Identification of a UDP-Gal: GlcNAc-R galactosyltransferase activity in Escherichia coli VW187

Article abstract of DOI:10.1016/j.bmcl.2004.11.077

A novel acceptor substrate for galactosyltransferase was synthesized containing GlcNAcα-pyrophosphate, covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc α-O-PO₃-PO₃-(CH ₂)₁₁-O-Phenyl). The new subs

Full text of DOI:10.1016/j.bmcl.2004.11.077

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1205–1211
Identification of a UDP-Gal: GlcNAc-R
galactosyltransferase activity in Escherichia coli VW187
Pedro J. Montoya-Peleaz,a John G. Riley,a,b Walter A. Szarek,a Miguel A. Valvano,c
John S. Schutzbachb and Inka Brockhausenb,*
aDepartment of Chemistry, QueenÕs University, Kingston, Ontario, Canada K7L 3N6
bDepartment of Medicine, Department of Biochemistry, The Arthritis Centre and Human Mobility Research Centre,
QueenÕs University, Kingston General Hospital, Kingston, Ontario, Canada K7L 2V7
cDepartment of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1
Received 18 September 2004; revised 29 November 2004; accepted 30 November 2004
Available online 24 December 2004
Abstract—A novel acceptor substrate for galactosyltransferase was synthesized containing GlcNAca-pyrophosphate, covalently
bound to a hydrophobic phenoxyundecyl moiety (GlcNAc a-O-PO3-PO3-(CH2)11-O-Phenyl). The new substrate was used to develop
an assay for a galactosyltransferase activity from Escherichia coli strain VW187 that is involved in lipopolysaccharide synthesis and
has not been studied by others. We showed that Gal was transferred from UDP-Gal to the novel acceptor substrate. This was a sig-
nificant improvement over our previous preliminary assays of the enzyme using endogenous substrate, and showed that these synthetic
substrates are useful for assaying enzymes that utilize lipid-bound substrates in O-chain synthesis in Gram-negative bacteria.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
quate amounts of endogenous sugar-PP-Und substrate
and unavailability of defined and stable synthetic
substrates.3 Our preliminary assays using endogenous
acceptor substrate for galactosyltransferase from VW187
called for improved and defined assay conditions. The
enzyme has not been studied by others.
The O-specific polysaccharide (O antigen) is the outer-
most component of the lipopolysaccharide (LPS), a
major constituent of the outer membrane in Gram-
negative bacteria. The outer O-chain of the LPS of Esch-
erichia coli strain VW187 (O7:K1) consists of repeats of
3-VioNAc b1–2 [Rha a1–3]Man a1–4Gal b1–3 GlcNAc
a1, where VioNAc is 4-acetamido-4,6-dideoxy-D-glu-
cose.1 The assembly of the repeat unit is thought to
occur at the cytosolic face of the plasma membrane
and involves a lipid-linked intermediate containing
GlcNAca1-pyrophosphoryl-undecaprenol (GlcNAc-PP-
Und).2 Additional sugars are added sequentially to the
GlcNAc-PP-Und intermediate to complete the forma-
tion of the O7 subunit. Subsequently, the subunits are
polymerized and then linked to lipopolysaccharide A
to form LPS. These reactions are catalyzed by specific
glycosyltransferases, which are either soluble enzymes
or associated with the plasma membrane.
In this work, we have attempted to overcome some of
these problems by chemically synthesizing an analog
of the sugar-PP-Und acceptor. GlcNAc was the pre-
ferred sugar for this molecule given that it is widely used
by many bacteria for the initiation reactions required for
the synthesis of the O antigen repeating units.4,5 There-
fore, in this work, we report the chemical synthesis of
GlcNAca-pyrophosphate bound to a phenoxyundecyl
moiety (Fig. 1) in [GlcNAc a1-O-PO2-O-PO2-O-
(CH2)11-O-Phenyl]2ꢀ (GlcNAc-PP-PhU). This synthetic
substrate was then utilized in the development of an effi-
cient in vitro assay for a galactosyltransferase activity
present in E. coli VW187.
In the past, it has been very difficult to assay and
characterize these enzymes because of the lack of ade-
2. Materials
All reagents were of the highest grade available and were
from Sigma Chemical Co., St. Louis, MO, unless other-
wise indicated. Protein concentrations were determined
Keywords: O-Antigen synthesis; Galactosyltransferase; Enzyme assay.
*
Corresponding author. Tel.: +1 613 549 6666x6355; fax: +1 613 549
2529; e-mail: brockhau@post.queensu.ca
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.077

1206
P. J. Montoya-Peleaz et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1205–1211
OH
O
HO
HO
( )7
O
O
P
NH
O
O
P
O
O
-
O
-
O
GlcNAc-PP-Und
OH
O
HO
HO
O
O
P
NH
O
O
P
O
O
O
-
O
-
O
GlcNAc-PP-PhU
Figure 1. Chemical structures of the GlcNAca-pyrophosphoryl-undecaprenol (GlcNAc-PP-Und) and the novel synthetic GlcNAca-pyrophosphate-
phenoxyundecyl (GlcNAc-PP-PhU) acceptor substrates.
with the BioRad (Bradford) protein assay using
bovine serum albumin as a standard. Escherichia coli
strain VW187 (E. coli O7:K1) has been previously
described.4,6
OH
O
HO
HO
OH
OR4
NH
Br
10
3. Preparation of enzyme source
O
13 R4 = H
8
d
12 R4 = THP
Bacteria were grown at 37 ꢁC in LBmedium containing
100 lg/mL ampicillin, inoculated with 10 mL of an over-
night culture of VW187. Cultures were incubated at
37 ꢁC on a shaker at 250 rpm. After 2.5 h (A600 = 0.8)
the bacteria were harvested by centrifugation at
5000 rpm for 10 min (IEC MultiRF Rotor). Cells were
resuspended in 12 mL of PBS, pH 7.2/glycerol (v/v)
9:1, and were stored at ꢀ20 ꢁC. All additional proce-
dures were carried out at 4 ꢁC. The bacterial cells were
sedimented by centrifugation, washed with 3 mL of
PBS, pH 7.2, and then resuspended in 0.5 mL of
10 mM Tris/acetate, pH 8.5 containing 50 mM sucrose
and 1.2 mM EDTA. The cells were sonically ruptured
using a Sonic Dismembrator Model 100 (Fisher Scien-
tific) for 2 pulses of 15 s with 2 min intervals to allow
for cooling on ice. Protein concentrations ranged be-
tween 2 and 12 mg/mL.
a
79%
e
OR1
O
R1O
OR4
10
.
O
R1
O
R2
7 R1 = Ac, R2 = Bn
O
NH
11 R4 = THP
10 R4 = H
b
O
f
90%
6 R1 = Ac, R2 = H
88%
c
74%
O
c
OR1
O
R1O
R1
O
.
O
O
R3
O
P
P
O
NH
10
OR3
OR3
OR3
O
O
9 R3 = Bn
4 R3 = H
5 R1 = Ac, R3 = Bn
3 R1 = Ac, R3 = H
b
93%
b
96%
g
OR1
80%
O
R1O
R1
4. Synthesis of acceptor substrate
O
O
P
O
NH
O
O
h
O
The synthesis of the GlcNAc-PP-PhU acceptor is shown
in Scheme 1. All nonhydrolytic reactions were per-
formed in oven-dried glassware under a dry nitrogen
atmosphere. Dichloromethane and tetrahydrofuran
(THF) were dried over CaH2 and sodium, respectively,
P
O-
O
10
O
OH
X+
2 R1 = Ac, X+ = NH4
1 R1 = H , X+ = pyridinium
+
1
and then distilled. The H, 13C, and 31P NMR spectra
Scheme 1. Synthesis of the GlcNAca-P-P-phenoxyundecyl acceptor
substrate. Reagents and conditions: (a) 1. NaH, DMF, benzyl
bromide; 2. Pyridine, acetic anhydride, 0 ꢁC; (b) Pd/C, CH3OH, H2;
(c) i-Pr2NP(OBn)2, 1H-tetrazole, CH2Cl2, ꢀ30 ! 0 ꢁC, 0.5 h, then m-
CPBA, ꢀ40 ! 25 ꢁC, overnight; (d) Dihydropyran, CH2Cl2, TsOH,
0 ꢁC ! rt, 16 h; (e) Phenol, KOH, Aliquat 336, 85 ꢁC, overnight; (f)
CH3OH, TsOH, 1.5 h, rt; (g) 1. 4, 1,10-carbonyldiimidazole, THF,
1.5 h, rt then CH3OH; 2. 3, THF, 48 h, rt; (h) NaOCH3, CH3OH,
20 min, rt, quenched with pyridinium resin. The % yield for each step is
shown.
were recorded using Bruker Avance 300-, 400-, 500-,
and 600-MHz spectrometers. Chemical shifts (d) are
reported in parts per million (ppm), and signals are
described as s (singlet), d (doublet), dd (doublet of dou-
blets), t (triplet), q (quartet), quin (quintet), and m (mul-
tiplet). Proton chemical shifts are given relative to those
of internal standards CHCl3, CH3OD, or DMSO:
d = 7.26, 3.30, or 2.25 ppm, respectively. Carbon

P. J. Montoya-Peleaz et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1205–1211
1207
chemical shifts are given relative to those of CDCl3,
CD3OD, or DMSO: d = 77, 49, or 39.5 ppm, respec-
tively. Phosphorus chemical shifts are given relative to
that of 85% phosphoric acid: d = 0 ppm. Mass spectra
were obtained using an Applied Biosystems/MDS Sciex
QStar XL spectrometer. TLC was performed using
Department of Chemistry, QueenÕs University, Kings-
ton ON. The matrix substance was 2,5-dihydroxyben-
zoic acid.
6. The synthetic GlcNAc-PP-PhU is an excellent acceptor
substrate
glass- or aluminum-backed Silicyle Silica Gel 60 F254
.
Reactions were monitored by charring of the plates after
spraying with either 5% H2SO4 in ethanol, or phospho-
molybdic acid (PMA) in ethanol.
The novel acceptor substrate GlcNAc-PP-PhU has
amphipathic properties similar to those of the endoge-
nous substrate GlcNAc-PP-Und, is soluble in metha-
nol/water mixtures, and can be added directly to
the reaction mixtures. The chemical and spectrometric
analyses of synthetic GlcNAc a-PP-PhU showed that
nearly 100% of the a-anomer was synthesized. The com-
pound was stable at ꢀ20 ꢁC in methanol for at least
6 months. The pyridinium salt of GlcNAc a-PP-PhU
bound to C18 Sep-Pak cartridges and could be eluted
with methanol and subsequently analyzed by HPLC
(Fig. 2).
The GlcNAc sugar head group and the phospholipid tail
were assembled separately and then coupled using well-
established methodology. Thus, GlcNAc 8 (Scheme 1)
was selectively protected at the anomeric position with
a benzyl group,7 and the remaining hydroxyl groups
were acetylated. The anomeric position was then depro-
tected by hydrogenolysis, and the product was treated
with i-Pr2NP(OBn)2 (dibenzyl diisopropylphosphorami-
dite)8 to afford the glycosyl phosphite, which was oxi-
dized to produce dibenzyl phosphate 5.9,10 Synthesis of
the tail fragment required initial protection of the hy-
droxyl group of 13 with a THP group,11 attachment of
the phenoxy group,12 followed by the removal of the
THP group. The dibenzyl phosphate 9 was synthesized
using the same conditions as employed in the case of
compound 5. Both 5 and 9 were then debenzylated by
hydrogenolysis. Compound 4 was then activated by
forming the phosphorimidazolidate, and compound 3
was then added.10,13 Finally, the acetyl groups were re-
moved using NaOCH3 in methanol.14
Our results show that GlcNAc a-PP-PhU salts are excel-
lent substrates for galactosyltransferase from VW187
homogenates. These amphipathic compounds can easily
be synthesized. Other advantages are that the substrate
and the enzymatic reaction products are readily sepa-
rated by HPLC using a C18 column and acetonitrile/
water mixtures as the mobile phase, and that com-
pounds containing the phenyl group can be readily de-
tected with UV absorbance.
The Gal-transferase activity of strain VW187 catalyzed
galactosyl transfer from UDP-Gal to the GlcNAc a-
PP-PhU acceptor in standard assays. More than 95%
of the enzyme product was eluted in the first methanol
fraction from C18 Sep-Pak columns. HPLC separation
revealed the presence of a new enzyme product that
eluted approximately 5 min before the unmodified
acceptor substrate (Fig. 2). Gal-transferase activity
was linear with time for more than 10 min (Fig. 3A)
and was proportional to enzyme concentration up to
at least 5 lg protein per assay (Fig. 3B). Ion exchange
chromatography of the fractions that did not bind to
C18 Sep-Pak showed that more than 80% of the total
amount of UDP-Gal added was still present after the
10 min incubation.
5. Galactosyltransferase assay using synthetic acceptor
substrate
Standard assays for galactosyl transfer to exogenously
added substrate were carried out in reaction mixtures
of 40 lL total volume, containing 0.5 mM GlcNAc-
PP-PhU (pyridinium salt), 5 mM MnCl2, 75 mM MES
buffer pH 7, 0.5 mM UDP-[3H]Gal (800–4400 cpm/
nmol), and 20 lL of enzyme homogenate (3–12 lg pro-
tein). The reaction was stopped by the addition of
0.7 mL of ice cold water and the mixtures were applied
to a 1 mL C18 Sep-Pak column that had been pre-
washed with 4 mL methanol followed by 6 mL water.
Columns were then washed with 5 mL water and the
hydrophobic product was eluted with 5 mL methanol.
Fractions of 1 mL were collected and 0.5 mL samples
of each fraction were counted in 5 mL Ready Safe scin-
tillation fluid (Beckman). All assays were carried out in
duplicate. For further analysis of the enzyme product,
the first fraction eluted with methanol from the Sep-
Pak columns was concentrated and injected into HPLC,
using a C18 column and acetonitrile/water (15/85) at
1 mL/min flow rate.15 The absorbance at 195 nm of
the eluant was measured. Fractions of 2 mL were col-
lected and the radioactivity measured by scintillation
counting. The molecular weight of the enzyme product
isolated from HPLC was determined by Matrix-assisted
laser desorption ionization (MALDI) mass spectrome-
try in the negative reflectron mode (Applied Biosystems
Voyager DE-STR MALDI-TOF) by Dr. B. Keller at the
Enzyme product from assays using VW187 homo-
genates containing UDP-[3H]Gal as the donor substrate
and GlcNAc a-PP-PhU as the acceptor substrate was
also isolated by HPLC for structural analysis. The
MALDI mass spectrum showed a major [MꢀH]ꢀ peak
at 788.6 m/z, and a minor peak at 626.5 m/z (Fig. 4).
The major peak was consistent with the molecular
weight of the Gal-transferase product [3H]Gal-Glc-
NAc-O-PO3-PO3-(CH2)11-O-Phenyl (789 Da).
We therefore demonstrated that the newly assayed Gal-
transferase activity in sonicated whole bacteria transfers
Gal from UDP-Gal to GlcNAc-PP-PhU. GlcNAc-PP-
PhU is an excellent substrate, and appears to be highly
accessible to the enzyme, which is thought to be associ-
ated with the inner surface of the plasma membrane.

1208
P. J. Montoya-Peleaz et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1205–1211
Figure 2. HPLC analysis of Gal-transferase reaction product using GlcNAc-PP-PhU as an acceptor substrate. 0.5 mM GlcNAc-PP-PhU was
incubated for 10 min with homogenates from VW187 as described in Experimental Procedures. The reaction product was isolated on a C18 Sep-Pak
column, by sequential fractionation with water eluting unreacted UDP-Gal and free Gal, and methanol to elute the enzyme product. The methanol
fractions were concentrated, and aliquots analyzed by HPLC using a C18 column and acetonitrile/water (15/85) as the mobile phase at a flow rate of
1 mL/min. The absorbance at 195 nm was measured and the radioactivity in eluting fractions was counted. Standard compounds were GlcNAc, and
substrate GlcNAc-PP-PhU.
(B)
0.5
(A)
0.4
0.3
0.2
0.1
0
800
600
400
200
0
0
1
2
3
4
5
6
0
5
10
15
20
Protein ( g/assay)
Incubation time (min)
Figure 3. Initial rate of galactosyltransferase activity. Galactosyltransferase activity was assayed using UDP-[3H]Gal as the donor substrate and
GlcNAc-PP-UPh as the acceptor substrate at conditions described in Section 5. Enzyme product was isolated by C18 Sep-Pak columns. (A)
Dependence of activity on incubation time. (B) Dependence of activity on protein concentration.
However, the membrane association and localization of
the enzyme remains to be determined. Chen et al. syn-
thesized a series of lipid-linked oligosaccharide pyro-
phosphate substrates to measure the activity of a
GlcNAc-transferase involved in peptidoglycan synthesis
from E. coli by a coupled fluorescence assay.10 Com-
pounds with short isoprene chains as well as a reduced
aliphatic chain were superior to the natural substrate,
with the exception of a compound containing a trans-
configuration after the first isoprene unit. Moraprenol-
pyrophosphoryl-Glc has also been shown to be an
acceptor substrate for E. coli mannosyltransferase.16
Thus the topology and membrane association not only
of the enzymes but also of substrates are important
factors for the sugar transfer reaction in bacteria. Pyro-
phosphate-containing lipids with a phenoxyundecyl
group may also be acceptor substrates for other glyco-
syltransferases that catalyze the subsequent steps of O7
chain synthesis, and for glycosyltransferases of other
strains of Gram-negative bacteria.
7. Experimental procedures for the synthesis of GlcNAc-
PP-PhU
7.1. 2[(11-Phenoxyundecyl)oxy]tetrahydro-2H-pyran (11)
11-Bromo-1-undecanol (13) (7.54 g, 30.0 mmol) and 3,4
dihydro-2H-pyran (5 equiv, 12.62 g, 150 mmol) were
dissolved in dry CH2Cl2 (140 mL) and the solution
was cooled to 0 ꢁC. p-Toluenesulfonic acid monohy-
drate (0.01 equiv, 0.057 g, 0.3 mmol) was then added
and the solution was stirred for 10 min; it was then stir-
red at rt for 16 h. The mixture was partitioned between
ether and a solution made up of saturated NaCl
(70 mL), saturated aqueous NaHCO3 (70 mL), and

P. J. Montoya-Peleaz et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1205–1211
1209
O
P
O
P
O
O
O
(CH2
)
O
Gal(beta 1-3)GlcNAc(alpha)
11
MALDI mass spectra, negative mode, reflectron
OH
OH
[M-H]-
Voyager Spec #1[BP = 788.7, 7954]
788.6621
7954.2
100
90
80
70
60
50
40
30
20
10
0
- 162 Da = precursor without Gal
587.2738
789.6559
Sample
in
653.3266
588.2686
586.2563
DHB
626.4879
517.1955
652.3286
654.3271
790.6522
789.1158
533.2169
563.2716
561.2630
552.2666
554.2693
681.3498
683.3413
535.2474
534.2382
597.2998
603.2975
598.3106
627.4858
630.3431
624.3185
661.3166
651.3273
649.3297
527.2333
513.2131
576.2676
828.4461
723.3657
736.3700
673.2978
696.3423
763.3940
813.4361
0
500
570
640
710
780
850
Mass (m/z)
Voyager Spec #1[BP = 587.2, 5024]
587.2113
5024.0
100
90
80
70
60
50
40
30
20
10
0
DHB
563.2148
517.1496
515.1068
Matrix
blank
681.2614
588.2178
589.2332
533.1791
571.2183
549.1932
625.2526
623.2532
626.2541
619.2452
622.2438
547.1996
548.1810
544.1852
703.2958
682.2640
679.2693
671.2742
573.2260
576.2073
568.2113
518.1612
521.1763
653.2708
649.2508
648.2570
597.2275
594.2194
701.2902
699.2914
698.2781
761.3071
755.3276
723.3080
722.3004
522.1875
783.3451
827.3855
672.2655
806.3454
0
500
570
640
710
780
850
Mass (m/z)
Figure 4. MALDI spectrum of purified enzyme product. Gal-transferase enzyme product from GlcNAc a1-O-PO2-O-PO2-O-(CH2)11-O-phenyl
substrate was prepared using the wild type strain VW187 as the enzyme source. Enzyme product was purified on a C18 Sep-Pak column, and by
HPLC using a C18 column and acetonitrile/water 6/94. Mass spectrometry was carried out using MALDI in the negative reflectron mode. DHB, 2,5-
dihydroxybenzoic acid matrix substance.
water (140 mL). The organic phase was washed with
saturated NaCl (50 mL · 2), dried over MgSO4, and
concentrated to yield a clear oil quantitatively (10.0 g).
Compound 12 was used without further purification in
the following step.
1H), 3.97 (t, J = 6.6 Hz, 2H), 4.60 (m, 1H), 6.93 (m,
3H), 7.29 (m, 2H). 13C NMR (CDCl3) d 19.65, 25.47,
26.0, 26.2, 29.25, 29.35, 29.44, 29.49, 29.51, 29.71,
30.7, 62.3, 67.67, 67.82, 98.8, 114.4, 120.4, 129.4, 159.1.
7.2. 11-Phenoxy-1-undecanol (10)
Phenol (1 equiv, 0.941 g, 10 mmol), KOH powder
(1.25 equiv, 0.70 g, 12.5 mmol), and Aliquat 336
(0.02 equiv, 0.081 g, 0.2 mmol) were sonicated for
5 min at rt. The mixture was then heated to 85 ꢁC, stir-
red for 5 min, and compound 12 (3.35 g, 10 mmol) was
added. After 6 h at 85 ꢁC the solution was cooled; ether
(75 mL) was added and the mixture was sonicated for
5 min, passed through a Florosil plug, washed with
more ether, and the solvent was removed. The crude
product was purified by centrifugal chromatography
{7:1 (v/v) ethyl acetate–hexane} to yield a clear liquid
The protected alcohol 11 (2.75 g, 7.9 mmol) was dis-
solved in methanol (40 mL) and p-toluenesulfonic acid
(100 mg) and the mixture was stirred for 1.5 h. The meth-
anol was then removed by evaporation and the remai-
ning solid was dissolved in CHCl3 (50 mL); the solution
was washed with saturated aqueous NaHCO3
(50 mL · 2), and dried over MgSO4. Removal of the sol-
vent afforded the pure product as a cream solid (1.88 g,
90%). 1H NMR (CDCl3) d 1.28–1.38 (m, 12H), 1.47 (m,
2H), 1.59 (m, 2H), 1.80 (quin, J = 7.3 Hz), 3.66 (q,
J = 6 Hz, 2H), 3.98 (t, J = 6.6 Hz, 2H), 6.94 (m, 3H),
7.29 (m, 2H). 13C NMR (CDCl3) d 25.7, 26.0, 29.24,
1
(2.75 g, 79%). H NMR (CDCl3) d 1.25–1.90 (m, H),
3.40 (m, 1H), 3.52 (m, 1H), 3.76 (m, 1H), 3.90 (m,

1210
P. J. Montoya-Peleaz et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1205–1211
29.35, 29.45, 29.49, 29.52, 32.6, 63.0, 67.8, 114.4, 120.4,
129.3, 159.1. MS (+TOF) m/z 287 [M+Na]+; HRMS
[M+Na]+, found: 287.1986. C17H28O2Na requires m/z,
287.1981.
saturated Na2SO4 (50 mL · 3), water (50 mL · 2), and
saturated NaCl (50 mL · 2), dried over Na2SO4, and
concentrated to yield a yellow oil. The product was puri-
fied by column chromatography {3:1 (v/v) ethyl acetate–
petroleum ether, then 100% ethyl acetate} to give a clear
1
7.3. Dibenzyl 11-phenoxyundecyl phosphate (9)
oil (0.617 mg, 88%). H NMR (CDCl3) d 1.70 (s, 3H),
2.02 (m, 9H), 3.93 (dd, J = 12.5, 2.2 Hz, 1H), 4.00 (m,
1H), 4.13 (dd, J = 12.5, 3.9 Hz, 1H), 4.36 (m, 1H),
Compound 10 (508 mg, 1.92 mmol) and tetrazole
(2 equiv, 271 mg, 3.84 mmol) were dissolved in dry
CH2Cl2 (20 mL) and the solution was cooled to
5.10 (m, 6H), 5.66 (m, 2H), 7.34 (m, 10H). 13C NMR
4
(CDCl3) d 20.52, 20.58, 20.61, 22.7, 51.8 (d, JCP
=
7.6 Hz), 61.2, 67.3, 69.6, 69.9 (app t, JCP = 5.5 Hz),
3
ꢀ30 ꢁC.
Bis(benzyloxy)(diisopropylamino)phosphine
3
70.1, 96.2 (d, JCP = 6.6 Hz), 128.0, 128.1, 128.77,
(2 equiv, 1.219 g, 3.84 mmol) was then added drop wise
over a 2 min period. The mixture was then stirred at rt
for 15 h, cooled to ꢀ40 ꢁC, and 77% m-CPBA (5 equiv,
2.16 g, 9.6 mmol) was added. The solution was stirred
for 1 h at 0 ꢁC, then for a further hour at rt. After addi-
tion of ethyl acetate (100 mL) the solution was washed
with saturated aqueous Na2SO3 (50 mL · 3), water
(50 mL · 2), and saturated NaCl (50 mL · 2), dried over
Na2SO4, and concentrated to yield a yellow oil. The
product was purified by column chromatography {2:3
(v/v) ethyl acetate–petroleum ether} to yield a white
4
4
128.81, 128.9, 135.2 (d, JCP = 6.5), 135.3 (d, JCP
=
6.3 Hz), 169.1, 170.1, 170.5, 171.1. 31P NMR (CDCl3)
d ꢀ2.3. MS (+TOF) m/z 630 (M++Na); HRMS
[M+Na]+, found: 630.1700. C28H34NO12NaP requires
m/z, 630.1710.
7.6. 3,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-a-D-gluco-
pyranosyl dihydrogen phosphate (3)
Compound 5 (251 mg, 0.413 mmol) was dissolved in
deoxygenated methanol (15 mL) and 10% Pd/C
(300 mg) was added. The solution was stirred overnight
under an H2 atmosphere. The Pd/C was removed by fil-
tration through a Celite plug, and the solvent was evap-
1
solid (751 mg, 74%). H NMR (CDCl3) d 1.26–1.45 (m,
12H), 1.52 (m, 2H), 1.68 (m, 2H), 1.81 (m, 2H), 3.97–
4.04 (m, 4H), 5.14 (m, 4H), 6.97 (m, 3H), 7.30–7.45
(m, 12H). 13C NMR (CDCl3) d 25.2, 25.9, 29.0, 29.2,
3
1
29.26, 29.34, 29.4, 30.0, 67.8 (app t, JCP = 6.3 Hz),
68.9 (d, JCP = 5.6 Hz), 114.4, 120.3, 127.8, 128.3,
orated to yield a white solid (170 mg, 96%). H NMR
3
(CD3OD) d 1.96 (s, 3H), 2.01 (s, 3H), 2.03 (s, 3H),
2.07 (s, 3H), 4.14 (dd, J = 12.3, 2.1 Hz, 1H), 4.25 (m,
1H), 4.32 (m, 2H), 5.11 (t, J = 10 Hz, 1H), 5.31 (dd,
J = 10.9, 9.3 Hz, 1H), 5.60 (dd, 6.2, 3.3 Hz, 1H). 13C
NMR (CD3OD) d 20.57, 20.62, 20.7, 22.4, 53.1, 62.7,
69.7, 70.3, 71.5, 96.0 (d, J = 6.2 Hz), 171.2, 172.0,
172.4, 173.7. 31P NMR (CD3OD) d ꢀ2.0. MS (ꢀTOF)
m/z 426 [MꢀH]ꢀ; HRMS [MꢀH]ꢀ, found: 426.0805.
C14H22NO12P requires m/z, 426.0806.
4
128.4, 129.3, 135.9 (d, JCP = 7.0 Hz), 159.0. 31P NMR
(CDCl3) d ꢀ0.45. MS (+TOF) m/z 547 [M+Na]+;
HRMS [M+Na]+, found: 547.2570. C31H41O5PNa re-
quires m/z, 547.2583.
7.4. 11-Phenoxyundecyl dihydrogen phosphate (4)
Compound 9 (517 mg, 0.986 mmol) was dissolved in
deoxygenated methanol (15 mL) and 10% Pd/C
(200 mg) was added. The solution was stirred overnight
under a H2 atmosphere. The Pd/C was removed by fil-
tration through a Celite plug, and the solvent was evap-
7.7. Ammonium P1-3,4,6-tri-O-acetyl-2-acetamido-2-
deoxy-a-D-glucopyranosyl P2–11-phenoxyundecyl hydro-
gen diphosphate (2)
1
orated to yield a white solid (317 mg, 93%). H NMR
(CD3OD) d 1.34–1.52 (m, 12H), 1.68 (app. quin, 2H),
1.78 (app. quin, 2H), 3.97 (app. q, 4H), 6.91 (m, 3H),
7.24 (m, 2H). 13C NMR (CD3OD) d 26.7, 27.2, 30.35,
30.47, 30.53, 30.65, 30.69, 31.50, 31.58, 67.7 (d,
3JCP = 6.0 Hz), 68.9, 115.5, 121.5, 130.4, 160.6. 31P
NMR (CD3OD) d 0.33. MS (+TOF) m/z 383 [M+K]+;
HRMS [M+K]+, found: 383.1379. C17H29O5PK re-
quires m/z, 383.1384.
Both sugar head group 3 (250 mg, 0.589 mmol) and tail
4 (1.1 equiv, 223 mg, 0.648 mmol) were dried separately
by coevaporation (·3) from toluene (3 mL)–diisopropyl-
amine (3 drops). Compound 4 was left on a vacuum line
for another 30 min, then dry THF (15 mL) was
added followed by 1,10-carbonyldiimidazole (4.3 equiv,
417 mg, 2.53 mmol); the solution was stirred at rt for
1.5 h. Dry methanol (212 lL) was added, and the solu-
tion was stirred for a further hour. The solvent was
removed under reduced pressure and the residue was
kept on a vacuum line for 1 h (no bubbling). A solution
of compound 3 in dry THF (15 mL) was added, the
reaction was stirred for 48 h, and evaporated. The crude
product was purified by a Sephadex G-15 size-exclusion
column using methanol (0.1% NH4HCO3) as eluent to
yield a white solid (0.365 mg, 80 %). 1H NMR (CD3OD)
d 1.26–1.45 (m, 12H), 1.50 (m, 2H), 1.69 (m, 2H), 1.79
(m, 2H), 2.06 (m, 12H), 3.43 (m, 2H), 3.96 (m, 3H),
4.21 (m, 2H), 4.56–4.69 (m, 3H), 5.12 (t, J = 9.7 Hz,
1H), 5.33 (t, J = 9.7 Hz, 1H), 5.66 (dd, J = 3.3, 7.3 Hz,
1H), 6.92 (m, 3H), 7.25 (t, J = 8.3 Hz, 2H), 8.75 (d,
J = 8.6 Hz, 1H). 13C NMR (CD3OD) d 19.5, 20.6,
7.5. 3,4,6-Tri-O-acetyl-2-acetamido-1-O-[bis(benzyl-
oxy)phosphoryl]-2-deoxy-a-D-glucopyranose (5)
Compound 6 (0.508 mg, 1.16 mmol) and tetrazole
(10 equiv, 832 mg, 11.6 mmol) were dissolved in dry
CH2Cl2 (20 mL) and the solution was cooled to
ꢀ20 ꢁC.
Bis(benzyloxy)(diisopropylamino)phosphine
(5 equiv, 1.84 g, 5.8 mmol) was then added drop wise
over a 2 min period. The mixture was stirred at rt for
5 h, cooled to ꢀ40 ꢁC, and 77% m-CPBA (10 equiv,
2.6 g, 11.6 mmol) was added. The solution was stirred
for 30 min at 0 ꢁC, then overnight at rt. After addition
of ethyl acetate (100 mL) the solution was washed with

P. J. Montoya-Peleaz et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1205–1211
1211
22.8, 26.9, 27.2. 30.4, 30.5, 30.71, 30.74, 53.2, 62.8, 67.2,
68.8, 69.7, 69.8, 73.0, 95.8, 115.5, 121.4, 130.4, 160.5,
171.2, 171.8, 172.4, 174.0. 31P NMR (CD3OD) ꢀ12.4
(J = 22 Hz), ꢀ9.4 (J = 22 Hz). MS (ꢀTOF) m/z 752
ase assays. This work was supported by grants from the
Natural Science and Engineering Research Council of
Canada and The Arthritis Society of Canada.
[MꢀH]ꢀ;
HRMS
[MꢀH]ꢀ,
found:
752.2479.
C31H48NO16P2 requires m/z, 752.2453.
References and notes
7.8. Pyridinium P1-2-acetamido-2-deoxy-a-D-glucopyr-
anosyl P2-11-phenoxyundecyl hydrogen diphosphate (1)
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A solution of compound 2 (25.1 mg, 0.0325 mmol) in
0.0325 M NaOCH3 (4 mL) was stirred for 20 min at rt
until reaction was complete {thin layer chromatogra-
phy, 6:2.5:0.4 (v/v/v) CHCl3–methanol–H2O}; it was
then quenched with an excess of IR-120 resin (Aldrich)
in the pyridinium form, and the solution was stirred
for another 40 min. The reaction mixture was filtered
and the resin was washed with methanol. The filtrate
and washings were combined and concentrated under
vacuum to yield a white solid (20.1 mg, 88%). 1H
NMR (CD3OD) d 1.26–1.45 (m, 12H), 1.50 (m, 2H),
1.69 (m, 2H), 1.79 (m, 2H), 2.06 (s, 3H), 3.45 (m, 1H),
3.73 (m, 2H), 3.82 (m, 1H), 3.91–4.05 (m, 6H), 5.64
(br s, 1H), 6.92 (m, 3H), 7.25 (t, J = 8.3 Hz, 2H), 8.04
(br s, 3H), 8.56 (m, 1H), 8.96 (br s, 2H). 13C NMR
(methanol) d 19.2, 22.7, 26.6, 27.0, 30.2, 30.3, 30.5,
31.4, 55.1, 62.5, 67.8, 68.7, 71.73, 72.7, 75.0, 96.5,
115.4, 121.3, 128.0, 130.4, 143.7, 146.6, 160.4, 174.0.
31P NMR (methanol) d ꢀ11.7 (J = 16.4 Hz), ꢀ9.6
(J = 16.4 Hz). MS (ꢀTOF) m/z 626 [MꢀH]ꢀ; HRMS
[MꢀH]ꢀ, found: 626.2126. C25H42NO13P2 requires
m/z, 626.2131.
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Acknowledgements
The authors thank Bernd Keller for the mass spectro-
metric analyses, and Eddie Khoo for galactosyltransfer-
16. Jann, K.; Pillat, M.; Weisgerber, C.; Shibaev, V. N.;
Torgov, V. I. Eur. J. Biochem. 1985, 151, 393–397.