Efficient Determination of the Enantiomeric Purity and Absolute

Article pubs.acs.org/jnp

Efficient Determination of the Enantiomeric Purity and Absolute Configuration of Flavanones by Using (S)‑3,3′-Dibromo-1,1′-bi-2naphthol as a Chiral Solvating Agent Guoxin Du,†,‡ Yisu Li,‡,§ Shunan Ma,† Rui Wang,† Bo Li,*,‡ Fujiang Guo,*,† Weiliang Zhu,‡ and Yiming Li*,† †

School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, People’s Republic of China ‡ CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, People’s Republic of China § Nano Science and Technology Institute, University of Science and Technology of China, 166 Renai Road, Suzhou, 215123, People’s Republic of China S Supporting Information *

ABSTRACT: The enantiomeric purity and absolute configuration of flavanones were first determined using (S)-3,3′-dibromo1,1′-bi-2-naphthol as a chiral solvating agent by means of 1H NMR spectroscopy. The enantiomeric purity results closely matched those based on chiral HPLC analysis.

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circular dichroism (ECD), optical rotatory dispersion (ORD), and supercritical fluid chromatography (SFC).2,3 However, these techniques have limitations; for example, HPLC requires an expensive chiral chromatographic column, and XRD requires high-quality monocrystals.4 NMR spectroscopy is an alternative technique for determining the enantiomeric purity and absolute configuration of chiral molecules, offering several advantages such as simple operation, small sample size, and low cost.5 Three types of chiral auxiliaries, a chiral solvating agent (CSA), chiral lanthanide shift reagent (CLSR), and chiral derivatizing agent (CDA), are generally used in NMR spectroscopy to determine the enantiomeric purity and absolute configuration.6 CSA and CLSR form diastereomeric complexes with substrate enantiomers in situ through weak intermolecular interactions, leading to a chemical shift nonequivalence (Δδrac) in analogous nuclei, and thus may be used directly.7 The values of Δδrac show the chemical shift differences of the enantiomeric H-3a signals of racemic flavanones in the presence of chiral solvating agents. CDA

lavanones are polyphenol substances present in various plants. Flavanones exhibit various biological activities, such as antiangiogenic, anti-inflammatory, anticancer, antimutagenic, and antioxidant activities.1 The backbone of a flavanone comprises a 2,3-dihydro-2-phenylbenzopyran-4-one moiety with a single C-2 stereogenic center (Figure 1). Different

Figure 1. Chemical backbone of flavanones.

spatial arrangements cause enantiomers to have diverse bioactive, toxic, and environmental fates. Defining the enantiomeric purity and absolute configuration of flavanones rapidly and accurately is crucial to further study their structure−activity relationships. Numerous experimental techniques have been reported for chiral discrimination, such as chiral high-performance liquid chromatography (HPLC), X-ray diffraction (XRD), electronic © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 4, 2015

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DOI: 10.1021/acs.jnatprod.5b00690 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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forms discrete diastereomers with substrate enantiomers through covalent bonds and, thus, can be used for NMR analysis.8 Overall, CSA is generally more attractive because it avoids the shortcomings of CDA and CLSR, including destruction of the sample, the possibility of racemiszation, high cost, and complex operation. Various types of CSAs have been reported, such as Pirkle’s alcohols, binaphthyl compounds, molecular tweezers, natural products, cyclodextrins, synthetic macrocycles, and ionic liquids.9 The binaphthyl-type BINOLs and related compounds are atropisomeric compounds that are broadly applicable CSAs. The hydroxy groups of the BINOL can form hydrogen bonds, and the naphthyl rings cause shielding effects that account for enantiomeric discrimination.10 It has been reported that enantiopure BINOLs are good NMR CSAs for the determination of enantiomeric purity of different chiral compounds including sulfinimines,11 amines,12−14 carboxylic acids,15 phytoalexins,16 alkaloids,17 and some active pharmaceutical ingredients (API) including promethazine,18 omeprazole,19 and fenfluramine.20 In addition, BINOLs exhibit potential applications as CSAs for the assignment of the absolute configuration of sulfoxides,21 amino acids,22 hydroxy acids,23 and carboxylic acids.24 In this study, (S)-3,3′-dibromo-1,1′-bi-2-naphthol [(S)-1] was selected from (S)-1−6 and was used as a CSA for the chiral discrimination of racemic bavachinin 7 by employing 1H NMR spectroscopy. The ideal amount of (S)-1 required for the chiral discrimination of racemic bavachinin was investigated. A graphical model was proposed in which the use of the δR and δS parameters for H-3a permits the direct configurational assignment from the NMR data. Intermolecular NOE studies provided insight into the association of diastereomeric complexes between (S)-1 and the two bavachinin 7 enantiomers. Herein, assessment of the enantiomeric purity and absolute configuration of five natural flavanones (8−12) by using (S)-1 as a CSA is discussed. HPLC−UV−ECD analysis was used to validate the results.

Scheme 1. Different Substituted Binaphthol Chiral Compounds

ability of the amino group to form a hydrogen bond, and this was further verified when one of the amino groups in (S)-5 was substituted with a hydroxy group in NOBIN (S)-6 and the Δδrac value was 0.003 ppm for H-3a. Thus, the effects of (S)-1 and (S)-2 were superior to other compounds. However, signal overlap was observed in the two sets of double doublets of H3a and H-3b induced by (S)-2, which reduced the accuracy of the integral. Therefore, (S)-1 was used in subsequent experiments. Next, the effect of the amount of (S)-1 on signal separation (Figure S1, Supporting Information) was investigated. The addition of 1 equiv of (S)-1 resulted in baseline separation of the signals for H-3a (Table S2, Supporting Information). The gradual increase in the amount of (S)-1 resulted in shielding of the 1H NMR signal of H-3a and a gradual increase in the chemical shift difference between the two enantiomers. The addition of 18 equiv of (S)-1 resulted in nearly equivalent Δδrac values compared to those obtained when 12 equiv of (S)-1 was added. The addition of 1, 2, and 6 equiv of (S)-1 resulted in signal overlap, which rendered chiral discrimination difficult. Although the addition of 12 and 18 equiv of (S)-1 resulted in increased signal separation, the operation is complex and expensive. Therefore, the optimum amount of (S)-1 to be used for chiral discrimination of racemic bavachinin 7 was 3 equiv. When both enantiomers of a flavanone are associated with one enantiomer of the CSA through noncovalent bonds, two diastereomeric solvation complexes are formed, and, by comparing the chemical shifts of the corresponding nuclei in both solvation complexes, the absolute configuration may be assigned (δR < δS vs δR > δS).26 The experimental procedure comprises the mixing of (S)- and (R)-bavachinin with (S)-1 and the construction of the configurational assignment by comparing the chemical shifts of H-3a. The chemical shifts are affected by strong aromatic shielding effects or weak shielding effects produced by the auxiliary, and therefore they depend on the spatial location of that hydrogen atom with regard to the naphthyl rings. Thus, the signs of δR and δS correlate the absolute configuration in the auxiliary (known) with that in the substrate part (unknown). The models for the assignment of the absolute configuration of flavanones (Figure 3) indicated

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RESULTS AND DISCUSSION Four types of substituted binaphthols (CSAs), 1,1′-bi-2naphthol (BINOL) derivatives (S)-1−3, 3,3′-diphenyl-[2,2′binaphthalene]-1,1′-diol (VANOL) (S)-4, 2,2′-diamino-1,1′binaphthyl (BINAM) (S)-5, and 2-amino-2′-hydroxy-1,1′binaphthyl (NOBIN) (S)-6, were evaluated using racemic bavachinin 7 as a model substrate (Scheme 1). Mixtures of racemic bavachinin with BINOL derivatives (S)-1 and (S)-2 displayed dramatic changes in the chemical shift values of 1H NMR signals for H-3a (trans to H-2) and H-3b (cis to H-2) between diastereomeric complexes (Figure 2), with Δδrac values of 0.012 and 0.019 ppm for H-3a (Table S1, Supporting Information). In both cases, the H-3a and H-3b signals were shielded, which was likely due to the magnetic anisotropic effect of the binaphthyl skeleton.25 However, BINOL derivative (S)-3 induced no differentiation possibly because of the steric hindrance of the 3,3′-triphenylsilyl group that blocks the hydrogen bond association between the naphthol moiety and the substrate. In addition, the results obtained using (S)-1 and (S)-2 were more satisfactory than those obtained using (S)-4, where the Δδrac value for H-3a was only 0.006 ppm. Thus, different binaphthyl skeletons resulted in different magnetic anisotropic effects on the substrate. Furthermore, BINAM (S)5 had no chiral discrimination effect, with a Δδrac value of 0 ppm for H-3a. This result possibly contributed to the weak B



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Figure 2. Overlaid 1H NMR spectra (600 MHz in CDCl3 at 298 K) of racemic bavachinin 7 in the presence of 3 equiv of (S)-1−6.

To elucidate the association of the complexes of (S)-1 and (S)- and (R)-bavachinin, NOE spectroscopy was used.28 Correlation peaks between several S-(1) aryl signals and the H-3a signals of (S)- and (R)-bavachinin were clearly detected, thus indicating a close intermolecular association (Figure 5). The observation of the intermolecular NOE between these hydrogen atoms confirmed the association of (S)-1 and bavachinin enantiomers. The difference in the chemical shift values of the corresponding H-3a of two bavachinin enantiomers 7 in the presence of (S)-1 inspired us to investigate the enantiomeric discriminating ability of (S)-1 with some other flavanones. We chose natural bavachinin 8, bavachin 9, farrerol 10, hesperetin 11, and naringenin 12 to screen the potential of (S)-1 as a CSA by using 1H NMR spectra. In addition, 1H NMR analysis and conventional chiral HPLC equipped with UV and ECD detectors were compared for the determination of enantiomeric purity and absolute configuration.29 Flavanones 9−12 are polar and thus are difficult to dissolve in a nonpolar solvent, such as CDCl3. The influence of the solvent on the chiral recognition was evaluated. When DMSOd6 and acetone-d6 were used as solvents, the 1H NMR signals of H-3a did not split. When acetonitrile-d3 was used as the solvent, the splitting of the 1H NMR signals of bavachin 9 was strongly reduced (Figure S4, Supporting Information). This result is consistent with a weak H-bonding of flavanones to (S)-1, which can be displaced in the presence of a polar solvent. To obtain the ideal splitting of the 1H NMR signals, we recorded 1H NMR spectra of the mixtures of flavanones 9−12 and (S)-1 in a CDCl3/acetonitrile-d3 mixture. The results indicated that the

that H-3a in (R)-bavachinin is more strongly shielded than that in (S)-bavachinin under the influence of the cone of one of the naphthyl rings.27 The results and graphics of mixing (S)- and (R)-bavachinin with (R)-1 are shown in Figure S2 (Supporting Information). Thus, when (S)- and (R)-flavanone are associated with (S)-1 as a CSA, the chemical shift value of H-3a of (S)-flavanone is greater than that of H-3a of (R)flavanone (H-3a: δR < δS). On the contrary, when (S)- and (R)flavanone are associated with (R)-1 as a CSA, the chemical shift value of H-3a of (R)-flavanone is greater than that of H-3a of (S)-flavanone (H-3a: δR > δS). Conversely, when one enantiomer of a flavanone is associated with two enantiomers of CSA through noncovalent bonds, a comparison of the chemical shift values of H-3a for two diastereomeric complexes determines their absolute configuration (ΔδRS or ΔδSR). The values of ΔδRS or ΔδSR show the chemical shift differences of the enantiomeric H-3a signals of enantiopure (S)- and (R)-flavanone in the presence of chiral solvating agents. The experimental procedure comprises the mixing of (S)-bavachinin with (S)-1 and (R)-1, individually, and the construction of the configurational assignment by comparing the chemical shifts of H-3a (Figure 4). The experimental results and graphics of mixing (R)-bavachinin with (S)-1 and (R)-1 are shown in Figure S3 (Supporting Information). According to the results, when (S)-flavanone is associated with (S)-1 and (R)-1, respectively, as a CSA, the chemical shift value of H-3a of (S)-flavanone and complex (S)1 is greater than that of (S)-flavanone and complex (R)-1 (H3a: ΔδRS < 0). C



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Figure 3. Models for the assignment of the absolute configuration of flavanones from the δR and δS signs of H-3a: (a) 1:3 mixture of (S)-bavachinin and (S)-1; (b) (R)-bavachinin with (S)-1. The black dashed line represents a H-bond.

Figure 4. Models for the assignment of the absolute configuration of flavanones from the ΔδRS signs of H-3a: (a) 1:3 mixture of (S)-bavachinin and (S)-1; (b) (S)-bavachinin with (R)-1. The black dashed line represents a H-bond.

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Figure 5. (a) Structures of bavachinin and (S)-1. The arrows represent the observed intermolecular NOEs. (b) Portion of the 2D NOESY spectrum of a 1:3 mixture of (S)-bavachinin and (S)-1 and that of (c) (R)-bavachinin and (S)-1 (600 MHz in CDCl3 at 298 K).

In conclusion, commercially available (S)-1 when associated with racemic bavachinin 7 permitted enantiodifferentiation via 1 H NMR spectroscopy. The formation of H-bond complexes of (S)- and (R)-bavachinin with (S)-1 was supported by the NOE results. The absolute configuration of bavachinin enantiomers was assigned by comparing the corresponding δR and δS with the models in Figure 3. In addition, this approach provided a simple general means to determine the enantiomeric purity and absolute configuration of flavanones. The technique described herein provided comparable enantiomeric purity and absolute configuration of five natural flavanones (8−12) with those obtained using conventional chiral HPLC.

amount of (S)-1 required increased with an increase in the acetonitrile-d3/CDCl3 ratio (Table 1). Notably, higher numbers of hydroxy groups in the flavanone structure require higher amounts of (S)-1. The enantiomeric purity of flavanones 8−12 was determined by integrating the corresponding 1H NMR H-3a signals and HPLC-UV absorption peaks (280 nm)30 for the S and R configuration, respectively. The NMR method yielded enantiomeric purities of 62.7:37.3, 48.6:51.4, 63.5:36.5, 51.9:48.1, and 48.6:51.4, and the HPLC method yielded enantiomeric purities of 63.2:36.8, 49.9:50.1, 63.4:36.6, 52.9:47.1, and 49.5:50.5 (Table 1). Thus, the results obtained using the NMR method closely matched those obtained using chiral HPLC analysis, and these two methods were mutually complementary. As mentioned previously, we assigned the S and R configurations of flavanones 8−12 according to the NMR method (Figure 3) and HPLC-ECD analyses (300 nm) according to a previous study that flavanones with the 2S configuration exhibit a negative Cotton effect in the π → π* region at 280−300 nm.31,32 According to the NMR results, H3a signals of (S)-flavanones are deshielded relative to the (R)flavanones (Table 1) and, according to the HPLC-ECD results, showed a negative Cotton effect (Figures S5−S9, Supporting Information) in the 280−300 nm region. Thus, the results obtained using NMR were consistent with those obtained using HPLC-ECD.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR experiments were performed on a Bruker Avance III 600 spectrometer equipped with a cryoprobe and Bruker AV500 spectrometer. The samples were prepared by dissolving racemic bavachinin, (S)-bavachinin, (R)bavachinin, and natural bavachinin 8 (1 mg) with 3−18 equiv of (S)-1−6 in 0.5 mL of CDCl3 and then transferred to 5 mm NMR tubes. In the chiral discrimination of the natural flavanone experiment, samples were prepared by dissolving natural flavanones 9−12 (0.5 mg) with 9−15 equiv of (S)-1 in 0.5 mL of a mixture of CDCl3 and acetonitrile-d3. All chemical shifts were measured with reference to the internal standard, TMS (δH = 0.000 ppm). Chemicals. Racemic bavachinin, (S)-bavachinin and (R)-bavachinin, were prepared according to the method published previously.33 E



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Table 1. Comparison of NMR Spectra and Chiral HPLC Analysis of Flavanones 8−12

a 8 in the presence of 3 equiv of (S)-1 was redissolved in CDCl3 600 MHz; 9 in the presence of 9 equiv of (S)-1 was redissolved in CDCl3/ acetonitrile-d3 (9:1) 600 MHz; 10 in the presence of 15 equiv of (S)-1 was redissolved in CDCl3/acetonitrile-d3 (9:1) 500 MHz; 11 in the presence of 15 equiv of (S)-1 was redissolved in CDCl3/acetonitrile-d3 (9:1) 600 MHz; 12 in the presence of 15 equiv of (S)-1 was redissolved in CDCl3/ acetonitrile-d3 (8.5:1.5) 500 MHz. b8 using a Chiral CD-Ph column, and the mobile phase is CH3OH/H2O (90:10); 9 using a Chiral CD-Ph column, and the mobile phase is CH3OH/H2O (85:15); 10 using a Chiral CD-Ph column, and the mobile phase is CH3OH/H2O (85:15); 11 using a Chiral CD-Ph column, and the mobile phase is CH3OH/H2O (85:15); 12 using a Chiral OD-RH column, and the mobile phase is CH3OH/H2O (60:40).

Bavachinin 8, bavachin 9, farrerol 10, hesperetin 11, and naringenin 12 were obtained from Usea Biotech Company (Shanghai, People’s Republic of China). (S)-1−6 were obtained from Daicel Company (Shanghai, People’s Republic of China). CDCl3 and acetonitrile-d3 (NMR quality) were obtained from J&K Scientific (Beijing, People’s Republic of China).

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ASSOCIATED CONTENT

configuration of flavanones from the ΔδRS signs of the H3a; 1H NMR spectra of a 1:3 mixture of natural bavachin and (S)-1 in acetonitrile-d3; HPLC-UV-ECD chromatogram of natural flavanones 8−12 (PDF)

AUTHOR INFORMATION

Corresponding Authors

S Supporting Information *

*Tel: +86 021 50806600-5318. Fax: +86 021 50807088. Email: [email protected] (B. Li). *Tel: +86 021 51322181. Fax: +86 021 51322193. E-mail: gfj@ shutcm.edu.cn (F.-J. Guo). *Tel: +86 021 51322191. Fax: +86 021 51322193. E-mail: [email protected] (Y.-M. Li).

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00690. 1 H NMR chemical shift nonequivalences (Δδrac) of racemic bavachinin in the presence of 3 equiv of (S)-1− 6; 1H NMR chemical shift nonequivalences (Δδrac) of racemic bavachinin in the presence of (S)-1; overlaid 1H NMR spectrum of racemic bavachinin in the presence of (S)-1; models for the assignment of the absolute configuration of flavanones from the δR and δS signs of the H-3a; models for the assignment of the absolute

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81173518), the Eastern Scholar Tracking F



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(32) Antus, S.; Baitz-Gacs, E.; Kajtar, J.; Snatzke, G.; Tokes, A. L. Liebigs Ann. Chem. 1994, 1994, 497−502. (33) Du, G. X.; Feng, L.; Yang, Z.; Shi, J. Y.; Huang, C.; Guo, F. J.; Li, B.; Zhu, W. L.; Li, Y. M. Bioorg. Med. Chem. Lett. 2015, 25, 2579− 2583.

Program of Shanghai Municipal Education Commission (201290), the National High Technology Research and Development Program of China (863 program, 2013AA093003), International Collaboration Project (2014DFA31130), and the program of Shanghai E-Institute of bioactive constituents in Traditional Chinese Medicine.

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Efficient Determination of the Enantiomeric Purity and Absolute

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