この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました。p-Coumaryl alcohol was dehydrogenated with peroxidase and H_2O_2 system. Five dilignols, p-coumarylresinol (I), dehydrodi-p-coumaryl alcohol (II), p-hydroxyphenyl-glycerol-β-coumaryl ether (III), monoepoxylignan (IV) and 5-5'-dilignol (V) were identified and determined by both gas chromatography and NMR spectrometry, and the ratio of the amounts of the three main dilignols (I, II and III) was 31 : 49 : 20. The dilignol (V) was trace in amount (0.6%), and 1, 2-diarylpropane-l, 3-diol (VI) could not be found. The ratio of the racemoid and mesoid couplings at C-β and C-β' carbons was about 9.4 : 1, and the dilignol (III) was a mixture consisting of erythro and threo isomers (1:4.7) whose ratio was determined by gas chromatography. From these results, it was concluded that coniferyl and p-coumaryl alcohols had almost the same reactivity on enzymic dehydrogenation

HIGUCHI, Takayoshi

NAKATSUBO, Fumiaki

Kyoto University Research Information Repository

TitleEnzymic Dehydrogenation of p-Coumaryl Alcohol. : III.Analysis of Dilignols by Gas Chromatography and NMRSpectrometryAuthor(s)NAKATSUBO, Fumiaki; HIGUCHI, TakayoshiCitationWood research : bulletin of the Wood Research Institute KyotoUniversity (1975), 58: 12-19Issue Date1975-03-31URL http://hdl.handle.net/2433/53381RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityEnzymic Dehydrogenation of p-Coumaryl Alcohol.III. Analysis of Dilignols by GasChromatography and NMR Spectrometry*Fumiaki NAKATSUBO** and Takayoshi HIGUCHI**Abstract--p-Coumaryl alcohol was dehydrogenated with peroxidase and HzOz system.Five dilignols, p-coumarylresinol (I), dehydrodi-p-coumaryl alcohol (II), p-hydroxyphenyl-glycerol-J3-p-coumaryl ether (III), monoepoxylignan (IV) and 5-5/-dilignol (V) wereidentified and determined by both gas chromatography and NMR spectrometry, and theratio of the amounts of the three main dilignols (I, II and III) was 31 : 49 : 20. The dilignol(V) was trace in amount (0.6 %), and 1, 2-diarylpropane-1, 3-diol (VI) could not be found.The ratio of the racemoid and mesoid couplings at C-J3 and CoW carbons was about 9.4 : 1,and thedilignol (III) was a mixture consisting of erythro and threo isomers (1: 4.7)whose ratio was determined by gas chromatography. From these results, it was concludedthat coniferyl and p-coumaryl alcohols had almost the same reactivity on enzymicdehydrogenation.IntroductionIn 1951, Freudenberg et at. D reported that p-coumaryl alcohol produced a verysimilar dehydrogenation polymer (DHP) to that of coniferyl alcohol based on thehydrogen uptake by the both DHP's and their elementary analysis. Later, Bland et at. 2)reported that an artificial lignin prepared from p-coumaric acid on potato parenchymaand Sphagnum MWL were highly condensed polymers containing double condensationsat C-3 and C-5 of the p-hydroxyphenyl ring, and suggested the different reactivitybetween p-coumaryl and coniferyl alcohols on dehydrogenation. Recently, Yamasakiet at. 3) reported that no difference of condensation pattern between p-coumaryl andconiferyl alcohols occured in their DHP formation from the yield of the condensed andnoncondensed type compounds obtained by permanganate and hydrogen peroxide oxi-dation of both the methylated DHP's.It seems that this problem is solved more clearly from the yield of the dilignolsof both the alcohols. In the previous paper, the isolation and identification of the fourdilignols of p-coumaryl alcohol, p-coumarylresinol (I), dehydrodi-p-coumaryl alcohol(II), p-hydroxyphenylglycerol-fi-p-coumaryl ether (III) and monoepoxylignan (IV)4\ and* Partly presented at the 23th Meeting of Japan Wood Research Society, Kyoto, April 1973.** Division of Lignin Chemistry.This paper is dedicated to Prof. Dr. Karl Kratzl, Organisch-Chemischen Institut der Un i-versiUit Wien for his 60th birthday.- 12-NAKATSUBO, HIGUCHI: Enzymic Dehydrogenation of p-Coumaryl Alcohl. III.wCH OH CH=CH-CH20H12~1CH ::.....I6-0OHIIvillVTthe trans configuration of the coumarane ring of the dilignol (11)5) were reported.In this paper, configuration of the dilignol (III) and the yields of these dilignolsdetermined by gas chromatography and NMR spectrometry are reported.ExperimentalPreparation of authentic dilignols (I), (II), (III), (IV), (V) and (VI)Dilignols (1), (II), (III) and (IV) were prepared by dehydrogenation of p-coumarylalcohol with peroxidase and HzOz system as reported recently4), and dilignols (II) and(III) were hydrogenated with 5 % Pd-C and hydrogen in a mixed solvent of dioxane/ethanol (2: 1).Tetrahydro-5-5'-dilignol (V) was obtained by dehydrogenation of dihydro-p-cou-maryl alcohol with peroxidase and HzOz system. The spot of this compound on asilica gel TLC plate (Merck Kieselgel PFZ54) showed a sky blue color under UV lamp(TOSHIBA Fl-3-S-type), and easily isolated by preparative TLC. NMR 0 (ppm:CDCl3) of tetra acetate: 1.99 (6 H, s, r, r'-acetyl groups), 2.01 (6 H, s, phenolic acetylgroups), 1.80,......,2.30 (4 H, m, (3, (3'-methylene protons), 2.69 (4 H, t, J =6.5, a, a'-methyleneprotons), 4.01 (4H, t, J=6.5, r, r'-methylene protons), 6.90,......,7.30 (8H, m, aromaticprotons). It is characteristic of 5-5'-dilignol that the phenolic acetyl protons give thepeaks at almost the same field with r, r'-primary alcoholic acetyl protons by theshielding effects of each aromatic rings. MS (70 eV) : 320 (M+, 100), 256 (66.7), 239(57.2), 211 (35), 197 (33.4).1, 2-Diarylpropane-1, 3-diol (VI) was synthesized by condensation of benzyl p-hy-- 13-WOOD RESEARCH No. 58 (1975)droxybenzaldehyde and benzyl p-hydroxybenzoic acid methyl ester and subsequenthydrogenation with 5 % Pd-C and reduction with lithium aluminum hydride. Thissynthetic method and the determination of configuration of the products will bereported elsewhere. NMR 0 (ppm: CD3COCD3) : 2.70,......,3.20 (l H, m, ,B-methine proton),3.50,......,4.20 (2 H, m, r-methylene protons), 4.89 (l H, d, J =9.0, a-methine proton of threoisomer), 5.02 (1 H, d, J =5.5, a-methine proton of erythro isomer), 6.55,......,7.10 (8 H, m,aromatic protons).Dehydrogenation of p-coumaryl alcoholDehydrogenation of p-coumaryl alcohol was carried out by the method reportedpreviously4), and the dilignols were extracted with ethyl acetate and then hydrogenatedwith 5 % Pd-C and hydrogen in dioxane/ethanol (2 : 1). A colorless foaming product,hydro-dilignol fraction (about 5 mg) was dissolved in pyridine (0.1 ml) and thenhexamethyl disilazane (0.1 ml) and trimethylchlorosilane (0.05 ml) were added, suc-cessively. The reaction mixture was shaken vigorously for 1 min. and after 5 min.keeping at room temperature, it was evaporated to dryness in a vacuum desiccatorcontaining PZ0 5• The residue was dissolved in CCl4 (0.5 ml) and analyzed by gaschromatograph-mass spectrometer. The amounts of diligonls were calculated from thepeak area on the chromatogram by using calibration curves prepared previously forthe respective authentic compounds. Alternatively the hydro-dilignol fraction wasanalyzed by NMR spectrometer.NIHONDENSHI J.G.C-750 gas chromatograph with a flame ionization detector anda SHIMAZU-LKB 9000 gas chromatograph-mass spectrometer were used for analysis ofthe trimethylsilyl ethers of the hydro-dilignol fraction at the following condition.Stainless steel column (2 m, 3 mm ID) packed with 2 % OV-17 on chromosorb AW.Column temperature: 220°C. Injector temperature: 250°C. Carrier gas: helium,2 kg/cmz. Mass spectra were taken by the use of a glass column at the same condition,and relative abundance of each peak was designated in parentheses. NMR spectrawere taken by the use of a R-22 HITACHI high resolution NMR spectrometer (90 MHz)with TMS internal standard. Chemical shifts and coupling constants were given ino-values and Hz, respectively.Results and DiscussionConfiguration of the dilignol (III)Recently guaiacylglycerol-,B-guaiacyl ether, the model compound of arylglycerol-,B-aryl ether structure in lignin was synthesized in high yield by the condensationreaction between benzyl vanillin and ethyl 2-methoxyphenoxy acetate and subsequentreduction with 5 % Pd-C and lithium aluminum hydride. The ratio of two isomers(erythro/threo) was about 3: 16). These configurations were determined by compari-- 14-NAKATSUBO, HIGUCHI: Enzymic Dehydrogenation of p-Coumaryl Alcohl. III.tlJreo isomermVo 46 50Time(min)/56Fig. 1. Gas chromatogram of TMS derivatives of dihydro-dilignol (III). Column: 2 %OV-17 on chromosorb A W, 2 m-glass column, 200°e. Carrier gas: helium, 28 ml/min.son with the results reported by Miksche et aI7). In NMR spectra of these acetates,the chemical shifts and coupling constants of a-methine protons were 0 6.12 (1 H, d,J=5.0, erythro isomer) and 0 6.17 (lH, d, J=6.2, threo isomer), respectively and adoublet peak of a-CH of erythro isomer appeared in higher field and gave a smallercoupling constant than that of threo isomer. On the other hand, NMR spectrum ofthe dihydro acetyl derivative of the dilignol (III) gave two doublet peaks at 0 6.09(l H, d, J=5.0) and 06.13 (1 H, d, J=6.2) whose ratio was about 1: 5, and this resultwas also supported by gas chromatography as shown in Fig. 1. The retention timesof TMS derivatives of erythro and threo dihydrodilignols (III) were 47.1 and 49.8min., respectively and the ratio of the peak areas was about 1: 4.7. Thus, it wasconcluded that the dilignol (III) was a mixture consisting of erythro and threoisomer whose ratio was 1: 4.7.Analysis of the dilignols by gas chromatography and NMR spectrometryA dilignol fraction was converted to its hydro-dilignol fraction by catalytichydrogenation with 5 % Pd-C and hydrogen in dioxane/ethanol (2: 1) This catalytic- 15-WOOD RESEARCH No. 58 (1975)hydrogenation was indispensable from the following two reasons.First, the peak area of the propenol dilignols, e. g., dilignol (II) and (III) etc.,on gas chromatogram are not proportional to the amounts of the compounds injected,but only when the propenol side chains are reduced to the propanol side chains, thepeak area of the dilignols are almost proportional to their amounts.Second, the 5-5'-dilignol (V) is seemed to be stable when its propenol side chainsare converted to the propanol side chains by reductions, as found for the coniferyl5-5'-dilignoI8) .FurtherrrlOre, Since the formation of the ring opened compound has been reportedby catalytic hydrogenation of the dilignol (II) in methanol, the mixed solvent, dioxane/methanol (2: 1) which avoided the ring opemng had to be used5).8mV2monoepoxy'i9T7o 10 20 30 40 70Time(min)Fig. 2. Gas chromatogram of TMS derivatives of hydro-dilignol fraction obtained bydehydrogenation of p-coumaryl alcohol with HzOz and peroxidase system. Column:2 % OV-17 on chromosorb A W, 2 m, 220°C. Peak 3: tetrahydro dilignol (V), Peak5: dihydro erythro dilignol (III), Peak 6: dihydro threo dilignol (III), Peak 7 :monoepoxylignan (IV), Peak 8: dihydro dilignol (II), Peak 10: dilignol (I).Figure 2 shows the gas chromatogram of TMS derivatives of the hydro-dilignolfraction. Six of ten peaks were identified by comparison of the retention times andmass fragmentation patterns of authentic dilignols. The amounts of the dilignols arecalculated from the peak areas, and summarized in Table 1. In the Table, column(A) is the retention times of each peak, and (B), (C) and (D) are peak areas, ratio of- 16-NAKATSUBO, HIGUCHI: Enzymic Dehydrogenation of p·Coumaryl Alcohl. III.&5.50 84.96 84.687.0 6.0 4.0 1.0PPM(S)Fig. 3. NMR Spectrum of hydro·dilignol fraction obtained by dehydrogenation ofp·coumaryl alcohol.the peak areas and ratio of the amounts of three mam dilignols, respectively. Column(F) shows the ratio of the amounts of three main dilignols of coniferyl alcohol whichwas reported earlier9).The NMR spectrum of the hydro·dilignol fraction is shown in Fig. 3. a-Methineprotons of the three main dilignols (I), (II) and (III) give the peak at (54.68 (2 H, d,J=4.0), (5 5.50 (l H, m) and (5 4.69 (l H, d, J=6.0), respectively and these peaks arenot interfered with other peaks. Therefore, the ratio of the amounts of these dilignolsTable 1. Estimation of dilignois by gas chromatography and NMR spectrometry.Peak number* A(min.) B(cm2) C(%) D(%) E(%) F(%)123456789107.711. 214.215.820.021. 524.431. 042.067.51.75.01.00.530.35.276.31.448.31.03.00.60.318.03.045.00.828.31.09.4204931204832195427A: retention time, B: peak area, C: ratio of the peak area, D: ratio of three maindilignols, E: ratio of three main diligonls obtained by NMR analysis, F: ratio of threemain dilignols of coniferyl alcohol 9).* peak number corresponds to those of the compounds in Fig. 2.- 17-WOOD RESEARCH No. 58 (1975)will be determined by the integration curve of their a-methine peaks. The result ISgiven in the column (E) of the Table 1.The following results were obtained from these data. TMS derivative of synthetic1, 2-diarylpropane-l, 3-diol (VI) gives a peak at 5.8 min. on gas chromatogram, butthe hydro-dilignol fraction did not give any peak at the same retention time (Fig. 2).Furthermore, p, p'-dihydroxystilbene which was synthesized from the dilignol (VI) byalkali degradation could not be found in the alkali degradation products of DHP anddilignol fraction. Therefore, the dilignol (VI) seems to be formed at a later dehydro-genation stage. Only 0.6 % of 5-5'-dilignol (V) was detected by gas chromatography,and then the double condensation at C-3 and C-5 reported by Bland et al. 2) may notbe possible at this dehydrogenation stage and also at DHP's stage3). The ratio of thep-coumarylresinol (I) and monoepoxylignan (IV) which were formed on the racemoidand mesoid coupling at C-f3 and C-f3' carbons, respectively, was 9.4: 1. Thus, it isexpected that coniferyl and sinapyl alcohols give the corresponding monoepoxylignanswith the same ratio on dehydrogenation. Investigation on this point, is now progres-sing. The ratio of the amounts of the three main dilignols (I), (II) and (III) was31 : 49 : 20, respectively by gas chromatography, and the same result was obtained byNMR analysis as shown in column (E) of Table 1. The ratio of the three coniferyldilignols corresponding to the dilignols (I), (II) and (III) formed on dehydrogenationof coniferyl alcohol9) is shown in column (F) of Table 1. The most reactive radicalof the four resonance radicals of both the alcohols is f3-radical because the three maindilignols are not formed without any participation of f3-radical of the side chain, andthe second reactive one is the radical at 5-position of aromatic ring because the yieldsof the coumarane type dilignols are larger than that of f3-ether type dilignols.Both p-hydroxy cinnamyl alcohols, p-coumaryl and coniferyl alcohols have a similarreactivity on enzymic dehydrogenation as described above, but a typical differenceappears on the amounts of the coumaranes. That is, the percentage of dehydrodi-coniferyl alcohol (54 %) is larger than that of dehydrodi-p-coumaryl alcohol, dilignol(II) (49 %), indicating the radical activating effect of the methoxyl group at 3-positionof aromatic ring.AcknowledgementThis investigation was supported in part by a Scientific Research Fund of theMinistry of Education.References1) K. FREUDENBERG and G. GEHRKE, Chern. Ber., 84, 443 (1951).- 18-NAKATSUBO, HIGUCHI: Enzymic Dehydrogenation of p-Coumaryl Alcohl. III.2) D. E. BLAND and A. F. LOGAN, Biochem. J., 95, 515 (1965).3) T. YAMASAKI, K. HATA and T. HIGUCHI, Mokuzai Gakkaishi, 18, 361 (1972).4) F. NAKATSUBO and T. HIGUCHI, Holzforschung in press.5) F. NAKATSUBO and T. HIGUCHI, Holzforschung in press.6) F. NAKATSUBO, K. SATO and T. HIGUCHI, Holzforschung In press.7) G. E. MIKSCHE, J. GRATZL and M. FRIED-MATZKA, Acta Chern. Scand, 20, 1038 (1966).8) K. FREUDENBERG and KLAUS-CHRISTIAN RENNER, Chern. Ber., 98, 1979 (1965).9) K. V. SARKANEN and C. H. LUDWIG, Lignins, 140 (1971), John Wiley Sons, Inc., New York.- 19-

Enzymic Dehydrogenation of p-Coumaryl Alcohol. : III. Analysis of Dilignols by Gas Chromatography and NMR Spectrometry

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Enzymic Dehydrogenation of p-Coumaryl Alcohol. : III. Analysis of Dilignols by Gas Chromatography and NMR Spectrometry

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