Structure elucidation of a novel trichothecene glycoside using1H and13C nuclear magnetic resonance spectroscopy

摘要

J. CHEM. SOC. PERKIN TRANS. i 1985 Structure Elucidation of a Novel Trichothecene Glycoside using IH and I3C Nuclear Magnetic Resonance Spectroscopy Charles P. Gorst-Allman," Pieter S. Steyn, and Robert VleggaarNational Chemical Research Laboratory, Council for Scientific and Industrial Research, P. 0.Box 395, Pretoria 000 I, Republic of South Africa Christiaan J. Rabie National Research Institute for Nutritional Diseases, Medical Research Council, P. 0.Box 70, Tygerberg7505, Republic of South Africa The structure elucidation of a novel trichothecene glycoside, 15-acetoxy-3a- hydroxy-4P- (a-D-glucopyranosy1oxy)-1 2,13-epoxytrichothec-9-ene, is based on a detailed study of its one- and two- dimensional 'H and 13C n.m.r. spectra, and chemical reactions. The metabolite displays a reduced toxicity when compared with other trichothecene metabolites in several biological systems.The trichothecenes form one of the most diverse and important families of mycotoxins and are produced by numerous species of Cephalosporium, Fusarium, Myrothecium, Stachybotrys, and Trichoderma.' As a consequence of the wide geographical distribution of these fungi, the mycotoxins have been implicated in a variety of human and animal diseases,2 and as such have been the subject of numerous toxicological report^.^ More than 60 trichothecenes are known at present. In this paper we describe the isolation and structure elucidation of the first naturally occurring trichothecene glycoside, an a-glucopy-ranoside derivative of monoacetoxyscirpenol, produced by a strain of Fusarium sulphureum, isolate MRC 514.The structure of the metabolite was elucidated as (1) by 'H and I3C high-field n.m.r. spectroscopy, chemical reactions, and a knowledge of the related trichothecene metabolites produced by this strain of F. sulphure~m.~Fast-atom bombardment (FAB) mass spectrometry indicated a molecular mass of 486, in accord with the molecular formula C23H3401 1. Absorption in the i.r. region at 1 720 cm-' and 3 340 cm-' was attributed to the presence of carbonyl and hydroxy groupings in compound (1). Addition of D,O to the sample caused the resonances at 6, 5.197, 4.875, 4.770, 4.764, and 4.501 in the 'H n.m.r. spectrum to disappear, thus identifying five hydroxy groups.The presence of a 12,13-epoxytrichothecenemoiety in the molecule was suggested by the characteristic doublets at 6 2.904 and 2.688 (J 4.2 Hz) assigned to the C-13 geminal protons. Two-dimensional (lH,l H) chemical shift correlation spectroscopy using the COSY-45 pulse sequence established the ('H,'H) connectivity pattern, and this, coupled with chemical shift criteria and coupling constant values, permitted the assignment of the complete 'H n.m.r. spectrum of compound (1) (Table). Thus the anomeric proton (6 4.717) was used as the starting point in the assignment of the protons of the glucosyl ring; the olefinic proton, 10-H (6 5.325), in the assignment of 11-H and 16-H; and the hydroxy proton, (3-OH) (6 5.197), in the assignment of 3-H, 2-H, and 4-H. The identification of the remaining protons, 7-H, 8-H, 14-H, and 15-H, follows from their chemical shift values.The 13C n.m.r. data for compound (l),collated in the Table, were obtained from proton-decoupled and single-frequency nuclear Overhauser-enhanced (n.0.e.) "C n.m.r. spectra, and revealed that the 23 carbon resonances observed in the n.m.r. spectrum of (1) are due to 3 methyl, 5 methylene, 10 methine, and 5 quaternary carbon atoms. Chemical shift criteria dictate that the resonances at 6, 170.20, 138.83, and 119.04 p.p.m. must be attributed to the acetate carbonyl carbon atom and the olefinic carbon atoms C-9 and C-10, respectively, whereas the resonance at 6, 99.71 p.p.m. is typical of the anomeric carbon atom in a-glycosides6 H 6' and must thus be attributed to C-1'.The remaining eight methine carbon atoms directly bonded to one oxygen atom resonate between 6, 86.96-67.07 p.p.m. (Table) and were individually assigned by two-dimensional ('H,I3C) chemical shift correlation ~pectroscopy.~~~~ Collectively they represent C-2, C-3, C-4, C-11, C-2', C-3', C-4', and C-5'. The methylene carbon atoms, C-7, C-8, C-13, C-15, and C-6', may be assigned as follows. The carbocyclic carbon atoms, C-7 and C-8, resonate at 6,20.67 and 27.54 p.p.m., respectively: the high-field signal is assigned to C-7 by analogy with the 13C n.m.r. spectrum of diacetoxyscirpenol,8 in which the corres- ponding resonances are at 6,21.1 and 27.9 p.p.m., respectively. The methylene carbon atom of the oxirane ring, C-13, resonates at 6,46.12 p.p.m., which value is characteristic of the chemical shift found for this carbon atom in other trichothecenes.8 The remaining methylene carbon signals, C-15 and C-6', appear at 6, 63.26 and 60.91 p.p.m., respectively, and were differentiated by correlation of the carbon resonances with the un-ambiguously assigned proton signals.The three methyl carbon signals appear at 6,6.86,20.82, and 22.85 p.p.m. The highest field signal is assigned to C-14 by comparison with the corresponding signal in other tricho- thecenes.8 In the coupled 13Cn.m.r. spectrum of (1) the quartet centred at 6, 22.85 p.p.m. shows additional fine structure in that each leg of the quartet is split into a doublet (J5.8 Hz) arising from allylic coupling with 10-H.Thus this signal corresponds to C-16. The signal at 6, 20.82 p.p.m. is therefore assigned to the acetate methyl carbon atom. 1554 ~~ ~~ Table. 'H and I3C N.m.r. data for compounds (1) and (2)" amp;'.d Atom (1) (2) (1) 2 3.302 3.248 78.81 D 3 4.071 4.000 77.42 D -3-OH 5.197 5.070 4 4.035 4.278 86.96 D 5 --49.09 S 6 --43.29 S 1.805 20.67 T 1.92 1.721 1.625 1.973 27.54 T 1.539 9 --138.83 S 10 5.325 5.285 119.04 D 11 3.836 3.783 67.07 D 12 --64.54 s 13(a) 2.904 2.867 46.12 T (b) 2.688 2.659 14 0.7 19 0.763 6.86 Q 15(a) 4.026 3.465 63.26 T (b) 3.789 3.242 15-OH -4.463 16 1.625 1.607 22.85 Q 1' 4.717 4.776 99.71 D 2' 3.215 3.21 3 72.12 D 2'-OH 4.501 4.373 3' 3.399 3.407 73.07 D 3'-OH 4.764 4.766 4' 3.070 3.055 70.32 D 4-OH 4.875 4.874 5' 3.572 3.562 73.07 D 6'(d 3.440 3.421 60.91 T (b) 3.665 3.693 6'-OH 4.770 4.820 G== --170.20 S -CH3 1.985 20.82 Q "Recorded on a Bruker WM-500 spectrometer. *Relative to 2H6Me2S0 at 6, 2.490.'Relative to-2H6Me2S0 at 6, 39.50 p.pm. Capital letters refer to multiplicities arising from coupling with directly bonded protons. S =singlet, D =doublet, T =triplet, Q =quartet. The remaining signals in the spectrum appear at tic 64.54, 49.09, and 43.29 p.p.m. and correspond to C-12, C-5, and C-6, respectively. Chemical shift criteria dictate that the low-field signal be assigned to the carbon atom directly bonded to oxygen, C-12, while C-5 and C-6 are assigned by comparison with the values obtained for the corresponding carbon atoms in diacetoxyscirpenol (6,49.1 and 44.2 p.p.m., respectively).The ambiguities remaining in the structure related to the nature and position of the sugar moiety, and the position of the hydroxy and acetoxy functions on the trichothecene nucleus. Additions of D,O to a sample of compound (1) in 'H6di- methyl sulphoxide removed a 4.5 Hz coupling from the 3-H signal in the 'H n.m.r. spectrum, thus locating the hydroxy function at C-3 of the trichothecene nucleus. Treatment of compound (1) with ~M-HC~ at 100 "C for 1 h with subsequent t.1.c. against various standards (glucose, galactose, mannose, xylose, rhamnose, and fructose) in four different developers established that the sugar was gluco~e.~ This was confirmed by the vicinal coupling constants in the 'H n.m.r.spectrum of compound (1) (Figure), where the large values encountered are indicative of a diaxial antiperiplanar arrangement for 2'-H, 3'-H, 4-H, and 5'-H. The a-glycoside linkage was inferred from the vicinal coupling constant observed for 1'-H and 2'-H (3.8 Hz), J. CHEM. SOC. PERKIN TRANS. I 1985 Figure. 'H Coupling constants (Hz) for compound (1). The coupling constants to the protons of the hydroxy groups have been omitted. These are (3-H, 3-OH) 4.5 Hz; (2'-H, 2'-OH) 5.6 Hz; (3'-H, 3'-OH) 5.1 Hz; (4-H, 4-OH) 5.3 Hz; and (6'-H, 6'-OH) 5.2 Hz and by the chemical shift value of C-1' in the 13C n.m.r.spectrum of glycoside (1) (6,99.71 p.p.m.).6 Finally, base hydrolysis of compound (1) with methanolic potassium hydroxide gave the de-acetylated product (2). The upfield shift of the C-15 protons in the 'H n.m.r. spectrum of compound (2) (A6 0.561 and 0.547p.p.m.), when compared with their chemical shift values in the acetate (l), placed the acetoxy function at C-15, and thus the glucose group must be located at (2-4. Biological evaluation of compound (1) gave several interest- ing results. In contrast to other 12,13-epoxytrichothecenes the molecule shows no significant dermatotoxic effects, nor is it lethal to brine shrimps at levels up to 200 times greater than those found for other trichothecenes. However, in vivo the metabolite is toxic to rats at levels below 90 mg kg-', presumably due to hydrolysis of the glucoside in the stomach.ExperimentalI. r. spectra were measured on a Perkin-Elmer 257 spectro-photometer using KBr discs, optical rotations on a Perkin- Elmer 241 polarimeter, and U.V. absorptions on a Unicam SP8- 100 spectrophotometer, both for solutions in methanol. T.1.c. was carried out on Merck precoated silica gel plates (coating thickness 0.25 mm). For column chromatography Merck silica gel, particle size 0.063--0.200 mm, and octadecyl Porasil B, prepared according to the method of Kingston and Gerhardt," were used. Isolation of Glycoside (l).-The procedures followed in the extraction and partition of maize meal (5 kg) infected with Fusarium sulphureum MRC 514 were identical with those described in ref.4. The aqueous extract (300 g) was purified further by chromatography on SiO, (2 kg) with methanol- ethyl acetate (1:9, v/v) as eluant. After elution of the four trichothecenes characterized previously from this train,^ an additional fraction giving a blue colour reaction with the 4-(p- nitrobenzy1)pyridine spray reagent ''was eluted. This material (450 mg) was purified on reversed-phase silica gel (100 g) with methanol-water (1 :1, v/v) as eluant to yield glycoside (1) (182 mg) as a pale yellow gum, CI~*~+79.5" (c 0.585);A,,,,,. end-absorption only; v,,,,,. 3 340,2 890,l 720,l 230, and 1 015 cm-'; m/z (FAB) 509 M +Na'. Acid HydroZysis Glycoside (l).-A solution of glycoside (1) (1 1 mg) in water (2 ml) was treated with hydrochloric acid (2M; J.CHEM. SOC. PERKIN TRANS. I 1985 2 ml) and kept at 100 ldquo;Cfor 1 h. The solution was cooled and extracted with ethyl acetate (2 x 5 ml). The aqueous layer was compared with standards of glucose, galactose, mannose, xylose, rhamnose, and fructose by t.1.c. in the following solvent systems: (a) n-butanol-acetic acid-water (4: 1 :5, v/v/v), (b) n-butanokthanol-water (4 :1 :2.2 v/v/v), (c) n-butanol-benzene- pyridinewater (5 :1 :3 :3, v/v/v/v), and (d) phenol saturated with water. In all cases the R, value of the unknown sugar was identical with that of glucose. Base Hydrolysis of Glycoside (1)-A solution of potassium hydroxide (3 mg) in methanol (0.5 ml) was added to a stirred solution of glycoside (1) (20 mg) in methanol (2 ml).After 5 h at 20deg;C, the solvent was removed in a stream of N,, and the resultant gum was purified on SiO, (10 g) with methanol- chloroform (1 :4, v/v) as eluant, to yield the hexaol(2) (8 mg) as a gum. References 1 Y. Ueno, Pure Appl. Chem., 1977,49,1737. 2 A. Ciegler, J. Food Protection, 1978,41,399. 3 Y. Ueno, Fundam. Appl. Toxicol., 1984, 4, S124; Y. Ueno, lsquo;Trichothecenes: Chemical, Biological, and Toxicological Aspects,rsquo; Elsevier, Amsterdam, 1983;M. Ohta, K. Ishii, and Y. Ueno, J. Biochem. (Tokyo), 1977,82,1591. 4 P. S. Steyn, R. Vleggaar, C. J. Rabie, N. P. J. Kriek, and J. R. Harington, Phytochemistry, 1978,17,949. 5 (a) A. Bax, lsquo;Two-dimensional Nuclear Magnetic Resonance in Liquids,rsquo; Delft University Press, Delft, 1982;(b) A. Bax, R. Freeman, and G. Morris, J. Mugn. Reson., 1981, 42, 164,A. Bax and R. Freeman, ibid., 1981,44,542. 6 J. B. Stothers, lsquo;Carbon-13 NMR Spectroscopy,rsquo; Academic Press, New York, 1972. 7 G. Bodenhausen and R. Freeman, J. Magn. Reson., 1977,28,471;A. Bax and G. Morris, ibid., 1981,42,501. 8 R. J. Cole and R. H. Cox, lsquo;Handbook of Toxic Fungal Metabolites,rsquo; Academic Press, New York, 1981. 9 J. B. Harborne, lsquo;Phytochemical Methods,rsquo; Chapman and Hall, London, 1973. 10 D. G.I. Kingston and B. B. Gerhart, J. Chromatogr., 1976,116,182. 11 S. Takitani, Y. Asabe, T. Kato, M. Suzuki, and Y. Ueno, J. Chromatogr., 1978,172,335. Received 5th December 1984; Paper 4/2061