zyxw PHYTOCHEMICAL ANALYSIS, VOL. 7,245-252 (1906) zyxwvuts 600 MHz 'H and 13CNMR Full Assignments of Two Saponins from Nothapodytes foetidat Luisella Verotta* and Simona Caldiroli Dipartimento de Chimica Organica e Industriale, Universitl degli Studi di Milano, via Venezian 21, 20133 Milano, Italy. Pierluigi Gariboldi Dipartimento di Scienze Chimiche, Universitl di Camerino, via S. Agostino 1, 62032 Camerino (MC), Italy Marco Tat& Pharmacia, BiopharmaceuticallStructural Biochemistry, via Giovanni XXIII 23, 2001 4 Nerviano (MI), Italy zyxwvutsrqpo zyxwvuts The application of one- and two-dimensional nuclear magnetic resonance techniques to the structure elucidation of two saponins from Nothapodytes foetidu is reported. Detailed structural information about the sapogenin, protobassic acid, and the oligosaccharidic chains, including sugar sequence and position of glycosidation, is provided. Keywords: Norhupodytes foetida; nuclear magnetic resonance spectroscopy; selective excitation; saponins: Mi-saponins A and B; protobassic acid 3-0-/3-D-glucopyranoside. two-dimensional (2D)-techniques. A related saponin 2 was also isolated from the same plant. INTRODUCTION Saponins are a large class of natural compounds, widespread in the vegetable kingdom, which contain a triterpenoid core to which, in one or more positions, a number of sugar units are bound. Because of their important biological activities their structures have been extensively studied and several hundreds of saponin structures have been elucidated (Mahato and Nandy, 1991). Such structural investigations are often very time consuming and tiresome, mainly due to the presence of the sugar moieties for which the site(s) of binding to the triterpenoid, the inter-glycosidic bonds and their conformation have to be assessed. This is generally achieved by chemical degradation leading to partial or total cleavage of the sugar moieties which could then be identified by classical methods. The nuclear magnetic resonance (NMR) analysis of the intact molecules has often been hampered by the extensive overlapping of most sugar signals. A relatively easier task is the identification of the terpenoid portion of the saponins for which much NMR information is available in the literature. This combination of chemical reactions and NMR analysis, however, requires some hundreds of milligrams of pure saponin which is often not available. In this paper we describe the structure and the full 'Hand I3C NMR assignments of a bidesmosidic saponin 1 isolated from Nothupodyfes foetidu (Wight) Sleum. (Icacinaceae) by the aid of a high field (600 MHz) spectrometer and the combination of several one-dimensional (1D)- and EXPERIMENTAL Plant material. Nothupodytes foeridu was collected on the mountains of Tamil Nadu (India) in June 1993 and identified by U. Boni (Indena SPA, Settala, Italy): a voucher specimen is deposited at Indena SPA. The extraction was as reported by Pirillo et al. (1995). zyxwv General. Thin layer chromatography (TLC) was conducted on precoated Kieselgel 60F2,, or RPI 8 (Merck, Dermstadt, Germany) plates using, respectively, the solvent systems: CHC1,:MeOH:nPrOH:H,O (5:6:1.4; organic layer) or CHCI,:MeOH:H20 (5:3:3; organic layer). Compounds were detected by spraying with H2S0,: MeOH ( I :9) followed by heating. Column chromatography was performed using Kieselgel 60 (0.063-0.200 mm; Merck; product number 7734. High speed counter current chromatography (HSCCC) was performed on a CCC 1000chromatograph (PharmaTech Research Corp., Baltimore, USA) equipped with a three multilayer coiled column (total volume 350 mL) rotating at 1032 r.p.m., a SSI liquid chromatography pump (model 300), a PTRC speed controller and a Rheodyne injection valve. Samples were filtered on Millex-HV filters (0.45 pm; Millipore, Bedford, MA, USA) prior to injection. Sephadex LH 20 (Pharmacia, Uppsala, Sweden) was used for gel filtration. Chromatographic separations were as reported in Pirillo et al. (1995). Fast atom bombardment (FAB) mass spectra were obtained on a Finnigan MAT (Bremen, Germany) VG 7070 mass spectrometer using NBA as matrix. zyxwvu zyxwv zyxwvutsrq zyx * Author to whom correspondence should be addressed. t This paper is dedicated to the memory of Professor P. Gariboldi who passed away on August 27, 1995. CCC 0958-0344/96/050245-08 0 1996 by John Wiley & Sons, Ltd NMR experiments. All spectra were measured on samples of about 10 mg dissolved in 750 pL of pyridine-d,, plus one drop of Received 10 August 1995 Accepted (revised) I0 April 1996 246 zyxwvutsr zyxwvut zyxwvutsrqponm L. VEROTTA ETAL. zy zyxwvutsrqpo zyx i e subspectra above. Figure 1. I D TOCSY spectra of saponin 1: the total spectrum is shown at the bottom of the panel witt E.COSY F l 4 - (ppmk 1.21 :3 1.4: 1.5 1 6 l;izyxwvutsrqpon 1 9 ? O 2. $3 22A-21A ?-3 22A-21 zyxwvutsrqponmlkjihgfedcb 2 2 2 : 2 0 1’3 1 8 t > 1 7 1 6 i - I . 1 ) 1 . 1 (iliini) Figure 2. Selected zone (2.4 to 1.1 p.p.m.) of the E-COSY spectrum of saponin 1. : zyxwvutsrq zyxwvutsrqponml NMR OF SAPONINS 247 spectra (Greisinger et al., 1988) were acquired with an 80 ms mixing time, an MLEV-17 spin-lock field (Bax and Davis, 1985) of 10 kHz flanked by two 2 ms trim pulses, 1024 points in F2,256 complex increments in FI, 8 scans per increment and a final data matrix of 2 x Ik points. The ROESY spectra (Kessler et al., 1987) were acquired with a 400 ms mixing time, an MLEV- 17 spin-lock field of 3 kHz obtained with small flip-angle pulses (30"), 1024 points in F2,256 complex increments in FI, 8 scans per increment and a final data matrix of 2 X Ik points. The E-COSY spectra (Griesinger et al., 1985) were acquired with 4096 points in F2, 1024 complex increments in FI, 32 scans per increment and a final data matrix of 8 x 4 k points. The HMQC spectra (Bax and Subramanian, 1986) were acquired with a nulling time of 300 ms, CD,OD, in 5 mm tubes. Spectra were measured in the phase sensitive mode at 28°C on a Varian Unity 600 spectrometer, operating at 599.919 MHz for 'H and at 150.858 for '.'C, equipped with a triple resonance indirect detection probe, a waveform generator on both the observing and the decoupling channel, and running Varian Software Vnmr 4.3a. The ID-experiments were run using the States-Haberkon method (States et al., 1982) whilst the 2D-experiments utilised the Hypercomplex method. ID- and 2Dspectral widths were 6000 Hz for 'H NMR and 15300 Hz at "C NMR. A11 spectra were referenced to TMS through solvent signals. The DQF-COSY (Rarnce ef al., 1983) spectra were acquired with 2048 points in F2, 512 complex increments in FI, 16 scans per increment and a final data matrix of 4 X 2k points. The TOCSY zyxwvutsrq zyxwvutsr zyxwvutsrqpon zyxwvuts z zyxwvutsrq Glcl-3 Xyll-Rhal4 0 . zyx R h a 1 1 ~ 2 c Apll-Rhal3 c. Rhall?;Xyl3 4 4,: I I10 , , , , , , 105 I 100 , , , , , 95 , , , , 85 90 F1 , , , ~ I 80 , , , -1 75 -~ ~ 1 7 1 70 , 0 I I , , I I 1 I 65 lppm) R = CHPOH Figure3. The HMBC (J=8Hz) experiment with the sugar linkages shown on the spectrum: arrows indicate significant 'C-H connectivities in structures 1 and 2. 248 zyxwvutsrqponm zyxwvutsr zyxwvutsrq zyxwvutsr zyxwvutsrq zyxwvuts zyxwv L. VEROTTA ETAL. 1024 data points in F2, 256 complex increments in FI, 8 scans per increment, a final data matrix of 2 X Ik points and a MPF7 waveform generator based "C-decoupling sequence during the acquisition (Fujiara et al., 1993). The HMBC spectra (Summers et al., 1986) were acquired with 1024 points in F2, 256 complex increments in FI, 32 cans per increment and a final data matrix of 2 x Ik points. For each sample one HMBC spectrum was optimized for a ''JC." of 8 Hz and another for a nJC.H of 4 Hz, with n=2-4. All 2D spectra were transformed with a cosine squared weighting function in both dimensions except for the HMBC spectra where a sinebell function was applied in F2 and a cosine squared in FI, together with a mixed mode display of the spectra (magnitude mode in F2 and phase sensitive mode in FI). Selective excitation spectra, ID-TOCSY (Kessler et al., 1986) were acquired using waveform generator based BURP shaped pulses (Geen and Freeman, 1991), mixing times ranging from 80 to 150 ms and an MLEV-17 spin-lcok field of 10 kHz preceded by a 2 ms trim pulse. The repetition rates for all spectra were about 1.5 S. (1 mL) were collected according to their composition (monitored by TLC) to yield 1 (47 mg) and 2 (57 mg). Compound 1. 3-0-/3-~-glucopyranosy1-28-0{ 3-O-/3-~-apiofuranosyl-4-O[a-~-rhamnopyranosyl ( I-3-P~-xylopyranosyl]a-L-rhamnopyranosyl (I-.2)-a-~-arabinopyranosyl)-protobassic acid. C,,H,,,O,,; colourless needles; m.p. 250°C (d)(EtOH); [a]?43.6 (MeOH, c 0.47) (literature values, m.p. 250-253"C, [a]g-45.0°; Kitagawa et al., 1975); 'H and "C NMR see Tables I , 2, and 3; FAB MS (negative mode) m/z 1377 [M+Na-HI-. zyxwvutsrq zyxwv Isolation of compounds 1 , 2 and 4. Fraction 5 (260 mg) from the medium pressure liquid chromatographic (MPLC) purification of crop 4 (Pirillo et aL, 1995) was submitted to HSCCC using the solvent system CHCI,:MeOH:n-PrOH:H,O (5:6: 1 :4; aqueous phase) as mobile phase at a flow-rate of 1 mUmin. Fractions 3 4 3 2 3 0 Compound 2. 3-O-~-o-gtucopyranosyt-28-0[a-~-rhamnopyranosyl (1 -+3)-/3-~-xylopyranosyI ( I -.4)-a-~-rharnnopyranosyl ( 1-.2)-a-~-arabinopyranosyl]-protobassic acid. C58H94027; amorphous white powder; m.p. 235-236°C (4 (EtOH); [a]2-3ISo (MeOH, c 1) (literature values, m.p. 235-238", [a]?-33.Io; Kitagawa et al., 1975); 'H and I3C NMR see Nigam et al. (1992); FAB MS (negative mode) d z 1221 [M-HI-; (positive mode) m/z 1245 [M+Na]'. Hydrolysis of the saponins. A mixture of 1 + 2 (52 mg) was refluxed with 5% .HCI:MeOH for 3 h. The solution was made neutral with NaHCO,, concentrated and extracted with n-butanol. The residue (23 mg) was purified by HSCCC using the solvent zyxwvuts 2 8 2 6 2 4 2 2 2 c F2 1 8 1 6 1 c 1 6 'ppm) Figure 4. Selected zone (3.4 to 0.8 p.p.m.) of the ROESY spectrum of saponin 1. 1 c i, t zyxwvutsrq zyxwvut zyxwvutsrqponm zyxwvutsrq zyxwvut NMR OF SAPONINS system CHCI,:MeOH:H20 (5:3:3; aqueous phase), as mobile phase at a flow-rate of 1 mL/min) yielding 3 (5.8 mg) which showed the same physico-chemical characteristics as reported by Nigarn et af. (1992). 249 viral activities (Pirillo et al., 1995). During the chemical investigation of the methanol extract, the saponins 1 and 2 were isolated by a combination of normal and reversed phase MPLC and counter current chromatography (CCC) techniques. The isolated amounts of the two saponins were low (about 40mg each, see Experimental section) and so the compounds had to be directly analysed by spectroscopic techniques. Compound 1 showed a quasi molecular peak at d z 1377 (M +Na - H)- in the FAB mass spectrum corresponding to The chromatographic a molecular formula of C63H10203,. behaviour on TLC (close Rf values and identical colour yields after spraying with sulphuric acid) and the I3C NMR spectrum of 1 strongly indicated a strong similarity to the saponins recently isolated by us from Crossopteryx febrifuga (Gariboldi et al., 1990). The main differences were found in the I3C NMR signals of the oxygenated carbons, suggesting a different sugar composition, or connection among them, together with the lacking of a hydroxyl group on the aglycone. Six anomeric protons were easily identified in 1 at 6 6.49 (d, J=2.5 Hz), 6.11 (bs),5.89 (d, 5=4.4 Hz), 5.55 (bs), 5.25 (d, J=8.1 Hz) and 5.17 ( d , J=8.3 Hz) correlating to carbons at S 93.03. 102.85, 112.02, 101.18, 104.97 and 105.67 respectively. The structure of the oligosaccharidic chain was assigned using the following argument. The isolated 'H NMR signals resonating in the uncrowded regions of the spectrum, between 5.1 and 6.6 p.p.m. were the starting point for the ID TOCSY (Kessler et al., 1986) experiments (Fig. 1). Because of the selectivity of the multi-step coherence transfer, the ID zyxwvutsrqpo zyxwvutsrqp zyxwvutsr zyxwvutsrq Compound 3. 3-O-P-~-glucopyranosyl protobassic acid. C,,H,,O,,; FAB MS (positive mode, glycerol) dz: 705 [M+K]'; 689 [M + Na]' ; 'H NMR (pyridine-d5-CD,OD; 599.9 19 MHz) S 5.62 (r, J=4.5 Hz, H-12), 5.21 (bs, H-I Clc), 5.17 (bs, H-6), 4.91 (ddd, J=7.6, 7.0, 4.1 Hz, H-2), 4.45 (d, J = 12.4 Hz, H-23A), 4.43 (d, J = 11.2 Hz, H-6A Glc), 4.31 (f, J z 8 . 8 Hz, H-3 Glc), 4.30 (2H, m, H-3+H-6B Glc), 4.20 (r, J=9.1 Hz, H-4 Glc), 4.15 ( r , 5=8.4Hz, H-2 Glc), 4.07 (d, J=12.4 Hz, H-2B), 4.01 (m,H5 Glc), 3.52 (bd, H-18), 2.51 (m, H-l5A), 2.36 (2H, m, H-IE+H-IIA), 2.12 (s, CH,-25), 1.92 (s, CH,-24), 1.75 ( h ~CH,, 26), 1.34 (s, CH,-27), 1.19 (2H, m, H-2lE+H-l5E), 1.13 (<, CH,-30), 0.93 (s, CH,-29); "C NMR see Table 3, glucose signals: 6 105.64 (d, C-I), 75.85 (d, C-2), 78.66 (d, C-3), 71.83 (d, C-4), 78.49 (d, C-5), 62.8 1 (t, C-6). RESULTS AND DISCUSSION Nothapodytes foetida is known to contain camptothecin and its 9-methoxy derivative (Govindachari and Viswanathan, 1972; Broglia et al., 1995). Recently, Pirillo et al. (1995) reported the isolation of foetidin I and mappicine glycosides from a polar extract of the trunk bark of the plant. Foetidin I was claimed to possess interesting anti-tumor and anti- Table 1. 'H NMR (600 MHz) chemical shifts of saponin 1 as determined by E-COSY experiment Proton 1E 1A 2 3 5 6 7A 7E 9 11A 11E 12 15A 15E 16A 16E 18 19A 19E 21A 21E 22A 22E 23A 238 24 25 26 27 29 30 MH)" 2.32 1.32 4.83 4.31 1.89 5.13 2.01 1.85 1.86 2.34 2.08 5.51 2.19 1.25 2.08 1.95 3.33 1.80 1.28 1.39 1.19 2.06 1.69 4.49 3.99 1.97 2.19 1.65 1.26 0.92 1.04 Significant crass-peak correlations in the ROESY spectrum multiplicity, J (Hz) dd, 1E-lA=11.0 dd, IA-lE=11.0 ddd, 2-lEz7.0, 2-1A=7.6 d, 3-2 = 4.1 d, 6 - 5 ~ 8 . 6 ddd, 6-7A=7.2, 6-7E =5.7 bd, 7A-7E=10.4 bd, 7E-7Azl0.4 dd,9-11A=11.9,9-11E=6.9 ddd, 1I A-11E= 17.0, 11A-9 = 6.9 ddd, 11E - l l A = 17.0, 11E-9= 11.6 f, 12-11A=4.5, 12-11E=4.5 rn rn m rn dd, 18-19A= 14.2, 18-19E=5.5 dd, 19A-18 = 14.2,19A-l9E = 14.0 dd, 19E-18=5.5 m rn rn rn zyxwvut d, 23A-238 = 11.O d, 236-23A= 11.O S S S S S S H-2, H-I 1E H-3, H-5, H-9 H-IE, H-I GIC H-IA, H-5, H-1 G l C H-lA, H-3, H-7A, H-6 H-7A, H-5 H-5, H-9, H-27 H-15E, H-26 H-27 H-25 H-1 E H-IlA, H-11E, H-18 H-26 H-7 E H-19A H-15A, H-22E H-lgE, H-22A, H-30 H-I6A, H-21A H-18, H-29, H-30 H-16A, H-19A H-22A, H-29, ti-30 H-21 E, H-30 H-16E H-24 H-24 H-25 H-24, H-26 H-25, H-1 Ara H-19A H-I9A, E, H-21A, E, H-30 H-18, H-29 p.p.m. from internal standard TMS:pyridine-d, and CD,OD. 250 zyxwvutsrqponm zyxwvut zyxwvuts zyxwvuts zyxwvutsrqponm zyxwvutsrq zyxwvutsrq zyxwvuts L. VEROTTA ETAL. Table2. ‘H and I3C assignments and significant BC-H connectivities (as determined by an HMBC experiment)of the sugar moiety of saponin 1. Sugar Position 3-OGlc 1 2 3 4 5 6A 6B 1 2 3 4 5A 58 1 2 3 4 5 CH3 1 2 3 4 5A 5B 1 2 3 4 5 28-OAra Rha I Rha II Api CH, 1 2 3 4A 4B 5A 58 MHIa 5.17 4.02 4.14 4.19 3.88 4.44 4.31 6.49 4.45 4.51 4.37 4.56 3.94 5.55 4.68 4.36 4.44 4.31 1.70 5.25 3.89 4.21 4.06 4.12 3.37 6.11 4.71 4.54 4.26 4.90 1.63 5.89 4.74 4.56 4.17 4.06 4.02 multiplicity J (Hz) MCI” d, 8.3 t, 8.4 t, 8.9 t, 9.0 m m, JA.,=11.6 m, JA.,=11.6 d, 2.5 bd, 5.0 bt, 3.1 dd, 9.5, 2.5 J,= 10.4 105.67 75.49 78.49 7 1.68 78.08 62.68 bs 101.18 71.62 82.22 75.68 68.72 18.44 104.97 75.69 83.42 69.68 66.95 bt, 3.0 bd, 11.5 bt, 9.4 d9, 6.4, 2.0 d,6.4 d, 8.1 t, 8.4 t, 8.9 m dd, 11.0, 5.0 t, 11.0 bs dd, 4.0, 3.0 dd, 10.4, 4.0 t, 10.0 dq, 6.4.2.2 d, 6.4 d, 4.4 d, 4.4 - d, 9.6 d, 9.6 d, 11.0 d, 11.0 93.03 75.40 69.24 65.54 62.32 102.85 72.37 72.59 74.1 1 70.67 18.55 112.02 77.67 79.88 74.68 Connected protons Glc-1, Glc-3, Glc-4, Glc-5 Glc-3 Glc-I Ara-2, Ara-3, Ara-5 Rha 1-1 Rha 1-3, Rha 1-2, Rha 1-5 Api-I Xyl-1 Rha 1-5 Xyl-I Xyl-2, Rha 11-1 Xyl-3 Xyl-3, Xyl-4 Xyl-3, Rha 11-3 Rha 11-3, Rha 11-4 Rha 11-3 Rha 1-3, Api-4 Api-I Api-I, Api-3 64.63 a6 in p.p.m. TOCSY subspectra of the single monosaccharide unit could be extracted from the crowded overlapping region between 3.8 and 5.0p.p.m. Each subspectra could be attributed to one set of coupled protons such as H-C( 1) to H-C(5) or HC(6) of a carbohydrate moiety. Moreover the ID TOCSY subspectra obtained by irradiating at S 5.13 (ddd) and 84.83 (ddd) respectively, recognized these protons as belonging to the triterpenoid skeleton, as they showed a set of coupled protons in the low frequency region (above 2.4 p.p.m.). The irradiation of the signal at 6 5.55 (bs), integrating for two protons, showed a set of coupled resonances both among sugar (3.5-4.5 p.p.m.) and low frequency protons (above 2.4 p.p.m.). This allowed assignment both to the anomeric proton of one monosaccharide and to an olefinic proton of the triterpenoid aglycone (Fig. 1). Using a combination of 1D and 2D TOCSY (Griesinger et al., 1988) and 2D DQF-COSY (Rance et al., 1983) experiments, the ID TOCSY subspectra of the six monosaccharidic units could easily be interpreted and, at the same time, the type of sugar, its configuration and conformation assigned. The identification of the Pbapiofuranosyl unit was made possible with the aid of carbon resonances as the coherence transfer stopped at the quaternary carbon (C-3). Table 2 reports proton chemical shifts and coupling constants of the sugar units as determined from the ID and 2D TOCSY and 2D DQF COSY experiments. The assignment of all of the proton resonances for the sugar moieties immediately allowed the assignment of the resonances of the linked carbon atoms, and, once the carbon spectrum had been completely assigned, an unambiguous determination of the interglycosidic linkages could be obtained from the long-range C-H correlation (HMBC) spectrum (Summers er al., 1986). The 2D ROESY experiment (Kessler et al., 1987) confirmed the interglycosidic linkages showing all ROE crosspeaks among protons spatially related. Of the six monosaccharide units, five appeared connected together but one (the p-D-glucopyranosyl unit) showed correlations (2D ROESY and HMBC experiments) only with the aglycone signals. The I3Cresonances of the aglycone were easily identified by subtracting the sugar carbon resonances from the total spectrum of 1. Some resonances were specific for a 3p hydroxy A’* oleanane skeleton carrying a carboxylic function in position 28. Three more oxygenated carbons were present ( 6 69.91, 67.60 and 65.54) which correlated, respectively, to protons at S 4.83, ddd, J=7.0, 7.6, 4.1, S 5.13, ddd, 3=7.2, 5.7, 8.6 and 64.49 and 3.99 (AB system). zyxwvuts zyxwvu zy NMR OF SAPONINS Their location in the molecular was deduced by a combination of 2D DQF COSY, 2D ROESY, HMQC (Bax and Subramanian, 1986) and HMBC experiments, thus assigning the oxygenated positions as 2p, 6/3, 23a. All of these experiments were self consistent with the proposed structure of protobassic acid. Owing to the complexity of the spin systems, the determination of both proton chemical shifts and coupling constants of the aglycone was achieved with E-COSY (exclusive correlation spectroscopy; Greisinger et al., 1985). This experiment favours the diagonal peaks in the diagonal multiplets, allowing the analysis of cross-peak multiplets even closer to the diagonal than for 2D-filtered COSY spectra (Fig. 2). Finally, the HMBC and 2D ROESY spectra were useful in the determination of the linkages of the sugar moieties to the aglycone. They identified the P-Dglucopyranosyl unit linked to C-3, while the five-membered oligosaccharidic chain is bonded, via an ester linkage, to C28 through the a-L-arabinopyranosyl unit (Fig. 3). From these considerations, 1 was assigned the structure of 3-0-p~-glucopyranosyl-28-0-{ 3-O-~-~-apiofuranosy1-4-0-[~u-r.rhamnopyranosyl ( l+3)-~-~-xylopyranosyl]-ff-~-rhamnopyranosyl(1+2)-c~-~-arabinopyranosyl) -protobassic acid, a saponin previously isolated from the seed kernels of Madhuca longifolia (Kitagawa et al., 1975) but never 25 1 described spectroscopically. Compound 1 was accompanied by a less polar compound 2 which showed in the 'H and I3C NMR spectra superimposable resonances for the aglycone but the absence of the apiosyl unit. Sophisticated NMR experiments were not repeated for compound 2, as the NMR spectra were easily interpreted with the aid of the previously reported data for 1. Compound 2 showed physico-chemical characteristics identical to those reported for Mi-saponin A, isolated from Madhuca longifolia (Kitagawa et al., 1975), Madhuca butyracea (Nigam et al., 1992) and Clerodendrum wildii (Toyota et al., 1990). Both saponins 1 and 2, when submitted to acid hydrolysis, gave protobassic acid 3-0-/3D-glucopyranoside (3,as already observed by Nigam et al. (1992). The study reported here demonstrates that the rapid and unambiguous structure elucidation of complex saponins and their full NMR assignments may be performed using very low amounts of sample (a few milligrams) with a combination of sequential 1D and inverse-detected 2D (Martin and Crouch, 1991) NMR techniques. The complete analysis, however, of COSY spectra of saponins is often difficult because of peak overlap. Therefore it is very useful to be able to relay coupling information from an isolated proton (such as the anomeric hydrogen of saccharides) which, when propertly excited, transfers the relayed coherence to coupled protons. This transfer is blocked by a quaternary carbon or by a heteronucleus, thus each network of mutually coupled protons can be detected by tracing the cross peaks from certain specific protons in a TOCSY experiment, or by reading each 1D subspectrum as an isolated spin system through a series of ID TOCSY experiments (Fig. 1). Moreover, the correct choice of mixing times, together with the peculiar in-phase multiplet structure (which contrasts with the anti-phase structure in the COSY spectra) permits the use of 1D TOCSY experiments to read correctly vicinal couplings which are too low to be determined by the less resolved COSY experiment. TOCSY experiments are frequently used for the determination of the amino acid constituents of peptides in which the amino acid units are separated by amide carbonyl atoms (Williamson, 1993, Barboni et al., 1994). The application of this technique to oligosaccharides is, however, rarely reported (Wessel er al., 1991; Willker and Leibfritz, 1992; Vasquez et al., 1992; Orsini et al., 1994; Gariboldi et al., 1995). The advantage of the E-COSY experiment is a reduction of multiplet lines in the cross peak thus allowing easy measurement of active constants and a very accurate measurement of passive coupling constants. E-COSY is thus among the most appropriate experiments in order to obtain the largest number of couplings with the greatest accuracy possible (Eberstadt et al., 1995), and becomes the experiment of choice when overlapped signals occur endowed with multiple couplings. It has, however, been rarely used for the assignment of triterpene protons (Gariboldi et al., 1995; Willker and Leibfritz, 1992). zyxwvuts zyxwvutsr zyxw zyxwvutsr ~~ Table3. I3C NMR assignments of the aglycone portion of 1 and 3, and 'C-H connectivities as determined by the HMBC experiment Compound Carbon 1 2 3 4 5 6 7 a 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a 6 in p.p.m. 18 46.44 69.91 83.22 44.00 48.86 67.60 41.15 39.57 49.23 36.99 24.01 123.35 143.71 42.97 28.25 23.37 47.65 41 .a5 46.65 31.12 34.24 32.79 65.54 16.75 19.02 18.55 26.22 176.42 33.19 23.72 Compound 3' Connected protons 46.86 H-25 71.10 H-3 84. I4 H-lGlc, H-23A, H-23B, H-24 44.39 H-2, H-3, H-24 49.95 H-25 68.42 41.51 H-26 39.7% H-26, H-27 49.21 H-25, H-26 37.35 H-5, H-25 25.05 122.47 146.57 H-27 43.80 H-26, H-27 29.32 H-27 24.82 48.35 41.51 46.86 H-29, H-30 31.86 H-29, H-30 35.68 H-29, H-30 34.45 65.64 H-3 17.37 H-3, H-5 19.76 H-5 19.61 27.16 H-22A 176.95 H - I Ara (only in I), 34.45 H-30 24.98 H-29 zyxwvu Acknowledgements This work was supported (40% and 60% funds) by Minister0 dell'Universiti e della Ricerca Scientifica e Technologica (MURST). 252 zyxwvutsrqponml zyxwvutsr zyxwv zyxwvuts zyxwvutsrqpo zyxwvuts zyxwvuts zyxwvut L. VEROTTA ETAL. REFERENCES Barboni, L., Gariboldi, P., Torregiani, E. and Verotta, L. (1994). Cyclopeptide alkaloids from Zizipbus mucronata. Pbytocbemistry35, 1579-1582. Bax, A. and Davis, D. G. (1985). MILEV-17-Based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65,355-360. Bax, A. and Subramanian, S. (1986). 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