Reversible and efficient inhibition of UDP-galactopyranose mutase by electrophilic, constrained and unsaturated UDP-galactitol analogues

Article abstract of DOI:10.1002/chem.201202302

A series of UDP-galactitols were designed as analogues of high-energy intermediates of the UDP-galactopyranose mutase (UGM) catalyzed furanose/pyranose interconversion, an essential step of Mycobacterium tuberculosis cell wall biosynthesis. The final compounds structurally share the UDP and the galactitol substructures that were connected by four distinct electrophilic connections (epoxide, lactone and Michael acceptors). All molecules were synthesized from a common perbenzylated acyclic galactose precursor that was derivatized by alkenylation, alkynylation and cyclopropanation. The inhibition study against UGM could clearly show that slight changes in the relative orientation of the UDP and the galactitol moieties resulted in dramatic variations of binding properties. Compared to known inhibitors, the epoxide derivative displayed a very tight, reversible, inhibition profile. Moreover, a time-dependent inactivation study showed that none of these electrophilic structures could react with UGM, or its FAD cofactor, the catalytic nucleophile of this still intriguing reaction. Shedding inhibitors: UDP-Galactopyranose mutase (UGM) is a validated target for treating tuberculosis. Its mechanism involves formation of a key covalent FAD-substrate intermediate, 1. A series of electrophilic UDP-galactitols were synthesized and assayed as inhibitors or inactivators of UGM. Strong inhibitions were observed, especially with epoxide 2. Interestingly, none of the molecules displayed an irreversible inhibition mode. Copyright

Full text of DOI:10.1002/chem.201202302

FULL PAPER
DOI: 10.1002/chem.201202302
Reversible and Efficient Inhibition of UDP-Galactopyranose Mutase by
Electrophilic, Constrained and Unsaturated UDP-Galactitol Analogues
Christophe Ansiaux, Inꢀs N’Go, and Stꢁphane P. Vincent*[a]
Abstract: A series of UDP-galactitols
were designed as analogues of high-
energy intermediates of the UDP-gal-
actopyranose mutase (UGM) catalyzed
furanose/pyranose interconversion, an
essential step of Mycobacterium tuber-
culosis cell wall biosynthesis. The final
compounds structurally share the UDP
and the galactitol substructures that
were connected by four distinct elec-
trophilic connections (epoxide, lactone
and Michael acceptors). All molecules
were synthesized from a common per-
benzylated acyclic galactose precursor
that was derivatized by alkenylation,
alkynylation and cyclopropanation.
The inhibition study against UGM
could clearly show that slight changes
in the relative orientation of the UDP
and the galactitol moieties resulted in
dramatic variations of binding proper-
ties. Compared to known inhibitors,
the epoxide derivative displayed a very
tight, reversible, inhibition profile.
Moreover, a time-dependent inactiva-
tion study showed that none of these
electrophilic structures could react with
UGM, or its FAD cofactor, the catalyt-
ic nucleophile of this still intriguing re-
action.
Keywords: carbohydrates · flavoen-
zymes · inhibitors · phosphonates ·
tuberculosis
Introduction
mitted that the FAD cofactor plays the very unusual role of
catalytic nucleophile in this mechanistically complex isomer-
ization reaction. Interestingly, structural data of the enzyme
alone or in complex with UDP and UDP-galactopyranosides
are now available for different UGMs, including important
human pathogens such as Trypanosoma cruzi, Aspergillus
fumigatus and Mycobacterium tuberculosis.[12] In parallel to
biochemical studies,[13] inhibitors have been designed, syn-
thesized and assayed against different UGM: these mole-
cules are either substrate analogues (carbohydrates[14] and
nucleotide–sugars[15]) or heterocyclic compounds derived
from inhibitor screenings and diversity-based approaches for
the discovery of novel antitubercular agents.[16] The descrip-
tion of non-natural substrates of UGM has also provided
key information about the interactions between the enzyme
and its substrate as well as the involvement of an intermedi-
ate oxycarbenium species.[17]
Thus, UGM catalyzes an isomerization through a multi-
step complex mechanism involving several high-energy in-
termediates and transition states. Despite the knowledge re-
cently accumulated, more inhibition studies are still necessa-
ry to obtain a clear picture of the UGM relative binding
modes to its substrate along the entire reaction coordinate.
This would allow, along with structural studies, the discovery
of, for instance, the rate limiting mechanistic steps and the
whole conformational itinerary of the galactose residue. Our
laboratory has focused on the synthesis of C-glycosidic ana-
logues of UDP-galactose, both in its furanose and its pyra-
nose forms, to generate the most complete family of mole-
cules mimicking the substrate in its ground state conforma-
tions or as a mechanistic intermediate (Figure 1). We first
probed the conformational itinerary of the sugar moiety
during its isomerization, thanks to the inhibition study of
Despite the availability of apparently potent drugs and vac-
cines, tuberculosis (TB) remains a major threat for public
health worldwide. Indeed, the epidemic is still growing and
the number of deaths caused by TB (around 10 million per
year) keeps increasing.[1] Moreover, the emergence of multi-
drug resistant strains have prompted the scientific communi-
ty to explore novel strategies to fight this major human
pathogen.[2]
In this context, the inhibition of the TBꢀs characteristic
and unique cell wall biosynthesis has become an attractive
strategy.[3] In fact, two glycoconjugates are the major compo-
nents of TB cell wall: mycolylarabinogalactan (mAG)[4] and
lipoarabinomannan (LAM),[5] and both play a pivotal role in
the virulence and the viability of TB.[6] Among the enzymes
that have been recently validated as targets for the discov-
ery of novel drugs against TB,[7] UDP-galactopyranose
mutase (UGM)[8] has also attracted the attention of mecha-
nistic enzymologists because of its very peculiar mecha-
nism.[9] UGM catalyzes the interconversion of UDP-galacto-
pyranose (Galp) into UDP-galactofuranose (Galf) by ex-
ploiting its FAD cofactor in an absolutely non-conventional
manner for a flavoenzyme.[10] Indeed, thanks to a pioneering
study performed by Kiessling in 2004,[11] it is now well-ad-
[a] Dr. C. Ansiaux, I. N’Go, Prof. S. P. Vincent
Dꢁpartement de Chimie, Laboratoire de Chimie Bio-Organique
University of Namur (FUNDP), Acadꢁmie Louvain
rue de Bruxelles 61, 5000 Namur (Belgium)
Fax : (+32)81-72-45-17
E-mail: stephane.vincent@fundp.ac.be
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201202302.
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Figure 1. Isomerization catalyzed by UGM, known C-glycosidic inhibitors 4–9 and novel generation of UDP-galactitols 10–13.
UGM by compounds 4–6, which led us to conclude that the
galactose unit adopts a 1,4B boat conformation in the catalyt-
ic site.[15b] This conformation may be achieved directly from
the pyranose for 1, or by the acyclic intermediate 2.
epoxides 10. This study describes the synthetic efforts to
achieve the preparation of compounds 10–13 as well as their
inhibition properties against UGM.
We then synthesized a series of exo-glycals (7–9) that dis-
played
interesting
time-dependent
inactivation
of
Results and Discussion
UGM.[15c,d] In contrast to its corresponding furanosides 8
and 9, exo-glycal 7 displayed a reversible mode of inhibition.
A little later, acyclic analogues mimicking intermediate 2
were synthesized and assayed by Liu et al.[15e] as well as by
our group.[14g] Since the inhibition profiles of acyclic galac-
tose derivatives (more precisely d-galactitols) gave very
promising results, we decided to explore further this design
and prepare compounds 10–13 (Figure 1). These four mole-
cules structurally share the UDP and the galactitol substruc-
tures. For all molecules, the linkage between the UMP and
galactitols was designed in such a way that the number of
atoms between the two subunits is always the same and cor-
responds to the natural substrate UDP-galactose. Within
this family, structural variations arise from the connection
between the phosphonate and the polyol. Alkyne 11 and
alkene 13 have been prepared to assess the effect of chang-
ing the relative orientation between the two key binding
subunits. In compounds 12 and 10 (prepared and assayed as
a mixture of two trans diastereomers), we added an electro-
philic functional group to probe the FAD cofactor nucleo-
philicity. For synthetic reasons that will be discussed below,
it was not possible to synthesize the Z stereoisomer of
alkene 13, or the cis stereoisomers of cyclopropane 12 and
Synthesis of alkyne analogue 11: The synthesis of all the tar-
geted compounds began with the preparation of the acyclic
derivative 14 through the well-established formation of an
intermediate dithioacetal followed by
a perbenzylation
(Scheme 1). Aldehyde 14 was transformed in the corre-
sponding terminal alkyne in 89% yield thanks to a one-step
alkynilation procedure involving a key diazo-phosphonate
reagent.[18] Alkyne 15 could be deprotonated and coupled to
dibenzyl chlorophosphate to yield phosphonate 16.[19] The
[20]
removal of all protecting groups with BCl3 followed by a
coupling with UMP preactivated as an imidazolide[21] afford-
ed the first analogue 11 in 31% yield after size-exclusion
chromatography and semipreparative HPLC purification.
Synthesis of alkene analogue 13: The same acyclic perbenzy-
lated d-galactose derivative 14 could be engaged in a
Horner–Wadsworth–Emmons olefination with bisphospho-
nate 18[22] that yielded phosphonate (E), with an expected
high diastereoselectivity (Scheme 2). This intermediate
could be fully deprotected in 95% yield and coupled, as for
the preceding alkyne, with UMP-imidazolide, thus affording
the second target compound, 13. We naturally attempted
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À
À
Scheme 1. Synthesis of alkyne analogue 11. a) HS
d) MeC(O)CH2P(O)(OMe)2, p-TolSO2N3, K2CO3, CH3CN/MeOH (59%); e) n-BuLi, ClP(O)
Im2CO, DMF/pyridine (31%).
ACHTUNGTRENNUNG
N
ACHTUNGTRENNUNG
Scheme 2. Synthesis of vinylphosphonate 13. a) 18, NaH, THF (75%); b) Br, PPh3CH2Br, tBuOK, THF (81%); c) PdACHTNUTGRENNUG(OAc)2, dppf HP(O)ACHTUGNTREN(NUGN OBn)2, THF
(69%); d) BCl3, DCM (95%); e) UMP, Im2CO, DMF/pyridine (48%).
the synthesis of the corresponding Z analogue through a
slightly different pathway (Scheme 2). Aldehyde 14 was first
converted into bromoalkenes 20 through an efficient Wittig
protocol. (Z)-Phosphonate 21 was thus obtained thanks to a
Pd catalyzed cross-coupling with dibenzylphosphite.[23] Inter-
mediate 21 could then be deprotected as previously descri-
bed. However, we never managed to obtain the desired
UDP-galactitol 13-Z in a pure form. Indeed, a quick intra-
molecular cyclization (Scheme 2) occurs during the reaction
and the purification of 13-Z. Depending on the UMP-cou-
pling protocols, we could observe 13-Z in crude NMR spec-
tra, sometimes in substantial proportions, but never as a
pure molecule.
We had faced the same difficulties with the corresponding
aminophosphonates previously described.[14g] This side-reac-
tion is, in fact, a general trend of this kind of molecule. We
also observed it for the cis isomers of UDP-galactitol epox-
ides 10 and cyclopropane 12 (see discussion below).
Synthesis of epoxides 10: To achieve the synthesis of target
compound 10, it was tempting to try a direct epoxidation
protocol from phosphonylated alkenes 19. However, after a
broad screening of peracids and hydroperoxides, even under
harsh conditions, we could never isolate the corresponding
cis- or trans-epoxides in reasonable yields. We, thus, devel-
oped a stepwise epoxidation protocol (Scheme 3). The phos-
phonylated (E)-alkene 19 could be dihydroxylated by using
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S. P. Vincent et al.
the desired UDP-galactitols as
a mixture of trans-epoxides in
order to validate the design.
Thus, the synthesis of the final
nucleotide sugars 10 could be
completed by debenzylating in-
termediate 22 under hydrogena-
tion conditions followed by
UMP coupling. As detailed for
(Z)-alkenes, the UDP-galactitol
displaying a cis-epoxide (gener-
ated from 25) could not be pro-
duced in a pure form due to an
intramolecular cyclization simi-
lar to the reaction described in
Scheme 2.
Demonstration of the absolute
configuration of intermediate
diols and epoxides: To demon-
strate the absolute configura-
tion of all intermediates, we
took advantage of the elegant
debenzylative cycloetherifica-
tion developed by Nicotra to
Scheme 3. Synthesis of epoxide 10. a) K2OsO4·2H2O (0.05 equiv), AD-Mix a, MeSO2NH2, tBuOH/H2O, RT
(59%); b) K2OsO4·2H2O (0.20 equiv), NMO (1.1 equiv), citric acid, tBuOH/H2O, RT (70%); c) Tf2O
(1.15 equiv), 4-DMAP (6.0 equiv), DCM 08C (76%); d) H2, Pd/C, H-cube (68%); f) UMP, CDI, DMF/Pyr,
RT (34%).
synthesize C-glycosides.[24]
A
Sharpless conditions to yield diol 24 as a single diastereom-
er. It should be noted that the diastereoselectivity is only in-
duced by the sugar moiety: the same selectivity is always ob-
served when AD-Mix a, AD-Mix b or no chiral inducer are
used. Under triflation conditions, diol 24 could be directly
transformed into cis-epoxide 25 in 74% yield. The cis con-
figuration was highly expected and could be demonstrated
similar approach had been exploited by Sinaꢃ[25] and by
Cumpstey et al. for the construction of novel C-galactofura-
nosides.[26] Upon acidic or electrophilic activation, perbenzy-
lated acyclic polyols give selectively tetrahydrofurans
through a 5-exo cylization, implying a nucleophilic attack of
the O-4 atom and its debenzylation.[27] Indeed, when epox-
ide 25 was activated under acidic conditions, the C-furano-
side 26 was formed as the major product (Scheme 4). 1H
1
by analyzing the H NMR spectra. Indeed, the syn-diol 24
obtained from the dihydroxyla-
tion of a (E)-alkene will give a
cis-epoxide through an intramo-
lecular SN2 reaction. The dem-
onstration of the absolute con-
figurations will be detailed
below.
The same dihydroxylation
procedures were then applied
on (Z)-alkene 21 but were sur-
prisingly not diastereoselective.
The yield in diols 22 could be
improved by using a slightly dif-
ferent dihydroxylation protocol.
However, the resulting 1:1 mix-
ture of anti diols 22a and 22b
could not be separated. The tri-
flation protocol yielded the two
trans-epoxides in 81% yield but
once again, as a non-separable
mixture. Given the importance
of the targeted enzyme, we de-
Scheme 4. Demonstration of the absolute configurations of epoxides 25, 23a and 23b. Reagents and condi-
cided to pursue the synthesis of tions: a) HClO4·H2O/THF; b) Ac2O, DMAP, Pyr.
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Inhibition of UDP-Galactopyranose Mutase
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and 13C NMR spectra of 26 and its acetate 27 were charac-
teristic of a furanoside (for instance the weak J2,3 coupling
constant) as compared with very similar structures that were
synthesized in our laboratory[15b,28] or by other groups.[25,26,29]
Having also in hand the corresponding a- and b-phosphony-
lated C-galactopyranosides,[15g] we could definitely conclude
from the NMR spectroscopy data that compounds 26–31 are
furanosides.
Moreover, nOe experiments clearly showed a strong
through-space interaction between protons H-1 and H-4,
proving that 27 is “a” configured. Since the starting epoxide
25 was cis, this a configuration implies that the exocyclic
stereogenic center (numbered C-1’) must be S. The two pre-
ceding steps operating through a net inversion of configura-
tions, epoxide 25 is 1’S,1R and its precursor, the syn-diol 24,
is 1’R,1R. The same debenzylative sequence could be ap-
plied to the mixture of trans epoxides 23a/b, which lead to a
mixture of two C-galactofuranosides 28 and 30 that could be
separated only after acetylation. A similar analysis (a/b as-
sessment, analysis of coupling constants and nOe experi-
ments) allowed us to demonstrate the absolute configura-
tions of all intermediate of this synthesis.
of the cyclopropanation originates only from the protected
polyol of alkenes 33. As demonstrated below, the two dia-
stereomeric products happened to be cis/trans isomers.
The cis/trans configuration assignments were confirmed
by the cyclizations observed in the following steps. Indeed,
when cyclopropane 34-trans was debenzylated, a spontane-
ous lactonization occurred giving 35-trans in 98% yield, and
thus demonstrated the cis relationship of the ester and the
polyol side-chain. This cylization was ascertained by NMR
spectroscopy experiments: the H-2 and C-2 signals of lac-
tone 35-trans were shifted downfield in the corresponding
1H and 13C NMR spectra. Such a lactonization was not ob-
served from cyclopropane 34-cis. However, the tentative
coupling of deprotected phosphonate 35-cis and UMP-mor-
pholidate yielded exclusively the cyclized oxaphospholane
36. On the other hand, the coupling of phosphonate 35-
trans with UMP-morpholidate provided the desired nucleo-
tide–sugar analogue 12 in a very satisfactory 80% yield.
Inhibition study against UGM: Several techniques have
been developed for the evaluation of the binding affinity of
small compounds towards UGM.[12e,15e,16b] The most usual
assay consists of following, by HPLC, the conversion of the
substrate UDP-Galf (3) (synthesized following standard pro-
cedures)[31] into UDP-Galp (2) in the presence and absence
of an inhibitor.[14c,f,15d,f,16a,17b] This technique has the draw-
back of being technically demanding, tedious and slow, but
has the huge advantage of being the only available competi-
tion assay against the enzymeꢀs natural substrate.
Interestingly, significant differences of inhibition percen-
tages were measured under these conditions and allowed us
to rank the binding affinities of these four nucleotide–sugar
analogues (10>11>12>13). Epoxide 10 was, by far, the
best inhibitor of this series. Gratifyingly, compared to all
Synthesis of cyclopropane 12: The synthesis of target cyclo-
propane 12 began with a Knoevenagel condensation[30] of al-
dehyde 14 and phosphonoacetate 32, which yielded trisubsti-
tuted alkenes 33 in 71% yield as a Z/E 2:1 diastereomeric
mixture (Scheme 5). The subsequent Corey–Chaykovsky cy-
clopropanation proceeded smoothly on both isolated al-
kenes 33-(E) and 33-(Z), with a slightly better yield starting
from the E isomer. Interestingly, the reactions from 33-(E)
and 33-(Z) yielded the same 1:1 mixture of two identical
stereomeric cyclopropanes 34 among the four possible dia-
stereoisomers. This result showed that the stereoselectivity
Scheme 5. Synthesis of lactone 12. a) TiCl4, NMO, THF (71%, Z/E 2:1); b) IMe3SO, NaH, cis/trans 1:1 (from 33-(E) 89%, from 33-(Z) 68%); c) i) H2,
Pd/C, Et3N; ii) Dowex 50WX8 H+ (35-trans 98%, 35-cis quant.); d) UMP-morpholidate, 1H-tetrazole (80%).
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S. P. Vincent et al.
UDP-galactose phosphonates 5–13 synthesized in our labo-
ratory, compound 10 was found to be the best inhibitor of
UGM. Comparison of inhibition percentages of alkyne 11
and alkene 13 also revealed that the connectivity between
the UDP and the galactitol substructures plays a key role in
the binding strength of the inhibitors. The inhibition by the
constrained cyclopropane derivative 12 was found to be in-
termediate between the alkyne and the alkene. UDP, a
UGM inhibitor the Kd (14 mm) of which has been measured
by several techniques,[15e] has become a reference inhibitor
to calibrate assays and allow comparison with other binding/
inhibition studies. Under the conditions described above
(Table 1), the inhibition percentage of UDP was 18% at
1 mm (at 250 mm, the inhibition percentage was !10%).
This signifies that epoxide 10 is a very tight binder of UGM
and that alkyne 11 and cyclopropane 12 are slightly better
than UDP.
scale of the inactivation studies. Therefore, the high-level of
UGM inhibition by epoxide 10 results from a non-covalent
and reversible tight binding process.
Conclusion
In conclusion, a series of UDP-galactitols were designed as
analogues of high-energy intermediates of the UGM cata-
lyzed furanose/pyranose interconversion, an essential step of
M. tuberculosis cell wall biosynthesis. The final molecules
structurally share the UDP and the galactitol substructures
that were connected by four distinct electrophilic connec-
tions (epoxide, lactone and Michael acceptors). All com-
pounds were synthesized from a common perbenzylated
acyclic galactose precursor that was derivatized by alkenyla-
tion, alkynylation and cyclopropanation. The inhibition
study against UGM clearly showed that slight changes in the
relative orientation of the UDP and galactitol moieties re-
sulted in dramatic variations of binding properties. Com-
pared to known inhibitors, the epoxide derivative displayed
a very tight, reversible, inhibition profile. Moreover, a time-
dependent inactivation study showed that none of these
electrophilic structures could react with UGM, or its FAD
cofactor, the catalytic nucleophile of this still intriguing re-
action.
Table 1. Inhibition percentages of UDP-galactitols.
10
11
12
13
inhibition [%][a]
69
18
10
5
[a] All inhibitors were assayed at 250 mm against UDP-Galf (105 mm) in
the presence of reduced E. coli UGM (22 nm).
Inactivation assays: It is now well-admitted that UGM cata-
lyzes the pyranose/furanose interconversion through an un-
usual covalent mechanism, in which the FAD cofactor plays
the role of catalytic nucleophile. UDP-galactitols 10–13 have
also been designed to probe FADꢀs nucleophilicity since
they all bear an electrophilic functionality at the vicinity of
the “anomeric” position. Indeed, Michael acceptors, such as
alkene 13 and alkyne 11, are known to react with nucleo-
philic amino acids within catalytic binding pockets.[32] More-
over, epoxides such as 10, are very often used as reactive
compounds in chemical biology and in medicinal chemis-
try.[33] The strained lactone functionality in analogue 12 may
also be a competent electrophile within a binding cavity dis-
playing a catalytic nucleophile. However, none of these mol-
Experimental Section
The detailed synthetic procedures for molecules 10–35 are gathered in
the Supporting Information along with their analytical data and the bio-
chemical procedures.
Acknowledgements
This work was funded by an ARC grant from the Acadꢁmie Louvain
(08/13-012), by the FNRS (FRFC grant 2.4.625.08.F) and by the Universi-
ty of Namur.
ecules displayed
a time-dependent inactivation profile
(under reducing conditions); this result contrasts with the in-
hibition properties of exo-glycals[15c,d] and fluorinated UDP-
Galf analogues[17b] under non-reducing conditions. Indeed, if
inhibitors 10–13 were preincubated with UGM before start-
ing the reaction kinetics by adding UDP-Galf, no significant
change in inhibition percentage could be measured, whatev-
er the incubation time and the relative inhibitor/UGM con-
centrations. Intrigued by the high level of inhibition of com-
pound 10, we attempted to evidence a covalent UGM–inac-
tivator or FAD–inactivator by mass spectrometry (under
electrospray and MALDI conditions). We could never ob-
serve any covalent adducts even in trace amount. As a con-
trol experiment, we followed the stability of compounds 10–
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Received: June 28, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
7
&
ÞÞ
These are not the final page numbers!

S. P. Vincent et al.
Shedding inhibitors: UDP-Galactopyr-
anose mutase (UGM) is a validated
target for treating tuberculosis. Its
mechanism involves formation of a
key covalent FAD–substrate inter-
mediate, 1. A series of electrophilic
UDP-galactitols were synthesized and
assayed as inhibitors or inactivators of
UGM. Strong inhibitions were
observed, especially with epoxide 2.
Interestingly, none of the molecules
displayed an irreversible inhibition
mode.
Inhibitors
C. Ansiaux, I. N’Go,
S. P. Vincent* .................. &&&& — &&&&
Reversible and Efficient Inhibition of
UDP-Galactopyranose Mutase by
Electrophilic, Constrained and Unsatu-
rated UDP-Galactitol Analogues
&
8
&
www.chemeurj.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!