UDP-N-trifluoroacetylglucosamine as an alternative substrate in N- acetylglucosaminyltransferase reactions

Article abstract of DOI:10.1016/S0008-6215(97)10033-7

The synthesis of UDP-N-trifluoroacetylglucosamine [uridine 5'-(2- trifluoroacetamido-2-deoxy-α-D-glucopyranosyl diphosphate, UDP-GlcNAc-F₃] is reported. The compound is found to serve as a substrate for the 'core-2' GlcNAc transferase (EC 2.4.1

Full text of DOI:10.1016/S0008-6215(97)10033-7

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Carbohydrate Research 306 1998 127–136
UDP-N-trifluoroacetylglucosamine as an alternative
substrate in N-acetylglucosaminyltransferase
reactions
a
b
b
Rafael F. Sala , Shawna L. MacKinnon , Monica M. Palcic ,
a,)
Martin E. Tanner
a
Department of Chemistry, UniÕersity of British Columbia, VancouÕer, British Columbia, Canada
V6T 1Z1
b
Department of Chemistry, UniÕersity of Alberta, Edmonton, Alberta, Canada T6G 2G2
Received 11 September 1997; accepted 14 September 1997
Abstract
The synthesis of UDP-N-trifluoroacetylglucosamine uridine 5 - 2-trifluoroacetamido-2-de-
X
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x
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oxy-a-D-glucopyranosyl diphosphate, UDP-GlcNAc-F3 is reported. The compound is found
to serve as a substrate for the ‘core-2’ GlcNAc transferase EC 2.4.1.102 that is involved in
the biosynthesis of O-linked glycoproteins and for the GlcNAcT-V transferase EC 2.4.1.155
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.
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.
that is a key biosynthetic enzyme controlling the branching pattern of cell surface complex
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.
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Asn-linked oligosaccharides. The trisaccharide b-D-Gal p- 1™3 - b-D-GlcpNAc-F3 1™6 -
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.
x
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.
a-D-GalpNAc-OR R s CH2 8CO2 Me was prepared from b-D-Galp- 1 ™ 3 -a-D-
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GalpNAc-OR using the ‘core-2’ GlcNAc transferase. The tetrasaccharide b-D-GlcpNAc- 1
.
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.
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x
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.
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™2 - b-D-GlcpNAc-F3- 1™6 -a-D-Manp- 1™6 -b-D-Glcp-OR Rs CH2 7CH3 was
.
.
.
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prepared from b-D-GlcpNAc- 1™2 -a-D-Manp- 1™6 -b-D-Glcp-OR Rs CH2 7CH3
using the GlcNAcT-V transferase. Removal of the trifluoroacetyl group was achieved under
mild basic conditions to give the corresponding glucosamine containing tetrasaccharide.
These examples demonstrate the feasibility of introducing masked forms of glucosamine
residues into oligosaccharides using GlcNAc-specific transferases. The requirement for the
trifluoroacetamido group as a specific recognition element was evident in the observation that
neither UDP-glucosamine nor UDP-glucose served as a donor substrates for the ‘core-2’
GlcNAc transferase. q 1998 Elsevier Science Ltd. All rights reserved.
Keywords: N-Acetylglucosaminyltransferase; UDP-sugar analog; Oligosaccharide synthesis
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1. Introduction
cell surfaces 1 . Compounds that interfere with these
recognition processes are candidates as new drug
targets in a variety of diseases. For this reason,
strategies for the synthesis of oligosaccharides have
received a great deal of attention in recent years
Cellular recognition in nature is often mediated via
binding events involving complex carbohydrates on
) Corresponding authors.
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2–7 . One promising method involves the use of
0008-6215r98r$19.00 q 1998 Elsevier Science Ltd All rights reserved.

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128
R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
transferase enzymes that employ sugar-nucleotides as
common components of natural glycoconjugates that
are generated by a large class of transferases that
utilize UDP-GlcNAc as a donor. The first of such
enzymes to be discussed is the UDP-D-GlcpNAc:b-
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x
the carbohydrate donors 8–10 . The main advantage
to this approach is that the enzymes are highly re-
giospecific and stereospecific with regard to directing
the activated donor carbohydrate to the appropriate
hydroxyl group of the acceptor, thereby eliminating
the need for multiple protection–deprotection steps
required in non-enzymatic syntheses. One of the
drawbacks of this strategy, is that the specificity of
these enzymes precludes the use of alternative donors
that differ significantly from the natural donors. This
makes the enzymatic synthesis of some unnatural
oligosaccharides very difficult.
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.
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.
D-Gal p- 1™3 -a-D-GalpNAc GlcNAc to GalNAc
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.
b- 1™6 -GlcNAc transferase that is also referred to
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as the ‘core-2’-GlcNAc transferase GlcNAcT, EC
.
2.4.1.102 and is involved in the biosynthesis of
O-linked glycoproteins in mammals 11,12 . This en-
zyme transfers a GlcNAc residue to the O-6 of
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x
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.
GalNAc in the disaccharide b-D-Galp- 1™3 -a-D-
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GalpNAc-OR –OR can be either a hydroxyl residue
of a serine or threonine in a protein or a simple aryl
Ž
.
.
N-Acetylglucosamine GlcNAc residues are very
or alkyl aglycon . In this paper, we describe the
Scheme 1.

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R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
129
Scheme 2.
synthesis of UDP-N-trifluoroacetylglucosamine
demonstrated the feasibility of this method by the
enzymatic synthesis of the tetrasaccharide 12 fol-
lowed by the removal of the trifluoroacetyl group to
generate the glucosamine containing tetrasaccharide
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.
UDP-GlcNAc-F3 and demonstrate that it can serve
as an unnatural donor in the GlcNAcT reaction
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.
Scheme 1 . We also describe its recognition by the
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.
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.
UDP-D-GlcpNAc:a-D-Manp- 1 ™ 6 -b-D-GlcpNAc
13 Scheme 2 .
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.
transferase V GlcNAcT-V, EC 2.4.1.155 . This en-
zyme transfers a GlcNAc residue to the O-6 of Man
Ž
.
in the trisaccharide b-D-GlcpNAc- 1 ™ 2 -a-D-
2. Results and discussion
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.
Ž
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.
. Ž
.
Manp- 1™6 -b-D-Glcp-OR Rs CH2 7CH3 11
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.
Scheme 2 and controls the branching pattern of
The synthesis of UDP-N-trifluoroacetylglucosa-
mine began with the preparation of the glycosyl
bromide 1 from an arb mixture of 1,3,4,5-tetra-O-
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asparagine-linked oligosaccharides 13,14 .
The ease of removal of the trifluoroacetyl group
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under relatively mild conditions 15–17 effectively
permits the introduction of glucosamine residues into
oligosaccharides that normally contain GlcNAc. This
could lead to the preparation of a variety of unnatural
compounds with potential bioactive properties as well
as provide a nucleophilic functionality to which other
molecules can be appended using reductive amination
chemistry. The direct transfer of UDP-glucosamine
was not expected to be feasible because the ac-
etamido functionality of the natural substrate donor
was being replaced by a charged ammonium group.
Indeed, we have shown that the ‘core-2’-GlcNAcT
accepts neither UDP-glucose nor UDP-glucosamine
as a donor substrate. This indicates the trifluoroac-
etamido group served as a necessary recognition ele-
ment in the transferase reaction. In addition, we have
acetyl-2-deoxy-2-trifluoroacetamido-D-glucose 18
Ž
.
Scheme 1 . Treatment of 1 with silver oxide in 10:1
acetonitrile:water gave 3,4,6-tri-O-acetyl-2-deoxy-2-
trifluoroacetamido-a-D-glucose 4 in 57% yield. The
relatively low yield was due to the formation of the
peracetylated glucal 2 and oxazoline 3 as by-products
in yields of 7 and 12%, respectively. These products
have been obtained previously upon treatment of 1
with MeOH under Koenigs–Knorr conditions 19 .
Compound 4 was phosphorylated by treatment with
diphenylchlorophosphate and 4-N, N-dimethyl-
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x
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.
aminopyridine DMAP in CH2Cl2 at y10 8C. These
conditions are known to produce anomerically en-
riched a-glycosyl phosphate diphenyl esters for a
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number of acetylated hexopyranoses 20 . Indeed the
a-diphenylphosphate 5 was obtained in 52% yield

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130
R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
Table 1
1
a
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x
Selected H NMR data for compounds 9 25 and 10
a Numbers in parentheses give coupling constants in Hz.
and no b-anomer was isolated upon purification of
phates. Cleavage of the O-acetyl groups in the pres-
ence of the base-sensitive trifluoroacetamido group
was accomplished by careful treatment with NaOMe
the reaction product by conventional chromatogra-
1
Ž
.
phy. A by-product ;15% , whose H NMR and
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.
mass spectra were consistent with that expected for
in MeOH to give 7 68% .
Ž
the corresponding a-glycosyl chloride 3,4,6-tri-O-
In further work, a more efficient method for the
preparation of compound 7 was developed. Treatment
of the peracetylated b-N-trifluoroacetylglucosamine
acetyl-2-deoxy 2-triflouroacetamido-a-D-gluco-
.
pyranosyl chloride , was also obtained. The forma-
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tion of glycosyl chlorides has been observed during
the diphenylphosphorylation of sugars when n-butyl-
18 with neat phosphoric acid at 60 8C the MacDon-
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ald procedure 23 , followed by deacetylation with
sodium methoxide in MeOH gave a 27% yield of 7.
The GlcNAc-F3 a-phosphate 7 was coupled to
UMP using standard Khorana conditions with UMP-
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lithium was used as the base 21 . The phenyl groups
in the phosphate triester were removed by hydrogena-
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tion over platinum oxide catalyst 22 to give 6. Low
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.
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yields ;30% were observed, presumably due to
morpholidate in dry pyridine 24 . The product UDP-
acid catalyzed decomposition of the glycosyl phos-
GlcNAc-F3 8, was isolated as its dilithium salt in
Table 2
1
a
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Selected H NMR data for compounds 11 26 , 12 and 13
a Numbers in parentheses give coupling constants in Hz.

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R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
131
44% yield following purification by anion exchange
3. Experimental
and size exclusion chromatographies.
‘Core-2’-GlcNAc-transferase was obtained from
mouse kidney acetone powder by extraction and di-
gestion with trypsin followed by a single step affinity
chromatography on UDP-hexanolamine Sepharose
General methods.—Melting points were recorded
on a Reichert melting point apparatus and were un-
corrected. Optical rotations were measured with a
Perkin–Elmer 241 MC polarimeter. TLC was per-
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.
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.
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.
see Section 3 . Incubation of b-D-Galp- 1™3 -a-D-
formed on Silica Gel 60F254 E. Merck, Darmstadt
with detection by charring after spraying with 5%
H2SO4 or by spraying with a solution containing
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GalpNAc-OR 9 25 with a portion of this enzyme
extract, in the presence of the donor UDP-GlcNAc-F3,
resulted in the formation of the trisaccharide product
10 in a 62% isolated yield.
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.
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.
H2SO4 31 mL ammonium molybdate 21 g , and
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.
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.
cerium sulfate 1 g in water 500 mL and then
Ž .
The structure of 10 was assigned on the basis of
the 1H NMR data showed in Table 1. The new
b-glycosidic linkage was established by the H-1 dou-
heating 110 8C for 5 min . Flash column chromatog-
Ž
raphy was performed using Silica Gel 60 230–400
mesh . All reagents were commercially available un-
less otherwise stated. The disaccharide b-D-Galp- 1
.
Ž
.
Ž
9
blet J1,2 8.5 Hz at d 4.63 ppm. This was in
agreement with a previous assignment of the N-
.
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.
x
Ž .
™ 3 -a-D-GalpNAc-OR R s CH2 8CO2 Me
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x
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.
.
acetylglucosamine analog of this trisaccharide 11 .
High resolution electrospray ionization mass spec-
and the trisaccharide b-D-GlcpNAc- 1 ™ 2 -a-D-
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.
w
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x
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.
Manp- 1™6 -b-D-Glcp-OR Rs CH2 7CH3 11
Ž
.
trometry HRESIMS confirmed the composition and
were respectively synthesized as previously described
Ž
w
xq
.
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size of 10 MqNa s833.3173 . The presence of
the N-trifluoroacetyl group was confirmed by the
singlet at d y76.1 ppm in the 19F NMR.
The trifluoro donor was also incubated with a
portion of cloned GlcNAcT-V transferase and accep-
tor 11. The resultant tetrasaccharide 12 was obtained
25,27 . UDP-GlcNAc was obtained from Sigma St.
3
.
w
Ž .
x
Ž
Louis, MO . UDP- 6- H N GlcNAc specific activ-
.
ity, 60 Cirmmol was from American Radiolabeled
Chemicals St. Louis, MO . Liquid scintillation
Ž
.
counting was performed with a Beckman LS-1801
Ž
.
instrument using Ecolite q cocktail from ICN Ra-
1
Ž .
diochemicals Costa Mesa, CA . Iatrobeads refers to
in a 56% isolated yield. From the H NMR data
Ž .
presented in Table 2, the new b-glycosidic linkage of
a beaded Silica Gel No. 6RS-8060 manufactured by
Ž
.
Ž
.
12 was confirmed by the H-1 doublet J1,2 8.4 Hz at
d 4.64 ppm. The composition of 12 was confirmed
Iatron Laboratories Tokyo . Ion exchange and gel
chromatographic supports were from BioRad Labora-
by the MqNa q peak at 937.3622 in the HRESIMS
spectrum. The presence of the N-trifluoroacetyl group
was confirmed by the singlet at d y75.5 in the 19F
NMR.
tories Missisauga, ON . Pyridine, acetonitrile,
CH2Cl2, and Et3N were distilled under N2 from
calcium hydride. Methanol was distilled under N2
from magnesium methoxide. All reactions performed
w
x
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.
1
The preparation of 10 and 12 clearly demonstrates
the feasibility of using enzymes to introduce a pro-
tected form of glucosamine into an oligosaccharide.
In order to show that the trifluoroacetamido group
was a specific recognition component in the enzy-
matic reaction, the alternative substrates UDP-gluco-
in organic solvents were carried out under argon. H
Ž
NMR spectra were recorded at 400 MHz Bruker
.
Ž
.
WH-400 or at 600 MHz Varian spectrometer in
solutions in deuterium oxide with acetone as the
Ž
.
Ž
external standard d 2.23 ppm and at 30 8C for
compounds 9 and 13 . 13C NMR spectra were
.
recorded at 75.43 MHz Varian XL-300 , 31P NMR
Ž
Ž
.
samine bearing a small positively charged group at
spectra were recorded at 81.0 MHz and 19F NMR at
.
Ž
C-2 and UDP-glucose bearing a small neutral group
at C-2 were tested as substrates. In either case, no
.
Ž
.
188.0 MHz with proton decoupling Bruker AC-200 .
The 31P chemical shifts are expressed relative to
detectable transfer was observed after eight days. The
presence of an acetamido moiety at C-2 was therefore
required for the transfer to occur; as one might expect
for these highly specific transferases.
external phosphoric acid 0.00 ppm . The 19F chemi-
cal shifts are expressed relative to CFCl3 for solu-
Ž
.
Ž
.
tions in CDCl3 0.00 ppm or AcOH for solutions in
Ž
.
Deprotection of tetrasaccharide 12 was performed
deuterium oxide y76.53 ppm . Signals upfield of
CFCl3 were assigned negative values. Desorption
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using a previously published procedure 17 which
employed potassium carbonate in aqueous MeOH.
These mild conditions allowed the isolation of the
desired tetrasaccharide 13 after purification by re-
versed-phase chromatography.
Ž
.
chemical ionization DCI mass spectra were recorded
on a Delsi Nermag R10-10C mass spectrometer using
ammonia as the chemical ionization gas. High resolu-
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.
tion liquid secondary ion HRLSI mass spectra were

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132
R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
.
Ž
recorded on a Kratos Concept II HQ mass spectrome-
JC,F 37.5 Hz, COCF3 , 115.53 q, JC,F 286.4 Hz,
Ž
.
.
Ž
.
Ž
.
ter. High resolution electrospray ionization HRESI
CF3 , 141.07 C-1 128.34 C-2 73.19, 66.40, 66.32,
60.92 C-3, C-4, C-5, C-6 20.82, 20.79, 20.75 CH3,
Ž
.
.
Ž
mass spectrometry of 10 was obtained using a Micro-
mass ZabSpec Hybrid Sector-TOF spectrometer us-
19
.
Ž
3Ac ; F NMR CDCl3 : d y76.4; DCIMS mrz
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.
Ž
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.
M q NH4 , 100% . Anal. Calcd. for
ing a 1% solution of ArOH in water:MeOH 1:1 as
401
Ž
.
liquid carrier. Millex-GV 0.22 mm filters units were
from Millipore, and C18 Sep-Pak sample-preparation
cartridges were from Waters Associates. Microanaly-
sis were carried out by Mr. Peter Borda in the
microanalytical laboratory at UBC.
C14H16O8NF3: C, 43.87; H, 4.21; N, 3.65. Found: C,
43.58; H, 4.28; N, 3.90.
2-Trifluoromethyl- 3,4,6-tri-O-acetyl-1,2-dideoxy-a-
D-glucopyrano 2,1-D -D -oxazo line 3 .—Obtained
(
2
)[
]
Ž .
22
w x
.
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as a clear oil as described above; a q3.68 c 2.2,
D
.
Ž
w
.
x
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3,4,6-Tri-O-acetyl-2-deoxy-2-trifluoroacetamido-a-
CHCl3 , lit. q5.58 c 2, CHCl3 18 ; Rf 0.32 7:3
1
Ž .
.
Ž
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D-glucose 4 .—3,4,6-Tri-O-acetyl-2-deoxy-2-trifluo-
benzene–ether ; H NMR CDCl3 : d 6.27 d, 1 H,
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.
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roacetamido-a-D-glucopyranosyl bromide 1 1.08 g,
2.3 mmol was dissolved in 10 mL of dry acetonitrile
J1,2 7.6 Hz, H-1 , 5.31 dd, 1 H, J2,3 sJ3,4 2.4 Hz,
H-3 , 4.93 ddd, 1 H, J2,4 1.3, J4,5 8.2 Hz, H-4 , 4.36
.
.
Ž
.
.
X
Ž
.
Ž
.
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Ž
.
Ž
containing silver oxide 0.50 g , water 1 mL was
added, and the mixture was stirred for 2 h in the dark.
The resulting suspension was filtered, concentrated,
and separated on a silica gel column using 1:3
EtOAc–hexanes. Fractions of higher mobility were
segregated for further purification. Later fractions
were pooled and evaporated to give 4 as a white
m, 1 H, H-2 4.28–4.15 m, 2 H, H-6, H-6 , 3.63
m, 1 H, H-5 , 2.10, 2.08, 2.06 3s, each 3, 3Ac ; 13C
.
Ž
.
Ž
.
Ž
Ž
NMR CDCl3 : d 170.60, 169.33, 169.02 COCH3,
3Ac , 156.22 q, JC,F 41.0 Hz, CNCF3 , 115.90 q,
JC,F 273.0 Hz, CF3 , 102.22 C-1 , 68.77, 68.60,
67.51, 63.97, 63.20 C-2, C-3, C-4, C-5, C-6 20.77,
.
.
Ž
.
Ž
.
Ž
.
19
Ž
.
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.
20.63, 20.62 CH3, 3Ac ; F NMR CDCl3 : d
Ž
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xq
.
Ž
.
solid. 0.53 g, 57% : mp 174 8C, lit. 173–174 8C
y71.3; DCIMS mrz 401 MqNH4 , 100% . Anal.
Calcd. for C14H16O8NF3: C, 43.87; H, 4.21; N, 3.65.
Found: C, 44.11; H, 4.19; N, 3.57.
Diphenyl 3, 4, 6 - tri - O - acetyl - 2 - deoxy - 2 -
trifluoroacetamido- a - D - glucopyranosyl phosphate
5 .—A mixture of 4 1.24 g, 3.1 mmol and DMAP
0.87 g, 7.1 mmol in 25 mL of dry CH2Cl2 was
stirred at room temperature for 10 min and then
cooled to y10 8C. Diphenylchlorophosphate 1.4
mL, 7.1 mmol was added dropwise and the solution
was stirred at this temperature for 2 h. The mixture
was then diluted with CH2Cl2 30 mL and washed
with ice-cold water, ice-cold 0.5 M HCl and saturated
NaHCO3. After drying the organic phase over
MgSO4, it was concentrated to a small residue which
was purified by column chromatography using 2:3
22
w
x w x
Ž
.
Ž
18 ; a q20.08 c 0.5, CHCl3 , lit. q25.08 c 1,
D
w
CHCl3 18 ; Rf 0.16 1:1 EtOAc–hexanes ; 1H
.
x
Ž
.
Ž
.
Ž
(
NMR CDCl3 : d 6.64 br d, 1 H, J2,NH 9.1 Hz,
.
Ž
.
)
NH , 5.34 dd, 1 H, J2,3 9.9, J3,4 9.6 Hz, H-3 , 5.33
d, 1 H, J1,2 3.3 Hz, H-1 , 5.13 dd, 1 H, J4,5 9.6 Hz,
Ž
.
Ž
Ž .
Ž
Ž
.
.
Ž
.
Ž
.
H-4 , 4.29 ddd, H-2 4.25–4.08 m, 3 H, H-5, H-6,
H-6 2.08, 2.03, 2.00 3s, each 3 H, 3Ac ; 13C NMR
X
.
Ž
.
Ž
.
Ž
Ž
.
Ž
CDCl3 : d 171.40, 171.05, 169.48 COCH3, 3Ac ,
157.30, q, COCF3, JC,F 37.7 Hz , 115.53 q, CF3,
JC,F 287.7 Hz , 90.87 C-1 , 70.46 C-5 , 67.82 C-3 ,
.
Ž
Ž
.
.
Ž
.
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
67.71 C-4 , 61.89 C-6 , 52.76 C-2 , 20.77, 20.60,
19
Ž
.
Ž
.
20.48 CH3, 3Ac ; F NMR CDCl3 : d y76.6;
Ž
w
xq
.
DCIMS mrz 419 MqNH4 , 100% . Anal. Calcd.
for C14H18O9NF3: C, 41.90; H, 4.52; N, 3.49. Found:
C, 42.15; H, 4.32; N, 3.29.
3, 4, 6 - Tri - O - acetyl - 5 - anhydro - 2 - deoxy - 2 -
EtOAc–hexanes to afford the title compound as white
20
Ž .
Ž
.
w x
Ž
trifluoroacetamido-D-arabino-hex-1-enitol 2 .—Ob-
crystals 1.02 g, 52% : mp 65 8C; a q60.08 c 1,
D
1
.
Ž
.
tained in 7% yield as a by-product of the chromato-
graphic purification of 4. Further purification by flash
chromatography using 9:1 benzene–ether was re-
CHCl3 ; Rf 0.27 2:3 EtOAc–hexanes ; H NMR
Ž
.
Ž
.
Ž
CDCl3 : d 7.40–7.08 m, 10 H, 2Ph , 6.88 br d, 1
.
Ž
H, J2,NH 8.7 Hz, NH , 6.01 dd, 1 H, JH,P 6.0 Hz,
J1,2 3.2 Hz, H-1 , 5.32 dd, 1 H, J3,4 9.9, J2,3 11.3
.
Ž
quired in order to separate this compound from the
22
w x
Ž
.
.
Ž
.
Ž
oxazoline 3. Clear oil, a
y46.58 c 1, CHCl3 ,
Hz, H-3 , 5.20 dd, 1 H, J4,5 9.7 Hz, H-4 , 4.41 ddd,
D
X
Ž
.
w
x
Ž
.
Ž
.
lit. y458 c 1, CHCl3 18 ; Rf 0.25 7:3 benzene–
1 H, H-2 , 4.20–3.88 m, 3 H, H-5, H-6, H-6 , 2.03,
1
13
.
Ž
Ž
.
.
Ž
Ž
.
Ž
.
Ž
.
ether ; H NMR CDCl3 : d 8.19 br s, 1 H, NH ,
2.01, 1.99 3s, each 3 H, 3Ac ; C NMR CDCl3 : d
170.92, 170.54, 169.21 COCH3, 3Ac , 157.77 q,
JC,F 38.4 Hz, COCF3 , 129.96, 129.89, 125.85,
125.80, 119.91, 119.85, 119.82, 119.76 2Ph , 115.45
q, JC,F 287.6 Hz, CF3 , 96.12 d, JC,P 7 Hz, C-1 ,
70.03 C-5 , 69.39 C-3 , 67.20 C-4 , 60.91 C-6 ,
Ž
.
Ž
7.69 s, 1 H, H-1 , 5.22 dd, 1 H, J3,4 sJ4,5 3.6 Hz,
H-4 , 5.15 dd, 1 H, J3,5 1.1 Hz, H-3 , 4.45–4.40
ddd, 1 H, J5,6 7.9, J5,6 4.1 Hz, H-5 , 4.35 dd, 1 H,
Jgem 11.9 Hz, H-6 , 4.22 dd, 1 H, H-6 2.12, 2.10,
2.08 3s, each 3 H, 3Ac ; C NMR CDCl3 : d
172.66, 170.48, 169.54 COCH3, 3Ac , 154.73 q,
.
Ž
.
.
Ž
.
Ž
X
Ž
.
X
.
Ž
.
Ž
.
Ž
.
.
13
Ž
.
Ž
.
Ž
Ž
.
Ž
.
Ž
.
Ž
2
Ž
.
Ž
.
52.48 d, JC,P 8.4 Hz, C-2 , 20.53, 20.45, 20.24

(
)
R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
133
19
CH3, 3Ac ; F NMR CDCl3 : d y76.1; 31P NMR
2
60.50 C-6 , 54.65 d, JC,P 8.0 Hz, C-2 ; 19F NMR
Ž
Ž
.
Ž
.
Ž
.
Ž
.
31
.
Ž
w
xq
Ž
.
Ž
CDCl3 : d y14.39; DCIMS mrz 651 MqNH4
,
deuterium oxide : d y75.7; P NMR deuterium
.
.
Ž
100% . Anal. Calcd. for C26H27O12 NPF3: C, 49.30;
H, 4.30; N, 2.21. Found: C, 49.08; H, 4.41; N, 2.22.
Monotriethylammonium salt of 3,4,6-tri-O-acetyl-2-
deoxy - 2 - trifluoroacetamido - a - D - glucopyranosyl
oxide : d y1.33; HRLSIMS mrz 354.0189 calcd.
.
for C8H12O9NPF3: 354.0202 .
Preparation of
7
by the MacDonald
Ž
procedure.—Crystalline phosphoric acid 500 mg,
5.1 mmol was dried in vacuo over phosphorus pen-
toxide for 12 h. Solid 1,3,4,6-tetra-O-acetyl-2-deoxy-
2-trifluoroacetamido-b-D-glucopyranose 280 mg,
Ž .
Ž
.
.
phosphate 6 .—A solution of 5 250 mg, 0.4 mmol
Ž
.
Ž
in 1:1 EtOAc–MeOH 10 mL was hydrogenated 4
atm in the presence of platinum oxide catalyst 25
mg . After 4 h, the catalyst was removed by filtration
and Et3N 2 mL was added to the filtrate. Evapora-
tion of the solvent afforded a syrup that was purified
by preparative thin layer chromatography using
10:10:1 CHCl3–MeOH–water to give the free acid
form of 6 as a white hygroscopic solid 60 mg, 32% ,
this was converted to the monotriethylammonium salt
by passing through a small column 3 cm of Dowex
50W-X8 triethylammonium form : Rf 0.6 10:10:1
CHCl3–MeOH–water ; H NMR deuterium oxide :
d 5.53 dd, 1 H, J1,P 7.1 Hz, J1,2 3.1 Hz, H-1 , 5.42
.
Ž
Ž
.
.
0.63 mmol was added and the mixture was heated at
Ž
.
60 8C in vacuo. The formation of a melt and the
evolution of CH3COOH vapors was observed. After
2 h, heating was ceased and the resulting dark black
mixture was dissolved in anhydrous THF 5 mL .
The solution was cooled to 0 8C and concentrated
Ž
.
Ž
.
Ž
.
ammonium hydroxide 0.5 mL was added. The pre-
cipitate of ammonium phosphate was filtered off and
Ž
.
Ž
.
Ž
Ž
Ž
.
washed with THF 20 mL . The combined filtrate and
washings were evaporated to give a syrupy residue
that was applied to two plates of silica gel 2 mm
1
.
.
Ž
.
Ž
Ž
.
Ž
.
dd, 1 H, J2,3 10.4, J3,4 9.8 Hz, H-3 , 5.13 dd, 1 H,
thickness and eluted with 10:10:1 CHCl3–MeOH–
.
Ž
J4,5 9.8 Hz, H-4 , 4.50–4.14 m, 4 H, H-2, H-5, H-6,
water. Isolation of the corresponding zone gave crude
6 as a light brown solid. This material was O-
deacetylated and purified as described above to give
H-6 2.14, 2.10, 2.02 3s, each 3 H, 3Ac ; 13C NMR
X
.
Ž
.
Ž
Ž
.
deuterium oxide :
COCH3, 3Ac , 159.64 q, JC,F 38.4 Hz, COCF3 ,
115.86 q, JC,F 284.5 Hz, CF3 , 92.79 d, JC,P 5.5
Hz, C-1 , 71.17 C-5 , 68.63 C-3 , 68.31 C-4 ,
d
174.07, 173.43, 173.14
.
Ž
.
Ž
.
7 73 mg, 27% .
Dilithium uridine 5 - 2-deoxy-2-trifluoroacetamido-
X
Ž
.
Ž
(
.
Ž
.
Ž
.
Ž
.
)
Ž .
a-D-glucopyranosyl diphosphate 8 .—A solution of
2
4-morpholine N, NX-dicyclohexylcarboxamidinium
Ž
.
Ž
.
62.05 C-6 , 52.57 d, JC,P 8.4 Hz, C-2 , 20.41,
19
X
Ž
.
Ž
Ž
.
.
20.37, 20.20 CH3, 3Ac ; F NMR deuterium ox-
uridine 5 -phosphomorpholidate 210 mg, 0.31 mmol
31
.
Ž
.
Ž
ide : d y75.4; P NMR deuterium oxide : d
in anhydrous pyridine 5–7 mL was concentrated to
dryness in vacuo. The process of dissolution in fresh
dry pyridine and concentration was repeated twice,
dry air being admitted into the flask after each con-
centration. Separately, an aqueous solution of the
Ž
y1.07; HRLSIMS mrz: 480.0522 calcd. for
.
C14H19O12 NPF3, 480.0519 .
Monopyridinium salt of 2 - deoxy - 2 - trifluoroac-
etamido-a-D-glucopyranosyl phosphate 7 .—Com-
pound 6 180 mg, 0.4 mmol was dissolved in 3 mL
of dry MeOH. The solution was cooled to 0 8C and
0.50 mL of NaOMe in MeOH 1 M was added.
After stirring for 20 min, the reaction was quenched
using Dowex 50W-X8 resin pyridinium form, previ-
ously washed with MeOH . The resin was filtered off
and the methanolic solution was evaporated. The
residue was dissolved in a small amount of water,
passed through a small column 3 cm of Dowex
50W-X8 pyridinium form resin and lyophilized to
give 7 as the monopyridinium salt 120 mg, 68% :
H NMR deuterium oxide : d 5.48 dd, 1 H, J1,P
7.2, J1,2 3.2 Hz, H-1 , 4.04 dd, 1 H, J2,3 10.6 Hz,
H-2 , 3.93–3.72 m, 4 H, H-3, H-5, H-6, H-6 , 3.53
Ž .
Ž
.
Ž
monopyridinium salt of sugar phosphate 7 100 mg,
0.22 mmol was treated similarly. A solution of the
.
Ž
.
residue in pyridine was added to the dry phospho-
morpholidate described above. This solution was con-
centrated in vacuo and redissolved in ;5 mL of dry
pyridine. The resulting solution was sealed, kept at
room temperature for 5 days and then concentrated.
The residual syrup was redissolved in 10 mL of
water, applied onto a column of Dowex 1X4-200
Ž
.
Ž
.
Ž
.
Ž
.
chloride form, strongly basic , and eluted with a
Ž
.
Ž
.
gradient of lithium chloride 0.1–1 M at pH 3.5. The
sugar nucleotide containing fractions were pooled,
concentrated to a small volume, and desalted by
passing through a column of Bio-Gel P-2 column
2.5=45 cm eluted with water. The appropriate
fractions were pooled and lyophilized to give 67 mg
1
Ž
.
Ž
.
Ž
X
.
Ž
.
13
Ž
.
Ž
Ž
.
dd, 1 H, J3,4 sJ4,5 9.6 Hz, H-4 ; C NMR de-
.
Ž
.
uterium oxide : d 159.61 q, JC,F 37.6 Hz, COCF3 ,
1
Ž
.
Ž
Ž
Ž
.
Ž
.
115.96 q, JC,F 284.7 Hz, CF3 , 93.12 d, JC,P 5.8
Hz, C-1 , 73.04 C-5 , 70.20 C-3 , 69.80 C-4 ,
44% of 8 as its dilithium salt dihydrate : H NMR
deuterium oxide : d 7.92 d, 1 H, J5,6 8.1 Hz, H-6 ,
.
Ž
.
Ž
.
Ž
.
.
Ž
.

(
)
134
R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
X
Ž
.
Ž
.
X
X
Ž
.
5.93 d, 1 H, J1 ,2 4.9 Hz, H-1 , 5.91 d, 1 H, H-5 ,
;119 mUr6 mL were used for preparative glyco-
sylations.
Radiochemical assay for GlcNAcT.—GlcNAcT
activity was measured using a modified version of a
Y
Ž
.
Y
Y
Y
5.57 dd, 1 H, J1 X,P 7.1, J1 ,2 3.3 Hz, H-1 , 4.34–
4.28 m, 2 H, H-2 , H-3 , 4.25–4.12 m, 3 H, H-4 ,
H-5 a, H-5 b , 3.95–3.72 m, 4 H, H-2 , H-5 , H-6 a,
H-6 b, H-3 , 3.54 dd, 1 H, J3 ,4 sJ4 ,5 9.6 Hz,
H-4 ; C NMR deuterium oxide : d 160.4 q, JC,F
X
Y
Ž
.
Ž
X
X
Y
Y
Y
.
Ž
Y
Y
.
Ž
Y
Y
Y
Y
w x
previously published procedure 11 . Each assay tube
contained: 1.6 mL of 500 mM GlcNAc solution, 1.0
mL of 10 mM UDP-GlcNAc, 1.5 mL of a 3.6 mM
solution of acceptor 1, 0.5 mL of UDP-6- H GlcNAc
Y
13
.
Ž
.
.
Ž
Ž
.
Ž
.
37.7 Hz, COCF3 , 167.2 C-4 , 152.7 C-2 , 142.5
3
Ž
.
Ž
Ž
.
Ž
.
w
x
C-6 , 116.6 q, JC,F 284.4 Hz, CF3 , 103.5 C-5 ,
94.6 d, JC,P 5.9 Hz, C-1 , 89.3 C-1 , 84.1 d, JC,P
9.2 Hz, C-4 , 74.7 C-3 , 73.9 C-5 , 71.2 C-2 ,
Y
X
.
Ž
Ž
.
.
Ž
Ž
Ž
.
Ž
;40 000 dpm , and 7.5 mL of HEPES buffer 0.2
X
X
Y
X
.
Ž
.
.
.
.
mM HEPES, pH 6.5; 2% Triton X-100 . To this total
of 12.1 mL was added 7.9 mL of crude or diluted
enzyme sample. The mixture was incubated for 90
min at 37 8C, diluted with approximately 3 mL of
water and loaded onto a C18 Sep-Pak cartridge which
had been pre-equilibrated with water. The cartridge
was washed with 50 mL of water and the product
eluted into a scintillation vial with 4 mL of MeOH.
The radioactivity of the eluants were quantitated by
liquid scintillation counting using 10 mL of scintilla-
tion cocktail.
Y
Y
X
Ž
.
Ž
70.5, 70.3 C-3 , C-4 , 65.8 d, JC,P 5.5 Hz, C-5 ,
61.1 C-6 , 55.3 d, JC 8.8 Hz, C-2 ; 19F NMR
Y
2
Y
Ž
.
Ž
.
,
P
31
Ž
.
Ž
deuterium oxide : d y74.9; P NMR deuterium
.
Ž
.
Ž
oxide : d y11.01 d, J 20.3 Hz, Pb , y12.90 d, J
.
Ž
20.3 Hz, Pa ; HRLSIMS mrz 674.07928 calcd. for
.
C17H23O17N3P2F3Li2, 674.0775 . Anal. Calcd. for
C17H22 N3O17P2 F3Li2 P2H2O: C, 28.79; H, 3.70; N,
5.92. Found: C, 28.68; H, 3.79; N, 5.71.
Preparation of the glycosyltransferase ‘core - 2’ -
(
(
)
GlcNAcT UDP-D-GlcpNAc:b-D-Gal- 1™3 -a-D-
(
)
(
(
)
GalpNAc - b - 1™6 - GlcNAc transferase EC
Enzymatic conÕersion of b-Gal- 1™3 -a-GalNAc-
))
Ž
(
.
(
) [ )]
(
2.4.1.102 .—Mouse kidney acetone powder 1 g,
OR 9 into b-Gal- 1™3 - b-GlcpNAc-F3 1™6 -
.
Ž
Ž .
a - GalpNAc - OR 10 .—UDP-N-trifluoroacetylglu-
Sigma was suspended in 25 mL NaOAc buffer A:
0.1 M NaOAc, pH 6.0; 0.2 M NaCl, 0.01 M EDTA
.
Ž . Ž
cosamine 0.46 mg, 0.64 mmol , 9 0.8 mg, 1.45
. Ž
containing 12 mg trypsin and stirred vigorously
overnight at 4 8C. The slurry was centrifuged at
6000=g for 20 min at 4 8C. Soybean trypsin in-
hibitor 5 mg was added to the supernatant which
was stored at 4 8C. The pellet was resuspended in 25
mL of buffer A containing 12 mg of trypsin and
stirred vigorously overnight at 4 8C. This preparation
was centrifuged at 6000=g for 20 min at 4 8C. The
addition of 5 mg of soybean trypsin inhibitor to the
combined supernatants was followed by the overnight
dialysis verses 1 L of HEPES N-2-hydroxyethyl-
piperazine-N -2-ethanesulfonic acid buffer B: 0.05
M HEPES, pH 6.5; 0.005 M manganese II chloride,
0.1% 2-mercaptoethanol . Dialysis tubing with a
molecular weight cutoff of 12 000–14 000 Da was
used. The dialysate was centrifuged at 100 000=g
for 30 min at 4 8C and the supernatant was then
mmol , bovine serum albumin 25 mL of a 100
mgrmL aqueous solution and alkaline phosphatase
from calf intestine 1 U , were added to a 1.5 mL
.
Ž
.
Ž
.
microcentrifuge tube. This was followed by the addi-
Ž
.
tion of 490 mL of GlcNAcT 14 mU and 474 mL of
50 mM HEPES buffer, pH 6.5 containing 0.1% 2-
mercaptoethanol. The reaction mixture was incubated
at room temperature. Additional donor 0.38 mg, 0.53
mmol was added after 48 h. After 96 h donor 0.3
Ž
.
Ž
.
Ž
.
mg, 0.42 mmol and alkaline phosphatase 0.5 U
Ž
were added to the reaction. After 8 days, the reaction
was worked up by loading it onto two pre-washed
C18 Sep-Pak cartridges. The cartridges were washed
with 30 mL of water and the crude product eluted
with 30 mL of MeOH. The MeOH eluant was evapo-
rated to dryness and the residue chromatographed on
an Iatrobead-column with 30:20:1 CH2Cl2–MeOH–
water. The product fractions were pooled, concen-
trated, dissolved in water and filtered through a 0.22
X
.
Ž
Ž .
.
Ž
.
assayed for GlcNAcT activity ;170 mU . This
crude extract was then loaded, at a flow rate of 0.15
mLrmin, onto a 20 mL UDP-hexanolamine
mm filter before lyophilization. Trisaccharide 10 was
obtained in 62% 0.73 mg isolated yield. Partial H
NMR assignments are reported in Table 1. HRESIMS
1
Ž
.
Ž
.
Sepharose 4B gel 3.4 mmolrmL column which had
previously been equilibrated with buffer B. The col-
Ž
.
Ž
umn was then washed with buffer B 40 mL and
subsequently eluted with 30 mL of 0.5 M NaCl
mrz 833.3173 calcd. for C32 H53N2O18F3Na:
.
833.3143 .
UDP-glucose and UDP-glucosamine were used as
donors in the above reaction but no transfer was
Ž
buffer C: 0.05 HEPES, pH 6.5; 0.5 M NaCl, 0.1%
.
Ž
.
2-mercaptoethanol . Fractions 1 mL were collected
in plastic tubes and assayed for ‘core-2’ activity. The
fractions containing the highest transferase activity
observed after 8 days.
[
Glucosyltransferase GlcNAcT - V UDP - D -

(
)
R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
135
(
)
GlcpNAc:a - D - Manp - 1™6 - b - D - GlcpNAc - V
Acknowledgements
(
)]
transferase GlcNAcT- V, EC 2.4.1.155 .—The 97
kDa GlcNAcT-V transferase was cloned from rat
This work was supported by the Natural Sciences
and Engineering Research Council of Canada.
w
x
kidney 27 and was a generous gift from Dr. M.
Pierce, Department of Biochemistry, University of
Georgia.
Radiochemical assay for GlcNAcT - V.—Enzyme
activity was monitored using a modified version of a
References
w
x
previously published procedure 28 . Each assay tube
contained: 8.0 mL of 1.38 mM UDP-GlcNAc, 1.0
mL of a 0.8 mM solution of acceptor 11, 1.0 mL of
w x
1 N. Sharon and L. Halina, Scientific American, Jan-
Ž
.
uary 1993 pp. 82–89.
3
5
w
x
Ž
.
UDP-6- H GlcNAc ;10 dpm , and 5.0 mL of
w x
2 S.J. Danishefsky and M.T. Bilodeau, Angew. Chem.
Ž
sodium cacodylate buffer 100 mM sodium cacody-
Ž
.
Int. Ed. Engl., 35 1996 1380–1419.
w x
3 F. Barresi and O. Hindsgaul, J. Carbohydr. Chem.,
late, pH 6.5; 20 mM EDTA; 40% glycerol; 2 mgrmL
Ž
.
18 1995 1043–1087.
4 K. Toshima and K. Tatsuta, Chem. ReÕ., 93 1993
.
bovine serum albumin . The work-up of the incu-
w x
Ž
.
bated samples was performed in the same manner as
given previously for the radiochemical assay for Glc-
NAcT.
1503–1531.
w x
5 J. Banoub, P. Boullanger, and D. Lafont, Chem. ReÕ.,
Ž
.
92 1992 1167–1195.
(
)
)
w x
Ž
.
Enzymatic conÕersion of b-D-GlcpNAc- 1™2 -a-D
6 H. Paulson, Angew. Chem. Int. Ed. Engl., 29 1990
823–839.
(
)
(
(
)
Ž
.
-Manp- 1™6 -b-D-Glcp-OR Rs CH2 7 CH3 11
w x
Ž
.
7 R.R. Schimdt, Angew. Chem. Int. Ed. Engl., 25 1986
(
) [ )]
(
into b-D-GlcpNAc- 1™2 - b-D-GlcpNAc-F3- 1™6 -
212–235.
(
)
(
(
)
)
a-D-Manp- 1™6 -b-D-Glcp-OR Rs CH2 7 CH3
w x
8 C.-H. Wong, R.L. Halcomb, Y. Ichikawa, and T.
Ž
.
Ž
12 .—UDP-N-trifluoroacetylglucosamine 2.4 mg,
Ž
.
Kajimoto, Angew. Chem. Int. Ed. Engl., 34 1995
.
Ž
.
3.34 mmol , 11 4.2 mg, 6.39 mmol , 500 mL of 50
mM sodium cacodylate buffer, pH 6.5 containing 10
521–546.
w x
Ž
.
Ž
9 O. Hindsgaul, Seminars Cell Biol., 2 1991 319–326.
10 D.G. Dreuckhammer, W.J. Hennen, R.L. Pedersen,
w
x
mM EDTA, 20% glycerol and 1 mgrmL bovine
C.F. Barbas III, C.M. Gautheron, T. Krach, and C.-H.
.
Ž
.
serum albumin and 50 mL of GlcNAcT-V 23 mU
Ž
.
Wong, Synthesis, 1991 449–525.
11 R. Oehrlein, O. Hindsgaul, and M.M. Palcic, Carbo-
hydr. Res., 244 1993 149–159.
12 H. Schachter, Curr. Opin. Struc. Biol., 1 1991
were added to a 1.5 mL microcentrifuge tube. The
reaction mixture was kept at room temperature. Addi-
w
w
w
w
w
x
Ž
.
Ž
.
x
Ž
.
tional donor 1.9 mg, 2.64 mmol was added after 72
h. After 2 weeks, the reaction was worked up using
the same C18 Sep-Pak procedure that was employed
previously for the GlcNAcT-reaction. The dried
MeOH residue was chromatographed on an Ia-
trobead-column eluted with 65:35:3 CHCl3–
MeOH–water. The pooled product fractions were
concentrated, dissolved in water and filtered through
a 0.22 mm filter before lyophilization. Tetrasaccha-
755–765.
x
13 R.D. Cummings, I.S. Trowbridge, and S. Kornfeld, J.
Biol. Chem., 257 1982 13421–13427.
14 H. Schachter, Biochem. Cell Biol., 64 1986 163–
Ž
.
x
Ž
.
181.
x
15 M. Imazawa and F. Eckstein, J. Org. Chem., 44
Ž
.
1979 2039–2041.
w
w
x
Ž
.
16 H. Newman, J. Org. Chem., 30 1965 1287–1288.
x
17 R.J. Bergeron, J.R. Garlich, and N.J. Stolowich, J.
Ž
.
Org. Chem., 49 1984 2997–3001.
Ž
.
ride 12 was obtained in a 56% 3.26 mg isolated
w
w
w
w
w
w
w
w
x
18 M.L. Wolfrom and P.J. Conigliaro, Carbohydr. Res.,
1
yield. Partial H NMR assignments are reported in
Ž
.
207 1990 63–76.
Ž
x
19 W. Meyer zu Reckendorf and N. Wassiliadau-Micheli,
Table 2. HRESIMS mrz 937.3622 calcd. for
Ž
.
Chem. Ber., 103 1970 1792–1796.
.
C36H61N2O21F3Na: 937.3617 .
x
Ž
.
20 S. Sabesan and S. Neira, Carbohydr. Res., 223 1992
(
) [
Deprotection of b - D - GlcpNAc - 1™2 - b - D -
169–185.
(
)]
(
)
GlcpNAc-F3- 1™6 -a-D-Manp- 1™6 -b-D-Glcp-OR
x
21 S.-C. Hung and C.-H. Wong, Tetrahedron Lett., 37
(
(
)
)
Ž
.
Rs CH2 7 CH3 12 .—Deprotection of tetrasaccha-
Ž
.
1996 4903–4906.
Ž
.
ride 12 0.5 mg, 0.55 mmol was successfully accom-
x
Ž
.
22 E.W. Putman, Methods Carbohydr. Chem., 2 1963
261–270.
plished using potassium carbonate in water–MeOH
x
23 C.D. Warren, Y. Konami, and R.W. Jeanloz, Carbo-
hydr. Res., 30 1973 257–279.
24 S. Roseman, J.J. Distler, J.G. Moffat, and H.G. Kho-
Ž
.
1:15 . The reaction was complete after 1 week at 40
Ž
.
8C. A C18 Sep-Pak work-up was used to obtain the
deprotected tetrasaccharide 13. Partial H NMR as-
x
1
Ž
.
rana, J. Am. Chem. Soc., 83 1961 659–663.
signments are reported in Table 2. HRESIMS mrz
x
25 R.M. Ratcliffe, D.A. Baker, and R.U. Lemieux, Car-
Ž
.
Ž .
bohydr. Res., 93 1981 35–41.
819.3984 calcd. for C34H63N2O20: 819.3974 .

(
)
136
R.F. Sala et al.rCarbohydrate Research 306 1998 127–136
w
w
x
w
x
26 O.P. Srivastava, O. Hindsgaul, M. Shoreibah, and M.
28 O. Hindsgaul, K.J. Kaur, G. Srivastava, M.
Ž
.
Pierce, Carbohydr. Res., 179 1988 137–161.
Blaszczyk-Thurin, S.C. Crawley, L.D. Heerze, and
x
Ž
.
27 M. Shoreibah, G.S. Perng, B. Adler, J. Weinstein, R.
Basu, R. Cupples, D. Wen, J.K. Browne, P. Buck-
haults, N. Fregien, and M. Pierce, J. Biol. Chem., 268
M.M. Palcic, J. Biol. Chem., 266 1991 17858–
17862.
Ž
.
1993 15381–15385.