Biphasic catalysis with disaccharide phosphorylases: Chemoenzymatic synthesis of α- D -glucosides using sucrose phosphorylase

Article abstract of DOI:10.1021/op400302b

Thanks to its broad acceptor specificity, sucrose phosphorylase (SP) has been exploited for the transfer of glucose to a wide variety of acceptor molecules. Unfortunately, the low affinity (K_m > 1 M) of SP towards these acceptors typically urges the addition of cosolvents, which often either fail to dissolve sufficient substrate or progressively give rise to enzyme inhibition and denaturation. In this work, a buffer/ethyl acetate ratio of 5:3 was identified to be the optimal solvent system, allowing the use of SP in biphasic systems. Careful optimization of the reaction conditions enabled the synthesis of a range of α-d-glucosides, such as cinnamyl α-d-glucopyranoside, geranyl α-d-glucopyranoside, 2-O-α-d-glucopyranosyl pyrogallol, and series of alkyl gallyl 4-O-α-d-glucopyranosides. The usefulness of biphasic catalysis was further illustrated by comparing the glucosylation of pyrogallol in a cosolvent and biphasic reaction system. The acceptor yield for the former reached only 17.4%, whereas roughly 60% of the initial pyrogallol was converted when using biphasic catalysis.

Full text of DOI:10.1021/op400302b

Article
pubs.acs.org/OPRD
Biphasic Catalysis with Disaccharide Phosphorylases:
Chemoenzymatic Synthesis of α‑D‑Glucosides Using Sucrose
Phosphorylase
Karel De Winter,*,† Tom Desmet,† Tim Devlamynck,† Lisa Van Renterghem,† Tom Verhaeghe,†
Helena Pelantova,‡ Vladimír Kren,§ and Wim Soetaert†
́
̌
†Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Biosciences
Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
§
‡Laboratory of Molecular Structure Characterization and Laboratory of Biotransformation, Institute of Microbiology, Academy of
̌ ́
Sciences of the Czech Republic, Vídenska 1083, 142 20 Prague, Czech Republic
S
* Supporting Information
ABSTRACT: Thanks to its broad acceptor specificity, sucrose phosphorylase (SP) has been exploited for the transfer of glucose
to a wide variety of acceptor molecules. Unfortunately, the low affinity (Km > 1 M) of SP towards these acceptors typically urges
the addition of cosolvents, which often either fail to dissolve sufficient substrate or progressively give rise to enzyme inhibition
and denaturation. In this work, a buffer/ethyl acetate ratio of 5:3 was identified to be the optimal solvent system, allowing the use
of SP in biphasic systems. Careful optimization of the reaction conditions enabled the synthesis of a range of α-D-glucosides, such
as cinnamyl α-D-glucopyranoside, geranyl α-D-glucopyranoside, 2-O-α-D-glucopyranosyl pyrogallol, and series of alkyl gallyl 4-O-
α-D-glucopyranosides. The usefulness of biphasic catalysis was further illustrated by comparing the glucosylation of pyrogallol in a
cosolvent and biphasic reaction system. The acceptor yield for the former reached only 17.4%, whereas roughly 60% of the initial
pyrogallol was converted when using biphasic catalysis.
addition of cosolvents such as DMSO or methanol. However,
low concentrations of these cosolvents often fail to dissolve
sufficient substrate, whereas high concentrations progressively
give rise to enzyme inhibition and denaturation.20,21
1. INTRODUCTION
Numerous biologically active molecules exist as glycosides in
nature.1 Glycosylation can expand structural diversity,2 induce
targeting of drugs to specific organs and tissues,3 and improve
the solubility of hydrophobic compounds.4 Furthermore,
glycosylation is known to drastically extend the stability of
labile molecules5 and mediate the controlled release of flavors
and fragrances.6 Unfortunately, large-scale chemical synthesis of
these molecules is limited by low yields, the use of toxic
catalysts, and the generation of waste.7,8 Biocatalytic
approaches, allowing one-step reactions with high regio- and
stereoselectivity, have therefore attracted much attention over
the past decade.9
Enzymatic glycosylation is typically performed with glyco-
syltransferases (GTs) or glycoside hydrolases (GH). However,
the former requires relatively expensive nucleotide-activated
sugars,10 while the latter suffers from low yields when used in
the synthetic direction.11 Less research has been done with
disaccharide phosphorylases, although they show potential as
biocatalysts for glycoside synthesis.12−14 Indeed, these enzymes
use a glycosyl phosphate donor, which is much cheaper than
the activated sugar nucleotides required by GTs and can be
synthesized by phosphorolysis of their natural substrates.14
Sucrose phosphorylase (SP) catalyzes the reversible
phosphorolysis of sucrose into α-D-glucose 1-phosphate
(αG1P) and D-fructose. Its broad acceptor specificity has
been exploited for the transfer of glucose to a wide variety of
acceptor molecules, such as polyols,15 phenolics,16,17 hydrox-
yfuranones,18 and stilbenoids.19 Unfortunately, the low affinity
(Km > 1 M) of SP towards these acceptors typically urges the
The use of liquid−liquid biphasic systems containing water
and a water-immiscible organic solvent, however, provides an
interesting alternative. The aqueous phase contains the
enzymes and water-soluble substrates, while hydrophobic
substrates are dissolved in the organic phase. Stirring or
shaking will transfer these substrates from the organic to the
aqueous phase, where they can be converted by the enzymes.
Improved enzyme stability and ease of product recovery, while
avoiding substrate and product inhibition, are among the major
advantages of biphasic systems.22,23 The usefulness of this type
of catalysis has been widely illustrated for numerous
applications, including the synthesis of oligosaccharides24 and
glycosides25,26 with glycosidases.
Although SP has recently been successfully applied for
glycosylation reactions in ionic liquids19 and supercritical
carbon dioxide,27 no reports on its use in biphasic reaction
systems are available to date. In this work, we describe the
glycosylation of various acceptors with the SP from
Bifidobacterium adolescentis in biphasic systems.
Special Issue: Biocatalysis 14
Received: October 23, 2013
Published: February 20, 2014
© 2014 American Chemical Society
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2. RESULTS AND DISCUSSION
Article
amounts of acceptor, without impairing the glucosylation
reaction catalyzed by the enzyme. Various solvents were
supplemented with 50 mg/mL cinnamyl alcohol and reacted
under the optimal conditions. TLC analysis revealed the
highest conversion when using ethyl acetate (EtOAc).
However, SP-mediated glucosylation of cinnamyl alcohol was
also observed when butyl acetate (BuOAc), methyl-tert-
butylether (MTBE), octane, diethyl ether, cyclohexene, and
pentane were used (Figure S.2 of SI).
Next, the solubility of different acceptors was evaluated in the
most promising solvents (Table S.1 of SI). Although the
solubility of all compounds was found to be very low in octane,
the majority of the evaluated acceptors could be dissolved in
BuOAc, MTBE, and EtOAc. The latter was found to be less
deleterious for the glycosylation activity of SP and was
therefore used in all further experiments (Figure S.2 of SI).
Finally, the concentration of cinnamyl alcohol in the organic
phase was varied to reveal optimal glucoside formation at 100
mg/mL (data not shown).
The glycosylation potential of SP was then evaluated by
incubating various acceptors under optimal glucosylation
conditions. Semiquantative data for a large number of acceptors
was obtained trough TLC analysis (Table 2), while HPLC
experiments allowed detailed analysis of a limited amount of
reactions (Table 3).
These results illustrate the versatile applicability of biphasic
catalysis for the production of glycosides with SP. The structure
of numerous glucosides was confirmed by NMR spectroscopy.
Aliphatic alcohols could be glycosylated up to octanol, but the
obtained product concentrations decreased with increasing
chain lengths. Surprisingly, secondary alcohols were found to
The β-D-glucoside of cinnamyl alcohol has recently gained
attention from the pharmaceutical industry due to its claimed
antifatigue, antiaging, antioxidant, and immune enhancing
properties.28−30 The enzymatic glucosylation of cinnamyl
alcohol has already been reported in an IL based cosolvent
system using SP from B. adolescentis.19 Unfortunately, the latter
synthesis suffered from low yields and labor-intensive product
recovery. Consequently, the use of a cosolvent was avoided by
reacting 250 μL of the alcohol with 250 μL MES buffer at pH
6.5 supplemented with 2 M sucrose and 50 U/mL SP. TLC
analysis of the reaction mixture after 48 h incubation at 60 °C
revealed a clear new spot having Rf higher than that of sucrose
and lower than that of cinnamyl alcohol (Figure S.1 of the
Supporting Information [SI]). The product was purified by
silica gel chromatography and identified to be cinnamyl α-D-
glucopyranoside by NMR spectroscopy.
Encouraged by these results, conditions enabling the efficient
enzymatic glucosylation with B. adolescentis SP were further
optimized. Indeed, a number of factors are known to influence
the glycosylation efficiency, including the pH, the type and
concentration of both donor and acceptor, and the reaction
temperature.31 A full factorial design experiment was performed
to determine their relative importance on the glucosylation of
cinnamyl alcohol. The cheap and readily available donor
sucrose was used, and the temperature was fixed at 37 °C
(Table 1).
Table 1. Optimization of SP-catalyzed synthesis of cinnamyl
α-D-glucopyranoside in a biphasic system: 23 factorial
design; the reaction mixtures were incubated during 48 h at
37 °C
Table 2. Glycosylation potential of the SP from B.
adolescentis in a biphasic reaction system; TLC analysis was
performed after 48 h incubation at 50 °C
run pH [sucrose] (M) buffer/acceptor ratio product spot intensity
1
2
3
4
5
6
7
8
6
0.5
0.5
2
5:1
1:1
5:1
1:1
5:1
1:1
5:1
1:1
++
+
6
product spot
intensity
product spot
intensity
6
+++
++++
++
acceptor
acceptor
6
2
a
a
pentanol
+++
++
+
cinnamyl alcohol
+++
−
++++
7.5
7.5
7.5
7.5
0.5
0.5
2
a
b
hexanol
menthol
+
a
b
heptanol
saligenin
+++++
++++
a
b
octanol
+
p-nitrophenol
+
2
a
b
nonanol
−
−
−
++++
+++
−
phenol
−
+
a
b
decanol
hydroquinone
The glucosylation yield was found to be strongly influenced
by the pH and buffer/acceptor (solvent) ratio, while high
sucrose concentrations were generally required to obtain
proper glucosylation. Indeed, a product concentration of 4.8
g/L was achieved when using 2 M sucrose at pH 7.5 with a 5:1
solvent ratio. Next, the pH (5−9) and solvent ratio (10:1 to
1:5) were varied to identify optima at pH 7.5 and a ratio of 5:3
(data not shown). Although higher sucrose concentrations were
not feasible due to viscosity limitations, the glycosylation
efficiency could be further improved by increasing the reaction
temperature to 50 °C. Interestingly, using αG1P as donor
resulted in significantly less product formation, confirming
sucrose to be the preferred donor substrate32 (data not shown).
Finally, the optimal reaction conditions were found to be a
solvent ratio of 5:3, 2 M sucrose, pH 7.5, and 50 °C, resulting
in the production of 6.4 g/L cinnamyl α-D-glucopyranoside.
Although these results confirmed the SP-mediated transfer of
a glucose moiety using biphasic catalysis, a suitable water-
immiscible solvent is required to allow glycosylation of solid
acceptors. The latter solvent should be able to dissolve high
a
b
dodecanol
catechol
+++
+++
+++++
++++
a
b
cyclohexanol
resorcinol
a
b
2-hexanol
pyrogallol
a
linalool
gallic acid methyl
b
ester
a
b
eugenol
++
gallic acid ethyl ester
++++
+++
a
nerolidol
−
gallic acid propyl
b
ester
a
β-citronellol
+
gallic acid lauryl
−
b
ester
a
geraniol
++
salicylic acid methyl
+
a
ester
a
b
2-phenylethanol
+
+++++
+++++
++
curcumin
−
+
a
b
1R-phenylethanol
resveratrol
a
b
1S-phenylethanol
quercetin
+
a
b
benzyl alcohol
vanillin
++
+++
a
b
anisyl alcohol
+
vanillyl alcohol
a
Glycosylation was performed using the acceptor as the organic phase.
Glycosylation was performed using EtOAc supplemented with 100
b
mg/mL acceptor as the organic phase.
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Table 3. Glycosylation of various acceptors with the SP from B. adolescentis; HPLC analysis was performed after 48 h incubation
at 50 °C
d
acceptor
[glucoside] (mM)
[glucoside] (g/L)
glucoside (g)
yield acceptor (%)
yield donor (%)
a
geraniol
1R-phenylethanol
20.4
133.2
126.7
21.5
16.2
2.6
6.5
37.9
36.0
6.4
0.65
3.79
3.60
0.64
0.48
0.07
0.74
5.06
3.24
3.02
1.83
0.92
0.9
4.3
1.6
10.7
10.1
1.7
a
a
1S-phenylethanol
4.1
a
cinnamyl alcohol
cinnamyl alcohol
0.8
b
4.8
5.8
1.3
b
hydroquinone
0.7
0.7
0.2
b
resorcinol
27.1
175.8
112.6
87.4
50.9
24.7
7.4
7.9
2.2
b
pyrogallol
50.6
32.4
30.2
18.3
59.1
75.7
42.9
26.9
14.0
14.1
9.0
c
pyrogallol
b
gallic acid methyl ester
7.0
b
gallic acid ethyl ester
4.0
b
gallic acid propyl ester
9.2
1.9
a
b
Glycosylation was performed using the acceptor as the organic phase. Glycosylation was performed using EtOAc supplemented with 100 mg/mL
c
d
acceptor as the organic phase. Glycosylation was performed using EtOAc supplemented with 50 mg/mL acceptor as the organic phase. Glucoside
produced in a 100 mL reaction.
Figure 1. HPLC chromatogram showing the glucosylation of gallic acid propyl ester and formation of glucobiose products with SP. The sample was
analyzed after 24 h incubation under optimal biphasic reaction conditions at 50 °C: gallic acid propyl ester (4.9 min), propyl gallyl 4-O-α-D-
glucopyranoside (8.2 min), D-fructose (21.7 min), D-glucose (23.2 min), sucrose (28.3 min), and glucobiose products (30.2−31.9 min).
be more easily glycosylated compared to primary alcohols.
Indeed, 48 h incubation under optimal reaction conditions
resulted in glucoside concentrations of 37.9 and 36.0 g/L for
1R- and 1S-phenylethanol, respectively, while only traces of
product were detected when reacting 2-phenylethanol. The
structure of R- and S-1-phenylethyl α-D-glucopyranoside was
confirmed by NMR spectroscopy. Also, SP from B. adolescentis
was found to glycosylate a range of structurally diverse
compounds with olfactory properties. Examples include the
monoterpenoids geraniol and β-citronellol, aromatic alcohols
such as benzyl alcohol, anisyl alcohol, cinnamyl alcohol and
vanillyl alcohol, as well as the phenylpropanoid eugenol, and
the phenolic aldehyde vanillin. Although these glycosides were
generally produced in significantly lower concentrations
compared to secondary alcohols (Table 3), we were able to
confirm the formation of geranyl α-D-glucopyranoside and
cinnamyl α-D-glucopyranoside by NMR spectroscopy.
However, we were able to couple a glucose moiety to
hydroquinone, catechol, resorcinol, and pyrogallol. Indeed,
the formation of 1-O-α-D-glucopyranosyl hydroquinone (0.7 g/
L), 1-O-α-D-glucopyranosyl resorcinol (7.4 g/L) and 2-O-α-D-
glucopyranosyl pyrogallol (50.6 g/L) was confirmed by NMR
spectroscopy. In addition, glucosylation of a series of gallic acid
esters was observed, as confirmed by NMR spectroscopy.
These potent antioxidants, including ethyl gallate (E313) and
propyl gallate (E310), are commonly applied in foods,
cosmetics, and hair products.33,34 The methyl ester glucoside
could be produced up to 30 g/L, while longer alkyl chains were
found to result in lower glucoside concentrations (Table 3).
In conclusion, a wide variety of glucosides could be
synthesized using the SP from B. adolescentis. Industrially
relevant glucoside concentrations of several g/L were obtained
for a number of acceptors.35,36 Not surprisingly, the acceptor
yields were much lower when using the pure acceptor as
organic phase (Table 3). The latter, however, could be
increased from 0.8 to 5.8% for cinnamyl alcohol by dissolving
In contrast to earlier work with the SP from L.
mesenteroides,16 we failed to obtain any glycosides of phenol.
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100 mg/mL of the acceptor in EtOAc rather than using the
alcohol in its undiluted form. Moreover, the acceptor yield for
pyrogallol could be improved from 59.1 to 75.7% by decreasing
the acceptor concentration from 100 to 50 mg/mL. However,
these increased yields were achieved at the expense of lower
product concentrations (Table 3).
3. CONCLUSIONS
Following the work on SP-mediated glycoside synthesis in
conventional solvents,32 ILs,19 and supercritical carbon
dioxide,27 this is the first report on the use of disaccharide
phosphorylases in biphasic reaction systems. Careful optimiza-
tion allowed the glucosylation of aliphatic alcohols, mono-
terpenoids, aromatic alcohols and phenolics. In addition a series
of alkyl gallyl 4-O-α-D-glucopyranosides were successfully
synthesized up to 30 g/L. These glucosylations were achieved
by reacting 62.5% aqueous MOPS buffer at pH 7.5 containing 2
M sucrose and 50 U/mL SP, and 37.5% EtOAc supplemented
with 100 mg/mL acceptor. Also, the production of 2-O-α-D-
glucopyranosyl pyrogallol was compared in cosolvent and
biphasic systems. The transfer ratio was found to be 5 times
higher when using the latter system, resulting in less hydrolysis
and formation of glucobiose side product. Consequently, the
use of biphasic catalysis was identified to be a valuable
alternative for glycoside synthesis with disaccharide phosphor-
ylases.
The donor yields, on the contrary, were less than 20% for all
acceptors (Table 3). Indeed, higher concentrations of sucrose
were added to stabilize the enzyme,19 and push the conversion
in the synthesis direction. Therefore, considerable amounts of
sucrose remained present at the end of the reaction (Figure 1).
Moreover, SP is known to exhibit an undesirable hydrolytic side
reaction, yielding D-glucose and D-fructose as products. The
liberated glucose can then be glucosylated by SP, resulting in
the formation of ‘glucobiose’ products.32,37 Unfortunately, this
competition between D-glucose and the acceptor compound
was found to further decrease the donor yield (Figure 1).
Finally, the performance of biphasic catalysis was compared
with a recently reported cosolvent system,19 using the
glycosylation of pyrogallol as a case study. Therefore, the
water-immiscible EtOAc was replaced by the IL AMMOENG
101, which has been successfully applied for the SP-mediated
synthesis of 3-O-α-D-glucopyranosyl-(E)-resveratrol.19 All other
parameters were identical for both reactions (Figure 2).
4. EXPERIMENTAL SECTION
4.1. Materials, Enzymes, and Instruments. The IL
AMMOENG 101 was kindly provided by Evonik Industries
AG, and ethyl acetate was bought from Fiers. All other
chemicals were analytical grade and purchased from Sigma-
Aldrich. The recombinant SP from B. adolescentis was produced
and partially purified by heat treatment as described recently.38
The activity of the enzyme was determined as published
earlier,19 and one unit of SP was defined as the activity that
corresponds to the release of 1 μmol fructose from 100 mM
sucrose in 100 mM phosphate buffer at pH 7 and 37 °C. NMR
spectra were measured on a Bruker AVANCE III 600 MHz
spectrometer (600.23 MHz for 1H, and 150.94 MHz for 13C) in
CD3OD at 25 °C. HPLC measurements were performed on a
Varian Prostar.
4.2. Glycosylation Reactions with SP. Small-scale
biphasic glycosylation reactions were performed in a
thermoshaker (Eppendorf) at 1400 rpm. Varying amounts of
different water-immiscible acceptors were added to 500 μL
aqueous buffer in a 1.5 mL Eppendorf. Alternatively, water-
immiscible organic solvents containing 100 mg/mL acceptor
were used. The acidity of the aqueous phase was varied by
using a citrate-phosphate buffer (pH 5−5.4), MES buffer (pH
5.5−6.5), MOPS buffer (pH 6.6−7.5), or tricine buffer (pH
7.6−9). Unless stated otherwise, the reactions were incubated
at 50 °C in the presence of 2 M sucrose and 50 U/mL SP. The
acceptor (YieldAcceptor) and donor yield (YieldDonor) are defined
as the ratio of the amount of product formed to the amount of
acceptor and donor added to the reaction, respectively (mol/
mol).
4.3. Solubility Measurements. The dissolution of various
acceptor molecules in different water-immiscible solvents was
performed in a water bath at 50 °C with 0.1 °C accuracy.
Varying amounts of acceptor were added to a 2 mL Eppendorf,
and solvent was added to 1 mL. Next, the samples were
vortexed multiple times and allowed to equilibrate for 48 h,
after which the samples were checked for remaining particles.
4.4. TLC Analysis. TLC analysis was performed on Merck
Silica gel 60 F254 precoated plates. The eluens was a mixture of
EtOAc/methanol/water (30:5:4 by volume), and spots were
visualized by UV detection at 254 nm, or charring with 10% (v/
v) H2SO4. All TLC plates of a single experiment were
Figure 2. Synthesis of 2-O-α-D-glucopyranosyl pyrogallol in a
○
●
cosolvent ( ) and biphasic ( ) reaction system. The reaction mixture
consisted of 62.5% aqueous MOPS buffer at pH 7.5 containing 2 M
sucrose and 50 U/mL SP, and 37.5% EtOAc supplemented with 100
mg/mL pyrogallol. For the cosolvent system, EtOAc was replaced by
the IL AMMOENG 101.
Interestingly, the acceptor yield for the cosolvent system
reached only 17.4%, while roughly 60% of the initial pyrogallol
was converted when using a biphasic system. This significant
difference can be explained by the transfer ratio of the enzyme
under the operational conditions. Indeed, a ratio of 5.7 for the
biphasic system, compared to 1.1 for the cosolvent system,
indicates that less hydrolysis (and synthesis of glucobioses)
occurs upon application of biphasic catalysis. In addition, the
course of product formation reveals the absence of secondary
hydrolysis (i.e., the degradation of 2-O-α-D-glucopyranosyl
pyrogallol), as previously reported by the Nidetzky group for α-
glucosyl glycerol15 (Figure 2).
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compared, thereby rating the intensity of product spots
between +++++ and + for the highest intensity and a barely
visible spot, respectively. Both the aqueous and the solvent
phase were spotted on TLC. The intensity of both product
spots was combined to yield a single rating.
signals of solvent were used as internal standards (δH 3.330
ppm, δC 49.30 ppm), and digital resolution enabled us to report
δH to three and δC to two decimal places. The proton spin
systems were assigned by COSY, and then the assignment was
transferred to carbons by HSQC. HMBC experiments enabled
assigning quaternary carbons and joining individual spin
systems together. Chemical shifts are given in δ-scale [ppm],
and coupling constants in Hz.
4.5. HPLC Analysis. HPLC analysis was performed on an
X-bridge amide column (250 mm × 4.6 mm, 3.5 μm, Waters,
U.S.A.) with Milli-Q water (solvent A) and acetonitrile (solvent
B), both containing 0.2% triethylamine, as the mobile phase.
The flow rate and temperature were set at 1.0 mL/min and 30
°C, respectively. The gradient elution was as follows: 95% of
solvent A (0−12 min), 5− 25% solvent B (12−15 min), 25%
solvent B (15−40 min), 25 to 5% solvent B (40−41 min) and
95% solvent A (41−50 min). Adequate detection was obtained
with an Alltech 2000ES evaporative light-scattering detector
(ELSD). The tube temperature, gas flow, and gain were set at
30 °C, 1.5 L/min, and 1, respectively. Homogeneous samples
containing both phases were obtained after intensive mixing.
These samples were then diluted in DMSO, and subjected to
HPLC analysis. The resulting concentrations thus refer to the
amount of product present in the total reaction volume. The
obtained peaks were calibrated using standard curves prepared
in Milli-Q water or methanol. All HPLC analyses were
performed in triplicate.
4.6. Comparison of Biphasic and Cosolvent Glyco-
sylation Reactions Catalyzed by SP. The glycosylation of
pyrogallol was carried out at 10 mL scale. The reaction mixture
consisted of 62.5% aqueous MOPS buffer at pH 7.5 containing
2 M sucrose and 50 U/mL SP, and 37.5% EtOAc
supplemented with 100 mg/mL pyrogallol. Alternatively,
EtOAc was replaced by the IL AMMOENG 101. Reactions
mixtures were incubated in a thermoshaker (Eppendorf) at 750
rpm and 50 °C. The concentrations of 2-O-α-D-glucopyranosyl
pyrogallol and fructose were determined by means of HPLC
analysis. The transfer ratio is defined as the ratio of the amount
of glucose transferred to the acceptor over the sum of the free
glucose and the glucose incorporated in glucobioses. The
former was calculated on the basis of the glucoside
concentration, while the latter was obtained by subtracting
the amount of glucoside from the obtained fructose
concentration.
4.7. Production and Purification of Glucosides. The
glycosylations of hydroquinone, resorcinol, pyrogallol, methyl
gallate, ethyl gallate, and propyl gallate were carried out at 100
mL scale in magnetically stirred flasks. Biphasic reaction
mixtures were created by adding 62.5 mL aqueous buffer to
37.5 mL organic solvent. The aqueous buffer consisted of 50
mM MOPS at pH 7.5 containing 2 M sucrose and 50 U/mL
SP. EtOAc supplemented with 100 mg/mL hydroquinone,
resorcinol, pyrogallol, methyl gallate, ethyl gallate, or propyl
gallate was used as organic phase. Alternatively, glycosylation of
1-phenylethanol, geraniol, and cinnamyl alcohol was performed
by substituting the EtOAc by the pure acceptor. Reactions were
terminated after 48 h incubation at 50 °C, after which the
reaction mixtures were heated (10 min at 95 °C) and
centrifuged (12000g, 4 °C, 15 min) to remove debris. The
samples were then evaporated in vacuo, and the residue was
purified by column chromatography (silicagel, EtOAc-meth-
anol−water (30:5:4 by volume)).
4.8.1. 1-O-α-D-Glucopyranosyl hydroquinone: 1H NMR
(600.23 MHz, CD3OD, 25 °C): δ 3.427 (1H, dd, J = 9.7, 8.9
Hz, H-4), 3.543 (1H, dd, J = 9.8, 3.7 Hz, H-2), 3.722 (1H, dd, J
= 11.3, 4.9 Hz, H-6u), 3.752 (1H, ddd, J = 9.7, 4.9, 2.0 Hz, H-
5), 3.791 (1H, dd, J = 11.3, 2.0 Hz, H-6d), 3. 863 (1H, dd, J =
9.8, 8.9 Hz, H-3), 5.310 (1H, d, J = 3.7 Hz, H-1), 6.720 (2H, m,
ΣJ = 8.9 Hz, H-meta), 7.026 (2H, m, ΣJ = 8.9 Hz, H-ortho).
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 62.73 (C-6),
71.92 (C-4), 73.73 (C-2), 74.50 (C-5), 75.28 (C-3), 100.85 (C-
1), 116.99 (C-meta), 120.17 (C-ortho), 152.22 (C-ipso), 154.14
(C-para).
4.8.2. 1-O-α-D-Glucopyranosyl resorcinol: 1H NMR
(600.23 MHz, CD3OD, 25 °C): δ 3.462 (1H, dd, J = 9.9, 8.9
Hz, H-4), 3.569 (1H, dd, J = 9.8, 3.7 Hz, H-2), 3.671 (1H, ddd,
J = 9.9, 4.4, 2.7 Hz, H-5), 3.728 (1H, dd, J = 12.0, 4.4 Hz, H-
6u), 3.762 (1H, dd, J = 12.0, 2.7 Hz, H-6d), 5.457 (1H, d, J =
3.7 Hz, H-1), 6.548 (1H, ddd, J = 8.1, 2.3, 0.8 Hz, H-6′), 6.636
(1H, dd, J = 2.3, 2.3 Hz, H-2′), 6.662 (1H, ddd, J = 8.2, 2.3, 0.8
Hz, H-4′), 7.087 (1H, dd, J = 8.2, 8.1 Hz, H-5′).
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 62.58 (C-6),
71.75 (C-4), 73.63 (C-2), 74.58 (C-5), 75.26 (C-3), 99.52 (C-
1), 105.85 (C-2′), 109.57 (C-4′), 110.83 (C-6′), 131.14 (C-5′),
159.90 (C-3′), 160.09 (C-1′).
4.8.3. 2-O-α-D-Glucopyranosyl pyrogallol: 1H NMR
(600.23 MHz, CD3OD, 25 °C): δ 3.441 (1H, dd, J = 10.1,
8.9 Hz, H-4), 3.604 (1H, dd, J = 9.6, 3.9 Hz, H-2), 3.771 (1H,
dd, J = 11.8, 5.5 Hz, H-6u), 3.881 (1H, dd, J = 9.6, 8.9 Hz, H-
3), 3.902 (1H, dd, J = 11.8, 2.4 Hz, H-6d), 4.234 (1H, ddd, J =
10.1, 5.5, 2.4 Hz, H-5), 5.024 (1H, d, J = 3.9 Hz, H-1) 6.385
(2H, d, J = 8.2 Hz, H-meta), 6.813 (1Ht, J = 8.2 Hz, H-para).
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 62.60 (C-6),
71.41 (C-4), 73.66 (C-2), 75.30 (C-3), 75.56 (C-5), 106.01 (C-
1), 108.95 (C-meta), 126.47 (C-para), 136.12 (C-ipso), 152.27
(C-ortho).
1
4.8.4. Methyl gallyl 4-O-α-D-glucopyranoside: H NMR
(600.23 MHz, CD3OD, 25 °C): δ 3.482 (1H, dd, J = 10.1, 9.2
Hz, H-4), 3.619 (1H, dd, J = 9.6, 3.8 Hz, H-2), 3.796 (1H, dd, J
= 11.9, 4.9 Hz, H-6u), 3.860 (3H, s, H-1′), 3.868 (1H, dd, J =
11.9, 2.5 Hz, H-6d), 3.902 (1H, dd, J = 9.6, 9.2 Hz, H-3), 4.243
(1H, ddd, J = 10.1, 4.9, 2.5 Hz, H-5), 5.149 (1H, d, J = 3.8 Hz,
H-1), 7.063 (2H, s, H-meta).
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 52.88 (C-1′),
62.41 (C-6), 71.18 (C-4), 73.66 (C-2), 75.22 (C-3), 75.46 (C-
5), 105.45 (C-1), 110.34 (C-meta), 128.10 (C-para), 139.75
(C-ipso), 152.31 (C-ortho), 168.60 (CO).
4.8.5. Ethyl gallyl 4-O-α-D-glucopyranoside: 1H NMR
(600.23 MHz, CD3OD, 25 °C): δ 1.372 (3H, t, J = 7.1 Hz,
H-2′), 3.479 (1H, dd, J = 10.1, 9.2 Hz, H-4), 3.619 (1H, dd, J =
9.6, 3.8 Hz, H-2), 3.793 (1H, dd, J = 11.8, 5.0 Hz, H-6u), 3.872
(1H, dd, J = 11.8, 2.5 Hz, H-6d), 3.901 (1H, dd, J = 9.6, 9.2 Hz,
H-3), 4.242 (1H, ddd, J = 10.1, 5.0, 2.5 Hz, H-5), 4.317 (1H, q,
J = 7.1 Hz, H-1′), 5.146 (1H, d, J = 3.8 Hz, H-1), 7.068 (2H, s,
H-meta).
4.8. Structure Elucidation of Glucosides. The structures
of the newly formed glucosides were determined by a
combination of 1D NMR (1H NMR and 13C NMR) and 2D
NMR (gCOSY, gHSQC and gHMBC) spectroscopy. Residual
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 14.85 (C-2′),
62.36 (C-1′), 62.45 (C-6), 71.22 (C-4), 73.67 (C-2), 75.24 (C-
785
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Article
3), 75.50 (C-5), 105.51 (C-1), 110.31 (C-meta), 128.47 (C-
para), 139.72 (C-ipso), 152.29 (C-ortho), 168.13 (CO).
4.8.6. Propyl gallyl 4-O-α-D-glucopyranoside: 1H NMR
(600.23 MHz, CD3OD, 25 °C): δ 1.038 (3H, t, J = 7.4 Hz, H-
3′), 1.778 (2H, tq, J = 7.4, 6.6 Hz, H-2′), 3.485 (1H, dd, J =
10.0, 9.2 Hz, H-4), 3.622 (1H, dd, J = 9.6, 3.8 Hz, H-2), 3.798
(1H, dd, J = 11.9, 4.9 Hz, H-6u), 3.872 (1H, dd, J = 11.9, 2.5
Hz, H-6d), 3.906 (1H, dd, J = 9.6, 9.2 Hz, H-3),4.223 (2H, t, J
= 6.6 Hz, H-1′), 4.246 (1H, ddd, J = 10.0, 4.9, 2.5 Hz, H-5),
5.153 (1H, d, J = 3.8 Hz, H-1), 7.070 (2H, s, H-meta).
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 11.07 (C-3′),
23.40 (C-2′), 62.41 (C-6), 67.92 (C-1′), 71.17 (C-4), 73.64 (C-
2), 75.21 (C-3), 75.45 (C-5), 105.44 (C-1), 110.28 (C-meta),
128.39 (C-para), 139.69 (C-ipso), 152.28 (C-ortho), 168.16
(CO).
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 16.79 (3′-Me),
18.04 (C-9′), 26.17 (C-8′), 27.74 (C-5′), 41.01 (C-4′), 62.96
(C-6), 65.11 (C-1′), 72.12 (C-4), 73.85 (C-2), 73.97 (C-5),
75.49 (C-3), 99.17 (C-1), 121.95 (C-2′), 125.36 (C-6′), 132.82
(C-7′), 141.86 (C-3′).
ASSOCIATED CONTENT
* Supporting Information
■
S
TLC plates showing the biphasic glycosylation of cinnamyl
alcohol (Figure S.1 and S.2) and the solubility of various
acceptors in different water-immiscible solvents (Table S.1).
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +3292649921. Fax: +3292646032. E-mail: karel.
dewinter@ugent.be.
■
4.8.7. 1R-Phenylethyl α-D-glucopyranoside: 1H NMR
(600.23 MHz, CD3OD, 25 °C): δ 1.493 (3H, d, J = 6.6 Hz,
H-2′), 3.304 (1H, dd, J = 9.8, 8.9 Hz, H-4), 3.333 (1H, dd, J =
9.8, 3.8 Hz, H-2), 3.709 (1H, m, H-6u), 3.735 (1H, m, H-5),
3.775 (1H, dd, J = 9.7, 8.9 Hz, H-3), 3.886 (1H, m, H-6d),
4.626 (1H, d, J = 3.8 Hz, H-1), 4.909 (1H, q, J = 6.6 Hz, H-
1′),7.272 (1H, m, H-para), 7.345 (2H, m, H-meta), 7.455 (2H,
m, H-ortho).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 24.92 (C-2′),
63.13 (C-6), 72.34 (C-4), 73.78 (C-2), 74.19 (C-5), 74.59 (C-
1′), 75.40 (C-3), 97.14 (C-1), 128.19 (C-ortho), 128.90 (C-
para), 129.71 (C-meta), 144.30 (C-ipso).
The authors thank the Fund for Scientific Research-Flanders
(FWO-Vlaanderen, doctoral scholarship for K.D.W.), the
European Commission (FP7-project ‘Novosides’, Grant
265854) and Ghent University (Multidisciplinary Research
Partnership ‘Ghent Bio-Economy’) for financial support.
K.D.W. is an aspirant of the Fund for Scientific Research-
Flanders (FWO-Vlaanderen).
4.8.8. 1S-Phenylethyl α-D-glucopyranoside: 1H NMR
(600.23 MHz, CD3OD, 25 °C): δ 1.499 (3H, d, J = 6.5 Hz,
H-2′), 3.323 (1H, dd, J = 10.0, 8.5 Hz, H-4), 3.362 (1H, ddd, J
= 10.0, 4.3, 2.3 Hz, H-5), 3.429 (1H, dd, J = 9.7, 3.8 Hz, H-2),
3.435 (1H, dd, J = 11.8, 2.3 Hz, H-6u), 3.543 (1H, dd, J = 11.9,
4.3 Hz, H-6d), 3.664 (1H, dd, J = 9.7, 8.5 Hz, H-3), 4.808 (1H,
q, J = 6.5 Hz, H-1′), 5.058 (1H, d, J = 3.8 Hz, H-1), 7.254 (1H,
m, H-para), 7.326 (2H, m, H-meta), 7.427 (2H, m, H-ortho).
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 22.44 (C-2′),
62.33 (C-6), 71.81 (C-4), 73.88 (C-2), 75.38 (C-3), 77.17 (C-
1′), 78.42 (C-5), 99.34 (C-1), 127.73 (C-ortho), 128.65 (C-
para), 129.54 (C-meta), 145.60 (C-ipso).
REFERENCES
■
(1) Kren, V.; Martinkova, L. Curr. Med. Chem. 2001, 8, 11.
(2) Kren, V.; Rezanka, T. FEMS Microbiol. Rev. 2008, 32, 5.
(3) Wong, A.; Toth, I. Curr. Med. Chem. 2001, 8, 9.
(4) Kren, V., Glycoside vs aglycon: The role of glycosidic residue in
biological activity. In Glycoscience; Fraser-Reid, B., Tatsuta, K., Thiem,
J., Eds.; Springer: Berlin, 2008; pp 2589−2644.
(5) Yamamoto, I.; Muto, N.; Nagata, E.; Nakamura, T.; Suzuki, Y.
Biochim. Biophys. Acta 1990, 1035, 1.
1
4.8.9. Cinnamyl α-D-glucopyranoside: H NMR (600.23
(6) Nakagawa, H.; Yoshiyama, M.; Shimura, S.; Kirimura, K.; Usami,
S. Biosci., Biotechnol., Biochem. 1998, 62, 7.
(7) Zhu, X.; Schmidt, R. R. Angew. Chem., Int. Ed. 2009, 48, 11.
(8) Muthana, S.; Cao, H. Z.; Chen, X. Curr. Opin. Chem. Biol. 2009,
13, 5−6.
(9) Desmet, T.; Soetaert, W.; Bojarova, P.; Kren, V.; Dijkhuizen, L.;
Eastwick-Field, V.; Schiller, A. Chem.Eur. J. 2012, 18, 35.
(10) Palcic, M. M. Curr. Opin. Chem. Biol. 2011, 15, 2.
(11) Desmet, T.; Soetaert, W. Biocatal. Biotrans. 2011, 29.
(12) Kitaoka, M.; Hayashi, K. Trends Glycosci. Glycotechnol. 2002, 14,
75.
MHz, CD3OD, 25 °C): δ 3.325 (1H, dd, J = 10.0, 8.9 Hz, H-4),
3.446 (1H, dd, J = 9.7, 3.7 Hz, H-2), 3.661 (1H, ddd, J = 10.0,
5.7, 2.3 Hz, H-5), 3.707 (1H, dd, J = 11.7, 5.7 Hz, H-6u), 3.714
(1H, dd, J = 9.7, 8.9 Hz, H-3), 3.851 (1H, dd, J = 11.7, 2.3 Hz,
H-6d), 4.240 (1H, ddd, J = 12.8, 6.6, 1.4 Hz, H-1′u), 4.407
(1H, ddd, J = 12.8, 5.8, 1.6 Hz, H-1′d), 4.922 (1H, d, J = 3.7
Hz, H-1), 6.410 (1H, ddd, J = 16.0, 6.6, 5.8 Hz, H-2′), 6.714
(1H, ddd, J = 16.0, 1.6, 1.4 Hz, H-3′), 7.242 (1H, m, H-para),
7.321 (2H, m, H-meta), 7.439 (2H, m, H-ortho).
13C NMR (150.94 MHz, CD3OD, 25 °C): δ 63.03 (C-6),
69.44 (C-1′), 72.18 (C-4), 73.87 (C-2), 74.15 (C-5), 75.44 (C-
3), 99.61 (C-1), 126.89 (C-2′), 127.82 (C-ortho), 129.01 (C-
para), 129.89 (C-meta), 134.21 (C-3′), 138.52 (C-ipso).
4.8.10. Geranyl α-D-glucopyranoside: 1H NMR (600.23
MHz, CD3OD, 25 °C): δ 1.630 (3H, s, H-9′), 1.695 (3H, q, J =
1.0 Hz, H-8′), 1.715 (3H, s, 3′-Me), 2.067 (2H, m, H-4′), 2.141
(2H, m, H-5′), 3.316(1H, dd, J = 9.9, 8.8 Hz, H-4), 3.403 (1H,
dd, J = 9.7, 3.8 Hz, H-2), 3.608 (1H, ddd, J = 9.9, 5.5, 2.4 Hz,
H-5), 3.658 (1H, dd, J = 9.7, 8.8 Hz, H-3), 3.704 (1H, ddd, J =
11.8, 5.5 Hz, H-6u), 3.824 (1H, dd, J = 11.8, 2.4 Hz, H-6d),
4.121 (1H, dd, J = 12.1, 7.5 Hz, H-1′u), 4.242 (1H, dd, J = 12.1,
6.4 Hz, H-1′d), 4.838 (1H, d, J = 3.8 Hz, H-1), 5.128 (1H, m,
H-6′), 5.409 (1H, m, H-2′).
(13) Luley-Goedl, C.; Nidetzky, B. Biotechnol. J. 2010, 5, 12.
(14) Desmet, T.; Soetaert, W. Process Biochem. 2012, 47, 1.
(15) Goedl, C.; Sawangwan, T.; Mueller, M.; Schwarz, A.; Nidetzky,
B. Angew. Chem., Int. Ed. 2008, 47, 52.
(16) Kitao, S.; Sekine, H. Biosci., Biotechnol., Biochem. 1994, 58, 1.
(17) Kitao, S.; Ariga, T.; Matsuda, T.; Sekine, H. Biosci., Biotechnol.,
Biochem. 1993, 57, 12.
(18) Kitao, S.; Matsudo, T.; Sasaki, T.; Koga, T.; Kawamura, M.
Biosci., Biotechnol., Biochem. 2000, 64, 1.
(19) De Winter, K.; Verlinden, K.; Kren, V.; Weignerova, L.;
Soetaert, W.; Desmet, T. Green Chem. 2013, 15, 7.
(20) Bertrand, A.; Morel, S.; Lefoulon, F.; Rolland, Y.; Monsan, P.;
Remaud-Simeon, M. Carbohydr. Res. 2006, 341, 7.
(21) Cerdobbel, A.; De Winter, K.; Aerts, D.; Kuipers, R.; Joosten,
H.-J.; Soetaert, W.; Desmet, T. Protein Eng. Des. Sel. 2011, 24, 11.
786
dx.doi.org/10.1021/op400302b | Org. Process Res. Dev. 2014, 18, 781−787

Organic Process Research & Development
Article
(22) Carrea, G.; Cremonesi, P. Methods Enzymol. 1987, 136, 4.
(23) Eggers, D. K.; Blanch, H. W.; Prausnitz, J. M. Enzyme Microb.
Technol. 1989, 11, 2.
(24) Wang, K.; Lu, Y.; Liang, W. Q.; Di Wang, S.; Jiang, Y.; Huang,
R.; Liu, Y. H. J. Agric. Food Chem. 2012, 60, 15.
(25) Hansson, T.; Andersson, M.; Wehtje, E.; Adlercreutz, P. Enzyme
Microb. Technol. 2001, 29, 8.
(26) Kwon, S. J.; Jung, H.-C.; Pan, J.-G. Appl. Environ. Microbiol.
2007, 73, 7.
(27) Shin, M. H.; Cheong, N. Y.; Lee, J. H.; Kim, K. H. Food Chem.
2009, 115, 3.
(28) Tayade, A. B.; Dhar, P.; Sharma, M.; Chauhan, R. S.; Chaurasia,
O. P.; Srivastava, R. B. J. Food Sci. 2013, 78, 3.
(29) Mishra, K. P.; Ganju, L.; Singh, S. B. Immunopharm.
Immunotechnol. 2012, 34, 3.
(30) Mook-Jung, I.; Kim, H.; Fan, W. Z.; Tezuka, Y.; Kadota, S.;
Nishijo, H.; Jung, M. W. Biol. Pharm. Bull. 2002, 25, 8.
(31) van Rantwijk, F.; Oosterom, M. W. V.; Sheldon, R. A. J. Mol.
Catal. B: Enzym. 1999, 6, 6.
(32) Goedl, C.; Sawangwan, T.; Wildberger, P.; Nidetzky, B. Biocatal.
Biotrans. 2010, 28, 1.
(33) Becker, L. Int. J. Toxicol. 2007, 26, 3.
(34) Kalaivani, T.; Rajasekaran, C.; Mathew, L. J. Food Sci. 2011, 76,
6.
̀
(35) Auriol, D.; ter Halle, R.; Lefevre, F., Industrial production of
caffeic acid-α-D-O-glucoside. In Practical Methods for Biocatalysis and
Biotransformations 2; Whittall, J., Sutton, W. P., Eds.; Wiley:
Chichester, 2012; pp 232−235.
(36) Buchholz, K.; Seibel, J. Carbohydr. Res. 2008, 343, 12.
(37) Kitao, S.; Yoshida, S.; Horiuchi, T.; Sekine, H.; Kusakabe, I.
Biosci., Biotechnol., Biochem. 1994, 58, 4.
(38) De Winter, K.; Soetaert, W.; Desmet, T. Int. J. Mol. Sci. 2012,
13, 9.
787
dx.doi.org/10.1021/op400302b | Org. Process Res. Dev. 2014, 18, 781−787