Alkoxyamino glycoside acceptors for the regioselective synthesis of oligosaccharides using glycosynthases and transglycosidases

Article abstract of DOI:10.1016/j.bmcl.2012.11.065

Alkoxyamino derivatives of oligosaccharides have been synthesized by enzymatic synthesis using a glycosynthase and a transglycosidase. The chemoselective assembly of unprotected oligosaccharides bearing glucose at the reducing end with N-alkyl-O-benzylhydroxylamine provides sugar derivatives that are good acceptors for enzymatic synthesis using either glycosynthase or transglycosidase. Furthermore, this method affords the possibility of controlling the regioselectivity of coupling depending on the nature of the alkoxyamino substituent and provides high-yield coupling of sugars without the need for complex protecting group chemistry.

Full text of DOI:10.1016/j.bmcl.2012.11.065

Bioorganic & Medicinal Chemistry Letters 23 (2013) 448–451
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Alkoxyamino glycoside acceptors for the regioselective synthesis of
oligosaccharides using glycosynthases and transglycosidases
⇑
David Teze, Michel Dion, Franck Daligault, Vinh Tran, Corinne André-Miral , Charles Tellier
LUNAM Université, Université de Nantes, CNRS FRE n°3478, UFIP, 2 rue de la Houssinière, F-44322 Nantes cedex 03, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2012
Revised 13 November 2012
Accepted 18 November 2012
Available online 29 November 2012
Alkoxyamino derivatives of oligosaccharides have been synthesized by enzymatic synthesis using a gly-
cosynthase and a transglycosidase. The chemoselective assembly of unprotected oligosaccharides bearing
glucose at the reducing end with N-alkyl-O-benzylhydroxylamine provides sugar derivatives that are
good acceptors for enzymatic synthesis using either glycosynthase or transglycosidase. Furthermore, this
method affords the possibility of controlling the regioselectivity of coupling depending on the nature of
the alkoxyamino substituent and provides high-yield coupling of sugars without the need for complex
protecting group chemistry.
Keywords:
Transglycosidases
Glycosynthases
Ó 2012 Elsevier Ltd. All rights reserved.
Oligosaccharide synthesis
Alkoxyaminoglucoside
Regioselectivity
The important role of oligosaccharides and their conjugates in
biology has been increasingly recognized in recent years. Unfortu-
nately, despite the considerable development of efficient methods
in this field, the assembly of oligosaccharides remains a substantial
challenge. Glycosidic bond formation requires meticulous control
of both regio- and stereoselectivity. The regioselectivity is extre-
mely challenging because of the similar reactivities of the hydroxyl
groups. In order to circumvent these difficulties, it is often neces-
sary to employ extensive protecting group chemistry with all its
attendant problems.
Chemo-enzymatic glycosylation has become a powerful tool for
the stereoselective synthesis of various glycosidic compounds. In
this way, complete control over both stereo- and regioselectivity
can be attained. Glycosyl transferases are useful in this regard as
they catalyze the formation of glycosidic bonds with both high
yield and selectivity. Unfortunately, glycosyl transferase methodol-
ogy is limited for the large-scale production of oligosaccharides be-
cause of the difficulty in accessing nucleotide sugar donors.
Engineered retaining glycosidases have provided an efficient ap-
proach for the synthesis of oligosaccharides. A first methodology,
using site-directed mutagenesis, was developed by Withers1–7
and co-workers on exoglycosidases and Planas and co-workers
on endoglycosidases.8–12 This approach was based on the mutation
of the catalytic nucleophile (aspartate or glutamate) by a neutral
amino acid (glycine, serine or alanine) and the use of donors with
an anomeric configuration opposite to that of the original substrate
(such as a-glycosyl fluorides), which yielded new enzymes called
glycosynthases.13 The mutant glycosidases provided high transgly-
cosylation yields because of the inability to hydrolyze the transgly-
cosylation product. A second approach, which was developed
recently in our laboratory, was based on the directed evolution of
glycosidases into transglycosidases.14–16 The mutant enzymes have
almost lost their hydrolytic activity while keeping their transgly-
cosidase activity.
One of the main drawbacks of the glycosynthase and transgly-
cosidase approaches applied to exoglycosidases is often the need
for a phenyl ring at the (+2) subsite to ensure the correct position-
ing of acceptors in the active site.14 Such an aromatic group pro-
vides stacking interaction with aromatic active-site residues and
leads to improved regioselectivity and yield in the oligosaccharide
synthesis. However, these substituents need to be eliminated in or-
der to functionalize the anomeric position for the subsequent syn-
thesis of glycoconjugates. Deprotection of aryl groups is difficult
without partial hydrolysis of glycosidic bonds, whereas alternative
phenyl thio acceptors have been shown to be excellent substrates
which can be easily deprotected by N-bromosuccinimide. Further-
more, all these approaches rely on the organic synthesis of aro-
matic substrate acceptors, which limits the applicability of
enzyme synthesis and its use in sustainable chemistry.
Recently, anomeric modification, such as oxime ligation, which
enables the formation of glycosyl derivatives without using
protecting groups, has been exploited for the synthesis of various
glycoconjugates.18–20 This chemoselective conjugation provides
predominantly a b-pyranoside for reducing terminal gluco-config-
uration, which can be subsequently cleaved in weakly acidic water
⇑
Corresponding author.
E-mail address: corinne.miral@univ-nantes.fr (C. André-Miral).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.065

D. Teze et al. / Bioorg. Med. Chem. Lett. 23 (2013) 448–451
449
Scheme 1. Chemoselective synthesis of acceptors 3a–c, 4 and 5. Reagents and conditions: (a) AcONa, H2O/MeOH 4/1, aldehyde or ketone (3 equiv), 95–97%; (b) NaCNBH3,
EtOH, HCl, 80–89%; (c) THF/AcOH 75/25, 43–86%.
solutions. This Letter presents the synthesis of aromatic glycocon-
jugates using oxime ligation and their use as acceptors in the enzy-
matic synthesis of oligosaccharides. This approach is validated
with TtbGly, for which both glycosynthase and transglycosidase
mutants have been cloned and over-expressed in our labora-
tory.14–17
Mutant TtbGly efficiently catalyzes the synthesis of b-(1 ? 3)-
disaccharides. Previous work has shown that the glycosynthase
E338G can act as an efficient catalyst for the regioselective synthe-
sis of the b-(1 ? 3)-disaccharides when the acceptor has a b-O-aryl
group and, more precisely, an aromatic aglycon group.17,21 For
example, the E338G mutant can produce a nearly quantitative
a correct positioning of the acceptor in the active site.17 Oxyamino
groups have usually been described in the literature for their use in
the synthesis of neoglycopeptides by chemoselective ligation with
native reducing sugars.20,22–24 The most attractive of these chemo-
selective reactions involves aminooxy-derivatized peptides with
natural, unmodified reducing sugars. It has also been shown that
the use of the oxy-methylamino group allows the preservation of
the pyranose cyclic form of the linked sugar.25,26
For the synthesis of acceptor compounds 3a–c, commercially
available formaldehyde, acetaldehyde or propionaldehyde was
reacted with O-benzylhydroxylamine hydrochloride in the pres-
ence of sodium acetate in H2O/methanol (4/1) at rt for 2 h, afford-
ing the corresponding O-benzyl-oximes 1a–c with 95–97% yield.
The oximes were reduced to the corresponding N-methyl-O-ben-
zylhydroxylamines 2a–c by treatment with NaCNBH3 and hydro-
chloric acid at rt for 30 min (80–89% yield). The hydroxylamines
yield (96%) of phenyl b-
oside with the most favorable b-
viously shown that the replacement of b-
D
-glucopyranosyl-(1 ? 3)-b-
-GlcPh acceptor.17 We have pre-
-GlcPh by b- -GlcBn as
D-glucopyran-
D
D
D
an acceptor results in a drop in the regioselectivity of the enzyme
leading to a mixture of (1 ? 3) and (1 ? 6)-disaccharides (ratio 55/
45).17 However, substitution of the benzyl aglycon group by a
biphenyl group allows partial recovery of the b-(1 ? 3) regioselec-
tivity, suggesting that modulation of the size of the aglycon group
can control the regioselectivity of the disaccharide synthesis.17 Fol-
lowing these observations, we present here an alternative that re-
places the aglycon of the sugar acceptor by an alkoxyamino group,
which can be easily introduced at the anomeric position and sub-
sequently cleaved. Furthermore, this aglycon modification can
accommodate various bulky modifications (1a–c, Scheme 1).
The choice of an N-alkyl-O-benzylhydroxylamine group at the
anomeric position for the acceptor was made since we have al-
ready shown that the presence of a b-O-aryl anomeric group is nec-
essary for either the catalytic activity of wild glycosynthase from
Thermus thermophilus or for the derived glycosynthases to ensure
2 and D-glucose were then reacted in THF/AcOH (75/25) at rt for
20 h affording compounds 3a–c in 89%, 43% and 86% yield, respec-
tively.25,26 Using the same procedure, compounds 4 and 5 were
synthesized from the N-methyl-O-benzylhydroxylamine 2a using
D-lactose and D-maltose, respectively, in an aqueous acetate buffer
(0.1 M, pH 4.5) at rt for 24 h, in 98% and 95% yield, respectively.
Compounds 3a–c, 4 and 5 (50 mM) were then tested for their
ability to react with
a
-galactosyl fluoride27,28 (50 mM) as donor
in the presence of the mutant glycosynthase, E338G, at 55 °C and
pH 7.8 (0.15 M NaHCO3) as shown in Scheme 2. The kinetics of
the transglycosylation reaction were followed by TLC until the
fluoride donor was completely consumed (16–20 h) and the reac-
tion was stopped. The components were separated by silica gel col-
umn chromatography and the structures of the products were
determined by standard one- and two-dimensional 1H and 13C
Scheme 2. Transglycosylation reaction of compound 3a with
7.8, 55 °C, glycosynthase E338G, 87%.
a-galactosyl fluoride as donor. Reagents and conditions: aqueous ammonium hydrogen carbonate buffer at pH

450
D. Teze et al. / Bioorg. Med. Chem. Lett. 23 (2013) 448–451
Table 1
Yields and regioselectivity obtained with the glycosynthase (E338G) for the trans-
glycosylation reaction of compounds 3a–c,
4
and
5
(50 mM) with
a-galactosyl
fluoride (50 mM) as donor
Acceptor
Regioselectivity
Yielda
BnON(Me)-b-
D
-Glc (3a)
b-(1 ? 3), >95%
b-(1 ? 2), 76%
b-(1 ? 3), 21%
b-(1 ? Xc), 3%
b-(1 ? 3), 74%
b-(1 ? 2), 26%
87%
84%
BnON(Et)-b-
D-Glc (3b)
BnON(nPr)-b-
D
-Glc (3c)
74%
BnON(Me)-b-
BnON(Me)-b-
D
-Lac (4)
-Mal (5)
ndb
nd
D
a
b
c
Overall yield of transglycosylation.
nd, Not detected.
Other unidentified regioisomer.
NMR. The overall yields were established after purification (See
Table 1).
Acceptors 3a,b,c were very good substrates for the glycosynth-
ase and di- or trisaccharides were obtained in high yield. With
acceptor 3a, a b-(1 ? 3) regioisomer was obtained as the major
regioisomer, in contrast with acceptors containing a benzyl group
at the anomeric position which gave a mixture of regioisomers.17
These results suggest that, compared to the benzyl aglycon, the
additional bond between the aryl group and the anomeric position
provides fewer constraints for acceptors at the +1 and +2 sites to fit
into the active site and give unique regioselectivity. Surprisingly,
the use of a bulky alkoxyamino group at the anomeric position of
the acceptor resulted in a modification of the regioselectivity, since
the ethyl derivative gave a b-(1 ? 2) regioisomer as the major
product and the n-propyl derivative a b-(1 ? 3) regioisomer as
the major product. In agreement with previous experiments using
other aglycons, lactose and maltose derivatives (compounds 4 and
5) were not good acceptors for the mutant as no transglycosylation
product was detected in the reaction medium.17 Finally, the depro-
tection of the anomeric position in aqueous solution at pH 2 affor-
ded the corresponding oligosaccharides nearly quantitatively.
Transglycosidases, which are an alternative to glycosynthases,
have also been shown to be suitable for oligosaccharide synthe-
sis.14,16 Although the directed evolution of glycosidases can pro-
vide enzymes with high transglycosidase activity, they often
suffer from a lack of strict regioselectivity. As an example, the
transglycosidase mutant F401S of TtbGly in the presence of oNPGal
as donor and GlcSPh as acceptor catalyzed the formation of a com-
plex mixture of disaccharides resulting from self-condensation of
Figure 2. Time course of 6a formation (O) in the presence of TtbGly mutant
transglycosidase F401S with oNPGal (15 mM) as donor, and 3a (15 mM) as acceptor
in 100 mM phosphate buffer pH 7.0 at 40 °C. Time course of the formation of a
minor regioisomer N(Me)-O-Bn-N-(b-D-galactopyranosyl(1 ? X)b-D-glucopyrano-
syl)hydroxylamine (4) with oNPGal as donor. The progress of the reaction was
followed by capillary electrophoresis.
the donor (b-
D
-Galp-(1 ? X)- b-
D-Glcp-oNP) and transglycosylation
products, b-
D
-Galp-(1 ? X)-b-
D
-Glcp-S-Ph (Fig. 1).29
Interestingly, when we used the same F401S mutant with 3a as
acceptor and the same donor, we obtained the disaccharide 6a as a
major product (identified as the b-(1 ? 3) regioisomer) and only
another minor regioisomer (<5%) and a donor self-condensation
product (<8%) (Fig. 1).
In conditions where the donor/acceptor ratio was 1 (15 mM), a
maximum yield of 80% was obtained for the major transglycosyla-
tion product 6a (Fig. 2). Interestingly, at long incubation times, the
transglycosylation yield barely leveled off due to a slow hydrolysis
of the transglycosylation product, which makes this mutant
transglycosidase as effective as the glycosynthase approach. This
result demonstrates that substrate engineering at the +2 site of
glycosidases can be a complementary approach to afford high
regioselectivity.
In conclusion, these results further demonstrate that combined
chemical and genetic engineering approaches are fruitful to
expand the library of accessible oligosaccharide structures by
chemo-enzymatic synthesis. Moreover, the use of a protecting
Figure 1. Capillary electrophoresis analysis of the product mixture obtained after 5 h of reaction with mutant F401S with (A) oNPGal (25 mM) as donor and GlcSPh (25 mM)
as acceptor, and (B) oNPGal (15 mM) as donor and 3a (15 mM) as acceptor.

D. Teze et al. / Bioorg. Med. Chem. Lett. 23 (2013) 448–451
451
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This research was supported by a grant from the Region Pays de
la Loire, France (AAP GlycoNet). We gratefully thank David Ropartz
for mass spectrometry analyses and data interpretation. These
analyses were performed within the BIBS platform located at INRA
Nantes (INRA, UR1268 Biopolymères Interactions Assemblages F-
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Supplementary data (detailed procedures for the preparation of
key intermediates, characterization data) associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmcl.2012.11.065.
References and notes
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