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Chinese Chemical Letters 19 (2008) 76–78 www.elsevier.com/locate/cclet

Two new coumarin glucosides biosynthesized by transgenic hairy roots of Polygonum multiflorum
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 multiflorum. 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 identified 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 multiflorum; 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 confined 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 significant roles in pharmaceutical utilization by means of biotransformation of coumarins. Hairy roots of Polygonum multiflorum, 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. multiflorum 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 flasks, respectively, which contain suspension cultures of P. multiflorum 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 five 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

calcd.38 110.70 (m) 3.0942. 1. The result Fig. 1H. 3H.20 (C-6). 1H.39 76.70 157.03 101.90 (3-CH3) 19.M.3) 10.1102).70 12. In the 1H and 13C NMR spectra of 4.72 (m) 3.65 77.4].52 (d.R.50 156. –CH3) 6.48 154. 7. 2. 4: R = –CH3. compound 3 was identified as 7-hydroxy-4-methylcoumarin 5-O-b-D-glucopyranoside (Fig. d ppm) 4 dH (J = Hz) 2. 3H. The base peak ([M–glc]+ at m/z 206. 2. 368.35 (d. aglycon) in the EI-MS indicated that product 3 should be a molecular which is a sugar attached to a phenolic group of substrate 1.82 156. 1H.58 (s. 1H.71 23. which is a new compound. 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. 3H.3) 10. aglycon) in the EI-MS suggested that product 4 should be a molecular which is a sugar attached to a phenolic group of substrate 2.53 (4-CH3) 4.37 (d.02 (s.39 (s. 1H.92 (H-10 ) and the carbon resonance at d 157.32 60. 1H. 1).16 103.2) 3. 2.99 116. respectively. 1H.49 160. The biosynthesis pathway of compounds 3 and 4 and their key HMBC (H C). 2.55 (s. 3 dH (J = Hz) 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 5.30–3.73 96. 7.64 77.20 159. Compound 3 was isolated as colorless needle crystals (CH3OH).1) dC 160. 3: R = H.30–3. Thus.29–3. –CH3) 2.91 (d. / Chinese Chemical Letters 19 (2008) 76–78 Table 1 1 H NMR (400 MHz) and No.30 99.82 (C-8) and 99. Yu et al.72 (m) 4.42 76.33 148. 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.96 69. –OH) 6.29–3.30–3. 354.85(4-CH3) 13 77 C NMR (100 MHz) data of compounds 3 and 4 (in DMSO-d6.29–3. –CH3) 6.73 (C-5).50 101. –OH) 6.95 (s.93 69. In the 1H and 13C NMR spectra of 3 (Table 1). Each signal in the NMR spectra of 3 was assigned by HMBC analysis and comparison with similar compounds [3.72 (m) 3.03 99.30 (C-5). A correlation was observed in the HMBC spectrum between the proton signal at d 4.29–3.1103.70 (m) 3. 1.17 104.72 (m) 3.26 73.70 (m) 3.70 (m) chromatographed on repeated silica gel columns using petroleum ether–ethyl acetate and ethyl acetate–methanol as solvent systems to afford 30 mg of 3 and 35 mg of 4.44 73.30–3. These results indicated that the b-D-glucopyranosyl residue was attached to 5-OH of 5. 1H) 2. 1H.56 (s. And the base peak ([M–glc]+ at m/z 192. .58 (7-OH). mp 301–302 8C and assigned the molecular formula C17H20O9 by HR-MS ([M]+ m/z 368.92 (d.53 (d. 2. calcd. and the carbon resonance at d 96.91 (H-10 ) and the carbon resonance at d 156. Compound 4 was isolated as white amorphous powder (CH3OH).37 154.7-dihydroxy-4-methylcoumarin.30–3.70 (m) 3. Correlations were observed in the HMBC spectrum between the proton signal at d 4.89 96.30 60. mp 294–295 8C and assigned the molecular formula C16H18O9 by HR-MS ([M]+ m/z 354.29–3.0945).2) 3.3) dC 161.72 (m) 3. and between the proton signal at d 10.

Fan. Dongbo Yu of University of Texas Southwestern Medical Center. Yin. Wang. J. W. J. Biotech. Acknowledgments This research was financially supported by Natural Sciences Foundation of Guangdong (No. Zhao. 3 (3) (2005) 141. F. Z. References [1] [2] [3] [4] L. Chin. C.C. Yan.D. N.Q.M. R. B.M. Soc. Ye. Chin. Ma. Cheng. Y. Biotech. H. S. H. We thank Dr.L.Y. 22 (4) (2006) 619 (English edition). 1) [3–4]. J. Thus. Yu. K. Am.X. C.Y. for his check in our manuscript. Yu et al. which is also a new compound. compound 4 was identified as 7-hydroxy-3. Y. / Chinese Chemical Letters 19 (2008) 76–78 showed that the b-D-glucopyranosyl residue was attached to 5-OH of substrate 2. . Zhao. Trost. 18 (1) (2002) 69. J. Nat. Med. Each signal in the NMR spectra of 4 was assigned by HMBC analyses and comparison with product 3. Wang. K. Greenman.4dimethylcoumarin 5-O-b-D-glucopyranoside (Fig. 125 (2003) 4518. R. Yu. 04010461). Zhang. Toste. Zhang. Chin.78 R. Chem.M.M. USA.D.