Chen Yongkuan¹, Kong Ningchuan¹, Li Cong², Wang Hanqing³
(¹ Yunnan Reascend Tobacco Technology Co. LTD., Kunming, 650106,China; ²Department of Application Chemistry, Yunnan University, Kunming, 650091, China; ³State Key Laboratory of OSSO, Lanzhou Institute of Chemistry Physics, Chinese Academy of Sciences, Lanzhou, 730000, China)

Received Aug. 20, 2007.

Abstract A C₁₂-polyhydroxy derivative of 1,4-diazine isolated from Yunnan flue-cured tobacco was identified as 2-(1',2',3',4'-tetrahydroxybutyl)-6-(2",3",4"-trihydroxybutyl)-pyrazine. Its structure was determined by the spectral data and synthetic method.
Keywords flue-cured tobacco; polyhydroxy pyrazine derivative; isolation; synthesis

A large number of the volatile flavor constituents of tobacco have been thought to be relative to pyrolysis of precursors during heating and burning of tobacco. In contrast with many examinations of precursors, reports having been published mainly discussed nor-carotenoids and flavonoids. A C₁₃-nor-carotenoid glucoside and a C₁₁-nor-carotenoid glucoside from flue-cured tobacco had been isolated and identified^[1,2]. However, very few reports on the precursors of polyhydroxy derivatives of pyrazines have been published. In this paper, the authors first report the isolation and identification of a polyhydroxy pyrazine from Yunnan flue-cured tobacco as 2-(1',2',3',4'-tetrahydroxybutyl)-6-(2",3",4"-trihydroxybutyl)-pyrazine. Its structure was identified by the spectral data and synthetic method.
    The isolation procedure for polyhydroxy pyrazine I was illustrated in Fig.1. The isolation procedure was improved upon the method described in the literature¹. The MeOH No.3 fraction (960mg) was obtained from flue-cured tobacco(3kg). Further, the MeOH No.3 fraction was re-crystallized. After successively crystallization twice, polyhydroxy pyrazine I was obtained as a white powder sample(18mg) with [a]²⁰ _D=-94.6 (1.15×10^-3, H₂O) and mp 380^oC(decomposing). The molecular weight of polyhydroxy pyrazine I was determined to be 304 by FAB mass spectrometry. The strong absorption at 275nm in the UV spectra was due to the conjugated aromatic ring, and the stretching vibration broad band at 3308cm^-1 in the infrared spectra showed that there associated with hydroxyl group in the structure. The ¹HNMR with D₂O and DOCD₃ as solvent at 20^oC showed that the compound has 13 non-active protons, and two of them are in low field, indicated that the protons attached to the aromatic ring. When DMSO and Eu(dpm) ₃ were substituted D₂O and DOCD₃ as solvent at 100^oC, 7 active protons clearly showed up. It is indicated that there are 7 hydroxy protons in the compound. The ¹³CNMR of DEPT demonstrated only 10 carbons with missing 2 carbons in low field. This implicated that the 12 carbons in this compound were different: 3 of them were secondary carbons (CH₂), 7 tertiary (CH) and 2 quarternary (C). Furthermore, the two tertiary carbons in low field for the 3-C and 5-C, together with missing two for the 2-C and 6-C, strongly suggested that the title compound was C₁₂H₂₀N₂O₇ with a ring and double bond 4. The structure of the compound (Fig.2) has been determined by the ¹H-¹H COSY, ¹³C-¹H COSY and its chemical shifts listed in Table 1. Among them, the long range heteronuclear correlation between 1'-H and 2-C and between 1'-H and 3-C indicated the 1'-C bonding to 2-C directly. Similarly, the long range heteronuclear correlation between 1"-H and 6-C and between 1"-H and 5-C indicated the 6-C bonding to 1"-C directly. The HMBC point of 5-C with 3-H, and of 3-C with 5-H, clearly showed the 2,6-bissubstituted pyrazine.
    To confirm the above structural elucidation, polyhydroxy pyrazine II was prepared by the Maillard reaction^[3] (Fig.3). D-glucose(1.8g), ammonium formate(4.5g) and 10 ml H₂O were mixed. After the mixture was refluxed at 100^oC for 6h, it was concentrated under reduced pressure in a water bath temperature not exceeding 45^oC. And MeOH (10ml) was added to the concentration. The filtrate was cooled down to 0^oC and placed for 24h. A buff crystal can be obtained. After successively crystallization three times with water, polyhydroxy pyrazine II was obtained as a white powder sample. The ¹HNMR, ¹³CNMR and IR spectra of the prepared polyhydroxy pyrazine II were identical with those of polyhydroxy pyrazine I obtained from Yunnan flue-cured tobacco. The absolute configuration of the polyhydroxy pyrazine ,however, could not be determined owing to the small amount of the sample. Up to now, there has been no clear explanation for the formation of tobacco polyhydroxy pyrazines. It needs our further researches whether or not the polyhydroxy pyrazines are considered to be a precursor of volatile pyrazines constituents.

Fig.1 Isolation Procedure of Polyhydroxy Pyrazine I

Table 1 The assignment of C, H of the title compound isolated from Yunnan tobacco

s=single; d=double; m=multiple

EXPERIMENTAL                                     
Chemicals. The following compounds were obtained commercially: D-glucose, ammonium formate, n-hexane , methanol(Beijing chemical plants). The solvents were distilled before use.
Instrumental analysis. Mass spectra was obtained with VG AutoSpec-3000. Infrared spectra was obtained with Bio-Rad FtS-135. Specific rotation was measured with a JASCO DIN-370. ¹HNMR and ¹³CNMR spectra were obtained Bruke AM400 and DRX-500. Melting point was obtained with a XRC-1. The chemical shifts are as ppm field from Me₄Si as internal standard.
Isolation of polyhydroxy pyrazine I. The isolation procedure was shown in Fig.1. polyhydroxy pyrazine I was obtained as white powder crystals. [a]²⁰ _D=-94.6 (1.15×10^-3, H₂O) and mp 380^oC(decomposing). IR(cm^-1): 3308. MS(m/z): 304. UV(nm): 375. ¹HNMR(δ): 2.94(m), 3.15(m), 3.62(m), 3.63(m), 3.65(m), 3.76(m), 3.79(m), 3.81(m), 3.83(m), 4.02(m), 5.15, 5.16(d), 8.22(s) , 8.41(s). ¹³CNMR(δ): 38.5, 63.3, 63.9, 71.8, 72.0, 72.2, 72.3, 75.2, 141.1, 144.3, 154.8, 156.9.
Synthesis of polyhydroxy pyrazine II. The possible synthetic processing was shown in Fig.3. Polyhydroxy pyrazine II was obtained as white powder crystals. [a]²⁰ _D=-94.8 (1.15×10^-3, H₂O) and mp 380℃(decomposing). IR(cm^-1): 3307. MS(m/z): 304. UV(nm): 375. ¹HNMR(δ): 2.95(m), 3.16(m), 3.61(m), 3.62(m), 3.63(m), 3.78(m), 3.79(m), 3.82(m), 3.83(m), 4.00(m), 5.14, 5.15(d), 8.20(s), 8.40(s). ¹³CNMR(δ): 38.7, 63.5, 64.0, 72.1, 72.2, 72.4, 74.4, 75.4, 141.2, 144.4, 155.0, 157.0.

Fig.3 The possible synthetic processing of the title compound

REFERENCES
[1]Hisashi Kodama, Takane Fujimori, Kunio Kato. Agric.Biol.Chem.,1982,46(5): 1409-1411.
[2] Hisashi Kodama, Takane Fujimori, Kunio Kato. Agric.Biol.Chem.,1982,45(3): 941-943.
[3]Li H, Li PW, Yang ZH. Chemistry of heterocyclic compounds in flavors and aroms. 1^st edition. Beijing: China Light Industry Press, 1992(in Chinese).

云南烤烟中2-(1',2',3',4'-四羟基丁基)-6-(2",3",4"-三羟基丁基)吡嗪的分离与合成
陈永宽¹，孔宁川¹，李聪²，汪汉卿³
（¹ 云南瑞升烟草技术（集团）有限公司，昆明650106；²云南大学应用化学系，昆明650091；³中科院兰州化学物理研究所OSSO国家重点实验室，兰州730000)
摘要 从云南烤烟中提取分离得到了一个具有C₁₂结构的1,4-吡嗪的多羟基衍生化合物，并采用光谱技术鉴定其为2-(1',2',3',4'-四羟基丁基)-6-(2",3",4"-三羟基丁基)吡嗪。同时，采用合成技术对其结构进行了确认。
关键词 烤烟；多羟基吡嗪衍生物；分离；合成

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