Letter Cite This: Org. Lett. 2018, 20, 312−314
pubs.acs.org/OrgLett
N‑Glucosides of Fairy Chemicals, 2‑Azahypoxanthine and 2‑Aza-8oxohypoxanthine, in Rice Jae-Hoon Choi,†,‡,§ Jing Wu,‡ Azusa Sawada,† Syougo Takeda,† Hirohide Takemura,† Kaoru Yogosawa,§ Hirofumi Hirai,†,‡,§ Mitsuru Kondo,‡ Kunihisa Sugimoto,∥ Tomohiro Asakawa,⊥ Makoto Inai,# Toshiyuki Kan,# and Hirokazu Kawagishi*,†,‡,§ †
Graduate School of Integrated Science and Technology, ‡Research Institute of Green Science and Technology, and §Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan ∥ Research & Utilization Division, Japan Synchrotron Radiation Research Institute, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan ⊥ Tokai University Institute of Innovative Science and Technology, 4-1-1 Kitakaname, Hiratsuka City, Kanagawa 259-1292, Japan # School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan S Supporting Information *
ABSTRACT: Plant growth stimulators, 2-azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), were isolated from the fairy-ring-forming fungus, Lepista sordida, and AHX-treated rice, respectively. Further metabolites of AHX were detected in AHX-treated rice by HPLC, and the metabolites 1−4 were isolated from the rice. The structures of 1−4 were determined by spectroscopic analysis and synthesis. Compounds 1−4 exhibited no significant activity against rice, indicating that rice regulates the activity of AHX and AOH by converting them into their glucosides.
T
First, we investigated whether AHX and AOH were converted to further metabolites after these were absorbed into rice seedlings or not. HPLC profiles of AHX and AOH metabolites were very similar to each other, and characteristic spectral properties of the metabolites from HPLC with photodiode-array detection were very similar to those of AHX and AOH (Figure S1, Supporting Information). In order to obtain the metabolites, the extracts of the AHX-treated seedlings were fractionated by ODS gel flash column chromatography followed by HPLC, leading to the purification of the novel compounds 1−4 (Figure 1). Compound 1 was purified as a white amorphous material. The molecular formula was determined as C10H13N5O7 by HRESIMS (m/z 314.0724 [M − H]−; calcd for C10H12N5O7, 314.0737), indicating the presence of seven degrees of unsaturation in the molecule. The structure of 1 was elucidated by interpretation of NMR spectra including DEPT, COSY, HMQC, and HMBC (Figure 2). The DEPT experiment indicated the presence of a methylene, five methines, and four quaternary carbons. 13C NMR data (δC 114.0, 142.1, 150.5, 154.8) indicated that 1 had the same skeleton as that of AOH (δC 113.2, 142.3, 148.6, 152.6) and possessed an additional six carbons compared with AOH. The signals in the region of δH 3.0−6.1 in the 1H NMR and δC 60−85 in the 13C NMR, the COSY correlations (H-1′/H-2′; H-2′/H-3′; H-3′/H-4′; H-4′/
he rings, ribbons, or arcs of regulated plant growth, which often occur on the floor of woodlands and grasslands, are commonly called “fairy rings”, owing to the interaction between a fungus and a plant.1 This phenomenon had been attributed to unknown “fairies” before our chemical disclosure. We found that 2-azahypoxanthine (AHX) and imidazole-4-carboxamide (ICA) are plant growth regulators produced by one of the fairyring forming fungi, Lepista sordida.2 Furthermore, we isolated a common metabolite of AHX in plants, 2-aza-8-oxohypoxanthine (AOH).3 We named the three compounds “fairy chemicals” (FCs) after the title of the article in Nature.4 FCs regulated the growth of all of the plants tested regardless of their species, and various examinations indicated that plants developed tolerance to various and continuous stress (low or high temperature, salt, or drought stress, etc.) from the environment by treatment with FCs, resulting in the growth promotion. Furthermore, FCs increased the yields of rice, wheat, and other varied crops in greenhouse and/or field experiments.2,3,5 We have also reported the endogenous presence of AHX and AOH in plants and the discovery of a new route in purine metabolic pathway in which AHX and AOH are biosynthesized.3,6 Based on the above results, we hypothesize that FCs are a new family of hormones in plants. To certify the hypothesis, it is necessary to know the further metabolism of those compounds. Herein, we describe the isolation and structure determination of metabolites 1−4 of AHX and AOH in rice. © 2017 American Chemical Society
Received: December 1, 2017 Published: December 13, 2017 312
DOI: 10.1021/acs.orglett.7b03736 Org. Lett. 2018, 20, 312−314
Letter
Organic Letters
compared with those of O-glycosides7 indicated that the βglucopyranosyl group was connected to the N1 position. The complete assignment of all the proton and carbon signals of NMR was accomplished as shown in Table 1. Compounds 2 and 3 were purified as white amorphous powders. The molecular formulas of 2 and 3 were determined as C10H13N5O7 by HRESIMS (2; m/z 314.0723 [M − H]− and 3; m/z 314.0724 [M − H]−; calcd for C10H12N5O7, 314.0737), indicating that both compounds were isomers each other and had seven degrees of unsaturation in the molecules. The structures of 2 and 3 were elucidated by interpretation of NMR spectra including DEPT, COSY, HMQC, and HMBC (Figure 2). The 1H and 13C NMR data indicated that 2 and 3 were also AOH-glucosides like 1 (Table 1). The positions of the bonds between the sugar part and aglyconewere determined by HMBC correlations (2; H-1′/C-4, C-8, 3; H-1′/C-5, C-8). The structure of 2 was confirmed by X-ray crystallography analysis (Figure S6, Supporting Information). Compound 4 was purified as a white amorphous material. The molecular formula was determined as C10H13N5O6 by HRESIMS (m/z 298.0778 [M − H]−; calcd for C10H12N5O6, 298.0788), indicating the presence of seven degrees of unsaturation in the molecule. The structure of 4 was elucidated by interpretation of NMR spectra including DEPT, COSY, HMQC, and HMBC (Figure 2). The DEPT experiment indicated the presence of a methylene, six methines, and three quaternary carbons. 13C NMR data (δC 120.2, 146.0, 151.6, 154.1) indicated that 4 possessed the same skeleton as that of AHX. The NMR data of the sugar moiety in 4 were very similar to those of 1−3. Confirmation of the stereochemistry of 4 including absolute configuration was performed by comparison with the synthetic sample. As shown in Scheme 1, the synthesis of 4 was performed by regio- and diastereoselective incorporation of a glucose unit into N1 positon of AHX. After regioselective incorporation of a diphenyl urea group at the N9 position, condensation with glucose unit was carried out by treatment of 5 with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (6) in the presence of CsCO2 to provide 7. Presumably, this glycosylation reaction would proceed through the neighboring participation of the acetoxy group at C-2 position of glucose to afford 7 in a diastereoselective manner. Treatment of 7 with ammonia in methanol and simultaneous removal of diphenylurea and an acetyl group proceeded smoothly to give 4. For the detailed synthetic procedure, see the SI. The NMR data and the specific rotation ([α]D25 −20, c
Figure 1. Structures of AHX, AOH, and their metabolites.
Figure 2. COSY and HMBC correlations of 1−4.
H-5′), and the HMBC correlations (H-1′/ C-2′, C-3′, C-5′; H2′/C-1′, C-3′; H-3′/C-2′, C-4′, C-5′; H-4′/C-3′,C-5′, C-6′; H5′/C-3′, C-4′, C-6′; H-6′/C-4′, C-5′) suggested that the additional part was a hexose. In addition, the vicinal diaxial coupling constants between H-1 and H-2 (9.2 Hz), H-2 and H3 (9.5 Hz), H-3 and H-4 (9.2 or 9.5 Hz), and H-4 and H-5 (9.5 or 9.2 Hz) indicated that the sugar part was a β-glucopyranose (Table 1 and Figure 2). The HMBC cross-peak (H-1′/C-6) and the relatively high field chemical shift at C-1′ (δC 84.1) Table 1. 1H and 13C NMR Data for 1−4 1 (in D2O) 1
position 4 5 6 8 1’ 2’ 3′ 4’ 5′ 6’a 6’b
H
δH (mult, J, Hz)
6.06 4.18 3.65 3.50 3.67 3.66 3.82
(d, 9.2) (dd, 9.2, 9.5) (m) (dd, 9.5, 9.2) (m) (m) (dd, 11.0, 4.5)
2 (in D2O) 13
1
C
δC, type 142.1, 114.0, 150.5, 154.8, 84.1, 71.3, 77.0, 69.8, 79.7, 61.1,
C C C C CH CH CH CH CH CH2
H
δH (mult, J, Hz)
5.46 4.44 3.62 3.58 3.62 3.70 3.83
(d, 9.5) (dd, 9.5, 9.0) (m) (m) (m) (dd, 12.5, 5.5) (dd, 12.5, 2.0)
3 (in CD3OD) 13
1
C
δC, type 142.5, 114.4, 150.8, 153.1, 82.6, 69.9, 77.0, 69.6, 79.5, 61.1,
C C C C CH CH CH CH CH CH2
313
H
δH (mult, J, Hz)
5.49 4.39 3.52 3.49 3.52 3.68 3.87
(d, 9.0) (dd, 9.0, 8.5) (m) (m) (m) (dd, 12.0, 5.5) (dd, 12.0, 5.2)
4 (in D2O) 13
1
C
δC, type 143.9, C 113.7, C 150.2, C 153.5, C 84.2, CH 72.6, CH 78.6, CH 71.0, CH 80.9, CH 62.9 CH2
H
δH (mult, J, Hz)
8.27 6.05 4.17 3.61 3.45 3.61 3.62 3.75
(s) (d, 9.2) (dd, 9.2, 8.5) (m) (dd, 9.5, 9.0) (m) (m) (dd, 10.0, 1.5)
13
C
δC, type 151.6, C 120.2, C 154.1, C 146.0, CH 83.9, CH 71.3, CH 77.0, CH 69.8, CH 79.6, CH 61.0 CH2
DOI: 10.1021/acs.orglett.7b03736 Org. Lett. 2018, 20, 312−314
Organic Letters
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Scheme 1. Synthesis of Compound 4 from AHX
Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kunihisa Sugimoto: 0000-0002-0103-8153 Toshiyuki Kan: 0000-0002-9709-6365 Hirokazu Kawagishi: 0000-0001-5782-4981 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partially supported by Grants-in-Aid for Young Scientists (Start-up; 26892013, and A, 16H06192) from MEXT. We thank K. Okamoto (Ushio ChemiX Co. Ltd.) for providing AHX. The synchrotron radiation X-ray powder diffraction measurements were carried out at BL02B1/SPring-8 in Japan (Proposal No. 2016B1508).
0.26 MeOH) of synthetic 4 were completely identical with those of natural 4, indicating that the absolute configuration of 4 was as shown. Compounds 1−3 were also isolated from AOH-treated rice. The CD spectra for 1−3 were very similar to those of synthetic 4 (Figure S7, Supporting Information). D-Glucose oxidase oxidized the acid hydrolysate of 2 and 3, indicating that the sugars in 2 and 3 were D-Glc (Experimental Section). Compounds 1−4 were examined for growth regulatory activity toward rice. All the N-glucosides exhibited no statistically significant activity (Figure S8, Supporting Information). One of the plant hormones, cytokinin, has a purine skeleton like AHX and AOH and is also converted into Nglucosides and O-glucosides. Those glucosides are more stable metabolically against cytokinin degradation enzymes than the corresponding free bases.8 In terms of their physiological activity, the O-glucosides and the N7- and N9-glucosides exhibit little or no activity against plants, suggesting that they are inactive forms of the plant horomone.9 The N7- and N9glucosides are usually resistant to glucosidases; thus, they cannot be converted into active cytokinins.10 To investigate the further conversion of N-glucosides 1−4, rice was treated with 1−4. The conversions from 1 and 4 into AOH were observed in both the shoot and root (Figure S9, Supporting Information). However, 2 and 3 were stable and did not convert into AOH in rice. The glucosides (2 and 3) could not be hydrolyzed by β-glucosidase like in the case of cytokinins. Thus, the modification is thought to be an irreversible inactivation. The results suggested that the plant regulates the activity of AHX and AOH by forming their glucosides. In conclusion, we succeeded in the isolation and purification of novel N-glucosides of fairy chemicals (1−4) from AHXtreated rice. N-Glucosides exhibited no statistically significant activity against rice. These results suggest that metabolites 1−4 play an important role in the regulation of endogenous FCs homeostasis.
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REFERENCES
(1) (a) Couch, H. B. Diseases of Turfgrasses, 3rd ed.; Krieger: Malabar, FL, 1995; pp 181. (b) Smith, J. D.; Jackson, N.; Woolhouse, A. R. Fungal Diseases of Amenity Turf Grasses, 3rd ed.; E. and F. N. Spon: London, U.K., 1989; p 339. (c) Shantz, H. L.; Piemeisel, R. L. J. Agric. Res. 1917, 11, 191. (d) Evershed, H. Nature 1884, 29, 384. (e) Ramsbottom, J. Nature 1926, 117, 158. (2) (a) Choi, J.-H.; Fushimi, K.; Abe, N.; Tanaka, H.; Maeda, S.; Morita, A.; Hara, M.; Motohashi, R.; Matsunaga, J.; Eguchi, Y.; Ishigaki, N.; Hashizume, D.; Koshino, H.; Kawagishi, H. ChemBioChem 2010, 11, 1373. (b) Choi, J.-H.; Abe, N.; Tanaka, H.; Fushimi, K.; Nishina, Y.; Morita, A.; Kiriiwa, Y.; Motohashi, R.; Hashizume, D.; Koshino, H.; Kawagishi, H. J. Agric. Food Chem. 2010, 58, 9956. (3) Choi, J.-H.; Ohnishi, T.; Yamakawa, Y.; Takeda, S.; Sekiguchi, S.; Maruyama, W.; Yamashita, K.; Suzuki, T.; Morita, A.; Ikka, T.; Motohashi, R.; Kiriiwa, Y.; Tobina, H.; Asai, T.; Tokuyama, S.; Hirai, H.; Yasuda, N.; Noguchi, K.; Asakawa, T.; Sugiyama, S.; Kan, T.; Kawagishi, H. Angew. Chem., Int. Ed. 2014, 53, 1552. (4) Mitchinson, A. Nature 2014, 505, 298. (5) (a) Asai, T.; Choi, J.-H.; Ikka, T.; Fushimi, K.; Abe, N.; Tanaka, H.; Yamakawa, Y.; Kobori, H.; Kiriiwa, Y.; Motohashi, R.; Deo, V. P.; Asakawa, T.; Kan, T.; Morita, A.; Kawagishi, H. JARQ 2015, 49, 45. (b) Tobina, H.; Choi, J.-H.; Asai, T.; Kiriiwa, Y.; Asakawa, T.; Kan, T.; Morita, A.; Kawagishi, H. Field Crop Res. 2014, 162, 6. (6) Suzuki, T.; Yamamoto, N.; Choi, J.-H.; Takano, T.; Sasaki, Y.; Terashima, Y.; Ito, A.; Dohra, H.; Hirai, H.; Nakamura, Y.; Yano, K.; Kawagishi, H. Sci. Rep. 2016, 6, 39087. (7) Yoshinari, T.; Sakuda, S.; Furihata, K.; Furusawa, H.; Ohnishi, T.; Sugita-Konishi, Y.; Ishizaki, N.; Terajima, J. J. Agric. Food Chem. 2014, 62, 1174. (8) McGaw, B. A.; Horgan, R. Planta 1983, 159, 30. (9) (a) Letham, D. S.; Palni, L. M. S. Annu. Rev. Plant Physiol. 1983, 34, 163. (b) Skoog, F.; Armstrong, D. J. Annu. Rev. Plant Physiol. 1970, 21, 359. (c) Hecht, S. M.; Frye, R. B.; Werner, D.; Hawrelak, S. D. J. Biol. Chem. 1975, 250, 7343. (10) Mok, D. W. S.; Mok, M. C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52, 89.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03736. Experimental section; synthesis; HPLC profiles; 1D NMR spectra; X-ray crystallographic data for 3; CD spectra; metabolism of 1−4 (PDF) 314
DOI: 10.1021/acs.orglett.7b03736 Org. Lett. 2018, 20, 312−314
