Preparation of fluorinated galactosyl nucleoside diphosphates to study the mechanism of the enzyme galactopyranose mutase

Article abstract of DOI:10.1039/a701630a

A novel latent→active phosphorylation strategy has been employed for the preparation of two fluorinated nucleoside diphosphates (compounds I and II). The strategy is based on the isomerisation of substituted allyl to vinyl glycosides which were subsequently phosphorylated by treatment with dibenzyl hydrogen phosphate, N-iodosuccinimide and a catalytic amount of trimethylsilyl triflate. This methodology is very suitable for the preparation of nucleoside diphosphates that have a modification in the saccharide moiety since the allyl moiety serves first as an anomeric protecting group, allowing for protecting-group manipulation and functionalisation of the sugar ring, but after isomerisation to the corresponding vinyl glycoside it acts as an anomeric leaving group. The 2-F and 4-F Gal-UDP derivatives I and II do not inhibit the enzyme galactopyranose mutase in the direction pyranose → furanose but both compounds have been found to inhibit the reverse reaction.

Full text of DOI:10.1039/a701630a

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Preparation of fluorinated galactosyl nucleoside diphosphates to
study the mechanism of the enzyme galactopyranose mutase†
Andrew Burton,a Paul Wyatt b and Geert-Jan Boons*,a
a
School of Chemistry, The University of Birmingham, Edgbaston, Birmingham, UK B15 2TT
b Glaxo Wellcome, Medicines Research Centre, Gunnels Wood Road, Stevenage,
Hertfordshire, UK SG1 2NY
A novel latent→active phosphorylation strategy has been employed for the preparation of two fluorinated
nucleoside diphosphates (compounds I and II). The strategy is based on the isomerisation of substituted
allyl to vinyl glycosides which were subsequently phosphorylated by treatment with dibenzyl hydrogen
phosphate, N-iodosuccinimide and a catalytic amount of trimethylsilyl triflate. This methodology is
very suitable for the preparation of nucleoside diphosphates that have a modification in the saccharide
moiety since the allyl moiety serves first as an anomeric protecting group, allowing for protecting-group
manipulation and functionalisation of the sugar ring, but after isomerisation to the corresponding vinyl
glycoside it acts as an anomeric leaving group. The 2-F and 4-F G al-U D P derivatives I and II do not
inhibit the enzyme galactopyranose mutase in the direction pyranose → furanose but both compounds
have been found to inhibit the reverse reaction.
HO
Introduction
OH
O
N
O
F
+
+
-Galactose in the furanose form is found in oligo- and poly-
saccharides and glycoconjugates of certain bacteria, protozoa
and fungi,1 however it is not a constituent of mammalian sac-
charides. The first polysaccharide found to contain galacto-
furanose was galactocarolose which is an extracellular β--
(1→5)-linked polygalactofuranose produced by Penicillium
charlesii.2 Galactofuranoside is also present in the oligosac-
charide core of glycoinositol phospholipids of Trypanosoma
cruzi which is the infective agent of Chagas’ disease.3 Mycobac-
terium tuberculosis is another pathogen that contains this rather
unusual sugar, and an arabinogalactan consisting of ~30 α--
galactofuranose and ~60 α--arabinofuranose residues which is
linked to a peptidoglycan via a unique diglycosyl phosphoryl
bridge has been isolated from this organism.4
The restriction of the five-membered-ring configuration of
galactose to parasites makes the galactofuranose biosynthetic
pathway an important target for the design of novel anti-
bacterial, antiprotozoal and antifungal drugs. However, a
detailed knowledge of the biosynthesis of galactofuranose-
containing oligosaccharides is needed.
NH4
NH4
HO
NH
O –
O –
O
O
O
O
P
P
O
O
O
OH OH
I
F
OH
+
O
O
+
NH4
NH4
HO
NH
O –
O –
HO
O
O
N
O
O
O
P
P
O
O
OH OH
II
Synthetic targets: UDP-2-deoxy-2-fluorogalactose I and UDP-4-deoxy-
4-fluorogalactose II
It has been proposed that the activated sugar for incorpor-
ation of α--galactofuranosides into oligosaccharides is uridine
5Ј-diphosphate (UDP)-galactofuranose which is probably
derived via the ring contraction of a galactopyranose derivative.
Recently, a galactopyranose mutase enzyme that catalyses the
interconversion of UDP-galactopyranose and UDP-galacto-
furanose has been cloned and overexpressed.5 The equilibrium
facilitated by this enzyme is biased in favour of the pyranose
isomer, and at equilibrium 10% of the galactopyranose is con-
verted into the furanose form. The reaction mechanism of ring
contraction is not yet known but it has been proposed that the
2-hydroxy group of galactose is involved.6
In this paper we report the synthesis of fluorinated UDP-
galactopyranose derivatives I and II which will be valuable
compounds in an investigation, at a molecular level, of the
enzymic interconversion of UDP-galactopyranose and UDP-
galactofuranose. The galactopyranose nucleoside diphosphate I,
having a fluorine atom at the 2-position of galactose, may facili-
tate mechanistic investigation of the action of galactopyranose
mutase and help establish the involvement of the 2-hydroxy
group in the ring contraction. Furthermore, the fluorine label
will enable any interconversion to be monitored by the use of
19F NMR spectroscopy. The derivative II has been substituted
with a fluorine atom at the 4-position of galactose and this
modification should have a very minor effect on the structural
and electronic properties of the molecule. Therefore, it is to be
expected that compound II will be recognised by the galacto-
pyranose mutase and act as an inhibitor of the enzyme, since
the 4-hydroxy group is required for the furanose ring to be
formed.
Results and discussion
It has been found that vinyl glycosides are efficient glycosyl
donors for the synthesis of O-glycosidic linkages.7 Recently
we described a new, latent→active anomeric phosphorylation
strategy based on the isomerisation of substituted allyl to
† Financial support for this project was provided by the Engineering
and Physical Sciences Research Council and Glaxo-Wellcome (Student-
ship for A. B.).
J. Chem. Soc., Perkin Trans. 1, 1997
2375

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vinyl glycosides which were subsequently phosphorylated by
treatment with dibenzyl hydrogen phosphate, N-iodosuc-
cinimide (NIS) and a catalytic amount of trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf).8,9 In this strategy, the allyl
moiety serves two purposes: (i) it acts as an anomeric protecting
group, allowing for protecting group manipulation and func-
tionalisation of the sugar ring and (ii) it functions as a latent
leaving group, since isomerisation gives a vinyl glycoside which
can be used as a glycosyl donor.
AcO
O
AcO
AcO
O
AcO
11
85%
(a), (b)
O
Ph
O
O
O
RO
RO
12 R = H
13 R = Bn
It was envisaged that the features of allyl and vinyl glycosides
would make them versatile synthons for the preparation of gly-
cosyl phosphates 9 and 18 (Schemes 1 and 2) and, after depro-
(c) 99%
(d) 70%
OAc
AcO
AcO
OR
O
F
OBn
O
OBn
O
O
(a), (b)
60%
(f)
O
O
O
O
RO
BnO
O
BnO
74%
HO
O
AcO
1
BnO
2 R = H
3 R = Tr
BnO
(c) 62%
16
14 R = H
15 R = Tf
(e) 100%
(g)
74%
(d), (e)
97%
12
F
OTr
O
OTr
OBn
O
F
O
OBn
O
(h)
OR
O
F
O
(g)
54%
O
O
O
BnO
O
O
BnO
33%
BnO
6
BnO
OBn
OBn
O
4 R = H
5 R = Tf
P
(f) 100%
(h), (i)
85%
O
17
18
OBn
OBn
BnO
BnO
BnO
(i), (j)
90%
(j)
O
F
O
O
O
BnO
78%
F
F
7
8
OH
O
+
(k)
60%
(k) 85% (α:β = 5.8:1)
Et3NH
HO
HO
OH
O –
O –
HO
BnO
OBn
O
O
F
P
OH
O
+
(l), (m)
O
Et3NH
+
O
+
O
HO
BnO
NH4
99%
NH4
O –
19
+
O–
F
HO
NH
F
OBn
OBn
Et3NH
O–
O
O –
10
9
O
HO
P
+
P
O
O
N
O
O
Et3NH
P
P
O
O
O
O
O
(n)
82%
HO
OH
OH OH
O
O
F
+
+
NH4
NH4
II
HO
NH
Scheme 2 Synthesis of diammonium 4-deoxy-4-fluoro-α--galacto-
pyranosyl uridin-5Ј-yl diphosphate II. Reagents and conditions: (a)
KOtBu, MeOH; (b) benzaldehyde dimethyl acetal, acetonitrile, CSA;
(c) benzyl bromide, NaH, DMF; (d) TFA, triethylsilane, DCM
(Ϫ20 ЊC→room temp.); (e) Tf2O, pyridine, DCM (Ϫ78 ЊC→room
temp.); (f) TBAF, THF; (g) Rh(PPh3)3Cl, BuLi, THF; (h) (BnO)2PO2H,
NIS, TMSOTf, DCM, molecular sieves (4 Å); (i) 10% Pd/C, H2, ethyl
acetate–propan-2-ol–water (4:6:2 v/v); (j) DOWEX 1-X8[Et3NHϩ]; (k)
uridine 5Ј-monomorpholidophosphate, pyridine.
O –
O –
O
O
N
O
O
P
P
O
I
O
Tr = triphenylmethyl
Tf = trifluoromethanesulfonate
O
OH OH
Scheme 1 Synthesis of diammonium 2-deoxy-2-fluoro-α--galacto-
pyranosyl uridin-5Ј-yl diphosphate I. Reagents and conditions: (a)
KOtBu, MeOH; (b) 2,2-dimethoxypropane, acetonitrile, CSA; (c) trityl
chloride, pyridine; (d) (i) oxalyl dichloride, DMSO, DCM (Ϫ78 ЊC), (ii)
Et3N; (e) LiAlH4, THF; (f) Tf2O, pyridine, DCM (Ϫ78 ЊC→room
temp.); (g) TEAF, acetonitrile (50 ЊC); (h) AcOH, aq. TFA; (i) benzyl
bromide, NaH, DMF; (j) Rh(PPh3)3Cl, BuLi, THF; (k) (BnO)2PO2H,
NIS, TMSOTf, DCM, molecular sieves (4 Å); (l) 10% Pd/C, H2, ethyl
acetate, propan-2-ol, water (4:6:2 v/v); (m) DOWEX 1-X8[Et3NHϩ];
(n) uridine 5Ј-monomorpholidophosphate, pyridine.
glycosides does not affect the chemistry, it complicates the
interpretation of the NMR spectra. Multi-gram quantities of
optically pure but-3-en-2-ol, however, can easily be obtained by
a literature procedure.11 Deacetylation of compound 1 with
potassion tert-butoxide in methanol, followed by treatment
with acetone, 2,2-dimethoxypropane and a catalytic amount of
acid yielded the partially protected allyl glycoside 2 in good
yield. Subsequent regioselective tritylation with trityl chloride
in pyridine gave compound 3, the hydroxy group of which was
substituted by a fluorine atom (→ 6) in a double-inversion
procedure. Thus, Swern oxidation of secondary alcohol 3 gave
the corresponding ketone which, after purification using silica
gel column chromatography, was reduced with LiAlH4 in tetra-
hydrofuran (THF ) to provide, in almost quantitative yield,
tection, it should be easy to convert these anomeric phosphates
into the corresponding nucleotide diphosphates I and II via a
standard method.10
Glycosyl donor 8, which is the precursor of the anomeric
phosphate 9, was prepared from the readily available allyl galac-
toside 1.8 Racemic but-3-en-2-ol was used for the preparation
of compound 1, resulting in the formation of a mixture of
diastereoisomers. While the diastereoisomeric nature of the
2376
J. Chem. Soc., Perkin Trans. 1, 1997

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1
the talose derivative 4. The H NMR spectrum of compound
Having successfully prepared the UDP-2-deoxy-2-fluoro-
4 showed that none of the isomeric galactoside was present,
and the coupling constant J1,2 1.8 Hz, typical of a 1,2-cis link-
age, confirmed the stereochemistry of the product. Next, fluor-
ine was introduced, first by treatment of compound 4 with
trifluoromethanesulfonic anhydride and pyridine in dichloro-
methane (DCM) to give the corresponding triflate 5 which was
immediately substituted by reaction with tetraethylammonium
fluoride (TEAF ) in acetonitrile at 50 ЊC to furnish the galacto-
side derivative 6. It is important to note that the isopropylidene
moiety of the intermediate triflate prevents a competing elimin-
ation reaction.12 Compound 6 was converted into the benzyl-
ated allyl glycoside 7 by treatment with a mixture of acetic acid
and trifluoroacetic acid (TFA) in water followed by benzylation
with benzyl bromide and sodium hydride in dimethylform-
amide (DMF ). The allyl moiety of compound 7 remained
intact throughout the synthetic manipulations described above,
but was now readily converted into an efficient anomeric leav-
ing group by isomerisation to give the vinyl glycoside 8. The
isomerisation of compound 7 was conveniently performed with
a catalytic amount of Wilkinson’s catalyst, treated with BuLi
in refluxing THF 13 and, after a reaction time of 30 min, com-
pound 8 was isolated in 78% yield. Under these isomerisation
conditions, we believe that Wilkinson’s catalyst is converted
into a rhodium hydride species which is superior for isomeris-
ation. It is also noteworthy that these isomerisation conditions
are compatible with the presence of a fluorine atom in the
glycoside.
The anomeric phosphate was introduced by reaction of a
solution of compound 8 in DCM with dibenzyl hydrogen
phosphate, NIS and a catalytic amount of TMSOTf. When the
reaction was performed at Ϫ20 ЊC, the anomeric phosphate 9
was isolated in a good yield but the undesired β-anomer was
the main product (65%; α:β = 1:2). Fortunately, an acceptable
α-selectivity could be achieved by performing the reaction at
room temperature (85%; α:β = 5.8:1) and the anomers could be
separated by flash chromatography. It is conceivable that when
the reaction is performed at room temperature, the initially
produced β-anomer may anomerise to give the thermo-
dynamically more stable α-anomer. However, attempted isom-
erisation of the β-phosphate by treatment with a catalytic
amount of hydrochloric acid failed and starting material was
recovered together with some trehalose. It should be noted that
others have successfully isomerised related compounds under
similar conditions.9a,b
galactose I, we focused our attention on the synthesis of the
UDP-galactose derivative having a fluorine at the 4-position
of the galactose. In this case, the allyl glucoside 11 was sel-
ected as the starting material and it was envisaged that the
galactose configuration could be obtained by conversion of
the 4-hydroxy group of a glucoside into a good leaving group
followed by substitution with a fluoride nucleophile. The glu-
coside 14 is the appropriate precursor for this transformation
and was obtained by a four-step procedure. Thus, deacetyl-
ation of compound 11 with KOtBu in methanol followed by
treatment with benzaldehyde dimethyl acetal and a catalytic
amount of camphor-10-sulfonic acid (CSA) gave regioselec-
tively the partially protected saccharide 12. Benzylation of
compound 12 under standard conditions afforded the fully
protected species 13, the benzylidene acetal of which was re-
ductively opened with triethylsilane and TFA 14 to yield the
required compound 14. The fluorine of intermediate target 16
was conveniently introduced by treatment of compound 14
with trifluoromethanesulfonic anhydride and pyridine in DCM,
to give triflate 15 and immediate nucleophilic displacement with
tetrabutylammonium fluoride (TBAF ) in THF. The anomeric
phosphate was obtained by isomerisation of the allyl moiety of
compound 16 to give the vinyl glycoside 17, which was phos-
phorylated by reaction with dibenzyl hydrogen phosphate, NIS
and a catalytic amount of TMSOTf. The success of this phos-
phorylation also depended strongly on the reaction temper-
ature. When the phosphorylation was performed at Ϫ20 ЊC,
compound 18 was isolated as a single anomer but in a disap-
pointing yield of 35%. The yield could be improved to 54% by
performing the reaction at room temperature. A substantial
amount of the undesired β-anomer is probably formed at low
reaction temperatures and which, in this case, hydrolyses during
the work-up and purification procedure. As for vinyl glycoside
8, the phosphorylation of compound 17 at a higher temper-
ature gave better α-selectivity which here is reflected in a higher
yield. Having compound 18 in hand, the synthesis of target II
was completed as performed for compound I. Thus, hydro-
genolysis of the benzyl ethers of compound 18 yielded com-
pound 19, which was condensed with uridine 5Ј-monomorpho-
lidophosphate under standard conditions to give, after puri-
fication by ion-exchange chromatography, the requisite UDP-
4-fluoro-4-deoxygalactose II. Compound II was also found to
be contaminated with a small amount of side-product (UPPU)
and the spectral data obtained were in accord with those
published previously.15
Next, the anomeric phosphate 9 was debenzylated by cata-
lytic hydrogenation over Pd/C in the presence of sodium acet-
ate, and the phosphate 10 was obtained as a triethylammonium
salt in a good yield after treatment of the product with Dowex
1-X8[Et3NHϩ] and purification by size-exclusion column
chromatography (LH-20). Finally, condensation of compound
10 with uridine 5Ј-monomorpholidophosphate in pyridine for
5 days gave, after ion-exchange column chromatography, the
requisite UDP-derivative I in a very pleasing yield of 82%. The
high yield in this coupling may be attributed to the stabilising
effect of the fluorine atom on the anomeric linkage. The spec-
tral analysis obtained for this compound confirmed it to be
the target compound I. The fluorine coupling constants in the
1H and 13C NMR spectra were consistent with a 2-deoxy-2-
fluoro glycoside and the α-glycosidic linkage was confirmed by
the anomeric coupling constants; J1,P 7.4 Hz, J1,2 3.7 Hz and
J1,F < 0.5 Hz. The presence of two multiplets of similar inten-
sity in the 31P NMR spectrum confirmed the presence of a
diphosphate linkage and matrix-assisted laser desorption/
ionisation time-of-flight (MALDI-TOF ) spectrometry gave a
negative ion that corresponded in m/z to the singly protonated
diphosphate. The presence of a small amount of side-product
was also observed in the sample of compound I and it is thought
that this corresponded to the symmetric by-product P1,P2-bis-
5Ј-uridine diphosphate (UPPU).
Preliminary inhibition studies5 show that derivatives I and II
do not inhibit the enzyme galactopyranose mutase in the direc-
tion pyranose → furanose but both compounds were found
to inhibit the reverse reaction. At concentrations of 166 µ
of inhibitor and 35 µ UDP-galactofuranose, 55 and 48%
inhibition of the transformation furanose → pyranose was
observed for compounds I and II respectively. The Km-value for
the enzyme is 1 m. Currently, we are examining whether com-
pound I can be converted by the enzyme into the analogous
furanose derivative and this experiment will firmly establish the
importance of the 2-hydroxy group for the enzymic transform-
ation. The full experimental detail of the inhibition studies will
be reported elsewhere.
Conclusions
We have described the preparation of two modified UDP-
galactosyluridine diphosphates (I and II) using a novel latent→
active phosphorylation strategy. A latent anomeric allyl moiety
functioned as an efficient anomeric protecting group but could
be converted into a leaving group by a Rh-catalysed isomeris-
ation to give a vinyl glycoside. The vinyl glycoside underwent
clean phosphorylation and the anomeric outcome of the reac-
tion could be controlled by the reaction temperature. The ano-
J. Chem. Soc., Perkin Trans. 1, 1997
2377

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meric phosphates 9 and 18 were converted by a known pro-
cedure into target nucleoside diphosphates I and II.
Initial biochemical investigations have revealed that deriva-
tives I and II do not inhibit the enzyme galactopyranose mutase
in the direction pyranose → furanose but both compounds
were found to inhibit the reverse reaction.
(C-2, -3, -4 and CHCH3), 73.7–73.5 (C-5 and -6), 28.1 and 26.3
(CH3CO2) and 21.6 and 20.4 (CHCH3); FAB m/z 297
([M ϩ Na]ϩ) (Found: C, 57.00; H, 8.13. C13H22O6 requires C,
56.91; H, 8.08%).
(R/S)-But-3-en-2-yl 3,4-O-isopropylidene-6-O-trityl-â-D-
galactopyranoside 3
Compound 2 (2.45 g, 9.27 mmol) was dissolved in pyridine (10
ml) and trityl chloride (2.58 g, 9.27 mmol) was added. The
mixture was stirred under argon and TLC analysis (acetone–
DCM, 1:49 v/v) after 16 h showed the conversion of the start-
ing material (Rf 0.07) into a major product (Rf 0.34). The sol-
ution was concentrated to dryness in vacuo, the residue was re-
dissolved in ethyl acetate (20 ml), and the resulting solution was
washed successively with saturated aq. sodium hydrogen car-
bonate (2 × 10 ml) and brine (2 × 10 ml). The organic phase
was dried (MgSO4), and concentrated to dryness in vacuo to
yield a sticky yellow solid. Purification using flash chromato-
graphy (100 g SiO2; eluent ethyl acetate–light petroleum, 1:3
v/v) and concentration of the appropriate fractions yielded
compound 3 (2.96 g, 62%) as a solid, δH (300 MHz; CDCl3) 7.52–
7.20 (m, 15 H, ArH), 6.07–5.52 (m, 1 H, CH᎐CH ), 5.28–5.04
Experimental
Reagents were purchased from Aldrich, Fluka and Lancaster.
All reaction solvents were distilled prior to use: DCM,
acetonitrile, pyridine, diethyl ether and DMF from calcium
hydride, THF from lithium aluminium hydride, acetone from
potassium carbonate and methanol from its magnesium Grig-
nard. Flash silica gel column chromatography was carried out
on silica gel (Merck 9385). Thin layer chromatography (TLC)
analysis was carried out on silica gel-coated aluminium plates
(Merck 12.05554 Kieselgel 60 F254). Compounds were visual-
ised by UV light (254 nm) and/or by dipping in H2SO4–
methanol (1:10 v/v) and subsequent charring. NMR spectra
were recorded on Bruker AC 300 and 250 spectrometers
equipped with Aspect 3000 computers. For H and 13C spectra,
1
᎐
2
chemical shifts are given in parts per million (δ) relative to
tetramethylsilane. For 19F spectra, chemical shifts are given in
parts per million (δ) relative to CFCl3, and for 31P spectra,
chemical shifts are given in parts per million (δ) relative to
H3PO4. Presaturation of water signals was used on all D2O
spectra. J-values are given in Hz. Fast-atom bombardment
(FAB) mass spectra were recorded using a VG Zabspec spec-
trometer with m-nitrobenzyl alcohol as matrix. Chemical ion-
isation (CI) mass spectra were recorded on a VG Prospec spec-
trometer using ammonia as reagent gas. Negative-ion MALDI-
TOF mass spectra were recorded using a Kratos Kompact
instrument using an indole–acrylic acid matrix. Light petrol-
eum refers to the 40–60 ЊC fraction.
(m, 2 H, CH᎐CH ), 4.44–4.23 (m, 1 H, CHCH ), 4.20 (2 d, J
᎐
2 3 1,2
8.5, 1 H, H-1), 4.18–3.98 (m, 2 H, H-3 and -4), 3.77–3.68 (m, 1
H, H-5), 3.60–3.34 (m, 3 H, H-2 and H2-6), 1.55–1.52 (2 s, 3 H,
CH3CO2), 1.39–1.25 (2 s, 3 H, CH3CO2) and 1.24–1.12 (m, J 6,
3 H, CHCH3); δC(75 MHz; CDCl3) 144.0 (ArC quaternary),
140.1 and 138.9 (CH᎐CH ), 128.8–127.0 (ArCH), 117.6 and
᎐
2
115.2 (CH ᎐CH), 110.1 (CH CO ), 100.6 and 98.9 (C-1), 86.7
᎐
2
3
2
(Ph3CO), 78.8–72.6 (C-2, -3, -4, -5 and CHCH3), 62.9 (C-6),
25.6 and 25.5 (CH3CO2) and 21.1 and 20.2 (CHCH3); FAB m/z
539 ([M ϩ Na]ϩ) (Found: C, 74.26; H, 6.91. C32H36O6 requires
C, 74.39; H, 7.02%).
(R/S)-But-3-en-2-yl 3,4-O-isopropylidene-6-O-trityl-â-D-
talopyranoside 4
(R/S)-But-3-en-2-yl 3,4-O-isopropylidene-â-D-galactopyranoside
2
To a cooled (Ϫ60 ЊC) solution of oxalyl dichloride (0.55 ml,
6.33 mmol) in DCM (5 ml) was added dimethyl sulfoxide
(DMSO) (0.88 ml, 10.23 mmol). After 10 min, a solution of
compound 3 (2.85 g, 5.52 mmol) in DCM (10 ml) was added
dropwise and the solution was stirred under argon at reduced
temperature (Ϫ60 ЊC). After 2 h, triethylamine (5 ml) was
added and TLC analysis (acetone–DCM, 1:49 v/v) showed the
conversion of starting material (Rf 0.34) into an intermediate
product (Rf 0.48). The solution was concentrated to dryness in
vacuo and ethyl acetate (20 ml) was added. The resulting solu-
tion was washed successively with saturated aq. sodium hydro-
gen carbonate (2 × 10 ml) and brine (2 × 10 ml). The organic
phase was dried (MgSO4), and concentrated to dryness in vacuo
to yield a sticky yellow solid. Purification using flash chrom-
atography (100 g SiO2; eluent ethyl acetate–light petroleum, 1:3
v/v) and concentration of the appropriate fractions gave a solid,
which was dissolved in THF (5 ml) and to the resulting, stirred
solution was added lithium aluminium hydride (725 mg, 19.0
mmol). TLC analysis (acetone–DCM, 1:24 v/v) immediately
after the addition showed the conversion of the starting
material (Rf 0.76) into a major product (Rf 0.67). Ethyl acetate
(20 ml) was added to the reaction mixture and the resulting
solution was washed successively with 1  aq. NaOH (2 × 10
ml) and brine (2 × 10 ml). The organic phase was dried
(MgSO4), and concentrated to dryness in vacuo to yield a waxy
yellow solid. Purification using flash chromatography (100 g
SiO2; eluent ethyl acetate–light petroleum, 1:3 v/v) and concen-
tration of the appropriate fractions yielded compound 4 (2.76 g,
97%) as a solid, δH (300 MHz; CDCl3) 7.53–7.20 (m, 15 H,
Potassium tert-butoxide (0.56 g, 5.00 mmol) was added to a
stirred solution of (R/S)-but-3-en-2-yl 2,3,4,6-tetra-O-acetyl-β-
-galactopyranoside 1 (6.00 g, 14.93 mmol) in dry methanol (20
ml), and the resulting mixture was stirred at room temperature
under argon. After 3.5 h, TLC analysis (acetone–DCM, 1:19
v/v) showed the complete conversion of the starting material
(Rf 0.47) into a product (Rf 0.00). The reaction mixture was
neutralised with Dowex 1-X8[Hϩ] and, after filtration, concen-
trated to dryness in vacuo to yield a solid, which was dissolved in
acetone (10 ml) and to the resulting solution was added 2,2-
dimethoxypropane (3.0 ml, 24.4 mmol), followed by a catalytic
amount of CSA (~5 mg). After stirring of the mixture for 2 h
under argon, TLC analysis (methanol–DCM, 1:9 v/v) showed
the conversion of the starting material (Rf 0.06) into a major
product (Rf 0.48). The reaction was quenched by the addition
of pyridine (0.30 ml) and the reaction mixture was concentrated
to dryness in vacuo to give a yellow oil, which was redissolved in
ethyl acetate (30 ml) and the solution was washed successively
with saturated aq. sodium hydrogen carbonate (2 × 10 ml) and
brine (2 × 10 ml). The organic phase was dried (MgSO4), and
concentrated to dryness in vacuo to yield a yellow oil. Purific-
ation using flash chromatography (100 g SiO2; eluent ethyl
acetate–light petroleum, 3:7 v/v) and concentration of the
appropriate fractions yielded title compound 2 as a solid (2.45
g, 60%), δ (300 MHz; CDCl ) 6.01–5.62 (m, 1 H, CH᎐CH ),
᎐
H
3
2
5.23–5.03 (m, 2 H, CH᎐CH ), 4.38–4.17 (m, 1 H, CHCH ), 4.23
᎐
2
3
(2 d, J1,2 8.1, 1 H, H-1), 4.13–3.02 (m, 2 H, H-3 and -4), 3.97–
3.86 (m, 1 H, H-2), 3.84–3.69 (m, 2 H, H2-6), 3.56–3.48 (m, 1 H,
H-5), 1.50–1.46 (2 s, 3 H, CH3CO2), 1.39–1.25 (2 s, 3 H,
CH3CO2) and 1.24–1.12 (m, J 6, 3 H, CHCH3); δC(75 MHz;
ArH), 6.07–5.60 (m, 1 H, CH᎐CH ), 5.28–5.02 (m, 2 H,
᎐
2
CH᎐CH ), 4.57 (2 d, J 1.8, 1 H, H-1), 4.42–4.08 (m, 3 H, H-
᎐
2
1,2
3, -4 and CHCH3), 3.78–3.68 (m, 2 H, H2-6), 3.62–3.30 (m, 2
H, H-2 and -5), 1.60–1.52 (2 s, 3 H, CH3CO2), 1.40–1.30 (2 s, 3
H, CH3CO2) and 1.27–1.12 (m, J 6, 3 H, CHCH3); δC(75 MHz;
CDCl3) 140.6 and 138.7 (CH᎐CH ), 117.7 and 115.0
᎐
2
(CH ᎐CH), 110.4 (CH CO ), 101.0 and 98.9 (C-1), 79.1–75.0
᎐
2
3
2
2378
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
CDCl ) 144.0 (ArC quaternary), 140.5 and 139.1 (CH᎐CH ),
mixture was poured onto crushed ice (5 g), extracted with
᎐
3
2
128.8–127.1 (ArCH), 117.4 and 114.8 (CH ᎐CH), 110.0
diethyl ether (20 ml), and the extract was washed successively
with saturated aq. sodium hydrogen carbonate (2 × 10 ml) and
brine (2 × 10 ml). The organic phase was dried (MgSO4), and
concentrated to dryness in vacuo to yield a sticky yellow solid.
Purification using flash chromatography (30 g SiO2; eluent ethyl
acetate–light petroleum, 1:5 v/v) and concentration of the
appropriate fractions yielded compound 7 (382 mg, 85%) as a
solid, δH (300 MHz; CDCl3) 7.37–7.20 (m, 15 H, ArH), 6.02–
᎐
2
(CH3CO2), 98.0 and 96.6 (C-1), 86.8 (Ph3CO), 79.7–71.8 (C-2,
-3, -4, -5 and CHCH3), 63.1 (C-6), 25.6 and 25.5 (CH3CO2) and
21.1 and 20.2 (CHCH3); FAB m/z 539 ([M ϩ Na]ϩ) (Found: C,
74.14; H, 7.15. C32H36O6 requires C, 74.39; H, 7.02%).
(R/S)-But-3-en-2-yl 2-deoxy-2-fluoro-3,4-O-isopropylidene-6-O-
trityl-â-D-galactopyranoside 6
Compound 4 (2.68 g, 5.18 mmol) was dissolved in DCM (20
ml), and pyridine (3 ml) was added. The stirred solution was
cooled (Ϫ78 ЊC) under argon and trifluoromethanesulfonic
anhydride (Tf2O) (0.78 ml, 1.30 g, 5.20 mmol) was added. The
reaction mixture was allowed to warm to room temperature
and TLC analysis (acetone–DCM, 1:49 v/v) after 40 min
showed the complete conversion of starting material (Rf 0.32)
into an intermediate product (Rf 0.76). The reaction mixture
was diluted with DCM (50 ml) and washed successively with
saturated aq. sodium hydrogen carbonate (2 × 10 ml) and brine
(2 × 10 ml). The organic phase was dried (MgSO4), and concen-
trated to dryness in vacuo to yield a sticky yellow solid. Purific-
ation using flash chromatography (SiO2 100 g; eluent ethyl
acetate–light petroleum, 1:3 v/v) and concentration of the
appropriate fractions gave compound 5 as a solid. The solid was
immediately dissolved in acetonitrile (30 ml), and the solution
was stirred under argon and heated (50 ЊC). TEAF hydrate
(3.00 g, 20.10 mmol) was added to the solution and TLC analy-
sis (acetone–DCM, 1:49 v/v) after 2 h showed the conversion
of compound 5 (Rf 0.76) into a major product (Rf 0.71). The
reaction mixture was concentrated to dryness in vacuo, the resi-
due was re-dissolved in ethyl acetate (30 ml) and the solution
was washed successively with saturated aq. sodium hydrogen
carbonate (2 × 10 ml) and brine (2 × 10 ml). The organic phase
was dried (MgSO4), and concentrated to dryness in vacuo to
yield a sticky yellow solid. Purification using flash chrom-
atography (50 g SiO2; eluent ethyl acetate–light petroleum, 1:3
v/v) and concentration of the appropriate fractions yielded com-
pound 6 (887 mg, 33%) as a solid, δH (300 MHz; CDCl3)
5.62 (m, 1 H, CH᎐CH ), 5.27–5.03 (m, 2 H, CH᎐CH ), 4.96–
᎐ ᎐
2 2
4.23 (m, 7 H, H-1, -2, CHCH3 and ArCH2), 3.95–3.90 (m, H-5),
3.66–3.50 (m, 4 H, H-3, -4 and H2-6) and 1.36–1.21 (2 d, J 6, 3
H, CHCH ); δ (75 MHz; CDCl ) 140.2 and 139.1 (CH᎐CH ),
᎐
3
C
3
2
138.4–137.9 (ArC quaternary), 128.4–127.6 (ArCH), 117.0 and
115.1 (CH ᎐CH), 99.3 and 98.4 (d, J 23.2, C-1), 92.0 (d, J2,F
᎐
2
1,F
183.7, C-2), 80.3 (d, J3,F 7.9, C-3), 77.5 and 73.6 (C-4 and -5),
74.7–72.7 (ArCH2), 68.7 (C-6) and 21.7 and 20.4 (CHCH3);
δF (282 MHz; CDCl3) Ϫ205.5 (ddd, J2,F 52.14, J3,F 12.71,
J1,F < 1); FAB m/z 529 ([M ϩ Na]ϩ) (Found: C, 73.41; H, 7.11.
C31H35FO5 requires C, 73.49; H, 6.96%).
(E/Z)-But-2-en-2-yl 3,4,6-tri-O-benzyl-2-deoxy-2-fluoro-â-D-
galactopyranoside 8
Butyllithium (1.5  in THF; 1000 µl, 1.5 mmol) was added to a
stirred, orange solution of Wilkinson’s catalyst (500 mg, 0.05
mmol) in THF (1 ml) under argon. The resulting brown solu-
tion was stirred at 20 ЊC for 5 min and was then transferred via
syringe into a refluxing solution of compound 7 (175 mg, 0.35
mmol) in THF (10 ml) under argon. TLC analysis (acetone–
DCM, 1:49 v/v) after 30 min showed complete conversion of
starting material (Rf 0.73) into a product (Rf 0.75). The reaction
mixture was diluted with DCM (20 ml) and concentrated to
afford an orange-red residue, which was purified by flash chrom-
atography (20 g SiO2; eluent DCM) to afford the isomeric
compound 8 (136 mg, 78%) as a solid, δH (300 MHz; CDCl3)
7.37–7.20 (m, 15 H, ArH), 4.97–4.37 (m, 9 H, H-1, -2, ArCH2
and C᎐CH), 3.99–3.92 (m, 1 H, H-5), 3.68–3.57 (m, 4 H, H-3, -4
᎐
and H2-6), 1.92–1.82 (m, 3 H, CH3CO) and 1.67–1.49 (m, 3 H,
7.53–7.20 (m, 15 H, ArH), 6.07–5.65 (m, 1 H, CH᎐CH ),
CH CH); δ (75 MHz; CDCl ) 151.6 and 149.5 [OC(CH )᎐C],
᎐
3 C 3 3
᎐
2
5.32–5.05 (m, 2 H, CH᎐CH ), 4.46–4.18 (m, 5 H, H-1, -2, -3, -4
138.4–137.8, (ArC quaternary), 138.8–127.6 (ArCH), 106.7,
᎐
2
and CHCH3), 3.76–3.69 (m, 1 H, H-5), 3.56–3.32 (m, 2 H, H2-
6), 1.60–1.52 (2 s, 3 H, CH3CO2), 1.40–1.30 (2 s, 3 H, CH3CO2)
and 1.27–1.12 (m, J 6, 3 H, CHCH3); δC(75 MHz; CDCl3) 143.9
98.9 (᎐CHCH ), 98.8 and 98.1 (d, J 24.3, C-1), 92.4, 98.1 (d,
᎐
3
1,F
J2,F 183.7, C-2), 80.3 (2 d, J3,F 11.3, C-3), 74.2 and 73.8 (C-4 and
-5), 74.8–72.8 (ArCH2), 68.5 (C-6), 18.6 and 15.6 (CH3CO) and
11.9 and 10.3 (CHCH3); δF (282 MHz; CDCl3) Ϫ204.3 and
Ϫ204.7 (2 ddd, J4,F 52.1, J3,F 14.0, J5,F 12.7); FAB m/z 529
([M ϩ Na]ϩ) (Found: C, 73.59; H, 6.89. C31H35FO5 requires C,
73.49; H, 6.96%).
(ArC quaternary), 140.0 and 138.8 (CH᎐CH ), 128.8–125.2
᎐
2
(ArCH), 117.4 and 115.4 (CH ᎐CH), 110.6 (CH CO ), 96.8 (d,
᎐
2
3
2
J1,F 23.7, C-1), 93.5 (d, J2,F 184.8, C-2), 86.8 (Ph3CO), 77.8 and
74.9 (C-4 and -5), 72.4 (d, J3,F 12.4, C-3), 62.7 (C-6), 25.6 and
25.5 (CH3CO2) and 21.1 and 20.2 (CHCH3); δF (282 MHz;
CDCl3) Ϫ218.6 (ddd, J2,F 48.3, J3,F 22.9, J1,F 3.8); FAB m/z
541 ([M ϩ Na]ϩ) (Found: C, 74.02; H, 6.87. C32H35FO5 requires
C, 74.11; H, 6.80%).
Dibenzyl 3,4,6-tri-O-benzyl-2-deoxy-2-fluoro-á-D-galacto-
pyranosyl phosphate 9
A mixture of vinyl glycoside 8 (135 mg, 0.24 mmol) in DCM
(1 ml) and molecular sieves (100 mg, 4 Å powdered) were stirred
for 1 h under argon. NIS (200 mg, 0.88 mmol) was suspended in
DCM (2 ml) by sonication in a separate reaction vessel. Diben-
zyl hydrogen phosphate (225 mg, 0.81 mmol) was added to the
suspension and the mixture was allowed to equilibrate under
argon for 5 min. TMSOTf (18.7 µl, 0.094 mmol) was then added
and the suspension was stirred for a further 30 s. The resulting
solution (1.1 ml) was then added by syringe to the ready pre-
pared solution of the vinyl glycoside 8. Immediate TLC analysis
(acetone–DCM, 1:99 v/v) of the reaction mixture showed the
conversion of the starting material (Rf 0.70) into a major prod-
uct (Rf 0.47). The reaction mixture was neutralised by the add-
ition of triethylamine, and ethyl acetate (20 ml) was added. The
resulting solution was washed successively with aq. sodium
metabisulfate (2 × 5 ml) and brine (2 × 5 ml). The combined
organic phases were dried (MgSO4), concentrated to dryness in
vacuo, and the resulting oil was purified using flash chroma-
tography (25 g SiO2; eluent THF–toluene, 1:9 v/v). Concentra-
tion of the appropriate fractions yielded compound 9 (121 mg,
(R/S)-But-3-en-2-yl 3,4,6-tri-O-benzyl-2-deoxy-2-fluoro-â-D-
galactopyranoside 7
To a solution of compound 6 (460 mg, 0.86 mmol) in acetic
acid–water (10 ml, 4:1 v/v) was added TFA dropwise until a
yellow colour persisted. TLC analysis (methanol–DCM, 1:9
v/v) showed the complete conversion of starting material (Rf
0.98) into an intermediate product (Rf 0.21). Water (50 ml) was
added and the resulting solution was concentrated in vacuo,
with continual addition of water, until no acid persisted; the
solution was then concentrated to dryness. Purification using
flash chromatography (50 g SiO2; eluent ethyl acetate) and con-
centration of the appropriate fractions gave a solid, which was
dissolved in DMF (2 ml), and sodium hydride (120 mg; 60%
dispersion in oil; 2.98 mmol) was added to the cooled (0 ЊC)
solution, followed by benzyl bromide (0.29 ml, 2.45 mmol).
After stirring of the mixture under argon for 3 h, TLC analysis
(acetone–DCM, 1:49 v/v) showed the complete conversion of
starting material (Rf 0.00) into a product (Rf 0.68). The reaction
J. Chem. Soc., Perkin Trans. 1, 1997
2379

View Article Online
71%) as a clear oil [a significant amount of the β-phosphate (21
mg, 14%) was also isolated], δH (300 MHz; CDCl3) 7.41–7.17
(m, 25 H, ArH), 5.95 (dd, J1,P 6.3, J1,2 3.7, 1 H, H-1), 5.18–4.83
(m, 1 H, H-2), 5.10–5.05 (m, JH ,P 7.4, 4 H, ArCH2OP), 4.80–
4.73 (m, 6 H, ArCH2), 4.12 (ddd, J2,3 = J3,F = 6.6, J3,4 < 1, 1 H,
H-3), 4.02 (dd, J4,5 2.9, 1 H, H-4), 3.95 (dt, 1 H, H-5, J5,6 10.3)
and 3.60–3.40 (m, 2 H, H2-6); δC(75 MHz; CDCl3) 139.1–138.6
(ArC quaternary), 128.6–127.8 (ArCH), 96.3 (C-1, JC-1,P 5.7,
JC-1,F 23.18), 88.5 (C-2, JC-4,F 188.8, JC-2,P 9.0), 76.4 (C-3, JC-3,F
17.5), 74.9 (C-4, JC-3,F 7.9), 72.0 (C-5), 75.2, 73.5 and 73.0
(ArCH2), 69.4–68.1 (ArCH2OP, JC,P 5.1) and 68.1 (C-6); δF (282
MHz; CDCl3) Ϫ206.7 (ddd, J2,F 49.6, J3,F 6.6, J5,F < 1); δP(121
MHz; CDCl3) Ϫ2.0 (1H decoupled); FAB m/z 735 ([M ϩ Na]ϩ)
(Found: Mϩ, 735.2482. C41H42FNaO8P requires m/z 735.2499).
tion mixture was neutralised with Dowex 1-X8[Hϩ] and, after
filtration, concentrated to dryness in vacuo to yield a solid. The
intermediate product was dissolved in acetonitrile (10 ml), and
benzaldehyde dimethyl acetal (2.40 ml, 15.78 mmol) was added
followed by a catalytic amount of CSA (~5 mg). After stirring
of the mixture for 2 h under argon, TLC analysis (methanol–
DCM, 1:9 v/v) showed complete conversion of the starting
material (Rf 0.05) into a product (Rf 0.32). The reaction was
quenched by the addition of pyridine (0.30 ml) and the result-
ing solution was concentrated to dryness in vacuo. The result-
ing yellow oil was re-dissolved in ethyl acetate (30 ml) and the
solution was washed successively with saturated aq. sodium
hydrogen carbonate (2 × 10 ml) and brine (2 × 10 ml). The
organic phase was dried (MgSO4), and concentrated to dryness
in vacuo to yield a yellow oil. Purification using flash column
chromatography (100 g SiO2; eluent ethyl acetate–light petrol-
eum, 3:7 v/v) and concentration of the appropriate fractions
yielded compound 12 (4.08 g, 85%) as a solid, δH (300 MHz;
CDCl3) 7.53–7.26 (m, 5 H, ArH), 5.98–5.15 (m, 1 H,
Bis(triethylammonium) 2-deoxy-2-fluoro-á-D-galactopyranosyl
phosphate 10
Compound 9 (120 mg, 0.17 mmol) was dissolved in a solvent
mixture of ethyl acetate–propan-2-ol–water (10 ml, 4:6:2
v/v/v), and sodium acetate (78 mg, 0.95 mmol) was added. 10%
Pd/C (100 mg) was added and hydrogen was bubbled through
the suspension for 24 h. The solution was filtered, then con-
centrated to dryness in vacuo, the residue was re-dissolved in
methanol (10 ml), and the solution was treated with Dowex 1-
X8[Et3NHϩ] and concentrated to dryness in vacuo. Purification
of the residue on a Sephadex LH-20 column (100 g; eluent
MeOH–water, 4:1 v/v) yielded compound 10 (77 mg, 99%) as a
solid, δH (300 MHz; D2O) 5.61 (dd, J1,P 7.72, J1,2 3.7, 1 H, H-1),
4.76–4.74 (m, 1 H, H-4), 4.58 (ddd, J2,F 49.6, J2,3 9.6, 1 H, H-2),
4.15–3.95 (m, 2 H, H-3 and -5), 3.65–3.60 (m, 2 H, H2-6), 3.13
(q, J 5.9, 12 H, CH2CH3) and 1.22 (t, 18 H, CH3CH2); δC(75
MHz; D2O) 95.7 (C-1, JC-1,P 5.7, JC-1,F 22.6), 91.5 (C-2, JC-2,F
178.0, JC-2,P 9.0), 70.8 (C-3, JC-3,F 17.5), 72.9 (C-4, JC-4,F 8.5),
69.8 (C-5) and 56.7 (C-6); δP(121 MHz; CDCl3) Ϫ0.75 (1H
decoupled).
CH᎐CH ), 5.52 (s, 1 H, ArCHO ), 5.32–5.11 (m, 2 H,
᎐
2
2
CH᎐CH ), 4.47 (d, J 8, 1 H, H-1), 4.42–4.27 (m, 2 H, H-4,
᎐
2
1,2
CHCH3), 3.87–3.53 (m, 2 H, H-2 and -3), 3.61–3.29 (m, 3 H,
H-5, H2-6) and 1.38–1.30 (2 d, J 6, 3 H, CHCH3); δC(75 MHz;
CDCl ) 139.7 and 138.6 (CH᎐CH ), 137.0 (ArC quaternary),
᎐
3
2
129.3–126.3 (ArCH), 117.7, 115.5 (CH ᎐CH), 101.9
᎐
2
(ArCHO2), 101.4 and 100.0 (C-1), 80.6–66.3 (CHCH3, C-2, -3,
-4 and -5), 68.7 (C-6) and 21.6 and 20.2 (CHCH3); FAB m/z 345
([M ϩ Na]ϩ) (Found: C, 63.33, H, 6.79. C17H22O6 requires C,
63.34; H, 6.88%).
(R/S)-But-3-en-2-yl 2,3-di-O-benzyl-4,6-O-benzylidene-â-D-
glucopyranoside 13
Sodium hydride (1.00 g; 60% dispersion in oil; 24.8 mmol) was
added to a cooled solution (0 ЊC) of compound 12 (3.06 g, 9.50
mmol) in DMF (20 ml). Benzyl bromide (2.43 ml, 20.45 mmol)
was added and after stirring of the mixture under argon for 3 h,
TLC analysis (acetone–DCM, 1:49 v/v) showed the complete
conversion of starting material (Rf 0.00) into product (Rf 0.48).
Methanol (30 ml) was added to the reaction mixture and
stirring was continued for a further 30 min. The mixture was
concentrated to dryness in vacuo, the resulting solid was dis-
solved in diethyl ether (50 ml), and the solution was washed with
brine (2 × 50 ml). The organic phase was dried (MgSO4), and
concentrated to dryness in vacuo to yield a yellow oil. Purific-
ation using flash chromatography (100 g SiO2; eluent ethyl
acetate–light petroleum, 1:4 v/v) and concentration of the
appropriate fractions yielded compound 13 (4.76 g, 99%) as a
solid, δH (300 MHz; CDCl3) 7.52–7.20 (m, 15 H, ArH), 6.00–
Diammonium 2-deoxy-2-fluoro-á-D-galactopyranosyl uridin-5Ј-
yl diphosphate I
Compound 10 (77 mg, 0.16 mmol) and uridine 5Ј-
monomorpholidophosphate (100 mg, 0.15 mmol) were separ-
ately dried by co-evaporation from pyridine twice (2 × 5 ml).
They were then dissolved in pyridine (5 ml each) and the solu-
tions combined. The resulting reaction mixture was stirred
under argon for 5 days before being concentrated to dryness in
vacuo and the resulting syrup was diluted with water (10 ml),
applied to a column of Dowex 1-X8[HCO2Ϫ] (1 × 12 cm) and
eluted with a linear gradient of aq. NH4HCO3 (0–1 ). The
appropriate fractions were combined and lyophilised to yield
title compound I (82 mg, 82%) as a solid, δH (300 MHz; D2O)
7.89 (d, J5,6 8.1, 1 H, U :H-6), 5.91–5.85 (m, 2 H, rib :H-1Ј,
U :H-5), 5.75 (dd, J1,P 7.4, J1,2 3.7, J1,F < 0.5, 1 H, gal:H-1),
4.65 (ddd, J2,F 45, J2,3 9.2, 1 H, gal:H-2), 4.38–3.77 (m, 8 H,
gal, H-3, -4, -5 and rib-H-2Ј, -H-3Ј, -H-4Ј and -H2-5Ј) and
3.72–3.65 (m, 2 H, H2-6); δC(75 MHz; D2O) 169.9 (U :C-2),
155.4 (U :C-4), 145.2 (U :C-6), 105.9 (U :C-5), 96.0 (gal:C-1,
JC-1,P 5.7, JC-1,F 22.0), 91.7 (rib :C-1Ј), 91.3 (gal:C-2, JC-2,F
185.9, JC-2,P 7.4), 86.4 (rib :C-4Ј, JC-4Ј,P 10.2), 76.9 and 72.8
(rib :C-2Ј, -3Ј), 75.0 (gal:C-5), 70.7 (gal:C-3, JC-3,F 17.0), 68.0
(gal:C-6) and 63.8 (rib :C-5Ј, JC-5Ј,P 5.9); δP(121 MHz; CDCl3)
Ϫ10.6 and Ϫ12.2 (2 m) (1H decoupled); MALDI-TOF m/z
567 [M]Ϫ.
5.67 (m, 1 H, CH᎐CH ), 5.56 (s, 1 H, ArCHO ), 5.29–5.10 (m, 2
᎐
2
2
H, CH᎐CH ), 4.95–4.74 (m, 4 H, ArCH ), 4.61–4.54 (2 d, J1,2 8,
᎐
2
2
1 H, H-1), 4.39–4.26 (m, 2 H, H-4 and CHCH3), 3.83–3.64 (m, 3
H, H-3 and H2-6), 3.49 (dd, J2,3 8, 1 H, H-2), 3.41–3.32 (m, 1 H,
H-5) and 1.37–1.32 (2 d, J 6, 3 H, CHCH3); δC(75 MHz; CDCl3)
142.8 and 140.0 (CH᎐CH ), 138.3–137.3 (ArC quaternary),
᎐
2
128.0–126.0 (ArCH), 117.3 and 115.3 (CH ᎐CH), 100.8
᎐
2
(ArCHO2), 101.1 and 102.2 (C-1), 82.2–81.1 (CHCH3, C-2, -3,
-4 and -5), 77.4–68.8 (ArCH2), 66.0 (C-6) and 21.6 and 20.4
(CHCH3); FAB m/z 525 ([M ϩ Na]ϩ) (Found: C, 73.93; H, 6.69.
C31H34O6 requires C, 74.07; H, 6.82%).
(R/S)-But-3-en-2-yl 2,3,6-tri-O-benzyl-â-D-glucopyranoside 14
Compound 13 (4.75 g, 9.46 mmol) was dissolved in DCM (30
ml) and the solution was cooled (Ϫ20 ЊC) and stirred under
argon. Subsequently, triethylsilane (7.80 ml, 47.3 mmol) and
TFA (3.80 ml, 47.3 mmol) were added dropwise. The reaction
mixture was allowed to warm to room temperature and stirring
was continued for 2 h. TLC analysis (acetone–DCM, 1:49 v/v)
showed the complete conversion of starting material (Rf 0.81)
into product (Rf 0.45). Ethyl acetate (50 ml) was added to the
reaction mixture and the resulting solution was washed succes-
(R/S)-But-3-en-2-yl 4,6-O-benzylidene-â-D-glucopyranoside 12
Potassium tert-butoxide (0.56 g, 5.00 mmol) was added to a
stirred solution of (R/S)-but-3-en-2-yl 2,3,4,6-tetra-O-acetyl-β-
-glucopyranoside 11 (6.00 g, 14.93 mmol) in dry methanol (20
ml), and the resulting reaction mixture was stirred at room
temperature, under argon. After 3.5 h, TLC analysis (acetone–
DCM, 1:19 v/v) showed the complete conversion of the start-
ing material (Rf 0.47) into an intermediate (Rf 0.00). The reac-
2380
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
sively with saturated aq. sodium hydrogen carbonate (2 × 10
ml) and brine (2 × 10 ml). The organic phase was dried
(MgSO4), and concentrated to dryness in vacuo to yield a sticky
yellow solid. Purification using flash chromatography (100 g
SiO2; eluent ethyl acetate–light petroleum, 1:3 v/v) and concen-
tration of the appropriate fractions yielded compound 14 (3.37
g, 70%) as a solid, δH (300 MHz; CDCl3) 7.38–7.21 (m, 15 H,
and concentrated to afford an orange-red residue. The residue
was purified by flash chromatography (20 g SiO2; eluent DCM)
to afford the required compound 17 (1.23 g, 74%) as a solid,
δH (250 MHz; CDCl3) 7.42–7.10 (m, 15 H, ArH), 4.98–4.48 (m,
9 H, H-1, -4, ArCH , C᎐CH), 3.89–3.42 (m, 5 H, H-2, -3,
᎐
2
-5 and H -6), 1.92–1.82 (m, 3 H, CH C᎐CH) and 1.67–1.49
᎐
3
2
(m, 3 H, CH3CH); δC(63 MHz; CDCl3) 151.5 and 149.1
[OC(CH )᎐C], 138.4–137.8 (ArC quaternary), 128.4–127.8
ArH), 6.00–5.67 (m, 1 H, CH᎐CH ), 5.27–5.04 (m, 2 H,
᎐
᎐
3
2
CH᎐CH ), 4.98–4.47 (m, 7 H, H-1 and ArCH ), 4.42–4.26 (m, 1
(ArCH), 105.4 and 100.1 (᎐CHCH ), 100.3 and 98.1 (C-1),
᎐
᎐
2
2
3
H, CHCH3), 3.78–3.33 (m, 6 H, H-2, -3, -4, -5 and H2-6) and
1.37–1.31 (2 d, J 6, 3 H, CHCH3); δH (75 MHz; CDCl3) 140.3
85.8 (d, JC-4,F 183, C-4), 78.9 (d, JC-3,F 18, C-3), 78.9 (CHCH3),
77.2 (C-2), 72.2 (d, JC-5,F 22, C-5), 72.0 (C-6), 75.5–73.2
and 139.1 (CH᎐CH ), 138.7–138.0 (ArC quaternary), 128.6–
(ArCH ), 18.6 and 15.6 (CH C᎐CH) and 11.9 and 10.3
᎐
2 3
᎐
2
127.7 (ArCH), 117.2 and 115.0 (CH ᎐CH), 101.9 and 100.5 (C-
(CHCH3); δF (282 MHz; CDCl3) Ϫ194.9 (2 ddd, J4,F 48, J3,F 28,
J5,F 20); CI m/z 524 ([M ϩ NH4]ϩ) (Found: C, 73.52; H,
7.01 C31H35FO5 requires C, 73.49; H, 6.96%).
᎐
2
1), 84.3–71.8 (CHCH3, C-2, -3, -4 and -5), 75.3–73.6 (ArCH2),
70.4 (C-6) and 21.6 and 20.4 (CHCH3); FAB m/z 527
([M ϩ Na]ϩ) (Found: C, 73.72, H, 7.15. C31H36O6 requires C,
73.78; H, 7.19%).
Dibenzyl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-á-D-
galactopyranosyl phosphate 18
(R/S)-But-3-en-2-yl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-â-D-
galactopyranoside 16
A mixture of vinyl glycoside 17 (125 mg, 0.22 mmol) in DCM
(1 ml) and molecular sieves (100 mg; 4 Å powdered) was stirred
for 1 h. In a separate reaction vessel, NIS (200 mg, 0.88 mmol)
was suspended in DCM (2 ml) by sonication. Dibenzyl hydro-
gen phosphate (225 mg, 0.81 mmol) was added to the suspen-
sion and the mixture was allowed to equilibrate under argon
for 5 min. TMSOTf (18.7 µl, 0.094 mmol) was then added and
the suspension was stirred for a further 30 s. The resulting
suspension (1.1 ml) was then added by syringe to the ready
prepared solution of the vinyl glycoside 17. Immediate TLC
analysis (acetone–DCM, 1:99 v/v) of the reaction mixture
showed the conversion of the starting material (Rf 0.79) into a
major product (Rf 0.50). The reaction mixture was neutralised
by the addition of triethylamine and diluted with ethyl acetate
(20 ml). The resulting mixture was filtered, and washed succes-
sively with aq. sodium metabisulfite (2 × 5 ml) and brine (2 × 5
ml). The organic phase was dried (MgSO4), concentrated to
dryness in vacuo, and the resulting oil was purified using flash
chromatography (25 g SiO2; eluent acetone–DCM, 1:49 v/v).
Concentration of the appropriate fractions yielded compound
18 (89 mg, 54%) as a clear oil, δH (300 MHz; CDCl3) 7.60–7.03
(m, 25 H, ArH), 5.95 (dd, J1,P 6.6, J1,2 3.3, 1 H, H-1), 5.11–4.99
(m, JH ,P 7.4, 4 H, ArCH2OP), 4.89 (ddd, J4,F 50, J3,4 2.2,
J4,5 < 1, 1 H, H-4), 4.80–4.73 (m, 4 H, ArCH2), 4.48 (s, 2 H,
ArCH2), 4.09 (dt, J5,F 29.4, J5,6 6.6, 1 H, H-5), 3.99 (ddd, J2,3
9.6, J2,P 2.6, 1 H, H-2), 3.83 (ddd, J3,F 27.6, 1 H, H-3) and 3.69–
3.47 (m, 2 H, H2-6); δC(75 MHz; CDCl3) 137.8–135.8 (ArC
quaternary), 128.5–127.8 (ArCH), 96.2 (C-1, JC-1,P 6.2), 86.8
(C-4, JC-4,F 183.4), 75.1 (C-3, JC-3,F 18.0), 75.1 (C-2, JC-2,P 8.5),
70.1 (C-5, JC-5,F 18.1), 73.7, 73.6 and 72.5 (ArCH2), 69.4–69.2
(ArCH2OP, JC᎐P 5.8 and 5.1) and 67.5 (C-6, JC-6,F 5.7); δF (282
MHz; CDCl3) Ϫ218.6 (ddd, J4,F 48, J3,F 29.3, J5,F 27.6); δP(121
MHz; CDCl3) Ϫ1.8 (1H decoupled); FAB m/z 735 ([M ϩ Na]ϩ)
(Found: [M ϩ Na]ϩ, 735.2482. C41H42FNaO8P requires m/z,
735.2499).
To a solution of compound 14 (2.50 g, 4.96 mmol) in DCM (20
ml) was added pyridine (3 ml). The resulting solution was
cooled (Ϫ78 ЊC) and stirred under argon, and Tf2O (0.74 ml,
4.96 mmol) was added. The reaction mixture was allowed to
warm to room temperature and, after 40 min, TLC analysis
(ethyl acetate–cyclohexane, 3:7 v/v) showed complete conver-
sion of starting material (Rf 0.66) into compound 15 (Rf 0.88).
The reaction mixture was diluted with DCM (50 ml) and was
washed successively with saturated aq. sodium hydrogen car-
bonate (2 × 10 ml) and brine (2 × 10 ml). The organic phase
was dried (MgSO4), and concentrated to dryness in vacuo to
yield compound 15 as a sticky yellow solid. Compound 15 was
immediately dissolved in THF (20 ml) stirred under argon, and
TBAF solution (1.1  in THF, dried over molecular sieves 4 Å;
4.5 ml, 4.95 mmol) was added. A colour change from yellow to
deep red was observed and immediate TLC analysis (ethyl
acetate–cyclohexane, 3:7 v/v) showed complete conversion of
the intermediate (Rf 0.88) into a major product (Rf 0.63). The
reaction mixture was concentrated to dryness in vacuo, the
resulting brown solid was dissolved in DCM (50 ml), and the
solution was washed with brine (2 × 10 ml). The organic phase
was dried (MgSO4), and concentrated to dryness in vacuo to
yield a sticky yellow solid. Purification using flash chrom-
atography (100 g SiO2; eluent ethyl acetate–cyclohexane, 1:9
v/v) and concentration of the appropriate fractions yielded
compound 16 (1.75 g, 70%) as a solid, δH (250 MHz; CDCl3)
7.42–7.22 (m, 15 H, ArH), 6.02–5.63 (m, 1 H, CH᎐CH ), 5.24–
᎐
2
5.02 (m, 2 H, CH᎐CH ), 4.98–4.47 (m, 7 H, H-1 and ArCH ),
᎐
2
2
4.86 (ddd, J3,4 4, J4,5 < 0.5, J4,F 48, 1 H, H-4), 4.48–4.42 (2 d, J1,2
8, H-1), 4.42–4.23 (m, 1 H, CHCH3), 3.79–3.37 (m, 5 H, H-2,
-3, -5 and H2-6) and 1.36–1.21 (2 d, J 6, 3 H, CHCH3); δC(63
MHz; CDCl ) 140.2 and 139.0 (CH᎐CH ), 138.4–137.7 (ArC
᎐
3
2
quaternary), 128.4–127.7 (ArCH), 117.0 and 114.4 (CH ᎐CH),
᎐
2
101.7 and 100.2 (C-1), 86.0 (2 d, JC-4,F 183, C-4), 79.0 (d, JC-3,F
19, C-3), 78.9 (CHCH3), 77.2 (C-2), 72.1 (d, JC-5,F 18, C-5),
67.7 (C-6), 75.3–72.2 (ArCH2) and 21.8 and 20.4 (CHCH3); CI
m/z 524 ([M ϩ NH4]ϩ) (Found: C, 73.80; H, 6.99. C31H35FO5
requires C, 73.50; H, 6.96%).
Bis(triethylammonium) 4-deoxy-4-fluoro-á-D-galactopyranosyl
phosphate 19
Compound 18 (170 mg, 0.24 mmol) was dissolved in a mixture
of ethyl acetate–propan-2-ol–water (10 ml; 4:6:2 v/v/v), and
sodium acetate (78 mg, 0.95 mmol) was added. 10% Pd/C (100
mg) was added and hydrogen was bubbled through the suspen-
sion for 24 h. The solution was filtered, then concentrated to
dryness in vacuo, the residue was re-dissolved in methanol (10
ml) and treated with Dowex 1-X8[Et3NHϩ], and the mixture
was concentrated to dryness in vacuo. Purification on a Sepha-
dex LH-20 column (100 g; eluent MeOH–water, 4:1 v/v)
yielded compound 19 (98 mg, 90%) as a solid, δH (300 MHz;
D2O) 5.51 (dd, J1,P 6,8, J1,2 3.7, 1 H, H-1), 4.89 (ddd, J4,F 50, J3,4
2.2, J4.5 < 1, 1 H, H-4), 4.15 (dt, J5,F 31.6, J5,6 5.9, 1 H, H-5),
3.97 (ddd, J2,3 9.6, J2,P 2.6, 1 H, H-2), 3.82 (ddd, J3,F 27.6, 1 H,
H-3), 3.74–3.72 (m, 2 H, H2-6), 3.13 (q, J 5.9, 12 H, CH2CH3)
(E/Z)-But-2-en-2-yl 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-â-D-
galactopyranoside 17
Butyllithium (1.5  in THF; 1 ml, 1.5 mmol) was added to a
stirred, orange solution of Wilkinson’s catalyst (500 mg, 0.50
mmol) in THF (1 ml) which was placed under argon. The
resulting brown solution was stirred at 20 ЊC for 5 min and was
then transferred via syringe into a refluxing solution of com-
pound 16 (1.66 g, 3.28 mmol) in THF (10 ml) under argon.
TLC analysis (acetone–DCM, 1:49 v/v) after 30 min showed
complete conversion of starting material (Rf 0.71) into product
(Rf 0.79). The reaction mixture was diluted with DCM (20 ml)
J. Chem. Soc., Perkin Trans. 1, 1997
2381

View Article Online
and 1.22 (t, 18 H, CH3CH2); δC(75 MHz; D2O) 97.4 (C-1, JC-1,P
6.2), 92.9 (C-4, JC-4,F 183.4), 72.8 (C-3, JC-3,F 18.1), 71.2 (C-2,
JC-2,P 8.5), 70.5 (C-5, JC-5,F 18.1) and 62.5 (C-6); δP(121 MHz;
CDCl3) Ϫ0.22 (1H decoupled).
References
1 R. M. de Lederkremer and W. Colli, Glycobiology, 1995, 5, 547.
2 W. N. Haworth, H. Raistrick and M. Stacey, Biochem. J., 1937, 31,
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3 R. M. Lederkremer, C. Lima, M. Ramirez, M. A. J. Ferguson,
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Diammonium 4-deoxy-4-fluoro-á-D-galactopyranosyl uridin-5Ј-
yl diphosphate II
Compound 19 (89 mg, 0.19 mmol) and uridine 5Ј-mono-
morpholidophosphate (130 mg, 0.19 mmol) were separately
dried by co-evaporation from pyridine (2 × 5 ml). They were
each dissolved in pyridine (10 ml) and the solutions were com-
bined. The reaction mixture was stirred under argon for 5 days
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syrup was diluted with water (10 ml) and applied to a column of
Dowex 1-X8[HCO2Ϫ] (1 × 12 cm) and eluted with a linear gra-
dient of aq. NH4HCO3 (0–1 ). The appropriate fractions were
combined and lyophilised to yield compound II (69 mg, 60%)
as a solid, δH (300 MHz; D2O) 7.91 (d, 1 H, U :H-6, J5,6 8.1),
5.95–5.91 (m, 2 H, rib :H-1Ј, U :H-5), 5.63 (dd, J1,P 7.0, J1,2 3.3,
1 H, gal:H-1), 4.88 (ddd, J4,F 50, J3,4 2.2, J4.5 < 1, 1 H, gal:H-4),
4.05 (dt, J5,F 31.6, J5,6 5.9, 1 H, gal:H-5), 3.82 (ddd, J3,F 27.6, 1
H, gal:H-3), 4.38–3.77 (m, 6 H, gal:H-2, rib :H-2Ј, -3Ј, -4Ј and
H2-5Ј) and 3.80–3.70 (m, 2 H, H2-6); δC(75 MHz; D2O) 169.1
(U, C-2), 154.7 (U, C-4), 144.3 (U, C-6), 105.4 (U, C-5), 98.3
(gal:C-1, JC-1,P 7.3), 92.9 (gal:C-4, JC-4,F 183.4), 91.2 (rib :C-1Ј),
85.8 (rib :C-4Ј, JC-4Ј,P 8.5), 76.5 and 72.3 (rib :C-2Ј and -3Ј), 73.3
(gal:C-3, JC-3,F 18.1), 71.0 (gal: C-2, JC-2,P 8.5), 70.7 (gal:C-5,
JC-5,F 18.1), 67.6 (gal:C-6) and 62.5 (rib :C-5Ј, JC-5Ј,P 5.9);
δP(121 MHz; CDCl3) Ϫ10.3 and 11.9 (2 d, JP, P 19.4) (1H
decoupled); MALDI-TOF m/z 566 [M]Ϫ.
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Acknowledgements
Paper 7/01630A
Received 7th March 1997
Accepted 23rd April 1997
The inhibition studies were performed by Professor M. McNeil
(University of Colorado, USA).
2382
J. Chem. Soc., Perkin Trans. 1, 1997