The Role of Diglycosides as Tea Aroma Precursors: Synthesis of Tea Diglycosides and Specificity of Glycosidases in Tea Leaves

Article abstract of DOI:10.1021/jf960852b

Two general synthetic routes were established in order to synthesize two diglycosides, primeverosides (1) and vicianosides (2), found in tea leaves. Procedure 1 is based on the Koenig-Knorr type of condensation of aglycon alcohols and 1-α-bromohexabenzoylprimeverose (6) and is suitable for the condensation of primary alcohols. Procedure 2 is to combine tribenzoyl-β-D-glucoside (8) and 1-α-bromotribenzoylxylose (4). The primeveroside of a tertiary alcohol was synthesized by this method which is also applicable to the synthesis of vicianosides. The hydrolysis rate of each of the 12 synthesized glycosides by a crude tea enzyme was evaluated, which suggest that the main glycosidase is primeverosidase and the enzyme mixture shows substrate specificity to both the carbohydrate and aglycon moieties.

Full text of DOI:10.1021/jf960852b

2674
J. Agric. Food Chem. 1997, 45, 2674−2678
Th e Role of Diglycosid es a s Tea Ar om a P r ecu r sor s: Syn th esis of Tea
Diglycosid es a n d Sp ecificity of Glycosid a ses in Tea Lea ves
Sachiko Matsumura,† Shunya Takahashi,‡ Mariko Nishikitani,† Kikue Kubota,† and
Akio Kobayashi*,†
Laboratory of Food Chemistry, Department of Nutrition and Food Science, Ochanomizu University,
2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112, J apan, and RIKEN (The Institute of Physical and Chemical
Research), Wako-shi, Saitama 351-01, J apan
Two general synthetic routes were established in order to synthesize two diglycosides, primeverosides
(1) and vicianosides (2), found in tea leaves. Procedure 1 is based on the Koenig-Knorr type of
condensation of aglycon alcohols and 1-R-bromohexabenzoylprimeverose (6) and is suitable for the
condensation of primary alcohols. Procedure 2 is to combine tribenzoyl-â-D-glucoside (8) and 1-R-
bromotribenzoylxylose (4). The primeveroside of a tertiary alcohol was synthesized by this method
which is also applicable to the synthesis of vicianosides. The hydrolysis rate of each of the 12
synthesized glycosides by a crude tea enzyme was evaluated, which suggest that the main glycosidase
is primeverosidase and the enzyme mixture shows substrate specificity to both the carbohydrate
and aglycon moieties.
Keyw or d s: Tea; flavor precursor; synthesis; primeveroside; vicianoside
INTRODUCTION
tigate the character and specificity of the glycosidase
in tea leaves have shown that the main glycosidase was
primeverosidase (Guo et al., 1995). However, the pri-
meverosides used in that study as enzyme substrates
were natural products separated from tea leaves in such
small amounts that limited their use in further inves-
tigations to clarify the formation mechanism of tea
aroma from the glycosidically bound aroma compounds.
It thus became necessary to obtain greater amounts of
various glycosides by synthetic methods.
p-Nitrophenyl-â-D-primeveroside has been synthe-
sized for biochemical studies on xyloglucan (Sone and
Masaki, 1986), and only two primeverosides, moridone
6-O-â-primeveroside (Vermes et al., 1980) and neohan-
cosides (Konda et al., 1996), have been synthesized to
elucidate the chemical structure of the natural products.
We tried to find a more general synthetic method for
obtaining diglycosides, primeverosides and vicianosides,
having typical tea aroma compounds as their aglycons,
to clarify the enzyme specificity for these glycosides in
tea leaves, as well as to elucidate their chemical
structures.
Our synthetic strategy toward primeverosides is
based on the Koenig-Knorr reaction. The first route
involves combining the primeverose moiety with aglycon
alcohol, and the second one combines the xylose moiety
with the already prepared glucoside. The former route
proved to be convenient for primary alcohols (procedure
1) and the latter for the less reactive tertiary alcohols
such as linalool as aglycon (procedure 2). As a variation
of latter procedure, the arabinose moiety substituted for
xylose was condensed with glucoside to give several
vicianosides in reasonable yield.
Aroma is one of the important factors to determine
the character and quality of various teas. In a recent
review of tea research (Teranishi and Hornstein, 1995),
more than 600 volatile compounds concerned with tea
aroma were described, among which aliphatic alcohols
and mono- and sesquiterpene alcohols were the main
constituents of the aroma volatiles of black and oolong
teas. These teas are called fermented and semifer-
mented teas, respectively, because their manufacture
involves the enzymatic activity in fresh tea leaves to
different extents. Biochemical aroma formation is
thought to proceed by two different processes.
One type follows normal biosynthetic routes such as
the formation of (Z)-3-hexenol from linolenic acid via
peroxidation by lipoxygenase and cleavage of the C-C
bond by a lyase (Hatanaka, 1993) or the formation of
various terpene alcohols via the mevalonate pathway
(Erman, 1985).
The other type involves the hydrolysis of glycosides
having aroma compounds as their aglycons. Various
glycosides have recently been identified from fruits and
other parts of plants (Schreier and Winterhalter, 1993).
In fresh tea leaves, benzyl-â-D-glucoside (Yano et al.,
1991) and (Z)-3-hexenyl-â-D-glucoside (Kobayashi et al.,
1994) have been identified as precursors of tea aroma,
as well as diglycosides, such as 6-O-â-xylopyrasyl-â-D-
glucopyranosides (primeverosides, 1) of benzyl alcohol,
2-phenylethanol and (S)-linalool (Guo et al., 1994) and
of some linalool oxides (Moon et al., 1994) from fresh
leaves of an oolong tea cultivar. Geranylvicianoside
(2c), the 6-O-R-L-arabinopyranosyl-â-D-glycopyranoside,
has also recently been identified in fresh tea leaves of
a green tea cultivar (Nishikitani et al., 1996). Studies
on the chemical structures of these glycosides to inves-
MATERIALS AND METHODS
An a lytica l In str u m en ts. GC and GC-MS Analyses.
Quantitative analysis of the volatiles was conducted with
Shimadzu GC-7A gas chromatograph equipped with FID. The
GC conditions were as follows: column, 50 m × 0.25 mm (i.d.)
fused silica capillary type, coated with PEG-20M (CP-Wax, film
thickness, 0.25 µm); N2 carrier gas flow rate, 1.1 mL/min; split
ratio, 35:1; column temperature, held at 60 °C for 4 min and
* Author to whom correspondence should be ad-
dressed (telephone 81-3-5978-2557; fax 81-3-5978-5759;
e-mail koba@comfort.eng.ocha.ac.jp).
† Ochanomizu University.
‡ RIKEN.
S0021-8561(96)00852-7 CCC: $14.00
© 1997 American Chemical Society

Glycosides as Tea Aroma Precursors
J. Agric. Food Chem., Vol. 45, No. 7, 1997 2675
F igu r e 1. General synthetic procedures for primeverosides and vicianosides.
raised to 180 °C at 2 °C/min; injection temperature, 170 °C.
Identification and structural investigation were carried out by
GC-MS using a Hewlett-Packard 5972 mass spectrometer
combined with a Hewlett-Packard 5890 GC. The GC condi-
tions were the same as those for the corresponding GC
analyses, except that He was used as the carrier gas. HR-
FABMS data were obtained with a J EOL J MS-AX505WA
instrument under the following conditions: acceleration volt-
age, 3.0 kV; matrix, glycerol; gas, Xe; standard, polyethylene
glycol.
NMR Analysis. PMR and CMR spectra were collected with
a J EOL-J NM-GX-270 (270 MHz) spectrometer, each sample
being dissolved in CDCl3 containing TMS as the internal
standard.
TLC Analysis. TLC was performed with precoated plates
of silica gel and a solvent system of 12:3:3:2 v/v (EtOAc/AcOH/
MeOH/H2O). To detect the glycosidic fraction, 0.2% (v/v)
naphthoresorcinol/EtOH:H2SO4 (19:1 v/v) was used before
heating to 110 °C.
h at room temperature, the reaction mixture was poured onto
ice-water and extracted with dichloromethane. After succes-
sively washing with a sat. NaHCO3 solution and water, and
then drying with MgSO4, the solvent was distilled off under
reduced pressure.
The residue was treated with ether to give 2,3,4,2′,3′,4′-hexa-
O-benzoyl-R-D-primeverosyl bromide (6, 480 mg, 33.3% yield)
as a solid material (mp 178 °C dec) which was used for the
next step without purification.
To a stirred mixture of an aglycon alcohol (0.9 mmol) and 6
(350 mg, 0.3 mmol) dissolved in acetonitrile (3 mL) was added
mercuric cyanide (250 mg, 0.9 mmol) at room temperature
while stirring, which was continued for 3 h. The reaction
mixture was diluted with dichloromethane and filtered. The
filtrate was successively washed with a saturated NaHCO3
solution and water, dried with MgSO4, and concentrated under
reduced pressure. The residue was chromatographed on silica
gel and eluted with toluene-ethyl acetate (20:1 v/v) to give
7a -c. The solid material was recrystallized from benzene-
cyclohexane (1:1 v/v).
The synthesized hexa-O-benzoyl-â-D-primeverosides (7a -
c, 0.2 mmol) were dissolved in methanol (10 mL). A 0.1 M
sodium methoxide solution (1.2 mmol) was added to metha-
nolyze the ester linkage. After stirring for 15 h at room
temperature, the mixture was neutralized with H+ type Dowex
50W-X8 ion exchange resin (Dow Chemical Co., Midland, MI).
The resulting neutral mixture was filtered and the methanol
was evaporated to give a syrup (1a -c), which was purified by
HPLC with a solvent system of acetonitrile-water (2:3 v/v).
Procedure 2. (R,S)-Linalyl-2,3,4-tri-O-benzoyl-â-D-glucopy-
ranoside (8d ) was prepared from glucose and (R,S)-linalool by
the same method described in the literature (Sone and Misaki,
1986). The Koenig-Knorr reaction was performed under the
same conditions as those used in procedure 1, 0.5 g of 8d (0.8
mmol) and 1.3 g of 4 (2.5 mmol) being dissolved in 10 mL of
acetonitrile, 0.65 g of mercuric cyanide (2.6 mmol) being added,
and stirring being continued for 3 h. After being diluted with
dichloromethane and after the organic layer was washed and
dried, the solvent was distilled off by following procedure 1.
The eluate from the silica gel column with toluene-ethyl
acetate (20:1 v/v) gave 416 mg of (R,S)-linalyl-2,3,4-tri-O-
Gen er a l Syn th etic Meth od for P r im ever osid es. All
reagents are purchased from Sigma-Aldrich J apan and are
used without any purification.
Procedure 1. 1,2,3,4-Tetra-O-benzoyl-â-D-glucose (3, mp
179.5 °C; Peter and Per, 1986) was prepared by following the
known method for preparing 1,2,3,4-tetra-O-acetyl-â-D-glucose
(Reynolds and Evans, 1953) using benzoyl chloride.
To a mixture of 8.0 g of 3 (13.4 mmol) and 10 g (19.0 mmol)
of freshly prepared 2,3,4-tri-O-benzoyl-R-D-xylosyl bromide (4,
mp 137 °C; Fletcher et al., 1947) dissolved in acetonitrile was
added 7.5 g of mercuric cyanide (30 mmol) in one portion while
stirring, which was continued for 3 h at room temperature.
The reaction mixture was then diluted with dichloromethane,
successively washed with a saturated NaHCO3 solution and
water, dried with MgSO4, and concentrated in vacuo. The
residue was chromatographed on silica gel and eluted with
toluene-ethyl acetate (20:1 v/v) to give 1,2,3,4-tetra-O-benzoyl-
6-(2,3,4-tri-O-benzoyl-D-xylopyranosyl)-â-D-glucose (5), which
was then recrystallized from ether; mp 181 °C.
To 1.5 g of 5 (1.4 mmol) dissolved in dichloromethane-
ethylacetate (9:1 v/v, 20 mL), titanium tetrabromide (1.1 g,
3.0 mmol) was added under N2 gas flow. After stirring for 1

2676 J. Agric. Food Chem., Vol. 45, No. 7, 1997
Matsumura et al.
Ta ble 1. HRMS Da ta for th e Syn th esized Diglycosid e
°C, stirring and cooling being continued for 1.5 h. The mixture
was poured onto 50 mL of ice-water, the organic layer
separated was washed and dried, and the solvent was distilled
off. The residue crystallized from methanol had mp 146 °C,
the yield of 9 being 3.01 g (57.3%). A mixed solution of 2,3,4-
tetra-O-benzoyl-â-D-glucosides (8a -d , 1.6 mmol) and 9 (0.3
mmol) in acetonitrile was condensed by the Koenig-Knorr
reaction in the presence of 0.35 g of mercuric cyanide (1.3
mmol) as described for procedure 2. Debenzoylation of vi-
cianoside hexabenzoates (10a -d ) was conducted with sodium
methoxide/methanol as described for procedure 2 to yield
vicianosides (2a -d ) in over 90% yield respectively.
observed ∆mmu
molecular formula
primeveroside type
benzyl alcohol (1a )
403.1605 +0.1 C18H27O10 [M + H]+
2-pehnylethanol (1b) 417.1798 +3.7 C19H29O10 [M + H]+
geraniol (1c)
linalool (1d )
471.2227 +2.1 C21H36O10Na [M + Na]+
471.2227 +2.1 C21H36O10Na [M + Na]+
vicianoside type
benzyl alcohol (2a )
425.1463 +4.0 C18H27O10Na [M + Na]+
2-phenylethanol (2b) 417.1768 +0.7 C19H29O10 [M + H]+
geraniol (2c)
linalool (2d )
471.2227 +0.6 C21H36O10Na [M + Na]+
471.2227 +0.6 C21H36O10Na [M + Na]+
P r ep a r a tion of th e Cr u d e En zym e Solu tion . Acetone
powder was prepared from fresh tea leaves of Camellia
sinensis var. sinensis Okumusashi, as previously described
(Yano et al., 1990). In 60 mL of a 50 mM citrate buffer
solution, 3.0 g of the acetone powder, 0.3 g of ascorbic acid,
and 0.5 g of Polyclar AT (General Anilin & Film Corp. New
York) were homogenized twice for 30 s while cooling at 0 °C.
After centrifuging at 10000g for 20 min at -5 °C, the resulting
supernatant was used as the crude enzyme solution.
benzoyl-6-â-(2,3,4-tri-O-benzoylxylopyranosyl)glucopyra-
noside (7d ). To a methanolic solution (10 mL) of 215 mg of
7d (0.2 mmol) was added 1.2 mL of 0.1 M sodium methoxide
while stirring. After 15 h of stirring at room temperature, the
solution was neutralized with Dowex-50W, filtered, and con-
centrated in vacuo to a syrupy material. Under the same
HPLC conditions as those already described, 77 mg of (R,S)-
linalyl-1-â-D-primeveroside (1d ) was obtained.
Gen er a l Syn th etic Meth od for Vicia n osid es. Benzyl-,
phenylethyl-, geranyl-, and (R,S)-linalyl-2,3,4-tetra-O-benzoyl-
â-D-glucopyranosides (8a -d ) and 2,3,4-tri-O-benzoyl-â-L-ara-
binopyranosyl bromide (9) were prepared under the same
conditions as those used for procedure 2.
H yd r olysis of Glycosid es w it h t h e Cr u d e E n zym e
Solu tion . Each synthetic glycoside (12 µM) was dissolved in
4.9 mL of a 50 mM sodium citrate buffer solution (pH ) 5.0).
After adding 100 µL of an acetone solution of nonanol (7 ppm)
as internal standard, the solution was incubated with 100 µL
of the crude enzyme solution for 15 min at 37 °C. The freed
aglycons were extracted with ether and analyzed by GC and
To a stirred chloroform solution (50 mL) of 1,2,3,4-tetra-O-
benzoyl-â-L-arabinopyranose (6.23 g, 10 mmol) was added 22.3
mL of a 33% HBr/AcOH solution dropwise during 15 min at 0
Ta ble 2. CMR Da ta for th e Syn th esized Disglycosid es
1a
1b
1c
1d
2a
2b
2c
2d
aglycon
1
2
3
4
5
6
7
8
139.7
130.0
130.0
129.4
130.0
130.0
72.8
139.5
129.7
129.4
129.3
129.4
129.7
35.9
66.5
121.6
142.0
40.7
115.9,115.2
144.4
137.5
132.0
129.6
129.0
132.0
132.0
73.9
139.7
129.9
129.5
127.4
129.5
129.9
36.1
66.5
120.0
142.0
40.0
116.0,116.2
144.3
81.5
42.7,42.5
23.6
125.7,125.6
132.0
81.6,81.5
42.7,42.5
23.7,23.6
125.8,125.7
132.1
27.4
26.8
125.1
132.6
25.9
125.1
131.6
26.0
73.4
25.9
73.2
25.6
9
17.8
17.8
18.0
17.7
10
16.6
23.3
16.6
23.2
glucose
1′
2′
3′
4′
5′
6′
104.0
75.6
78.7
71.9
76.5
70.6
105.0
75.7
78.4
72.1
76.7
70.8
102.8
74.9
77.9
71.4
76.9
69.7
99.6,99.3
75.2,75.0
78.1
71.4
76.5
102.2
74.0
76.6
71.6
75.9
70.3
103.1
73.9
76.3
71.5
75.9
70.2
101.7
73.9
76.8
70.1
75.8
69.2
99.8,100.0
75.1,75.0
78.2
72.4
75.8
69.8,69.6
68.7
xyl or ara
1′′
2′′
3′′
4′′
5′′
106.3
75.8
77.8
72.2
67.7
106.2
75.7
77.3
71.6
67.6
105.5
74.8
77.7
71.1
66.9
105.4
74.9
77.6
71.2
66.8
104.6
72.4
73.1
69.2
67.1
104.6
71.8
73.2
69.2
67.1
104.5
71.6
73.2
69.0
67.1
104.1
71.8
74.2
69.4
66.1

Glycosides as Tea Aroma Precursors
J. Agric. Food Chem., Vol. 45, No. 7, 1997 2677
Ta ble 3. Hyd r olysis Ra tes (%) of Tw elve Glycosid es by
th e Cr u d e En zym e Solu tion
GC-MS. The amounts of the aglycons were determined from
the peak area ratio to that of nonanol and transformed into a
mol ratio. In order to compare the enzyme activity toward
each glycoside, the percentage of each mol yield to the highest
one was used as the hydrolysis ratio (%).
glycosides
glucoside
vicianoside
primeveroside
The aqueous layer after the ether extraction was concen-
trated in vacuo, and the resulting sugars were determined
qualitatively by TLC. Solutions without enzyme or without
substrate were also incubated at the same time as blanks.
aglycons
benzyl-
2-phenylethyl-
geranyl-
linalyl-
4.1
5.4
44.5
22.2
1.2
1.2
3.0
9.3
11.9
9.5
100.0
36.7
RESULTS AND DISCUSSION
among a series of diglycosides with the same aglycon,
the hydrolysis rates of primeveroside are high without
exception. This specificity of the tea glycosidases does
explain our previous experimental results in which the
composition of volatiles freed from the glycoside fraction
of tea leaves by crude tea glycosidase differed greatly
from that obtained with commercial â-glucosidase or a
nonspecific glycosidase (Morita et al., 1994).
By comparing the hydrolysis rate of each glycoside
with the same glycoside moiety but different aglycons,
those of the geranyl- and linalyl-diglycosides were much
higher than those of the benzyl and 2-phenylethyldi-
glycosides. It may be concluded that the primeverosi-
dase in tea leaves was specific not only to the glycoside
but also to the aglycon, and the double specificity of the
enzyme may explain the formation of a large amount
of terpene alcohols with a low content of aromatic
alcohols during black tea fermentation, in spite of the
high content of benzyl-â-D-glucoside in fresh tea leaves
(Morita et al., 1994).
The species of mono- or disaccharoses after the
enzymatic hydrolysis were determined by TLC. A main
spot (Rf ) 0.23) was identical to synthetic primeverose
and there were two minor spots of glucose and xylose;
therefore, the main glycosidase in tea leaves really is
â-primeverosidase, as claimed by Guo et al., 1995).
Regarding the stereostructure of the aglycons, racemic
linalool yielded a diastereomeric mixture of a glycoside.
The NMR spectra of 1d and 2d show anomeric protons
in the glucose moiety at 4.31 ppm (J ) 7.8 Hz) and at
4.33 ppm (J ) 7.7 Hz) with the same intensity. The
stereospecificity of the primeverosidase will be discussed
elsewhere as well as the synthesis of (R)- and (S)-
linalylprimeverosides and the naturally occurring pri-
meverosides with optically active linalool in fresh tea
leaves.
Syn th etic P r oced u r es (F igu r e 1). To prepare
primeverosides with various aglycons by a simple
synthetic procedure, the Koenig-Knorr reaction between
the halogenated primeverose moiety and an aglycon
alcohol was thought to be the most suitable for a simple
and effective preparation of diglycosides, as well as for
the formation of a â-glycoside linkage. The traditional
method used acetyl protective groups for the glycoside
moiety but orthoester formation occurred during the
glycosidation reaction. We found that the benzoate
derivatives did not exhibit such a side reaction and,
moreover, were more favorable for 1-R-halogenation.
The bromination of heptabenzoylprimeverose (5) with
the usual brominating reagent of HBr/CH3COOH failed,
because of cleavage of the internal glycosidic linkage,
while bromination with titanium tetrabromide gave
stable bromide 6. The most suitable Koenig-Knorr
reaction conditions for the synthesis of these diglyco-
sides were provided by using mercuric cyanide as the
condensing agent in acetonitrile. The hexabenzoylpri-
meverosides (7a -c) were also stable and their stereo-
structures were confirmed by NMR. In particular, the
anomeric protons in the xylose and glucose moieties
appeared at 4.94 ppm (J ) 6.1 Hz) and 4.83 ppm (J )
7.9 Hz), respectively, showing that both the glycosidic
linkages had the â-configuration.
As linalool is a tertiary alcohol, which is less reactive
toward glycosylation, others (Konda et al., 1996) have
recommended primeverosyl fluoride as Koenig-Knorr
reagent; however, we prefer linalyl tribenzoylglucoside
(8d ) as the condensing alcohol in the Koenig-Knorr
reaction with xylosyl bromide (4), because procedure 2
is expected to be applicable for the general synthesis of
vicianosides and the starting material 8d is readily
obtainable by a known synthetic method (Sone and
Misaki, 1986). With procedure 2, the newly formed
glycosidic linkage was proved to have the â-configura-
tion by NMR as described later.
LITERATURE CITED
All the hexabenzoylprimeverosides were hydrolyzed
with sodium methoxide in methanol with good yields.
After the synthetic products were purified by HPLC,
their molecular structures were confirmed by FABMS
and NMR spectrometry. The FABMS data are sum-
marized in Table 1, showing the observed millimass
values to be in excellent agreement with the calculated.
The CMR data, summarized in Table 2, are consistent
with those of natural products in the literature (Otsuka
et al., 1990; Inagaki et al., 1995; Guo et al., 1993, 1994;
Pabst et al., 1991; Watanabe et al., 1994; Nishikitani et
al., 1996). Thus the chemical structures of these
natural products are confirmed by synthesis.
En zym a tic Hyd r olysis. The hydrolysis rates (%) of
twelve glycosides by the crude enzyme solution are
summarized in Table 3. Geranyl primeveroside has the
highest rate ()100), as well as the relatively high rates
for the other primeverosides, supports the assertion that
primeverosidase is the main glycosidase in tea leaves
(Guo et al., 1995). The data in Table 3 also show that,
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