Biosynthesis of the dialkylmaleic anhydride-containing antibiotics, tautomycin and tautomycetin

摘要

J. CHEM. SOC. PERKIN TRANS. 1 1995 Biosynthesis of the dialkylmaleic anhydride-containing antibiotics, tautomycin and tautomycetin Makoto Ubukata," Xing-Chun Cheng,b Jun Uzawa and Kiyoshi Isono Biotechnology Research Center, Toyama Prefectural University, Kosugi-machi, Imizu-gun, Toyarna 939-03, Japan The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 35I-01, Japan Department of Marine Science, School of Marine Science and Technology, Tokai University, 3-20-1, Orido, Shimizu, Shizuoka 424, Japan The biosynthetic origins of tautomycin and tautomycetin produced by Streptomyces spirovrrticillatus and Streptomyces griseochromogenus, respectively, have been studied by feeding experiments with 3C labelled precursors. The left half of tautomycin and tautomycetin are synthesized from one propionate and a C-5 unit. The latter is formed from three acetate units with decarboxylation.The labelling pattern from I3C-acetates indicated that a-keto glutarate or an equivalent may be a precursor. The results of a feeding experiment of 1,2-' 3C,glutamate afforded the direct proof for this idea. The right half of tautomycin is biosynthesized by a polyketide pathway which starts with isobutyrate followed by introduction of a glycolate, five acetate and five propionate units. The terminal methyl carbon originates from an acetate-methyl probably formed by decarboxylation of the intermediate terminal P-keto carboxylate anion. The right half of tautomycetin is formed via a polyketide pathway which starts with acetate followed by introduction of three acetate and four propionate units and one butyrate unit.The antifungal antibiotics, tautomycin and tautomycetin were found to be produced by Streptomyces spiroverticillutus and Stwptomyces griseochromogenus, respectively. Both antibiotics have similar structures and biological activities. These two antibiotics have not only a unique 2-(l-hydroxy-2- carboxyethyl)-3-methylmaleicanhydride struct~re,~,~ but also have unique biological activity. On further investigation, it was found that tautomycin and tautomycetin induced blebs on the cell surface of human leukemia cell K562.' More recently it was found that tautomycin inhibits protein phosphatase activity in a cell-free system,6 -* and competes with the specific binding of C3Hlabe1led okadaic acid to a particulate fraction prepared from mouse skin.Because of the increasing importance of tautomycin as a strong specific inhibitor of protein phosphatases 1 and 2A, its stereochemistry was elucidated,' and a total synthesis of tautomycin has been recently completed." This paper deals with the biosynthesis of these unique polyketide antibiotics, tautomycin and tautomycetin (Fig. I). Results and discussion Biosynthesis of tautomycin In the feeding experiments for a tautomycin-producing strain, Streptoniyces spiroverticillutus sp. JC-84-44, sodium 1-'"1-acetate, sodium 2-' 3Cacetate, sodium 1,2- "C,acetate, sodium 1-' 3Cpropionate, sodium 2-13Cpropionate, so-dium 3-13Cpropionate, L-methyl-13Cmethionine,sodium 1-' 3Cisobutyrate, 1,2-' 3C,glycine and sodium 1,2-'3C,glutamate were used as '3C-labelled precursors for tautomycin.The detailed '3C enrichment data of tautomycin are listed in Table 1. Feeding experiments of sodium 1-'3Cacetate showed that C-4, C-8, C-10, C-16, C-20, C-1' and C-7' were derived from sodium l-13Cacetate. 13C Enrichments were observed at C-1, (2-2, C-3, C-5, C-6, C-7, C-9, C-11, C-12, C-13, C-14, C-15, C-17, C-18, C-19, C-21, C-2', C- 3', C-4', C-5', C-6', 3-Me, 7-Me, 13-Me, 15-Me, 19-Me, 25-Me and 5'-Me as a result of the feeding experiment with sodium 2-13Cacetate. Relatively high enrichment ratios at C-I, C-5, C-9, C-11, C-17, C-21, C-2' and C-4' among them were observed as shown in Table 1.These results are consistent with the results of the feeding experiment of sodium l-' 3Cacetate and indicated that seven acetate units were incorporated into tautomycin. The lower enrichment values at C-2, C-3, C-6, C-7, C-12, C-13, C-14, C-15, C-18, C-19, C-5', C-6', 3-Me, 7-Me, 13- Me, 15-Me, 19-Me and 5'-Me indicated randomization of sodium 2-' 3Cacetate to 1,2,3- "CJpropionate presumably by multiple passages through the Krebs cycle followed by conversion of succinate into propionate by methylmalonyl Co-A mutase as already observed in cationomycin '' and cytovaricin biosynthesis. ' In the feeding experiments using 13C propionate, C-2, C-6, C-12, C-14, C-18 and C-6' were derived from sodium l -l 3Cpropionate and 3-Me, 7-Me, 13-Me, 15-Me, 19-Me and 5'-Me were derived from sodium 3-13Cpropionate as shown in Table 1.These results indicated that six propionate units were incorporated into tautomycin. Additional evidence for the incorporation of 3C propionate was obtained in the feeding experiment using sodium 2-' 3Cpropionate. which showed 13C enrichment at C-3, C-7, C-13, C-15, C-19 and C-5'. The feeding experiment using sodium I1-l 'Clisobutyrate showed that only C-24 was derived from C-l of isobutyrate. In the feeding experiment using ~-methyl-'~Cmethionine,high enrichment was observed at the methoxy methyl at C-23. In the 2-D INADEQUATE13 spectrum of tautomycin labelled with sodium 1,2-' 3C,acetate, the pattern of carbon- carbon correlation observed by cross peaks between the coupled carbons due to the randomization of sodium 1,2-' 3C,acetate revealed most of carbon connectivities of tautomycin. Although no enrichment could be detected on C-22 and C-23 in the 1D 13C NMR of tautomycin labelled with sodium l -'3Cacetate, sodium C2-l 3Cacetate or sodium 1,2-"C2acetate, the rather weak cross peaks of C-22 (66.4 ppm) and C-23 (80.6 ppm) were observed in the 2-D INADEQUATE spectrum of tautomycin labelled with sodium I ,2-'3C,acetate.3 The low incorporation of sodium 1,2-' 3C2acetate may indicate that acetate was metabolized to isocitrate which was converted into glyoxylate, followed by conversion into glycolate, namely hydroxyacetyl-Co A, then introduced into C-22 and C-23.In the leucomycin biosynthetic J. CHEM. SOC. PERKIN TRANS. 1 1995 0 Tau tomyan OH 0 Me OH 0 OH Me 2'6 Tautomycetin Fig. 1 Structures of tautomycin and tautomycetin Table 1 13C Chemical shifts and isotopic incorporation into tautomycin Relative enrichment ~ ~~~ Carbon 1-13C-2-13C-L-M~-'~C-I-"C-c2-139-3-13C -No. S( "C) Acetate Acetate Methionine Propionate Propionate Propionate 1 28.0 -3.5 2 213.0 -1.9 3 47.4 -1.6 4 29.1 3.6 -5 30.7 -3.7 6 74.3 -1.8 7 34.9 -2.0 8 28.1 3.0 -9 36.1 -3.9 -10 95.4 3.5 11 30.2 -3.9 12 26.7 -2.4 13 27.6 -2.7 14 74.8 -1.9 15 34.8 2.6 16 27.4 3.3 17 31.4 -3.3 18 74.3 -1.8 19 52.4 -1.6 20 215.0 4.4 -21 45.8 -4.4 22 66.4 23 80.6 24 76.5 --25 28.7 26 19.4 -1.53 -1' 196.6 5.7 2' 40.9 -6.3 3' 64.0 2.7 4' 142.1 -3.2 5' 142.9 -I .8 6' 165.8 -2.3 -7' 164.8 2.7 3-Me 16.2 -2.5 7-Me 17.9 -2.1 13-Me 10.9 -1.9 15-Me 16.7 -2.1 19-Me 13.7 -1.9 25-Me 17.8 -2.1 5'-Me 10.2 -2.6 23-OMe 59.I a Relative enrichments were normalized to peak intensities for the 23-OMe signal.Enrichment ratio of the peak of tautomycin enriched by l-13Cisobutyrate was 5.2. 'Relative enrichment of 23-OMe was normalized to peak intensity for the C-20 signal. study, Omura et al. also observed that sodium l-13Cacetate glycolate pathway." To test this possibility, a feeding and 2-'3Cacetate were not incorporated into a C, unit experiment with l ,2-'3C2glycine which gives glyoxylate by corresponding to C-3 and -4 to which a hydroxyl and a methoxy transamination was performed.This I3C double labelled unit group are attatched, re~pectively.'~ They postulated that the C2 was highly incorporated into C-22 and C-23 (Jcc = 42.4 Hz) of unit of leucomycin are derived from glycerol via glycine-tautomycin. This result indicated that glycine was incorporated J. CHEM. SOC. PERKIN TRANS. I 1995 200 153 '00 50 ~ ~ I i I I Fig. 2 PFG 2-D INADEQUATE spectrum of tautomycin labelled with 1,2-'3C,glycine. JEOL 01-400FT NMR spectrometer, 35 mg 140 mm-3 using micro NMR tube, T = 5.8 ms, measurement time; 16 h. into C-22 and C-23 via the glyoxylate-glycolate pathway. The '3C-13C labelling pattern detected by the pulsed field gradient (PFG) 2-D INADEQUATE l6 technique using a micro NMR tube is shown in Fig.2. 1,2-13C2Glycine was also incorporated into six acetate units, C-4 and C-5, C-8 and C-9, C- 10 and C-1 1, C-16 and C- 17, C-20 and C-2 1, C-1 ' and C-2', probably through the glycine reductase pathway. However, the possibility of indirect incorporation of glycine via the glycine reductase pathway through acetate into C-22 and C-23 could be ruled out, because the higher enrichment at C-22 and C-23 were observed even in 1D 13C NMR. The low level incorporation into 2,3-13C,propionate units, C-3 and 3-Me, C-7 and 7-Me, C-13 and 13-Me, C-15 and 15-Me, C-19 and 19-Me, are reasonably explained by second cycle passage of acetate through the Krebs cycle followed by the methyl malonate- propionate shunt via succinate.'' On the basis of the above data, we conclude that the right half chain of the tautomycin molecule is synthesized by a polyketide pathway which starts with isobutyrate followed by introduction of a glycolate unit, and then five acetate and five propionate units. The terminal methyl carbon (C-I) was derived from 2-' 3Cacetate which may be metabolized to P-keto carboxylic acid, after which the terminal methyl ketone is formed by decarboxylation. The left half of the antibiotic, the dialkylmaleic anhydride moiety is synthesized from one propionate and a C-5 unit. Although the biosynthesis of 2-butyl-3-methylmaleic anhydride from the condensation of a hexanoyl derivative with oxalacetic acid has been reported,' there is no biosynthetic precedent for the 24 1-hydroxy-2-carboxyethyl)-3-methylmaleicanhydride path.As we have described above, C-1' and -7' were derived from sodium 1-13Cacetate and carbons 2', 3' and 4' were derived from sodium 2-l3C)acetate. It is to be noted that the enrichment ratios at C-1' and -2' derived from sodium 1-"Clacetate and 2-13Cacetate are twice as high as those at C-7' and C-4'. This labelling pattern suggests strongly that the C-5 unit may come from a-keto glutarate which is formed from acetate through Krebs cycle (Fig. 4).The clear evidence of this hypothesis was finally obtained from the feeding experiment of 2401 165 '60 155 1so 145 t' I 7' C-4' Fig.3 PFG 2-D INADEQUATE spectrum of tautomycin labelled with L- I ,2-' 3C,glutamate. JEOL a-400 FT NMR spectrometer, 29 mg 140 mm-3 using micro NMR tube, T = 5.8 ms, measurement time; 22 h. L- 1,2-' 3C2glutamate which is a direct precursor of a-1,2-13C2keto glutarate (transamination). Only C-4' and C-7' (.Icc 61.5 Hz) in tautomycin were enriched with ~-1,2-'~C~- glutamate. The PFG 2-D INADEQUATE spectrum of tautomycin labelled with L- 1,2-l 3C2glutamate are shown in Fig. 3. This result directly evidenced that the C-5 unit was derived from a-keto glutarate. The dialkylmaleic anhydride moiety may be biosynthesized via an aldol type condensation of the ketone of x-keto glutarate with the active methylene of propionyl CoA, followed by dehydration and then hydration of the resulting allylic position at C-3'.Thus, the biosynthesis of tautomycin is depicted in Fig. 4.It is noteworthy that this experiment affords the first clear evidence of the biosynthesis of a C-5 unit which has been deduced from the distribution of label from '3C acetate for the biosynthesis of domoic acid * and others.' Biosynthesis of tautomycetin Feeding experiments with sodium l-13Cacetate showed that carbons 4, 10, 14, 18, 1' and 7' were derived from sodium I -l 3Cacetate. Feeding experiments of sodium 2-' 3Cacetate showed that carbons 1,5,6,7,8,9, 11, 15, 19,2', 3', 4', 5', 7-Me, 9-Me, 13-Me, 17-Me and 5'-Me were enriched. These results indicated randomization of 2-'3Cacetate to I ,2,3-' 3C3- propionate as already observed and discussed with tautomycin.In the experiments using 13C propionate, carbon 6, 8, 12, 16 and 6' were derived from sodium l-'3Cpropionate and carbons 7-Me, 9-Me, 13-Me, 17-Me and 5'-Me were derived from sodium 3-' 3Cpropionate. These results indicated that five propionate units were incorporated into tautomycetin. In the case of sodium 2-' 3Cpropionate, '3C enrichment at carbons 7,9, 13, 17 and 5' were observed. The experiment using sodium l-13Cbutyrate showed that only the carbon 2 was derived from C-1 of butyrate (Table 2). The 13C NMR and Echo-type 2D INADEQUATE 13*20 spectra of tautomycetin labelled with 1,2-' 3C2acetate are shown in Fig. 5. Five pairs of cross peaks observed between C-4 and C-5 (Jcc 53.8 Hz), C-10 and C-I1 (Jcc 29.4 Hz), C-14 and 2402 J.CHEM. SOC. PERKIN TRANS. I 1995 isocitrna 'COOH COSCoAI -glycincpropime . OH 0 OH 0 0 mcthimint mthyl # Biosynthesis of tautomycin. 0:derived from C-2 methyl carbon of 2-13Cacetate. 13C Enrichment ratios at C-1' and C-2' are higher than Fig. 4 those at C-4'and C-7'. Fig. 5 Echo-type 2D INADEQUATE spectrum of tautomycetin labelled with 1,2-' 'CJacetate. JEOL GX-400 FT NMR spectrometer, t = 5 ms, measurement time; 37 h. C-15(Jcc39.2Hz),C-18and 18-Me(Jcc41.9Hz)andC-1' and C-2' (Jcc 57.4 Hz) confirmed the five acetate units. No radomization occurred and cross peaks between C-4' and C-7' could not be detected in this case.On the basis of the above data, the biosynthetic origin of tautomycetin can be summarized as follows. The right half chain of the tautomycetin molecule is formed via the polyketide pathway which starts with acetate followed by introduction of three acetate and four propionate units and one butyrate unit. The terminal methyl carbon (C-1) was derived from 2-13Cacetate. The data suggests that the terminal methylene are formed by decarboxylation of the corresponding P-keto carboxylate or P-hydroxy carboxylate anion. The left half dialkylmaleic anhydride moiety has the same structure as that of tautomycin and gave a similar enrichment pattern. As described above, C-1' and -7' were derived from l -l 3Cacetate and C-2', -3' and -4' were derived from 2-13Cacetate.This labelling pattern suggested that the anhydride moiety was formed by condensation of one molecule of propionate with the C-5 unit derived from m-keto glutarate as in the case of tautomycin (Fig. 6). Since the absolute configuration of the dialkylmaleic anhydride moiety of tautomycetin is identical with that of tautomycin,? both the moieties may be biosynthesized by similar enzyme systems. t CD curve of the trimethyl ester derived from the dialkylmaleic anhydride moiety of tautomycin was identical with that of tautomycetin. J. CHEM. SOC. PERKIN TRANS. 1 1995 2403 Table 2 3C Chemical shifts and isotopic incorporation into tautomycetin Relative enrichment a Carbon 1-'3C- 2-'3C- 1-'3C- I-'"- 2- 3C - 3-'3C- No.6( 3C) Acetate Acetate Butyrate Propionate Propionate Propionate 1 120.0 1.6 3 I 139.0 - 3 156.0 ~~ 4 126.0 - 5 201.o 1.9 6 52.8 1.8 7 27.1 1.4 8 44.9 1.4 9 29.8 1.5 10 32.5 - 11 31.7 1.6 12 73.6 I .6 13 52.5 1.8 14 215.0 - 15 46.6 1.4 16 66.0 - 17 42.8 1.3 18 73.4 - 19 18.4 1.4 1' 170.0 - 3' 40.6 1.3 3' 63.0 1.3 4' 142.2 1.7 5' 142.9 1.5 6' 165.7 - 7' 164.8 - 7-Me 20.0 1.4 9-Me 19.2 1.5 13-Me 28.7 1.4 17-Me 10.3 2.0 5'-Me 10.2 2.0 1 " 2" 20.5 13.9 -- ~ ~~ ~ Relative enrichments were normalized to peak intensities for the C-2" signal. Experimental 3C NM R spectra were measured on a JEOL FX-100FT NMR spectrometer.2D INADEQUATE spectra were recorded on JEOL GX-400 FT NMR. Pulsed field gradient (PFG) 2D- INADEQUATE spectra were recorded on a JEOL CX-400 FT NMR spectrometer with a PFG unit. Micro NMR tubes were made by Shigemi Ltd, Japan. The 13C labelled acetate and propionate (90 atom) were purchased from MSD ISOTOPES, Canada. 1-'3CButyrate (99 atom) was purchased from ICON, U.S.A. ~-rnethyl-'3CMethionine (98.2 atom) was purchased from MSD ISOTOPES, Canada. I ,2-13C,Glycine (99 atom) was purchased from ISOTEC, U.S.A. L-1,2-' 3C,Glutamic acid (99 atom) was purchased from EURISO-TOP, France. Culture of the '3C-labelled tautomycin-producing strain Streptomyces spiroverticillatus sp.JC-84-44 grown on starch- yeast agar was inoculated into a 400 cm3 cylindrical flask containing 70 cm3 of a medium composed of glucose 2, soluble starch 1, meat extract O.l, dry yeast 0.4, soybean flour 2.5, NaClO.25 and K,HPO, 0.005, and cultured at 28 "C for 48 h on a rotary shaker. Aliquots (0.5cm3) of the seed culture was inoculated into the same medium. Fermentation was carried out on a rotary shaker at 28 "C. After 24 h of fermentation, a '3C-labelled precursor was added to the culture at a concentration of 0.6 (w/v), and fermentation was continued for an additional 48 h. In the cases of 1,2-'3C,glycine and L-sodium 1,2-' 3C2glutamate prepared from L- 1,2-'3C2glutamic acid neutralized with 0.1 mol dm-13 aq. NaOH, precursors were added in doses of 0.007 (w/v) at 24, 30, 36 and 46 h after inoculation, and the cultures were further incubated for 24 h.Isolation of 13C-labelledtautomycin Each fermentation broth (490 cm3; pH 7.2; 350 cm3 in the cases of 1,2-13C,glycine and L-sodium 1,2-13C,glutamate) was filtered; the filtrate was extracted with EtOAc and the mycelium cake was extracted with acetone. The acetone extract was concentrated under reduced pressure to give an aqueous solution, which was then extracted with EtOAc. Both the EtOAc extracts were combined and evaporated to dryness under reduced pressure. The residue was chromatographed on a silica gel column with CHC1,-MeOH (4: 1). The eluate was monitored with silica gel TLC, and then further purified by reverse phase HPLC Senshu pak ODS-H column with MeOH- H,O-buffer (1 diethylamine-formic acid, pH 7.3), 8 :1 : 1 as a solvent system. The fraction were adjusted to pH 4 with 1 mol dm-3 HCl and concentrated to give an aqueous solution, which was then extracted with EtOAc and evaporated under reduced pressure to give pure tautomycin.From each 490 cm3 of the culture broth supplemented with 3C-labelled precursors, 20- 45 mg of purified l3C-labe1led tautomycin was obtained. The yields of 1 ,2-'3C,glycine labelled and L-I ,2-'3C2glutamate labelled tautomycins were 35.7 mg and 29.9 mg, respectively. Culture of I3Clabelled tautomycetin-producing strain and isolation of I3Clabelled tautomycetin For preparation of labelled tautomycetin, Streptomyces griseochrornogenus sp.JC-84-1223 was used. The cultural J. CHEM. SOC. PERKIN TRANS. 1 1995 bsol;COOHr ICOSCoA pmpionna DOH OH 0 OH 0 0 O0 k 0 Fig. 6 Biosynthesis of tautomycetin. a:derived from C-2 methyl carbon of 2-' Tlacetate. conditions and isolation procedure were the same as those for 3C labelled tautomycin, except that the volume of the fermentation broth of tautomycetin was increased to 980 cm3 and that MeOH-buffer for HPLC was adjusted to pH 4.0. Acknowledgements This work was supported by a Grant-in-Aid for Scientific Reserch from the Ministry of Education, Science and Culture of Japan. References 1 X.-C. Cheng, T. Kihara, H. Kusakabe, J. Magae, Y. Kobayashi, R.-P. Fang, Z.-F.Ni, Y.-C. Sheng, K. KO, I. Yamaguchi and K. Isono, J. Anfibiot., 1987,40, 907. 2 X.-C. Cheng, T. Kihara, X. Ying, M. Uramoto, H. Osada, H. Kusakabe, B. N. Wang, Y. Kobayashi, K. KO, I. Yamaguchi, Y.-C. Sheng and K. Isono, J. Antihiot., 1989,42, 141. 3 (a)M. Ubukata, X.-C. Cheng and K. Isono, J. Chem. Soc., Chern. Commun., 1990, 244; (h)X.-C. Cheng, M. Ubukata and K. 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Tsuzuki, A. Nakagawa and G. Lukacs, J. Antibiot., 1983,36, 61 1. 16 Field gradient 2-D INADEQUATE techique for detection of natural abundance "C 13C coupling will be reported elsewhere. 17 (a) C. E. Moppett and J. K. Sutherland, J. Chem. Soc., Chem. Commun., 1966, 772; (h) J. L. Bloomer, C. E. Moppett and 3. K. Sutherland, J. Chem. Soc., Chem Commun., 1965,619. 18 D. J. Douglas, U. P. Ramsey, J. A. Walter and J. L. C. Wright, J. Chem. Soc., Chrm. Commun., I 992, 714. 19 T. Hemscheidt and E. D. Spenser, J. Am. Chem. Soc., 1993,115,3020. 20 D. L. Turner, J. Mugn. Reson., 1982,49, 175. Paper 5/03359D Received 25th May 1995 Accepted 6th June 1995