Nucleoside triphosphate mimicry: A sugar triazolyl nucleoside as an ATP-competitive inhibitor of B. anthracis pantothenate kinase

Article abstract of DOI:10.1039/b909729e

The synthesis of a library of nucleoside triphoshate mimetics is described where the Mg²⁺ chelated triphosphate sidechain is replaced by an uncharged methylene-triazole linked monosaccharide sidechain. The compounds have been evaluated as inhib

Full text of DOI:10.1039/b909729e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Nucleoside triphosphate mimicry: a sugar triazolyl nucleoside as an
ATP-competitive inhibitor of B. anthracis pantothenate kinase†
a
b
a
a
b
Andrew S. Rowan, Nathan I. Nicely,‡ Nicola Cochrane, Wjatschesslaw A. Wlassoff, Al Claiborne and
Chris J. Hamilton*c
Received 18th May 2009, Accepted 29th June 2009
First published as an Advance Article on the web 27th July 2009
DOI: 10.1039/b909729e
2
+
The synthesis of a library of nucleoside triphoshate mimetics is described where the Mg chelated
triphosphate sidechain is replaced by an uncharged methylene-triazole linked monosaccharide
sidechain. The compounds have been evaluated as inhibitors of Bacillus anthracis pantothenate kinase
and a competitive inhibitor has been identified with a K
i
that is 3-fold lower than the K value of ATP.
m
Background
donor substrate, uridine-5¢-diphosphogalactose (UDP-Gal) 1 is
replaced with a bridging 1,4-b-D-glucose linkage (Fig. 1). Lac-
5
Nucleoside triphosphates (NTPs) play a critical role in a vast range
of biological processes ranging from protein phosphorylation
and cell signalling to DNA replication. A major challenge for
the design of NTP mimetics as inhibitors of NTP-dependent
enzymes (eg. kinases, DNA/RNA polymerases) is the replacement
of the tetra-anionic triphosphate sidechain with a neutral, or
significantly less charged, motif that will facilitate cell permeability
whilst ensuring they still bind to their biological target(s) with
comparable or greater efficiency than the native substrate. Most
kinases/polymerases accept their incoming NTP substrates pre-
coordinated to a divalent magnesium cation which is essential
for substrate binding and catalysis. The divalent cation facilitates
substrate binding via coordination to both the triphosphate
ligand and carboxylate sidechains (usually aspartates) in the NTP
binding domain. The design of uncharged bioisosteres of the
tosyl uridine, 2, proved to be a competitive inhibitor of 1,4-GalT
activity from leukemia ascites fluid with a K
to K of the native substrate 1 implying that the central glucose
moiety of 2 is isosteric with a pyrophosphate-metal ion complex
Fig. 1). More recently, the inhibitory effect of 2 (500 mM) against
i
value comparable
m
(
bovine b-1,4-galactosyltransferase was shown to be <10% in the
6
presence of K
m
concentrations of UDP-Gal (120 mM). This may
be attributed to structural differences in GalTs from different
species and in particular the gross binding conformation of their
donor substrates.
Pyrophosphate mimicry by hexoses is also observed in
nature. The dolichol-pyrophosphate-a-D-GlcNAc-synthase in-
hibitor tunicamycin uses an L-rhamnose-configured monosaccha-
2+
ride to mimic the pyrophosphate-Mn complex of the natural
7,8
dolichol-pyrophospho-GlcNAc substrate (Fig. 1). The stereo-
1,2
triphosphate sidechain has so far proven to be challenging.
configuration and substitution patterns of the central sugar motifs
in 2 and tunicamycin significantly differ (Fig. 1). Evidently, no sin-
gle monosaccharide motif can operate as a generic pyrophosphate-
metal ion isostere due to different ligand-binding requirements of
sugar nucleotides with different enzymes. How a neutral monosac-
charide effectively mimics a charged pyrophosphate-metal ion
chelate could be a combination of appropriate spacing/orientation
of the terminal nucleoside and sugar motifs and interactions of the
bridging glucose motif with the pyrophosphate-binding pocket.
Substrate-binding interactions between the pyrophosphate-metal
ion and aspartate sidechains in the pyrophosphate binding pocket
could perhaps be replaced by hydrogen bonding interactions with
one or more hydroxyl groups of a monosaccharide. We were
curious to see if any such examples of pyrophosphate-metal
ion mimicry would translate into the design of NTP mimics by
replacing the terminal b,g-pyrophosphate motif with a neutral
monosaccharide.
However, conformationally rigidified ATP mimetics have recently
been prepared using a bicyclic (ethylene bridged) oxazolidine
ring structure to mimic the enzyme-bound conformation of
the Mg -complexed triphosphate sidechain. In this case, an
inhibitor of human tyrosine kinase (HER-2) was developed with
2
+
3
a K
i
value of 88 mM (compared to K (ATP) of 5 mM). NTPs
m
adopt different conformations when bound to different enzymes.
Molecules that mimic specific enzyme-bound conformation(s) of
2
+
NTP-Mg could help to confer inhibitor selectivity for individual
(
or sub-populations of) NTP-dependent enzymes.
In the sugar nucleotide arena, there have been numerous
attempts to prepare uncharged pyrophosphate isosteres, but with
4
limited success. One notable exception is lactosyl uridine 2, where
2
+
the pyrophosphate-Mn complex of the galactosyltransferase
a
School of Chemistry and Chemical Engineering, David Keir Building,
Stranmillis Road, Queen’s University Belfast, Belfast, BT9 5AG
We recently reported the preparation of sugar nucleoside
monophosphates (sugar-NMPs) such as 3 and 4, which could
b
Centre for Structural Biology, Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157, USA
9
c
School of Chemical Sciences and Pharmacy, University of East Anglia,
potentially serve as significantly less charged ATP mimics (Fig. 2).
Norwich, Norfolk NR4 7TJ
However, these sugar-NMPs still retain a charged and hydrolyti-
cally unstable glycosyl phosphate linkage. Replacing the remaining
phosphate with a readily accessible O-methylene-triazole linker
†
Electronic supplementary information (ESI) available: Additional exper-
imental procedures for compound syntheses and characterisation data are
provided in the supplementary information. See DOI: 10.1039/b909729e
(
Fig. 2) would resolve these polarity/stability concerns. The
‡
Current address: Duke Human Vaccine Institute, 450 Research Drive,
Durham, NC 27708, USA.
viability of the triazole group as a bioisostere for the phosphate
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2+
Fig. 1 Examples of pyrophosphate-M mimicry by monosaccharides.
Fig. 2 Proposed NTP mimics.
group in the sugar-phosphate backbone of nucleic acids has
10
recently been demonstrated. Herein, we describe the synthesis
and evaluation of a parallel library of sugar-triazole-nucleosides
(
STNs) as inhibitors of the type-III pantothenate kinase from
Scheme 1 Reagents and conditions: (i) PPh , LiN , CBr , DMF, r.t., 3 days;
3
3
4
Bacillus anthracis (BaPanK). From this library, a competitive
(ii) conc. NH
4
OH, MeOH, r.t., 6 days; (iii) PPh
3
, DIAD, DPPA, THF, r.t.,
◦
6
8 h; (iv) TFA/H
2
O (9:1), 0 C–r.t., 25 min.
inhibitor is identified whose K
i
is 3-fold lower than K
m
of the
competing substrate (ATP).
Similar procedures were use to access 5¢-azido cytidine 11
4
11,12
from the N -benzoyl protected derivative 9.
The N-benzoyl
Results and discussion
protecting group was employed to prevent intermolecular alkyla-
tion of the nucleobase via reaction with the 5¢-bromo nucleoside
intermediate, which is generated in situ prior to reaction with
lithium azide. 5¢-Azido adenosine 14 has previously been prepared
Chemistry
AMP-glucose 3 and AMP-galactose 4 were prepared (as anomeric
mixtures) using reported methods. For construction of the sugar-
triazole-nucleoside (STN) library, 5¢-azido uridine 7 and 5¢-azido-
9
6
13
in five steps from N -benzoyl adenosine. Herein, a more succinct
route involved treatment of 2¢,3¢-O-isopropylidene adenosine 12
with diphenyl phosphoryl azide, diisopropylazodicarboxylate and
2
-deoxythymidine 8 were prepared, in good yield, by treatment
14
of the free nucleosides, 5 and 6 with triphenylphosphine, carbon
tetrabromide and lithium azide (Scheme 1).
triphenylphosphine to give 13. The isopropylidene protecting
group served to enhance the solubility of 13, which facilitated
11
4
030 | Org. Biomol. Chem., 2009, 7, 4029–4036
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purification by column chromatography prior to its removal under
acidic conditions to give 14. A variety of methods were employed
to access the different propargyl glycosides. Alpha-propargyl
15
16
17
glycosides of D-Glc 15, D-GlcNAc 16, D-Man 17 and D-Xyl
9 were all obtained using acid catalysed Fischer glycosylation
1
conditions (Scheme 2). The same conditions were used to prepare
O-propargyl derivatives of L-Ara 18 and D-Gal 20 as anomeric
mixtures, which were inseparable by chromatography. However,
for 20 the 4,6-O-benzylidene acetal derivatives, 21 and 22, were
separable by chromatography enabling access to the desired
a-galactoside 23.
Scheme 3 Reagents and conditions: (i) propargyl alcohol, BF
3
2
·OEt ,
◦
CH
2
Cl
2
, 0–20 C; (ii) NaOMe, MeOH.
Scheme 2 Reagents and conditions: (i) propargyl alcohol, AcCl (cat.),
Scheme 4 Reagents and conditions: (i) propargyl alcohol, AgOTf, 4A˚
◦
◦
1
8
00–120 C, 1–3 days; (ii) PhCH(OMe)
0 C.
2
, TsOH, MeCN; (iii) AcOH(aq),
molecular sieves, CH Cl , 0 C, 2 h; (ii) NaOMe, MeOH; (iii) TiBr ,
2
2
4
◦
◦
2 2 4 2
CH Cl /EtOAc; (iv) Zn dust, NH Cl, EtOH/H O, 90 C.
Treatement of peracetylated D-Rib 24, D-Gal 27a and D-Xyl
(a:b ratio = 1:1.1). Subsequent deacetylation under Zemplen
18
21
2
8a with propargyl alcohol in the presence of BF
3
·OEt was used
conditions in a sonicator rapidly proceeded to completion in
to access the b-propargyl glycosides 26, 27c, 28c (Scheme 3).
The b-propargyl glycoside of GlcNAc 31 was accessed via silver
triflate promoted glycosylation of propargyl alcohol with glycosyl
just three minutes.
Amino-sugar 36 could not be accessed by hydrogenation of
azido-sugar 35 as this would also have reduced the alkyne, hence
alternative methods were explored. Under Staudinger conditions
(with tributylphosphine) the azide moiety of 35 was not reduced
and only unreacted starting material was recovered. However
19
bromide 29 (Scheme 4a). Similar procedures were employed to
access glucosamine derivative 35 (Scheme 4b). Preparation of
2
-azido-2-deoxy-a-D-glucopyranosyl bromide 33 by treatment of
peracetylated precursor 32 with HBr in glacial acetic acid was
22
treatment with zinc and ammonium chloride reduced the azide
whilst leaving the alkyne aglycone intact.
unsuccessful, providing a complex mixture of products (by TLC).
20
An alternative treatment with titanium(IV) bromide gave the
desired beta-anomer in 31% yield (40% of unreacted starting
material was also recovered during chromatography). Reaction of
The thymidine STNs 49–59 were the first to be prepared using
the copper-(I)-catalysed [3 + 2] azide-alkyne cycloaddition (Cu-
ACC) reaction between the aforementioned propargyl glycosides
and azido-nucleoside 8 in the presence of copper sulfate and
3
3 with propargyl alcohol in the presence of silver triflate provided
propargyl glycoside 34 in good yield as an anomeric mixture
23
sodium ascorbate (Scheme 5, Table 1). Under these conditions,
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Table 1 Compounds prepared and screened as inhibitors of BaPanK
%
enzyme
% enzyme
Yield (%) activity
% enzyme
Yield (%) activity
% enzyme
Yield (%) activity
a
a
a
a
Yield (%) activity
1
6
a-D-Glc
37
38
39
40
41
42
43
44
45
46
47
(72)
(85)
(84)
(82)
(86)
(88)
(85)
(80)
(53)
(84)
(71)
(48)
93
95
97
4
84
90
90
95
87
76
95
93
49
50
51
52
53
54
55
56
57
58
59
(61)
(88)
(68)
(54)
(62)
(60)
(57)
(82)
(49)
(58)
(81)
98
91
60
61
62
63
64
65
66
67
68
69
70
(74)
(91)
(81)
(87)
(80)
(94)
(94)
(90)
(76)
(81)
(81)
96
99
105
105
94
91
91
71
72
73
74
75
76
77
78
79
80
81
(60)
(80)
(67)
(70)
(76)
(82)
(81)
(83)
(68)
(80)
(70)
90
b-D-Glc
104
b
16
c
a-D-Gal
80
—
c
b-D-Gal
—
86
105
102
100
109
a-D-Man
a-D-Xyl
108
102
100
92
98
104
100
b-D-Xyl
16
a-D-GlcNAc
b-D-GlcNAc
L-Ara (a/b = 1:3)
b-D-Rib
102
110
c
—
c
—
91
107
83
D-GlcN (a/b = 1.1:1) 48
a
Assays carried out in the presence of inhibitor (1 mM), ATP (125 mM) and pantothenate (250 mM). Percentage activity is relative to the same assay
carried out in the absence of inhibitor. No further decrease in activity was observed when inhibitor concentration was increased to 2 mM. False positives
ie. inhibition of the PK/LDH coupled enzymes) in this assay.
b
c
(
Scheme 5 Reagents and conditions: (i) 8, CuSO
ascorbate (10 mol %), TBTA (5 mol %), MeOH/H
4
(5 mol %), sodium ascorbate (10 mol %), MeOH/H
O/MeCN (1:1:1), r.t., sonication.
2
O (3:1); (ii) 7 or 11 or 14, CuBr (5 mol %), sodium
2
reactions took 1–3 days to reach completion (by TLC). For
the adenosine, uridine and cytidine derivatives 37–48 and 60–
8
1, alternative reaction conditions using copper(I) bromide, tris-
24
21
(
benzyltriazolylmethyl)amine (TBTA), under sonication gave
improved yields and shorter reaction times (30–40 minutes).
Enzyme assays
The type-III pantothenate kinase from Bacillus anthracis
(
BaPanK) was used to study the NTP mimicry of all the STNs that
were made. Pantothenate kinase (PanK) catalyses the first obligate
step in Coenzyme A biosynthesis, namely the ATP-dependent
phosphorylation of pantothenic acid (Scheme 6a). To date, the
crystal structures of type-III PanK enzymes from Thermotoga
Scheme 6 (a) BaPanK catalysed phosphorylation of pantothenate;
b) coupled assay used to monitor BaPanK activity; abbreviations:
(
ADP = adenosine triphosphate, PK = pyruvate kinase, LDH = lactate
25,26
27
maritima (TmPanK),
Pseudomonas aeruginosa (PaPanK) and
dehydrogenase, PEP = phosphoenol pyruvate, Pyr = pyruvate.
28
BaPanK have been reported, but co-crystallistaion of any of
2
+
2+
these Mg -dependent enzymes with ATP-Mg has not yet been
accomplished. Type-III PanK enzymes exhibit unusually high
27
PaPanK and the structure of the ADP:pantothenate ternary
29,25,27
26
K
m
values of 3–10 mM
compared to K
m
(ATP) values of
complex with TmPanK. For the studies reported herein, BaPanK
<
200 mM generally observed with PanK types I and II. Some
activity/inhibition was monitored using a coupled assay as
outlined in Scheme 6b.
BaPanK is able to utilise a range of NTPs as phosphate
type III panKs also display a broad tolerance for other NTPs as
alternative phosphate donor substrates.
27,29
For Type-III PanKs,
the nucleobase end of the ATP binding site is open to the solvent
and doesn’t support tight binding interactions with the adenosyl
nucleobase, as shown by active site docking of the inert substrate
mimic adenosine 5¢-(b,g-imido)triphosphate (AMPPNP) with
donors (Table 2). The catalytic efficiencies (kcat/K ) of the purine
NTPs (ATP, dATP, dGTP) were 5–10-fold greater than their
pyrimidine counterparts. This is primarily due to their lower
m
K
m
values whereas the steady state turnover rates (kcat) of the
4
032 | Org. Biomol. Chem., 2009, 7, 4029–4036
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Table 2 NTP substrate kinetics with BaPanK
-
1
-1
-1
Substrate
K
m
(mM)
k
cat (s )
k
m
cat/K (s mM )
ATP
510 (± 19)
138 (± 6)
117 (± 13)
1759 (± 302)
839 (± 91)
723 (± 62)
1715 (± 155)
1.5 (± 0.02)
0.9 (± 0.01)
0.8 (± 0.02)
1.5 (± 0.15)
0.5 (± 0.02)
0.4 (± 0.01)
1.7 (± 0.06)
2.9 (± 0.2)
6.6 (± 0.4)
6.5 (± 0.9)
0.9 (± 0.2)
0.6 (± 0.1)
0.5 (± 0.1)
1.0 (± 0.1)
dATP
dGTP
CTP
dCTP
dTTP
UTP
pyrimidines were much closer to (and in some cases better than)
those of the purine NTPs. The K (ATP) for BaPanK (510 mM)
is significantly lower than those reported for other type-III PanKs
m
2
5–27,29
(
2
k
(
3–10 mM).
It is interesting to note that removal of the
¢-hydroxyl on the ribose ring leads to reductions in both K and
cat, but with no significant overall reduction in catalytic efficiency
). The K value for pantothenate is 275 (± 11) mM. All
m
kcat/K
m
m
of the NTPs in Table 2 have previously been evaluated as
substrates for PaPanK where a preference for purine NTPs was
18
also observed. However, a direct qualitative comparison cannot
be made because the NTP specificity of PaPanK was determined
from relative enzyme activity measurements in the presence of a
fixed concentration of the different NTPs (250 mM). Not all type-
III enzymes share the same NTP substrate promiscuity. As well as
ATP, HpPanK can also utilise GTP and CTP (but not UTP),
whereas BsPanK cannot utilise any of these three alternative
Fig. 3 Compound 40 as a competitive inhibitor of BaPanK. Double
reciprocal plot of initial rate against ATP concentration in the presence
of compound 40 at 0 mM (open triangles), 150 mM (closed circles) and
4
00 mM (open squares).
the adenine nucleobase also makes a notable contribution to the
efficacy of 40. In the absence of a type-III PanK structure co-
20
2+
phosphate donors.
crystallised with ATP, Mg and the additional monovalent cation,
Compounds 37–81 were screened (at 1 mM concentrations)
for BaPanK inhibition in the presence of ATP (125 mM) and
reasons for inhibition by the b-galacto-configured STN 40 are not
immediately clear. The structure of the ADP:pantothenate ternary
2
+
pantothenate (250 mM). High inhibitor and low (<K
m
) ATP
complex with TmPanK shows how ATP-Mg must bind in an
26
concentrations were chosen to ensure that even moderate ATP-
competitive inhibitors wouldn’t go unnoticed. Under these condi-
tions the majority of the STNs displayed no significant (ie. <20%)
inhibition (Table 1). Compounds 52, 69, 73 and 79 proved to
be false positives due to inhibition of the coupled enzymes (PK
and/or LDH). Similar inhibitory effects on the coupled enzymes
were also observed for the sugar-NMPs 3 and 4.
The b-galacto-configured adenosyl STN 40 displayed 96%
inhibition of BaPanK activity under these assay conditions, but
did not inhibit the PK/LDH coupled enzymes during control
experiments. Detailed analysis showed 40 to be a competitive
extended conformation during the catalytic pathway. The loss of
inhibitory activity going from the b-galactoside 40 to its a-anomer
39 could be the consequence of 40 being able to adopt such an
extended conformation whereas its a-configured diastereoisomer
39 would be bent into a disfavourable orientation at the anomeric
linkage. Evidently, the axial hydroxyl of the galactose portion of 40
plays a significant role in inhibitor recognition. Using Autodock
22
(4.0.1), it has not been possible to predict a satisfactory binding
19
orientation for 40 with the BaPanK apo-enzyme where inversion
of the axial hydroxyl (ie. to 38) results in a significant loss of
binding interactions and/or steric clashing that could plausibly
account for the significant loss of inhibitor activity with 38 (data
not shown). For future consideration, co-crystallisation studies of
40 with BaPanK would help establish the binding orientation of
40 as well as if/how the b-linked galacto-motif is able to mimic
the b,g-pyrophosphate portion of ATP with this enzyme.
inhibitor of BaPanK (with respect to ATP) with a K
Fig. 3). This suggests that, for this enzyme, 40 operates as an
ATP mimetic, which, despite lacking all of the negative charges
of the parent triphosphate, has a K that is 3-fold lower than
the K value of the competing substrate (ATP). The anomeric
i
of 164 mM
(
i
m
configuration of the sugar-triazole sidechain is important as
the a-galacto-configured adenosyl STN 39 shows no inhibitory
activity. The axial configuration of the 4-hydroxyl of the galactose
moiety is also of great importance as the gluco-configured epimer
Conclusions
This study demonstrates how effective ATP mimics can be
prepared where the charged triphosphate sidechain is replaced
by an uncharged monosaccharide and that small variations in the
monosaccharide stereoconfiguration lead to dramatic changes in
inhibitor potency. It will be interesting to evaluate and compare
the structure–activity relationship of this STN library with other
NTP-dependent enzymes. More detailed mechanistic studies with
BaPanK, and other kinases, are ongoing and will be reported in
due course.
38 was inactive. The anomeric mixture of L-arabino-configured
ATP mimic 46 (with an axial 4-hydroxyl group) displayed 24%
inhibition at 1 mM concentrations (Table 1). Based on the
structure–activity data for 38–40, it is likely that the b-anomer
is the active component of 46. The K
and dTTP are, at most, only 3.5-fold greater than that of ATP
Table 2). Hence, the lack of any significant activity with the
m
values for UTP, CTP
(
b-galacto-configured pyrimidines 52, 63, 74 (Table 1) suggests that
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Experimental
(C-2¢¢), 71.91 (C-3¢), 70.57 (C-4¢¢), 63.03 (CH
2
), 62.67 (C-6¢¢), 52.01
-
+
(
C-5¢); HRMS (ES ) m/z calc. for C19
H
25
+
N
8
O
9
(M - H ): 509.1744;
General
found 509.1758; m/z 509 (5%, M - H ).
1
13
H and C NMR spectra were obtained using Bruker AV 300,
5
¢-Deoxy-5¢-[4-(L-arabinopyranosyloxymethyl)-1,2,3-triazol-1-
DPX 300 or DRX 500 NMR spectrometers. Chemical shifts are
quoted in parts per million (d) using the residual solvent peak as
an internal reference. IR spectra were recorded on a Perkin-Elmer
yl]adenosine (46). Was prepared from an anomeric mixture of
prop-2-ynyl-a/b-L-arabinopyranoside 18 (27.4 mg, 0.146 mmol)
and 5¢-azido-5¢-deoxyadenosine 14 (46.8 mg, 0.160 mmol)
as described for 40. Following automated chromatography
RX I FT-IR system. Optical rotations ([a]
D
) were obtained with a
Perkin-Elmer Model 341 Polarimeter, using the specified solvent
(
EtOAc/MeOH 7:3 as eluent), 46 (59 mg, 84%) was obtained
-
1
2
-1
and concentration, and are quoted in units of 10 deg cm g . Thin
layer chromatography (TLC) was carried out on Macherey-Nagel
SIL G-25 UV254 glass-backed silica plates, which were visualised
using a UV lamp, basic potassium permanganate solution or
sulfuric acid (10% (v/v) in ethanol). Flash chromatography was
carried out using Fluorochem silica gel for flash chromatography.
Automated flash chromatography was performed on a Biotage
Horizon or Biotage SP1 HPFC system using Si 12 + M or Si
20
as a white solid with an a/b ratio of 1:3: [a]
DMSO); d (500 MHz, DMSO-d ) 8.15, 8.14, 8.06, 7.95, 7.92 (5
s, 6H, H-2a, H-2b, H-8a, H-8b, H-5¢¢¢a, H-5¢¢¢b), 5.89 (d, 2H,
H-1¢a, H-1¢b), 4.80–4.67 (m, 6H, H-1¢¢b, H-5¢aa, H-5¢ab, H-5¢aa,
H-5¢ba, OCH a), 4.57 (d, 1H, J 12.5, OCH b), 4.53 (t, 2H,
J 5.0, H-2¢a, H-2¢b), 4.48 (d, 1H, J 12.5, OCH a), 4.42 (d,
H, J 12.0, OCH b), 4.30–4.20 (m, 4H, H-3¢a, H-3¢b, H-4¢a,
H-4¢b), 4.17–4.13 (m, 1H, H-1¢¢a), 3.72–3.50 (m, 6H, H-2¢¢b, H-
D
= +92.2 (c = 1.19,
H
6
¥
A
H
B
A
H
B
A
H
B
1
A
H
B
4
0 + M pre-packed cartridges. Dry pyridine was purchased from
3
3
(
1
1
(
7
(
¢¢b, H-4¢¢a, H-4¢¢b, H-5¢¢aa, H-5¢¢b), 3.44–3.38 (m, 1H, H-5¢¢bb),
.38–3.32 (m, 1H, H-5¢¢ba), 3.32–3.28 (m, 2H, H-2¢¢a, H-3¢¢a); d
) 156.06 (C-6), 153.10 (C-2), 149.47 (C-4a),
Fluka, and other solvents were dried using a Braun 1 solvent
purification system. Sonication-mediated reactions were carried
out using a Branson model 2510 sonicator bath operating at a
C
125 MHz, DMSO-d
6
49.42 (C-4b), 143.99 (C-4¢¢¢b), 143.89 (C-4¢¢¢a), 140.16 (C-8a),
40.02 (C-8b), 125.34 (C-5¢¢¢), 119.09 (C-5), 102.56 (C-1¢¢a), 99.17
C-1¢¢b), 87.97 (C-1¢b), 87.85 (C-1¢a), 82.56 (C-4¢a), 82.41 (C-4¢b),
2.85 (C-2¢b), 72.76 (C-2¢a), 72.53 (C-2¢¢a), 70.94 (C-3¢a), 70.90
C-3¢b), 70.56 (C-3¢¢a), 69.02 (C-2¢¢b), 68.64 (C-4¢¢b), 68.44 (C-
¢¢b), 67.71 (C-4¢¢a), 65.55 (C-5¢¢a), 63.37 (C-5¢¢b), 61.14 (CH a),
0.37 (CH
calc. for C18
24
frequency of 40 kHz. TBTA was made as previously described.
Propyl-2-ynyl-b-D-glucopyranoside was purchased from Aldrich.
Recombinant BaPanK was overexpressed and purified as previ-
28
ously described. Rabbit muscle pyruvate kinase (PK) and rabbit
muscle lactate dehydrogenase (LDH) were purchased from Sigma.
Enzyme assays were carried out using either a PerkinElmer UV
Lambda 25 spectrophotometer or a Tecan Saffire microplate
3
6
2
+
2
b), 51.49 (C-5¢a), 51.36 (C-5¢b); HRMS (ES ) m/z
+
H
25
N
8
O
8
+
(M + H ): 481.1795; found 481.1787; m/z
1
1
11
11
14
18
18
19
reader. Compounds 7, 8, 11, 13 27a, 27b, 29 and
+
+
9
4
83 (44%, 2M + Na ), 961 (8%, 2M + H ), 503 (32%, M + Na ),
81 (100%, M + H ).
30
32
were prepared following published methods. Representative
procedures are described below for the preparation of STNs 40, 46,
0, 65 and 78. Experimental methods and characterisation data
+
5
5¢-Deoxy-5¢-[4-(b-D-glucopyranosyloxymethyl)-1,2,3-triazol-1-
for other STNs and all other synthetic intermediates are provided
in the supplementary material.†
yl]thymidine (50). Prop-2-ynyl-b-D-glucopyranoside (23.5 mg,
0.108 mmol) and 5¢-azido-5¢-deoxythymidine 8 (31.7 mg,
0
.119 mmol) were dissolved in MeOH (2 mL). Aqueous CuSO
4
5
¢-Deoxy-5¢-[4-(b-D-galactopyranosyloxymethyl)-1,2,3-triazol-
-yl]adenosine (40). Prop-2-ynyl-b-D-galactopyranoside 27c
28.5 mg, 0.131 mmol) and 5¢-azido-5¢-deoxyadenosine 14 (42 mg,
.144 mmol) were measured into a sample vial, followed by
solution (0.05 M, 110 mL, 5.5 mmol) was added followed by
aqueous sodium ascorbate solution (0.1 M, 110 mL, 11.0 mmol).
This was stirred for 44 hrs then concentrated in vacuo and purified
1
(
0
by automated chromatography (EtOAc/MeOH 3:1 as eluent) to
copper(I) bromide (1 mg, 6.6 mmol). Aqueous sodium ascorbate
solution (40 mM, 332 mL, 13.3 mmol) and a solution of TBTA
in acetonitrile (20 mM, 333 mL, 6.6 mmol) was added, followed
by methanol (700 mL), acetonitrile (367 mL) and water (368 mL).
The sample vial was sealed, the lid pierced with a syringe needle,
and the reaction mixture was sonicated for 30 minutes. This was
concentrated in vacuo and purified by automated chromatography
20
give 50 (46 mg, 88%) as a white solid: [a]
MeOH); d (500 MHz, CD
.0, H-6), 6.19 (t, 1H, J 7.0, H-1¢), 4.97 (d, 1H, J 12.5, OCH
.77 (d, 1H, J 12.5, OCH ), 4.78 (dd, 1H, J 14.5, 4.0, H-5¢a),
D
= +23.7 (c = 0.99,
H
3
OD) 8.03 (s, 1H, H-5¢¢¢), 7.24 (d, 1H, J
),
1
4
4
4
1
1
9
A
H
B
A
H
B
.70 (dd, 1H, J 14.5, 6.5, H-5¢b), 4.41 (dt, 1H, J 6.0, 5.0, H-3¢),
.37 (d, 1H, J 7.5, H-1¢¢), 4.15 (dt, 1H, J 7.0, 4.5, H-4¢), 3.88 (dd,
H, J 12.5, 2.0, H-6¢¢a), 3.66 (dd, 1H, J 12.0, 6.0, H-6¢¢b), 3.34 (t,
H, J 9.0, H-3¢¢), 3.29–3.26 (m, 2H, H-4¢¢, H-5¢¢), 3.20 (dd, 1H, J
.0, 7.5, H-2¢¢), 2.27 (dd, 2H, J 6.5, 6.0, H-2¢), 1.89 (d, 3H, J 1.0,
(
EtOAc/MeOH 7:3 as eluent) to give 40 (55 mg, 82%) as a white
solid: [a]
2
0
D
= +2.3 (c = 1.48, DMSO); d
H
(500 MHz, CD OD)
3
8
.17 (s, 1H, H-2), 7.86, 7.85 (2 ¥ s, 2H, H-8, H-5¢¢¢), 5.99 (d, 1H,
J 4.5, H-1¢), 4.96 (d, 1H, J 12.0, OCH ), 4.88 (dd, 1H, J 14.5,
.5, H-5¢a), 4.79 (dd, 1H, J 14.5, 3.5, H-5¢b), 4.67 (d, 1H, J 12.0,
OCH ), 4.44 (t, 1H, J 5.5, H-3¢), 4.38 (m, 1H, H-4¢), 4.34 (d,
H, J 8.0, H-1¢¢), 4.29 (t, 1H, J 5.0, H-2¢), 3.84 (dd, 1H, J 3.5,
.0, H-4¢¢), 3.78 (dd, 1H, J 11.5, 7.0, H-6¢¢a), 3.72 (dd, 1H, J 11.5,
.0, H-6¢¢b), 3.58 (dd, 1H, J 9.5, 7.5, H-2¢¢), 3.55 (ddd, 1H, J 7.0,
CH
3
); d
C
(125 MHz, CD
3
OD) 166.38 (C-4), 152.22 (C-2), 145.83
A
H
B
(
C-4¢¢¢), 138.28 (C-6), 126.56 (C-5¢¢¢), 112.02 (C-5), 103.66 (C-1¢¢),
4
8
7
3
7.00 (C-1¢), 85.47 (C-4¢), 78.11, 78.03 (C-3¢¢, C-5¢¢), 75.06 (C-2¢¢),
A
H
B
2.40 (C-3¢), 71.67 (C-4¢¢), 63.03 (CH ), 62.92 (C-6¢¢), 52.59 (C-5¢),
2
1
1
5
4
+
9.56 (C-2¢), 12.56 (CH
3
); HRMS (ES ) m/z calc. for C19
H
28
+
N
5
O
10
+
(
(
M + H ): 486.1831; found 486.1833; m/z 524 (19%, M + K ), 508
32%, M + Na ).
+
.5, 1.0, H-5¢¢), 3.50 (dd, 1H, J 10.0, 3.5, H-3¢¢); d
OD) 157.30 (C-6), 154.19 (C-2), 150.60 (C-4), 145.77 (C-4¢¢¢),
41.25 (C-8), 127.13 (C-5¢¢¢), 120.29 (C-5), 104.51 (C-1¢¢), 90.10
C-1¢), 83.54 (C-4¢), 76.93 (C-5¢¢), 74.85, 74.81 (C-2¢, C-3¢¢), 72.39
C
(125 MHz,
CD
3
5¢-Deoxy-5¢-[4-(a-D-xylopyranosyloxymethyl)-1,2,3-triazol-1-
1
(
yl]uridine (65). Was prepared from prop-2-ynyl-a-D-xylopyra-
noside 19 (27.7 mg, 0.147 mmol) and 5¢-azido-5¢-deoxyuridine 7
4
034 | Org. Biomol. Chem., 2009, 7, 4029–4036
This journal is © The Royal Society of Chemistry 2009

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(
43.6 mg, 0.162 mmol) as described for 40. Following automated
linear region of product formation and K
i
values were determined
chromatography (EtOAc/MeOH 4:1 as eluent), 65 (63 mg, 94%)
by weighted non-linear regression analysis of the hyperbola plot of
rate against substrate concentration using the equation for linear
competitive inhibition in Grafit version 5 (Erithacus Software
Ltd).
2
0
was obtained as a white solid: [a]
(500 MHz, CD OD) 8.04 (s, 1H, H-5¢¢), 7.40 (d, 1H, J 8.0,
H-6), 5.73–5.70 (m, 2H, H-1¢, H-5), 4.86 (d, 1H, J 3.5, H-1¢¢), 4.82
D
= +107.7 (c = 0.76, DMSO);
d
H
3
(
(
(
1
2
CD
1
(
(
dd, 1H, J 14.5, 3.5, H-5¢a), 4.80 (d, 1H, J 12.5, OCH
dd, 1H, J 15.0, 7.0, H-5¢b), 4.64 (d, 1H, J 12.5, OCH
dt, 1H, J 6.5, 3.5, H-4¢), 4.19 (dd, 1H, J 5.5, 4.0, H-2¢), 4.11 (t,
A
H
H
B
), 4.73
), 4.25
NTP substrate kinetics were carried out in a final volume of
A
B
300 mL in a 96-well microplate. For the K
m
determination of
ATP, each reaction mixture contained NADH (150 mM), PEP
(2 mM), Pan (250 mM), BaPanK (2.5 mg), PK (2.5 units), LDH
(5 units), and varying concentrations of ATP substrate. Reactions
were initiated by addition of BaPanK. The same conditions were
used for alternative NTP substrates except 12.5 units of PK was
used to ensure that PK was not the rate limiting step in these
coupled assays (as was observed when 2.5 units of PK were used).
Kinetic data were analysed (by non-linear regression) using Grafit.
H, J 6.0, H-3¢), 3.59–3.53 (m, 2H, H-3¢¢, H-5¢¢a), 3.52–3.43 (m,
H, H-4¢¢, H-5¢¢b), 3.38 (dd, 1H, J 9.0, 3.5, H-2¢¢); d (125 MHz,
OD) 166.15 (C-4), 152.08 (C-2), 145.72 (C-4¢¢¢), 143.44 (C-6),
26.62 (C-5¢¢¢), 103.12 (C-5), 100.06 (C-1¢¢), 93.41 (C-1¢), 83.00
C
3
C-4¢), 75.16 (C-3¢¢), 74.19 (C-2¢), 73.59 (C-2¢¢), 71.95 (C-3¢), 71.56
-
C-4¢¢), 63.34 (C-5¢¢), 61.60 (CH
2
), 52.52 (C-5¢); HRMS (ES ) m/z
+
calc. for C17
H
22
+
N O10 (M-H ): 456.1367; found 456.1364; m/z 456
5
(
100%, M - H ).
5
¢-Deoxy-5¢-[4-(2-acetylamino-2-deoxy-a-D-glucopyranosy-
Acknowledgements
loxymethyl)-1,2,3-triazol-1-yl]cytidine (78). Was prepared from
prop-2-ynyl-2-acetylamino-2-deoxy-a-D-glucoside 16 (31.2 mg,
.120 mmol) and 5¢-azido-5¢-deoxycytidine
.132 mmol) as described for 40. Following automated chromatog-
We are thankful for financial support from the Wellcome Trust
(grant ref. 067525/Z/02/Z and VS/07/QUEB/A5) (WAW, CJH,
0
0
1 (35.5 mg,
NC), the Department of Employment and Learning (ASR), the
National Institutes of Health (grant ref. GM-35394 (NIN, AC))
and a grant from the Southeast Regional Centre of Excellence
for Biodefense and Emerging Infections. We also thank the En-
gineering and Physical Sciences Research Council (EPSRC) Mass
Spectrometry Service Centre, Swansea, for invaluable support.
raphy (EtOAc/MeOH 7:3 as eluent), 78 (53 mg, 83%) was obtained
as a white solid: d (500 MHz, CD OD) 8.02 (s, 1H, H-5¢¢¢), 7.35
d, 1H, J 7.5, H-6), 5.87 (d, 1H, J 7.5, H-5), 5.71 (d, 1H, J 3.5,
H-1¢), 4.87 (d, 1H, J 3.5, H-1¢¢), 4.84 (dd, 1H, J 14.5, 3.5, H-5¢a),
.81 (d, 1H, J 12.5, OCH
.62 (d, 1H, J 12.5, OCH
.21 (dd, 1H, J 6.0, 3.5, H-2¢), 4.08 (dd, 1H, J 7.0, 6.0, H-3¢), 3.91
dd, 1H, J 11.0, 3.5, H-2¢¢), 3.83 (dd, 1H, J 12.0, 2.0, H-6¢¢a), 3.69
dd, 1H, J 12.0, 5.5, H-6¢¢b), 3.66 (dd, 1H, J 10.5, 8.5, H-3¢¢), 3.62
ddd, 1H, J 9.5, 5.5, 2.0, H-5¢¢), 3.36 (dd, 1H, J 10.0, 9.0, H-4¢¢),
H
3
(
4
4
4
(
(
(
1
(
9
7
6
A
H
B
), 4.74 (dd, 1H, J 15.0, 7.0, H-5¢b),
A
H
B
), 4.26 (dt, 1H, J 7.0, 3.5, H-4¢),
Notes and references
1
2
3
4
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3
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C
(125 MHz, CD
3
OD) 173.79 (C=O), 167.78
C-4), 158.15 (C-2), 145.42 (C-4¢¢¢), 143.67 (C-6), 126.50 (C-5¢¢¢),
8.06 (C-1¢¢), 96.37 (C-5), 94.83 (C-1¢), 82.76 (C-4¢), 74.70 (C-2¢),
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¢¢), 61.27 (CH
calc. for C20
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H
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N
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O
10 (M + H ): 528.2054; found 528.2051; m/z
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5
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◦
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1
initiated by the addition of BaPanK. Inhibitor stock solutions
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m
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4
036 | Org. Biomol. Chem., 2009, 7, 4029–4036
This journal is © The Royal Society of Chemistry 2009