Use of apple seed meal as a new source of β-glucosidase for enzymatic glucosylation of 4-substituted benzyl alcohols and tyrosol in monophasic aqueous-dioxane medium

Article abstract of DOI:10.1016/j.bmcl.2004.02.042

A facile method for enzymatic glycosylation of 4-substituted benzyl alcohols and tyrosol with glucose in a monophasic aqueous-dioxane medium was reported, using a crude meal of apple seed as a new catalyst. The corresponding β-D-glucosides were synthesized in moderate yields (13.1-23.1%), among which the salidroside was obtained in 15.8% yield.

Full text of DOI:10.1016/j.bmcl.2004.02.042

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2095–2097
Use of apple seed meal as a new source of b-glucosidase for
enzymatic glucosylation of 4-substituted benzyl alcohols and
tyrosol in monophasic aqueous-dioxane medium
a
Ai Min Tong, Wen Ya Lu, Jian He Xu and Guo Qiang Lin
b
a,
b
*
a
Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering,
East China University of Science and Technology, Shanghai 200237, China
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
b
Received 31 December 2003; revised 9 February 2004; accepted 10 February 2004
Abstract—A facile method for enzymatic glycosylation of 4-substituted benzyl alcohols and tyrosol with glucose in a monophasic
aqueous-dioxane medium was reported, using a crude meal of apple seed as a new catalyst. The corresponding b-D-glucosides were
synthesized in moderate yields (13.1–23.1%), among which the salidroside was obtained in 15.8% yield.
ꢀ
2004 Elsevier Ltd. All rights reserved.
As a part of a program to search for novel glycoside
compounds with biological activities, we are interested
in synthesizing a series of 4-substituted benzyl glucosides
attractive, but the use of an expensive glycosyl donor
increased the cost of the synthesis. There had been rel-
atively few examples of reverse hydrolysis, although it is
the simplest and the most cost-effective process. In order
to favor the formation of b-glucosides in higher yields,
liquid-state alcohols were preferentially used both as
and salidroside (2-(4-hydroxyphenyl)ethyl b-D-gluco-
side). Glycosidases can catalyze the synthesis of ano-
merically pure glycosides in one step using a reverse
hydrolysis process or a transglycosidation procedure,
while chemical synthesis of anomerically pure glucosides
is circuitous and expensive. This problem in chemical
synthesis has stimulated the development of enzymatic
3
substrates and as solvents. For instance, Vic and Crout
had synthesized benzyl b-D-glucopyranoside in 40%
yield by this method. However, the need to use a very
high concentration of the alcohols clearly limits the
scope and application of this reaction. Moreover, when
an alcohol is a solid, it is necessary to use a solvent in the
system to dissolve the substrate. We found that dioxane-
aqueous medium was more effective for our reaction
1
approaches.
Almond b-glucosidase has been widely used to catalyze
the synthesis of various alkyl b-glucopyranosides in or-
2
–6
ganic media. For the large-scale production of alkyl-
b-glucopyranosides, however, it will be necessary to find
easily available and relatively cheap catalysts. Many
reports described a transglycosylation procedure where
an expensive and activated glycosyl donor was used. For
3
than the traditional tert-butanol-aqueous or CH CN-
aqueous system. In this communication, therefore,
we describe the synthesis of seven b-glucopyranosides
(Scheme 1) through a reverse hydrolysis process
starting directly from D-glucose and the corresponding
alcohols (in either liquid or solid state) in a monophasic
2
example, Akita et al. synthesized a series of b-gluco-
sides using p-nitrophenyl glucopyranoside (PNPG) as a
glucosyl donor in an aqueous medium and 4-meth-
oxybenzyl b-D-glucopyranoside was formed in 25%
yield. The result of the transglucosylation procedure was
1
2
3
4
n=1, X=H
n=1, X=OCH
3
OH
X
n=1, X=CH
3
O
n=1, X=NO
2
HO
HO
O
5 n=1, X=F
n
OH
6
n=1, X=Cl
Keywords: b-
D-Glucoside; Enzymatic synthesis; Apple seed meal;
7 n=2, X=OH
b-Glucosidase; Salidroside.
*
0
Corresponding author. Tel.: +86-21-6425-2498; fax: +86-21-6425-
250; e-mail: jianhexu@ecust.edu.cn
Scheme 1. Structure of 4-substituted benzyl b-D-glucopyranosides and
salidroside.
2
960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.042

2096
A. M. Tong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2095–2097
7
buffer-dioxane medium. Scigelova et al. screened gly-
cosidases from several enzyme preparations and found a
preparation from apples had glucosidase activity.
However, to the best of our knowledge, our work first
applied the crude meal of apple seeds as a biocatalyst in
the synthesis of glycosides 1, 2, and 7. Furthermore, the
synthesis of novel glycoside compounds 3–6 are reported
for the first time.
determined from both of the forward and reverse reac-
tions), a sharp decrease in reaction rate was observed
around 20h. As compared with the time course of the
reaction by almond b-glucosidase, it is clear that the
crude enzyme from the apple seed meal showed a very
similar profile. Therefore, the crude enzyme from apple
seeds could become a good complement to the source of
the almond b-glucosidase. It is expected that the reverse
hydrolysis reactions catalyzed by glycoside hydrolases
might also be applied for the glycosylation of other
solid-state alcohols.
In the above-mentioned monophasic medium system,
the ratio of dioxane to buffer was found to be a sensitive
parameter affecting both the reaction rate and the equ-
librium yield, and the highest yield was obtained at
an optimum ratio of 10:1 (v/v). Under the optimal
condition, seven 4-substituted benzyl or phenylethyl
All the 4-substituted benzyl alcohols were obtained from
Aldrich Chemical Co. Tyrosol was from TCI. All other
chemicals were of the highest purity commercially
available and used without further purification. Almond
b-glucosidase (EC 3.2.1.21) was purchased from Sigma
Chemical Co. (3.8 U/mg). Apples were purchased from
the local market. NMR spectra were recorded on a
Bruker DRX-500 spectrometer or Bruker AM-300
spectrometer. Mass spectra were recorded on a Bruker
APEXIII 7.0TESLA FTMS using ESI mode. IR spec-
tra were recorded on a Digital FTIR instrument. Optical
rotations were measured using Perkin–Elmer 241 MC
polarimeter.
b-D-glucopyranosides were successfully synthesized in
moderate yields (Table 1) by using the crude meal of
apple seeds as a new source of b-glucosidase. It should
be noticed that for alcohols 1, 2, and 5, which are in
liquid state, the yields were relatively higher (>18%),
perhaps due to the better mixing effect between liquid–
liquid than that between solid–liquid and for tyrosol 7,
the product of glucosylation, salidroside, was also
obtained in 15.8% yield.
The time course of the glucosylation reaction of 4-ni-
trobenzyl alcohol was monitored by HPLC, as shown in
Figure 1. Since the reaction at 20h had been
approaching the equilibrium conversion (ca. 13.3%, as
2
D
0
Benzyl b-
MeOH); H NMR d (D
D
-glucopyranoside (1): ½aŠ )55.5 (c 0.885,
1
2
O, ppm) 3.26 (dd, 1H, J ¼ 8:7,
8.4 Hz), 3.33 (m, 3H), 3.67 (dd, 1H, J ¼ 5:6, 12.4 Hz),
3
4
7
.87 (dd, 1H, J ¼ 1:9, 12.4 Hz), 4.46 (d, 1H, J ¼ 8:0Hz),
.69 (d, 1H, J ¼ 11:6 Hz), 4.88 (d, 1H, J ¼ 11:6 Hz),
13
.34 (m, 5H); C (DMSO): d 61.2 (C-6), 69.4 (C-4), 70.2
Table 1. Synthesis of b-glucopyranosides by apple seed meal
0
(
C-1 ), 73.5 (C-2), 76.8 (C-3 or C-5), 76.9 (C-3 or C-5),
102.1 (C-1), 127.2, 127.5, 128.0, 138.0 (C-aryl); FTIR
À1
(KBr, cm ): 3502, 3427, 3062, 2931, 2883, 1497, 1455,
416, 1382, 1370, 1318, 1280, 1242, 1210, 1157, 1108,
081, 1051, 1031, 993, 891, 753, 735, 697, 585; m=z
a
Entry
n
X
Alcohol state [Glucose]
0
/M Yield (%)
1
2
3
4
5
6
7
1
1
1
1
1
1
2
–H
Liquid
Liquid
Solid
0.25
0.25
0.25
0.25
0.25
0.25
0.25
20.4
23.1
16.7
13.1
18.1
16.1
15.8
–CH
–CH
3
O
1
1
3
–NO
–F
2
Solid
þ
þ
(ESI ) C13
H
18
O
6
Na (M+Na ), 293.0.
Liquid
Solid
Solid
–Cl
–OH
20
-glucopyranoside (2): ½aŠ )59.4 (c
4
0
-Methoxybenzyl b-
.88, MeOH); H NMR d (D
D
D
O, ppm) 3.25 (dd, 1H,
1
2
a
Isolated yield.
J ¼ J ¼ 8:5 Hz), 3.33 (m, 3H), 3.69 (dd, 1H, J ¼ 5:6,
1
2
1
1
2.4 Hz), 3.76 (s, 3H), 3.88 (d, 1H, J ¼ 12:2 Hz), 4.42 (d,
H, J ¼ 8:0Hz), 4.60(d, 1H, J ¼ 11:3 Hz), 4.81 (dd, 1H,
15
J ¼ 11:6 Hz), 6.92 (d, 4H, J ¼ 8:6 Hz), 7.33 (d, 4H,
13
J ¼ 8:6 Hz); C (DMSO): d 55.5 (C-Me), 61.7 (C-6),
0
6
9.7 (C-4), 70.1 (C-1 ), 73.5 (C-2), 77.3 (C-3 or C-5), 77.4
C-3 or C-5), 102.5 (C-1), 114.0, 129.9, 130.3, 159.2 (C-
(
aryl); FTIR (KBr, cm ): 3543, 3337, 2955, 2926, 2857,
1
0
5
0
À1
1
8
613, 1586, 1513, 1367, 1306, 1248, 1165, 1075, 1029,
þ
þ
20, 766, 709; m=z (ESI ) C14
20 7
H O Na (M+Na ),
found: 323.1100, calcd for: 3323.1101.
2
0
4
0
-Methylbenzyl b-
D
-glucopyranoside (3): ½aŠ )60.3 (c
D
1
.98, MeOH); H NMR d (D O, ppm) 2.33 (s, 3H), 3.25
2
(dd, 1H, J
J ¼ 5:6, 12.4 Hz), 3.88 (dd, 1H, J ¼ 1:6, 12.3 Hz), 4.47
d, 1H, J ¼ 8:9 Hz), 4.68 (d, 1H, J ¼ 11:5 Hz), 4.86 (d,
1
¼ J
2
¼ 8:7 Hz), 3.34 (m, 3H), 3.68 (dd, 1H,
0
20
40
60
80
Reaction time (h)
(
1
H, J ¼ 11:5 Hz), 7.26 (d, 2H, J ¼ 7:8 Hz), 7.33 (d, 2H,
Figure 1. Time-course of 4-nitrobenzyl glucopyranoside formation in
buffer-dioxane (1:10, v/v) catalyzed by crude meal of apple seed (j),
compared to that by almond b-glucosidase (s). In the reaction system,
the crude meal of apple seed added was 50mg/ml and the almond
b-glucosidase added was 5 mg/ml.
13
J ¼ 7:9 Hz); C (DMSO): d 20.9 (C-Me), 61.4 (C-6),
0
69.5 (C-4), 70.4 (C-1 ), 73.7 (C-2), 77.0(C-3 or C-5), 77.1
(C-3 or C-5), 102.1 (C-1), 127.9, 128.9, 135.1, 136.7
À1
(C-aryl); FTIR (KBr, cm ): 3356, 2955, 3001, 2925,

A. M. Tong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2095–2097
2097
2
1
855, 1519, 1463, 1410, 1364, 1311, 1168, 1132, 1112,
þ
J ¼ 1:9, 12.3 Hz), 3.96 (m, 1H), 4.34 (d, 1H, J ¼ 8:0Hz),
6.75 (d, 2H, J ¼ 8:5 Hz), 7.11 (d, 2H, J ¼ 8:5).
079, 1037, 1015, 897, 799, 748, 650, 613; m=z (ESI )
þ
C H O Na (M+Na ), found: 307.1139, calcd for:
3
14
20
6
07.1152.
The fresh apple seeds were peeled off and powdered,
washed three times with ethyl acetate, and two times
with acetone. The powder was dried in a vacuum des-
iccator and stored at 4 ꢁC.
2
0
4
1
3
1
-Nitrobenzyl b-
.03, MeOH); H NMR d (D
D
-glucopyranoside (4): ½aŠ )49.5 (c
D
O, ppm) 3.32 (m, 4H),
1
2
.69 (dd, 1H, J ¼ 5:8, 12.3 Hz), 3.88 (dd, 1H, J ¼ 2:0,
2.3 Hz), 4.51 (d, 1H, J ¼ 7:9 Hz), 4.82 (d, 1H,
General procedure for the enzymatic preparation of
glucosides: To 0.5 ml solution containing 1.25 mmol
glucose and 0.07 M Na HPO –KH PO (pH 6.0), was
2 4 2 4
J ¼ 13:1 Hz), 4.98 (d, 1H, J ¼ 13:1 Hz), 7.57 (d, 2H,
13
J ¼ 8:6 Hz), 8.14 (d, 2H, J ¼ 8:7 Hz); C (DMSO): d
0
6
1.2 (C-6), 68.5 (C-4), 70.1 (C-1 ), 73.5 (C-2), 76.7 (C-3
or C-5), 77.0(C-3 or C-5), 1 02 .5 (C-1), 123.3, 128.1,
added 250mg crude enzyme. The reaction was started
by the addition of benzyl alcohol (15.0mmol) in 5 ml
dioxane and the mixture was shaken at 160rpm and
50 ꢁC for 72 h. The reaction was quenched by addition
of 10ml methanol. Then the crude enzyme was filtered
off and washed with methanol (5 ml · 2). The filtrate was
À1
1
3
1
46.4, 146.7 (C-aryl); FTIR (KBr, cm ): 3539, 3351,
250, 3001, 2951, 2925, 2866, 1602, 1514, 1442, 1398,
348, 1290, 1153, 1080, 1040, 985, 896, 833, 736, 613;
þ
þ
m=z (ESI ) C13
calcd for: 338.0846.
H O NaN (M+Na ), found: 338.0835,
17 8
2 4
dried over Na SO and evaporated under reduced
pressure. The residue was purified by flash chromato-
graphy on Silica Gel 60 (100–200 mesh) with ethyl
acetate/methanol (12:1) as elute, giving b-pyranogluco-
sides (Table 1) as amorphous solid. Aliquots (50 ll) were
removed at time intervals and quenched by addition of
950 ll methanol. The samples were then analyzed by
HPLC using YWG-C18-5l column, eluted with MeOH/
2
D
0
4
0
-Fluorobenzyl b-
D
-glucopyranoside (5): ½aŠ )55.7 (c
1
2
.98, MeOH); H NMR d (D O, ppm) 3.26 (dd, 1H,
J ¼ 9:0, 8.1 Hz), 3.34 (m, 3H), 3.69 (dd, 1H, J ¼ 5:6,
1
2.3 Hz), 3.88 (dd, 1H, J ¼ 2:0, 12.5 Hz), 4.47 (d, 1H,
J ¼ 8:0Hz), 4.67 (d, 1H, J ¼ 11:6 Hz), 4.86 (d, 1H,
13
J ¼ 11:6 Hz), 7.10(m, 2H), 7.41 (m, 2H); C (DMSO):
0
d 61.5 (C-6), 69.1 (C-4), 70.5 (C-1 ), 73.8 (C-2), 77.1 (C-3
or C-5), 77.3 (C-3 or C-5), 102.4 (C-1), 115.1, 115.3,
H O (40:60) and detected at 254 nm.
2
À1
1
3
1
C
3
34.6, 163.2, 163.5 (C-aryl); FTIR (KBr, cm ): 3542,
345, 2859, 2789, 1901, 1604, 1511, 1446, 1368, 1220,
076, 1031, 896, 829, 776, 617; m=z (ESI )
Acknowledgements
þ
þ
13
H
11.0901.
17
O
6
NaF (M+Na ), found: 311.0896, calcd for:
This research was financially supported by National
Natural Science Foundation of China (Grant No.
203900506). The authors are grateful to Dr. Pei-Ran
Chen for his helpful discussion and technical assistance.
2
D
0
4
0
-Chlorobenzyl b-
D
-glucopyranoside (6): ½aŠ )47.7 (c
1
.90, MeOH); H NMR d (D O, ppm) 3.26 (dd, 1H,
2
J ¼ 8:9, 8.3 Hz), 3.34 (m, 3H), 3.68 (dd, 1H, J ¼ 5:7,
1
2.3 Hz), 3.88 (dd, 1H, J ¼ 2:0, 12.4 Hz), 4.47 (d, 1H,
J ¼ 8:0Hz), 4.69 (d, 1H, J ¼ 11:8 Hz), 4.87 (d, 1H,
References and notes
13
J ¼ 11:8 Hz), 7.39 (m, 4H); C (DMSO): d 61.4 (C-6),
0
6
8.9 (C-4), 70.4 (C-1 ), 73.7 (C-2), 76.9 (C-3 or C-5), 77.2
C-3 or C-5), 102.4 (C-1), 128.4, 129.6, 132.1, 137.4 (C-
1
. Rantwijk, F. V.; Oosteram, M. W.; Sheldon, R. A. J. Mol.
Catal. B Enzym. 1999, 6, 511.
2. Akita, H.; Kurashima, K.; Nakamura, T.; Kato, K.
(
aryl); FTIR (KBr, cm ): 3349, 2932, 2874, 1685, 1594,
À1
1
1
(
493, 1467, 1402, 1362, 1307, 1283, 1208, 1167, 1131,
Tetrahedron: Asymmetry 1999, 10, 2429.
3
. Vic, G.; Crout, D. H. G. Carbohydr. Res. 1995, 4, 315.
4. Ducret, A.; Trani, M.; Lortie, R. Biotechnol. Bioeng. 2002,
7, 752.
111, 1079, 1042, 1014, 897, 832, 810, 799, 763, 637; m=z
þ þ
ESI ) C H O NaCl (M+Na ), found: 327.0605, calcd
13 17 6
7
for: 327.0606.
5
6
7
. Hansson, T.; Andersson, M.; Wehtje, E.; Adlercreutz, P.
Enzyme Microb. Technol. 2001, 29, 527.
. Andersson, M.; Adlercreutz, P. J. Mol. Catal. B Enzym.
2
0
4
)
2
-Hydroxyphenylethyl b-
D
-glucopyranoside (7): ½aŠ
D
1
30.1 (c 0.50, MeOH); H NMR d (D O, ppm) 2.76 (m,
2
2
001, 14, 69.
H), 3.11 (m, 1H), 3.24 (m, 3H), 3.55 (dd, 1H, J ¼ 5:8,
. Scigelova, M.; Singh, S.; Crout, D. H. G. J. Mol. Catal. B
Enzym. 1999, 6, 483.
1
2.4 Hz), 3.74 (dd, 1H, J ¼ 3:0, 7.0 Hz), 3.78 (dd, 1H,