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3-Methylbutanoyl and 3-methylbut-2-enoyl disaccharides from green coffee beans (Coffea arabica)
Phytochemistry 60 (2002) 409–414 www.elsevier.com/locate/phytochem
3-Methylbutanoyl and 3-methylbut-2-enoyl disaccharides from green coffee beans (Coffea arabica) Bernhard Weckerlea, Tama´s Ga´tib, Ga´bor To´thb,1, Peter Schreiera,* a Lehrstuhl fu¨r Lebensmittelchemie, Universita¨t Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany Technical Analytical Research Group of the Hungarian Academy of Sciences, Institute for General and Analytical Chemistry, Budapest University of Technology and Economics, Szent Gelle´rt te´r 4, H-1111 Budapest, Hungary
b
Received 26 November 2001; received in revised form 23 January 2002
Abstract Three 3-methylbutanoyl and 3-methylbut-2-enoyl disaccharides isolated from green coffee beans (Coffea arabica) were identified as 3-methylbutanoyl-1-O-b-d-glucopyranosyl-b-d-apiofuranoside, 3-methylbutanoyl-6-O-a-d-glucopyranosyl-b-d-fructofuranoside, and 3-methylbut-2-enoyl-1-O-b-d-glucopyranosyl-b-d-apiofuranoside. The structures were established by one- and twodimensional 1H and 13C NMR spectra as well as by ESI MS/MS spectra. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Coffea arabica; Flavour precursors; Green coffee; 3-Methylbutanoyl-1-O-b-d-glucopyranosyl-b-d-apiofuranoside; 3-Methylbutanoyl-6O-a-d-glucopyranosyl-b-d-fructofuranoside; 3-Methylbut-2-enoyl-1-O-b-d-glucopyranosyl-b-d-apiofuranoside; HPLC–ESI–MS/MS; 1H NMR; 13 C NMR; Sugar conjugates
1. Introduction In the past, a great deal of attention has been focused on the analysis of volatile flavour compounds of green and roasted coffee (Czerny and Grosch, 2000; Mayer and Grosch, 2001; Clarke and Vitzthum, 2001), information about their biogenetic sugar-bound precursors in green coffee, however, is surprisingly scarce. Whereas our knowledge about flavour progenitors in plant tissues has been extended tremendously in the last decade (Winterhalter and Skouroumounis, 1997; Winterhalter et al., 1999), for coffee the studies about glycoconjugated precursors are restricted to diterpenoid glycosides (Maier and Wewetzer, 1978; Bradbury and Balzer, 1999). In most of the bound flavour constituents characterized to date glycosidic linkages prevail. The rare information about sugar esters comprises few substances, for instance, the precursor of marmelo lactones in quince (Winterhalter et al., 1991), glucopyranosyl anthranilate from pinuela fruit (Parada et al., 1996), carbohydrate * Corresponding author. Tel.: +49-931-888-5481; fax: +49-931888-5484. E-mail addresses: [email protected] (P. Schreier), [email protected] (G. To´th). 1 Tel.: +36-1-463-3411; fax: +36-1-463-3408.
esters of cinnamic acid from various fruits (Latza et al., 1996), and the glucose ester of (E)-2,6-dimethyl-6hydroxyocta-2,7-dienoic acid from Riesling wine (Winterhalter et al., 1997). In this paper, we describe the isolation and identification of novel 3-methylbutanoyl and 3-methylbut-2-enoyl disaccharides from green coffee beans (Coffea arabica).
2. Results and discussion From a water-methanol extract of grounded and defatted green coffee beans (Coffea arabica, provenience Ethiopia) a fraction containing flavour precursors was obtained by chromatography on Amberlite XAD. The screening for sugar conjugates in the methanolic fraction after Amberlite XAD chromatography was performed by enzymatic hydrolysis of the lyophylized extracts with subsequent HRGC–MS analysis. Among the liberated volatiles, 3-methylbutanoic acid and 3methylbut-2-enoic acid were identified, both serving as target compounds for the following separation steps, which comprised several fractionations by preparative and analytical reversed phase liquid chromatography on glass columns and HPLC columns, finally yielding the sugar esters 1–3 in pure forms (cf. Experimental). The
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(02)00042-0
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screening for sugar conjugates in the fractions was carried out by HPLC coupled with an evaporative light scattering detector (ELSD) using an RP-18 column. In addition, the fractions obtained in the course of the clean-up procedure and the purified compounds 1–3 were analysed by HPLC–ESI–MS/MS (negative mode) to get first structural information. The recorded spectral data are outlined in Table 1. The data reveal that compounds 1–3 are acyl disaccharides with molecular masses of 426, 396 and 394, respectively (quasimolecular ions [MH] at m/z 424.9, 394.9 and 392.9 for compounds 1–3, respectively). Additional signals at m/z 322.9 (compound 1), 292.9 (compounds 2 and 3) and m/z 161.1 (compounds 1–3) indicated that the substructural sugar units consist of two hexose moieties in case of compound 1 and each of one hexose and one pentose unit for compounds 2 and 3, respectively. Finally, structural evaluation was performed by a number of NMR experiments. The structures of 1–3 (Fig. 1) were determined by 1D and 2D NMR studies using DEPT, COSY, sel-1D TOCSY (Stott et al., 1995), HSQC and HMBC techniques including gradient selection and linear prediction. Despite of severe signal overlap even at 600 MHz, we achieved a complete assignment not only for the carbon but also for the proton signals. The 1H and 13C chemical shifts and couplings of compounds 1–3 are summarized in Table 2 (1H NMR) and Table 3 (13C NMR). The 1H NMR spectra of 2 and 3 showed unusually low chemical shift for the anomeric protons. Whereas in b-d-glucosides the anomeric proton resonates around 4.5 ppm, the anomeric proton in 2 and 3 showed a downfield shift and resonated at 5.34 ppm and 5.36 ppm indicating ester linkages (Loveys and
Millborrow, 1981). In case of compound 1 the protons 60 a and 60 b of one of the hexose moieties showed a downfield shift ( 3.83 ppm and 3.65 ppm) in comparison with a terminal glucose moiety, indicating an ester linkage at position 6 of the hexose unit. For compounds 2 and 3 the hexose unit could be identified as a glucose moiety (J20 ,30 – J30 ,40 – J40 ,50 – 9.0 Hz) exhibiting b-configuration, indicated by the coupling constant J10 ,20 =8.2 and 8.4 Hz, respectively. As to the acyl units, the data of 1 were in agreement with those of 2 ( 171.1 ppm and 172.3 ppm for the C=O, 42.5 and 42.3 ppm for C-2, 25.0 and 25.1 for C-3 and 22.2 and 22.1 for the terminal methyl groups), confirming the results of HRGC–MS analysis. In the 1H NMR spectrum of 1 the H-4 and H-5 protons are accidentally isochronous, whereas in compound 2 the terminal methyl groups show a small difference ( 0.90 ppm and 0.91 ppm, respectively). Intensive HMBC connectivities between the protons of 2 H-10 ( 5.34 ppm) and of 1 H-60 a ( 4.24 ppm) and H-60 b ( 4.01 ppm) and the carbonyl carbons ( 171.1 ppm and 172.3 ppm, respectively) proved the linkage between the acid and the sugar moieties at the particular positions. The linkage at position 6 of the glucose moiety of 1 caused a strong downfield shift of C-60 and an upfield shift of C50 , which is in agreement with literature data (Yoshimoto et al., 1980). In contrast, the 13C NMR data of 3 with chemical shifts of 164.2 ppm (C=O), 115.1 ppm (C-2) and 159.2 ppm of C-3 pointed out the presence of an a,b-unsaturated ester substructure (Friebolin, 1999). The data of 3 in the 1H NMR spectrum showed for the methyl protons of the acyl moiety two distinct singlets at 1.91 ppm and 2.13 ppm as well as a singlet of an olefinic proton at 5.72 ppm. In addition, there was a
Table 1 Main fragments of ESI mass spectra [achieved in MS/MS negative ion mode with argon as collision gas and a water (5 mM NH4OAc)–acetonitrile (5 mM NH4OAc)-gradient] of compounds 1–3 m/z
1
2
3
485.2 455.3 453.3 424.9 394.9 392.9 322.9 292.9 161.1 148.9
[Ma+AcOHb–H] – – [Ma–H]
Ø [Ma+AcOHb–H] – – [Ma–H] – – [Ma–Ac–H] [Hexd–H2O–H] [Pene–H]
Ø Ø [Ma+AcOHb–H] – – [Ma–H] – [Ma–Ac–H] [Hexd–H2O–H] [Pene–H]
– [Ma–Ac–H] – [Hexd-H2O-H]
a
M=total molecule. AcOH=acetic acid. c A=acyl moiety (for 1 and 2: 3-methylbutanoic acid, for 3: 3-methylbut-2-enoic acid). d Hex=hexose moiety. e Pen=pentose moiety. b
Fig. 1. Structures of compounds 1–3.
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B. Weckerle et al. / Phytochemistry 60 (2002) 409–414 Table 2 1 H chemical shifts of compounds 1–3, measured in DMSO-d6, compared with literature data Position
1
Referencea
2
3
Referenceb
Acyl moiety
3-Methylbutanoyl
Octanoyl
3-Methylbutanoyl
3-Methylbut-2-enoyl
Aglycone linalool
1cis 1trans 2 3 4 5 6 7 8 9 10
– – 2.19 (m) 1.99 (m) 0.90 (d; 6.7) 0.90 (d; 6.7)
– – 2.4–2.6 (m) 1.5–1.7 (m) 1.2 j j 1.4 0.7–1 (m)
– – 2.24 (d; 7.0) 2.00 (m) 0.90 (d; 7.0)c 0.91 (d; 7.0)c
– – 5.72 (s) – 1.91 (s) 2.13 (s)
5.19 (d; 10.5) 5.26 (d; 18.0) 5.85 (dd; 10.5; 18.0) – 1.64 (m) 1.99 (m) 5.18 (m)
5.34 (d) 3.49 (dd; J10 ,20 3.7; J20 ,30 9.8) 3.71 (t) 3.38 (t; J30 ,40 9.8) 3.95–4.04 (m) 4.20 (dd; J50 ,60 a 5.2; J60 a,60 b 12.1) 4.37 (dd; J50 ,60 b 5.2)
5.34 (d; J10 ,20 8.2) 3.09 (dd; J20 ,30 – 9.0) 3.23 (t; J30 ,40 – 9.0) 3.06 (t; J40 ,50 – 9.0) 3.36 (m) 3.81 (dd; J50 ,60 a 2.0; J60 a,60 b 10.0) 3.38 (m)
1.67 (s) 1.59 (s) 1.36 (s)
Glucose moiety 10 20 30 40 50 60 a
5.17 (d; J10 ,20 3.7) 3.20 (dd; J20 ,30 9.7) 3.48 (t; J30 ,40 9.7) 3.06 (t; J40 ,50 9.7) 3.90 (m) 4.24 (dd; J50 ,60 a 1.0 J60 a,60 b 10.0 ) 4.01 (dd; J50 ,60 b 6.0)
60 b
Fructose moiety 00
1 1ab00 200 300 400 400 a 400 b 500 500 a 500 b 600 ab a b c
– 3.38 (m) – 3.88 (d; J300 ,400 8.0) 3.73 (m) – – 3.58 (m) – – 3.55 (m)
5.36 (d; J10 ,20 8.4) 3.05 (dd; J20 ,30 – 9.0) 3.23 (t; J30 ,40 – 9.0) 3.11 (t; J40 ,50 – 9.0) 3.37 (m) 3.83 (dd; J50 ,60 a 1.0; J60 a,60 b 10.0) 3.39 (dd; J50 ,60 b 5.0)
4.5 (d; 7.9) 3.2 (t; 8.4) 3.4 (t; 8.9) 3.34 (t; 9.0) 3.49 (m) 3.35 (m) 3.78 (m)
4.79 (d; J100 ,200 3.1) – 3.75 (d) – – 3.84 (d; J400 a,400 b 9.3) 3.57 (d) – 3.35 (d; J500 a,500 b 11.6) 3.38 (d) –
5.06 (d; 3.0) – 3.95 (d; 3.0) – – 3.86 (2d; 10.2) 4.03 (2d; 10.2) – 3.64 (s) 3.64 (s) –
Apiose moiety – 3.57–3.63 (m) – 4.16 (d) 3.97 (t; J300 ,400 8.8) – – 3.79–3.88 (m) – 3.72–3.79 (m)
4.80 (d; J100 ,200 3.0) – 3.75 (d) – – 3.83 (d; J400 a,400 b 9.2) 3.57 (d) – 3.32 (d; J500 a,500 b 11.3) 3.30 (d) –
6-O-Octanoylsucrose, measured in D2O at 300 MHz (Thevenet et al., 1999). Linalyl-3-O-b-d-apiofuranosyl-(1–6)-b-d-glucopyranoside, measured in D2O at 360 MHz (Mbairaroua et al., 1994). Signals are interchangeable.
NOE effect between H-2 and H-4 noticed. The linkage between the acid and the sugar unit of 3 via the carbon C-1 of the glucose moiety was proven by an HMBC connectivity between proton H0 1 ( 5.36 ppm; d; J10 ,20 8.4) and the carbonyl carbon of the acid. With regard to the second sugar units of compounds 1–3 (hexose for 1, pentose for 2 and 3) the 1H and 13C NMR data of 2 agreed with those of compound 3. These pentose moieties were identified as b-d-apiofuranose. The coupling constants of the terminal apiose unit between the anomeric protons H-100 and H-200 showed values of 3.0 Hz and 3.1 Hz, respectively, indicating bconfiguration. Configuration of C-300 was determined by NOESY experiments. Between protons H-100 and 500 a and 500 b, respectively, there was not any NOE con-
nectivity detectable, indicating trans-constitution of proton H-100 and the C-500 methylene group. Additionally, the data of the apiose moieties were in good agreement with those of literature (see Table 2 and 3). The data of the complete sugar moiety of 1 were in accurate consistence with those of an acyl-6-O-sucrose (Mbairaroua et al., 1994). The coupling constant J10 ,20 of the glucose moiety with 3.7 Hz confirmed a-configuration. By means of TOCSY experiments (irradiating 5.17 ppm, mix 120 ms and irradiating 3.73 ppm, mix 120 ms) a complete assignment for the proton signals of the glucose and the fructose moiety was achieved. In addition, an HMBC connectivity between H-10 and C-200 was detected. The coupling constants J20 ,30 , J30 ,40 and J40 ,50 (all 9.7 Hz) as well as J300 ,400 and J400 ,500 (both 8.0 Hz) are in
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Table 3 13 C NMR chemical shifts of compounds 1–3, measured in DMSO-d6, compared with literature data Position
1
Referencea
2
3
Referenceb
Acyl moiety
3-Methylbutanoyl
Octanoyl
3-Methylbutanoyl
3-Methyl-2-butenoyl
Aglycone linalool
C=O 1 2 3 4 5 6 7 8
172.3 – 42.4 25.1 22.1 22.1
177.1 – 34.1 31.4 28.6 28.4 24.7 22.4 13.7 – –
171.1 – 42.5 25.0 22.2 22.2 – – – – –
164.2 – 115.1 159.2 27.0 20.1 – – – – –
– 117.00 142.40 82.30 40.90 22.60 125.20 134.30 25.60 17.70 23.00
93.9 (1JCH=161 Hz) 72.4 76.3 69.7 76.2 67.3
93.5 (1JCH=161 Hz) 72.3 76.4 69.7 76.2 67.4
98.00
109.1 75.7 78.8 73.2 63.0 –
109.80 77.50 80.10 74.30 64.40
22.1 – – – Glucose moiety
10
91.5
92.3
20 30 40 50 60
71.5 72.7 70.1 70.0 63.4
71.4 72.7 70.0 70.7 61.8
Fructose moiety 100 200 300 400 500 600
62.2 103.9 76.9 74.5 82.7 62.6 a b
74.00 76.60 70.70 75.20 68.60
Apiose moiety 63.1 104.1 76.7 74.5 81.9 63.6
109.1 (1JCH=170 Hz) 75.8 78.8 73.3 63.1 –
6-O-Octanoylsucrose, measured in D2O at 75 MHz (Thevenet et al., 1999). Linalyl-3-O-b-d-apiofuranosyl-(1–6)-b-d-glucopyranoside, measured in D2O at 360 MHz (Mbairaroua et al., 1994).
accordance with the diaxial arrangement of the corresponding protons. The data of the sucrose moiety were in good agreement with those of literature (see Tables 2 and 3). In conclusion, three new sugar esters were isolated from green beans of Coffea arabica and identified as 3methylbutanoyl-6-O-a-d-glucopyranosyl-b-d-fructofuranoside (1), 3-methylbutanoyl-1-O-b-d-glucopyranosylb-d-apiofuranoside (2) and 3-methylbut-2-enoyl-1-O-bd-glucopyranosyl-b-d-apiofuranoside (3). These compounds supplement information about the structural composition of acyl disaccharides in plant tissues and extend our knowledge on sugar-bound flavour precursors.
3. Experimental 3.1. Extraction and isolation Green coffee beans (Coffea arabica) (1 kg; origin Ethiopia) were grounded and defatted by extraction
with petroleum ether. After drying the remaining powder was extracted with 2.5 l of a methanol-water mixture (7:1, v/v) for 24 h at ambient temperature. The filtrate was concentrated under reduced pressure to approximately 200 ml and the remaining aqueous phase was lyophilized. After redilution with 15 ml of water the aqueous extract was applied onto a glass column (1080 cm) filled with Amberlite1 XAD-2 (Supelco, Bellefonte, USA). After washing with water (1.5 l) and subsequently with diethyl ether (1 l) the sugar conjugates were eluted with methanol (1.5 l). The eluate was concentrated under reduced pressure and the residue lyophilized to yield 16 g dry powder. The powder was suspended in chloroform (25 ml) and applied onto a glass column (875 cm) filled with silica gel 60. The elution was performed using stepwise each of 1 l-mixtures of hexane-chloroform (90:10; 70:30; 50:50, v/v), 750 ml-mixtures of dichloromethane-ethyl acetate (75:25; 60:40; 25:75, v/v), and 800 ml-mixtures of methanol-ethyl acetate (20:80; 30:70; 50:50; 75:25, v/ v), collecting 200 ml-fractions at a flow rate of 15 ml
B. Weckerle et al. / Phytochemistry 60 (2002) 409–414
min1. The fractions were checked by HPLC–ELSD and, after enzymatic hydrolysis, by HRGC–MS analysis. The target acids, 3-methylbutanoic acid and 3methylbut-2-enoic acid, were detected in fractions 36–39 eluted by the 50:50 methanol–ethyl acetate mixture. After solvent separation from these combined fractions under reduced pressure, lyophilization of the residue yielded 3.7 g of a white powder. The powder, redissolved in 5 ml of water, was fractionated by LC on a RP-18 column (2.520 cm; LiChrospher1, Merck, Darmstadt, Germany) using a methanol-water gradient starting with 100% water (250 ml) and increasing the proportion of methanol in steps of 10% (each step 250 ml) to end up with 100% methanol. Twenty milliliter fractions were collected at a flow rate of 5 ml min1 and analyzed by HPLC–ELSD as well as, after enzymatic hydrolysis, by HRGC–MS. Fractions 34–37 (containing 3-methylbutanoic acid after hydrolysis) and 38–42 (containing 3-methylbut-2-enoic acid after hydrolysis) each were combined, concentrated under reduced pressure and lyophilized, yielding 150 mg (combined fractions; 1A) and 120 mg (combined fractions; 1B) of white powders, respectively. Further purification of fraction 1A, redissolved in 5 ml of water, was performed by HPLC on a preparative RP-18 column using acetonitrile-water (10:90, v/v) at a flow rate of 6 ml min1 and using a split valve for synchronous ELSD detection to collect detected signals in different flasks. Per injection 7 mg of the extract were fractionated; the procedure was repeated 21 times. Fractions of the same composition were combined, concentrated under reduced pressure and lyophilized. Enzymatic hydrolysis with subsequent HRGC-MS analysis revealed two eluates containing 3-methylbutanoic acid (2A=compound 1: yield 7 mg; 3A=compound 2: yield 2.5 mg) which were separately subjected to final spectroscopic analyses. For the purification of fraction 1B two more fractionation steps were necessary. The first fractionation of the powder redissolved in 5 ml of water was done by HPLC on a preparative RP-18 column using a linear gradient of acetonitrile-water from 1% acetonitrile (t=0 min) to 20% acetonitrile (t=45 min) at a flow rate of 6 ml min1 and using a split valve for synchronous ELSD detection to collect detected signals in different flasks. Per injection 8 mg of the extract were fractionated; the procedure was repeated 15 times. Fractions of the same composition were combined, concentrated under reduced pressure and lyophilized. These extracts were checked, after enzymatic hydrolysis, by HRGC–MS for their content of 3-methylbut-2-enoic acid. One fraction was obtained containing a precursor (2B) from which the acid was liberated after enzymatic hydrolysis. For final purification fractionation was done by HPLC on an analytical RP-18 column using a linear gradient of acetonitrile-water (flow rate 1 ml min1) from 1% ace-
413
tonitrile (t=0 min) to 50% acetonitrile (t=45 min) and using a split valve for synchronous ELSD detection to collect the detected signals in different flasks. Per injection 0.35 mg of the extract were fractionated; the procedure was repeated 20 times. Fractions of the same composition were combined, concentrated under reduced pressure and lyophilized. These extracts were checked, after enzymatic hydrolysis, by HRGC–MS for their content of 3-methylbut-2-enoic acid. One fraction (3B=compound 3: yield 1.2 mg) was obtained containing a precursor from which the acid was liberated by enzymatic hydrolysis. 3.2. Enzymatic hydrolysis Aliquots (1–10 mg) of the lyophilized extracts were incubated in 5 ml of an 0.1 M phosphate buffer (pH 5.7) for 24 h at 37 C using Rohapect VRF1 (Ro¨hm, Darmstadt). After acidification to pH 2, the suspensions were extracted twice with 5 ml diethyl ether, the combined organic phases were dried over sodium sulphate and carefully concentrated to approximately 0.5 ml by distillation on a Vigreux column. These organic extracts were analyzed by HRGC–MS. 3.3. NMR spectroscopy NMR spectra were recorded in CD3OD using Bruker Avance DRX-600 and DRX-500 spectrometers. Chemical shifts are given on the -scale and were referenced to TMS. In the 1D and 2D NMR experiments pulse programs were taken from the Bruker software library. 3.4. HPLC–ELSD HPLC–ELSD analysis was performed with a Knauer HPLC Maxi Star including Eurochrom 2000 software and a Sedere Evaporative Light Scattering Detector (ELSD, model Sedex 55). The temperature of the detector was set to 40 C and compressed air was used at a pressure of 240 kPa. HPLC was carried out (i) on an analytical Eurospher 100 C-18 column (2504.6 mm I.D., 5 mm) (Knauer) and (ii) a preparative Eurospher 100 C-18 column (25016 mm I.D., 5 mm) (Knauer) with water and acetonitrile as solvents (conditions cf. section 3.1). 3.5. HPLC–ESI MS/MS Mass spectrometry was performed on a Finnigan triple-stage quadrupole TSQ 7000 HPLC–MS/MS system (Finnigan MAT, Bremen, Germany) with electrospray ionization (ESI) as interface in negative mode. For HPLC, an Applied Biosystems (Foster City, CA, USA) Model 140B pump was used. For ESI in the negative ion mode the spray capillary voltage was set to 3.0 kV
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and the temperature of the heated inlet capillary was set to 230 C. Nitrogen served both as sheath gas (482 MPa) and as auxiliary gas. HPLC was carried out on an Eurospher 100 C-18 column (1002 mm I.D., 5 mm) (Knauer) with a linear gradient of water (5 mM NH4OAc)–acetonitrile (5 mM NH4OAc) at a flow rate of 0.2 ml min1 from 1 to 30% acetonitrile in 30 min. Negative ions were detected scanning from m/z 200 to 1500 with a total scan duration of 1.0 s. The MS/MS experiments were performed at a collision energy of 15– 25 eV with argon (0.24 Pa) serving as collision gas, scanning a mass range from m/z 20 to 800. 3.6. HRGC–MS A Fisons Instrument GC 8000 Series gas chromatograph with split injection (220 C; 1:20) was directly coupled to a Fisons Instrument MD 800 mass spectrometer. The GC was equipped with a J&W DB-Wax fused silica capillary column (30 m0.25 mm i.d., df=0.25 mm). The conditions were as follows: carrier gas flow (He), 1.6 ml min1 (at 50 C); temperature program, 3 min isothermal at 50 C, then 50–220 C at 4 C/min; temperature of ion source, 220 C and that of the connecting parts, 230 C. The electron energy for the EI mass spectra was 70 eV and the cathodic current was 4.1 mA. Spectra were recorded with a mass range of m/z 35 to 250. Identifications were carried out by comparison of chromatographic retention and mass spectral data of target compounds with that of authentic reference samples. 3.7. Elemental analysis The data for compounds 1, 2 and 3 are as follows: compound 1 (found: C, 47.8%; H, 7.0%. C17H30O12 requires: C, 47.9%; H, 7.1%). Compound 2 (found: C, 48.6%; H, 7.2%. C16H28O11 requires: C, 48.5%; H, 7.1%). Compound 3 (found: C, 48.8%; H, 6.9%. C16H26O11 requires: C, 48.7%; H, 6.7%).
Acknowledgements G.T. thanks the A. v. Humboldt Foundation for a visiting grant (July 2000, University of Wu¨rzburg). Illy Caffee S.p.A., Trieste (Italy), and the Fonds der Chemischen Industrie, Frankfurt, are thanked for financial support. We are grateful to Professor Dr. O. Vitzthum, Bremen, for helpful discussions.
References Bradbury, A.G.W., Balzer, H.H., 1999. Carboxyatractyligenin and atractyligenin glycosides in coffee. Colloq. 18th Sci. Int. Cafe´, 71–77. Clarke, R.J., Vitzthum, O.G., 2001. Coffee: Recent Developments. Blackwell, Oxford. Czerny, M., Grosch, W., 2000. Potent odorants of raw Arabica coffee. J. Agric. Food Chem. 48, 868–872. Friebolin, H., 1999. Basic one- and two-dimensional NMR spectroscopy, 3rd Edition. Wiley-VCH, Weinheim. Latza, S., Gansser, D., Berger, R.G., 1996. Carbohydrate esters of cinnamic acid from fruits Physalis peruviana, Psidium guajava and Vaccinium vitis-idaea. Phytochemistry 43, 481–485. Loveys, B.R., Millborrow, B.V., 1981. Isolation and characterization of 10 -O-abscisic acid-b-d-glucopyranoside from fruit of Lupinus luteus. Agr. Biol. Chem. 8, 571–589. Maier, H.G., Wewetzer, H., 1978. Determination of diterpene glycosides in coffee. Z. Lebensm. Unters.-Forsch. 167, 105–107. Mayer, F., Grosch, W., 2001. Aroma simulation on the basis of the odorant composition of roasted coffee headspace. Flav. Fragr. J. 16, 180–190. Mbairaroua, O., Thon-That Thang, Tapiero, C., 1994. Synthe`ses de 6-O-b-d-apiofuranosyl-b-d-glucopyranosides de monoterpe´nyle. Carbohydr. Res. 253, 79–99. Parada, F., Krajewski, D., Duque, C., Ja¨ger, E., Herderich, M., Schreier, P., 1996. 1-O-b-d-Glucopyranosyl anthranilate from pinuela (Bromelia plumieri) fruit. Phytochemistry 42, 871–873. Stott, K., Stonehouse, J., Keeler, J., Hwang, T.L., Shaka, A.J., 1995. Excitation sculpting in high-resolution nuclear magnetic resonance spectroscopy: application to selective NOE experiments. J. Am. Chem. Soc. 117, 4199–4200. Thevenet, S., Wernicke, A., Belniak, S., Descotes, G., Bouchu, A., Queneau, R., 1999. Esterification of unprotected sucrose with acid chlorides in aqueous medium: kinetic reactivity versus acyl- or alkyloxycarbonyl-group migrations. Carbohydr. Res. 318, 52–66. Winterhalter, P., Skouroumounis, G.K., 1997. Glycoconjugated aroma compounds: occurrence, role and biotechnological transformation. In: Scheper, T. (Ed.), Advances in Biotechnology Engineering Biotechnology. Springer, Heidelberg, Berlin, New York, pp. 73–105. Winterhalter, P., Lutz, A., Schreier, P., 1991. Isolation of a glucosidic precursor of isomeric marmelo lactones from quince fruit. Tetrahedron Lett. 32, 3669–3670. Winterhalter, P., Messerer, M., Bonnla¨nder, B., 1997. Isolation of the glucose ester of (E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid from Riesling wine. Vitis 36, 55–56. Winterhalter, P., Knapp, H., Straubinger, M., 1999. Water soluble aroma precursors. Analysis, structure and reactivity. In: Teranishi, R., Wick, E.L., Hornstein, I. (Eds.), Flavor Chemistry—Thirty Years of Progress. Kluwer Academic/Plenum Publishers, New York, pp. 255–264. Yoshimoto, K., Itatani, Y., Shibata, Y., Tsuda, Y., 1980. Utilization of sugars in organic synthesis. Part IV. Carbon-13 nuclear magnetic resonance (NMR) spectra of O-acylglucoses. Additivity of shift parameters and its application to structure elucidations. Chem. Pharm. Bull. 28, 208–219.
3-Methylbutanoyl and 3-methylbut-2-enoyl disaccharides from green coffee beans (Coffea arabica) Bernhard Weckerlea, Tama´s Ga´tib, Ga´bor To´thb,1, Peter Schreiera,* a Lehrstuhl fu¨r Lebensmittelchemie, Universita¨t Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany Technical Analytical Research Group of the Hungarian Academy of Sciences, Institute for General and Analytical Chemistry, Budapest University of Technology and Economics, Szent Gelle´rt te´r 4, H-1111 Budapest, Hungary
b
Received 26 November 2001; received in revised form 23 January 2002
Abstract Three 3-methylbutanoyl and 3-methylbut-2-enoyl disaccharides isolated from green coffee beans (Coffea arabica) were identified as 3-methylbutanoyl-1-O-b-d-glucopyranosyl-b-d-apiofuranoside, 3-methylbutanoyl-6-O-a-d-glucopyranosyl-b-d-fructofuranoside, and 3-methylbut-2-enoyl-1-O-b-d-glucopyranosyl-b-d-apiofuranoside. The structures were established by one- and twodimensional 1H and 13C NMR spectra as well as by ESI MS/MS spectra. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Coffea arabica; Flavour precursors; Green coffee; 3-Methylbutanoyl-1-O-b-d-glucopyranosyl-b-d-apiofuranoside; 3-Methylbutanoyl-6O-a-d-glucopyranosyl-b-d-fructofuranoside; 3-Methylbut-2-enoyl-1-O-b-d-glucopyranosyl-b-d-apiofuranoside; HPLC–ESI–MS/MS; 1H NMR; 13 C NMR; Sugar conjugates
1. Introduction In the past, a great deal of attention has been focused on the analysis of volatile flavour compounds of green and roasted coffee (Czerny and Grosch, 2000; Mayer and Grosch, 2001; Clarke and Vitzthum, 2001), information about their biogenetic sugar-bound precursors in green coffee, however, is surprisingly scarce. Whereas our knowledge about flavour progenitors in plant tissues has been extended tremendously in the last decade (Winterhalter and Skouroumounis, 1997; Winterhalter et al., 1999), for coffee the studies about glycoconjugated precursors are restricted to diterpenoid glycosides (Maier and Wewetzer, 1978; Bradbury and Balzer, 1999). In most of the bound flavour constituents characterized to date glycosidic linkages prevail. The rare information about sugar esters comprises few substances, for instance, the precursor of marmelo lactones in quince (Winterhalter et al., 1991), glucopyranosyl anthranilate from pinuela fruit (Parada et al., 1996), carbohydrate * Corresponding author. Tel.: +49-931-888-5481; fax: +49-931888-5484. E-mail addresses: [email protected] (P. Schreier), [email protected] (G. To´th). 1 Tel.: +36-1-463-3411; fax: +36-1-463-3408.
esters of cinnamic acid from various fruits (Latza et al., 1996), and the glucose ester of (E)-2,6-dimethyl-6hydroxyocta-2,7-dienoic acid from Riesling wine (Winterhalter et al., 1997). In this paper, we describe the isolation and identification of novel 3-methylbutanoyl and 3-methylbut-2-enoyl disaccharides from green coffee beans (Coffea arabica).
2. Results and discussion From a water-methanol extract of grounded and defatted green coffee beans (Coffea arabica, provenience Ethiopia) a fraction containing flavour precursors was obtained by chromatography on Amberlite XAD. The screening for sugar conjugates in the methanolic fraction after Amberlite XAD chromatography was performed by enzymatic hydrolysis of the lyophylized extracts with subsequent HRGC–MS analysis. Among the liberated volatiles, 3-methylbutanoic acid and 3methylbut-2-enoic acid were identified, both serving as target compounds for the following separation steps, which comprised several fractionations by preparative and analytical reversed phase liquid chromatography on glass columns and HPLC columns, finally yielding the sugar esters 1–3 in pure forms (cf. Experimental). The
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(02)00042-0
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screening for sugar conjugates in the fractions was carried out by HPLC coupled with an evaporative light scattering detector (ELSD) using an RP-18 column. In addition, the fractions obtained in the course of the clean-up procedure and the purified compounds 1–3 were analysed by HPLC–ESI–MS/MS (negative mode) to get first structural information. The recorded spectral data are outlined in Table 1. The data reveal that compounds 1–3 are acyl disaccharides with molecular masses of 426, 396 and 394, respectively (quasimolecular ions [MH] at m/z 424.9, 394.9 and 392.9 for compounds 1–3, respectively). Additional signals at m/z 322.9 (compound 1), 292.9 (compounds 2 and 3) and m/z 161.1 (compounds 1–3) indicated that the substructural sugar units consist of two hexose moieties in case of compound 1 and each of one hexose and one pentose unit for compounds 2 and 3, respectively. Finally, structural evaluation was performed by a number of NMR experiments. The structures of 1–3 (Fig. 1) were determined by 1D and 2D NMR studies using DEPT, COSY, sel-1D TOCSY (Stott et al., 1995), HSQC and HMBC techniques including gradient selection and linear prediction. Despite of severe signal overlap even at 600 MHz, we achieved a complete assignment not only for the carbon but also for the proton signals. The 1H and 13C chemical shifts and couplings of compounds 1–3 are summarized in Table 2 (1H NMR) and Table 3 (13C NMR). The 1H NMR spectra of 2 and 3 showed unusually low chemical shift for the anomeric protons. Whereas in b-d-glucosides the anomeric proton resonates around 4.5 ppm, the anomeric proton in 2 and 3 showed a downfield shift and resonated at 5.34 ppm and 5.36 ppm indicating ester linkages (Loveys and
Millborrow, 1981). In case of compound 1 the protons 60 a and 60 b of one of the hexose moieties showed a downfield shift ( 3.83 ppm and 3.65 ppm) in comparison with a terminal glucose moiety, indicating an ester linkage at position 6 of the hexose unit. For compounds 2 and 3 the hexose unit could be identified as a glucose moiety (J20 ,30 – J30 ,40 – J40 ,50 – 9.0 Hz) exhibiting b-configuration, indicated by the coupling constant J10 ,20 =8.2 and 8.4 Hz, respectively. As to the acyl units, the data of 1 were in agreement with those of 2 ( 171.1 ppm and 172.3 ppm for the C=O, 42.5 and 42.3 ppm for C-2, 25.0 and 25.1 for C-3 and 22.2 and 22.1 for the terminal methyl groups), confirming the results of HRGC–MS analysis. In the 1H NMR spectrum of 1 the H-4 and H-5 protons are accidentally isochronous, whereas in compound 2 the terminal methyl groups show a small difference ( 0.90 ppm and 0.91 ppm, respectively). Intensive HMBC connectivities between the protons of 2 H-10 ( 5.34 ppm) and of 1 H-60 a ( 4.24 ppm) and H-60 b ( 4.01 ppm) and the carbonyl carbons ( 171.1 ppm and 172.3 ppm, respectively) proved the linkage between the acid and the sugar moieties at the particular positions. The linkage at position 6 of the glucose moiety of 1 caused a strong downfield shift of C-60 and an upfield shift of C50 , which is in agreement with literature data (Yoshimoto et al., 1980). In contrast, the 13C NMR data of 3 with chemical shifts of 164.2 ppm (C=O), 115.1 ppm (C-2) and 159.2 ppm of C-3 pointed out the presence of an a,b-unsaturated ester substructure (Friebolin, 1999). The data of 3 in the 1H NMR spectrum showed for the methyl protons of the acyl moiety two distinct singlets at 1.91 ppm and 2.13 ppm as well as a singlet of an olefinic proton at 5.72 ppm. In addition, there was a
Table 1 Main fragments of ESI mass spectra [achieved in MS/MS negative ion mode with argon as collision gas and a water (5 mM NH4OAc)–acetonitrile (5 mM NH4OAc)-gradient] of compounds 1–3 m/z
1
2
3
485.2 455.3 453.3 424.9 394.9 392.9 322.9 292.9 161.1 148.9
[Ma+AcOHb–H] – – [Ma–H]
Ø [Ma+AcOHb–H] – – [Ma–H] – – [Ma–Ac–H] [Hexd–H2O–H] [Pene–H]
Ø Ø [Ma+AcOHb–H] – – [Ma–H] – [Ma–Ac–H] [Hexd–H2O–H] [Pene–H]
– [Ma–Ac–H] – [Hexd-H2O-H]
a
M=total molecule. AcOH=acetic acid. c A=acyl moiety (for 1 and 2: 3-methylbutanoic acid, for 3: 3-methylbut-2-enoic acid). d Hex=hexose moiety. e Pen=pentose moiety. b
Fig. 1. Structures of compounds 1–3.
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B. Weckerle et al. / Phytochemistry 60 (2002) 409–414 Table 2 1 H chemical shifts of compounds 1–3, measured in DMSO-d6, compared with literature data Position
1
Referencea
2
3
Referenceb
Acyl moiety
3-Methylbutanoyl
Octanoyl
3-Methylbutanoyl
3-Methylbut-2-enoyl
Aglycone linalool
1cis 1trans 2 3 4 5 6 7 8 9 10
– – 2.19 (m) 1.99 (m) 0.90 (d; 6.7) 0.90 (d; 6.7)
– – 2.4–2.6 (m) 1.5–1.7 (m) 1.2 j j 1.4 0.7–1 (m)
– – 2.24 (d; 7.0) 2.00 (m) 0.90 (d; 7.0)c 0.91 (d; 7.0)c
– – 5.72 (s) – 1.91 (s) 2.13 (s)
5.19 (d; 10.5) 5.26 (d; 18.0) 5.85 (dd; 10.5; 18.0) – 1.64 (m) 1.99 (m) 5.18 (m)
5.34 (d) 3.49 (dd; J10 ,20 3.7; J20 ,30 9.8) 3.71 (t) 3.38 (t; J30 ,40 9.8) 3.95–4.04 (m) 4.20 (dd; J50 ,60 a 5.2; J60 a,60 b 12.1) 4.37 (dd; J50 ,60 b 5.2)
5.34 (d; J10 ,20 8.2) 3.09 (dd; J20 ,30 – 9.0) 3.23 (t; J30 ,40 – 9.0) 3.06 (t; J40 ,50 – 9.0) 3.36 (m) 3.81 (dd; J50 ,60 a 2.0; J60 a,60 b 10.0) 3.38 (m)
1.67 (s) 1.59 (s) 1.36 (s)
Glucose moiety 10 20 30 40 50 60 a
5.17 (d; J10 ,20 3.7) 3.20 (dd; J20 ,30 9.7) 3.48 (t; J30 ,40 9.7) 3.06 (t; J40 ,50 9.7) 3.90 (m) 4.24 (dd; J50 ,60 a 1.0 J60 a,60 b 10.0 ) 4.01 (dd; J50 ,60 b 6.0)
60 b
Fructose moiety 00
1 1ab00 200 300 400 400 a 400 b 500 500 a 500 b 600 ab a b c
– 3.38 (m) – 3.88 (d; J300 ,400 8.0) 3.73 (m) – – 3.58 (m) – – 3.55 (m)
5.36 (d; J10 ,20 8.4) 3.05 (dd; J20 ,30 – 9.0) 3.23 (t; J30 ,40 – 9.0) 3.11 (t; J40 ,50 – 9.0) 3.37 (m) 3.83 (dd; J50 ,60 a 1.0; J60 a,60 b 10.0) 3.39 (dd; J50 ,60 b 5.0)
4.5 (d; 7.9) 3.2 (t; 8.4) 3.4 (t; 8.9) 3.34 (t; 9.0) 3.49 (m) 3.35 (m) 3.78 (m)
4.79 (d; J100 ,200 3.1) – 3.75 (d) – – 3.84 (d; J400 a,400 b 9.3) 3.57 (d) – 3.35 (d; J500 a,500 b 11.6) 3.38 (d) –
5.06 (d; 3.0) – 3.95 (d; 3.0) – – 3.86 (2d; 10.2) 4.03 (2d; 10.2) – 3.64 (s) 3.64 (s) –
Apiose moiety – 3.57–3.63 (m) – 4.16 (d) 3.97 (t; J300 ,400 8.8) – – 3.79–3.88 (m) – 3.72–3.79 (m)
4.80 (d; J100 ,200 3.0) – 3.75 (d) – – 3.83 (d; J400 a,400 b 9.2) 3.57 (d) – 3.32 (d; J500 a,500 b 11.3) 3.30 (d) –
6-O-Octanoylsucrose, measured in D2O at 300 MHz (Thevenet et al., 1999). Linalyl-3-O-b-d-apiofuranosyl-(1–6)-b-d-glucopyranoside, measured in D2O at 360 MHz (Mbairaroua et al., 1994). Signals are interchangeable.
NOE effect between H-2 and H-4 noticed. The linkage between the acid and the sugar unit of 3 via the carbon C-1 of the glucose moiety was proven by an HMBC connectivity between proton H0 1 ( 5.36 ppm; d; J10 ,20 8.4) and the carbonyl carbon of the acid. With regard to the second sugar units of compounds 1–3 (hexose for 1, pentose for 2 and 3) the 1H and 13C NMR data of 2 agreed with those of compound 3. These pentose moieties were identified as b-d-apiofuranose. The coupling constants of the terminal apiose unit between the anomeric protons H-100 and H-200 showed values of 3.0 Hz and 3.1 Hz, respectively, indicating bconfiguration. Configuration of C-300 was determined by NOESY experiments. Between protons H-100 and 500 a and 500 b, respectively, there was not any NOE con-
nectivity detectable, indicating trans-constitution of proton H-100 and the C-500 methylene group. Additionally, the data of the apiose moieties were in good agreement with those of literature (see Table 2 and 3). The data of the complete sugar moiety of 1 were in accurate consistence with those of an acyl-6-O-sucrose (Mbairaroua et al., 1994). The coupling constant J10 ,20 of the glucose moiety with 3.7 Hz confirmed a-configuration. By means of TOCSY experiments (irradiating 5.17 ppm, mix 120 ms and irradiating 3.73 ppm, mix 120 ms) a complete assignment for the proton signals of the glucose and the fructose moiety was achieved. In addition, an HMBC connectivity between H-10 and C-200 was detected. The coupling constants J20 ,30 , J30 ,40 and J40 ,50 (all 9.7 Hz) as well as J300 ,400 and J400 ,500 (both 8.0 Hz) are in
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Table 3 13 C NMR chemical shifts of compounds 1–3, measured in DMSO-d6, compared with literature data Position
1
Referencea
2
3
Referenceb
Acyl moiety
3-Methylbutanoyl
Octanoyl
3-Methylbutanoyl
3-Methyl-2-butenoyl
Aglycone linalool
C=O 1 2 3 4 5 6 7 8
172.3 – 42.4 25.1 22.1 22.1
177.1 – 34.1 31.4 28.6 28.4 24.7 22.4 13.7 – –
171.1 – 42.5 25.0 22.2 22.2 – – – – –
164.2 – 115.1 159.2 27.0 20.1 – – – – –
– 117.00 142.40 82.30 40.90 22.60 125.20 134.30 25.60 17.70 23.00
93.9 (1JCH=161 Hz) 72.4 76.3 69.7 76.2 67.3
93.5 (1JCH=161 Hz) 72.3 76.4 69.7 76.2 67.4
98.00
109.1 75.7 78.8 73.2 63.0 –
109.80 77.50 80.10 74.30 64.40
22.1 – – – Glucose moiety
10
91.5
92.3
20 30 40 50 60
71.5 72.7 70.1 70.0 63.4
71.4 72.7 70.0 70.7 61.8
Fructose moiety 100 200 300 400 500 600
62.2 103.9 76.9 74.5 82.7 62.6 a b
74.00 76.60 70.70 75.20 68.60
Apiose moiety 63.1 104.1 76.7 74.5 81.9 63.6
109.1 (1JCH=170 Hz) 75.8 78.8 73.3 63.1 –
6-O-Octanoylsucrose, measured in D2O at 75 MHz (Thevenet et al., 1999). Linalyl-3-O-b-d-apiofuranosyl-(1–6)-b-d-glucopyranoside, measured in D2O at 360 MHz (Mbairaroua et al., 1994).
accordance with the diaxial arrangement of the corresponding protons. The data of the sucrose moiety were in good agreement with those of literature (see Tables 2 and 3). In conclusion, three new sugar esters were isolated from green beans of Coffea arabica and identified as 3methylbutanoyl-6-O-a-d-glucopyranosyl-b-d-fructofuranoside (1), 3-methylbutanoyl-1-O-b-d-glucopyranosylb-d-apiofuranoside (2) and 3-methylbut-2-enoyl-1-O-bd-glucopyranosyl-b-d-apiofuranoside (3). These compounds supplement information about the structural composition of acyl disaccharides in plant tissues and extend our knowledge on sugar-bound flavour precursors.
3. Experimental 3.1. Extraction and isolation Green coffee beans (Coffea arabica) (1 kg; origin Ethiopia) were grounded and defatted by extraction
with petroleum ether. After drying the remaining powder was extracted with 2.5 l of a methanol-water mixture (7:1, v/v) for 24 h at ambient temperature. The filtrate was concentrated under reduced pressure to approximately 200 ml and the remaining aqueous phase was lyophilized. After redilution with 15 ml of water the aqueous extract was applied onto a glass column (1080 cm) filled with Amberlite1 XAD-2 (Supelco, Bellefonte, USA). After washing with water (1.5 l) and subsequently with diethyl ether (1 l) the sugar conjugates were eluted with methanol (1.5 l). The eluate was concentrated under reduced pressure and the residue lyophilized to yield 16 g dry powder. The powder was suspended in chloroform (25 ml) and applied onto a glass column (875 cm) filled with silica gel 60. The elution was performed using stepwise each of 1 l-mixtures of hexane-chloroform (90:10; 70:30; 50:50, v/v), 750 ml-mixtures of dichloromethane-ethyl acetate (75:25; 60:40; 25:75, v/v), and 800 ml-mixtures of methanol-ethyl acetate (20:80; 30:70; 50:50; 75:25, v/ v), collecting 200 ml-fractions at a flow rate of 15 ml
B. Weckerle et al. / Phytochemistry 60 (2002) 409–414
min1. The fractions were checked by HPLC–ELSD and, after enzymatic hydrolysis, by HRGC–MS analysis. The target acids, 3-methylbutanoic acid and 3methylbut-2-enoic acid, were detected in fractions 36–39 eluted by the 50:50 methanol–ethyl acetate mixture. After solvent separation from these combined fractions under reduced pressure, lyophilization of the residue yielded 3.7 g of a white powder. The powder, redissolved in 5 ml of water, was fractionated by LC on a RP-18 column (2.520 cm; LiChrospher1, Merck, Darmstadt, Germany) using a methanol-water gradient starting with 100% water (250 ml) and increasing the proportion of methanol in steps of 10% (each step 250 ml) to end up with 100% methanol. Twenty milliliter fractions were collected at a flow rate of 5 ml min1 and analyzed by HPLC–ELSD as well as, after enzymatic hydrolysis, by HRGC–MS. Fractions 34–37 (containing 3-methylbutanoic acid after hydrolysis) and 38–42 (containing 3-methylbut-2-enoic acid after hydrolysis) each were combined, concentrated under reduced pressure and lyophilized, yielding 150 mg (combined fractions; 1A) and 120 mg (combined fractions; 1B) of white powders, respectively. Further purification of fraction 1A, redissolved in 5 ml of water, was performed by HPLC on a preparative RP-18 column using acetonitrile-water (10:90, v/v) at a flow rate of 6 ml min1 and using a split valve for synchronous ELSD detection to collect detected signals in different flasks. Per injection 7 mg of the extract were fractionated; the procedure was repeated 21 times. Fractions of the same composition were combined, concentrated under reduced pressure and lyophilized. Enzymatic hydrolysis with subsequent HRGC-MS analysis revealed two eluates containing 3-methylbutanoic acid (2A=compound 1: yield 7 mg; 3A=compound 2: yield 2.5 mg) which were separately subjected to final spectroscopic analyses. For the purification of fraction 1B two more fractionation steps were necessary. The first fractionation of the powder redissolved in 5 ml of water was done by HPLC on a preparative RP-18 column using a linear gradient of acetonitrile-water from 1% acetonitrile (t=0 min) to 20% acetonitrile (t=45 min) at a flow rate of 6 ml min1 and using a split valve for synchronous ELSD detection to collect detected signals in different flasks. Per injection 8 mg of the extract were fractionated; the procedure was repeated 15 times. Fractions of the same composition were combined, concentrated under reduced pressure and lyophilized. These extracts were checked, after enzymatic hydrolysis, by HRGC–MS for their content of 3-methylbut-2-enoic acid. One fraction was obtained containing a precursor (2B) from which the acid was liberated after enzymatic hydrolysis. For final purification fractionation was done by HPLC on an analytical RP-18 column using a linear gradient of acetonitrile-water (flow rate 1 ml min1) from 1% ace-
413
tonitrile (t=0 min) to 50% acetonitrile (t=45 min) and using a split valve for synchronous ELSD detection to collect the detected signals in different flasks. Per injection 0.35 mg of the extract were fractionated; the procedure was repeated 20 times. Fractions of the same composition were combined, concentrated under reduced pressure and lyophilized. These extracts were checked, after enzymatic hydrolysis, by HRGC–MS for their content of 3-methylbut-2-enoic acid. One fraction (3B=compound 3: yield 1.2 mg) was obtained containing a precursor from which the acid was liberated by enzymatic hydrolysis. 3.2. Enzymatic hydrolysis Aliquots (1–10 mg) of the lyophilized extracts were incubated in 5 ml of an 0.1 M phosphate buffer (pH 5.7) for 24 h at 37 C using Rohapect VRF1 (Ro¨hm, Darmstadt). After acidification to pH 2, the suspensions were extracted twice with 5 ml diethyl ether, the combined organic phases were dried over sodium sulphate and carefully concentrated to approximately 0.5 ml by distillation on a Vigreux column. These organic extracts were analyzed by HRGC–MS. 3.3. NMR spectroscopy NMR spectra were recorded in CD3OD using Bruker Avance DRX-600 and DRX-500 spectrometers. Chemical shifts are given on the -scale and were referenced to TMS. In the 1D and 2D NMR experiments pulse programs were taken from the Bruker software library. 3.4. HPLC–ELSD HPLC–ELSD analysis was performed with a Knauer HPLC Maxi Star including Eurochrom 2000 software and a Sedere Evaporative Light Scattering Detector (ELSD, model Sedex 55). The temperature of the detector was set to 40 C and compressed air was used at a pressure of 240 kPa. HPLC was carried out (i) on an analytical Eurospher 100 C-18 column (2504.6 mm I.D., 5 mm) (Knauer) and (ii) a preparative Eurospher 100 C-18 column (25016 mm I.D., 5 mm) (Knauer) with water and acetonitrile as solvents (conditions cf. section 3.1). 3.5. HPLC–ESI MS/MS Mass spectrometry was performed on a Finnigan triple-stage quadrupole TSQ 7000 HPLC–MS/MS system (Finnigan MAT, Bremen, Germany) with electrospray ionization (ESI) as interface in negative mode. For HPLC, an Applied Biosystems (Foster City, CA, USA) Model 140B pump was used. For ESI in the negative ion mode the spray capillary voltage was set to 3.0 kV
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and the temperature of the heated inlet capillary was set to 230 C. Nitrogen served both as sheath gas (482 MPa) and as auxiliary gas. HPLC was carried out on an Eurospher 100 C-18 column (1002 mm I.D., 5 mm) (Knauer) with a linear gradient of water (5 mM NH4OAc)–acetonitrile (5 mM NH4OAc) at a flow rate of 0.2 ml min1 from 1 to 30% acetonitrile in 30 min. Negative ions were detected scanning from m/z 200 to 1500 with a total scan duration of 1.0 s. The MS/MS experiments were performed at a collision energy of 15– 25 eV with argon (0.24 Pa) serving as collision gas, scanning a mass range from m/z 20 to 800. 3.6. HRGC–MS A Fisons Instrument GC 8000 Series gas chromatograph with split injection (220 C; 1:20) was directly coupled to a Fisons Instrument MD 800 mass spectrometer. The GC was equipped with a J&W DB-Wax fused silica capillary column (30 m0.25 mm i.d., df=0.25 mm). The conditions were as follows: carrier gas flow (He), 1.6 ml min1 (at 50 C); temperature program, 3 min isothermal at 50 C, then 50–220 C at 4 C/min; temperature of ion source, 220 C and that of the connecting parts, 230 C. The electron energy for the EI mass spectra was 70 eV and the cathodic current was 4.1 mA. Spectra were recorded with a mass range of m/z 35 to 250. Identifications were carried out by comparison of chromatographic retention and mass spectral data of target compounds with that of authentic reference samples. 3.7. Elemental analysis The data for compounds 1, 2 and 3 are as follows: compound 1 (found: C, 47.8%; H, 7.0%. C17H30O12 requires: C, 47.9%; H, 7.1%). Compound 2 (found: C, 48.6%; H, 7.2%. C16H28O11 requires: C, 48.5%; H, 7.1%). Compound 3 (found: C, 48.8%; H, 6.9%. C16H26O11 requires: C, 48.7%; H, 6.7%).
Acknowledgements G.T. thanks the A. v. Humboldt Foundation for a visiting grant (July 2000, University of Wu¨rzburg). Illy Caffee S.p.A., Trieste (Italy), and the Fonds der Chemischen Industrie, Frankfurt, are thanked for financial support. We are grateful to Professor Dr. O. Vitzthum, Bremen, for helpful discussions.
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