C13-norisoprenoid glucoconjugates from lulo (Solanum quitoense L.) leaves

Article abstract of DOI:10.1021/jf9807364

With the aid of multilayer coil countercurrent chromatography, subsequent acetylation, and liquid chromatographic purification of a glycosidic mixture obtained from lulo (Solanum quitoense L.) leaves, three C₁₃-norisoprenoid glucoconjugates were isolated in pure form. Their structures were elucidated by NMR, MS, and CD analyses to be the novel (6R,9R)-13-hydroxy-3-oxo-α-ionol 9-O-β-D-glucopyranoside (4a), the uncommon (3S,5R,8R)-3,5-dihydroxy-6,7-megastigmadien-9-one 5-O-β-D-glucopyranoside (citroside A) (5a), and the known (6S,9R)-vomifoliol 9-O-β-D-glucopyranoside (6a). Enzymatic treatment of compound 5a showed the formation of 3-hydroxy- 7,8-didehydro-β-ionone (7), an important lulo peeling volatile, which in its turn after chemical reduction and heated acid catalyzed rearrangement generates β-damascenone (9) and 3-hydroxy-β-damascone (10).

Full text of DOI:10.1021/jf9807364

J. Agric. Food Chem. 1999, 47, 1641−1645
1641
C13-Nor isop r en oid Glu cocon ju ga tes fr om Lu lo
(Sola n u m qu itoen se L.) Lea ves
Coralia Osorio,† Carmenza Duque,*,† and Yoshinori Fujimoto‡
Departamento de Qu´ımica, Universidad Nacional de Colombia, AA 14490, Bogota´, Colombia, and
Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, J apan
With the aid of multilayer coil countercurrent chromatography, subsequent acetylation, and liquid
chromatographic purification of a glycosidic mixture obtained from lulo (Solanum quitoense L.) leaves,
three C13-norisoprenoid glucoconjugates were isolated in pure form. Their structures were elucidated
by NMR, MS, and CD analyses to be the novel (6R,9R)-13-hydroxy-3-oxo-R-ionol 9-O-â-D-
glucopyranoside (4a ), the uncommon (3S,5R,8R)-3,5-dihydroxy-6,7-megastigmadien-9-one 5-O-â-D-
glucopyranoside (citroside A) (5a ), and the known (6S,9R)-vomifoliol 9-O-â-D-glucopyranoside (6a ).
Enzymatic treatment of compound 5a showed the formation of 3-hydroxy-7,8-didehydro-â-ionone
(7), an important lulo peeling volatile, which in its turn after chemical reduction and heated acid
catalyzed rearrangement generates â-damascenone (9) and 3-hydroxy-â-damascone (10).
Keyw or d s: Lulo; Solanum quitoense; (6R,9R)-13-hydroxy-3-oxo-R-ionol 9-O-â-D-glucopyranoside;
new C13-norisoprenoid glucoconjugate; (3S,5R,8R)-3,5-dihydroxy-6,7-megastigmadien-9-one 5-O-â-
D-glucopyranoside; (6S,9R)-vomifoliol 9-O-â-D-glucopyranoside; aroma precursors; 3-hydroxy-7,8-
didehydro-â-ionone; â-damascenone; 3-hydroxy-â-damascone
INTRODUCTION
C13-norisoprenoids (Osorio and Duque, 1995). This paper
describes, for the first time, the isolation and charac-
terization of the novel glucoconjugated C13-noriso-
prenoid (6R,9R)-13-hydroxy-3-oxo-R-ionol 9-O-â-D-glu-
copyranoside (4a ) as well as the presence of the
uncommon (3S,5R,8R)-3,5-dihydroxy-6,7-megastigma-
dien-9-one 5-O-â-D-glucopyranoside (5a ) and its role as
precursor in the formation of 3-hydroxy-7,8-didehydro-
â-ionone, â-damascenone, and 3-hydroxy-â-damascone
and the known (6S,9R)-vomifoliol 9-O-â-D-glucopyrano-
side (6a ).
Lulo plant (Solanum quitoense L.), native to South
America, is an important fruit species in Colombia due
to the pleasant and delicate aroma of its fruit. During
recent years, its cultivation has been increasing signifi-
cantly because the Colombian government included this
species in a particular fruit export promotion program.
After our first studies on the aroma composition of
the fruit pulp and peelings (Sua´rez and Duque, 1991;
Sua´rez et al., 1993), further investigations reported on
the glycosidically bound aroma substances (Sua´rez et
al., 1991). Our latter research revealed the presence of
the glycoconjugated aroma compounds 1-3 as precur-
sors of monoterpenols in the peelings of this fruit
(Wintoch et al., 1993; Duque et al., 1993).
EXPERIMENTAL PROCEDURES
Gen er a l. NMR spectra were taken on a Fourier transform
J EOL J NM-EX500 spectrometer and on a Bruker AC 500
spectrometer with CDCl3 as solvent and Me4Si as internal
standard. UV spectra were obtained with a Merck Hitachi
diode array detector L-4500. CD spectra were recorded on a
J ASCO J -500C polarimeter. Fast atom bombardment mass
spectrometry (FAB-MS) was carried out with a J EOL J MS-
AX505HA spectrometer using glycerol matrix and ion source
temperature at 305 °C. Positive ions over a range m/z 10-
650 were scanned. For chemical ionization mass spectrometry
(CI-MS), a Shimadzu 9020 DF mass spectrometer was used
(reactant gas, isobutane). Positive ions over a range of m/z 70-
700 were scanned. For flash chromatography Merck silica gel
60 (0.032-0.063 nm) was employed. All solvents used were of
high purity at purchase (Merck) and were redistilled before
use.
P la n t Ma ter ia l. Fresh lulo (S. quitoense L.) leaves came
from a commercial orchard located in the rural area La Vega,
Cundinamarca, Colombia. Voucher specimens were coded COL
352780 at the Instituto de Ciencias Naturales de la Univer-
sidad Nacional de Colombia.
As a continuation to the above-mentioned studies, we
performed a preliminary screening of bound volatiles
in lulo leaves, which revealed a high amount of bound
Isola tion of a Glycosid ic Extr a ct. Lulo leaves (10 kg)
were cut blended with 1 L of methanol (adjusted to pH 7.0).
After centrifugation (10000g, 30 min), the supernatant was
concentrated under reduced pressure (rotavapor) and then
extracted with petroleum ether and diethyl eter to eliminate
* Author to whom correspondence should be addressed (e-
mail cduqueb@ciencias.ciencias.unal.edu.co; fax 571-3165220).
† Universidad Nacional de Colombia.
‡ Tokyo Institute of Technology.
10.1021/jf9807364 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/12/1999

1642 J. Agric. Food Chem., Vol. 47, No. 4, 1999
Osorio et al.
Ta ble 1. MS Sp ectr a l Da ta for Acetyla ted Glu cosid e 4b
Ta ble 3. 13C NMR Sp ectr a l Da ta for Acetyla ted Glu cosid e
4b (CDCl3, 125 MHz, δ Rela tive to TMS)
fragment FAB-MS CI-MSa EI-MSb
assignmenta
(m/z)
(%)
(%)
(%)
interpretation
1b δ
DEPT
597
331
271
7
17
2
6
100
14
[M + H]+
20.56-20.69
20.86
CH3
CH3
CH3
CH3
C
CH3CO (5×)
70 [(hexose + 4 acetyl - H2O) + H]+
10 [(hexose + 4 acetyl - H2O -
AcOH) + H]+
C10
26.89b
27.67c
36.25
C11
C12
C1
C2
C6
C6′
C13
C4′
C2′
C5′
C3′
C9
C1′
C4
C7
C8
249
211
207
169
14
2
4
78
3
7
20 [(aglycon + acetyl - H2O) + H]+
8
[331 - 2 AcOH]+
48.01
51.16
61.87
64.06
68.26
71.56
71.86
72.73
CH2
CH
CH2
CH2
CH
CH
CH
CH
CH
CH
CH
CH
CH
C
C
C
C
C
C
C
10 [(aglycon - H2O) + H]+
100 [(211 - acetyl) + H]+
19
28
a
b
Reactant gas isobutane. Electron impact at 20 eV.
Ta ble 2. 1H NMR Sp ectr a l Da ta for Acetyla ted Glu cosid e
4b (CDCl3, 500 MHz, Cou p lin g Con sta n ts in Her tz, δ
Rela tive to TMS)
76.52
99.61
δ
signal
6 H, 2s
J
assignmenta
0.99/1.04
1.24
H3 C11/H3 C12
H3 C10
H3 acetates (5×)
Ha C2
Hb C2
H C6
123.63
127.21
136.28
157.44
169.14
169.31
170.12
170.28
170.55
198.43
3 H, d
6.4
2.01-2.12
15 H, 5 s
1 H, d
1 H, d
2.15
17.4
17.4
9.0
C5
2.40
2.60
CH3CO
CH3CO
CH3CO
CH3CO
CH3CO
C3
1 H, d
3.65
4.08
4.23
4.25
1 H, dxdxd
1 H, dxd
1 H, dxd
1 H, quintet
1 H, d
10.1/4.1/2.3
12.0/2.3
12.0/4.1
6.4
H C5′
Ha C6′
Hb C6′
H C9
H C1′
H2 C13
H C2′
H C4′
H C3′
H C7
H C8
4.58
8.3
a
Assignments were based on a 1H-13C COSY experiment.
4.60
4.99
5.10
5.20
5.60
5.67
6.04
2 H, br s
1 H, dxd
1 H, dxd
1 H, dxd
1 H, dxd
1 H, dxd
1 H, br s
b,c Interchangeable values.
10.1/8.3
10.1/9.6
10.1/9.6
15.1/9.0
15.1/6.4
For glycoside 5b the following spectral data were obtained:
UV (Et2O) λmax 227 nm; FAB-MS, m/z (%) 597 (8, [M + H]+),
331 (6, [hexose + 4 acetyl - H2O + H]+), 249 (5, [aglycon +
1
acetyl - H2O + H]+); H NMR (400 MHz, CDCl3) δ 1.15 (3H,
H C4
s, CH3-11), 1.31 (3H, s, CH3-12), 1.42 (1H, dd, Ha-2)*, 1.42 (3H,
s, CH3-13), 1.47 (1H, t, J ) 13.3 Hz, Ha-4), 2.00-2.10 (15H,
5s, 5 acetates), ∼2.00 (1H, Hb-2)*, 2.18 (3H, s, CH3-10), 2.47
(1H, dd, J ) 13.3, 3.2 Hz, Hb-4), 3.66 (1H, ddd, J ) 9.8, 5.9,
2.9 Hz, H-5′), 4.08 (1H, dd, J ) 12.0, 5.9 Hz, Hb-6′), 4.17 (1H,
dd, J ) 12.0, 2.9 Hz, Ha-6′), 4.78 (1H, d, J ) 7.8 Hz, H-1′),
5.01 (1H, dd, J ) 9.8, 7.8 Hz, H-2′), 5.02 (1H, dd, J ) 9.8, 9.7
Hz, H-4′), 5.16 (1H, m, H-3), 5.21 (1H, t, J ) 9.8 Hz, H-3′),
5.91 (1H, s, H-8) (asterisks indicate that the signals are
obscured due to partial overlapping); 13C NMR (100 MHz,
CDCl3) δ 20.62-20.75 (5 acetates), 25.81 (CH3-13), 26.62 (CH3-
10), 28.79 (CH3-12), 31.81 (CH3-11), 35.76 (C-1), 43.22 (C-4),
44.54 (C-2), 62.22 (C-6′), 66.98 (C-3), 68.51 (C-4′), 71.13 (C-
2′), 71.90 (C-5′), 72.90 (C-3′), 77.81 (C-5), 95.29 (C-1′), 100.88
(C-8), 116.87 (C-6), 169.46-170.49 (5 acetates), 197.68 (C-7),
210.74 (C-9). Signals were assigned on the basis of H-H and
H-C COSY spectra.
a
Assignments were based on a 1H-1H COSY experiment.
chlorophyll pigments and remaining volatiles (Humpf and
Schreier, 1991). The aqueous residue was divided in five
portions, and each was passed through an Amberlite XAD-2
column (45 × 800 mm, 10 mL/min) (Gunata et al., 1985). After
washing with 2 L of distilled water, elution was performed
with 1.5 L of methanol. The combined methanol eluates were
concentrated to dryness under vaccuum and lyophilyzed to
afford 15 g of crude glycosides.
Mu ltilayer Coil Cou n ter cu r r en t Ch r om atogr aph y (ML-
CCC). Portions of ∼1.5 g of glycosidic extract were placed in
an Ito multilayer countercurrent chromatograph (P.C. Inc.,
Potomac, MD) with 75 m × 2.6 mm i.d. PTFE tubing (total
volume ) 400 mL) for separation of glycosides. The MLCCC
apparatus was operated at a rotation speed of 800 rpm, using
CHCl3/MeOH/H2O (7:13:8) as solvent system with the less
dense layer as mobile phase at a flow rate of 1 mL/min. Fifty
fractions (each 10 mL) were collected.
Acetyla tion a n d P u r ifica tion of Acetyla ted Glyco-
sid es. Combined MLCCC fractions (13-16) were concentrated
under reduced pressure to dryness. The dry residue (1 g) was
acetylated with 20 mL of Ac2O/pyridine and 500 mg of
4-(dimethylamino)pyridine at room temperature overnight.
The peracetylated glycosides were further separated by flash
chromatography using 150 mL of hexane/diethyl ether (1:5)
as solvent. Combined fractions (10 mL each) 71-77, 89-103,
and 119-130 were concentrated in vacuo to 1 mL for subse-
quent HPLC purification.
P r ep a r a tive HP LC. The semipurified acetylated glycosides
4b-6b obtained above were finally purified by preparative
HPLC using an Ultrasphere Si column (5 µm, 250 mm × 4.6
i.d., Beckman), with diethyl ether as mobile phase at a flow
rate of 1 mL/min, thus yielding pure compounds as white
crystals: 4b (30 mg), 5b (2 mg), and 6b (26 mg).
The spectral data of glycoside 6b were as follows: UV (Et2O)
λmax 230 nm; CI-MS, m/z (%) 555 (5, [M + H]+), 537 (2, [M -
H2O + H]+), 331 (100), 271 (16), 207 (66), 206 (12), 169 (32),
1
150 (24); H and 13C NMR data were in good agreement with
those showed by an authentic sample of peracetylated (6S,9R)-
vomifoliol 9-O-â-D-glucopyranoside. CD data of the glucoside
6a correspond to those published by Otsuka et al. (1995),
showing 6S as the absolute configuration at C-6.
Dea cetyla tion a n d En zym a tic Hyd r olysis. To 1 mg of
4b in 5 mL of MeOH was added 5 mL of 0.02 M NaOMe
solution. After 12 h, the mixture was neutralized by adding
50 mg of Amberlite IR-120 (15-50 mesh, H+ form). After
removal of the ion-exchange resin by filtration, the solvent was
evaporated in vacuo, and the deacetylated glycoside was
dissolved in 5 mL of H2O. To this solution was added 10 mL
of citric acid-phosphate buffer (pH 5.5) as well as 500 µg of a
commercially available â-glucosidase (almond, emulsine), and
the mixture was incubated over 24 h at 37 °C. The liberated
aglycon (woody, balsamic, and pleasant odor) was extracted
with diethyl ether and subjected to HRGC/and HRGC/MS
analyses: Ri (DB-5) 1785, Ri (HP1) 1704; EI-MS (70 eV), m/z
(%) 224 (M, 1), 209 (M - CH3, 1), 206 (M - H2O, 1), 191 (1),
181 (1), 168 (1), 167 (3), 163 (3), 150 (3), 125 (11), 109 (5), 107
Glycoside 4b showed the following spectral data: UV (Et2O)
λmax 228 nm; MS, cf. Table 1; H NMR, cf. Table 2; 13C NMR,
1
cf. Table 3; CD spectrum showed a positive maximum at 240
nm (∆ꢀ ) +17.7, MeOH).

Glucoconjugates as Aroma Precursors in Tropical Fruits
J. Agric. Food Chem., Vol. 47, No. 4, 1999 1643
(9), 95 (19), 83 (25), 81 (11), 79 (12), 69 (5), 67 (7), 55 (19), 53
(8), 45 (19), 43 (100), 41 (30), 39 (19). In the same way, after
deacetylation and enzymatic hydrolysis, compound 5b yielded
an aglycon (green-woody odor), which was also analyzed by
HRGC and HRGC/MS: Ri (HP1) 1609, Ri (DB-5) 1689, Ri (DB-
Wax) 2682; EI-MS (70 eV), m/z (%) 206 (M+, 29), 191 (26), 173
(58), 147 (16), 131 (14), 130 (10), 129 (9), 119 (27), 115 (11),
105 (14), 91 (31), 79 (13), 78 (8), 77 (21), 65 (10), 63 (9), 55 (5),
53 (11), 51 (12), 45 (2), 43 (100), 41 (22), 39 (22). MS and 1H
NMR data were in good agreement with those published by
Sannai et al. (1984). In the same way as described above,
compound 6b was also deacetylated and subjected to enzymatic
hydrolysis, and the liberated aglycon (sweet, woody, and fruity
odor) was analyzed by HRGC and HRGC/MS: Ri (DB-5) 1806;
Ri (HP1) 1749; EI-MS (70 eV) data as reported by Achenbach
et al., (1981).
program starting at 50 °C, raised to 300 °C at 5 °C/min, and
kept at 300 °C for 10 min. Injector and detector temperatures
were kept at 300 °C. The flow rate for the carrier gas was 1.1
mL/min He, the flow rate for the make up gas was 30 mL/
min, and those for the detector gases were 30 mL/min H2 and
30 mL/min air. Split injection 1:10 and 2 µL injection volumes
were used. The linear retention index (Ri) is based on a series
of n-hydrocarbons.
Ca p illa r y Ga s Ch r om a t ogr a p h y/Ma ss Sp ect r om et r y
(HRGC/MS). A Hewlett-Packard 5890 gas chromatograph
with split injector (1:10), equipped with a selective mass 5970
Hewlett-Packard detector, was used. The same type of columns
and temperature conditions as mentioned above for HRGC
were used, temperature of ion source and all connecting parts,
300 °C; electron energy, 70 eV; mass range, 30-350.
Isola tion of Na tu r a l Com p ou n d 5a . Five grams of crude
glycosidic extract was subjected to MLCCC using CHCl3/
MeOH/H2O (7:13:8) as mentioned above. Combined fractions
(13-16) was refractionated by MLCCC using EtOAc/n-BuOH/
H2O (3:2:5) as solvent system with the more dense layer acting
as mobile phase at a flow rate of 1 mL/min. Fifty-five fractions
(each 5 mL) were collected. Subfractions (21-28) were finally
purified by subsequent preparative HPLC on RP-18 and
Lichrosorb Diol columns with methanol (flow rate ) 1 mL/
min) and hexane/n-butanol/methanol/water (65:25:9:1) (flow
rate ) 1 mL/min) as solvent systems, respectively, to yield 1
mg of pure 5a . Glucoside 5a showed the following spectral
data: UV (MeOH) λmax 227 nm; 1H NMR (500 MHz, CD3OD) δ
1.12 (3H, s, CH3-11), ∼1.31 (1H, Ha-2)*, 1.34 (3H, s, CH3-12),
∼1.42 (1H, Ha-4)*, 1.43 (3H, s, CH3-13), 1.88 (1H, dd, J ) 11.5,
3.9 Hz, Hb-2), 2.16 (3H, s, CH3-10), 2.45 (1H, dd, J ) 11.3, 3.9
Hz, Hb-4), 3.10 (1H, dd, J ) 8.5, 7.8 Hz, H-2′), 3.15-3.19 (2H,
m, H-4′ and H-5′), 3.21 (1H, dd, J ) 8.5, 8.5 Hz, H-3′), 3.57
(1H, dd, J ) 11.6, 5.2 Hz, Hb-6′), 3.77 (1H, dd, J ) 11.7, 1.8
Hz, Ha-6′), 4.29 (1H, tt, J ) 11.3, 3.9 Hz, H-3), 4.48 (1H, d, J
) 7.8 Hz, H-1′), 5.86 (1H, s, H-8) (asterisks indicate that the
signals are obscured due to partial overlaping). In the same
way as described above, natural glucoside 5a was enzymati-
cally hydrolyzed with emulsin (â-glucosidase), producing com-
pound 7 as hydrolysis product. This time compound 7 was
characterized by HRGC and HRGC/MS. The data obtained
were in good agreement with the data mentioned above for
this compound.
Mod el Rea ction s. (a) Reduction of 3-Hydroxy-7,8-didehy-
dro-â-ionone. To 2 mg of 3-hydroxy-7,8-didehydro-â-ionone (7)
dissolved in 5 mL of ethanol was added 2 mg of NaBH4, and
the reaction was allowed to continue for 24 h. After extraction
with ethyl acetate, the organic phase was dried and concen-
trated, yielding 1 mg of 3-hydroxy-7,8-didehydro-â-ionol (8),
which was characterized by HRGC and HRGC/MS: Ri (DB-
Wax) 2763; EI-MS (70 eV), m/z (%) 208 (M+, 9), 193 (13), 175
(9), 149 (7), 133 (8), 131 (11), 121 (6), 119 (8), 105 (18), 93 (7),
91 (20), 79 (12), 77 (15), 69 (16), 67 (5), 65 (9), 55 (14), 53 (10),
45 (10), 43 (100), 41 (27), 39 (19). The chromatographic and
mass spectral data are in agreement with those showed by an
authentic sample.
(b) Acid Treatment of 3-Hydroxy-7,8-didehydro-â-ionol. A
solution of 0.5 mg of compound 8 dissolved in 10% aqueous
ethanol was adjusted to pH 3.0 and placed in a sealed ampule
for 1 day at 100 °C (Sefton et al., 1989). The reaction products
were extracted with ethyl ether and characterized by HRGC
and HRGC/MS as â-damascenone (9) (Ri DB-Wax 1817) and
3-hydroxy-â-damascone (10) (Ri DB-Wax 2554) in a 13:87 ratio.
The mass spectral data for compounds 9 and 10 are in good
agreement with those of authentic samples.
Ca p illa r y Ga s Ch r om a togr a p h y (HRGC). A Hewlett-
Packard 5890 gas chromatograph with FID equipped with a
J &W DB-5 fused silica capillary column (30 m × 0.31 mm i.d.;
film thickness ) 0.52 µm) was used. A Hewlett-Packard HP1
fused silica capillary column (12 m × 0.2 mm i.d.; film
thickness ) 0.33 µm) was also used. The DB-5 column was
operated with a temperature program starting at 60 °C, raised
to 300 °C at 5 °C/min, and kept at 300 °C for 10 min. The
conditions for the HP1 column were as follows: temperature
RESULTS AND DISCUSSION
Isola tion of Glycosid es. A glycosidic extract from
lulo leaves was obtained by Amberlite XAD-2 adsorption
(Gunata et al., 1985) and methanol elution. It was
subsequently subjected to a prefractionation using ML-
CCC (Roscher and Winterhalter, 1993). Monitoring of
separated MLCCC fractions by TLC revealed major
products in MLCCC fractions 13-16, which after acetyl-
ation and flash chromatography gave three semipurified
fractions 71-77, 89-103, and 119-130. Each of the
latter mentioned fractions was finally purified by HPLC
on SiO2 to afford pure peracetylated glucosides 4b, 5b,
and 6b, respectively.
Ch a r a cter iza tion of Glu cosid e 4a . The character-
ization was made in its acetate 4b form by UV and MS
as well as by 1H and 13C NMR mono- and bidimensional
spectroscopy. Compound 4b showed a UV absorption
maximum at 228 nm, indicating an enone structure
(Hesse et al., 1987). FAB-MS as well as CI-MS data (cf.
Table 1) yielded a molecular mass of 597 and diagnostic
ions m/z 331 and 271, thus indicating a monosacharide
1
(hexose) as sugar moiety. From both H (cf. Table 2) and
13C NMR data (cf. Table 3) the presence of a glucose
moiety was confirmed. The 1H NMR exhibited a doublet
at δ 4.58 (J ) 8.3 Hz) for the anomeric proton, indicating
a â-glycosidic linkage. Furthermore, from these NMR
data obtained for the sugar moiety was evident a
coincidence with data published for per-O-acetylated
â-glucoside (Guldner and Winterhalter, 1991).
The 1H NMR data (Table 2) analysis led us to
conclude that the aglycon part of 4b resembles the
3-oxo-R-ionol structure except for the absence of the
CH3-13 signal and the presence of an oxymethylene
signal at δ 4.60 (br s), which correlates with the olefinic
H-4 at δ 6.04 in the H-H COSY experiment. The latter
correlates in its turn with the signal at δ 64.06 in the
H-C COSY experiment. This analysis indicates that
compound 4b holds an acetylated hydroxyl group at
C-13. The 13C NMR data (Table 3) are also in good
agreement with the structure proposed for 4b, that is,
the peracetylated 13-hydroxy-3-oxo-R-ionol â-D-glucopy-
ranoside. The chemical shift of H-9 resonating upfield
(∼1.1 ppm) in glucoside 4b from the corresponding
signal for acetylated hydroxyl group (Neugebauer et al.,
1995) clearly indicates that the sugar is attached to the
hydroxyl group in the C-9 position. Deacetylation of 4b
and subsequent treatment with â-glucosidase (emulsine)
led to the release of the corresponding aglycon, the
structure of which was confirmed as 13-hydroxy-3-oxo-
R-ionol by HRGC/MS analysis. Among the two stereo-
genic centers found in 4b, absolute stereochemistry at
C-6 was determined on the basis of CD. Compound 4b

1644 J. Agric. Food Chem., Vol. 47, No. 4, 1999
Osorio et al.
exhibited a positive maximum at 240 nm (∆ꢀ ) +17.7,
MeOH), thus establishing its 6R absolute stereochem-
istry, which correlates well with the data published for
the (6R)-3-oxo-R-ionol isomer (Pabst et al., 1992). The
chemical shift of C-9 (δ 76.52, Table 3) in glucoside 4b
clearly indicates R configuration for this stereogenic
center. Pabst et al. (1992) reported the 13C NMR
chemical shifts of the â-D-glucopyranoside of (9R)- and
(9S)-3-oxo-R-ionol to be δ 77.0 and 74.7, respectively.
On the basis of the overall results for compound 4b,
we assign the structure (6R,9R)-13-hydroxy-3-oxo-R-
ionol 9-O-â-D-glucopyranoside for compound 4a . As far
as we know, this is the first time that compound 4a has
been found in nature. Experiments to investigate the
role of this glucoside as precursor of the C13-noriso-
prenoids present in lulo aroma are under way.
Ch a r a cter iza tion of 5a a n d 6a . This characteriza-
tion was also performed in their acetate 5b and 6b
forms.
In the case of 5b, FAB-MS indicates a molecular
weight of 596 and the presence of a peracetylated hexose
as sugar unit. 1H NMR suggested the presence of a
tetraacetyl-â-D-glucopyranosyl moiety, with data in good
agreement with those reported for other â-glucopyra-
nosides (Guldner and Winterhalter, 1991). Furthermore,
the 1H NMR data showed the signals assignable to three
methyl singlets (δH 1.15, 1.31, 1.42), an acetyl (δH 2.18
and δC 26.62 and 210.74), and two oxymethines, one
being tertiary at δC 77.81 and the other secondary,
presumably in the acetate form, at δH 5.16 and δC
66.98. These facts, the signal at δ 5.91 correlating with
the signal at δ 100.88 in the H-C COSY experiment
and the further signals in the 13C NMR spectrum at δ
116.87 and 197.68, clearly indicated the presence of an
allenic group in the aglycon part of 5b. The position of
the glucose moiety was settled as follows. The 3-O- and
5-O-glucosides of 5a are known compounds, and their
13C NMR data were reported (Miyase et al., 1987;
Umehara et al., 1988). The former showed C-3 and C-5
signals at δ 72.0 and 71.3, respectively, while the latter
exhibited them at δ 62.5 and 78.1, respectively. Taking
into consideration an acylation shift (downfield shift
would be several parts per million), the data of com-
pound 5b (δ 66.98 and 77.81) are much more consistent
with those of an acetylated 5-O-glucoside. Thus, com-
pound 5b has been established to be the pentaacetate
of 5-O-â-D-glucopyranoside of 3,5-dihydroxy-6,7-me-
gastigmadien-9-one. The stereochemistry of the three
chiral centers of this compound was determined as
follows. The 1H NMR spectrum of 5b clearly showed
axial configuration (δ 5.16, multiplet, W1/2 ) 21 Hz) for
H-3. Irradiation of protons of CH3-12 (δ 1.31) produced
an NOE on the signal at δ 5.16, indicating a CH3-12
axial configuration. Further NOE experiments irradiat-
ing CH3-13 (δ 1.42) did not have any effect on the signal
of H-3 at δ 5.16, thus demonstrating an equatorial
position for this methyl group. Assuming that the
aglycon of compound 5a is being formed by biodegrada-
tion of allenic carotenoids such us fucoxanthine or
neoxanthine (Isoe et al., 1971), the stereochemistry at
C-3 must be S. This fact and the results of the above-
mentioned NOE experiments clearly showed (3S,5R)
stereochemistry for compound 5b. In relation to the
other chiral center, we assigned R configuration at C-8
on the basis of the NMR data in deuteropyridine of the
natural 5a , which were in good agreement with those
published by Umehara et al. (1988) for the (3S,5R,8R)-
F igu r e 1. Structures of the major glucoconjugated C13
norisoprenoids identified in lulo (S. quitoense L.) leaves.
-
F igu r e 2. Generation of 3-hydroxy-7,8-didehydro-â-ionone (7),
â-damascenone (9), and 3-hydroxy-â-damascone (10) from
(3S,5R,8R)-3,5-dihydroxy-6,7-megastigmadien-9-one 5-O-â-D-
glucopyranoside (5a ).
isomer. On the basis of the above results, we have
assigned the structure of (3S,5R,8R)-3,5-dihydroxy-6,7-
megastigmadien-9-one 5-O-â-D-glucopyranoside for com-
pound 5a .
Deacetylation of 5b and subsequent treatment with
emulsin led to the liberation of the 3-hydroxy-7,8-
didehydro-â-ionone (7) (Figure 2), the structure of which
1
was confirmed by HRGC and HRGC/MS and H NMR
analyses. As per our observations compound 5b is quite

Glucoconjugates as Aroma Precursors in Tropical Fruits
J. Agric. Food Chem., Vol. 47, No. 4, 1999 1645
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labile. Thus, one could think that the acetylenic com-
pound could come directly as a result of base treatment
of 5b. For this reason, we isolated pure glycoside 5a
from lulo leaves and submitted it to enzymatic hydroly-
sis with emulsin, finding 3-hydroxy-7,8-didehydro-â-
ionone (7) as reaction product. Additional model reac-
tions depicted in Figure 2 (NaBH4 reduction of compound
7 and its further heated acid catalyzed rearrangement)
showed compound 5a as a new precursor of â-dama-
scenone (9) and 3-hydroxy-â-damascone (10), important
industrial flavor compounds. It is also important to point
out that 3,5-dihydroxy-6,7-megastigmadien-9-one 5-O-
â-D-glucopyranoside (5a ) is the true precursor of 3-hy-
droxy-7,8-didehydro-â-ionone (7), one of the major vol-
atiles found in lulo peelings (Sua´rez et al., 1993).
Finally, it is important to mention that glucoside 5a
(citroside A) has been reported only in Citrus unshius
(Umehara et al., 1988) leaves. In contrast, the peracetyl-
ated 5b derivative has never been reported.
1
Analyses of the spectral data (UV, CI-MS, H NMR
and 13C NMR mono- and bidimensional experiments)
of 6b and comparison with data for authentic sample
allowed us to establish for this glycoside the structure
of peracetylated (6S,9R)-vomifoliol 9-O-â-D-glucopyra-
noside. Glucoside 6a is a ubiquitous natural compound
that has been detected in various fruit species (Winter-
halter and Schreier, 1994). Its role in the formation of
theaspirones (Winterhalter and Schreier, 1994), key
components of black tea aroma, has been established.
The role of 6a as an intermediate in the generation of
vitispiranes has also been studied (Winterhalter and
Schreier, 1988).
ACKNOWLEDGMENT
We are indebted to Mr. N. Hara (Tokyo Institute of
Technology) for measuring FAB-MS spectra. We thank
Instituto de Inmunolog´ıa del Hospital San J uan de Dios,
Santafe´ de Bogota´, for NMR analyses of compound 6b.
We also thank Prof. Dr. M. Tagawa from Aichi Medical
College, J apan, for generously providing synthetic com-
pound 6b.
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Received for review J uly 8, 1998. Revised manuscript received
December 28, 1998. Accepted December 29, 1998. We grate-
fully acknowledge grants from the EU (CI1-CT92-0019), COL-
CIENCIAS, and IPICS, Uppsala University, Sweden.
J F9807364