Substrate specificity of N-Acetylglucosaminyl(diphosphodolichol) N-acetylglucosaminyl transferase, a key enzyme in the dolichol pathway

Article abstract of DOI:10.1016/S0968-0896(00)00334-5

N-Acetylglucosaminyl(diphosphodolichol) N-acetylglucosaminyl transferase, also known as Enzyme II, is the second enzyme in the dolichol pathway. This pathway is responsible for the assembly of the tetradecasaccharide pyrophosphate dolichol, which is the substrate for oligosaccharyl transferase. In order to study the specificity of Enzyme II, four unnatural dolichol diphosphate monosaccharides were synthesized, with the C-2 acetamido group in the natural substrate Dol-PP-GlcNAc 1a replaced by fluoro, ethoxy, trifluoroacetamido, and amino functionalities. These analogues 1b-e were evaluated as glycosyl acceptors for Enzyme II, which catalyzes the formation of dolichol diphosphate chitobiose (Dol-PP-GlcNAc₂) from UDP-GlcNAc and Dol-PP-GlcNAc. Enzyme II from pig liver was found to be highly specific for its glycosyl acceptor and the acetamido group shown to be a key functional determinant for this glycosylation reaction.

Full text of DOI:10.1016/S0968-0896(00)00334-5

Bioorganic & Medicinal Chemistry 9 (2001) 1133±1140
Substrate Speci®city of N-Acetylglucosaminyl(diphosphodolichol)
N-Acetylglucosaminyl Transferase, a Key Enzyme
in the Dolichol Pathway
Vincent W.-F. Tai, Mary K. O'Reilly and Barbara Imperiali*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received 6 November 2000; accepted 30 November 2000
AbstractÐN-Acetylglucosaminyl(diphosphodolichol) N-acetylglucosaminyl transferase, also known as Enzyme II, is the second
enzyme in the dolichol pathway. This pathway is responsible for the assembly of the tetradecasaccharide pyrophosphate dolichol,
which is the substrate for oligosaccharyl transferase. In order to study the speci®city of Enzyme II, four unnatural dolichol diphos-
phate monosaccharides were synthesized, with the C-2 acetamido group in the natural substrate Dol-PP-GlcNAc 1a replaced by
¯uoro, ethoxy, tri¯uoroacetamido, and amino functionalities. These analogues 1b±e were evaluated as glycosyl acceptors for
Enzyme II, which catalyzes the formation of dolichol diphosphate chitobiose (Dol-PP-GlcNAc2) from UDP-GlcNAc and Dol-PP-
GlcNAc. Enzyme II from pig liver was found to be highly speci®c for its glycosyl acceptor and the acetamido group shown to be a
key functional determinant for this glycosylation reaction. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
chemoenzymatic synthesis via Enzyme II (Fig. 1).2 As
part of our ongoing program on studies of OT, several
dolichol-linked disaccharides with substitutions at the
C-2 position (3b±e) are required. This prompted us to
explore the possibility of preparing these derivatives
3b±e via a chemoenzymatic approach employing Enzyme
II. The study of the glycosyltransferases along the doli-
chol pathway is also interesting, as they may be useful
for the preparation of modi®ed oligosaccharides and
glycoproteins.
Asparagine-linked glycosylation, the ®rst committed
step in N-linked glycoprotein biosynthesis, is essential
for structural and functional integrity of eukaryotic
cells. This modi®cation occurs cotranslationally with
the transfer of a common tetradecasaccharide from
dolichol diphosphate-GlcNAc2Man9Glc3 toan aspara-
gine residue of a nascent polypeptide chain. The tetra-
decasaccharide donor is assembled in a stepwise fashion
via a series of glycosyltransferases along the dolichol
pathway.1 Enzyme II in the pathway (N-acetyl-
glucosaminyl(diphosphodolichol) N-acetylglucosaminyl
transferase, EC 2.4.1.141) catalyzes the transfer of a
N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to
dolichol diphosphate N-acetylglucosamine (Dol-PP-
GlcNAc). The resulting dolichol diphosphate chitobiose
(Dol-PP-GlcNAc-b-1,4-GlcNAc) is further glycosylated
togive Dol-PP-GlcNAc 2Man9Glc3, the glycosyl donor
for N-linked glycosylation. In vitro, Dol-PP-GlcNAc2 is
often used as a substrate for oligosaccharyl transferase
(OT) due to the diculty in isolation of the tetra-
decasaccharide donor. Radiolabeled Dol-PP-GlcNAc-
(3H)-GlcNAc 3a was previously prepared by an ecient
Early studies on Enzyme II were focused mainly on the
characterization of the product from this enzymatic
reaction and the application of this enzyme to prepare
Dol-PP-GlcNAc2. Enzyme II from di€erent sources
such as yeast,3À6 mung bean seedling,7 rat8,9 and pig
livers10,11 has been documented. Compared with other
enzymes along the dolichol pathway such as Enzyme I
(and OT), reports on the puri®cation of this enzyme are
scarce. To the best of our knowledge, only two reports
concerning the puri®cation and biochemical studies of
Enzyme II have appeared.3,7 Enzyme II activity from
yeast and mung bean seedling was stimulated by the
addition of divalent metals such as Mg2+ and Mn2+
.
Furthermore, uridine nucleotides, especially UDP and
UDP-Glc, were found to inhibit Enzyme II from mung
bean seedlings.7 Herein, the chemical synthesis of four
unnatural dolichol-linked monosaccharides (1b±1e) are
*Corresponding author. Tel.: +1-617-253-1838; fax: +1-617-452-
2419; e-mail: imper@mit.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00334-5

1134
V. W.-F. Tai et al. / Bioorg. Med. Chem. 9 (2001) 1133±1140
Figure 1. Enzymatic reaction catalyzed by Enzyme II.
described together with the evaluation of these com-
pounds as substrates for Enzyme II from pig liver.
can be obtained by the phosphorylation of saccharides
6a±c with electrophilic phosphorylating reagents or
from oxazoline 7 with nucleophilic phosphorylating
reagents.
Results and Discussion
Planning
Synthesis of Dol-PP-2DFGlc 1b
3,4,6-Tri-O-acetyl-2-deoxy-2-¯uoro-a,b-d-glucopyranose
6b was prepared in quantitative yield from chemoselec-
tive deacetylation of the known tetraacetate 815À17 with
ammonium carbonate in DMF (Scheme 1). Phos-
phorylation of reducing sugar 6b with DMAP/diphenyl
chlorophosphate18 gave a separable mixture of ano-
meric diphenyl phosphates 9a and 9b in 74% yield
(a/b=2/1). The stereochemistry of the products was
The key step for the preparation of dolichol diphos-
phate sugars involves the condensation of a protected
a-glycosyl phosphate 4a±d with dolichol monophos-
phate (Dol-P, 5) (Fig. 2). Twostrategies have been
developed in the literature.12À14 The ®rst method
involves the activation of Dol-P 5 via the mixed
anhydride or the phosphoroimidazolidate followed by
the nucleophilic attack of glycosyl phosphate.13 Alter-
natively, a-glycosyl phosphate 4a±d can be activated as
the phosphoroimidazolidate followed by the attack of
Dol-P 5.14 The second method was applied throughout
these synthesis. Acetate protecting groups have been
used exclusively for the sugar portion due to the special
characteristic of the dolichol pyrophosphate moiety.
Speci®cally, the presence of a large number of double
bonds in dolichol does not allow the use of protecting
groups that require hydrogenolysis for deprotection.
Moreover, the acid labile pyrophosphate group also
limits the use of protecting groups that require cleavage
under acidic conditions. The a-glycosyl phosphate 4a±c
1
assigned based on their H NMR signatures: H-1 of 9a
resonates as a doublet of doublets at 6.13 ppm
(J1,2=3.5 Hz, J1,P=6.7 Hz). Interestingly, H-1 of 9b
shows an extra coupling with the C-2 ¯uorine atom at
5.50 ppm (dt, J1,2=J1,P=7.5 Hz, J1,F=2.9 Hz). In
Scheme 1. Reagents and conditions: (i) (NH4)2CO3, DMF; (ii) (1)
DMAP, CH2Cl2, rt; (2) diphenyl chlorophosphate, À12 ! 0 ꢀC; (iii)
H2, Pt2O, EtOH/EtOAc; (iv) (1) CDI, DMF; (2) Dol-P 5; (v) NaOMe,
CH2Cl2/MeOH.
Figure 2. Retrosynthetic analysis of dolichol diphosphate mono-
saccharides.

V. W.-F. Tai et al. / Bioorg. Med. Chem. 9 (2001) 1133±1140
1135
addition, the C-2 ¯uorine atom also coupled with the
H-2 (J2,F ꢁ 50 Hz), and H-3 (J3,F ꢁ 12±14 Hz) protons.
31P NMR shows the corresponding phosphate peaks at
ca. À12 ppm for both compounds. Catalytic hydro-
genolysis of a-diphenyl phosphate 9a with platinum
(IV) oxide gave 89% yield of a-phosphate 4b isolated as
the triethylamine salt. Dolichol monophosphate (Dol-P)
5 was prepared according to the Danilov modi®ed Cra-
mer procedure.19 Activation of a-phosphate 4b with
1,10-carbonyldiimidazole (CDI) followed by coupling
with Dol-P 5 gave the desired protected Dol-PP-
2DFGlc 10b in 45% yield. 31P NMR of 10b showed the
corresponding peaks at À10.6 and À13.3 ppm, respec-
tively. Removal of the acetate groups in 10b gave the
desired Dol-PP-2DFGlc 1b in 86% yield.
reducing sugar 6c in 70% yield as a mixture of a- and b-
anomers (a/b ꢁ1/1 as determined by 1H NMR in
CDCl3). Similar to the phosphorylation of ¯uorinated
analogue 6b, compound 6c gave a mixture of a- and b-
diphenyl phosphates 13a and 13b in 85% yield (a/b
ꢁ1.8/1) employing the phosphorochloridate method
(DMAP/diphenyl chlorophosphate). a-Diphenyl phos-
phate 13a was readily transformed to the desired
Dol-PP-2OEtGlc 1c via the coupling of Dol-P 5 with
a-phosphate 4c followed by subsequent deprotection.
Synthesis of Dol-PP-GlcNTFA 1d and Dol-PP-GlcN 1e
3,4,6-Tri-O-acetyl-2-deoxy-2-tri¯uoroacetamido-a-d-
glucopyranosyl phosphate 4d has recently been pre-
26
pared by twoindependent groups.
The a-phosphate
4d was prepared using the phosphorochloridate method
of the corresponding reducing sugar in 52% yield by the
Tanner group.26 This compound 4d was further trans-
formed to UDP-GlcNTFA and proven to be a substrate
of `core-2' GlcNAc transferase. On the other hand,
Busca and Martin showed that both the a- and b-phos-
phates could be obtained in high yields from the ring
opening of oxazoline 7 with dibenzyl phosphate under
di€erent conditions.27 Following the Martin protocol,
ring opening of the known oxazoline 727,28 with diben-
zyl phosphate in re¯uxing 1,2-dichloroethane gave the
thermodynamic product 14 in 65% yield (Scheme 3).27
The stereochemistry of 14 was con®rmed to be a-based
on the coupling constant of H-1±H-2 protons (J1,2=3.3
Hz) in the 1H NMR spectrum. Catalytic hydrogenolysis
of 14 with 10% palladium on charcoal gave quantitative
yield of a-phosphate 4d, which was then condensed
with Dol-P 5 to give 63% yield of the protected Dol-PP-
GlcNTFA 10d. Chemoselective deprotection of the
acetates in 10d with guanidine/guanidinium nitrate
solution29 gave Dol-PP-GlcNTFA 1d in 47% yield after
silica gel chromatography. On the other hand, treat-
ment of 10d with lithium hydroxide (10 equiv) gave the
amine derivative Dol-PP-GlcN 1e in 63% yield.
Synthesis of Dol-PP-2OEtGlc 1c
The preparation of Dol-PP-2OEtGlc 1c followed a
similar strategy to that of Dol-PP-2DFGlc 1b. A pro-
tected glucose derivative with a free hydroxyl group at
C-2 is required to incorporate the ethoxy functionality.
However, methylation of 1,3,4,6-tetra-O-acetyl a-d-glu-
copyranose with Purdie's reagent (MeI, Ag2O) was
known to result in 1,2-acyl migration.20,21 Glucosamine
11 has been shown earlier to give good yields for
methylation and benzylation, and can be removed easily
under acidic conditions.22À24 The known N-(3,4,6-tri-O-
acetyl-b-d-glucopyranosyl)-piperidine 11 was prepared
from commercially available b-d-glucose pentaacetate
according to the Hodge procedure.22 Alkylation of
compound 11 with ethyl iodide and freshly prepared
silver oxide (Ag2O)25 gave 90% yield of the desired 2-O-
ethyl glucose derivative 12 (Scheme 2). Removal of the
piperidine group in 12 with acetic acid (HOAc)23 gave
Scheme 2. Reagents and conditions: (i) EtI, Ag2O, PhH; (ii) AcOH,
H2O, acetone; (iii) (1) DMAP, CH2Cl2, rt; (2) diphenyl chlorophos-
phate, À10 ꢀC ! rt; (iv) H2, Pt2O, EtOH/EtOAc; (v) (1) CDI, DMF;
(2) Dol-P 5; (vi) NaOMe, CH2Cl2/MeOH.
Scheme 3. Reagents and conditions: (i) dibenzyl phosphate, 1,2-
dichloroethane, Á; (ii) H2, 10% Pd/C, MeOH; (iii) (1) CDI, DMF;
(2) Dol-P 5; (iv) guanidine/guanidinium nitrate, CH2Cl2/MeOH;
(v) LiOH, CH2Cl2/MeOH.

1136
V. W.-F. Tai et al. / Bioorg. Med. Chem. 9 (2001) 1133±1140
surprising that even a slight modi®cation at the C-2
position diminishes the activity of this enzyme. The
electronic e€ects of the substituents at the C-2 position
should induce minimal e€ects on the nucleophilicity of
the C-4 hydroxyl group. The acetamido group is postu-
lated to act as a functional determinant for this trans-
ferase reaction through highly speci®c hydrogen
bondings of the N±H and CˆO groups to the enzyme.
Neither the 2-deoxy-2-¯uoro analogue 1b nor the 2-O-
ethyl analogue 1c function as substrates, perhaps due to
the lack of the proposed essential acetamido group. The
low transferase activity of the tri¯uoroacetamido ana-
logue 1d may be accounted for by the strong electronic
e€ects of the tri¯uoromethyl group rendering the car-
bonyl a less ecient hydrogen bond acceptor. The fact
that the amine analogue Dol-PP-GlcN 1e, without a
carbonyl group, is a better substrate than 1d suggests
that the minimal recognition element for this transferase
reaction is an N±H unit. It is puzzling at this stage why
the tri¯uoroacetamido analogue 1d is such a poor sub-
strate since the more acidic N±H proton should be a
better hydrogen bond donor compared to an acetamido
group. On the other hand, the product inhibition of
Dol-PP-GlcNTFA-(3H)-GlcNAc 3d has not yet been
examined. It is possible that 3d inhibits the transferase
reaction through a stronger hydrogen bonding via the
more acidic proton of the tri¯uoroacetamido group.
The availability of Dol-PP-GlcNTFA-GlcNAc will
allow competition studies with the natural substrate 1a
which may give further insights tothe present ®ndings.
The substrate speci®city observed for Enzyme II is dif-
ferent from that of `core-2' GlcNAc transferase reported
by Tanner.26 UDP-GlcNTFA was found to be a sub-
strate for `core-2' GlcNAc transferase but neither the
2-amino (UDP-GlcN) nor the 2-hydroxyl (UDP-Glc)
derivatives were. In addition, the N-acetylgalactosamine
derivative Dol-PP-GalNAc, with an axial hydroxyl
group at C-4, was also not a substrate for Enzyme II.31
Figure 3. Percent conversion of 1a, 1d, and 1e.
Biological evaluation
Unnatural dolichol diphosphate monosaccharides (1b±e)
were evaluated as glycosyl acceptors for Enzyme II
from partially puri®ed pig liver microsomes using radio-
labeled UDP-(3H-GlcNAc) as the donor. The formation
of dolichol-linked disaccharides was quanti®ed by mea-
suring the amount of tritium incorporated into the
organic layer (unreacted UDP-(3H-GlcNAc) partitions
into the aqueous layer) after successive aqueous-organic
extractions.2 Unnatural analogues Dol-PP-2DFGlc 1b
and Dol-PP-2OEtGlc 1c showed no transferase activity
even at high concentrations and long reaction times.30
On the other hand, the amine derivatives Dol-PP-
GlcNTFA 1d and Dol-PP-GlcN 1e were shown to be
extremely poor and moderate substrates for Enzyme II
(Fig. 3), respectively. Even at higher concentrations of
1d (1.5 mM) and 1e (600 mM), only 13 and 41% con-
versions were observed after 14 h, respectively. Deriva-
tives 1b±d were further shown to be inhibitors when
studied in competition with the natural substrate 1a. As
shown in Table 1, the percent conversions of Dol-PP-
GlcNAc 1a were reduced to68±73% when equivalent
amounts of the unnatural derivatives 1b±d were added.
Conclusion
In summary, Enzyme II utilizes highly speci®c glycosyl
acceptors as substrates. Slight modi®cations remote
from the reaction center result in loss of transferase
activity. The acetamido group of the natural substrate
Dol-PP-GlcNAc 1a is proposed to function as a switch
for this glycosylation through highly speci®c hydrogen
bondings of the N±H and CˆO moieties to com-
plementary residues of the enzyme while the minimum
recognition element is an N±H unit (Fig. 4).
It is most likely that the unnatural analogues 1b±d act as
competitive inhibitors of Enzyme II by binding speci®-
cally to the Dol-PP-GlcNAc binding site, however
detailed kinetic studies tocon®rm this have not been
carried out at the current time due to diculties asso-
ciated with the discontinuous HPLC-based assay. It is
Table 1. Relative percent conversion of 1a when competed with 1b±d
Compoundsa
Relative % conversionb
1a
1
1a+1bc
1a+1c
1a+1d
0.68
0.73
0.68
a150 mM of each compound and 5 mL of pig liver microsomes were
used.
bPercent conversion for 10 min of reaction.
c10 mL of pig liver microsomes were used in this case.
Figure 4. Substrate speci®city of glycosyl acceptor for Enzyme II.

V. W.-F. Tai et al. / Bioorg. Med. Chem. 9 (2001) 1133±1140
1137
Experimental
General methods for synthesis
General procedure for coupling of sugar phosphate with
dolichol monophosphate.14 To a solution of the sugar
phosphate (0.04 mmol) in DMF (1 mL) at room tem-
perature was added 1,10-carbonyldiimidazole (CDI, 0.2
mmol) in one portion. After 2 h, MeOH (0.36 mmol)
was added (toquench excess CDI) tothe mixture and
stirred for 30 min. Dolichol monophosphate 519 (tri-N-
butyl ammonium form, 0.028 mmol) in CH2Cl2 (1 mL)
was then added and the mixture was allowed to stir for
3 days at room temperature. The crude mixture was
concentrated followed by chromatography with DE-52
(acetate form, eluted with increasing concentration of
NH4OAc in CHCl3, MeOH (2:1 v/v)). The products
were concentrated and further puri®ed by silica gel
chromatography.
Melting points were uncorrected. Optical rotations were
measured with a JASCO DIP-1000 automatic digital
polarimeter operating at 589 nm at 25 ꢀC and are
reported in degrees. Concentration (c) is indicated as
units of 10 mg/mL. IR spectra were recorded on a
Perkin-Elmer 1600 FT-IR spectrometer. NMR spectra
were measured on a Varian 500, Bruker AM-500
1
(500.15 MHz for H, 125 MHz for 13C), or General
Electric QE300 (75 MHz for 13C). All chemical shifts
were recorded in ppm down®eld from tetramethylsilane
on the d scale. 31P NMR chemical shifts are reported in
ppm relative to 85% phosphoric acid external standard.
Multiplicities are reported in the following abbrevia-
tions: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (doublet of doublets), etc. Mass spectra
were obtained at the Mass Spectrometry Laboratory,
operated by the College of Chemistry, California Insti-
tute of Technology, Pasadena, CA 91125, USA or UCR
Mass Spectrometry Facility, Department of Chemistry,
University of California, Riverside, CA 92521, USA.
All reactions were monitored by analytical thin-layer
chromatography (TLC) on glass precoated with silica
gel 60F254 (E. Merck) and compounds were visualized
with 20% w/v dodecamolybdophosphoric acid in ethanol
and subsequent heating. Phosphorus compounds were
visualized with molybdenum blue spray reagent. All
columns were packed wet with E. Merck silica gel 60
(230±400 mesh) as the stationary phase and eluted by
¯ash chromatography. All solvents were reagent grade.
Methylene chloride and triethylamine were distilled
from calcium hydride. Tetrahydrofuran and toluene
were distilled from sodium/benzophenone ketyl. Other
reagents were purchased from commercial suppliers
(Aldrich, Sigma) and used without further puri®cation.
General procedure for deprotection of peracetylated
dolichol-linked saccharides
Method A. To a stirred solution of peracetylated doli-
chol-linked saccharide (3 mmol) in CH2Cl2 (2.5 mL) at
room temperature was added a solution of NaOMe (4
mg) in MeOH (0.5 mL). After 30 min, cation exchange
resin (Dowex 50Â8, pyridinium form, 200 mg) was
added and the mixture was stirred for 10 min. The resin
was then ®ltered and washed with CHCl3/MeOH (2/1
v/v). Concentration of the solvent gave dolichol-linked
saccharide in the pyridinium form.
Method B. To a stirred solution of peracetylated doli-
chol-linked saccharide (3 mmol) in CH2Cl2 (1.5 mL) at
room temperature was added a solution of guanidine/
guanidinium nitrate (1 mL). After 20 min, cation
exchange resin (Dowex 50Â8, pyridinium form, 200 mg)
was added and the mixture was stirred for 10 min. The
resin was then ®ltered and washed with CHCl3/MeOH
(2/1 v/v). Concentration of the solvent followed by col-
umn chromatography gave dolichol-linked saccharide.
(Stock solution of the guanidine/guanidinium nitrate
reagent was prepared by dissolving guanidinium nitrate
(122.1 mg, 1 mmol) and sodium methoxide (10.8 mg, 0.2
mmol) in MeOH/CH2Cl2.)
General procedure for phosphorylation. To a solution of
the reducing sugar (0.42 mmol) in CH2Cl2 (2.5 mL) at
room temperature was added DMAP (0.92 mmol) in
one portion. The mixture was stirred for 15 min and
cooled to À12 ꢀC. Diphenyl chlorophosphate (0.92
mmol) was then added and the mixture was allowed to
warm slowly to 0 ꢀC over 2 h. The mixture was diluted
with CH2Cl2 (80 mL) and washed successively with ice
water (25 mL), aq HCl (1 N, 25 mL), and a saturated
aqueous solution of NaHCO3 (25 mL). The organic
extracts were dried (Na2SO4), ®ltered, and concentrated.
The anomers were then separated by ¯ash column
chromatography.
Method C. To a stirred solution of peracetylated doli-
chol-linked saccharide (1.1 mmol) in CH2Cl2/MeOH (0.3
mL, 5/1 v/v) at room temperature was added aqueous
lithium hydroxide solution (3 M, 11 mmol). After 20
min, the mixture was worked up as in method B.
3,4,6-Tri-O-acetyl-2-deoxy-2-¯uoro-ꢀ,ꢁ-D-glucopyranose
(6b). A solution of tetraacetate 817 (356 mg, 1.02 mmol)
and ammonium carbonate (977 mg, 10.2 mmol) was
stirred in DMF (10 mL) at room temperature for 26 h.
The mixture was diluted with EtOAc (80 mL) and
washed with water (80 mL), and brine (80 mL). The
organic layer was dried (Na2SO4), ®ltered, and con-
centrated to give compound 6b (316.8 mg, quantitative)
as a mixture of a- and b-anomers. MS (DCI, NH3): 326
(MNH+4 ); HRMS: calcd for C12H21FNO8: m/z
326.125120; found: 326.123990.
General procedure for removal of protecting groups on
dialkyl phosphates. A solution of the protected phos-
phate (0.16 mmol) and Pt2O (10 mol%, for diphenyl
phosphate) (or 10% Pd/C (20 mg, for dibenzyl phos-
phate)) was stirred under H2 in alcoholic solvent (8
mL) until nostarting material as shown by TLC. The
catalyst was ®ltered thorugh a pad of Celite and
washed with EtOH (15 mL). Et3N (1 mL) was added
followed by the evaporation of solvent in vacuo to
give the mono-(triethylammonium) salt of the phos-
phate.
Diphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-¯uoro-ꢀ-D-gluco-
pyranosyl phosphate (9a), diphenyl 3,4,6-tri-O-acetyl-2-

1138
V. W.-F. Tai et al. / Bioorg. Med. Chem. 9 (2001) 1133±1140
deoxy-2-¯uoro-ꢁ-D-glucopyranosyl
phosphate
(9b).
67.4, 67.8 (d, J=7 Hz, C-4), 71.0 (d, J=19 Hz, C-3),
87.5 (dd, J=9, 193 Hz, C-2), 91.5 (dd, J=5, 20 Hz),
169.6, 170.0, 170.7; 19F NMR (CDCl3, 470 MHz) d
À200.8 (dd, J=12, 50 Hz); 31P NMR (CDCl3, 162
MHz) d 0.6; [a]D +83.3ꢀ (c 1, CHCl3); MS (Àve FAB):
387 (MÀ); HRMS: calcd for C12H17O11FP: m/z
387.049253; found: 387.049400.
Compound 6b (129.3 mg, 0.4195 mmol) gave after
column chromatography (n-hex, EtOAc (3/2 v/v)) a
mixture of a- and b-diphenyl phosphates (167.3 mg,
74%, a/b ꢁ2/1). The two compounds were further
separated by chromatography (Et2O, n-hex (2/1 v/v)) to
give a-diphenyl phosphate 9a (94.9 mg, 42%) as an oil
followed by b-diphenyl phosphate 9b (39.8 mg, 18%) as
a white solid, mp 73±75 ꢀC. Data for a-diphenyl phos-
phate 9a: IR (neat) cmÀ1 1754, 1590, 1488, 1368, 1292,
P1-Dolichyl P2-[3,4,6-tri-O-acetyl-2-deoxy-2-¯uoro-ꢀ-D-
glucopyranosyl] diphosphate (10b). 19F NMR (CDCl3,
CD3OD (5/1 v/v), 470 MHz) d À201.5 (brd, J=48 Hz);
31P NMR (CDCl3, CD3OD (5/1 v/v), 162 MHz) d
À10.6, À13.3; MS (Àve ESI): 779.1, 813.2, 847.2, 881.3,
915.3, 949.3, 983.4 (m/2 for n=12±18).
1
1220, 1187, 1162, 1083, 1042, 962, 776, 689; H NMR
(CDCl3, 500 MHz) d 1.99 (s, 3H, Ac), 2.03 (s, 3H, Ac),
2.09 (s, 3H, Ac), 3.80 (dd, 1H, J=3.9, 12.6 Hz, H-6a),
4.00 (ddd, 1H, J=2.1, 3.9, 10.4 Hz, H-5), 4.15 (dd, 1H,
J=3.9, 12.6 Hz, H-6b), 4.61 (ddt, 1H, J=3.5, 9.6, 48
Hz, H-2), 5.07 (t, 1H, J=10 Hz, H-4), 5.55 (dt, 1H,
J=9.6, 11.8 Hz, H-3), 6.13 (dd, 1H, J=3.5, 6.7 Hz, H-
1), 7.20±7.39 (m, 10H, Ph); 13C NMR (CDCl3, 125
MHz) (d 20.4, 20.5, 20.6, 60.8, 66.9 (d, J=7 Hz, C-4),
69.5, 69.9 (d, J=19 Hz, C-3), 86.4 (dd, J=8, 196 Hz, C-
2), 94.6 (dd, J=5, 22 Hz, C-1), 120.0, 120.03, 120.3
(2Â), 125.7 (2Â), 129.8, 129.9, 150.1 (d, J=7.5 Hz),
150.2 (d, J=7 Hz), 169.3, 169.8, 170.3; 31P NMR
(CDCl3, 162 MHz) d À12.0; [a]D +100.6ꢀ (c 0.8,
CHCl3); MS (FAB): 541 (MH+), 563 (MNa+); HRMS:
calcd for C24H26O11FNaP: m/z=563.109448; found:
563.109000.
P1-Dolichyl P2-[2-deoxy-2-¯uoro-ꢀ-D-glucopyranosyl] di-
phosphate (1b). MS (Àve ESI): 716.1, 750.2, 784.2,
818.2, 852.3, 885.8, 920.9 (m/2 for n=12±18).
3,4,6-Tri-O-acetyl-1-deoxy-2-O-ethyl-1-piperidino-ꢁ-D-
glucopyranose (12). A mixture of compound 1122 (730.6
mg, 1.9566 mmol), iodoethane (1.56 mL, 19.566 mmol)
and silver oxide (680 mg, 2.935 mmol) was stirred vig-
orously in benzene (7.3 mL) for 1 day. The solid was
®ltered through a pad of Celite and washed with toluene
(8 mL). The solvent was removed under high vacuum to
give the 2-ethoxy compound 12 (751.1 mg, 90%) as a
white solid, mp 99±101 ꢀC; IR (neat) cmÀ1 935, 1749,
Data for b-diphenyl phosphate 9b: IR (neat) cmÀ1 1750,
1590, 1489, 1368, 1294, 1225, 1187, 1163, 1081, 1036,
1010, 957, 775, 689, 668; 1H NMR (CDCl3, 500 MHz) d
2.03 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.07 (s, 3H, Ac), 3.82
(ddd, 1H, J=2.1, 4.7, 10.1 Hz, H-5), 4.02 (dd, 1H,
J=2.1, 12.5 Hz, H-6a), 4.21 (dd, 1H, J=4.7, 12.5 Hz,
H-6b), 4.44 (ddd, 1H, J=7.5, 9, 50 Hz, H-2), 5.07 (t,
1H, J=9.7 Hz, H-4), 5.32 (dt, 1H, J=9.2, 14.2 Hz, H-
3), 5.50 (dt, 1H, J=2.9, 7.5 Hz, H-1), 7.19±7.37 (m,
10H, Ph); 13C NMR (CDCl3, 125 MHz) d 20.5 (2Â),
20.6, 61.3, 67.5 (d, J=7 Hz, H-4), 72.2 (d, J=19 Hz, H-
3), 72.7, 89.0 (dd, J=10, 192 Hz, H-2), 96.5 (dd, J=5,
25 Hz, H-1), 120.1, 120.2, 120.3, 120.4, 125.7 (2Â),
129.7, 129.8, 150.1 (d, J=7 Hz), 150.2 (d, J=7 Hz),
169.4, 169.7, 170.4; 19F NMR (CDCl3, 470 MHz) d
À200.4 (dd, J=13.6, 50 Hz); 31P NMR (CDCl3, 162
MHz) d À12.1; [a]D +43.4ꢀ (c 1, CHCl3); MS (FAB):
563 (MNa+); HRMS: calcd for C24H26O11FNaP: m/z
563.109448; found: 563.110600.
1442, 1367, 1234, 1111, 1089, 1048, 993, 907, 861; H
1
NMR (CDCl3, 500 MHz) d 1.12 (t, 3H, J=7.0 Hz,
OCH2CH3), 1.45±1.60 (m, 6H, CH2), 2.01 (s, 3H, Ac),
2.05 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.61 (m, 2H, NCHH),
2.86 (m, 2H, NCHH), 3.48 (t, 1H, J=9.2 Hz, H-2),
3.48±3.53 (m, 2H, OCHHCH3, H-5), 3.86 (dq, 1H,
J=7.0, 9.2 Hz, OCHHCH3), 3.91 (d, 1H, J=9.2 Hz, H-1),
4.06 (dd, 1H, J=2.3, 12.0 Hz, H-6a), 4.21 (dd, 1H,
J=4.9, 12.0 Hz, H-6b), 4.89 (t, 1H, J=9.7 Hz, H-3/H-4),
5.07 (t, 1H, J=9.4 Hz, H-4/H-3); 13C NMR (CDCl3,
125 MHz) d 15.6, 20.6, 20.7 (2Â), 24.6, 26.1, 49.1, 62.6,
67.2, 69.1, 72.7, 74.9, 75.4, 95.9, 169.8, 170.0, 170.6; [a]D
+27.1ꢀ (c 0.9, CHCl3); MS (FAB): 402 (MH+); HRMS:
calcd for C19H32NO8: m/z 402.212792; found: 402.211900.
3,4,6-Tri-O-acetyl-2-O-ethyl-ꢀ,ꢁ-D-glucopyranose (6c).
To a solution of the 2-ethoxy compound 12 (494.9 mg,
1.2328 mmol) in acetone (15 mL) was added water (200
mL) and acetic acid (100 mL) at room temperature. The
mixture was heated under re¯ux for 2 days. The solvent
was removed under reduced pressure and the crude
product was chromatographed (EtOAc, n-hex (1/1 v/v))
to give compound 6c (290 mg, 70%); MS (FAB): 357
(MNa+); HRMS: calcd for C14H22O9Na m/z
357.116152; found: 357.116300.
Mono(triethylammonium) salt of 3,4,6-tri-O-acetyl-2-
deoxy-2-¯uoro-ꢀ-D-glucopyranosyl phosphate (4b). a-Di-
phenyl phosphate 9a (87.1 mg, 0.1612 mmol) underwent
hydrogenolysis to give a-phosphate 4b (70.1 mg, 89%)
as the mono(triethylammonium) salt; IR (neat) cmÀ1
3401, 2906, 2687, 1746, 1646, 1456, 1229, 1162, 1039,
1
957, 917, 836, 772; H NMR (CDCl3, 500 MHz) d 1.27
Diphenyl 3,4,6-tri-O-acetyl-2-O-ethyl-ꢀ-D-glucopyrano-
syl phosphate (13a) and diphenyl 3,4,6-tri-O-acetyl-2-O-
ethyl-ꢁ-D-glucopyranosyl phosphate (13b). Phosphoryla-
tion of compound 6c (219.2 mg, 0.6557 mmol) gave
after column chromatography (CHCl3, CH3CN (15/1
v/v) followed by (10/1 v/v)) a-diphenyl phosphate 13a
(204.9 mg, 55%) and b-diphenyl phosphate 13b (112.2
mg, 30%) both as oils. Data for a-diphenyl phosphate
13a: IR (neat) cmÀ1 2977, 1750, 1590, 1489, 1367, 1291,
(t, 9H, J=7.2 Hz, N(CH2CH3)3), 1.97 (s, 3H, Ac), 2.00
(s, 3H, Ac), 2.01 (s, 3H, Ac), 3.01 (q, 6H, J=7.2 Hz,
N(CH2CH3)3), 4.11 (dd, 1H, J=1.5, 12.5 Hz, H-6a),
4.24 (dd, 1H, J=3.2, 12.5 Hz, H-6b), 4.34 (brd, 1H,
J=10 Hz, H-5), 4.44 (ddt, 1H, J=3.4, 9.6, 50 Hz, H-2),
5.02 (t, 1H, J=10 Hz, H-4), 5.50 (dt, 1H, J=9.1, 11.5
Hz, H-3), 5.83 (dd, 1H, J=3.4, 6.4 Hz, H-1); 13C NMR
(CDCl3, 125 MHz) d 8.4, 20.5, 20.6, 20.7, 45.4, 61.5,

V. W.-F. Tai et al. / Bioorg. Med. Chem. 9 (2001) 1133±1140
1139
1222, 1188, 1163, 1111, 1049, 1010, 959, 915, 776, 690;
1H NMR (CDCl3, 500 MHz) d 1.08 (t, 3H, J=7.0 Hz,
OCH2CH3), 1.99 (s, 3H, Ac), 2.00 (s, 3H, Ac), 2.06 (s,
3H, Ac), 3.48 (dq, 1H, J=7.0, 9.2 Hz, OCHHCH3),
3.55 (dt, 1H, J=3.3, 9.9 Hz, H-2), 3.67 (dq, 1H, J=7.0,
9.2 Hz, OCHHCH3), 3.75 (dd, 1H, J=2.1, 12.6 Hz, H-
6a), 3.91 (ddd, 1H, J=2.1, 3.9, 10.3 Hz, H-5), 4.14 (dd,
1H, J=3.9, 12.6 Hz, H-6b), 5.04 (t, 1H, J=9.9 Hz, H-3/
H-4), 5.36 (t, 1H, J=9.8 Hz, H-4/H-3), 6.05 (dd, 1H,
J=3.3, 6.9 Hz, H-1), 7.19±7.37 (m, 10H, Ph); 13C NMR
(CDCl3, 125 MHz) d 15.2, 20.5 (2Â), 20.7, 61.1, 67.0,
67.5, 69.5, 71.0, 76.8 (d, J=7 Hz), 96.0 (d, J=6 Hz),
120.0, 120.1 (2Â), 120.6, 120.7 (2Â), 125.5, 129.8, 129.9,
150.3, 150.4, 169.5, 170.0, 170.5; 31P NMR (CDCl3, 162
MHz) d À11.8; [a]d +98.7ꢀ (c 1.3, CHCl3); MS (FAB):
589 (MNa+); HRMS: calcd for C26H13O12NaP: m/z
589.145085; found: 589.146200.
(5/1 v/v) 162 MHz) d À10.0, À12.4; MS (Àve ESI):
1652.4, 1720.5, 1788.6, 1857.0, 1924.5 (MÀ for n=13±17).
P1-Dolichyl P2-[2-O-ethyl-ꢀ-D-glucopyranosyl] diphos-
phate (1c). MS (Àve ESI): 1526.1, 1594.2, 1662.3,
1730.7, 1798.2 (MÀ for n=13±17).
Mono(triethylammonium) salt of 3,4,6-tri-O-acetyl-2-
deoxy-2-tri¯uoroacetamido-ꢀ-D-glucopyranosyl phosphate
(4d).27 Hydrogenolysis of a-dibenzyl phosphate 1427
(145.2 mg, 0.2195 mmol) gave a-phosphate 4d (135.5
mg, quantitative) as a foam; IR (neat) cmÀ1 3411, 2995,
1748, 1560, 1458, 1369, 1224, 1189, 1157, 1040, 962,
921, 840, 718; 1H NMR (CDCl3, 500 MHz) d 1.08
(t, 9H, J=7.3 Hz, N(CH2CH3)), 1.75 (s, 3H, Ac), 1.79
(s, 3H, Ac), 1.85 (s, 3H, Ac), 2.87 (q, 6H, J=7.3 Hz,
N(CH2CH3)), 3.90 (dd, 1H, J=2.0, 12.4 Hz, H-6a), 4.03
(dd, 1H, J=3.4, 12.4 Hz, H-6b), 4.07 (dt, 1H, J=2.9,
10.2 Hz, H-5), 4.14 (dt, 1H, J=2.6, 10.5 Hz, H-2), 4.92
(t, 1H, J=9.6 Hz, H-3/H-4), 5.19 (t, 1H, J=9.6 Hz, H-
4/H-3), 5.32 (dd, 1H, J=3.3, 6.8 Hz, H-1); 13C NMR
(CDCl3, 125 MHz) d 7.9, 19.8, 20.0, 20.1, 45.7, 52.3 (d,
J=7.5 Hz), 61.3, 67.9 (2Â), 68.0, 70.6, 92.8, 157.9 (q,
J=37 Hz), 169.7, 170.4, 171.0; 31P NMR (CDCl3, 162
MHz) d À0.9; [a]D +33.9ꢀ (c 0.9, CHCl3); MS (Àve
FAB): 480 (MÀ); HRMS: calcd for C14H18NO12F3P: m/
z 480.051874; found: 480.051500.
Data for b-diphenyl phosphate 13b: IR (neat) cmÀ1
2978, 1749, 1590, 1489, 1367, 1295, 1226, 1189, 1163,
1
1102, 1072, 1043, 1026, 1010, 956, 773, 690, 668; H
NMR (CDCl3, 500 MHz) d 0.99 (t, 3H, J=7.0 Hz,
OCH2CH3), 2.01 (s, 3H, Ac), 2.02 (s, 3H, Ac), 2.04 (s,
3H, Ac), 3.42 (dd, 1H, J=7.8, 9.0 Hz, H-2), 3.46 (dq,
1H, J=7.0, 9.4 Hz, OCHHCH3), 3.54 (dq, 1H, J=7.0,
9.4 Hz, OCHHCH3), 3.77 (ddd, 1H, J=2.3, 4.7, 9.9 Hz,
H-5), 3.99 (dd, 1H, J=2.3, 12.4 Hz, H-6a), 4.21 (dd,
1H, J=4.7, 12.4 Hz, H-6b), 5.04 (t, 1H, J=9.5 Hz, H-3/
H-4), 5.12 (t, 1H, J=9.2 Hz, H-4/H-3), 5.35 (t, 1H,
J=7.2 Hz, H-1), 7.18±7.36 (m, 10H, Ph); 13C NMR
(CDCl3, 125 MHz) d 15.3, 20.6 (3Â), 61.6, 68.0, 68.3,
72.4, 73.7 (d, J=2 Hz), 79.1 (d, J=9 Hz), 99.1 (d, J=6
Hz), 119.9, 120.0, 120.4 (2Â), 125.5, 129.6, 129.7, 150.3
(d, J=6 Hz), 150.4 (d, J=6 Hz), 169.6, 169.8, 170.5; 31P
NMR (CDCl3, 162 MHz) d À12.1; [a]D +29.1ꢀ (c 1.0,
CHCl3); MS (FAB): 589 (MNa+); HRMS: calcd for
C26H13O12NaP: m/z 589.145085; found: 589.145400.
P1-Dolichyl P2-[3,4,6-tri-O-acetyl-2-deoxy-2-tri¯uoroacet-
amido-ꢀ-D-glucopyranosyl] diphosphate (10d). 19F NMR
(CDCl3, 470 MHz) d À76.4; 31P NMR (CDCl3, 162
MHz) d À10.1, À12.4; MS (Àve ESI): 825.7, 859.8,
893.8, 927.9, 961.9, 995.9, 1030.0 (m/2 for n=12±18).
P1-Dolichyl P2-[2-deoxy-2-tri¯uoroacetamido-ꢀ-D-gluco-
pyranosyl] diphosphate (1d). MS (Àve ESI): 1593.1,
1661.2, 1729.6, 1797.7, 1865.2 (MÀ for n=13±17).
Mono(triethylammonium) salt of 3,4,6-tri-O-acetyl-2-O-
ethyl-ꢀ-D-glucopyranosyl phosphate (4c). Hydro-
genolysis of a-diphenyl phosphate 13 (89.1 mg, 0.1573
mmol) gave the mono(triethylammonium) salt of a-phos-
phate 4c (79.5 mg, 98%); IR (neat) cmÀ1 3402, 2980,
1747, 1451, 1368, 1234, 1166, 1107, 1044, 957, 921,
P1-Dolichyl P2-[2-amino-ꢀ-D-glucopyranosyl] diphosphate
(1e). MS (Àve ESI): 1498.5, 1566.6, 1634.6, 1702.7,
1736.7 (MÀ for n=13±17).
Biological methods
1
840; H NMR (CDCl3, CD3OD (2/1 v/v) 500 MHz d)
Crude porcine liver microsomes were prepared from
fresh pig liver based on a protocol developed for rat
liver.32,33 Brie¯y, a 100 g sample of fresh pig liver typi-
cally a€ords 2.5±5 mL of a microsomal preparation
(ꢁ100 mg/mL protein) that can be stored at À80 ꢀC in
the presence of 30% glycerol. This material can be used
for several months without signi®cant loss of transferase
activity. The natural substrate Dol-PP-GlcNAc 1a was
prepared according to literature procedure.2 Radio-
labeled uridine diphosphate N-acetyl-d-glucosamine-6-3H
(UDP-(3H)-GlcNAc 2, speci®c activity of 60 Ci/mmol,
concentration=1 mCi/mL) was obtained from American
Radiolabeled Chemicals, Inc., St. Louis, MO, USA.
0.96 (t, 3H, J=7.0 Hz, OCH2CH3), 1.14 (t, 9H, J=7.3
Hz, N(CH2CH3)), 1.85 (s, 3H, Ac), 1.87 (s, 3H, Ac),
1.89 (s, 3H, Ac), 2.92 (dq, 6H, J=3.3, 7.3 Hz,
N(CH2CH3)), 3.30 (dt, 1H, J=2.9, 10 Hz, H-2), 3.33
(dt, 1H, J=7.0, 9.5 Hz, OCHHCH3), 3.56 (dq, 1H,
J=7.0, 9.5 Hz, OCHHCH3), 3.94 (brd, 1H, J=10.5 Hz,
H-6a), 4.05±4.09 (m, 2H, H-5, H-6b), 4.88 (t, 1H, J=9.6
Hz, H-3/H-4), 5.15 (t, 1H, J=9.7 Hz, H-4/H-3), 5.53
(dd, 1H, J=3.3, 6.9 Hz, H-1); 13C NMR (CDCl3,
CD3OD (2/1 v/v) 75 MHz) d 7.8, 17.8, 20.0, 20.1, 20.2,
45.2, 61.4, 66.1, 67.5, 67.9, 71.4, 77.2 (d, J=8 Hz), 92.3
(d, J=6 Hz), 169.8, 170.4, 171.0; 31P NMR (CDCl3,
CD3OD (2/1 v/v) 162 MHz) d 0.5; [a]D +79.2ꢀ (c 1.1,
CHCl3); MS (Àve FAB): 413 (MÀ); HRMS: calcd for
C14H22O12P: m/z 413.084890; found: 413.085900.
Enzyme II assay
The assay bu€er consisted of 50 mM Tris acetate, pH
7.0, 3 mM dithiothreitol (DTT), 5 mM MgCl2, 0.25 M
sucrose and 1% Nonidet P-40. Dolichol-linked mono-
P1-Dolichyl P2-[3,4,6-tri-O-acetyl-2-O-ethyl-ꢀ-D-gluco-
pyranosyl] diphosphate (10c). 31P NMR (CDCl3, CD3OD

1140
V. W.-F. Tai et al. / Bioorg. Med. Chem. 9 (2001) 1133±1140
saccharides (1a±e) were aliquoted from a chloroform/
methanol stock solution into an eppendorf, and the
solvent was evaporated under a stream of nitrogen. To
this eppendorf was added UDP-(3H)-GlcNAc (60 Ci/
mmol for the study of Dol-PP-2DFGlc 1b, and 0.6 Ci/
mmol for the other analogues, ®nal concentra-
tion=0.167 mM) in ethanol/water, and the solvent was
again evaporated under nitrogen. Assay bu€er was
added (®nal volume=100 mL) tothe eppendorf and
vortexed vigorously. The reaction was initiated by the
addition of freshly thawed pig liver microsomes (5 or 10
mL) and shaken at 180 rpm. Aliquots (10 mL) of the
reaction mixture were quenched with 0.6 mL of chloro-
form/methanol/2.5 mM MgCl2 (3/2/1 v/v) at di€erent
times. The upper aqueous phase was removed and the
lower phase re-extracted twice with TUP (theoretical
upper layer, chloroform/methanol/water 2.75/44/53.25
v/v, 100 mL). The combined aqueous phases were then
mixed with Ecolite (5.5 mL) and counted on a Beckman
LS-5000TD scintillation counter to give the amount of
unreacted UDP-(3H)-GlcNAc. The organic layer was
mixed with Betamax (5.5 mL) and counted as well. The
percent conversion of dolichol-linked disaccharide was
based on [3Horg/(3Haq+3Horg)Â100].
7. Kaushal, G. P.; Elbein, A. D. Plant Physiol. 1986, 81, 1086.
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Acknowledgements
This research was supported by the National Institute of
Health (GM39334).
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Dol-PP-GlcNAc was observed.
31. Tai, V. W.-F. Mechanistic Studies of Oligosaccharyl
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Â
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