NMR Spectroscopic Method for the Assignment of 3,5-Dioxygenated

Article pubs.acs.org/jnp

NMR Spectroscopic Method for the Assignment of 3,5-Dioxygenated Aromatic Rings in Natural Products Ya-Nan Yang, Hui Zhu, Zhong Chen, Fu Liu, Ya-Wen An, Zi-Ming Feng, Jian-Shuang Jiang, and Pei-Cheng Zhang* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: In recent years, certain “new” naturally occurring compounds (1−28) with 3,5-dioxygenated aromatic rings have been reported. A comparison of the NMR data of these compounds with the data of four model compounds (A−D) indicated that the structures of these “new” compounds were erroneous. The reason for the incorrect elucidation of the structures of 1−28 was attributed to “deceptively simple” 1H NMR spectra, which displayed two broad singlets with integrations of 1:2 for H-2 and H-5, H-6, respectively. To expose the misleading results from the spectra, serial 1H NMR experiments on compounds A−D were performed using various deuterated solvents and temperatures. The results revealed separated proton signals for the ABX system in certain deuterated solvents. Additionally, the characteristic differences between 3,4- and 3,5-dioxygenated aromatic rings in their 13C NMR spectra are summarized based on our experiment and data reported. This approach is useful for analyzing the patterns of dioxygenated aromatic rings in natural products, especially when “deceptively simple” 1H NMR spectra are displayed. ignans and flavonoids are among the most common secondary metabolites in plants. According to their biosynthetic origin, the phenylpropanoid unit is one of the precursors. The aromatic rings of the phenylpropanoid units exhibit various oxygenation patterns. The dioxygenation patterns may involve the 3,4-, 2,4-, 2,5-, and 2,6-positions but seldomly the 3,5-positions. However, certain “new” naturally occurring compounds with 3,5-dioxygenated aromatic rings have been reported in recent years (Figure 1),1−22 including flavanones, dihydroflavonols, flavans, isoflavones, tetrahydrofuran lignans, furofuran lignans, and other compounds. NMR spectroscopy is a useful technique for identifying and analyzing naturally occurring compounds. The coupling constants in a 1H NMR spectrum can be exploited as powerful evidence to verify protons that are coupled, especially for definition of the oxygenation patterns of aromatic rings. For example, a typical ABX spin system resulting from a 3,4dioxygenated aromatic ring is consistent with the m-coupling constant of H-2 (d, J = 2.0 Hz), the o-coupling constant of H-5 (d, J = 7.5 Hz), and the o, m-coupling constants of H-6 (dd, J = 7.5, 2.0 Hz), while the resonances for H-2, H-4, and H-6 in 3,5dioxygenated aromatic rings typically show singlets or mdoublets and m-triplets with small coupling constants (J = 2.0 Hz). Careful NMR data analyses of these “new” compounds attracted our attention. The structural information from the 1H NMR spectrum appeared consistent with the characteristics of

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© XXXX American Chemical Society and American Society of Pharmacognosy

a 3,5-dioxygenated aromatic ring; however, the chemical shifts of the oxygen-bearing carbons at δ 144−153 in the 13C NMR spectra were inconsistent (Figure 1). In 1994, huazhongilexone, a flavanone with a 3,5-dihydroxy B-ring, was isolated from Ilex centrochinensis by Lin et al.23 However, in 1998, Anthoni et al.24 demonstrated that the substituted pattern of the B-ring was incorrect by synthesizing racemic 3′,5,5′,7-tetrahydroxyflavanone using 2-hydroxy-4,6diisopropyloxyacetophenone and 3,5-diisopropyloxybenzaldehyde. Since then, at least 27 “new” natural compounds with 3,5dioxygenated aromatic rings have been reported.1−4,6−22 These articles were cited by many researchers who ignored the incorrect elucidation of the 3,5-dioxygenated aromatic ring.25−51 These findings inspired us to investigate this problem more thoroughly and to clarify the difference between 3,4- and 3,5-dioxygenated aromatic rings using NMR experiments.

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RESULTS AND DISCUSSION Recently, the chemical constituents of some Traditional Chinese Medicines (TCM), including Rhodiola crenulata,52 the fruits of Arctium lappa,53 Sophora f lavescens,54 the root bark of Lycium barbarum, and Polygonum cuspidatum,55 were investigated by us, and many lignans and flavonoids with the Received: November 2, 2014

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DOI: 10.1021/np5008679 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Previously reported “new” natural compounds with 3,5-dioxygenated aromatic rings.

3,4-dioxygenated aromatic pattern were isolated. During the 1H NMR spectroscopic analysis of these compounds, the phenomenon of a “deceptively simple” proton spectrum that exhibited two broad singlets with approximate integrations of 1:2 for H-2 and for H-5, H-6, respectively, was found. The four compounds A−D, i.e., a benzofuran-type neolignan, a tetrahydrofuran-type lignan, a dihydroflavonol, and an isoflavone, were used as examples, and their 1H NMR spectra were recorded in DMSO-d6 (Figure 2). Owing to the overlapping signals of H-5 and H-6, it was difficult to confirm the substitution patterns of the aromatic rings of these four compounds, even using 2D NMR data. Under these conditions,

the 3,4-dioxygenated aromatic ring might be erroneously elucidated as a 3,5-dioxygenated aromatic ring. Comparison of the NMR spectra of compound D with those of compounds 8−12 gave similar 1H NMR and 13C NMR data, especially the two broad proton singlets at δ 7.05 and 6.96 with an integration of 1:2 for three protons and two carbon signals at δ 147.6 and 146.1 for oxygen-bearing aromatic carbons of the B-ring (Figures S18 and S43, Supporting Information). The results suggest that the assignment of compounds 8−12 as possessing 3,5-dioxygenated B-rings is questionable. Furthermore, comparing the NMR data of compounds 1−3, compounds 4 and 5, and compounds 6 and 7 with the data B



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Figure 2. “Deceptively simple” proton spectra of compounds A−D in DMSO-d6.

Figure 3. Influence of temperature on the 1H NMR spectra of compound A in D2O and B in DMSO-d6.

C



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Figure 4. Influence of deuterated solvent on the 1H NMR spectra of compounds A−D.

of 3′,4′-dihydroxy-7-methoxyflavanone,56 compound C, and dulcisflavan,57 respectively, and the data of compounds 13−28 with the data of compounds A and B, the structure elucidation of the 3,5-dioxygenated aromatic rings is clearly incorrect. Temperature and solvent effects are two important factors that may affect the proton signals in the 1H NMR experiments. Therefore, we examined the 1H NMR spectra of compounds A−D using various temperatures and deuterated solvents to gain insight into the reported “deceptively simple” proton spectra. Compound A was selected as a model compound for the 1H NMR experiments. Temperature effects were investigated by obtaining measurements at five different temperatures in D2O, ranging from 283 to 323 K (Figure 3). The results showed only a small change in the coupling pattern of the B-ring proton resonances with decreasing temperature, clearly demonstrating the m-coupling constant of H-2, the o-coupling constant of H-5, and the o-, m-coupling constants of H-6 in a typical ABX system were challenging. Next, when 1H NMR experiments on compound B were performed in DMSO-d6 at different temperatures (293−323 K), a trend similar to that found in compound A was observed, as shown in Figure 3. On the basis of these analyses, the temperature only slightly improved the “deceptively simple” proton spectrum. To experimentally validate the effect of deuterated solvents on the couplings of protons with compounds A−D, we used a series of deuterated solvents, including protic solvents (D2O, methanol-d4, and acetic acid-d4) and aprotic solvents (DMSOd6, acetone-d6, and pyridine-d5). The 1H NMR spectra of compounds A−D in various deuterated solvents are shown in

Figure 4. For compound D in DMSO-d6 and methanol-d4, two singlets with the approximate integration of 1:2 for H-2 and H5, H-6, respectively, were obtained. A typical “deceptively simple” spectrum misled previous researchers to ascribe a 3,5dioxygenated aromatic ring to compound D.58 When acetoned6, pyridine-d5, and acetic acid-d4 were used, a set of proton signals was obtained with a m-coupling constant for H-2 (d, J = 2.5 Hz), an o-coupling constant for H-5 (d, J = 8.5 Hz), and o-, m-coupling constants for H-6 (dd, J = 8.5, 2.5 Hz). Subsequently, 1H NMR experiments on compounds A−C in different deuterated solvents were also performed (Figure 4). These compounds exhibited separated proton signals for the ABX system in certain deuterated solvents. According to our investigation, the phenomenon of a “deceptively simple” spectrum frequently occurred in DMSO-d6, which is a commonly used solvent, particularly for flavonoids. These results indicate that 1H NMR experiments are markedly influenced by the solvent due to the dipole moments and dielectric permittivity, magnetic permeability, and other physicochemical properties of the solvents, which could affect the molecular interaction between solvent and solute. The 13C NMR data should also be analyzed carefully when the 1H NMR data display a “deceptively simple” spectrum. To clarify the characteristic differences between 3,4- and 3,5dioxygenated aromatic rings, 13C NMR experiments were conducted on six model compounds (E−J), which were identified by comparison with reported spectroscopic data.59−64 A comparison of their carbon signals with the signals of Syn-1−6, containing verified 3,5-dioxygenated aromatic rings,24,65−69 yielded some notable differences (Figure 5). The D



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Figure 5. Differences between a 3,5-dioxygenated aromatic ring and a 3,4-dioxygenated aromatic ring by 13C NMR spectroscopy.

In the “deceptively simple” proton spectra of compounds A− D in DMSO-d6, three different aromatic protons appear as two singlets, which should have resonated as an ABX system. It was easy to reach an incorrect conclusion regarding the presence of a 3,5-dioxygenated aromatic ring without considering the 13C NMR data. A systematic study of the influence of solvent and temperature during 1H NMR experiments revealed that such a misleading conclusion could be prevented by changing the deuterated solvent. Furthermore, the characteristic differences between 3,4- and 3,5-dioxygenated aromatic rings in the 13C NMR spectra are summarized based on the experimental and reported data, which are useful for analyzing the patterns of dioxygen-substituted aromatic rings in natural products.

chemical shifts of C-3 and C-4 in E−J were at δ 144−153, which is significantly shielded compared to the chemical shifts of C-3 and C-5 in Syn-1−6 at δ 158−162. A major difference revealed an obviously incorrect elucidation of the 3,5dioxygenated aromatic rings with chemical shifts of C-3,5 at δ 144−153 in certain “new” compounds. Meanwhile, the shielding of C-2,4,6 in Syn-1−6 compared to C-2,5,6 in E−J was also observed, particularly for the remarkably shielded C-4 in 3,5-dioxygenated aromatic rings. Additionally, analyses of the chemical shifts of C-1 in these 12 compounds revealed an approximate 10 ppm chemical shift difference of C-1 between the 3,4- and 3,5-dioxygenated aromatic rings when C-1 carried the same substituent (i.e., saturated or unsaturated carbon). When the saturated carbon was connected at C-1, the C-1 chemical shifts were δ 128−138 and 141−146 in the 3,4- and 3,5-dioxygenated aromatic rings, respectively. When the unsaturated carbon was connected at C-1, the C-1 chemical shifts were δ 120−129 and 132−136 in the 3,4- and 3,5dioxygenated aromatic rings, respectively. These differences were primarily caused by the effects of donation of e′-density of ortho- and/or para-oxygen-bearing substituents.70 Considering the solvent effect on the carbon signals, 13C NMR experiments were conducted on compounds B and C in various deuterated solvents (Figures S33−S42, Supporting Information). The maximum Δδ value was approximately 3 ppm in any two different deuterated solvents, which was within the range of the shift differences previously summarized.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR experiments were performed using a Bruker Avance 500 MHz NMR spectrometer (Bruker-Biospin, Billerica, MA, USA) using a broadband probe equipped with a z-gradient coil. All the NMR samples had a volume of 500 μL and were run in standard 5 mm NMR tubes. Pulse programs used for the Bruker Avance 500 MHz NMR spectrometer were standard sequences obtained from the Bruker Topspin 2.1 pulse sequence library. 1H and 13C NMR chemical shifts were calibrated relative to the solvents and TMS. Preparative HPLC was performed using a Shimadzu LC-6AD instrument with an SPD-20A detector, using a YMC-Pack ODS-A column (250 mm × 20 mm, 5 μm; YMC Corp., Japan). Flash chromatography was conducted using Combiflash RF200 (Teledyne Isco Corp., Lincoln, NE, USA). HPLC-DAD analysis was performed using an Agilent 1200 series system (Agilent E



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Technologies, Waldbronn, Germany) with an Apollo C18 column (250 mm × 4.6 mm, 5 μm; Alltech Corp., Nicholasville, KY, USA). Column chromatography was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China) and Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden). DMSO-d6, acetone-d6, pyridine-d5, methanol-d4, acetic acid-d4, and D2O were purchased from Cambridge Isotope Laboratories (Cambridge, MA, USA). Compound C (taxifolin, ID: 111816-201102) and compound I (quercetin, ID: 100081-201408) were purchased from The National Institutes for Food and Drug Control of China. Racemic J was synthesized by using 2,4,6-trihydroxyacetophenone and 3,4dimethoxybenzaldehyde.71 Plant Material. The roots of Rhodiola crenulata (ID-S-2599) and Polygonum cuspidatum (ID-S-2593) were purchased from Pushenglin Pharmaceutical Co., Ltd., in Beijing in July 2007 and December 2009, respectively. The roots of Sophora f lavescens (ID-S-2438) were collected in Weichang Town, Hebei Province, China, in July 2010. The fruits of Arctium lappa (ID-S-2434) were collected in Wuchang Town, Heilongjiang Province, China, in October 2011. The root barks of Lycium barbarum (ID-S-2592) were collected in Zhongning Town, Ningxia Province, China, in March 2012. All of the plant materials were identified by Prof. Lin Ma. Their voucher specimens were deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China. Extraction and Isolation. Compounds A52 and B53 were isolated from the roots of Rhodiola crenulata and the fruits of Arctium lappa, respectively. Compound D was isolated from the roots of Sophora f lavescens. The air-dried powdered plant material (40 kg) was extracted with 70% EtOH. After the solvent was evaporated under reduced pressure, the resulting crude extract of the plant was extracted sequentially with petroleum ether, EtOAc, and n-butanol. The EtOAc fraction (1200 g) was subjected to chromatography on silica gel and continuously eluted with petroleum ether, EtOAc, acetone, and MeOH, to afford fractions A−D. Fraction B (120 g) was chromatographed over silica gel, eluting with CDCl3−MeOH (from 100:0 to 0:100), to give fractions B1−B20. Fraction B4 (1.2 g) was further separated by Sephadex LH-20 with H2O−MeOH (from 75:25 to 0:100) to afford 40 fractions (fractions B4-1−40). Fraction B4-18 was purified by preparative HPLC, using MeOH−H2O (60:40) as the mobile phase, to yield compound D (55 mg), which was identified as 7,3′-dihydroxy-4′-methoxyisoflavone.72 Compounds E and F were isolated from the root barks of Lycium barbarum. The plant material (100 kg) was extracted with 80% EtOH. The resulting crude extract of the plant was extracted sequentially with petroleum ether, EtOAc, and n-butanol. The EtOAc fraction (420 g) was subjected to flash chromatography elution with petroleum ether−EtOAc (from 100:0 to 0:100) to yield fractions 1−96. Fraction 75 was further separated by preparative HPLC, using MeOH−H2O (50:50) as the mobile phase, to afford compounds E (52 mg) and F (39 mg). Compound G was isolated from the rhizome of Polygonum cuspidatum. This plant material (20 kg) was extracted with 80% EtOH. After the solvent was evaporated under reduced pressure, the residue was suspended in H2O and partitioned successively with petroleum ether, EtOAc, and nBuOH. The EtOAc-soluble portion was subjected to chromatography on silica gel and continuously eluted with CHCl3, EtOAc, acetone, and MeOH, to afford fractions A−D. Fraction D (80 g) was chromatographed over Sephadex LH-20 eluting with H2O−MeOH in gradient to yield fractions D1−D56. Fraction D21 was subjected to preparative HPLC, using MeOH−H2O (35:65) as the mobile phase, to afford compound G (32 mg). Compound H was isolated from the fruits of Arctium lappa. The plant material (100 kg) was extracted with 80% EtOH. The residue was partitioned sequentially with petroleum ether, EtOAc, and n-butanol. The EtOAc fraction was subjected to chromatography on silica gel eluting with a petroleum ether−EtOAc gradient (from 100:0 to 0:100) to yield compound H (160 mg).

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ASSOCIATED CONTENT

S Supporting Information *

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H and 13C NMR spectra for compounds A−J are provided free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-63165231. Fax: +86-10-63017757. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research described in this publication was supported by the National Natural Science Foundation of China (No. 81303207) and the Beijing Natural Science Foundation (No. 7144227).

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