Carbon-13 Nuclear Magnetic Resonance Spectra of Phenolic Glycosides Isolated from Chestnut Galls*

Transcription

1 Agric. Biol. Chem., 43 (6), , Carbon-13 Nuclear Magnetic Resonance Spectra of Phenolic Glycosides Isolated from Chestnut Galls* Tetsuo OZAWA and Yoshinori TAKINO Institute of Applied Biochemistry, The University of Tsukuba, Sakura-mura, Niihari-gun, Ibaraki-ken Received September 20, 1978 The 13C-NMR spectra of five phenolic glycosides, dimetbyl crenatin (2), cretanin (4), neocretanin (5), chesnatin (8), and chestanin (9) isolated from galls were compared and the structures 8 and 9 were confirmed as proposed in the previous papers. In the course of biochemical studies on chestnut galls, we have reported isolation and structural elucidation of five characteristic phenolic glycosides, crenatin (1), cretanin (4), neocretanin (5), chesnatin (8), and chestanin (9).14) In the last two compounds, however, the anomeric configuration of glycosidic linkage and the positions of glucose and dehydro -digallic acid on the 3,4,5-trihydroxybenzyl alco hol moiety remained to be solved. Attempts to determine the anomeric configuration by 'H-NMR spectroscopy were unsuccessful, because the anomeric proton peak of glucose was overlapped with the methylene proton peak of the trihydroxybenzyl alcohol moiety. However, comparison of the 13C-NMR spectra of these related compounds gave suggestive infor mation for the structures of chesnatin (8) and chestanin (9). The present paper describes the structural confirmation of the phenolic compounds isolated from chestnut galls by 13C-NMR spectroscopy. The schematic 13C- NMR spectra and the spectral data of the compounds are shown in Fig. 2 and Table I, respectively. Dimethyl crenatin (2) was used in this ex periment, since crenatin (1) itself was hardly obtained as a pure sample. The spectrum of 2 was assigned by the use of off-resonance decoupling experiment and substituent chemi cal shift theory." The sugar carbon signals were observed at 62.5, 71.2, 75.6, 77.7, 78.1, * Biochemical Studies on Chestnut Galls. Part VII. For Part VI see ref. 4. and ppm corresponding to C-6', C-4', C-2', C-5', C-3', and C-l', respectively. 1,7) The signals of C-1' and aromatic methine resonance appeared at almost the same region (105.4 and ppm). However, double peak height of the two overlapping aromatic methine resonances at ppm enabled to assign the former to C-1'. The anomeric carbon signal having such a chemical shift value indicates the Ĉ-configuration of the glucosidic linkage. The peak at 64.9 ppm was assigned to the methylene carbon (C-7) of the hydroxybenzyl alcohol moiety. The aromatic ring signals of 2 appeared as four peaks instead of six and the intensity of the signals at and ppm were approximately twice as strong as those at and ppm. This indicates a symmetrical substitution pattern of the ring. According to substituent chemical shift theory, the chemical shifts of the aromatic ring signals of 2 would be as follows, C-2 (105.1), C-4 (129.2), C-1 (134.6), and C-3 (145.7 ppm). Therefore, the quaternary carbon signals at 135.1, 139.5, and ppm are assigned to C-4, C-1, and C-3 (C-5), respectively, and the aromatic methine signal observed at ppm is attributed to C-2 (C-6). The above data are consistent with the proposed structure of crenatin (1)." The signals of cretanin (4) were assigned by comparing the spectrum with those of 2 and methyl gallate (3). The chemical shifts of the sugar carbons of 4 were almost identical with those of 2. However, the C-1' signal was ob-

2 1174 T. OZAWA and Y. TAKINO FIG. 1. Structures of Phenolic Glycosides Isolated from Chestnut Galls and Related Compounds. served at ppm, though the glucosidic carbons in Ĉ-linkage were generally to appear at the range of ppm. The peaks at and ppm were considered to be due to C-4 and C-1, respectively, by compari son with the spectrum of 2. However, their assignments may be reversed. Esterification of the alcoholic hydroxyl group in the benzyl alcohol moiety with gallic acid gives an upfield shift for C-1 and a downfield shift for C-7. The upfield shift of C-3 in 4 as compared with 2 is explained by the effect of demethylation. The ester carbonyl signal of the galloyl group is observed at ppm. The structural characteristics of neocre tanin (5) among the related phenolic glucosides isolated from chestnut galls are the presence of a formyl group in the molecule and the linkage of a galloyl group to C-6' of glucose. The 13C-NMR spectrum of 5 also corroborated its structure. The signal at ppm is at tributed unequivocally to the formyl carbon by its large chemical shift value and splitting to a doublet in the off-resonance spectrum. According to the substituent chemical shift theory, the aromatic ring carbon of the hydroxybenzaldehyde moiety were estimated to reson ate at 111.0, 132.4, 140.2, and ppm for C-2, C-1, C-4, and C-3, respectively. The substitution of the formyl group for the hy droxymethyl group resulted in the upfield shift of C-1, C-3, and C-4, and downfield shift of C-2. The signals due to the galloyl group were assigned by analogy with the spectra of methyl gallate (3) and cretanin (4). However, signals of C-3" and C-4" could not be assigned unambiguously. The signals of glucose carbons showed a characteristic 1,6-disubstituted pat-

3 13C -NMR Spectra of Phenolic Glycosides from Chestn ut Galls 1175 FIG. 2. The Schematic 13C-NMR Spectra of Dimethyl Crenatin (2), Methyl Gallate (3), Cretanin (4), Neocretanin (5), Dimethyl pentamethyl-dehydrodigallate (7), Chesnatin (8), and Chestanin (9). tern. The peak at ppm is attributed to the C-1' signal in fl-linkage and the peak at 64.8 ppm can be assigned to C-6' carrying a galloyl group based on the acylation effects." Moreover, upfield shift of the C-5' peak is explained by the fl-effect of the C-6' substitu tion. In the spectrum of dimethyl pentamethyl - dehydrodigallate (7), the signals at 52.2 and 52.3 ppm are assigned to two ester methyl carbons. The methoxy-methyl carbons are observed as four signals at 56.3, 61.1, 61.3, and 61.4 ppm instead of five. However, judg ing from the intensity of the signals, the peak at 56.3 ppm corresponds to two carbons. The resonances at 107.3, 108.8, and ppm are

4 1176 T. OZAWA and Y. TAKJNO TABLE I. 13C-NMR DATA a The numbering system is shown in Fig. 1. b Measured in D,O using dioxane (5, 67.4 ppm) as internal standard and converted to TMS scale. c Measured in CDCl3 d The letters s, d, t, and q showed the singlet, doublet, triplet, and quartet, respectively, in the off-re sonance 1H-decoupled spectra.e The assignments of signals marked with * may be reversed. f The assignments of individual signals remain obscure.g Methoxy- and ester-methyl carbons of 7. due to aromatic methine carbons. The signals at and ppm corresponding to the aromatic carbons bearing an ester group are attributed to C-la"' and C-lb"', respectively, based on the a substitution effect. The seven peaks at 142.0, 142.2, 147.0, 147.3, 150.4, 152.2, and ppm are due to the oxygenated aromatic carbons. Two ester-carboxyl car bons are observed at and ppm. Though all carbon atoms were detected, it was difficult to assign the signals individually. Chesnatin (8) is composed of each one mole

5 13C-NMR Spectra of Phenolic Glycosides from Chestnut Galls 1177 of glucose, 3,4,5-trihydroxybenzyl alcohol and dehydrodigallic acid (6).3' The signals of its ISC-NMR spectrum can be classified into these three moieties by comparing the spectrum with those of cretanin (4) and dimethyl pentamethyldehydrodigallate (7). The peaks corresponding to the glucose and 3,4,5-trihydroxybenzyl alcohol groups could be assigned by compari son of the chemical shifts with those of the crenatin moiety in the spectrum of 4. It resulted that the 4-hydroxyl group of trihydroxy benzyl alcohol is glucosylated with Ĉ-linkage, indicating that chesnatin (8) has the partial structure of crenatin (1). All carbon signals (14 peaks) corresponding to the dehydrodi gallic acid moiety were observed as shown in the case of dimethyl pentamethyl-dehydro digallate (7). The presence of a carbonyl carbon in the slightly lower field (170.0 ppm) as compared with an ester-carbonyl carbon (166.7 ppm) suggests that one of the carboxyl groups in the dehydrodigallic acid moiety is free. The differences in the chemical shifts between the dehydrodigalli cacid moiety of chesnatin (8) and dimethyl pentamethydehydrodigallate are mainly attributed to the methylation of hydroxyl groups. The 13C-NMR spectrum of chestanin (9) gave the similar chemical shifts to those of chesnatin (8). However, it has two remarkable characters, one of which is the presence of five pairs of signals consisting of very close peaks at 66.9 vs. 67.2, vs , vs , vs , and vs ppm. They are assigned to the signals belonging to 3,4,5- trihydroxybenzyl alcohol by comparing with those in the spectrum of 8. The other character is the upfield shift of one carbonyl-carbon signal belonging to dehydrodigallic acid to ppm from ppm. These facts indi cate that chestanin (9) has two moles of 3,4,5- trihydroxybenzyl alcohol which possess the same symmetrical substitution pattern as in the case of 8, and that two carboxyl groups of the dehydrodigallic acid moiety are combined with 3,4,5-trihydroxybenzyl alcohol through an ester linkage. Though the signals corres ponding to the glucose carbons were not split into a pair of peaks different from the 3,4,5- trihydroxybenzyl alcohol moiety, the molar quantity of glucose in chestanin (9) was confirmed to be 2 by quantitative acid hydrolysis." Moreover, the peak at ppm was regarded due to a Ĉ-glucosidic carbon. From the results described above, the struc tures of chesnatin and chestanin are considered to be 8 and 9, respectively, as proposed in the previous papers."' However, it could not be confirmed in this experiment which carboxyl group of dehydrodigallic acid in chesnatin (8) was esterified with crenatin. EXPERIMENTAL The 13C-NMR spectra were measured on a JEOL JMN-FX 100 Fourier-transform NMR spectrometer at room temperature. The spectra were recorded at a spectral width of 6000 Hz and 8192 data points. The pulse widths were 4 `6 Đsec (45 pulse) and the repeti tion time between pulses 1-3 sec. The chemical shifts were expressed in ppm downfield from TMS as internal standard. Unless otherwise noted, all spectra were obtained in CD3OD solutions. To exchange labile hydroxyl protons with deuterons, the samples were dissolved in deuterium oxide, and then freeze-dried. Acknowledgment. We thank gratefully Mr. Tetsu Hinomoto, JEOL Ltd., for kindly measuring the 13C- NMR spectra. REFERENCES 1) T. Ozawa, D. Kobayashi and Y. Takino, Agric. Biol. Chem., 41, 1257 (1977). 2) Idem., ibid., 42, 1213 (1978). 3) T. Ozawa, K. Haga, N. Arai and Y. Takino, ibid., 42, 1511 (1978). 4) T. Ozawa, N. Arai and Y. Takino, ibid., 42, 1907 (1978). 5) G. C. Levy and G. L. Nelson, "Carbon-13 Nuclear Magnetic Resonance for Organic Chemi sts," Wiley-Interscience, New York, 1972, p. 79; J. B. Stothers, "Carbon-13 NMR Spectroscopy," Academic Press, New York, 1972, p ) T. E. Walker, R. E. London, T. W. Whaley, R. Barker and N. A. Matwiyoff, J. Ain. Chem. Soc., 98, 5807 (1976). 7) T. Usui, N. Yamaoka, K. Matsuda, K. Tsuzi mura, H. Sugiyama and S. Seto, J. Chem. Soc. Perkin Trans. I, 1973, 2425.