Synthesis and analysis of substrate analogues for UDP-galactopyranose mutase: Implication for an oxocarbenium ion intermediate in the catalytic mechanism

Article abstract of DOI:10.1021/ol0631408

(Chemical Equation Presented) UDP-D-galactofuranose (2), which is essential for both cell growth and virulence in many pathogenic microorganisms, is converted from UDP-D-galactopyranose (UDP-Galp, 1) by the flavin adenine dinucleotide (FAD)-dependent enzyme UDP-galactopyranose mutase (UGM). Here, we report the synthesis of UDP-GalOH (13) and show it as an inhibitor for UGM with a binding affinity similar to that of 1. These results are more consistent with a mechanism involving an oxocarbenium ion intermediate in UGM catalysis.

Full text of DOI:10.1021/ol0631408

ORGANIC
LETTERS
2007
Vol. 9, No. 5
879-882
Synthesis and Analysis of Substrate
Analogues for UDP-Galactopyranose
Mutase: Implication for an
Oxocarbenium Ion Intermediate in the
Catalytic Mechanism
Kenji Itoh,† Zhishu Huang,†,‡ and Hung-wen Liu*
DiVision of Medicinal Chemistry, College of Pharmacy and Department of Chemistry
and Biochemistry, UniVersity of Texas at Austin, Austin, Texas 78712
h.w.liu@mail.utexas.edu
Received December 28, 2006
ABSTRACT
UDP-
D
-galactofuranose (2), which is essential for both cell growth and virulence in many pathogenic microorganisms, is converted from
-galactopyranose (UDP-Galp, 1) by the flavin adenine dinucleotide (FAD)-dependent enzyme UDP-galactopyranose mutase (UGM). Here,
UDP-
D
we report the synthesis of UDP-GalOH (13) and show it as an inhibitor for UGM with a binding affinity similar to that of 1. These results are
more consistent with a mechanism involving an oxocarbenium ion intermediate in UGM catalysis.
The emergence of multidrug resistant strains of human
pathogens is a major concern. The prevalence of these
resistant strains has compelled researchers to investigate new
targets/approaches for antimicrobial drug design. The path-
way for D-galactofuranose (Galf) biosynthesis is one potential
target for chemotherapeutic development.1 Galf is an essential
component in the glycoconjugates of many pathogenic
microorganisms1 but has not been found in mammals. In the
Galf biosynthetic pathway, UDP-D-galactopyranose (UDP-
Galp, 1) is converted to UDP-D-galactofuranose (UDP-Galf,
2) by UDP-galactopyranose mutase (UGM) (Scheme 1), and
Galf is subsequently transferred from 2 to various glyco-
conjugates by the appropriate galactofuranosyl transferase.2
Because UGM is pivotal for mycobacterial cell growth and
infection, UGM inhibitors may be potential antimicrobial
agents.2a
UGM is a homodimeric enzyme, which binds 1 equiv of
flavin adenine dinucleotide (FAD) per monomer and cata-
lyzes the reversible conversion of UDP-Galp (1) and UDP-
Galf (2).3 The mechanism for the reaction catalyzed by UGM
has been the subject of much discussion, and several
(2) (a) Pan, F.; Jackson, M.; Ma, Y. F.; McNeil, M. J. Bacteriol. 2001,
183, 3991-3998. (b) Kremer, L.; Dover, L. G.; Morehouse, C.; Hitchin,
P.; Everett, M.; Morris, H. R.; Dell, A.; Brennan, P. J.; McNeil, M. R.;
Flaherty, C.; Duncan, K.; Besra, G. S. J. Biol. Chem. 2001, 276, 26430-
26440.
(3) (a) Nassau, P. M.; Martin, S. L.; Brown, R. E.; Weston, A.; Monsey,
D.; McNeil, M. R.; Duncan, K. J. Bacteriol. 1996, 178, 1047-1052. (b)
Sanders, D. A.; Staines, A. G.; McMahon, S. A.; McNeil, M. R.; Whitfield,
C.; Naismith, J. H. Nat. Struct. Biol. 2001, 8, 858-863.
† Authors contributed equally to this study.
‡ Current address: Laboratory of Bioorganic and Medicinal Chemistry,
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou
510080, China.
(1) Pedersen, L. L.; Turco, S. J. Cell Mol. Life Sci. 2003, 60, 259-266.
10.1021/ol0631408 CCC: $37.00
© 2007 American Chemical Society
Published on Web 02/01/2007

the reduced FAD (3) to the oxocarbenium ion intermediate
(4) is also feasible.4c,d In this mechanism, a SET is followed
by a radical recombination between the resulting substrate
radical (11) and the FAD semiquinone (12) to give the
FAD-substrate adduct (6/7/8).4d As delineated in Scheme
1, this FAD-substrate adduct may play a central role to
facilitate the opening and recyclization of the galactose ring.
Formation of an oxocarbenium ion intermediate has been
implicated in the mechanism of numerous chemical and
enzymatic reactions.8 The intermediacy of this ionic species
has prompted the design of oxocarbenium mimics as transi-
tion-state inhibitors for many enzymes.9 Although such an
oxocarbenium ion is not an intermediate in the SN2 mech-
anism for UGM catalysis, it must exist if the reaction
proceeds via either an SN1 or SET mechanism. To design
effective inhibitors for this promising drug target, detailed
knowledge about UGM reaction intermediates and/or transi-
tion states is essential. Herein, we report the chemical
synthesis and enzymatic analysis of a linear substrate
analogue, UDP-galactitol (UDP-GalOH, 13), to probe the
possible involvement of an oxocarbenium ion intermediate
in UGM catalysis. Galactose (Gal), galactose-1-phosphate
(Galp-P), UMP, and UDP were also used, along with 13, to
study the binding of a substrate/inhibitor to UGM.
Scheme 1
UDP-GalOH (13) was synthesized according to Scheme
2.10 Methyl R-D-galactopyranoside (14) was used as the
starting material and was converted to 2,3,4,6-tetra-O-benzyl-
galactopyranose (16) following a literature procedure in two
steps.11 The linear alcohol 17 was readily obtained by
reduction of 16 with NaBH4. Selective protection of the
primary alcohol by a tert-butyldimethylsilyl group followed
by blocking of the 5-OH by a benzyl group gave 19. Partial
deprotection of the tert-butyldimethylsilyl group occurred
under the benzylation conditions, which resulted in the low
yield (33%) of 19. After deprotection of the tert-butyldi-
methylsilyl group, compound 20 was treated with dibenzyl
chlorophosphonate12 to give the dibenzyl phosphate deriva-
tive 21. The benzyl groups of 21 were then removed by
hydrogenolysis on catalytic amounts of palladium hydroxide
to give the phosphonate derivative 22. The final product
UDP-GalOH (13) was prepared by incubating 22 and uridine
phosphomorpholidate in anhydrous pyridine in the presence
of 1H-tetrazole for 4 days.13 The desired product was purified
possibilities have been proposed.4 In an early report, a
pathway involving a 1,4-anhydrogalactopyranose intermedi-
ate (5) was postulated (Scheme 1).4a,b This mechanism was
based on a positional isotope exchange experiment which
indicated cleavage of the anomeric C-O bond of UDP-Galp
occurred during turnover.4a However, this mechanism is not
consistent with recent experiments showing that incubation
of synthetic 5 with UGM and UDP does not yield 1 and 2
as products.4d,5
Because the catalytic efficiency (kcat/Km) of UGM is
increased by more than 2 orders of magnitude under reducing
conditions,3b,6 several alternative mechanisms involving the
reduced FAD cofactor (3) have since been proposed (Scheme
1). One possible route involving the formation of a FAD-
substrate adduct (7)4d is supported by experiments trapping
the adduct (7) by chemical reduction and verification of the
reduced product (9) using mass spectroscopy.7 The adduct
formation may be initiated by an SN2 attack of N5 of the
reduced FAD (3) on C-1 of the substrate (1 in the forward
direction) to give 6/7/8. It is also possible that the attack
proceeds in an SN1 fashion at C-1 where an oxocarbenium
ion intermediate (4 in the forward direction) is generated
after the cleavage of the UDP group (1 f 4 f 6/7/8).7 A
redox process involving a single-electron transfer (SET) from
(8) (a) Woods, R. J.; Andrews, C. W.; Bowen, J. P. J. Am. Chem. Soc.
1992, 114, 859-864. (b) Banait, N. S.; Jencks, W. P. J. Am. Chem. Soc.
1991, 113, 7951-7958. (c) Richard, J. P.; Huber, R. E.; Heo, C.; Amyes,
T. L.; Lin, S. Biochemistry 1996, 35, 12387-12401. (d) Scheuring, J.;
Schramm, V. L. Biochemistry 1997, 36, 4526-4534. (e) Namchuk, M. N.;
McCarter, J. D.; Becalski, A.; Andrews, T.; Withers, S. G. J. Am. Chem.
Soc. 2000, 122, 1270-1277. (f) Withers, S. G. Carbohydr. Polym. 2001,
44, 325-337. (g) Unrau, P. J.; Bartel, D. P. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 15393-15397.
(4) (a) Barlow, J. N.; Girvin, M. E.; Blanchard, J. S. J. Am. Chem. Soc.
1999, 121, 6968-6969. (b) Zhang, Q.; Liu, H.-w. J. Am. Chem. Soc. 2001,
123, 6756-6766. (c) Fullerton, S. W.; Daff, S.; Sanders, D. A.; Ingledew,
W. J.; Whitfield, C.; Chapman, S. K.; Naismith, J. H. Biochemistry 2003,
42, 2104-2109. (d) Huang, Z.; Zhang, Q.; Liu, H.-w. Bioorg. Chem. 2003,
31, 494-502.
(9) (a) Boutellier, M.; Horenstein, B. A.; Semenyaka, A.; Schramm, V.
L.; Ganem, B. Biochemistry 1994, 33, 3994-4000. (b) Chen, X. Y.; Link,
T. M.; Schramm, V. L. J. Am. Chem. Soc. 1996, 118, 3067-3068. (c) Allart,
B.; Gatel, M.; Guillerm, D.; Guillerm, G. Eur. J. Biochem. 1998, 256, 155-
162. (d) Shi, W.; Li, C. M.; Tyler, P. C.; Furneaux, R. H.; Grubmeyer, C.;
Schramm, V. L.; Almo, S. C. Nat. Struct. Biol. 1999, 6, 588-593.
(10) See Supporting Information for details.
(5) Caravano, A.; Sinay, P.; Vincent, S. P. Bioorg. Med. Chem. Lett.
2006, 16, 1123-1125.
(11) Koto, S.; Morishima, N.; Miyata, Y.; Zen, S. Bull. Chem. Soc. Jpn.
1976, 49, 2639-2640.
(6) Zhang, Q.; Liu, H.-w. J. Am. Chem. Soc. 2000, 122, 9065-9070.
(7) Soltero-Higgin, M.; Carlson, E. E.; Gruber, T. D.; Kiessling, L. L.
Nat. Struct. Mol. Biol. 2004, 11, 539-543.
(12) Macher, I. Carbohydr. Res. 1987, 162, 79-84.
(13) Wittmann, V.; Wong, C. H. J. Org. Chem. 1997, 62, 2144-2147.
880
Org. Lett., Vol. 9, No. 5, 2007

involved. As shown in Scheme 3, formation of 9 may result
in UGM inactivation due to covalent modification of the
flavin coenzyme. A subsequent C1-N5 bond scission to
eliminate compound 24 as a turnover product, however, could
regenerate the oxidized flavin coenzyme, which may be
readily reduced in the presence of excess dithionite and made
available for another round of catalysis.
Scheme 2
To test the validity of UDP-GalOH (13) as a mechanistic
probe for UGM, the binding affinity of UDP-GalOH to
UGMred, the active form of UGM, was first determined.
Because the bound FADred of UGMred gave emission at 527
nm upon excitation at 373 nm in 100 mM KH2PO4 buffer
(pH 7.5),14 monitoring the change of fluorescence upon
substrate binding should enable us to determine if UGMred
binds various substrate analogues. Consequently, fluores-
cence titrations of UGMred with the natural substrate UDP-
Galp (1) and the synthesized UDP-GalOH (13) were carried
out in the presence of 5 mM sodium dithionite. In both cases,
the intensity of the fluorescence increases and shows
saturation at high substrate concentrations.10 Clearly, both
UDP-Galp and UDP-GalOH can bind to the active site of
UGMred. These titration curves were fitted to eq 1 to give
dissociation constants (Kd) of 52 µM for UDP-Galp (1) and
of 46 µM for UDP-GalOH (13).10
by Bio-Gel P2 size exclusion chromatography and MonoQ
ion-exchange FPLC. The structure of the final product 13
1
was confirmed by H, 13C, and 31P NMR spectroscopy as
well as high-resolution FABMS.10
F ) F0 +
The design of UDP-GalOH (13) as a mechanistic probe
is based on the fact that it carries a good leaving group, UDP,
at C-1, and is a mimic of the putative linear intermediate 7
formed during catalysis. Thus, it is reasonable to assume that
UDP-GalOH will be recognized by UGM. Subsequent
reaction of 13 with the reduced flavin (3) in the enzyme
active site will lead to the elimination of UDP and the
formation of a covalent adduct 9 (Scheme 3). For this
([E] + [S] + K ) - ([E] + [S] + K )2 - 4[E] [S]
x
0
d
0
d
0
Fc
(1)
2[E]0
The Kd values for UMP, UDP, Galp-P, and Gal were also
determined under identical conditions. As summarized in
Table 1, the binding affinities of UMP and UDP are
Table 1. Dissociation Constants of Substrate Analogues
Scheme 3
compound
Kd (µM)a
% relative activityb
UDP-Galp (1)
UDP-GalOH (13)
UDP
UMP
Galp-P
52 ( 9
46 ( 8
14 ( 3
27 ( 7
730 ( 80
680 ( 60
100
45.9
49.4
50.6
100
Gal
97.5
a Determined by fluorescence titration in the presence of Na2S2O4.
b Determined by dividing the % conversion of 2 to 1 in the presence of vs
in the absence of the compound listed.
comparable to those of UDP-Galp and UDP-GalOH. In
contrast, Galp-P and Gal, which lack a uridine moiety, bind
UGMred poorly. These observations indicate that the uridine
moiety plays a significant role in binding, whereas the sugar
moiety makes only a minimal contribution. The fact that
UDP-arabinose and UDP-N-acetylgalactose are substrates for
substitution reaction to proceed through the SN1 or SET
mechanism, formation of a cation intermediate (such as 23)
is required. However, the corresponding primary cation 23
is much less stable than 4 and 10, where the positive charge
can be delocalized across both the ring oxygen and the
anomeric carbon (C-1). Hence, if UGM recognizes and
processes UDP-GalOH, the reaction more likely proceeds
via an SN2 mechanism where no cation intermediate is
(14) The fluorescence spectrum of UGMred is similar to that of other
reduced flavoenzymes reported in the literature (Ghisla, S.; Massey, V.;
Lhoste, J. M.; Mayhew, S. G. Biochemistry 1974, 13, 589-597). See Figure
1 in Supporting Information.
Org. Lett., Vol. 9, No. 5, 2007
881

UGM is consistent with this conclusion.15 A previous X-ray
crystal structure and modeling studies showed that the uridine
moiety is close to a conserved Trp156 residue, suggesting
that hydrophobic stacking with UDP is a crucial binding
interaction between UGM and UDP-sugars.3b,16 Binding and
modeling studies performed by STD-NMR17 also showed
that UDP binds to the active site of UGM in a manner
analogous to the natural substrate UDP-Galp. Taken together,
these results strongly indicated that UMP, UDP, UDP-Galp
(1), and UDP-GalOH (13) all bind to the same site of UGMred
and are likely positioned similarly within this site.
Having the proper binding of UDP-GalOH (13) in the
active site of UGM established, we investigated the catalytic
competence of UGM on 13. UDP-GalOH was incubated with
UGMred in 100 mM KH2PO4 buffer (pH 7.5) at 37 °C for 2
min, and the reaction mixture was analyzed by HPLC (Figure
1). The analysis showed that UDP-GalOH is stable under
conditions. The extent of inhibition by these compounds was
evaluated by comparing UGMred activities in the absence and
presence of the inhibitors (Table 1). Among the analogues
examined, UDP is known to be an inhibitor for UGM.18 As
expected, we observed inhibition of UGMred to 49% by UDP
under our assay conditions. Because the previous STD-
NMR study had demonstrated competition between UDP and
UDP-Galp (1) in binding to UGMred,17 the observed inhibition
of UGMred by UDP based on the activity assay is most likely
competitive. Galp-P and Gal exhibited no/little detectable
inhibition, whereas UDP-GalOH (13) and UMP exhibited
levels of inhibition comparable with UDP. Because only
compounds containing a UDP/UMP group are inhibitors for
UGMred, these results once again demonstrate that the uridine
moiety plays a major role in substrate recognition and
binding. More importantly, the correlation between the extent
of inhibition and the binding affinity determined by fluo-
rescence titration strongly suggests that UDP-GalOH and
UMP, behaving the same as UDP, are also competitive
inhibitors for UGMred.
In summary, the above results demonstrated that UDP-
GalOH (13) binds to the active site of UGM in a manner
similar to that of UDP and UDP-Galp (1). The inability of
UDP-GalOH (13) to be processed by UGMred is likely a
reflection of its inability to form a reactive oxocarbenium
ion intermediate or to stabilize an oxocarbenium transition
state. The fact that UDP-GalOH binds closely to the reduced
flavin (3) but fails to react with it to form a covalent adduct
(9) argues against an SN2 mechanism for UGM catalysis.
Thus, the available evidence, although indirect, is more
consistent with a mechanism involving an oxocarbenium ion
intermediate (4 or 10) which is susceptible to attack by a
nucleophile,8b,e,19 such as the reduced flavin (3), to form the
FAD-substrate adduct (6/7/8). The electron-deficient nature
of the oxocarbenium ions could also facilitate electron
transfer from FADred to form a radical pair, such as 11 and
12, followed by covalent bond formation. Attempts to resolve
whether the interconversion catalyzed by UGM proceeds via
an SN1 or an SET pathway are in progress.
Figure 1. HPLC analysis of UGM reactions.10 (a) UDP-GalOH
(13) in the absence of UGMred, (b) UGMred + UDP-GalOH, (c)
UGMred + UDP-Galf (2).
the reaction conditions (Figure 1a), but it is not a substrate
for UGMred (Figure 1b). As a control, almost 60% of UDP-
Galf (2) was converted to UDP-Galp (1) by UGMred under
the identical conditions (Figure 1c). A longer incubation
period (1 h) at 37 °C did not produce any product either.
UDP-GalOH (13) was then examined as a potential
inhibitor for UGM by incubating it (2.5 mM) with UGMred
(8.2 nM) for 10 min at room temperature prior to the addition
of the natural substrate UDP-Galf (2) (50 µM). After that,
the reaction was continued for an additional 2 min at 37 °C.
The competence of UDP, UMP, Galp-P, and Gal as
substrates or inhibitors was also tested under identical
Acknowledgment. This work was supported by the NIH
grant GM54346 and the Welch Foundation grant F-1511.
Supporting Information Available: Preparation of 13
and analysis of binding of various substrate analogues to
UGM (PDF) are presented. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL0631408
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882
Org. Lett., Vol. 9, No. 5, 2007