Environmentally benign glycosylation of aryl pyranosides and aryl/alkyl furanosides demonstrating the versatility of thermostable CGTase from Thermoanaerobacterium sp.

Article abstract of DOI:10.1039/c4gc00631c

An extensive study on the specificity of transglycosylation and disproportionation of Thermoanaerobacterium sp. cyclodextrin glucosyltransferases against aryl glucopyranosides or furanosides is reported. While a mixture of maltoside and isomaltoside was obtained respectively using p-nitrophenyl glucopyranoside as an acceptor, only one regioisomer, namely p-nitrophenyl α-d-Glcp-(1,3)-α-l-Araf, was isolated using p-nitrophenyl arabinofuranoside as an acceptor. Interestingly, similar outcomes were found when using p-nitrophenyl galactofuranoside. Furthermore, activation by microwave irradiation resulted in faster reaction times and higher yields and led to glucosidic oligosaccharides with up to 70% conversion. The influence of the anomeric and C-4 configurations of the glycosidic acceptors on the transglycosylation, previously stated for the CGTase family, was not observed and unconventional substrate specificity towards alkyl furanosides was highlighted. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4gc00631c

Green Chemistry
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Environmentally benign glycosylation of aryl
pyranosides and aryl/alkyl furanosides
demonstrating the versatility of thermostable
CGTase from Thermoanaerobacterium sp.†
Cite this: Green Chem., 2014, 16,
803
3
a,b
a,b
c
a,b
Alizé Pennec, Laurent Legentil,* Luis Herrera-Estrella, Vincent Ferrières,
c
a,b
Anne-Laure Chauvin* and Caroline Nugier-Chauvin
An extensive study on the specificity of transglycosylation and disproportionation of Thermoanaero-
bacterium sp. cyclodextrin glucosyltransferases against aryl glucopyranosides or furanosides is reported.
While a mixture of maltoside and isomaltoside was obtained respectively using p-nitrophenyl glucopyra-
noside as an acceptor, only one regioisomer, namely p-nitrophenyl α-D-Glcp-(1,3)-α-L-Araf, was isolated
using p-nitrophenyl arabinofuranoside as an acceptor. Interestingly, similar outcomes were found when
using p-nitrophenyl galactofuranoside. Furthermore, activation by microwave irradiation resulted in faster
reaction times and higher yields and led to glucosidic oligosaccharides with up to 70% conversion. The
influence of the anomeric and C-4 configurations of the glycosidic acceptors on the transglycosylation,
previously stated for the CGTase family, was not observed and unconventional substrate specificity
towards alkyl furanosides was highlighted.
Received 8th April 2014,
Accepted 14th May 2014
DOI: 10.1039/c4gc00631c
www.rsc.org/greenchem
side acceptors and in particular the selective formation of
alkyl glucopyranosyl-furanoside conjugates by enzymatic
Introduction
Glycoconjugates incorporating rare furanosides are nowadays means have still to be developed. Such a scaffold can be found
8
gaining momentum for detergent applications and also as new in the exopolysaccharide expressed by Lactobacillus rhamnosus
9
therapeutics. Indeed, while alkyl C-glycofuranosides were and some strains of Streptococcus thermophilus or as a disac-
sought for their amphiphilic properties, oligomers of L-arabino- charide unit grafted onto some natural flavonoids. Such
furanose were described as potential immunomodulators as architectures found applications in the food industry as
1
10
they stimulate the secretion of various cytokines by macro- thickening agents for example.
2
phages. Furthermore, alkyl derivatives like octyl β-D-galacto-
Intermolecular transglycosylation catalyzed by cost-effective
3
–5
furanoside have exhibited mycobacteriostatic properties and catalysts is a key process for the synthesis of high value carbo-
are detrimental for the survival of Leishmania donovani and hydrate-based substrates from donors extracted from biomass.
6
infection of macrophages by the parasite. Recently, we Within the available catalysts, cyclodextrin glucosyltransferase
described the access to such monosaccharides from renewable enzymes (CGtase, E.C. 2.4.1.19) are starch degrading enzymes
resources thanks to one-pot biocatalyzed furanosylation of belonging to the important family of endo-glycosyl trans-
1
1
various alcohols in the presence of an arabinofuranosidase of ferases. Further hydrolysing starch [α-1,4-hydrolysis], the
7
the GH 51 family. Such a strategy used furanosyl donors for enzyme also catalyzes linear oligosaccharide disproportiona-
transglycosylation. However, the transglycosylation on furano- tion as well as cyclic and linear dextrin coupling, depending
1
2
on the bacterial source involved (Fig. 1).
Substrate specificity of CGtases has been extensively
studied and demonstrated the efficiency of CGtases to trans-
glycosylate several glucosyl units onto analogs of glucopyrano-
a
Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, 11 allée de
Beaulieu, CS 50837, 35708 Rennes Cedex 7, France.
E-mail: laurent.legentil@ensc-rennes.fr; Tel: +33 223 238 140
1
3
b
side. Additionally, it was highlighted that derivatization of
the acceptor at C-1 did not exhibit significant differences in
activity whereas changing the configuration of the C-2 and
C-4 hydroxyl groups resulted in serious loss of the CGTase
Université européenne de Bretagne,
c
Laboratorio Nacional de Genómica para la Biodiversidad (Langebio),
CINVESTAV-IPN, Km 9.6 Lib. Nte, Carr. Irapuato – León, CP36821, Irapuato,
México. Fax: +52 462 607 8276; Tel: +52 462 166 3000
†
Electronic supplementary information (ESI) available: Experimental details,
1
4
1
13
efficiency. Nakamura et al. showed also by analogy with
α-amylase that a pNP group on the anomeric position of the
analytical data as well as H and C NMR data for all new compounds. See DOI:
0.1039/c4gc00631c
1
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considered (Fig. 2). The method was eventually extended to
alkyl furanosides 5 to 11 as acceptors owing to their potential
as therapeutics and surfactants.
Results and discussion
To evaluate the scope of glucosyl transfer, the reaction was
first performed with UV-active pyranosides and furanosides 1
to 4 (Fig. 2). Recently, Yoon and Robyt demonstrated that the
acceptor/donor ratio influences the degree of polymerization
(
DP) of the oligoglucanes generated and that a ratio higher
than 3 favoured transglycosylation/disproportionation reaction
1
7
over cyclization. Consequently, all acceptors 1 to 4 and
β-cyclodextrin, in a ratio of 7/2, were incubated for 72 hours in
5
0 mM phosphate buffer pH 6 at 50 °C in the presence of
−
1
Toruzyme® 3.0 L (0.05 mg mL ). These conditions were
described as optimum for CGTase glucosylation.
1
8
Fig. 1 Reactions catalyzed by cyclodextrin glucosyltransferases.
After 72 hours, the reaction mixture was analyzed by liquid
chromatography coupled to mass spectrometry (LC-MS). For
each substrate, the chromatogram showed a succession of
peaks with decreasing intensities (see ESI†). The peaks with
the shortest retention times correspond to the remaining start-
ing pNP glycosides according to mass analysis (peak at
1
5
acceptor is not detrimental to the catalysis. Finally, trans-
glycosylation on dodecyl maltoside was recently performed
and allowed the access to alkyl oligoglucosides with non-ionic
1
6
surfactant properties. Such results demonstrated the high
versatility of the CGTase family towards various acceptors.
Nevertheless its potential for glucosylation of alkyl furanosides
has still to be evaluated.
3.80 min for 1, 3.64 min for 2, 3.38 min for 3 and 3.22 min for
4
). The MS spectra also indicated a mass difference of 162
between each major consecutive peak which is characteristic
of a molecule of glucose minus one molecule of water. This
confirmed the successive transfer of one glucosyl unit to the
Thus, in this study the intermolecular transglycosylation
and disproportionation reactions of the CGTase of Thermo-
anaerobacterium sp. (EC 2.4.1.19) or Toruzyme® 3.0 L, against
pyranosidic and furanosidic acceptors, were compared. In
addition, conventional and microwave-assisted reaction (MAR)
conditions were evaluated with both substrate types. To
achieve this goal, readily available, low cost and bearing a
chromogenic anomeric group compounds, p-nitrophenyl α/β-D-
glucopyranoside (pNP α-Glcp 1 and pNP β-Glcp 2) and furano-
side targets, p-nitrophenyl α-L-arabinofuranoside (pNP α-Araf
n
pNP glycoside backbone to form pNP Glcp -glycoside. Further-
more, with compound 1, an additional signal at m/z 139
corresponding to the p-nitrophenol group was present. It is
most likely due to the hydrolysis of compound 1 or its use as a
donor by the CGTase. Indeed 1 had the same α-configuration
as cyclodextrin or malto-oligosaccharides, both substrates of
1
9
CGTase.
Integration of relevant peaks in the chromatograms led to
estimation of the percentage of the remaining starting accep-
tors, the oligosaccharides formed and the subsequent hydro-
lysis (Table 1). Overall transglycosylation reached 67, 89 and
3
) and p-nitrophenyl β-D-galactofuranoside (pNP β-Galf 4), were
59% with acceptors 2, 3 and 4, respectively (entries 2 to 4), but
only 23% with 1 (entry 1). These data corroborated the signifi-
cant hydrolysis of the α-D-glycosidic bond (55%) by the
CGTase. Nevertheless, oligomers with a degree of polymeriz-
ation up to 10 could be detected in the reaction mixture what-
ever the nature of the substrate. The di- and trisaccharides
were the main products from the reaction, corresponding to
the transfer of one or two glucosyl entities from the cyclo-
dextrin. It highlighted the prevalence of transglycosylation
over disproportionation.
To confirm these initial results, the kinetic profiles of the
enzymatic reactions with each substrate were performed
(
Fig. 3). As expected, the plots showed a decrease in the
amount of acceptor over time. The pyranosidic substrates were
rapidly consumed after only 2 hours whereas less than 40% of
pNP Araf 3 and 25% of pNP Galf 4 were glycosylated after 5 h
Fig. 2 p-Nitrophenyl and alkyl glycosides used as acceptors.
3804 | Green Chem., 2014, 16, 3803–3809
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Table 1 Conversion yield of the reaction between cyclodextrin and pNP α/β-glycoside in the presence of CGTase from Thermoanaerobacterium sp.
of conversion
%
Remaining
acceptor (%)
Hydrolysis
(%)
trans-
Glycosylation (%)
pNP Glcp-
glycoside
pNP Glcp
glycoside
2
-
pNP Glcp
glycoside
3
-
4
pNP Glcp -
glycoside
Entry
Acceptors
1
2
3
4
pNP α-D-Glcp (1)
pNP β-D-Glcp (2)
pNP α-D-Araf (3)
pNP β-D-Galf (4)
22
28
11
40
55
0
0
23
67
89
59
10
36
41
32
7
18
21
16
6
9
13
7
Trace
4
9
4
1
Fig. 3 Kinetic profile of the reaction catalyzed by CGTase between cyclodextrin and 1 (A), 2 (B) 3 (C) and 4 (D).
indicating a slower reaction rate for the furanosidic vs. pyrano-
The main products of all reactions were isolated for identifi-
sidic acceptors. When 1 was used as an acceptor, the trans- cation. In the chromatograms of the reaction on pNP β-Glcp 2,
glycosylation rate reached a maximum after just 2 h, and then the presence of a minor peak at 7.43 minutes next to the
the amount of all resulting oligosaccharides decreased over major peak at 6.64 minutes was pointed out. Both peaks had a
2
time. Again, this was proof that the pNP α-D-Glcp and the mass of 463 corresponding to a disaccharide (pNP β-Glcp ).
resulting pNP oligosides were acceptors and donors of the After isolation and NMR analysis, it was established that the
CGTase. With 2, 3 and 4 as acceptors, the amount of all reac- peak at 6.64 min coincided with the pNP β-maltoside 12
tion products reached a steady state after 30 h and the global (anomeric proton at 5.21 and 5.09 ppm and anomeric carbons
1
13
conversion remained stable. Therefore, the resulting pNP at 102.9 and 102.7 ppm in the H and C NMR spectra,
2
0,21
glycosides were not consumed during the course of the reac- respectively)
and the peak at 7.43 minutes resulted from
tion and thus are not themselves donors for the CGTase. the transglycosylation of a glucose unit in an α-1,6-fashion pro-
Furthermore such a plateau is characteristic of an acceptor/ ducing the pNP β-isomaltoside 13 (Fig. 4) as confirmed by the
donor ratio below 3 where cyclization of linear maltosides peaks at 5.05 and 4.80 ppm, and at 101.7 and 100.7 ppm in
1
7
1
13
22
back to cyclodextrin is favoured over transglycosylation. The the H and C NMR spectra, respectively. The formation of
furanosides 3 and 4 demonstrated a good response to trans- the isomaltoside was also noticeable in the HPLC chromato-
glycosylation, and the sugar cyclic size did not affect the reac- gram of the reaction on pNP α-Glcp 1 but to a lesser extent. As
tion yield. This result confirmed the large reaction scope of for the pNP arabinoside acceptor 3, fractions containing pNP
the CGTase towards pento- and hexofuranosidic acceptors.
Glcp-Araf and pNP Glcp -Araf were isolated after reaction on a
2
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α-D-Glcp-(1,4)-α-D-Glcp-(1,3)-β-D-Galf 17 were identified as the
major products of the glucosylation of pNP Galf (Fig. 4). Here
the yields were rather low and other regioisomers [pNP α-D-
Glcp-(1,6)-β-D-Galf principally] were detected. In conclusion,
the CGTase action on furanosides was slower than on pyrano-
sides but the regioselectivity for α-(1,3) ligation was higher in
particular with pNP Araf. However, substitution at C-5 of the
furanose led to a drop in the transglycosylation yield and the
regioselectivity.
In order to further optimize the action of the CGTase onto
the substrates, microwave activation was attempted to (i) accel-
erate the reaction and (ii) improve the transglycosylation
2
3
yields. Such a protocol should not degrade the enzyme as
CGTase of Thermoanaerobacterium sp. has the highest thermo-
2
4,25
stability within the CGTase family.
The initial investigation
of the microwave radiation (MAR) activation was performed on
pNP α-Glcp 1, and later extended to the other glycosides in
order to investigate the effect of the anomeric configuration
and the ring size (Table 2). Firstly, the microwave assisted reac-
tion, by drastically reducing the hydrolysis of pNP α-Glcp 1
Fig. 4 Isolated products and subsequent yields.
preparative scale. While the chromatogram also showed an otherwise observed, increased the transglycosylation yield
additional peak at the base of the major one, both isolated from 23% up to 76% with a reaction time of 10 seconds
fractions contained only one regioisomer. The isolated disac- instead of 48 hours (entries 1 and 2). Secondly, the amount of
charide presented an anomeric proton at 5.71 ppm associated products resulting from the transfer of 1, 2 or 3 glucosyl resi-
with a carbon at 107.7 ppm. The coupling constant JH1,H2 dues to 1 was almost tripled (29, 24 and 15% compared to 10,
reaches 1.2 Hz, a typical value for an anomeric proton of a fur- 7 and 6%, respectively). Furthermore, disproportionation reac-
anoside in the 1,2-trans configuration. The second anomeric tion was also affected as the ratio disaccharides/tri- and tetra-
position showed a proton at 4.96 ppm and a carbon at saccharides decreased under MAR conditions. In addition, by
1
01.8 ppm representative of a pyranosidic anomeric position increasing the MAR power from 500 W (entry 2) to 1575 W
with a JH1–H2 of 4 Hz characteristic of a glycopyranosidic bond (entry 4), no notable variation in the transglycosylation yield
1
in the 1,2-cis configuration. 2D- H NMR HMBC experiments was detected. The CGTase of Thermoanaerobacterium sp. was
p f
exhibited also a long range correlation between H -1 and C
-3 denatured above 1575 W microwave power as only starting
indicating a (1,3)-glycosidic bond (see ESI†). These data materials were detected. Finally, by raising the reaction time
pointed unambiguously towards pNP α-D-Glcp-(1,3)-α-L-Araf 14 from 10 seconds to 30 seconds, the transglycosylation yields
as the sole disaccharide isolated from the action of CGTase on dropped from 74% to 52% (entry 3). The same conditions were
3
. Similarly, characterisation of the fraction corresponding to applied with the other pNP glycosides. With the β-anomer 2,
pNP Glcp -Araf allowed the identification of the trisaccharide the transglycosylation yield was slightly improved and again
2
pNP α-D-Glcp-(1,4)-α-D-Glcp-(1,3)-α-L-Araf 15 as the unique con- the tri- and tetrasaccharide amounts were higher (entries 5 to
stituent of this fraction. Isolated yields reach 17% for the dis- 7). Surprisingly no by-products resulting from the transglycosy-
accharide and 10% for the trisaccharide. Those values were lation of a glucosyl unit in an α-1,6-fashion were observed. As
slightly higher than the ones obtained after the action of the for the pNP furanosides 3 and 4, no improvement in terms of
CGTase on 2. Similarly, pNP α-D-Glcp-(1,3)-β-D-Galf 16 and pNP conversion and selectivity was observed. Those findings estab-
Table 2 Microwave-power effect on the transglycosylation yields of pNP glycosides with the CGTase, over a 10 s period
Conversion (%)
Microwave
power (W)
trans-Glycosylation
(%)
Hydrolysis
(%)
Entry
Acceptor
pNP Glcp
2
pNP Glcp
3
pNP Glcp
4
pNP Glcp
5
1
2
3
4
5
6
7
pNP α-Glcp 1
—
500
500
1575
—
500
1575
23
74
52
78
67
80
78
55
0
0
0
0
0
0
10
29
nd
28
36
30
28
7
24
nd
25
18
24
24
6
15
nd
17
9
17
17
Trace
6
nd
8
4
9
9
a
b
b
b
b
pNP β-Glcp 2
a
b
Reaction performed over a 30 second period. nd: not determined.
3806 | Green Chem., 2014, 16, 3803–3809
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Table 3 Extension of the glucosylation catalyzed by CGTase to alkyl furanosides
Products (yield, %)
1
2
Entry
Series
Alkyl furanoside
R
R
Conversion (%)
n = 0
n = 1
1
2
3
4
5
6
7
α-D-Araf
5
6
7
8
9
10
11
H
H
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
68
62
73
82
88
58
16
18 (14)
19 (25)
20 (21)
21 (19)
22 (19)
23 (25)
24 (5)
25 (11)
26 (14)
27 (13)
28 (12)
29 (11)
30 (10)
31 (3)
CH
2
(CH
(CH
(CH
(CH
(CH
2
2
2
2
2
)
2
)
3
)
4
)
5
)
7
β-D-Galf
2
CH OH
lished that microwave-assisted enzymatic reaction did not isolate and identify the different glucosylation products
modify the selectivity and the substrate specificity of the (Table 3). Only the di- and trisaccharides were characterized
CGTase Therm. It enabled the successful synthesis of both thanks to NMR experiments. With alkyl arabinofuranosides
targets pNP glucosides in shorter time and higher yields and 11–16, similar isolated yields than with pNP substrates were
thus would provide a practical synthetic method for the gluco- obtained. The di- and tri-saccharides represented an average of
sylation of specific maltoside derivatives. It also demonstrated 30% of the oligosaccharides and were thus the major products
the importance of microwave activation in reducing the pres- of the reaction. The nature of the glycosidic bond was eluci-
ence of 1,6-linked byproducts. In addition, this process due to dated thanks to HMBC experiments. As previously stated, the
the sole use of aqueous solvents and no hazardous substances α-(1,3) bond between the first glucosyl unit and the furanosyl
can be considered environmentally friendly.
residue was favoured. In contrast, the reaction with octyl Galf
Finally, the replacement of anomeric nitrophenoxy by an 17 as an acceptor yielded a lower amount of di- and trisacchar-
alkoxy chain on Araf and Galf could allow the biocatalyzed syn- ides confirming the lower affinity of the galactofuranoside for
thesis of relevant alkyl oligosaccharides with both pyranose the enzyme. Anyway, in both cases, in only two steps and
and furanose rings. It would provide an attractive alternative to without protecting group manipulation, oligosaccharides
the chemical synthesis of the α-D-Glcp-(1,3)-furanoside bond incorporating an α-D-Glcp-(1,3)-furanoside link could be
2
6
often tricky to obtain selectively. Alkyl arabino- and galacto- obtained. Such a pattern could be found for example as part of
furanosides, having a chain length varying from methyl to the exopolysaccharide expressed by some strains of Streptococ-
2
7
hexyl for the arabinofuranoside and octyl for the galacto- cus pneumonia. or associated with triterpenoidal saponin
2
8
furanoside, were thus considered as acceptors (Table 3). They extracted from Meryta denhamii seem. This last compound
were readily obtained following a known procedure by glycosy- showed some antihyperglycemic effect and hence demon-
lation of arabinan in the presence of an arabinofuranosidase strated the therapeutic potential of those scaffolds.
7
and a suitable alcohol. Alkyl furanosides and cyclodextrin
were incubated in the presence of Toruzyme® 3.0 L for
7
2 hours according to the protocol previously established. The
Conclusions
resulting crude mixture was analyzed by UPLC-MS spec-
−
trometry. [M − H] masses corresponding to the different pro- In conclusion, this paper reported an extensive study of the
ducts of transglycosylation were deduced from the mass versatility of CGTase Therm. against acceptors having either a
spectrum allowing the neat visualization of glycoconjugates pyranosyl or a furanosyl conformation. Our methodology
with up to 4 residues of glucose on the alkyl furanoside (see enabled the enzymatic transglycosylation reaction of a glucosyl
ESI†). However, a certain amount of the starting material unit onto a range of carbohydrate acceptors using readily avail-
remained but no hydrolysis could be observed. So the tested able donors and biocatalysts, in water and under microwave
alkyl furanosides were confirmed as acceptors of the CGTase irradiation, following an environmentally benign procedure. It
even if they did not bear an aromatic ring at the anomeric demonstrated that the enzyme conserved its catalytic activity
position. Preparative scale-up was implemented in order to independently of the substrate form. Additionally, the appli-
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cation of microwave irradiation showed a positive effect on 1 : 1) from 90 : 5 to 60 : 40. Elution of the products was moni-
yields, reaction time and byproduct formation without de- tored by UV detection and by TLC. Fractions corresponding
naturing the enzyme when pNP pyranoside was used as a sub- to individual products were collected, evaporated and
strate. Interestingly our methodology enabled the enzymatic lyophilized.
transglycosylation reaction of a glucosyl unit onto a range of
alkyl furanoside acceptors to selectively obtain alkyl α-Glcp-
(
1,3)-Araf or Galf. Such scaffolds need yet to be evaluated for
Acknowledgements
potential surfactant properties or as bacteriostatics.
We are grateful to Novozymes A/S (Bagvaerd, Denmark) for the
gift or Toruzyme® 3.0 L, to Rennes Metropole for a mobility
fellowship to A. P. and to “Région Bretagne” and CNRS for
financial support.
Experimental section
Materials
All reagents were purchased from commercial sources and
were used without further purification unless noted. Thermo-
anaerobacter sp. CGTase (Toruzyme® 3.0 L) was a gift from
Novozymes A/S (Bagvaerd, Denmark).
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−
1
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