Synthesis of UDP-glucose derivatives modified at the 3-OH as potential chain terminators of β-glucan biosynthesis

Article abstract of DOI:10.1016/j.carres.2008.01.031

A series of UDP-d-glucose derivatives and precursors that have been modified at C-3 were synthesised from d-glucose as potential chain terminators of β-glucan biosynthesis. None of the UDP-derivatives or the precursors tested displayed significant anti-fungal activity in a series of germination assays on the dermatophyte Trichophyton rubrum.

Full text of DOI:10.1016/j.carres.2008.01.031

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1012–1022
Synthesis of UDP-glucose derivatives modified at the 3-OH
as potential chain terminators of b-glucan biosynthesis
Ramona Danac,a Lucy Ball,b Sarah J. Gurrb and Antony J. Fairbanksa,*
aChemistry Research Laboratory, Oxford University, Mansfield Road, Oxford OX1 3TA, UK
bDepartment of Plant Sciences, Oxford University, South Parks Road, Oxford OX1 3RB, UK
Received 13 December 2007; received in revised form 17 January 2008; accepted 22 January 2008
Available online 2 February 2008
Abstract—A series of UDP-D-glucose derivatives and precursors that have been modified at C-3 were synthesised from D-glucose as
potential chain terminators of b-glucan biosynthesis. None of the UDP-derivatives or the precursors tested displayed significant
anti-fungal activity in a series of germination assays on the dermatophyte Trichophyton rubrum.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Anti-fungal agents; b-Glucan; Biosynthesis; Oligosaccharides; Chain termination
1. Introduction
thesis by the synthesis and use of carbohydrate deriva-
tives that have been modified at the hydroxyl group at
which subsequent units would be added after their
incorporation into the growing oligosaccharide chain.
Chain-termination processes involving modified
monosaccharide derivatives have previously been
implicated in the biological effects of some monosaccha-
ride derivatives on mammalian glycoconjugate4 and
glycosoaminoglycan biosynthesis.5 However, a chain-
termination approach has not yet been more widely
investigated, particularly as a strategy for the develop-
ment of new classes of inhibitors of the biosynthesis of
pathogenic oligosaccharides. This ‘oligosaccharide’ situ-
ation contrasts with the widespread use of chain termi-
nation of oligonucleotide synthesis, ranging from its
original development by Sanger et al.6 as a means of
DNA sequencing, to several anti-viral therapies curr-
ently in clinical use, perhaps most pre-eminently in
the case of AZT.7 Moreover, the growing body of data
demonstrating that many glycosyl transferases do pro-
cess activated donor substrates that have been modified
in a minimal way at a single hydroxyl group8,9 augurs
well for a chain-termination approach.
Oligomeric carbohydrate structures that are specific to
pathogenic organisms, and which are vital for their sur-
vival, represent particularly attractive targets for thera-
peutic intervention.1 Over recent years, substantial
effort has been expended in the search for potent inhi-
bitors of glycosyl transferases, both as a means of treat-
ing human disease and, more relevantly, to disrupt
pathogen-specific biosynthetic pathways.2 However,
the design of such inhibitors can be problematic, since
in many cases little is known about the precise enzymes
involved. Moreover, such inhibitors in general have to
mimic a reaction transition state which effectively
involves three distinct species—the glycosyl acceptor,
the nucleoside di-phosphate, and the glycosyl donor.
The net result is that specifically designed glycosyl trans-
ferase inhibitors have yet to find clinical application.3
This lack of success invokes the need for alternative
approaches to be considered. In particular, where the
targeted oligosaccharide consists of multiple monosac-
charide repeat units, an alternative strategy to enzymatic
inhibition may prove useful. Such an approach would be
to invoke chain termination of oligosaccharide biosyn-
As part of a program10 aimed at investigating the po-
tential opportunities that chain termination of oligosac-
charide biosynthesis offers to disease control, we became
*
Corresponding author. E-mail: antony.fairbanks@chem.ox.ac.uk
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.01.031

R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
1013
interested in the rational design of novel anti-fungal
O
O
O
O
agents. Fungal infections represent a serious hazard to
human and animal health,11 and, just as for bacterial
infection, drug resistance to current therapies is increas-
ing.12 The fungal cell wall consists of large sections of
oligosaccharide materials including chitin, and b-glucan.
The biosynthesis of these non-mammalian oligosaccha-
rides could be potentially targeted13 using a chain-termi-
nation approach. The synthesis and anti-fungal activity
of a variety of N-acetylglucosamine derivatives, which
were rationally designed as potential chain terminators
of chitin biosynthesis, have already been reported.10a,b
The other potential target, b-glucan, is assembled step-
wise by the b-(1?3) glucan synthases,14 which transfer
single glucose residues to a growing oligomeric chain;
the donor substrate for these enzymes being UDP-D-glu-
cose. Potential chain terminators of this process are,
therefore, D-glucose residues in which the 3-OH has
been modified. If such materials are processed by a
b-(1?3) glucan synthase, then their transfer to the
terminus of a growing glucan chain will result in a
chain-termination step since the required 3-OH at which
subsequent units would be added will now be lacking.
The most obvious approach was that of synthesising
and testing the modified UDP-glucose donors them-
selves, since these are the actual substrates processed
by b-(1?3) glucan synthases, although previous work
implied that the high polarity of such compounds may
compromise their ability to penetrate intracellularly
and thus produce any anti-fungal effects.10b Alterna-
tively, potential pro-drug molecules could be synthes-
ised, which may then be converted to the active UDP-
donors once inside the fungal cell. Following on from
the previous work,10a,b which had identified GlcNAc
derivatives that displayed anti-fungal activity, isosteric
replacement of the 3-OH of glucose was envisaged by
replacement of the OH group by –H, –OMe, –F,
–NHAc and N3.
O
O
c
O
O
O
O
HO
MeO
d,e
1
3a
a,b
O
O
O
O
O
O
O
O
O
O
HO
F
2
3b
g,h
f
O
O
O
O
O
O
O
O
O
O
N3
3c
3d
˚
Scheme 1. Reagents and conditions: (a) PCC, CH2Cl2, 4A molecular
sieves; (b) NaBH4, EtOH, H2O, 68% over two steps; (c) KOH, MeI,
Bu4NBr, acetone, 0 °C to rt; 86%; (d) NaH, THF, imidazole, then CS2,
then MeI; (e) BuSn3H, toluene, reflux, 62% over two steps; (f) DAST,
2,4,6-collidine, CH2Cl2, ꢁ20 °C to rt, 61%; (g) Tf2O, pyridine, CH2Cl2,
ꢁ20 °C; (h) NaN3, DMF, 50 °C, 93% over two steps.
of configuration. Thus, the usual oxidation/reduction
sequence led readily to diacetone allose 2, which was
then converted to fluoride 3c by treatment with diethyl-
amino sulfur trifluoride (DAST) and collidine in dichlo-
romethane, and to azide 3d by a triflation/azide
displacement sequence (Scheme 1).
Attention then turned to conversion of these furanose
derivatives into the corresponding pyranose peracetates,
glycosyl phosphates and UDP-derivatives (Scheme 2). A
parallel series of transformations ultimately allowed
access to the UDP-derivatives 9a–d, and in addition pro-
vided potential pro-drug intermediates, which were also
tested for anti-fungal activity. Treatment of furanose
diacetonides 3a–d with aqueous trifluoroacetic acid
followed by complete acetylation of the crude products
by treatment with acetic anhydride and pyridine
produced the pyranose acetates 4a–d as mixtures of
anomers. It was notable that the yield of 3-deoxy acetate
4b was considerably lowered by the formation of fura-
nose by-products.16 Selective removal of the anomeric
acetate of 4a–d was achieved using a mixture of ethylene
diamine and acetic acid in THF, leading to hemiacetals
5a–d. Phosphorylation with tetrabenzyl pyrophos-
phate17 gave the benzyl protected anomeric phosphates
6a–d as anomeric mixtures in which the desired a-ano-
mers were predominant (a:b ratios ꢀ5:1), and which in
all the cases were readily separated by silica gel flash
chromatography. Removal of the benzyl protecting
groups was achieved by catalytic hydrogenation in
methanol in the presence of palladium hydroxide
providing phosphates 7a–c, which were converted to
the corresponding triethylammonium salts by the addi-
tion of triethylamine, and which were then used in the
2. Results and discussion
Selective access to the 3-OH for modification was
achieved using diacetone glucose 1 as the starting mate-
rial. A variety of standard synthetic procedures were
undertaken to achieve the desired functional group
inter-conversions (Scheme 1). Methylation by treatment
of 1 with potassium hydroxide and methyl iodide in
acetone in the presence of tetrabutylammonium bro-
mide as a phase transfer catalyst15 produced methyl
ether 3a, whilst conversion to an intermediate xanthate
ester by sequential treatment of 1 with sodium hydride,
carbon disulfide and methyl iodide in THF, followed by
tributyl tin hydride mediated free radical reduction led
to deoxy compound 3b. Introduction of either nitrogen
or fluorine at C-3 of glucose required a double inversion

1014
R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
O
O
OAc
O
OAc
O
OAc
O
O
a,b
c
d
O
AcO
AcO
AcO
R
R
R
O
OAc
AcO
AcO
AcO OH
O
R
O P OBn
OBn
3a R=OMe
3b R=H
4a R=OMe
4b R=H
5a R=OMe
6a R=OMe
6b R=H
5b R=H
5c R=F
5d R=N3
3c R=F
4c R=F
6c R=F
3d R=N3
4d R=N3
6d R=N3
e
O
OH
O
OH
O
OAc
NH
HO
HO
O
R
AcO
f
g
R
R
O
O
O
O
N
O
O
HO
HO
AcO
O P O P O
O P O
O P O
. 2 Et3NH+
. 2 Et3NH+
O
O
O
O
. 2 Et3NH+
HO
OH
8a R=OMe
8b R=H
7a R=OMe
7b R=H
9a R=OMe
9b R=H
8c R=F
7c R=F
8d R=NHAc
7d R=NHAc
9c R=F
9d R=NHAc
Scheme 2. Reagents and conditions: (a) CF3CO2H, H2O, AcOH, rt; (b) Ac2O, pyridine; 4a, 86%; 4b 20%; 4c 61%; 4d 80%; (c) (CH2NH2)2, AcOH,
THF, rt; 5a, 70%; 5b 70%; 5c 65%; 5d 67%; (d) LDA, [(BnO)2P(O)]2O, THF, ꢁ78 to 0 °C; 6aa, 68%; 6ba, 52%; 6ca, 74%; 6da, 73%; (e) H2, Pd(OH)2,
MeOH, (and Ac2O for 7d); (f) Et3N, MeOH, H2O, rt; 8a, 89%; 8b, 92%; 8c, 90%; 8d, 93%; (g) UMP-morpholidate, tetrazole, pyridine, rt; 9a, 51%; 9b,
52%; 9c, 51%; 9d, 54%.
next step without further purification or characterisa-
tion. In the case of phosphate 6d, it was envisaged that
hydrogenation would cause concomitant reduction of
the azide group and acetic anhydride was added to the
reaction mixture before hydrogenation, which resulted
in the formation of acetamide 7d. Deacetylation of the
crude glycosyl phosphate triacetates 7a–d was achieved
by treatment with a mixture of triethylamine, MeOH
and water to produce the deprotected glycosyl phos-
phates 8a–d as their triethylammonium salts. Finally
coupling of 8a–d with UMP-morpholidate provided
the corresponding UDP-derivatives 9a–d (Scheme 2).
Due to the simultaneous reduction of the azide func-
tionality during the removal of the benzyl protecting
groups observed above, a different reaction sequence
was undertaken to access the 3-azido UDP-derivative
9e (Scheme 3). Phosphorylation18 of hemiacetals 5d with
o-phenylene phosphorochloridate 10, followed by the
removal of the o-phenylene protecting group using lead
tetraacetate in dioxane, gave the azido glycosyl phos-
phate 7e. Acetate deprotection, again by treatment with
a mixture of triethylamine, MeOH and water, produced
the deprotected azido glycosyl phosphate 8e. Finally,
coupling with UMP-morpholidate provided the corres-
ponding UDP-derivative 9e (Scheme 3).
compounds that were tested 4a–d, 5a–d 6a–d, 8a–e and
9a–e, only compound 6a (ꢀ40% inhibition of germina-
tion at a 1 mM concentration) displayed significant
biological activity in the 1–1000 lM range that was
examined. These results contrast somewhat with bio-
logical test data previously obtained10a,b for a series of
OAc
O
O
Cl P O
O
AcO
N3
+
OH
AcO
10
5d
a,b
OH
O
OAc
O
HO
N3
AcO
N3
c
O
O
HO
AcO
O P O
O
O P O
8e
7e
O
. 2 Et3NH+
. 2 Et3NH+
d
OH
O
NH
O
HO
N3
O
O
O
N
O
HO
O P O P O
O
O
Potential anti-fungal action of the putative chain-
termination compounds was assessed using an assay
which measured the effect of compounds on germination
of fungal spores/germlings of the dermatophyte19 Trich-
ophyton rubrum, a fungal pathogen that causes superfi-
cial skin diseases in humans and animals. Of the
. 2 Et3NH+
HO
OH
9e
Scheme 3. Reagents and conditions: (a) collidine, 10, THF, 0 °C to rt;
(b) Pb(OAc)4, dioxane, 12 °C to rt; (c) Et3N, MeOH, H2O, rt, 46%
over three steps; (d) UMP-morpholidate, pyridine, rt, 55%.

R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
1015
protected GlcNAc derivatives, which had been modified
at C-4 as potential chain terminators of chitin bio-
synthesis. Based on previous experience, the lack of
in vivo bioactivity of the UDP-derivatives 9a–e and
glycosyl phosphates 8a–e was unsurprising; poor cellular
penetration of these highly polar compounds probably
limited their availability to interfere with b-glucan
biosynthesis. However, the lack of activity of acetates
4a–d/5a–d, which may have been expected to act as
potential pro-drugs,20 was more thought provoking.
Whilst it is perhaps unreasonable to speculate too deeply
since the mode of action of the anti-fungal GlcNAc
compounds has not been thoroughly investigated, it is
possible to imagine that perhaps the intracellular
machinery of b-glucan biosynthesis is less tolerant than
that of chitin biosynthesis, and potential chain termina-
tors may not be processed through the required bio-
synthetic steps. Alternatively pro-drug protecting
group identity may also play an important role in
producing bioactivity.21
were recorded on a Walters 2790-Micromass LCT
electrospray ionisation mass spectrometer, using either
electrospray ionisation (NH3, Cl) techniques as stated.
m/z Values are reported in Daltons and are followed
by their percentage abundance in parentheses. Optical
rotations were measured on a Perkin–Elmer 241 polar-
imeter with a path length of 1 dm. Concentrations are
given in g/100 mL. Microanalyses were performed by
the Inorganic Chemistry Laboratory Elemental Analysis
service, Oxford University, UK. Thin layer chromato-
graphy (TLC) was carried out on Merck Kieselgel
60F254 pre-coated glass-backed plates. Visualisation of
the plates was achieved using a UV lamp (kmax = 254
or 365 nm), and/or ammonium molybdate (5% in 2 M
sulfuric acid), or sulfuric acid (5% in ethanol). Flash
column chromatography was carried out using Sorbsil
C60 40/60 silica. Dichloromethane was distilled from
calcium hydride, or dried on an alumina column. An-
hydrous THF, DMF, pyridine, MeOH and toluene were
purchased from Fluka over molecular sieves. ‘Petrol’
refers to the fraction of light petrol ether boiling in the
range of 40–60 °C. CMAW (chloroform/MeOH/acetic
acid/water) used as eluant was prepared in the following
ratio (CHCl3/MeOH/AcOH/H2O, 60:30:3:5). Purifica-
tion of the UDP-derivatives was performed as previ-
ously described.10b Compounds 2,22,23 3a,15 3b,24 3c,25
3d,22a 4a,26 4b,27 4c,28 4d,29 5a30 and 5c18 were prepared
using the routes shown in Schemes 1 and 2, and exhi-
bited spectroscopic data in agreement with those
reported previously.
3. Conclusions
A series of D-glucose acetates, glycosyl phosphates and
UDP-derivatives that have been modified at C-3 have
been synthesised as potential inhibitors of b-(1?3)-
glucan biosynthesis. Compounds were tested for anti-
fungal activity in a spore germination assay on the
fungus T. rubrum. Although one of the test compounds
did show moderate anti-fungal activity at a concentra-
tion of 1 mM, the general conclusion reached was that
this series of compounds did not display significant
anti-fungal activity. Further investigations into the
potential uses of carbohydrate chain terminators as the
basis for novel strategies against a variety of other
infective agents are in progress, and the results will be
reported in due course.
4.2. 2,4,6-Tri-O-acetyl-3-deoxy-D-ribo-hexopyranose (5b)
Glacial acid acetic (0.50 g, 8.42 mmol, 1.4 equiv) was
added drop-wise to a stirred solution of ethylenediamine
(3.10 g, 0.051 mol, 1.2 equiv) in THF (50 mL). Tetraace-
tate 4b (2 g, 6.0 mmol, 1 equiv) was added, and the
resulting mixture was then stirred for 48 h at rt. After this
time water (50 mL) was added, and the mixture was
extracted with CH2Cl2 (3 ꢂ 50 mL). The combined
organic extracts were washed with aqueous HCl
(2.0 M, 50 mL), saturated aqueous NaHCO3 (50 mL)
and then concentrated in vacuo. Purification by flash
chromatography (petrol/ethyl acetate 1:1) yielded triace-
tate 5b (1.22 g, 70%) as a colourless oil as a mixture of
anomers (a:b ratio approximately 2:1 by integration over
selected parts of the 1H NMR spectrum); mmax (thin film)
3362 (br, OH), 1750 (br s, C@O) cmꢁ1; dH (400 MHz,
CDCl3) 1.63 (1H, aq, J 11.2 Hz, H-3ab), 1.99 (1H, aq,
J 11.2 Hz, H-3aa), 2.03, 2.04, 2.07 (6 ꢂ H, 3 ꢂ s,
6 ꢂ OCCH3), 2.27 (1H, adt, J 4.8 Hz, J 11.3 Hz, H-
3ea), 2.51 (1H, adt, J 4.8 Hz, J 11.9 Hz, H-30b), 3.71
(1H, adt, J 4.1 Hz, J 9.5 Hz, H-5b), 4.12–4.19 (5H, m,
H-5a, H-6, H-60), 4.67 (1H, d, J1,2 7.8 Hz, H-1b), 4.70–
4.87 (4H, m, H-4, H-2), 5.32 (1H, d, J1,2 3.1 Hz, H-1a);
dc (100.6 MHz, CDCl3) 20.7, 20.8, 20.9, 21.0
4. Experimental
4.1. General
Melting points were recorded on a Kofler hot block and
are uncorrected. Proton and carbon nuclear magnetic
resonance (dH, dC) spectra were recorded on Bruker
DPX 250 (250 MHz), Bruker DPX 400 (400 MHz), Bru-
ker DQX 400 (400 MHz), Bruker AVC 500 (500 MHz)
or Bruker AMX 500 (500 MHz) spectrometers. All
chemical shifts are quoted on the d-scale in ppm using
residual solvent as an internal standard. Low resolution
mass spectra were recorded on a Micromass Platform 1
spectrometer using electrospray ionisation in either posi-
tive or negative polarity (ES+ or ESꢁ), or using a VG
Micromass spectrometer. High resolution mass spectra

1016
R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
(6 ꢂ C@OCH3), 28.3 (C-3a), 32.9 (C-3b), 62.5 (C-6a),
62.6 (C-6b), 65.8 (C-4b), 65.9 (C-4a), 67.6 (C-5a), 68.2
(C-2a), 70.2 (C-2b), 75.2 (C-5b), 89.3 (C-1a), 96.9 (C-
1b), 169.6, 169.7, 170.5, 170.8, 171.0, (6 ꢂ CH3C=O);
m/z (ES+) 272 (MꢁH2O+H+, 100%), 313 (M+Na+,
49%); HRMS (ES+) calcd for C12H18O8Na (MNa+)
313.0826; found 313.0888. (Found: C, 49.63; H, 6.20.
C12H18O8 requires C, 49.65; H, 6.25).
of NH4Cl (100 mL) was added, and the mixture was
extracted with CH2Cl2 (3 ꢂ 100 mL). The combined
organic extracts were dried (Na2SO4), filtered and
concentrated in vacuo. The residue was purified by flash
column chromatography (EtOAc/petrol, 6:4) to afford
the two anomers of dibenzylphosphate 6a (1.22 g of
the a-anomer 6aa, 68% and 0.38 g of the b-anomer
20
6ab, 21%) as pale yellow oils. 6aa: ½aꢃD +74 (c 1.0,
CHCl3); mmax (thin film) 1749 (br s, C@O); dH
(400 MHz, CDCl3) 1.92, 1.98, 2.10 (3 ꢂ 3H, 3 ꢂ s,
3 ꢂ COCH3), 3.67 (1H, at, J 9.7 Hz, H-3), 3.94 (1H,
dd, J6,5 2.3 Hz, J6,6 12.4 Hz, H-6), 4.01 (1H, ddd, J5,6
4.3. 2,4,6-Tri-O-acetyl-3-azido-3-deoxy-D-glucopyranose
(5d)
0
Glacial acid acetic (3.98 g, 0.066 mol, 1.4 equiv) was
added drop-wise to a stirred solution of ethylenediamine
(3.42 g, 0.057 mol, 1.2 equiv) in THF (300 mL). Tetra-
acetate 4d (17.7 g, 0.047 mol, 1 equiv) was added, and
the resulting mixture was then stirred for 48 h at rt. After
this time water (300 mL) was added, and the mixture was
extracted with CH2Cl2 (3 ꢂ 300 mL). The combined
organic extracts were washed with aqueous HCl
(2.0 M, 400 mL), saturated aqueous NaHCO3 (400 mL)
and then concentrated in vacuo. Purification by flash
chromatography (petrol/ethyl acetate, 1:1) yielded tri-
acetate 5d (10.5 g, 67%) as a colourless oil as a mixture
of anomers (a:b ratio approximately 4:1 by integration
over selected parts of the 1H NMR spectrum); mmax (thin
film) 3441 (br, w, OH), 2111 (s, N3), 1747 (br s, C@O), dH
(400 MHz, CDCl3) 2.06, 2.11, 2.14 (3 ꢂ 6H, 3 ꢂ s,
6 ꢂ OCCH3), 3.66 (1H, at, J 10.1 Hz, H-3b), 3.71 (1H,
m, H-5b), 4.03 (1H, at, J 10.4 Hz, H-3a), 4.06 (1H, dd,
2.3 Hz, J5,6 4.5 Hz, J5,4 10.1 Hz, H-5), 4.13 (1H, dd,
J6 ,5 4.5 Hz, J6 ,6 12.4 Hz, H-60), 4.85 (1H, adt, J
3.3 Hz, J 9.6 Hz, H-2), 5.03 (1H, at, J 10.1 Hz, H-4),
5.05–5.11 (4H, m, 2 ꢂ PhCH2), 5.86 (1H, dd, J1,P
5.9 Hz, J1,2 3.3 Hz, H-1), 7.35–7.38 (10H, m, 10 ꢂ Ar-
H); dC (100.6 MHz, CDCl3) 20.5, 20.6, 20.7
(3 ꢂ COCH3), 60.4 (OCH3), 61.6 (C-6), 68.7 (C-4),
69.6 (d, JC,P 6.0 Hz, 2 ꢂ PhCH2), 69.7 (C-5), 71.7 (d,
JCꢁ2,P 7.0 Hz, C-2), 77.6 (d, C-3), 94.2 (d, JCꢁ1,P
6.0 Hz, C-1), 128.0, 128.7, 128.8, 135.3, 135.3, 135.4
(12 ꢂ Ar-C), 169.3, 169.8, 170.6 (3 ꢂ CH3CO); dP
(162 MHz, CDCl3) ꢁ2.51 (1H decoupled); m/z (ES+)
602 (M+Na+, 100%); HRMS (ES+) calcd for
C27H33O12PNa (MNa+) 603.1607; found 603.1602.
(Found: C, 55.79; H, 5.67; P, 5.25. C27H33O12P requires
C, 55.86; H, 5.73; P, 5.34). 6ab: dH (400 MHz, CDCl3)
1.98, 1.99, 2.11 (3 ꢂ 3H, 3 ꢂ s, 3 ꢂ COCH3), 3.43 (3H,
s, OCH3), 3.51 (1H, at, J 9.6 Hz, H-3), 3.72 (1H, ddd,
0
0
0
0
J6,5 3.8 Hz, J6,6 9.9 Hz, H-6a), 4.14–4.20 (4H, m,
J5,6 2.5 Hz, J5,6 5.0 Hz, J5,4 9.9 Hz, H-5), 4.10 (1H,
H-60a, H-5a, H-6b, H-60b), 4.66 (1H, d, J1,2 7.8 Hz,
H-1b), 4.72 (1H, dd, J2,1 3.5 Hz, J2,3 10.6 Hz, H-2a),
4.77 (1H, at, J 8.1 Hz, H-2b), 4.92 (2H, m, H-4a,
H-4b), 5.39 (1H, d, J1,2 3.3 Hz, H-1a); dc (100.6 MHz,
CDCl3) 20.6, 20.7, 20.9 (6 ꢂ C@OCH3), 60.6 (C-3a),
62.1 (C-6), 63.8 (C-3b), 67.1 (C-5a), 68.4 (C-4), 71.9
(C-2a), 72.6 (C-5b), 73.2 (C-2b), 89.5 (C-1a), 95.6
(C-1b), 169.4, 169.5, 170.1, 170.5, 170.9, 171.1
dd, J6,5 2.5 Hz, J6,6 12.4 Hz, H-6), 4.20 (1H, dd, J6 ,5
0
0
5.0 Hz, J6 ,6 12.4 Hz, H-60), 5.01–5.14 (6H, m, H-4,
0
2 ꢂ PhCH2, H-2), 5.30 (1H, at, J 7.8 Hz, H-1), 7.33–
7.37 (10H, m, 10 ꢂ Ar-H); dC (100.6 MHz, CDCl3)
20.6, 20.7, 20.8 (3 ꢂ COCH3), 59.3 (OCH3), 61.8 (C-6),
68.5 (C-4), 69.6, 69.7 (2 ꢂ d, JC,P 6.0 Hz, 2 ꢂ PhCH2),
71.9 (d, JCꢁ2,P 8.0 Hz, C-2), 72.8 (C-5), 80.9 (C-3),
96.5 (d, JCꢁ1,P 4.0 Hz, C-1), 127.8, 127.9, 128.4, 128.5,
128.6, 135.1, 135.2, 135.3 (12 ꢂ Ar-C), 169.2, 169.3,
170.6 (3 ꢂ CH3CO); dP (162 MHz, CDCl3) ꢁ3.16 (1H
decoupled).
(6 ꢂ CH3C=O); m/z (ES+) 349 (M+NH þ, 100%), 314
4
(MꢁH2O+H+, 60%), 354 (M+Na+, 42%); HRMS
(ES+) calcd for C12H17O8N3Na (MNa+) 354.0913; found
354.0908. (Found: C, 43.45; H, 5.73; N, 12.61.
C12H17O8N3 requires C, 43.51; H, 5.71; N, 12.68).
4.5. Dibenzylphosphate-2,4,6-tri-O-acetyl-3-deoxy-D-
ribo-hexopyranoside (6b)
4.4. Dibenzylphosphate-2,4,6-tri-O-acetyl-3-O-methyl-D-
glucopyranoside (6a)
LDA (0.73 mL of a 2.0 M solution in THF/heptane/
ethylbenzene, 1.4 mmol, 1.1 equiv) was added slowly
to a stirred solution of triacetate 5b (0.4 g, 1.3 mmol,
1 equiv) in THF (20 mL) at ꢁ78 °C under argon. After
LDA (1.7 mL of a 2.0 M solution in THF/heptane/
ethylbenzene, 3.4 mmol, 1.1 equiv) was added slowly
to a stirred solution of triacetate 5a (1 g, 3.1 mmol,
1 equiv) in THF (40 mL) at ꢁ78 °C under argon. After
15 min,
a solution of tetrabenzyl pyrophosphate
(0.96 g, 1.8 mmol, 1.3 equiv) in THF (10 mL) was
added, and the reaction mixture was allowed to warm
to 0 °C. After 5 h at 0 °C a saturated aqueous solution
of NH4Cl (100 mL) was added, and the mixture was
extracted with CH2Cl2 (3 ꢂ 100 mL). The combined
15 min,
a solution of tetrabenzyl pyrophosphate
(2.18 g, 4.03 mmol, 1.3 equiv) in THF (10 mL) was
added, and the reaction mixture was allowed to warm
to 0 °C. After 5 h at 0 °C a saturated aqueous solution

R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
1017
organic extracts were dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by flash
column chromatography (EtOAc/petrol, 6:4) to afford
the two anomers of dibenzylphosphate 6b (0.4 g of the
a-anomer 6ba, 52% and 0.18 g of the b-anomer 6bb,
and 0.34 g of the b-anomer 6cb, 18%) as colourless oils.
20
6ca: ½aꢃD +69 (c 1.0, CHCl3); mmax (thin film) 1748 (br
s, C@O); dH (400 MHz, CDCl3) 1.96, 2.01, 2.11
(3 ꢂ 3H, 3 ꢂ s, 3 ꢂ C@OCH3), 3.92–3.99 (2H, m, H-5,
H-6), 4.16 (1H, dd, J6 ,5 4.5 Hz, J6 ,6 12.6 Hz, H-60),
4.72 (1H, adt, J3,F 53.0 Hz, J 9.3 Hz, H-3), 4.99–5.11
(5H, m, H-2, 2 ꢂ PhCH2), 5.19–5.27 (1H, m, J 12.9 Hz,
J 10.4 Hz, J 9.11 Hz, H-4), 5.89 (1H, adt, J1,P 6.8 Hz, J
3.5 Hz, H-1), 7.34–7.39 (10H, m, 10 ꢂ Ar-H); dC
(100.6 MHz, CDCl3) 20.4, 20.5, 20.6 (3 ꢂ C@OCH3),
61.0 (C-6), 68.0 (d, JCꢁ4,F 18.0 Hz, C-4), 69.0 (d, JCꢁ5,F
7.0 Hz, C-5), 69.4 (d, JC,P 5.0 Hz, 2 ꢂ PhCH2), 67.0
(dd, JCꢁ2,F 17.0 Hz, JCꢁ2,P 7.0 Hz, C-2), 88.5 (d, JCꢁ3,F
188 Hz, C-3), 94.0 (dd, JCꢁ1,F 9.0 Hz, JCꢁ1,P 5.0 Hz,
C-1), 128.0, 128.6, 128.7, 128.8 (12 ꢂ Ar-C), 169.1,
169.7, 170.5 (3 ꢂ CH3C@O); dP (162 MHz, CDCl3)
0
0
20
24%) as colourless oils. 6ba: ½aꢃD +84 (c 1.0, CHCl3);
m
max (thin film) 1748 (br s, C@O); dH (400 MHz, CDCl3)
1.90, 1.98 (2 ꢂ 3H, 2 ꢂ s, 2 ꢂ C@OCH3), 2.01–2.05 (1H,
m, H-3a), 2.06 (3H, s, C@OCH3), 2.32 (1H, adt, J
4.8 Hz, J 11.4 Hz, H-3e), 3.98–4.02 (2H, m, H-5, H-6),
4.16 (1H, dd, J6 ,5 5.0 Hz, J6 ,6 12.9 Hz, H-60), 4.82–
4.94 (2H, m, H-2, H-4), 5.07–5.12 (4H, m, 2 ꢂ PhCH2),
5.81 (1H, dd, J1,2 3.0 Hz, J1,P 6.3 Hz, H-1), 7.34–7.38
(10H, m, 10 ꢂ Ar-H); dC (100.6 MHz, CDCl3) 20.5,
20.6, 20.8 (3 ꢂ C@OCH3), 28.4 (C-3), 61.8 (C-6), 65.0
(C-4), 67.0 (d, JCꢁ2,P 7.0 Hz, C-2), 69.4 (d, JC,P
5.0 Hz, 2 ꢂ PhCH2), 69.6 (C-5), 93.4 (d, JCꢁ1,P 6.0 Hz,
C-1), 127.9, 128.6, 128.7, 135.4, 133.5 (12 ꢂ Ar-C),
169.4, 169.7, 170.6 (3 ꢂ CH3C@O); dP (162 MHz,
CDCl3) ꢁ2.07 (1H decoupled); m/z (ES+) 609
(M+MeCN=NH4þ, 100%), 573 (M+Na+, 55%), 568
(M+NH4þ, 20%), 551 (M+H+, 15%); HRMS (ES+)
calcd for C26H31O11PNa (MNa+) 573.1501; found
573.1496. (Found: C, 56.70; H, 5.67; P, 5.52.
C26H31O11P requires C, 56.73; H, 5.68; P, 5.63). 6bb:
dH (400 MHz, CDCl3) 1.63 (1H, aq, J 11.8 Hz, H-3a),
1.89, 1.94, 2.02 (3 ꢂ 3H, 3 ꢂ s, 3 ꢂ C@OCH3), 2.57
(1H, adt, J 5.0 Hz, J 12.3 Hz, H-3e), 3.80 (1H, m,
H-5), 4.16 (2H, m, H-6, H-60), 4.80–4.90 (2H, m, H-2,
H-4), 5.00–5.08 (4H, m, 2 ꢂ PhCH2), 5.32 (1H, at, J
6.8 Hz, H-1), 7.29–7.32 (10H, m, 10 ꢂ Ar-H); dC
(100.6 MHz, CDCl3) 20.6, 20.7, 20.8 (3 ꢂ C@OCH3),
32.4 (C-3), 62.1 (C-6), 65.0 (C-4), 68.4 (d, JCꢁ2,P
9.0 Hz, C-2), 69.5 (d, JC,P 7.0 Hz, 2 ꢂ PhCH2), 75.8
(C-5), 97.7 (d, JCꢁ1,P 5.0 Hz, C-1), 127.7, 127.9, 128.0,
128.3, 128.5, 128.6, 128.7 (12 ꢂ Ar-C), 169.3, 169.4,
170.5 (3 ꢂ CH3C@O); dP (162 MHz, CDCl3) ꢁ3.20
(1H decoupled).
0
0
ꢁ2.62 (1H decoupled); dF (376 MHz, CDCl3) ꢁ200.9
(1H decoupled); m/z (ES+) 627 (M+MeCN=NH4
,
þ
100%), 586 (M+NH4þ, 40%), 569 (M+H+, 15%);
HRMS (ES+) calcd for C26H30O11FPNa (MNa+)
591.1408; found 591.1402. (Found: C, 54.91; H, 5.33;
P, 5.29. C26H30O11F P requires C, 54.93; H, 5.32; P,
5.45). 6cb: dH (400 MHz, CDCl3) 1.98, 2.02, 2.12
(3 ꢂ 3H, 3 ꢂ s, 3 ꢂ C@OCH3), 3.74 (1H, m, H-6), 4.12
(1H, dd, J6,5 2.0 Hz, J6,6 12.4 Hz, H-60), 4.24 (1H, dd,
0
J6 ,5 4.8 Hz, J6 ,6 12.4 Hz, H-60), 4.59 (1H, adt, J3,F
51.8 Hz, J 9.1 Hz, H-3), 5.01–5.10 (4H, m, 2 ꢂ PhCH2),
5.25–5.31 (3H, m, H-4, H-2, H-1), 7.31–7.36 (10H, m,
10 ꢂ Ar-H); dC (100.6 MHz, CDCl3) 20.4, 20.6
(3 ꢂ C@OCH3), 61.1 (C-6), 67.6 (d, JCꢁ4,F 19.0 Hz,
C-4), 69.7 (d, JC,P 6.0 Hz, 2 ꢂ PhCH2), 71.2 (dd, JCꢁ2,F
19.0 Hz, JCꢁ2,P 9.0 Hz, C-2), 71.7 (d, JCꢁ5,F 8.0 Hz,
C-5), 90.6 (d, JCꢁ3,F 193 Hz, C-3), 95.7 (dd, JCꢁ1,F 12.0
Hz, JCꢁ1,P 5.0 Hz, C-1), 127.8, 127.9, 128.0, 128.4,
128.5, 128.7, 128.9 (12 ꢂ Ar-C), 169.0, 169.1, 170.5
(3 ꢂ CH3C@O); dP (162 MHz, CDCl3) ꢁ3.17 (1H decou-
pled); dF (376 MHz, CDCl3) ꢁ196.4 (1H decoupled).
0
0
4.7. Dibenzylphosphate-2,4,6-tri-O-acetyl-3-azido-3-
deoxy-D-glucopyranoside (6d)
4.6. Dibenzylphosphate-2,4,6-tri-O-acetyl-3-deoxy-3-
fluoro-D-glucopyranoside (6c)
LDA (1.7 mL of a 2.0 M solution in THF/heptane/
ethylbenzene, 3.3 mmol, 1.1 equiv) was added slowly
to a stirred solution of triacetate 5d (1 g, 3.02 mmol,
1 equiv) in THF (40 mL) at ꢁ78 °C under argon. After
LDA (1.8 mL of a 2.0 M solution in THF/heptane/ethyl-
benzene, 3.6 mmol, 1.1 equiv) was added slowly to a stir-
red solution of triacetate 5c (1 g, 3.24 mmol, 1 equiv) in
THF (40 mL) at ꢁ78 °C under argon. After 15 min, a
solution of tetrabenzyl pyrophosphate (2.27 g,
4.22 mmol, 1.3 equiv) in THF (10 mL) was added, and
the reaction mixture was allowed to warm to 0 °C. After
5 h at 0 °C a saturated aqueous solution of NH4Cl
(100 mL) was added, and the mixture was extracted with
CH2Cl2 (3 ꢂ 100 mL). The combined organic extracts
were dried (Na2SO4), filtered and concentrated in vacuo.
The residue was purified by flash column chromatogra-
phy (EtOAc/petrol, 1:1) to afford the two anomers of
dibenzylphosphate 6c (1.27 g of the a-anomer 6ca, 74%
15 min,
a solution of tetrabenzyl pyrophosphate
(2.11 g, 3.91 mmol, 1.3 equiv) in THF (10 mL) was
added, and the reaction mixture was allowed to warm
to 0 °C. After 5 h at 0 °C a saturated aqueous solution
of NH4Cl (100 mL) was added, and the mixture was
extracted with CH2Cl2 (3 ꢂ 100 mL). The combined
organic extracts were dried (Na2SO4), filtered and
concentrated in vacuo. The residue was purified by flash
column chromatography (EtOAc/petrol, 1:1) to afford
the two anomers of dibenzylphosphate 6d (1.29 g of
the a-anomer 6da, 73% and 0.31 g of the b-anomer

1018
R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
20
6db, 17.5%) as pale yellow oils. 6da: ½aꢃD +84 (c 1.0,
CHCl3); mmax (thin film) 2110 (br s, N3), 1750 (br s,
C@O); dH (400 MHz, CDCl3) 1.96, 2.00, 2.12 (3 ꢂ 3H,
3 ꢂ s, 3 ꢂ C@OCH3), 4.80 (1H, at, J 10.1 Hz, H-3),
3.92 (1H, dd, J6,5 2.3 Hz, J6,6 12.4 Hz, H-6), 3.97 (1H,
2.1 equiv) was then added and the resulting mixture was
concentrated in vacuo to afford triethylammonium salt
7b (0.27 g, 88%) as a colourless oil, which was used in
the next step without further purification.
0
ddd, J5,6 2.3 Hz, J5,6 4.0 Hz, J5,4 10.1 Hz, H-5), 4.12
4.10. Phosphate-2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-a-D-
glucopyranoside, ditriethylammonium salt (7c)
(1H, dd, J6 ,5 4.0 Hz, J6 ,6 12.4 Hz, H-60), 4.78 (1H,
ddd, J2,3 10.1 Hz, J2,P 2.8 Hz, J2,1 3.3 Hz, H-2), 4.95
(1H, at, J 10.1 Hz, H-4), 5.05–5.15 (4H, m, 2 ꢂ PhCH2),
5.85 (1H, dd, J1,P 6.8 Hz, J1,2 3.3 Hz, H-1), 7.36–7.39
(10H, m, 10 ꢂ Ar-H); dC (100.6 MHz, CDCl3) 20.4,
20.6 (3 ꢂ C@OCH3), 60.1 (C-3), 61.3 (C-6), 67.4 (C-4),
69.7 (C-5), 69.7, 69.8 (d, JC,P 6.0 Hz, 2 ꢂ PhCH2), 70.6
(d, JCꢁ2,P 7.0 Hz, C-2), 93.3 (d, JCꢁ1,P 5.0 Hz, C-1),
128.0, 128.5, 128.7, 128.8, 128.9 (12 ꢂ Ar-C), 169.1,
169.6, 170.5 (3 ꢂ CH3C@O); dP (162 MHz, CDCl3)
ꢁ2.50 (1H decoupled); m/z (ES+) 614 (M+Na+,
100%), 650 (M+MeCN=NH4þ, 18%); HRMS (ES+)
calcd for C26H30O11N3PNa (MNa+) 614.1515; found
614.1510. (Found: C, 52.59; H, 5.08; N, 7.03; P, 5.15.
C26H30O11N3P requires C, 52.79; H, 5.11; N, 7.10; P,
5.24). 6db: dH (400 MHz, CDCl3) 1.99, 2.01, 2.14
(3 ꢂ 3H, 3 ꢂ s, 3 ꢂ C@OCH3), 3.67 (1H, at, J 10.1 Hz,
0
0
Pd(OH)2 (0.08 g, 20% w/w) was added to a solution of
compound 6c (0.4 g, 0.70 mmol, 1 equiv) in dry MeOH
(10 mL), and the resulting solution was stirred under
an atmosphere of hydrogen at rt. After 12 h, the
solution was filtered through CeliteÒ. Et3N (0.20 mL,
1.48 mmol, 2.1 equiv) was then added and the resulting
mixture was concentrated in vacuo to afford triethyl-
ammonium salt 7c (0.42 g, 90%) as a colourless oil,
which was used in the next step without further
purification.
4.11. Phosphate-2,4,6-tri-O-acetyl-3-acetamido-3-deoxy-
a-D-glucopyranoside, ditriethylammonium salt (7d)
Pd(OH)2 (0.08 g, 20% w/w) and Ac2O (0.13 mL,
1.34 mmol, 1.5 equiv) were added to a solution of com-
pound 6d (0.4 g, 0.67 mmol, 1 equiv) in dry MeOH
(10 mL), and the resulting solution was stirred under
an atmosphere of hydrogen at rt. After 12 h, the solution
was filtered through CeliteÒ. Et3N (0.20 mL, 1.41 mmol,
2.1 equiv) was then added and the resulting mixture was
concentrated in vacuo to afford triethylammonium salt
7d (0.37 g, 86%) as a colourless oil, which was used in
the next step without further purification.
0
H-3), 3.78 (1H, m, H-5), 4.10 (1H, dd, J6,5 2.5 Hz, J6,6
0
0
12.6 Hz, H-6), 4.21 (1H, dd, J6 ,5 5.0 Hz, J6 ,6 12.6 Hz,
H-60), 5.05–5.09 (6H, m, H-4, H-2, 2 ꢂ PhCH2), 5.33
(1H, at, J 7.6 Hz, H-1), 7.30–7.37 (10H, m, 10 ꢂ Ar-
H); dC (100.6 MHz, CDCl3) 20.4, 20.6 (3 ꢂ C@OCH3),
61.5 (C-6), 63.7 (C-3), 67.8 (C-4), 69.7, 69.8 (2 ꢂ d,
JC,P 6.0 Hz, 2 ꢂ PhCH2), 70.9 (d, JCꢁ2,P 9.0 Hz, C-2),
73.5 (C-5), 96.4 (d, JCꢁ1,P 4.0 Hz, C-1), 127.8, 127.9,
128.0, 128.4, 128.5, 128.6 (12 ꢂ Ar-C), 169.0, 169.1,
170.5 (3 ꢂ CH3C@O); dP (162 MHz, CDCl3) ꢁ3.21
(1H decoupled).
4.12. Phosphate-3-O-methyl-a-D-glucopyranoside, ditri-
ethylammonium salt (8a)
4.8. Phosphate-2,4,6-tri-O-acetyl-3-O-methyl-a-D-gluco-
pyranoside, ditriethylammonium salt (7a)
Compound 7a (0.39 g, 0.64 mmol, 1 equiv) was dis-
solved in a mixture of MeOH (5 mL), H2O (2 mL) and
Et3N (1 mL) and the resulting solution was stirred under
argon at room temperature for 6 days. The mixture was
then concentrated in vacuo, and the residue was applied
to a DEAE Sephadex A25 anion exchange column using
a triethylammonium bicarbonate buffer gradient (0.2–
0.35 M) to afford triethylammonium salt 8a (0.27 g,
89%) as a white solid; dH (400 MHz, D2O) 1.15 (18H,
t, J 7.3 Hz, NCH2CH3), 3.07 (12H, q, NCH2), 3.35–
3.47 (3H, m, H-4, H-3, H-2), 3.50 (3H, s, OCH3), 3.63
Pd(OH)2 (0.08 g, 20% w/w) was added to a solution of
compound 6a (0.4 g, 0.69 mmol, 1 equiv) in dry MeOH
(10 mL), and the resulting solution was stirred under an
atmosphere of hydrogen at rt. After 12 h, the solution
was filtered through CeliteÒ. Et3N (0.20 mL, 1.44 mmol,
2.1 equiv) was then added and the resulting mixture was
concentrated in vacuo to afford triethylammonium salt
7a (0.39 g, 94%) as a colourless oil, which was used in
the next step without further purification.
0
(1H, dd, J6,6 12.4 Hz, J6,5 4.8 Hz, H-6), 3.72 (1H, dd,
J6 ,6 12.4 Hz, J6 ,5 1.5 Hz, H-60), 3.75 (1H, m, H-5),
5.33 (1H, dd, J1,P 7.1 Hz, J1,2 3.2 Hz, H-1); dC
(100.6 MHz, D2O) 8.6 (NCH2CH3), 46.9 (NCH2CH3),
60.4 (OCH3), 60.6 (C-6), 69.2 (C-4), 71.6 (d, JCꢁ2,P
8.0 Hz, C-2), 72.7 (C-5), 83.0 (C-3), 94.8 (d, JCꢁ1,P
6.0 Hz, C-1); dP (162 MHz, D2O) ꢁ0.21 (1H decoupled);
m/z (ESꢁ) 273 (MꢁH+, 100%); HRMS (ESꢁ) calcd for
C7H14O9P (MꢁH+) 273.0380; found 273.0370.
0
0
4.9. Phosphate-2,4,6-tri-O-acetyl-3-deoxy-a-D-ribo-hexo-
pyranoside, ditriethylammonium salt (7b)
Pd(OH)2 (0.06 g, 20% w/w) was added to a solution of
compound 6b (0.3 g, 0.54 mmol, 1 equiv) in dry MeOH
(10 mL), and the resulting solution was stirred under an
atmosphere of hydrogen at rt. After 12 h, the solution
was filtered through CeliteÒ. Et3N (0.16 mL, 1.14 mmol,

R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
1019
4.13. Phosphate-3-deoxy-a-D-ribo-hexopyranoside,
ditriethylammonium salt (8b)
under argon at room temperature for 6 days. The mix-
ture was then concentrated in vacuo, and the residue
was applied to a DEAE Sephadex A25 anion exchange
column using a triethylammonium bicarbonate buffer
gradient (0.2–0.35 M) to afford triethylammonium salt
8d (0.27 g, 93%) as a white solid; dH (400 MHz, D2O)
1.15 (18H, t, J 7.3 Hz, NCH2CH3), 1.93 (3H, s,
NHCOCH3), 3.08 (12H, q, J 7.3 Hz, NCH2), 3.36 (1H,
at, J 9.9 Hz, H-4), 3.48 (1H, adt, J 10.6 Hz, J 2.8 Hz,
Compound 7b (0.27 g, 0.47 mmol, 1 equiv) was dis-
solved in a mixture of MeOH (5 mL), H2O (2 mL) and
Et3N (1 mL), and the resulting solution was stirred
under argon at room temperature for 6 days. The mix-
ture was then concentrated in vacuo, and the residue
was applied to a DEAE Sephadex A25 anion exchange
column using a triethylammonium bicarbonate buffer
gradient (0.2–0.35 M) to afford triethylammonium salt
8b (0.19 g, 92%) as a white solid; dH (400 MHz, D2O)
1.15 (18H, t, J 7.3 Hz, NCH2CH3), 1.70 (1H, aq, J
11.6 Hz, H-3a), 2.04 (1H, m, H-3e), 3.07 (12H, q, J
7.3 Hz, NCH2), 3.50 (1H, m, H-4), 3.56–3.72 (4H, m,
H-5, H-2, H-6, H-60), 5.26 (1H, dd, J1,P 5.3 Hz, J1,2
3.3 Hz, H-1); dC (100.6 MHz, D2O) 8.6 (NCH2CH3),
34.3 (C-3), 47.0 (NCH2CH3), 60.9 (C-6), 64.4 (C-4),
67.2 (d, JCꢁ2,P 8.0 Hz, C-2), 73.2 (C-5), 93.4 (d, JCꢁ1,P
5.0 Hz, C-1); dP (162 MHz, D2O) 0.54 (1H decoupled);
m/z (ESꢁ) 243 (MꢁH+, 100%), 487 (2MꢁH+, 50%),
731 (3MꢁH+, 25%); HRMS (ESꢁ) calcd for
C6H12O8P (MꢁH+) 243.0275; found 243.0267.
0
H-2), 3.65 (1H, dd, J6,6 12.4 Hz, J6,5 4.3 Hz, H-6),
3.71 (1H, dd, J6 ,6 12.4 Hz, J6 ,5 2.3 Hz, H-60), 3.79
(1H, m, H-5), 3.99 (1H, at, J 10.4 Hz, H-3), 5.38 (1H,
dd, J1,P 7.1 Hz, J1,2 3.5 Hz, H-1); dC (100.6 MHz,
D2O) 8.6 (NCH2CH3), 22.6 (NHCOCH3), 47.0
(NCH2CH3), 54.2 (C-3), 60.6 (C-6), 67.9 (C-4), 71.6
(d, JCꢁ2,P 9.0 Hz, C-2), 73.1 (C-5), 94.6 (d, JCꢁ1,P
5.0 Hz, C-1); 172.9 (NHCOCH3); dP (162 MHz, D2O)
ꢁ1.05 (1H decoupled); m/z (ESꢁ) 300 (MꢁH+, 100%),
601 (2MꢁH+, 30%); HRMS (ESꢁ) calcd for
C8H15NO9P (MꢁH+) 300.0489; found 300.0479.
0
0
4.16. Phosphate-3-azido-3-deoxy-a-D-glucopyranoside,
ditriethylammonium salt (8e)
4.14. Phosphate-3-deoxy-3-fluoro-a-D-glucopyranoside,
ditriethylammonium salt (8c)
A solution of azide 5d (1.0 g, 3.02 mmol, 1 equiv) in dry
THF (25 mL) was added to a stirred solution of o-
phenylene phosphorochloridate 10 (1.72 g, 9.06 mmol,
3 equiv) and sym-collidine (1.28 g, 10.0 mmol, 3.5 equiv)
in THF (25 mL) at 0 °C. The reaction mixture was stir-
red at 0 °C for 30 min, and then filtered to remove the
precipitate. Water (0.4 mL) and sym-collidine (1.28 g)
were added to the filtrate and the reaction mixture was
stirred for a further 30 min at room temperature. The
solution was then cooled to 0 °C and re-filtered before
the solvent was removed in vacuo. The product was then
dried thoroughly under vacuum. The residue was
dissolved in a minimal amount of dioxane (ꢀ5 mL),
cooled to 12 °C, lead tetraacetate (2.67 g, 6.02 mmol,
2 equiv) was added and the reaction mixture was then
stirred for 3 h at room temperature. The solvent was
then removed and the residue of crude 7e was dissolved
in a mixture of MeOH (10 mL), Et3N (2 mL) and water
(4 mL). The resulting mixture was stirred under argon at
room temperature for 6 days. The reaction mixture was
then concentrated in vacuo, and the residue was applied
to a DEAE Sephadex A25 anion exchange column using
a triethylammonium bicarbonate buffer gradient (0.2–
0.35 M) to afford triethylammonium salt 8e (0.67 g,
46%) as a white solid; dH (400 MHz, D2O) 1.16 (18H,
t, J 7.2 Hz, NCH2CH3), 3.08 (12H, q, J 7.2 Hz,
NCH2), 3.38 (1H, at, J 10.2 Hz, H-4), 3.47 (1H, adt, J
10.2 Hz, J 2.7 Hz, H-2), 3.59–3.67 (2H, m, H-3, H-6),
Compound 7c (0.42 g, 0.71 mmol, 1 equiv) was
dissolved in a mixture of MeOH (5 mL), H2O (2 mL)
and Et3N (1 mL), and the resulting solution was stirred
under argon at room temperature for 6 days. The mix-
ture was then concentrated in vacuo, and the residue
was applied to a DEAE Sephadex A25 anion exchange
column using a triethylammonium bicarbonate buffer
gradient (0.2–0.35 M) to afford triethylammonium salt
8c (0.29 g, 90%) as a white solid; dH (400 MHz, D2O)
1.15 (18H, t, J 7.3 Hz, NCH2CH3), 3.08 (12H, q, J
7.3 Hz, NCH2), 3.59– 3.76 (4H, m, H-4, H-2, H-6,
H-60), 3.78 (1H, m, H-5), 4.53 (1H, adt, J3,F 54.6 Hz, J
9.1 Hz, H-3), 5.39 (1H, m, H-1); dC (100.6 MHz, D2O)
8.6 (NCH2CH3), 46.9 (NCH2CH3), 60.3 (C-6), 68.0 (d,
JCꢁ4,F 18.0 Hz, C-4), 70.5 (dd, JCꢁ2,F 17.0 Hz, JCꢁ2,P
8.0 Hz, C-2), 72.1 (d, JCꢁ5,F 7.0 Hz, C-5), 95.2 (d, JCꢁ3,F
178.0 Hz, C-3), 94.7 (dd, JCꢁ1,F 10.0 Hz, JCꢁ1,P 5.0 Hz,
C-1); dP (162 MHz, D2O) ꢁ0.08 (1H decoupled); dF (376
MHz, CDCl3) ꢁ200.5 (1H decoupled); m/z (ESꢁ) 261
(MꢁH+, 100%), 523 (2MꢁH+, 37%), 1046 (3MꢁH+,
10%); HRMS (ESꢁ) calcd for C6H11FO8P (MꢁH+)
261.0180; found 261.0170.
4.15. Phosphate-3-acetamido-3-deoxy-a-D-glucopyran-
oside, ditriethylammonium salt (8d)
3.72 (1H, dd, J6 ,6 12.6 Hz, J6 ,5 1.7 Hz, H-60), 3.77
(1H, m, H-5), 5.35 (1H, dd, J1,P 6.8 Hz, J1,2 3.4 Hz,
H-1); dC (100.6 MHz, D2O) 8.6 (NCH2CH3), 47.0
(NCH2CH3), 60.4 (C-6), 66.0 (C-3), 68.4 (C-4), 70.7
0
0
Compound 7d (0.37 g, 0.59 mmol, 1 equiv) was dis-
solved in a mixture of MeOH (5 mL), H2O (2 mL) and
Et3N (1 mL), and the resulting solution was stirred

1020
R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
(d, JCꢁ2,P 9.6 Hz, C-2), 72.7 (C-5), 94.5 (d, JCꢁ1,P
6.4 Hz, C-1); dP (162 MHz, D2O) ꢁ1.39 (1H decoupled);
m/z (ESꢁ) 284 (MꢁH+, 30%), 569 (2MꢁH+, 100%), 854
(3MꢁH+, 80%), 1138 (4MꢁH+, 35%); HRMS (ESꢁ)
calcd for C6H12N3O8P (MꢁH+) 284.0289; found
284.0281.
1.67 (1H, aq, J 11.6 Hz, glu: H-3ax), 2.05 (1H, adt, J
11.4 Hz, J 4.5 Hz, glu: H-3eq), 3.02 (16H, q, J 7.3 Hz,
NCH2), 3.47–3.72 (5H, m, glu: H-4, H-2, H-5, H-6,
H-60), 3.90–4.25 (5H, m, rib: H-5, H-50, H-4, H-3, H-2),
5.39 (1H, dd, J1,2 3.3 Hz, J1,P 7.1 Hz, glu: H-1), 5.82
(2H, m, rib: H-1, U: H-5), 7.84 (1H, d, J5,6 8.1 Hz, U:
H-6); dC (100.6 MHz, D2O) 8.55 (NCH2CH3), 34.5
(glu: C-3), 46.9 (NCH2CH3), 60.7 (glu: C-6), 64.1 (glu:
C-4), 65.2 (d, JCꢁ5,P 6.0 Hz, rib: C-5), 67.0 (d, JCꢁ2,P
8.0 Hz, glu: C-2), 69.9 (rib: C-3), 73.7 (glu: C-5), 74.1
(rib: C-2), 83.6 (d, JCꢁ4,P 10.0 Hz, rib: C-4), 88.8 (rib:
C-1), 94.8 (d, JCꢁ1,P 7.0 Hz, glu: C-1), 102.9 (U: C-5),
142.0 (U: C-6), 151.1 (U: C-4), 166.5 (U: C-2); dP
(162 MHz, D2O) ꢁ11.2, ꢁ12.5 (1H decoupled); m/z
(ESꢁ) 549 (MꢁH+, 100%); HRMS (ESꢁ) calcd for
C15H23N2O16P2 (MꢁH+) 549.0522; found 549.0524.
4.17. Uridinediphosphoryl-3-O-methyl-a-D-glucopyran-
oside, ditriethylammonium salt (9a)
4-Morpholine-N,N0-dicyclohexylcarboxamidinium uri-
dine 50-monophosphormorpholidate (0.43 g, 0.63 mmol,
2 equiv) and tetrazole (0.066 g, 0.94 mmol, 3 equiv) were
added to a solution of compound 8a (0.15 g, 0.31 mmol,
1 equiv) in dry pyridine (10 mL), and the resulting solu-
tion was stirred at room temperature. After 6 days the
solution was concentrated in vacuo. The residue was
applied to a DEAE Sephadex A25 anion exchange
column using a triethylammonium bicarbonate buffer
gradient (0.35–0.5 M) to afford UDP-derivative 9a
4.19. Uridinediphosphoryl-3-deoxy-3-fluoro-a-D-gluco-
pyranoside, ditriethylammonium salt (9c)
22
(0.13 g, 51%) as a white solid, ½aꢃD +20 (c 1.25, MeOH);
4-Morpholine-N,N0-dicyclohexylcarboxamidinium uri-
dine 50-monophosphormorpholidate (0.44 g, 0.64 mmol,
2 equiv) and tetrazole (0.068 g, 0.97 mmol, 3 equiv) were
added to solution of compound 8c (0.15 g, 0.32 mmol,
1 equiv) in dry pyridine (10 mL) and the resulting solu-
tion was stirred at room temperature. After 6 days, the
solution was concentrated in vacuo. The residue was
applied to a DEAE Sephadex A25 anion exchange
column using a triethylammonium bicarbonate buffer
gradient (0.35–0.5 M) to afford UDP-derivative 9c
d
H (400 MHz, D2O) 1.11 (18H, t, J 7.3 Hz, NCH2CH3),
3.03 (12H, q, J 7.3 Hz, NCH2CH3), 3.35–3.47 (3H, m,
glu: H-4, H-3, H-2), 3.48 (3H, s, OCH3), 3.62 (1H, dd,
0
0
J6,5 4.5 Hz, J6,6 12.4 Hz, glu: H-6), 3.70 (1H, dd, J6 ,5
2.3 Hz, J6 ,6 12.4 Hz, glu: H-60), 3.77 (1H, m, glu:
0
H-5), 3.91–4.24 (5H, m, rib: H-5, H-50, H-4, H-3,
H-2), 5.45 (1H, dd, J1,P 7.1 Hz, J1,2 3.0 Hz, glu: H-1),
5.80 (2H, m, rib: H-1, U: H-5), 7.82 (1H, d, J 8.1 Hz,
U: H-6); dC (100.6 MHz, D2O) 8.5 (NCH2CH3), 46.9
(NCH2CH3), 60.4 (glu: C-6), 60.5 (OCH3), 65.3 (d,
JCꢁ5,P 5.0 Hz, rib: C-5), 68.9 (glu: C-4), 70.0 (rib: C-3),
71.6 (d, JCꢁ2,P 9.0 Hz, glu: C-2), 73.1 (glu: C-5), 74.1
(rib: C-2), 83.1 (glu: C-3), 83.6 (d, JCꢁ4,P 10.0 Hz, rib:
C-4), 88.7 (rib: C-1), 96.0 (d, JCꢁ1,P 7.0 Hz, glu: C-1),
102.9 (U: C-5), 141.9 (U: C-6), 152.1 (U: C-4), 167.5
(U: C-2); dP (162 MHz, D2O) ꢁ11.1, ꢁ12.9 (1H decou-
pled); m/z (ESꢁ) 579 (MꢁH+, 100%); HRMS (ESꢁ)
calcd for C16H25N2O17P2 (MꢁH+) 579.0628; found
579.0629.
22
(0.12 g, 51%) as a white solid, ½aꢃD +9.1 (c 1.0, MeOH);
d
H (400 MHz, D2O) 1.13 (18H, t, J 7.5 Hz, NCH2CH3),
3.04 (12H, q, J 7.5 Hz, NCH2), 3.61–3.83 (5H, m, glu:
H-5, H-6, H-60, H-4, H-2), 3.92–4.27 (5H, m, rib: H-5,
H-50, H-4, H-3, H-2), 4.50 (1H, adt, J3,F 54.3 Hz, J
9.2 Hz, glu: H-3), 5.52 (1H, m, glu: H-1), 5.80–5.85
(2H, m, rib: H-1, U: H-5), 7.85 (1H, d, J 7.8 Hz, U:
H-6); dC (100.6 MHz, D2O) 8.6 (NCH2CH3), 46.9
(NCH2CH3), 60.1 (glu: C-6), 64.1 (d, JCꢁ5,P 5.0 Hz,
rib: C-5), 66.2 (d, JCꢁ4,F 17.0 Hz, glu: C-4), 69.7 (dd,
JCꢁ2,F 19.0 Hz, JCꢁ2,P 6.0 Hz, glu: C-2), 70.2 (rib:
C-3), 72.9 (d, JCꢁ5,F 6 Hz, glu: C-5), 74.2 (rib: C-2),
84.1 (d, JCꢁ4,P 8.0 Hz, rib: C-4), 88.7 (rib: C-1), 94.1
(dd, JCꢁ1,P 5.0 Hz, JCꢁ1,F 9.0 Hz, glu: C-1), 95.0 (d,
JCꢁ3,F 184 Hz, glu: C-3), 102.9 (U: C-5), 142.2 (U:
C-6), 152.2 (U: C-4), 166.6 (U: C-2); dP (162 MHz,
D2O) ꢁ11.2, ꢁ13.1 (1H decoupled); dF (376.6 MHz,
D2O) ꢁ200.4 (1H decoupled); m/z (ESꢁ) 567 (MꢁH+,
100%); HRMS (ESꢁ) calcd for C15H22FN2O16P2
(MꢁH+) 567.0428; found 567.0431.
4.18. Uridinediphosphoryl-3-deoxy-a-D-ribo-hexopyran-
oside, ditriethylammonium salt (9b)
4-Morpholine-N,N0-dicyclohexylcarboxamidinium uri-
dine 50-monophosphormorpholidate (0.30 g, 0.44 mmol,
2 equiv) and tetrazole (0.047 g, 0.67 mmol, 3 equiv) were
added to a solution of compound 8b (0.10 g, 0.22 mmol,
1 equiv) in dry pyridine (8 mL) and the resulting solu-
tion was stirred at room temperature. After 6 days, the
solution was concentrated in vacuo. The residue was
applied to a DEAE Sephadex A25 anion exchange
column using a triethylammonium bicarbonate buffer
gradient (0.35–0.5 M) to afford UDP-derivative 9b
4.20. Uridinediphosphoryl-3-acetamido-3-deoxy-a-D-
glucopyranoside, ditriethylammonium salt (9d)
22
(0.09 g, 52%) as a white solid, ½aꢃD +19 (c 0.5, MeOH);
4-Morpholine-N,N0-dicyclohexylcarboxamidinium uri-
dine 50-monophosphormorpholidate (0.41 g, 0.59 mmol,
d
H (400 MHz, D2O) 1.11 (18H, t, J 7.3 Hz, NCH2CH3),

R. Danac et al. / Carbohydrate Research 343 (2008) 1012–1022
1021
2 equiv) and tetrazole (0.062 g, 0.88 mmol, 3 equiv) were
added to a solution of compound 8d (0.15 g, 0.30 mmol,
1 equiv) in dry pyridine (10 mL) and the resulting solu-
tion was stirred at room temperature. After 6 days, the
solution was concentrated in vacuo. The residue was
applied to a DEAE Sephadex A25 anion exchange
column using a triethylammonium bicarbonate buffer
gradient (0.35–0.5 M) to afford UDP-derivative 9d
(d, JCꢁ5,P 6.0 Hz, rib: C-5), 67.6 (glu: C-3), 68.9 (glu:
C-4), 70.2 (rib: C-3), 71.9 (d, JCꢁ2,P 8.0 Hz, glu: C-2),
73.6 (glu: C-5), 74.7 (rib: C-2), 84.1 (d, JCꢁ4,P 6.5 Hz,
rib: C-4), 88.8 (rib: C-1), 95.7 (d, JCꢁ1,P 4.0 Hz, glu:
C-1), 102.3 (U: C-5), 141.7 (U: C-6), 159.7 (U: C-4),
165.2 (U: C-2); dP (162 MHz, CD3OD) ꢁ11.3, ꢁ13.1
(1H decoupled); m/z (ESꢁ) 590 (MꢁH+, 100%); HRMS
(ESꢁ) calcd for C17H22N5O16P2 (MꢁH+) 590.0542;
found 590.0542.
22
(0.13 g, 54%) as a white solid, ½aꢃD +13 (c 0.75, MeOH);
d
H (400 MHz, D2O) 1.09 (18H, t, J 7.3 Hz, NCH2CH3),
1.88 (3H, s, C@OCH3), 3.00 (12H, q, J 7.3 Hz,
NCH2CH3), 3.34 (1H, at, J 10.1 Hz, glu: H-4), 3.46
4.22. Germination assays
0
(1H, m, glu: H-2), 3.63 (1H, dd, J6,5 4.5 Hz, J6,6
The effect of compounds 4a–d, 5a–d, 6a–d, 8a–e and
9a–e was assessed on the germination of T. rubrum
microconidia as follows. T. rubrum conidia were
harvested, and the spore suspensions were adjusted to
the appropriate density. Test compounds were made
up into solution and added to the spore suspension to
obtain final concentrations of 0 lM (control), 1 lM,
10 lM, 100 lM and 1mM of each test compound. The
resulting suspensions were pipetted with the appropriate
number of replicates onto 0.1% keratin agar and
incubated for 24 h at 30 °C. After incubation, the images
of the germinated spores were taken to allow assessment
of germ tube formation. A sample of 30 sets of 10 spores
was counted to quantify germ tube initiation. The
number of spores out of 300 counted per treatment
was recorded as a proportion and plotted. Error bars
were estimated as the standard deviation within treat-
ments. Compound 6a showed 40% inhibition of germ
tube formation with respect to the relevant control at
a concentration of 1mM. No other compounds showed
significant inhibition of germination at any of the
concentrations tested.
0
0
12.4 Hz, glu: H-6), 3.69 (1H, dd, J6 ,5 2.0 Hz, J6 ,6
12.4 Hz, glu: H-60), 3.80 (1H, m, glu: H-5), 3.93–4.24
(6H, m, glu: H-3, rib: H-2, H-3, H-4, H-5, H-50), 5.49
(1H, dd, J1,P 7.3 Hz, J1,2 3.5 Hz, glu: H-1), 5.79 (2H,
m, rib: H-1, U: H-5), 7.79 (1H, d, J 8.1 Hz, U: H-6);
dC (100.6 MHz, D2O) 8.5 (NCH2CH3), 22.2 (C@OCH3),
46.9 (NCH2CH3), 54.4 (glu: C-3), 60.6 (glu: C-6), 65.3
(d, JCꢁ5,P 5.0 Hz, rib: C-5), 67.9 (glu: C-4), 70.0 (rib:
C-3), 71.8 (d, JCꢁ2,P 7.0 Hz, glu: C-2), 73.3 (glu: C-5),
74.2 (rib: C-2), 83.6 (d, JCꢁ4,P 9.0 Hz, rib: C-4), 88.8
(rib: C-1), 95.3 (d, JCꢁ1,P 4.0 Hz, glu: C-1), 102.7 (U:
C-5), 141.9 (U: C-6), 151.5 (U: C-4), 169.6 (U: C-2),
178.2 (CH3C=O); dP (162 MHz, D2O) ꢁ11.2, ꢁ12.8
(1H decoupled); m/z (ESꢁ) 606 (MꢁH+, 100%); HRMS
(ESꢁ) calcd for C17H26N3O17P2 (MꢁH+) 606.0737;
found 606.0729.
4.21. Uridinediphosphoryl-3-azido-3-deoxy-a-D-gluco-
pyranoside, ditriethylammonium salt (9e)
4-Morpholine-N,N0-dicyclohexylcarboxamidinium uri-
dine 50-monophosphormorpholidate (0.26 g, 0.37 mmol,
1.1 equiv) was added to a solution of compound 8e
(0.16 g, 0.34 mmol, 1 equiv) in dry pyridine (3 mL) and
the resulting solution was stirred at room temperature.
After 10 days, the solution was concentrated in vacuo.
The residue was applied to a DEAE Sephadex A25
anion exchange column using a triethylammonium
bicarbonate buffer gradient (0.35–0.5 M) to afford
uridinediphosphoryl-3-azido-3-deoxy-a-D-glucopyran-
oside, ditriethylammonium salt 9e (0.15 g, 55%) as a
Acknowledgement
The authors gratefully acknowledge the European
Union (Marie Curie Intra-EU Fellowship to R.D.).
References
22
white solid, ½aꢃD +29 (c 0.5, MeOH); dH (400 MHz,
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´
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