The first chemical synthesis of UDP-α-D-galactofuranose

Article abstract of DOI:10.1039/b000210k

The chemical synthesis of uridine 5′-diphosphate α-D-galactofuranose (UDP-Galf) was examined. UDP-Galf was synthesized from α-D-galactofuranosyl phosphate and uridine 5′-monophosphate. The potential use of UDP-Galf as a biochemical donor of D-galactofuran

Full text of DOI:10.1039/b000210k

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The first chemical synthesis of UDP-ꢀ-D-galactofuranose
Yury E. Tsvetkov and Andrei V. Nikolaev*
Department of Chemistry, University of Dundee, Dundee, UK DD1 4HN
Received (in Cambridge, UK) 11th January 2000, Accepted 10th February 2000
1
Uridine 5Ј-diphosphate α--galactofuranose, which is likely to be a biochemical donor of -galactofuranosyl
residues in Nature, is synthesized from α--galactofuranosyl phosphate and uridine 5Ј-monophosphate.
Introduction
-Galactofuranose (-Galf ) is distributed in Nature as a com-
ponent of immunologically important glycoconjugates from
bacteria, protozoa and fungi, including some clinically signifi-
cant pathogens. -Galf residues are structural blocks of the cell
wall of Mycobacterium tuberculosis and other mycobacteria1
and principal constituents of the glycosylphosphatidylinositol
(GPI) anchor of Leishmania lipophosphoglycans.2 They are
also present in lipopeptidophosphoglycans of Trypanosoma
cruzi,3 in glycosylinositolphospholipids of Leishmania4 and
Endotrypanum schaundinni5 and in a complex and unique
peptidophosphogalactomannan of Penicillium charlesii.6 In
addition, they are found in N-linked glycoproteins of Crithidia
fasciculata, Crithidia harmonosa, Leishmania samueli, Herp-
etomonas samuelpessoai and Trypanosoma cruzi.3 -Galf-
containing conjugates are vital for the survival of parasites and
bacteria. Since this monosaccharide is not present in humans,
the enzymes involved in the biosynthesis and transfer of
-galactofuranose become important drug targets.
It was suggested7,8 (but not strictly proved) that uridine 5Ј-
diphosphate α--galactofuranose (UDP-Galf ) 1 is expected to
be a substrate for -galactofuranosyl transferases responsible
for the transfer of -Galf units in the biosynthesis of various
Scheme 1 Reagents: i, 1,1Ј-carbonyldiimidazole, DMF; ii, DMF as
solvent.
glycoconjugates. Compound 1 was first prepared recently9 by
enzymic methods from UDP-α--galactopyranose in 3% yield
using the enzyme UDP-galactopyranose mutase. The authors
claimed the procedure is able ‘to produce about 0.5 mg of the
UDP-Galf in a week’.9 Obviously, greater amounts of the com-
pound, required for biochemical studies, could be produced by
chemical synthesis. In this paper we report the first chemical
synthesis of the nucleotide sugar 1.
Results and discussion
UDP-Galf 1 was prepared from α--galactofuranosyl phos-
phate 2 and uridine 5Ј-monophosphate 3 (Scheme 1). Com-
pound 2, in turn, was synthesized starting from methyl α,β--
galactofuranoside 5,10 which was converted to the known11
galactofuranosyl dibenzyl phosphate 9 (Scheme 2) by consecu-
tive benzoylation, acetolysis (→ 6), 1-bromination (→ 8) and
glycosylation of dibenzyl hydrogen phosphate in the presence
of triethylamine. This approach appeared to be more reliable in
our hands than the preparation of 1,2,3,5,6-penta-O-benzoyl-
α,β--galactofuranose 7,12,13 as a starting substance, by direct
benzoylation of -galactose. Two-step deprotection of the
phosphotriester 9 was performed as described11 to produce the
glycosyl phosphate 2 (88%) isolated as a triethylammonium
salt.
Scheme 2 Reagents: i, (a) PhCOCl, pyridine; (b) H2SO4, Ac2O; ii, HBr,
AcOH–CH2Cl2; iii, (BnO)2PO2H, Et3N, toluene; iv, (a) H2, Pd(OH)2/C,
ethyl acetate–Et3N; (b) Et3N–MeOH–water.
reported16 to be equally effective as the phospho-morpholidate
approach for O-deprotected derivatives. Before the conden-
sation, the nucleotide 3 (triethylammonium salt) was converted
to the activated UMP-imidazolide 4 by the reaction with 1,1Ј-
carbonyldiimidazole. The reaction of compounds 2 and 4 in
dimethylformamide (DMF) was monitored by 31P NMR,
which revealed the greatest proportion of the product 1 (corre-
sponding to 45–50% yield) in the mixture at 18–19 h. Basic
degradation of UDP-Galf 1 (δP Ϫ9.9, d and Ϫ10.7, d, JP, P
20.9 Hz) seemed to be the major side reaction, decreasing the
yield and resulting in α--galactofuranose 1,2-cyclophosphate
The phospho-imidazolidate activation14–16 was chosen for
the preparation of the pyrophosphate 1. This method was
DOI: 10.1039/b000210k
J. Chem. Soc., Perkin Trans. 1, 2000, 889–891
This journal is © The Royal Society of Chemistry 2000
889

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(δP 17.2)† and UMP 3 (δP 1.6). The nucleotide 3 then reacted
with the imidazolide 4 (δP Ϫ8.7), which is in excess, to give sym-
metrical diuridine diphosphate (δP Ϫ9.6, d, JP, P 21.5 Hz). After
the work-up, UDP-Galf 1 (a dipotassium salt) was isolated in
23% yield using gel-chromatography on a column of Sephadex
G-15 followed by reversed-phase C-18 HPLC.
It is noteworthy that coupling of the galactosyl phosphate
2 and UMP-morpholidate in pyridine in the presence of
1H-tetrazole, which was reported18 to be an efficient catalyst in
phospho-morpholidate condensations, produced the nucleotide
sugar 1 in 12% yield. The highest proportion of the product
(corresponding to 25–30% yield) in the reaction mixture was
observed at 15–17 h using 31P NMR spectroscopy.
minϪ1. Reversed-phase HPLC was done on a semipreparative
C-18 column (Chromsphere 5, 25 × 1 cm) using 0.02 mol dmϪ3
aq. KH2PO4 as eluent at 3 cm3 minϪ1 with UV-monitoring at
270 nm. Anhydrous pyridine and DMF were purchased from
Aldrich. Dichloromethane, toluene and pyridine were freshly
distilled from CaH2. All the solutions containing UDP-Galf
1 were concentrated in vacuo (oil pump, ≈0.01 mmHg) at
18–20 ЊC.
Uridine 5Ј-monophosphate disodium salt (Sigma) was con-
verted to the triethylammonium salt 3 (containing 1.42 equiv.
1
of Et3N, detected by H NMR spectrometry) by passing its
aq. solution through a column (15 × 2.5 cm) of Dowex
50W × 8 (triethylammonium form); the column was washed
with water, then appropriate fractions were pooled and
freeze-dried.
The structure of compound 1 was unambiguously confirmed
by NMR and mass spectrometry. The 31P NMR spectrum
exhibited two doublet signals (δP Ϫ10.1, d, Pα and Ϫ11.4, d, Pβ,
JP, P 20.9 Hz), which are characteristic for nucleoside diphospho-
sugars.9,14–16,18 The presence of the (1→5)-pyrophosphate bridge
was confirmed by the C-1, C-2, H-1 and H-2 signals of the
-galactofuranose residue and C-4 and C-5 signals of the
-ribofuranose residue (see Experimental section). These
signals were shifted as a result of the α- and β-effects of phos-
phorylation and coupled with phosphorus. The α-configuration
of the -galactofuranosyl phosphate fragment was evident
from the value of JH1,H2 4.2 Hz, which was close to that pub-
lished11 for the α-phosphate 2. The coupling constant value
for the corresponding isomeric β-phosphate was reported11 to
Dibenzyl 2,3,5,6-tetra-O-benzoyl-ꢀ-D-galactofuranosyl
phosphate 9
1-O-Acetyl-2,3,5,6-tetra-O-benzoyl-α,β--galactofuranose
6
{[α]D25 ϩ9.5 (c 1, CHCl3); δH (CDCl3) 2.10 (s, Acα), 2.22 (s, Acβ),
4.69–4.86 (3 H, m, 4-H and 6-H2), 5.64 (d, J2,3 1.4, 2-Hβ), 5.75
(dd, J3,4 4.8, 3-Hβ), 5.79 (dd, J2,3 7.1, 2-Hα), 5.91 (dt, J4,5
=
J5,6a = 6.0, J5,6b 3.8, 5-Hα), 6.12 (dt, J4,5 = J5,6a = 4.0, J5,6b 7.0,
5-Hβ), 6.27 (dd, J3,4 6.3, 3-Hα), 6.55 (s, 1-Hβ), 6.70 (d, J1,2 4.8,
1-Hα) and 7.15–8.20 (20 H, m, 4 × Ph); α:β = 1:3.7} was
prepared from methyl α,β--galactofuranoside 510 by conven-
tional benzoylation with benzoyl chloride in pyridine followed
by acetolysis with 1.4% H2SO4 in Ac2O (v/v).
1
be about 1.7 Hz. In general, our H, 13C and 13P NMR data
were in agreement with those published9 for UDP-Galf 1,
apart from distinctive assignments of signals for C-2, C-3 and
C-4 of the -ribofuranose residue, which were amended in the
light of the 13C NMR data of uridine 5Ј-monophosphate 3.19
The molecular mass of compound 1 was confirmed by electro-
spray mass spectrometry. The signals in the mass spectrum (see
Experimental section) corresponded to the pseudo-molecular
ions for the uridine diphosphohexose (M, 566.055 for the free
acid 1).
To a stirred solution of the tetrabenzoate 6 (3.13 g, 4.9 mmol)
in CH2Cl2 (30 cm3) cooled to 0 ЊC was added a 33% solution (10
cm3) of HBr in AcOH. After storage for 2 h at 0 ЊC and an
additional 2 h at rt, the mixture was concentrated to dryness
and the residue was azeotroped with toluene (3 × 30 cm3) to
give the galactofuranosyl bromide 8.
To a stirred mixture of dibenzyl hydrogen phosphate (1.91 g,
6.86 mmol) and Et3N (0.97 cm3, 6.95 mmol) in toluene (10 cm3)
was added a solution of the prepared bromide 8 in the same
solvent (25 cm3) and the stirring was continued for a further
16 h. The solid (Et3NHBr) was filtered off and the solvent was
removed under reduced pressure. FCC (toluene–ethyl acetate,
9:1 v/v) of the residue produced the protected α--galacto-
furanosyl phosphate 9 (2.48 g, 59%) as a solid, mp 110–111 ЊC;
[α]D26 ϩ51.3 (c 1, CHCl3); δP Ϫ2.35 {lit.,11 mp 112–113 ЊC; [α]D
ϩ54.6 (c 1, CHCl3); δP Ϫ3.66}. The 1H and 13C NMR data were
virtually identical to those published.11
It should be noted that UDP-Galf 1 is rather unstable: it was
hydrolysed (≈95% extent), forming -galactose and uridine 5Ј-
diphosphate in 0.015 mol dmϪ3 aq. solution after 24 h at 22 ЊC.
Nevertheless, a frozen solution of the compound was stored
at Ϫ80 ЊC for several months without detectable degradation.
A purity control could be properly performed using reversed-
phase C-18 HPLC.
The nucleotide sugar 1 is currently being tested as a substrate
for the Leishmania β--galactofuranosyl transferase and the
results will be published in due course.
ꢀ-D-Galactofuranosyl bis(triethylammonium) phosphate 2
The galactosyl phosphate 9 (1.82 g, 2.13 mmol) was depro-
tected by successive hydrogenation over palladium hydroxide
(20 wt% Pd on carbon, contains ≈50% of water; 1 g, ≈0.94
mmol Pd) in ethyl acetate–triethylamine (10:1) and debenzoyl-
ation with MeOH–water–triethylamine (5:2:1 v/v/v) following
the published11 procedure. The resulting syrup was dissolved
in water and applied to a column (12 × 2.5 cm) of Amberlite
IRA-420 (HCO3Ϫ-form) resin. The column was first washed
with water and then with a gradient of 0.1→0.4 mol dmϪ3 aq.
triethylammonium hydrogen carbonate. Appropriate fractions
were freeze-dried to give the phosphate 2 (0.865 g, 88%) as a
hygroscopic amorphous solid, [α]D26 ϩ44.5 (c 1, water); δP 0.43
{lit.,11 for the bis(cyclohexylammonium) salt [α]D ϩ41 (c 1,
Experimental
General procedures
Mps were determined on a Reichter hot-plate apparatus and
are uncorrected. Optical rotations were measured with a
Perkin-Elmer 141 polarimeter; [α]D-values are given in units
of 10Ϫ1 deg cm2 gϪ1. NMR spectra (1H at 300 MHz, 13C at
75 MHz, and 31P at 121 MHz) were recorded with a Bruker
DPX-300 spectrometer for solutions in deuterium oxide, unless
otherwise indicated. Chemical shifts (δ in ppm) are given
relative to those for Me4Si (for H and 13C) and external aq.
1
85% H3PO4 (for 31P); J-values are given in Hz. ES mass spectra
were recorded with a Micromass Quattro system (Micromass
Biotech, UK). Flash-column chromatography (FCC) was
performed on Kieselgel 60 (0.040–0.063 mm) (Merck). Gel-
1
water); δP 2.50}. The H NMR data were nearly identical to
those published.11
filtration chromatography was performed on
a column
(95 × 2.5 cm) of Sephadex G-15 in water, flow rate 1.5 cm3
Uridine 5Ј-(ꢀ-D-galactofuranosyl diphosphate), dipotassium salt 1
To a solution of uridine 5Ј-monophosphate 3 (93 mg, 0.20
mmol of the triethylammonium salt containing 1.42 equiv. of
Et3N) in DMF (1.5 cm3) was added 1,1Ј-carbonyldiimidazole
(97 mg, 0.60 mmol) and the mixture was kept for 3 h at rt.
† The 13P NMR chemical-shift value is characteristic for similar cyclic
phosphates:17 for uridine 2Ј,3Ј-cyclophosphate, δP 17.6; for 4-nitro-
phenyl α--galactopyranoside 3,4-cyclophosphate, δP 16.8.
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Methanol (0.05 cm3) was added to destroy the excess of reagent
and the reaction mixture was concentrated. The residue was
azeotroped with pyridine (3 × 2 cm3), then dried in vacuo for
15–20 min to produce the imidazolide 4. α--Galactofuranosyl
phosphate 2 (58 mg, 0.126 mmol) was dried by evaporation
of pyridine (3 × 2 cm3). A solution of the imidazolide 4 in
pyridine (2 cm3) was added and the resulting mixture was
taken to dryness again. The residue was dissolved in DMF
(1 cm3) and the solution was kept under argon at rt while the
reaction was monitored by 31P NMR spectroscopy. After 19 h,
the reaction mixture was diluted with water, washed with
chloroform and the aqueous phase was concentrated. Gel
chromatography of the residue (with monitoring by analytical
HPLC on C-18 column) provided UDP-Galf-containing
fractions, which were pooled, concentrated and repurified using
preparative HPLC to separate the product 1 from UMP 3 and
uridine 5Ј-diphosphate (the retention times were 4.50 min, 5.20
min and 4.15 min, respectively). The appropriate fractions were
desalted twice by gel filtration to produce UDP-Galf 1 (18.4
mg, 22.7%) as an amorphous solid, [α]D22 ϩ18.5 (c 0.75, water);
δH 3.49 (1 H, dd, J5,6a 7.0, J6a,6b 11.6, 6-Ha, Gal), 3.58 (1 H, dd,
J5,6b 4.1, 6-Hb, Gal), 3.64 (1 H, ddd, 5-H, Gal), 3.69 (1 H, dd,
J4,5 5.1, 4-H, Gal), 4.03 (1 H, ddd, J2,3 8.5, J2,P 2.2, 2-H, Gal),
4.07–4.17 (3 H, m, 4-H and 5-H2, Rib), 4.11 (1 H, dd, J3,4 7.1, 3-
H, Gal), 4.24 (2 H, m, 2- and 3-H, Rib), 5.51 (1 H, dd, J1,2 4.2,
J1,P 5.5, 1-H, Gal), 5.83 (1 H, d, J5,6 8.7, 6-H, uracil), 5.85 (1 H,
d, J1,2 4.9, 1-H, Rib) and 7.83 (1 H, d, 5-H, uracil); δC 62.6 (C-6,
Gal), 65.3 (d, J5,P 5.1, C-5, Rib), 70.1 (C-3, Rib), 72.5 (C-5,
Gal), 73.9 (C-3, Gal), 74.2 (C-2, Rib), 77.0 (d, J2,P 8.0, C-2,
Gal), 82.2 (C-4, Gal), 83.6 (d, J4,P 9.5, C-4, Rib), 88.7 (C-1,
Rib), 98.0 (d, J1,P 5.5, C-1, Gal), 103.1 (C-6, uracil) and 142.0
(C-5, uracil); δP Ϫ10.1 (d, JP, P 20.9, Pα) and Ϫ11.4 (d, Pβ);
ESMS(Ϫ) m/z 564.61 (65%, [M Ϫ H]Ϫ), 586.66 (100, [M Ϫ 2
H ϩ Na]Ϫ) and 602.60 (27, [M Ϫ 2 H ϩ K]Ϫ) (free acid C15H24-
N2O17P2 requires M, 566.055) {lit.,9 δP Ϫ10.5 (d, JP, P 20.2, Pα)
and Ϫ11.8 (d, Pβ); ESMS(Ϫ) m/z 565.1 (100%, [M Ϫ H]Ϫ),
602.9 (20, [M Ϫ 2 H ϩ K]Ϫ) and 640.8 (10, [M Ϫ 3 H ϩ 2
K]Ϫ)}.
Acknowledgements
This work and Y. E. T. were supported by a Wellcome Trust
International Grant. The research of A. V. N. was supported by
an International Research Scholar’s award from the Howard
Hughes Medical Institute. We are indebted to Professor M. A. J.
Ferguson (University of Dundee) for his interest.
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