 mg, 0.96 mmol), and eluates no longer contained 2a. he HC1 eluate was concentrated by Hg(CN)2 (5.34 mg, 2.1 1 mmol) yielded 474 mg (66%) of 1Oa: 'H rotary evaporation at ambient temperature, and the remaining solution NMR lacked the 6: 6.56 doublet, and the 6 5.93 triplet was a doublet was lyophilized. Recrystallization of the lyophilized residue from (J = 9.5 Hz); I3cNMR 6: 85.96 (bt). EtOH gave pure 2a dihydrochloride (234 mg, 5.97 x lo6 dpdmmol, 85% yield). P-Cytosylglucose, 8 Sodium (2 mg, 0.08 mmol) in a 25-mL round-bottom flask was Preparation of [l'-14~]-cytosinine,3a washed with hexane, dried in a stream of N2, and reacted with MeOH A sample of [1'-14c]-1 hydrochloride (320 mg, 0.70 mmol, 3.85 x (2 mL) under a N2 atmosphere.