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Chinese Chemical Letters 19 (2008) 7678 www.elsevier.com/locate/cclet

Two new coumarin glucosides biosynthesized by transgenic hairy roots of Polygonum multiorum
Rong Min Yu a,*, Liang Bin Zhou a, Chun Yan Yan a, Guo Yan Duan a, Yu Zhao b
a College of Pharmacy, Jinan University, Guangzhou 510632, China Department of Traditional Chinese Medicines and Natural Drug Research, College of Pharmaceutical Sciences, Zhejiang University, 310031 Hangzhou, China b

Received 27 August 2007

Abstract The glycosylation of hydroxylcoumarin was investigated by using suspension cultures of hairy roots of Polygonum multiorum. Two new coumarin glucosides (3 and 4) were biosysthesized by regioselectively glycosylation of corresponding substrates (1 and 2) in the system. The structures of two products were identied as 7-hydroxy-4-methylcoumarin 5-O-b-D-glucopyranoside and 7hydroxy-3,4-dimethylcoumarin 5-O-b-D-glucopyranoside on the ground of chemical and spectroscopic methods, respectively. # 2007 Rong Min Yu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Polygonum multiorum; Hairy roots; Glycosylation; 7-Hydroxy-4-methylcoumarin 5-O-b-D-glucopyranoside; 7-Hydroxy-3,4dimethylcoumarin 5-O-b-D-glucopyranoside

Glycosylation, an important reaction in plant secondary metabolism, regioselectively and stereoselectively biotransforms xenobiotics. Glycosides, products of glycosylation, differentiate much in the aspects of physicochemical property and bioactivity, such as enhancement of solubility and stability. Coumarins, one of the most important secondary metabolites in the plant kingdom, have been reported to show various biological and pharmacological activities. However, the pharmaceutical application of most coumarins is conned by their low water solubility. Since their glycosides are more soluble in water and blood plasma, it is relatively easy to transport in biological systems and play signicant roles in pharmaceutical utilization by means of biotransformation of coumarins. Hairy roots of Polygonum multiorum, induced by Ri plasmid in Agrobacterium rhizogenes, are chosen to be the biotransformation system, with characteristics of fast growth, stable genetic traits, high content of bioactive constituents and so on [1]. Suspension cultures of P. multiorum hairy roots were induced and cultured by our research group as described previously [1,2]. Substrates 1 (100 mg) and 2 (100 mg), both were synthesized by our research group (purity >98%), were administered to 20 asks, respectively, which contain suspension cultures of P. multiorum hairy roots. Coculture was proceeded at 25 8C for 4 days on a rotary shaker (110 rpm) in the dark. The dried cultures were extracted with methanol for ve times at 50 8C. The extract was combined and evaporated to dryness in vacuo. The residue was

* Corresponding author. E-mail address: tyrm@jnu.edu.cn (R.M. Yu). 1001-8417/$ see front matter # 2007 Rong Min Yu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2007.10.047

R.M. Yu et al. / Chinese Chemical Letters 19 (2008) 7678 Table 1 1 H NMR (400 MHz) and No. 3 dH (J = Hz) 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 5.95 (s, 1H) 2.55 (s, 3H, CH3) 6.52 (d, 1H, 2.3) 10.58 (s, 1H, OH) 6.37 (d, 1H, 2.3) dC 161.38 110.37 154.70 157.30 99.20 159.89 96.82 156.16 103.50 101.26 73.39 76.93 69.64 77.32 60.71 23.85(4-CH3)
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C NMR (100 MHz) data of compounds 3 and 4 (in DMSO-d6, d ppm) 4 dH (J = Hz) 2.02 (s, 3H, CH3) 2.56 (s, 3H, CH3) 6.53 (d, 1H, 2.3) 10.39 (s, 1H, OH) 6.35 (d, 1H, 2.1) dC 160.99 116.33 148.50 156.73 96.49 160.03 99.48 154.17 104.03 101.44 73.42 76.96 69.65 77.30 60.70 12.90 (3-CH3) 19.53 (4-CH3)

4.92 (d, 1H, 7.2) 3.303.72 (m) 3.303.72 (m) 3.303.72 (m) 3.303.72 (m) 3.303.72 (m)

4.91 (d, 1H, 7.2) 3.293.70 (m) 3.293.70 (m) 3.293.70 (m) 3.293.70 (m) 3.293.70 (m)

chromatographed on repeated silica gel columns using petroleum etherethyl acetate and ethyl acetatemethanol as solvent systems to afford 30 mg of 3 and 35 mg of 4, respectively. Compound 3 was isolated as colorless needle crystals (CH3OH), mp 294295 8C and assigned the molecular formula C16H18O9 by HR-MS ([M]+ m/z 354.0942, calcd. 354.0945). And the base peak ([Mglc]+ at m/z 192, aglycon) in the EI-MS indicated that product 3 should be a molecular which is a sugar attached to a phenolic group of substrate 1. In the 1H and 13C NMR spectra of 3 (Table 1), the coupling pattern of the sugar proton signals and the chemical shifts of the sugar carbon signals indicated that the sugar component in 3 was b-glucose. Correlations were observed in the HMBC spectrum between the proton signal at d 4.92 (H-10 ) and the carbon resonance at d 157.30 (C-5), and between the proton signal at d 10.58 (7-OH), and the carbon resonance at d 96.82 (C-8) and 99.20 (C-6). These results indicated that the b-D-glucopyranosyl residue was attached to 5-OH of 5,7-dihydroxy-4-methylcoumarin. Each signal in the NMR spectra of 3 was assigned by HMBC analysis and comparison with similar compounds [3,4]. Thus, compound 3 was identied as 7-hydroxy-4-methylcoumarin 5-O-b-D-glucopyranoside (Fig. 1), which is a new compound. Compound 4 was isolated as white amorphous powder (CH3OH), mp 301302 8C and assigned the molecular formula C17H20O9 by HR-MS ([M]+ m/z 368.1103, calcd. 368.1102). The base peak ([Mglc]+ at m/z 206, aglycon) in the EI-MS suggested that product 4 should be a molecular which is a sugar attached to a phenolic group of substrate 2. In the 1H and 13C NMR spectra of 4, the coupling pattern of the sugar proton signals and the chemical shifts of the sugar carbon signals demonstrated that the sugar component in 4 was b-glucose. A correlation was observed in the HMBC spectrum between the proton signal at d 4.91 (H-10 ) and the carbon resonance at d 156.73 (C-5). The result

Fig. 1. The biosynthesis pathway of compounds 3 and 4 and their key HMBC (H C). 1, 3: R = H; 2, 4: R = CH3.

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showed that the b-D-glucopyranosyl residue was attached to 5-OH of substrate 2. Each signal in the NMR spectra of 4 was assigned by HMBC analyses and comparison with product 3. Thus, compound 4 was identied as 7-hydroxy-3,4dimethylcoumarin 5-O-b-D-glucopyranoside (Fig. 1) [34], which is also a new compound. Acknowledgments This research was nancially supported by Natural Sciences Foundation of Guangdong (No. 04010461). We thank Dr. Dongbo Yu of University of Texas Southwestern Medical Center, USA, for his check in our manuscript. References
[1] [2] [3] [4] L. Wang, R.M. Yu, H. Zhang, K.D. Cheng, Chin. J. Biotech. 18 (1) (2002) 69. R.M. Yu, N. Ma, C.Y. Yan, Y. Zhao, Chin. J. Biotech. 22 (4) (2006) 619 (English edition). B.M. Trost, F.D. Toste, K. Greenman, J. Am. Chem. Soc. 125 (2003) 4518. Y.Y. Zhang, W.C. Ye, C.L. Fan, Z.Q. Yin, H. Wang, S.X. Zhao, Chin. J. Nat. Med. 3 (3) (2005) 141.