CHIRALITY 26:189–193 (2014)

Determination of the Absolute Stereochemistry of Two NewAristophyllene Sesquiterpenes: A Combined Theoretical and

Experimental InvestigationRUI-BO WU, ZHONG-BIN CHENG, QING-HUA HAN, TING-TING LIN, JING-WEI ZHOU, GUI-HUA TANG, AND SHENG YIN*

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China

© 2014 Wiley Perio

ABSTRACT Aristoyunnolins G (1) and H (2), two new diastereoisomeric sesquiterpenesfeaturing a rare aristophyllene skeleton, were isolated from the traditional Chinese medicineAristolochia yunnanensis. Their absolute stereochemistry involving three chiral centers wasdetermined by combined chemical, spectral, and Density Functional Theory (DFT) calculationmethods. Chirality 26:189–193, 2014. © 2014 Wiley Periodicals, Inc.

KEY WORDS: aristophyllene sesquiterpenes; absolute configuration; ECD; DFT

*Correspondence to: Sheng Yin, School of Pharmaceutical Sciences, Sun Yat-senUniversity, Guangzhou, Guangdong 510006, People’s Republic of China.E-mail: yinsh2@mail.sysu.edu.cnThe first two authors contributed equally to this work.Received for publication 11 November 2013; Accepted 12 December 2013DOI: 10.1002/chir.22294Published online 10 February 2014 in Wiley Online Library(wileyonlinelibrary.com).

INTRODUCTIONThe complete structure elucidation of a newly isolated

natural compound may require considerable effort andcomprise many different methods. Conventional approachesto determine the absolute configuration (AC) of chiralsubstances include X-ray diffraction, electronic circulardichroism (ECD), nuclear magnetic resonance (NMR), analy-sis of Mosher esters and related covalent derivatives, totalsynthesis, and chemical degradation.1 These approaches areoften time-consuming and costly and may require significantamounts of analyte. The comparison of computational andexperimental spectroscopic data, such as ECD, vibrationalcircular dichroism (VCD), optical rotation (OR), NMR,ultraviolet (UV), in contrast, can provide a streamlinedapproach that, at a minimum, significantly reduces the numberof possible stereoisomers.2 In recent years, a number of usefultheoretical methods, such as the configuration interaction (CI)method, Density Functional Theory (DFT), Time-DependentDensity Functional Theory (TDDFT), and time-dependentHartree-Fock (TDHF), have been established for computedspectra.3 Among them, DFT and TDDFT, which have provento be reliable and feasible approaches, are perhaps the mostwidely used ab initio methodology in the field of chiropticalproperties prediction,4 particularly for the prediction of ECDspectra. Many successful applications of CD spectra predictionby TDDFT are being reported in the literature.5–10

Aristophyllene sesquiterpenoids represent a very rare skele-ton only found in the genusAristolochia (Aristolochiaceae). Theyare structurally related to elemane-type sesquiterpenoids,11 withthe C11-C12-C13 fragment in elemane being rearranged from C-7to C-6 (Fig. 1). As aristophyllene contains three chiral centers(C-5, 10, and 12), it may give rise to eight stereoisomers, whichcan be grouped into four pairs of enantiomers. Although theAC of the oxymethine carbon (C-12) could be determined byMosher’s method, the ACs of C-5 and C-10 could not be deter-mined simply by correlating with that of C-12 using traditionalNMR methods due to the flexibility of the C11-C12-C13 ”tail”.Thus, the hurdle in solving the ACs of aristophyllenes lies indetermination of the AC of C-5 or C-10. So far, only sixaristophyllenes (3�8, see Fig. 1) have been reported,11,12 theACs of four (5�8) were assigned using the CD exciton chiralitymethod by making use of their aristolochyl structural moiety,while the other two still remained unsolved. However, since ex-citon coupling requires two or more similar interacting

dicals, Inc.

chromophores in the same molecular, the method cannot beapplied in a straightforward manner to molecules which haveonly a single site for derivatization, such as 1�4. And the appli-cation of CD exciton chirality method in models possessingthree different chromophores such as 5�8 may complicate theinterpretation of Cotton effects and lead to a dubious conclusion.During our search for discovering structurally interestingmetabolites from medicinal plants, two diastereoisomericaristophyllene sesquiterpenoids, aristoyunnolins G (1) and H(2) (Fig. 1), were isolated from the stems of Aristolochiayunnanensis Franch. Herein, we reported the determination ofthe absolute stereochemistry of these two sesquiterpenes usingcombined theoretical and experimental methods.

MATERIALS AND METHODSGeneral

Optical rotations were measured on a Rudolph Autopol I automaticpolarimeter. IR spectra were determined on a Bruker Tensor 37 infraredspectrophotometer. NMR spectra were measured on a Bruker AM-400spectrometer at 25°C. Electrospray ionization, mass spectrometry(ESIMS) was measured on a Finnigan LCQ Deca instrument, and high-resolution ESIMS (HRESIMS) was performed on a Waters MicromassQ-TOF. A Shimadzu LC-20 AT equipped with an SPD-M20A PDA detectorwas used for high-performance liquid chromatography (HPLC). A YMC-pack ODS-A column (250 × 10mm, S-5 μm, 12 nm) was used forsemipreparative HPLC separation. Silica gel (300�400 mesh, QingdaoHaiyang Chemical) and C18 reversed-phase silica gel (12 nm, S-50 μm,YMC) were used for column chromatography. All solvents used were ofanalytical grade (Guangzhou Chemical Reagents).

Plant MaterialStems of A. yunnanensis. were collected in October 2010 from

Yunnan Province, P.R. China, and were identified by Prof. You-KaiXu of Xishuangbanna Tropical Botanical Garden, Chinese Academyof Sciences. A voucher specimen (accession number: NMX201010)has been deposited at the School of Pharmaceutical Sciences, SunYat-sen University.

Fig. 1. Structures of aristoyunnolins G (1) and H (2) and related analogs.

WU ET AL.190

Extraction and IsolationFraction IIIc (1.9 g)13 was subjected to C18 reversed-phase (RP-18) sil-

ica gel CC (MeOH/H2O, 6:4 10:0) to give four fractions (IIIc1�IIIc4);fraction IIIc2 (200mg) was further purified on silica gel CC (PE/acetone,60:1 10:1) to afford 1 (34mg) and 2 (28mg). 1:colorless oil; [α]25D +235.5(c 0.10, CHCl3); UV (MeOH) λmax (log ε) 218 (3.66) nm; CD (c 6.3 × 10�4

M, CH3CN), λmax (Δε) 253 (�0.39), 335 (+0.22); IR (KBr) νmax 3403, 2967,2927, 1693, 1454, and 1363 cm�1; 1H NMR (CDCl3, 400 MHz) δ 5.87(1H, dd, J =17.6, 10.9 Hz, H-1), 5.11 (1H, d, J = 17.6 Hz, H-2b), 5.02(1H, d, J = 10.9 Hz, H-2a), 6.26 (1H, s, H-3b), 6.23 (1H, s, H-3a), 3.44(1H, br s, H-5), 5.71 (1H, t, J = 3.4 Hz, H-7), 2.13 (2H, m, H-8), 1.43(2H, m, H-9), 1.83 (2H, m, H-11), 3.82 (1H, m, H-12), 1.13 (3H, d, J = 6.1Hz, H-13), 9.64 (1H, s, H-14), 0.75 (3H, s, H-15); 13C NMR data, seeTable 1; positive ESIMS m/z 257.2 [M+Na]+; HRESIMS m/z 257.1519[M+Na]+ (calcd. for C15H22O2Na, 257.1512). 2:colorless oil; [α]25D

TABLE 1. The experimental and calculated chemical shifts at the muum model with chloro

1 13C NMR B1(cal.) 13C NMR Δδ

1 145.7 147. 4 1.72 112.0 110.9 �1.13 137.3 138.6 1.34 150.6 151.0 0.45 42.0 42.6 0.66 134.6 136.4 1.87 126.8 125.8 �1.08 22.7 24.8 2.19 27.6 28.5 0.910 38.2 42.1 3.911 46.3 44.4 �1.912 64.3 66.5 2.213 22.6 22.4 �0.214 194.1 190.6 �3.515 26.2 19.8 �6.4

The calculated chemical shifts were obtained after corrections using the slope andarticle (δ = (187.0716- σ)/1.0623; δ is the corrective computational chemical shifts (ppthe relative errors between experimental observation and theoretical prediction).

Chirality DOI 10.1002/chir

�183.8 (c 0.08, CHCl3); UV (MeOH) λmax (log ε) 218 (3.67) nm; CD(c 7.5 × 10�4 M, CH3CN), λmax (Δε) 256 (+0.28), 335 (�0.15); IR (KBr)νmax 3416, 2963, 2928, 1697, 1457, and 1384 cm�1; 1H NMR (CDCl3, 400MHz) δ 5.85 (1H, dd, J =17.5, 10.9 Hz, H-1), 5.03 (1H, d, J = 17.5 Hz, H-2b), 4.96 (1H, d, J = 10.9 Hz, H-2a), 6.21 (1H, s, H-3b), 6.19 (1H, s, H-3a), 3.35 (1H, br s, H-5), 5.64 (1H, t, J = 3.6 Hz, H-7), 2.07 (2H, m, H-8),1.42 (2H, m, H-9), 1.91 (2H, m, H-11), 3.78 (1H, m, H-12), 1.09 (3H, d,J = 6.2 Hz, H-13), 9.61 (1H, s, H-14), 0.71 (3H, s, H-15); 13C NMR data,see Table 1; positive ESIMS m/z 257.2 [M+Na]+; HRESIMS m/z257.1515 [M+Na]+ (calcd. for C15H22O2Na, 257.1512).

Preparation of (R)- and (S)-MTPA Esters of 1 and 2Sesquiterpenoid (4 mg) was separated into two equal portions and

dried completely under high vacuum. Freshly distilled anhydrous pyri-dine (250 μL) was added to each portion. The reaction mixtures werestirred at room temperature for 10 min. Either (R)- or (S)-MTPA-Cl (12μL) was added to each vial. Completion of the reaction was monitoredby thin-layer chromatography (TLC). The reaction mixture was evapo-rated in vacuo to provide the reaction crude, which was purified by C18

RP-HPLC using a gradient of MeOH/H2O to afford (S)-MTPA ester and(R)-MTPA ester (80:20→ 100:0, 20 min, 3.0 mL/min, tR of derivatives wereca. 19 min), respectively.S-MTPA-ester of 1 (1a). colorless oil; 1H NMR (CDCl3, 400MHz) δ 5.81(1H, dd, J =17.6, 10.8 Hz, H-1), 5.00 (1H, dd, J = 17.6, 1.0 Hz, H-2b), 4.94(1H, dd, J = 10.8, 1.0 Hz, H-2a), 6.07 (1H, s, H-3b), 5.90 (1H, s, H-3a),3.27 (1H, br s, H-5), 5.51 (1H, t, J = 3.3 Hz, H-7), 1.95 (2H, m, H-8), 1.29(2H, m, H-9), 2.12 (1H, ddd, J = 15.5, 9.4, 0.9, H-11b), 1.82 (1H, dd,J = 15.5, 1.2, H-11a), 5.28 (1H, m, H-12), 1.29 (3H, d, J = 6.2 Hz, H-13),9.59 (1H, s, H-14), 0.70 (3H, s, H-15); 13C NMR (CDCl3, 100 MHz) δ145.2 (CH, C-1), 111.7 (CH2, C-2), 137.2 (CH2, C-3), 150.2 (C, C-4), 43.1(CH, C-5), 132.1 (C, C-6), 125.0 (CH, C-7), 22.5 (CH2, C-8), 27.9 (CH2,C-9), 38.0 (C, C-10), 41.6 (CH2, C-11), 71.3 (CH, C-12), 20.3 (CH3, C-13),193.8 (CH, C-14), 25.5 (CH3, C-15); ESIMS m/z 473.1 [M+Na]+, 468.1[M+NH4]

+.R-MTPA-ester of 1 (1b). colorless oil; 1H NMR (CDCl3, 400MHz) δ 5.81(1H, dd, J =17.6, 10.8 Hz, H-1), 5.00 (1H, dd, J = 17.6, 1.0 Hz, H-2b), 4.94(1H, dd, J = 10.8, 1.0 Hz, H-2a), 6.07 (1H, s, H-3b), 5.90 (1H, s, H-3a),3.27 (1H, br s, H-5), 5.51 (1H, t, J = 3.3 Hz, H-7), 1.95 (2H, m, H-8), 1.29(2H, m, H-9), 2.12 (1H, ddd, J = 15.5, 9.4, 0.9, H-11b), 1.82 (1H, dd,J = 15.5, 1.2, H-11a), 5.28 (1H, m, H-12), 1.29 (3H, d, J = 6.2 Hz, H-13),9.59 (1H, s, H-14), 0.70 (3H, s, H-15); 13C NMR (CDCl3, 100 MHz) δ145.0 (CH, C-1), 111.8 (CH2, C-2), 137.2 (CH2, C-3), 150.3 (C, C-4), 43.0(CH, C-5), 132.7 (C, C-6), 125.9 (CH, C-7), 22.6 (CH2, C-8), 27.8 (CH2,

PW1PW91-SCRF/6-311+G(2d,p) level in PCM solvent contin-form as a solvent

2 13C NMR B9(cal.) 13CNMR Δδ

145.6 147. 6 2.0111.8 110.8 �1.0137.0 138.7 1.7150.9 151.4 0.543.5 43.2 �0.3

134.7 136.4 1.7125.6 125.2 �0.422.9 24.7 1.828.0 28.7 0.738.3 42.3 4.046.6 44.6 �2.066.2 66.0 �0.222.8 24.4 1.6

194.2 190.7 �3.525.6 19. 6 �6.0

intercept obtained from linear regression analysis as suggested in the reviewm), is the computational isotropic shielding constants for each C atom, Δδ is

Fig. 2. The optimized structures of B1 and B9 at the B3LYP/6-31+G(d,p) levelof theory in the gas phase, as well as important NOE correlations are labeled.

Fig. 3. Comparison of experimental (in acetonitrile) and calculated ECDspectra of 1 (B1) and 2 (B9). The calculated ECD spectra were obtained fromTDDFT calculation at the PBE0-SCRF/6-311++g(2d,2p) theoretical level in thePCM solvent continuum model with acetonitrile as a solvent based on the op-timized structures in the gas phase. UV-shift = 52 nm, σ = 0.9 eV for B1 andσ = 0.8 eV for B9 were assigned to match the experimental spectra band.

Fig. 4. The HOMO-LUMO plot and energy gap of 1 (B1) and 2 (B9).

TWO NEW ARISTOPHYLLENE SESQUITERPENES 191

C-9), 38.1 (C, C-10), 41.8 (CH2, C-11), 71.8 (CH, C-12), 20.13 (CH3, C-13),193.9 (CH, C-14), 25.9 (CH3, C-15); ESIMS m/z 473.1 [M+Na]+, 468.1[M+NH4]

+.S-MTPA-ester of 2 (2a). colorless oil; 1H NMR (CDCl3, 400MHz) δ 5.80(1H, dd, J =17.6, 10.8 Hz, H-1), 5.00 (1H, dd, J = 17.6, 1.0 Hz, H-2b), 4.93(1H, dd, J = 10.8, 1.0 Hz, H-2a), 6.20 (1H, s, H-3b), 6.16 (1H, s, H-3a),3.31 (1H, br s, H-5), 5.57 (1H, t, J = 3.4Hz, H-7), 2.02 (2H, m, H-8), 1.36(2H, m, H-9), 2.15 (1H, dd, J = 14.6, 4.8, H-11b), 1.94 (1H, dd, J = 14.6,7.5, H-11a), 5.15 (1H, m, H-12), 1.27 (3H, d, J = 6.2 Hz, H-13), 9.64 (1H,s, H-14), 0.73 (3H, s, H-15); 13C NMR (CDCl3, 100 MHz) δ 145.4 (CH,C-1), 111.9 (CH2, C-2), 136.9 (CH2, C-3), 150.1 (C, C-4), 43.3 (CH, C-5),132.3 (C, C-6), 126.1 (CH, C-7), 22.7 (CH2, C-8), 28.4 (CH2, C-9), 38.0(C, C-10), 41.9 (CH2, C-11), 71.9 (CH, C-12), 19.2 (CH3, C-13), 193.8(CH, C-14), 25.1 (CH3, C-15); ESIMS m/z 468.2 [M+NH4]

+, 923.3[2M+Na]+.R-MTPA-ester of 2 (2b). colorless oil; 1H NMR (CDCl3, 400MHz) δ 5.84(1H, dd, J =17.5, 10.9Hz, H-1), 5.03 (1H, d, J = 17.5Hz, H-2b), 4.97 (1H, d,J = 10.8 Hz, H-2a), 6.20 (1H, s, H-3b), 6.18 (1H, s, H-3a), 3.34 (1H, br s, H-5), 5.64 (1H, t, J = 3.4Hz, H-7), 2.08 (2H, m, H-8), 1.41 (2H, m, H-9), 2.22(1H, dd, J = 14.7, 5.7, H-11b), 2.01 (1H, dd, J = 14.7, 7.2, H-11a), 5.18 (1H,m, H-12), 1.20 (3H, d, J = 6.2 Hz, H-13), 9.64 (1H, s, H-14), 0.74 (3H, s,H-15); 13C NMR (CDCl3, 100 MHz) δ 145.1 (CH, C-1), 112.0 (CH2, C-2),136.9 (CH2, C-3), 150.1 (C, C-4), 43.2 (CH, C-5), 132.6 (C, C-6), 125.8(CH, C-7), 22.8 (CH2, C-8), 28.1 (CH2, C-9), 38.2 (C, C-10), 42.0 (CH2, C-11), 72.0 (CH, C-12), 19.0 (CH3, C-13), 193.9 (CH, C-14), 25.3 (CH3, C-15); ESIMS m/z 473.1 [M+Na]+, 468.2 [M+NH4]

+, 923.1 [2M+Na]+.

COMPUTATIONAL SECTIONAll the theoretical calculations including geometry optimiza-

tion and frequency analysis, as well as 13C NMR and ECDspectrum prediction, were carried out with DFT and TDDFTmethods in Gaussian 09.14,15 The geometry optimizations wereperformed at the B3LYP/6-31+G (d, p) level in the gas phase.Based on the final optimized structure, all the NMR data andECD spectra were calculated at the mpw1pw91-SCRF/6-311+G (2d, p) level and PBE0-SCRF/6-311++g (2d, 2p) level withthe Polarizable Continuum Model (PCM) solvent continuummodels, respectively. All the chosen theoretical methods andbasis set in optimization and spectra calculation wererecommended in previous studies.16–20 The computational13C NMR were finally obtained by the linear regression analy-sis method.21 The theoretical predicted ECD spectra werefitted in the SpecDis software package.22,23

RESULTS AND DISCUSSIONCompound 1, a colorless oil, had the molecular formula

C15H22O2, as established by HRESIMS at m/z 257.1519[M+Na]+ (calcd. 257.1512). The NMR spectra of 1 werealmost identical to those of mandolin R (3) (maximum ΔδC 0.2ppm), a known compound isolated from Aristolochia mollissimawith the ACs undetermined. The 2D NMR experiment furtherconfirmed that 1 and 3 shared the same planar structure. How-ever, the optical rotation of 1 was far different from that of 3 (1:[α]D +235; 3: [α]D�6311), indicating they might be enantiomersand the latter one was partially racemic. The NMR spectra of 2bore a resemblance to those of 1 with the maximum ΔδC occur-ring at C-12 and C-5 (ΔδC=1.9 and 1.5 ppm, respectively). The2D NMR experiment confirmed that 1 and 2 shared the sameplanar structure. The obvious differences of 1 and 2 on theTLC behavior and optical rotation implied that they were diaste-reoisomers. As the C-12 of 1 and 2 were hydroxylated methine,the AC of C-12 was determined by the modified Mosher’smethod, which gave R configuration in both compounds. Tosolve the relative configuration of C-5 and C-12, the NOESYexperiments of 1 and 2 were performed, which gave two similar

NOESY spectra. However, the key NOESY correlations of H-3/H-9, H-5/CH3-15, and H-5/H-12 from both spectra could only as-sign the pseudoaxial orientation of the acrylaldehyde groups onthe cyclohexene ring but failed to determine the equatorial or ax-ial orientation of Me-15, as H-5 may have correlations with either

Chirality DOI 10.1002/chir

Fig. 5. Regression analysis of experimental vs. calculated 13C NMR chemical shifts (ppm).

WU ET AL.192

cis or trans Me-15. Thus, the relative configuration of thecyclohexene ring still remained ambiguous.As CD spectrum is very sensitive to the AC and conforma-

tion of molecules with chromophores adjoining the chiral cen-ter, the predicted CD spectra of all possible configurations for1 and 2 were generated using the DFT or TDDFT methodsto compare with the experimental data. Taking into consider-ation all R/S chiral centers and rotatable single bond in thearistophyllene sesquiterpenoids, there are hundreds ofconformations that could exist theoretically. Fortunately,many conformers, especially those arising from the flexibilityof C11-C12-C13 side-chain, could be excluded by the experi-mental NOESY correlations (H-3/H-9, H-5/CH3-15, and H-5/H-12) and the modified Mosher’s results (R configurationfor C12). As a result, all the possible structures (32 in total)were considered with configuration isomers (α or β for C5and C10 side-chain, so 22 = 4) and conformation isomers(due to the rotatable single bond C1-C10/C4-C5/C4-C14, so23 = 8). And all of the computational models are shown inS15 and S16 (see Supplementary Material).All the computational predicted ECD spectra of the 32 con-

formers were compared with the two experimental ECD spec-tra. However, only B1 and B9 (Fig. 2) could reproduce all ofthe experimental Cotton effects of 1 and 2, respectively. Asshown in Figure 3, the experimental and simulated spectrashared a similar tendency on Cotton effects and close λmax ateach peak, while the differences were attributed to the absorp-tion intensity. Moreover, the theoretically predicted HOMO-LUMO gap for B1 and B9 is 4.97 and 4.90 ev, correspondingto the absorption bands at ~253 and ~257 nm, respectively(Fig. 4). These predicted absorption bands were very close tothe negative peak at ~253 nm for 1 and positive peak at ~256nm for 2,. Therefore, the second Cotton effect most probablyoriginated from the π→ (HOMO)→ π* (LUMO) excitation whilethe first Cotton effect (peak around 334 ~ 338 nm) was presumablyattributed to n→ π* transitions. Furthermore, as shown in Figure 5and Table 1, the linear correlation of the experimental and computa-tional 13C NMR data was very satisfactory. Therefore, we couldconclude that both the theoretically predicted 13C NMR data andECD spectra were good enough to assign the ACs of the two newaristophyllene sesquiterpenoids.

CONCLUSIONSTwo new diastereoisomeric sesquiterpenes, aristoyunnolins,

G (1) and H (2), featuring a rare aristophyllene skeleton, were

Chirality DOI 10.1002/chir

isolated from the traditional Chinese medicine A. yunnanensis.Their structures were established on the basis of extensive spec-troscopic analysis. The ACs of 1 (5R, 10R, 12R) and 2 (5S, 10S,12R) were determined by comparison of their calculated CDspectra and 13C NMR data with the experimental results. Boththe theoretically predicted 13C NMR data and ECD spectra weregood enough to assign the ACs of the two new aristophyllenesesquiterpenoids in comparison to the experimental data.

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Chirality DOI 10.1002/chir