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431
(J. Appl. Glycosci., Vol. 46, No. 4, p. 431-437 (1999) )
Practical Enzymatic Synthesis of Primeverose and Its Glycoside
Takeomi Murata, Mutsumi Shimada, Naoharu Watanabe,
Kanzo Sakata** and Taichi Usui*
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University
(836, Ohya, Shizuoka 422-8529, Japan)
ƒÀ-D-Xylosidase from Aspergillus pulverulentus regioselectively induced a ƒÀ-D-xylosyl transfer reac-
tion from 4-0-ƒÀ-D-xylopyranosyl-D-xylopyranose (xylobiose) to the primary hydroxyl group of D-
glucose. The 6-0-ƒÀ-D-xylopyranosyl-ƒÀ-D-glucopyranose (primeverose, 1) produced was isolated by
chromatography on a column of charcoal-Celite in a 29% overall yield based on the donor. In the same
way, when p-nitrophenyl ƒÀ-D-glucopyranoside was used as an acceptor instead of D-glucose, the enzyme
predominated p-nitrophenyl 6-0-ƒÀ-D-xylopyranosyl-ƒÀ-D-glucopyranoside (pNP ƒÀ-primeveroside, 2) to
its isomers, pNP 4-0-ƒÀ-D-xylopyranosyl-ƒÀ-D-glucopyranoside and pNP 3-O-ƒÀ-D-xylopyranosyl-ƒÀ-D-
glucopyranoside. Three transfer products were easily separated from one another by Toyopearl HW-
40S column chromatography and the desired compound, 2, was obtained in a 13% yield based on the
acceptor added. These reactions were efficient enough. to allow one-pot preparations of 1 and 2.
Most plant aroma constituents are present as
aroma precursors. The mechanism for aroma
formation has been proved to be oxidative,
peroxidative, thermal degradation, and en
zymatic hydrolysis.1,2) Sakata and coworkers
have already isolated ƒÀ-primeverosides as
aroma precursors of monoterpene and aromatic
alcohol from tea leaves3-5) and Jasminum sarn-
bac flowers.6,7) They have also isolated a ƒÀ-
primeverosidase concerned with aroma forma
tion during tea processing.8-10) However, bio-
logical functions of primeverose have not been
clarified because of the difficulty of supplying a
large amount of 1. Compound 2 has been used
as an exogenous substrate for ƒÀ-primeverosi
dase assay.10) Therefore, there is presently a
great interest in developing synthetic routes to 1
and its glycosides. The former one has been
obtained only by enzymatic hydrolysis of some
natural glycosides from plant sources. An
organic chemical method for obtaining 2 has
been developed," but it involves various elabo
rate procedures for protection, glycosylation,
and deprotection. From a practical viewpoint,
the use of ƒÀ-glycosidase-mediated transglycosy
lation is attractive for oligosaccharide synthe-
sis. It has been shown that ƒÀ-xylosidases from
different sources have strong transfer activity
from xylooligosaccharides to various alcoh
ols,12-15) phenolic compounds, 15,16) monosaccha
rides, and disaccharides.17-21) The object of the
present investigation is to develop a synthetic
route for the selective transfer of xylosyl resi
due onto 0-6 of glycosyl residue.
This paper shows the practical enzymatic synthesis of 1 and 2 through R-xylosidasemediated transglycosylation.
MATERIALS AND METHODS
Materials. Pectinase GTM from Aspergillus
pulverulentus was kindly supplied , by Amano
Pharmaceutical Co., Ltd. (Nagoya, Japan). p-
Nitrophenyl ƒÀ-D-xylopyranoside (XylƒÀ-pNP)
and PNP ƒÀ-D-glucopyranoside (GlcƒÀ-pNP)
were from Sigma Chemical CO., Ltd. ('St. Louis,
MO, USA).. Xylobiose was prepared from
Xylooligo 95P (Suntory Ltd., Osaka, Japan)
* To whom correspondence should be addressed .* * Present address: Biomolecular Conversion 1, Insti
tute for Chemical Research, Kyoto University (Uji, Kyoto 611-0011, Japan)
432 J. Appl. Glycosci., Vol. 46, No. 4 (1999)
by charcoal-Celite chromatography. All other
chemicals were obtained from commercial
sources.
Enzyme assay. ƒÀ-Xylosidase activity was
assayed as follows: A mixture containing 2 mM
Xylf-pNP in 0.9 mL of 50 mM sodium acetate
buffer (pH 4.0) and an appropriate amount of
enzyme in a total volume of 0.1 mL was incubat
ed for 10 min at. 40•Ž. The reaction was
stopped by adding 0.5 mL of 1.0 M Na2CO3, and
then the liberated p-nitrophenol was measured
spectrophotometrically at 405 nm. One unit of
enzyme was defined as the amount hydrolyzing
1,a mole of XylƒÀ-pNP per minute.
Analytical method. HPAEC-PAD analysis was conducted on a DX-300 Bio-LC system equipped with a pulsed amperometric detector (Dionex, Osaka, Japan). Oligosaccharides were separated on a CarboPac P-1 column (Dionex, q 4 x 250 mm) at a flow rate of 1 mL/ min at room temperature. The elution was affected by the following conditions: eluent A, H2O; eluent B, 100 mM NaOH; and eluent C, 100 mM NaOH containing 1 M sodium acetate. The elution program began with 80% eluent A and 20% eluent B, followed by a gradient of up to 100% eluent B in 15 min. Then, eluent B maintained for 5 min, followed by a gradient of up to 30% eluent C in 20 min. All solvents were degassed before use and continuously bubbled with helium gas.
HPLC was done with a TSKgel ODS-80•Žs
column (ƒÓ4.6 x 75 mm) on a Hitachi 6000 Series
liquid chromatograph equipped with an L-4000
ultraviolet detector. Elution of the column was
affected with H2O-MeOH of 88: 12. The flow
rate was 0.8 mL/min at 40•Ž and monitored by
measuring absorbance at 300 nm.
1H- and 13C -NMR spectra were recorded on a
JEOL JNM-LA 500 or a JNM-EX 270 spectrom
eter at 25•Ž. Chemical shifts are expressed in s
relative to sodium 3- (trimethylsilyl) propionate
as the external standard.
Partial purification of ƒÀ-xylosidase from
Pectinase GTM. Pectinase GTM (200 g) was dis
solved in 2 L of 20 mM sodium phosphate
buffer (pH 6.0). To the enzyme solution, solid
ammonium sulfate was added to give a 90
saturation. After the precipitation was cen
trifuged off, solid ammonium sulfate was added to give 95% saturation. The precipitation formed was collected and dissolved in 20 mM sodium phosphate buffer (pH 6.0), and then dialyzed against the same buffer. The dialyzed solution was lyophilized for the enzymatic syn-thesis.
Hydrolytic reaction of ƒÀ-xylosidase on pri
meverose (1) and xylobiose. The relative rates
of attack of ƒÀ-xylosidase on 1 and xylobiose
were measured by incubating a mixture (1 mL)
containing 2 mM of substrates in 50 mM sodium
acetate buffer (pH 4.0) with 0.6 U or 6 U of ƒÀ-
xylosidase (Pectinase GTM) at 40•Ž. Samples
(101a L) were taken at 5-min intervals during
the reaction (0, 5, 10, 15 and 20 mm). After
inactivation of each sample by boiling for 5 min,
the liberated xylose and glucose were measured
by HPAEC-PAD. The reaction was linear from
5 to 20 min. The rate of attack on 1 was
arbitrarily set at 1.
Preparation of primeverose (1). Pectinase
GTM was used for the synthesis of primeverose
without purification. Xylobiose (3 g) and glu
cose (9.6 g) were dissolved in 5 mL of 100 mM
sodium acetate buffer (pH 4.0), followed by
17.5 U of ƒÀ-xylosidase (Pectinase GTM). The
mixture was incubated for 157 h at 40•Ž and the
reaction was terminated by boiling for 5 min.
The reaction mixture was loaded onto a
charcoal-Celite column (~ 5 x 50 cm) equilibrat
ed with H2O. The column was washed with 600
mL of H2O, and then eluted with 0 (3 L) -30 (3
L) % EtOH of linear gradient. The effluent
solution was monitored by measuring the absor
bance at 485 nm (neutral sugar content, phenol-
sulfuric acid method). As shown in Fig. 1, the
chromatogram showed two peaks (F-1, tubes
10-50; F-2, tubes 94-127). The second peak was
presumed to contain a transfer product. The F-
1 contained xylose, which was hydrolyzed from
xylobiose. The F-2 fraction was concentrated,
and crystallized from EtOH to afford 1.1 g (29
yield based on the donor) of 1 as crystal recrys
tallized from EtOH.
Preparation of p-nitrophenyl ƒÀ primeveroside
(2). Pectinase GTM contains significant amounts
of ƒÀ-D-glucosidase activity, which degrades
G1cf-pNP when used as the acceptor substrate.
433Practical Enzymatic Synthesis of Primeverose and Its Glycoside
Fig. 1. Charcoal-Celite chromatography of transglyco
sylation product formed from xylobiose and n-
glucose by ƒÀ-xylosidase from Aspergillus pul
verulentus.
•œ absorbance at 485 nm. F-1, xylose; F-2, prime
verose (1).
Therefore, partially purified enzyme was pre
pared for the synthesis as mentioned above.
Xylobiose (506 mg) and Glcf-pNP (177 mg)
were dissolved in 1 mL of 100 mM sodium ace
tate buffer (pH 4.0), followed by 10 U of the ƒÀ-
xylosidase. The mixture was incubated for 6 h
at 40•Ž and the reaction was terminated by
boiling for 5 min. The reaction mixture was
loaded onto a Chromatorex ODS DM1020•Ž
column (~ 3 x 50 cm) equilibrated with 20% of
McOH in aqueous solution. The column was
eluted with the same solution. The eluate frac
tion (1640 mL), which displayed coincident
absorptions of 300 and 485 nm, was concen
trated to a small volume (15 mL). The solution
was loaded onto a Toyopearl HW-40S column
(~ 4 x 90 cm) equilibrated with 20% of McOH in
an aqueous solution, and the effluent solution
was monitored by measuring the absorbance at
485 nm (neutral sugar content, phenol-sulfuric
acid method). As shown in Fig. 2, the chro
matogram showed six main peaks (F-1, tubes
140-147; and F-2, tubes 148-156; F-3, tubes 169-
190; F-4, tubes 211-220; F-5, tubes 221-231; and
F-6, tubes 234-257). The F-3, F-4, and F-5
peaks were concentrated and lyophilized to
afford compounds 2 (33.7 mg), 3 (9.4 mg), and 4
(8.3 mg), respectively. The F-1 and F-2 peaks
were identified pNP 6-O-ƒÀ-D-glucopyranosyl-
ƒÀ-D-glucopyranoside and pNP 3-0-ƒÀ-D-gluco-
pyranosyl-ƒÀ-D-glucopyranoside, respectively, by 1H : and 13C-NMR (data not shown). The F-6
peak contained GlcƒÀ-pNP used as the acceptor
substrate.
Fig. 2. Toyopearl HW-40S chromatography of transglycosylation products formed
from xylobiose and GlcƒÀ-pNP by ƒÀ-xylosidase from Aspergillus pulverulentus.
•œ absorbance at 485 nm; •›, absorbance at 300 nm. F-1, Glcfl-6Glcf-pNP; F-2,
GlcƒÀ1-3G1cƒÀ-pNP; F-3, XylƒÀ1-6G1cf-pNP (2); F=4, Xylf1-4G1cf-pNP (3); F-5, Xyll1-
3Glcp-pNP (4); F-6, Glcf-pNP.
434 J. Appl. Glycosci., Vol. 46, No. 4 (1999)
Table 1. 1H- and 13C-NMR data for primeverose (1) in D20.
Table 2. 1H- and 13H-NMR data for compounds of
2, 3, and 4 in D2O.
aND, not determined. 2, p-nitrophenyl 6-O-ƒÀ-D-xylo-
pyranosyl-R-D-glucopyranoside. 3, p-nitropheny14-O-
l-D-xylopyranosyl-f-D-glucopyranoside. 4, p-nitro-
phenyl 3- O-Q-D-xylopyranosyl-ƒÀ-D-glucopyranoside.
RESULTS AND DISCUSSION
Characterization of transfer products. The positive ion mode FAB-MS spectrum of 1
showed a molecular ion at m/z 313 ([M+ H] +)
with a fragment ion at m/z 133 (fragment from
pentose). It indicates that 1 has a sequence of
Pen-Hex. The 1H-NMR signals of 1 were easily
assigned by DQF-COSY (Table 1). 13C-NMR
spectrum of 1 using HSQC provided useful
information on the composition and sugar
sequence. All of the different carbon lines were
resolved using carbon-protons shift correlation
(Table 1). The NMR and FAB-MS analyses
revealed that 1 is a 6- 0- f3-D-xylopyranosyl-ƒÀ-D-
glucopyranose: [ a ] D5 -3.3•K(c 1.0, H20) [Ref.
(22) [a]20D -3.4•K (c 2.5, H2O)]; mp 190-191•Ž
(from EtOH) [Ref. (22) mp 194-197•Ž].
In the same way, the structures of compounds
2, 3, and 4 were similarly characterized. Phys
iological data of 2 were almost identical to
those of pNP 6- 0-ƒÀ-D-xylopyranosyl-ƒÀ-D-gluco
pyranoside reported previously." Each posi
tive ion mode FAB-MS spectrum of 3 and 4
showed a molecular ion at m/z 456 ([M+
Na] +) , suggesting that both compounds were
disaccharide Pen-Hex-OC6H4N02. Based on
the sugar sequence, the structures of the trans
fer products were elucidated by their 1H- and 13C -NMR spectra as in Table 2 . Compounds 3
and 4 were shown to be pNP 4-O-ƒÀ-D-xylopyra-
nosyl-ƒÀ-D-glucopyranoside and pNP 3-O-ƒÀ-D-
xylopyranosyl-ƒÀ-D-glucopyranoside, respective-
ly.
Preparation of primeverose (1). In general, primeverose is a common carbo
hydrate unit of primeverosides, which exist as aroma precursors of plants in a very small amount. Therefore, it's very difficult to obtain
primeverose from natural sources. Enzymatic methods utilizing glycosidase-catalyzed trans
glycosylation would facilitate large-scale preparation of primeverose in as short a pathway as possible, because glycosidase and xylosyl donors such as xylobiose are commercially available in large amounts. In this study, com-
pound 1 was obtained at the gram-scale through f3-D-xylosidase-catalyzed transxylosylation. It was easily obtained on charcoal-Celite chromatography, and crystallization from EtOH, with a 29% yield based on xylobiose used as the donor.
435Practical Enzymatic Synthesis of Primeverose and Its Glycoside
Fig. 3. Time course of the formation of primeverose
(1) and the degradation of xylobiose by ,ƒÀ-xylo
sidase from Aspeygillus pulverulentus.
The enzyme reaction was performed with xylobiose
(300 mg), glucose (958 mg), and ƒÀ-xylosidase .(5.8 U) at
40•Ž in 0.5 mL of 100 mM sodium acetate (pH 4.0). The
amounts of 1 (•œ and xylobiose (•›) were analyzed by
the H PAEC-PAD method.
Fig. 4. Effects of organic solvents on the formation of
p-nitrophenyl, ƒÀ-primeveroside (2) by ,ƒÀ-xylo
sidase from. Aspergillus pulverulentus.
The enzyme reactions were performed as described in "Materials and Methods" section except for the solvent
system:•¡, buffer only; •›, 25% acetone;•œ 25% DMSO;
• , 25% ethanol. The amounts of 2 were analyzed by
HPLC.
Production of 1 reached a maximum at 100 h and its concentration varied little during subse
quent reactions (Fig. 3). On the other hand, a significant amount of xylobiose still remained at that time. The unreacted xylobiose was com
pletely degraded at 200-h incubation. This prolonged incubation was helpful for the selective hydrolysis of xylobiose from the reaction mixture, because it was very difficult to separate 1 from xylobiose by charcoal-Celite column chromatography. In a separate experiment, the relative rate of hydrolysis of xylobiose with 1
(1.0) was a 130-fold difference. Thus, xylobiose was a much better substrate than 1 under hydrolytic conditions. These results suggest that once 1 is formed, it is not significantly hydrolyzed by the enzyme and the amount increases gradually and exclusively with time. As a result, the prolonged enzyme reaction allowed the selective removal of xylobiose from the reaction mixture.
Preparation of p-nit rophenyl ƒÀ primeveroside
(2) and its positional analogues (3 and 4). The enzyme used in this synthesis was almost
devoid of ƒÀ-D-glucosidase activity, which
degrades the acceptor substrate G1cf-pNP
(vide infra). Thus, the R-D-xylosidase fraction
from Pectinase GTM was 'fractionated by 90-95
saturated ammonium sulfate precipitation in
order to separate R-D-xylosidase and fl-D-
glucosidase activities. When GlcƒÀ-pNP was
used as the acceptor substrate instead of
glucose, the enzyme formed three transfer
products, compound 2 and its isomers 3 and 4,
with a 20.2% total yield based on the acceptor
added and in a ratio of 78:13: 9. These com-
pounds were readily separated by Toyopearl
HW-40S chromatography (Fig. 2). Replace
ment of Glc%3-pNP by glucose acceptor did not
significantly alter the direction of the xylosyla
tion: about four-fifths of the xylosylation occur-
red at 0-6 and one-fifth at 0-3 and 0-4; the
enzyme predominated 2 to its isomers 3 and 4.
The solvent effects at different concentra
tions of various organic solvents on ƒÀ-D-xylo-
sidase-mediated transglycosylation were
examined as shown in Fig. 4. Time course of 2
was analyzed by the HPLC method using an
ODS column (Fig. 4). Maximum production of
2 at 25% acetone was observed after 60 h and its
concentration varied little during the subse
quent reaction. In the absence of organic sol-
vent, the amount of maximum production,
which was reached at 26 h, was almost equal to
that at 25% acetone, but its concentration de
436 J. Appl. Glycosci., Vol. 46, No. 4 (1999)
creased to three-fourths its maximum during
the subsequent reaction. The use of 25% eth
anol appears to considerably decrease trans
glycosylation activity. This might be due to the
formation of ƒÀ-D-xylosidase-mediated ethanoly
sis as previously reported.13,15) In general, the
use of an organic co-solvent in transfer reaction
utilizing glycosidase not only ensured the
sufficient solubility of hydrophobic substrate,
but also resulted in the high yields of transfer
product. However, the present co-organic sol-
vent system was not significantly effective for
the production of 2.
In conclusion, we developed practical enzy
matic methods for obtaining 1 and 2 through
,Q-D-xylosidase-mediated transglycosylation. These compounds would be useful as tools for
the physiological investigation of plants.
We thank Amano Pharmaceutical Co., Ltd. for the
gift of Pectinase GTM. This work was supported by the Research Grant for Leading Research Utilizing Potential of the Regional Science and Technology from the Science and Technology Agency, Japan.
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(Received July 30, 1999; Accepted September 14, 1999)
プ リメベ ロース とその配糖体 の実践的酵素合成
村 田健 臣,島 田 睦,渡 辺修治
坂 田完三,碓 氷泰市
静岡大学農学部応用生物化学科(422-8529静 岡市
大谷836)
Aspergillus puiverulentus由 来 のβ-キ シ ロ シター ゼ
は,4-O-β-D-キ シ ロ ピラ ノ シルーD-キ シ 「ロピ ラ ノー ス
(キ シ ロ ビオー ス)か らD-グ ル コー ス の1級 水 酸 基 へ
の高 位 置選 択 的 キ シ ロ シル 移 転 反 応 を触 媒 し,6-O-
β-D-キ シ ロ ピラ ノ シ ルーD-グ ル コ ピ ラ ノー ス(プ リメ
ベ ロー ス,1)を 生 成 し た.本2糖 は活性 炭-セ ライ ト
カ ラ ム ク ロマ トグ ラ フ ィー に よ り29%の 収 率 で容 易
に 単 離 で き た.同 様 に して,D-グ ル コー ス の 代 わ り
に ρ-ニ トロ フェ ニ ル β-D-グ ル コ ピ ラ ノシ ドを受 容 体
基 質 と し て 用 い た 場 合,本 酵 素 に よ って ρ-ニ ト ロ
フ ェ ニ ル6-O-β-D-キ シ ロ ピ ラ ノ シル ーβ-D-グル コ ピ
ラ ノシ ド(ρNPβ-プ リメベ ロ シ ド,2)が 優 先 的 に 生
成 し,そ の 構 造 異 性 体 で あ るpNP4-O-β-D-キ シ ロ
ピラ ノシ ルβ-D-グ ル コ ピ ラ ノ シ ドと ρNP3-O-β-D-
キ シロ ピ ラ ノ シルーβ-D-グ ル コ ピ ラ ノ シ ドも同 時 に 生
成 した.3種 類 の 転 移 生 成 物 は ト ヨパ ー ルHW-40S
カ ラム クロマ トグ ラ フ ィー に よっ て容 易 に分 離 され,
目的 化 合 物2を 受 容 体 基 質 当 り13%の 収 率 で得 た.
これ らの糖 転 移 反 応 は,1お よ び2の ワ ン ポ ッ ト合 成
法 と して有 効 であ った.