Chemical synthesis of uridine 5′-diphospho-α-D-xylopyranose

Article abstract of DOI:10.1016/j.tetasy.2004.11.073

Uridine 5′-diphospho-α-d-xylopyranose, which donates d-xylose during glycoconjugate biosynthesis, was chemically synthesized from α-d-xylose 1-phosphate and uridine 5′-monophosphoimidazolide.

Full text of DOI:10.1016/j.tetasy.2004.11.073

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 309–311
Chemical synthesis of uridine 50-diphospho-a-D-xylopyranose
Takeshi Ishimizu, Takashi Uchida, Kyoko Sano and Sumihiro Hase*
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan
Received 15 November 2004; accepted 29 November 2004
Available online 22 December 2004
Abstract—Uridine 50-diphospho-a-D-xylopyranose, which donates D-xylose during glycoconjugate biosynthesis, was chemically
synthesized from a-D-xylose 1-phosphate and uridine 50-monophosphoimidazolide.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
port the chemical synthesis of 1, using the phospho-
imidazolidate activation method.8
D-Xylose is widely distributed in nature and is a
component of various glycoconjugates, such as proteo-
glycans1 and O-glucose type glycans2 of animal cells;
polysaccharides of plant cell walls;3 N-glycans4 of plants
and some invertebrates; and capsular polysaccharides5
of fungi. Uridine 50-diphospho-a-D-xylopyranose 1 is a
substrate for xylosyltransferase, which transfers the D-
xylose to glycoconjugates. Biosynthesis of 1 is mediated
by UDP-glucuronic acid decarboxylase with UDP-a-D-
glucuronic acid as the substrate.6 Compound 1 was
enzymatically prepared using UDP-glucuronic acid
decarboxylase in vitro.6 By regioselective attack of the
1,2-anhydro sugar (D-xylal) by the nucleoside diphos-
phate moiety, UDP-D-xylose was chemically synthe-
sized;7 however, it was an a/b anomeric mixture (a/
b = 5:3). Preparation of pure 1 is critical for glycoconju-
gate biosynthetic studies and for structural, functional,
and kinetic studies of xylosyltransferases. Here, we re-
2. Results and discussion
Compound 1 was prepared from the activated com-
pound, uridine 50-monophosphate imidazole 2 and the
tri-n-octylammonium salt of D-xylopyranose 1-phos-
phate 3 (Scheme 1). Compound 2 was prepared by reac-
tion of the UMP free acid 5 and N,N0-carbon-
yldiimidazole9 (Scheme 2). Compound 310 was used for
the coupling reaction with 2 because 3 is soluble in pyr-
idine, whereas other salts (ammonium, monocyclohexyl-
ammonium, pyridinium, and triethylamine) of a-D-
xylopyranose 1-phosphate are not. The reaction11 was
monitored by TLC. The best yield occurred after a reac-
tion time of 5 days. The product was purified first using
Bio-gel P2 gel-filtration chromatography and then Wako-
Sil-II 5C30 reverse-phase HPLC. The yield was 34–36%.
O
O
O
O
N
HO
HO
N
HO
O
P
O
P
O-
P
(C8H17)3N+
O-
P
HO
OH
OH
N
O
N
N
O
O
O
O
O
O
O
N
O
O-
(C8H17)3N+
+
O
O
O
O
(i)
OH
OH
OH
OH
1
3
2
Scheme 1. Reagents and conditions. (i) DMF:pyridine (35:8, v/v), 5 days.
*
Corresponding author. Tel.: +81 6 6850 5380; fax: +81 6 6850 5383; e-mail: suhase@chem.sci.osaka-u.ac.jp
0957-4166/$ - see front matter ꢀ 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.073

310
T. Ishimizu et al. / Tetrahedron: Asymmetry 16 (2005) 309–311
O
O
N
N
ONa
P
OH
P
N
O
N
O
O
O
ONa
O
O
OH
O
O
2
(i)
(ii)
OH
OH
OH
OH
4
5
Scheme 2. Reagents and conditions. (i) Dowex H+; (ii) N,N0-carbonyldiimidazole, DMF, 25 ꢁC, 15 h.
1
The structure of 1 was confirmed using H NMR spec-
2. (a) Hase, S.; Kawabata, S.; Nishimura, H.; Takeya, H.;
Sueyoshi, T.; Miyata, T.; Iwanaga, S.; Takao, T.; Shi-
monishi, Y.; Ikenaka, T. J. Biochem. 1988, 104, 867–868;
(b) Hase, S.; Nishimura, H.; Kawabata, S.; Iwanaga, S.;
Ikenaka, T. J. Biol. Chem. 1990, 265, 1858–1861.
3. OÕNeill, M.; York, W. S. The Plant Cell Wall; Blackwell:
Oxford, 2003; pp 1–54.
4. (a) Ishihara, H.; Takahashi, N.; Oguri, S.; Tejima, S. J.
Biol. Chem. 1979, 254, 10715–10719; (b) van Kuik, J. A.;
van Halbeek, H.; Kamerling, J. P.; Vliegenthart, J. F.
J. Biol. Chem. 1985, 260, 13984–13988.
5. (a) Blandamer, A.; Danishefsky, I. Biochim. Biophys. Acta
1966, 117, 305–313; (b) Bhattacharjee, A. K.; Bennett, J.
E.; Glaudemans, C. P. Rev. Infect. Dis. 1984, 6, 619–624.
6. (a) Ankel, H.; Feingold, D. S. Biochemistry 1965, 4, 2468–
2475; (b) Bar-Peled, M.; Griffith, C. L.; Doering, T. L.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12003–12008.
7. Ernst, C.; Klaffke, W. J. Org. Chem. 2003, 68, 5780–5783.
8. (a) Cramer, F.; Schaller, H.; Staab, H. A. Chem. Ber. 1961,
94, 1612–1621; (b) Schaller, H.; Staab, H. A.; Cramer, F.
Chem. Ber. 1961, 94, 1621–1635; (c) Zhao, Y.; Thorson, J.
S. J. Org. Chem. 1998, 63, 7568–7572.
troscopy and mass spectrometry. The anomeric configu-
00 00
1
ration of 1 is a, because J1 ,2 is 3.3 Hz. The H NMR
spectrum of 1 is essentially superimposable with that
of enzymatically prepared 1.6b The mass spectrum signal
for 1 ([M+H]+, 537.0) corresponds to that calculated for
uridine diphosphopentose ([M+H]+, 537.3).
Compound 1 is a substrate for b-D-glucoside a-1,3-xylo-
syltransferase,12 which is involved in biosynthesis of the
Xyla1-3Xyla1-3Glcb1-O-Ser conjugate of the epidermal
growth factor-like domain of coagulation factor VII and
IX.2 When 2-[(2-pyridyl)amino]ethyl b-D-glucopyrano-
side (Glcb-R) was used as the acceptor in an a 1,3-xylo-
syltransferase assay with 1 as the D-xylose donor, Xyla1-
3Glcb-R was generated (Fig. 1).13
9. The UMP disodium salt 4 (90 mg, 244 lmol) was dis-
solved in water (3 cm3). Dowex 50W-X2 (H+ form) was
added to bring the pH of the solution to 1. The mixture
was stirred at ambient temperature for 1 h and then
poured into a glass column (0.5 · 10 cm). The resin was
washed with 5-column volumes of water. The appropriate
fractions were pooled and lyophilized. N,N0-carbonyldi-
imidazole (75 mg, 463 lmol) and 5 were dissolved in DMF
(4.5 cm3) and the solution stirred at room temperature for
15 h under nitrogen (Scheme 2). The reaction was mon-
itored using TLC silica gel 60 F254 coated glass plates
(1.5 · 3.5 cm) with chloroform-methanol–0.5 M ammo-
nium bicarbonate (5:5:1, v/v/v) as the mobile solvent and
visualized using UV light at 310 nm. Methanol (0.03 cm3)
was added to destroy excess N,N0-carbonyldiimidazole
and the mixture was stirred for an additional 30 min. The
mixture was concentrated and azeotroped with pyridine
(2 cm3) three times in vacuo to remove water and the
residue used as 2.
10. The di(monocyclohexylammonium) salt of a-D-xylose
1-phosphate (80 mg, 187 lmol) was dissolved in water
(1.5 cm3) and chromatographed over Dowex 50W-X2
(pyridinium form; 0.5 · 10 cm column). The resin was
washed with water (5 cm3) and the eluent concentrated to
3 cm3. Pyridine (9 cm3) and tri-n-octylamine (0.09 cm3)
were added to the concentrate, producing 3. The solution
was concentrated and azeotroped with pyridine (1 cm3)
three times.
Figure 1. b-D-Glucoside:a 1,3-xylosyltransferase assay using 1 synthe-
sized chemically as described herein. (A) A 12-h incubation in the
presence of enzyme. (B) A 12-h incubation mixture in the absence of
enzyme. Arrowheads indicate the elution positions of 1,2-[(2-pyr-
idyl)amino]ethyl b-D-glucopyranoside (Glcb-R) and 2, Xyla1-3Glcb-
R.
11. Compounds 2 and 3 were dissolved in a mixture of DMF–
pyridine (35:8, v/v, 4.3 cm3) and the solution stirred at
room temperature for 5 days. The reaction was monitored
using TLC silica gel 60 F254 coated glass plates
(1.5 · 3.5 cm) with 2-propanol–1 M ammonium bicarbon-
ate (2:1, v/v) as the mobile solvent and visualized using UV
References
´
1. (a) Gregory, J. D.; Laurent, T. C.; Roden, L. J. Biol.
Chem. 1964, 239, 3312–3320; (b) Wilson, I. B. H. Cell Mol.
Life Sci. 2004, 61, 794–809.

T. Ishimizu et al. / Tetrahedron: Asymmetry 16 (2005) 309–311
311
00 00
5.79 (d, 1H, H-5), 7.79 (d, 1H, H-6) ppm; J1 ,2 = 3.4Hz,
light at 310 nm. The reaction was stopped by addition of
water (4.3 cm3). Then, 100 mM ammonium bicarbonate
(2 cm3) was added and excess tri-n-octylamine removed by
extraction with diethylether (4.3 cm3). The sample was
concentrated and applied to Bio-gel P2 (2 · 160 cm
column) and eluted with 250 mM ammonium bicarbonate.
Product 1 was detected by measuring the absorbance at
260 nm. The appropriate fractions were concentrated and
further purified using Wako Sil-II5C30-AR reverse-phase
HPLC (0.46 · 25 cm column) with 50 mM ammonium
acetate buffer, pH 4.5, as the eluent and a flow rate of
1 mL/min. The fractions, containing 1, were pooled and
desalted. 1H NMR (600 MHz, D2O, Me4Si): d 3.34(dt,
1H, H-200), 3.44 (td, 1H, H-400), 3.53 (t, 1H, H-300), 3.58 (m,
2H, H-500), 4.04 (m, 2H, H-50), 4.11 (m, 1H, H-30) 4.20 (d,
2H, H-20nd H-40), 5.37 (dd, 1H, H-100), 5.81, (d, 1H, H-10),
00
J1 ,P = 7.0 Hz, J2 ,3 = 9.6 Hz, J2 ,P = 3.4Hz, J3 ,4 = 9.3
00 00
00
00 00
0
0
Hz, J1 ,2 = 4.1 Hz, J5,6 = 8.3 Hz; MS (MALDI-TOF):
m/z = 537.0 ([M+H]+).
12. Omichi, K.; Aoki, K.; Minamida, S.; Hase, S. Eur. J.
Biochem. 1997, 245, 143–146.
13. A bovine liver microsomal fraction, 7 mM Glcb-R, and
13 mM 1 were incubated in 20 mM HEPES–NaOH,
pH 7.2, 150 mM NaCl, 0.1% TritonX-100, 20 mM MnCl2
at 37 ꢁC for 12 h. Then, the mixture was chromatographed
over WakoSil-II 5C18-HG (0.6 · 15 cm column). The
product was eluted with 50 mM ammonium acetate,
pH 4.5 at a flow rate of 2 mL/min. The elution was
monitored by measuring the fluorescence at 400 nm
(excitation at 320 nm). Structural analysis of the product,
Xyla1-3Glcb-R, has been described.12