practical enzymatic synthesis of primeverose and its glycoside

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.


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.


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


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.

REFERENCES

1) R. Teranishi, G. R. Takeoka and M. Guntert eds. :

Flavor precursors-Thermal and enzymatic conver

sions-, in ACS Symposium Series 490, American

Chemical Society, Washington, D.C. (1992).

2) N. Watanabe : Plant aroma precursors and their

utilization, New Food Ind., 37, 8-16 (1995) (in

Japanese).

3) W. Guo, K. Sakata, N. Watanabe, R. Nakajima, A.

Yagi, K. ma and S. Luo : Geranyl 6-O-ƒÀ-D-xylopyra

nosyl-ƒÀ-D-glucopyranoside isolated as an aroma

precursor from tea leaves for oolong tea. Phyto

chemistry, 33, 1373-1375 (1993).

4) J.-H. Moon, N. Watanabe, K. Sakata, A. Yagi, K.

ma and S. Luo : trans- and cis-Linalool 3,6-oxide

6-0-ƒÀ-D-xylopyranosyl-ƒÀ-D-glucopyranosides iso

lated as aroma precursors from leaves for oolong

tea. Biosci. Biotechnol. Biochem., 58, 1742-1747

(1994)

5) J.-H. Moon, N. Watanabe, Y. Ijima, A. Yagi and K.

Sakata : cis- and trans-Linalool 3,7-oxides and

methyl salicylate glycosides and (Z) -3-hexenyl ƒÀ-D-

glucopyranoside as aroma precursors from tea

leaves for oolong tea. Biosci. Biotechnol. Biochem.,

60, 1815-1819 (1996).

6) N. Watanabe, R. Nakajima, S. Watanabe, J.-H.

Moon, J. Inagaki, K. Sakata, A. Yagi and K. Ina•F

Linalyl and bornyl disaccharide glycosides from Gardenia jasminoides flowers. Phytochemistry, 37, 457-459 (1994).

7) J. Inagaki, N. Watanabe, J.-H. Moon, A. Yagi, K.

Sakata, K. ma and S. Luo : Glycosidic aroma pre

cursors of 2-phenylethyl and benzyl alcohol from

Jasminum sambac flowers. Biosci. Biotechnol. Bio-

chem., 59, 738-739 (1995).

8) W. Guo, K. Yamauchi, N. Watanabe, T. Usui, S.

Luo and. K. Sakata : A primeverosidase as a main

glycosidase concerned with the alcoholic aroma

formation in tea leaves. Biosci. Biotechnol. Biochem.,

59, 962-964 (1995).

9) W. Guo, K. Ogawa, K. Yamauchi, N. Watanabe, T.

Usui, S. Luo and K. Sakata : Isolation and charac

terization of a ƒÀ-primeverosidase concerned with

alcoholic aroma formation in tea leaves. Biosci.

Biotechnol. Biochem., 60, 1810-1814 (1996).

10) K. Ogawa, Y. Ijima, W. Guo, N. Watanabe, T. Usui,

S. Dong, Q. Tong and K. Sakata : Purification of a

ƒÀ- primeverosidase concerned with alcoholic aroma

formation in tea leaves (Cv. Shuixian) to be proces

sed to oolong tea. J. Agric. Food Chem., 45, 877-882

(1997).

11) Y. Sone and A. Misaki : Syntheses of p-nitrophenyl

6-O-ƒÀ- and 6-O-ƒÀ-D-xylopyranosyl-ƒÀ-D-glucopyrano

side. J. Carbohydr. Chem., 5, 671-682 (1986).

12) F. Deleyn, M. :Claeyssens and C. K. De Bruyne :

Transfer reactions catalyzed by ƒÀ-D-xylosidase

from Penicillium wortmanni. Can. J. Biochem. Cell

Biol., 63, 204-211 (1985).

13) H. Shinoyama, Y. Kamiyama and T. Yasui : Enzy

matic synthesis of alkyl ƒÀ-xylosides from xylobiose

by application of the transxylosyl reaction of

Aspergillus niger ƒÀ-xylosidase. Agric. Biol.' Chem.,

52, 2197-2202 (1988).

14) H. Shinoyama and T. Yasui : Superiority of Asper

gillus niger ƒÀ-xylosidase for the enzymatic synthe

sis of alkyl ƒÀ-xylosides in the presence of a variety

of alcohols. Agric. Biol. Chem., 52, 2375-2377 (1988).

15) J. Sulistyo, Y. Kamiyama and T. Yasui : Purifica

tion and some properties of Aspergillus pulverulen

tus ƒÀ-xylosidase with transxylosylation capacity. J.

Ferment. Bioeng., 79, 17-22 (1995).

16) J. Sulistyo, Y. Kamiyama, H. Ito and T. Yasui :

Enzymatic synthesis of hydroquinone ƒÀ-xyloside

from xylooligosaccharides. Biosci. Biotechnol. Bio

chem., 58, 1311-1313 (1994).

17) M. Matsuo, A. Fujie, M. Win and T. Yasui : Four

types of ƒÀ-xylosidases from Penicillium wortmanni

IFO 7237. Agric. Biol. Chem., 51, 2367-2379 (1987).

18) T. Yasui, H. Kizawa, Y. Masada and H. Shino

yama : A novel nonreducing disaccharide, O-ƒÀ-D-

xylopyranosyl- (1•¨1') -ƒÀ-D-xylopyranose, produced

from xylobiose by transxylosylation with Aspergil

lus niger ƒÀ-xylosidase. Agric. Biol. Chem., 53, 3381-

3382 (1989).


19) H. Kizawa, H. Shinoyama and T. Yasui : The syn

thesis of new xylosyloligosaccharides by transxylo

sylation with Aspergillus niger ƒÀ-xylosidase. Agric.

Biol. Chem., 55, 671-678 (1991).

20) M. Kurakake, S. Osada and T. Komaki : Trans

xylosylation of ƒÀ-xylosidase from Aspergillus awa

mori K4. Biosci. Biotechnol. Biochem., 61, 2010-2014

(1997) .

21) A. Vetere, M. Bosco and S. Paoletti : Enzymatic

synthesis and characterization of 6-O-ƒÀ-D-xylopyra

nosyl-2-acetamido-2-deoxy-D-glucopyranose, a

structural analog of primeverose. Carbohydr. Res.,

311, 79-83 (1998).

22) Dictionary of Carbohydrates on CD-ROM, Chapman

& Hall (1998).

(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.

practical enzymatic synthesis of primeverose and its glycoside

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