Chemical synthesis of UDP-β-L-arabinofuranose and its turnover to UDP-β-L-arabinopyranose by UDP-galactopyranose mutase

Article abstract of DOI:10.1016/S0960-894X(00)00616-8

Uridine-5′-diphospho-β-L-arabinofuranose, a possible donor of L-arabinofuranose residues in plants, was synthesized. This compound, in the presence of UDP-galactopyranose mutase, underwent interconversion with UDP-β-L-arabinopyranose that is a likely precursor of L-arabinofuranose in vivo. This result provided a working model for the biogenesis of arabinofuranose in plants.

Full text of DOI:10.1016/S0960-894X(00)00616-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 145±149
Chemical Synthesis of UDP-ꢀ-L-Arabinofuranose and its
Turnover to UDP-ꢀ-L-Arabinopyranose by
UDP-Galactopyranose Mutase
Qibo Zhanga and Hung-wen Liua,b,Ã
aDepartment of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
bDivision of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry,
University of Texas, Austin, TX 78712, USA
Received 1 September 2000; accepted 26 October 2000
AbstractÐUridine-50-diphospho-b-l-arabinofuranose, a possible donor of l-arabinofuranose residues in plants, was synthesized.
This compound, in the presence of UDP-galactopyranose mutase, underwent interconversion with UDP-b-l-arabinopyranose that
is a likely precursor of l-arabinofuranose in vivo. This result provided a working model for the biogenesis of arabinofuranose in
plants. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
greatly favors the thermodynamically more stable pyr-
anose form 2.5 The catalytic mechanism has recently
been shown to involve cleavage of the anomeric C±O bond
to form a bicyclo-acetal intermediate (3, Scheme 1).9 While
enzymes responsible for the biosynthesis and transfer of
d-Galf have attracted much attention due to their
potential as drug targets for bacteria, fungi, and para-
sites, less is known about the biosynthesis of arabino-
furanose (4, Araf) at enzymatic level. Structurally,
l-arabinofuranose di€ers from d-galactofuranose only
by a shorter side chain at C-4. It is thus conceivable that
biosynthesis of l-arabinofuranose follows a path similar
to that of d-galactofuranose, involving an enzyme cata-
lyzed ring contraction of l-arabinopyranose. Indeed,
preliminary data have shown that the source of l-
arabinofuranose (Araf ) residues of the plant poly-
saccharides is UDP-l-arabinopyranose (UDP-l-Arap,
5, see Scheme 3),10,11 which may be the substrate of the
putative mutase. In order to test the viability of this
hypothesis, we have chemically synthesized UDP-l-
Araf (4) and examined its competence as a substrate for
UDP-galactopyranose mutase, a homologue of the
putative arabinopyranose mutase. Reported in this
paper are the results of these experiments.
Glycoconjugates containing furanose residues are
ubiquitous in nature.1 Among the known naturally
occurring furanose sugars, galactofuranose and
arabinofuranose are most frequently found. They are
especially abundant in the surface constituents of
microorganisms, such as bacterial O-antigens, fungal
exopolysaccharides, protozoal glycoproteins, and the
cell walls of mycobacteria. For example, all of the
galactose and arabinose units in two major myco-
bacterial cell wall polysaccharides, arabinogalactan
(AG) and lipoarabinomannan (LAM), have been shown
to exist in the furanose form.2 Arabinofuranose is also a
common structural component in plant cell walls.1,3 Its
occurrence in plant arabinan, rhamnogalacturonan,
arabinogalactan, and arabinogalactan proteins has been
well documented, in which arabinofuranose is typically
coupled to other sugar components via an a-l-(1!5),
a-l-(1!2), or a-l-(1!3) linkage.1
The precursor of d-galactofuranose (d-Galf ) found in
various glycoconjugates is UDP-d-Galf (1), which is
biosynthetically derived from the corresponding galac-
topyranose (2, UDP-d-Galp) via an unusual ring con-
traction reaction catalyzed by UDP-galactopyranose
mutase.4 8 This enzyme mediated interconversion
Results and Discussion
The synthesis of UDP-b-l-arabinofuranose (4) was
accomplished by a sequence of reactions delineated in
Scheme 2. l-Arabinose was converted, according to a
ÃCorresponding author. Tel.: +512-232-7811; fax: +512-471-2746; e-
mail: h.w.liu@mail.utexas.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00616-8

146
Q. Zhang, H. Liu / Bioorg. Med. Chem. Lett. 11 (2001) 145±149
one-pot procedure developed by Ichikawa et al.,12 to
tetra-O-acetyl-l-arabinofuranose (6) as a 1:1 mixture of
a,b isomers. Subsequent preparation of b-l-arabinofur-
anosyl phosphate followed a strategy of de Lederkremer
and co-workers.13 As shown in Scheme 2, upon treat-
ment with bromotrimethylsilane, tetra-acetate 6 was
converted to the corresponding tri-O-acetyl-l-arabino-
furanosyl bromide (7), which was chromatographically
unstable on silica gel. Hence, without puri®cation,
compound 7 was reacted directly with excess triethyl-
ammonium dibenzyl phosphate in anhydrous toluene
overnight. The resulting mixture was ®ltered and the
®ltrate was chromatographed on silica gel (ethyl ace-
tate/hexanes, 3/5) to yield the desired product 8 as a
single isomer. This product was isolated as a syrup. The
1-H signal in the 1H NMR spectrum appeared as a
doublet of doublets (J1,2=4.5, JH-1,P=5.4), and those of
C-1 and C-2 in the 13C NMR spectrum, due to coupling
with 31P, resonated as a doublet at d 97.8 (J=5) and d
75.9 (J=5), respectively. The observed coupling constant
between 1-H and 2-H clearly indicates a cis relationship
between the two hydroxyl groups at C-1 and C-2.14 The
product 8 was therefore characterized as dibenzyl (2,3,5-
tri-O-acetyl-b-l-arabinofuranosyl) phosphate. The
corresponding a isomer was not observed in this case.
Compound 8 was debenzylated by catalytic hydrogena-
tion over 10% palladium on charcoal in the presence of
triethylamine to a€ord the phosphate 9 as a mono-
triethylammonium salt in good yield (92%). Deacetyla-
tion of compound 9 was readily achieved by treatment
with methanol/water (5/2) in the presence of triethyl-
amine, providing the desired b-l-arabinofuranosyl
phosphate (10) as its bistriethylammonium salt in
quantitative yield. The ®nal product UDP-l-Araf (4)
was synthesized from 10 and uridine phosphomorpholi-
date in anhydrous pyridine in the presence of 1H-tetra-
zole.15,16 The reaction mixture was separated ®rst by
size exclusion chromatography (Sephadex LH-20,
methanol), and then by HPLC on a semi-preparative
C18 column (1.5% acetonitrile in 50 mM triethyl-
ammonium acetate) to give nearly homogeneous UDP-
l-Araf (4) as the bistriethylammonium salt in 42%
yield. The structure of the puri®ed product was con-
®rmed spectroscopically by NMR (1H, COSY, 13C, and
31P) and high-resolution ESI-MS. The presence of two
doublets of similar intensity in the proton decoupled 31P
NMR con®rmed the presence of a diphosphate link-
age.17 The availability of this furanose derivative is
important for the bioassay. Since the equilibrium of the
interconversion greatly favors the pyranose form, little
Scheme 1.

Q. Zhang, H. Liu / Bioorg. Med. Chem. Lett. 11 (2001) 145±149
147
turnover would be seen if UDP-l-arabinopyranose was
used as the substrate.
tioned conditions. As expected, this compound was
identi®ed as UDP-l-Arap (5) by NMR and MS analy-
sis,19 and by comparison to reported data.20 The kinetic
analysis was carried out by the same discontinuous
HPLC assay. The product and substrate ratios were
calculated from the integration of the corresponding
peaks from the HPLC chromatogram. The Km and kcat
for UDP-l-Araf were determined to be 0.6 mM and
12 s 1, respectively. The kcat value is comparable to that
(kcat=27 s 1) found for UDP-d-Galf, but the Km value
is nearly 30-fold greater than that of UDP-d-Galf
(Km=22 mM). These results suggest that signi®cant
interactions exist between the hydroxymethyl side chain
of UDP-d-Galf and the surrounding amino acid resi-
dues in the enzyme active site. Nevertheless, UDP-l-
Araf is clearly a reasonable substrate for UDP-galacto-
pyranose mutase.
To demonstrate that UDP-l-Araf (4) can undergo
interconversion to generate UDP-l-Arap (5) in the
presence of a mutase, the synthetic product 4 was incu-
bated with Escherichia coli UDP-galactopyranose
mutase puri®ed from an overproducing recombinant
strain, E. coli BL-21 (DE3)/pQZ-1.18 An assay solution
consisting of 2 mM UDP-l-Araf (4) and 72 mM mutase
in 30 mL of 100 mM potassium phosphate bu€er (pH
7.5) was incubated for 10 min at 37 ꢀC. The resulting
mixture was analyzed by reversed-phase HPLC (C18
column, 1.5% acetonitrile in 50 mM triethylammonium
acetate). The detector was set at 262 nm and the ¯ow
rate was 1.0 mL/min. Under these conditions, a new
peak with a retention time of 5.9 min was observed,
whereas UDP-l-Araf was eluted at 8.2 min (Fig. 1).
More than 90% conversion of 4 to this new product
could be achieved by prolonged incubation or by
including 20 mM sodium dithionite in the reaction since
The work presented here is a proof of concept that
UDP-l-Araf can be converted from UDP-l-Arap by the
action of a mutase. On the basis of our data, this con-
version can be catalyzed by a single enzyme, UDP-
galactopyranose mutase, which works on both the
d-Galp/d-Galf and l-Arap/l-Araf series. However, so
the mutase is more reactive in its reduced form.18
A
preparative incubation allowed this product to be pur-
i®ed by semi-preparative HPLC under the aforemen-
Scheme 2.

148
Q. Zhang, H. Liu / Bioorg. Med. Chem. Lett. 11 (2001) 145±149
References and Notes
1. Brett, C. T.; Waldron, K. W. Physiology and Biochemistry
of Plant Cell Walls; Chapman and Hall: London, 1996.
2. Brennan, P. J.; Nikaido, H. Annu. Rev. Biochem. 1995, 64,
29.
3. Fincher, G. B.; Stone, B. A.; Clarke, A. E. Annu. Rev. Plant
Physiol. 1983, 34, 47.
4. Nassau, P. M.; Martin, S. L.; Brown, R. E.; Weston, A.;
Monsey, D.; McNeil, M. R.; Duncan, K. J. Bacteriol. 1996,
178, 1047.
Scheme 3.
5. Koplin, R.; Brisson, J.-R.; Whit®eld, C. J. Biol. Chem.
1997, 272, 4121.
6. Lee, R.; Monsey, D.; Weston, A.; Duncan, K.; Rithner, C.;
McNeil, M. Anal. Biochem. 1996, 242, 1.
7. Weston, A.; Stern, R. J.; Lee, R. E.; Nassau, P. M.; Mon-
sey, D.; Martin, S. L.; Scherman, M. S.; Besra, G. S.; Duncan,
K.; McNeil, M. R. Tuberc. Lung Dis. 1998, 78, 123.
8. Chen, P.; Bishai, W. Infect. Immun. 1998, 66, 5099.
9. Barlow, J. N.; Girvin, M. E.; Blanchard, J. S. J. Am. Chem.
Soc. 1999, 121, 6968.
10. Bolwell, G. P.; Northcote, D. H. Biochem. J. 1983, 210,
497.
11. Rodgers, M. W.; Bolwell, G. P. Biochem. J. 1992, 288,
817.
Figure 1. HPLC chromatogram of an incubation mixture of UDP-l-
Araf and UDP-galactopyranose mutase.
12. Ichikawa, Y.; Manaka, A.; Kuzuhara, H. Carbohydr. Res.
1985, 138, 55.
13. de Lederkremer, R. M.; Nahmad, V. B.; Verela, O. J. Org.
Chem. 1994, 59, 690.
14. Bundle, D. R.; Lemieux, R. U. Methods Carbohydr.
Chem. 1976, 7, 79.
far all the arabinofuranose units found in the surface
constituents of microorganisms are d-arabinose,21,22
and the precursor of d-arabinofuranose in microorgan-
isms has been established to be a b-d-arabinofuranosyl-
1-monophosphoryldecaprenol. This notion is supported
by many lines of evidence,23 25 including the incor-
poration of chemically synthesized decaprenol phos-
phoarabinose into the oligosaccharide products in the
presence of a membrane preparation from myco-
bacteria.26 Therefore, in microorganisms it is likely that
two distinct mutases are required to catalyze these two
related ring contraction reactions, if d-arabinofuranose
is indeed derived from d-arabinopyranose in its phos-
phoryldecaprenol form.27 On the contrary, the arabino-
furanose units found in plant polysaccharides are l-
arabinose, where the scenario of one enzyme to catalyze
the conversion of 2 to 1 and 5 to 4 is de®nitely a possi-
bility. The ready availability of UDP-l-Araf (4) through
the chemical synthesis reported herein will certainly
facilitate the detection and isolation of the desired
mutase in plants. It would be interesting to compare
whether the putative UDP-l-Arap mutase and UDP-d-
Galp mutase are the same enzyme or are evolutionarily
related, and whether the Arap mutase can also accept
Galp/Galf as substrates. Additionally, with large quan-
tities of 4 available, one could also attempt to isolate the
arabinofuranosyl transferase and study how nature
incorporates arabinofuranose into biological molecules.
15. Wittman, V.; Wong, C.-H. J. Org. Chem. 1997, 62, 2144.
Speci®cally, the triethylammonium salt of b-l-arabinofur-
anosyl phosphate (10, 400 mg, 0.9 mmol) was dried by co-eva-
porating with anhydrous pyridine (4 mL) a few times. To this
residue was added UMP-morpholidate (1.6 g, 2.1 mmol) in
anhydrous pyridine (3 mL) and the solution was evaporated
again to dryness under vacuum. This was followed by the
addition of 1H-tetrazole (200 mg, 2.8 mmol) in anhydrous
pyridine (5 mL) and the resulting solution was stirred at room
temperature for 36 h. Removal of the solvent under vacuum
gave a solid residue that was dissolved in methanol and chro-
matographed on a Sephadex LH-20 column (2.5Â120 cm)
using methanol as the eluent. Fractions containing the desired
product, judging by TLC analysis (EtOH/NH4OH/H2O, 5/3/1),
were pooled and evaporated to dryness under reduced pres-
sure. Compound 4 (42% yield) was further puri®ed by HPLC
using a C18 column (Alltech, 10Â250 mm). The eluent was
1.5% acetonitrile in 50 mM triethylammonium acetate bu€er,
pH 6.8, and the ¯ow rate was 5 mL/min. The retention time of
4 under these conditions was 8.2 min.
16. See Tsvetkov, Y. E.; Nikolaev, A. V. J. Chem. Soc., Perkin
Trans. 1 2000, 889 for an alternate method to make UDP-sugar.
17. All compounds a€orded satisfactory NMR (1H, 13C, 31P,
and COSY) and high-resolution mass spectral charac-
terization. Spectra data of 4 (bistriethylammonium salt): 1H
NMR (500 MHz, D2O) d 1.30 (18H, t, J=7.5 Hz, Et3N-Me),
3.23 (12H, q, J=7.5, Et3N-CH2), 3.72 (1H, dd, J=13.0, 6.0, 5-
H), 3.82 (1H, dd, J=12.5, 3.0, 5-H), 3.94 (1H, ddd, J=7.5,
6.0, 3.0, 4-H), 4.16 (2H, m, 2-H, 3-H), 4.22 (1H, ddd, J=11.5,
6.0, 3.0, 50-H), 4.26 (1H, ddd, J=12.0, 5.0, 2.0, 50-H), 4.30
(1H, m, 40-H), 4.40 (2H, m, 20-H, 30-H), 5.66 (1H, dd, J=6.0,
3.5, 1-H), 6.00 (1H, d, J=8.5, 500-H), 6.01 (1H, d, J=4.5,
10-H), 7.99 (1H, d, J=8.5, 600-H); 13C NMR (125 MHz, D2O) d
9.8 (Et3N-Me), 48.2 (Et3N-CH2), 63.8 (C-5), 66.5 (d, J=6,
C-50), 71.3 (C-20), 74.8 (C-3), 75.4 (C-30), 78.3 (d, J=8, C-2),
84.3 (C-4), 84.9 (d, J=9, C-40), 89.9 (C-10), 99.3 (d, J=6, C-1),
104.2 (C-500), 143.2 (C-600), 153.4 (C-200), 167.8 (C-400); 31P
NMR (121 MHz, D2O) d 12.3 (d, J=20.8), 10.9 (d,
Acknowledgements
This work was supported in part by a grant from the
National Institutes of Health (GM54346). H.-w. L. also
thanks the National Institute of General Medical
Sciences for a MERIT Award.

Q. Zhang, H. Liu / Bioorg. Med. Chem. Lett. 11 (2001) 145±149
149
J=20.8). Negative ion high-resolution ESI-MS calcd for
C14H21N2O16P2 [M H] 535.0372, found m/z 535.0370.
18. Zhang, Q.; Liu, H.-W. J. Am. Chem. Soc. 2000, 122, 9065.
19. Spectra data of 5 (bistriethylammonium salt): 1H NMR
(500 MHz, D2O) d 1.30 (18H, br, Et3N-Me), 3.23 (12H, br,
Et3N-CH2), 3.74 (1H, dd, J=12.5, 2.0 Hz, 5-H), 3.83 (1H, dt,
J=10.0, 3.5, 2-H), 3.95 (1H, dd, J=10.0, 3.5, 3-H), 4.05 (1H,
m, 4-H), 4.15 (1H, d, J=12.5, 5-H), 4.22 (1H, ddd, J=12.0,
5.5, 2.5, 50-H), 4.27 (1H, ddd, J=12.0, 4.5, 2.5, 50-H), 4.31
(1H, quint, J=3.0, 40-H), 4.40 (2H, m, 20-H, 30-H), 5.63 (1H,
dd, J=7.5, 3.5, 1-H), 6.00 (1H, d, J=8.5, 500-H), 6.01 (1H, d,
J=5.0, 10-H), 7.99 (1H, d, J=8.0, 600-H); 13C NMR (75 MHz,
D2O) d 9.8 (Et3N-Me), 48.3 (Et3N-CH2), 65.6 (C-5), 66.5 (d,
J=6, C-50), 70.0 (d, J=9, C-2), 70.3 (C-3), 70.5 (C-4), 71.3 (C-
20), 75.4 (C-30), 84.9 (d, J=9, C-40), 89.9 (C-10), 97.8 (d, J=6,
C-1), 104.3 (C-500), 143.2 (C-600), 153.4 (C-200), 167.9 (C-400);
31P NMR (121 MHz, D2O) d 12.3 (d, J=21), 10.7 (d,
J=21). Negative ion high-resolution ESI-MS calcd for
C14H21N2O16P2 [M H] 535.0372, found m/z 535.0340.
20. Pauly, M.; Porchia, A.; Olsen, C. E.; Nunan, K. J.;
Scheller, H. V. Anal. Biochem. 2000, 278, 69.
21. Wolucka, B. A.; McNeil, M. R.; de Ho€man, E.; Choj-
nacki, T.; Brennan, P. J. J. Biol. Chem. 1994, 269, 23328.
22. D'Souza, F. W.; Ayers, J. D.; McCarren, P. R.; Lowary,
T. L. J. Am. Chem. Soc. 2000, 122, 1251.
23. Lee, R. E.; Mikusova, K.; Brennan, P. J.; Besra, G. S. J.
Am. Chem. Soc. 1995, 117, 11829.
24. Scherman, M. S.; Kalbe-Bournonville, L.; Bush, D.; Xin,
Y.; Deng, L.; McNeil, M. J. Biol. Chem. 1996, 271, 29652.
25. Xin, Y.; Lee, R. E.; Scherman, M. S.; Khoo, K.-H.; Besra,
G. S.; Brennan, P. J.; McNeil, M. Biochim. Biophys. Acta
1997, 1335, 231.
26. Lee, R. E.; Brennan, P. J.; Besra, G. S. Glycobiology 1997,
7, 1121.
27. However, the possible involvement of a nucleotidyl
diphopho-d-arabinofuranose as the d-Araf donor in the bio-
synthesis of the microbial oligosaccharides has also been
speculated.26