6-Azido d-galactose transfer to N-acetyl-d-glucosamine derivative using commercially available β-1,4-galactosyltransferase

Article abstract of DOI:10.1016/j.tetlet.2008.02.018

A new strategy to tag glycoproteins carrying terminal GlcNAc was developed using commercially available bovine β-1,4-galactosyltransferase (GalT) and UDP-6-azidogalactose. The azide function was then allowed to react via a biotinylated Staudinger-Bertozzi probe demonstrating the usefulness of such a procedure to tag any glycoprotein possessing a N-acetylglucosamine terminal residue from any type of cell lysate.

Full text of DOI:10.1016/j.tetlet.2008.02.018

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 2294–2297
6
-Azido D-galactose transfer to N-acetyl-D-glucosamine derivative
using commercially available b-1,4-galactosyltransferase
a,
b,
b
a
Micha e¨ l Bosco , Sophie Le Gall , Christophe Rihouey , Samuel Couve-Bonnaire ,
b
b
a,
*
Muriel Bardor , Patrice Lerouge , Xavier Pannecoucke
a
UMR-CNRS 6014, IRCOF, INSA et Universit e´ de Rouen, rue tesni e` re, 76131 Mont Saint Aignan, France
UMR-CNRS 6037, IFRMP23, UFR des Sciences, Universit e´ de Rouen, 76821 Mont Saint Aignan, France
b
Received 21 December 2007; revised 31 January 2008; accepted 4 February 2008
Available online 8 February 2008
Abstract
A new strategy to tag glycoproteins carrying terminal GlcNAc was developed using commercially available bovine b-1,4-galactosyl-
transferase (GalT) and UDP-6-azidogalactose. The azide function was then allowed to react via a biotinylated Staudinger–Bertozzi
probe demonstrating the usefulness of such a procedure to tag any glycoprotein possessing a N-acetylglucosamine terminal residue from
any type of cell lysate.
Ó 2008 Elsevier Ltd. All rights reserved.
Many proteins are subject to post-translational glycosyl-
ation, which in turn generates structural diversity required
to perform necessary physiological task. Proteomic strate-
enables glycoprotein isolation from crude preparations.
This two step labeling process can be used to outfit a target
molecule for detection or for isolation, depending on the
nature of the probes.
1
gies typically do not distinguish among glycoforms, and
simply catalog the abundance of protein, whether modified
or not. Identification of glycosylated proteins requires that
they could be separated from complex mixtures that con-
tain both unglycosylated and heterogeneously glycosylated
In this Letter, we report a new strategy, readily adaptable
to any cell or tissue type to tag glycoprotein carrying termi-
nal GlcNAc on their O- or N-glycans using commercially
available b-1,4-galactosyltransferase (GalT). These Glc-
NAc-terminated proteins can be found in all higher eukary-
otic organisms and are particularly abundant in N-linked
2
,3
proteins.
Historically, physical separation has been
accomplished by affinity chromatography using lectins or
4
17
antibodies that are specific for special glycan epitopes.
glycans in plant proteins. This is a post-lysis method
In newer methods, labels can be introduced either by the
exploitation of glycoprotein-specific reactions, that is, the
reaction of 1,2 diol with boronic acid to form boronic dies-
involving biosynthetic and chemical manipulations that
are completed only after cell lysis, introducing a modified
carbohydrate bearing a chemical reporter. As a chemical
reporter, azido group was chosen for its stability and com-
plete lack of reactivity toward all functional groups present
5
,6
ters
functional group on glycoprotein of interest
or enzymatical introduction of a bioorthogonal
7
–11
or by using
After chemical
1
2–16
18
the cell’s own biosynthetic machinery.
in biomolecules. Although kinetically stable, azides are
tagging, covalent modification with a highly selective probe
predisposed to unique modes of reactivity owing to their
large intrinsic energy content. Bioorthogonal reactions have
thus been developed, including the Staudinger ligation of
*
Corresponding author. Tel.: +33 2 35 52 24 27; fax: +33 2 35 52 29 62.
19
azides with functionalized phosphines and the [3+2]
E-mail address: xavier.pannecoucke@insa-rouen.fr (X. Pannecoucke).
The two first authors participated equally to this work by being,
respectively, involved in the synthesis of activated probes and their
application to oligosaccharide derivation.
2
0,21
cycloaddition of azides with activated alkynes.
For our purpose, we chose to introduce the azide func-
tionality at the C-6 position of the galactose ring because
0
040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.02.018

M. Bosco et al. / Tetrahedron Letters 49 (2008) 2294–2297
2295
Eling and co-workers showed that a biotin moiety
appended to the C-6 position of UDP-D-Gal could serve
as a substrate for human GalT. Thus, sugar nucleotide 1
AcO
AcO
N3
AcO
AcO
N3
O
a
O
9
I
AcO
AcO
OAc
of 6-azido-6-deoxy D-galactose was synthesized
Fig. 1).
(see
5
6
Two strategies could be envisioned to prepare pyrophos-
AcO
AcO
N3
+
NH+
3
Et NH Et
3
O
-
-
phate 1 (UDP-GalN ). The first one, essentially developed
O
O
P
3
O
O
O
2
2
O
P
O
by Hindsgaul’s group, is based on the reaction between
AcO
2
3
O
O
b
N
NH
an activated galactose moiety with uridine diphosphate.
In the second methodology, first developed by Moffatt
HO OH
O
2
4
7
β
and Khorana, galactose-1-phosphate derivatives would
0
react with uridine-5 -phosphoromorpholidate 10. Even if
Scheme 2. Synthesis of b-anomer of UDP derivative. Reagents and
conditions: (a) TMSI, BiI , CH Cl , 0 °C, 30 min; (b) (i) (Bu N) –UDP;
(ii) HPLC C18, 4% (2 steps).
the first methodology was often lower yielding, desired
products as anomeric mixtures could be more rapidly
obtained.
3
2
2
4
2
First, 6-azido-6-deoxygalactose 3 was prepared, starting
from diisopropylidene galactose and following modified
diate iodide. More stable protecting group acetate was then
tried (Scheme 2). In this case, iodide 6 could be isolated
27
2
5
Hindsgaul and co-workers procedure (Scheme 1). In
our case, we could not use benzyl as hydroxyl protective
group due to deprotection conditions which would affect
the azide function. Hydroxyl groups were temporarily pro-
tected as their trimethylsilyl ethers. Anomeric formation of
the iodide and reaction with tetrabutylammonium salt of
uridine diphosphate followed by trimethylsilyl deprotec-
tion and purification delivered, in our hands, GalT-sub-
strate 1 in poor yield and as a mixture of a,b-anomers.
One of the problems, in the previous synthesis, is the
extreme lability of the trimethylsilylether protection, ren-
dering impossible purification and isolation of the interme-
and reaction with tetrabutylammonium salt of uridine
diphosphate afforded sugar nucleotide 7 as a unique b-ano-
mer. This anchimeric assistance surprised us under the
slightly basic reaction conditions used, but at the same time
Jakeman and co-workers observed the same feature in the
fucose and mannose series. Even if the b stereoselectivity
observed was total, it was useless for our purpose as the
GalT could only use a-anomer as the substrate.
28
2
6
We thus turned our attention toward the second meth-
odology, developed by Moffatt and Khorana and improved
24
by Wong et al. The first part of this pathway was the syn-
thesis of the azidogalactosyl phosphate 9 (Scheme 3).
Depending on the phosphorylating agent used, it would
be possible to favor one of the anomers of 9. Using 2-
HO
HO
N
3
29
+
Et NH
chloro-4H-benzodioxaphosphorin-4-one 8 or tricoordi-
3
O
+
Et NH
30,31
3
nated phosphorous (PIII) reagent,
the configuration
O- O-
O
HO
O
O
of the anomeric centre could be controlled and almost only
O
P
P
a-anomer of 9 was isolated. 9a was then reacted with uri-
O
O
O
N
NH
0
dine-5 -phosphoromorpholidate 10 to yield after purifica-
HO OH
O
tion GalT-substrate 1 exclusively under its a anomeric
form.
1
α
Fig. 1. Sugar nucleotide 1 of 6-azido-6-deoxy D-galactose.
AcO
AcO
N
3
O
OH
O
O
N
a,b,c
8
+
d,e
10
HO
HO
3
Et NH
3
5
1α
a,b,c
d,e
-
O
O
P
O
AcO
O
N
OH
O
O
OH
9
HO
O
2
3
O
O-
O
P
O
O
Me SiO
N
3
O
3
O
P
O
f,g
O
N
NH
1
α,β
Me SiO
3
Cl
O
I
Me SiO
OH
3
HO
O
8
4
10
Scheme 1. Synthesis of the mixture of anomers of sugar nucleotide 1.
Reagents and conditions: (a) MsCl, DMAP, CH Cl , 0 °C; (b) NaN
Bu NBr, DMF, 110 °C, 5 d, 87% (2 steps); (c) HCl, 0.06 M 80 °C, 16 h,
5%; (d) TMSCl, pyridine, 0 °C, 1 h; (e) TMSI, CH Cl , 0 °C, 30 min; (f)
Bu N) –UDP, overnight; (g) (i) Bu NF, H O, 90 min; (ii) alkaline
phosphatase, overnight; (iii) HPLC C18, 2. 5% (4 steps).
Scheme 3. Synthesis of a-anomer of UDP analogue 1. Reagents and
conditions: (a) CH COONH , DIEA, DMF, 2 d, 85%; (b) (i) 2-chloro-4H-
benzodioxaphosphorin-4-one 8, Et N, dioxanne, THF, 0 °C, 20 min; (ii)
O, 74% (2 steps); (c) t-BuOOH (2 equiv), I cat., THF, 2 d, 74%; (d) 10,
N, NH HCO , CH OH, 1 d; (ii)
2
2
3
,
3
4
4
3
8
(
2
2
H
2
2
4
2
4
2
1H-tetrazole, pyridine, 5 d, 46%; (e) (i) Et
3
4
3
3
HPLC C18, 53%.

2296
M. Bosco et al. / Tetrahedron Letters 49 (2008) 2294–2297
O
O
O
O
O
O
OH
OH
OH
HO
HO
O
a
O
b
O
HO
HO
HO
HO
HO
HO
O
O
O
O
O
O
HO
O
HO
NH
1α
NH
13
NH
N3
NH
O
1
O
O
1
12
O
O
P
H
Ph
N
O
O
O
Ph
O
O
P
HN
O
NH
H
O
H
OMe
Ph
H
H
HN NH
N
O
O
N
S
O
14
H
H
NH
O
O
Ph
1
3
S
O
Scheme 4. Coupling of 6-azido-6-deoxy D-galactose to a GlcNAc-terminated substrate 11 and activation through a Staudinger ligation. Reagents and
conditions: (a) 1a, 11, b-1,4-galactosyltransferase from bovine milk, alkaline phosphatase, MnCl
rt, overnight.
2 2
, glycine buffer, 37 °C, 2 d; (b) 13, THF/H O (5/95, v/v),
Supplementary data
Supplementary data (experimental part and spectra)
associated with this article can be found, in the online
version, at doi:10.1016/j.tetlet.2008.02.018.
References and notes
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2
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3
substrate such as 4-methyl umbelliferyl N-acetyl b-D-glu-
3
2
cosaminide 11 (MU-GlcNAc). Using wild-type bovine
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8
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4
Staudinger ligation with a biotinylated phosphine 13.
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derivative 14 (Scheme 4 and Fig. 2) in mild conditions as
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1
2376.
To conclude, we have developed a new strategy, to tag
glycoprotein carrying terminal GlcNAc. Using commer-
cially available bovine b-1,4-galactosyltransferase (GalT),
we could transfer 6-azidogalactose onto the N-acetylglu-
cosamine residue of MU-GlcNAc. The azide function
was then allowed to react via a Staudinger–Bertozzi rear-
rangement with a biotinylated probe demonstrating the
usefulness of such a procedure to tag any glycoprotein pos-
sessing a N-acetylglucosamine terminal residue from any
type of cell lysate.
11. Boeggeman, E.; Ramakrishnan, B.; Kilgore, C.; Khidekel, N.; Hsieh-
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Acknowledgment
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The authors want to thank the ANR (Agence Nationale
de la Recherche) for financial support (ANR-05-JCJC-
0
190-01).

M. Bosco et al. / Tetrahedron Letters 49 (2008) 2294–2297
2297
2
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Disaccharide 12 was purified on a carbograph LudgerClean EB10
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2 3
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2
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33. MALDI mass spectrometric analysis: Labelling and coupling reac-
tions were monitored on an Applied Biosystems Voyager-DE Pro
time-of-flight mass spectrometer. The accelerating voltage was 20 kV,
guide wire 0.002%, delay time 100 ns and grid voltage 80%. The
instrument was operated in reflector mode under positive ion
conditions. A nitrogen laser was used at 337 nm with 300 laser shots
averaged per spectrum. 2,5-dihydroxybenzoic acid (Aldrich chemi-
cals) was used as the matrix for all experiments. The matrix
concentration was 5 mg/mL in acetonitrile/trifluoroacetic acid 0.1%
(3/7). Sample (1 lL) was spotted onto the MALDI target followed by
1 lL of matrix. The mixture was then allowed to air dry prior to
analysis. Calibration was performed using instrument default settings
and data analysis was carried out using Data Explorer software.
34. Staudinger ligation between azido-labelled disaccharide and biotinyl-
ated phosphine: 200 lg of biotinylated phosphine 13 was incubated
overnight at rt with azido-labelled disaccharide 12 in a water/THF
mixture (95/5, v/v).
2
2
2
2
2
6
054–6065.
0. Oscarson, S.; Sehgelmeble, F. W. Tetrahedron: Asymmetry 2005, 16,
21–125.
3
1
3
3
1. Lazarevic, D.; Thiem, J. Carbohydr. Res. 2002, 337, 2187–2194.
2. In vitro labelling of MU-GlcNAc with 6-azido D-galactose: 200 lg of
UDP 6-azido D-galactose 1a was incubated in 100 mM glycine buffer,
2
pH 8.4, containing 10 mM MnCl with 10 lg of MU-GlcNAc 11