Synthesis of l-rhamnose and n-acetyl-d-glucosamine derivatives entering in the composition of bacterial polysaccharides by use of glucansucrases

Article abstract of DOI:10.1080/07328300902755796

Transglucosylation reactions using sucrose as glucosyl donor and either N-acetyl-D-glucosamine, L-rhamnose, or methyl -L-rhamnopyranoside as acceptors were carried out with recombinant glucansucrases from families 70 and 13 of glycoside-hydrolases. Depend

Full text of DOI:10.1080/07328300902755796

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Synthesis of L-Rhamnose and N -
Acetyl-D-Glucosamine Derivatives
Entering in the Composition of
Bacterial Polysaccharides by Use of
Glucansucrases
a b c
a b c
d
Elise Champion
, Isabelle André
, Laurence A. Mulard ,
a b c e
a b c
a
Pierre Monsan
, Magali Remaud-Siméon
& Sandrine Morel
b c
a
Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de
Rangueil, F-31077, Toulouse, France
b
CNRS, UMR5504, F-31400, Toulouse, France
c
INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés,
F-31400, Toulouse, France
d
Institut Pasteur, Unité de Chimie des Biomolécules, CNRS URA
2
128, 28 rue du Dr. Roux, F-75015, Paris, France
e
Institut Universitaire de France, 103 Boulevard Saint-Michel,
F-75005, Paris, France
Published online: 01 Apr 2009.
To cite this article: Elise Champion , Isabelle André , Laurence A. Mulard , Pierre Monsan , Magali
Remaud-Siméon & Sandrine Morel (2009): Synthesis of L-Rhamnose and N -Acetyl-D-Glucosamine
Derivatives Entering in the Composition of Bacterial Polysaccharides by Use of Glucansucrases,
Journal of Carbohydrate Chemistry, 28:3, 142-160
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Journal of Carbohydrate Chemistry, 28:142–160, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328300902755796
Synthesis of L-Rhamnose
and N-Acetyl-D-Glucosamine
Derivatives Entering in the
Composition of Bacterial
Polysaccharides by Use
of Glucansucrases
1
1
2
Elise Champion, Isabelle Andr e´ , Laurence A. Mulard, Pierre
3
1
1
Monsan, Magali Remaud-Sim e´ on, and Sandrine Morel
1Universit e´ de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077
Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France; and INRA, UMR792
Ing e´ nierie des Syst e` mes Biologiques et des Proc e´ d e´ s, F-31400 Toulouse, France
2
Institut Pasteur, Unit e´ de Chimie des Biomol e´ cules, CNRS URA 2128, 28 rue du Dr.
Roux, F-75015 Paris, France
3
Universit e´ de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-
1077 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France; INRA, UMR792
3
Ing e´ nierie des Syst e` mes Biologiques et des Proc e´ d e´ s, F-31400 Toulouse, France; and
Institut Universitaire de France, 103 Boulevard Saint-Michel, F-75005 Paris, France
Transglucosylation reactions using sucrose as glucosyl donor and either N-acetyl-D-
glucosamine, L-rhamnose, or methyl α-L-rhamnopyranoside as acceptors were car-
ried out with recombinant glucansucrases from families 70 and 13 of glycoside-
hydrolases. Depending on the enzyme specificity, various carbohydrate structures
were synthesized and characterized including α-D-glucopyranosyl-(1→6)-N-acetyl-D-
glucosamine, α-D-glucopyranosyl-(1→4)-N-acetyl-D-glucosamine, α-D-glucopyranosyl-
(
1→1)-β-L-rhamnopyranoside, α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→1)-
β-L-rhamnopyranoside, methyl α-D-glucopyranosyl-(1→4)-α-L-rhamnopyranoside, and
methyl α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside. Disaccharides were ob-
tained with yields going up to 64%. The structural diversity generated as well as the
obtained yields appear to be related to enzyme active site architecture, which can be
modulated and improved by enzyme engineering. Several of the obtained disaccharides
Received November 13, 2008; accepted January 16, 2009.
Address correspondence to Sandrine Morel, Universit e´ de Toulouse, INSA, UPS, INP,
LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; CNRS, UMR5504, F-31400
Toulouse, France; and INRA, UMR792 Ing e´ nierie des Syst e` mes Biologiques et des
Proc e´ d e´ s, F-31400 Toulouse, France. E-mail: sandrine.morel@insa-toulouse.fr
1
42

Synthesis of L-rhamnose and N-acetyl-D-glucosamine Derivatives
1
43
enter in the composition of surface polysaccharides of pathogenic bacteria, among which
is Shigella flexneri. Our results outline the potential of glucansucrases in the chemo-
enzymatic synthesis of complex carbohydrates of biological interest whose chemical
synthesis may be seen as a limitation.
Keywords
Glucansucrase, Enzymatic glucosylation, L-Rhamnose, N-Acetyl-D-
glucosamine, Bacterial polysaccharide
INTRODUCTION
Two categories of natural enzymes catalyze glycosyl transfer onto sugar
acceptors: glycosyltransferases of the Leloir pathway, which use expensive
nucleotide-activated sugars as glycosyl donor, and transglycosidases, often as-
sociated with low yields of the glycosylated targets. For both types, reactivity
toward acceptors is strictly controlled by the enzyme selectivity and specificity.
In this regard, glucansucrases (GS, EC 2.4.1), which are transglycosidases
found in glycoside hydrolases (GH) families 13 and 70 according to the CAZY
[
1]
classification, are very attractive synthetic tools. They naturally catalyze
glucan synthesis and the concomitant release of fructose from sucrose donor, a
largely available and low-cost substrate. The type, number, and organization of
the glucosidic linkages displayed in the glucan polymer is strongly dependant
[
2–10]
on glucansucrase specificities.
An interesting particularity of glucansu-
crases is their ability to catalyze the glucosylation of nonnatural acceptors
(Fig. 1). Glucansucrases are indeed able to transfer glucosyl residues onto
Figure 1: Reactions catalyzed by glucansucrases. G: glucosyl; F: fructosyl or fructose;
AccOH: nonnatural acceptor.

44 E. Champion et al.
1
a large variety of acceptors, including sugar[11–18] and nonsugar[11,16,19–24]
molecules. In this process, acceptor recognition, glucosylation yields, and re-
sulting products are strongly dependent on glucansucrase stereo- and re-
giospecificities.
In the present study, we have investigated the glucosylation of commer-
cially available N-acetyl-D-glucosamine and L-rhamnose, as well as of methyl
[
25,26]
α-L rhamnopyranoside,
prepared in one step from the latter, by use of sev-
eral recombinant or native glucansucrases, displaying distinct specificities (Ta-
ble 1). The interest of using enzymatically produced disaccharides as starting
building blocks for the synthesis of fragments of the O-antigen part of various
Shigella flexneri lipopolysaccharides is highlighted.
RESULTS AND DISCUSSION
Glucansucrases are α-retaining transglucosidases that follow a double dis-
placement mechanism, in which a β-D-glucosyl enzyme covalent intermedi-
ate is first formed from sucrose substrate.[
27–29]
In a second step, the glucosyl
moiety is transferred to an acceptor, which can be either (1) water leading to
the production of glucose, (2) fructose to form sucrose isomers, or (3) the glu-
cose moiety released from sucrose hydrolysis to form soluble oligosaccharides
and α-glucans. The major reaction of these enzymes is the synthesis of glucan
polymer. However, an exogenous hydroxylated acceptor well recognized by the
enzyme may be glucosylated at the detriment of polymer synthesis (reaction
4
in Fig. 1). Depending on the enzyme regiospecificity, distinct types of gluco-
sidic linkage are found in the formed polymer or in the nonnatural glucosyl
derivative (Fig. 1).
In order to take advantage of the ability of glucansucrases to glucosylate
nonnatural acceptors in a regio- and stereospecific manner, five recombinant
glucansucrases were tested for the glucosylation of N-acetyl-D-glucosamine
(
D-GlcpNAc), L-rhamnose (L-Rhap), and methyl α-L-rhamnopyranoside (α-L-
RhapOMe). As shown in Table 1, these enzymes catalyze the synthesis of di-
verse types of glucosidic linkage and were chosen on purpose to attempt the
synthesis of structurally distinct disaccharides. They correspond to engineered
forms of the dextransucrase from Leuconostoc mesenteroides NRRL B-512F
[
30,31]
(
DSR-S1, DSR-S2, and DSR-S3),
the truncated alternansucrase from L.
and the amylosucrase from Neisseria
[
32]
mesenteroides NRRL B-1355 (ASR),
[
7]
polysaccharea (AS). Notably, mutant enzymes DSR-S2 and DSR-S3 carry mu-
tations in the region downstream the second aspartic acid of the catalytic triad,
known to participate in the distortion of the glucosyl enzyme and in the posi-
tioning of the acceptor, which could potentially alter the enzyme stereo- and
[
31]
regiospecificity.

L.
L.
ie
ides
o
o
ployschrea
m
n
e
msnter
N.
p
p
p
1
45

46 E. Champion et al.
1
Figure 2: Analysis of D-GlcpNAc glucosylation by four different GH70 glucansucrases.
Comparison of HPLC chromatograms (with UV
detection) at the starting time of the
λ=220nm
ꢁ
reaction (t ) and after total sucrose consumption (t =24h); P1 and P1 : acceptor reaction
0
f
products.
Glucosylation of N-acetyl-D-glucosamine
Enzyme Screening
Acceptor reactions were first carried out using sucrose as donor and D-
GlcpNAc as acceptor in equimolar ratio. The HPLC profiles of the acceptor
reaction products synthesized by DSR-S1, its two variants (DSR-S2and DSR-
S3),and ASR were all highly similar (Fig. 2). It can thus be assumed that all
tested GH70 glucansucrases produced the same compound, herein called P1
(
tR = 8.2 min). Interestingly, the main acceptor reaction product obtained by
action of AS, product P2 (tR = 8 min), displayed a retention time slightly dif-
fering from that of P1, indicating that a structurally distinct product was syn-
thesized by transglucosylation (Fig. 3). Other glucosylation products were de-
ꢁ
ꢁ
tected (P1 and P2 in Fig. 2 and 3, respectively), although in lower amounts
that did not allow further characterization.
Structural Characterization
The structure of P1, which was synthesized using DSR-S2mutant, was an-
alyzed by HRMS and NMR (supporting materials 1 and 5; details given in
Experimental section).
HRMS analysis of P1 indicated a molecular weight of 383 Da, correspond-
1
ing to a monoglucosylated form of D-GlcpNAc.The H NMR spectrum of P1
shows two doublets at 4.87 ppm and 4.88 ppm, assigned to the anomeric proton

Synthesis of L-rhamnose and N-acetyl-D-glucosamine Derivatives
1
47
Figure 3: Analysis of D-GlcpNAc glucosylation by GH13 glucansucrase, amylosucrase from
N. polysaccharea. Comparison of HPLC chromatograms (with UV
detection) at the
λ=220nm
ꢁ
starting time of the reaction (t ) and after total sucrose consumption (t f = 24 h); P2 and P2 :
0
acceptor reaction products.
of the D-glucosyl residue, and two doublets at 4.66 ppm and 5.13 ppm, which
by comparison with the D-GlcpNAc spectrum were assigned to the anomeric
proton of β- and α-D-GlcpNAc, respectively. Signal integration indicates a dis-
accharide structure in good agreement with MS data. Moreover, both H1Glc
doublets exhibit weak J1−2coupling constants (3.4 and 3.5 Hz), which are char-
acteristic of an α-D-glucosidic linkage, in agreement with the known stere-
ospecificity of the DSR-S2mutant. Besides, H-1Glc:C-6GlcNAcand H-6GlcNAc:C-1Glc
cross-peaks in the HMBC spectrum ascertain the presence of an α-(1→6) link-
1
13
age. All H and C signal assignments are listed in the Experimental section.
From these assignments, it can be concluded that P1 is a new disaccharide
corresponding to α-D-glucopyranosyl-(1→6)-N-acetyl-D-glucosamine (Fig. 4).
Analogously, with a molecular weight of 383 Da determined by HRMS
analysis, P2 is also a disaccharide (supporting materials 2 and 6; details given
in Experimental section). Moreover, NMR analysis indicates two major differ-
ences in the 13C spectra of P2 in comparison to that of P1. Differences in-
volve δC6, lowered by 4.5 ppm, and δC4, which is increased by at least 7.2 ppm.
Taking into account the H-1Glc:C-4GlcpNAc and H-4GlcpNAc:C-1Glc cross-peaks in
the HMBC spectrum, all data converge to suggest that P2 corresponds to
the yet unknown regioisomer, namely α-D-glucopyranosyl-(1→4)-N-acetyl-D-
glucosamine (Fig. 4).
Glucosylation Reaction
As shown in Table 2, for all tested enzymes, D-GlcpNAc conversion de-
grees were composed between 6% and 64% with yields of monoglucosylated
D-GlcpNAc products ranging from 4% to 61% based on quantitative HPLC

48 E. Champion et al.
1
Figure 4: Structure of disaccharides P1 and P2 obtained by glucansucrase-mediated
glucosylation of N-acetyl-D-glucosamine.
analysis. Acceptor glucosylation, which is in competition with polymer synthe-
sis, is thus highly dependent on acceptor recognition by the enzyme. Notably,
conversion degrees and monoglucosylated acceptor yields are almost equal, in-
dicating that P1 and P2 are the two major products formed, respectively.
D-GlcpNAc glucosylation by the native alternansucrase from L. mesen-
teroides NRRL B-1355 was previously reported.[
11]
The acceptor conversion
degree was very low and the glucosylation products were not characterized.
We showed here that all tested GH family 70 enzymes produced the same α-
(
1→6)-linked disaccharide P1 upon D-GlcpNAc glucosylation. These enzymes
thus conserve their same natural regiospecificity toward D-GlcpNAc. DSR-S1
and its two variants were the most efficient enzymes for the synthesis of P1.
Interestingly, mutations introduced downstream the second aspartic acids of
the catalytic triad of the enzymes did not alter the enzyme regiospecificity,
but had a beneficial effect on the conversion degree, which increased by more
Table 2: Transglucosylation of D-GlcpNAc using different recombinant
glucansucrases at acceptor:sucrose molar ratio of 1:1 (146 mM).
Recombinant enzyme
DSR-S
1
DSR-S
2
DSR-S
3
ASR
14
13 (P1)
AS
D-GlcpNAc conversion
26
54
64
6
a
degree (%)
Monoglucosylated
24 (P1)
54 (P1)
61 (P1)
4 (P2)
b
D-GlcpNAc yield (%)
At final time (24 h), sucrose is fully consumed by all variants.
a
The conversion degree of the acceptor was calculated using the formula: ([D-GlcpNAc]
initial
–
[D-GlcpNAc]
) / [D-GlcpNAc]
× 100.
t24h
initial
b
%
Monoglucosylated D-GlcpNAc = [Monoglucosylated D-GlcpNAc]t /[consumed sucrose]
f
t × 100.
f

Synthesis of L-rhamnose and N-acetyl-D-glucosamine Derivatives
1
49
than twofold for both DSR-S2 and DSR-S3mutants compared to DSR-S1. Lo-
cated close to the catalytic residues, mutations introduced in DSR-S2 and DSR-
S3may favor binding of the nonnatural acceptor, suggesting that engineering
of glucansucrases can greatly enhance the ability of these enzymes for the syn-
thesis of novel oligosaccharides.
Glucosylation of L-rhamnose
Enzyme Screening
Acceptor reactions were carried using an equimolar L-rhamnose:sucrose
ratio (Table 3). Among family 70 glucansucrases, only DSR-S2 and ASR were
able to glucosylate L-rhamnose. Interestingly, they synthesized several coprod-
[
31–33]
ucts, notably leucrose and isomaltose,
synthesis was not in favor of the disaccharide formation (supporting material
0). Consequently, the conversion degree and the yield of each independent
but the competition with polymer
1
product were very low, thus preventing further characterization. Regarding
glucosylation reactions carried out with AS, two major products were obtained
P3 and P4) concomitantly with turanose and trehalulose products[ (sup-
34]
(
porting material 9). P3 and P4 were produced in larger amounts and purified
for structural characterization.
Optimization of P3 and P4 synthesis by AS
To improve P3 and P4 synthesis, the influence of acceptor:sucrose ratio
on the conversion degree was first studied at two sucrose concentrations (146
mM and 292 mM) (Table 4). In both cases, increasing the acceptor concen-
tration favors acceptor glucosylation at the cost of polymer formation, and
yields higher amounts of products. However, L-rhamnose conversion degree
decreases concomitantly. Maintaining similar acceptor:sucrose ratio but vary-
ing the initial sucrose concentration did not significantly affect the glucosyla-
tion yields. Therefore, in search for the best compromise between glucosylation
yield and L-rhamnose conversion degree, we retained a sucrose concentration
of 292 mM and an acceptor:sucrose molar ratio of 2.5 for preparative scale-up.
Table 3: Transglucosylation of L-rhamnose using different recombinant
glucansucrases at acceptor:sucrose ratio of 1:1 (146 mM).
Recombinant enzyme
DSR-S
1
DSR-S
2
DSR-S
3
ASR
AS
6.5
2 (P3 + P4)
L-rhamnose conversion
<1
10
<1
6
a
degree (%)
Number of new products
a
0
5
0
4
The conversion degree of the acceptor was calculated using the formula ([Acceptor]
–
initial
[Acceptor]
) / [Acceptor]
× 100. At final time (24 h), sucrose was fully consumed.
t24h
initial

50 E. Champion et al.
1
Table 4: Effect of the acceptor:sucrose molar ratio (A:S) on L-rhamnose
glucosylation catalyzed by amylosucrase.
A:S Ratio ([S]) = 146 mM)
A:S Ratio ([S] = 292 mM)
1
2.5
5
10
0.5
1
2.5
5
L-Rhamnose (mM) a 146
365
730
1460
146
292
730
1460
Conversion degree
6.5
4.4
4.4
8.4
3.1
2.1
8.0
2.7
6.6
4.8
8.9
3.1
(
%)
Monoglucosylated
12.6
17.5
4.1
4.5
12.3
2.9
L-rhamnose (P3)
b
yield (%)
Diglucosylated
2.1
2.6
3.1
1.4
2.1
3.0
L-rhamnose (P4)
c
yield (%)
%
%
a
L-Rhap converted
4.4
2.1
3.4
1.0
2.5
0.6
1.7
0.4
5.3
2.7
4.5
2.1
3.6
1.2
2.5
0.6
d
into P3
L-Rhap converted
into P4e
The conversion degree of the acceptor was calculated using the formula: ([Acceptor]
–
initial
[
Acceptor]
)/[Acceptor]
× 100.
t24h
initial
b
Monoglucosylated rhamnose yield is the molar ratio: [Glc-Acceptor]t24h/[consumed
sucrose]
× 100. At final time, sucrose is fully consumed.
× 100. At final time, sucrose is fully consumed.
t24h
c
Di-glucosylated rhamnose yield is the molar ratio: [Glc -Acceptor]t24h/[consumed
2
sucrose]
t24h
d
e
%
%
L-Rhap converted into P3 is the molar ratio: [Glc-Acceptor]
L-Rhap converted into P4 is the molar ratio: [Glc -Acceptor]
/[Acceptor] × 100.
t24h
t24h
t0
t0
/[Acceptor] × 100.
2
Structural Characterization of P3 and P4
P3 and P4 were separated by preparative HPLC. HRMS data of P3 indi-
cates a molecular weight of 326 Da corresponding to the mass of a monogluco-
sylated L-rhamnose (supporting materials 3 and 7; details given in Experimen-
tal section). The 1D and 2D L-rhamnose NMR spectra were used as reference
1
(
data not shown). L-Rhamnose is found in a C4 pyranose ring conformation,
as revealed by a weak J1,2 coupling constant. Signals at 4.76 ppm and 5.00
ppm were assigned to the anomeric proton in β and α form, respectively.
1
Two anomeric signals of equal intensity, only, are observed in the H NMR
spectrum of P3. Aided by the chemical shifts (see Experimental section) and
1
1
JC,H values of the anomeric carbons, signals at 5.19 ppm ( JC1,H1 = 173.9 Hz)
1
and 4.78 ppm ( JC1,H1 = 161.3 Hz) were assigned to the anomeric protons of
D-glucosyl and L-rhamnosyl residues in α and β configuration, respectively.
No signal corresponding to the α/β-anomerization of a reducing disaccharide
1
3
could be found. In contrast, the chemical shift attributed to C-1Rha in the
C
NMR spectrum suggested that the two sugar moieties were linked by their
anomeric carbons through an (α1→ β1) linkage, as confirmed by HMBC anal-
ysis showing two inter-residual scalar couplings, C-1Rha:H-1 and C-1 :H-
Glc
Glc
1Rha, respectively.
In agreement with MS data, which strongly suggest that P4 is a trisac-
1
charide of 488 Da made of two hexoses and a deoxyhexose, the H NMR

Synthesis of L-rhamnose and N-acetyl-D-glucosamine Derivatives
1
51
Figure 5: Structures of disaccharide P3 and trisaccharide P4 obtained by AS-mediated
glucosylation of L-rhamnose.
spectrum of P4 indicates three doublets of equal intensity in the anomeric
region (supporting materials 4 and 8; details given in Experimental section).
In support of this assumption, the 13C NMR spectrum showed a single sig-
nal in the C-6Rha region (16–17 ppm), and two signals in the C-6Glc region
(60–61 ppm), indicating the presence of one L-rhamnosyl ring and two D-
glucosyl rings. The HMBC spectrum showed strong cross-peaks between an
H-1Glc and a C-4Glc, and between an H4Glc and a C1Glc, respectively, indicat-
ing the presence of an α-(1→4)-linkage between the two glucosyl residues.
Besides, as for P3, NMR analysis revealed that the glucosyl moiety incorpo-
rated first was linked to the L-rhamnosyl residue through an (α1→ β1) link-
1
13
age. The whole sets of P3 and P4 H and C chemical shifts are given in the
experimental section and in supporting materials 3–4 and 7–8. Finally, P3 and
P4 are new oligosaccharides identified as the α-D-glucopyranosyl-(1→1)-β-L-
rhamnopyranoside and α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→1)-
β-L-rhamnopyranoside, respectively (Fig. 5).
Glucosylation of methyl α-L-Rhamnopyranoside
The acceptor reaction was carried out using an equimolar acceptor:sucrose
ratio and a sucrose concentration of 146 mM. HPLC analysis of the reaction
mixtures revealed the presence of two main products (P5 and P6) (support-
ing materials 11 and 12). NMR data of these compounds were in agreement
with those of chemically synthesized methyl α-D-glucopyranosyl-(1→4)-α-
[
35,36]
L-rhamnopyranoside (P5)
rhamnopyranoside (P6)
7
and methyl α-D-glucopyranosyl-(1→3)-α-L-
used as reference compounds. Using the family
0 glucansucrases, methyl α-L-rhamnopyranoside conversion degrees rang-
ing from 3% to 17% were obtained, indicating a poor recognition of this
[
37]

52 E. Champion et al.
1
Table 5: Transglucosylation of α-L-RhapOMe using different recombinant
glucansucrases at acceptor:sucrose ratio of 1 (146 mM).
Recombinant enzyme
DSR-S
1
DSR-S
2
DSR-S
3
ASR
AS
<1
0
Conversion degreea
3
0
5
4
6
3
17
3
α−D-Glcp-(1→3)-α-L-RhapOMe (P6)
b
yield (%)
α−D-Glcp-(1→4)-α-L-RhapOMe (P5)
<1
<1
3
10
0
b
yield (%)
a
The conversion degree of the acceptor was calculated according to the formula
(
[Acceptor]
– [Acceptor]
) / [Acceptor]
initial
× 100.
initial
t24h
b
The percentage of monoglucosylated acceptor was determined by the ratio [Glc-
Acceptor] / [consumed sucrose]
× 100. At final time, sucrose was totally consumed.
t24h
t24h
acceptor. The best conversion was obtained for ASR, forming preferentially
methyl α-D-glucopyranosyl-(1→4)-α-L-rhamnopyranoside and, to a lesser
extent, methyl α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside. Two addi-
tional peaks present on the chromatogram were putatively assigned to the ho-
mologous trisaccharides but were not further characterized. Noteworthy, AS
was unable to use methyl α-L-rhamnopyranoside as an acceptor. Indeed, the
anomeric hydroxyl group, which was the preferred AS-mediated glucosylation
site of L-rhamnose, is blocked in methyl α-L-rhamnopyranoside, thus prevent-
ing the enzyme from transferring the glucosyl residue onto it, or onto any other
hydroxyl groups of α-L-RhapOMe (Table 5).
We have shown in this paper that glucosylation of N-acetyl-D-glucosamine
and L-rhamnose catalyzed by glucansucrases leads to the synthesis of various
disaccharides, and even trisaccharides. To our knowledge, these oligosaccha-
rides were never reported before. All involve the formation of 1,2-cis gluco-
sides, which are difficult to obtain by chemical synthesis due to lack of efficient
control of the stereochemical outcome of the glucosylation step. Providing an
enzymatic access to such compounds is thus of interest.
A number of studies have shown that introducing an enzymatic step in
the synthesis of complex oligosaccharides could be of great advantage. The
potency of such alternatives has been demonstrated in bacterial oligosac-
[
38–40]
charide synthesis.
glycovaccines.
Occasionally, the strategy was used in the field of
The disaccharides that were more specifically targeted
[
41–44]
in this work were α-D-glucopyranosyl-(1→6)-N-acetyl-D-glucosamine, α-D-
glucopyranosyl-(1→4)-N-acetyl-D-glucosamine, and a set of α-D-glucosyl-α-L-
rhamnopyranose. Noteworthy, such disaccharides are components of various
S. flexneri O-antigen repeating units.[
45–47]
Oligosaccharide fragments of the O-antigens of S. flexneri serotypes 5a, 2a,
and 3a, among others, have been chemically synthesized,[
36,37,48,49]
a number
as their methyl glycosides using an appropriately protected [α-D-Glcp-(1→3)]-
[
50,51]
α-L-Rhap-OMe acceptor.

Synthesis of L-rhamnose and N-acetyl-D-glucosamine Derivatives
1
53
In the case of S. flexneri 2a, several of those synthetic haptens were shown
to be immunogenic in an animal model when administered as oligosaccharide-
[
52]
protein conjugates.
With the view of providing alternative synthetic path-
ways to complex oligosaccharides, the enzymatically produced disaccharides
could serve as building blocks involved at an early stage of a chemo-enzymatic
synthesis of selected S. flexneri oligosaccharides. Demonstrating the feasibil-
ity of such strategy may open the way to a new diversity of potent chemo-
enzymatic routes integrating an enzymatic step at the early stage of the syn-
thesis of glycosylated targets, as nicely illustrated for the synthesis of sialy-
[
53–55]
lated oligosaccharides and conjugates.
However, to be of interest in a
chemo-enzymatic pathway, enzymatic transglucosylation must be efficient and
high yielding. With DSR-S1, ASR, and AS recombinant glucansucrases tested
in our work, we have obtained low yields of transglucosylation. Considering the
case of N-acetyl-D-glucosamine glucosylation, the use of DSR-S1 led to the syn-
thesis of α-D-glucopyranosyl-(1→6)-N-acetyl-D-glucosamine with a conversion
degree of 24% yield. Noteworthy, the use of the triple mutant DSR-S3, mutated
in the region close to the catalytic residues, allowed increasing the conversion
degree up to 64%, demonstrating that enzyme engineering significantly im-
proved the transglucosylation activity toward the poorly recognized nonnatu-
ral acceptor. In particular, amino acids in close vicinity with catalytic residues
appear to be key mutagenesis targets. Overall, data presented here serve as
a basis to apply semi-rational engineering to selected glucansucrases in order
to modulate their selectivity and/or regioselectivity and identify new variants
able to produce the appropriate glucosylated building blocks in amounts com-
patible with their use in multistep synthesis.
EXPERIMENTAL
Materials
Choice of Recombinant Glucansucrases
For DSR-S and ASR glucansucrases, the enzyme constructs chosen for this
study are, respectively, DSR-S vardel ꢀ4N (DSR-S1)and ASR C-del APY (ASR).
Both enzymes are truncated variants from L. mesenteroides NRRL B-512F dex-
transucrase and from L. mesenteroides NRRL B-1355 alternansucrase. They
were constructed to reduce the glucansucrase degradation occurring during
heterologous enzyme expression by Escherichia coli and have previously been
shown to display the same behavior than the wild-type enzyme in terms of
[
30,32]
specificity and synthesized products.
DSR-S vardel ꢀ4N variants (DSR-S2:ꢀ4N SEV663YDA, DSR-S3:DSR-
S SEV663NNS[ ) are mutated in the active site downstream the catalytic
aspartic acid D662. These mutants were designed by sequence alignment
31]

54 E. Champion et al.
1
analyses with glucansucrases of various linkage specificities, and described to
have a particular influence on acceptor binding, as they preferentially catalyze
the formation of disaccharides to the detriment of dextran formation.
All recombinant enzymes were produced in E. coli as reported
[
7,30,32]
elsewhere.
Nonpurified DSR-S and ASR (sonication supernatants)
◦
[7,30,32]
stored at –20 C were used for enzymatic assays. Purified AS,
at –80 C, was used for enzymatic reactions.
conserved
◦
Chemical Material
Sucrose, L-rhamnose, and N-acetyl-D-glucosamine were purchased from
Sigma-Aldrich. Methyl α-L-rhamnopyranoside[
25,26]
and reference compounds
[
37]
methyl α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside and methyl α-D-
[
35]
glucopyranosyl-(1→4)-α-L-rhamnopyranoside
were synthesized chemically
at the Unit e´ de Chimie des Biomol e´ cules - Institut Pasteur.
Acceptor Reaction Assay
The glucosylation reaction was performed in the enzyme optimal buffer: in
Tris-HCl (50 mM, pH = 7.5) for AS assays, in sodium acetate buffer (AcONa)
(
20 mM, pH = 5.4) for ASR assays, and in AcONa (50 mM, [CaCl2] = 0.05 g/L,
pH = 5.2) for DSR-S assays. The reaction mixture (1 mL) was carried out at
◦
3
0 C with sucrose and acceptor in equimolar ratio (146 mM). Enzymes were
used at 1 U/mL. For DSR-S and ASR, one unit is defined as the amount of en-
◦
zyme that catalyzes the formation of 1 µmol of fructose/min at 30 C, in enzyme
buffer and 292 mM sucrose. For AS, activity was determined under the same
conditions, except that sucrose was used at a concentration of 146 mM. All
the acceptor reactions were stopped after total sucrose consumption by heat-
◦
ing at 95 C for 5 min. The final mixture was centrifuged at 18,000 g for 10 min
and filtered on a 0.22-µm membrane before HPLC analysis.
Glucosyl Acceptor Production
In order to characterize glucosylated products of L-rhamnose and N-acetyl-
D-glucosamine, acceptor reactions were conducted at preparative scale.
P1 from N-acetyl-D-glucosamine glucosylation by DSR-S2 (0.8 U/mL) was
synthesized in 50 mL mixture reaction (500 mM in sucrose, 100 mM in accep-
tor); P2 from N-acetyl-D-glucosamine glucosylation by purified AS (1 U/mL)
was produced in 100 mL mixture reaction (292 mM in sucrose, 730 mM in ac-
ceptor). P3 and P4 from L-rhamnose glucosylation by purified AS (1 U/mL)
were produced in 60 mL mixture reaction (292 mM in sucrose, 730 mM in
◦
acceptor). After a 24 h reaction time at 30 C, the media were centrifugated
◦
at 4800 rpm for 20 min at 4 C to remove proteins and filtered for a better
clarification. The purification of the glucosylated products was performed on
a preparative octadecyl reverse-phase chromatography column (C18 column)

Synthesis of L-rhamnose and N-acetyl-D-glucosamine Derivatives
1
55
(Bischoff Chromatography). Ultra-pure water was used as eluent at a constant
flow rate of 50 mL/min. Glucosyl detection was carried out with a refractome-
ter, and each peak was collected separately, concentrated, and reinjected into
an analytical HPLC system to check the purity of the compounds. Purified P1,
P2, P3, and P4 were used as reference to determine yield of products obtained
from glucansucrases (Tables 2 and 4).
Analytical Methods
High-Performance Liquid Chromatography (HPLC)
The HPLC analysis device consisted of a Dionex P 680 series pump, a
Shodex RI 101 series refractometer, a Dionex UVD 340 UV/Vis detector, and an
autosampler HTC PAL. Five columns were employed to separate the acceptor
reaction products and to determine the acceptor conversion degree and product
yields: (i) Biorad HPLC Carbohydrate Analysis columns: AMINEX HPX-87C
◦
at 80 C (elution with ultra-pure water at 0.6 mL/min); (ii) HPX-87K columns
◦
(
300 × 7.8 mm) at 65 C (elution with ultra-pure water at 0.6 mL/min); (iii) C18
column Bischoff Prontosil Eurobond, 5 µm (elution with ultra-pure water at rt
and 1 mL/min); (iv) C30: Bischoff Prontosil Eurobond, 5 µm, 250 × 4.0 mm
(
elution with ultra-pure water at rt and 1 mL/min); and (v) C18RP: Sinergi
Fusion RP Phenomenex, 4 µm, 250 × 4.6 mm (elution with ultra-pure water
at rt and 1 mL/min).
High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic
Resonance (NMR)
Accurate mass determination was carried out using an Autospec mass
spectrometer arranged in an EBE geometry (Micromass, Manchester, UK). The
instrument was operated at 8 kV accelerating voltage in positive mode. The ce-
sium gun was set to 35 keV energy and 1 µL of sample was mixed in the tip of
the probe with a glycerol or dithiothreitol/dithioerythritol matrix. NMR analy-
1
13
ses: H (400.130 MHz), C (100.612 MHz); HSQC and HMBC were registered
on a Bruker-ARX 400 spectrometer equipped with an ultra-shim system. Sam-
ples were dissolved in deuterium oxide at c.a. 80 g/L and experiments were
performed at 300K.
α-D-glucopyranosyl-(1→6)-N-acetyl-D-glucosamine (P1)
+
HRMS (FAB): Calcd for C14H26NO11: 384.1506 [MH ], Found: 384.1518
1
β-anomer: H NMR (400.130 MHz, D2O) δ: 1.96 (s, 3H, COCH3), 3.32 (t, 1H,
H-4 ), 3.46 (m, 1H, H-2 ), 3.49 (m, 1H, H-3), 3.58–3.92 (m, 8H, H-2, H-4, H-5,
H-6, H-3 , H-5 , H-6 ), 4.66 (d, 1H, J1,2 = 11.2 Hz, H-1), 4.87 (d, 1H, H-1 )
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ

56 E. Champion et al.
1
1
3
ꢁ
C NMR (100.612 MHz) δ: 22.16 (CH3), 56.58 (C2), 60.43 (C6 ), 65.65 (C6),
ꢁ
ꢁ
ꢁ
ꢁ
6
9.47 (C4 ), 69.97 (C4), 71.45 (C2 ), 71.78 (C5 ), 73.03 (C3 ), 74.08 (C3), 74.30
ꢁ
(C5), 95.05 (C1), 97.97 (C1 ), 174.47 (CO)
1
α−anomer: H NMR (400.130 MHz, D2O) δ: 1.96 (s, 3H, COCH3), 3.32 (t, 1H,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
H-4 ), 3.46 (m, 1H, H-2 ), 3.48 (m, 1H, H-3), 3.65–3.92 (m, 9H, H-5 , H-3 , H-6 ,
ꢁ
H-5, H-2, H-4, H-6), 4.88 (d, 1H, H-1 ), 5.13 (d, 1H, J1,2 = 4.8 Hz, H-1)
1
3
ꢁ
C NMR (100.612 MHz) δ: 22.62 (CH3), 57.09 (C2), 60.97 (C6 ), 61.00 (C6),
ꢁ
ꢁ
ꢁ
ꢁ
6
9.78 (C4 ), 72.06 (C2 ), 73.14 (C5 ), 73.27 (C3 ), 74.79 (C3), 75.06 (C5), 77.22
ꢁ
(C4), 90.91 (C1), 97.91 (C1 ), 175.22 (CO)
α−D-glucopyranosyl-(1→4)-N-acetyl-D-glucosamine (P2)
+
HRMS (FAB): Calcd for C14H25NO11Na: 406.1325 [M+Na] , Found: 406.1356
1
β-anomer: H NMR (400.130 MHz, D2O) δ: 2.02 (s, 3H, COCH3), 3.40 (t, 1H,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
H-4 ), 3.56 (m, 1H, H-2 , H-5), 3.66–3.79 (m, 5H, H-3 , H-2, H-4, H-5 , H-3), 3.82
(
ꢁ
m, 4H, H-6, H-6 ), 4.70 (d, 1H, J1,2 = 8 Hz, H-1β), 5.39 (d, 1H, H-1 )
1
3
ꢁ
C NMR (100.612 MHz) δ: 22.62 (CH3), 57.09 (C2), 60.97 (C6 ), 61.00 (C6),
ꢁ
ꢁ
ꢁ
ꢁ
6
9.78 (C4 ), 72.06 (C2 ), 73.14 (C5 ), 73.27 (C3 ), 74.79 (C3), 75.06 (C5), 77.22
ꢁ
(C4), 95.25 (C1), 99.87 (C1 ), 175.22 (CO)
1
α-anomer: H NMR (400.130 MHz, D2O) δ: 2.02 (s, 3H, COCH3), 3.40 (t, 1H,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
H-4 ), 3.56 (m, 1H, H-2 ), 3.66–3.82 (m, 7H, H-4, H-3 , H-5 , H-6, H-6 ), 3.89
(
dd, 1H, H-2), 3.94 (m, 1H, H-5), 4.00 (dd, 1H, H-3), 5.18 (d, 1H, J1,2 = 3.6 Hz,
ꢁ
H-1α), 5.40 (d, 1H, H-1 )
1
3
ꢁ
C NMR (100.612 MHz) δ: 22.34 (CH3), 54.36 (C2), 60.97 (C6 ), 61.00 (C6),
ꢁ
ꢁ
ꢁ
ꢁ
6
9.78 (C4 ), 70.57 (C5), 71.62 (C3), 72.17 (C2 ), 73.14 (C5 ), 73.30 (C3 ), 77.94
ꢁ
(C4), 91.13 (C1), 100.15 (C1 ), 174.98 (CO)
α−D-glucopyranosyl-(1→1)-β-L-rhamnopyranoside (P3)
+
HRMS (FAB): Calcd for C12H22O10K: 365.0850 [M+K] , Found: 365.0861
1
H NMR (400.130 MHz, D2O) δ: 1.23 (d, 3H, J5,6 = 6.7 Hz, H-6), 3.32–3.33 (m,
ꢁ
2
H, H-4, H-5), 3.35 (d, 1H, J3,4 = 10 Hz, H-4 ), 3.49 (ddd, 1H, J2,3 = 10 Hz,
ꢁ
ꢁ
ꢁ
ꢁ
H-2 ), 3.55 (m, 1H, H-5 ), 3.58 (m, 1H, H-3), 3.64 (dd, 1H, H-3 ), 3.65 (dd, 1H,
ꢁ
H-6 ), 3.76 (dd, 1H, H-6 ), 3.94 (d, 1H, J2,3 = 3,2 Hz, H-2), 4.78 (d, 1H , J1,2 =
ꢁ
1
.2 Hz, H-1), 5.19 (d, 1H, J1,2 = 3.7 Hz, H-1 )
1
3
ꢁ
ꢁ
C NMR (100.612 MHz) δ: 16.82 (C6), 60.63 (C6 ), 69.55 (C4 ), 71.00 (C2), 71.13
ꢁ
ꢁ
ꢁ
(
9
C2 ), 72.16 (C4), 72.62 (C5), 72.67 (C3), 72.73 (C5 ), 72.93 (C3 ), 94.91 (C1),
ꢁ
5,23 (C1 )

Synthesis of L-rhamnose and N-acetyl-D-glucosamine Derivatives
1
57
α−D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→1)-β-L-rhamnopyranoside
(
P4)
+
HRMS (FAB): Calcd for C18H32O15K: 527.1378 [M+K] , Found: 527.1366
1
H NMR (400.130 MHz, D2O) δ: 1.22 (d, 3H, J5,6 = 6.7 Hz, H-6), 3.31–3.32 (m,
ꢁ
ꢁ
ꢁꢁ
ꢁ
ꢁꢁ
ꢁꢁ
3H, H-4, H-5, H-4 ), 3.47–3.61 (m, 6H, H-2 H-2 H-3, H-3 H-4 ,H-5 ), 3.70–
ꢁ
ꢁ
ꢁꢁ
ꢁꢁ
3
.74 (m, 5H, H-5 , H-6 , H-6 ), 3.93 (m, 2H, H-2, H-3 ), 4.79 (d, 1H, J1,2 = 1.2
ꢁ
ꢁꢁ
Hz, H-1), 5.20 (d, 1H, J1,2 = 3.7 Hz, H-1 ), 5.31 (d, 1H, J1,2 = 3.6 Hz, H-1 )
1
3
ꢁ
C NMR (100.612 MHz) δ: 16.77 (C6), 60.64 (C6, C6 ), 69.47 (C4?), 70.97 (C2),
ꢁ
ꢁ
ꢁꢁ
71.00 (C2 ), 71.12 (C5 ), 71.86 (C2 ), 72.15 (C4), 72.69 (C5), 72.72 (C3), 72.87
ꢁ
ꢁꢁ
ꢁ
ꢁ
(
(
C3 ), 73.02 (C5 ), 73.42 (C3?), 76.78 (C4 ), 94.96 (C1), 95.05 (C1 ), 99.83
C1 ) (C4), 91.13 (C1), 100.15 (C1 ), 174.98 (CO)
ꢁꢁ
ꢁ
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