Synthesis of a novel UDP-carbasugar as UDP-galactopyranose mutase inhibitor

Article abstract of DOI:10.1021/ol500848q

The multistep synthesis of a novel UDP-C-cyclohexene, designed as a high energy intermediate analogue of the UDP-galactopyranose mutase (UGM) catalyzed isomerization reaction, is reported. The synthesis of the central carbasugar involved the preparation of a galactitol derivative bearing two olefins necessary for the construction of the cyclohexene ring by a ring-closing metathesis as a key step. Further successive phosphonylation, deprotection, and UMP coupling provided the target molecule. The final molecule was assayed against UGM and compared with UDP-C-Galf, the C-glycosidic UGM substrate analogue.

Full text of DOI:10.1021/ol500848q

Letter
pubs.acs.org/OrgLett
Synthesis of a Novel UDP-carbasugar as UDP-galactopyranose
Mutase Inhibitor
Sandy El Bkassiny,† Ines N’Go,† Charlotte M. Sevrain,† Abdellatif Tikad,† and Stephane P. Vincent*,†
̀
́
†University of Namur, Dep
́
artement de Chimie, Laboratoire de Chimie Bio-Organique, rue de Bruxelles 61, B-5000 Namur, Belgium
S
* Supporting Information
ABSTRACT: The multistep synthesis of a novel UDP-C-cyclohexene, designed as a high energy intermediate analogue of the
UDP-galactopyranose mutase (UGM) catalyzed isomerization reaction, is reported. The synthesis of the central carbasugar
involved the preparation of a galactitol derivative bearing two olefins necessary for the construction of the cyclohexene ring by a
ring-closing metathesis as a key step. Further successive phosphonylation, deprotection, and UMP coupling provided the target
molecule. The final molecule was assayed against UGM and compared with UDP-C-Galf, the C-glycosidic UGM substrate
analogue.
Here we describe the synthesis and biochemical evaluation of a
novel UDP-cyclitol 7 designed as a transition-state analogue of
the UGM-catalyzed ring contraction. Our multistep synthesis
involves the construction of a key cyclohexene scaffold through a
ring-closing metathesis reaction.
ecause of the emergence of extremely resistant strains,
1
Btuberculosis still threatens the world population. Thus,
novel strategies to fight Mycobacterium tuberculosis have recently
been developed.2 Among them, UDP-galactopyranose mutase
(UGM)3 has been validated as a new target because of its
involvement in the biosynthesis of the mycobacterial cell wall.4
Indeed, some UGM inhibitors have been shown to kill the
bacterium,5 a result that confirmed that the enzyme is essential
for the survival of the pathogen.4 UGM catalyzes the
interconversion of UDP-galactopyranose (UDP-Galp) 1 into
UDP-galactofuranose (UDP-Galf) 2 (Figure 1), the biosynthetic
precursor of all galactofuranose-containing eukaryotic and
prokaryotic glycoconjugates.6,7 UGMs from major pathogens
have been identified,6a,8 but interestingly, this enzyme is absent in
mammals, which may favor the discovery of selective therapeutic
agents.
As highlighted above, the UGM-catalyzed interconversion of
UDP-Galp 1 into UDP-Galf 2 involves three important
intermediates: the acyclic adduct 5 and the oxycarbeniums 3
and 4. In order to obtain high-affinity mechanistic probes, several
groups have designed UDP-galactose analogues mimicking
either the charged intermediate 414a,b,15 or UDP coupled to
acyclic galactitols to mimic intermediate 5.9h,14g,h From these
studies it can be concluded that UGM has a much greater affinity
for the furanose Galf compared to the pyranose. In addition, the
cationic character of the intermediates is probably not a critical
parameter to mimic given the fact that cationic UDP-Galf
analogues never displayed strong inhibition profiles.14a,15 On the
other hand, the UDP-galactitol (acyclic form) derivatives such as
molecules 89h and 914h showed strong inhibition profiles against
UGM.
We thus reasoned that UDP-galactose mimicking both the
acyclic intermediate 5 and the galactofuranose substructure of 6
may display potent inhibition profiles and would be interesting to
cocrystallize with UGMs. We thus designed UDP-carbasugar 7 as
an inhibitor of UGM (Figure 1). Compared to UDP-galactitols 8
and 9, the cyclohexene ring has a similarity to a galactofuranose,
thanks in part to the geometry of the exocyclic C5−C6 diol, while
maintaining some similarity with the acyclic intermediate 5.
Moreover, the cyclohexene, with two sp2 hybridized carbons,
may partially mimic the conformation of the oxycarbenium 4.
Globally, cyclohexene 7 gathers some important structural
features of the transformation of the galactitol 5 to the furanose 6.
Moreover, UGM is a unique flavoenzyme whose structure(s)
and mechanism have been extensively studied.6b,9 Surprisingly, it
was discovered that the FAD cofactor plays the role of catalytic
nucleophile yielding covalent intermediates 5 and 6 (Figure 1)
after the release of the UDP moiety.6b,9i,10 Several crystal
structures of UGM have been obtained, some of them in the
presence of UDP or UDP-Galp.9b,11 However, many questions
remain unsolved regarding both the mechanism and the binding
modes of UGM with its substrate UDP-Galf and the key high-
energy intermediates. For instance, the SN1/SN2 nature of the
substitutions occurring during this isomerization is still under
debate,6b,9h,i,10,12 with some experiments suggesting a cationic
intermediate such as 4 and other data indicating a concerted
process.9e,13 Therefore, the design of new mechanistic probes
that mimic UDP-Galf or the transition states of this
enzyme9g,h,13,14 may help us to better understand its mechanism
and the structural requirements for tight binding to this
important therapeutic target.
Received: March 21, 2014
Published: April 18, 2014
© 2014 American Chemical Society
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Figure 1. Mechanism of UGM and design of the new high energy intermediate analogue 7.
Our retrosynthetic analysis of 7 is outlined in Figure 2. The key
cyclohexene 10 would be obtained by ring-closing metathesis of
Scheme 1. Synthesis of Intermediate 16
Figure 2. Retrosynthetic analysis.
the diene 11 which could possibly be prepared from the
protected D-galactonolactone 13 via successive Wittig and
Grignard reactions. We anticipated that lactone 13 would be
easily accessible in a few steps from D-galactose.
PMB group, the addition of 4-nitrophenylhydrazine was required
to remove the byproduct 4-methoxybenzaldehyde which was
found to be inseparable from alcohol 16.
Our first objective was the generation of diene 11, precursor of
the cyclohexene 10. The synthesis began with oxidation of D-
galactose in the presence of bromine followed by protection of
the resulting D-galactono-1,4-lactone as an acetonide to afford
the known lactone 1416 which, after benzylation, provided
intermediate 13 in 61% yield (Scheme 1). The lactone 13 was
then transformed into diol 15 in four steps. The reduction of the
lactone by DIBAL-H was directly followed by a Wittig olefination
without purification. A p-methoxybenzyl group was then
installed prior to standard dihydroxylation resulting in the
formation of epimeric diols 15 (in a ratio 87:13) in 84% yield
over four steps after a single purification by silica gel
chromatography.
In order to synthesize compound 16, the diol 15 was
regioselectively homologated: the silylation of the primary
alcohol followed by Dess−Martin oxidation afforded the
corresponding ketone. Olefination of the resulting carbonyl
group under classical Wittig conditions, and subsequent
deprotection of the p-methoxybenzyl ether with DDQ in
DCM/H2O gave 16 in 68% yield over four steps, with a single
purification (Scheme 1). Noteworthy, after the cleavage of the
The intermediate 16 was oxidized by Dess−Martin period-
inane (DMP) to give the ketone 12, which was then treated by
allylmagnesium bromide to provide a mixture of two
diastereoisomers 11a and 11b in a ratio 33:67 with 62% yield
over two steps (Scheme 2). This stereoselectivity was in
accordance with Mulzer’s study in which the same ratio was
observed for the addition of allylmagnesium bromide (reagent)
on related acyclic ketones bearing two different stereogenic
centers on each side.17 In this study, Mulzer had shown that the
choice of protecting groups around the ketone strongly
influenced the stereoselectivity of the Grignard addition. The
absolute configurations in molecules 11a and 11b were at first
assigned based on Mulzer’s model17 and were confirmed by
NOE experiments performed on the cyclized derivatives 17a and
17b.
The second key step of this synthesis was the generation of the
cyclohexene ring by ring-closing metathesis. While this reaction
has been already used for a general access to various
carbasugars,18 including cyclohexenes,19 the synthesis of
hindered trisubstituted cyclohexenes remains complex and
poorly described in the literature. Recently, Kiessling et al.
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the formation of 10 in 53% isolated yield, along with the expected
byproduct (Me)P(O)(OMe)2. Compound 10 was then
deprotected efficiently with an excess of TMSBr in CH2Cl2
followed by hydrogenolysis catalyzed by Pd(OH)2. The desired
phosphonate 19 was quantitatively obtained after maximum 20
min of reaction time to avoid the double bond saturation.
The cyclohexene phosphonate 19 was then coupled to an
activated form of UMP to generate UDP-carbasugar 7, which was
obtained in 14% yield after size-exclusion chromatography and
reversed-phase HPLC. As for the synthesis of UDP-galactofur-
anose 2,22 the activation of UMP as a N-methylimidazolium salt
gave the best results. The low yield can be explained by the fact
that once formed, the UDP-carbasugar can decompose into a
cyclic phosphonate and UMP.23 This intramolecular reaction
occurs not only during the reaction but also during the
purification steps. This side reaction was also observed for
other phosphonate analogues of UDP-galactose 1 and 2.14e,24
In order to study the inhibition profile of UDP-cyclohexene 7,
we used UDP-C-α-Galf 2024 (the C-glycosidic analogue of
UGM’s substrate UDP-α-Galf) and UDP 21 for comparison
purposes (Table 1). Indeed, UDP is often used in the literature as
Scheme 2. Synthesis of the Cyclohexene Ring
have reported the synthesis of epimers of shikimic acid using this
powerful procedure.19a We thus investigated this approach
starting from the mixture of epimeric dienes 11(a,b), and it was
found that 0.12 equiv of Hoveyda−Grubbs (II) catalyst and a
portionwise addition were necessary for the reaction to go to
completion. After 6 h, cyclohexenes 17a and 17b were isolated in
21% and 63% yields, respectively (Scheme 2).
Table 1. Inhibition Percentages and Kd of 7, 20, and 21
The structures of the diastereoisomers 17a and 17b were
confirmed by NOE experiments performed on product 17b in
CDCl3 which revealed different correlations between H5 and
H3, H4′b and H2, and finally the two correlations H4′a−H3 and
H4′a−H5. These results confirmed the absolute configuration of
compound 17b at C-4.
With the cyclohexene scaffold in hand, we focused on the
synthesis of the target nucleotide-sugar analogue 7. Cleavage of
the silyl ether in 17b with TBAF in THF gave the alcohol 18 in
84% yield (Scheme 3). Initial investigations on Michaelis−
a
b
inhibitor
% inhibition (HPLC)
Kd (μM, FP)
UDP (21)
39.1 8.0
10.3 6.1
29.4 5.7
45 1.1
517 1.4
870 1.4
20
7
a
Inhibition assay conditions: [Inhibitor] = 1 mM, reduced enzyme
[UGMKp] = 12 nM, [UDP-Galf ] = 105 μM, [dithionite] = 12.5 mM.
Fluorescence polarization assay conditions: [fluorescent probe] = 15
b
nM, [I] = 10 μM to 2 mM, nonreduced enzyme [UGMKp] = 500 nM.
Scheme 3. Synthesis of Final UDP-carbasugar 7
a control inhibitor.9h The comparison with 20 was also useful
because the replacement of the exo-anomeric oxygen atom by a
methylene group may affect the mode of interactions with
UGM.11a
Two complementary enzymatic assays were carried out on
UGM of Klebsiella pneumoniae (UGMKp) which is the most
studied UGM enzyme for inhibition studies.9h,14b,15,25 UGM is a
flavoenzyme whose cofactor must be reduced to be kinetically
competent. Thus, we realized first a competition assay against the
substrate UDP-Galf 2 under reducing conditions.9h In this assay,
the conversion of 2 into its isomer 3 is monitored by HPLC in
presence and absence of inhibitors. The decreases in reaction
rates are translated into inhibition percentages that have been
gathered in Table 1.
From this first series of experiments, we noticed that the
carbasugar 7 has an inhibition level intermediate between UDP
21 and UDP-C-Galf 20. This result shows that when UGM is
catalytically active, the enzyme has a stronger affinity for the
cyclohexene analogue 7 than for the galactofuranose subunit of
20. Therefore, these results indicate that, to mimic high-energy
intermediates of the UGM catalyzed reaction, it is appropriate, to
design molecules in which the galactose unit is indeed between a
furanose state as in 6 (Figure 1) and an acyclic state as in 5.
However, to obtain a more complete picture of the binding
properties of carbasugar 7, we also measured the fluorescence
Becker reaction between a sulfonate or a bromide, generated
from 18, and diethyl phosphite failed under various experimental
conditions.20 Fortunately, the Michaelis−Arbusov reaction was
successful, but only when the specific conditions recently
reported in the literature were applied:21 the treatment of 18
with trimethyl phosphite and zinc bromide as Lewis acid under
microwave irradiation led to a complete conversion after 2 h and
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polarization (FP)26 on UGMKp under nonreducing conditions.
This assay is easier to perform, but it only gives an estimation of
the affinities of the molecules because the enzyme is not in its
catalytically active state. The inhibition profile has been found in
the following order: UDP > UDP-C-Galf > 7, thus showing a
reversal of affinities compared to the HPLC assay. The latter
result was not surprising since major affinity changes of UDP-
galactose analogues toward UGM as a function of the redox state
of the enzyme have been observed several times in the
literature.9e,24,25,14i
In summary, we have explored a new synthetic pathway for the
generation of the UDP-carbasugar 7 starting from D-galactose
and by using ring closing-metathesis as a key step for the
transformation of acyclic galactitol into the cyclohexene ring.
When assayed against the reduced enzyme, the final cyclohexene
7 displayed a better affinity than the galactofuranoside 20, which
constitutes important information for the future design of
transition-state analogues.
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AUTHOR INFORMATION
Corresponding Author
■
*Fax: +32 81 72 45 17. E-mail: stephane.vincent@unamur.be.
Notes
The authors declare no competing financial interest.
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14860−14866. (i) N’Go, I.; Golten, S.; Arda, A.; Canada, J.; Jimenez-
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ACKNOWLEDGMENTS
This work was funded by an ARC grant from the Academ
Louvain (A.R.C. 08/13-012 for S.B. and I.G.) and by FNRS
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