The present work aims at clearing the structure of some p-menthenediols, which are already known to be present in musts and wines as glycosides, referred to as isomers 1, 2 and 3. Isomers 2 and 3 correspond to cis-p-menth-1-ene-6,8-diol and p-menth-1-ene-7,8-diol; isomer 1, which is only found in traces, is probably identical with trans- p-menth-1-ene-6,8-diol. Their concentration is reported for both free and bound forms in musts from some varieties and compared with the alpha-terpineol and 2-exo-hydroxy-1,8-cineole concentrations.  In general, the free forms were scarcely present; among the bound forms p-menth-1-ene-7,8-diol first and then cis-p-menth-1-ene-6,8-diol prevailed. Besides improvement of varietal classification, the compounds identified give evidence of allylic hydroxylase activity in grape berries

Mandery, H.

Rapp, A.

Reniero, F.

Versini, G.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 30, 143-149 (1991) Structural identification and presence of some p-menth-1-enediols in grape products by G. VERSINil), A. RAPP2), F. RENIER01) and H. MANDERY3) l) Istituto Agrario Provinciale, Laboratorio di Analisi e di Ricerca, San Michele all'Adige, Italia 2) Bundesanstalt für Züchtungsforschung im Wein- und Gartenbau, Institut für Rebenzüchtung, Geilweilerhof, Siebeldingen, BR Deutschland 3) BASF AG, Ludwigshafen/Rh., BR Deutschland Aufklärung der Struktur und Vorkommen von p-Menth-1-endiolen in Traubenerzeugnissen Z u s a m m e n f a s s u n g : In der vorliegenden Arbeit wird die Strukturaufklärung einiger p-Menthendiole gegeben, die bereits als Glycoside in Traubenmosten und Weinen nachgewiesen und als Isomere 1, 2 und 3 bezeichnet wurden. Es zeigte sich, daß cis-p-Menth-l-en-6,8-diol (ds-Sobrerol) und p-Menth-l-en-7,8-diol mit den Isomeren 2 und 3 übereinstimmen und trans-p-menth-l-en-6,8-diol (trans-Sobrerol), das nur in geringen Spuren gefunden wurde, vermutlich mit dem Isomer 1 identisch ist. In Traubenmosten verschiedener Rebsorten wurden die Gehalte dieser Verbindungen sowohl in der freien als auch in der gebundenen Form ermittelt und mit den Gehal-ten von a-Terpineol und 2-0xo-hydroxy-1,8-cineol verglichen. Die freien Komponenten Jagen im allgemeinen nur in geringer Konzentration vor. Bei den gebundenen Formen war p-Menth-1-en-7,8-diol vorherrschend, gefolgt von cis-p-Menth-1-en-6,8-diol. Die identifizierten Verbindungen erlauben eine bessere Sortencharakterisierung, darüber hinaus läßt das Vorkommen dieser Kom-ponenten auf eine Allylhydroxylase-Aktivität in Weinbeeren schließen. K e y wo r d s : must, flavour, terpene, free and bound constituents, analysis, variety of vine, classification, allylic hydroxylase. Introduction Recent research showed the presence of some p-menthenediols in must and/or wines either as a-terpineol metabolites of Botrytis cinerea (MANDERY 1986; RAPP and MANDERY 1988) or as typical grape compounds either in free form, such as two isomers of p-menth-8-ene-1,2-diol (BAUMES et al. 1986) or as glycosides. The latter include the p-menth-1-ene-8,9-diol (WINTERHALTER et al. 1990) and the so called p-menthenediols -isomers 1 and 2 (spectral data in STRAUSS et al. 1987) or isomers 1, 2 and 3 (WILLIAMS et al. 1989; WINTERHALTER et al. 1990). No spectral data are reported for WILLIAMS' isomer 1, whereas isomers 2 and 3 correspond to 1 and 2 reported by STRAUSS, from the same working group. These are not yet defined as concerns the position of the double bound as well as the position of at least one of the two alcoholic functions, the other one being likely in the position C 8 because of the relevant MS fragment at m/, 59. These com-pounds or some of them have been found in Rhine Riesling musts and wines as agly-cons after enzymatic hydrolysis or in other varieties after chemical hydrolysis. In this study the undefined structures of the two most relevant above mentioned isomers are cleared, a probable structure of the third one, present only in traces, is pre-sented, and the concentrations of these compounds in some varietal musts are exem-plified. 144 G. VERSINI, A. RAPP, F. RENIERO and H. MArw&RY Materialsand methods Materials Must samples of different grape varieties from the vine collection of the Istituto Agrario Provinciale or from vineyards of the same area were an1:1lyzed. Reference compounds 1. trans p-menth-1-ene-6,8-diol ( trans sobrerol) was purchased from Aldrich. 2. cis p-menth-l--ene-6,8-diol (cis sobrerol) was synthesized from (-)carveol (isomer mixture from Aldrich) by hydroxylation in water at pH 1, uri.der stirring for 15 h at room temperature and subsequent extraction of the raw reaction products with pen-tane: methylene chloride, 2:1 in vol., and purification by preparative TLC silica gel (ethyl acetate: toluene, 9:1 in vol.). In this way, it was also possible to separate par-tially (80 % ) the cis isomer from the trans form (yield of ca. 8 °/0 ). Mass spectral data (m/, (%)): 152 (4), 137 (21), 119 (3), 109 (62)• 94 (57), 79 (86), 69 (30), 59 (100), 55 (25), 43 (79), 41 (38), 31 (28), similar to those already reported by MANDERY (1986). 1H and 13C NMR spectra data (o, ppm): 1H NMR (300 MHz, (CD3)2CO): 1.17 (s,6,8-CH3), 1.34 (m,1,4-H), 1.74 (m,3,7-H), 1.85 (m,1,5-H), 2.24 (m, 3_}1), 3.28 (s,1,8-0H), 3.72 (d,l,J = 6.1 Hz, 6-0H), 4.11 (m,1,6-H), 5.43 (m,1,2-H); 13C NMR (75 MHz, (CD3)2CO): 20.92 (C-7), 26.92 (C-3), 27.79 (C-9), 28.11 (C-10), 35.69 (C-5). 45.66 (C-4), 68.77 (C-6), 71.58 {C-8), 123.53 (C-2), 136.0 (C-1). 3. p-menth-1-ene-7,8-diol was synthesized from (S) (-)perillyl alcohol (Aldrich) by using the same cited reaction and purification processes (yield of ca. 5 % ). Mass spectral data: (m/, (%)): 152 (11), 137 (2), 121 (12), t09 (24), 94 (20), 93 (25), 91 (24), 81 (18), 79 (94), 67 (16), 59 (100), 55 (19), 43 (64), 41 (30). 31 (32). 1H and 13C NMR (o, ppm, CDC13) spectral data accord with tlJOSe partially published by BLUTHE et al. (1980). except for 3.93 (s,2,7-H). Methods 100 ml of must, after addition of 2-octanol as internal standard (23 µg), clarification with bentonite (700 mg/l) and centrifugation, were subjected to liquid chromatography on Amberlite XAD-2 adsorbent {0.1-0.2 mm; Serva) using 1:1 glass column (15 x 120 mm) (GUNATA et al. 1985). The free and bound compounds were separately eluted as previously reported (VERSINI et al. 1988). The bound fraction was enzymatically hydro-lyzed (RohapectC (Röhrn), buffer Citrate at pH 5, 15 hat 40 °c); after addition of the same quantity of internal standard, it was extracted three tiifles by hand with pen-tane : methylene chloride, 2:1 in vol., concentrated and quantified by capillary gas chromatography. Two C.Erba HRGC 5300 gas chromatographs with FID were used. One was equipped with two fused silica WCOT capillary columns: He'W'lett-Packard HP-20 M (30 m; 0.32 mm i.d.; d.f. = 0.5 µm) and J and W DB-WAX (30 tn; 0.32 mm i.d.; d.f. 0.3 µm). The other gas chromatograph had a fu.sed silica S\l.pelco SPBtm-5 (60 m; 0.32 mm i.d.; d.f. = 0.25 µm) WCOT capillary column. The respective temperature programmes were: 5 min at 55 °C, 2.5 °C/min up to 193 °C, 50 min at 193 °C; Carrier gas (H2)-flow: 25 ml/ min; t.inj = t.det = 250 °C; split injection. 5 min at 80 °C, 2 °C/min up to 260 °C, 50 min at 260 °C; carrier gas (H2)-flow: 1.5 ml/ min; t.inj = t.det = 280 °C; split injection. p-Menth-1-enediols in grape products 145 Electron impact mass spectra were recorded at 70 eV with a 5979 Hewlett-Packard mass detector connected to a 5980 Hewlett-Packard gas chromatograph equipped with a HP-20M fused silica WCOT capillary column (25 m; 0.2 mm i.d.; d.f. = 0.2 µm); He as carrier gas, splitless injection; t.inj = 220 "C, t.source = 200 °C, and programmed tem-perature: 1 min at 60 "C; 10 °C/min up to 80 "C, 0.5 min at 80 °C, 2.5 °C/min up to 190 °C, 30 min at 190 °C. The 1H and 13C NMR spectra were recorded with a Broker AC 300 instrument. Results and discussion 1. Identification of p-menth-l-ene-7,8-diol and of cis and trans p-menth-1 ene-6,8-diols An allylic hydroxylase activity on monoterpenic compounds is known to be present in several plants (KARP and CROTEAU 1988). Thus, the synthese of trans 2,6-dimethy-locta-2, 7-diene-1,6-diols from linalool by suspension cells of Nicotiana tabacum (SuGA et al. 1976) and - with some cis isomer, too - by cultured Botrytis cinerea (RAPP et al. 1986) can be explained. Both the same compounds were found as free and bound forms and in different ratios in grapes (RAPP et al. 1986; STRAUSS et al. 1988; VERSINI et al. 1988). When starting from a.-terpineol, some p-menthenediols were produced by Botrytis cinerea (MANDERY 1986; RAPP and MANDERY 1988) and p-menth-l-ene-7,8-diol, cis and transp-menth-1-ene-6,8-diols by Nicotiana tabacum callus suspension cells (SUGA et al. 1980). These compounds have been searched for also in grapes or in grape products, assuming that a similar biotransformation of a.-terpineol takes place. The three com-pounds identified on tobacco were found mostly as glycosides (Fig. 2); their retention 8 7 1 1 '1 50 6 1 5 1 3 1 l u~~u 1 UßWJ~ 4 60 70 min l ~~ 80 Fig.1: Part of a FID-chromatogram (DB-Wax) of an extract of Silvaner must including p-menth-1-ene-diols aglycons. - 1 = p-menth-1-ene-7,8-diol, 2 cis sobrerol, 3 = trans geranic acid, 4 = trans sobrerol, 5 = cis 8-hydroxy linalool + hydroxy geraniol, 6 = trans 8-hydroxy linalool, 7 = 3,7-dimethyl-1,5-diene-3,7-diol ( = terpendiol I), 8 = 2-phenylethanol. Ausschnitt aus dem FID-Chromatogramm (DB-Wax) eines Extraktes aus Silvanermost mit dem Bereich der p-Menth-1-en-diol-Aglykone. 146 g 100 90 80 70 60 50 G. VERSINI, A. RAPP, F. RENIERO and H. MANDERY 43 Average of 44.23 to 44.46 min. 5 79 ~ ~H .g 40 ::1 ..Cl <{ QI u c 0 "Cl c .6 <{ 30 109 93 20 67 152 10 137 O-<--.......ll.1111.1-+uuµll..l.f-..L.µJwµ.l..,-llljJU~..,w.'l'--,--~f---.--'TW....~-.-'-r--r--r' 1001 90 80 70 60 50 40 30 20 40 60 80 100 120 140 Moss I Charge Fig. 2: Mass spectrum of p-menth-l-ene-7,8-diol of grape must. Massenspektrum von p-Menth-1-en-7 ,8-diol aus Traubenmost. 40 Average of 40.40 to 40.66 min. 59 94 69 60 80 100 Moss I Charge HOA ~H 109 137 120 140 Fig. 3: Mass spectrum of transp-menth-1-ene-6.B-diol ( = trans-sobrerol) of grape must. Massenspektrum von trans-p-Menth-1-en-6,8-diol ( = trans Sobrerol) aus Traubenmost. p-Menth-1-enediols in grape products 147 times on capillary columns with different polarity (HP-20M, DB-WAX and SPBtm-5) and mass spectral data were either the same as with the reference compounds or simi-lar for the most relevant fragments i.e. for trans p-menth-1-ene-6,8-diol. Fig. 1 shows a short section of a chromatogram of an extract from Silvaner must. The identified compounds (p-menth-1-ene-7,8-diol, cis and trans p-menth-1-ene-6,8-diol (cis and trans sobrerol)) together with few other aroma compounds (i.e. 2-phenyle-thanol, trans geranic acid, terpendiol I) are marked. The mass spectra of p-menth-1-ene-diols identified in grape must are shown in Fig. 2 (p-menth-1-ene-7,8-diol) and Fig. 3 ( cis p-menth-1-ene-6,8-diol). These spectra correspond with those of the refer-ence substances. So it was possible to ascertain that the already mentioned p-menthe-nediol isomers 1 and 2 of STRAUSS et al. (1987) or 2 and 3 of WILLIAMS et al. (1989) correspond respectively to the cis p-menth-1-ene-6,8-diol and to the p-menth-1-ene-7,8-diol, whereas the third or isomer 1, reported by WILLIAMS et al. (1989), probably cor-responds to trans p-menth-1-ene-6,8-diol. The p-menth-1-ene-7,8-diol was also reported as an unidentified compound of a-terpineol metabolite of Botrytis cinerea (MANDERY 1986; RAPP and MANDERY 1988). 2. Presence of identified compounds in musts of some varieties In the table the concentrations of free and bound forms of the above components as well as those of a-terpineol and of 2-exo-hydroxy-1,8-cineole, another probable a-terpineol derivative recently identified (BITTEUR et al. 1990), are reported for musts from different varieties. Contents (ppb) of free and bound a-terpineol and its derivatives in different varietal musts Gehalte an freiem und gebundenem a-Terpineol und daraus resultierenden Derivaten (ppb) in Mosten verschiedener Rebsorten a-Terpi- 2-exo-OH- trans cis p-Menth-1-Varieties neo! 1,8-Cineole Sobrerol Sobrerol ene-7 ,8-diol b b b b b Chardonnay + 0.4 + + Teroldego• 0.4 + + + + + 0.4 Pinotblanc + 0.9 + 0.5 + 1 + 9 Pinotnoir* + 3.3 + 1.3 + 0.4 + 1.5 1 17 Sauvignon + 13 1.1 3.5 + + 2 0.6 30 blanc Sylvaner + 56 + 20 0.4 + 8 2.7 102 Rhine Ries- 3.0 51 1.7 10.5 + 0.5 0.4 12.6 2.8 89 ling Gewürz- + 16.5 + 7 + + + 6.5 + 22 traminer Yellow Muscat 32.5 59 3.1 4.8 0.6 0.9 + 2.5 3.5 25 ofTrentino Rose Muscat* 5.5 47.5 1.1 10 0.4 0.8 0.7 20 8 200 f = Freeform. -: <0.1 ppb. b = Bound form. +: 0.1-0.3 ppb. . = Red grape variety. 148 G. VERSINI, A. RAPP, F. RENIERO and H. MANDERY Generally, they are present as glycosides andin varieties with a content approxi-mately higher than 1 ppb of bound a-terpineol. Among the other p-menthenediols, p-menth-1-ene-7,8-diol prevails in all cases, similar as with the free compounds meta-bolized by tobacco cells, but in grape must the ratio between cis and trans p-menth-1-ene-6,8-diol concentrations is inverted, i.e. the cis isomer is much more abundant than the trans one, which is only present in traces or not detectable. A higher level of bound cis p-menth-1-ene-6,8-diol has recently been found as typi-cal for grapes and its derivatives from the Traminer family (Traminer, Gewiirztraminer and Savagnin) (VERSINI et al. 1990). Summary The present work aims at clearing the structure of some p-menthenediols, which are already known to be present in musts and wines as glycosides, referred to as iso-mers 1, 2 and 3. Isomers 2 and 3 correspond to cis p-menth-1-ene-6,8-diol and p-menth-1-ene-7,8-diol; isomer 1, which is only found in traces, is probably identical with trans p-menth-1-ene-6,8-diol. Their concentration is reported for both free and bound forms in musts from some varieties and compared with the a-terpineol and 2-exo-hydroxy-1,8-cineole concentrations. In general, the free forms were scarcely present; among the bound forms p-menth-1-ene-7,8-diol first and then cis p-menth-1-ene-6,8-diol prevailed. Besides improvement of varietal classification, the compounds identified give evidence of allylic hydroxylase activity in grape berries. Financial support of Progetto Strategico: „lnnovazione produttiva nelle piccole e medie imprese", sottoprogetto: „Qualita e tipicita degli alimenti: metodologie innovative d'indagine" -C.N.R. (Rome) is acknowledged. Literature BAUMES, R.; CoRDONNIER, R.; Nrrz, S.; DRAWERT, F.; 1986: Identification and determination of volatile constituents in wines from different wine cultivars. J. Sei. Food Agricult. 37, 927-943. BrrrEUR, S. M.; BAUMES, R. L.; BAYONOVE, C. L.; VERSINI, G.; MARTIN, C. A.; DALLA SERRA, A.; 1990: 2-exo-hydroxyl-1,6-cineole: a new component from grape var. Sauvignon. J. Agricult. Food Chem. 38, 1210-1213. BLUTHE, N.; Ecaro, J.; FETIZON, M.; LAZARE, S.; 1980: Cyclobutane ring opening of pin-2(10)-ene with mercury (II) salts. A new, high-yield synthesis of p-mentha-1,8-diene-7-ol. J. Chem. Soc. Per-kin l, 1747-1751. GUNATA, Y. Z.; BAYONOVE, c. L.; BAUMES, R. L.; CoRDO!'/NIER, R. E.; 1985: The aroma of grapes. I. Extrac-tion and determination of free and glycosidically bound fractions of some grape aroma com-ponents. J. Chromatogr. 331, 83-90. KARP, F.; CROTEAu, R.; 1986: Role of hydroxylases in monoterpene biosynthesis. In: ScHREIER, P. (Ed.): Bioflavour 87, 173-198. de Gruyter, Berlin, New York. MANDERY, H.; 1986: Einfluß von Botrytis cinerea auf die Monoterpenfraktion des Weinaromas. Diplomarbeit, Univ. Karlsruhe. RAPP, A.; MA1''DERY, H.; 1988: Influence of Botrytis cinerea on the monoterpene fraction of wine aroma. In: SCHREIER, P. (Ed.): Bioflavour 87, 445-452. de Gruyter, Berlin, New York. - - ; --; N1EBERGALL, H.; 1986: Neue Monoterpendiole in Traubenmost und Wein sowie in Kul-turen von Botrytis cinerea. Vitis 25, 79-84. STRAuss, C. R.; GOOLEY, P. R.; WILSON, B.; WILLIAMS, P. J.; 1987: Application of droplet countercurrent chromatography to the analysis of conjugated forms of terpenoids, phenols, and other consti-tuents of grape juiee. J. Agricult. Food Chem. 35, 519-524. p-Menth-1-enediols in grape products 149 - - ;--; W1LSON, B.; WILLIAMS, P. J.; 1988: Novel monoterpene diols and diol glycosides in Vitis vinifera grapes. J. Agricult. Food Chem. 36, 569-573. SUGA, T.; AOK!, T.; HIRATA, T.; LEE, Y. S.; NISHIMURA, O.; UTSUMI, M.; 1980: Biotransformation of for-eign substrates with callus tissues. Transformation of terpineols with tobacco suspension cells. Chem. Lett. 15, 229-230. - - ; HIRATA, T.; H1RAN0, Y.; ITo, T.; 1976: Biotransformation of monoterpenes by tobacco tissue cultures. Selective hydroxylation of trans-methyl group in isopropyridene group. Chem. Lett. 11, 1245---1248. VERSINI, G.; DALLA SERRA, A.; DELL'EvA, M.; SCIENZA, A.; RAPP, A.; 1988: Evidence of some glycosidi-cally bound new monoterpenes and norisoprenoids in grapes. In: SCHREIER, P. (Ed.): Biofla-vour 87, 161-170. de Gruyter, Berlin, New York. - - ; --; Sc1ENZA, A.; BARCHETTI, P.; 1990: Particolaritä compositive dell'uva e de! vino Traminer aromatico: confronto fra cultivar e variazioni a livello terpenico in fermentazione e nell'invec-chiamento. Proc. Symp. Gewürztraminer, Bolzano, May 22nd (in press). WILLIAMS, P. J.; SEITON, M. A.; WILSON, B.; 1989: Nonvolatile conjugates of secondary metabolites as precursors of varietal grape flavor components. In: TERANISHI, R.; BUTTERY, R. G.; SHAHIDI, F. (Eds.): Flavor Chemistry: Trends and Developments, 35---48. ACS Symp. Ser. No. 388. Ameri-can Chemical Society, Washington, DC. WINTERHALTER, P.; SEITON, M.A.; WILLlAMS, P.J.; 1990: Two-dimensional GC-DCCC analysis of the glycoconjugates of monoterpenes, norisoprenoids, and shikimate-derived metabolites from Riesling wine. J. Agricult. Food Chem. 38, 1041-1048. Received, 21. 3. 1991 Dr. G. VERSINI lstituto Agrario Provinciale Laboratorio di Analisi e di Ricerca 1-38010 San Michele all'Adige (Trento) Italia Prof. Dr. A. RAPP Bundesanstalt für Züchtungsforschung im Wein- und Gartenbau Institut für Rebenzüchtung Geilweilerhof D-6741 Siebeldingen BR Deutschland 

Structural identification and presence of some p-menth-1-enediols in grape products

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Structural identification and presence of some p-menth-1-enediols in grape products

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