Enzymatic synthesis of nucleobase-modified UDP-sugars: Scope and limitations

Article abstract of DOI:10.1016/j.carres.2014.12.005

Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields. Attempts to effect 4″-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4″-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.

Full text of DOI:10.1016/j.carres.2014.12.005

Carbohydrate Research 404 (2015) 17–25
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Enzymatic synthesis of nucleobase-modified UDP-sugars: scope and
limitations
Ben A. Wagstaff a, , Martin Rejzek a, , Thomas Pesnot b, Lauren M. Tedaldi c, Lorenzo Caputi a,d
,
Ellis C. O’Neill a, Stefano Benini d, , Gerd K. Wagner b,c, , Robert A. Field a,
⇑
⇑
⇑
a Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
b School of Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
c King’s College London, Faculty of Natural & Mathematical Sciences, Department of Chemistry, Britannia House, 7 Trinity Street, London SE1 1DB, UK
d Bioorganic Chemistry and Bio-Crystallography laboratory, Faculty of Science and Technology, Free University of Bolzano, Piazza Università 5, 39100 Bolzano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2014
Received in revised form 11 December 2014
Accepted 12 December 2014
Available online 31 December 2014
Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found
to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galact-
ose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast,
UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly
effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of
gluco-configured 5-substituted UDP-sugar derivatives in good yields. Attempts to effect 400-epimerization
of these 5-substituted UDP-glucose with UDP-glucose 400-epimerase from yeast were unsuccessful, while
use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only
5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the estab-
lished potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access
to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.
Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
Keywords:
Sugar nucleotide
Modified nucleobase
Enzymatic synthesis
Pyrophosphatase
Epimerase
1. Introduction
UDP-sugars with a fluorogenic substituent at position 5 of the
uracil base (Fig. 1).10,11 In a proof of concept study, Wagner et al.
Glycosyltransferases (GTs) are a large class of carbohydrate
active enzymes that are involved in numerous important biological
processes, with impact in cellular adhesion, carcinogenesis and
neurobiology, amongst many others.1–3 As such, GTs have enor-
mous potential as targets for drug discovery. For the full realization
of this potential, both chemical inhibitors, and operationally sim-
ple and generally applicable GT bioassays, especially for high-
throughput inhibitor screening, are indispensable tools.4 Many
GTs use UDP-sugars as their donor substrates, and non-natural
derivatives of these sugar-nucleotides are therefore of considerable
interest as GT inhibitor candidates and assay tools.5 Wagner et al.
have recently described 5-substituted UDP-sugars (Fig. 1) as a new
class of GT inhibitors with a unique mode of action.6–9 Depending
on the nature of the 5-substituent, these 5-substituted UDP-sugars
also exhibit useful fluorescent properties,10–12 and we have
recently reported a series of novel auto-fluorescent derivatives of
demonstrated that fluorescence emission by 5-formylthienyl-
UDP- -galactose (1f) is quenched upon specific binding to
a
-D
several retaining galactosyltransferases (GalTs), and that this effect
can be used as a read-out in ligand-displacement experiments.11
To date, such 5-substituted UDP-sugar probes had to be prepared
using chemical synthesis (reviewed in Ref. 5). For instance, Wagner
et al. showed that it is possible to directly transform 5-iodo-
UDP-a-D-Gal (1b) into 5-formylthienyl-UDP-a-D-Gal (1f) using
Suzuki coupling under aqueous conditions.6 The aim of the current
work was to explore alternative methods for the preparation of
5-substituted UDP-sugars (1–4) using chemo-enzymatic
approaches (reviewed in Ref. 13) starting from 5-substituted UTP
derivatives 5b–f.12
2. Results and discussion
Access to gluco- and galacto-configured UDP-sugars lies at the
heart of this study. In brief, enzymatic synthesis approaches to
such compounds may employ a number of different enzymes,
either affording the required sugar nucleotide via pyrophosphate
⇑
Corresponding authors. Tel.: +44 1603 450720; fax: +44 1603 450018 (R.A.F.).
E-mail addresses: stefano.benini@unibz.it (S. Benini), gerd.wagner@kcl.ac.uk
(G.K. Wagner), rob.field@jic.ac.uk (R.A. Field).
These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.carres.2014.12.005
0008-6215/Ó 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

18
B. A. Wagstaff et al. / Carbohydrate Research 404 (2015) 17–25
O
N
2.1. Enzymatic synthesis of 5-substituted UDP-Gal derivatives
using a GalU-GalPUT protocol
OH
O
N
HO
5
4''
X
X
O
NH
NH
1''
HO
O
O
O
O
O
O
O
R
2.1.1. One-pot GalU-GalPUT reactions
O P O P O
OH
HO P O P O P O
OH OH
O
O
1'
In an attempt to generate 5-substituted UDP-Gal derivatives
1b–f, a multienzyme approach was assessed (Scheme 1).14,15 This
protocol employs UTP (5a) and glucose-1-phosphate with
UDP-glucose pyrophosphorylase (GalU, EC 2.7.7.9) to generate
UDP-Glc (2a) in situ. Galactose-1-phosphate uridylyltransferase
(GalPUT, EC 2.7.7.12) then catalyses the reaction of UDP-Glc (2a)
OH
OH
OH OH
OH OH
5
1 4'' ax, R = OH
2
R
4'' eq, = OH
3 4'' eq, R = NH2
a
b
c
d
e
f
4
R
4'' eq, = NHAc
O
S
and
a-D-galactose-1-phosphate, giving the corresponding
H
I
X =
UDP-Gal (1a). UDP-Glc (2a) is only produced in catalytic quantity
(typically 0.5 mol % related to sugar-1-phosphate) as it is continu-
ously recycled, via Glc-1-P, by the action of GalU (Scheme 1). In this
reaction, inorganic pyrophosphate is released and inorganic pyro-
phosphatase (IPP) is employed to achieve its hydrolysis, driving
the overall equilibrium of the multi-enzyme reaction towards the
formation of the desired UDP-Gal (1a) sugar nucleotide.
In a control experiment, Gal-1-P was converted into UDP-Gal
(1a) using an equimolar quantity of UTP (5a) and a catalytic
amount of UDP-Glc (2a). The transformation reached a complete
conversion (by SAX HPLC) within 1 h (data not shown). Next, the
5-substituted UTP derivatives 5b–f were used in combination with
CHO
OMe
Figure 1. Target nucleobase-modified UDP-sugar derivatives (1)–(4) and UTP
precursors (5).
bond formation [action of uridylyltransferase (GalPUT) or pyro-
phosphorylase (GalU)], or by epimerization of the C-400 stereo-
chemistry of the pre-formed sugar nucleotide [action of
epimerase (GalE)] (Scheme 1).13 We have employed both the
former and latter approaches in syntheses of natural14 and
non-natural14–17 sugar nucleotides.
O
X
NH
O
O
N
O
O
HO P O P O P O
O
OH
OH OH
OH OH
IPP
PPi
2 Pi
UTP (5a)
GalU
O
N
OH
X
OH
O
O
NH
HO
HO
HO
HO
O
O
O
HO
O
P
O P O P O
OH OH
OH
O
O
OH
OH OH
OH
Glc-1-P
UDP-Glucose (2a)
GalPUT
OH
HO
HO
O
N
OH
O
HO
HO
O
X
NH
O
P
OH
O
O P O P O
OH
O
O
HO
O
OH
O
OH
Gal-1-P
OH
OH OH
UDP-Galactose (1a)
GalE
Scheme 1. Strategies for enzymatic preparation of based-modified UDP-Glucose and UDP-Galactose. X is as outlined in Figure 1; GalPUT = galactose-1-phosphate
uridylyltransferase; GalU = UDP-glucose pyrophosphorylase; GalE = UDP-Galactose 400-epimerase; IPP = inorganic pyrophosphatase. Arrows in black indicate a one pot
reaction; arrows in grey indicate a separate reaction.

B. A. Wagstaff et al. / Carbohydrate Research 404 (2015) 17–25
19
Gal-1-P in an attempt to generate the corresponding 5-substituted
UDP-Gal derivatives 1b–f (Fig. 1; Scheme 1). In all cases, reaction
with the 5-substituted UTPs was slower than with the parent
compound. After 24 h incubation, formation of product was
detected in the case of 5-(4-methoxyphenyl)-UDP-Gal (1d) (5%)
and 5-(2-furyl)-UDP-Gal (1e) (23%) and the products were isolated
and characterized. The formation of 5-(4-methoxyphenyl)-UDP-
Glc (2d) and 5-(2-furyl)-UDP-Glc (2e) as intermediates in the reac-
tion is implicit, but their presence in the reaction mixture was not
detected. The 5-iodo-UTP (5b) and the 5-(5-formyl-2-thienyl)-UTP
(5f) derivatives were not converted into the corresponding sugar
nucleotides at all; in the case of the 5-phenyl-UTP (5c), the conver-
sion was less than 5% and the product 1c was not isolable. In order
to assess which of the enzymes, GalU or GalPUT, is failing to use
these latter base-modified compounds as substrates, a series of
reverse reactions and inhibition experiments was performed.
pete with UDP-Gal (1a) to bind in the active site of GalPUT. This
implies that the formylthienyl substitution of the uracil base pre-
vents the corresponding sugar nucleotides from binding to GalPUT
and explains the observed lack of conversion of 2f into 1f in the
one-pot GalU-GalPUT protocol. However, the lack of conversion
might also be due to a lack of tolerance of GalU for 5-substitution
of the uracil ring of UTP.
2.1.4. Competing 5-substituted-UTP and unsubstituted UTP as
GalU substrates
A series of experiments were conducted to assess the flexibility
of GalU towards 5-substitution of its UTP substrate. A control
experiment [GalU, Glc-1-P, UTP (5a)] showed the rapid conversion
of UTP (5a) into UDP-Glc (2a), as indicated by the diagnostic uracil
H6 signals in 1H NMR spectra (Fig. 3A). In a competition experi-
ment employing Glc-1-P, UTP (5a) and 5-iodo-UTP (5b) in molar
ratio 1:1:5, UTP (5a) remained almost completely intact (Fig. 3B).
Instead, 5-iodo-UTP (5b) was rapidly converted into 5-iodo-UDP-
Glc (2b), as shown by a new H6 signal (Fig. 3B) and confirmed by
LC–MS: a molecular ion for 5-iodo-UDP-Glc (2b) ([MꢀH]ꢀ m/z
691) was detected, but one for UDP-Glc (2a) ([MꢀH]ꢀ m/z 565)
was absent. These data suggest that, if used in excess, 5-iodo-
UTP (5b) can out-compete UTP (5a), the natural substrate of GalU,
indicating some degree of relaxed GalU substrate specificity. The
fact that no formation of 5-iodo-UDP-Gal (1b) was observed in
the multi-enzyme transformation (Scheme 1) suggests that
although a small quantity of 5-iodo-UDP-Glc (2b) may have been
formed in that reaction, it could not be further processed by
GalPUT.
2.1.2. The GalPUT reaction in reverse
In the presence of excess Glc-1-P, GalPUT can be used to run the
reverse conversion, UDP-Gal (1a) into UDP-Glc (2a). As shown in
Figure 2A, after 1 h the conversion of substrate into product is
nearly complete, as judged by 1H NMR analysis of the diagnostic
anomeric signals (dd) of the sugar phosphates. When a synthetic
sample of 5-(5-formyl-2-thienyl)-UDP-Gal (1f) was subjected to
equivalent conditions, no conversion was observed after 1 h (data
not shown). After extended incubation (24 h) only traces of
5-(5-formyl-2-thienyl)-UDP-Glc (2f) and Gal-1-P were detectable
by 1H NMR (Fig. 2B). This result suggests that 5-(5-formyl-2-
thienyl)-UDP-Gal (1f) either does not bind to the GalPUT active site
or that it might bind in a non-productive way. If the latter were
true, 1f should act as a GalPUT inhibitor.
2.2. Enzymatic synthesis of 5-substituted UDP-Glc 2b–f using
GalU
2.1.3. 5-(5-Formyl-2-thienyl)-UDP-Gal as a GalPUT inhibitor
When UDP-Gal (1a), Glc-1-P and 1f were mixed in a molar ratio
1:10:3.5, the conversion to UDP-Glc (2a) after 30 min was the same
as in the absence of 1f (not shown), implying that 1f does not com-
2.2.1. GalU reactions with substituted UTPs
The results presented above indicate that GalU possesses a
degree of substrate flexibility regarding 5-substitution of UTP,
O
N
O
N
OH
large excess
OH
O
HO
HO
X
X
O
NH
NH
HO
HO
OH
O
OH
HO
HO
1"
1"
O
O
O
O
O
O
O
HO
HO
HO
HO
O P O P O
OH
O P O P O
OH
O
P
GalPUT
O
P
O
O
+
OH
+
OH
O
OH
OH
OH
O
OH
OH OH
OH OH
OH
OH
1a
1f
2a
2f
X = H (UDP-Gal)
X = 5-formyl-2-thienyl
Glc-1-P
X = H (UDP-Glc)
X = 5-formyl-2-thienyl
Gal-1-P
Figure 2. Reverse action of GalPUT. (A) Incubation of UDP-Gal (1a) and Glc-1-P. (B): Incubation of 5-(5-formyl-2-thienyl)-UDP-Gal (1f) and Glc-1-P.

20
B. A. Wagstaff et al. / Carbohydrate Research 404 (2015) 17–25
O
O
N
OH
NH
NH
O
HO
HO
6
O
O
O
O
O
N
O
O
OH
HO P O P O P O
OH OH OH
O
O
P O P O
OH OH
O
O
OH OH
OH OH
GalU
5a
2a
O
O
Glc-1-P
+
OH
I
I
NH
NH
O
HO
HO
O
O
O
N
O
O
O
O
N
O
O
PPi
HO
HO P O P O P O
OH OH OH
P O P O
OH OH
OH OH
OH OH
5b
2b
Figure 3. UTP (5a) and 5I-UTP (5b) competition in GalU mediated conversion of Glc-1-P into the corresponding sugar nucleotides. (A) control with UTP (5a) only. (B) UTP (5a)
and large excess 5I-UTP (5b).
O
N
O
N
OH
X
X
NH
NH
O
HO
HO
O
O
O
O
O
O
O
GalU
HO
Glc-1-P
HO P O P O P O
OH OH
+
O
P O P O
OH
O
O
OH
OH
equimolar
OH OH
OH OH
PPi
IPP
5a-f
2a-f
2 Pi
Figure 4. GalU-mediated formation of 5-substituted UDP-Glc derivatives in the absence of IPP (time point 120 min). X indicates: a = H, b = I, c = Ph, d = 4-MeO-Ph, e = 2-furyl,
f = 5-formyl-2-thienyl.

B. A. Wagstaff et al. / Carbohydrate Research 404 (2015) 17–25
21
potentially offering easy access to 5-substituted UDP-Glc (2b–f)
derivatives. This was indeed the case when 5-substituted UTP
derivatives 5b–f and an equimolar amount of Glc-1-P were
subjected to GalU (Fig. 4). Conversions to the corresponding sugar
nucleotides 2b–f ranged from 9% to 54% after 120 min. Unsurpris-
ingly, the lowest conversion was detected for the bulky 5-(5-for-
myl-2-thienyl)-derivative 2f. A control reaction of UTP (5a) with
Glc-1-P under the same conditions gave 57% conversion to
UDP-Glc (2a). When inorganic pyrophosphatase (IPP) was added
to reactions, the conversions could be further improved (Fig. 4).
Under these conditions, the conversion of 5f into the
5-(5-formyl-2-thienyl)-derivative 2f was a tolerable 21%. GalU also
showed remarkable substrate flexibility towards the configuration
of sugar-1-phosphates18—a feature that it has in common with
other pyrophosphatases, such as RmlA.19,20 When UTP (5a) was
employed as a co-substrate, GalU proved capable of accepting
1H NMR spectra showed that only 5-iodo-UDP-Glc (2b) was
formed and no trace of 5-iodo-UDP-Gal (1b) was detected, even
after 120 min (not shown).
From the above data, it is evident that GalU is not able to
simultaneously bind both Gal-1-P and 5-iodo-UTP (5b), although
both in their own right are productive substrates in the presence
of alternative co-substrates. It may be that a conformational
change is required in order to enable co-substrates to bind to GalU
in a productive manner, but this is either too slow, or it does not
happen at all, when Gal-1-P and 5-iodo-UTP are employed. Further
structural analyses are required in order to address this point.
2.3. Enzymatic epimerization of 5-iodo-UDP-Glc (2b) to give the
corresponding 5-iodo-UDP-Gal (1b) using GalE
a
-
D
-glucosamine-1-phosphate (GlcN-1-P) and N-acetyl-
a-D-gluco-
As noted above, GalU successfully produces a range of base-
modified gluco-configured UDP-sugars but fails to produce the
corresponding galacto-configured compound. The one-pot, GalU-
GalPUT protocol showed some flexibility, producing galacto-
configured analogues 1d and 1e in low yield, but 1b, 1c and 1f
were not accessible by this route. An alternative approach to the
galacto-configured series is an epimerization of 400-OH in the
base-modified UDP-Glc derivatives. Uridine-50-diphosphogalactose
400-epimerase (GalE, E.C. 5.1.3.2) is an enzyme known to catalyse
the conversion of UDP-Gal (1a) into UDP-Glc (2a), with the equilib-
rium favouring the latter over the former (ca 1:4).21 Previous work
suggested that 5-formylthienyl-UDP-Gal (1f) is not a substrate for
Streptococcus thermophilus GalE.22 Therefore GalE from two further
organisms was assessed: galactose-adapted yeast (ScGalE)23 and
Erwinia amylovora (EaGalE).24
As control experiments, the conversion of UDP-Gal (1a) into
UDP-Glc (2a) was achieved using both ScGalE and EaGalE and the
progress of the epimerization was followed by 1H NMR. Under
the condition employed, the equilibrium reaction mixtures were
reached within 10 min and the ratio between galacto-/gluco-con-
figured products were approximately 1:4, as expected (Fig. 7).
Treatment of 5-formylthienyl-UDP-Gal (1f) with ScGalE and EaGalE
did not show any 400-OH epimerization by 1H NMR, even after pro-
longed incubation (120 min). Similarly, when 5-iodo-UDP-Gal (1b)
was used as a substrate, ScGalE failed to effect conversion, even
after extended incubation (120 min).
samine-1-phosphate (GlcNAc-1-P), as well as -galactose-1-
a-D
phosphate (Gal-1-P) (Fig. 5). Conversions to the corresponding
UDP-sugars 1a, 3a and 4a, respectively, reached 39–48% after
120 min (Fig. 5). With 5-iodo-UTP (5b) the GalU-mediated conver-
sions were lower in the case of GlcN-1-P (42%) and GlcNAc-1-P
(20%); disconcertingly, no conversion at all was detected in the
case of Gal-1-P (Fig. 5).
2.2.2. The mutual incompatibility of 5-iodo-UTP and Gal-1-P as
co-substrates for GalU
The lack of GalU-mediated conversion of Gal-1-P with 5-iodo-
UTP was somewhat unexpected and warranted further analysis.
First, it was shown that in the presence of a high concentration
of inorganic pyrophosphate (PPi), GalU can perform the reverse
conversion from 5-iodo-UDP-Glc (2b) to Glc-1-P and 5-iodo-UTP
(5b). The conversion was complete, as judged by 1H NMR [anomer-
ic proton resonances (dd) were used as diagnostic peaks], within
10 min with 10 mM PPi (Fig. 6). When 5-iodo-UDP-Gal (1b) was
subjected to analogous conditions no conversion was observed
even after incubation for 60 min (data not shown). To see whether
the lack of conversion of the galacto-configured substrates was
down to lack of binding or to non-productive binding of the sub-
strates, inhibition experiments employing 5 equiv of 5-iodo-UDP-
Gal (1b), 1 equiv of 5-iodo-UDP-Glc (2b) and excess PPi were con-
ducted. By 1H NMR, 5-iodo-UDP-Glc (2b) was fully converted into
Glc-1-P and 5-iodo-UTP (5b) within 10 min as in the no inhibitor
control reaction (Fig. 6). No conversion 5-iodo-UDP-Gal (1b) was
detected, which suggests that 5-iodo-UDP-Gal (1b) does not bind
to the active site of GalU. A competition experiment was designed
to show whether a large excess of Gal-1-P (5 equiv) can outcom-
pete the natural acceptor Glc-1-P (1 equiv) in a GalU mediated
conversion of 5-iodo-UTP (5b) (1 equiv) into 5-iodo-UDP-sugar.
However, in contrast, EaGalE showed rapid epimerization of 5-
iodo-UDP-Gal (1b) into 5-iodo-UDP-Glc (2b) and the transforma-
tion reached equilibrium after about 30 min giving mixed
galacto-/gluco-configured products in the ratio 3:7. The reverse
conversion of 2b into 1b using EaGalE was also shown to achieve
a ca 7.5:2.5 equilibrium mixture of gluco-/galacto-configured sugar
nucleotides after 30 min (Fig. 7).
O
O
OH
HO
4''
X
X
O
NH
NH
HO
O
O
O
O
O
N
O
N
O
R
GalU
O
P O P O
OH OH
HO P O P O P O
OH OH
+
sugar-1-P
O
O
OH
equimolar
OH OH
OH OH
PPi
1a 4'' ax, R = OH, X = H, 40%
5a X = H (= UTP)
5b X = I
1b
3a
4'' ax, R = OH, X = I, 0%
4'' eq, R = NH2, X = H, 48%
sugar-1-P = GlcN-1-P, GlcNAc-1-P or Gal-1-P
3b 4'' eq, R = NH2, X = I, 42%
4a
4b
4'' eq, R = NHAc, X = H, 39%
4'' eq, R = NHAc, X = I, 20%
Figure 5. GalU mediated transformations of UTP (5a) and 5I-UTP (5b) with three different sugar-1-phosphates (Gal-1-P, GlcN-1-P or GlcNAc-1-P) to the corresponding sugar
nucleotides 1, 3 and 4 at time point 120 min.

22
B. A. Wagstaff et al. / Carbohydrate Research 404 (2015) 17–25
O
OH
HO
HO
I
NH
O
O
O
N
O
HO
O
P O P O
OH OH
O
OH OH
1b
O
N
O
N
OH
OH
I
I
NH
NH
O
O
HO
HO
HO
HO
1''
1
O
O
O
O
O
O
O
O
GalU
HO
HO
+
O
P O P O
OH
PPi
O
P OH
OH
+
HO P O P O P O
O
O
OH OH
OH
OH
OH OH
OH OH
2b
Glc-1-P
5b
Figure 6. Reverse action of GalU in the presence of excess 10 mM PPi. (A) Conversion of 5-iodo-UDP-Glc (2b) into Glc-1-P. (B) The same as A, but in the presence of excess
(5 equiv) 5-iodo-UDP-Gal (1b).
galactose. Given the established potential for Pd-mediated cross-
coupling of 5-iodo-UDP-sugars, the enzymatic procedures elabo-
rated in this study provide useful additions to the repertoire of
transformation available for the production of novel sugar
nucleotides.
3. Conclusions
5-Substituted gluco- and galacto-configured UDP-sugars are
versatile tools for glycoscience research. To date, access to such
compounds has relied on chemical synthesis approaches. Here
we have investigated enzymatic synthesis routes to such com-
pounds, relying either on pyrophosphate bond formation [action
of uridylyltransferase (GalPUT) or pyrophosphorylase (GalU)] or
epimerization of the C-400 stereochemistry of the pre-formed sugar
nucleotide [action of epimerase (GalE)]. These studies demonstrate
that the one-pot combination of glucose-1-phosphate uridylyl-
transferase (GalPUT) and UDP-glucose pyrophosphorylase (GalU)
is able to catalyse the conversion of 5-substituted UTP derivatives
into the corresponding 5-substituted UDP-galactose derivatives in
a number of instances, albeit in poor yield (<5–23% isolated yield).
It appears that the specificity of GalPUT is a limiting factor in the
utility of this reaction. In contrast, GalU in conjunction with inor-
ganic pyrophosphatase was able to convert 5-substituted UTP
derivatives plus a range of gluco-configured sugar-1-phosphates
into the corresponding sugar nucleotides in practical yields
(20–98%). Subsequent attempts to convert these gluco-configured
compounds to the corresponding galacto-isomers proved problem-
atic, with UDP-glucose 400-epimerase (GalE) from both yeast and
Erwinia proving ineffective for bulky 5-aryl derivatives. However,
in contrast to the yeast enzyme, the Erwinia GalE proved effective
with 5-iodo-UDP-glucose, readily converting it to 5-iodo-UDP-
4. Experimental
4.1. General methods
4.1.1. Chemicals
All chemicals and reagents were obtained commercially and
used as received unless stated otherwise. The identity of products
from our control experiments (1a, 2a and 4a) was confirmed by
comparison of 1H NMR spectra and/or HPLC retention times of
authentic samples. 5-Substituted UTP derivatives 5b–f12 and
5-iodo-UDP-Gal (1b), 5-iodo-UDP-Glc (2b) and 5-(5-formyl-2-thie-
nyl)-UDP-Gal (1f)7 were prepared by chemical synthesis following
published procedures. The identity of the following known
compounds were confirmed by comparison of analytical data with
published literature: (1b),7 (1d),7 (1e),7 (2b),10 (2c),10 (2d),10 (2e),10
(3a),25 (4b).6
4.1.2. Spectroscopy
1H NMR spectra were recorded in D2O on a Bruker Avance III
spectrometer at 400 MHz and chemical shifts are reported with

B. A. Wagstaff et al. / Carbohydrate Research 404 (2015) 17–25
23
O
N
O
OH
HO
OH
X
X
NH
NH
O
O
HO
HO
HO
O
O
O
O
O
N
O
EaGalE
HO
HO
O
P O P O
OH OH
O
P O P O
OH OH
O
O
OH OH
OH OH
2a
2b
1a
1b
X = H (UDP-Glc)
X = I
X = H (UDP-Gal)
X = I
Figure 7. EaGalE mediated epimerization. (A) UDP-Gal (1a) into UDP-Glc (2a) (30 min). (B) 5-Iodo-UDP-Glc (2b) into 5-iodo-UDP-Gal (1b) (30 min).
respect to residual HDO at dH 4.70 ppm. High resolution accurate
mass spectra were obtained using a Synapt G2 Q-Tof mass
spectrometer using negative electrospray ionization. Low resolu-
tion mass spectra were obtained using either a Synapt G2 Q-Tof
or a DecaXPplus ion trap in ESI negative mode by automated direct
injection.
(EMD4Biosciences, Germany) were transformed with the pETM-
30::GalE construct for expression of the recombinant GST-fusion
protein. Cells containing the construct were grown overnight in
10 mL 2 ꢁ YT medium containing Kanamycin (30
l
g mLꢀ1) at
37 °C. The starter culture was used to seed 1 L of medium (1:100
dilution) and the culture was grown at 37 °C for 3 h (O.D. 0.8).
The temperature was then decreased to 18 °C and the culture
was left to equilibrate for 1 h before induction with 1 mM IPTG
for 16 h. Cells were harvested by centrifugation at 4500g for
15 min at 4 °C, re-suspended in 50 mL ice cold PBS containing
0.2 mg mLꢀ1 lysozyme and protease inhibitors, stirred for 30 min
at room temperature and lysed by sonication (Soniprep, MSE,
UK) on ice for 2 min using 2 s cycles (15.6 MHz). After centrifuga-
tion at 18000g for 20 min at 4 °C the supernatant was filtered and
loaded onto a GSTrap HP 5 mL column (GE Healthcare, Sweden)
equilibrated with PBS at a flow rate of 1.5 mL minꢀ1. The column
was then washed with PBS until the A280 reached the baseline
and the enzyme was eluted with 10 mM reduced glutathione in
50 mM TRIS–HCl buffer at pH 8.0. The eluted protein was dialysed
against 50 mM TRIS–HCl buffer at pH 8.0 containing 10% glycerol,
concentrated to 0.1 mg mLꢀ1 and stored at ꢀ20 °C. Protein purity
was confirmed by SDS–PAGE.
4.1.3. Enzymes
Galactose-1-phosphate
uridylyltransferase
(GalPUT,
EC
2.7.7.12) from Escherichia coli was over-expressed and purified as
described earlier.14 Glucose-1-phosphate uridylyltransferase
(GalU) from Escherichia coli was over-expressed and purified as
described earlier.26 Inorganic pyrophosphatase (IPP) from
Saccharomyces cerevisiae was purchased from Sigma–Aldrich.
Uridine-50-diphosphogalactose
400-epimerase
(GalE)
from
galactose-adapted yeast (Saccharomyces cerevisiae, ScGalE) was
purchased from Sigma–Aldrich. Uridine-50-diphosphogalactose
400-epimerase (GalE) from Erwinia amylovora (EaGalE) was cloned,
overexpressed and purified as detailed below.
4.1.4. Erwinia amylovora (EaGalE)
The GalE gene (ENA accession number FN666575.1) was ampli-
fied by PCR from genomic DNA isolated from E. amylovora strain
Ea273 (ATCC 49946) using the following primers: GalE-F 50-CGAT-
CACCATGGCTATTTTAGTCACGGGGG and GalE-R 50-CGATCACTC-
4.2. Sugar nucleotide purification methods
4.2.1. Purification method 1
GAGTCAACTATAGCCTTGGGG. These primers included NcoI and
XhoI restriction sites, respectively (underlined). The PCR product
was purified from agarose gel using a QIAquick gel extraction kit
(Qiagen, Germany) and treated for 3 h at 37 °C with NcoI and XhoI
(NEB, USA) for double digestion. After purification using QIAquick
PCR Purification Kit (Qiagen, Germany), the digested PCR product
was ligated into pETM-30 vector.27 The construct was propagated
in Escherichia coli NovaBlue cells (EMD4Biosciences, Germany),
purified using a DNA miniprep kit (Sigma, USA) and sequenced
by Microsynth AG (Switzerland) to test the correctness of the gene
sequence. E. coli BL21 (DE3) chemically competent cells
Strong anion-exchange (SAX) HPLC on Poros HQ 50. An aqueous
solution of a sample was applied on a Poros HQ 50 column (L/D 50/
10 mm, CV = 3.9 mL). The column was first equilibrated with 5 CV
of 5 mM ammonium bicarbonate buffer, followed by a linear gradi-
ent of ammonium bicarbonate from 5 mM to 250 mM in 15 CV,
then hold for 5 CV, and finally back to 5 mM ammonium bicarbon-
ate in 3 CV at a flow rate of 8 mL/min and detection with an on-line
detector to monitor A265. After multiple injections, the column was
washed with 3 CV of 1 M ammonium bicarbonate followed by 3 CV
of MQ water.

24
B. A. Wagstaff et al. / Carbohydrate Research 404 (2015) 17–25
4.2.2. Purification method 2
(Purification method 1) giving bisammonium salt of 2f. 1H NMR
(400 MHz, D2O): d 9.69 (1H, s, CHO), 8.32 (1H, s, H-6), 7.92 (1H, d,
Reverse phase (RP) C18 purification. The purification was
performed on a Dionex Ultimate 3000 instrument equipped with
UV/vis detector. A solution of a sample in water was applied on a
3
3JTh3,Th4 = 4.2 Hz, Th), 7.64 (1H, d, JTh3,Th4 = 4.3 Hz, Th), 6.01 (1H, d,
3
3
3
J1 ,2 = 4.7 Hz, H-10), 5.51 (1H, dd, J1 ,2 = 3.4 Hz, J1 ,Pb = 7.3 Hz, H-100).
0
0
00 00
00
Phenomenex Luna
5
l
m
C18(2) column (L/D 250/10 mm,
HRMS, ESI negative: m/z calcd for
675.0304, found: 675.0304.
C
20H25N2O18P2Sꢀ [MꢀH]ꢀ:
CV = 19.6 mL) and eluted isocratically with 50 mM Et3NHOAc, pH
6.8 with 1.5% CH3CN in 8 CV at a flow rate of 5 mL/min and
detection with on-line UV detector to monitor A265. Fractions
containing the sugar nucleotide were pooled and freeze-dried.
4.3.3.2. 5-Iodo-UDP-
pound 3b was prepared from 5-iodo-UTP (5b, 0.5 mg, 0.56
and GlcN-1-P (0.15 mg, 0.56 mol) as described in General proce-
a-
D-glucosamine (3b).
The title com-
lmol)
l
4.3. Enzymatic transformations
dure 2 and the product was isolated using Purification method 1
followed by Purification method 2. The title compound 3b eluted
at Rf = 12.9 min and was obtained after freeze-drying as a triethyl-
4.3.1. General procedure 1 (GalU-GalPUT-IPP)
ammonium salt (3b ꢁ 0.5 Et3N, 0.1 mg, 20 %). 1H NMR (400 MHz,
UTP analogue (5a–f, 0.5 mg, 1 equiv),
phate (1 equiv) and UDP-Glc (2a, 0.5 mol-%) were dissolved in
buffer (500 L, 50 mM HEPES pH 8.0, 5 mM KCl, 10 mM MgCl2).
A small sample (50 L) was separated for no enzyme control. Then
enzymes were added to give final concentration of GalU
(137 g/mL), IPP (1.4 U/mL) and GalPut (329 g/mL) in a final
volume of 700 L. The mixture was incubated at 37 °C with gentle
shaking. At time points analytical samples were separated (50 L)
and MeOH was added (50 L) to precipitate the enzymes. The
a-D-galactose-1-phos-
3
D2O): d 8.13 (1H, s, H-6), 5.85 (1H, d, J1 ,2 = 3.7 Hz, H-10),
5.66–5.60 (1H, m, H-100), 4.31–4.27 (2H, m, H-20, H-30), 4.21–4.12
(3H, m, H-5a0, H-5b0, H-40), 3.87–3.66 (5H, m, H-200, H-300, H-500,
0
0
l
l
3
H-6a00, H-6b00), 3.45–3.39 (1H, m, H-400), 3.19 (3H, q, JCH2,CH3 = 6.8
3
l
l
Hz, (CH3CH2)3N), 1.17 (4.5H, t, JCH2,CH3 = 6.8 Hz, (CH3CH2)3N).
l
HRMS, ESI negative: m/z calcd for C15H23IN3O16Pꢀ2 [MꢀH]ꢀ:
l
689.9604, found: 689.9596.
l
sample was vortexed for 1 min, centrifuged (10,000 rpm for
5 min) and the supernatant was filtered through a disc filter
Acknowledgements
(0.45 lm). The filtrate was analysed by SAX HPLC (10 lL injection,
This work was supported by the UK BBSRC Institute Strategic
Programme Grant on Understanding and Exploiting Metabolism
(MET) [BB/J004561/1] and the John Innes Foundation, with
additional support by the EPSRC (First Grant EP/D059186/1, to
G.K.W.) and the MRC (Grant No. 0901746, to G.K.W.). We thank
Dr. M. Malnoy for providing E. amylovora genomic DNA. Plasmid
pETM-30 was obtained from the European Molecular Biology
Laboratory under a signed Material Transfer Agreement. Part of
this work (cloning, overexpression and purification of EaGalE)
was supported by the Autonomous Province of Bolzano grant: ‘A
structural genomics approach for the study of the virulence and
pathogenesis of Erwinia amylovora’ and the Free University of
Bolzano grant: Galactose and glucuronic acid metabolism in
Erwinia spp. (GAMEs).
Purification method 1). After 24 h the reaction was quenched by
addition of MeOH (the same volume as the sample volume) and
processed as indicated for the analytical sample. Products were
isolated using SAX HLC (Purification method 1). Pooled fractions
containing the sugar nucleotide were freeze-dried. When
necessary, Purification method 2 was also applied.
4.3.2. General procedure 2 (GalU)
UTP analogue (5a–f, 0.5 mg, 1 equiv) and sugar-1-phosphate
(1 equiv) were dissolved in deuterated buffer (660
HEPES pD 8.0, 5 mM KCl, 10 mM MgCl2) and 1H NMR was acquired
(100 scans) of the no enzyme control. GalU (40 L, final
c = 0.14 mg/mL) was added to total 0.7 mL. Where indicated IPP
(10
L, final c = 1.4 U/mL) was added to this mixture. 1H NMR
lL, 50 mM
l
l
spectra were acquired at time points 10, 30, 60, 120 min to monitor
reaction progress. The reaction was quenched by addition of an
equal volume of methanol (0.7 mL), and the resulting solution
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