Probing the transferase activity of glycosidases by means of in situ NMR spectroscopy

Article abstract of DOI:10.1016/S0957-4166(99)00297-9

This paper describes the conditions in which in situ NMR spectroscopy is a suitable technique to use when following the course of enzymatic transglycosylation reactions. Using this methodology, the reactions must be carried out in D₂O. Our experiments indicate that the rate of the transglycosylation reaction is reduced in this solvent while the rate of the hydrolysis of the disaccharides produced is enhanced depending on the nature of the anomeric substituent. However, this undesirable effect is generally weak because the rates of the transglycosylation reactions are always faster than the rates of the hydrolysis whatever the solvent. The great advantage of NMR spectroscopy lies in its potential to detect, in a single experiment, all the components of the reaction without any disturbance of the reaction medium.

Full text of DOI:10.1016/S0957-4166(99)00297-9

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2905–2912
Probing the transferase activity of glycosidases by means of
in situ NMR spectroscopy
∗
P. Spangenberg, V. Chiffoleau-Giraud, C. André, M. Dion and C. Rabiller
Unité de Recherches en Biocatalyse (UPRES no. 2161), Faculté des Sciences et des Techniques, 2, rue de la Houssinière,
BP 92208, F-44322 Nantes cedex 3, France
Received 11 June 1999; accepted 14 July 1999
Abstract
This paper describes the conditions in which in situ NMR spectroscopy is a suitable technique to use when
following the course of enzymatic transglycosylation reactions. Using this methodology, the reactions must be
carried out in D2O. Our experiments indicate that the rate of the transglycosylation reaction is reduced in this
solvent while the rate of the hydrolysis of the disaccharides produced is enhanced depending on the nature of the
anomeric substituent. However, this undesirable effect is generally weak because the rates of the transglycosylation
reactions are always faster than the rates of the hydrolysis whatever the solvent. The great advantage of NMR
spectroscopy lies in its potential to detect, in a single experiment, all the components of the reaction without any
disturbance of the reaction medium. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
The key role played by carbohydrates in biological mechanisms has led to the rapid development of
sugar chemistry. On the one hand, the regioselective synthesis of the glycosidic bond may be achieved
1
by means of the standard chemical approach which usually requires cumbersome and sophisticated
protection–deprotection multisteps. On the other hand, the enzymatic synthesis of the saccharides has
2
become, over the last 10 years, a very powerful alternative. Two kinds of enzymes are able to catalyse
the formation of the glycosidic bond: the glycosyltransferases and the glycosylhydrolases. The former
generally lead to high yields and total regio- and stereoselectivities but need very expensive sugar
3–5
nucleotides as activated donors.
their use.
Furthermore, their high cost and low stability have greatly limited
The latter can also catalyse the formation of the glycosidic linkage not only via the reverse reaction
but also via the transferase activity. Due to their stability and low cost, these enzymes are very attractive
∗
Corresponding author. Tel: 33 (0)2 51 12 57 32; fax: 33 (0)2 51 12 57 32; e-mail: claude.rabiller@chimbio.univ-nantes.fr
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00297-9

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P. Spangenberg et al. / Tetrahedron: Asymmetry 10 (1999) 2905–2912
especially since they also induce a high stereoselectivity.6–9 Their main disadvantages come from their
rather low regioselectivity and the moderate yields usually obtained since the transferase activity remains
in competition with the hydrolysis of the substrate and of the glycosides synthesised. In addition, other
products may be synthesised since the autocondensation of the activated glycoside donor can occur.10 To
improve the yields in such reactions, suitable experimental conditions and/or new enzymatic activities
must be found which can enhance the concentration of the transglycosylation products at the expense
1
0,11
of the hydrolysis and can provide a greater regioselectivity.
We have shown, for instance, the
possibility of performing such enzymatic reactions in ice.11 In order to avoid the hydrolysis of the
glycosides produced, a very elegant method has also been developed by Withers et al. Their strategy,
which includes the directed mutation of β-glycosidases, gives very powerful enzymes producing high
12
yields of transglycosylation products.
Since transglycosylation reactions are under kinetic control, it is of great interest to know the time
when the highest concentration of a given disaccharide is present in the reaction medium in order to
quench the conversion before any significant hydrolysis takes place. The aim of this paper is to present
the conditions in which proton NMR spectroscopy allows the direct measurement of each component in
such reactions.
2. Results and discussion
Several analytical methods are available for this purpose, particularly chromatographical techniques.
For instance, the relative concentrations of all the compounds involved in the reaction described in
Scheme 1 can be measured by means of HPLC. Capillary electrophoresis also seems to be a powerful
13,14
method
for the kinetic study of such reactions. Another approach is proton NMR spectroscopy.
This technique has the advantage of giving signals where intensities are directly proportional to the
concentrations of the substances. Furthermore, this non-invasive spectroscopy enables the study of the
enzymatic reactions thus suppressing the possible side-effects arising from the taking of aliquots. In
addition, the resonance region between 4.0 and 6.0 ppm, despite being rather narrow, allows a clear
separation (at 500 MHz) of all the anomeric protons of the saccharides present in the reaction medium
(see Figs. 1 and 5) and the resolution is quite good although the enzyme is present in the solutions.
However, when carried out in an NMR tube, the course of the enzymatic reaction has to be studied at
a very high D O/H O ratio since the residual water protons present their resonance absorption around
2
2
4
.6–4.8 ppm. Thus, the question is whether the substitution of H O by heavy water exerts an influence on
2
the glycosidase activity and consequently on the rates of the reactions. In order to investigate the kinetics
of two reactions were first studied:
–
Reaction A: autocondensation of 2-nitrophenyl β-D-galactopyranoside (o-NP-β-Gal) in the presence
of a thermostable β-glycosidase from Thermus thermophilus (Tt β-gly), (Scheme 1).
Reaction B: autocondensation of 4-nitrophenyl α-D-galactopyranoside (p-NP-α-Gal) in the presence
of the α-galactosidase from green coffee beans (Scheme 2).
–
Each reaction was performed in the same conditions (temperature, concentration, enzymatic activity,
see experimental section) either in H O or in D O. In the first case, samples were taken of the reaction
2
2
mixture and analysed by means of NMR spectroscopy (after quenching the enzymatic activity by
adding 1 M NaOH) while in the second, the NMR tube was the reactor. The major kinetic compounds
obtained in these two already known reactions are the 2-nitrophenyl β-D-galactopyranosyl-[1→3]-
β-D-galactopyranoside 3β and the 4-nitrophenyl α-D-galactopyranosyl-[1→3]-α-D-galactopyranoside
10,15–17
3α
(Schemes 1 and 2). The resonances of all the anomeric α and β protons located between 4.2

P. Spangenberg et al. / Tetrahedron: Asymmetry 10 (1999) 2905–2912
2907
Scheme 1. Reaction A: autocondensation of the 2-nitrophenyl β-D-galactopyranoside catalysed by a β-glycosidase (Tt β-gly)
from the thermophilic species Thermus thermophilus
Scheme 2. Reaction B: autocondensation of the 4-nitrophenyl α-D-galactopyranoside catalysed by the α-galactosidase from
green coffee beans
and 6 ppm are sufficiently separated to allow the integration of the resonances corresponding to each
component as shown in Fig. 1.
The results of the kinetic studies are shown in Fig. 2 (reaction A) and Fig. 3 (reaction B). A comparison
between the kinetic profiles obtained in H O and D O indicates some differences. The most important
2
2
observation is that the hydrolytic activity of the glycosidases is enhanced in deuterium oxide while the
transglycosylation rates are reduced in this solvent. For instance, in the case of reaction A, after 1.75 h of
incubation, the galactose concentrations are 27 mM and 38 mM, respectively, in H O and D O while in
2
2
both cases very low amounts of the donor 1β remain. A similar effect is observed in the case of reaction
B: an incubation of 3–4 h produces galactose concentrations of 32 mM (in H O) and 41 mM (in D O).
2
2
In each reaction, the rate of hydrolysis of the major disaccharide is higher in deuterium oxide solutions.
As a consequence, the time at which the maximum concentrations for 3β or 3α obtained in D O is
2
higher than in H O and thus leads to lower concentrations of the disaccharides in deuterium oxide. This
2
is particularly true with reaction B where the highest concentrations in H O and in D O for 3α are 19.2
2
2
mM (within 2 h) and 13.4 mM (within 2.5 h), respectively. For reaction A, this effect is less important
because the rate of the transglycosylation reaction is very fast whatever the solvent. Thus, the highest
concentrations for compound 3β in D O and in H O are, respectively, 10.3 mM (1 h of incubation)
2
2
and 12.4 mM (0.5 h of incubation). These results show that proton NMR spectroscopy is a suitable

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P. Spangenberg et al. / Tetrahedron: Asymmetry 10 (1999) 2905–2912
1
Figure 1. Typical H NMR spectra (500 MHz) of the mixture of compounds in reaction A carried out in the NMR tube after 0.5
h of incubation (solvent D O, buffer pD 7). Only the anomeric region is shown
2
Figure 2. Kinetic study of reaction A at T=25°C. Left curves: in situ NMR experiment in D
the maximum concentrations for the disaccharide 3β are, respectively, 10.3 mM and 12.4 mM corresponding to yields of 36
and 43%)
2 2
O, right curves: reaction in H O
(
technique to use when following the course of the glycosidase transferase activity only if the rate of
transglycosylation is much faster than the rate of hydrolysis of the products which seems to be enhanced
in D O. This undesirable effect is highly reduced when the reactions are performed in the presence of
2
an excess of acceptor. For instance, the transglycosylation C was performed using 1α (1 equivalent) as
a donor and α-methyl galactoside (3 equivalents) as an acceptor in the presence of the α-galactosidase
from green coffee beans. Fig. 4 shows the kinetic profiles in H O and in D O. It is clear that the rate
2
2
of the reaction is once again less rapid in the latter solvent. The difference from the previous reactions
discussed in this work lies in the fact that the hydrolysis of the disaccharides bearing the methoxy group
at the anomeric position is much slower than that of the disaccharides substituted with a nitrophenoxy
group. As a result, after the same incubation time (5.3 h), the concentrations of the major disaccharide
3
α(X) are 33 mM in H O and 28 mM in D O while the concentrations of the remaining donor 1α were,
2
2
respectively, 8 mM and 18 mM (Fig. 4). In our laboratory, a systematic study of the reactions catalysed
by glycosidases
18,19
also indicated similar trends thus showing that in situ NMR can be used to optimise
such reactions, taking into account the fact that a small reduction in the rates of the reactions occurs in
the presence of D O.
2

P. Spangenberg et al. / Tetrahedron: Asymmetry 10 (1999) 2905–2912
2909
Figure 3. Kinetic study of reaction B at T=37°C. Left curves: in situ NMR experiment in D
2
O, right curves: reaction in H
2
O
(
the maximum concentrations for the disaccharide 3α are, respectively, 13.4 mM and 19.2 mM corresponding to yields of 30
and 43%)
2 2
Figure 4. Kinetic study of reaction C at T=37°C. Left curves: in situ NMR experiment in D O, right curves: reaction in H O.
3
α(X) is the methyl α-D-galactopyranosyl-[1→3]-α-D-galactopyranoside and 4α(X) the corresponding [1→4] regioisomer.
Small amounts of other disaccharides were also present
In order to exemplify the power of in situ NMR for studying these very complex transformations,
Fig. 5 shows the spectra of mixtures obtained with the above reaction catalysed by two different
α-galactosidases at T=25 and T=37°C. At the higher temperature, the water resonance moves to
high fields, thus allowing the analysis of a part of the spectrum completely masked by the water
resonance at 25°C. Incidentally, an advantage of NMR spectroscopy over other techniques is clearly
shown in the autocondensation experiments (reactions A and B) which can also be considered as the
enzymatic hydrolysis of the activated donors 1β and 1α. The corresponding phenol thus liberated
can be measured by means of visible spectroscopy. Usually, this technique is used to determine the
glycosidase activity of an enzymatic preparation. Obviously, vis is not suitable for this purpose since
significant autocondensation takes place and thus the concentration of phenol is not identical to the
concentration of nitrophenylgalactoside which has really been transformed, particularly at the beginning
of the transformation.

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P. Spangenberg et al. / Tetrahedron: Asymmetry 10 (1999) 2905–2912
Figure 5. Proton NMR spectra at 500 MHz (only the anomeric protons are shown) of mixtures obtained with reaction C (con-
densation of 4-nitrophenyl α-D-galactopyranoside 1α with methyl α-D-galactopyranoside) catalysed with the α-galactosidases
Raf A19 at 37°C (left spectrum) and Aga B at 25°C (right spectrum). Lines: a: represents the resonances of H-1 (3α and 4α),
19
0
0
0
b: H-1 (1α), c: H-1 (2α), d: H-1 (3α), f: H-1 [4α(X)], g: H-1 (α-methyl galactopyranoside), h: H-1 (4α), i: H-1 (2β). Other
disaccharides are also present
3. Experimental
3.1. General
The crude extract cells of E. coli cells (BL21pETBg8) in which the β-glycosidase from Ther-
mus thermophilus had been overexpressed, were used as a biocatalyst and prepared according to the
14
literature. The α-galactosidase from green coffee beans was purchased from Sigma. The chemicals
supplied by Aldrich were used without further purification. The α-galactosidases Aga B (from Bacillus
stearothermophilus) and Raf A (from E. coli) were supplied by Prof. R. Mattes (Institute of Industrial
Genetics, University of Stuttgart). The D O was purchased from Eurisotop (isotopic purity 99.9%).
2
1
13
The complete analysis of the NMR H and C resonances and subsequent structure assignment were
made using standard 2D sequences (COSY HH and HCOOR correlations) and by comparisons with
8
,10,11
previous data.
The spectra were recorded with a Bruker AX500 spectrometer operating at 500 MHz
1
for H (solvent D O) and chemical shifts in ppm quoted from the resonance of the methyl groups of
2
(trimethylsilyl)-3-propansulfonic acid was used as an internal reference.
3.2. Kinetic study of the autocondensation reaction of o-NP-β-Gal catalysed by Tt β-Gly (reaction A)
Solvent H O at 25°C: 6.1 mL of the phosphate buffer containing 12.1 mg (55.7 µM) of the reference
2
was prepared and adjusted to pH 7. In a separate experiment, 57 µL of the crude extract of E. coli
cells (BL21pETBg8) containing 1.8 units (calculated from the hydrolysis of o-NP-β-Gal at 25°C) of the
Thermus thermophilus β-glycosidase were concentrated overnight in a desiccator (P O ) and redissolved
2
5
in 0.6 mL of the buffer solution. To this solution, 110 mg (0.365 mmol) of 2-nitrophenyl β-D-galactoside
dissolved in 5.5 mL of the buffer and 0.35 mL of DMF (used as a co-solvent to dissolve all the
components of the mixture, particularly the large amounts of 2-nitrophenol synthesised at the end of
the experiment) was added. The solution was allowed to proceed at 25°C. Aliquots of 540 µL of the
reaction mixture were taken at different times. For each sample, the reaction was quenched by addition
of 15 µL of 1 M NaOH. The water was then removed under reduced pressure and the resulting mixture

P. Spangenberg et al. / Tetrahedron: Asymmetry 10 (1999) 2905–2912
2911
was dried in a desiccator (P O ). The powders obtained were redissolved in D O and the solution was
2
5
2
1
submitted to H NMR quantitative analysis.
Solvent D O at 25°C (kinetics in the NMR tube): 0.83 mL of 0.1 M, pH 7 phosphate buffer was
2
lyophilised, dissolved in 0.5 mL of D O, lyophilised once more and redissolved in 0.83 mL of D O
2
2
buffer. Then, to 0.5 mL of the D O buffer, 15 mg (50 µmol) of 2-nitrophenyl galactoside, 1.7 mg (7.6
2
µmol) of the reference (trimethylsilyl)-3-propansulfonic acid and 48 µL of DMF-d were added. The
6
presence of DMF was necessary to dissolve the phenol synthesised. In a separated experiment, 59.7 µL of
10
the crude extract of E. coli cells (BL21pETbg8) containing 0.25 units (calculated from the hydrolysis of
o-NP-β-Gal at 25°C) were concentrated overnight in a desiccator (P O ) and dissolved in the remaining
2
5
0.13 mL phosphate buffer at pD 7.0. This enzymatic solution was added to the reactant solution. The
resulting mixture was immediately filtered in an NMR tube and the reaction was allowed to proceed in
the probe of the magnet of the spectrometer at 25°C.
3
.3. Kinetic study of the autocondensation reaction of p-NP-α-Gal catalysed by α-galactosidase from
green coffee beans (reaction B)
Solvent H O at 37°C: 130 µL of the Sigma preparation of the α-galactosidase from green coffee beans
2
containing 15.7 units were dried in a desiccator containing P O . The resulting powder was redissolved
2
5
into 0.72 mL from the 8.72 mL of the 0.1 M phosphate buffer containing the silylated reference (9.16
mM). The pH of this solution was adjusted to 7 prior to the introduction of the enzyme. 4-Nitrophenyl-
β-D-galactoside (236.6 mg, 0.785 mmol) was redissolved in 8 mL of the above phosphate buffer and
the enzyme preparation was added. The reaction was allowed to proceed at 37°C. Aliquots of 360 µL
of the reaction mixture were taken off at different times. For each sample, the reaction was quenched by
addition of 10 µL of 1 M NaOH. The water was then removed under reduced pressure and the resulting
mixture was dried in a desiccator (P O ). The powders obtained were redissolved in D O and the solution
2
5
2
1
was submitted to H NMR quantitative analysis.
Solvent D O at 37°C (kinetic study in the NMR tube): 21 mg (70 µmol) of 4-nitrophenyl α-D-
2
galactoside and the amount of salts necessary for 0.78 mL from the 0.1 M (pH 7) phosphate buffer
were lyophilised, dissolved in D O and lyophilised once more. The buffer reference mixture containing
2
the silylated reference (0.78 mL) was prepared as above and adjusted to pD 7. The α-galactosidase from
green coffee beans (Sigma) was dried in a desiccator containing P O and redissolved in 0.18 mL of the
2
5
phosphate buffer reference mixture. The solutions were warmed to 37°C and the enzymatic preparation
was added to the reactant solution. The resulting mixture was immediately filtered in an NMR tube and
the reaction was allowed to proceed in the magnet of the spectrometer at 37°C.
3
.4. Kinetic study of the condensation reaction of p-NP-β-Gal with methyl α-D-galactopyranoside
catalysed by α-galactosidase from green coffee beans (reaction C)
The procedure and the amounts of all the components were identical to those used in the above
reactions apart from the presence of the α-methylgalactoside (3 equivalents for 1 of p-NP-α-Gal).

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P. Spangenberg et al. / Tetrahedron: Asymmetry 10 (1999) 2905–2912
Acknowledgements
Thanks are due to the French Ministry of Education and Research for a grant to one of us (V.C.G.) and
for financially supporting this work. R. Mattes (Institute of Industrial Genetics, University of Stuttgart)
is gratefully acknowledged for providing the α-galactosidases Raf A and Aga B.
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