Study of Uridine 5′-Diphosphate (UDP)-Galactopyranose Mutase Using UDP-5-Fluorogalactopyranose as a Probe: Incubation Results and Mechanistic Implications

Article abstract of DOI:10.1021/acs.orglett.6b01618

Uridine 5′-diphosphate-5-fluorogalactopyranose (UDP-5F-Galp, 7) was synthesized, and its effect on UDP-Galp mutase (UGM) was investigated. UGM facilitated the hydrolysis of 7 to yield UDP and 5-oxogalactose (24), but no 11 was detected. ¹⁹F NMR and trapping experiments demonstrated that the reaction involves the initial formation of a substrate-cofactor adduct followed by decomposition of the resulting C5 gem-fluorohydrin to generate a 5-oxo intermediate (10). The results support the current mechanistic proposal for UGM and suggest new directions for designing mechanism-based inhibitors.

Full text of DOI:10.1021/acs.orglett.6b01618

Letter
pubs.acs.org/OrgLett
Study of Uridine 5′-Diphosphate (UDP)-Galactopyranose Mutase
Using UDP-5-Fluorogalactopyranose as a Probe: Incubation Results
and Mechanistic Implications
Geng-Min Lin,† He G. Sun,‡ and Hung-wen Liu*,†,‡
†Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
‡Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, Texas
78712, United States
S
* Supporting Information
ABSTRACT: Uridine 5′-diphosphate-5-fluorogalactopyra-
nose (UDP-5F-Galp, 7) was synthesized, and its effect on
UDP-Galp mutase (UGM) was investigated. UGM facilitated
the hydrolysis of 7 to yield UDP and 5-oxogalactose (24), but
no 11 was detected. 19F NMR and trapping experiments
demonstrated that the reaction involves the initial formation of
a substrate−cofactor adduct followed by decomposition of the
resulting C5 gem-fluorohydrin to generate a 5-oxo intermediate
(10). The results support the current mechanistic proposal for
UGM and suggest new directions for designing mechanism-based inhibitors.
ridine 5′-diphosphate (UDP)-galactopyranose mutase
(UGM) is a flavoenzyme that catalyzes the redox-neutral
formation of UDP-Galf (6 → 2) or UDP-Galp (4 → 1) as
the product (Scheme 1). At equilibrium, the ratio of UDP-Galp
U
interconversion of UDP-galactopyranose (UDP-Galp, 1) and
UDP-galactofuranose (UDP-Galf, 2).1 This is an important
enzyme for many pathogenic bacteria, including Mycobacterium
tuberculosis, the causative agent of tuberculosis, since UDP-Galf
(2) is the precursor of Galf residues found in their cell
surfaces.2 The emergence of multidrug-resistant strains of M.
tuberculosis has prompted the search for new biomedical
approaches to combat this life-threatening disease.3 The
absence of UGM in mammalian cells has made inhibition of
UGM to disrupt this biosynthetic pathway a promising target in
the development of new antimicrobial agents. Indeed, the
inhibition of UGM has been demonstrated to adversely affect
mycobacterial cell growth.4
Scheme 1. Current Mechanistic Model of UGM Catalysis
In addition to its therapeutic potential, the unique catalytic
mechanism of UGM has also attracted much attention. It has
been shown that UGM is catalytically active only under
reducing conditions, where its flavin adenine dinucleotide
(FADred, 3) coenzyme remains reduced throughout this overall
redox-neutral reaction.5 The reduced FAD 3 acts as a
nucleophile to displace the UDP moiety from 1 or 2 to form
a covalent linkage between N5 of FAD and C1 of Galp (1 → 4)
or Galf (2 → 6).6 Subsequent scission of the C1−O5 or C1−
O4 bond is assisted by the lone pair on N5 of FAD to yield
acyclic iminium ion intermediate 5, which was first detected by
trapping with hydride reagent7 and was observed in a recent
crystal structure of a UGM mutant.8 Recyclization of 5
produces the furanosyl ring of Galf (5 → 6) or the pyranosyl
ring of Galp (5 → 4). This recyclization reaction is followed by
the elimination of reduced FAD, which may occur concurrently
with nucleophilic attack at C1 by UDP, leading to the
to UDP-Galf is approximately 10 to 1.5 A series of FAD
analogues were used to verify the role of FADred in this
isomerization reaction. The data supported a chemical
mechanism for UGM involving an SN2-type displacement of
UDP from UDP-Galp/Galf by N5 of FADred.6c,i
In an effort to learn more about the catalytic properties of
UGM and to develop new mechanism-based inhibitors
targeting UGM, UDP-5F-Galp (7) was recognized as a
promising core structure. It was expected that 7 would react
Received: June 4, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b01618
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with UGM to form cofactor−substrate adduct 8. Subsequent
ring opening of 8 to form the iminium ion intermediate would
result in a gem-fluorohydrin moiety at C5 (9) that should
undergo rapid dehydrofluorination9 to afford 10 (Scheme 2).
Scheme 3. Synthesis of UDP-5F-Galp (7)
Scheme 2. Predicted Reaction of UGM with UDP-5F-Galp
(7) Based on the Working Mechanistic Model
The absence of C5−OH in 10 would prevent cyclization of 10
to regenerate the pyranosyl ring but would still allow C1−O4
bond formation to yield UDP-5-oxo-Galf (11). The 5-oxo
group in 11 may react with a nucleophilic residue in the active
site to form a covalent adduct and thus inhibit the enzyme. In
addition, further modification of the C6 hydroxyl group to a
better leaving group in 7 could enhance the nucleophilic
susceptibility at C6 in 10 or 11 and promote enzyme
modification and inactivation.
appearance of two new peaks in the HPLC traces of the
reaction workups, one at 24.1 min and the other at 30.5 min,
was also noted (Figure 1, trace c). The species responsible for
To test these premises, we prepared the targeted compounds
and investigated their effects on the activity of UGM. Reported
herein are the chemical syntheses of 7 along with its 6-deoxy-6-
fluoro derivative (26), characterization of their reactions with
UGM, and the mechanistic implications of the incubation
outcomes.
The epoxide fluoridolysis strategy developed by Coward and
co-workers10 was applied to synthesize 7. As depicted in
Scheme 3, the reaction was initiated by derivatization of the C6
hydroxyl group of methyl α-D-galactopyranoside (12) with
triphenylmethyl chloride (12 → 13). Benzyl protection of the
remaining hydroxyl groups followed by acid hydrolysis
selectively exposed the C6 hydroxyl (13 → 15),11 which was
then phenylselenylated via bromination and substitution (15 →
17). Deprotection of the anomeric hydroxyl group of 17 and
subsequent reaction with freshly prepared dibenzyl phosphoro-
chloridate gave α-phosphate 19 exclusively. Oxidation of 19
and thermal decomposition of the resulting selenoxide
produced exo-olefin 20.12 Epoxidation using dimethyldioxirane
(DMDO) generated in situ13 and subsequent ring opening
using hydrogen fluoride10 gave the desired fluorohydrin 21 as
the major product along with its L isomer (section S2 in the
Supporting Information (SI)). Global benzyl deprotection and
coupling with uridine 5′-monophosphate (UMP)14 provided
UDP-5F-Galp (7).
Incubation of 7 (200 μM) with UGM (less than 1 μM) was
carried out at 37 °C for 5 min in 50 μL of 100 mM potassium
phosphate (KPi) buffer (pH 7.5) in the presence of 20 mM
Na2S2O4.5 No consumption of 7 was apparent as monitored by
high-performance liquid chromatography (HPLC) (see the SI
for HPLC methods). However, depletion of 7 (200 μM) was
observed when the enzyme concentration and reaction time
were increased to 20 μM and 1 h, respectively. Meanwhile, the
Figure 1. HPLC traces (method A; see the SI) of the incubation of
UDP-5F-Galp (7) with UGM: (a) standards of related uridine-
containing species; (b) synthetic 7; (c, d) reactions of 200 μM 7 with
(c) 20 μM UGM for 1 h and (d) buffer for 24 h.
these new peaks were determined to be UDP and FAD on the
basis of coelution with standards and the mass of each species
as verified by mass spectrometry (section S3). While UDP was
derived from 7, FAD was detected as a result of its dissociation
from UGM during the workup. In the absence of enzyme, the
formation of UDP and consumption of 7 were also observable,
but only over an extended period of time (24 h; Figure 1, trace
d). No reaction product consistent with 11 was detected under
any of the HPLC conditions tested.
To assess whether the hydrolysis of UDP-5F-Galp to release
UDP is catalyzed by UGM, UGM at various concentrations
(0.0, 0.8, 2, and 5 μM) was incubated with 200 μM UDP-5F-
Galp (7) anaerobically at 37 °C. The consumption of 7 and
formation of UDP were followed up to 24 h, as shown in Figure
S5. Except for UDP, no other uridine-containing product was
detected in the reaction (section S4). The apparent first-order
hydrolysis rate of 7 increased from 0.112 0.003 h−1 in the
B
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absence of UGM to 1.108 0.008 h−1 with 5 μM UGM. It is
thus clear that UGM can accelerate the hydrolysis of 7. A
comparison was also made using assay mixtures containing 7
and apo-UGM or apo-UGM reconstituted with either FAD or
5-deaza-FAD. Only incubation with the FAD-reconstituted
UGM showed significant hydrolysis activity compared with the
no-enzyme control (section S5). A reductant such as Na2S2O4
is also required for the hydrolysis (section S5). These results
demonstrate that reduced FAD (3) plays a direct role in UGM-
catalyzed hydrolysis of 7.
(section S8). The identification of 24 as the turnover product
in this experiment suggested that 10 was hydrolyzed to
regenerate the reduced FAD and the active enzyme (Scheme
4).
Scheme 4. Proposed Hydrolysis of 7 by UGM through the
Intermediacy of 10
To study the catalytic function of 3, the reaction mixture was
treated with NaBH3CN in order to trap the putative Schiff base
adduct formed between the reduced FAD and 7.7 Two new
species were indeed detected by liquid chromatography−
electrospray ionization mass spectrometry (LC-ESI-MS): 22
(m/z 948.2 for [M − H]− and 473.6 for [M − 2H]2−) and 23
(m/z 950.2 for [M − H]− and 474.6 for [M − 2H]2−) (Figure
2A and section S6). Hence, the reduced FAD (3) acts as a
The reason that 10 cannot cyclize to form the furanosyl ring
via the C4−OH is not clear. One possible scenario is that the
C5 carbonyl group of 10 may be involved in a hydrogen-
bonding network that hinders the proper alignment of the C4
hydroxyl group to reach C1 of 10 in the active site. The
extended lifetime of 10 would result in its hydrolysis. In fact, we
did observe that UGM could catalyze the hydrolysis of UDP-
Galp (1) at high enzyme concentration and extended
incubation time (section S9). It is thus likely that hydrolysis
of the Schiff base intermediate (5 or 10) is an inherent side
activity of UGM but is typically suppressed by minimizing the
lifetime of the intermediate.
The formation of the C5-oxo-bearing intermediate 10 during
the enzymatic reaction prompted the design and synthesis of
UDP-[5,6-F2]-Galp (26), which is expected to react with UGM
similarly to generate 6-deoxy-6-fluoro-10, whose α-fluoro
carbonyl functionality might be susceptible to modification by
an active-site residue. As shown in Scheme 5, the hydroxyl
Figure 2. (A) ESI-MS (negative-ion mode) of the adducts 22 and 23
trapped from reactions of UGM with UDP-5F-Galp (7) in the
presence of NaBH3CN. (B) 19F NMR spectra of the reaction of 7 with
UGM acquired every 15 min for 12 h. Shown here are the spectra for
the first 135 min.
Scheme 5. Synthesis of UDP-[5,6-F2]-Galp
nucleophile to displace UDP of UDP-5F-Galp (7) as it does
during the catalysis of the UDP-Galp/UDP-Galf isomerization
reaction. When the reaction of 7 and UGM in KPi buffer (in
D2O) was monitored using 19F NMR spectroscopy, a time-
dependent reduction of the 5-F triplet signal of 7 (at −119
ppm) was observed, while a new singlet signal appeared at
−122 ppm (Figure 2B and section S7). The chemical shift of
the latter peak is consistent with the reported value for free
fluoride.15 These results suggest that the reaction between 7
and UGM proceeds at least up to 9, followed by its
decomposition to 10 as shown in Scheme 2. However, the
reaction ensues no further than 10 since UDP-5-oxo-Galf (11),
the predicted product of the reaction of 7 with UGM, was not
detected under the HPLC conditions examined. The fact that
UGM does not lose activity during incubation with 7 (data not
shown) indicates that the reduced FAD could somehow be
regenerated from 10.
To further characterize the turnover product from the
reaction of 7 with UGM, the reaction mixture after
lyophilization was incubated with O-(2,3,4,5,6-
pentafluorobenzyl)hydroxylamine (PFBHA) in pyridine, fol-
lowed by the treatment with acetic anhydride.16 LC-ESI-MS
analysis of the workup revealed the occurrence of peaks at m/z
564.1 and 759.1, consistent with mono- and di-O-pentafluor-
obenzyl oxime acetates of 5-oxo-D-galactose (24), respectively
group at C6 of 21 was subjected to fluorination using
diethylaminosulfur trifluoride (DAST) to generate 25. The
resulting product was hydrogenated to remove the benzyl
protecting groups followed by coupling with UMP to give 26.
Although facilitated hydrolysis of 26 at C1 to yield UDP and
release of fluoride were noted in the presence of UGM as
observed for 7, no apparent decrease in the activity of UGM
was observed when 2 μM enzyme was preincubated with 200
μM 26 for up to 24 h (data not shown).
In summary, the C5-fluorinated substrate analogue 7 was
prepared, and its reaction with UGM was fully characterized.
Release of UDP from 7 is UGM-dependent, and compound 24
was identified as the turnover product. Our results clearly
revealed the intermediacy of 5 (or 9/10) in the catalytic
mechanism of UGM and lend further credence to the currently
accepted mechanism of UGM. They also suggest a more
associative mechanism (SN2-like) during substrate−FAD
C
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
■
Corresponding Author
*h.w.liu@mail.utexas.edu
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes
of Health (GM035906) and the Welch Foundation (F-1511).
■
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