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Structure elucidation of the antibiotic demalonylniphimycin by two-dimensional NMR techniques
V. IVANOVA & R. SCHLEGEL* Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev-Str., 26, 1113 Sofia, Bulgaria and *Hans-Knoll-Institut fur Naturstoff-Forschung, Beutenbergstr. 11, D-07745 Jena, Germany
Code Number: AC97001
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ABSTRACT. The structure of the antibiotic demalonylniphimycin has been determined by two-dimensional NMR techniques. Mass spectroscopy indicated a MH^+ of 1056. The molecular formula was determined as C56H101N3O15 (MW. 1055). All other structural information was obtained from ^1H and ^13C NMR, ^1H - ^1H double-quantum filtert, phase sensitive COSY experiments, while the correlation between proton and carbon chemical shifts was performed by the ^1H - ^13C COSY experiment. Demalonylniphimycin is active against Gram- positive bacteria and fungi.
Non-polyene antibiotics, described in the literature, such as azalomycins F^3a, F^4a and F^5a (Iwasaki et al., 1982a, 1982b), scopafungin (Samain et al., l982), niphimycin (Bassi et al., 1983; Keller-Schierlein et al., 1983; Gassmann et al., 1984), copiamycin (Fukushima et al., 1982), guanidylfungins A and B (Takesako et al., 1984) and others have similar chemical structures and antimicrobial spectrum. Their structures show a macrocyclic polyhydroxyl lactone and a 6-membered inner-molecular hemiketal ring. They are malonyl monoesters with a side chain with a terminal guanidine. The demalonyl derivatives of methyl-azalomycin F4, methyl-scopafungin, methyl-copiamycin and methyl-guanidylfungin A, obtained by a semisynthetic pathway, showed a higher activity against bacteria and fungi than the parent compounds (Takesako et al., 1985, 1986).
Demalonylniphimycin (antibiotic 165-3) was first isolated from the culture broth of Streptomyces hygroscopicus strain 111-81. The strain was isolated from a soil sample from Bulgaria (Ivanova et al., 1983, 1985). Another fermentation product, devoid of a monoester group, demalonylcopiamycin, was isolated from S.hygroscopicus var. crystallogenes (Fukai et al., 1986). Strain DSM 3816 of Streptomyces sp. also produces demalonylniphimycin (amycin B) (Hammann et al., 1986; Grabley et al., 1990).
In the present article we report the isolation of demalonylniphimycin from Streptomyces hygroscopicus 111-81 and the structure elucidation by two-dimensional NMR techniques.
MATERIALS and METHODS
Organism and growth conditions. Streptomyces hygroscopicus, strain 111-81 was obtained from the Bulgarian National Collection of Industrial Microorganisms and Cell Cultures. Growth conditions have been previously described (Ivanova et al., 1983; 1985).
Isolation of demalonylniphimycin. The culture broth of strain 111-81 (5.0 litres) was centrifuged at pH 7.0-7.2. The mycelium was extracted separately with 3 volumes of methanol and the filtrate with n-butanol (2:1). The active solvent extracts were combined and evaporated to dryness under reduced pressure. The crude extracts were dissolved in a small amount of methanol, filtered and precipitated with acetone-ether (10:1, v/v). The precipitate was allowed to stand for 24 hrs at 5-10 C, filtered and washed with acetone and ether. Five grams of crude powder were obtained from 5 litres of culture broth. A methanolic solution of the powder (1.0 g) was chromatographed on a silica gel 60 (70-325 mesh) column. Demalonylniphimycin was eluted with the lower phase of a mixture of chloroform/methanol/water (175:100:50). The active eluates were combined and evaporated in vacuo to dryness. A methanolic solution was precipitated with acetone-ether (10:1, v/v) and filtered to give 200 mg of the antibiotic. Demalonylniphimycin was further purified by preparative high performance liquid chromatography (HPLC).
Preparative HPLC of demalonylniphimycin. This was carried out as follows. Column: Lichroprep RP18 (250 x 5 mm). Mobile phases: gradient of 40 to 70% acetonitrile in 0.01M sodium phosphate buffer, pH 4.0; 0.01M sodium phosphate buffer, pH 4.0 / acetonitrile (35:65). Column: Sephadex LH-20. Eluent: methanol. Detection: 220nm.
After concentration and drying, 48 mg of pure demalonylniphimycin were obtained. Analytical HPLC. Demalonylniphimycin was analysed by HPLC using a HP 85 apparatus with a HP 1040 A UV detector. Column: Lichrospher RP 18 (125x4 mm). Mobile phases: 0.01M sodium phosphate buffer, pH 4.0 / acetonitrile (35:65; 40:60). Flow rate: 1.20 ml/min. Detection: 220 nm. Rt = 4.86; 6.28.
Thin-layer chromatography (TLC). TLC of demalonylniphimycin was carried out on silica gel plates (Merck 60, F254) with the following mobile phases: chloroform/methanol/water (2:2:1) lower phase, Rf = 0.30; chloroform/methanol/acetic acid (15:5:1), Rf = 0.32; n-butanol/ethanol/water (5:1:4), Rf = 0.57. The chromatographic spot was visualised by spraying with a 3% (w/v) solution of vanillin/ethanol + 1.50 ml sulphuric acid, the reagents of Dragendorf, Sakaguchi and by heating at 110 C for 3-5 min.
General experimental procedures. UV spectra in methanol were scanned on a Perkin-Elmer (Lambda 9) UV-spectrometer; IR spectrum was analysed on a Bruker (IFS-66) FT-IR spectrometer in KBr tablets. Fast atom bombardment (FAB-MS) was carried out on a Varian Mat 311-A mass spectrometer. ^1H and ^13C NMR: spectra were measured in MeOD on a Bruker AMX-400 NMR spectrometer at 400 MHz for ^1H and 100.62 for ^13C. ^13C multiplicity data were obtained from JMOD experiments. Chemical shifts were expressed as values (ppm) with TMS as an internal standard. The ^1H - ^1H correlation spectrum (COSY) was recorded in phase-sensitive, double quantum filtert mode, using the standard pulse sequence in MeOH-d4. The ^1H - ^13C correlation spectrum (COSY) was recorded in MeOH-d4, using the standard pulse sequence. The optical rotation was measured at 25 C using a Perkin-Elmer 141 polarimeter with a 1-dm cell. Mp. determined on a Kofler hot-stage apparatus are uncorrected.
Demalonylniphimycin. Colourless powder; Mp. 126-128 C; [a]^25D + 35.9 (c = 0.5, methanol); UVlmax (methanol) 204, 226, 233, 240nm, IRnmax 3350, 2960, 2925, 2880, 1720 (C=O), 1635 (C=C), 1375, 1320, 1240, 1130, 1060, 985cm^-1; FAB-MS (glycerol) m/z 1056 (M+H)^+, C56H101N3O15, 1038 (MH-H20)^+, 448, 386, 294, 282, 252, 224, 210, 185. ^1H NMR (400MHz) and ^13C NMR (100.62) in Tables 1 and 2. Table 1. ^1H-NMR data of demalonylniphimycin (MeOH-d4, 400 MHz)
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Proton ^1H (ppm) Proton ^1H (ppm)
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H-2 2.43 (1H,m) H-29 4.06 (1H,m)
H-3 4.07 (1H,m) H-30 5.66 (1H,dd,J=10.7,15 Hz)
H-4 5.42 (1H,dd,J=8.7,14.8 Hz) H-31 6.18 (1H,dd,J=10.6,15 Hz)
H-5 5.71 (1H,dd,J=10.3,14.9 Hz) H-32 6.06 (1H,dd,J=10.7,15.2 Hz)
H-6 2.30 (1H,m) H-33 5.51 (1H,dd,J=8.8,15.2 Hz)
H-7 3.74 (1H,m) H-34 2.53 (1H,m)
H-8 1.51/1.71 (2H,m) H-35 4.76 (1H,dd,J=3.8,8.8 Hz)
H-9 3.74 (1H,m) H-36 1.91 (1H,m)
H-10 1.51 (1H,m) H-37 0.96/1.35 (2H,m)
H-11 3.87 (1H,m) H-38 1.61 (1H,m)
H-12 1.39/1.63 (2H,m) H-39 1.33/1.61 (2H,m)
H-13 1.06/1.39 (2H,m) H-40 1.33/1.97 (2H,m)
H-14 1.58 (1H,m) H-41 1.97 (2H,m)
H-15 4.03 (1H,m) H-42 5.49 (1H,dt)
H-16 1.73/1.91 (2H,m) H-43 5.44 (1H,dt)
H-18 3.33 (1H,dd,J=9.33 Hz) H-44 2.07 (2H,m)
H-19 3.88 (1H,m) H-45 1.65 (2H,m)
H-20 1.33/1.90 (2H,m) H-46 3.15 (2H,m)
H-21 4.16 (1H,m) H-47 1.01 (dd,J=6.85 Hz)
H-22 1.45/1.62 (2H,m) H-48 1.07 (dd,J=6.87 Hz)
H-23 4.02 (1H,m) H-49 0.87 (dd,J=6.80 Hz)
H-24 1.45/1.62 (2H,m) H-50 0.90 (dd,J=6.80 Hz)
H-25 3.87 (1H,m) H-51 0.85 (dd,J=6.80 Hz)
H-26 1.45/1.65 (2H,m) H-52 1.02 (dd,J=6.85 Hz)
H-27 4.12 (1H,m) H-53 0.92 (dd,J=6.80 Hz)
H-28 1.56 (1H,m) H-54 0.91 (dd,J=6.80 Hz)
H-56 2.84 (3H,s)
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Table 2. ^13C-NMR data of demalonylniphimycin (MeOH-d4, 100.62 MHz)
-------------------------------------------------------------------------- Carbon ^13C JMOD Carbon ^13C JMOD -------------------------------------------------------------------------- C-1 176.63 s C C-29 75.95 d CH-O- C-2 47.90 d CH C-30 135.02 d C= C-3 75.53 d CH-O- C-31 132.04 d C= C-4 132.31 d C= C-32 131.80 d C= C-5 136.46 d C= C-33 137.04 d C= C-6 43.48 d CH C-34 40.82 d CH C-7 76.12 d CH-O- C-35 79.75 d CH-O- C-8 39.19 t CH^2 C-36 32.48 d CH C-9 75.15 d CH-O- C-37 42.57 t CH^2 C-10 44.49 d CH C-38 40.55 d CH C-11 72.13 d CH-O- C-39 40.65 t CH^2 C-12 33.45 t CH^2 C-40 27.81 t CH^2 C-13 37.53 t CH^2 C-41 33.87 t CH^2 C-14 30.66 d CH C-42 132.85 d C= C-15 66.44 d CH-O- C-43 129.80 d C= C-16 41.99 t CH^2 C-44 30.62 t CH^2 C-17 100.01 s C C-45 29.87 t CH^2 C-18 77.55 d CH-O- C-46 41.99 t CH^2 C-19 69.66 d CH-O- C-47 15.06 q CH^3 C-20 41.30 t CH^2 C-48 16.92 q CH^3 C-21 65.29 d CH-O- C-49 10.48 q CH^3 C-22 44.77 t CH^2 C-50 14.61 q CH^3 C-23 66.54 d CH-O- C-51 20.37 q CH^3 C-24 44.77 t CH^2 C-52 17.88 q CH^3 C-25 72.34 d CH-O- C-53 14.44 q CH^3 C-26 43.19 t CH^2 C-54 11.22 q CH^3 C-27 69.52 d CH-O- C-55 158.20 s C C-28 45.21 d CH C-56 28.30 q N-CH^3 --------------------------------------------------------------------------- RESULTS AND DISCUSSION
Demalonylniphimycin (Fig. 1) is a non-polyenic antibiotic. It is a white amorphous powder, soluble in alcohols, pyridine, dimethylsulfoxide, N,N-dimethylformamide and glacial acetic acid. It does not dissolve in acetone, ethyl acetate, chloroform, ether, n-hexane and water. On thin-layer plates the antibiotic demonstrated positive colour reactions with the reagents of Sakaguchi, Dragendorf, iodine vapour, conc. sulphuric acid, 3% vanillin-sulphuric acid solution.
Demalonylniphimycin differs from niphimycin A by 86 mass-units (-OCCH2 COOH). The ^13C-NMR spectrum showed the following structural elements. In the 10 to 21 ppm range the peaks (quartets) of eight methyl groups are recorded. The signal at 28.30 ppm corresponds to a -N-CH3 group. The range from 27 to 48 ppm (doublets and triplets) indicated the presence of 8 methine and 15 methylene carbon atoms, while that from 66 to 80 ppm (doublets), the presence of 13 -CH-O- groups. The signal at 100.01 ppm (singlet) showed the presence of a 6-membered inner-molecular hemiketal ring. In the 130 to 138 ppm range 8 signals of olefinic carbon atoms are observed. The singlet at 158.20 ppm is related to the guanidine-C-atom, while the singlet at 176.63 ppm indicated the presence of a lactone. By NMR analysis the complete structure of demalonylniphimycin was determined. The assignment of the ^1H-NMR spectrum of demalonylniphimycin was based on the homonuclear double-quantum filtert, phase sensitive COSY experiments, while the correlation between proton and ^13C chemical shifts was performed by means of the ^1H - ^13C COSY experiment. On the COSY spectrum the resonances of the protons in the part of the dienic system were at d 6.18 (1H,dd,31-H), 6.06 (1H,dd,32-H), 5.66 (1H,dd,30-H), 5.51 (1H,dd,33-H). The other four olefinic protons were distinguishable at d 5.71 (1H,dd,5-H), 5.42 (1H,dd,4-H), 5.49 (1H,dt,42-H) and 5.44 (1H,dt,43-H).
The protons, 34-H, 29-H, 6-H, 3-H, 44-H and 41-H, directly coupled to the olefinic ones, could be assigned, without ambiguity, as well. The proton 34-H at d 2.53 was clearly coupled to the methyl group at d 1.02 (52-H) and to the methine signal that, on the basis of the chemical shift at d 4.76 and the lack of other couplings, was assigned to the 35-H adjacent to the carboxylic group. The proton of the 35-H showed a coupling with 36-H (d 1.91,-CH-) which was coupled to 53-H (d 0.92,CH3). On the other side of the dienic system, the proton 29-H (d 4.06,-CH-O-) showed a coupling with 28-H (d 1.56,-CH-) which was coupled to 51-H (d 0.85,CH^3) and to 27-H (d 4.12,-CH-O-). The proton 6-H at d 2.30 was coupled to the methyl group at d 1.07 (48-H) and to 7-H (d 3.74,-CH-O-). 3-H (d 4.07, -CH-O-) showed a coupling with 2-H (d 2.43,-CH-), which was coupled to 47-H (d 1.01,CH^3). The proton 41-H at d 1.97 was clearly connected to the -CH^2- at d 1.33/1.97 (40H). 44-H (d 2.07,-CH2-) was coupled with 45-H(d 1.65,-CH2-), which was connected to the -CH2- at d 3.15 (46-H). Determining the remaining part of the structure by NMR presents problems because of the signal overlapping in two narrow regions (-OH and -CH2-). The ^1H - ^13C heteronuclear COSY experiment was used to complete the structure because the proton chemical shifts of the remaining 10 out of 15 methylene groups could be determined. The proton 27-H was connected to the methylene group at d 1.45 (26-H); 26-H was clearly connected to the -CH-O- at d 3.87 (25-H); 25-H was coupling with -CH2- at d 1.45/1.62 (24-H); 24-H was connected to the -CH-O- at d 4.02 (23-H), which was connected to another methylene group at d 1.45/1.62 (22-H); 22-H was coupled with the proton of -CH-O- at d 4.16 (21-H) ; 20-H (d 1.33/1.90,-CH2-) was connected to 21-H and to the -CH-O- at d 3.88 (19-H) which was coupled with the proton of -CH-O- at d 3.33 (18-H).
On the other side of the carbonyl group the proton 7-H showed a coupling with 8-H (d 1.51/1.71,-CH2-); 8-H was connected to the -CH-O- at d 3.74 (9-H) which was coupled to the 10-H (d 1.51,-CH-).
The methine hydrogen at d 1.51 (10-H) was coupled in addition to the methyl group at d 0.87 (49-H) and to the -CH-O- (d 3.87,11-H), which was connected to the methylene group (d 1.39/1.63,12-H), 13-H (d 1.06/1.39,-CH2) was connected to 12-H and to the methine hydrogen at d 1.58 (14-H), which was coupled to 50-H (d 0.90,CH3); 14-H was connected to the -CH-O- at d 4.03 (15-H), which was connected to the methylene group (d 1.73/1.91,16-H). Furthermore the 36-H exhibited a connectivity with another -CH2- in the upper part of the homonuclear COSY spectrum at d 0.96/1.35 (37-H) which was connected to the methine hydrogen at d 1.61 (38-H) which was coupled to 54H (d 0.91,CH3) and to 39-H (d 1.33/1.61,-CH2-). All the proton and carbon assignments of demalonylniphimycin are reported in Tables 1 and 2.
Demalonylniphimycin is a natural product, directly isolated from the culture broth of Streptomyces hygroscopicus strain 111-81 and has no malonate groups in its structure (Fig. 1). The antibiotic shows activity against the following test cultures: Staphylococcus aureus 209, Bacillus subtilis L-2, Bacillus mycoides, Bacillus idosus, Candida albicans, Candida utilis, Candida krusei, Candida tropicalis, Fusarium culmorum, Botrytis cinerea.
ACKNOWLEDGEMENTS. The authors are grateful to Mrs.M.Ponelle, Sandoz AG, Basle, Switzerland for helpful discussions on the structure determination by two-dimensional NMR analysis.
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Iwasaki, S., M.Namikoshi, K.Sasaki, M.Amana, K.Fukushima, S.Nozoe. & S.Okuda (1982). Studies on macrocyclic lactone antibiotics. III. Skeletal structure of azalomycin F^4a. Chem. Pharm. Bull., 30: l669-1673
Keller-Schierlein, W., B.Joos, H.-P.Kaiser, P.Gassmann (1983). Versuche zur Strukturaufklarung von Niphimycin. 2. Die Konstitution von Desmalonylniphimycin. Helv. Chim. Acta, 66: 226-258
Samain, D. , J. C.Cook & K.L.Rinehart (1982). Structure of scopafungin, a potent nonpolyene antifungal antibiotic. J. Am. Chem. Soc., 104: 4129-4141 Takesako, K. & T.Beppu (1984). Studies on new antifungal antibiotics, guanidylfungins A and B. II. Structure elucidation and biosynthesis. J. Antibiotics, 37: 1170-1186 Takesako, K., T.Beppu, T.Nakamura & A.Obayashi (1985). Demalonyl derivatives of guanidylfungin A and copiamycin: Their synthesis and antifungal activity. J. Antibiotics, 38: 1363-1370 Takesako, K., T.Nakamura, A.Obayashi, S.Iwasaki, M.Namikoshi, S.Okuda & T.Beppu (1986). Demalonyl derivatives of azalomycin F^4 and scopafungin. J. Antibiotics, 39: 713-716 Copyright 1997 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia The following images related to this document are available:Line drawing images[ac97001b.gif] [ac97001a.gif] |
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