HETEROCYCLES

Paper | Special issue | Vol. 80, No. 1, 2010, pp. 281-288
Received, 17th February, 2009, Accepted, 11th March, 2009, Published online, 17th March, 2009.
DOI: 10.3987/COM-09-S(S)11

■ M10709, a New Cyclic Peptide Antibiotic from Clinically Isolated Streptomyces sp.

Takuya Kumamoto, Hiroyuki Koshino, Daisuke Watanabe, Yuko Matsumoto, Kazuki Aoyama, Ken-ichi Harada, Tsutomu Ishikawa, and Yuzuru Mikami*

Chiba University, , Japan

Abstract

A new cyclic heptapeptide, M10709, was isolated from the mycelial cake of a clinically isolated Streptomyces sp. IFM 10709. The planar structure of M10709 was determined on the basis of IR, MS, NMR, and UV spectra. The absolute configuration of α-position of amino acid residues in the acid hydrolysate was determined by advanced Marfey’s method to be L except that of tryptophan-derived amino acid. The compound showed antibacterial activity against Micrococcus luteus IFM 2066.

INTRODUCTION
New sources of bioactive metabolites such as marine microorganisms1 and new microbes from different ecological niches have promoted recent advances in the discovery of new drugs.2 However, such attention has never been paid to pathogenic microbes. We were interested in biologically active compounds produced by pathogenic microorganisms, especially from pathogenic actinomycetes3 and fungi4 because (i) pathogenic microorganisms should have unique competition mechanisms between pathogenic microbes and other microbes, plants, and animals, and (ii) pathogenic microbes should also have different metabolic pathways from those of non pathogenic microbes. During continuing studies on the search of biologically active compounds from clinical isolates of Streptomyces and Nocardia species,5-7 one antimicrobial compound named M10709 (1) was isolated as a metabolite of the mycelial cake of a clinically isolated Streptomyces sp. IFM 10709.

RESULTS AND DISCUSSION
The physico-chemical properties of M10709 (1) are summarized in Table 1. It has molecular formula C53H78N8O10 confirmed by HRESIMS m/z 1009.5721 (calcd for C53H78N8NaO10: 1009.5739). In IR spectrum, absorption bands at 3600-3250 cm-1 and 1639 cm-1 suggested the presence of hydroxy, amide N-H and carbonyl groups, respectively. The existence of a hydroxy group was also supported from the result of FABMS; the characteristic fragment ion at 969 (MH+-H2O) was observed as a base peak. The 13C NMR spectrum of 1 indicated the presence of thirteen methyls, four methylenes, twenty-two methines, and twelve quaternary carbons including seven amide carbonyl carbons (Table 2). Analysis of 1H NMR spectrum (Table 2) by 2D DQFCOSY and TOCSY revealed the presence of five amide NH signals and two N-methyl signals. This evidence together with information of seven carbonyl carbons suggested that M10709 (1) is a heptapeptide. UV absorption at 221 and 283 nm suggested the existence of tryptophan or related indole ring system.8 The physical properties and spectral data were compared with those of known cyclic peptide cyclomarin C (2) (Figure 1),9-11 isolated as a metabolite of marine Streptomyces strain CNB-982 by Fenical and Clardy (Table 1, 2) and these compounds were found to have similar IR and UV spectra and NMR resonance except characteristic signals of 2-amino-3,5-dimethylhex-4-enoic acid (ADH) residue of cyclomarin C (2).
Careful interpretation of 2D NMR spectra including DQFCOSY, TOCSY, 1H-13C HSQC, 1H-13C HMBC,

and 1H-15N HSQC revealed that the seven amino acid residues are N-(1,1-dimethylallyl)tryptophan, N-methyl-γ-hydroxyleucine, alanine, β-methoxyphenylalanine, N-methylleucine, and two valines. Six amino acid residues except for one valine are same components as those of cyclomarin C. Sequential assignments of seven amino acid residues of 1 were performed by analyses of 1H-13C and 1H-15N HMBC (Figure 2). Connectivity between N-(1,1-dimethylallyl)tryptophan and one valine residue was confirmed by NOE between H-7 of indole ring portion and Me-20 of the valine based on NOESY data (Figure 3). Other H-C connectivity was determined by 1H-13C long range correlations of HMBC

spectrum. Presence of reverse N-prenylated tryptophan and two N-methylated amino acid residues were supported by 1H-15N HMBC data (Figure 2).

For the confirmation of the stereochemistry on M10709 (1), advanced Marfey’s method utilizing both enantiomers of 1-fluoro-2,4-dinitrophenyl-5-leucinamide (FDLA) was applied.12,13 Acid hydrolysate of M10709 (1) was treated with d- and l-FDLA, respectively, and the each mixture of DLA derivatives of amino acids was subjected to LC/MS. Among the amino acids assigned by NMR analysis, five amino acids, i.g. alanine, β-methoxyphenylalanine, N-methyl-γ-hydroxyleucine, N-methylleucine, and valine, were detected as the corresponding DLA derivatives with the correct molecular weight. Tryptophan derivative was not detected provably due to the lability of tryptophan and/or 1,1-dimethylallyl parts in acidic condition. As to the determination of absolute configuration, the l-DLA derivatives of l-amino acids generally shows shorter retention time in HPLC analysis than corresponding d-DLA derivatives.12 As a result, peaks derived from five detectable l-DLA derivatives were observed in shorter retention time than those of the corresponding d-derivatives. Thus, the absolute configurations of α-position of these amino acids were determined as l.14 Even though some differences were observed on the chemical shifts on 1H NMR [H-2, H-7, and H-15 on N-(1,1-dimethylallyl)tryptophan residue; H-45, H-46, H-47, and NMe-8 on N-methyl-γ-hydroxyleucine], they showed quite similar NMR data, implying that M10709 (1) could have the same stereochemistry as that of cyclomarin C (2).
In vitro antibacterial activities of M10709 (1) are shown in Table 3. The compound showed antibacterial activity only against Micrococcus luteus IFM 2066, and its MIC value was 5.0 µg/mL. The compound did not show antibacterial activities against other gram-positive and negative bacteria such as Staphylococcus aureus and Escherichia coli. M10709 (1) did not show antifungal activities against Candida albicans and Aspergillus niger.

EXPERIMENTAL
Methods
Melting point was measured on a micro melting-point hot stage (Yanaco) and uncorrected. IR spectrum was recorded on a JASCO Infrared spectrophotometer FT/IR-300E. NMR spectra were recorded on a JEOL ECA-600 spectrometer at 600 and 150 MHz for 1H and 13C, respectively. UV spectra were recorded on a JASCO UV/VIS spectrophotometer V-530. ESIMS was recorded on a Shimadzu LCMS-2010EV mass spectrometer with ESI/APCI Dual source. FABMS was recorded on a JEOL JMS-SX102 mass spectrometer. Acid hydrolysis (reaction time: 9 h) and conversion of the hydrolysate to the corresponding DLA derivatives were carried out according to the reported procedure.12,13 The separation of d- and l-DLA derivatives of amino acids was performed using an Agilent Model 1100 liquid chromatograph system. The separations were carried out on a YMC-Pack Pro C18 (100 × 4.6 mm i.d., 3 μm, YMC) column at 40 °C. Acetonitrile (MeCN)–water containing 5 mM of ammonium formate and 5 mM of formic acid was used as the mobile phase under a linear gradient elution mode (MeCN, 30 %-60 %, 20 min) at a flow rate of 1.0 mL/min, with UV detection at 340 nm and 210-600 nm by photodiode array detection. The ESIMS for LC/MS of Marfey’s method were recorded on an Agilent LC/MSD mass spectrometer using a Model 1100 HPLC system. Peaks of each DLA derivatives were detected on the following retention time (d- and l-DLA derivatives, respectively, unit: min): N-methyl-γ-hydroxyleucine (7.5, 6.9), alanine (7.8, 5.4), β-methoxyphenylalanine (13.5, 8.8), N-methylleucine (15.6, 13.2), valine (12.4, 7.6), N-(1,1-dimethylallyl)tryptophan (not detected). ORD was recorded on a JASCO J-720WI spectropolarimeter.
Taxonomic position of producing strain
Chemotaxonomic studies15 on Streptomyces sp. IFM 10709 strain confirmed that it belongs to a genus Streptomyces. Sequence analysis studies of 16S rRNA gene showed that Streptomyces sp. IFM 10709 strain showed high similarity to Streptomyces rectiverticillatus NRBC 13709 with the similarity value of 99.85%.
Fermentation and isolation of active compound
The seed broth was prepared by inoculating mycelial elements of the producing strain (Streptomyces sp. IFM 10709, a clinical isolate from a stomach of a 85 years old patient with stomach cancer in Japan) grown on Brain heart infusion (BHI) agar (Difco, Detroit) in 10 mL of BHI broth with 2% glucose in 50-mL Erlenmeyer shake flask. The culture was incubated on a rotary shaker at 250 rpm for 96 h. Ten percent inoculum was transferred to a 500-mL Erlenmeyer flask containing 150-mL of the production medium composed of meat extract 0.5%, peptone 0.5%, glucose 1%, starch 1.0% and antifoam 0.005%. The pH was adjusted to 7.4, and the culture was incubated on a rotary shaker at 250 rpm for 6 days. After the incubation, the broth was filtered and mycelial cake was extracted with MeOH. The broth and the mycelial extract were combined and concentrated in vacuo. The crude (2.07 g) was dissolved in H2O (300 mL) and was extracted with AcOEt (1 x 900 mL). The organic layer was evaporated and the residue was suspended in H2O - MeCN, 1 : 1 (v/v). The mixture was centrifuged (3000 rpm, 10 min) and the supernatant was concentrated in vacuo and subjected to preparative HPLC [column: nacalaitesque, Cosmosil 5C18-AR-II, 250 x 10 mm i.d., eluent: H2O - MeCN, 40 : 60 (v/v), 4.0 mL/min] to give pale pink powder (1.4 mg), which was washed with hexane - AcOEt, 10 : 1 (v/v) to give M10709 (1) as colorless powder (1.2 mg).
Antimicrobial activities
Antimicrobial activities of the compound were determined by microbroth dilution method using BHI broth for bacteria and Sabouraud dextrose broth (Difco, Detroit) for fungi, respectively.

ACKNOWLEDGMENTS
Ms. Yuriko Nozawa, Taisho Pharmaceutical Co. Ltd., was acknowledged for performing the advanced Marfey’s method.

References

1. G. M. Konig and A. D. Wright, “Trends in marine biotechnology”, in ‘Drug discovery from nature’, ed. by G. Grabley and R. Thiericke, Springer, Berlin, 1999, pp. 180-185.
2. U. Graefe and Y. Mikami, “Novel antibacterial drugs from microorganisms”, in ‘Drug discovery from nature’, ed. by G. Grabley and R. Thiericke, Springer, Berlin, 1999, pp. 281-296.
3. Y. Mikami, Actinomycetologica, 2007, 21, 46. CrossRef
4. S. Nakadate, K. Nozawa, H. Horie, Y. Fujii, M. Nagai, T. Hosoe, K. Kawai, T. Yaguchi, and K. Fukushima, J. Nat. Prod., 2007, 70, 1510. CrossRef
5. H. Shigemori, H. Komaki, K. Yazawa, Y. Mikami, A. Nemoto, Y. Tanaka, T. Sasaki, Y. In, T. Ishida, and J. Kobayashi, J. Org. Chem., 1998, 63, 6900. CrossRef
6. A. Nemoto, Y. Hoshino, K. Yazawa, A. Ando, Y. Mikami, H. Komaki, Y. Tanaka, and U. Grafe, J. Antibiot., 2002, 55, 593.
7. A. Nemoto, Y. Tanaka, Y. Karasaki, H. Komaki, K. Yazawa, Y. Mikami, T. Tojo, K. Kadowaki, M. Tsuda, and J. Kobayashi, J. Antibiot., 1997, 50, 18.
8. E. Pretsch, W. Simon, J. Sebl, and T. Clerc ‘Tables of spectral data for structure determination of organic compounds’, Springer, Berlin, 1989.
9. M. K. Renner, Y.-C. Shen, X.-C. Cheng, P. R. Jensen, W. Frankmoelle, C. A. Kauffman, W. Fenical, E. Lobkovsky, and J. Clardy, J. Am. Chem. Soc., 1999, 121, 11273. CrossRef
10. For the total synthesis of cyclomarin C: a) S.-J. Wen, T.-S. Hu, and Z.-J. Yao, Tetrahedron, 2005, 61, 4931; CrossRef b) S.-J. Wen and Z.-J. Yao, Org. Lett., 2004, 6, 2721. The optical rotation of the synthetic samples ([α]D -72.8) was reported to be higher than that of the natural material. CrossRef
11. For the biosynthesis of cyclomarins and isolation of cyclomarin D from the marine bacterium Salinispora arenicola CNS-205: A. W. Schultz, D.-C. Oh, J. R. Carney, R. T. Williamson, D. W. Udwary, P. R. Jensen, S. J. Gould, W. Fenical, and B. S. Moore, J. Am. Chem. Soc., 2008, 130, 4507. CrossRef
12. K. Fujii, Y. Ikai, T. Mayumi, H. Oka, M. Suzuki, and K.-i. Harada, Anal. Chem., 1997, 69, 3346. CrossRef
13. K. Fujii and K.-i. Harada, Anal. Chem., 1997, 69, 5146. CrossRef
14. Partial epimerization of N-methylleucine (L : D = ca. 6 : 4) during the acid hydrolysis was observed, even in the shorter period for hydrolysis (30 min).
15. S. Miyadoh, “Identification procedure at the genus level”, in ‘Identification manual for actinomycetes’, eds. by S. Miyadoh et al., Business Center for Academic Societies, Tokyo, 2001, pp. 9-19.