Probing myo-inositol 1-phosphate synthase with multisubstrate adducts

Article abstract of DOI:10.1039/c2ob26577j

The synthesis of a series of carbohydrate-nucleotide hybrids, designed to be multisubstrate adducts mimicking myo-inositol 1-phosphate synthase first oxidative transition state, is reported. Their ability to inhibit the synthase has been assessed and resu

Full text of DOI:10.1039/c2ob26577j

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
PAPER
Probing myo-inositol 1-phosphate synthase with
multisubstrate adducts†
Cite this: Org. Biomol. Chem., 2012, 10,
9601
Rania M. Deranieh,a Miriam L. Greenberg,a Pierre-B. Le Calvez,b Maura C. Mooneyc
and Marie E. Migaud*b,d
Received 9th August 2012,
Accepted 24th October 2012
The synthesis of a series of carbohydrate–nucleotide hybrids, designed to be multisubstrate adducts
mimicking myo-inositol 1-phosphate synthase first oxidative transition state, is reported. Their ability to
inhibit the synthase has been assessed and results have been rationalised computationally to estimate
their likely binding mode.
DOI: 10.1039/c2ob26577j
www.rsc.org/obc
subsequent enolisation, proposed to be substrate-assisted, pre-
cedes the intramolecular aldol condensation and the sub-
Introduction
myo-Inositol 1-phosphate synthase (mIPS) catalyses the first sequent reduction of the resulting inosose-2-phosphate to mIP
step in inositol biosynthesis, and carries out the conversion of with the concomitant oxidation of NADH to NAD.9
D-glucose 6-phosphate (G6P) into myo-inositol 1-phosphate
While this catalytic sequence has been validated, the crys-
(mIP).1 The structural gene encoding mIP synthase (INO1) has tallographic analyses of eukaryotic mIPS with various inhibi-
been identified, isolated and cloned from several prokaryotic tors,1,4,10 substrate analogues11 and cofactors12 have provided
and eukaryotic microorganisms.2,3 The gene sequence of INO1 structural information which have raised more questions than
exhibits a high degree of homology amongst eukaryotes which anticipated. For instance, mIPS isolated from Saccharomyces
drops when compared to prokaryotes.2,4 Stoker et al. have cerevisiae was co-crystallised in the presence of NAD with
demonstrated that
a mutant Mycobacterium tuberculosis 2-deoxyglucitol 6-phosphate (1, Fig. 1) and with 2-deoxy-D-
lacking INO1 is only viable in the presence of extremely high glucitol 6-(E)-vinylhomophosphonate (2, Fig. 1), respectively
levels of exogenous inositol.5 The limited degree of homology and the mechanistic consequences rationalised.1 Both G6P
and the innate need for mIPS activity in bacterial strains like analogues are potent competitive inhibitors of mIPS; however
M. tuberculosis renders mIPS an attractive therapeutic target their binding modes were very distinct. 2-Deoxy glucitol
for the identification of novel antiproliferative agents. Simi- 6-phosphate (1) is a competitive inhibitor of mIPS with Ki in
larly, mIPS has been proposed as possible target for bipolar the mid-micromolar range against eukaryotic mIPS, binding
drugs.6 As such, a carefully rationalised understanding of the the NAD-mIPS complex in almost a head-to-tail type binding
eukaryotic mIPS’ mechanism and its divergence from the pro- (Type 1 binding, Fig. 2). 2-Deoxy-D-glucitol 6-(E)-vinylhomophos-
karyotic mIPS, is crucial if one is to facilitate such endeavours. phonate, the most potent mIPS competitive inhibitor at sub-
From a mechanistic perspective,7,8 mIPS converts G6P into micromolar concentration is a slow-binding inhibitor and
mIP (Scheme 1) in 4 steps within one catalytic cycle, using when compared to the binding mode of (1), (2) binds in what
NAD as a prosthetic group. The mIPS/NAD complex catalyses is anticipated to be a substrate-like binding mode (Type 2
the oxidation of the C5 hydroxyl of G6P presented in its binding, Fig. 2). Additionally and unlike (1), the glucitol (2)
opened form in the cofactor-bound enzyme catalytic pocket was shown to be oxidised by mIPS/NAD to yield a mIPS/NADH/
with the concomitant reduction of NAD to NADH. The enone phosphonate complex, mimic of the mIPS/NADH/
5-ketoglucose 6-phosphate productive complex A (Scheme 1),
further supporting the substrate-like binding mode of (2).
aCollege of Liberal Arts and Sciences, Biological Sciences, Wayne State University,
These results, along with the crystallographic analyses strongly
5047 Gullen Mall, Detroit, MI 48202, USA
supported the proposal that 2-deoxy-D-glucitol 6-(E)-vinylhomo-
phosphonate binding mode was best at mapping mIPS cataly-
tic and binding sites, both for NAD and its natural substrate,
glucose 6-phosphate. Yet, the limited and controversial nature
of the chemical and structural data available on mIPS catalytic
site, compounded by the major induced-fit movements occur-
ring within mIPS upon the binding of NAD to the apo-mIPS
bCentre for Cancer Research and Cell Biology, Queen’s University Belfast, 97 Lisburn
Road, Belfast BT9 7BL, UK
cSchool of Chemistry and Chemical Engineering, Queen’s University, Belfast, David
Keir Building, Stranmillis Road, Belfast, BT9 5AG, NI, UK
dSchool of Pharmacy, Medical Biology Centre, Queen’s University Belfast, 97 Lisburn
Road, Belfast BT9 7BL, UK. E-mail: m.migaud@qub.ac.uk
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c2ob26577j
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9601

Paper
Organic & Biomolecular Chemistry
View Article Online
Scheme 1 mIPS catalytic cycle.
Fig. 1 2-Deoxy-D-glucitol 6-phosphate (1) and 2-deoxy-D-glucitol 6-(E)-vinyl-
homo-phosphonate (2), low micromolar inhibitors of eukaryotic mIPS.
and the binding of G6P to the mIPS/NAD complex, implies
that new approaches to designing high affinity mIPS ligands
capable of providing structural information both at the NAD
and the G6P binding sites would be highly valuable.
We decided to employ a multisubstrate adduct (MA)
approach to the design of such potential ligands and use the
transition state of the first oxidative step of the catalytic cycle
as template. The design of the multisubstrate adducts was
based on the transition state for which the C-5 hydrogen of
G6P is transferred as hydride to NAD.8,13 The selection of syn-
thetic targets was then guided by chemical feasibility. To
support the MA approach, it must be noted that mIPS/NAD
Fig. 2 Depiction of head-to-tail binding, through backbone superimposition of
1rm0 and 1jki mIPs structures.
exchange studies on a mixture of 5-2H-G6P/G6P have shown
that tightening of the complex occurs in the early stages of the
catalysis, with no scrambling of the deuterium atom upon
9602 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
turnover to L-2-2H-mIP and L-mIP respectively (Fig. 1).14 There-
fore, we proposed to generate a class of multisubstrate adducts
where the carbohydrate moiety of G6P was to be linked to an
aromatic ring incorporated in a nucleosidic linkage with a
riboside moiety. Such a molecule would possess the features of
the phosphosugar substrate and of the nucleotide cofactor,
both required for a multisubstrate type binding to mIPS. It is
anticipated that upon mIPS binding, such MA would offer
high levels of stabilisation through interactions with the indi-
vidual substrate and cofactor binding pockets’ residues, as
well as high level of specificity. Ideally, an adduct of this type
could incorporate the adenosine moiety of NAD+ as the
adenine nucleobase which has been shown to provoke a major
conformational shift upon binding of NAD+ through specific
and selective interactions and as such being deeply buried
into the NAD+ binding pocket.12 However, adenosine diphos-
phate ribose (ADPR) was found to be a poor inhibitor of mIPS,
thus indicating the importance of the ribosyl nicotinamide
moiety for tight binding and potency. Additionally, since it is
thought that it is the open form of G6P, and not the cyclic
anomer,7 which binds NAD+-bound mIPS, the carbohydrate
moiety most likely to provide the best binding potential with
mIPS as part of an adduct structure is therefore the reduced
sugar form, i.e. glucitol. Finally, on all occasions when the
2-deoxy sugar series were synthesised and evaluated as mIPS’s
inhibitors, increased potency was achieved over the hydroxyl-
ated parent series. It is therefore reasonable to consider also a
set of multisubstrate adducts where the 2-deoxy series can be
assessed and compared for potency against the hydroxylated
sugar parents.
Fig. 3 Rationale for MAs design.
structural flexibility between the two multisubstrate adduct
subunits while offering H-bond interactions and metal coordi-
nation opportunities.1 Finally, since (1) and (2) adopt opposite
binding conformations inside the mIPS active site, and that
the initial binding conformation of the substrate remains
unknown, the target MAs were designed to address such
binding versatility. Therefore, the structure of the multisub-
strate adducts was devised to include a glucitol or 2-deoxyglu-
citol moiety which incorporated a phosphoryl moiety on the
primary alcohols of the modified glucitol, so to provide oppor-
tunities for the “head-to-tail binding” like for 2-deoxy glucitol
6-phosphate (1) or for the productive binding of 2-deoxy-D-
glucitol 6-(E)-vinylhomophosphonate (2) with NAD-mIPS.
Therefore, a phosphate monoester group was introduced at the
positions X, Y and W (Fig. 3) to generate the series of mono-
and di-phosphorylated MAs.
The multisubstrate adduct was to also incorporate features
from NAD, in particular that of the nicotinamide nucleotide
part of the cofactor, which was to be linked by a triazole
moiety to the glucitol moieties.15,16 A methylenoxy was chosen
Results and discussion
Synthesis
to bridge the glycon units and to compensate for the spatial In order to synthesise the glucitol based MA series, we first
and rotational restrictions introduced by the triazolide moiety. focused on accessing two types of precursors, the alkynes (7)
As such, the selected triazolide-type linkage (Fig. 2) provided and (9) (Scheme 2) and the furanosyl azides (11) and (14)
Scheme 2 Synthesis of glucitol precursors of MAs.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9603

Paper
Organic & Biomolecular Chemistry
View Article Online
Scheme 3 Synthesis of furanosyl azide precursors of MAs.
(Scheme 3). These two families of precursors were then respectively. Both intermediates (6) and (8) were phosphory-
coupled via copper(I)-catalysed triazolide chemistry leading to lated via a Mitsunobu-type reaction using the free acid of
the fully-protected MA precursors, which were subsequently dibenzylphosphate diester, leading to the two glucitol precur-
deprotected. The protecting group strategy adopted for this sors (7) and (9) in good 80% and 74% yields, respectively.
synthetic sequence aimed at accessing the pure MAs from the Other phosphorylation methods including phosphochloridite
pure benzylated parents, so that a quantitative deprotection and phosphoramidite P(III) chemistry and phosphochloridate
step by hydrogenolysis could be conducted.
and pyrophosphate P(V) chemistry proved either unsuccessful
The alkyne intermediate (5), precursor of (7) and (9), was or extremely low yielding.
obtained via a 7-step route, starting from an α/β D-glucose
Two furanosyl azide units were synthesized with the aim to
mixture. The protection of the anomeric hemiacetal with an incorporate a phosphate moiety at the C5 position of the fura-
allyl group under Fisher conditions was followed by the protec- nosyl moiety. Thus, by treatment of (10) with azido-trimethyl-
tion of the C6-position with trityl chloride affording allyl 6-O- silane with AlCl3 in diethyl ether, the first furanosyl azide unit
trityl-D-glucopyranoside intermediate in 52% overall yield.17 (11) was obtained quantitatively.16 After removal of the benzo-
The remaining free hydroxyl groups were then benzylated ate groups, the secondary hydroxyl groups were protected by
giving allyl-2,3,4-tri-O-benzyl-6-O-trityl-D-glucopyranoside (3) in using 2,2-dimethoxypropane in acetone, leading to (12) in 87%
excellent 92% yield.18 After the removal of the allyl group, the yield over two steps. The phosphate moiety was then intro-
reduction of the pyranose ring yielded 2,3,4-tri-O-benzyl-6-O- duced using the Mitsunobu protocol, and the isopropylidene
trityl-D-glucytol (4) almost quantitatively. tert-Butyldiphenylsilyl moiety removed in the presence of TFA to facilitate purification
chloride was used to selectively protect the primary alcohol in and affording the diol intermediate in 67% yield over 2 steps.
84% yield and the propargylation of the secondary alcohol at The furanosyl hydroxyl groups had to be acetylated to form
the C5 position was achieved in 72% yield by using propargyl (14), as its solubility in organic solvents had a dramatic effect
bromide and sodium hydride in THF at reflux. The propargyl on the reaction rates during the triazolide formation by click
intermediate (5) was then selectively deprotected at the C6 and chemistry.
C1 positions by treatment with AlCl3 in diethyl ether to access
The cyclisation between the different precursors (5), (7), (9)
the C6-deprotected propargylated glucitol (6), or by treatment and (11), (14) occurred upon treatment with a catalytic amount
of (5) with TBAF in THF to access the C1-deprotected parent (8), of copper sulfate and sodium ascorbate in a tBuOH–H2O
Table 1 Synthesis of fully-protected MAs (15)–(19) by click chemistry
Alkyne
R6
R1
Azide
R5
R
Product
Yield
(5)
(5)
(7)
(9)
(9)
Tr
TBDPS
(11)
(14)
(14)
(11)
(14)
Bz
Bz
H
Ac
Bz
Ac
(15)
(16)
(17)
(18)
(19)
(62%)
(37%)
(73%)
(58%)
(62%)
Tr
TBDPS
P(O)(OBn)2
P(O)(OBn)2
Bz
Tr
P(O)(OBn)2
TBDPS
TBDPS
P(O)(OBn)2
P(O)(OBn)2
P(O)(OBn)2
9604 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
Table 2 Synthesis of MAs (21)–(24) from the fully protected MA precursors (16)–(19)
Substrate
Product
R6
R5
R
R1
R6′
R5′
R1′
(%)a
16
17
18
19
Tr
P(O)(OBn)2
P(O)(OBn)2
Bz
Ac
Ac
Bz
Ac
TBDPS
H
P(O)(OH)2
P(O)(OH)2
H
H
23b (84%)
24c (94%)
21b (81%)
22b (73%)
Tr
P(O)(OBn)2
TBDPS
H
P(O)(OH)2
P(O)(OBn)2
P(O)(OBn)2
P(O)(OH)2
P(O)(OH)2
H
H
P(O)(OBn)2
TBDPS
P(O)(OH)2
a Overall isolated yield. b Deprotection with Method A. c Deprotection with Method B.
mixture, heated at 40 °C for a period of 6–8 hours (Table 1) in (Table 2, Method A). For compounds (15), (18) and (19), the
low (e.g. (16) in 37%) to good yields (e.g. (17) in 73%). In order acyl moieties were first removed with sodium methoxide in
to force this reaction, the furanosyl azide was used in slight methanol, and the TBDPS was then removed using TBAF in
excess.
THF. Water was added prior to the removal of THF to prevent
It is anticipated that the lower yields observed for the for- product decomposition and allow for the extraction of the
mation of (16) are to be associated with the likely hydrophilic respective products in DCM. The untimely removal of THF
solvation level of the partially protected highly polar phospho- under reduced pressure and increased ionic strength caused
sugar, thus limiting its interactions with the more lipophilic the loss of the phosphate tri-ester moiety at the C6 position.
glucitol propargyl reagents. Similar results were obtained for After purification by chromatography column, the partially
the deoxy-glucitol series, e.g. compound (35).
The deprotection sequence proved to be crucial to the suc- lysis yielding (21) and (22) in good (73–94%) overall yields.
cessful completion of this class of MAs’ synthesis. Initially, the The trityl protected MAs (16) and (17) were deprotected via
protected phosphorylated intermediates underwent hydrogeno-
following sequence was selected; ester deprotection, followed a two-step process consisting in the use of sodium methoxide
by trityl or TBDPS deprotection followed by hydrogenolysis. in methanol followed by hydrogenolysis (Table 2, Method B).
However, this strategy required workup and purification by While the trityl moiety could be deprotected under mild acidic
column chromatography at each step, resulting in noticeable conditions in (16),19 these conditions also yielded the removal
mass losses. Crucially, it was noticed that compounds bearing of the phosphate tri-ester group at the C1 position in the pro-
a TBDPS group and those bearing a trityl moiety did not gener- tected MA (17). This phosphate hydrolysis was assumed to
ate the same type of sugar-based impurities, each class leading occur via an intramolecular nucleophilic substitution initiated
to
a different type of disproportionation, in particular by the initial release of C6–OH group and acid catalysed acti-
with regards to phosphate moiety losses. Therefore, the vation of the PvO bond.20 The trityl group removal was there-
specific sequence was developed for the TBDPS-protected MAs fore carried out concurrently to the removal of the benzyl
Scheme 4 Synthesis of 2-deoxy glucitol precursors of MAs.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9605

Paper
Organic & Biomolecular Chemistry
View Article Online
groups of the phosphoesters at the hydrogenolysis step leading
The 2-deoxy progargylated glucitol (30) was selectively
to (24) in excellent 94% overall yield. The removal of the first deprotected at the C6 and C1 positions by treatment with alu-
benzyl moiety of the phosphate group is fast, thus generated a minium trichloride in diethyl ether and TBAF in THF, to yield
more stable phosphodiester intermediate. The in situ gener- the propargylated glucitols (31) and (32), respectively
ation of the phosphoric acid diester is then sufficient to (Scheme 5). Both intermediates (31) and (32) were phosphory-
initiate the acid-catalysed removal of the trityl group, revealing lated via a Mitsunobu-type reaction using dibenzylphosphate
the C6–OH moiety which remains unreactive towards the par- leading to the two glucitol precursors (33) and (34), respecti-
tially and fully deprotected phosphate ester moieties.
vely. At this stage, problems were encountered during purifi-
Triacetylated D-glucal (25) was used as starting material for cation, in particular for the compound (33). Both the by-
the preparation of the 2-deoxy series of MAs, as 2-deoxy-D- product DIHD and the C6-phosphorylated alkyne (33)
glucose was not suitable as starting material for its implemen- co-eluted regardless of the eluent system used. Therefore, iso-
tation in the strategy developed for the D-glucose series lation of compound (33) as a pure product was not possible
(Scheme 4). As such, the partially protected glycal (26) was under standard silica chromatographic methods. Nonetheless,
obtained following removal of the acetate groups by treatment the diisopropyl hydrazodicarboxylate (DIHD) contaminated
with sodium methoxide in methanol and selective protection material could be used in the subsequent steps.
of the primary hydroxyl with trityl chloride. Following the ben-
The coupling between the different types of propargylated
zylation of (26) in 53% yield, the fully protected glycal (27) was precursors (30), (33) and (34) and the azido-furanosides pre-
subjected to a mercuration–reductive demercuration sequence viously synthesised occurred by using catalytic amount of
in order to convert the D-glucal derivative into its 2-deoxy-D- copper sulfate and sodium ascorbate in a tBuOH–H2O mixture
glucose analogue (28). This was achieved in 51% yield. From at 40 °C for 6–8 hours (Table 3) in presence of a slight excess
this stage onwards, the same synthetic sequence as that devel- of the azido reagent. While facile isolation of the cyclised pro-
oped for the D-glucose series was implemented.
ducts was achieved for most reactions, it was necessary to first
Scheme 5 Selective deprotection and phosphorylation of the precursors to the 2-deoxy glucitol MAs series.
Table 3 Synthesis of fully-protected 2-deoxy glucitol MAs (35)–(39) by click chemistry
Alkyne
R6
R1
Azide
Product
R
R5′
30
33
33
34
34
Tr
TBDPS
14
14
11
14
11
35a (28%)
36 (39%)
37 (71%)
38 (62%)
39 (44%)
H
P(O)(OBn)2
P(O)(OBn)2
Bz
P(O)(OBn)2
Bz
P(O)(OBn)2
TBDPS
Ac
Bz
Ac
Bz
P(O)(OBn)2
Tr
Tr
TBDPS
P(O)(OBn)2
P(O)(OBn)2
a To facilitate subsequent purification, the acetylated triazolide product obtained from (30) and (14) had to be deprotected following
work up using MeOH/MeONa.
9606 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
Table 4 Synthesis of MAs (40)–(44) from the fully protected MA precursors (35)–(30)
Substrate
Product
R6
R5
R
R1
R6′
R5′
R1′
(%)a
35
36
37
38
39
Tr
P(O)(OBn)2
P(O)(OBn)2
Bz
P(O)(OBn)2
Bz
H
TBDPS
H
P(O)(OH)2
P(O)(OH)2
H
P(O)(OH)2
H
H
42b (75%)
43b (76%)
44b (68%)
40c (90%)
41c (87%)
P(O)(OBn)2
Ac
Bz
Ac
Bz
TBDPS
P(O)(OH)2
H
P(O)(OBn)2
Tr
Tr
TBDPS
P(O)(OBn)2
P(O)(OBn)2
P(O)(OH)2
H
H
H
P(O)(OH)2
P(O)(OH)2
a Overall isolated yield. b Deprotection with Method A. c Deprotection with Method B.
Fig. 4 mIPS inhibition by MAs assayed at 100 and 200 micromolar concentrations.
remove the acetate groups to isolate the corresponding pro- and the reaction mixture was incubated for 1 h at 37 °C. The
tected MA for the 2-deoxy glucitol adduct (35).
remaining mIP synthase activity was determined by the rapid
The same sequence of deprotection as that developed for colorimetric method of Barnett et al.22 with minor
the glucitol series was applied to the tritylated and silylated modifications.
protected 2-deoxyglucitol MAs to yield the fully deprotected
All MA compounds thus synthesised and assayed against
2-deoxy parent series, compounds (40)–(44). Similar yields to mIPS displayed some level of inhibition, even though modest,
those obtained for the glucitol MAs series were obtained for at high micromolar levels in a concentration-dependent
the preparation of the 2-deoxy MAs (Table 4).
manner (Fig. 4).
Table 5 provides a summary of the inhibitory properties of
the series of MAs thus far synthesised, with the remaining
activity being measured at two concentrations (100 and
200 μM) of MAs in the enzymatic assay solution. As indicated
in Table 5, compounds (40) and (41), both 2-deoxy glucitol
based MAs, lack inhibitory properties. This would indicate
that in the case of the deoxy series, the phosphate positioned
Biological evaluation
All MAs were evaluated against purified yeast recombinant
mIPS enzyme as inhibitors of G6P conversion to mIP. Purified
protein was suspended in the reaction buffer and incubated in
the presence of the MAs in various concentrations (0, 100 μM
and 200 μM). Glucose 6-phosphate (5 mM) was then added
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9607

Paper
Organic & Biomolecular Chemistry
View Article Online
Table 5 Inhibitory properties of the schematically presented phosphorylated MAs
Inhibitor
a
a
a
a
a
Series
concentration MAs
%
MAs
22
%
MAs
23
%
MAs
%
MAs
21
%
Glucitol series
100 μM
200 μM
100 μM
200 μM
24
40
81
67
109
84
90
81
81
67
93
92
95
93
99
93
76
63
2-Deoxy-glucitol
series
43
42
41
101
90
44
a % mIPS activity remaining upon incubation.
next to the 2-deoxy carbon has a detrimental effect on recog- effectively with NAD, which possesses a Km in the micromolar
nition by mIPS. However, a phosphate group located at the C6 range. In an attempt to address some of these aspects of MAs’
position of the 2-deoxyglucitol end of the MAs restores inhi- binding to mIPS, we conducted pre-incubation experiments,
bition, whether or not the riboside end is phosphorylated, as whereby apo-mIPS and the MAs were pre-incubated for 15 min
demonstrated by the inhibitory properties displayed by (43) prior to the addition of NAD, the cofactor and G6P, the sub-
and (44). For comparison, 2-deoxy glucitol 6-phosphate was strate. No difference in the overall remaining enzyme activity
also incubated under the same conditions, and less than 5% was observed, indicative of a truly reversible equilibrium and
mIPS activity was observed at these concentrations, consistent the absence of long-lived enzyme inhibitor complex (i.e. low
with the fact that it is a potent inhibitor of mIPS.
Koff). This also indicated that even if the flexibility of the MAs
was detrimental to recognition by mIPS, it was not a limiting
factor with regards to mIPS inhibition. When the 5-phosphate
riboside is the only anionic moiety in the MAs (i.e. (23) and
(42)), no concentration-dependent inhibition is observed,
indicative that the remaining sugar moiety has no impact on
mIPS binding. On the other hand, when there is no phosphate
on the furanoside moiety, then the hexose series display a con-
sistent inhibitory trend. This would indicate that the recog-
nition process is glucitol phosphate-driven rather than
riboside phosphate-driven.
Graphical representation using (24) as example:
We conducted some docking experiments using 1rm0 and
1jki crystallographic data,1,11 for which mIPS was either co-
crystallised in the presence of 2-deoxy-D-glucitol 6-(E)-vinyl-
homophosphonate or 2-deoxy glucitol 6-phosphate, respect-
ively. On all accounts, the docking scores were better when
1rm0 was used. Importantly, with 1jki, the glucitol end of all
the MAs, except (41), appears to dock preferentially in the NAD
binding pocket, with the riboside end being accommodated
in the glucose 6-phosphate binding pocket. With 1rm0, all
MAs tested show binding of the riboside moiety in the
NAD pocket for nicotinamide riboside with the (2-deoxy)-
glucitol moiety fitting in the substrate binding site. To illus-
trate this, the docked poses of (43) in 1rm0 and 1jki are found
in Fig. 5.
Importantly, the introduction of the riboside adduct at the
C5-position of the glucitol 6-phosphate moiety has a detrimen-
tal impact on the overall inhibitory activity of this class of com-
pounds. This might be due to the removal of the C5-hydroxyl-
mIPS hydrogen bonding observed between 2-deoxy glucitol
6-phosphate and mIPS, reducing the overall mIPS binding
affinity for the MAs. Alternatively, the flexibility of the MAs
might be a major limiting factor with regards to the initial
enzyme–inhibitor recognition. It must also be noted that NAD
is thought to bind the apo-enzyme, which then becomes con-
formationally “ready” for G6P binding. In a standard assay,
the multisubstrate adducts might not be able to compete
Importantly, the same residues involved in the interaction
between the two known potent inhibitors of mIPS remain
apparently involved in the preferred coordination of the MAs
when high docking scores are observed. Additionally, the
9608 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
Fig. 5 Interaction of compound (43) with mIPS, using the conformation adopted in the crystal structure (a) 1rm0 and (b) 1jki.
compounds which appear to dock best in the anticipated compounded by the high MAs flexibility is likely to disfavour
binding mode (i.e. occupancy of both the nicotinamide ribo- strong binding affinity with the mIPS binding pocket, and
side binding pocket and the glucose 6-phosphate binding might be a leading factor for the overall poor potency amongst
pocket by the corresponding MAs moieties), are also the com- the MA inhibitors.
pounds which display the best inhibition against mIPS, thus
supporting the overall observation that 1rm0 provides a more
informative and reliable crystallographic starting point of the
mIPS binding pocket. The poses also indicate that to occupy
Conclusion
both pockets, the MAs ether linker adopts a highly strained
conformation which pushes the triazolide ring out of the
hydrophobic pocket which surrounds the nicotinamide ring in
NAD, adopting an almost perpendicular conformation. This
We have synthesised a series of carbohydrate–nucleotide
hybrids, designed to be multisubstrate adducts, mimicking
myo-inositol 1-phosphate synthase first oxidative transition
state. We have shown that these compounds inhibited the
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9609

Paper
Organic & Biomolecular Chemistry
View Article Online
synthase at high micromolar levels, on par with the Km of
The synthetic procedures to access compounds (3), (4), (11),
glucose 6-phosphate (Km ∼ 1 mM) and have used known crys- (25), (26), (27), (28), and (29) and their respective characteris-
tallographic information to rationalize computationally their ation have been reported in the ESI.†
likely binding mode. It is possible that the stereochemistry at
General procedures
the C5-position of the hexitol derivatives, imposed by the use
of a D-sugar as starting material, has had a detrimental effect
PROCEDURE A: PHOSPHORYLATION WITH DIBENZYLPHOSPHATE. To a sol-
of the overall stability of the enzyme–inhibitor complex, due to ution of TPP (3.0 equiv.) in dry THF (C ∼ 1.0 M) at 0 °C, DIAD
the adoption of an unfavourable conformation while fitting (3.0 equiv.) was added and the mixture was stirred until a
the carbohydrate/nucleotide binding pockets. The route to the yellow solid had precipitated. Dibenzylphosphate (1.5 equiv.)
C5-epimers of (24), (43) and (44), from (4) and (29) are now was then added to the mixture and after being stirred at 0 °C
under investigation.
for 5 minutes, a solution of substrate in dry THF (C ∼ 0.01 M)
was slowly added. The reaction mixture was stirred and
allowed to warm to rt over a period of 12 hours. The THF was
then removed under reduced pressure and the residue was dis-
solved in Et2O (C ∼ 1 mg mL−1). The organic solution was
Experimental
All reactions requiring anhydrous or inert conditions were washed with a saturated NaHCO3 solution (3 × 1/3 VEt2O mL),
carried out under a positive atmosphere of argon in oven-dried water (3 × 1/3 VEt2O mL), brine (1/3 VEt2O mL) and dried over
glassware. Solutions or liquids were introduced in the round MgSO4. The filtrate was kept in a sealed flask at rt overnight.
bottom flask using oven-dried syringes or cannula through The white precipitate was filtered off and purified by flash
rubber septa. All reactions were stirred magnetically using column chromatography.
Teflon-coated stir bars unless otherwise specified. In the case
PROCEDURE B: TRIAZOLE FORMATION BY CLICK CHEMISTRY. To a flask
requiring heating, the reactions were warmed using an electri- containing a suspension of alkyne (1.0 equiv.), the appropriate
cally-heated silicon oil bath, and the stated temperature is the azide (1.1 equiv.) and a water–tBuOH mixture (1 : 1 v/v, C ∼
temperature of the bath. In the cases requiring −78 °C cooling, 0.05 mmol mL−1) was added a pre-mixed copper sulfate (0.2
the reactions were chilled with a dry ice/acetone bath. Organic equiv.) and sodium ascorbate (0.8 equiv.) aqueous solution (in
solutions obtained after an aqueous work-up and washed with a minimum of water). The resulting yellow reaction mixture
brine (saturated solution of NaCl) were dried over MgSO4. was heated at 40 °C until the media turned light blue
Removal of solvents was accomplished using a rotary evapor- (6–12 hours). After being cooled, the reaction mixture was
ator at water aspirator pressure or under high vacuum diluted with water (V = V water–tBuOH mL) and the aqueous
(0.5 mmHg). Tetrahydrofuran and diethyl ether were distilled suspension was extracted with EtOAc (3 × 1/3 V mL). The
under argon from sodium benzophenone. Dichloromethane organic layer was then washed with water (3 × V mL) and then
and pyridine were distilled over calcium hydride. N,N- brine (V mL) and dried over MgSO4. After removal of EtOAc
Dimethylformamide (DMF) was distilled over barium oxide under reduced pressure, the crude product was purified by
under reduced pressure or purchased from Sigma-Aldrich. All silica column chromatography.
the solvents were stored under argon over activated Linde 4 Å
PROCEDURE C (METHOD B IN TABLES 2 AND 4): DEPROTECTION OF FULLY
molecular sieves. All other solvents and reagents were used as PROTECTED MULTISUBSTRATES INCORPORATING A TR MOIETY. To a flask
received from commercial suppliers or purified according to: containing the appropriated fully protected and phosphory-
Perrin, D.D.; Armarego, W.L.F.; Perrin, D.R. Purification of Lab- lated multisubstrate (1.0 equiv.) in MeOH (C ∼ 0.01 M) was
oratory Chemicals; Pargamon Press; New York, 1980. Chemicals added MeONa (0.1 equiv., 25% wt in MeOH) at rt. The reaction
were purchased from Sigma-Aldrich Chemical Company, Lan- mixture was stirred until the TLC showed that reaction reached
caster, Alfa Aesar, Maybridge or ACROS. Solvents for extrac- completion (0.5–2 hours). MeOH was then removed under
tions and chromatography were of technical grade. Solvents reduced pressure and the residue was dissolved in EtOAc (V =
used in reactions were freshly distilled from appropriated 2 × V MeOH mL). The organic solution was washed with water
drying agents before use. All other reagents were recrystallised until pH 7. EtOAc was then removed under reduced pressure
or distilled as necessary. Flash chromatography was carried and the crude residue was dissolved in MeOH (C′ ∼ 0.01 M).
out using Merck Silica (40–60 μ) and acid washed sand. 1H, To the organic solution was added Pd/C (10% of mass of start-
13C (decoupled) and 2D (H-COSY: 512 × 512, HMQC: 128 × ing material) and the reaction was stirred under a H2 atmos-
2048) NMR spectra were all recorded on Brüker avance DPX phere (balloon) overnight. MeOH was then removed under
500 and 31P spectra were recorded on Brüker avance DPX 300. reduced pressure and the residue was dissolved in water (V′ =
1
TMS (0 ppm, H NMR), CDCl3 (77 ppm, 13C NMR) and HMPA V′ MeOH mL) and the aqueous layer was washed with EtOAc
(26.7 ppm, 31P NMR) were used as internal references. High- (3 × 1/2 V′ mL). The resulting aqueous solution was then
resolution mass spectrometry (HRMS) was recorded on a VG freeze-dried to give the corresponding deprotected multisub-
Quattro Triple Quadropole Mass Spectrometer (ES). Optical strate as a pure compound.
rotations were measured on a Perkin-Elmer 341 polarimeter.
PROCEDURE D (METHOD A IN TABLES 2 AND 4): DEPROTECTION OF FULLY
IR data were recorded on a Perkin Elmer 1720X IR Fourier PROTECTED MULTISUBSTRATES INCORPORATING A TBDPS MOIETY. To a
transform spectrometer.
flask containing the appropriated fully protected and
9610 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
phosphorylated multisubstrate (1.0 equiv.) in THF (C ∼ 0.01 further 8 hours at reflux, the reaction was carefully quenched
M) was added a solution of TBAF (1.1 equiv., 1 M in THF) with water (10 mL) and concentrated under reduced pressure.
dropwise. The reaction was monitored by TLC until it was The residue was then dissolved with EtOAc (10 mL) and the
shown to have reached completion. The reaction mixture was organic phase was isolated and washed with water (3 × 5 mL),
diluted with water (V = 2 × VTHF mL) and extracted with EtOAc brine (5 mL) and dried with MgSO4. The dried extract was con-
(3 × 1/2 V mL). The organic layer was concentrated under centrated under reduced pressure. The crude product was puri-
reduced pressure and the residue was carefully purified by fied by silica column chromatography (Hex/EtOAc/TEA: 9/0.9/
flash column chromatography (CHCl3/EtOH: 1/0–8/2 v/v). The 0.1–8/1.9/0.1 v/v/v) to give 3 (0.50 g, 72%) as a colourless oil.
pure fractions containing products were concentrated and 1H (500 MHz CDCl3) δ 7.66, 7.62 (4H, m, HAr), 7.47–7.44 (6H,
then dissolved in MeOH (C ∼ 0.01 M) and a solution of m, HAr), 7.42–7.36 (2H, m, HAr), 7.34–7.15 (24H, m, HAr),
MeONa in MeOH (0.1 equiv., 25% wt in MeOH) was added 7.13–7.00 (2H, m, HAr), 6.89–6.87 (2H, m, HAr), 4.71–4.57 (4H,
dropwise. The reaction was stirred until completion m, CH2–Ph), 4.56–4.40 (2H, m, CH2–Ph), 4.29–4.25 (1H, dd, J =
(1–2 hours). MeOH was then removed under reduced pressure 2.4 Hz, 15.5 Hz, CH2–alkyne), 4.08 (1H, dd, J = 2.4 Hz, 15.5 Hz,
and the residue was dissolved in EtOAc (V = 2 × V MeOH mL). CH2–alkyne), 3.94–3.79 (6H, m, H2, H3, H4, H5, H1), 3.49–3.48
The organic solution was washed with water until pH 7. EtOAc (1H, dd, J = 2.4 Hz, 10.4 Hz, H6), 3.31–3.27 (1H, dd, J = 5.6 Hz,
was removed under reduced pressure and the crude material 10.5 Hz, H6), 2.26 (1H, t, J = 2.4 Hz, CHuC), 1.01 (9H, s,
was dissolved in MeOH (C′ ∼ 0.01 M). To the organic solution C(CH3)3). 13C (125 MHz CDCl3) δ 143.9(3) (CAr Tr), 138.8, 138.7,
was added Pd/C (10% of mass of starting material) and the 138.4 (CAr Bn), 135.7, 135.6 (CHAr TBDPS), 133.6, 133.5 (CAr
reaction was stirred under a H2 atmosphere (balloon) over- TBDPS), 129.6(2) (CHAr TBDPS), 128.8, 128.2, 128.1, 127.9,
night. The MeOH was then removed under reduced pressure 127.8, 127.6(2), 127.3(2), 127.1, 126.9 (CHAr), 86.8 (C(Ph)3),
and the residue was dissolved in water (V′ = V′MeOH mL). The 80.6 (C2), 80.3 (CuCH), 78.9 (C5), 78.4 (C3), 78.1 (C4), 74.4
aqueous solution was washed with EtOAc (3 × 1/2 V′ mL), and (CH2–Ph), 74.3 (CHuC), 73.7 (CH2–Ph), 73.2 (CH2–Ph), 64.2
was then freeze-dried to give the corresponding unprotected (C1), 63.2 (C6), 57.7 (CH2–alkyne), 26.9 (CH3), 19.2 (C(CH3)3).
multisubstrate as a pure 1H and 13C NMR compound.
2,3,4-Tri-O-benzyl-1-O-tert-butyldiphenylsilyl-5-O-propagyl-6-O-
HRMS ES+-TOF: Calculated for C65H66O6SiNa [M + Na]+
993.4526, found [M + Na]+ = 993.4537. FTIR (cm−1): 3292
=
trityl-D-glucitol (5). To a solution of (3) (8.00 g, 11.5 mmol) and (νC–H alkyne), 3031 (νC–HAr), 2856 (νC–H alkyl), 2360
4-DMAP (0.28 g, 2.3 mmol) in dry DCM (100 mL) was added (νCuC), 1975 (νCuC–H), 1105 (νC–O), 700 (δC–H alkyne).
dry TEA (4.79 mL, 34.5 mmol). After stirring for 20 minutes, [αD] = 7.95, C = 1.51 mg mL−1 in CHCl3.
TBDPSCl (3.3 mL, 12.7 mmol) was syringed to the reaction
2,3,4-Tri-O-benzyl-5-O-propargyl-6-O-trityl-D-glucitol (6). To a
mixture. The reaction was stirred overnight and then concen- solution of (5) (0.50 g, 0.52 mmol) in THF (18 mL) was added a
trated under reduced pressure. The residue was directly puri- TBAF (1.04 mL, 1.0 mmol, in THF C ∼ 1 M) dropwise. The reac-
fied by silica column chromatography (Hex/EtOAc/TEA: 9/0.9/ tion mixture was heated at reflux for 4 hours, cooled and
0.1 v/v/v) to give 2,3,4-tri-O-benzyl-1-O-tert-butyldiphenylsilyl-6- diluted with water (20 mL). The aqueous mixture was extracted
O-trityl-D-glucitol intermediate (9.00 g, 84%) as a white foam. with EtOAc (3 × 10 mL). The organic extracts were then washed
1H (500 MHz, CDCl3) δ 7.62–7.61 (4H, m, HAr), 7.45–7.42 (6H, with water (3 × 5 mL), brine (5 mL) and dried over MgSO4. The
m, HAr), 7.41–7.36 (2H, m, HAr), 7.32–7.12 (26H, m, HAr), dried extract was concentrated under reduced pressure to give
6.94–6.92 (2H, m, HAr), 4.63 (1H, d, J = 11.4 Hz, CH2–Ph), 4.59 a yellow oil which was purified by silica column chromato-
(1H, d, J = 11.4 Hz, CH2–Ph), 4.54 (1H, d, J = 11.4 Hz, CH2–Ph), graphy (Hex/EtOAc/TEA: 9/1.9/0.1 v/v/v) to give (6) (0.28 g, 74%)
4.51 (1H, d, J = 11.4 Hz, CH2–Ph), 4.32 (2H, s, CH2–Ph), as a colourless oil. 1H (500 MHz CDCl3) δ 7.46–7.44 (6H, m,
3.99–3.95 (1H, m, H5), 3.89–3.76 (5H, m, H1, H2, H3, H4), 3.34 HAr), 7.32–7.21 (22H, m, HAr), 7.07–7.05 (2H, m, HAr), 4.71
(1H, dd, J = 3.6 Hz, 9.5 Hz, H6), 3.25 (1H, dd, J = 5.7 Hz, 9.5 (1H, d, J = 11.4 Hz, CH2–Ph), 4.67 (1H, d, J = 11.4 Hz, CH2–Ph),
Hz, H6), 3.01 (1H, d, J = 4.8 Hz, OH), 1.03 (9H, s, 3 × CH3 4.62 (1H, d, J = 11.4 Hz, CH2–Ph), 4.56 (1H, d, J = 11.4 Hz,
TBDPS). 13C (125 MHz, CDCl3) δ 143.9(3) (CAr Tr), 138.4, 138.0, CH2–Ph), 4.55 (2H, d, J = 11.4 Hz, CH2–Ph), 4.31 (1H, dd, J =
137.9 (CAr Bn), 135.6, 133.4 (CHAr TBDPS), 133.3(2) (CAr 2.3 Hz, 15.6 Hz, CH2–alkyne), 4.13 (1H, dd, J = 2.3 Hz, 15.6 Hz,
TBDPS), 129.6(2) (CHAr TBDPS), 128.7, 128.3, 128.2(2), 128.1, CH2–alkyne), 3.99–3.97 (1H, m, H2), 3.95–3.92 (1H, m, H5),
127.9, 127.8, 127.6, 127.4(2), 127.0 (CHAr), 86.5 (C2), 79.6 (C5), 3.89–3.86 (1H, m, H3), 3.77–3.72 (1H, m, H1), 3.78 (1H, qd, J =
78.0 (C4), 74.0 (C3), 73.1, 72.9, 71.1 (CH2–Ph), 64.8 (C1), 63.4 4.9 Hz, H4), 3.63–3.59 (1H, dd, J = 4.9 Hz, 10.1 Hz, H1), 3.49
(C6), 26.9 (CH3), 19.1 (C(CH3)3). HRMS ES+-TOF: Calculated for (1H, dd, J = 3.1 Hz, 10.5 Hz, H6), 3.34 (1H, dd, J = 5.5 Hz, 10.5
C62H64O6SiNa [M + Na]+ = 955.4370, found [M + Na]+
955.4371. FTIR (cm−1): 3434 (νO–H), 3290 (νC–H alkyne), 3031 143.8(3) (CAr Tr), 138.3, 138.3, 138.2 (CAr Bn), 128.7, 128.3,
(νC–HAr), 2875 (νC–H alkyl), 1071 (νC–O), 736 (δCsp33–H).
128.2, 128.0(2), 127.9(2), 127.7, 127.6, 127.5, 127.3, 126.6
=
Hz, H6), 2.36 (1H, t, J = 2.3 Hz, CHuC). 13C (125 MHz CDCl3) δ
To a solution of 2,3,4-tri-O-benzyl-1-O-tert-butyldiphenyl- (CHAr), 86.9 (C(Ph)3), 80.7 (CuCH), 79.2 (C2), 79.0 (C5), 78.5
silyl-6-O-trityl-D-glucitol (0.65 g, 0.70 mmol) in THF (20 mL) was (C3), 78.2 (C4), 74.9 (CH2–Ph), 74.8 (CuCH), 75.8, 73.5 (CH2–
added sodium hydride (0.028 g, 1.9 mmol, 60% in oil) portion- Ph), 62.9 (C6), 61.9 (C1), 57.7 (CH2–alkyne). HRMS ES+-TOF:
wise. After 30 minutes at reflux, propargyl bromide (0.1 mL, Calculated for C49H48O6Na [M + Na]+ = 755.3348, found [M +
80% in toluene, 0.77 mmol) was added to the reaction. After a Na]+ = 755.3349. FTIR (cm−1): 3447 (νO–H), 3032, 3029 (νC–
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9611

Paper
Organic & Biomolecular Chemistry
View Article Online
HAr), 2885 (νC–H alkyl), 2361 (νCuC), 1956 (νCuC–H), 1090 1.4 mmol) was phosphorylated and purified by silica column
(νC–O), 746 (δCsp3–H), 699 (δC–H alkyne).
2,3,4-Tri-O-benzyl-1-O-(dibenzylphosphatyl)-5-O-propargyl-6-O-
chromatography (Hex/EtOAc: 9/1–7/3 v/v) to give (9) (1.00 g,
74%) as a colourless oil. H (500 MHz CDCl3) δ 7.69–7.65 (5H,
1
trityl-D-glucitol (7). Using procedure A, (6) (0.25 g, 0.34 mmol) m, HAr), 7.42–7.16 (30H, m, HAr), 5.07–5.03 (4H, m, CH2–OP),
was phosphorylated and purified by silica column chromato- 4.73–4.53 (6H, m, CH2–Ph), 4.48–4.44 (1H, m, H5), 4.25–4.20
graphy (Hex/EtOAc/TEA: 9/0.9/0.1–7/2.9/0.1 v/v/v) to give (7) (1H, m, H2), 4.15 (1H, dd, J = 2.4 Hz, 15.8 Hz, CH2–alkyne),
(0.27 g, 80%) as a colourless oil. 1H (500 MHz CDCl3) 4.04 (1H, dd, J = 2.4 Hz, 15.8 Hz, CH2–alkyne), 3.95 (1H, dd, J =
δ 7.47–1.46 (7H, m, HAr), 7.32–7.15 (31H, m, HAr), 6.99–6.97 3.3 Hz, 10.7 Hz, H1), 3.92–3.88 (3H, m, H1, H6), 3.86–3.83 (1H,
(2H, m, HAr), 4.99–4.96 (4H, m, CH2–OP), 4.68–4.45 (6H, m, m, H4), 3.82–3.78 (1H, m, H3), 2.23 (1H, t, J = 2.4 Hz, CHuC),
CH2–Ph), 4.35–4.33 (1H, m, H5), 4.30 (1H, dd, J = 2.4 Hz, 15.6 1.08 (9H, s, C(CH3)3). 13C (125 MHz CDCl3) δ 138.5, 138.3,
Hz, CH2–alkyne), 4.22–4.09 (2H, m, H2, CH2–alkyne), 138.0 (CAr Bn), 135.7 (d, J = 6.9 Hz, CAr), 135.5 (CHAr TBDPS),
4.01–3.99 (1H, m, H3), 3.94–3.87 (3H, m, H4, H1), 3.52 (1H, 133.3, 133.2 (CAr TBDPS), 133.1 (d, J = 5.9 Hz, CAr), 129.5(2)
dd, J = 2.6 Hz, 10.5 Hz, H6), 3.31 (1H, dd, J = 5.1 Hz, 10.5 Hz, (CHAr TBDPS), 128.4, 128.3, 128.1(2), 128.0(2), 127.8(2), 127.7,
H6), 2.33 (1H, t, J = 2.3 Hz, CHuC). 13C (125 MHz CDCl3) 127.6(2), 127.4, 127.0 (CHAr), 80.2 (C2), 79.6 (CuCH), 78.3 (d,
δ 143.8(3) (CAr Tr), 138.3, 138.2, 138.1 (CAr Bn), 135.8 (d, J = J = 7.3 Hz, C5), 78.2 (C4), 77.8 (C3), 74.3, 73.9, 73.0 (CH2–Ph),
7.1 Hz, CAr), 133.7 (d, J = 5.3 Hz, CAr), 128.7, 128.4, 128.3, 69.8 (CHuC), 69.1 (d, J = 5.5 Hz, CH2–OP), 66.8 (d, J = 5.5 Hz,
128.2, 128.1, 128.0, 127.9, 127.8(2), 127.7, 127.5, 127.4, 127.2, C6), 63.7 (C1), 57.2 (CH2–alkyne), 26.7 (CH3), 19.0 (C(CH3)3).
126.9 (CHAr), 86.8 (C(Ph)3), 80.1 (CuCH), 78.6 (C5), 78.4 (d, J = 31P (77 MHz CDCl3) δ −0.01 (s, decoupled). HRMS ES+-TOF:
7.7 Hz, C2), 77.9 (C3), 77.6 (C4), 74.5 (CHuC), 74.1, 73.5, 73.2 Calculated for C60H65O9SiPNa [M + Na]+ = 1011.4033, found
(CH2–Ph), 69.1, (d, J = 5.8 Hz, CH2–OP), 68.2 (d, J = 5.8 Hz, [M + Na]+ = 1011.4040. FTIR (cm−1): 3447 (νO–H), 3063, 3030
CH2–OP), 64.7 (d, J = 5.4 Hz, C1), 63.0 (C6), 57.7 (CH2–alkyne). (νC–HAr), 2856 (νC–H alkyl), 2360 (νCuC), 1958 (νCuC–H),
31P (77 MHz CDCl3) δ −0.53 (s, decoupled). HRMS ES+-TOF: 1112 (νP–O–C), 1071 (νC–O), 741 (δCsp3–H), 699 (δC–H
Calculated for C63H61O9PNa [M + Na]+ = 1014.4366, found alkyne).
[M + Na]+ = 1014.4355. [αD] = −4.49, C = 0.89 mg mL−1 in
Azido-2,3-isopropylidene-β-D-ribofuranoside (12). To a suspen-
CHCl3.
2,3,4-Tri-O-benzyl-1-O-tert-butyldiphenylsilyl-5-O-propargyl-D-
sion of (11) (4.00 g, 8.2 mmol) in methanol (50 mL) at rt,
MeONa (1.77 g, 32.8 mmol, 25% wt in MeOH) was added drop-
wise. After being stirred for 3 hours at rt, the reaction mixture
was neutralised by successive filtrations through a pad of
Dowex (R) HCR-W2 until pH 7. Methanol was then removed
under reduced pressure and the residue was dissolved in
acetone (50 mL) and 2,2-dimethoxypropane (0.94 g, 9.0 mmol)
was added dropwise to the solution. A catalytic amount of con-
centrated sulfuric acid (0.1 mL) was added and the reaction
was stirred for a further 4 hours. The reaction was quenched
with a saturated NaHCO3 solution (50 mL). The acetone was
then removed under reduced pressure and the residue was
diluted with water (100 mL). The aqueous solution was
extracted with EtOAc (3 × 50 mL), and the combined extracts
were washed with water (n × 25 mL) until neutral pH and then
with brine (25 mL). The organic solution was dried over
MgSO4 and concentrated under reduced pressure. The crude
product was purified by column chromatography (Hex/EtOAc:
9/1–75/25) to give (12) (1.50 g, 87% overall) as a colourless oil.
1H (500 MHz, CDCl3), δ (ppm) 5.54 (1H, m, H1), 4.77 (1H,
dd, J = 0.8 Hz, 5.9 Hz, H2), 4.53 (1H, d, J = 5.9 Hz, H3), 4.41
(1H, m, H4), 3.79–3.75 (1H, m, H5), 3.71–3.66 (1H, m, H5),
2.33–2.30 (1H, m, OH), 1.49 (3H, s, CH3), 1.32 (3H, s, CH3).
13C (500 MHz, CDCl3), δ (ppm) 113.4 (C isopropylidene), 98.5
(C1), 89.0 (C4), 86.3 (C3), 82.1 (C2), 64.1 (C5), 26.9, 25.3 (CH3).
glucitol (8). To a solution of (5) (0.040 g, 0.041 mmol) in Et2O
(2 mL) was carefully added aluminium trichloride powder
(0.016 mg, 0.12 mmol) portion-wise. After 5 hours at rt, the
reaction mixture was diluted with Et2O (5 mL) and carefully
quenched with water (5 mL). The organic phase was washed
with a saturated NaHCO3 solution (5 mL), water (3 × 5 mL),
brine (5 mL) and was dried over MgSO4. The dried filtrate was
concentrated under reduced pressure to give a crude yellow oil.
This residue was purified by silica column chromatography
(Hex/EtOAc: 75/25 v/v) to give (8) (0.020 mg, 67%) as colourless
oil. 1H (500 MHz CDCl3) δ 7.69–7.60 (4H, m, HAr), 7.47–7.23
(21H, m, HAr), 4.73–4.69 (2H, m, CH2–Ph), 4.60 (1H, d, J = 11.4
Hz, CH2–Ph), 4.54 (1H, d, J = 11.4 Hz, CH2–Ph), 4.02 (2H, dd,
J = 2.3 Hz, 16.0 Hz, CH2–alkyne), 3.96–3.92 (2H, m, H6, H5),
3.88–3.73 (6H, m, H6, H1, H2, H4), 3.69 (1H, quadruplet, J =
4.3 Hz, H3), 2.32 (1H, t, J = 2.3 Hz, CHuC), 2.28 (1H, t, J =
6.3 Hz, HO), 1.08 (9H, s, C(CH3)3). 13C (125 MHz CDCl3)
δ 138.5, 138.3, 138.2 (CAr Bn), 135.7, 135.6 (CHAr TBDPS),
133.4, 133.3 (CAr TBDPS), 130.4, 130.35 (CHAr TBDPS), 129.8,
129.7, 129.6, 128.6, 128.4, 128.3, 128.2, 128.0, 127.9(2), 127.8,
127.7, 127.5, 127.3 (CHAr), 80.0 (C2), 79.9 (CuCH), 79.7 (C5),
78.9 (C3), 78.4 (C4), 74.7 (CH2–Ph), 74.6 (CHuC), 75.5, 73.0
(CH2–Ph), 63.3 (C1), 60.9 (C6), 56.8 (CH2–alkyne), 26.7 (CH3),
19.1 (C(CH3)3). HRMS ES+-TOF: Calculated for C46H52O6SiNa
[M + Na]+ = 751.3434, found [M + Na]+ = 751.3431. FTIR
(cm−1): 3440 (νO–H), 3033, 3029 (νC–HAr), 2886 (νC–H alkyl),
HRMS ES+-TOF: Calculated for C8H14N3O4 [M
216.0984, found [M + H]+ = 216.0982.
+ =
H]+
Azido-2,3-O-acetyl-5-O-(dibenzylphosphate)-β-D-ribofuranoside
2360 (νCuC), 1963 (νCuC–H), 1089 (νC–O), 745 (δCsp3–H), (14). Using procedure A, (12) (1.44 g, 6.7 mmol) was phos-
698 (δC–H alkyne).
2,3,4-Tri-O-benzyl-6-O-(dibenzylphosphatyl)-1-O-tert-butyldiphenyl-
phorylated and then purified by flash chromatography (Hex/
EtOAc 75/25) to give azido-2,3-isopropylidene-5-(dibenzylpho-
silyl-5-O-propargyl-D-glucitol (9). Using procedure A, (8) (1.00 g, sphate)-β-D-ribofuranoside. The collected fractions containing
9612 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
the desired product were concentrated and dissolved in a MeOH (3 mL) and MeONa (0.1 mL 25% wt in MeOH). The
mixture of TFA–water (50 mL, 8 : 2 v/v) and stirred for 8 hours reaction mixture was stirred for 20 minutes and concentrated.
at rt. The reaction mixture was then extracted with EtOAc The crude was then purified by silica column chromatography
(3 × 10 mL). The combined organic extracts were washed with (Hex/EtOAc/TEA: 8/1.9/0.1–0/0.9/0.1 v/v/v) to give (16) (0.026 g,
an aqueous saturated solution of NaHCO3 until pH over 7, 37% over 2 steps) as a colourless oil. 1H (500 MHz CDCl3)
then with water (until neutral pH), brine (10 mL) and then δ 7.70 (1H, s, H triazole), 7.66–7.60 (5H, m, HAr), 7.43–7.20
dried over MgSO4. The organic layer was concentrated under (43H, m, HAr), 7.00–6.97 (2H, m, HAr), 5.81 (d, J = 1.8 Hz, H1′),
reduced pressure and the crude material was purified by 5.11–5.02 (4H, m, CH2–OP), 4.85 (1H, d, J = 11.5 Hz, CH2–Ph),
column chromatography (Hex/EtOAc: 75/25–0/1) to give the 4.78 (1H, d, J = 11.8 Hz, CH2–triazole), 4.65–4.63 (5H, m, CH2–
corresponding diol intermediate (1.95 g, 67% overall) as col- Ph), 4.62 (1H, d, J = 11.8 Hz, CH2–triazole), 4.51 (2H, m, H5′),
ourless oil.
4.30–4.25 (1H, m, H4′), 4.20–4.17 (1H, m, H2′), 4.05–4.01 (1H,
1H (500 MHz, CDCl3), δ (ppm) 7.28–7.26 (10H, s, HAr), 5.27 m, H5), 4.00–3.95 (5H, m, H1, H3, H4, H3′), 3.50 (1H, dd, J =
(1H, d, J = 1.8 Hz, H1), 5.20 (1H, dd, J = 4.9 Hz, 6.0 Hz, H3), 2.4 Hz, 10.5 Hz, H6), 3.30 (1H, dd, J = 5.6 Hz, 10.5 Hz, H6),
5.04 (1H, dd, J = 1.8 Hz, 4.9 Hz, H2), 4.99 (4H, m, CH2–Ph), 1.04 (9H, s, C(CH3)3). 13C (125 MHz CDCl3) δ 46.2 (C triazole)
4.22–4.20 (1H, m, H4), 4.13–4.09 (1H, m, H5), 4.04–4.01 (1H, 143.3(3) (CAr Tr), 137.6(2), 136.7 (CAr Bn), 135.6 (CHAr), 135.5
m, H5). 13C (125 MHz, CDCl3), δ (ppm) 135.9 (d, J = 5.9 Hz, (d, J = 7.0 Hz, CAr), 133.4 (d, J = 3.3 Hz, CAr), 130.1, 129.4,
CAr), 135.6 (d, J = 5.4 Hz, CAr), 128.6, 127.9, 127.8(2) (CHAr), 128.5, 128.3, 128.2, 128.1, 127.9, 127.8(2), 127.7(2), 127.6,
93.4 (C1), 78.5 (d, J = 8.3 Hz, C4), 72.4 (C2), 68.2 (C3), 69.4, 127.5, 127.4 (CHAr), 122.7 (CH triazole), 89.8 (C1′), 86.7
67.9 (d, J = 5.5 Hz, CH2–Ph,), 66.2 (d, J = 5.5 Hz, C5). 31P (C(Ph)3), 81.3 (d, J = 7.6 Hz, C4′), 80.0 (C4), 78.6 (C2), 77.7 (C5),
(77 MHz, CDCl3), δ (ppm) −0.68 (s, decoupled). HRMS ES+- 77.3 (C3), 74.0, 73.6, 73.4 (CH2–Ph), 72.3 (C2′), 69.5(2) (d, J =
TOF: Calculated for C17H18N3O7P [M + H]+ = 436.1274, found 3.7 Hz, CH2–OP), 68.2 (C3′), 66.1 (d, J = 6.1 Hz, C5′), 64.7 (CH2–
[M + H]+ = 436.1270. FTIR (cm−1): 3031 (νC–HAr), 2946 (νC–H triazole), 63.6 (C6), 63.0 (C1), 26.0 (CH3)3, 19.1 (C(CH3)3). 31P
alkyl), 2112 (νN3), 1456 (νC–O), 1246 (νC–N), 1020 (νP–O–C), (77 MHz CDCl3) δ −0.11 (s, decoupled). HMRS ES+-TOF: Calcu-
739 (νCsp3–H). [αD] = −104.7, (c 0.42 mg mL−1 CHCl3).
lated for C84H88N3O13PSiNa [M + Na]+ = 1428.5728, [M + Na]+ =
This pure product was then acetylated using acetic an- 1428.5728.
hydride (0.6 mL, 6.18 mmol) in pyridine (50 mL) and the reaction
mixture was stirred for 6 hours at rt. The pyridine was partially
1-[(2,3-Di-O-Acetyl-5-O-dibenzylphosphatyl)-β-D-ribofuranosyl]-
4-methylenoxy-[5-(2R,3S,4R,5R)-2,3,4-tris-O-benzyl-1-O-(dibenzyl-
removed in vacuo. The residue was dissolved in EtOAc (50 mL) phosphatyl)-6-O-trityl-hexanyl]-1H-1,2,3-triazole (17). Using pro-
and the organic layer was washed with a saturated copper cedure B, alkyne (7) (0.046 g, 0.046 mmol) was mixed with (14)
sulfate solution until the blue colour remained unchanged. (0.026 g, 0.051 mmol) in a water–tBuOH (1 mL) mixture. A pre-
The organic solution was washed with water (3 × 20 mL), brine mixed copper sulfate (0.005 g, 0.022 mmol), sodium ascorbate
(20 mL) and dried over MgSO4. The organic solution was con- (0.007 g, 0.044 mmol) aqueous solution (1 mL) was added to
centrated under reduced pressure and the crude material was the suspension. After 8 hours and removal under vacuum of
purified by silica column chromatography (Hex/EtOAc: 75/25 the solvents, the crude material was purified by silica column
v/v) to give (14) (1.05 g, 80%) as a waxy colourless oil.
chromatography (Hex/EtOAc/TEA: 1/0.9/0.1–0/0.9/0.1 v/v/v) to
1H (500 MHz, CDCl3) δ 7.27–7.25 (10H, m, HAr), 5.28 (1H, give 13 (0.047 g, 73%) as a colourless oil. H (500 MHz CDCl3)
d, J = 1.8 Hz, H1), 5.21 (1H, dd, J = 4.9 Hz, 6.0 Hz, H3), 5.03 δ 7.71 (1H, s, H triazole), 7.44–7.42 (6H, m, HAr), 7.28–7.11
(1H, dd, J = 1.8 Hz, 4.9 Hz, H2), 4.98 (4H, m, CH2–Ph), (42H, m, HAr), 6.93–6.92 (2H, m, HAr), 5.05 (1H, d, J = 3.8 Hz,
4.22–4.19 (1H, m, H4), 4.12–4.08 (1H, m, H5), 4.04–4.00 (1H, H1′), 5.78 (1H, dd, J = 3.8 Hz, 5,2 Hz, H2′), 5.66 (1H, t, J =
m, H5), 2.02 (3H, s, CH3), 1.94 (3H, s, CH3). 13C (125 MHz, 5.2 Hz, H3′), 5.00–4.91(8H, m, CH2–OP), 4.78 (1H, d, J =
CDCl3) δ 169.3, 169.2 (CvO Ac), 135.9 (d, J = 6.7 Hz, CAr), 135.6 11.9 Hz, CH2–triazole), 4.63–4.40 (8H, m, CH2–Ph, H4′, CH2–
(d, J = 4.9 Hz, CAr), 128.6, 127.9, 127.8(2) (CHAr), 92.9 (C1), 79.9 triazole), 4.33–4.29 (1H, m, H1), 4.22–4.10 (3H, m, H1, H5′),
(d, J = 8.3 Hz, C4), 74.4 (C2), 70.2 (C3), 69.4, 68.5 (d, J = 5.5 Hz, 3.98–3.96 (1H, m, H3), 3.90–3.87 (1H, m, H5), 3.86–3.81 (2H,
CH2–Ph), 66.2 (d, J = 5.5 Hz, C5), 20.4, 20.3 (CH3). 31P m, H4, H2), 3.60 (1H, dd, J = 2,7 Hz, 10.5 Hz H6), 3.32 (1H, dd,
(77 MHz, CDCl3) δ −0.36 (s, decoupled). HRMS ES+-TOF: Cal- J = 5.2 Hz, 10.5 Hz H6), 2.09 (3H, s, CH3), 2.06 (3H, s, CH3).
culated for C24H22N3O9PNa [M + Na]+ = 542.1304, found 13C (125 MHz CDCl3) δ 169.2, 169.0 (CvO Ac), 145.7 (C tria-
1
[M + Na]+ = 542.1356. [αD] = −69.1 (c 0.68 mg mL−1 CHCl3).
1-[5-O-(Dibenzylphosphatyl)-β-D-ribofuranosyl]-4-methylenoxy-
[5-(2R,3S,4R,5R)-2,3,4-tris-O-benzyl-1-O-(tert-butyldiphenylsilyl)-
zole), 143.8(3) (CAr Tr), 138.2, 138.1, 138.0 (CAr), 135.8(2), 135.4
(2) (CAr, d, J = 6.5 Hz), 128.7, 128.5(2), 128.4, 128.2(2), 128.0(2),
127.9(2), 127.8, 127.5(2), 127.3, 127.0(2) (CHAr), 122.3 (CH tri-
6-O-trityl-hexanyl]-1H-1,2,3-triazole (16). Using procedure B, (5) azole), 89.7 (C1′), 86.8 (CPh3), 81.2 (d, J = 7.6 Hz, C4′), 79.6
(0.051 g, 0.052 mmol) was mixed with (14) (0.030 g, (C4), 78.2 (d, J = 7.6 Hz, C2), 77.7 (C5), 77.2 (C3), 74.1 (C2′),
0.057 mmol) in a water–tBuOH (1 mL) mixture. A pre-mixed 74.0, 73.4, 73.1 (CH2–Ph), 70.7 (C3′), 69.5(2) (d, J = 3.7 Hz,
copper sulfate (0.006 g, 0.021 mmol), sodium ascorbate CH2–OP), 68.2 (d, J = 2.1 Hz, CH2–OP), 68.2 (d, J = 6.1 Hz, C5′),
(0.008 mg, 0.042 mmol) aqueous solution (1 mL) was added to 66.4 (d, J = 5.1 Hz, C1), 64.1 (CH2–triazole), 63.0 (C6), 20.4,
the previous suspension. After 6 hours and removal under 20.3 (CH3). 31P (77 MHz CDCl3) δ 0.19 (s, decoupled), −0.09
vacuum of the solvents, the crude material was dissolved in (s, decoupled). LRMS: Calculated MALDI-TOF: Calculated for
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9613

Paper
Organic & Biomolecular Chemistry
View Article Online
C86H87N3O18P2Na [M + Na]+ = 1534.5, found [M + Na]+
1534.3.
=
73.0 (CH2–Ph), 74.2 (C2′), 70.7 (C3′), 69.5, 69.1 (CH2–OP), 67.2
(d, J = 5.9Hz, C5′), 66.4 (d, J = 5.2 Hz, C6), 63.9 (CH2–Triazole),
63.7 (C1), 26.1 (CH3), 20.4, 20.3 (CH3), 19.1 (C(CH3)3). 31P
(77 MHz CDCl3) δ 0.17 (s, decoupled), 0.01 (s, decoupled).
LRMS MALDI-TOF: Calculated C83H91N3O18P2SiNa [M + Na]+ =
1530.5, found [M + Na]+ = 1530.4.
1-[(2,3,5-Tri-O-benzoyl)-β-D-ribofuranosyl]-4-methylenoxy-[5-
(2R,3S,4R,5R)-2,3,4-tris-O-benzyl-6-O-(dibenzylphosphatyl)-1-O-
(tert-butyldiphenylsilyl))-hexanyl]-1H-1,2,3-triazole (18). Using
procedure B, (9) (0.043 g, 0.043 mmol) was mixed with (11)
(0.023 g, 0.047 mmol) in a water–tBuOH (1 mL) mixture. A pre-
1-[(5-O-Phosphatyl)-β-D-ribofuranosyl]-4-methylenoxy-[5-(2S,3R,-
mixed copper sulfate (0.004 g, 0.022 mmol), sodium ascorbate 4R,5R)-hexan-1,2,3,4,6-pentaolyl]-1H-1,2,3-triazole (23). Using
(0.007 g, 0.044 mmol) aqueous solution (1 mL) was added to procedure D, compound (16) (48 mg, 0.034 mmol) was depro-
the previous suspension. After 12 hours and removal under tected and purified to give (23) (14 mg, 84% overall) as a white
1
vacuum of the solvents, the crude material was purified by gum. H (500 MHz D2O) δ 8.20 (1H, s, H triazole), 5.98 (1H, d,
silica column chromatography (Hex/EtOAc: 1/1–0/1 v/v) to give J = 5.0 Hz, H1′), 4.47 (1H, t, J = 4.5 Hz, H2′), 4.27 (1H, t, J =
(18) (0.037 g, 58%) as a colourless oil. 1H (500 MHz CDCl3) 4.5 Hz, H3′), 4.19–4.16 (1H, m, H4′), 3.88–3.80 (3H, m, H5′,
H5), 3.74–3.70 (1H, d, J = 12.1 Hz, CH2–triazole), 3.61–3.47
δ 8.02–7.94 (6H, m, HAr), 7.68 (1H, s, H triazole), 7.66–7.12
(44H, m, HAr), 6.25 (1H, dd, J = 2.8 Hz, 5.2 Hz, H2′), 6.26 (1H, (7H, m, H6, CH2–triazole, H2, H3, H4, H1), 3.38 (1H, dd, J =
t, J = 6.3 Hz, H3′), 6.16 (1H, d, J = 2.8 Hz, H1′), 5.00–4.98 (4H, 6.6 Hz, 11.7 Hz, H6). 13C (125 MHz D2O) δ 143.6 (C triazole),
123.1 (CH triazole), 92.1 (C1′), 84.7 (d, J = 5.7 Hz, C4′), 78.9
CH2–triazole, H5, H4), 4.24–4.19 (1H, m, H5′), 3.98–3.78 (H6, (C5), 75.2 (C4), 72.8 (C3), 70.5 (C2′), 69.5 (C2), 69.4 (C3′), 64.1
H1, H2, H3), 1.04 (9H, s, C(CH3)3). 13C (125 MHz CDCl3) (d, J = 4.3 Hz, C5′), 62.2 (C6), 62.2 (C1), 59.4 (CH2–triazole). 31
m, CH2–OP), 4.87–4.84 (1H, m, H4′), 4.73–4.42 (10H, CH2–Ph,
P
(77 MHz D2O) δ 2.08 (s, decoupled). HRMS ES−-TOF: Calcu-
δ 166.0, 165.0, 164.8 (CvO Bz), 145.7 (C triazole), 138.5, 138.3,
138.1 (CAr Bn), 135.8 (d, J = 7.0 Hz, CAr), 135.6 (CHAr), 133.3 (d, lated for C14H25N3O13P [M − H]− = 474.1125, found [M − H]− =
J = 3.4 Hz, CAr), 129.8, 129.7, 129.6(2) (CHAr), 129.3, 128.7, 474.1110.
128.6 (CAr), 128.5(2), 128.4 (CHAr), 128.4(2), 128.2 (CAr Bz),
128.1, 127.8(2), 127.7, 127.6(2), 127.5, 127.4 (CHAr), 122.7 (CH 4R,5R)-1-O-(phosphatyl)-hexan-2,3,4,6-tetraolyl]-1H-1,2,3-triazole
1-[(5-O-Phosphatyl)-β-D-ribofuranosyl]-4-methylenoxy-[5-(2S,3R,-
(24). Using procedure D, compound (17) (85 mg, 0.060 mmol)
was deprotected purified to give (24) (31 mg, 94% overall) as a
white gum. H (500 MHz D2O) δ 8.22 (1H, s, H triazole), 6.10
triazole), 90.1 (C1′), 80.8 (C4′), 80.2 (C4), 79.5 (d, J = 6.9 Hz,
C5), 78.2 (C2), 77.9 (C3), 75.2 (C2′), 74.3, 73.9, 73.0 (CH2–Ph),
71.8 (C5′), 69.1(2) (d, J = 3.1 Hz, CH2–OP), 67.2 (d, J = 5.9 Hz,
C5), 63.9 (CH2–triazole), 63.8 (C6), 26.9 (CH3), 19.1 (C(CH3)3).
31P (77 MHz CDCl3) δ 0.18 (s, decoupled). HRMS: Calculated
1
(1H, d, J = 4.5 Hz, H1′), 4.66 (1H, t, J = 4.8 Hz, H2′), 4.30 (1H, t,
J = 4.8 Hz, H3′), 4.20–4.18 (1H, m, H4′) 4.06–3.65 (12H, m, H1,
H2, H3, H4, H5, H6, H5′,CH2–triazole). 13C (125 MHz D2O)
δ 144.5 (C triazole), 123.6 (CH triazole), 92.1 (C1′), 84.7 (d, J =
7.2 Hz, C4′), 77.9 (C5), 75.5 (C4), 73.4 (C3), 71.7 (d, J = 6.2 Hz,
C2), 70.9 (C2′), 69.6 (C3′), 65.2 (d, J = 4.7 Hz, C5′), 63.4 (d, J =
5.0 Hz, C1), 62.8 (C6), 62.7 (CH2–triazole). 31P (77 MHz D2O)
δ 5.68 (s, decoupled), 5.30 (s, decoupled). HRMS: Calculated
ES−-TOF for C14H26N3O16P2 [M − H]− = 554.0788, found
[M − H]− = 554.0878.
ES+-TOF: Calculated for C86H87N3O16P2Na [M
1476.5593, found [M + H]+ = 1476.5659.
+ =
H]+
1-[2,3-Di-O-acetyl-5-O-(dibenzylphosphatyl)-β-D-ribofuranosyl]-
4-methylenoxy-[5-(2R,3S,4R,5R)-2,3,4-tris-O-benzyl-6-O-(dibenzyl-
phosphatyl)-1-O-(tert-butyldiphenylsilyl))-hexanyl]-1H-1,2,3-triazole
(19). Using procedure B, alkyne (9) (0.055 g, 0.055 mmol) was
mixed with (14) (0.032 g, 0.061 mmol) in a water–tBuOH
(1 mL) mixture.
A pre-mixed copper sulfate (0.006 g,
0.021 mmol), sodium ascorbate (0.009 g, 0.042 mmol)
1-(β-D-Ribofuranosyl)-4-methylenoxy-[5-(2S,3R,4R,5R)-6-O-phos-
aqueous solution (1 mL) was added to the previous mixture. phatyl-hexan-1,2,3,4-tetraolyl]-1H-1,2,3-triazole (21). Using pro-
cedure C, compound (18) (120 mg, 0.078 mmol) was
After 8 hours and removal under vacuum of the solvents, the
crude material was purified by silica column chromatography deprotected and purified to give compound (21) (30 mg, 81%
(Hex/EtOAc: 1/1–0/1 v/v) to give (19) (0.051 g, 62%) as a colour- overall) as a white gum. 1H (500 MHz D2O) δ 8.19 (1H, s, H
1
triazole), 6.05 (1H, dd, J = 1.7 Hz, 3.9 Hz, H1′), 4.59 (1H, dt, J =
less oil. H (500 MHz CDCl3) δ 7.66–7.61 (5H, H triazole, HAr),
7.40–7.14 (38H. m, HAr), 7.11–7.09 (2H, m, HAr), 5.93 (1H. d, 1.7 Hz, 5.0 Hz, H2′), 4.33 (1H, dt, J = 1.7 Hz, 5.0 Hz, H3′),
J = 3.8 Hz, H1′), 5.76 (1H, dd, J = 3.8 Hz, 5.2 Hz, H2′), 5.64 (1H, 4.19–4.13 (2H, m, CH2–triazole, H4′), 4.00–3.96 (2H, m, CH2–
triazole, H5), 3.77–3.57 (8H, m, H1, H2, H3, H4, H5′, H6),
t, J = 5.2 Hz, H3′), 5.02–4.95 (4H, m, CH2–OP), 4.67 (1H. d, J =
11.8 Hz, CH2–Ph), 4.64–4.38 (10H, m, H5′, H4′, CH2–triazole, 3.50–3.46 (1H, ddd, J = 1.7 Hz, 6.6 Hz, 11.7 Hz, H6). 13C
CH2–Ph), 4.21–4.14 (2H, m, H6), 3.95 (1H, dd, J = 2.9 Hz, (125 MHz D2O) δ (ppm): 144.0 (C triazole), 123.9 (CH triazole),
92.0 (C1′), 85.1 (C4′), 77.4 (d, J = 7.8 Hz, C5), 74.7 (C4), 72.8
10.8 Hz, H1), 3.87–3.77 (5H, m, H1, H2, H3, H4, H5), 2.08 (3H,
s, CH3), 20.6 (3H, s, CH3), 1.02 (9H, s, C(CH3)3). 13C (125 MHz (C3), 70.0 (C2′), 69.3 (C2), 69.0 (C3′), 63.7 (d, J = 4.9 Hz, C6),
CDCl3) δ 169.2, 168.9 (CvO, Ac), 145.5 (C triazole), 138.6, 62.2 (C1), 61.9 (C5′), 60.9 (CH2–triazole). 31P (77 MHz D2O)
δ
1.36 (broad, decoupled). HRMS ES−-TOF: Calculated
138.3, 138.0 (CAr Bn), 135.9(2) (d, J = 7.0 Hz, CAr), 135.6 (CHAr),
133.3(2) (d, J = 3.3 Hz, CAr), 129.6(2), 128.5(2), 128.3(2), 128.2, C14H25N3O13P [M − H]− = 474.1125, found [M − H]−
=
128.1(2), 127.9(2), 127.8(3), 127.7, 127.6, 127.5, 127.4(2) (CHAr), 474.1135.
122.6 (CH triazole), 89.7 (C1′), 81.2 (d, J = 7.6 Hz, C4′), 80.2
1-[(5-O-Phosphatyl)-β-D-ribofuranosyl]-4-methylenoxy-[5-(2S,3R,-
(C4), 79.5 (d, J = 7.1 Hz, C5), 78.2 (C2), 77.9 (C3), 74.3, 73.9, 4R,5R)-6-O-phosphatyl-hexan-1,2,3,4-tetraolyl]-1H-1,2,3-triazole
9614 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
(22). Using procedure D, compound (19) (91 mg, 0.065 mmol) m, Ph), 4.72 (1H, d, J = 11.4 Hz, CH2–Ph), 4.63 (1H, d, J = 11.4
was deprotected and purified to yield (22) (27 mg, 73% overall) Hz, CH2–Ph), 4.59 (2H, s, CH2–Ph), 4.13–4.12 (2H, m, CH2–
1
as a pure white gum. H (500 MHz D2O) δ 8.24 (1H, s, H tria- alkyne), 3.97–3.75 (5H, m, H-1, H-3, H-4, H-5), 3.73–3.71 (1H,
zole), 6.08 (1H, d, J = 4.6 Hz, H1′), 4.56 (1H, t, J = 4.7 Hz, H2′), m, H-6), 3.67–3.64 (1H; m, H-6), 2.39 (1H, t, J = 2.4 Hz,
4.37 (1H, t, J = 4.7 Hz, H3′), 4.29–4.28 (1H, m, H4′), 4.18–4.14 CHuC), 1.89–1.81 (2H, m, H-2), 1.07 (9H, s, (CH3)). δC
(1H, m, H5), 4.06–4.02 (2H, m, H5′), 3.99–3.95 (2H, m, CH2– (125 MHz; CDCl3; Me4Si) 138.5, 138.0, 135.6, 135.5, 133.6(2),
triazole), 3.75–3.65 (5H, m, H1, H2, H3, H4), 3.59 (1H, dd, J = 129.6, 128.3, 128.1, 127.7, 127.6(2), 80.1, 80.0 (CuCH), 79.4
3.06 Hz, 11.8 Hz, H6), 3.47 (1H, dd, J = 6.6 Hz, 11.8 Hz, H6). (C-3), 75.6 (C-4), 74.7 (CHuC), 74.3, 72.7 (CH2–Ph), 61.0 (C-6),
13C (125 MHz D2O) δ 143.8 (C triazole), 123.9 (CH triazole), 60.2 (C-1), 57.0 (CH2–alkyne), 33.6 (C-2), 26.9 (CH3), 19.2
92.8 (C1′), 84.6 (d, J = 8.6 Hz, C4′), 77.9 (d, J = 7.3 Hz, C5), 75.5 (C–(CH3)3). HRMS (ES) calculated [M + H]+ = 623.3193; found
(C4), 73.4 (C3), 70.8 (C2′), 70.7 (C2), 69.7 (C3′), 69.4 (d, J = 7.7 [M + H]+ = 623.3181.
Hz, C6), 65.0 (d, J = 4.5 Hz, C5′), 62.8 (C1), 62.7 (CH2–triazole).
31P (77 MHz D2O) δ 1.20 (broad, decoupled). HRMS ES−-TOF:
Calculated for C14H26N3O16P2 [M − H]− = 554.0788, found
[M − H]− = 554.0775.
3,4-Di-O-benzyl-5-O-propargyl-6-O-trityl-2-deoxy-D-glucitol (32).
To a solution of (30) (0.11 g, 0.13 mmol) in THF (10 mL) was
added a TBAF (0.15 mL, 0.15 mmol, in THF C ∼ 1 M). The reac-
tion mixture was stirred overnight at rt then diluted with water
(20 mL) and extracted with EtOAc (3 × 10 mL). The organic
layer was washed with water (3 × 10 mL), brine (10 mL) and
dried over MgSO4. EtOAc was removed under reduced pressure
until dry. The crude product was purified by silica column
chromatography (Hex/EtOAc/TEA: 80/19/1 v/v/v) to yield (32)
(0.067 g, 79%) as a white foam. δH (500 MHz; CDCl3; Me4Si)
7.46–7.44 (5H, m, Ph), 7.27–7.19 (18H, m, Ph), 7.10–7.08 (2H,
m, Ph), 4.65 (1H, d, J = 11.4 Hz, CH2–Ph), 4.57–4.49 (3H, m,
CH2–Ph), 4.34 (1H, dd, J = 2.3 Hz, 15.6 Hz, CH2–alkyne), 4.20
(1H, dd, J = 2.3 Hz, 15.6 Hz CH2–alkyne), 3.92–3.90 (1H, m,
H-5), 3.82–3.77 (2H, m, H3, H-1), 3.63–3.61 (2H, m, H4, H-1),
3.47 (1H, dd, J = 3.1 Hz, 10.5 Hz, H-6), 3.34 (1H, dd, J = 5.5 Hz,
10.5 Hz, H-6), 2.36 (1H, t, J = 2.3 Hz, HCuC), 1.77–1.74 (2H,
m, H-2). δC (125 MHz; CDCl3; Me4Si) 143.8(3), 138.4, 138.3,
128.7, 128.3, 128.1, 128.0, 127.9, 127.8, 127.6(2), 127.4, 127.0,
86.9 (C–(Ph)3), 80.9 (C-5), 80.1 (CuCH), 78.6 (C-3), 77.7 (C-4),
74.4 (CHuC), 74.0, 73.0 (CH2–Ph), 63.2 (C-1), 60.3 (C-6), 57.8
3,4-Di-O-benzyl-1-O-tert-butyldiphenylsilyl-5-O-propargyl-6-O-
trityl-2-deoxy-D-glucitol (30). To a solution of (29) (0.12 g,
0.15 mmol) in dry THF (5 mL) was added sodium hydride
(0.06 g, 0.15 mmol, 60% wt in oil) at rt. The reaction mixture
was stirred for 20 minutes at rt and propargyl bromide
(0.018 mL, 0.16 mmol, 80% wt in toluene) was added. After
8 hours at rt, the reaction mixture was diluted with water
(10 mL) and the aqueous mixture was extracted with EtOAc
(3 × 5 mL). The combined extracts were washed with water (3 ×
5 mL), brine (5 mL) and dried over MgSO4. EtOAc was removed
under reduced pressure and the crude product was purified by
silica column chromatography (Hex/EtOAc/TEA: 9/0.9/0.1 v/v/v)
to yield (30) (0.097 g, 77%) as colourless oil. δH (500 MHz;
CDCl3; Me4Si) 7.68–7.67 (4H, m, Ph), 7.46–7.35 (5H, m, Ph),
7.32–7.20 (24H, m Ph), 7.09–7.07 (2H, m, Ph), 4.60–4.45 (4H,
m, CH2–Ph), 4.21 (1H, d, J = 2.3 Hz, CH2–alkyne), 4.16 (1H, d,
J = 2.3 Hz, CH2–alkyne), 4.02–3.87 (3H, m, H-5, H-1), 3.81–3.66
(2H, m, H-3, H-4), 3.54 (1H, dd, J = 2.4 Hz, 10.5 Hz, H-6), 3.34
(1H, dd, J = 5.6 Hz, 10.5 Hz, H-6), 2.36 (1H, t, J = 2.4 Hz,
CHuC), 1.87–1.81 (2H, m, H-2), 1.09 (9H, s, (CH3). δC
(CH2–alkyne), 34.2 (C-2). HRMS (ES) calculated [M + Na]+
=
649.2930, found [M + Na]+ = 649.2940.
3,4-Di-O-benzyl-1-O-(dibenzylphosphatyl)-5-O-propargyl-6-O-
(125 MHz; CDCl3; Me4Si) 143.9(3), 138.5, 138.3, 133.6, 133.5, trityl-2-deoxy-D-glucitol (34). Using the procedure B, (32)
129.6, 128.7, 128.3(2), 128.1, 128.0(2), 127.9, 127.8, 127.6(2), (0.067 g, 0.11 mmol) was phosphorylated and purified by
127.4, 127.0, 86.8, 80.8 (C-5), 80.0 (CuCH), 79.5 (C-3), 76.4 column chromatography (Hex/EtOAc/TEA: 9/0.9/1–8/1.9/0.1 v/v/v)
(C-4), 74.5 (CHuC), 74.2, 73.0 (CH2–Ph), 63.1 (C-1), 60.4 (C-6), to yield (34) (0.060 g, 63%) as a colourless oil. δH (500 MHz;
57.3 (CH2–alkyne), 33.2 (C-2), 26.8 (CH3), 19.4 (C–(CH3)3). CDCl3; Me4Si) 7.42–7.40 (7H, m, Ph), 7.27–7.14 (29H, m, Ph),
HRMS (ES) calculated [M + Na]+ = 887.4108; found [M + Na]+ = 4.96 (2H, s, CH2–OP), 4.95 (2H, m, CH2–OP), 4.51 (1H, d, J =
887.4127. FTIR (cm−1): 3294 (νC–H alkyne), 3030 (νC–HAr), 11.4 Hz, CH2–Ph), 4.44 (1H, d, J = 3.4 Hz, CH2–Ph), 4.41 (1H,
2857 (νC–H alkyl), 2361 (νCuC), 1978 (νCuC–H), 1100 (νC–O), d, J = 3.4 Hz, CH2–Ph), 4.38 (1H, d, J = 11.4 Hz, CH2–Ph), 4.27
702 (νC–H alkyne). [α]2D2 +15.3 (c 0.39 mg mL−1 in CHCl3).
3,4-Di-O-benzyl-1-O-tert-butyldiphenylsilyl-5-O-propargyl-2-
(1H, dd, J = 2.4 Hz, 15.5 Hz, CH2–alkyne), 4.07 (1H, dd, J =
2.4 Hz, 15.5 Hz, CH2–alkyne), 4.00 (2H, quad., J = 6.5 Hz, H-1),
deoxy-D-glucitol (31). To a solution of (30) (0.12 g, 0.14 mmol) 3.84 (1H, td, J = 2.6 Hz, 5.9 Hz, H-5), 3.79–3.76 (1H, m, H-3),
in Et2O (20 mL) was added aluminium trichloride (0.036 g, 3.67 (1H, dd, J = 4.2 Hz, 5.9 Hz, H-4), 3.44 (1H, dd, J = 2.6 Hz,
0.27 mmol) portion-wise at rt. The reaction was stirred for 10.6 Hz, H-6), 3.25 (1H, dd, J = 5.6 Hz, 10.6 Hz, H-6), 2.29 (1H,
30 minutes and then quenched with water (5 mL). The organic t, J = 2.4 Hz, CHuC), 1.83–1.79 (2H, m, H-2). δC (125 MHz;
layer was washed with a saturated NaHCO3 solution until pH CDCl3; Me4Si) 143.6(3), 138.5, 138.2, 135.6 (d, J = 6.8 Hz), 133.4
7, then water (3 × 5 mL) and brine (5 mL). The organic layer (d, J = 5.1 Hz), 128.8 128.7, 128.4, 128.1(2), 128.0, 127.9, 127.8
was dried over MgSO4 and then concentrated under reduced (2), 127.7, 127.5, 127.4, 127.2, 126.9, 86.7, 80.0 (CuCH), 78.5
pressure. The crude product was purified by silica column (C-5), 77.7 (C-3), 77.5 (C-4), 74.5 (CHuC), 73.6, 73.0 (CH2–Ph),
chromatography (Hex/EtOAc: 8/2–75/25 v/v) to yield (31) 69.0 (d, J = 5.8 Hz, CH2–OP), 68.3 (d, J = 5.8 Hz, CH2–OP), 66.7
(0.060 g, 71.2%) as white foam. δH (500 MHz; CDCl3; Me4Si) (d, J = 5.1 Hz, C1), 63.5 (C6), 57.6 (CH2–alkyne), 35.4 (d, J =
7.67–7.65 (4H, m, Ph), 7.45–7.35 (7H, m, Ph), 7.32–7.26 (9H, 7.1 Hz, C-2). δp (121 MHz; CDCl3; Me4Si) 0.46 (s, decoupled).
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9615

Paper
Organic & Biomolecular Chemistry
View Article Online
HRMS (ES) calculated [M + Na]+ = 909.3532, found [M + Na]+ = (d, J = 6.7 Hz), 135.5, 135.4 (d, J = 6.7 Hz), 128.7, 128.4(2),
909.3565.
128.2, 128.1, 127.9(2), 127.8(2), 127.3(2), 126.9, 122.3 (CH tri-
azole), 89.7 (C-1′), 86.9 (C–(Ph)3), 81.2 (d, J = 7.6 Hz, C-4′), 80.1
(C-4), 79.6 (C-5), 75.4 (C-2′), 75.3 (C-3), 73.7, 72.6 (CH2–Ph),
1-[2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl]-4-methylenoxy-[5-(3S,4R,-
5R)-3,4-di-O-benzyl-1-O-(dibenzylphosphatyl)-6-O-trityl-hexanyl]-
1H-1,2,3-triazole (37). Using procedure C, alkyne (32) (0.23 g, 70.7 (C-3′), 69.5, 69.0 (t, J = 5.2 Hz, CH2–OP), 66.4 (d, J = 5.3
0.27 mmol) was mixed with azido-2,3,5-tri-O-benzoyl-β-D-ribo- Hz, C-5′), 64.9 (d, J = 5.9 Hz, C-1), 64.3 (CH2–triazole), 63.4
furanoside (11) (0.14 g, 0.30 mmol) in a water–tBuOH (5 mL) (C-6), 30.2 (d, J = 7.1 Hz, C-2), 20.4, 20.3 (CH3). δp (121 MHz;
mixture. A pre-mixed copper sulfate (0.027 g, 0.11 mmol), CDCl3; Me4Si) 0.15 (s, decoupled), −0.01 (s, decoupled). HRMS
sodium ascorbate (0.043 g, 0.22 mmol) aqueous solution (ES) calculated [M + H]+ = 1406.5120, found [M + H]+
(5 mL) was added to the previous suspension. After 8 hours, 1406.5144.
=
the crude material was purified by silica column chromato-
graphy (Hex/EtOAc/TEA: 75/24/1–1/0.9/0.1 v/v/v) to give (37)
(0.24 g, 71%) as a colourless oil. δH (500 MHz; CDCl3; Me4Si)
8.01–7.94 (8H, m, Ph), 7.75 (1H, s, H triazole), 7.59–7.53 (3H,
m, Ph), 7.48–7.33 (20H, m, Ph), 7.38–7.28 (17H, m, Ph),
7.09–7.07 (2H, m, Ph), 6.27 (1H, d, J = 3.1 Hz, H-1′), 6.23 (1H, t,
J = 3.1 Hz, H-2′), 6.14 (1H, t, J = 6.0 Hz, H-3′), 5.00–4.95 (1H, m,
CH2–OP), 4.89–4.84 (3H, m, CH2–OP), 4.75 (1H, dd, J = 3.4 Hz,
12.2 Hz, CH2–triazole), 4.65–4.43 (7H, m, CH2–Ph, CH2–
triazole, H-1), 3.86–3.79 (3H, m, H-5′, H-5), 3.73 (1H, t, J = 5.5
Hz, H-4), 3.66–3.63 (1H, m, H-3), 3.54 (1H, dd, J = 3.1 Hz, 10.4
Hz, H-6), 3.38 (1H, dd, J = 5.7 Hz, 10.4 Hz, H-6), 1.81–1.68 (2H,
m, H-2). δC (125 MHz; CDCl3; Me4Si) 166.1, 165.1, 165.0
(CvO), 146.2 (C triazole), 143.9(3), 138.5, 138.3, 135.6 (d, J =
6.8 Hz), 133.8, 133.6, 133.3 (d, J = 3.3 Hz) 129.8(2), 129.7,
129.3, 128.7, 128.7, 128.6, 128. 5(2), 128.4, 128.3, 128.1(2),
128.0, 127.9, 127.8(2), 127.6, 127.4, 127.0, 122.1 (CH triazole),
90.2 (C-1′), 87.0 (C–(Ph)3), 81.0 (C-4′), 80.6 (C-4), 80.0 (C-5),
77.6 (C-2′), 75.2 (C-3), 74.0, 73.9 (CH2–Ph), 71.7 (C-3′), 69.2 (d,
J = 1.9 Hz), 69.1 (d, J = 1.9 Hz), 64.3 (CH2–triazole), 63.8 (C-5′),
63.6 (C-6), 60.0 (C-1), 34.1 (C-2). δp (121 MHz; CDCl3; Me4Si)
1-[2,3,5-Tri-O-benzoyl-β-D-ribofuranosyl]-4-methylenoxy-[5-(3S,4R,-
5R)-3,4-di-O-benzyl-6-O-(dibenzylphosphatyl)-1-O-(tert-butyl-
diphenylsilyl)-hexanyl]-1H-1,2,3-triazole (39). Using the pro-
cedure A, the alkyne precursor (0.31 g, 0.50 mmol) was
phosphorylated and the crude passed through a silica column
chromatography (Hex/EtOAc: 9/1–8/2 v/v) to give the phos-
phorylated intermediate (34) as a crude colourless oil. Using
procedure B, the whole quantity of this crude was mixed with
azido-2,3,5-tri-O-benzoyl-β-D-ribofuranoside (11) (0.27 g,
0.55 mmol) in a water–tBuOH mixture (5 mL, 1 : 1 v/v). A pre-
mix aqueous solution (5 mL) of copper sulfate (0.050 g,
0.20 mmol) and sodium ascorbate (0.079 g, 0.40 mmol) was
added to the suspension. After 6 hours, the crude product was
purified by column chromatography (Hex/EtOAc: 75/25 v/v) to
give (39) (0.30 g, 44% overall) as a colourless oil. δH (500 MHz;
CDCl3; Me4Si) 8.02–7.99 (4H, m, Ph), 7.96–7.94 (2H, m, Ph),
7.72 (1H, s, H triazole), 7.65–7.62 (5H, m, Ph), 7.44–7.22 (3H,
m, Ph), 7.20–7.10 (31H, m, Ph), 6.25 (1H, dd, J = 2.8 Hz,
5.5 Hz, H-2′), 6.19 (1H, d, J = 5.5 Hz, H-3′), 6.16 (1H, d, J =
2.8 Hz, H-1′), 5.04–5.02 (4H, dd, J = 5.7 Hz 7.9 Hz, CH2–OP),
4.87–4.84 (1H, m, H-4′), 4.72 (1H, dd, J = 3.95 Hz, 12.1 Hz,
CH2–triazole), 4.68–4.65 (1H, d, J = 12.1 Hz, H-5′), 4.65–4.62
(2H, m, CH2–triazole, H-5′), 4.55–4.53 (4H, m, CH2–Ph),
4.44–4.41 (1H, dd, J = 2.1 Hz, 6.0 Hz, H-6), 4.26–4.21 (1H, dd,
J = 6.0 Hz, 10.5 Hz, H-6), 3.97–3.94 (1H, m, H-3), 3.87–3.85 (1H,
m, H-5), 3.78–3.74 (1H, m, H-1), 3.70–3.65 (2H, m, H-1, H-4),
1.91–1.84 (1H, m, H-2), 1.82–1.76 (1H, m, H-2), 1.05 (9H, s,
(CH3). δC (125 MHz; CDCl3; Me4Si) 166.0, 165.0, 164.9 (CvO),
145.7 (C triazole), 138.5, 138.0, 135.9 (d, J = 2.1 Hz), 135.8 (d,
J = 2.1 Hz), 135.6, 133.8, 133.7, 133.5, 133.2, 133.7(2), 129.8(2),
129.6(2), 129.4, 128.8, 128.7, 128.5(2), 128.4(2), 128.3(2), 127.9,
127.8(2), 127.8(3), 127.6(3), 127.5, 122.7 (CH triazole), 90.2
(C-1′), 80.9 (C-4′), 79.3 (C-4), 79.0 (d, J = 7.0 Hz, C-5), 75.5 (C-3),
75.2 (C-2′), 73.9, 72.6 (CH2–Ph), 71.8 (C-3′), 69.2 (t, J = 4.9 Hz,
CH2–OP,), 67.4 (d, J = 5.8 Hz, C-6), 64.0 (C-5′), 63.9 (CH2–tri-
azole), 60.4 (C-1), 33.5 (C-2), 26.9 CH3), 19.2 (C–(CH3)3). δp
(121 MHz; CDCl3; Me4Si) 0.10 (s, decoupled). HRMS (ES) calcu-
lated [M + H]+ = 1370.5175, found [M + H]+ = 1370.5187.
0.15 (s, decoupled). HRMS (ES) calculated [M + H]+
1374.5092, found [M + H]+ = 1374.5063.
=
1-[2,3-Di-O-acetyl-5-O-(diphenylphosphatyl)-β-D-ribofuranosyl]-
4-methylenoxy-[5-(3S,4R,5R)-3,4-di-O-benzyl-1-O-(dibenzylphosphatyl)-
6-O-trityl)-hexanyl]-1H-1,2,3-triazole (38). Using procedure C,
(34) (0.24 g, 0.27 mmol) was mixed with azido-2,3-di-O-acetyl-
5-O-(diphenylphosphatyl)-β-D-ribofuranoside (14) (0.16 g,
0.30 mmol) in a water–tBuOH (5 mL) mixture. A pre-mixed
copper sulfate (0.028 g, 0.11 mmol), sodium ascorbate
(0.044 g, 0.22 mmol) aqueous soltion (5 mL) was added to the
previous suspension. After 6 hours, the crude material was
purified by silica column chromatography (Hex/EtOAc/TEA:
75/24/1–6/3.9/0.1 v/v/v) to give (38) (0.24 g, 62%) as a colourless
oil. δH (500 MHz; CDCl3; Me4Si) 7.74 (1H, s, H triazole),
7.44–7.42 (8H, m, Ph), 7.27–7.15 (44H, m, Ph), 7.02–7.01 (2H,
m, Ph), 6.01 (1H, d, J = 3.9 Hz, H-1′), 5.76 (1H, dd, J = 3.9 Hz,
5.4 Hz, H-2′), 5.63 (1H, t, J = 5.4 Hz, H-3′), 4.98–4.92 (8H, m,
CH2–OP), 4.83–4.80 (1H, d, J = 12.0 Hz, CH2–Ph), 4.55–4.51
(3H, t, J = 12.0 Hz, CH2–Ph), 4.42–4.36 (3H, m, H-4′, CH2–tria-
zole), 4.19–4.08 (3H, m, H-5′, H-1), 3.83–3.81 (1H, m, H-3),
1-[2,3-Di-O-acetyl-5-O-(diphenylphosphatyl)-β-D-ribofuranosyl]-
4-methylenoxy-[5-(3S,4R,5R)-3,4-di-O-benzyl-6-O-(dibenzylphos-
phatyl)-1-O-(tert-butyldiphenylsilyl)-hexanyl]-1H-1,2,3-triazole
3.79–3.75 (1H, m H-5), 3.68–3.66 (1H, m, H-4), 3.52 (1H, dd, J = (36). Using the procedure A, the alkyne precursor (0.31 g,
2.8 Hz, 10.5 Hz, H-6), 3.32 (1H, dd, J = 5.5 Hz, 10.5 Hz, H-6), 0.45 mmol) was phosphorylated and partially purified by
2.06 (3H, s, CH3), 2.03 (3H, s, CH3), 1.85–1.81 (1H, m, H-2), column chromatography (Hex/EtOAc: 9/1–8/2 v/v) to give phos-
1.79–1.74 (1H, m, H-2). δC (125 MHz; CDCl3; Me4Si) 169.2, phorylated intermediate (32) as a crude colourless oil. Using
168.9 (CvO), 145.9 (C triazole), 143.9(3), 138.4, 138.1, 135.8(2) procedure C, the whole quantity of this crude was mixed with
9616 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
azido-2,3-di-O-acetyl-5-O-(diphenylphosphatyl)-β-D-ribofurano- 68.7 (C-3′), 66.3 (d, J = 6.0 Hz, C-5′), 63.9 (CH2–triazole), 63.5
side (14) (0.28 g, 0.55 mmol) in a water–tBuOH mixture (5 mL, (C-6), 62.9 (C-1), 33.2 (C-2), 26.1 (CH3), 19.0 (C–(CH3)3). δp
1 : 1 v/v). A pre-mix aqueous solution (5 mL) of copper sulfate (121 MHz; CDCl3; Me4Si) −0.13 (s, decoupled). HRMS (ES) cal-
(0.049 g, 0.20 mmol) and sodium ascorbate (0.078 g, culated [M + Na]+ = 1322. 5303, found [M + Na]+ = 1322.5309.
0.40 mmol) was added to the suspension. After 8 hours, the
1-(β-D-Ribofuranosyl)-4-methylenoxy-[5-(3S,4R,5R)-1-O-(phos-
crude product was purified by column chromatography (Hex/ phatyl)-hexan-3,4,6-triolyl]-1H-1,2,3-triazole (41). Using pro-
EtOAc: 75/25 v/v) to give (36) (0.27 g, 39% overall) as a colour- cedure D, compound (39) (160 mg, 0.098 mmol) was
less oil. δH (500 MHz; CDCl3; Me4Si) 7.68 (1H, s, H triazole),
deprotected and purified to give compound (41) (39 mg, 87%
7.63–7.61 (4H, m, Ph), 7.40–7.20 (36H, m, Ph), 5.93 (1H, d, J = overall) as a white gum. δH (500 MHz; D2O; Me4Si) 8.11 (1H, s,
3.8 Hz, H-1′), 5.75 (1H, t, J = 5.2 Hz, H-2′), 5.63 (1H, t, J = H triazole), 6.00 (1H, d, J = 3.9 Hz, H-1′), 4.27 (1H, t, J = 5.2 Hz,
5,2 Hz, H-3′), 5.00–4.96 (8H, m, CH2–OP), 4.63–4.47 (6H, m,
H-2′), 4.07 (1H, q, J = 5.2 Hz, H-3′), 3.87–3.70 (5H, m, H-4,
CH2–Ph), 4.41–4.38 (2H, m, H-4′, H-5′), 4.23–4.10 (4H, m, H-6, CH2–triazole, H-5′, H-5, H-4′), 3.67 (1H, d, J = 3.2 Hz, CH2–tri-
H-5′), 3.93–3.89 (1H, m, H-3), 3.84–3.81 (1H, m, H-5), 3.75–3.67 azole), 3.60–3.58 (2H, m, H-1), 3.56–3.49 (2H, m, H-6, H-3),
(1H, m H-1), 3.66–3.62 (1H, m, H-1), 2.07 (3H, s, CH3), 2.06 3.41 (1H, dd, J = 2.0 Hz, 7.7 Hz, H-6), 1.73–1.64 (2H, m, H-2).
(3H, s, CH3), 1.78–1.71 (2H, m, H-2), 1.02 (9H, s, CH3). δC δC (125 MHz; D2O; Me4Si) 144.5 (C triazole), 124.3 (CH tria-
(125 MHz; CDCl3; Me4Si) 169.2, 169.1 (CvO), 145.6 (C tri- zole), 92.4 (C-1′), 85.5 (C-4′), 78.3 (C-5), 75.1 (C-4), 73.5 (C-3),
azole), 138.5, 138.0, 135.9, 135.8 (d, J = 2.8 Hz), 135.6(2), 135.5, 71.7 (C-2′), 66.8 (C-3), 63.6 (d, J = 4.9 Hz, C-1), 62.4 (C-5′), 61.5
133.7(2) (d, J = 5.3 Hz), 129.6(2), 128.5(2), 128.4(2), 128.3(2), (CH2–triazole), 60.0 (C-6), 34.1 (d, J = 6.9 Hz, C-2). δp
128.2(2), 128.0(2), 127.9(2), 127.8, 127.6(2), 127.5(2), 122.6 (CH (121 MHz; D2O; Me4Si) 1.16 (br, decoupled). HRMS (ES) calcu-
triazole), 89.7 (C-1′), 81.3 (d, J = 7.6 Hz, C-4′), 79.4 (C-4), 79.1 lated [M − H]− = 458.1176, found [M − H]− = 458.1164.
(d, J = 7.1 Hz, C-5), 75.5 (C-3), 75.2 (C-2′), 73.2, 72.6 (CH2–Ph),
70.7 (C-3′), 69.5 (t, J = 4.7 Hz, CH2–OP), 69.2 (t, J = 4.7 Hz,
CH2–OP,), 67.5 (d, J = 5.8 Hz, C-5,), 66.4 (d, J = 5.6 Hz, C-6),
63.9 (CH2–triazole), 60.4 (C-1), 33.5 (C-2), 26.9 (CH3), 20.4, 20.3
(CH3), 19.1 (C–(CH3)3). δp (121 MHz; CDCl3; Me4Si) 0.12
(s, decoupled), 0.02 (s, decoupled). HRMS (ES) calculated
[M + H]+ = 1402.5284, found [M + H]+ = 1402.5277.
1-[(5-O-Phosphatyl)-β-D-ribofuranosyl]-4-methylenoxy-[5-(3S,4R,-
5R)-1-O-(phosphatyl)-hexane-3,4,6-triolyl]-1H-1,2,3-triazole
(40). Using procedure D, compound (38) (52 mg, 0.037 mmol)
was deprotected to give (40) (18 mg, 90% overall) as a white
gum. δH (500 MHz; D2O; Me4Si) 8.21 (1H, s, H triazole), 6.10
(1H, d, J = 4.6 Hz, H-1′), 4.58 (1H, q, J = 4.6 Hz, H-2′), 4.38 (1H,
t, J = 4.6 Hz, H-3′), 4.30–4.27 (1H, m, H-4′), 4.06–3.93 (5H, m,
H-4, CH2–triazole, H-5′), 3.86–3.81 (2H, m, H-5, H-3), 3.67–3.55
(3H, m, H-1, H-6), 3.49–3.46 (1H, m, H-6), 1.80–1.71 (2H, m,
H-2). δC (125 MHz; D2O; Me4Si) 143.1 (C triazole), 123.7 (CH
triazole), 92.6 (C-1′), 84.4 (d, J = 8.5 Hz, C-4′), 79.5 (C-5), 75.4
(C-4), 71.8 (C-3), 70.6 (C-2′), 66.8 (C-3′), 65.0 (d, J = 4.6 Hz, C-1),
63.4 (d, J = 5.0 Hz, C-5′), 62.5 (CH2–triazole), 60.1 (C-6), 34.1 (d,
J = 6.8 Hz, C-2). δp (121 MHz; D2O; Me4Si) 4.20 (s, decoupled),
1-[5-O-(Dibenzylphosphatyl)-β-D-ribofuranosyl]-4-methylenoxy-
[5-(3S,4R,5R)-3,4-di-O-benzyl-1-O-(tert-butyldiphenylsilyl)-6-O-
trityl-hexanyl]-1H-1,2,3-triazole (35). Using procedure B, (30)
(0.060 g, 0.069 mmol) was mixed with azido-3,5-di-O-acetyl-5-
O-(dibenzylphosphatyl)-β-D-ribofuranoside (14) (0.040 g,
0.076 mmol) in a water–tBuOH (1 mL) mixture. A pre-mixed
copper sulfate (0.007 g, 0.030 mmol), sodium ascorbate
(0.011 mg, 0.060 mmol) aqueous solution (1 mL) was added to
the suspension. After 8 hours, the crude product was dissolved
in MeOH (3 mL) and MeONa (0.1 mL 25% wt in MeOH) was
3.12 (s, decoupled). HRMS (ES) calculated [M − H]−
=
538.0839, found [M − H]− = 538.0809.
1-(β-D-Ribofuranosyl)-4-methylenoxy-[5-(3S,4R,5R)-6-O-(phos-
(44). Using
trated. The crude material was purified by column chromato- procedure D, compound (37) (31 mg, 0.023 mmol) was depro-
tected and purified to give compound (44) (7 mg, 68% overall)
added dropwise and was stirred for 20 minutes, then concen- phatyl)-hexan-1,2,3,4-tetraolyl]-1H-1,2,3-triazole
graphy (Hex/EtOAc/TEA: 8/1.9/0.1–0/0.9/0.1 v/v/v) to give (35)
(0.025 g, 28% overall) as a colourless oil. δH (500 MHz; CDCl3; as a pure white gum. δH (500 MHz; D2O; Me4Si) 8.13 (1H, s, H
Me4Si) 7.65–7.60 (4H, m, Ph), 7.44–7.21 (44H, m, Ph), triazole), 5.88 (1H, dd, J = 1.8 Hz, 3.8 Hz, H-1′), 4.53 (1H, td, J =
1.8 Hz, 5.8 Hz, H-2′), 4.26 (1H, t, J = 51.8 Hz, H-3′), 4.67–4.62
7.00–6.97 (2H, m, Ph), 5.87 (1H, d, J = 1.8 Hz, H-1′), 5.09–5.00
(4H, m, CH2–OP), 4.81 (1H, d, J = 11.5 Hz, CH2–Ph), 4.79 (1H, (2H, m, H-3, H-4), 4.10–4.06 (2H, m, CH2–triazole), 3.91–3.87
d, J = 11.8 Hz, CH2–triazole), 4.65–4.62 (3H, m, CH2–Ph), 4.61 (1H, m, H-5), 3.79–3.77 (1H, m, H-4′), 3.70–3.64 (2H, m, H-5′,
H-6), 3.58–3.53 (3H, m, H-1, H-6, H-5′), 3.41–3.39 (1H, m, H-1),
(1H, d, J = 11.8 Hz, CH2–triazole), 4.53 (2H, m, H-5′), 4.31–4.27
(1H, m, H-4′), 4.20–4.18 (1H, m, H-2′), 4.05–4.01 (1H, m, H-5), 1.70–1.63 (1H, m, H-2), 1.56–1.53 (1H, m, H-2). δC (125 MHz;
4.01–3.96 (5H, m, H-1, H-3, H4, H-3′), 3.55 (1H, dd, J = 2.4 Hz, D2O; Me4Si) 143.2 (C triazole), 122.6 (CH triazole), 90.1 (C-1′),
83.3 (C-4′), 78.3 (d, J = 7.2 Hz, C-5), 75.4 (C-4), 71.7 (C-3), 70.7
10.5 Hz, H-6), 3.30 (1H, dd, J = 5.6 Hz, 10.5 Hz, H-6), 1.77–1.72
(2H, m, H-2), 1.08 (9H, s, (CH3). δC (125 MHz; CDCl3; Me4Si) (C-2′), 67.2 (C-3′), 64.5 (d, J = 3.5 Hz, C-6), 63.0 (CH2–triazole),
145.9 (C triazole), 143.4(3), 137.6, 136.7(2), 135.5, 135.3 (d, J = 60.4 (C-5′), 58.1 (C-1), 35.6 (C-2). δp (121 MHz; D2O; Me4Si) 1.78
(s, decoupled). HRMS (ES) calculated [M + Na]+ = 482.1152,
7.0 Hz), 133.2 (d, J = 3.3 Hz), 133.0, 132.9, 130.1, 129.4, 128.5,
128.3, 128.2, 128.1, 127.9, 127.8(2), 127.7(2), 127.6, 127.5, found [M + Na]+ = 482.1114.
127.4, 122.7 (CH triazole), 90.0 (C-1′), 86.6 (C–(Ph)3), 81.0 (d,
1-[(5-O-Phosphatyl)-β-D-ribofuranosyl]-4-methylenoxy-[5-(3S,4R,-
J = 7.4 Hz, C-4′), 80.2 (C-4), 78.5 (C-2), 77.6 (C-5), 77.1 (C-3), 5R)-6-O-(phosphatyl)-hexan-1,3,4-triolyl]-1H-1,2,3-triazole (43).
73.7, 73.0 (CH2–Ph), 72.4 (C-2′), 69.6(2) (d, J = 3.7 Hz, CH2–OP), Using procedure D, compound (36) (0.11 g, 0.077 mmol) was
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9617

Paper
Organic & Biomolecular Chemistry
View Article Online
deprotected and purified to give (43) (0.032 g, 76% overall) as (w/v) trichloroacetic acid and kept on ice for 10 min. The preci-
a white gum. δH (500 MHz; D2O; Me4Si) 8.19 (1H, s, H triazole), pitated protein was removed by centrifugation. The super-
6.00 (1H, d, J = 4.6 Hz, H-1′), 4.50 (1H, q, J = 4.9 Hz, H-2′), 4.31 natant (200 μL) was incubated with 200 μL of 0.2 M NaIO4 for
(1H, t, J = 4.6 Hz, H-3′), 4.25–4.23 (1H, m, H-4′), 4.13–4.06 (3H, 1 h. Na2SO3 (200 μL of 1 M) was then added to the supernatant
m, CH2–triazole, H-4), 3.98–3.87 (2H, m, H-5′), 3.81–3.76 (1H, to remove the excess NaIO4. For the measurement of phos-
m, H-5), 3.68–3.62 (1H, m, H-3), 3.57–3.53 (2H, m, H-1), phate, a 600 μL reagent mixture (240 μL of H2O, 120 μL of
3.43–3.37 (1H, m, H-6), 3.05–2.99 (1H, m, H-6), 1.22–1.04 (2H, 2.5% ammonium molybdate, 120 μL of 10% ascorbic acid, and
m, H-2). δC (125 MHz; D2O; Me4Si) 142.1 (C triazole), 121.9 120 μL of 6 N sulfuric acid) was added and incubated for 1 h at
(CH triazole), 91.3 (C-1′), 84.6 (d, J = 7.0 Hz, C-4′), 78.0 (d, J = 37 °C. The absorbance was measured at 820 nm, and activity
7.4 Hz, C-5), 75.3 (C-4), 71.5 (C-3), 70.8 (C-2′), 66.9 (C-3′), 65.0 was determined by the amount of inorganic phosphate liber-
(d, J = 3.0 Hz, C-6), 64.3 (CH2–triazole), 61.7 (d, J = 4.1 Hz, ated. For each assay, a second aliquot of the sample was
C-5′), 59.0 (C-1), 35.7 (C-2). δp (121 MHz; D2O; Me4Si) 1.36 (s, measured for phosphates not having been released by NaIO4
decoupled), 1.30 (s, decoupled). HRMS (ES) calculated [M − treatment to control for phosphatase activity or for any phos-
H]− = 538.0839, found [M − H]− = 538.0831.
1-[(5-O-Phosphatyl)-β-D-ribofuranosyl]-4-methylenoxy-[5-(3S,4R,- was subtracted from the experimental sample to obtain mIPS
phates that may have been released from the MAs. This value
5R)-hexan-1,3,4,6-tetraolyl]-1H-1,2,3-triazole (42). Using pro-
cedure D, compound (35) (170 mg, 0.13 mmol) was depro-
tected and purified to give (42) (45 mg, 75% overall) as a pure
white gum. δH (500 MHz; D2O; Me4Si) 8.19 (1H, s, H triazole),
6.01 (1H, d, J = 4.7 Hz, H-1′), 4.49 (1H, t, J = 4.8 Hz, H-2′), 4.30
(1H, t, J = 4.8 Hz, H-3′), 4.22–4.19 (1H, m, H-4′), 3.95–3.71 (6H,
m, H-5, H-5′, H-4, CH2–triazole), 3.58–3.47 (4H, m, H-6, H-3,
H-1), 3.39–3.35 (1H, m, H-6), 1.67–1.60 (1H, m, H-2), 1.57–1.51
(1H, m, H-2). δC (125 MHz; D2O; Me4Si) 142.9 (C triazole),
122.6 (CH triazole), 92.7 (C-1′), 84.7 (d, J = 8.2 Hz, C-4′), 79.5
(C-5), 75.6 (C-4), 72.0 (C-3), 70.9 (C-2′), 67.3 (C-3′), 64.9 (d, J =
4.7 Hz, C-5′), 62.7 (C-6), 60.2 (CH2–triazole), 60.0 (C-1), 35.8
(C-2). δp (121 MHz; D2O; Me4Si) 1.83 (s, decoupled). HRMS
activity. All results were normalised to 100% mIPS activity.
Data represents the average of three independent experiments
for the 100 μM set, and two independent experiments for the
200 μM set.
Docking experimental methods
MODEL PREPARATION. It is essential to prepare the crystal struc-
tures prior to ligand docking. Crystal structures of 1jki and
1rm0 were prepared using the Protein Preparation Wizard23 in
the Schrödinger Suite 2009. Default parameters were employed
for the initial preparation step followed by optimisation of the
hydrogen-bond network and a subsequent minimisation using
the OPLS-AA 2001 force field. Crystal ligands (1jki: NAD and
2-deoxy-glucitol-6-phosphate, 1rm0: NAD and 2-deoxy-D-
glucitol 6-(E)-vinylhomophosphonate) were finally omitted
from the complex.
(ES) calculated [M + Na]+ = 482.1152, found [M + Na]+
482.1143.
=
In vitro assay of isolated yeast mIPS enzyme
LIGAND BUILDING. All compounds were built and geometries
‘cleaned’ using the Build panel incorporated in the Maestro24
graphic interface of the Schrödinger Suite 2009.
PURIFICATION OF RECOMBINANT YEAST MIPS. Saccharomyces cerevi-
siae BY4741 ino1Δ mutant bearing the pRDINO1 construct was
used to express the recombinant 6xHis-tagged mIPS. Cells
were grown at 30 °C in synthetic minimal medium lacking
uracil. Galactose (2%) was used to induce over-expression of
the recombinant protein. Cell extracts were prepared by dis-
rupting the cells with glass beads. mIPS was purified by
affinity chromatography using ProBond nickel-chelating resin
to bind the protein. The resin was washed twice with 20 mM
then 60 mM imidazole in Tris buffer (50 mM Tris-Cl (pH 7.4)
and 300 mM NaCl). The protein was eluted with 250 mM imi-
dazole in Tris buffer, dialyzed, concentrated, and resuspended
in (50 mM Tris-Cl, 50 mM NaCl, 10 mM DTT). Protein concen-
tration was determined by the Bradford method using bovine
serum albumin as the standard.
MIPS ENZYME ASSAY. Purified mIPS enzyme activity was deter-
mined by the rapid colorimetric method of Barnett et al.21
with minor modifications. Purified protein was suspended in
the reaction buffer (100 mM Tris acetate (pH 8.0), 0.8 mM
NAD, 2 mM DTT, 14 mM NH4Cl) containing the inhibitor
present in various concentrations (0, 100 μM, and 200 μM).
LIGAND DOCKING. Prior to ligand docking the Glide25 software
requires generation of grids which describe the character of
the intended target site. In 1jki the grid box was centred on
residues N77, T247, G295, S296, Q325, D356, K369 and K412
and the box dimensions were 32 Å × 32 Å × 32 Å. In the ligand
docking step, compounds (21), (22), (23), (24), (40), (41), (42),
(43) and (44), were docked into the generated grids. The mid-
point of the compounds to be docked was enclosed in a box of
dimensions 12 Å × 12 Å × 12 Å, which incorporated the region
around the NAD and G6P binding site. The van der
Waal radius for ligand atoms was scaled to 0.8. In 1rm0 the
grid box was defined by residues N77, A245, S323, G324,
Q325, T326, D356, K369, K412 and D438. The remaining steps
for 1rm0 ligand docking are the same as those described
for 1jki.
Acknowledgements
After addition of 5 mM glucose 6-phosphate to a final volume We thank the Centre for Cancer Research and Cell Biology at
of 150 μL, the reaction mixture was incubated for 1 h at 37 °C. QUB for funding P.LeC.’s PhD project, the National Institutes
The reaction was terminated by the addition of 50 μL of 20% of Health [grant number DK081367] for funding M.L.G., and
9618 | Org. Biomol. Chem., 2012, 10, 9601–9619
This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry
Paper
View Article Online
the Wayne State University Biology Department Graduate
Enhancement grant for supporting R.M.D.
F. Movahedzadeh, N. G. Stoker and N. Q. McDonald, Struc-
ture, 2002, 10(3), 393–402.
11 X. S. Jin, K. M. Foley and J. H. Geiger, J. Biol. Chem., 2004,
279(14), 13889–13895.
12 X. S. Jin and J. H. Geiger, Acta Crystallogr., Sect. D: Biol.
Crystallogr., 2003, 59, 1154–1164.
13 S. M. Byun and R. Jenness, Biochemistry, 1981, 20(18),
5174–5177.
14 (a) M. W. Loewus and F. A. Loewus, Carbohydr. Res., 1980,
82(2), 333–342; (b) W. R. Sherman, A. Rasheed, L. A. Mauck
and J. Wiecko, J. Biol. Chem., 1977, 252(16), 5672–5676.
15 G. A. Patani and E. J. LaVoie, Chem. Rev., 1996, 96(8), 3147–
3176.
16 E. J. Amigues, E. Armstrong, M. Dvorakova, M. E. Migaud
and M. Huang, Nucleosides, Nucleotides Nucleic Acids, 2009,
28(3), 238–259.
References
1 J. H. Geiger and X. Jin, Sub-Cell. Biochem., 2006, 39, 157–
180.
2 N. Bachhawat and S. C. Mande, Trends Genet., 2000, 16(3),
111–113.
3 S. L. Ju, G. Shaltiel, A. Shamir, G. Agam and
M. L. Greenberg, J. Biol. Chem., 2004, 279(21), 21759–
21765.
4 K. GhoshDastidar, A. Chatterjee, A. Chatterjee and
A. L. Majumder, Sub-Cell. Biochem., 2006, 39, 315–340.
5 F. Movahedzadeh, D. A. Smith, R. A. Norman,
P. Dinadayala, J. Murray-Rust, D. G. Russell, S. L. Kendall,
S. C. G. Rison, M. S. B. McAlister, G. J. Bancroft,
N. Q. McDonald, M. Daffe, Y. Av-Gay and N. G. Stoker, Mol.
Microbiol., 2004, 51(4), 1003–1014.
6 (a) G. Agam, A. Shamir, G. Shaltiel and M. L. Greenberg,
Bipolar Disord., 2002, 4, 15–20; (b) H. Chen and L. Xiong,
J. Biol. Chem., 2010, 285(31), 24238–24247.
7 F. Tian, M. E. Migaud and J. W. Frost, J. Am. Chem. Soc.,
1999, 121(24), 5795–5796.
8 M. W. Loewus, F. A. Loewus, G. U. Brillinger, H. Otsuka
and H. G. Floss, J. Biol. Chem., 1980, 255(24), 1710–1712.
9 M. E. Migaud and J. W. Frost, J. Am. Chem. Soc., 1996, 118
(3), 495–501.
10 R. Kniewel, J. A. Buglino, V. Shen, T. Chadha, A. Beckwith
and C. D. Lima, J. Struct. Funct. Genomics, 2002, 2(3), 129–
134; R. A. Norman, M. S. B. McAlister, J. Murray-Rust,
17 M. M. Zhang, Y. G. Du and F. Z. Kong, Carbohydr. Res.,
2001, 330(3), 319–324.
18 D. L. J. Clive, X. He, M. H. D. Postema and
M. J. Mashimbye, J. Org. Chem., 1999, 64(12), 4397–4410.
19 A. K. Pathak, V. Pathak, L. E. Seitz, K. N. Tiwari,
M. S. Akhtar and R. C. Reynolds, Tetrahedron Lett., 2001, 42
(44), 7755–7757.
20 H. S. Cheon, Y. Lian and Y. Kishi, Org. Lett., 2007, 9(17),
3323–3326.
21 E. Bettelli, P. Cherubini, P. D’Andrea, P. Passacantilli and
G. Pianeateni, Tetrahedron, 1998, 54, 6011.
22 J. E. G. Barnett, R. E. Brice and D. L. Corina, Biochem. J.,
1970, 119, 183–186.
23 Protein Preparation Wizard, Schrödinger, LLC, New York,
NY, 2009.
24 Maestro, 9.0 ed, Schrödinger, LLC, New York, NY, 2009.
25 Glide, 5.5 ed, Schrödinger, LLC, New York, NY, 2009.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9601–9619 | 9619