Synthesis of UDP-GlcNAc derivatives modified at OH-4 as potential chain-terminators of chitin biosynthesis

Article abstract of DOI:10.1016/j.tetasy.2007.05.032

A series of UDP-GlcNAc derivatives and precursors that have been modified at the 4-position were synthesised from N-acetyl glucosamine as potential chain terminators of chitin biosynthesis. None of the UDP-derivatives or the precursors tested displayed an

Full text of DOI:10.1016/j.tetasy.2007.05.032

Tetrahedron: Asymmetry 18 (2007) 1299–1307
Synthesis of UDP-GlcNAc derivatives modified at OH-4
as potential chain-terminators of chitin biosynthesis
a
a
b
b
a,
*
Thierry Muller, Ramona Danac, Lucy Ball, Sarah J. Gurr and Antony J. Fairbanks
a
Chemistry Research Laboratory, Oxford University, Mansfield Road, Oxford OX1 3TA, UK
Department of Plant Sciences, Oxford University, South Parks Road, Oxford OX1 3RB, UK
b
Received 28 March 2007; accepted 29 May 2007
Abstract—A series of UDP-GlcNAc derivatives and precursors that have been modified at the 4-position were synthesised from N-acetyl
glucosamine as potential chain terminators of chitin biosynthesis. None of the UDP-derivatives or the precursors tested displayed any
significant anti-fungal activity in cell adhesion or germination assays on the dermatophyte Trichophyton rubrum.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
ing. Subsequently, chain-termination strategies have
found routine and widespread use as a means of interfering
with oligonucleotide synthesis, and, moreover, have be-
come the molecular basis of anti-viral therapies in clinical
As bacterial, viral and fungal drug resistance to currently
administered treatments increases, the need for the devel-
opment of new therapeutic strategies against infective
agents becomes more urgent. Crucial to the survival of
many pathogens are carbohydrate structures, which are
either themselves structurally unique or specific to non-
mammalian organisms. Indeed, inhibition or interference
with the correct biosynthesis of oligosaccharide materials
represents an attractive and potentially highly selective
strategy for the development of new classes of therapeutic
4
use, perhaps most pre-eminently in the case of AZT.
Although chain-termination processes have been impli-
cated in the biological affects of some monosaccharide
5
derivatives on mammalian glycoconjugate and glycoso-
6
aminoglycan biosynthesis, it is curious that a chain-termi-
nation approach has not been widely promulgated as a
strategy for the development of new classes of inhibitors
of the biosynthesis of pathogenic oligosaccharides.
1
agents.
Although there are potential pitfalls in this approach, it
is our considered opinion that this research route merits
further investigation. For example, in cases where multi-
ple repeat units of carbohydrate structures are essential
for pathogenic survival (e.g., in cell wall formation),
the statistical chances of incorporation leading to
chain-termination would be more favourable. Moreover,
there is now good evidence that glycosyl transferases
do process activated donor substrates that are modified
In recent years, substantial effort has been expended in the
search for inhibitors of particular glycosyl transferase and
glycosidase enzymes as a means of disrupting specific bio-
2
synthetic pathways. The design of such inhibitors can be
problematic, since in many cases little is known about the
precise enzymes involved. However, an alternative strategy,
and one that does not rely on precise structural informa-
tion about specific enzymes in particular biochemical path-
ways, is to exploit the structural analysis of the key
targeted oligosaccharide structure. This strategy is to
invoke chain-termination of oligosaccharide biosynthesis.
7
in a minimal way at a single hydroxyl group. Although
some of these compounds have been shown to act as en-
zyme inhibitors, many are good substrates that are read-
8
ily processed. Indeed, as alluded to above, there is also
Chain-termination of oligonucleotide synthesis was origi-
nally developed by Sanger as a means of DNA sequenc-
a literature precedent that chain-terminating modified
carbohydrates have already been incorporated into
3
5
mammalian glycoprotein oligosaccharides and glycos-
6
aminoglycans, with the net results of inhibition of their
*
0
Corresponding author. E-mail: antony.fairbanks@chem.ox.ac.uk
biosynthesis.
957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.05.032

1300
T. Muller et al. / Tetrahedron: Asymmetry 18 (2007) 1299–1307
As part of a program aimed at investigating the potential
opportunities that chain-termination of oligosaccharide
biosynthesis offers to disease control, we became interested
in the rational design of novel anti-fungal agents. Fungal
infections represent a serious hazard to human and animal
closed15 the synthesis of GlcNAc derivatives 1a–d, in which
OH-4 had been modified, and these were used as a starting
point for the synthesis of the corresponding glycosyl phos-
phates and UDP compounds. Removal of the allyl protec-
tion for methoxy-, deoxy- and fluoro-compounds 1a–c was
readily achieved by heating with tetrakistriphenylphos-
phine palladium in acetic acid. However, this procedure
was found to also reduce the azide functionality in com-
9
health, and, just as for bacterial infection, drug resistance
to current therapies is increasing.10 The fungal cell wall11
consists of large sections of oligosaccharide materials
including chitin, a polymer of b(1–4)N-acetylglucosamine
1
6
pound 1d. A variety of alternative deprotection condi-
(
GlcNAc), and also b-glucan. The biosynthesis of both of
tions were investigated until a successful procedure,
these non-mammalian oligosaccharides could be poten-
tially targeted using a chain-termination approach.
involving treatment with PdCl in methanol, was arrived
upon.
2
1
2
The present study focuses on the attempted inhibition of
chitin biosynthesis. Chitin is assembled stepwise by the
Subsequent conversion to the desired a-glycosyl phos-
phates 3a–d was achieved by treatment of the hemiacetals
with LDA and tetrabenzyl pyrophosphate in THF at
ꢀ78 ꢁC. Removal of the benzyl protecting groups by cata-
lytic hydrogenation in the presence of Pearlman’s catalyst,
allowed the formation of the corresponding glycosyl phos-
phates 4a–c as their triethylamine salts, and which were
used immediately in the next step after filtering over Cel-
1
3
enzyme chitin synthase, which transfers single GlcNAc
residues to a growing oligomeric chain; the donor substrate
for the enzyme being UDP-GlcNAc. Potential chain termi-
nators of this process are therefore GlcNAc residues in
1
4
which the 4-hydroxyl has been modified. If such materials
are processed by chitin synthase, then their transfer to the
terminus of the growing chitin chain results in a chain-
termination step since the required 4-hydroxyl at which
subsequent units would be added, will now be lacking.
The most obvious approach was that of synthesising and
testing the modified UDP-donors themselves, since these
are the actual substrates processed by the chitin synthase.
Alternatively potential pro-drug molecules could be syn-
thesised, which may then be converted to the active
ꢂ
ite . Alternative deprotection conditions were investigated
in the case of azido compound 3d which would not cause
simultaneous reduction of the azido group, but all of the
conditions investigated were found to be unsatisfactory.
Therefore, in this case, the catalytic hydrogenation was
run in an MeOH/acetic anhydride solvent mixture, which
led directly to the formation of the corresponding 4-NHAc
1
7
derivative 4d. Removal of the benzoyl protection of the 3-
and 6-hydroxyls then allowed access to the corresponding,
completely de-protected glycosyl phosphates 5a–d, which
were purified by anion exchange chromatography. Finally
the protracted reaction of the de-protected glycosyl phos-
phates 5a–d with UMP-morpholidate led to the formation
of the corresponding UDP-derivatives 6a–d, which were
also purified by anion exchange chromatography (Scheme
1
5
UDP-donor once inside the cell. Herein, we report the
synthesis and investigation of a variety of glycosyl phos-
phate and UDP-derivatives of GlcNAc which have been
modified at the 4-position.
2
. Results and discussion
1
).
As a first foray into this field, a decision was taken to access
compounds where the normal 4-hydroxyl of GlcNAc was
replaced by –H, –OMe, –F and –NHAc. We recently dis-
The anti-fungal action of the putative chain-termination
compounds 6a–d was assessed using two assays on micro-
OBz
O
OBz
O
OBz
O
OBz
O
a
b
c
R
R
R
R
BzO
BzO
BzO
O
P
BzO
O
P
AcHN
AcHN OH
AcHN
AcHN
O
O
OBn
O
O
.
2 Et3NH+
OBn
O
3
3
3
3
a R=OMe
b R=H
4a R=OMe
4b R=H
1
1
1
1
a R=OMe
b R=H
2a R=OMe
2b R=H
2c R=F
2d R=N3
c R=F
4c R=F
c R=F
d R=N3
4d R=NHAc
d
d R=N3
O
OH
O
OH
O
NH
R
e
R
HO
O
P
O
P
N
O
HO
O
O
P
O
AcHN
O
O
O
AcHN
O
O
. 2 Et3NH+
O
O
+
HO
OH
.
2 Et3NH
5
5
5
5
a R=OMe
b R=H
6
6
6
6
a R=OMe
c R=F
b R=H
c R=F
d R=NHAc
d R=NHAc
Scheme 1. Reagents and conditions: (a) (Ph
(BnO) P(O)]
O, THF, ꢀ78 to 0 ꢁC; 3a, 62%; 3b, 65%; 3c, 89%; 3d, 73%; (c) H
rt then Et N; 4d, 70%; (d) cyclohexylamine, MeOH, 70 ꢁC; 5a, 84%; 5b, 78%; 5c, 86%; 5d, 65%; (e) UMP-morpholidate, tetrazole, pyridine, rt; 6a, 65%; 6b,
5%; 6c, 47%; 6d, 67%.
3
P)
4
Pd, H
2
2
O, AcOH, 80 ꢁC; 2a, 83%; 2b, 81%; 2c, 79%; or PdCl , MeOH, rt; 2d, 73%; (b) LDA,
[
2
2
2
, Pd(OH) , MeOH then Et
2
3 2
N; 4a, 86%; 4b, 93%; 4c, 89%; or MeOH/Ac O,
3
6

T. Muller et al. / Tetrahedron: Asymmetry 18 (2007) 1299–1307
1301
conidia and germlings of the dermatophyte18 Trichophyton
rubrum, a fungal pathogen that causes superficial skin
diseases in humans and animals. These two assays measured
the effects of the compounds both on adhesion and germi-
nation of fungal spores/germlings. However, none of the
completely de-protected putative chain terminators 6a–d
displayed significant biological activity in the 1–1000 lM
range that was examined. Moreover the de-protected
glycosyl phosphates 5a–d were also investigated for any
anti-fungal activity, but again none of these compounds dis-
played any significant anti-fungal effects in the range tested.
theses. Optical rotations were measured on a Perkin–Elmer
241 polarimeter 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 Chromatogra-
phy (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 sulphuric acid), or sul-
phuric acid (5% in ethanol). Flash column chromatography
was carried out using Sorbsil C60 40/60 silica. Dichloro-
methane was distilled from calcium hydride, or dried on
an alumina column. Anhydrous THF, DMF, pyridine,
methanol and toluene were purchased from Fluka over
molecular sieves. ‘Petrol’ refers to the fraction of light
petroleum ether boiling in the range of 40–60 ꢁC. CMAW
(chloroform/methanol/acetic acid/water) used as eluant
These results are in contrast to the biological test data ob-
1
5
tained for a series of protected GlcNAc derivatives,
which had been modified at the 4-position. Moreover the
UDP-methoxy derivative 6a has recently been synthesised
by Finney, and has been reported to be a substrate for chi-
1
4
tin synthase. One rationalisation for the lack of activity
was prepared in the following ratio (CHCl /MeOH/
3
5
d
of the UDP derivatives, in line with previous hypotheses,
AcOH/H O, 60:30:3:5).
2
1
9
is the poor compound polarity profile which could mean
that these UDP derivatives are unable to effectively pene-
trate intracellularly.
+
A BioRad AG 50 W-X2 cation exchange column (NEt H -
3
form, typically 2 · 20 cm) was used for the formation of the
+
triethylamine salts. The NEt H -form of the resin was pre-
3
pared by passing 1 L of a 0.1 M aqueous solution of NEt3
through the column and then a minimum 1 L of water until
pH ꢁ 7 was reached; the product was eluted with water and
collected in 1–2 mL fractions (monitoring by TLC, eluting
3
. Conclusions
A series of UDP-GlcNAc derivatives that have been mod-
ified at the 4-position have been synthesised as potential
chain terminators of chitin biosynthesis. However, none
of the UDP-derivatives displayed any significant anti-fun-
gal activity in either cell adhesion or germination assays
on T. rubrum. It is possible that this lack of biological
activity is due to poor intracellular penetration of these
highly polar compounds, particularly since one had been
previously synthesised and in that study was demonstrated
to be a substrate for a fungal chitin synthase. Further
investigations into potential uses of carbohydrate chain ter-
minators as the basis for novel strategies against a variety
of other infective agents are currently in progress, and
the results will be reported in due course.
+
with CMAW). After the column, the H -form of the resin
was regenerated by passing 1 L of 1 M HCl and then 1 L
(minimum) of water until pH ꢁ 7 was again reached; the
resin could be used several times but must be stored in
+
the H -form. General procedure for the purification of
2
0
UDP-derivatives: after co-evaporating several times with
water, all the crude materials were submitted to a DEAE
Sephadex A25 anion exchange column (typical size
2 · 20 cm) using a triethylammonium bicarbonate buffer
(TEAB, gradient 0.35–0.5 M). The TEAB was obtained
by preparing a 2 M aqueous solution of NEt (not misci-
3
ble), and by adding dry ice until a clear solution was
obtained (this stock solution could be kept in the fridge
for several days, if it turned cloudy, dry ice could be added
until a clear solution was obtained again). The clean resin
was kept in 5 mM TEAB in the fridge. The column was
prepared by first eluting with 500 mL of a 350 mM solution
of TEAB under light pressure (ꢁ1 drop per second). The
crude reaction product was then applied and the fraction
collected whilst eluting consecutively with 100 mL of 350,
400, 450 mM TEAB and finally with 500 mM TEAB until
compound elution was complete. In cases where it was
difficult to visualise the product by TLC (CMAW) MS
4. Experimental
4
.1. General
Melting points were recorded on a Kofler hot block and are
uncorrected. Proton and carbon nuclear magnetic reso-
nance (d , d ) spectra were recorded on Bruker DPX 250
250 MHz), Bruker DPX 400 (400 MHz), Bruker DQX
00 (400 MHz), Bruker AVC 500 (500 MHz) or Bruker
H
C
(
4
ꢀ
(ES ) was useful to locate material in the different factions
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 positive or negative polar-
(fraction size ꢁ1–2 mL). It should be noted that the resin
can be regenerated up to 20 times by heating it for up to
2 h at 50 ꢁC in 2 M TEAB followed by washing with water
until pH ꢁ 7 is reached, and it can then be kept in 5 mM
TEAB in a fridge.
+
ꢀ
ity (ES or ES ), or using a VG Micromass spectrometer.
High resolution mass spectra were recorded on a Walters
4.2. 2-Acetamido-3,6-di-O-benzoyl-2-deoxy-4-O-methyl-D-
glucopyranose 2a
2
790-Micromass LCT electrospray ionisation mass spec-
trometer, using either electrospray ionisation (NH , Cl)
3
techniques as stated. m/z values are reported in Daltons
Tetrakis(triphenylphosphine)palladium (3.96 g, 3.44 mmol,
and are followed by their percentage abundance in paren-
0.5 equiv) was added to a solution of allyl glycoside 1a

1302
T. Muller et al. / Tetrahedron: Asymmetry 18 (2007) 1299–1307
(
3.32 g, 6.87 mmol, 1 equiv) in AcOH (40 mL) and the
4.4. 2-Acetamido-3,6-di-O-benzoyl-2,4-dideoxy-4-fluoro-D-
glucopyranose 2c
resulting mixture was heated at 80 ꢁC. After 4 h, the mix-
ture was evaporated to dryness. The residue was then puri-
fied by flash column chromatography (CH Cl /MeOH,
Tetrakis(triphenylphosphine)palladium
(0.897 g,
0.78
2
2
9
5:5) to afford hemiacetal 2a (2.53 g, 83%) as a white solid
mmol, 0.5 equiv) was added to a solution of allyl glycoside
1c (0.73 g, 1.55 mmol, 1 equiv) in AcOH (12 mL) and the
resulting mixture was heated at 80 ꢁC. After 2 h, the
mixture was evaporated to dryness. The residue was then
purified by flash column chromatography (CMAW) to
hemiacetal 2c (0.528 g, 79%) as a colourless solid and as
a mixture of anomers, a/b-ratio being approximately 25:1
and as a mixture of anomers, a/b-ratio being approxi-
mately 10:1 (by integration over selected parts of the spec-
trum); m
(KBr) 1722 (br s, C@O), 1658 (br w, C@O)
max
ꢀ
1
cm ; dH (400 MHz, CDCl ) 1.85 (6H, s, OCCH ), 3.43
3
3
(
6H, s, CH ), 3.64 (1H, at, J 9.6 Hz, H-4a), 3.88 (1H, at,
3
J 9.6 Hz, H-4b), 4.25 (1H, dt, J 2.8 Hz, J 10.0 Hz, H-5a),
0
4.37–4.43 (2H, m, H-2), 4.54–5.58 (4H, m, H-6, H-6 ),
5.26 (1H, d, J1,2 3.6 Hz, H-1a), 5.53 (1H, dd, J 9.2 Hz, J
10.8 Hz, H-3b), 5.64 (1H, dd, J 9.2 Hz, J 10.8 Hz, H-3a),
6.22 (1H, d, J2,NH 9.6 Hz, NHa), 6.33 (1H, d, J 9.6 Hz,
(by integration over selected parts of the spectrum); m
max
ꢀ
1
(KBr) 1724 (br s, C@O), 1660 (w, C@O) cm ; dH
(400 MHz, CDCl ) 1.83 (3H, s, CH ), 4.36–4.41 (1H, m,
3
3
0
H-5), 4.42–4.51 (3H, m, H-2, H-6, H-6 ), 4.76 (1H, dt,
J4,F 51.1 Hz, J 9.4 Hz, H-4), 5.24 (1H, t, J 3.6 Hz, H-1),
5.70–5.79 (1H, m, H-3), 6.16 (d, 1H, J2,NH 9.6 Hz), 7.41–
7.49 (4H, m, 4· Ar-H), 7.54–7.57 (2H, m, 2· Ar-H),
7.62–7.67 (4H, m, 4· H); d (100.6 MHz, CDCl ) 23.1
H-1b), 6.67 (1H, d, J 7.2 Hz, NHb), 7.41–7.50 (8H, m, 8·
Ar-H), 7.55–7.65 (4H, m, 4· Ar-H), 7.99–8.08 (8H, m, 8·
Ar-H); d (100.6 MHz, CHCl ) 23.1 (2· C@OCH ), 52.6
c
3
3
(
7
1
2· C-2), 60.7 (2· CH ), 63.2 (2· C-6), 68.9 (2· C-5),
3
C
3
3.9 (2· C-3), 78.1 (2· C-4), 91.7 (C-1a), 97.8 (C-1b),
28.5, 128.6, 128.7, 129.5, 129.7, 129.8 (12· Ar-CH, 4·
(2· CH ), 52.2 (d, J
7.1 Hz, 2· C-2), 62.5 (2· C-6),
3
2,F
67.1 (d, J
23.1 Hz, 2· C-5), 71.7 (d, J
18.1 Hz,
C-3,F
C-5,F
Ar-C), 132.1, 133.2 (4· Ar-CH), 166.4, 166.9 (4· PhC@O),
1
8
2· C-3), 87.2 (d, J
187.1 Hz, 2· C-4), 128.5, 128.7,
C-4,F
+
þ
,
70.5 (2· CH C@O); m/z (ES ) 502 (M þ MeCN=NH
129.3, 129.7, 129.9 (12· Ar-CH, 4 Ar-C), 133.5, 133.4 (8·
3
4
+
+
), 336 (100%); HRMS (ES ): (MNa ) calcd for
Ar-CH), 166.3, 166.8 (4· PhC@O), 170.4 (2· CH C@O);
3
+
C H NO Na, 466.1483; found, 466.1467. (C H NO
requires C, 62.30; H, 5.68; N, 3.16. Found: C, 62.42; H,
d
(376.6 MHz, CDCl ) ꢀ196.7 (m, F). m/z (ES ) 490
2
3
25
8
23 25
8
F
3
þ
+
(M þ MeCN=NH , 12), 337 (100%); HRMS (ES ):
4
+
5
.97; N, 3.38.)
(MNa ) calcd for C H FNO Na, 454.1270; found,
2
2
22
7
4
54.1273. (C H FNO requires C, 61.25; H, 5.14; N,
22 22 7
4.3. 2-Acetamido-3,6-di-O-benzoyl-2,4-dideoxy-D-xylo-
hexopyranose 2b
3.25. Found: C, 61.35; H, 5.15; N, 3.43.)
4.5. 2-Acetamido-3,6-di-O-benzoyl-2,4-dideoxy-4-azido-D-
glucopyranose 2d
Tetrakis(triphenylphosphine)palladium (4.84 g, 4.19 mmol,
0
.5 equiv) was added to a solution of allyl glycoside 1b
(
3.8 g, 8.38 mmol, 1 equiv) in AcOH (30 mL) and the
Palladium(II) dichloride (0.007 g, 0.040 mmol, 0.4 equiv)
was added to a solution of allyl glycoside 1d (0.05 g,
0.101 mmol, 1 equiv) in MeOH (3 mL) under argon atmo-
sphere and the resulting mixture was stirred at room tem-
perature for 14 h. The mixture was filtered through Celite
and concentrated in vacuo. The residue was then purified
by flash column chromatography (CH Cl /MeOH, 95:5)
to afford hemiacetal 2d (0.033 g, 73%) as a white solid
and as a mixture of anomers, a/b-ratio being approxi-
mately 6:1 (by integration over selected parts of the spec-
resulting mixture was heated at 80 ꢁC. After 2 h, the mix-
ture was evaporated to dryness. The residue was then puri-
fied by flash column chromatography (CH Cl /MeOH,
2
2
9
5:5) to afford hemiacetal 2b (2.8 g, 81%) as a colourless
solid as a mixture of anomers, a/b-ratio being approxi-
mately 10:1 (by integration over selected parts of the spec-
trum); mmax (KBr) 1653 (s, C@O) cm ; dH (400 MHz,
CDCl ) 1.84–1.93 (2H, m, H-4), 1.90 (3H, s, CH a), 1.96
(
1
2
2
ꢀ
1
3
3
3H, s, CH b), 2.26 (1H, ddd, J
12.4 Hz, J3;4
gem
0
5.0 Hz, J
3
gem
0
.8 Hz, H-4 b), 2.30 (1H, ddd, J
12.4 Hz, J3;4
0
5.0 Hz,
trum); mmax (KBr) 3423 (br s, OH), 2111 (s, N ), 1723 (br
3
0
ꢀ1
J 1.8 Hz, H-4 a), 3.92–3.99 (1H, m, H-2b), 4.33–4.42
s, C@O), 1659 (br w, C@O) cm ; dH (400 MHz, CDCl3)
1.85 (3H, s, OCCH a), 1.95 (3H, s, OCCH b), 3.87–3.99
0
(
5H, m, H-2a, H-6, H-6 ), 4.44–4.47 (1H, m, H-5b), 4.48–
3
3
4
.54 (1H, m, H-5a), 4.64 (1H, d, J1,2 8.4 Hz, H-1b), 5.25
(2H, m, H-4), 4.12 (1H, m, H-5b), 4.20 (1H, adt, J
2.8 Hz, J 10.3 Hz, H-5a), 4.43 (2H, atd, J 3.3 Hz, J
10.0 Hz, H-2a), 4.50 (1H, m, H-2b), 4.55 (1H, dd, J6,5
(
1H, td, J 5.4 Hz, J 10.8 Hz, H-3b), 5.36 (1H, d, J1,2
3
6
6
.6 Hz, H-1a), 5.46 (1H, td, J 5.2 Hz, J 11.2 Hz, H-3a),
.08 (1H, d, J2,NH 9.6 Hz, NHa), 6.64 (1H, d, J2,NH
.8 Hz, NHa), 7.40–7.44 (8H, m, 8· Ar-H), 7.53–7.57
3.6 Hz, J6;6
0
12.3 Hz, H-6), 4.66 (1H, dd, J
0
2.1 Hz, J
0
6 ;5
6 ;6
0
12.3 Hz, H-6 ), 5.26 (1H, d, J 9.5 Hz, H-1b), 5.30 (1H,
1
,2
(
4H, m, 4· Ar-H), 7.99–8.04 (8H, m, 8· Ar-H); d
d, J1,2 3.1 Hz, H-1a), 5.48 (1H, m, H-3b), 5.62 (1H, at, J
10.2 Hz, H-3a), 6.22 (1H, d, J2,NH 9.4 Hz, NHa), 6.66
(1H, d, J 7.1 Hz, NHb), 7.44–7.51 (8H, m, 8· Ar-H),
7.58–7.62 (4H, m, 4· Ar-H), 8.04–8.11 (8H, m, 8· Ar-H);
d (100.6 MHz, CHCl ) 23.1 (2· C@OCH ), 52.5 (2· C-
C
(
100.6 MHz, CDCl ) 23.0 (CH b), 23.3 (CH a), 52.6 (C-
3 3 3
2
6
1
1
a), 58.2 (C-2b), 65.8 (C-5a), 65.9 (C-6b), 66.3 (C-6a),
9.3 (C-3a), 69.5 (C-3b), 92.6 (C-1a), 98.1 (C-1b), 128.4,
28.5, 128.6, 129.7, 129.8, 129.9 (12· Ar-CH, 4· Ar-C),
33.3, 133.9 (8· Ar-CH), 166.5, 166.8 (4· PhC@O), 170.7
c
3
3
2), 60.9 (2· C-4), 63.3 (2· C-6), 68.3 (2· C-5), 72.3, 74.2
(C-3a, C-3b), 91.8 (C-1a), 97.8 (C-1b), 128.5, 128.6,
128.8, 129.7, 129.7, 129.8, 129.9130.1 (12· Ar-CH, 4· Ar-
+
+
(
2
2· CH C@O); m/z (ES ) 332 (M+H , 3), 331 (M, 21),
3
+
+
+
46 (MꢀH , 100); HRMS (ES ): (MNa ) calcd for
22 23 7 22 23 7
C H NO Na, 436.1372; found, 436.1367. (C H NO
C), 133.4, 133.7, 134.3 (4· Ar-CH), 166.3, 166.9 (4·
+
requires C, 63.91; H, 5.61; N, 3.39. Found: C, 63.67; H,
.63; N, 3.27.)
PhC@O), 170.6 (2· CH C@O); m/z (ES ) 513
3
þ
+
5
(M þ MeCN=NH4
,
100), 337 (42); HRMS (ES ):

T. Muller et al. / Tetrahedron: Asymmetry 18 (2007) 1299–1307
1303
+
(
4
MNa ) calcd for C H N O Na, 477.1386; found,
(1H, ddd, Jgem 12.8 Hz, J 4.8 Hz, J 1.8 Hz, H-4 ), 4.31–
4.32 (2H, m, H-6, H-6 ), 4.34–4.40 (1H, m, H-5), 4.47
2
2
22
4
7
eq
0
77.1382. (C H N O requires C, 58.15; H, 4.88; N,
2
2
22
4
7
1
2.33. Found: C, 57.95; H, 4.91; N, 11.93.)
(1H, ttd, J2,P 1.2 Hz, J1,2 3.2 Hz, J 10.0 Hz, H-2), 5.02–
5
.07 (4H, m, 2· PhCH ), 5.32 (1H, td, H-3), 5.81 (1H,
2
4
.6. Dibenzylphosphate-2-acetamido-3,6-di-O-benzoyl-2-
dd, J 6.0 Hz, H-1), 5.95 (1H, d, NH), 7.33–7.44 (14H,
1,P
deoxy-4-O-methyl-a-D-glucopyranoside 3a
m, 14· Ar-H), 7.51–7.58 (2H, m, Ar-H), 7.97–8.02 (4H,
m, Ar-H); dC (100.6 MHz, CDCl ) 22.9 (CH ), 32.7 (C-
3
3
LDA (0.218 mL, 1.8 M in THF–heptane–ethylbenzene,
4), 52.0 (d, JC-2,P 8.0 Hz, C-2), 65.5 (C-6), 68.1 (C-5),
0
.38 mmol, 1.1 equiv) was added dropwise to a solution of
hemiacetals 2a (0.158 g, 0.36 mmol, 1 equiv) in dry THF
6 mL) which was cooled to ꢀ78 ꢁC. After 15 min, a solu-
tion of tetrabenzyl pyrophosphate (0.25 g, 0.46 mmol,
.3 equiv) in dry THF (6 mL) was slowly added and the
68.2 (C-3), 69.8 (d, JC,P 6.0 Hz, PhCH ), 69.9 (d, J
2
C,P
6.0 Hz, PhCH ), 97.9 (d, J
7.0 Hz, C-1), 127.7, 128.1,
C-1,P
2
(
128.4, 128.5, 128.7, 128.8, 129.3, 129.5, 129.7, 129.8 (16·
Ar-CH), 133.2, 133.4 (4· Ar-CH), 135.3, 135.4 (4· Ar-C),
1
166.1, 166.4 (2· PhC@O), 170.5 (CH C@O);
d
P
3
1
+
mixture was then allowed to warm to 0 ꢁC. After 3 h, a sat-
urated solution of NH Cl (50 mL) was added and the aque-
ous phase was extracted with CH Cl (3 · 50 mL). The
combined organic layers were dried (Na SO ), filtered and
(162 MHz, CDCl ) ꢀ2.3 ( H decoupled); m/z (ES ) 732
3
þ
+
(M þ MeCN=NH , 100), 696 (M+Na , 3%); HRMS
4
4
+
+
(ES ): (MNa ) calcd for C H NO PNa, 696.1969;
36 36 10
2
2
found, 696.1966. (C H NO P requires C, 64.19; H,
2
4
36 36 10
concentrated in vacuo. The residue was purified by flash
column chromatography (EtOAc/petrol, 3:2) to afford a-
glycosyl phosphate 3a (0.155 g, 62%) as a white foam,
5.39; N, 2.08. Found: C, 64.33; H, 5.31; N, 2.24.)
4.8. Dibenzylphosphate-2-acetamido-3,6-di-O-benzoyl-2,4-
dideoxy-4-fluoro-a-D-glucopyranoside 3c
2
3
½
aꢂ ¼ þ46:3 (c 1.0, CHCl ); m
(KBr) 1723 (br s,
C@O), 1678 (br w, C@O) cm ; dH (400 MHz, CDCl3)
D
3
max
ꢀ1
1
9
2
4
5
1
5
.61 (3H, s, C@OCH ), 3.41 (3H, s, CH ), 3.64 (1H, at, J
LDA (0.436 mL, 1.8 M in THF–heptane–ethylbenzene,
0.78 mmol, 1.1 equiv) was added dropwise to a solution
of hemiacetals 2c (0.308 g, 0.71 mmol, 1 equiv) in dry
THF (12 mL) which had been cooled to ꢀ78 ꢁC. After
15 min, a solution of tetrabenzyl pyrophosphate (0.5 g,
0.93 mmol, 1.3 equiv) in dry THF (12 mL) was slowly
added and the mixture was allowed to warm to 0 ꢁC. After
3
3
.6 Hz, H-4), 4.08–4.12 (1H, m, H-5), 4.40 (1H, dd, J
5
0
,6
.0 Hz, J6;6
0
12.4 Hz, H-6), 4.44–4.53 (2H, m, H-2, H-6 ),
.47 (1H, ttd, J2,P 1.2 Hz, J1,2 3.2 Hz, J 10.0 Hz, H-2),
.02–5.14 (4H, m, 2· PhCH ), 5.32 (1H, dd, J 9.2 Hz, J
2
0.8 Hz, H-3), 5.73 (1H, dd, J 3.2 Hz, J 6.0 Hz, H-1),
1
,2
1,P
.88 (1H, d, J2,NH 9.6 Hz, NH), 7.34–7.47 (14H, m, 14·
Ar-H), 7.56–7.61 (2H, m, Ar-H), 8.01–8.10 (4H, m,
3 h, a saturated solution of NH Cl (50 mL) was added and
4
Ar-H); dC (100.6 MHz, CDCl ) 22.7 (C@OCH ), 52.1 (d,
the aqueous phase extracted with CH Cl (3 · 50 mL). The
3
3
2
2
JC-2,P 8.0 Hz, C-2), 60.9 (CH ), 62.5 (C-6), 69.8 (d, J
combined organic layers were dried over Na SO , filtered
2 4
3
C,P
6
7
1
1
.0 Hz, PhCH ), 69.9 (d, J
6.0 Hz, PhCH ), 71.1 (C-5),
and concentrated in vacuo. The residue was then purified
by flash column chromatography (EtOAc/petrol, 3:2) to
afford a-glycosyl phosphate 3c (0.44 g, 89%) as a white
2
C,P
2
2.3 (C-3), 77.3 (C-4), 96.6 (d, JC-1,P 6.0 Hz, C-1), 128.0,
28.1, 128.2, 123.3, 128.5, 128.6, 128.8, 128.9, 129.1,
29.6, 129.7, 129.8 (16· Ar-CH, 2· Ar-C), 133.3, 133.6
2
3
foam, ½aꢂ ¼ þ59:9 (c 1.0, CHCl ); m
(KBr) 1724 (br
s, C@O), 1661 (br, C@O) cm ; d (400 MHz, CDCl3)
D
3
max
ꢀ
1
(
4· Ar-CH), 135.3, 135.4 (2· Ar-C), 166.1, 166.7 (2·
H
PhC@O), 170.4 (CH C@O); d (162 MHz, CDCl ) ꢀ2.3
1.69 (3H, s, CH ), 4.28–4.32 (1H, m, H-5), 4.45–4.58
3
P
3
3
1
+
þ
0
(
H decoupled); m/z (ES ) 762 (M þ MeCN=NH
,
(3H, m, H-2, H-6, H-6 ), 4.78 (1H, dt, J
50.8 Hz, J
4
4,F
+
+
1
7
6
00%); HRMS (ES ): (MNa ) calcd for C H NO PNa,
9.4 Hz, H-4), 5.06–5.18 (4H, m, 2· PhCH ), 5.58–5.66
3
7
38
11
2
26.2075; found, 726.2054. (C H NO P requires C,
(1H, m, H-3), 5.72–5.73 (1H, m, H-1), 6.24 (1H, d, J
3
7
38
11
2,NH
3.15; H, 5.44; N, 1.99. Found: C, 63.10; H, 5.61; N, 1.89.)
9.6 Hz, NH), 7.33–7.45 (14H, m, 14· Ar-H), 7.56–7.60
(
2H, m, Ar-H), 8.01–8.06 (4H, m, Ar-H); d (100.6 MHz,
C
4
.7. Dibenzylphosphate-2-acetamido-3,6-di-O-benzoyl-2,4-
CDCl ) 22.6 (CH ), 52.1 (at, J 8.0 Hz, C-2), 61.8 (C-6),
3 3
dideoxy-a-D-xylo-hexopyranoside 3b
69.3 (d, JC-5,F 24.1 Hz, C-5), 69.9 (d, JC,P 6.0 Hz, PhCH2),
0.1 (d, JC,P 6.0 Hz, PhCH ), 70.3 (d, J 19.1 Hz, C-3),
7
2
C-3,P
LDA (3.67 mL, 2.0 M in THF–heptane–ethylbenzene,
86.3 (d, JC-4,F 188.1 Hz, C-4), 96.1 (d, JC-1,P 7.0 Hz, C-1),
128.1, 128.5, 128.6, 128.8, 128.9, 129.0, 129.3, 129.5,
129.7, 129.9 (16· Ar-CH, 2· Ar-C), 133.3, 133.7 (2·
Ar-CH), 135.2, 135.3 (2· Ar-C), 166.1, 166.7 (2· PhC@O),
7
.34 mmol, 1.1 equiv) was added dropwise to a solution
of hemiacetals 2b (2.76 g, 6.67 mmol, 1 equiv) in dry
THF (30 mL) which had been cooled to ꢀ78 ꢁC. After
1
1
8
5 min, a solution of tetrabenzyl pyrophosphate (4.67 g,
.68 mmol, 1.3 equiv) in dry THF (20 mL) was slowly
170.4 (CH C@O); d (162 MHz, CDCl ) ꢀ2.3 ( H decou-
3
P
3
+
pled); d (376.6 MHz, CDCl ) ꢀ196.5 (m, F); m/z (ES )
F
3
þ
+
+
added and the mixture was then allowed to be warmed to
ꢁC. After 3 h, a saturated solution of NH Cl (100 mL)
750 (M þ MeCN=NH , 100%); HRMS (ES ): (MNa )
4
0
calcd for C H FNO PNa, 714.1875; found, 714.1851.
4
36 35
10
was added and the aqueous phase extracted with CH Cl
(C H FNO P requires C, 62.52; H, 5.10; N, 2.03.
2
2
36 35 10
(
3 · 100 mL). The combined organic layers were dried over
Found: C, 62.54; H, 5.11; N, 1.99.)
Na SO , filtered and concentrated in vacuo. The residue
2
4
was then purified by flash column chromatography
4.9. Dibenzylphosphate-2-acetamido-3,6-di-O-benzoyl-2,4-
dideoxy-4-azido-a-D-glucopyranoside 3d
(
(
EtOAc/petrol, 3:2) to afford a-glycosyl phosphate 3b
23
2.392 g, 65%) as a white foam, ½aꢂ ¼ þ58:5 (c 1.0,
D
ꢀ
1
CHCl ); m
(KBr) 1653 (s, C@O) cm ; d (400 MHz,
LDA (0.12 mL, 2.0 M in THF–heptane–ethylbenzene,
0.39 mmol, 1.1 equiv) was added dropwise to a solution of
3
max
H
CDCl ) 1.68 (3H, s, CH ), 1.86–1.91 (1H, m, H-4 ), 2.32
3
3
ax

1304
T. Muller et al. / Tetrahedron: Asymmetry 18 (2007) 1299–1307
compound 2d (0.10 g, 0.22 mmol, 1 equiv) in dry THF
11 mL) which had been cooled to ꢀ78 ꢁC. After 15 min,
a solution of tetrabenzyl pyrophosphate (0.15 g, 0.28 mmol,
.3 equiv) in dry THF (5 mL) was slowly added and the
dry MeOH (8 mL) and the resulting solution stirred under
(
an H -atmosphere. After 3 h, the solution was filtered over
2
Celite, NEt (0.289 mL, 2.05 mmol, 2.1 equiv) wad added
3
1
and the resulting mixture was concentrated in vacuo to
afford triethylammonium salt 4c (0.538 g, 89%) as a white
solid which was used in the next step without further
purification.
mixture was allowed to warm to 0 ꢁC. After 3 h, a saturated
solution of NH Cl (50 mL) was added and the aqueous
4
phase extracted with CH Cl (3 · 50 mL). The combined
2
2
organic layers were dried over Na SO , filtered and concen-
2
4
trated in vacuo. The residue was then purified by flash
column chromatography (EtOAc/petrol, 3:2) to afford
a-glycosyl phosphate 3d (0.114 g, 73%) as a white foam,
4.13. Phosphate-2,4-diacetamido-3,6-di-O-benzoyl-2,4-di-
deoxy-4-a-D-glucopyranoside, ditriethylammonium salt 4d
2
0
½
aꢂ ¼ þ90:2 (c 0.5, MeOH); m
(KBr) 2112 (s, N ),
725 (br s, C@O), 1676 (br w, C@O) cm ; dH (400 MHz,
Pd(OH)2 20% (0.02 g, 20% w/w) and Ac O (0.66 mL,
D
max
3
2
ꢀ
1
1
6.95 mmol, 50 equiv) were added to a solution of dibenzyl-
phosphate 3d (0.10 g, 0.14 mmol, 1 equiv) in dry MeOH
(3 mL) and the resulting solution stirred under an
CDCl ) 1.52 (3H, s, C@OCH ), 3.76–3.83 (2H, m, H-4,
3
3
0
H-5), 4.29–4.42 (3H, m, H-6, H-6 , H-2), 4.90–5.04 (4H,
m, 2· PhCH ), 5.32 (1H, dd, J 9.4 Hz, J 10.8 Hz, H-3),
H -atmosphere. After 6 h, the solution was filtered over
2
2
5
.62 (1H, dd, J 3.2 Hz, J 6.0 Hz, H-1), 5.91 (1H, d,
Celite, NEt (0.04 mL, 0.29 mmol, 2.1 equiv) was added
1
,2
1,P
3
J2,NH 9.3 Hz, NH), 7.20–7.36 (14H, m, 14· Ar-H), 7.45–
and the resulting mixture was concentrated in vacuo to
afford triethylammonium salt 4d (0.07 g, 70%) as a white
solid which was used in the next step without further
purification.
7
.50 (2H, m, Ar-H), 7.90–7.96 (4H, m, Ar-H); d
C
(
8
100.6 MHz, CDCl ) 22.7 (C@OCH ), 52.0 (d, J
3
3
C-2,P
.0 Hz, C-2), 60.2 (C-4), 62.5 (C-6), 69.9 (d, JC,P 6.0 Hz,
PhCH ), 70.0 (d, J 6.0 Hz, PhCH ), 70.4 (C-5), 71.5 (C-
2
C,P
2
3
1
), 96.5 (d, JC-1,P 7.0 Hz, C-1), 128.1, 128.5, 128.6, 128.8,
28.9, 129.7, 129.9 (16· Ar-CH, 2· Ar-C), 133.4, 133.8
4.14. Phosphate-2-acetamido-2-deoxy-4-O-methyl-a-D-
glucopyranoside, ditriethylammonium salt 5a
(
(
4· Ar-CH), 135.1, 135.2, 135.3 (4· Ar-C), 166.0, 166.6
2· PhC@O), 170.4 (CH C@O); d (162 MHz, CDCl )
Cyclohexylamine (14.65 mL, 130.94 mmol, 100 equiv) was
added to a solution of dibenzoate 4a (0.95 g, 13.10 mmol,
1 equiv) in dry MeOH (20 mL) and the resulting solution
heated at 70 ꢁC. After 48 h, the solution was concentrated
in vacuo, the residue dissolved in water (50 mL) and
3
P
3
1
+
þ
ꢀ
2.3 ( H decoupled); m/z (ES ) 773 (M þ MeCN=NH
,
4
+
+
1
7
6
00%); HRMS (ES ): (MNa ) calcd for C H N O PNa,
3
6
35
4
10
37.1988; found, 737.1985. (C H N O P requires C,
3
6
35
4
10
0.50; H, 4.94; N, 7.84. Found: C, 60.22; H, 5.28; N, 7.29.)
washed with CHCl (3 · 50 mL) after which it was concen-
3
4
.10. Phosphate-2-acetamido-3,6-di-O-benzoyl-2-deoxy-4-
trated in vacuo by coevaporating MeOH. The residue was
O-methyl-a-D-glucopyranoside, ditriethylammonium salt 4a
applied to a BioRad AG 50W-X2 cation exchange column
+
(
NEt H -form) to afford diol salt 5a (0.57 g, 84%) as a
3
Pd(OH) 20% (0.108 g, 10% w/w) was added to a solution
white solid, dH (400 MHz, D O) 1.23 (18H, t, J 7.4 Hz,
2
2
of dibenzylphosphate 3a (1.08 g, 1.54 mmol, 1 equiv) in dry
MeOH (15 mL) and the resulting solution stirred under an
H -atmosphere. After 3 h, the solution was filtered over
2
NCH CH ), 2.01 (3H, s, C@OCH ), 3.15 (12H, q,
2
3
3
NCH ), 3.54 (3H, s, CH ), 3.71–3.75 (1H, m, H-6), 3.80–
2 3
0
3.84 (3H, m, H-3, H-5, H-6 ), 3.89 (1H, dt, J 3.3 Hz, J
1,2
Celite, NEt (0.453 mL, 3.23 mmol, 2.1 equiv) was added
10.8 Hz, H-2), 5.34 (1H, dd, J1,P 7.3 Hz, H-1); dC
(100.6 MHz, D O) 8.6 (NCH CH ) 22.3 (C@OCH ), 47.0
3
and the resulting mixture was concentrated in vacuo to
afford triethylammonium salt 4a (0.96 g, 86%) as a white
solid, which was used in the next step without further
purification.
2
2
3
3
(NCH CH ), 54.2 (d, J 8.0 Hz, C-2), 70.9, 71.9 (C-3,
2
3
C-2,P
C-5), 79.7 (CH ), 93.5 (d, J
6.0 Hz, C-1), 175.0
C-1,P
3
1
(CH C@O); d (162 MHz, D O) ꢀ0.7 ( H decoupled);
3
P
2
ꢀ
+
ꢀ
+
m/z (ES ) 314 (MꢀH , 100%); HRMS (ES ): (MꢀH )
4
.11. Phosphate-2-acetamido-3,6-di-O-benzoyl-2,4-dideoxy-
calcd for C H NO P, 314.0635; found, 314.0640.
9
17
9
a-D-xylo-hexopyranoside, ditriethylammonium salt 4b
4.15. Phosphate-2-acetamido-2,4-dideoxy-a-D-xylo-hexo-
Pd(OH) 20% (0.05 g, 10% w/w) was added to a solution of
pyranoside, ditriethylammonium salt 5b
2
dibenzylphosphate 3b (0.5 g, 0.74 mmol, 1 equiv) in dry
MeOH (7 mL) and the resulting solution stirred under an
Cyclohexylamine (2.54 mL, 22.7 mmol, 100 equiv) was
added to a solution of dibenzoate 4b (0.112 g, 0.23 mmol,
1 equiv) in dry MeOH (4 mL) and the resulting solution
heated at 70 ꢁC. After 48 h, the solution was concentrated
in vacuo, the residue dissolved in water (50 mL) and
H -atmosphere. After 3 h, the solution was filtered over
2
Celite, NEt (0.219 mL, 1.56 mmol, 2.1 equiv) was added
3
and the resulting mixture was concentrated in vacuo to
afford triethylammonium salt 4b (0.48 g, 93%) as a white
solid which was used in the next step without further
purification.
washed with CHCl (3 · 50 mL) after which it was concen-
3
trated in vacuo by coevaporating with MeOH. The residue
was applied to a BioRad AG 50 W-X2 cation exchange col-
+
4
4
.12. Phosphate-2-acetamido-3,6-di-O-benzoyl-2,4-dideoxy-
-fluoro-a-D-glucopyranoside, ditriethylammonium salt 4c
umn (NEt H -form) to afford diol triethylammonium salt
3
5b (0.086 g, 78%) as a white solid, dH (400 MHz, D O)
2
1
.13 (18H, t, J 7.4 Hz, NCH CH ), 1.40 (1H, aq, J
2 3
Pd(OH) 20% (0.108 g, 10% w/w) was added to a solution
12.2 Hz, H-4 ), 1.89–1.94 (1H, m, H-4 ), 1.93 (3H, s,
2
ax eq
of dibenzylphosphate 3c (0.678 g, 0.98 mmol, 1 equiv) in
C@OCH ), 3.05 (12H, q, NCH ), 3.46 (1H, dd, J
3
2
5,6

T. Muller et al. / Tetrahedron: Asymmetry 18 (2007) 1299–1307
1305
0
+
ꢀ
+
5
3
4
.7 Hz, J6;6
0
12.1 Hz, H-6), 3.55 (1H, dd, J5;6
0
3.2 Hz, H-6 ),
(MꢀH , 100%); HRMS (ES ): (MꢀH ) calcd for
.66 (1H, dt, J1,2 3.3 Hz, J 10.8 Hz, H-2), 3.89 (1H, td, J
.7 Hz, J 11.0 Hz, H-3), 4.00–4.05 (1H, m, H-5), 5.32
C H N O P, 341.0749; found, 341.0744.
10
18
2
9
(
(
(
(
(
(
1H, dd, J1,P 7.3 Hz, H-1); dC (100.6 MHz, D O) 8.5
4.18. Uridinediphosphoryl-2-acetamido-2,4-dideoxy-4-O-
methyl-a-D-glucopyranoside, ditriethylammonium salt 6a
2
NCH CH ) 22.4 (C@OCH3), 34.8 (C-4), 47.0
2
3
NCH CH ), 55.5 (d, J 8.0 Hz, C-2), 64.0 (C-6), 65.5
2
3
C-2,P
0
C-3), 69.9 (C-5), 94.5 (d, JC-1,P 6.3 Hz, C-1); d
4-Morpholine-N,N -dicyclohexylcarboxamidinium uridine
P
1
+
0
162 MHz, D O) ꢀ0.9 ( H decoupled); m/z (ES ) 344
5 -monophosphormorpholidate
(0.33 g,
0.49 mmol,
2
þ
ꢀ
+
M þ MeCN=NH , 100%); HRMS (ES ): (MꢀH ) calcd
2 equiv) and tetrazole (0.052 g, 0.74 mmol, 3 equiv) were
added to a solution of phosphate 5a (0.128 g, 0.25 mmol,
4
for C H NO P, 284.0530; found, 284.0520.
8
15
8
1
equiv) in dry pyridine and the resulting solution stirred
4
glucopyranoside, ditriethylammonium salt 5c
.16. Phosphate-2-acetamido-2,4-dideoxy-4-fluoro-a-D-
at rt. After 5 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 6a
Cyclohexylamine (5.45 mL, 47.63 mmol, 100 equiv) was
added to solution of dibenzoate 4c (0.34 g, 0.48 mmol,
1
8
(0.133 g, 65%) as a white solid, ½aꢂ ¼ þ40:8 (c 1.0,
D
1
equiv) in dry MeOH (10 mL) and the resulting solution
MeOH); dH (400 MHz, D O) 1.11 (18H, t, J 7.4 Hz,
2
was heated at 70 ꢁC. After 48 h, the solution was concen-
NCH CH ), 1.94 (3H, s, C@OCH ), 3.01 (12H, q,
2
3
3
trated in vacuo, and the residue was dissolved in water
NCH ), 3.46 (3H, s, CH ), 3.66 (1H, dd, J 4.0 Hz, J
0
2
3
5,6
6;6
0
(
50 mL) and washed with CHCl (3 · 50 mL) after which
12.6 Hz, glu: H-6), 3.77–3.83 (2H, m, glu: H-3, H-6 ),
3
it was concentrated in vacuo by coevaporating with
3.90 (1H, dt, J1,2 3.3 Hz, J 10.6 Hz, glu: H-2), 3.96–4.28
0
MeOH. The residue was applied to a BioRad AG 50 W-
(6H, m, glu: H-5, rib: H-2, H-3, H-4, H-5, H-5 ), 5.41
+
X2 cation exchange column (NEt H -form) to afford diol
(1H, dd, J1,P 7.3 Hz, glu: H-1), 5.80 (1H, d, J5,6 8.0 Hz,
U: H-5), 5.83–5.88 (1H, m, rib: H-1), 7.85 (1H, d, U: H-
6); d (100.6 MHz, D O) 8.5 (NCH CH ) 22.4 (C@OCH ),
3
triethylammonium salt 5c (0.209 g, 86%) as a white solid,
dH (400 MHz, D O) 1.13 (18H, t, J 7.4 Hz, NCH CH ),
2
2
3
C
2
2
3
3
1
.99 (3H, s, C@OCH ), 3.13 (12H, q, NCH ), 3.73–3.82
46.9 (NCH CH ), 54.1 (d, J
60.5 (glu: C-6, CH ), 65.2 (d, J
3 C-5,P
8.0 Hz, glu: C-2), 60.4,
5.0 Hz, rib: C-5), 69.8
3
2
2
3
C-2,P
0
(
(
2H, m, H-6, H-6 ), 3.90–3.96 (1H, m, H-5), 4.00–4.08
2H, m, H-2, H-3) 4.38 (1H, dt, J4,F 50.8 Hz, J 9.2 Hz,
(glu: C-5), 71.1 (glu: C-3), 72.3 (rib: C-3), 73.3 (rib: C-2),
74.1 (glu: C-4), 83.3 (d, JC-4,P 9.0 Hz, rib: C-4), 88.8 (rib:
C-1), 94.7 (d, JC-1,P 6.0 Hz, glu: C-1), 102.8 (U: C-5),
141.9 (U: C-6), 151.9 (U: C-4), 166.3 (U: C-2), 174.3
H-4), 5.35–5.39 (1H, m, H-1); dC (100.6 MHz, D O) 8.6
2
(
NCH CH ), 22.2 (C@OCH ), 46.9 (NCH CH ), 53.7 (at,
2
3
3
2
3
J 8.1 Hz, C-2), 60.0 (C-6), 69.2 (d, JC-3,F 19.1 Hz, C-3),
1
7
0.4 (d, JC-5,F 25.1 Hz, C-5), 89.4 (d, JC-4,F 180.1 Hz, C-
(CH C@O); d (162 MHz, D O) ꢀ11.5, ꢀ13.2 ( H decou-
3
P
2
ꢀ
+
ꢀ
4
), 93.5 (d, JC-1,P 5.0 Hz, C-1); dP (162 MHz, D O) 0.1
pled); m/z (ES ) 620 (MꢀH , 100%); HRMS (ES ):
2
1
+
(
(
H decoupled); d (376.6 MHz, D O) ꢀ197.9 (m, F); m/z
(MꢀH ) calcd for C H N O P , 620.0888; found,
F
2
18 28
3
17 2
ꢀ
+
ꢀ
+
ES ) 302 (MꢀH , 100%); HRMS (ES ): (MꢀH ) calcd
620.0888.
for C H FNO P, 302.0436; found, 302.0436.
8
14
8
4.19. Uridinediphosphoryl 2-acetamido-2,4-dideoxy-a-D-
4
pyranoside, ditriethylammonium salt 5d
.17. Phosphate-2,4-diacetamido-2,4-dideoxy-a-D-gluco-
xylo-hexopyranoside, ditriethylammonium salt 6b
0
4
-Morpholine-N,N -dicyclohexylcarboxamidinium uridine
0
Cyclohexylamine (1.37 mL, 11.95 mmol, 100 equiv) was
5 -monophosphormorpholidate
(0.18 g,
0.27 mmol,
added to a solution of dibenzoate 4d (0.09 g, 0.11 mmol,
2 equiv) and tetrazole (0.028 g, 0.41 mmol, 3 equiv) were
added to a solution of phosphate 5b (0.066 g, 0.13 mmol,
1 equiv) in dry pyridine and the resulting solution stirred
at rt. After 5 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 6b
1
equiv) in dry MeOH (4 mL) and the resulting solution
heated at 70 ꢁC. After 48 h, the solution was concentrated
in vacuo, the residue was dissolved in water (10 mL) and
washed with CHCl (3 · 10 mL) after which it was concen-
3
trated in vacuo by coevaporating with MeOH. The residue
was applied to a DEAE Sephadex A25 anion exchange col-
umn using a triethylammonium bicarbonate buffer gradient
1
8
(0.07 g, 65%) as a white solid, ½aꢂ ¼ þ40:2 (c 1.0, MeOH);
D
(
0.2–0.35 M) to afford diol triethylammonium salt 5d
d
(400 MHz, D O) 1.24 (18H, t, J 7.4 Hz, NCH CH ),
H 2 2 3
(
0.04 g, 65%) as a white solid, dH (400 MHz, D O) 1.16
1.49–1.56 (1H, m, glu: H-4 ), 1.87–1.94 (1H, m, glu: H-
2
ax
(
1
3
18H, t, J 7.3 Hz, NCH CH ), 1.92 (3H, s, C@OCH ),
4 ), 1.94 (3H, s, C@OCH ), 3.11 (12H, q, NCH ), 3.47
2
3
3
eq
3
2
.93 (3H, s, C@OCH ), 3.8 (12H, q, NCH , J 7.3 Hz),
(1H, dd, J
0
5;6
5.5 Hz, J6;6 12.4 Hz, glu: H-6), 3.67 (1H,
0
3
2
0
dd, J5;6 3.2 Hz, glu: H-6 ), 3.82 (1H, dt, J 3.3 Hz, J
0
1,2
.50 (1H, dd, J5,6 4.1 Hz, J6;6
0
12.4 Hz, H-6), 3.58 (1H,
0
dd, J5;6
3
0
1.7 Hz, J
0
12.4 Hz, H-6 ), 3.73–3.80 (3H, m, H-
10.6 Hz, glu: H-2), 3.97–4.35 (6H, m, glu: H-2, H-3, H-5,
6 ;6
0
, H-5, H-4), 3.86–3.91 (1H, dt, J2,1 3.2 Hz, J 10.0 Hz, H-
rib: H-2, H-5, H-5 ), 5.52 (1H, dd, J 7.3 Hz, glu: H-1),
1,P
2
), 5.35 (1H, dd, J1,P 7.3 Hz, J1,2 3.2 Hz, H-1); dC
5.91–5.96 (2H, m, rib: H-1, U: H-5), 7.93 (1H, d, J5,6
8.0 Hz, U: H-6); dC (100.6 MHz, D O) 8.6 (NCH CH )
(
100.6 MHz, D O) 8.6 (NCH CH ) 22.2, 22.3 (2·
2
2
3
2
2
3
C@OCH ), 47.0 (NCH CH ), 51.9 (C-4), 54.5 (d, J
9
22.5 (C@OCH ), 34.6 (glu: C-4), 47.0 (NCH CH ), 55.3
3
2
3
C-2,P
3
2
3
.0 Hz, C-2), 61.0 (C-6), 68.8 (C-5), 72.0 (C-3), 93.8 (d,
(d, JC-2,P 8.0 Hz, glu: C-2), 64.0 (glu: C-6), 65.3 (d, JC-5,P
5.0 Hz, rib: C-5), 65.8 (glu: C-3), 70.0 (glu: C-5), 70.3
(rib: C-3), 74.1 (rib: C-2), 83.5 (d, JC-4,P 9.5 Hz, rib: C-4),
JC-1,P 5.6 Hz, C-1), 174.9, 175.0 (2· CH C@O); d
3
P
1
ꢀ
(
162 MHz, D O) ꢀ1.37 ( H decoupled); m/z (ES ) 341
2

1306
T. Muller et al. / Tetrahedron: Asymmetry 18 (2007) 1299–1307
8
5
8.2 (rib: C-1), 95.6 (d, JC-1,P 5.0 Hz, glu: C-1), 103.0 (U: C-
), 142.0 (U: C-6), 151.6 (U: C-4), 169.8 (U: C-2), 175.3
(2· C@OCH ), 46.3 (NCH CH ), 52.4 (glu: C-4), 55.0 (d,
C-2,P
3
2
3
J
8.0 Hz, glu: C-2), 62.0 (glu: C-6), 65.3 (d, J
C-5,P
1
(
CH C@O); d (162 MHz, D O) ꢀ11.3, ꢀ12.8 ( H decou-
5.0 Hz, rib: C-5), 69.6 (glu: C-5), 70.4 (rib: C-3), 73.1
(glu: C-3), 74.6 (rib: C-2), 84.1 (d, JC-4,P 8.0 Hz, rib: C-4),
88.8 (rib: C-1), 95.4 (d, JC-1,P 6.0 Hz, glu: C-1), 102.3 (U:
C-5), 141.8 (U: C-6), 151.9 (U: C-4), 165.1 (U: C-2),
3
P
2
ꢀ
+
ꢀ
pled); m/z (ES ) 590 (MꢀH , 100%); HRMS (ES ):
+
(
5
MꢀH ) calcd for C H N O P , 590.0772; found,
1
7
26
3
16 2
90.0783.
1
73.2, 173.3 (2· CH C@O); d (162 MHz, MeOD) ꢀ11.4,
3
P
1
ꢀ
+
4
.20. Uridinediphosphoryl-2-acetamido-2,4-dideoxy-4-
ꢀ13.1 ( H decoupled); m/z (ES ) 647 (MꢀH , 100%);
ꢀ
19 29 4 17 2
+
fluoro-a-D-glucopyranoside, ditriethylammonium salt 6c
HRMS (ES ): (MꢀH ) calcd for C H N O P ,
6
47.1002; found, 647.1000.
0
4
5
2
-Morpholine-N,N -dicyclohexylcarboxamidinium uridine
0
-monophosphormorpholidate
equiv) and tetrazole (0.016 g, 0.23 mmol, 3 equiv) were
(0.106 g,
0.16 mmol,
Acknowledgments
added to a solution of phosphate 5c (0.04 g, 0.078 mmol,
equiv) in dry pyridine and the resulting solution stirred
1
The authors gratefully acknowledge the European Union
Marie Curie Intra-EU Fellowship to R.D.), the BBSRC
and Stiefel Laboratories Inc. (CASE award to L.B.), and
the EP Abraham Cephalosporin Fund for financial
support.
at rt. After 5 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 6c
(
1
D
8
(
d
0.03 g, 47%) as a white solid, ½aꢂ ¼ þ45:0 (c 0.1, MeOH);
(400 MHz, D O) 1.23 (18H, t, J 7.4 Hz, NCH CH ),
2 2 3
H
2
.04 (3H, s, C@OCH ), 3.151 (12H, q, NCH ), 3.74–4.38
3
2
0
References
(
10, m, glu: H-2, H-3, H-5, H-6, H-6 , rib: H-2, H-3, H-4,
0
H-5, H-5 ), 5.46–5.48 (1H, m, glu: H-1), 5.89 (1H, d, J
5
,6
1
. (a) Asano, N. Glycobiology 2003, 13, 93R–104R; (b) Sears, P.;
Wong, C.-H. Angew. Chem. 1999, 111, 2446–2471; Angew.
Chem., Int. Ed. 1999, 38, 2300–2324.
. Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357–2364.
. Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci.
U.S.A. 1977, 74, 5463–5467.
8
.0 Hz, U: H-5), 5.94–5.97 (1H, m, rib: H-1), 7.87 (1H, d,
U: H-6); dC (100.6 MHz, D O) 8.6 (NCH CH ) 23.6
2
2
3
(
2
C@OCH ), 47.0 (NCH CH ), 55.3 (at, J 8.1 Hz, glu: C-
3
2
3
2
3
), 59.3 (glu: C-6), 61.2 (d, JC-5,P 5.0 Hz, rib: C-5), 69.9
(
5
d, JC-3,F 18.1 Hz, glu: C-3), 71.4 (d, JC-5,F 24 Hz, glu: C-
), 72.3 (rib: C-3), 72.3 (rib: C-2), 84.5 (d, JC-4,P 9.0 Hz,
4. Goody, R. S.; M u¨ ller, B.; Restle, T. FEBS Lett. 1991, 291, 1–
5.
rib: C-4), 89.1 (rib: C-1), 89.4 (d, JC-4,F 83.4 Hz, glu: C-
), 94.5 (d, JC-1,P 6.0 Hz, glu: C-1), 103.1 (U: C-5), 141.6
4
5. (a) Dimitroff, C. J.; Bernacki, R. J.; Sackstein, R. Blood 2003,
101, 602–610; (b) Woynarowska, B.; Dimitroff, C. J.; Sharma,
M.; Matta, K. L.; Bernacki, R. J. Glycoconj. J. 1996, 13, 663–
674; (c) Woynarowska, B.; Skrincosky, D. M.; Haag, A.;
Sharma, M.; Matta, K. L.; Bernacki, R. J. J. Biol. Chem.
994, 269, 22797–22803; (d) Bernacki, R. J.; Korytnyk, W. In
The Glycoconjugates; Academic Press: New York, 1982; Vol.
, pp 245–263.
. (a) Berkin, A.; Szarek, W. A.; Kisilevsky, R. Glycoconj. J.
005, 22, 443–451; (b) Kisilevsky, R.; Szarek, W. A.; Ancsin,
(
U: C-6), 151.7 (U: C-4), 168.3 (U: C-2), 175.1 (CH C@O);
3
1
dP (162 MHz, D O) ꢀ11.3, ꢀ13.1 ( H decoupled); d
2
F
ꢀ
(
(
376.6 MHz, D O) ꢀ198.2 (m, F); m/z (ES ) 608
2
+
ꢀ
+
MꢀH , 100%); HRMS (ES ): (MꢀH ) calcd for
1
C H FN O P , 608.0689; found, 608.0686.
1
7
25
3
16 2
4
4.21. Uridinediphosphoryl-2,4-diacetamido-2,4-dideoxy-a-D-
glucopyranoside, ditriethylammonium salt 6d
6
2
J. B.; Elimova, E.; Marone, S.; Bhat, S.; Berkin, A. Am. J.
Pathol. 2004, 164, 2127–2137; (c) Berkin, A.; Szarek, W. A.;
Kisilevsky, R. Carbohydr. Res. 2002, 337, 37–44; (d) Thomas,
S. S.; Plenkiewicz, J.; Ison, E. R.; Bols, M.; Zou, W.; Szarek,
W. A.; Kisilevsky, R. Biochim. Biophys. Acta 1995, 1272, 37–
0
4
5
2
-Morpholine-N,N -dicyclohexylcarboxamidinium uridine
0
-monophosphormorpholidate
equiv) and tetrazole (0.015 g, 0.21 mmol, 3 equiv) were
(0.095 g,
0.14 mmol,
added to a solution of phosphate 5d (0.039 g, 0.07 mmol,
equiv) in dry pyridine (3 mL) and the resulting solution
stirred at rt. 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
4
8.
1
7
. See for example: (a) Sujino, K.; Uchiyama, T.; Hindsgaul, O.;
Seto, N. O. L.; Wakarchuk, W. W.; Palcic, M. M. J. Am.
Chem. Soc. 2000, 122, 1261–1269; (b) Kajihara, Y.; Endo, T.;
Ogasawara, H.; Kodama, H.; Hashimoto, H. Carbohydr. Res.
1
995, 269, 273–294; (c) Srivastava, G.; Hindsgaul, O.; Palcic,
1
D
8
derivative 6d (0.040 g, 67%) as a white solid, ½aꢂ
¼
M. M. Carbohydr. Res. 1993, 245, 137–144.
þ41:6 (c 0.5, MeOH); d (400 MHz, MeOD) 1.21 (18H,
8. There are more than 50 examples of modified donors acting
as substrates for glycosyl transferases. For a review, see:
Qian, X.; Sujino, K.; Palcic, M. M. In Carbohydrates in
Chemistry and Biology; Ernst, B., Hart, G. W., Sina y¨ , P.,
Eds.; Wiley VCH, 2000; Vol. 2, pp 685–703.
. Nucci, M.; Marr, K. A. Emerg. Infect. 2005, 41, 521–526.
0. Vanden Bossche, H.; Marichal, P.; Odds, F. C. Trends
Microbiol. 1994, 2, 393–400.
H
t, J 7.3 Hz, NCH CH ), 2.02, 2.08 (2· 3H, 2· s, C@OCH ),
2
3
3
2
J6;6
.94 (12H, q, J 7.3 Hz, NCH ), 3.57 (1H, dd, J 4.9 Hz,
2
6,5
0
12.4 Hz, glu: H-6), 3.63 (1H, dd, J
0
1.7 Hz, J
0
6 ;5
6 ;6
0
1
(
2.4 Hz, glu: H-6 ), 3.84 (2H, m, glu: H-4, H-5), 3.97
9
1
1H, m, glu: H-3), 4.05 (1H, m, glu: H-2), 4.14 (1H, m,
rib: H-4), 4.25–4.29 (3H, m, rib: H-2, H-5, H-5 ), 4.35
1H, at, J 4.8 Hz, rib: H-3), 5.59 (1H, dd, J1,P 7.4 Hz, J1,2
0
(
3
(
6
11. Bowman, S. M.; Free, S. J. BioEssays 2006, 28, 799–808.
12. Georgopapadadakou, N. H.; Tkacz, J. S. Trends Microbiol.
1995, 3, 98–104.
.1 Hz, glu: H-1), 5.85 (1H, d, J5,6 8.1 Hz, U: H-5), 5.99
1H, d, J 5.3 Hz, rib: H-1), 8.04 (1H, d, J 8.1 Hz, U: H-
); dC (100.6 MHz, MeOD) 9.0 (NCH CH ), 21.8, 22.1
13. Munro, C. A.; Gow, N. A. R. Med. Mycol. 2001, 39, 41–53.
2
3

T. Muller et al. / Tetrahedron: Asymmetry 18 (2007) 1299–1307
1307
1
4. For a similar recently published approach which demon-
strates that the methylated UDP-GlcNAc derivative 6a is a
substrate for chitin synthase, see: Chang, R.; Moquist, P.;
Finney, N. S. Carbohydr. Res. 2004, 339, 1531–1536.
5. Danac, R.; Muller, T.; Ball, L.; Gurr S. J.; Fairbanks A. J.,
ChemBioChem 2007, 8, doi:10.1002/cbic.200700234.
6. Initially, the 4-azido compound was the synthetic target, but
later problems with the selective benzyl removal forced a
change of plan and the 4-NHAc derivative was synthesised
and tested instead.
17. Some migration of benzoyl protecting groups also occurred
during this hydrogenation step, and a small quantity of the
corresponding the 4-BzNH derivative was isolated as a side
product.
18. Weitzman, I.; Summerbell, R. C. Clin. Microbiol. Rev. 1995,
8, 240–259.
19. For example: methylated UDP-derivative 6a, CLogP =
ꢀ4.5; fluoride UDP-derivative 6c, CLogP = ꢀ4.2.
20. Murphy, P. J. Organophosphorous Reagents; Oxford Univer-
sity Press: Oxford, UK, 2004; pp 264–266.
1
1