Isolation of 2-(1′,2′,3′,4′-tetrahydroxybutyl)-6-(2″,3″,4″-trihydroxybutyl)-Pyrazine from Yunnan flue-cured tobacco and its syntheses

Chen Yongkuan, Kong Ningchuan, Li Cong, Wang Hanqing
Yunnan Reascend Tobacco Technology Co. LTD., Kunming, 650106,China; Department of Application Chemistry, Yunnan University, Kunming, 650091, China; State Key Laboratory of OSSO, Lanzhou Institute of Chemistry Physics, Chinese Academy of Sciences, Lanzhou, 730000, China)

AbstractA 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.

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 literature. 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 [20=-94.6 (1.15×10-3, HO) and mp 380(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 HNMR with DO and DOCD as solvent at 20showed 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) were substituted DO and DOCD as solvent at 100, 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 (CH), 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 C1220 with a ring and double bond 4. The structure of the compound (Fig.2) has been determined by the H-H COSY, 13C-H 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 HO were mixed. After the mixture was refluxed at 100for 6h, it was concentrated under reduced pressure in a water bath temperature not exceeding 45. And MeOH (10ml) was added to the concentration. The filtrate was cooled down to 0and 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 HNMR, 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.
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Fig.1 Isolation Procedure of Polyhydroxy Pyrazine I

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

position 13   position 13  
1′ 72.2 5.14,5.15(d)   144.3 8.20(s)
2′ 74.2 3.82(m)   154.8
3′ 71.8 3.78(m) 1″ 38.5 2.95(m),3.16(m)
4′ 63.8 3.82(m),3.62(m) 2″ 72.0 4.00(m)
  156.8 3″ 75.1 3.62(m)
  141.1 8.40() 4″ 63.3 3.78(m),3.63(m)

s=single; d=double; m=multiple

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. HNMR 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 MeSi 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. [20=-94.6 (1.15×10-3, HO) and mp 380(decomposing). IR(cm-1): 3308. MS(m/z): 304. UV(nm): 375. HNMR(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. [20=-94.8 (1.15×10-3, HO) and mp 380℃(decomposing). IR(cm-1): 3307. MS(m/z): 304. UV(nm): 375. HNMR(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.2 The structure of the title compound


Fig.3 The possible synthetic processing of the title compound