6″-Azido-6″-deoxy-UDP-N-acetylglucosamine as a glycosyltransferase substrate

Article abstract of DOI:10.1016/j.bmcl.2010.12.090

6″-Azido-6″-deoxy-UDP-N-acetylglucosamine (UDP-6Az-GlcNAc) is a potential alternate substrate for N-acetylglucosaminyltransferases. This compound could be used to generate various glycoconjugates bearing an azide functionality that could in turn be subjected to further modification using Staudinger ligation or Huisgen cycloaddition. UDP-6Az-GlcNAc is synthesized from α-benzyl-N-acetylglucosaminoside in seven-steps with an overall yield of 6%. It is demonstrated to serve as a substrate donor for the glycosyl transfer reaction catalyzed by the human UDP-GlcNAc:polypeptidyltransferase (OGT) to the acceptor protein nucleoporin 62 (nup62).

Full text of DOI:10.1016/j.bmcl.2010.12.090

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1199–1201
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
600-Azido-600-deoxy-UDP-N-acetylglucosamine as a glycosyltransferase substrate
Alain Mayer a, Tracey M. Gloster b, Wayne K. Chou a, David J. Vocadlo b, Martin E. Tanner a,
⇑
a Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1
b Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada V5A 1S6
a r t i c l e i n f o
a b s t r a c t
600-Azido-600-deoxy-UDP-N-acetylglucosamine (UDP-6Az-GlcNAc) is a potential alternate substrate for N-
acetylglucosaminyltransferases. This compound could be used to generate various glycoconjugates bear-
ing an azide functionality that could in turn be subjected to further modification using Staudinger ligation
Article history:
Received 26 November 2010
Revised 16 December 2010
Accepted 16 December 2010
Available online 23 December 2010
or Huisgen cycloaddition. UDP-6Az-GlcNAc is synthesized from
a-benzyl-N-acetylglucosaminoside in
seven-steps with an overall yield of 6%. It is demonstrated to serve as a substrate donor for the glycosyl
transfer reaction catalyzed by the human UDP-GlcNAc:polypeptidyltransferase (OGT) to the acceptor
protein nucleoporin 62 (nup62).
Keywords:
Glycosyltransferase
Azide
Ó 2011 Elsevier Ltd. All rights reserved.
Sugar nucleotide
Click chemistry
UDP-N-acetylglucosamine
Carbohydrates bearing bioorthogonal chemical reporters have
been extensively used to generate glycoconjugates or polysaccha-
rides that can be probed or modified using specific chemical reac-
tions.1 In order to do this the modified sugar must first be activated
as a sugar nucleotide, which may then serve as an alternate sub-
UDP-N-acetylglucosamine 1 (UDP-6Az-GlcNAc) and to test its suit-
ability as an alternate N-acetylglucosaminyltransferase substrate
(Scheme 1). We reasoned that the replacement of a hydroxyl group
by an azide group is less sterically demanding than the replace-
ment of a hydrogen atom, and that the ‘remote’ positioning of
the label might be more forgiving in certain instances.
strate for
a glycosyltransferase. This has been accomplished
in vivo using free sugars lacking the nucleotide diphosphate that
are cell permeable.2–11 Upon entering the cell, these sugars are
converted into sugar nucleotide donors by the cell’s catalytic
machinery, and then transferred onto their appropriate cellular
targets. Transfer by a glycosyltransferase has also been accom-
plished in vitro using non-cell permeable sugar nucleotide donors
and either cellular lysates or purified substrates/enzymes.12–17,8
The most common chemical reporter that has been employed is
the azide functionality as it can be chemically modified via either
the Staudinger ligation or the Huisgen’s cyclization (click chemis-
try).18,19 Much of the work to date has focused on amino sugars,
such as N-acetylglucosamine and N-acetylneuraminic acid, as
these are common components of many biologically important
glycoconjugates. In these cases, the azide functionality has typi-
cally been incorporated into the N-acetamido functionality as an
N-azidoacetyl substituent.12,14,7–10 One potential problem with this
approach is that the N-acetamido functionality may serve as a key
recognition element for glycosyltransferases, and the extra steric
bulk of the azide group may preclude such sugar nucleotides from
acting as substrates. In order to expand the tool kit available to the
chemical glycobiologist we decided to prepare 600-deoxy-600-azido-
The glycosyltransferase reaction we chose to probe was that of
the human UDP-GlcNAc:polypeptidyltransferase (OGT).20–23 This
enzyme is responsible for the glycosylation of many cellular pro-
tein targets at specific serine and threonine residues with an O-
linked-N-acetylglucosamine residue (O-GlcNAc). This posttransla-
tional modification has been identified on a wide variety of pro-
teins including transcription factors, nuclear pore proteins, and
enzymes. In certain cases, the site of glycosylation coincides with
that of phosphorylation indicating that reversible glycosylation
may play a role in cellular signaling. The protein substrate we
chose to analyze is the nuclear pore protein p62 (nup62).24–26 It
is well established that this protein is extensively modified with
as many as 10 N-acetylglucosamine residues by the action of OGT.
We chose to use a purely chemical route for the synthesis of
UDP-6Az-GlcNAc 1 starting from the
a-benzylglycoside of N-ace-
tylglucosamine 2 (Scheme 1). A one-pot tosylation of the 6-hydro-
xyl followed by peracetylation gave compound 3 in a 75% yield.
The anomeric center of compound 3 was deprotected via hydrog-
enolysis and the crude product was immediately subjected to
phosphitylation with dibenzyl N,N-diisopropyl phosphoramidite
and then oxidation with mCPBA. This gave the
a-dibenzylphos-
phate 4 in an overall 70% yield. It should be noted that any b-dib-
enzylphosphate that forms in this procedure readily decomposes
due to neighboring group participation from the acetamido func-
⇑
Corresponding author. Tel.: +1 604 822 9453; fax: +1 604 822 2847.
E-mail address: mtanner@chem.ubc.ca (M.E. Tanner).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.090

1200
A. Mayer et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1199–1201
N3
OGT
O
N3
HO
+
nup62
nup62
O
HO
O
HO
HO
AcHN
O
OUDP
HO
AcHN
azidoglycosylated nup62
Ph2P
UDP-6Az-GlcNAc 1
O
HN
NH
OCH3
H
H
O
H
N
O
S
(CH2)4
O
NH
2
O
biotin phosphine probe
O
Ph2P
O
O
HN
NH
NH
HO
HO
H
H
O
H
N
nup62
O
AcHN
O
O
S
(CH2)4
NH
O
2
O
biotinylated nup62
Scheme 1.
Figure 1. OGT can use UDP-6Az-GlcNAc as a substrate donor to transfer 6Az-
GlcNAc onto the protein acceptor nup62. The azido moiety undergoes the
Staudinger ligation upon treatment with a biotin phosphine probe. The biotin
moiety can be detected by Western blot using horseradish peroxidase-conjugated
streptavidin.
tionality at C-2.27 Thus, it is not necessary to separate anomeric
phosphates. Dibenzylphosphate 4 was then converted to UDP-
6Az-GlcNAc 1 in a four-step procedure without purification of
the intermediate compounds. Hydrogenolysis followed by treat-
ment with triethylamine in water/methanol was used to deprotect
the phosphate and hydroxyl groups, respectively.28 Treatment
with sodium azide in DMF led to the displacement of the tosyl
group and incorporation of the azide at C-6. This was left to a late
stage in the synthesis to avoid problems with performing hydrog-
enolysis in the presence of an azide functionality. Finally, treat-
ment with UMP-morpholidate and tetrazole generated UDP-6Az-
GlcNAc 1.29 Following anion exchange and size exclusion chroma-
tography, a sample of compound 1 was obtained that contained an
impurity of 6Az-GlcNAc 1-phosphate due to incomplete coupling.
Treatment with alkaline phosphatase was used to dephosphorylate
the impurity and a second round of ion exchange chromatography
provided compound 1 in a pure form with an overall yield of 12%
from compound 4. It should be noted that a recent report has de-
scribed the chemoenzymatic synthesis of compound 1 from 6-
deoxy-6-azido-N-acetylglucosamine.30,31 Cai et al. used the N-ace-
tylglucosamine 1-kinase NahK and ATP to introduce the anomeric
phosphate and then used the N-acetylglucosamine 1-phosphate
uridyltransferase GlmU and UTP to introduce the UDP functional-
ity. While their chemoenzymatic approach is attractive due to
the facile introduction of the phosphate functionalities, our purely
synthetic approach may be preferable to researchers who do not
wish to clone and express the NahK and GlmU enzymes. Overall,
the two approaches require a similar number of overall steps and
result in a similar overall yield of pure product.
Figure 2. OGT can utilize UDP-6Az-GlcNAc as a substrate to modify nup62 with
6Az-GlcNAc in vitro. (a) Western blot analysis of OGT modification of nup62 with
UDP-6Az-GlcNAc was assessed for its ability to be used as a sub-
strate donor for OGT in vitro using nup62 as an acceptor. Assays
were performed at pH 7.4 and 37 °C and typically allowed to pro-
ceed for 1 h (over which time OGT activity has been shown to be
linear). Following incubation of UDP-6Az-GlcNAc with OGT and
nup62, the reaction was terminated, and then incubated with a
biotin phosphine probe (Fig. 1).10 The reactive azido group of the
6Az-GlcNAc undergoes the Staudinger ligation with the phosphine,
to generate biotin-labeled nup62. Western blot analysis, where the
biotin group could be probed with horseradish peroxidase-conju-
gated streptavidin, allowed visualization of the 6Az-GlcNAc trans-
fer onto nup62.
Western blot analysis demonstrated that OGT was capable of
utilizing UDP-6Az-GlcNAc as a substrate and does indeed transfer
6Az-GlcNAc onto nup62 (Fig. 2a), whereas in the absence of either
OGT or nup62 there is little or no signal. Given that no signal is
seen in lane 1 of Figure 2d (no nup62), the faint band in lane 2
of Figure 2a (no nup62) likely stems from trace sample spill-over
from lane 3. The signal, which correlates with the amount of
6Az-GlcNAc transferred, increases when a higher concentration
6Az-GlcNAc (at a concentration of 50 lM or 200 lM). (b) Western blot analysis of
OGT modification of nup62 with 6Az-GlcNAc at varying concentrations of OGT
(from 288 nM to 36 nM, left to right). OGT activity was terminated using 100 mM
UDP at either 0 h (left) or 2 h (right) prior to incubation with the biotin phosphine.
(c) Western blot analysis of OGT modification of nup62 with 6Az-GlcNAc at varying
concentrations of OGT (from 288 nM to 36 nM, left to right). OGT activity was
terminated by boiling the sample for 5 min at either 0 h (left) or 2 h (right) prior to
incubation with the biotin phosphine. (d) Western blot analysis of OGT modifica-
tion of nup62 with 6Az-GlcNAc to demonstrate it occurs in a time-dependent
manner. In all cases, the blots were probed with streptavidin, which binds to the
biotin; the biotin is conjugated to a phosphine probe, which had undergone the
Staudinger ligation with the azido group of the 6Az-GlcNAc prior to gel analysis.
of UDP-6Az-GlcNAc is used in the assay. Two ways to terminate
OGT activity prior to incubation with the biotin phosphine probe
were tested; by boiling the assay mixture for 5 min to precipitate
the proteins or by addition of 100 mM UDP (UDP has previously
been shown to be a good inhibitor of OGT, with a reported IC50
of 1.8 l
M).32 Both methods were shown to work well to terminate
OGT activity over a range of OGT concentrations (Fig. 2b,c, 0 h time
points), with no detectable signal following incubation with the

A. Mayer et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1199–1201
1201
biotin phosphine probe (which is performed at 37 °C for 1.5 h).
References and notes
Using these methods, we were able to demonstrate that the degree
of 6Az-GlcNAc modification was dependent on the enzyme con-
centration (Fig. 2b,c, 2 h time points). In addition, we were able
to show that the modification of nup62 with 6Az-GlcNAc by OGT
occurred in a time-dependent manner (Fig. 2d), with maximum
modification reached by approximately 70 min.
1. Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13.
2. Koenigs, M. B.; Richardson, E. A.; Dube, D. H. Mol. BioSyst. 2009, 5, 909.
3. Liu, F.; Aubry, A.; Schoenhofen, I. C.; Logan, S. M.; Tanner, M. E. ChemBioChem
2009, 107, 1317.
4. Dube, D. H.; Prescher, J. A.; Quang, C. N.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 4819.
5. Rabuka, D.; Hubbard, S. C.; Laughlin, S. T.; Argade, S. P.; Bertozzi, C. R. J. Am.
Chem. Soc. 2006, 128, 12078.
In summary, we were able to demonstrate that UDP-6Az-Glc-
NAc serves as an alternate substrate for a key human N-acetylgly-
cosaminyltransferase, OGT. This provides an attractive alternative
to the use of N-azidoacetamido-substituted sugars and could be
particularly useful in cases where such compounds do not serve
as substrates. While both types of azide-substituted sugar donors
are accepted by OGT, it is likely that this will not be the case for
all N-acetylglucosaminyltransferases, and that certain transferases
will not tolerate modifications to the acetamido moiety. In fact, ini-
tial results with certain glycosaminoglycan synthases have indi-
cated that UDP-6Az-GlcNAc serves as an alternate substrate,
whereas UDP-N-azidoacetylglucosamine (UDP-GlcNAz) does not
(P.L. DeAngelis and R.J. Linhardt, personal communication). One
disadvantage to the approach of installing the azide at the C-6 po-
sition, however, is that one cannot feed the free 6Az-GlcNAc to
mammalian cells and expect that the UDP moiety will be installed
in vivo. This is because the conversion of GlcNAc into UDP-GlcNAc
first requires a phosphorylation of the C-6 hydroxyl group by N-
acetylglucosamine 6-kinase. Thus, this technology will be largely
restricted to in vitro labeling studies.
6. Nandi, A.; Sprung, R.; Barma, D. K.; Zhao, Y.; Kim, S. C.; Falck, J. R.; Zhao, Y. Anal.
Chem. 2006, 78, 452.
7. Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 430, 873.
8. Vocadlo, D. J.; Hang, H. C.; Kim, E.-J.; Hanover, J. A.; Bertozzi, C. R. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 9116.
9. Hang, H. C.; Yu, C.; Kato, D. L.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 14846.
10. Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007.
11. Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125.
12. Clark, P. M.; Dweck, J. F.; Mason, D. E.; Hart, C. R.; Buck, S. B.; Peters, E. C.;
Agnew, B. J.; Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2008, 130, 11576.
13. Marchesan, S.; MacMillan, D. Chem. Commun. 2008, 4321.
14. Hang, H. C.; Yu, C.; Pratt, M. R.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126, 6.
15. Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 13132.
16. Tai, H.-C.; Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C. J. Am.
Chem. Soc. 2004, 126, 10500.
17. Khidekel, N.; Arndt, S.; Lamarre-Vincent, N.; Lippert, A.; Poulin-Kerstein, K. G.;
Ramakrishnan, B.; Qasba, P. K.; Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2003, 125,
16162.
18. Kohn, M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43, 3106.
19. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
20. Gloster, T. M.; Vocadlo, D. J. Curr. Signal Transduction Ther. 2010, 5, 74.
21. Hanover, J. A. FASEB J. 2001, 15, 1865.
22. Haltiwanger, R. S.; Holt, G. D.; Hart, G. W. J. Biol. Chem. 1990, 265, 2563.
23. Lubas, W. A.; Frank, D. W.; Krause, M.; Hanover, J. A. J. Biol. Chem. 1997, 272,
9316.
Acknowledgements
24. Hanover, J. A.; Cohen, C. K.; Willingham, M. C.; Park, M. K. J. Biol. Chem. 1987,
262, 9887.
This research was supported by the Canadian Institutes of
Health Research (CIHR) and the Natural Sciences and Engineering
Research Council of Canada (NSERC). A.M. was supported by a fel-
lowship from the Swiss National Science Foundation (grant No.
PBBEA-112767). T.M.G. is a Sir Henry Wellcome postdoctoral fel-
low and a Michael Smith for Health Research trainee award holder.
D.J.V. is a Canada Research Chair in Chemical Glycobiology and a
Scholar of the Michael Smith Foundation for Health Research.
25. Davis, L. I.; Blobel, G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7552.
26. Holt, G. D.; Snow, C. M.; Senior, A.; Haltiwanger, R. S.; Gerace, L.; Hart, G. W. J.
Cell Biol. 1987, 104, 1157.
27. Sim, M. M.; Kondo, H.; Wong, C.-H. J. Am. Chem. Soc. 1993, 115, 2260.
28. Yeager, A. R.; Finney, N. S. J. Org. Chem. 2005, 70, 1269.
29. Srivastava, G.; Alton, G.; Hindsgaul, O. Carbohydr. Res. 1990, 207, 259.
30. Guan, W.; Cai, L.; Fang, J.; Wu, B.; Wang, P. G. Chem. Commun. 2009, 6976.
31. Cai, L.; Guan, W.; Kitaoka, M.; Shen, J.; Xia, C.; Chen, W.; Wang, P. G. Chem.
Commun. 2009, 2944.
32. Dorfmueller, H. C.; Borodkin, V. S.; Blair, D. E.; Pathak, S.; Navratilova, I.; van
Aalten, D. M. Amino Acids, in press. doi:10.1007/s00726-010-0688-y.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.090.