RAPID COMMUNICATIONS I N MASS SPECTROMETRY, VOL. 5 , 534-537 (1991)
Structure Determination of the Antibiotic Desertomycin B by Fast-atom Bombardment Mass Spectrometric Techniques Zoltan Dinya,* Ferenc Sztaricskai* and GCza Horvath Research Group for Antibiotics of the Hungarian Academy of Sciences, University of Debrecen, PO Box 70, Debrecen, H-4010 Hungary SPONSOR REFEREE: Dr KBroly VCkey, Central Research Institute for Chemistry, Hungarian Academy of Sciences, Budapest, Hungary
By means of different fast-atom bombardment (FAB) mass spectrometric techniques (i.e., positive- and negative-ion FAB ionizations and high resolution measurements) we have determined the chemical structure of desertomycin B which is a novel representative of the family of macrolide antibiotics carrying a guanidino function in its side-chain.
Desertomycin, a broad-spectrum antibiotic, coproduced with flavofungin by the strain Streptomyces flauofungini’ (which was isolated from a soil sample collected in the Sahara desert) was first reported by Uri et al. ,* in 1958. In 1983 Dolak et al. ,3 also described the isolation of desertomycin from the culture of Streptomyces macronensis Dietz sp. nov. UC 8271 (NRRL 12566) and gave a (erroneous) molecular formula of Cs7H,09N024 for the antibiotic. Upon preliminary structural studies’-’ desertomycin appeared to be a large-ring macrolide compound, related to monazomycin,6 ~ c o p a f u n g i nprimycin,8 ,~ azalomycin F,9-” niphithricins A and B,” guanidylfungins A and B,’’-l4 copiamycin,” neocopiamycin16 and niphimy~in.’~ Because of the interesting biological properties of desertomycin,’8 such as blockage of the K+ channels in muscle fibers, cytotoxicity against leukemia cells, as well as the possibility of selective isolation of human-pathogenic fungi from ordinary culturesamples treated with desertomycin, we have undertaken the structure elucidation of desertomycins. The antibiotic, which behaved as a single compound on paper chromatography,’,’ was later found to be a mixture of at least three components. High-performance liquid chromatographic (HPLC) analysis of the pre-purified desertomycin (Fig. 1) showed that it contained two minor components (a and c) besides the major one (b). Since neither of these components was found to be convertible into the others, the chemical structure of the major compound (b), named desertomycin A , was first studied and determined (Fig. 2) in 1986 together with Bax et al.,’” by using modern two-dimensional NMR and fast-atom bombardment (FAB) mass spectrometric techniques. This structure was independently confirmed by the chemical and NMR investigations of the Bognar At that time, however, the chemical structure of the two minor components still remained unknown. During the present studies, the crude desertomycin complex was concentrated so as to enrich component c (this being the more abundant of the minor components present) the resulting mixture being enriched in * Authors to whom correspondence should be addressed 0951-4198/91/110534-04 $05.00 01991 by John Wiley & Sons, Ltd.
2.52
i
I
0
1
2
3
4
5
D
r e t e n t i o n time Inin.)
Figure 1. HPLC of the desertomycin complex. [Column: Hypersil ODS (100X2.1 mm; 5 p m ) ; mobile phase: 20:8 0.01 ~ a q u . NH,OAc-CH,CN; flow rate: 1 mL/min; detection: UV 228 nm].
this component by about 3 to 4-fold. The product prepared in this way was found to be suitable for FAB-mass spectrometric investigations, and the present paper deals with the structural elucidation of this minor component c (named desertomycin B) of the antibiotic complex.
MATERIALS AND METHODS Crude desertomycin, used for the investigations was produced by the original Streptomyces strain described by Uri.’,2.18The sample was obtained from Dr Istvan Bekesi (Institute of Biology, University Medical School of Debrecen, Hungary), and it was further purified by HPLC. Analytical HPLC was carried out on a reversed-phase Hypersil ODS column, 100 mm X 2.1 mm; 5 ym (Altex, Berkeley, CA, USA); mobile phase: 20 : 8 0.01 M aqu. NH,OAc-CH3CN; flow rate: 1 mL/min with Hewlett-Packard (Palo Alto, CA, Received 30 A U ~ U S 1991 I Arreprd 2 Sepremher 1991
STRUCTURE DETERMINATION O F DESERTOMYCIN B BY FABMS OH
0
OH
OH
535
OH
h:
0
OH
R DESERTOMYCIN A
:
R =NH2
D E S E R T O M Y C I N 6 : R = NH--CdNH \NH~
Figure 2. Structure of desertomycin A and B
USA) gradient HPLC system (HP 1090) employing UV detection at 228nm. A gradient of acetonitrile was applied over 60min to a final composition of 100% acetonitrile. Preparative reversed-phase liquid chromatography was carried out in a Waters Associates 500 instrument, using a Prep Pak 500/C,8 column [mobile phase: 1 : 4 0.01 M aqu. NH,OAc-MeOH; flow rate: 100 mL/min] and employing a refractometric detector.
HPLC grade acetonitrile and methanol were purchased from Mallinckrodt (Paris, NY, USA). Purified water was obtained from a Milli-Q purifying system (Millipore, Bedford, MA, USA). Purity check. TLC was performed on Kieselgel 60 F254(Merck, Darmstadt, Germany) pre-coated layer with 6: 4 :3 n-BuOH-pyridine-H,O (upper layer); R,: 0.68 for desertomycin A and 0.71 for desertomycin
.I
279.2
23!. I 385.1
389.1
288
- .
388
488
sBe
&RE
798
18B 98. 88. 78.
611. 58.
48.
38.
ee . 18.
81
A
888
38
I088
-
-
1188
1288
Figure 3. Positive-ion FAB mass spectrum of pure desertomycin A.
1388
1346.1
I489
STRUCTURE DETERMINATION OF DESERTOMYCIN B BY FABMS
536
98.
BE. 135.8 78.
l52.E 68. 58.
4s.
iaa 98. 68. 7%. 66
-
5E.
4E.
38.
2E
.
1192.?
18.
1346.1
e,.,
A
.
Figure 4. Positive-ion FAB mass spectrum of the purified desertomycin complex
B. Visualization of the spots was effected by iodine vapour. I3CN M R spectra. These were recorded on a Bruker WP 200 SY (I3C50.303 MHz) spectrometer.*' In the 13C NMR spectrum of the desertomycin sample obtained upon enrichment of desertomycin B, a characteristic signal at 153.1 ppm was detected after a larger acquisition time, which could not be assigned to any structural portion of desertomycin A. Mass spectrometry. Positive- and negative-ion FAB mass spectra were obtained on a ZAB-SE mass spectrometer (VG Analytical, Manchester, UK) operated at 8 keV accelerating potential with a resolving power of 1000 (10% valley definition) and a scan rate of 30s/decade from 1500 to 1OOu in the low-resolution mode. High-resolution mass measurements were carried out by the peak matching procedure at a resolving power of 8000. Data acquisition and processing were performed by using a VG 1112.50 data system. The samples were dissolved in a 3 :7 (by volume) mixture of dimethyl sulfoxide (DMSO) and 'magic bullet' (MB)23 (1:5 dithioerythritol-dithiothreitol) on a stainless-steel probe tip. For negative-ion measurements the matrix was acidified with 0.1 M IIC1. Operating conditions for the FAB gun (Ion Tech, Teddington, UK) on the instrument were 9 keV at 1.2 mA with xenon as the
FAB gas. The sample and the mass spectrometer ionsource were maintained at room temperature in each experiment. RESULTS The positive-ion FAB mass spectra of pure desertomycin A (Fig. 3) showed peaks at mlz 1346 [M+MB]', 1215 [M+Na]', 1193 [ M + H ] + , 1175 [M+H-H,O]+ 1157 [M+H-2H20]+, 1139 [ M + H - 3 H 2 0 j f , 103i [M + H-mannose (C6H,,O5)]+,1015 [M+ H-mannose (C6H,206)]+ and 997 [M + H-mannose-H,O]'. Below the mlz900 range only peaks arising from the matrix [MB]+ and the solvent [nDMSO+H]+ were detected. The high-resolution measurements unequivocally confirmed the previously established'" C61H1,0021N molecular formula for desertomycin A [high resolution FABMS, mlz: 1192.7558; calcd. u)]. 1192.7570 ( A = 1.2 X The positive-ion FAB mass spectrum of the sample enriched in desertomycin B is depicted in Fig. 4. Compared to the spectrum of pure desertomycin A , an additional, well-distinguishable peak (miz 1235) has appeared in the molecular-ion range. The top part of the positive- and negative-ion FAB mass spectra are shown in Fig. 5. The negative-ion data were used to
STRUCTURE DETERMINATION O F DESERTOMYCIN B BY FABMS
537
1215 0
10
-
1288
I
1227
0
1168 1180 1200 1220 1240 1260 1280 1300 1320 1340 1360 1380
Figure5 Molecular ion regions of positive- and negative-ion FAB mass spectrum of the pre-purified desertomycin complex (in MB with 0.1 M HCI) (a) positive-ion FAB; (b) negative-ion FAB.
confirm the positive-ion spectrum. The peaks characteristic of desertomycin A (at r n l z 1346 [M MB]+ and 1193 [M HI+) appeared two mass units lower in the negative-ion FAB spectrum (Fig. 5 , lower part) and the [M-HI- fragment was detected at rnlz 1191. The peaks rnlz 1227 and . d z 1288 corresponded to those of the [M C1]- and [M + DMSO - H,O]- fragments, respectively, of desertomycin A . The peak at rnlz 1235 in the positive-ion spectrum is assigned at rnlz 1271 in the negative-ion spectrum and this is related to an uptake of one molar equivalent of hydrochloric acid. All of these data indicated that the molecular mass of desertomycin B, present in the antibiotic complex, is 1234u. The peak at rnlz 1388 corresponded to the fragment [1234 MB]+ providing the evidence that the peak at mlz 1234 is indeed due to the molecular ion. The mass value of 1234.7772 u obtained by means of high resolution positive-ion FAB measurements is in best accord with an elemental composition C,,H,,,N,O,, (calcd. value: 1234.7788; A = 1.6 x u). According to these data the molecular formulae of desertomycin A and B thus differ by a CH,N, moiety. The present FABMS studies clearly show that desertomycin B (Fig. 2) carries a guanidino function in the side-chain. This is also supported by our previous I3C NMR studies which indicated an extremely weak signal at 156.2ppm in the spectrum of the crude antibiotic complex, corresponding to the carbon atom of the guanidino moiety. Thus the antibiotic desertomycin B is a novel representative of the family of macrolide antibiotics carrying a guanidino function in the sidechain.
+
+
+
+
Acknowledgements This work was supported by the Hungarian Academy of Sciences (OTKA Grant No. 298) and by the Reanal Fine Chemical Works (Budapest, Hungary). The authors are indebted to Dr I. Bekesi for
the samples of desertomycin. We wish to thank Professor K. L. Rinehart Jr (University of Illinois at Urbana-Champaign) for technical support of our FAB measurements.
REFERENCES 1. J. Uri, Acta Microbiol. Hungarica 33, 305 (1986). 2. J. V. Uri, R. Bognar, I. BCkCsi and B. Varga, Nature 182, 401 (1958). 3. L. A . Dolak, F. Reusser, L. Baczynskyj, S. A. Mizsak, 8. R. Hannon and T. M. Castle, J . Antibiotics 36, 13 (1983). 4. F. Sztaricskai and J. V. Uri, Microchim. Acta 3, 431 (1963). 5. R . Bognir, F. Sztaricskai, L. Somogyi, M. Puskas and S. Makleit, Acta Chim. Acad. Sci. Hung. 56, 53 (1968). 6. K. Akasaki, K. Karasawa, M. Watanabe, H. Yonehara and H . Umezawa, J . Antibiotics 26, 127 (1963). 7. D . Samain, J. C. Cook and K. L. Rinehart, Jr., J . A m . Chem. SOC. 104, 4120 (1982). 8. J. Aberhart, T. Fehr, R . C. Jain, P. Demayo, 0. Motl, L. Baczynskyj, D. E. F. Gracey. D . B. Maclean and I. Szilagyi, J . Am. Chem. Soc. 92, 5816 (1970). 9. M. Namikoshi, K. Sasaki, Y. Koioso, K. Fukushima, S. Iwasaki, S. Nozoe and S. Okuda, Chem. Pharm. Bull. 30, 1653 (1982). 10. M. Namikoshi, S. Iwasaki, K. Sasaki, M. Yano, K. Fukushima, S. Nozoe and S. Okuda, Chem. Pharm. Bull. 30, 1658 (1982). 11. S. Iwasaki, M. Namikoshi, K. Sasaki, M. Yano, K. Fukushima, S. Nozoe and S. Okuda, Chem. Pharm. Bull. 30, 1669 (1982). 12. H.-P. Fiedler, W. Worner, H. Zahner, H. P. Kaiser, W. Keller-Schierlein and A . Miiller, J . Antibiotics 34, 1107 (1981). 13. K. Takesako and T. Beppu, J . Antibiotics 37, 1161 (1984). 14. K. Takesako and T. Beppu, J . Antibiotics 37, 1170 (1984). 15. K. Fukushima, T . Arai, S. Iwasaki, M. Namikoshi and S . Okuda, J . Antibiotics 35, 1480 (1982). 16. T. Arai, J . Uno, I. Horimi and K. Fukushima, J . Antibiotics 37, 103 (1984). 17. P. Gassmann, L. Hagmann, W. Keller-Schierlein and D. Samain, Helu. Chim. Acta 67, 696 (1984). 18. J. V. Uri, Acta Microbiol. Hungarica 33, 271 (1986). 19. A. Aszalos and Z . Dinya (unpublished results). 20. A. Bax, A. Aszalos, Z . Dinya and K. Sudo, J . A m . Chem. SOC. 108, 8056 (1986). 21. R . Bognar, GY. Batta, Z . Dinya, I. PelyvAs and F. Sztaricskai, Acta Chim. Hungarica (in press). 22. G. Horvath., GY. Batta, R . Bognar and F. Sztaricskai, Acta Chim. Hungarica'(in press). 23. Cochran, R. L., Applied Spectrosc. Reu. 22, 137 (1986).
Structure Determination of the Antibiotic Desertomycin B by Fast-atom Bombardment Mass Spectrometric Techniques Zoltan Dinya,* Ferenc Sztaricskai* and GCza Horvath Research Group for Antibiotics of the Hungarian Academy of Sciences, University of Debrecen, PO Box 70, Debrecen, H-4010 Hungary SPONSOR REFEREE: Dr KBroly VCkey, Central Research Institute for Chemistry, Hungarian Academy of Sciences, Budapest, Hungary
By means of different fast-atom bombardment (FAB) mass spectrometric techniques (i.e., positive- and negative-ion FAB ionizations and high resolution measurements) we have determined the chemical structure of desertomycin B which is a novel representative of the family of macrolide antibiotics carrying a guanidino function in its side-chain.
Desertomycin, a broad-spectrum antibiotic, coproduced with flavofungin by the strain Streptomyces flauofungini’ (which was isolated from a soil sample collected in the Sahara desert) was first reported by Uri et al. ,* in 1958. In 1983 Dolak et al. ,3 also described the isolation of desertomycin from the culture of Streptomyces macronensis Dietz sp. nov. UC 8271 (NRRL 12566) and gave a (erroneous) molecular formula of Cs7H,09N024 for the antibiotic. Upon preliminary structural studies’-’ desertomycin appeared to be a large-ring macrolide compound, related to monazomycin,6 ~ c o p a f u n g i nprimycin,8 ,~ azalomycin F,9-” niphithricins A and B,” guanidylfungins A and B,’’-l4 copiamycin,” neocopiamycin16 and niphimy~in.’~ Because of the interesting biological properties of desertomycin,’8 such as blockage of the K+ channels in muscle fibers, cytotoxicity against leukemia cells, as well as the possibility of selective isolation of human-pathogenic fungi from ordinary culturesamples treated with desertomycin, we have undertaken the structure elucidation of desertomycins. The antibiotic, which behaved as a single compound on paper chromatography,’,’ was later found to be a mixture of at least three components. High-performance liquid chromatographic (HPLC) analysis of the pre-purified desertomycin (Fig. 1) showed that it contained two minor components (a and c) besides the major one (b). Since neither of these components was found to be convertible into the others, the chemical structure of the major compound (b), named desertomycin A , was first studied and determined (Fig. 2) in 1986 together with Bax et al.,’” by using modern two-dimensional NMR and fast-atom bombardment (FAB) mass spectrometric techniques. This structure was independently confirmed by the chemical and NMR investigations of the Bognar At that time, however, the chemical structure of the two minor components still remained unknown. During the present studies, the crude desertomycin complex was concentrated so as to enrich component c (this being the more abundant of the minor components present) the resulting mixture being enriched in * Authors to whom correspondence should be addressed 0951-4198/91/110534-04 $05.00 01991 by John Wiley & Sons, Ltd.
2.52
i
I
0
1
2
3
4
5
D
r e t e n t i o n time Inin.)
Figure 1. HPLC of the desertomycin complex. [Column: Hypersil ODS (100X2.1 mm; 5 p m ) ; mobile phase: 20:8 0.01 ~ a q u . NH,OAc-CH,CN; flow rate: 1 mL/min; detection: UV 228 nm].
this component by about 3 to 4-fold. The product prepared in this way was found to be suitable for FAB-mass spectrometric investigations, and the present paper deals with the structural elucidation of this minor component c (named desertomycin B) of the antibiotic complex.
MATERIALS AND METHODS Crude desertomycin, used for the investigations was produced by the original Streptomyces strain described by Uri.’,2.18The sample was obtained from Dr Istvan Bekesi (Institute of Biology, University Medical School of Debrecen, Hungary), and it was further purified by HPLC. Analytical HPLC was carried out on a reversed-phase Hypersil ODS column, 100 mm X 2.1 mm; 5 ym (Altex, Berkeley, CA, USA); mobile phase: 20 : 8 0.01 M aqu. NH,OAc-CH3CN; flow rate: 1 mL/min with Hewlett-Packard (Palo Alto, CA, Received 30 A U ~ U S 1991 I Arreprd 2 Sepremher 1991
STRUCTURE DETERMINATION O F DESERTOMYCIN B BY FABMS OH
0
OH
OH
535
OH
h:
0
OH
R DESERTOMYCIN A
:
R =NH2
D E S E R T O M Y C I N 6 : R = NH--CdNH \NH~
Figure 2. Structure of desertomycin A and B
USA) gradient HPLC system (HP 1090) employing UV detection at 228nm. A gradient of acetonitrile was applied over 60min to a final composition of 100% acetonitrile. Preparative reversed-phase liquid chromatography was carried out in a Waters Associates 500 instrument, using a Prep Pak 500/C,8 column [mobile phase: 1 : 4 0.01 M aqu. NH,OAc-MeOH; flow rate: 100 mL/min] and employing a refractometric detector.
HPLC grade acetonitrile and methanol were purchased from Mallinckrodt (Paris, NY, USA). Purified water was obtained from a Milli-Q purifying system (Millipore, Bedford, MA, USA). Purity check. TLC was performed on Kieselgel 60 F254(Merck, Darmstadt, Germany) pre-coated layer with 6: 4 :3 n-BuOH-pyridine-H,O (upper layer); R,: 0.68 for desertomycin A and 0.71 for desertomycin
.I
279.2
23!. I 385.1
389.1
288
- .
388
488
sBe
&RE
798
18B 98. 88. 78.
611. 58.
48.
38.
ee . 18.
81
A
888
38
I088
-
-
1188
1288
Figure 3. Positive-ion FAB mass spectrum of pure desertomycin A.
1388
1346.1
I489
STRUCTURE DETERMINATION OF DESERTOMYCIN B BY FABMS
536
98.
BE. 135.8 78.
l52.E 68. 58.
4s.
iaa 98. 68. 7%. 66
-
5E.
4E.
38.
2E
.
1192.?
18.
1346.1
e,.,
A
.
Figure 4. Positive-ion FAB mass spectrum of the purified desertomycin complex
B. Visualization of the spots was effected by iodine vapour. I3CN M R spectra. These were recorded on a Bruker WP 200 SY (I3C50.303 MHz) spectrometer.*' In the 13C NMR spectrum of the desertomycin sample obtained upon enrichment of desertomycin B, a characteristic signal at 153.1 ppm was detected after a larger acquisition time, which could not be assigned to any structural portion of desertomycin A. Mass spectrometry. Positive- and negative-ion FAB mass spectra were obtained on a ZAB-SE mass spectrometer (VG Analytical, Manchester, UK) operated at 8 keV accelerating potential with a resolving power of 1000 (10% valley definition) and a scan rate of 30s/decade from 1500 to 1OOu in the low-resolution mode. High-resolution mass measurements were carried out by the peak matching procedure at a resolving power of 8000. Data acquisition and processing were performed by using a VG 1112.50 data system. The samples were dissolved in a 3 :7 (by volume) mixture of dimethyl sulfoxide (DMSO) and 'magic bullet' (MB)23 (1:5 dithioerythritol-dithiothreitol) on a stainless-steel probe tip. For negative-ion measurements the matrix was acidified with 0.1 M IIC1. Operating conditions for the FAB gun (Ion Tech, Teddington, UK) on the instrument were 9 keV at 1.2 mA with xenon as the
FAB gas. The sample and the mass spectrometer ionsource were maintained at room temperature in each experiment. RESULTS The positive-ion FAB mass spectra of pure desertomycin A (Fig. 3) showed peaks at mlz 1346 [M+MB]', 1215 [M+Na]', 1193 [ M + H ] + , 1175 [M+H-H,O]+ 1157 [M+H-2H20]+, 1139 [ M + H - 3 H 2 0 j f , 103i [M + H-mannose (C6H,,O5)]+,1015 [M+ H-mannose (C6H,206)]+ and 997 [M + H-mannose-H,O]'. Below the mlz900 range only peaks arising from the matrix [MB]+ and the solvent [nDMSO+H]+ were detected. The high-resolution measurements unequivocally confirmed the previously established'" C61H1,0021N molecular formula for desertomycin A [high resolution FABMS, mlz: 1192.7558; calcd. u)]. 1192.7570 ( A = 1.2 X The positive-ion FAB mass spectrum of the sample enriched in desertomycin B is depicted in Fig. 4. Compared to the spectrum of pure desertomycin A , an additional, well-distinguishable peak (miz 1235) has appeared in the molecular-ion range. The top part of the positive- and negative-ion FAB mass spectra are shown in Fig. 5. The negative-ion data were used to
STRUCTURE DETERMINATION O F DESERTOMYCIN B BY FABMS
537
1215 0
10
-
1288
I
1227
0
1168 1180 1200 1220 1240 1260 1280 1300 1320 1340 1360 1380
Figure5 Molecular ion regions of positive- and negative-ion FAB mass spectrum of the pre-purified desertomycin complex (in MB with 0.1 M HCI) (a) positive-ion FAB; (b) negative-ion FAB.
confirm the positive-ion spectrum. The peaks characteristic of desertomycin A (at r n l z 1346 [M MB]+ and 1193 [M HI+) appeared two mass units lower in the negative-ion FAB spectrum (Fig. 5 , lower part) and the [M-HI- fragment was detected at rnlz 1191. The peaks rnlz 1227 and . d z 1288 corresponded to those of the [M C1]- and [M + DMSO - H,O]- fragments, respectively, of desertomycin A . The peak at rnlz 1235 in the positive-ion spectrum is assigned at rnlz 1271 in the negative-ion spectrum and this is related to an uptake of one molar equivalent of hydrochloric acid. All of these data indicated that the molecular mass of desertomycin B, present in the antibiotic complex, is 1234u. The peak at rnlz 1388 corresponded to the fragment [1234 MB]+ providing the evidence that the peak at mlz 1234 is indeed due to the molecular ion. The mass value of 1234.7772 u obtained by means of high resolution positive-ion FAB measurements is in best accord with an elemental composition C,,H,,,N,O,, (calcd. value: 1234.7788; A = 1.6 x u). According to these data the molecular formulae of desertomycin A and B thus differ by a CH,N, moiety. The present FABMS studies clearly show that desertomycin B (Fig. 2) carries a guanidino function in the side-chain. This is also supported by our previous I3C NMR studies which indicated an extremely weak signal at 156.2ppm in the spectrum of the crude antibiotic complex, corresponding to the carbon atom of the guanidino moiety. Thus the antibiotic desertomycin B is a novel representative of the family of macrolide antibiotics carrying a guanidino function in the sidechain.
+
+
+
+
Acknowledgements This work was supported by the Hungarian Academy of Sciences (OTKA Grant No. 298) and by the Reanal Fine Chemical Works (Budapest, Hungary). The authors are indebted to Dr I. Bekesi for
the samples of desertomycin. We wish to thank Professor K. L. Rinehart Jr (University of Illinois at Urbana-Champaign) for technical support of our FAB measurements.
REFERENCES 1. J. Uri, Acta Microbiol. Hungarica 33, 305 (1986). 2. J. V. Uri, R. Bognar, I. BCkCsi and B. Varga, Nature 182, 401 (1958). 3. L. A . Dolak, F. Reusser, L. Baczynskyj, S. A. Mizsak, 8. R. Hannon and T. M. Castle, J . Antibiotics 36, 13 (1983). 4. F. Sztaricskai and J. V. Uri, Microchim. Acta 3, 431 (1963). 5. R . Bognir, F. Sztaricskai, L. Somogyi, M. Puskas and S. Makleit, Acta Chim. Acad. Sci. Hung. 56, 53 (1968). 6. K. Akasaki, K. Karasawa, M. Watanabe, H. Yonehara and H . Umezawa, J . Antibiotics 26, 127 (1963). 7. D . Samain, J. C. Cook and K. L. Rinehart, Jr., J . A m . Chem. SOC. 104, 4120 (1982). 8. J. Aberhart, T. Fehr, R . C. Jain, P. Demayo, 0. Motl, L. Baczynskyj, D. E. F. Gracey. D . B. Maclean and I. Szilagyi, J . Am. Chem. Soc. 92, 5816 (1970). 9. M. Namikoshi, K. Sasaki, Y. Koioso, K. Fukushima, S. Iwasaki, S. Nozoe and S. Okuda, Chem. Pharm. Bull. 30, 1653 (1982). 10. M. Namikoshi, S. Iwasaki, K. Sasaki, M. Yano, K. Fukushima, S. Nozoe and S. Okuda, Chem. Pharm. Bull. 30, 1658 (1982). 11. S. Iwasaki, M. Namikoshi, K. Sasaki, M. Yano, K. Fukushima, S. Nozoe and S. Okuda, Chem. Pharm. Bull. 30, 1669 (1982). 12. H.-P. Fiedler, W. Worner, H. Zahner, H. P. Kaiser, W. Keller-Schierlein and A . Miiller, J . Antibiotics 34, 1107 (1981). 13. K. Takesako and T. Beppu, J . Antibiotics 37, 1161 (1984). 14. K. Takesako and T. Beppu, J . Antibiotics 37, 1170 (1984). 15. K. Fukushima, T . Arai, S. Iwasaki, M. Namikoshi and S . Okuda, J . Antibiotics 35, 1480 (1982). 16. T. Arai, J . Uno, I. Horimi and K. Fukushima, J . Antibiotics 37, 103 (1984). 17. P. Gassmann, L. Hagmann, W. Keller-Schierlein and D. Samain, Helu. Chim. Acta 67, 696 (1984). 18. J. V. Uri, Acta Microbiol. Hungarica 33, 271 (1986). 19. A. Aszalos and Z . Dinya (unpublished results). 20. A. Bax, A. Aszalos, Z . Dinya and K. Sudo, J . A m . Chem. SOC. 108, 8056 (1986). 21. R . Bognar, GY. Batta, Z . Dinya, I. PelyvAs and F. Sztaricskai, Acta Chim. Hungarica (in press). 22. G. Horvath., GY. Batta, R . Bognar and F. Sztaricskai, Acta Chim. Hungarica'(in press). 23. Cochran, R. L., Applied Spectrosc. Reu. 22, 137 (1986).
