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IR and Vibrational Circular Dichroism Spectroscopy of Matrine- and Artemisinin-Type Herbal Products: Stereochemical Characterization and Solvent Effects Yuefei Zhang,† M. Reza Poopari, Xiaoli Cai, Aliaksandr Savin, Zahra Dezhahang, Joseph Cheramy, and Yunjie Xu* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada, T6G 2G2 S Supporting Information *

ABSTRACT: Five Chinese herbal medicinesmatrine, oxymatrine, sophoridine, artemisinin, and dihydroartemisininwere investigated using vibrational circular dichroism (VCD) experiments and density functional theory calculations to extract their stereochemical information. The three matrine-type alkaloids are available from the dry roots of Sophora f lavescens and have long been used in various traditional Chinese herbal medicines to combat diseases such as cancer and cardiac arrhythmia. Artemisinin and the related dihydroartemisinin, discovered in 1979 by Professor Youyou Tu, a 2015 Nobel laureate in medicine, are effective drugs for the treatment of malaria. The VCD measurements were carried out in CDCl3 and DMSO-d6, two solvents with different dielectric constants and hydrogen-bonding characteristics. A “clusters-in-a-liquid” approach was used to model both explicit and implicit solvent effects. The studies show that effectively accounting for solvent effects is critical to using IR and VCD spectroscopy to provide unique spectroscopic features to differentiate the potential stereoisomers of these Chinese herbal medicines.

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dichroism (ECD) spectroscopy.8 Vibrational circular dichroism (VCD), the extension of ECD into the infrared region, has become an increasingly powerful method for unambiguous absolute configuration assignment.9 VCD measures the absorption intensity difference in the IR bands of a chiral molecule when left versus right circularly polarized light is used. Therefore, a VCD spectrum contains many well-resolved IR bands with unique signs and is highly sensitive not only to chirality but also to the conformational distribution of the molecule. In addition, density functional theory (DFT) simulations of VCD have advanced over the last 10 years or more and have become highly reliable because the calculations are based on the electronic ground state.10 By comparing the calculated and experimental VCD features, one can confidently determine both the conformation and absolute configuration of the molecular system.11 Unlike X-ray diffraction analysis, VCD does not require the presence of a heavy atom in the molecule or a single crystal. VCD spectra can be recorded for samples as neat liquids, as films, or in solution, and no derivatization of the molecule is needed prior to analysis. In 2015, Batista Jr. and coworkers reviewed recent advances in the use of vibrational optical activity spectroscopic methods for the stereochemical characterization of natural products and noted the potential for

any natural products have significant pharmacological and biological properties such as antitumor, antibacterial, and insecticidal activities.1,2 These products are a rich and important source of inspiration for new drug discovery. The significant current interest in this area is highlighted by a 2015 review article titled “The re-emergence of natural products for drug discovery in the genomics era”.3 Furthermore, the successful hit rate tends to be better with known medicinal plants, such as traditional Chinese and Indian herbal medicines, where an extensive traditional knowledge of their medicinal properties has been accumulated through a long history of usage.4 For example, artemisinin isolated from Artemisia annua has led to at least 10 new drugs currently on the market.4 Challenges in drug discovery research based on herbal medicines, such as quality assurance, the identification of active components, and an understanding of the molecular mechanisms of multicomponent herbal medicines, have been addressed by recent technical advances in analytical separation techniques, spectroscopy, and biotechnology. One important issue yet to be fully addressed is stereochemical characterizations. Molecular chirality, i.e., the spatial arrangement of atoms in a given molecule, is paramount in natural product research because the assignment of absolute configurations is essential to establish reliable structure−activity relationships. The main traditional methods for establishing absolute configuration include X-ray crystallography,5 stereocontrolled synthesis of the targeted molecule, Mosher’s ester NMR method,6 optical rotation (OR),7 and electronic circular © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 7, 2015

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DOI: 10.1021/acs.jnatprod.5b01082 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Five Chinese herbal compounds: matrine, sophoridine, oxymatrine, artemisinin, and dihydroartemisinin. The matrine framework is also included.

Figure 2. Experimental IR and VCD spectra of the five compounds studied in DMSO-d6. VCD spectra shown as dotted lines were measured using a 50 μm path length, whereas the others were measured with a 200 μm path length. See Experimental Section for details.

this technique to become more routinely used in the field of natural products.12 In the current study, the IR and VCD spectra of two important types of Chinese herbal medicinesmatrine-type alkaloids and artemisinin-type compoundsare analyzed, complemented by DFT-calculated spectra. The five Chinese herbal products studiedmatrine, oxymatrine, sophoridine, artemisinin, and dihydroartemisininare shown in Figure 1, along with the matrine skeleton. The first three matrine-type alkaloids can be extracted from the dry root of Sophora f lavescens, which is referred to as Kushen in Chinese and has long been used in a variety of traditional Chinese herbal medicines to combat diseases such as cancer and cardiac arrhythmia.13 In recent years, considerable research efforts have been directed toward the evaluation of the antitumor activities of matrine-type alkaloids in combination with other anticancer

drugs.14 The crystal structures of these three compounds have been reported by several groups.15−18 The discovery of artemisinin, also known as Qinghaosu in Chinese, and the related dihydroartemisinin by Prof. Youyou Tu and her colleagues19 revolutionized the treatment of malaria and garnered Prof. Tu the 2015 Nobel Prize in Physiology or Medicine. Tu’s team showed that artemisinin is a sesquiterpenoid lactone with an unusual peroxide bridge and that the endoperoxide functionality is responsible for its antimalarial activity.19 X-ray crystallography of artemisinin was redone in 1999 to determine its absolute configuration20 because the original structural analyses did not provide this information.19 The X-ray crystallographic study of dihydroartemisinin was reported in 1984,21 and its antimalarial activity has been inextricably linked to its unusual endoperoxide trioxane moiety. B



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in DMSO-d6 and in CDCl3, as well as some subtle variation in the related IR spectra, are noted. These points will be addressed later. Artemisinin has a sesquiterpenoid lactone structure, whereas dihydroartemisinin has essentially the same structure, except that the lactone group is replaced by a hemiacetal moiety. Although they share similar IR features in the 1500−1320 cm−1 range, artemisinin exhibits a strong CO stretching band at 1730 cm−1 that is absent in the spectrum of dihydroartemisinin and also some medium-to-strong bands that are much lower in intensity in the dihydroartemisinin spectrum. The chirality of these two compounds differs at C-10, where an additional stereogenic center is present in dihydroartemisinin. Therefore, as expected, their VCD features are similar in the 1500−1200 cm−1 region, although some small differences are evident. In addition, because of the extra OH group in dihydroartemisinin, the hydrogen-bonding interactions with a solvent such as DMSO should differ compared to those of artemisinin. It would be interesting to determine if molecular modeling can capture these differences well. Structural Properties of the Five Chinese Herbal Compounds. From a structural viewpoint, the matrine skeleton contains a quinolizidinic A/B ring system fused with a quinolizidinone C/D ring system. It has four stereogenic carbon centers at C-5, C-6, C-7, and C-11 and two nitrogen stereogenic centers at N-1 and N-16 (Figure 1), following the same numbering convention used in the literature.16 This arrangement theoretically results in 64 stereoisomers or 32 enantiomeric pairs for the matrine-type alkaloids, as originally proposed by Ibragimov and co-workers.28 Thus far, nine stereoisomers in the matrine family have been reported (assignments of the C stereogenic centers are given in parentheses): trans-matrine (SSSR),15,16 cis-matrine (RRRS),29 allomatrine (SRSR),30 sophoriridine (SRRS),18 isomatrine (RRRR),31 isosophoridine (SSRS),32 tetrahydroneosophoramine (SRRR),33 trans-neomatrine (SSRR), and cis-neomatrine (SSRR).34 Some confusion exists about the chirality labeling of the matrine compounds, especially at C-7. This issue was previously addressed by Ibragimov and co-workers.28 However, in a number of subsequent publications, the matrine configuration was cited or assigned as SSRR, such as in references 35 and 36. A closer examination of the reported crystal structures led to the conclusion that the reported transneomatrine and cis-neomatrine in the 2003 X-ray crystallographic study34 have the same absolute configuration at the four stereogenic carbon atoms as the reported trans-matrine.15,16,36 Moreover, the reported trans-neomatrine has the same A/B trans and C/D trans arrangement and the same configurational assignment S(SSSR)R at N-1, C-5, C-6, C-7, C-11, and N-16, respectively, which is the same configuration as the transmatrine reported in 2009.16 Note that the four middle chirality labels correspond to those of the stereogenic carbon centers and are enclosed in parentheses. Therefore, the reported transneomatrine34 is actually trans-matrine. The reported cisneomatrine, with A/B trans and C/D cis arrangement, has a chirality assignment of S(SSSR)S and should be called cismatrine. The previous cis-matrine reported by a Russian group29 has the chirality assignment of R(RRRS)R and is the mirror-image of the newly assigned cis-matrine. Therefore, among the 64 possible stereoisomers, eight of them have been identified thus far. Notably, in the above stereoisomer discussion, further conformational flexibility of the sixmembered rings has not been taken into account explicitly.

One goal is to evaluate how well IR and VCD spectroscopy, together with DFT calculations, can provide unique spectroscopic features to differentiate the potential stereoisomers. In contrast to NMR spectroscopy, VCD is an optical spectroscopic technique where the contribution from multiple conformers of a flexible molecule can be directly taken into account by their respective Boltzmann factors. Although the matrine framework (Figure 1), which has four stereogenic carbon centers, appears relatively rigid, chirality is also associated with the two bridgehead nitrogen atoms. Additional conformational flexibility associated with the deformation of the six-membered rings may also be expected in these molecules. In the current work, the central aim is to clarify these structural issues and compare the outcome with those obtained in the previous solid-state studies. Furthermore, a focus is placed on the solvent effectsboth implicit bulk solvent effects and explicit hydrogen-bonding interactions between a chiral solute and solvent moleculeson chiroptical spectroscopic features 9c,22,23 and, therefore, on the interpretation of the experimental data. In particular, the “clusters-in-a-liquid” approach24 has been applied to account for solvent effects. In this approach, the key hydrogen-bonded species in solution are first identified and then placed in the solvent modeled by the polarizable continuum model (PCM).25 Solvent effects have not been extensively discussed in the VCD literature of natural products, including the literature of herbal medicines.26 Two solvents, CDCl3 and DMSO-d6, which have very different dielectric constants of 4.7113 and 46.826, respectively, were used to compare the solvent effects. Both solvents are capable of hydrogen bonding to chiral solutes when suitable hydrogenbond donors or acceptors are available. Such solvent effects have been demonstrated to alter the preferred helicity and other conformational characteristics of chiral molecules when going from the solid to solution state.27

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RESULTS AND DISCUSSION Experimental IR and VCD Spectra of the Five Chinese Herbal Compounds. The experimental IR and VCD spectra of the three matrine-type compounds in the 1800−1100 cm−1 region in DMSO-d6 are compared in Figure 2, along with the corresponding spectra of the two artemisinin-type compounds. Chemically, matrine and sophoridine are a pair of diastereomers, and oxymatrine is the oxide of matrine. The strong similarity between their IR spectra is expected; however, their VCD spectra differ noticeably. In the carbonyl stretching region between 1650 and 1590 cm−1, the sophoridine VCD spectrum shows a negative peak, whereas the corresponding VCD band in matrine is a medium positive peak. In the next-lower region from 1500 to 1400 cm−1, a group of bands that mainly correspond to CH2 scissoring show dominant negative features for sophoridine, whereas matrine exhibits both positive and negative features. In the next region, 1400−1230 cm−1, which corresponds mostly to CH2 wagging and twisting, similar VCD features are observed for both compounds, except for an obvious bisignate feature with a negative peak at ∼1266 cm−1 and a positive peak at ∼1252 cm−1 in the matrine spectrum, which is absent in the spectrum of sophoridine. In the lowestwavenumber region of 1230−1100 cm−1, corresponding to the C−C stretching of the rings and also to CH bending, differences are again observed. By contrast, oxymatrine has the same stereogenic carbon labels as matrine, resulting in quite similar VCD spectroscopic features, except in the region below 1300 cm−1. Some obvious differences between the VCD spectra C



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Table 1. Predicted Structural and Energy Properties of the Most Stable Conformers of Matrine, Oxymatrine, and Sophoridine at the B3LYP/cc-PVTZ Level in the Gas Phase configuration conformer

ring A

ring B

ring C

ring D

A/B

C/D

A/C

B/C

ΔEb

Bf %c

ΔGb

Bf %c

trans-matrine cis-matrine trans-oxymatrine cis-oxymatrine sophoridine I sophoridine II sophoridine III sophoridine IV

chair chair chair chair chair chair chair chair

chair chair chair chair t-boata t-boat chair chair

chair chair chair chair t-boat t-boat t-boat boat

sofa sofa half-chair half-chair half-chair half-chair half-chair half-chair

trans trans trans trans trans trans trans cis

trans cis trans cis trans cis cis trans

cisoid cisoid cis cis trans trans trans trans

cisoid cisoid cis cis cis cis cis cis

0 4.34 0 4.12 0 2.69 5.12 6.31

85.2 14.8 84.1 15.9 64.8 21.9 8.2 5.1

0 5.16 0 4.85 0 3.85 7.56 7.59

88.9 11.1 87.6 12.4 76.7 16.2 3.6 3.6

“t-boat” denotes “twisted-boat”. bΔE and ΔG are the relative energies and Gibbs free energies in kJ/mol, respectively. cPercentage Boltzmann factors based on ΔE or ΔG at room temperature. a

Figure 3. Calculated IR and VCD spectra of a subset of eight stereoisomers with the matrine frame at the B3LYP/cc-pVTZ level. The chirality associated with C-5, C-6, C-7, and C-11 is indicated, whereas the A/B and C/D ring junctions were fixed in the trans arrangement.

that N-16 is of R chirality and A/B trans means that N-1 is of S chirality. The Boltzmann factors based on energy and free energy are, in general, quite similar for all of these conformers, and the Boltzmann factors based on free energies will be used for the remainder of the paper. Interestingly, in the most stable optimized geometry for each of the matrine-type compounds, the four atoms C−C(O)−N are nearly perfectly planar, with a dihedral angle of ∼178.5° to 180.0°. As a result, the nitrogen lone pair lies in the nodal plane of the π-system, and double-bond character exists between N16 and C-15. This situation is similar to that observed in the crystal structures of trans-matrine, oxymatrine, and sophoridine. Furthermore, the most stable conformer obtained in each of the three matrine-type compounds has essentially the same structure as that identified in the solid state. Notably, a second conformer with the C/D cis ring junction was identified for both matrine and oxymatrine, representing slightly more than 10% of the total population. In addition, the cis-matrine identified in the calculation is also consistent with the cismatrine obtained from the crystal structure, whereas no corresponding cis conformer has been reported in the crystal state for oxymatrine. For sophoridine, the next-higher energy conformer has a population of approximately 17%, although no corresponding structure has been identified in the crystal state either.

The conformational searches for matrine, sophoridine, and oxymatrine were performed using the Spartan software.37 Eight and nine low-energy conformers with some population at room temperature were identified for matrine and oxymatrine, respectively, in the initial searches. These structures were then reoptimized at the B3LYP/cc-pVTZ level. Notably, despite the extensive conformational searches, only two relevant conformers were identified for matrine and for oxymatrine at the B3LYP/cc-pVTZ level. All of the other conformers of matrine and oxymatrine are significantly less stable and do not meaningfully contribute to the total population, with a total sum substantially less than 1% at room temperature. The situation is somewhat different for sophoridine. Nineteen conformers of sophoridine were initially identified, and four relevant conformers at room temperature were obtained at the B3LYP/cc-PVTZ level. The calculated relative zero-point energy corrected energies, the calculated relative free energies at room temperature, and the associated Boltzmann factors at room temperature based on the free energies for all of the aforementioned conformers are listed in Table 1, together with their structural characteristics, i.e., ring conformations and ring−ring junction arrangements. The expression “cisoid” (“transoid”) in reference 38 has been adopted here to denote a junction dihedral angle larger (smaller) than 15° (165°). Please note that C/D trans means D



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Figure 4. Comparison of the experimental IR and VCD spectra in DMSO-d6 with the calculated Boltzmann-weighted IR and VCD spectra of matrine in the gas phase and in DMSO.

VCD spectra of the relevant conformers in the gas phase and in DMSO-d6 are depicted for matrine, oxymatrine, and sophoridine. The related relative energies and free energies of the associated conformers in DMSO-d6 are listed in Table S1, Supporting Information, as well as the corresponding Boltzmann factors. The Boltzmann factors for the two main conformers are very much the same in the gas phase as in DMSO. The inclusion of the bulk solvent shifts the CO stretching frequency substantially to the longer wavenumber region by approximately 60 to 70 cm−1, although no sign change is detected for the carbonyl VCD features in all cases. A much weaker frequency shift is observed for the lowerfrequency bands. Notably, the direction of the shift, i.e., red or blue shift, often differs for different vibrational bands when a solvent is included in the simulation. For example, this effect is clearly demonstrated in Figure S4, Supporting Information, when the IR spectra in the gas phase are compared with those in DMSO. Spectroscopic features in the 1550 to 1430 cm−1 region, mainly associated with the CH2 wagging motions, are greatly affected by the inclusion of bulk solvent. Not only do the relative IR intensities of the peaks vary in this region, but the associated VCD patterns are also noticeably modified. The IR and VCD patterns in the further-lower wavenumber region, by contrast, exhibit little change with respect to solvent. Similar examinations were also conducted for artemisinin and dihydroartemisinin, where bulk solvents exert less influence on both IR and VCD features. This observation is consistent with the facts that both compounds exhibit structural rigidity and that each has only one dominant conformer. The experimental and calculated Boltzmann-weighted IR and VCD spectra of matrine in the gas phase and in DMSO-d6 are compared in Figure 4. Although the strong CO stretching IR band and the weak positive VCD band are captured by the gasphase calculation, the PCM spectra give considerably better agreement in terms of the absolute and relative IR band positions and relative intensities as well as in terms of the signs and shapes of the VCD bands for the whole region. From Figure S2, Supporting Information, it is clear that the Boltzmann-weighted IR and VCD spectra are essentially the same as those obtained with just the most stable trans-matrine conformer, except for a slight increase in the carbonyl-stretch VCD intensity. For this reason, confirming the contribution of cis-matrine is difficult. However, the relatively stronger experimental VCD intensity of the carbonyl CO stretching compared to the predicted intensity suggests the existence of cis-matrine because cis-matrine features a substantially stronger

Similar conformational searches were carried out for artemisinin and dihydroartemisinin, where five and seven conformers with different ring conformations were identified, respectively. These conformers were further reoptimized at the B3LYP/cc-pVTZ level, and only one relevant conformer at room temperature was obtained for each, whereas the other conformers together represented less than 0.1% of the total population. An additional conformational search was also performed for dihydroartemisinin with regard to the relative orientation of its OH group. The potential energy scan carried out at the B3LYP/6-31+G(d,p) level is shown in Figure S1, Supporting Information. Two shallow minima, together with a deep global minimum, were tentatively identified. Because these possible shallow minima are much higher in energy than the predominant conformers and have no relevant population at room temperature, no further calculations were carried out for them. The identified dominant conformer is similar to the crystal structure observed for artemisinin, and the same is true for dihydroartemisinin. Comparison of the Experimental and Calculated IR and VCD Spectra of the Five Chinese Herbal Compounds in DMSO-d6. As indicated in the introduction, one goal is to evaluate whether IR and VCD data are sufficient to distinguish among stereoisomers, especially because VCD spectroscopic features contain unique chirality signatures. To this end, a discussion of the simulated IR and VCD spectra of a subset of eight fully optimized stereoisomers with the A/B trans and C/D trans arrangement and with different chirality labels associated with the four stereogenic carbon centers is essential. Some of the eight stereoisomers were previously investigated by DFT and NMR spectroscopy.38 The corresponding IR and VCD results are summarized in Figure 3. As is evident in this figure, the eight isomers exhibit distinct IR and VCD spectra. Although distinguishing among them using the experimental IR spectra alone may be difficult because of other potential complications arising from the solvent (vide infra), their very different VCD features allow them to be clearly differentiated. The solvent effects are addressed here. The measurements in DMSO-d6 can be first used to illustrate the bulk solvent effects on the IR and VCD spectra. DMSO is a polar aprotic solvent with no acidic hydrogen atoms but can act as a hydrogen-bond acceptor. Because matrine has no suitable protons to donate, it is not expected to form strong solute− solvent hydrogen bonds with DMSO. The same is true of oxymatrine and sophoridine in DMSO. In Figures S2−S4, Supporting Information, the simulated single-conformer IR and E



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Figure 5. Comparison of the experimental IR and VCD spectra in DMSO-d6 with the calculated Boltzmann-weighted IR and VCD spectra of oxymatrine in the gas phase and in DMSO.

Figure 6. Comparison of the experimental IR and VCD spectra in DMSO-d6 with the calculated IR and VCD spectra of the most stable conformer of sophoridine in the gas phase and in DMSO. Some features are labeled with numbers for easier comparison.

Figure 7. Optimized geometries of artemisinin, dihydroartemisinin, and the dihydroartemisinin-DMSO-d6 complex in DMSO-d6 calculated at the 631+G(d,p) level using the PCM of DMSO.

positive carbonyl VCD intensity than trans-matrine (Figure S1, Supporting Information). The simulated IR and VCD spectra of oxymatrine in the gas phase and the PCM of DMSO-d6 are compared with the experimental data in Figure 5. The situation is quite similar to the case of matrine. As expected, both the gas phase and the PCM give reasonably good agreement with the experimental results, although the latter improves the agreement with the experimental data. For example, the relative intensities of the IR bands in the 1550−1450 cm−1 region are better captured with the inclusion of solvent, as are the related VCD bands in the same region. Again, the Boltzmann-weighted IR and VCD spectra are essentially the same as those of trans-oxymatrine.

Although the aforementioned comparison confirms that transoxymatrine is by far the dominant conformer in DMSO, determining whether cis-oxymatrine contributes to the observed spectra is difficult. In the case of sophoridine, the situation differs somewhat from the aforementioned cases of matrine and oxymatrine. Sophoridine II exhibits a strong positive carbonyl VCD feature, in contrast to sophoridine I, which has a weak negative carbonyl VCD band (Figure S4, Supporting Information). Furthermore, the sophoridine II conformer was predicted to represent approximately 19% of the total population, in comparison to ∼72% for the sophoridine I conformer. As a result, the Boltzmann-weighted VCD spectrum includes a small positive F



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Figure 8. Comparison of the experimental and calculated IR and VCD spectra of artemisinin and dihydroartemisinin in DMSO-d6. The calculations were done at the 6-31+G(d,p) level in the PCM of DMSO-d6.

with the hydroxy bending motion. It can be hypothesized that hydrogen bonding between the OH group and DMSO may cause this band to shift in frequency or to diminish in intensity. This hypothesis will be discussed further in the next section. Consideration of Both Explicit and Implicit Solvent Effects in CDCl3 and DMSO-d6. CDCl3 is a nearly nonpolar solvent, and with three electron-withdrawing chlorine atoms, its hydrogen atom is more acidic than those of methane. A solute− solvent hydrogen-bonding interaction is expected when the hydrogen atom of a CDCl3 molecule is placed in close proximity to the oxygen atom(s) of matrine, oxymatrine, and sophoridine. This situation is in contrast to the case of DMSOd6, where no such explicit hydrogen-bonding interactions are anticipated in the case of the three matrine-type compounds. A comparison of the experimental IR and VCD spectra of matrine, oxymatrine, and sophoridine in CDCl3 and in DMSOd6 is shown in Figure S6, Supporting Information. Despite an overall similarity between the IR and VCD spectra recorded in the two solvents, some differences are evident. For example, in the highest wavenumber region, i.e., the carbonyl stretching region, matrine in DMSO-d6 shows one IR band, whereas the corresponding band in CDCl3 is broader and red-shifted by ∼12 cm−1 from that in DMSO-d6. Similar red shifts of ∼9 and ∼10 cm−1 are observed for sophoridine and oxymatrine when comparing their DMSO-d6 and CDCl3 spectra. In the 1500− 1250 cm−1 section, some relatively small intensity differences are observed between the DMSO-d6 and CDCl3 spectra. Are these differences mainly due to the very different dielectric constants of DMSO-d6 and CDCl3? The simulated singleconformer IR and VCD spectra of these three matrine-type compounds in CDCl3 using the PCM are also summarized in Figures S2−S4, Supporting Information, and their relative energies and Boltzmann factors are given in Table S1,

carbonyl VCD band, which is in disagreement with the negative carbonyl VCD band observed experimentally. This disagreement may reflect a deficiency in the DFT energy calculation. Because the Boltzmann-weighted spectra do not improve the agreement with the experimental data in other regions either, focus is placed only on sophoridine I for the remainder of the paper. The simulated IR and VCD spectra of the most stable conformer of sophoridine in the gas phase and with the PCM of DMSO-d6 are compared with the experimental spectra in Figure 6. Overall, both the IR and VCD spectroscopic features of sophoridine I agree well with the experimental data. One noticeable difference is the band labeled 6, where the negative VCD intensity from theory is exaggerated. In the case of artemisinin and dihydroartemisinin, each compound has only one dominant conformer in DMSO-d6 at room temperature, with more than 99.9% of the total population. Their respective geometries are given in Figure 7. The calculated IR and VCD spectra with the inclusion of the DMSO bulk solvent effect are compared with the experimental data in Figure 8. As is evident in this figure, the agreement between the experimental and calculated data is excellent for artemisinin. Although the main IR and VCD features are reproduced for dihydroartemisinin, some noticeable disagreements are evident. For example, the predicted IR band at approximately 1450 cm−1 (marked with an arrow in Figure 8) is not observed in the experimental spectrum. In addition, the corresponding predicted medium-strength negative VCD band is also not observed in the experimental spectrum. To ensure that these differences are not due to basis-set issues, calculations were also carried out for both artemisinin and dihydroartemisinin at the B3LYP/cc-pVTZ level, where similar results were obtained (Figure S5, Supporting Information). A closer examination reveals that this particular band is associated G



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Figure 9. Comparison of the experimental IR and VCD spectra of matrine in CDCl3 with the corresponding Boltzmann-weighted IR and VCD spectra of the 1:1 matrine−CDCl3 complex in CDCl3 using the PCM, of matrine in CDCl3 using the PCM, and of matrine in the gas phase.

Figure 10. Comparison of the experimental IR and VCD spectra in CDCl3 with the corresponding spectra of the dominant 1:2 oxymatrine− (CDCl3)2 complex (see text for details) in CDCl3. The Boltzmann-weighted IR and VCD spectra of five conformers of the 1:1 oxymatrine−CDCl3 complex in CDCl3 using the PCM; the Boltzmann-weighted IR and VCD spectra of the two relevant conformers of oxymatrine in CDCl3 calculated using the PCM and in the gas phase are also included.

corresponding to CH2 wagging and twisting and CH bending) are reproduced in the gas phase. With the inclusion of both implicit and explicit solvent effects, the relative IR intensities of the group of bands and the detailed VCD features in the 1520− 1450 cm−1 region are much better represented. Oxymatrine in CDCl3 demonstrates another interesting aspect of explicit solvation. The same 1:1 solute−solvent complex approach was first applied to account for the explicit solvent effects of oxymatrine in CDCl3. Because the structure of oxymatrine includes two hydrogen-bond acceptors, two types of explicit hydrogen bonds were considered: one to the carbonyl oxygen atom, as previously discussed, and the other to the oxygen atom of the N−O dative bond, which is partially negatively charged. The geometry search for the 1:1 hydrogenbonded complex resulted in five conformers of the oxymatrine−CDCl3 complex. Their single-conformer IR and VCD spectra are compared in Figure S8, Supporting Information, together with their respective Boltzmann factors. The hydrogen-bonding interaction at the N−O oxygen site is much stronger than that at the carbonyl oxygen site, resulting in just one dominant conformer (89.3%) of the 1:1 complex. Because the carbonyl group in this dominant conformer is not hydrogen-bonded to CDCl3, the red shift of the CO

Supporting Information. Not surprisingly, compared with DMSO-d6, the inclusion of the bulk CDCl3 solvent similarly affects the IR and VCD spectra, except that the induced vibrational frequency shifts are slightly smaller than with DMSO-d6. Therefore, in the following discussion, the focus will be placed on the different hydrogen-bonding capabilities of these two solvents and the combined explicit and implicit solvent approach. For matrine, the 1:1 matrine−CDCl3 complex in bulk CDCl3 can be used to model both the explicit and implicit solvent effects simultaneously. CDCl3 may form a hydrogen bond with the carbonyl oxygen atom of matrine by approaching either of the electron lone pairs, although CDCl3 approaching from the right side of the CO (see Figure 1) does not result in a stable conformer. Two conformers were identified; their singleconformer IR and VCD spectra are compared in Figure S7, Supporting Information, together with their Boltzmann percentages at room temperature. The experimental IR and VCD spectra of matrine in CDCl3 are compared with the Boltzmann-weighted simulated spectra in the gas phase, in the bulk solvent, and with the combined explicit and implicit solvation model in Figure 9. The main IR and VCD features in the CO stretching and 1400−1200 cm−1 regions (mainly H



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Figure 11. Comparison of the experimental IR and VCD spectra of sophoridine in CDCl3 and the corresponding IR and VCD spectra of the 1:1 sophoridine I−CDCl3 complex in CDCl3 using the PCM and sophoridine I in CDCl3 using the PCM and in the gas phase.

Figure 12. Comparison of the experimental IR and VCD spectra in DMSO-d6 with the corresponding calculated spectra of the 1:1 dihydroartemisinin−DMSO-d6 complex using the explicit model and the PCM of the DMSO solvent, as well as those with only the PCM.

magnitude of the carbonyl VCD features. Seven conformers were identified. Their single-conformer IR and VCD spectra are compared in Figure S9, Supporting Information, together with their Boltzmann percentages at room temperature. A superficial glance through the VCD spectra of the seven conformers leads to the same conclusion as before: sophoridine I is the dominant conformer, whereas the sophoridine II conformer can exist in only insignificant amounts, if at all. The experimental IR and VCD spectra of sophoridine in CDCl3 are therefore compared with the simulated spectra of the sophoridine I conformer in the gas phase, in the bulk solvent, and with the combined explicit and implicit solvation model in Figure 11. The combined explicit and implicit approach provides the best agreement with the experimental data. For example, the observed and calculated VCD bands now correlate reasonably well in the region most affected by the solvent, although the magnitude of the band labeled as 6 in Figure 6 is still exaggerated by theory. A further interesting point is that, with the combined explicit and implicit approach, the negative carbonyl VCD intensity increases substantially relative to the other main VCD features in comparison to the situation in the gas phase and in implicit CDCl3. It is noted that the carbonyl VCD intensity relative to the other main VCD features is larger in CDCl3 than in DMSO-d6. The cause of such an intensity enhancement is attributable to the explicit hydrogen-bonding interaction between sophoridine and CDCl3. As indicated above, explicit hydrogen-bonding interactions between dihydroartemisinin and DMSO molecules may be responsible for the poor agreement between theory and

stretching band is much less than those in the matrine−CDCl3 and sophoridine−CDCl3 complexes. The experimental data, however, clearly indicates that the CO bands of these three compounds in CDCl3 appear at almost the same wavenumber. Therefore, in the case of oxymatrine, to faithfully capture the explicit solute−solvent hydrogen-bonding interaction in CDCl3, a 1:2 complex in which both of the oxygen atoms in oxymatrine are hydrogen-bonded to CDCl3 simultaneously must be considered. The simulated IR and VCD spectra of the 1:2 complex between oxymatrine and CDCl3 are also included in Figure 10 for comparison. Indeed, whereas the Boltzmannweighted IR spectrum of the five conformers of the 1:1 complex show two carbonyl stretching bands with the dominant band at a much higher frequency than the experimentally observed band, the 1:2 complex displays a carbonyl stretching band similar to the matrine case and much closer to the experimental data. Overall, the IR bands of the 1:2 complex exhibit better agreement with the experimental data than the 1:1 complex. More importantly, the detailed VCD spectroscopic features in the 1350−1500 cm−1 region are much better reproduced by the 1:2 than the 1:1 complexes. This example showcases the effectiveness of the “clusters-in-a-liquid” approach to account for the protic solvent effects. In the case of sophoridine, as previously discussed, only the sophoridine I conformer appears to exist as the dominant conformer in DMSO-d6. For completeness, all four sophoridine conformers are utilized to construct possible sophoridine− CDCl3 complexes in bulk CDCl3 to establish whether the explicit hydrogen-bonding interactions change the sign or I



Article

multiple hydrogen-bond acceptor sites, which can greatly complicate the modeling process. In conclusion, the IR and VCD spectra of five Chinese herbal medicinesmatrine, sophoridine, oxymatrine, artemisinin, and dihydroartemisininwere measured in CDCl3 and DMSO-d6 to extract their stereochemical information, such as their absolute configurations and conformations. Molecular mechanics (MM) and DFT calculations were carried out to identify the dominant conformers and to simulate their IR and VCD spectra in solution for comparison with the experimental data. Their distinct IR and especially VCD features permit identification of a specific stereoisomer, although the solvent effects may mask such a clear identification. To effectively account for solvent effects and thereby improve the comparison with the experimental data, a general approach (i.e., the “clusters-in-a-liquid” approach) of including both the key explicit hydrogen-bonding interactions between a chiral solute and solvent molecules and the implicit solvation effects using the PCM was applied. Using this approach, substantial improvements in the agreement between theory and experiment were made, especially in the cases of oxymatrine in CDCl3 and dihydroartemisinin in DMSO-d6.

experiment for this compound when compared to the agreement observed for artemisinin. Dihydroartemisinin has a hydroxy group that is intramolecularly hydrogen-bonded to O11, with an OH···O bond length of 2.561 Å in the PCM of DMSO. This bond is classified as a weak hydrogen bond. No other geometries with a different OH orientation were identified in the extensive conformational searches or in the subsequent DFT calculations. It may be hypothesized that this intramolecular hydrogen bond could break to allow the formation of a stronger intermolecular hydrogen bond with DMSO. The optimized geometry of the 1:1 dihydroartemisinin−DMSO-d6 complex in the PCM of DMSO-d6 is given in Figure 7. The corresponding IR and VCD spectra of this complex are compared with the experimental data in Figure 12. Indeed, the complex captures the experimental IR patterns for the entire range. The O−H bend vibration is shifted to a higher frequency and blended into a group of other peaks to generate the broad band observed in the 1480−1420 cm−1 region. Furthermore, correlation of the calculated IR bands in the lower-wavenumber region is now straightforward. The corresponding calculated VCD features (indicated with an arrow), now without the additional medium-strength negative VCD band at ∼1450 cm−1, agree much better with the experimental spectrum in the wavenumber region beyond 1380 cm−1. Both the sign and relative intensity of the two negative VCD bands at 1279.4 and 1251.1 cm−1, labeled as 1 and 2, respectively, are well reproduced for the complex, as are those of the next three positive VCD bands in the 1250−1200 cm−1 region, labeled as 3, 4, and 5. The next three VCD bands in the region below 1200 cm−1, with +/−/+, labeled as 6, 7, and 8, are also better reproduced with the combined explicit and implicit solvent model than with just the PCM. In addition, similar analyses were performed for the IR, VCD, and dissymmetry factor spectra using the freely available program developed by Covington and Polavarapu;39 the results are summarized in Figure S10, Supporting Information. A comparison of the SimIR, SimVCD, and SimDF factors obtained for the experimental data versus the final simulated spectra using either the PCM or the explicit+PCM approach is shown. For example, for dihydroartemisinin in DMSO, the SimIR, SimVCD, and SimDF factors are all greater with the explicit+PCM approach than with the PCM alone, demonstrating better agreement between experiment and theory with the former rather than the latter. For systems with explicit hydrogen-bonding interactions, different IR bands often experience different frequency shifts. Therefore, a direct comparison of the similarity factors with the three solvation models may not be appropriate because the similarity program uses one frequency scaling factor to optimize overlap for the whole frequency region of interest. As demonstrated in this section, the “clusters-in-a-liquid” solvation approach significantly improves the agreement between the calculated and experimental IR and VCD spectra under several different conditions. Applying this approach to account for solvent effects improves the agreement between theory and experiment considerably, especially when a solvent may form hydrogen bonds with the chiral solute under investigation. In choosing a solvent for the VCD study, the hydrogen-bonding ability must be carefully examined, in addition to the usual criteria such as no strong solvent IR bands in the region of interest and good solubility and stability. For example, we avoided using CDCl3 as the solvent for artemisinin and dihydroartemisinin because both of them have

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EXPERIMENTAL SECTION

FTIR and VCD Measurements. Matrine, oxymatrine, sophoridine (HPLC > 98%), artemisinin, and dihydroartemisinin (HPLC > 98%) were purchased from Chendu Must Bio-Technology Co. Ltd. (Chengdu, SiChuan, China) and were used without further purification. DMSO-d6 and CDCl3 were purchased from SigmaAldrich (St. Louis, MO, USA) and used as solvents for the IR and VCD measurements. The experimental IR and VCD spectra were obtained using an FTIR spectrometer (Vertex 70, Bruker (Billerica, MA, USA)) equipped with a VCD module (PMA50, Bruker). To obtain reproducible and good-quality VCD spectra, the concentration and path length were optimized to ensure that the IR absorbance for most VA bands in the frequency region of interest was between 0.2 and 0.8. The samples were contained in a demountable IR cell with CaF2 windows. For the three matrine-type compounds, the upper frequency region was measured using a 0.05 mm PTFE spacer and at a concentration of 40 mg/mL. Because the absorption strength of the carbonyl stretching band was noticeably greater than those of the other vibrational bands, separate VCD measurements were carried out for the region below 1550 cm−1 using a 0.2 mm path length and the same concentration. For artemisinin, a concentration of 40 mg/mL in DMSO-d6 and a path length of 0.1 mm were used, whereas a concentration of 80 mg/mL in DMSO-d6 and a path length of 0.05 mm were used for dihydroartemisinin. All VCD spectra were measured with a total acquisition time of 3 h (3 × 1 h), or ∼13 000 scans, and with a resolution of 4 cm−1. The final IR and VCD spectra were obtained by subtracting the corresponding solvent spectra measured under identical conditions. MM and DFT Calculations. For the initial conformational searches, the “conformer distribution” option was employed as implemented in the Spartan program.37 A series of conformers were generated in the MM model using the MM force field. The relevant conformers at room temperature were reoptimized with the DFT approach described below. The Gaussian 09 package40 was used for all the geometry optimizations, harmonic frequency calculations, and calculations of the IR and VCD intensities at the B3LYP/cc-pVTZ and/or the B3LYP/6-31+G(d,p) level. A Lorentzian line shape with a half-width at half-height of 4 cm−1 was used for simulations of the IR and VCD spectra. The results obtained with the two basis sets were similar, and only one was included in the main text. The integral equation formalism version of the PCM25 using universal force field radii was applied to account for the effects of solvent molecules implicitly. For this purpose, the dielectric constants of 46.826 and 4.7113 were used for DMSO-d6 and CDCl3, respectively. Note that no J



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scaling factors were applied to allow easy observation of how implicit and explicit solvent effects influence the wavenumber of each vibrational band in the fingerprint region.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01082. Relative energies and free energies of the three matrinetype compounds; potential energy profile of dihydroartemisinin along the HOCO dihedral angle; single conformer IR and VCD spectra of all conformers of matrine, oxymatrine, and sophoridine in the gas phase, in CDCl3, and in DMSO-d6; comparison of the experimental and simulated IR and VCD spectra of artemisinin and dihydroartemisinin in DMSO-d6 at the B3LYP/ccpVTZ level; single-conformer IR and VCD spectra of all conformers of the complexes of matrine−CDCl3, oxymatrine−CDCl 3 , and sophoridine−CDCl 3 in CDCl3; and comparison of the experimental IR and VCD spectra of matrine, sophoridine, and oxymatrine in CDCl3 and in DMSO-d6 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (Y. Xu): +1 780 4941244. E-mail: [email protected]. Present Address

† School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, Hubei, People’s Republic of China, 430073.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by the University of Alberta, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, and the Alberta Enterprise and Advanced Education. We thank C. L. Covington for discussion of the similarity comparison programs. We also gratefully acknowledge access to the computing facilities of the Shared Hierarchical Academic Research Computing Network (SHARCNET: www.sharcnet.ca), the Western Canada Research Grid (Westgrid), and Compute/Calcul Canada. Y.Z. acknowledges the China Scholarship Council for a Visiting Professor Fellowship, X.C. thanks Wuhan Institute of Technology for an Exchange Student Fellowship, J.C. acknowledges the Government of Alberta for a Queen Elizabeth II Graduate Scholarship, and Y.X. holds a senior Canada Research Chair in Chirality and Chirality Recognition.

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