Complete ¹H- and 13C- Resonance Assignment of Methyl 2alpha, 3beta, 24-Tri-O-acetylurs-12-en-28-oate and Methyl 2alpha, 3beta, 24-Tri-O-acetylolean-12-en-28-oate by NMR Spectroscopy

Abstracts

The complete ¹H and 13C chemical shift assignments of extended hydrogen spin systems in triterpenoid derivatives (methyl 2alpha, 3beta, 24-tri-O-acetylurs-12-en-28-oate and methyl 2alpha, 3beta, 24-tri-O-acetylolean-12-en-28-oate) was accomplished making use of one and two dimensional NMR techniques (HMBC, HMQC, COSY and NOESY).

triterpenoids; complete ¹H and 13C-NMR signal assignments; 1D NMR; 2D NMR (¹H x 13C-HMBC, ¹H x 13C -HMQC, ¹H x ¹H-COSY, ¹H x ¹H-NOESY)

O trabalho descreve o estudo de dois triterpenos isômeros (1: 2alfa, 3beta, 24-tri-O-acetil-12-eno-28-ursolato de metila e 2: 2alfa, 3beta, 24-tri-O-acetil-12-eno-28-oleanato de metila) efetuando a completa atribuição dos deslocamentos químicos dos hidrogênios e carbonos. Foram utilizadas técnicas de ressonância magnética nuclear (¹H e 13C) uni- e bidimensionais empregando os seguintes passos: a) Análise comparativa dos espectros de RMN 13C-PND e RMN 13C-DEPT para identificação dos carbonos quaternários, metínicos, metilênicos, e metílicos; b) aplicação da técnica ¹H x 13C HMBC [acoplamento de higrogênio e carbono-13 via duas (²J CH) e três (³J CH) ligações] para atribuição dos deslocamentos químicos dos espectros de 13C; c) uso dos espectros ¹H x 13C HMQC [interação spin-spin de higrogênio e carbono-13 via uma (¹J CH) ligação] para determinar os deslocamentos químicos dos átomos de hidrogênio e para confirmar os dos carbonos hidrogenados; d) uso dos espectros bidimensionais de correlação homonuclear de hidrogênio e hidrogênio (¹H x ¹H-COSY), e de efeito nuclear Overhauser homonuclear de hidrogênio e hidrogênio (¹H x ¹H- NOESY) para confirmar os sinais de hidrogênios e para determinações configuracionais (alfa e beta) dos hidrogênios metilênicos e metínicos e, e) análise dos padrões de desdobramento (mutiplicidade e constante de acoplamento) nos espectros unidimensionais para confirmar os sinais de vários átomos de hidrogênio. Foram descritas as condições experimentais dos aparelhos utilizados (RMN 500 MHz, para hidrogênio e 125 MHz, para carbonos) e de isolamento dos constituintes 1 e 2. Todos os resultados são resumidos na forma de tabelas.

Article

Complete ¹H- and ¹³C- Resonance Assignment of Methyl 2a, 3b, 24-Tri-O-acetylurs-12-en-28-oate and Methyl 2a, 3b, 24-Tri-O-acetylolean-12-en-28-oate by NMR Spectroscopy

F.J.Q. Monte*^a, and J.P. Kintzinger^b

^a

Departamento de Química Orgânica e Inorgânica, Centro de Ciências, Universidade Federal do Ceará, 60021-970 Fortaleza - CE, Brazil;

^bLaboratoire de RMN et de Modelisation Moléculaire, UA 422 du CNRS, Faculté de Chimie, Université Louis Pasteur, 67008-Strasbourg, France

Received: August 1, 1997

O trabalho descreve o estudo de dois triterpenos isômeros (1: 2a, 3b, 24-tri-O-acetil-12-eno-28-ursolato de metila e 2: 2a, 3b, 24-tri-O-acetil-12-eno-28-oleanato de metila) efetuando a completa atribuição dos deslocamentos químicos dos hidrogênios e carbonos. Foram utilizadas técnicas de ressonância magnética nuclear (¹H e ¹³C) uni- e bidimensionais empregando os seguintes passos: a) Análise comparativa dos espectros de RMN ¹³C-PND e RMN ¹³C-DEPT para identificação dos carbonos quaternários, metínicos, metilênicos, e metílicos; b) aplicação da técnica ¹H x ¹³C HMBC [acoplamento de higrogênio e carbono-13 via duas (²J_CH) e três (³J_CH) ligações] para atribuição dos deslocamentos químicos dos espectros de ¹³C; c) uso dos espectros ¹H x ¹³C HMQC [interação spin-spin de higrogênio e carbono-13 via uma (¹J_CH) ligação] para determinar os deslocamentos químicos dos átomos de hidrogênio e para confirmar os dos carbonos hidrogenados; d) uso dos espectros bidimensionais de correlação homonuclear de hidrogênio e hidrogênio (¹H x ¹H-COSY), e de efeito nuclear Overhauser homonuclear de hidrogênio e hidrogênio (¹H x ¹H- NOESY) para confirmar os sinais de hidrogênios e para determinações configuracionais (a e b) dos hidrogênios metilênicos e metínicos e, e) análise dos padrões de desdobramento (mutiplicidade e constante de acoplamento) nos espectros unidimensionais para confirmar os sinais de vários átomos de hidrogênio. Foram descritas as condições experimentais dos aparelhos utilizados (RMN 500 MHz, para hidrogênio e 125 MHz, para carbonos) e de isolamento dos constituintes 1 e 2. Todos os resultados são resumidos na forma de tabelas.

The complete ¹H and ¹³C chemical shift assignments of extended hydrogen spin systems in triterpenoid derivatives (methyl 2a, 3b, 24-tri-O-acetylurs-12-en-28-oate and methyl 2a, 3b, 24-tri-O-acetylolean-12-en-28-oate) was accomplished making use of one and two dimensional NMR techniques (HMBC, HMQC, COSY and NOESY).

Keywords: triterpenoids, complete ¹H and ¹³C-NMR signal assignments; 1D NMR; 2D NMR (¹H x ¹³C-HMBC, ¹H x ¹³C -HMQC, ¹H x ¹H-COSY, ¹H x ¹H-NOESY)

Introduction

The extraordinary advances made in spectroscopic techniques have enormously accelerated the research in the field of isolation and structure elucidation of complex natural products. Of all the physical methods, the NMR technique has greatly changed during the last two decades mainly by introduction and development of multiple pulse and 2D NMR. Consequently, a large number of triterpenes have been examined by NMR spectroscopy and much chemical shift data has been accumulated and utilized in further investigations of these natural products. On the other hand, the use of these secondary metabolites as therapeutical agents has been the subject of extensive exploratory activities during recent years¹. The ¹H and ¹³C triterpene chemical shifts provided useful information concerning conformations and configurations of these complex organic derivatives and are also useful for the better understanding of the correlations between their molecular conformations and their biological activities².

In this paper we report an extensive NMR study of two isomeric (C₃₇H₅₆O₈) triterpenoids derivatives with their complete ¹H and ¹³C signal assignments, by application of 1D and 2D spectral experiments. The compounds investigated were methyl 2a,3b,24-tri-O-acetylurs-12-en-28-oate (1) and methyl 2a,3b,24-tri-O-acetylolean-12-en-28-oate (2) obtained after acetylation and methylation of a mixture isolated from Mentha villosa Huds³. This plant is used as a remedy in the treatment of amebiasis, giardiasis⁴ and shistosomiasis⁵.

This is the first report giving the complete assignment of these pentacyclic triterpenoid derivatives of ursolic and oleanolic acids. The following steps were concurrently employed: a) comparative analysis of the ¹³C-NMR-PND and ¹³C-NMR-DEPT for identification of quaternary, methine, methylene and methyl carbon atoms; b) application of the HMBC experiment to chemical shift assignment of the ¹³C spectra; c) use of the HMQC spectra to determine the chemical shifts of the hydrogen atoms and to confirm those of the hydrogenated carbons; d) use of hydrogen ¹H x ¹H - COSY and ¹H x ¹H - NOESY maps to confirm the ¹H assignments (and, indirectly, also the ¹³ C assignments) and to establish the configurational assignment (a and b) of all methylene and methine hydrogens and e) analysis of the splitting patterns (multiplicity and coupling constant) in the 1D NMR spectra to confirm the resonances (including the configurational assignment) of various hydrogen atoms.

Experimental

Plant material

Mentha villosa was collected in the "Horto de Plantas Medicinais" of the "Universidade Federal do Ceará", Fortaleza, Brazil. A voucher of the plant (N. 16.545) is deposited in the Herbarium "Prisco Bezerra" of the Departamento de Biologia of the Universidade Federal do Ceará.

Isolation procedure

The fraction obtained from the ETOH extract by partition with CHCl₃ was successively chromatographed on silica gel column to afford fraction E (eluted with hexane-CHCl₃ 2:8). Fraction E was methylated with CH₂N₂ and then acetylated with Ac₂O/pyridine in the usual manner to yield a product named E-MeAc. Silica gel preparative TLC of E-MeAc lead to a fraction (R_f 0.60, eluted with CH₂Cl₂), identified as a mixture of the triterpene derivatives (1 and 2) which were separated by preparative HPLC Waters model 6000A, detector R - 401 diferential refractometer; RP 18 (250 x 9.4 mm) column; mobile phase: methanol-water 98:2, flow-rate of 2 mL min^-1.

NMR spectra

¹

H- and

¹³C-NMR experiments were performed on a BRUKER ARX 500 spectrometer working at 500.1 MHz for hydrogen and 125.75 MHz for

¹³C carbon, using CDCl

₃ as solvent. Solutions were made from 0.35 ml of CDCl

₃ and 2-8 mg of triterpenes with TMS as the internal standard. For all experiments the temperature was stabilised at 298 K. For the NOESY experiments, the samples were degased by bubbling nitrogen through the solution and fitting a teflon serum cap. The 2D experiments were acquired and processed with the software provided by BRUKER on ASPECT X32.

Typical acquisition and processing conditions for COSY and NOESY experiments were: relaxation delay of 1 to 2 seconds, 512 to 1024 t₁ increments; 1024 to 2048 t₂ points; sweep width of 6 ppm. Sine bell squared and shifted (p/4, p/6 and p/8) apodization functions were used for processing. The mixing time in the NOESY experiments, generally set at 1.2-1.5 seconds, was also varied between 0.8 and 2 seconds, without substantial change in the results. For ¹H x ¹³C (¹³C detected) and ¹³C x ¹H (¹H detected) correlations, the same relaxation delay was used, 512 to 1024 t₁ increments, 1024 to 2048 t₂ points, the sweep width being respectively 7 ppm for ¹H and 180 ppm for ¹³C. Lorentzian and Gaussian deconvolution were generally used in the processing. The number of scans was set for an overall acquisition time of about 12 h to 16 h.

Results and Discussion

An essential prerequisite to the unambiguous assignment of ¹H chemical shifts from ¹³C - ¹H shift correlated spectra is to first unambiguously assign the ¹³C chemical shifts of protonated carbons^6,7.

The signals corresponding to quaternary, methine, methylene and methyl carbon atoms were identified by comparative analysis of the ¹³C-NMR - PND and ¹³C - NMR DEPT spectra. The ¹H x ¹³C - HMBC Heteronuclear Multiple Connectivity - coupling of hydrogen and carbon-13 via two (²J_CH) and three (³J_CH) bonds spectra were successfully used to attribute the chemical shifts of several protonated as well as, of almost all non-protonated carbons. For example, the singlet at d_H 4.20 in the ¹H-NMR spectrum of 1 (Fig. 1) was correlated with hydrogens attached at an oxygenated carbon. Assuming this resonance assignable to 2H-24, the application of the HMBC technique led to the assignment of CH-3, C-4, CH-5 and CH₃-23 by identifying ²J_CH and ³J_CH connectivities. Thus, the carbon atom C-4 (_C 43.00) is readily assigned as the only quaternary carbon of the four; C-3 (d_C 79.90) is an oxygenated carbon and C-5 (d_C 55.52) is distinguished by additional 3H-25 (d_H 1.07) cross peak (³J_CH) for the latter carbon, while CH₃-23 (d_CH 23.10) was identified as a methyl carbon. Working along the molecule in this fashion, using the hydrogens from only the six methyl resonances, allowed in addition unambiguous assignment of CH₂-1 (d_C 44.32), CH₂-7 (d_C 33.20), C-8 (d_C 39.30), CH-9 (d_C 47.60), C-10 (d_C 37.50), C-13 (d_C 138.60), C-14 (d_C 41.80), CH₂-15 (d_C 27.90), CH-18 (d_C 52.85), CH-19 (d_C 39.10), CH-20 (d_C 38.87), CH₂-21 (d_C 30.65) and CH₂-24 (d_C 65.50) (Table 1). The quaternary carbons C-8 (d_C 39.30) and C-14 (d_C 41.80) of 1 were distinguished from the earlier assignment of 3⁸ (Fig. 1) since the chemical shifts of these carbon atoms are almost invariant for 1 and 3. Thus, in the HMBC spectrum of 3, only the carbon C-8 (d_C 39.35) showed connectivities with the hydrogen atoms 2H-11 (d_H 1.90-1.85), while only the carbon C-14 (d_C 41.69) showed a correlation with the hydrogen atom H-12 (d_H 5.26). On the other hand, the H-18 doublet (d_H 2.24, J = 11.4 Hz) of 1 allowed the localization of H-19 (d_H 1.32) in the ¹H x ¹H - COSY spectrum which in turn determined the chemical shift of the CH-19 (d_H 39.35) through ¹H - ¹³C - HMQC Heteronuclear Multiple Quantum Coherence - spin-spin interaction of hydrogen and carbon-13 via one (¹J_CH) bond spectrum. Thus, the resonance at d_C 38.87 correlates to CH-20. Two methine (CH-2 and CH-12), four methylene (CH₂-6, CH₂-11, CH₂-16 and CH₂-22) and one quaternary (C-17) carbons showed no cross peak with methyl hydrogens in the HMBC experiment as expected. Nevertheless, all were unambiguously identified. CH-2 (d_C 69.28) is readily assigned as an oxygenated carbon and CH-12 (d_C 125.00) is a sp² carbon. The methylene carbons were identified by use of a COSY technique. For example, the signal of H-9 (d_H 1.57) allowed the localization of the 2H-11 (d_H 1.92) hydrogens which in turn, lead to assignment of CH₂-11 (d_C 23.30) from the HMQC spectrum. In this way, were also assigned the chemical shifts of the CH₂-16 (d_C 24.20) and CH₂-22 (d_C 36.60) carbons. The overcrowded region of the COSY map did not allow the characterization of the 2H-6 hydrogens with respect to 2H-7. By exclusion, the last signal correspondening to one methylene carbon was attributed to CH₂-6 (d_C 19.20). The remaining quaternary carbon C-17 (d_C 48.00) was identified by comparative analysis of ¹³C-NMR - PND and ¹³C-NMR - DEPT spectra.

Finally, all methyl ¹³C signals were assigned from their connectivities with assigned methyl ¹H signals. These methyl ¹H signals were identified and distinguished on the basis of the observed connectivities in the HMBC map with previously assigned carbon atoms (Table 1). In this way, only the signal of 3H-23 (d_H 1.03) showed a cross peak at the ¹³C frequency with the readily assigned CH-3 (d_C 79.90); 3H-25 (d_H 1.07) with CH₂-1 (d_C 44.32); 3H-26 (d_H 0.74) with CH₂-7 (d_C 33.20); 3H-27 (d_H 1.06) with C-13 (d_C 138.60); 3H-29 (d_H 0.84) with CH-18 (d_C 52.85) and 3H-30 (d_H 0.94) with CH₂-21 (d_C 30.65). The assignment of the carbon signals were then carried out by an HMQC experiment (Table 1).

The assignment of CH₂ hydrogens as a or b were obtained by analysis of the splitting patterns (multiplicity and coupling constant) in the 1D NMR, long range coupling⁹ (⁴J) in the ¹H x ¹H-COSY and especially ¹H x ¹H-NOESY spectra (Table 1). These spectra also indicated that 1 was in conformation 1a and confirmed the cis junction of rings D and E (see 4).

In a report of 3b-O-acetyl-27-norurs-13-en-28-oic acid¹⁰, the chemical shifts of the carbon atoms CH₃-29 (d_C 20.02) and CH₃-30 (d_C 17.66) were inconsistent with that of the same carbon atoms in compound 1. In the case of 1 the ¹H x ¹³C - HMBC spectrum allowed the obvious assignment of CH₃-29 (d_C 16.95) by connectivities of the resonance at d_H 2.24 (H-18) to the resonance at d_C 16.95 (CH₃-29) and of the resonance at d_C 52.85 (CH-18) to the resonance at d_H 0.84 (3H-29). Consequently, the signal at d_C 21.15 was attributed to CH₃-30.

Using the same procedure, the HMBC, COSY, NOESY and HMQC spectra furnished the ¹H and ¹³C chemical shifts of compound 2 (Fig. 1, Table 2); and also the molecular conformation 2a and cis ring jucntion as in 4.

Acknowledgments

The authors gratefully acknowledge Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazilian Agency for financial support and Laboratoire de RMN et de Modelisation Moléculaire, ULP, Strasbourg, France, for facilities provided. Special gratitude is extended to R. Graff for technical assistance with the NMR measurements.

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Publication Dates

• Publication in this collection
  17 Mar 2008
• Date of issue
  Feb 1998

History

• Received
  01 Aug 1997
• Accepted
  01 Aug 1997