Artificial N-functionalized UDP-glucosamine analogues as modified substrates for N-acetylglucosaminyl transferases

Article abstract of DOI:10.1016/j.carres.2006.01.017

Analogues of UDP-GlcNAc modified at the 2-acetamido group of the GlcNAc moiety were prepared in order to study their role in the mechanism of N-acetylglucosaminyl transferase mediated glycosylation reactions. The structural analogues with N-formyl-, N-pro

Full text of DOI:10.1016/j.carres.2006.01.017

Carbohydrate Research 341 (2006) 569–576
Artificial N-functionalized UDP-glucosamine analogues as
modified substrates for N-acetylglucosaminyl transferases
*
´
Daniel Lazarevic and Joachim Thiem
Institut fu¨r Organische Chemie, Universita¨t Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
Received 26 September 2005; received in revised form 6 December 2005; accepted 15 January 2006
Available online 30 January 2006
´
´
Dedicated to Professor Andras Liptak on the occasion of his 70th birthday
Abstract—Analogues of UDP-GlcNAc modified at the 2-acetamido group of the GlcNAc moiety were prepared in order to study
their role in the mechanism of N-acetylglucosaminyl transferase mediated glycosylation reactions. The structural analogues with N-
formyl-, N-propionyl-, N-butyryl- and N-isobutyryl-groups were synthesized, utilizing the morpholidate coupling method starting
from D-glucosaminyl-1-phosphate after selective N-acylation of its amino group with the appropriate N-acyloxysuccinimide esters
as well as a chlorinated formylformiate.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: GlcNAc-1-phosphate; Acylamidoglucose; UDP-glucosamine analogues; Morpholidate coupling; Carbohydrate-binding proteins
1. Introduction
ognized acceptor, eliminating the need for numerous
protection and deprotection steps. However, this meth-
odology suffers in the sense that these enzymes preclude
the use of alternative carbohydrate donors that differ
significantly in structure from their naturally occurring
counterparts, making enzymatic synthesis of unnatural
oligosaccharides a still challenging and difficult endeav-
our. By use of their naturally occurring glycosylation
donor substrate N-acetylhexosaminyl transferases take
part in the stepwise glycosylation towards glycoconju-
gates, among them sphingolipids and gangliosides, regu-
lating the essential lipid metabolism in human nervous
system cells. A number of disease phenotypes, based
on deficient degradation of specific gangliosides are
known, including the many well characterized hydro-
lases taking part in this process.23 Their counter parts,
the ganglioside generating and membrane bound trans-
ferases are so far less well investigated since they are
not applicable to common methods such as X-ray
crystallography and require alternative investigative
techniques based on affinity labelling and enzyme–sub-
strate interaction studies. Modified structural analogues
of UDP-GlcNAc applicable for this purpose are
described in this contribution.
Approaches for efficient syntheses of carbohydrate
building blocks up to complex oligosaccharides still
receive increasing attention. In addition to naturally
occurring carbohydrate structures displayed on cell sur-
faces and involved in molecular recognition processes
mediated by binding events1–5 unnatural oligosaccharide
target compounds became the goal of both chemical and
enzymatic approaches.6–13 Some of these compounds
show potential as drug candidates since they interfere
in cellular recognition either by modulation or inhibi-
tion and give rise to more effective artificial analogues
than their natural substrates. A promising and still
developing approach besides classical linear and conver-
gent glycosylation paths involves the use of transferases
that employ nucleoside diphosphohexoses (NDP
hexoses) as activated carbohydrate donors.14–22 Such
transferases of the Leloir pathway display high
stereospecificity and regioselectivity in directing an acti-
vated carbohydrate donor to a specific position of a rec-
*
Corresponding author. Tel.: +49 40 42838 4241; fax: +49 40 42838
4325; e-mail: thiem@chemieuni-hamburg.de
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.01.017

570
D. Lazarevic´, J. Thiem / Carbohydrate Research 341 (2006) 569–576
Natural and unnatural NDP-sugars have been synthe-
via azidonitration.29,30 After regioselective introduction
of the azide in equatorial manner into the 2-position
the 2-azidoglucopyranosyl nitrate obtained was sub-
jected to reductive hydrolysis yielding the benzylated
2-azidoglucose 2 after treatment with sodium nitrite in
an aqueous dioxane solution. The following stereoselec-
tive phosphorylation was carried out employing dibenz-
yl-N,N0-di-iso-propylphosphoramidite in the presence of
1H-tetrazole as catalyst via an initially formed phos-
phate. This was oxidized with meta-chloroperbenzoic
acid to the protected a-phosphate 3 in a one-pot reac-
tion, in which no formation of the corresponding
b-phosphate was observed. Complete debenzylation
and reduction of the azide functionality was carried
out by hydrogenolysis using 10% palladium on activated
charcoal affording glucosaminyl-1-phosphate (4) in one
step, which turned out to be the most critical of the
whole synthetic protocol regarding yield and completion
of deprotection. The same conditions [50 bar H2 atmo-
sphere, solvent system ethylacetate, methanol, water
(1:2:1) and excessive amount of palladium catalyst] pre-
viously reported by us for the synthesis of galactosamin-
yl-1-phosphate by a similar synthetic route were
applied leading to the unprotected amine 4 in 67% yield
(Scheme 1).
sized by the classical method24 and many more recent
variations such as by Wittmann and Wong.25 In our
previous work we reported the syntheses of several
structural analogues of UDP-GalNAc, displaying
2-amino-2-deoxy, 2-azido-2-deoxy, N-bromoacetyl, N-
propionyl and N-butyryl functionalities by N-acylation
of D-galactosaminyl-1-phosphate, in the latter three
cases with corresponding N-hydroxysuccinimide esters
with high N-selectivity and good yields.26 This concep-
tion was transferred to D-glucosaminyl-1-phosphate giv-
ing rise to N-acylamidoglucosyl phosphates all of which
were converted to uridine glucosaminyl nucleotides
under morpholidate coupling conditions.
Recently in the UDP-GlcNAc series, UDP-N-trifluor-
acetylglucosamine27 and 2-azido analogue of UDP-
GlcNAc were prepared.28 Attempts for their further
processing by UDP-GlcNAc transferases showed
success for the N-trifluoracetylated UDP derivative.
However, neither the corresponding non-acylated
UDP-2-aminoglucose nor the UDP-2-azidoglucose
sugar nucleotides proved to be transferable by trans-
ferases. This indicates the presence of an acylamido
group to be critical since it serves as a necessary recog-
nition element in order to be processed by GlcNAc
transferases. UDP-N-Formamido-, UDP-N-propionyl-
amido-, UDP-N-butyramido- and UDP-N-isobutyram-
idoglucose, described in this paper are therefore
expected to be potentially transferable in transferase
reactions to give oligosaccharide analogues with poten-
tial bioactive properties.
The subsequent selective N-acylation of the amino-
phosphate 4 utilizing N-propionyl-oxysuccinimide, N-
butyroxysuccinimide
and
N-isobutyrosuccinimide,
respectively, gave the 2-acylamidophosphates 6–8 in
yields ranging from 80% to 85% at pH 7.0 in a solvent
system of THF and water (1:10) dissolving both the
esters and the highly polar aminophosphate 4.
No hydrolysis of the phosphate group and no side
products arising from O-acylation at the unprotected
3-, 4- and 6-positions could be monitored as expected.
The appropriate N-acyloxysuccinimide esters were easily
prepared by reacting N-hydroxysuccinimide with either
propionyl chloride, butyryl chloride and pivaloyl chlo-
ride in the presence of triethylamine followed by column
chromatographic purification or recrystallization. N-
Formylation of the aminophosphate 4 was carried out
by use of 2,4,5-trichlorophenylformiate31 as formylating
reagent under improved conditions as described for the
preparation of the phosphates 6–8, thus leading to the
N-formamido modified aminoglucosyl-1-phosphate 5
in 78% yield after Biogel P2 purification procedures
and lyophilization in a DIPEA mediated transesterifica-
tion step. Foregoing attempts to prepare the N-formyl-
ated glucosaminyl-1-phosphate by use of the more
common formylating reagents such as formyl acetate
or p-nitrophenyl formiate were unsuccessful or led to
unsatisfactory yields and side products.
2. Results and discussion
2.1. Syntheses of phosphates
The selective N-acylation of unprotected aminosugars
with esters derived from N-hydroxysuccinimide as acti-
vated acylating agents has proven to be a versatile and
general approach to acylamidosugars, since such esters
display moderate reactivity making them ideal reactants
for N-nucleophiles. A wide range of acyl groups can be
introduced and many other functionalities such as labile
phosphate groups at the anomeric position within the
aminosugar are compatible to this reaction as we have
shown in our recent work on the synthesis of selectively
N-acylated galactosamines.26
For the syntheses of N-acylamidoglucopyranosyl
phosphates and further towards their corresponding uri-
dine diphospho derivatives D-glucosaminyl-1-phosphate
(4) was chosen as the building block and proved to be a
suitable common starting material for the structural
analogues of UDP-GlcNAc reported in this paper.
The synthesis of D-glucosaminyl-1-phosphate (4) started
from perbenzylated D-glucal (1) and followed a pathway
The acylamido phosphates 5–8 were isolated as
ammonium salts due to size exclusion chromatography
with 250 mM ammonium hydrogencarbonate solution
and were converted to triethylammonium salts by ion

D. Lazarevic´, J. Thiem / Carbohydrate Research 341 (2006) 569–576
OBn OBn
571
O
O
i, ii
iii, iv
BnO
BnO
BnO
BnO
N3
OH
1
2
OBn
OH
O
O
BnO
BnO
HO
HO
v
O
P
O
P
N3
H3N
O
O
OBn
OH
OBn
O
3
4
Scheme 1. Reagents and conditions: (i) CeIV(NH4)2(NO3)6, NaN3, MeCN, ꢀ25 ꢁC, 1 day; (ii) NaNO2, dioxane, water, 80 ꢁC, 1 day; (iii)
(iPr)2NP(OBn)2, 1H-tetrazole, CH2Cl2, 5 h; (iv) mCPBA, CH2Cl2, 0 ꢁC, 1 h, 3: 58%; (v) H2, Pd/C (10%), EtOAc/MeOH/water (1:2:1), 65 bar, rt, 2 d,
4: 67%.
exchange chromatography for solubility reasons in the
subsequent nucleotide coupling with UMP morphol-
idate (Scheme 2).
following rearrangement towards an aziridino-carboxo-
nium ion may be concluded (Scheme 3).
1H, 13C and 31P NMR data for the unequivocal struc-
tural assignments of the phosphates 5–8 in the form of
their ammonium salts are summarized in the upper half
of Tables 1 and 2.
2.2. Syntheses of UDP glucosamines
The syntheses of the UDP-N-acylamidoglucoses 9–12
from their triethylammonium phosphate precursors
5–8 were performed under modified Khorana condi-
tions24–28 with the 4-morpholine-N,N0-dicyclohexylcarb-
oxamidinium salt of uridine monophospho-morphol-
idate as the activated UMP source under argon
atmosphere in a solution of anhydrous DMF/anhydrous
pyridine (ꢁ2:1) at room temperature. The coupling reac-
tions were worked up after 5 days by evaporation of the
organic solvents without heating and subsequent freeze
drying, followed by size exclusion chromatography
and subsequent desalting on Biogel P2. The modified
unnatural UDP glucosamines were thus obtained in a
yield range around 30% as colourless ammonium salt
solids (Scheme 4).
By mass spectrometric investigation of the azidophos-
phate 3 an unusual fragmentation pattern by Maldi-
TOF analysis was observed. In addition to the expected
mass peaks 774 [M+K]+ and 758 [M+Na]+ further
masses at 746 [MꢀN2+K]+, 730 [MꢀN2+Na]+, 459
[MꢀO2P(OBn)2]+ and 430 [MꢀO2P(OBn)2ꢀN2]+
appeared. Very likely the latter four correspond to cat-
ionic species arising from 3 upon loss of either nitrogen
or a dibenzylphosphate group as well as both function-
alities. Thus a fragmentation path either by an initial
rearrangement of a nitrene via an imine and subsequent
dephosphorylation to an imino-carboxonium ion or vice
versa by initial phosphate loss via an aziridinium ion
OH
OH
O
O
HO
HO
HO
HO
O
P
i, ii
O
P
HN
O
H3N
O
O (HNEt3)
(HNEt3)
O
OH
R
O
O
4
6, R = CH2CH3
7, R = CH2CH2CH3
8, R = CH(CH3)2
iii
5, R = H
Scheme 2. Reagents and conditions: (i) 1.5–2.5 equiv R–CO2–succinimide, THF/water, pH 7.0–7.5, rt, 1–2 days; (ii) Dowex 50W-X8 (triethyl-
ammonium form), 6: 79%, 7: 86%, 8: 84%; (iii) 1 equiv 2,4,5-trichlorophenylformiate, 1.2 equiv di-iso-propylethylamine, DMF, rt, 1–2 days, 5: 78%.

Table 1. 1H and 31P NMR chemical shifts (d in ppm) and coupling constants (J in Hz) for compounds 5–12 in D2O as solvents
Compound Acylamido
Glucopyranose ring
Ribofuranose ring
H-20, H-30 H-40
Uracil
H-1
H-2
H-3
H-4
H-5
H-6a, H-6b
3.89, m
31P
1.58
H-10
H-50a, H-50b H-500
H-600
5
5.52, dd
4.01, m
3.83, dd
3.57, dd
3.97, m
J1,2 = 3.3 J2,3 = 10.3 J3,4 = 9.7 J4,5 = 9.6 J5,6a = 4.3 J6a,6b = 12.7
J1,P = 7.1
J5,6b = 2.6
6
7
8
2.51, q
1.28, t
5.55, dd
4.02, m
3.83, dd
3.57, dd
3.97, m
3.90, m
0.27
1.86
2.15
J1,2 = 3.3 J2,3 = 10.3 J3,4 = 9.7 J4,5 = 9.6 J5,6a = 5.3 J6a,6b = 12.9
J1,P = 7.1
5.52, dd
J1,2 = 3.3 J2,3 = 10.3 J3,4 = 9.7 J4,5 = 9.6 J5,6a = 4.1 J6a,6b = 12.9
J1,P = 7.1
5.53, dd
J5,6b = 2.7
3.96, dt
3.40, t
1.70, m
1.01, t
2.02, m
1.11, d
4.01, m
3.83, dd
3.59, dd
3.88, m
J5,6b = 2.7
3.97, ddd
4.02, m
3.86, dd
3.60, dd
3.92, m
J1,2 = 3.1 J2,3 = 10.3 J3,4 = 9.7 J4,5 = 9.6 J5,6a = 4.3 J6a,6b = 12.7
J1,P = 7.1
J5,6b = 2.7
9
5.53, dd
4.02, m
3.83, dd
3.59,
3.96, m
3.90, m
ꢀ10.4
5.98, d
4.37, m
4.37, m
4.37, m
4.36, m
4.30, m 4.24, m
5.98, d
7.96, d
7.95, d
7.98, d
7.99, d
0
0
00 00
J1,2 = 3.3 J2,3 = 10.5 J3,4 = 9.7 J4,5 = 9.6 J5,6a = 4.3 J6a,6b = 12.9 ꢀ12.1
J1 ;2 ¼ 4:1
J5 ;6 ¼ 8:1
J1,P = 7.3
5.53, dd
J5,6b = 2.5
3.95, m
10
11
12
2.50, q
1.27, t
4.00, m
3.82, dd
3.57,
3.89, m
ꢀ10.6
5.98, d
4.30, m 4.24, m
4.32, m 4.25, m
4.33, m 4.25, m
5.97, d
0
0
00 00
J1,2 = 3.3 J2,3 = 10.4 J3,4 = 9.7 J4,5 = 9.6 J5,6a = 4.3 J6a,6b = 12.7 ꢀ12.3
J1 ;2 ¼ 4:1
J5 ;6 ¼ 8:1
J1,P = 7.1
5.54, dd
J5,6b = 2.7
3.97, m
3.41, t
1.72, m
1.03, t
2.01, m
1.10, d
4.02, m
3.85, dd
3.59, m
3.90, m
ꢀ10.2
5.99, d
5.98, d
0
0
00 00
J1,2 = 3.3 J2,3 = 10.4 J3,4 = 9.7 J4,5 = 9.6 J5,6a = 4.3 J6a,6b = 12.7 ꢀ12.1
J1 ;2 ¼ 4:1
J5 ;6 ¼ 8:1
J1,P = 7.1
5.55, dd
J5,6b = 2.7
3.98, m
4.04, m
3.87, dd
3.61, dd
3.92, m
ꢀ10.3
6.00, d
5.98, d
0
0
00 00
J1,2 = 3.3 J2,3 = 10.1 J3,4 = 9.6 J4,5 = 9.6 J5,6a = 4.3 J6a,6b = 12.5 ꢀ12.3
J1 ;2 ¼ 4:0
J5 ;6 ¼ 8:1
J1,P = 7.3 J5,6b = 2.7

D. Lazarevic´, J. Thiem / Carbohydrate Research 341 (2006) 569–576
573
Table 2. 13C NMR chemical shifts (d in ppm) and coupling constants between carbon and phosphorus (J in Hz) for compounds 5–12 in D2O as
solvents
Compound
Acylamido
Glucopyranose ring
Ribofuranose ring
Uracil
C-1
179.1, CO 94.7, d
C-2
C-3 C-4 C-5 C-6 C-10 C-20 C-30 C-40
C-50
C-200 C-400 C-500 C-600
5
6
53.6, d
71.2 69.6 73.2 60.2
71.1 69.8 73.2 60.7
J1,P = 6.1 J2,P = 8.3
178.9, CO 94.7, d
53.7
29.6, CH2 J1,P = 5.7 J2,P = 8.1
9.9, CH3
7
8
178.1, CO 94.6, d
37.8, CH2 J1,P = 6.1 J2,P = 8.1
19.3, CH2
13.2, CH3
178.6, CO 94.7, d
53.7, d
71.0 69.7 73.1 60.5
71.2 69.9 73.1 60.6
53.9, d
28.7, CH J1,P = 6.1 J2,P = 8.3
12.6, CH3
9
179.2, CO 94.5, d
53.7, d
71.0 69.5 73.1 60.3 88.5 74.0 69.6 83.3, d
65.1, d
0
152.4 166.7 102.8 141.6
152.5 166.7 102.9 141.7
0
J1,P = 6.1 J2,P = 8.1
J4 ;P ¼ 9:1 J5 ;P ¼ 5:3
10
179.3, CO 94.8, d
53.9, d
29.7, CH2 J1,P = 6.1 J2,P = 8.3
9.9, CH3
71.1 69.7 73.3 60.6 88.7 74.2 69.8 83.5, d
65.2, d
0
0
J4 ;P ¼ 9:1 J5 ;P ¼ 5:3
11
12
177.8, CO 94.6, d
37.7, CH2 J1,P = 6.3 J2,P = 8.3
19.1, CH2
13.1, CH3
178.8, CO 94.6, d
53.7, d
71.0 69.7 73.1 60.4 88.5 74.1 69.8 83.4, d
65.1, d
0
152.3 166.9 103.0 141.6
152.2 166.4 103.2 141.5
0
J4 ;P ¼ 9:1 J5 ;P ¼ 5:1
53.8, d
71.1 69.7 73.1 60.5 88.4 74.2 69.9 83.4, d
65.1, d
0
0
28.8, CH J1,P = 6.3 J2,P = 8.5
12.7, CH3
J4 ;P ¼ 9:1 J5 ;P ¼ 5:1
OBn
OBn
O
O
∆
BnO
BnO
BnO
BnO
O
P
O
- N2
N3
N
P
O
O
~
OBn
OBn
OBn
OBn
Azide 3
Nitrene
OBn
OBn
O
- O2P(OBn)2
O
BnO
BnO
BnO
BnO
O
P
HN
HN
O
OBn
OBn
Imino-carboxonium ion
Imine
- O2P(OBn)2
OBn
O
OBn
~
O
BnO
BnO
BnO
BnO
N
H
N
Aziridino-carboxonium ion
Aziridinium ion
Scheme 3. Fragmentation pattern of the azidophosphate 3 observed by Maldi-TOF spectrometry in positive mode and DHB as matrix.
1H, 13C, 31P NMR and coupling constants data
of the UDP glucosamines 9–12 are summarized
in the lower half of Tables 1 and 2. Additional syn-
theses of novel and unnatural UDP-hexosamine
analogues are in progress and will be reported in
due course.

574
D. Lazarevic´, J. Thiem / Carbohydrate Research 341 (2006) 569–576
O
N
OH
OH
NH
O
O
HO
HO
HO
HO
i, ii
O
P
O
P
O
P
O
HN
O
HN
O
O
O
O
O
O (HNEt3)
(HNEt3)
R
R
O
O
OH
O
(NH4)
HO
OH
9, R = H
10, R = CH2CH3
5, R = H
6, R = CH2CH3
11, R = CH2CH2CH3
12, R = CH(CH3)2
7, R = CH2CH2CH3
8, R = CH(CH3)2
Scheme 4. Reagents and conditions: (i) 1.6 equiv 4-morpholine-N,N0-dicyclohexylcarboxamidinium UMP-morpholidate, DMF/pyridine (2:1), rt,
5 days; (ii) 1. Biogel P2, 250 mM NH4HCO3, 2. Biogel P2, deionized water, 9: 28%, 10: 30%, 11: 33%, 12: 30%.
3. Experimental
3.1. General methods
3.3. Procedure B: UMP morpholidate coupling of
glucosaminyl phosphates
The phosphate (0.18 mmol) obtained by procedure A
was dissolved in water (1 mL) and passed through a col-
umn (1 cm · 5.5 cm) of Dowex 50W-X8 (triethylammo-
nium form) to give the phosphate in form of the
corresponding triethylammonium salt in quantitative
yield. The phosphate was dissolved together with
uridine-50-monophosphomorpholidate (4-morpholine-
N,N0-dicyclohexyl carboxamidinium salt, 1.6 equiv,
0.22–0.29 mmol) in anhydrous pyridine (10 mL) and
concentrated to dryness without heating under reduced
pressure. Ventilation to normal pressure was carried
out with dry argon. After repeating this procedure three
times, the resulting syrup-like residue was dissolved in
anhydrous pyridine/anhydrous DMF (ꢁ3 mL, 1:2) and
stirred for 5 days at rt sealed under an argon atmo-
sphere. Removal of the solvents without heating under
reduced pressure followed by dissolving the residue in
water, filtration and subsequent separation on Biogel
P2 with first 250 mM NH4HCO3 solution and after-
wards for desalting purpose with deionized water as elu-
ent, yielded the uridine diphosphoglucosamine in the
form of its ammonium salt as a colourless and highly
hygroscopic powder.
TLC was performed on silica gel 60-coated aluminium
sheets (E. Merck) using the given eluent mixtures. Spots
were visualized under UV light at 366 nm and by spray-
ing with 10% H2SO4 in EtOH and subsequent heating.
Column chromatography was performed on silica gel
60 (230–240 mesh, grain size 0.040–0.063 nm, E. Merck).
Size exclusion chromatography and desalting proce-
dures were performed on Biogel P2 (Bio-Rad) either
with aqueous 250 mM NH4HCO3 solution or deionized
water as eluent. Optical rotations were measured on a
Perkin–Elmer Polarimeter 243, with [a]D values given
in units of 10ꢀ1 deg cm2 gꢀ1. NMR spectra were
recorded on a Bruker AMX-400 NMR spectrometer.
Chemical shifts are referred to the solvents used.
Maldi-TOF spectra were measured on a Bruker Biflex-
II spectrometer equipped with a N2 laser unit (337 nm)
with DHB as matrix both in positive and negative
modes.
3.2. Procedure A: selective N-acylation
D-Glucosamine-1-phosphate (0.1 mmol) was dissolved
in water (0.5 mL) and treated with a solution of the N-
acyloxysuccinimide (0.15 mmol) of choice in THF/water
(0.5 mL, 5:1). The pH was adjusted to 7.0 using 400 mM
solution of potassium hydroxide in water. After stirring
overnight at rt the resulting solution was if, necessary,
treated with some more N-acyloxysuccinimide (0.1
mmol) in THF/water (0.5 mL, 5:1) with subsequent
pH adjustment to 7.0 and stirring overnight. The result-
ing solution was purified on Biogel P2 with water as elu-
ent, and after freeze drying the product containing
fractions yielded 80–85% of the desired N-acylamido
glucosyl phosphate.
3.4. Synthesis of glycosyl phosphates
3.4.1. (2-Azido-3,4,6-tri-O-benzyl-2-deoxy-a-D-glucopyr-
anosyl) dibenzyl phosphate (3). 1H-Tetrazole (307 mg,
4.38 mmol) was suspended in dry dichloromethane
(3 mL) under argon and stirring at rt, followed by the
addition of dibenzyl-N,N0-diisopropylphosphoramidite
(0.73 mL, 745 mg, 2.59 mmol). Within 15 min the
suspension became a clear solution and 2-azido-3,4,5-
tri-O-benzyl-2-deoxy-a/b-D-glucopyranose29,30 (427 mg,
0.9 mmol, M = 475.54 g/mol) in dry dichloromethane

D. Lazarevic´, J. Thiem / Carbohydrate Research 341 (2006) 569–576
575
(3 mL) was added rapidly. After stirring for 4 h at rt the
solution was cooled to 0 ꢁC and 3-chloroperbenzoic acid
(617 mg, 2.5 mmol) was added in small doses, followed
by stirring for 1 h at rt. Removal of the solvent under
reduced pressure at 30 ꢁC bath temperature and purifi-
cation by column chromatography yielded compound
tion to give the title ammonium salt 5 (30 mg, 78%) as
a colourless foamy solid; ½aꢂD +66.5 (c 1.0 water).
20
3.4.4.
2-Deoxy-2-propionylamido-a-D-glucopyranosyl
phosphate (6). According to procedure A 2-amino-2-
deoxy-a-D-glucopyranosyl phosphate 4 (75 mg, 0.29
mmol) and N-propionyloxysuccinimide ester were
reacted to give the title compound 6 (80 mg, 79%) as a
20
3 (382 mg, 58%) as a colourless syrup. ½aꢂD ꢀ42.5 (c
1.0 CHCl3); Maldi-TOF (DHB, positive mode): m/z =
774 [M+K]+, 758 [M+Na]+, 746 [MꢀN2+K]+, 730
20
colourless hygroscopic powder; ½aꢂD +35 (c 1.0 water).
[MꢀN2+Na]+,
459
[MꢀO2P(OBn)2]+,
430
[MꢀO2P(OBn)2ꢀN2]+, IR (KBr): m = 2114 cmꢀ1 (N3);
1H NMR (acetone-d6): d 7.19 (m, 25H, 5 · Ph); 5.73
(dd, 1H, H-1, J1,2 = 3.3 Hz, J1,P = 6.1 Hz); 4.98, 4.95
(2 · d, 4H, 2 · POCH2Ph); 4.78, 4.76, 4.62, 4.46, 4.35,
4.28 (6 · d, 6H, 3 · OCH2Ph); 3.86 (m, 1H, H-5,
J4,5 = 9.8 Hz, J5,6a = 4.3 Hz, J5,6b = 2.7 Hz); 3.84 (dd,
1H, H-3, J2,3 = 10.7 Hz, J3,4 = 10.1 Hz); 3.74 (dd, 1H,
H-2); 3.71 (dd, 1H, H-4); 3.67 (m, 1H, H-6a,
J6a,6b = 13.1 Hz); 3.54 (m, 1H, H-6b); 13C NMR (ace-
tone-d6): d 97.8 (d, C-1, JC-1,P = 6.1 Hz), 81.7 (C-3);
75.8 (C-4); 74.0 (C-5); 69.2 (C-6); 65.1 (d, C-2, JC-
2,P = 8.3 Hz); 31P NMR (acetone-d6): d ꢀ0.87.
3.4.5. 2-Butyramido-2-deoxy-a-D-glucopyranosyl phos-
phate (7). According to procedure A 2-amino-2-
deoxy-a-D-glucopyranosyl phosphate 4 (73 mg, 0.28
mmol) and N-butyroxy-succinimide were converted to
the title compound 7 in form of a colourless hygroscopic
20
solid (88 mg, 86%); ½aꢂD +44 (c 1.0 water).
3.4.6.
2-Deoxy-2-isobutyramido-a-D-glucopyranosyl
phosphate (8). 2-Amino-2-deoxy-a-D-glucopyranosyl
phosphate 4 (72 mg, 0.28 mmol) was reacted with N-
iso-butyroxysuccinimide as described by procedure A
thus to give title compound 7 (85 mg, 84%) as a colour-
20
less hygroscopic powder; ½aꢂD +19.5 (c 1.0 water).
3.4.2. 2-Amino-2-deoxy-a-D-glucopyranosyl phosphate
(4). (2-Azido-3,4,6-tri-O-benzyl-2-deoxy-a-D-glucopyr-
anosyl) dibenzylphosphate (3, 379 mg, 0.52 mmol) was
dissolved in a mixture of water/MeOH/EtOAc (25 mL,
1:2:1). This solution was treated with palladium on acti-
vated charcoal (650 mg, 10%) and hydrogenated under
65 bar pressure for 3 days. After filtration and evapora-
tion of the organic solvents under reduced pressure fol-
lowed by lyophilization the crude product was purified
on Biogel P2 with water as eluent resulting in compound
3.4.7. Uridine 50-(2-deoxy-2-formylamido-a-D-glucopyran-
osyl diphosphate) (9). According to procedure B the
triethylammonium salt of 2-deoxy-2-formylamido-a-D-
glucopyranosyl phosphate (5, 38 mg, 78 lmol) was
reacted with the UMP morpholidate reagent (85 mg,
125 lmol) resulting in the title compound 9 (14 mg,
28%) as a colourless solid.
3.4.8. Uridine 50-(2-deoxy-2-propionylamido-a-D-gluco-
pyranosyl diphosphate) (10). 2-Propionylamido-2-
deoxy-a-D-glucopyranosyl phosphate (6) in form of its
triethylammonium salt (83 mg, 166 lmol) was reacted
with the UMP morpholidate coupling reagent (183 mg,
266 lmol) as described in procedure B to give com-
pound 10 (36 mg, 30%) as a colourless powder.
20
4 (89 mg, 67%) as a colourless foamy solid. ½aꢂD ꢀ36.5 (c
1.0 water); 1H NMR (D2O): d 5.77 (dd, 1H, H-1,
J1,2 = 3.5 Hz, J1,P = 6.6 Hz); 3.97 (dd, 1H, H-3,
J2,3 = 10.4 Hz, J3,4 = 9.4 Hz); 3.94 (m, 1H, H-5,
J4,5 = 9.9 Hz, J5,6a = 4.0 Hz, J5,6b = 2.3 Hz); 3.90 (dd,
2H, H-6a, J6a,6b = 12.5 Hz); 3.86 (dd, 2H, H-6b); 3.60
(dd, 1H, H-4); 3.43 (m, 1H, H-2, J2,P = 2,3 Hz). 13C
NMR (D2O): d 92.2 (d, C-1, J1,P = 6.1 Hz); 73.2 (C-5);
69.9 (C-3); 69.4 (C-4); 60.3 (C-6); 54.5 (dd, C-2,
J2,P = 9.1 Hz); 31P NMR (D2O): d 2.70.
3.4.9. Uridine 50-(2-butyramido-2-deoxy-a-D-glucopyran-
osyl diphosphate) (11). The triethylammonium salt of
2-butyramido-2-deoxy-a-D-glucopyranosyl phosphate
(7, 71 mg, 133 lmol) was reacted with the UMP
morpholidate reagent (147 mg, 215 lmol) according to
procedure B to give the title compound 11 (30 mg,
33%) as a colourless powder.
3.4.3. 2-Deoxy-2-formylamido-a-D-glucopyranosyl phos-
phate (5). The triethylammonium salt of 2-amino-2-
deoxy-a-D-glucopyranosyl
0.1 mmol) together with 2,4,5-trichlorophenylformiate
(27 mg, 0.1 mmol) and di-iso-propylethylamine
phosphate
4
(27 mg,
3.4.10. Uridine 50-(2-isobutyramido-2-deoxy-a-D-gluco-
pyranosyl diphosphate) (12). The triethylammonium
salt of 2-isobutyramido-2-deoxy-a-D-glucopyranosyl
phosphate (8, 60 mg, 113 lmol) was reacted with the
UMP morpholidate reagent (125 mg, 183 lmol) accord-
ing to procedure B to give the title compound 12 (23 mg,
30%) as a colourless powder.
(15.5 mg, 20.5 lL, 0.12 mmol) in 3 mL anhydrous
DMF were stirred for 2 days at room temperature.
Upon dilution with 50 mL deionized water the resulting
solution was neutralized with 1 M HCl solution, follow-
ing freeze drying and desalting on Biogel P2 with
250 mM aqueous ammonium hydrogencarbonate solu-

576
D. Lazarevic´, J. Thiem / Carbohydrate Research 341 (2006) 569–576
15. Wang, P.; Shen, G.-J.; Wang, Y.-F.; Ichikawa, Y.; Wong,
Acknowledgement
C.-H. J. Org. Chem. 1993, 58, 3985–3990.
16. Herrmann, G. F.; Wang, P.; Shen, G.-J.; Wong, C.-H.
Angew. Chem., Int. Ed. Engl. 1994, 33, 1241–1242.
17. Gygax, D.; Spies, P.; Winkler, T.; Pfaar, U. Tetrahedron
1991, 47, 5119–5122.
Support of this work by the Fonds der Chemischen
Industrie and the Deutsche Forschungsgemeinschaft
(SFB 470, A5) is gratefully acknowledged.
18. Look, G. C.; Ichikawa, Y.; Shen, G.-J.; Cheng, P.-W.;
Wong, C.-H. J. Org. Chem. 1993, 58, 4326–4330.
19. Wong, C.-H.; Haynie, S. L.; Whitesides, G. M. J. Org.
Chem. 1982, 47, 5416–5418.
References
1. Sharon, N.; Lis, H. Sci. Am. 1993, 82–89.
2. Sharon, N.; Lis, H. Eur. J. Biochem. 1993, 218, 1–27.
3. Kobata, A. Acc. Chem. Res. 1993, 26, 319–324.
4. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
5. Varki, A. Glycobiology 1993, 3, 97–130.
6. Danishefsky, S. J.; Bilodeau, M. T. Angew. Chem., Int. Ed.
Engl. 1996, 35, 1380–1419.
20. Heidlas, J. E.; Lees, W. J.; Pale, P.; Whitesides, G. M.
J. Org. Chem. 1992, 57, 146–151.
21. Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T.
Angew. Chem., Int. Ed. Engl. 1995, 34, 521–546.
22. Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic
Organic Chemistry; Elsevier: Oxford, 1994.
23. Sandhoff, K.; Kolter, T. Trends Cell Biol. 1996, 6, 98–101.
24. Moffat, J. G.; Khorana, H. G.; Roseman, S.; Distler, J. J.
J. Am. Chem. Soc. 1961, 83, 659–664.
7. Barresi, F.; Hindsgaul, O. J. Carbohydr. Chem. 1995, 18,
1043–1087.
8. Oberthur, M.; Leimkuhler, C.; Kahne, D. Org. Lett. 2004,
¨
25. Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62, 2144–
2147.
17, 2873–2876.
9. Toshima, K.; Tatsuta, K. Chem. Rev. 1993, 93, 1503–
´
26. Lazarevic, D.; Thiem, J. Carbohydr. Res. 2002, 337, 2187–
1531.
2194.
10. Banoub, J.; Boullanger, P.; Lafont, D. Chem. Rev. 1992,
27. Sala, R. F.; MacKinnon, S. L.; Palcic, M. M.; Tanner, M.
E. Carbohydr. Res. 1998, 306, 127–136.
28. Losey, H. C.; Jiang, J.; Biggins, J. B.; Oberthuer, M.; Ye,
X. Y.; Dong, S. D.; Kahne, D.; Thorson, J. S.; Walsh, C.
T. Chem. Biol. 2002, 9, 1305–1314.
92, 1167–1195.
11. Paulsen, H. Angew. Chem., Int. Ed. Engl. 1990, 29, 823–
839.
12. Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25,
212–235.
29. Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57,
1244–1251.
30. Grundler, G.; Schmidt, R. R. Liebigs Ann. Chem. 1984,
1826–1847.
13. Kunz, H. Angew. Chem., Int. Ed. Engl. 1987, 26, 294–
308.
14. Halcomb, R. L.; Huang, H.; Wong, C.-H. J. Am. Chem.
Soc. 1994, 116, 11315–11322.
31. Martinez, J.; Laur, J. Synthesis 1982, 979–981.