A fluoro analogue of UDP-α-d-glucuronic acid is an inhibitor of UDP-α-d-apiose/UDP-α-d-xylose synthase

Article abstract of DOI:10.1039/c1cc13140k

UDP-2F-glucuronic acid was synthesized and analyzed as a mechanistic probe to investigate the ring contraction step catalyzed by UDP-d-apiose/UDP-d-xylose synthase (AXS). The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1cc13140k

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COMMUNICATION
A fluoro analogue of UDP-a-D-glucuronic acid is an inhibitor of
UDP-a-D-apiose/UDP-a-D-xylose synthasewz
Sei-hyun Choi,a Mark W. Ruszczycky,b Hua Zhangb and Hung-wen Liu*ab
Received 27th May 2011, Accepted 20th July 2011
DOI: 10.1039/c1cc13140k
UDP-2F-glucuronic acid was synthesized and analyzed as a
mechanistic probe to investigate the ring contraction step catalyzed
by UDP-D-apiose/UDP-D-xylose synthase (AXS).
D-Apiose (3-C-hydroxymethyl-D-erythrose, 1) is a sugar found
in the pectic polysaccharides rhamnogalacturonan-II (RG-II)1
and apiogalacturonan,2 which are components of the plant
cell-wall. It is a five-carbon furanose sugar carrying a hydroxy-
methyl group at C-3. This branched-chained furanose structure is
rare in nature and has been observed in only a small number
monosaccharides, such as streptose and hamamelose, in addition
to apiose. The D-apiose residues are important for plant
growth and development, because they crosslink the RG-II
polysaccharides through the formation of a borate tetraester.3
The significance of such crosslinks is demonstrated by the
dwarfed phenotype of mur1 mutated plants, in which the
L-fucose residue within RG-II is replaced by L-galactose thereby
inhibiting the crosslinking of the polysaccharides.3
The building block of D-apiose in the cell-wall polysaccharides
is UDP-D-apiose (2),4,5 which is derived directly from
UDP-a-D-glucuronic acid (UDP-GlcA, 3). The conversion of
UDP-GlcA to UDP-apiose is catalyzed by a single enzyme,
UDP-D-apiose/UDP-D-xylose synthase (AXS).5 This transfor-
mation is NAD+-dependent and UDP-D-xylose (8) can also be
formed during the reaction.5 The proposed reaction sequence
(Scheme 1) involves oxidation of 3 by NAD+ to form UDP-
4-ketoglucuronate (4), which then undergoes decarboxylation
to afford a 4-ketoxylose intermediate (UDP-4-KX, 5). Subsequent
ring contraction (5 - 7) followed by reduction at C-30 by
NADH yields the UDP-D-apiose product.
Scheme 1 Two proposed mechanisms for the biosynthesis of UDP-D-
apiose catalyzed by AXS.
reduction of the resulting aldehyde moiety in 7 by NADH is
essential to drive the reaction to completion and to regenerate
the NAD+ coenzyme. The formation of UDP-apiose (2) and
UDP-xylose (8) during catalysis can be rationalized by the fact
that both C-3 and C-4 in 6 could act as the nucleophile in the
recyclization step.
Two plausible mechanisms for the rearrangement step have
been proposed.6 As shown in Scheme 1, bond-cleavage between
C-2/C-3 of 5 in a retroaldol reaction (mechanism A) could
yield an enediol intermediate (6), which, after recyclization
between C-4/C-2, would give 7. Since these steps are reversible,
A pathway involving a 1,2-bond shift (5 - 7, mechanism B)
is also conceivable for the ring contraction step. In contrast to
the stepwise retroaldol–aldol route, the C–C bond migration
in this concerted mechanism would require deprotonation of
O-3 and bypass formation of a discrete intermediate such as 6.
In both mechanisms, however, the formation of UDP-xylose (8)
could result from a premature ‘‘capture’’ of the decarboxylated
4-keto intermediate 5 by NADH, prior to the rearrangement step.
We envisioned that a fluorinated analogue of UDP-GlcA,
UDP-2-fluoro-2-deoxyglucuronic acid (UDP-2F-GlcA, 9),
could be a useful probe to study the mechanism of AXS. As
depicted in Scheme 2, upon incubation, compound 9 may be
a Department of Chemistry and Biochemistry, University of Texas at
Austin, Austin, Texas 78712, USA. E-mail: h.w.liu@mail.utexas.edu;
Tel: +1 512-232-7811
b Medicinal Chemistry Division, College of Pharmacy,
University of Texas at Austin, Austin, Texas 78712, USA
w This article is part of the ChemComm ‘Glycochemistry and glycobiology’
web themed issue.
z Electronic supplementary information (ESI) available: Full experi-
mental details and characterization. See DOI: 10.1039/c1cc13140k
c
10130 Chem. Commun., 2011, 47, 10130–10132
This journal is The Royal Society of Chemistry 2011

deprotected using NaOMe, and the resulting monophosphate
23 was coupled to UMP using UMP-morpholidate11 to
complete the synthesis of 9 (overall yield after 10 steps: 7%).
To test whether UDP-2F-GlcA (9) can be processed by
AXS, UDP-2F-GlcA (400 mM) was incubated with AXS
(10 mM) and NAD+ (400 mM) in 50 mM Tris buffer
(pH 8.0) at room temperature. To our disappointment, no
new peak was observed by HPLC over a 40 min incubation
period (Fig. S1, ESIz). To determine whether UDP-2F-GlcA
actually binds to AXS, a competition assay was performed, in
which UDP-GlcA (3, 400 mM) was incubated with AXS
(same conditions mentioned above) in the presence of two
different concentrations of 9 (400 mM and 4 mM). An identical
incubation without 9 was also carried out as a control. As
shown in Fig. S2–S4 (ESIz), UDP-2F-GlcA (9) showed inhibition
of AXS against 3 in a concentration-dependent manner. These
results suggest that compound 9 can be recognized by AXS
and likely binds to the same active site.
Scheme 2 Mechanistic scenario for UDP-2F-GlcA.
converted to a 4-ketointermediate (11) by oxidative decarboxyl-
ation. If AXS employs the 1,2-shift mechanism (mechanism B),
then compound 11 may be turned over to give UDP-2F-apiose (13)
as the product. However, if the retroaldol/aldol mechanism
(mechanism A) is operative, no apiose product is expected due
to the absence of a hydroxyl group at the C-2 position of 11.
A likely product in this case is the fluorinated xylose analogue
14. Thus, product analysis of the incubation mixture could
shed light on the mechanism of the AXS-catalyzed reaction.
Synthesis of compound 9 starting from the commercially
available tri-O-acetyl-D-glucal (16) was accomplished following
the reaction sequence shown in Scheme 3. According to a
literature procedure,8 the O-acetyl protecting groups in 16 were
first replaced by sterically larger pivaloyl groups (16 - 17) to
enhance the stereoselectivity of the subsequent C-2 fluorination
reaction, which was carried out using Selectfluor.9 A dibenzyl
phosphate group was then introduced at C-1 by treatment of 18
with dibenzyl N,N-diisopropylphosphoramidite and 3-chloro-
perbenzoic acid (m-CPBA).10 Selective deprotection of the
primary pivaloyl group of 19 by reduction with DIBAL-H,
followed by Jones oxidation and hydrogenation, yielded the
phosphate compound 22. The remaining pivaloyl groups were
The fact that UDP-2F-GlcA is not a substrate for AXS
makes it unsuitable for the proposed mechanistic studies of
AXS (Scheme 1). The inability of AXS to effect the initial
dehydrogenation of the C-4 hydroxyl group of 9 is likely a
consequence of inductive destabilization of the partial positive
charge developed at C-4 during the oxidation step by the
2-fluoro substituent, although we had hoped to avoid such a
destabilization by designing the 2-F rather than a 3-F analogue.
Poor positioning of 9 in the active site of AXS could also
prevent efficient hydride transfer.
Although no new product was found when 9 was incubated
with AXS, an apparent shift of reaction flux was noted when 9
was included in the incubation with UDP-GlcA (3). As shown
in Fig. 1, two product peaks could be detected by HPLC of the
reaction of AXS with 3. The peak with a retention time of
17 min corresponds to UMP, which derives from the decom-
position of UDP-D-apiose (2) to form apiose 1,2-cyclic phosphate
7
1
(15, t E 100 min). The second peak appearing at 25 min
2
Fig. 1 HPLC traces of the AXS reaction. (a) UDP-GlcA (3)
standard, (b) UDP-xylose (8) standard, (c) NAD+ standard, (d) the
reaction mixture containing 10 mM AXS, 400 mM NAD+ and 400 mM
UDP-GlcA (3) in 50 mM Tris buffer (pH 8.0) was incubated for 12 h,
(e) excess H2NOH was added to (d) and the mixture was incubated
for an additional 3 h. The 35 minute peak (*) was isolated and
analyzed by MS.
Scheme 3 Synthetic scheme for compound 9.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10130–10132 10131

In summary, we report the synthesis and analysis of UDP-
2F-GlcA (9) as a potential mechanistic probe to investigate the
mechanism of ring contraction catalyzed by AXS. Although
compound 9 turned out not to be a substrate for AXS, it was
identified as an inhibitor. The observed change in the distribution
of products and the 4-keto-intermediate (5) as a function of
inhibitor concentration is most likely the result of competitive
inhibition between 9 and both 5 as well as 3. This observation
is reminiscent of the ability of myo-inositol 1-phosphate
synthase to bind redox-altered mechanistic intermediates and
analogues with the unproductive redox form of the nicotinamide
cofactor to form dead-end complexes competitively versus the
substrate.15 Attempts to gain greater insight regarding the
mode of inhibition by UDP-2F-GlcA in order to better under-
stand the mechanism of the AXS-catalyzed reaction are in
progress.
Fig. 2 The ratio of 5/8 versus UMP produced by AXS as determined
by HPLC peak integrations is dependent on the concentration of the
inhibitor, UDP-2F-GlcA (black: 4 mM 9, grey: 400 mM 9, white: no
inhibitor).
We would like to thank Dr Yung-nan Liu for her technical
support. This work was supported in part by grants from the
National Institutes of Health (GM035906, GM054346 and
F32Al082906).
Notes and references
1 T. Ishii, T. Matsunaga, P. Pellerin, M. A. O’Neill, A. Darvill and
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4 Abbreviations: HPLC, high performance liquid chromatography; MS,
mass spectroscopy; NAD+, b-nicotinamide adenine dinucleotide;
NADH, b-nicotinamide adenine dinucleotide, reduced form; Tris,
tris(hydroxymethyl)aminomethane; UMP, uridine 50-monophosphate;
UDP, uridine 50-diphosphate.
5 J. M. Picken and J. Mendicino, J. Biol. Chem., 1967, 242, 1629;
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Scheme 4 Partitioning of the EꢀNADHꢀUDP-4-KX ternary complex.
corresponds to a mixture of three components: UDP-D-xylose (8),
the residual UDP-apiose (2), and UDP-4-KX (5), the latter of
which can be released from the active site during turnover.
A similar phenomenon has also been reported for several other
NAD+-dependent enzymes such as UDP-galactose 4-epimerase,12
CDP-tyvelose 2-epimerase,13 and RsU4kpxs from Ralstonia
solanacearum.14 Partial separation of the components of this
peak was achieved by treatment of the reaction mixture at the
end of incubation with hydroxylamine, which reacts with 5 to
afford the corresponding 4-oxime adduct (see Fig. 1e and
ESIz). Since 5 and 8 are relatively stable while 2 is readily
decomposed to UMP and 15, analysis of the peak integrations
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respectively. As shown in Fig. 2, the ratio of the two peaks at
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UDP-D-xylose (8) versus the rearrangement of UDP-4-KX to
UDP-apiose (2, Scheme 4, B vs. A). However, model analysis
indicates that it is more likely a consequence of simple
competitive inhibition by 9 versus both the normal substrate
(3) and the released UDP-4-KX (5) intermediate. In this
scenario, partitioning of the ternary complex containing
UDP-4-KX between release of UDP-4-KX versus formation
of products (Scheme 4, C vs. A,B) would be unaffected by the
presence of 9. However, this would not be true for the binding
back of free UDP-4-KX to complete the reaction (Scheme 4, D),
which would have to compete with 9 to do so. This would then
result in an increase in the HPLC peak ratio observed during
the time course of the experiment.
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c
10132 Chem. Commun., 2011, 47, 10130–10132
This journal is The Royal Society of Chemistry 2011