Chemoenzymatic synthesis of GDP-azidodeoxymannoses: Non-radioactive probes for mannosyltransferase activity

Article abstract of DOI:10.1039/b807016d

GDP-2-, 3-, 4- or 6-azidomannoses can be successfully prepared from the corresponding azidomannose-1-phosphates and GTP using the enzyme GDP-Mannosepyrophosphorylase (GDP-ManPP) from Salmonella enterica and may serve as useful probes for mannosyltransferase activity. The Royal Society of Chemistry.

Full text of DOI:10.1039/b807016d

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Chemoenzymatic synthesis of GDP-azidodeoxymannoses: non-radioactive
probes for mannosyltransferase activityw
Silvia Marchesan and Derek Macmillan*
Received (in Cambridge, UK) 25th April 2008, Accepted 2nd June 2008
First published as an Advance Article on the web 18th July 2008
DOI: 10.1039/b807016d
GDP-2-, 3-, 4- or 6-azidomannoses can be successfully prepared
from the corresponding azidomannose-1-phosphates and GTP
using the enzyme GDP-Mannosepyrophosphorylase (GDP-
ManPP) from Salmonella enterica and may serve as useful
probes for mannosyltransferase activity.
a-phosphate group, required for GDP-ManPP activity, to
yield 2-, 3-, 4- and 6-azidodeoxy mannose compounds 5–8
respectively.
To test 5–8 as unnatural substrates for GDP-ManPP, we
overexpressed the protein in E. coli (strain ER2566(DE3))
transformed with the plasmid pET16b-GDP-ManPP. Induc-
tion at OD600 = 0.6 with 1 mM IPTG at 30 1C for 5 h yielded
the poly-histidine tagged protein, which was purified from the
cell free extract by Nickel affinity chromatography. SDS-
PAGE and Western blot analysis (using an antibody raised
against the poly-histidine tag) confirmed the identity and
purity of GDP-ManPP. Typical yields of 1.1 mg LÀ1 GDP-
ManPP were obtained using this method. The enzyme was
concentrated and the solution exchanged against 50 mM Tris;
pH 7.6, 8 mM MgCl2, 1 mM DTT to test for activity, initially
with 6-azidomannose-1-phosphate (8). The reaction was mon-
itored by HPLC and the formation of the corresponding
GDP-6-azidodeoxy derivative 9 was observed (Fig. 1(a)).
After 60 h at 37 1C, the protein content was removed by
filtration of the reaction mixture through a 10 kDa molecular
weight cut-off membrane, and the resulting solution was
purified by anion-exchange HPLC in a gradient of ammonium
formate. The desired GDP-6-azidodeoxymannose 9 (33 mg,
The discovery of promiscuous carbohydrate processing en-
zymes has provided chemists with powerful tools to readily
access unnatural glycoconjugates with complete regio- and
stereoselectivity, in water, and with minimal protecting group
manipulation.1 It has also facilitated metabolic engineering
approaches employing tagged sugars displaying functional
groups, such as ketones, which may alter the properties of
various targeted cellular components.2 This methodology was
rapidly extended to sugars containing azides or acetylenes
combined with the Staudinger ligation, or ‘‘click’’ chemistry
for further manipulation.3 Such methods have also been used
to explore glycosyltransferase activity and substrate specificity
in vitro and in vivo. In most cases, it is the N-acetyl group of
glucosamine, mannosamine or galactosamine which carries
the azide functionality.4 Herein we report the first chemoenzy-
matic synthesis of four GDP-azidodeoxymannoses and
investigate their use as substrates for a mannosyltransferase
(ManT). Being a common constituent of N-linked glyco-
proteins, and a direct modification of mammalian glycopro-
teins in the extracellular matrix, it is clear that the availability
of tools to profile mannose transfer may have valuable appli-
cations in disease diagnosis, discovery of novel mannosyl-
transferase activities and in glycoprotein remodelling.5
1
63%), was confirmed by H, 13C and 31P NMR spectroscopy
and high-resolution mass spectrometry. Having demonstrated
enzymatic activity with 6-azidomannose-1-phosphate, we next
optimised a colourimetric activity assay in order to compare
the remaining isomers with the natural substrate in a high
A key requirement is access to activated sugar nucleotides.
We investigated the potential for GDP-mannose pyrophos-
phorylase (GDPManPP, EC. 2.7.7.13)6 to produce our desired
azidodeoxy products. Ultimately this provides the potential
for conducting GDP-azidomannose synthesis and glycosyl
transfer in one pot, which is not likely to be possible if they
were prepared synthetically.7 First, the azide was installed at
four positions on the pyranose ring (1–4), by using displace-
ment reactions of sodium azide on suitably protected tosylate
and triflate precursors (Scheme 1).8 Phosphoramidite chemis-
try at the anomeric centre allowed for the insertion of the
Department of Chemistry, University College London, 20 Gordon
Street, London, UK WC1H 0AJ. E-mail: d.macmillan@ucl.ac.uk;
Tel: 020 7679 4684
w Electronic supplementary information (ESI) available: Experimental
details for synthesis of compounds 1–9, expression, purification and
use of GDP-ManPP and ManT and for the enzymatic activity. See
DOI: 10.1039/b807016d
Scheme 1 Reagents and conditions: (a) i-Pr2NPO(OAll)2, 1H-tetra-
zole, DCM, À40 1C to room temp., 46–72%. (b) p-TolSO2Na,
Pd(PPh3)4, THF–MeOH, room temp.; then MeOH–H2O–Et3N
(5 : 2 : 1), 86–99%.
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Fig. 1 (a) Anion-exchange HPLC monitoring of the GDP-ManPP catalyzed synthesis of GDP-6-azido-6-deoxymannose 9 (peak at 13 min = 9,
peak at 22 min = GTP). (b) This methodology is applicable to the synthesis of all the azidodeoxymannose isomers as observed by a colourimetric
activity assay: B = blank, M1P = mannose-1-phosphate as substrate, 6/2-N3 = 6/2-azidodeoxy mannoses as substrates. (c) ESI-MS analysis of
the enzymatic reaction mixture shows that GDP-4-azidodeoxymannose is incorporated most readily by the a-1,2-ManT into the disaccharide 4-
AzMan(a-1,2)Man-a-OMe.
throughput fashion (Fig. 1(b)). The screen was based on the
inorganic pyrophosphatase-mediated hydrolysis of this reac-
tion byproduct, and subsequent formation of a complex
between phosphate, ammonium molybdate, and malachite
green dye, which shifted the dye absorbance maximum to
650 nm.9 Typically, the reagent was calibrated against a series
of phosphate standards and a standard curve was generated
against which 25 mL reaction samples were analysed at timed
intervals.8 Subsequently the 2-, 3- and 4-azido derivatives were
also isolated in 41, 52 and 55% yield, respectively, and
characterised by 1H, 13C and 31P NMR spectroscopy and
high-resolution mass spectrometry.
In conclusion, we have developed an effective chemoenzy-
matic route to four GDP-azidodeoxymannoses. The colouri-
metric GDP-ManPP activity assay proved to be a useful
analytical tool to monitor product formation and will facilitate
optimisation of the enzymatic syntheses of azide-containing
compounds. We also demonstrated that a recombinant a-1,2-
ManT from S. cerevisiae can successfully process GDP-4-
azidomannose. Consequently GDP-azidomannoses should
function as useful non-radioactive probes to investigate ManT
activity and, when successfully transferred to substrates, these
compounds provide useful synthetic handles for further en-
gineering via azide chemistry.11
If the GDP azidomannose products were to find utility, for
example, as probes for mannosyltransferase activity or glyco-
protein remodelling, it was essential to investigate whether
they can subsequently be transferred by ManTs to potential
substrates. In a model system we over-expressed the soluble
fragment of the S. cerevisiae a-1,2-ManT from E. coli.10 After
partial purification, the protein was concentrated (to 1 ml LÀ1
culture), exchanged against an appropriate buffer for subse-
quent MS analysis (100 mM ammonium formate, pH 7.5), and
characterised by SDS-PAGE and Western blot. The reaction
mixture containing the protein (107.2 mL), the GDP-mannose
donor (1.25 mM) and the a-methyl mannoside acceptor
(7.5 mM), in a total volume of 160 ml of 50 mM ammonium
formate buffer pH 7.5, 10 mM MnCl2, was incubated at 30 1C
for 24 h. The protein content was removed and the filtrate was
analysed by ESI-MS. Unsurprisingly, not all the isomers
appeared to be converted to the desired product, however
GDP-4-azidodeoxymannose was successfully incorporated
into the disaccharide 4-AzMan(a-1,2)Man-a-OMe (Fig.
1(c)). This preliminary observation opens the door to in vitro
profiling of mannosyltransferase activity and to the remodel-
ling of mannose-containing glycoconjugates such as glyco-
proteins by using the azide as a synthetic handle.
The authors acknowledge financial support from The Royal
Society, The University of Edinburgh and UCL. The RfbM
gene from S. enterica strain LT2, encoding the GDP-ManPP
was originally obtained as a gift from Prof. L. Elling.
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