Substrate specificity of the glycosyl donor for oligosaccharyl transferase

Article abstract of DOI:10.1021/jo0100345

Oligosaccharyl transferase (OT) catalyzes the co-translational transfer of a dolichol-linked tetradecasaccharide (Dol-PP-GlcNAc₂Man₉Glc₃, 1a) to an asparagine side chain of a nascent polypeptide inside the lumen of the endoplasmic reticulum (ER). The glycosyl acceptor requires an Asn-XaaThr/Ser sequon, where Xaa can be any natural amino acid except proline, for N-linked glycosylation to occur. To address the substrate specificity of the glycosyl donor, three unnatural dolichol-linked disaccharide analogues (Dol-PP-GlcNTFA-GlcNAc 1c, Dol-PP-2DFGlc-GlcNAc 1d, and Dol-PP GlcNac-Glc 1e were synthesized and evaluated as substrates or inhibitors for OT from yeast. The synthetic analogue Dol-PP-GlcNAc-Glc 1e, with substitution in the distal sugar, was found to be a substrate (K_mapp = 26 μM) for OT. On the other hand, the analogues Dol-PP-GlcNTFA-GlcNAc 1c (K_i = 154 μM) and Dol-PP-2DFGlc-GlcNAc 1d (K_i = 252 μM), with variations in the proximal sugar, were inhibitors for OT. The dolichol-linked monosaccharide Dol-PP-GlcNAc 3 was found to be the minimum unit for glycosylation to occur.

Full text of DOI:10.1021/jo0100345

J . Org. Chem. 2001, 66, 6217-6228
6217
Su bstr a te Sp ecificity of th e Glycosyl Don or for Oligosa cch a r yl
Tr a n sfer a se
Vincent W.-F. Tai and Barbara Imperiali*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
imper@mit.edu
Received J anuary 12, 2001
Oligosaccharyl transferase (OT) catalyzes the co-translational transfer of a dolichol-linked tetra-
decasaccharide (Dol-PP-GlcNAc2Man9Glc3, 1a ) to an asparagine side chain of a nascent polypeptide
inside the lumen of the endoplasmic reticulum (ER). The glycosyl acceptor requires an Asn-Xaa-
Thr/Ser sequon, where Xaa can be any natural amino acid except proline, for N-linked glycosylation
to occur. To address the substrate specificity of the glycosyl donor, three unnatural dolichol-linked
disaccharide analogues (Dol-PP-GlcNTFA-GlcNAc 1c, Dol-PP-2DFGlc-GlcNAc 1d , and Dol-PP-
GlcNAc-Glc 1e) were synthesized and evaluated as substrates or inhibitors for OT from yeast. The
synthetic analogue Dol-PP-GlcNAc-Glc 1e, with substitution in the distal sugar, was found to be
a substrate (Kmapp ) 26 µM) for OT. On the other hand, the analogues Dol-PP-GlcNTFA-GlcNAc
1c (Ki ) 154 µM) and Dol-PP-2DFGlc-GlcNAc 1d (Ki ) 252 µM), with variations in the proximal
sugar, were inhibitors for OT. The dolichol-linked monosaccharide Dol-PP-GlcNAc 3 was found to
be the minimum unit for glycosylation to occur.
In tr od u ction
peptides with threonine at the (i + 2) site are better
substrates than the corresponding serine-containing pep-
tides. Simple tripeptides with capped C- and N-termini
can be used as substrates for in vitro OT studies. Several
mechanisms have been proposed to explain the activation
of the seemingly unreactive carboxamide of asparagine
in N-linked glycosylation, and this topic has been
reviewed.3-5,7
Asparagine-linked glycosylation is essential for struc-
tural and functional integrity of many eukaryotic pro-
teins.1-7 This glycosylation is mediated by the membrane-
associated multimeric enzyme oligosaccharyl transferase
(OT, EC 2.4.1.119)† in the lumen of the endoplasmic
reticulum (ER). OT catalyzes the transfer of a dolichol-
linked tetradecasaccharide (Dol-PP-GlcNAc2Man9Glc3,
1a ) to an asparagine side chain within a nascent polypep-
tide chain as the peptide is translocated into the lumen
of the ER (Scheme 1). The resulting â-linked glycopeptide
2a is further processed by glycosidases and glycosyl-
transferases in the ER and Golgi apparatus to yield the
mature glycan structures that include other glycosyl
units such as galactose, fucose, and sialic acid. This
cotranslational event is the first committed step in the
biosynthesis of all N-linked protein glycoconjugates;
therefore, an understanding of this reaction is critical.
It has been well established that the minimum recog-
nition sequence for N-linked glycosylation is an Asn-Xaa-
Thr/Ser triad where Xaa can be any amino acid except
proline.8,9 Both in vitro and in vivo data showed that
Dolichol-linked tetradecasaccharide 1a , biosynthesized
via the dolichol pathway, is the preferred substrate for
OT both in vivo and in vitro. Exceptions occur in systems
such as trypanosomatid protozoa where one or more of
the glycosyltransferases are missing.10 As a result of the
poor availability of the full length substrate, most in vitro
studies have been carried out with truncated glycosyl
donors such as the chitobiosyl derivative, Dol-PP-
GlcNAc2, 1b. Early studies by Sharma et al. showed that
Dol-PP-GlcNAc2Man was also a substrate, whereas no
transfer was observed for Dol-PP-GlcNAc2Man9 and Dol-
PP-GlcNAc 3.11 Contrary to this result, Bause and co-
workers reported that Dol-PP-GlcNAc 3 was in fact a poor
substrate for OT,12 and this discrepancy will be addressed
later (inter infra). Using yeast genetics techniques, Aebi
et al. also showed that various truncated dolichol-linked
oligosaccharides were substrates for OT.13 In an attempt
to study the chain length effect of the polyisoprene,
Coward and co-workers showed that compounds with
shorter isoprenoid chains or saturated alkyl chains
attached to chitobiosyl diphosphate were poor substrates
for OT.14 The existence of the saturated isoprenoid unit
* Tel: (617) 253-1838. Fax: (617) 452-2419.
† Also known as dolichol-diphospho-oligosaccharide-protein glycosyl
transferase, abbreviated both as OT and OST in the literature.
(1) Kornfeld, R.; Kornfeld, S. Annu. Rev. Biochem. 1985, 54, 631-
664.
(2) Cummings, R. D. In Glycocojugates; Allen, H. J ., Kisailus, E.
C., Eds.; Marcel Dekker: New York, 1992; pp 333-360.
(3) Silberstein, S.; Gilmore, R. FASEB J . 1996, 10, 849-858.
(4) Imperiali, B.; Hendrickson, T. L. Bioorg. Med. Chem. 1995, 3,
1565-1578.
(5) Imperiali, B. Acc. Chem. Res. 1997, 30, 452-459.
(6) Imperiali, B.; O’Connor, S. E.; Hendrickson, T.; Kellenberger,
C. Pure Appl. Chem. 1999, 71, 777-787.
(10) Bosch, M.; Trombetta, S.; Engstrom, U.; Parodi, A. J . J . Biol.
Chem. 1988, 263, 17360-17365.
(11) Sharma, C. B.; Lehle, L.; Tanner, W. Eur. J . Biochem. 1981,
(7) Knauer, R.; Lehle, L. Biochim. Biophys. Acta 1999, 1426, 259-
273.
116, 101-108.
(8) Marshall, R. D. Annu. Rev. Biochem. 1972, 41, 673-702.
(9) Struck, D. K.; Lennarz, W. J . In The Biochemistry of Glycopro-
teins and Proteoglycans; Lennarz, W. J ., Ed.; Plenum Press: New York,
1980; pp 35-83.
(12) Bause, E.; Breuer, W.; Peters, S. Biochem. J . 1995, 312, 979-
985.
(13) Burda, P.; Aebi, M. Biochim. Biophys. Acta 1999, 1426, 239-
257.
10.1021/jo0100345 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/29/2001

6218 J . Org. Chem., Vol. 66, No. 19, 2001
Imperiali
Sch em e 1
Sch em e 2
F igu r e 1. Structures of dolichol-linked disaccharides 1b-e.
next to the pyrophosphate moiety as well as the presence
of the cis-isoprenoid units still remains to be addressed.
Previous efforts have focused mainly on the study of
the glycosyl acceptor; the investigation of the role of the
glycosyl donor and its leaving group will help to better
understand the complete process of N-linked glycosyla-
tion. The intriguing feature that N-acetylglucosamines
(GlcNAc) constitute the first two saccharides in the
glycosyl donor 1a prompted us to study the role of the
C-2 acetamido group in the first two GlcNAc units.
Herein, the chemical synthesis of three unnatural doli-
chol-linked disaccharides are described, together with the
evaluation of these compounds as substrates or inhibitors
of yeast OT.
reaction center should result in significant destabilization
of the oxocarbenium ion, thus inhibiting such a reaction
pathway. Withers and co-workers have demonstrated
that 2-deoxy-2-fluoroglycosides with good leaving groups
at the C-1 position can be used as mechanism-based
inhibitors of retaining â-glucosidases. In some cases,
2-deoxy-2-fluoroglycosyl-enzyme intermediates have been
isolated and characterized.16,17 So the study of analogue
1d will be particularly interesting, both in terms of
studying the mechanism of OT and trapping of any
glycosyl-enzyme intermediate. Additionally, Dol-PP-
GlcNAc-Glc 1e was included to address the role of the
acetamido group in the distal saccharide site (D-site).
Previously, radiolabeled Dol-PP-GlcNAc-(3H)-GlcNAc
4 was prepared by an efficient chemoenzymatic synthesis
of Dol-PP-GlcNAc 3 via Enzyme II from pig liver micro-
somes (Scheme 2).18 However, this general approach
failed to give sufficient amount of the unnatural deriva-
tives 1c and 1d .19 Our study showed that slight modifica-
Resu lts a n d Discu ssion
A substrate analogue approach was undertaken to
examine the substrate specificity of the glycosyl donor.
The trifluoroacetamido and 2-deoxy-2-fluoro derivatives
1c,1d (Figure 1) were designed to address the importance
of the C-2 acetamido group in the proximal saccharide
site (P-site). Previously, the trifluoroacetamido analogue
of UDP-GlcNAc was shown to be a substrate for “core-2”
GlcNAc transferase.15 On the other hand, substitution
with an electronegative fluorine atom adjacent to the
(16) McCarter, J . D.; Yeung, W.; Chow, J .; Dolphin, D.; Withers, S.
G. J . Am. Chem. Soc. 1997, 119, 5792-5797.
(17) Street, I. P.; Kempton, J . B.; Withers, S. G. Biochemistry 1992,
31, 9970-9978.
(18) Imperiali, B.; Zimmerman, J . W. Tetrahedron Lett. 1990, 31,
6485-6488.
(19) Tai, V. W.-F.; O’Reilly, M. K.; Imperiali, B. Bioorg. Med. Chem.
2001, in press.
(14) Fang, X. G.; Gibbs, B. S.; Coward, J . K. Bioorg. Med. Chem.
Lett. 1995, 5, 2701-2706.
(15) Sala, R. F.; MacKinnon, S. L.; Palcic, M. M.; Tanner, M. E.
Carbohydr. Res. 1998, 306, 127-136.

Oligosaccharyl Transferase Substrate Specificity
J . Org. Chem., Vol. 66, No. 19, 2001 6219
Sch em e 3
tion of the acetamido group in 3 resulted in significant
loss of Enzyme II activity, and therefore the C-2 aceta-
mido group was proposed to be an important functional
determinant for this transferase reaction. The failure of
the chemoenzymatic approach prompted us to prepare
compounds 1b-e via chemical synthesis.
13 can be readily prepared by the acyl migration of
chitobiosyl chloride 1225 by heating in water/acetone
(Scheme 4). Acylation of 13 with trifluoroacetic anhydride
(TFAA)/pyridine gave 97% yield of 14. The 1H NMR
spectrum of 14 looks similar to that of chitobiose octaac-
etate, while the 19F NMR spectrum shows a singlet at
-76.6 ppm corresponding to the newly incorporated
trifluoroacetate group. Bromination of compound 14
followed by oxazoline ring formation with 2,6-lutidine
gave oxazoline 15 in 61% over two steps.19,26 Oxazoline
15 shows the correct mass at 693 (MNa+) and H-1
appears as a doublet at 6.22 ppm (J 1,2 ) 7.4 Hz) in the
1H NMR spectrum. Ring opening of oxazoline 15 with
dibenzyl phosphate 16 in refluxing 1,2-dichloroethane26
gave solely R-dibenzyl phosphate 17 in 64% yield. The
stereochemistry of 17 was confirmed to be R on the basis
of the coupling constant of H-1-H-2 protons. H-1 appears
The preparation of dolichol-linked disaccharides 1b-e
followed a similar strategy as for the previous prepara-
tion of their monosaccharide counterparts.19 The key step
involves the condensation of R-linked disaccharide phos-
phates 5 with dolichol monophosphate 6 (Scheme 3). The
R-phosphates 5 can in turn be prepared from phospho-
rylation of the lactol (for Z ) OH in 7), bromide (for Z )
Br in 7) or oxazoline (for X, Z ) -NdC(CF3)-O- in 7).
Commercially available chitobiose octaacetate was em-
ployed for the preparation of Dol-PP-GlcNAc-GlcNAc 1b
and Dol-PP-GlcNTFA-GlcNAc 1c. The other unnatural
disaccharides were assembled by two different routes to
establish the â-1,4-linkages. The disaccharide in Dol-PP-
2DFGlc-GlcNAc 1d was prepared by glycosylation of the
trichloroacetimidate Schmidt donor 820 with glycosyl
acceptor 9. Glycosyl donor 8 was chosen because of its
high reactivity and good stereoselectivity for the â-prod-
uct.20 On the other hand, glycosylation of commercially
available bromoacetyl glucose 10 with the known accep-
tor 1121 afforded protected Glc-â-(1f4)-GlcNAc. This
glycosylation has previously been communicated by
O’Connor using a mixture of R- and â-benzyl glycosides
11.22 The in vitro substrate 1b was prepared as described
previously.23
as a doublet of doublets at 5.65 ppm (J 1,2 ) 4 Hz, J 1,P
)
6 Hz). Catalytic hydrogenolysis of R-dibenzyl phosphate
17 with 10% palladium on charcoal afforded a quantita-
tive yield of R-phosphate 18 isolated as its triethylamine
salt. Dolichol monophosphate (Dol-P) 6 was prepared
according to the Danilov modified Cramer procedure.27
Activation of R-phosphate 18 with 1,1′-carbonyldiimida-
zole (CDI) followed by coupling with Dol-P 6 gave
protected Dol-PP-GlcNTFA-GlcNAc 19 in 49% yield.
Chemoselective deprotection of the acetates in 19 with
guanidine/guanidinium nitrate solution28 afforded Dol-
PP-GlcNTFA-GlcNAc 1c in 39% yield after silica gel
chromatography.
Syn th esis of Dol-P P -2DF Glc-GlcNAc, 1d . As il-
lustrated in Scheme 5, glycosyl acceptor 9 was prepared
Syn th esis of Dol-P P -GlcNTF A-GlcNAc, 1c. Prepa-
ration of Dol-PP-GlcTFA-GlcNAc 1c begins with the
installation of the trifluoroacetamido group via the
known amine hydrochloride 13.24 The amine derivative
(24) Eshdat, Y.; Sharon, N. Methods Enzymol. 1977, 46, 403-414.
(25) Spinola, M.; J eanloz, R. W. J . Biol. Chem. 1970, 245, 4158-
4162.
(20) Kinzy, W.; Schmidt, R. R. Liebigs Ann. Chem. 1985, 1537-1545.
(21) Goss, P. E.; Baker, M. A.; Carver, J . P.; Dennis, J . W. Clin.
Cancer Res. 1995, 1, 935-944.
(22) O’Connor, S. E.; Imperiali, B. Chem. Biol. 1998, 5, 427-437.
(23) Lee, J .; Coward, J . K. J . Org. Chem. 1992, 57, 4126-4135.
(26) Busca, P.; Martin, O. R. Tetrahedron Lett. 1998, 39, 8101-8104.
(27) Danilov, L. L.; Maltsev, S. D.; Shibaev, V. N. Bioorg. Khim.
1988, 14, 1287-1289.
(28) Ellervik, U.; Magnusson, G. Tetrahedron Lett. 1997, 38, 1627-
1628.

6220 J . Org. Chem., Vol. 66, No. 19, 2001
Imperiali
Sch em e 4a
Sch em e 6a
a
Reagents and conditions: (a) H2O, acetone, ∆ (74%); (b) TFAA,
a
Reagents and conditions: (a) BF3‚Et2O, 4 Å molecular sieves,
pyridine, CH2Cl2, 0 °C (97%); (c) (1) 30% HBr/HOAc, CH2Cl2; (2)
2,6-lutidine, CH3CN (61% for two steps); (d) dibenzyl phosphate
16, 1,2-dichloroethane, ∆ (64%); (e) H2, 10% Pd/C, MeOH (quan-
titative); (f) (1) CDI, DMF; (2) Dol-P 6 (49%); (g) guanidine/
guanidinium nitrate, CH2Cl2/MeOH (39%).
CH2Cl2, -78 f 5 °C (83%); (b) (1) H2, Raney Ni, Ac2O, EtOH; (2)
H2, 10% Pd/C, MeOH; (3) Ac2O, pyridine (96%, R: â ) 2:1); (c) 30%
HBr/HOAc, CH2Cl2 (85%); (d) AgOTf, dibenzyl phosphate 16, THF
(60%, R: â ) 1.6:1); (e) H2, 10% Pd/C, MeOH (quantitative); (f) (1)
CDI, DMF; (2) Dol-P 6 (36%); (g) NaOMe, CH2Cl2/MeOH (73%).
Sch em e 5a
resonate at 5.15 ppm (J 1,2 ) 4 Hz) and 4.67 ppm (J 1,2
)
7.6 Hz, J 1,F ) 4 Hz), respectively. For simplicity in the
characterization of compounds at a later stage, the major
anomer 22b was chosen to carry on the synthesis.
Reductive ring opening of â-anomer 22b with sodium
cyanoborohydride [Na(CN)BH3] under acidic conditions
(HCl/Et2O)30 gave the glycosyl acceptor 9 with good
regioselectivity in 87% yield.
Glycosyl donor 8 was prepared according to the Schmidt
procedure via azidonitration31 of commercially available
tri-O-benzyl-D-glucal followed by subsequent imidate
formation with NaH/CCl3CN.20 Glycosylation of the
2-deoxy-2-fluoro derivative 9 with the Schmidt donor 8
under Lewis acidic conditions (BF3‚Et2O) gave 83% yield
of the perbenzylated disaccharide 23 (Scheme 6). The
stereochemistry of the newly formed glycosidic bond was
a
Reagents and conditions: (a) (1) NaOMe, MeOH; (2) Ph-
CH(OMe)2, p-TsOH, DMF, 50 °C (76%, R:â ) 1:1.8); (b) NaH, BnBr,
DMF, 0 °C (85%); (c) Na(CN)BH3, THF, HCl/Et2O, 0 °C (87%).
from the known 2-deoxy-2-fluoro-1,3,4,6-â-D-glucose tet-
raacetate 20.29 The benzylidene moiety was readily
installed by acidic benzylidenation of 2-deoxy-2-fluoro
glucose to give 76% yield of the reducing sugar 21.
Benzylation of the free hydroxyl groups in 21 gave a
separable mixture of R- and â-benzyl glucosides 22a and
22b in 85% combined yield (R:â ) 1:1.8). Both anomers
showed the correct mass at 451 (MH+), and the stereo-
chemistry was assigned on the basis of the H-1-H-2
coupling constants. The H-1 protons of 22a and 22b
1
confirmed to be â (J 1′,2′ ) 7.6 Hz) by H NMR analysis.
The benzyl and azido groups in 23 were successively
replaced by acetate groups to give disaccharide 24 in 96%
yield as a mixture of R- and â-anomers (R:â ≈ 2:1). The
mixture of acetates 24 was treated with 30% HBr in
acetic acid to give exclusively R-bromide 26 in 85%
isolated yield. Phosphorylation of R-bromide 26 with
(30) Garegg, P. J .; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982,
108, 97-101.
(31) Lemieux, R. U.; Ratcliffe, R. M. Can. J . Chem. 1979, 57, 1244-
(29) Kova´c, P. Carbohydr. Res. 1986, 153, 168-170.
1251.

Oligosaccharyl Transferase Substrate Specificity
J . Org. Chem., Vol. 66, No. 19, 2001 6221
Sch em e 7a
exclusively R-dibenzyl phosphate 34 in 75% yield over
two steps. Similarly, removal of the benzyl protecting
groups followed by condensation with Dol-P 6 gave
protected Dol-PP-GlcNAc-Glc 36 in 59% yield. Deprotec-
tion of 36 gave Dol-PP-GlcNAc-Glc 1e in 73% yield after
silica gel chromatography.
In summary, three unnatural dolichol-linked disac-
charides (1c-e) have been prepared from the condensa-
tion of activated sugar phosphates with Dol-P 6. These
analogues are stable in their acetate forms at -20 °C,
and only small amounts of material are deprotected just
1
prior to carrying out biological studies. Both the H and
13C NMR spectra of these compounds are dominated by
signals from the isoprene units of dolichol. In general,
these compounds are best characterized by electrospray
ionization (ESI) mass spectroscopy (negative mode),
which shows the typical Gaussian distribution pattern
due to the dolichol isomers with variable number of
isoprene units.
Biologica l Eva lu a tion
The unnatural dolichol-linked disaccharides (1c-e)
were first evaluated as glycosyl donor substrates for
oligosaccharyl transferase from yeast (Saccharomyces
cerevisiae). The standard OT assay, which uses radio-
labeled Dol-PP-GlcNAc-(3H)-GlcNAc 4 as substrate, is
not applicable for the present study. This method cannot
distinguish whether the unnatural analogues are sub-
strates or inhibitors because both will compete with 4
and result in an apparent decrease in (3H)-glycopeptide
formation. So a modified assay method based on a
radiolabeled peptide developed by Coward35 was em-
ployed for this study. As illustrated in Scheme 8, the
radiolabeled tetrapeptide Bz-Asn-Leu-Thr-Lys(3H-Ac)-
NH2 37r was chosen as the acceptor substrate as a result
of the ease of incorporating the tritium label at the final
stage of synthesis. Upon quenching the enzymatic reac-
tion, both the radiolabeled peptide substrate 37r and the
glycopeptide products partition into the aqueous phase
after successive aqueous-organic extractions. The amount
of product formed is quantified by scintillation counting
after HPLC separation of the (3H)-glycopeptide.
The tetrapeptide Bz-NLTK-NH2 42 was readily pre-
pared by standard Fmoc-based solid-phase peptide syn-
thesis (SPPS) (Scheme 9). Acetylation of Bz-NLTK-NH2
42 gave a quantitative yield of Bz-NLTK(Ac)-NH2 37.
Both tetrapeptides 42 and 37 gave the correct masses
and displayed consistent 1H NMR spectra. The radio-
labeled tetrapeptide Bz-NLTK(3H-Ac)-NH2 37r was pre-
pared by first treating 42 with 0.4 equiv of radioactive
acetic anhydride [(3H)-Ac2O, 100 mCi/mmol] followed by
capping with excess acetic anhydride. The specific activity
of tetrapeptide Bz-NLTK(3H-Ac)-NH2 37r was found to
be 29.3 µCi/µmol on the basis of the radioactivity from a
single peak eluted from HPLC. The Km for Bz-NLTK(Ac)-
NH2 37 was determined to be 4.2 µM (∼6-fold better
compared to the tripeptide Bz-NLT-NHMe).
a
Reagents and conditions: (a) AgOTf, CH2Cl2/toluene, -45 f
0 °C (84%); (b) (1) H2, 10% Pd/C, EtOH; Ac2O, DMAP (cat.),
pyridine, CH2Cl2 (74%); (c) hydrazine monoacetate, DMF (84%);
(d) (1) LiHMDS, THF, -68 °C; (2) tetrabenzyl pyrophosphate 33,
-68 f 0 °C (89%); (e) H2, 10% Pd/C, MeOH (quantitative); (f) (1)
CDI, DMF; (2) Dol-P 6 (59%); (g) NaOMe, CH2Cl2/MeOH (73%).
dibenzyl phosphate 16 using silver trifluoromethane-
sulfonate (AgOTf) as promoter gave 60% yield of a
separable mixture of R- and â-dibenzyl phosphates 27a
and 27b in the ratio of 1.6:1. The use of silver-promoted
phosphorylation on bromide 26 was necessary since the
preliminary attempts at phosphorylation of reducing
sugar 25 failed as a result of the highly insoluble nature
of this compound in solvents such as CH2Cl2, iPr2O, CH3-
CN, and THF. Similarly, R-dibenzyl phosphate 27a was
hydrogenated to give R-phosphate 28, which was further
condensed with Dol-P 6 to give protected Dol-PP-2DFGlc-
GlcNAc 29 in 36% yield. Deacetylation of 29 with sodium
methoxide gave Dol-PP-2DFGlc-GlcNAc 1d .
Syn th esis of Dol-P P -GlcNAc-Glc, 1e. Synthesis of
disaccharide 30 followed the preliminary account by
O’Connor.22 Glycosylation of commercially available bro-
moacetyl glucose 10 and readily available glycosyl ac-
ceptor 1121 with silver trifluoromethanesulfonate (AgO-
Tf)32 as promoter gave the desired disaccharide 30 in 84%
yield (Scheme 7). The linkage of the newly formed
1
glycosidic bond was assigned to be â on the basis of H
NMR analysis; H-1′ appeared as a doublet at 4.37 ppm
with a coupling constant of 8.1 Hz for H-1′-H-2′. The
benzyl groups were replaced with acetate groups by
hydrogenolysis and subsequent acetylation to give 74%
yield of peracetylated Glc-â-(1f4)-GlcNAc 31. The R-ano-
mer was found to be the major compound and could be
crystallized from MeOH/pentane to afford pure R-31, mp
221-223 °C. Chemoselective deacetylation of the ano-
meric acetate in 31 by hydrazine monoacetate followed
by phosphorylation of the reducing sugar with tetra-
benzyl pyrophosphate {[(BnO)2P(O)]2O, 33}33,34 gave
Unnatural dolichol-linked saccharides were first ex-
amined for substrate activity with OT using radiolabeled
(33) Bommer, R.; Kinzy, W.; Schmidt, R. R. Liebigs Ann. Chem.
1991, 425-433.
(34) Oltvoort, J . J .; van Boeckel, C. A. A.; de Koning, J . H.; van
Boom, J . H. Synthesis 1981, 305-308.
(32) Garegg, P. J .; Norberg, T. Acta Chem. Scand. B 1979, 33, 116-
118.
(35) Gibbs, B. S.; Coward, J . K. Bioorg. Med. Chem. 1999, 7, 441-
447.

6222 J . Org. Chem., Vol. 66, No. 19, 2001
Imperiali
Sch em e 8
Glycopeptides Bz-Asp(GlcNAc-â-(1f4)-GlcNAc)-Leu-Thr-
Lys(Ac)-NH2 38 and Bz-Asp(Glc-â-(1f4)-GlcNAc)-Leu-
Thr-Lys(Ac)-NH2 41 displayed the correct masses at
1048.77 (MNa+) and 1007.66 (MNa+), respectively. Masses
corresponding to glycopeptides 39 and 40 were not
detected. Surprisingly, other than the expected masses
(845.30, MNa+; 860.98, MK+) for Bz-Asp(GlcNAc)-Leu-
Thr-Lys(Ac)-NH2 43, a major peak corresponding to
glycopeptide 38 was also observed when using Dol-PP-
GlcNAc 3 as a substrate (43:38 ≈ 1:2 based on peak
intensity). This observation can be best explained by the
presence of endogenous Enzyme II in the microsomal OT
preparation, which biosynthesizes Dol-PP-GlcNAc-GlcNAc
1b when 3 is at a high concentration. This is the first
time that Dol-PP-GlcNAc 3 has been directly demon-
strated to be a substrate for OT, which is in agreement
with an earlier report by Bause12 stating that 3 is a poor
substrate (vide supra).
F igu r e 2. Glycopeptide formation over 2 and 9 h.
Sch em e 9
The kinetic parameters for substrates Dol-PP-GlcNAc-
GlcNAc 1b and Dol-PP-GlcNAc-Glc 1e are shown in
Table 1. Both the Kmapp and Vmaxapp of the in vitro
substrate 1b are in accord with the literature values.35
Interestingly, replacement of the acetamido group in the
distal sugar gave 2.5-fold improvement in binding; on the
other hand, Vmaxapp was lowered by 3.2-fold. Overall, the
Bz-Asn-Leu-Thr-Lys(3H-Ac)-NH2 37r as the acceptor.
Reactions were carried out for 2 and 9 h followed by
HPLC and radioactivity analyses. Generally, the peak
corresponding to the newly formed glycopeptide was not
directly observed by HPLC because of the sensitivity of
the UV detector but was readily revealed by radioactivity
monitoring. The differences in retention times (ca. 4 min)
between peptide 37r and glycopeptides allow easy and
accurate quantification of products. Preliminary screen-
ing results of the unnatural analogues are shown in
Figure 2. Dol-PP-GlcNAc-Glc 1e, with a modification in
the D-site, was found to be a substrate for OT with
comparable activity to the in vitro substrate 1b. Disac-
charide analogues 1c and 1d (50 µM), with modifications
in the P-site, showed no transferase activity over 2 h.
However, a 5-fold increase in concentration of Dol-PP-
GlcNTFA-GlcNAc 1c resulted in 1.7-fold increase in
glycopeptide formation over 9 h. Interestingly, the
monosaccharide derivative Dol-PP-GlcNAc 318 also showed
product formation (11% relative to 1b) by radioactivity
analysis. These experiments were repeated with non-
radiolabeled peptide acceptor 37 and fractions corre-
sponding to the apparent glycopeptide (tR ) 19-20 min)
were submitted for MALDI mass spectroscopy analysis.
unnatural substrate 1e is 76% as effective (Vmaxapp/Kmapp
)
as the in vitro substrate 1b. Because of the complicated
nature of the effects with Dol-PP-GlcNAc 3, further
analysis was not undertaken.
In contrast, analogues 1c and 1d , with a modified C-2
substituent in the proximal sugar, showed no apparent
transferase activity. These analogues were further ana-
lyzed as inhibitors for OT. The sensitivity of our method
allows kinetic studies using initial rates. Variable amounts
of unnatural analogues were competed against constant
amounts of the in vitro substrate 1b (22 µM) and
radiolabeled peptide 37r (21 µM). The approximate Ki’s
were calculated as described by Segel.36
The estimated Ki’s for analogues 1c and 1d are 2.4- to
3.9-fold the Kmapp of 1b. Replacement of the C-2 acetamido
group by the similarly sized trifluoroacetamido group
reduces the binding to the enzyme; however, no signifi-
cant transferase activity was observed. Further reduction
in binding was noted by the smaller 2-deoxy-2-fluoro
(36) Segel, I. H. In Enzyme Kinetics; J ohn Wiley & Sons: New York,
1975; pp 100-107.

Oligosaccharyl Transferase Substrate Specificity
J . Org. Chem., Vol. 66, No. 19, 2001 6223
Ta ble 1. Kin etic Con sta n ts for Com p ou n d s 1b-e
compound
Kmapp (µM)
Vmaxapp (pmol/min)
Vmaxapp/Kmapp (rel)
Ki (µM)
Dol-PP-GlcNAc-GlcNAc 1b
Dol-PP-GlcNAc-Glc 1e
64.3
26.0
10.3
3.2
1
0.76
Dol-PP-GlcNTFA-GlcNAc 1c
Dol-PP-2DFGlc-GlcNAc 1d
154 ( 14
252 ( 71
chemical shifts are reported in ppm relative to 85% phosphoric
acid external standard. Multiplicities are reported in the
following abbreviations: 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 Department of Chemistry, Massachusetts
Institute of Technology, Cambridge, MA 02139, or UCR Mass
Spectrometry Facility, Department of Chemistry, University
of California, Riverside, CA 92521. All reactions were moni-
tored by analytical thin-layer chromatography (TLC) on glass
precoated with silica gel 60 F254 (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 flash
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 and used without further purification.
Dol-PP-GlcNAc-GlcNAc 1b was prepared according to Cow-
ard’s procedure.23 Stock solutions of dolichol-linked saccharides
were quantified by the Barlett assay.37,38
Gen er a l P r oced u r e for Hyd r ogen olysis of Diben zyl
P h osp h a tes 17, 27a , a n d 34. A solution of dibenzyl phosphate
(0.16 mmol) and 10% Pd/C (20 mg) was stirred under H2 in
MeOH (8 mL) until no starting material remained as indicated
by TLC. The catalyst was filtered through a pad of Celite and
washed with MeOH (15 mL). Et3N (1 mL) was added followed
by evaporation of solvent in vacuo to give the mono(triethyl-
ammonium) salt of the phosphate.
Gen er a l P r oced u r e for Cou p lin g of Su ga r P h osp h a te
w ith Dolich ol Mon op h osp h a te.23 To a solution of the sugar
phosphate (0.04 mmol) in DMF (1 mL) at room temperature
was added 1,1′-carbonyldiimidazole (CDI, 0.2 mmol) in one
portion. After 2 h, MeOH (0.36 mmol) was added (to quench
excess CDI) to the mixture and stirred for 30 min. Dolichol
monophosphate 627 (tri-n-butylammonium form, 0.028 mmol)
in CH2Cl2 (1 mL) was then added, and the mixture was allowed
to stir for 3 d 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 combined and
further purified by silica gel chromatography.
2-Ac e t a m id o -3,4,6-t r i-O -a c e t y l-2-d e o x y -â-D -g lu c o -
pyr an osyl-(1f4)-1,3,6-tr i-O-acetyl-2-deoxy-2-tr iflu or oacet-
a m id o-r-D-glu cop yr a n ose 14. To a solution of the amine
hydrochloride 1324 (364.0 mg, 0.5424 mmol) in CH2Cl2 at 0 °C
were added pyridine (219.4 µL, 2.712 mmol) and trifluoroacetic
anhydride (TFAA) (114.9 µL, 0.8137 mmol). The mixture was
allowed to stir at room temperature for 2 h and poured into a
saturated aqueous solution of NH4Cl (5 mL). The organic phase
was separated, washed with brine (5 mL), dried (Na2SO4), and
filtered. Concentration of the filtrate followed by crystallization
from MeOH gave the desired product 14 (383.5 mg, 97%) as a
white solid, mp > 250 °C; IR (neat) cm-1 3295, 1745, 1667,
1558, 1370, 1227, 1157, 1033, 940, 754; 1H NMR (CDCl3, 500
MHz) δ 1.97 (s, 3H, Ac), 2.01 (s, 3H, Ac), 2.02 (s, 3H, Ac), 2.06
(s, 3H, Ac), 2.08 (s, 3H, Ac), 2.17 (s, 3H, Ac), 2.21 (s, 3H, Ac),
3.63 (ddd, 1H, J ) 2.3, 3.8, 9.8 Hz, H-5), 3.77 (t, 1H, J ) 9.5
Hz, H-4), 3.91-3.96 (m, 2H, H-2′, H-5′), 4.02 (dd, 1H, J ) 2.0,
12.5 Hz, H-6a), 4.19 (dd, 1H, J ) 1, 12.2 Hz, H-6a′), 4.32 (m,
F igu r e 3. Substrate specificity of glycosyl donor for oligosac-
charyl transferase.
derivative. Although the mode of inhibition was not
determined in this study, it is most likely that these
inhibitors act in a competitive fashion. The large experi-
mental deviation of the Ki value for 2-deoxy-2-fluoro
analogue 1d suggests that the mode of inhibition may
be more complicated.
The substrate specificity of glycosyl donor for yeast OT
is summarized in Figure 3. A minimum requirement for
glycosyl donors in OT catalysis is an N-acetylglucosamine
(GlcNAc) unit as demonstrated by the glycopeptide 43
formed from Dol-PP-GlcNAc 3. The efficiency of this
transferase process can be improved by the addition of a
second sugar. The requirement for an N-acetyl-glucos-
amine in the distal site is not absolute since substitution
with a glucose unit (as in Dol-PP-GlcNAc-Glc 1e) also
results in comparable transferase activity. The presence
of the distal sugar may simply assist the binding of the
glycosyl donor. On the other hand, the acetamido group
in the proximal sugar plays a significant role in OT
catalysis. Minor changes in the acetamido group resulted
in significant loss of activity as in the case of the
trifluoroacetamido derivative 1c or total loss of activity
as in 1d . The loss of transferase activity cannot be simply
explained by the reduced binding (2.4- to 3.9-fold less
than 1b) of the unnatural analogues, the acetamido group
is therefore proposed to be involved in the catalytic
machinery. The acetamido group may interact with the
enzyme through critical hydrogen bonding with the
catalytic site. Additionally, the low transferase activity
of 1c may be explained by the strong electron-withdraw-
ing effect of the trifluoromethyl group, rendering the
adjacent carbonyl a less efficient hydrogen bond acceptor.
Exp er im en ta l Section
Melting points were uncorrected. Optical rotations were
measured with a Perkin-Elmer 241 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 INOVA 500, Bruker AM-500
1
(500.15 MHz for H, 125 MHz for 13C), or Varian Mercury 300
(37) Barlett, G. R. J . Biol. Chem. 1959, 234, 466-468.
(38) New, R. R. C. In Liposomes, A Practical Approach; New, R. R.
C., Ed.; Oxford University Press: New York, 1990; pp 105-107.
(75 MHz for 13C). All chemical shifts were recorded in ppm
downfield from tetramethylsilane on the δ scale. 31P NMR

6224 J . Org. Chem., Vol. 66, No. 19, 2001
Imperiali
1H, H-2), 4.39 (dd, 1H, J ) 4.1, 12.5 Hz, H-6b), 4.47 (dd, 1H,
J ) 3.4, 12.2 Hz, H-6b′), 4.49 (d, 1H, J ) 8.2 Hz, H-1′), 5.06 (t,
1H, J ) 9.5 Hz, H-3′), 5.15 (dd, 1H, J ) 9.5, 10.3 Hz, H-4′),
5.29 (dd, 1H, J ) 9.1, 10.9 Hz, H-3), 5.88 (d, 1H, J ) 9.1 Hz,
NH), 6.18 (d, 1H, J ) 3.6 Hz, H-1), 6.59 (d, 1H, J ) 8.5 Hz,
NH); 13C NMR (CDCl3, 125 MHz) δ 20.33, 20.56, 20.59, 20.65,
20.91, 20.99, 23.19, 51.91, 54.44, 61.30, 61.61, 67.78, 70.06,
70.84, 71.98, 72.44, 75.66, 89.43, 101.78, 168.67, 169.28,
170.48, 170.54, 170.81, 171.24, 171.61; 19F NMR (CDCl3, 470.6
MHz) δ -76.6; [R]D +16.8° (c ) 0.7, CHCl3); MS (FAB) 731
(MH+); HRMS m/z calcd for C28H38F3N2O17 731.212258, found
731.211900.
17 (51.8 mg, 0.05460 mmol) gave R-phosphate 18 (51.7 mg,
100%) as the mono(triethylammonium) salt; IR (neat) cm-1
3303, 1745, 1705, 1662, 1374, 1225, 1166, 1048, 967; 1H NMR
[CDCl3, CD3OD (2:1v/v), 500 MHz] δ 1.10 (t, 9H, J ) 7.3 Hz),
1.67 (s, 3H, Ac), 1.80 (s, 3H, Ac), 1.81 (s, 3H, Ac), 1.81 (s, 3H,
Ac), 1.88 (s, 3H, Ac), 1.92 (s, 3H, Ac), 2.87 (q, 6H, J ) 7.3 Hz),
3.45 (dd, 1H, J ) 8.5, 10.3 Hz), 3.52 (ddd, 1H, J ) 2.2, 3.7,
10.0 Hz), 3.66 (t, 1H, J ) 9.6 Hz), 3.81 (dd, 1H, J ) 2.0, 12.4
Hz), 3.87 (dd, 1H, J ) 3.5, 12.2 Hz), 3.97 (brd, 1H, J ) 9.8
Hz), 4.06 (brd, 1H, J ) 12.1 Hz), 4.22 (hidden under DOH
signal), 4.28 (brd, 1H, J ) 12.1 Hz), 4.54 (d, 1H, J ) 8.3 Hz,
H-1′), 4.80 (t, 1H, J ) 9.5 Hz), 5.13 (dd, 1H, J ) 9.4, 10.4 Hz),
5.15 (dd, 1H, J ) 9.2, 10.5 Hz), 5.28 (dd, 1H, J ) 3.1, 7.3 Hz,
H-1); 13C NMR (CDCl3, 125 MHz) δ 7.99, 19.92, 19.94, 19.99,
20.05, 22.23, 22.03, 45.41, 52.64 (d, J ) 6.7 Hz), 54.74, 61.51,
68.16, 69.00, 70.87, 71.10, 71.86, 75.17, 92.55 (d, J ) 5.3 Hz),
100.09, 169.62, 170.35, 170.43, 170.70, 171.13, 171.44; [R]D
+23.0° (c ) 0.7, MeOH); MS (FAB) 767 (M-); HRMS m/z calcd
for C26H35F3N2O19P 767.152376, found 767.149500.
2-Aceta m id o-3,4,6-tr i-O-a cetyl-2-d eoxy-â-D-glu cop yr a -
n osyl-(1f4)-2-t r iflu or om e t h yl-(2-a ce t a m id o-3,6-d i-O-
a cetyl-1,2-d id eoxy-r-D-glu cop yr a n o)-[2,1,d ]-2-oxa zolin e
15. To a solution of compound 14 (352.0 mg, 0.4818 mmol) in
CH2Cl2 (10 mL) was added a solution of hydrogen bromide in
acetic acid (30%, 2.5 mL). The mixture was stirred at room
temperature for 3 h. The hydrogen bromide was then blown
off under N2, and the remaining acetic acid was coevaporated
twice with toluene. The crude bromide was stirred with 2,6-
lutidine (112 µL, 0.9636 mmol) in CH3CN (10 mL) at room
temperature for 13 h. Concentration of the solvent followed
by column chromatography [CHCl3, MeOH (96:4 v/v)] gave
oxazoline 15 (197.7 mg, 61%) as a white solid, mp 196s198
°C; IR (neat) cm-1 3276, 1741, 1664, 1555, 1370, 1229, 1166,
1044, 922; 1H NMR (CDCl3, 500 MHz) δ 1.93 (s, 3H, Ac), 2.01
(s, 3H, Ac), 2.02 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.11 (s, 3H, Ac),
2.12 (s, 3H, Ac), 3.44 (ddd, 1H, J ) 2.6, 4.7, 9.0 Hz, H-5), 3.64
(brd, 1H, J ) 9.2 Hz, H-4), 3.70-3.75 (m, 2H, H-2′, H-5′), 4.14
(dd, 1H, J ) 2.5, 12.3 Hz, H-6a′), 4.19-4.26 (m, 3H, H-6a,b,
H-6b′), 4.33 (brd, 1H, J ) 7.2 Hz, H-2), 4.89 (dd, 1H, J ) 8.4
Hz, H-1′), 5.07 (t, 1H, J ) 9.5 Hz, H-3′), 5.32 (dd, 1H, J ) 9.4,
10.4 Hz, H-4′), 5.71 (t, 1H, J ) 1.1 Hz, H-3), 5.77 (d, 1H, J )
8.3 Hz, NH), 6.22 (d, 1H, J ) 7.4 Hz, H-1); 13C NMR (CDCl3,
125 MHz) δ 20.62, 20.67, 20.71, 20.76, 20.89, 23.14, 55.01,
62.81, 62.84, 64.46, 68.36, 68.87, 69.59, 71.93, 72.23, 76.34,
101.60, 101.91, 169.03, 169.40, 170.54, 170.66, 170.80, 170.91;
[R]D +0.7° (c ) 1.2, CHCl3); MS (FAB) 693 (MNa+); HRMS m/z
calcd for C26H33F3N2O15Na 693.173073, found 693.475600.
Diben zyl 2-Aceta m id o-3,4,6-tr i-O-a cetyl-2-d eoxy-â-D-
g lu c o p y r a n o s y l-(1f4)-3,6-d i-O -a c e t y l-2-d e o x y -2-t r i-
flu or oa ceta m id o-r-D-glu cop yr a n osyl P h osp h a te 17. A
mixture of oxazoline 15 (178.3 mg, 0.2659 mmol) and dibenzyl
phosphate 16 (111 mg, 0.3989 mmol) was heated in 1,2-
dichloroethane at 80-90 °C for 5 h. The mixture was cooled
and diluted with CHCl3 (10 mL). The solution was washed
successively with saturated aqueous NaHCO3 solution (4 mL)
and brine (4 mL). The combined organic extracts were dried
(Na2SO4), filtered, and concentrated. Flash column chroma-
tography [CHCl3, MeOH (96:4 v/v)] of the crude product gave
R-dibenzyl phosphate 17 (161.6 mg, 64%) as a white solid, mp
164 °C (decompose): IR (neat) cm-1 3285, 1746, 1673, 1556,
P 1-[2-Acetam ido-3,4,6-tr i-O-acetyl-2-deoxy-â-D-glu copy-
r a n osyl-(1f4)-3,6-d i-O-a cet yl-2-d eoxy-2-t r iflu or oa cet a -
m id o-r-D-glu cop yr a n osyl] P 2-Dolich yl P yr op h osp h a t e
19. 19F NMR [CDCl3, CD3OD (5:1 v/v) 282 MHz] δ -76.7; 31P
NMR [CDCl3, CD3OD (5:1 v/v), 121 MHz] δ -10.5, -12.7; MS
(-ve ESI) 1003.3, 1037.4, 1071.4, 1105.4 (m/2 for n ) 13-16).
P 1-[2-Aceta m id o-2-d eoxy-â-D-glu cop yr a n osyl-(1f4)-2-
d eoxy-2-tr iflu or oa ceta m id o-r-D-glu cop yr a n osyl] P 2-Doli-
ch yl P yr op h osp h a te 1c. To a stirred solution of peracety-
lated Dol-PP-GlcNTFA-GlcNAc 19 (6.2 mg, 2.85 µmol) 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 50 × 8, pyridinium form, 200 mg) was
added, and the mixture was stirred for 10 min. The resin was
then filtered and washed with CHCl3/MeOH (2:1 v/v). Con-
centration of the solvent followed by column chromatography
[CHCl3, MeOH, H2O (72:21:2 v/v)] gave Dol-PP-GlcNTFA-
GlcNAc 1c (2.3 mg, 39%); MS (-ve ESI) 1798.7, 1865.2, 1933.7,
2001.8 (M- for n ) 13-16). [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 (9:1 v/v, 10 mL).]
4,6-Di-O-b en zylid en e-2-d eoxy-2-flu or o-r,â-D-glu cop y-
r a n ose 21. A solution of 2-deoxy-2-fluoro-1,3,4,6-â-D-glucose
tetraacetate 2029 (2.50 g, 7.14 mmol) and sodium methoxide
(193 mg, 3.57 mmol) was stirred in MeOH (50 mL) for 3 h
followed by neutralization with Dowex 50 W-X8 (H+) resin (1.7
g). The resin was filtered, and the solvent was removed in
vacuo to give 2-deoxy-2-fluoroglucose (1.4497 g). A solution of
the free sugar, benzaldehyde dimethyl acetal (1.18 mL, 7.85
mmol), and p-toluenesulfonic acid (136 mg) in DMF (1.5 mL)
was heated at 50 °C in a water bath at 20 mmHg. After 7.5 h,
NaHCO3 (180 mg, 2.142 mmol) was added followed by the
removal of solvent. To the crude product was added EtOAc/
MeOH (50 mL) and MgSO4. After 30 min, the suspension was
1
1370, 1229, 1157, 1044, 958, 745, 700; H NMR (CDCl3, 500
MHz) δ 1.92 (s, 3H, Ac), 1.99 (s, 3H, Ac), 2.01 (s, 3H, Ac), 2.02
(s, 3H, Ac), 2.06 (s, 3H, Ac), 2.09 (s, 3H, Ac), 3.61 (ddd, 1H, J
) 2, 4, 10 Hz), 3.73 (t, 1H, J ) 9.5 Hz), 3.78 (dt, 1H, J ) 8.5,
10.5 Hz), 3.91 (dt, 1H, J ) 2.5, 10.5 Hz), 3.99 (dd, 1H, J ) 2.0,
12.5 Hz), 4.05 (dd, 1H, J ) 2.0, 12.5 Hz), 4.27 (dd, 1H, J )
3.5, 12.5 Hz), 4.37 (dd, 1H, J ) 4, 12 Hz), 4.56 (d, 1H, J ) 8.0
Hz), 4.99-5.07 (m, 5H), 5.18 (dd, 1H, J ) 9.5, 10.5 Hz), 5.21
(dd, 1H, J ) 9.5, 10.5 Hz), 5.65 (dd, 1H, J ) 4.0, 6.0 Hz), 5.80
(d, 1H, J ) 9.0 Hz), 6.76 (d, 1H, J ) 9.0 Hz), 7.29-7.37 (m,
10H); 13C NMR (CDCl3, 125 MHz) δ 20.02, 20.32, 20.56, 20.59,
20.63, 20.88, 23.16, 52.73, 52.76 (d, J ) 7.5 Hz), 55.02, 61.10,
61.70, 68.04, 69.74, 70.14 (d, J ) 5.5 Hz), 70.25 (d, J ) 5.4
Hz), 70.74, 71.98, 72.28, 74.79, 75.18, 94.82 (d, J ) 5.6 Hz),
101.11, 128.12, 128.15, 128.77, 128.81, 129.03, 134.96, 135.01,
169.33, 170.32, 170.47, 170.67, 170.97 (2×); [R]D +32.1° (c )
1.1, CHCl3); MS (FAB) 971 (MNa+); HRMS m/z calcd for
filtered through
a pad of silica gel topped with Celite.
Concentration of the solvent followed by column chromatog-
raphy [n-hex, EtOAc (1:1 v/v)] gave the desired compound 21
(1.4692 g, 76%) as a white solid: selected peaks for 1H NMR
(CDCl3, 500 MHz) δ 4.95 (ddd, J ) 3.7, 5.8, 9.8 Hz, H-1 for
â-21), 5.47 (t, J ) 3.7 Hz, H-1 for R-21); 19F NMR (CDCl3, 282
MHz) δ -200.91 (dd, J ) 16, 52 Hz), -201.38 (dd, J ) 14, 49
Hz); MS (FAB) 271 (MH+); HRMS m/z calcd for C24H35FNO15
596.199073, found 596.200600.
1,3-Di-O-ben zyl-4,6-d i-O-ben zylid en e-2-d eoxy-2-flu or o-
r-D-glu cop yr a n ose 22a , a n d 1,3-Di-O-ben zyl-4,6-d i-O-ben -
zylid en e-2-d eoxy-2-flu or o-â-D-glu cop yr a n ose 22b. To a
stirred solution of reducing sugar 21 (200.0 mg, 0.7401 mmol)
in DMF (3 mL) at 0 °C was added NaH (60%, 74.0 mg, 1.8501
mmol) in one portion. After 10 min, benzyl bromide (264 µL,
2.2202 mmol) was added, and the mixture was allowed to stir
at 0 °C for 1 h. The mixture was diluted with EtOAc (50 mL)
and water (25 mL). The aqueous phase was separated and
extracted with EtOAc (2 × 25 mL). The combined organic
extracts were washed with brine (25 mL), dried (MgSO4),
C
40H48F3N2O19PNa 971.243871, found 971.246000.
Mon o(tr ieth yla m m on iu m ) Sa lt of 2-Aceta m id o-3,4,6-
tr i-O-a cetyl-2-d eoxy-â-D-glu cop yr a n osyl-(1f4)-3,6-d i-O-
a cet yl-2-d eoxy-2-t r iflu or oa cet a m id o-r-D-glu cop yr a n o-
syl P h osp h a te 18. Hydrogenolysis of R-dibenzyl phosphate

Oligosaccharyl Transferase Substrate Specificity
J . Org. Chem., Vol. 66, No. 19, 2001 6225
filtered, and concentrated. Flash column chromatography
[n-hex, EtOAc (9:1 v/v) followed by (7:1 v/v)] of the crude solid
gave first â-glucoside 22b (181.0 mg, 54%) followed by R-glu-
coside 22a (101.8 mg, 31%) both as white solids. Da ta for th e
r-glu cosid e 22a : mp 119-121 °C; IR (neat) cm-1 2915, 2865,
1453, 1370, 1352, 1178, 1162, 1135, 1085, 1067, 1054, 1027,
1H, J ) 1.8, 3.8, 9.8 Hz, H-5′), 3.28 (dd, 1H, J ) 8.2, 9.8 Hz,
H-4′), 3.32 (dd, 1H, J ) 7.8, 9.8 Hz, H-2′), 3.48 (ddd, 1H, J )
1.8, 3.8, 9.9 Hz, H-5), 3.55 (dd, 1H, J ) 4.0, 11 Hz, H-6a′),
3.61 (dd, 1H, J ) 2.0, 11 Hz, H-6b′), 3.65 (dd, 1H, J ) 8.5, 9.6
Hz, H-3′), 3.70 (dt, 1H, J ) 8.7, 15.2 Hz, H-3), 3.81 (dd, 1H, J
) 1.8, 11.1 Hz, H-6a), 3.88 (dd, 1H, J ) 3.8, 11.1 Hz, H-6b),
4.02 (t, 1H, J ) 9.4 Hz, H-4), 4.35 (d, 1H, J ) 7.6 Hz, H-1′),
4.38 (dt, 1H, J ) 8.4, 50.5 Hz, H-2), 4.39 and 4.43 (ABq, 2H,
J ) 12.2 Hz, OCH2Ph), 4.53 (dd, 1H, J ) 2.7, 7.8 Hz, H-1),
4.53 (d, 1H, J ) 12.5 Hz), 4.55 (d, 1H, J ) 11.0 Hz), 4.69 (d,
2H, J ) 12.1 Hz), 4.76 and 4.90 (ABq, 2H, J ) 11.9 Hz), 4.77
(d, 1H, J ) 11.1 Hz), 4.79 and 4.83 (ABq, 2H, J ) 10.8 Hz,
OCH2Ph), 4.94 (d, 1H, J ) 12.2 Hz), 7.16-7.39 (m, 30H,
OCH2Ph); 13C NMR (CDCl3, 125 MHz) δ 66.88, 68.01, 68.33,
70.65, 73.31, 73.44, 74.23, 74.74, 74.81, 75.03, 75.50, 76.03 (d,
J ) 8.1 Hz), 77.67, 81.07 (d, J ) 17.3 Hz), 83.13, 92.37 (d, J )
183 Hz), 99.17 (d, J ) 23.6 Hz), 100.84, 127.33, 127.47, 127.55,
127.62, 127.70, 127.77, 127.85, 127.90, 127.99, 128.09, 128.27,
128.40, 128.41, 128.46, 136.88, 137.80, 137.88, 127.92, 138.11,
138.55; [R]D -27.7° (c ) 1.1, CHCl3); MS (FAB) 933 (MNa+);
HRMS m/z calcd for C54H56FN3O9Na 932.389827, found
932.392900.
1
1017, 1000, 750, 732, 696, 660; H NMR (CDCl3, 500 MHz) δ
3.62 (dt, 1H, J ) 0.9, 9.5 Hz, H-4), 3.73 (t, 1H, J ) 10.4, H-6a),
3.93 (dt, 1H, J ) 4.9, 10.1 Hz, H-5), 4.17 (dt, 1H, J ) 9.5, 11.6
Hz, H-3), 4.24 (dd, 1H, J ) 4.9, 10.4 Hz, H-6b), 4.53 (ddd, 1H,
J ) 4.0, 8.8, 49 Hz, H-2), 4.64 and 4.78 (ABq, 2H, J ) 12.2
Hz, OCH2Ph), 4.82 and 4.87 (ABq, 2H, J ) 11.6 Hz, OCH2-
Ph), 5.56 (s, 1H, CHPh), 7.26-7.50 (m, 15H, Ph); 13C NMR
(CDCl3, 125 MHz) δ 62.56, 68.79, 69.87, 74.73, 76.73 (d, J )
18 Hz), 81.09 (d, J ) 8 Hz), 90.29 (d, J ) 195 Hz), 96.19 (d, J
) 20 Hz), 101.33, 126.00, 127.81, 127.90, 128.02, 128.22,
128.29, 128.49, 128.98, 136.65, 137.09, 138.23; 19F NMR
(CDCl3, 282 MHz) δ -200.88 (dd, J ) 13, 49 Hz); [R]D +70.0°
(c ) 1.4, CHCl3); MS (FAB) 451 (MH+); HRMS m/z calcd for
C27H28FNO5 451.192077, found 451.191800. Da ta for th e
â-glu cosid e 22b: mp 118-119 °C; IR (neat) cm-1 2872, 1451,
1369, 1174, 1092, 1067, 1010, 744, 697; 1H NMR (CDCl3, 500
MHz) δ 3.44 (dt, 1H, J ) 4.9, 10 Hz, H-5), 3.72 (t, 1H, J ) 9.5
Hz, H-4), 3.82 (t, 1H, J ) 10.5 Hz, H-6a), 3.84 (dt, 1H, J )
8.8, 15.3 Hz, H-3), 4.40 (dd, 1H, J ) 4.9, 10.5 Hz, H-6b), 4.45
(dt, 1H, J ) 7.9, 50 Hz, H-2), 4.67 (dd, 1H, J ) 4.0, 7.6 Hz,
H-1), 4.71 and 4.95 (ABq, 2H, J ) 12 Hz, OCH2Ph), 4.84 and
4.88 (ABq, 2H, J ) 11.9 Hz, OCH2Ph), 5.58 (s, 1H, CHPh),
7.26-7.51 (m, 15H, Ph); 13C NMR (CDCl3, 125 MHz) δ 66.17,
68.54, 71.16, 74.33, 78.84 (d, J ) 18 Hz), 80.37 (d, J ) 9 Hz),
92.84 (d, J ) 188 Hz), 99.89 (d, J ) 24 Hz), 101.30, 126.00,
127.75, 127.82, 127.92, 128.03, 128.25, 128.31, 128.49, 129.06,
136.54, 137.01, 137.89; 19F NMR (CDCl3, 282 MHz) δ -198.34
(dd, J ) 14, 47 Hz); [R]D -62.4° (c ) 1, CHCl3); MS (FAB) 451
(MH+); HRMS m/z calcd for C27H28FNO5 451.192077, found
451.191400.
2-Aceta m id o-3,4,6-tr i-O-a cetyl-2-d eoxy-â-D-glu cop yr a -
n osyl-(1f4)-1,3,6-tr i-O-a cetyl-2-d eoxy-2-flu or o-r,â-D-glu -
cop yr a n ose 24. A solution of disaccharide 23 (190.0 mg,
0.2088 mmol) and a catalytic amount of Raney nickel was
stirred under H2 in acetic anhydride (3 mL) and EtOH (10 mL)
for 6 h. The catalyst was filtered through a pad of Celite, and
the solvent was removed under reduced pressure. The crude
product was passed through a short plug of silica and washed
with CHCl3, MeOH (5:1 v/v) to give the acetamido compound.
This compound was hydrogenated with 10% Pd/C (110 mg) in
EtOH (25 mL) for 12 h. The catalyst was again filtered through
Celite and washed with MeOH (20 mL). Concentration of the
filtrate gave the free disaccharide (108.3 mg), which was
acetylated with Ac2O (2 mL) and DMAP (cat) in pyridine (8
mL) for 3 h. Concentration of the solvent followed by column
chromatography [CHCl3, MeOH (96:4 v/v)] gave the heptaac-
etate 24 (128.1 mg, 96%, R:â ≈ 2:1) as a white solid: IR (neat)
cm-1 1747, 1662, 1531, 1372, 1227, 1045; selected peaks for
1H NMR (CDCl3, 500 MHz) of R-anomer δ 6.35 (d, J ) 4.0 Hz,
H-1) and 4.64 (d, J ) 8.2 Hz, H-1′); â-anomer δ 5.71 (dd, J )
3.1, 8.2 Hz, H-1) and 4.77 (d, J ) 8.2 Hz, H-1′); MS (FAB) 660
(MNa+); HRMS m/z calcd for C26H36FNO16Na 660.191581,
found 660.190500.
1,3,6-Tr i-O-b e n zyl-2-d e oxy-2-flu or o-â-D -glu cop yr a -
n ose 9. To a mixture of benzylidene 22b (332.0 mg, 0.737
mmol) and sodium cyanoborohydride (695 mg, 11.05 mmol)
in THF (15 mL) at 0 °C was added HCl/Et2O solution until no
bubble was observed. After 2 h, brine (25 mL) and Et2O (100
mL) were added. The aqueous phase was separated and
extracted with Et2O (2 × 50 mL). The combined organic
extracts were dried, filtered, and concentrated. Flash column
chromatography [n-hex, EtOAc (3:1 v/v)] of the crude mixture
gave glycosyl acceptor 9 (293.8 mg, 88%) as an oil; IR (neat)
cm-1 3467, 3030, 2871, 1497, 1454, 1365, 1209, 1124, 1074,
1005, 737, 698; 1H NMR (CDCl3, 500 MHz) δ 2.55 (d, 1H, J )
2.2 Hz, OH), 3.46 (ddd, 1H, J ) 4.0, 5.2, 9.4 Hz, H-5), 3.56 (dt,
1H, J ) 8.5, 14.6 Hz, H-3), 3.66 (dt, 1H, J ) 2.2, 9.4 Hz, H-4),
3.72 (dd, 1H, J ) 5.2, 10.5 Hz, H-6a), 3.78 (dd, 1H, J ) 4.0,
10.5 Hz, H-6b), 4.40 (ddd, 1H, J ) 7.6, 8.5, 51 Hz, H-2), 4.56
(dd, 1H, J ) 3.1, 7.6 Hz, H-1), 4.62 and 4.57 (ABq, 2H, J )
12.2 Hz, OCH2Ph), 4.69 (d, 2H, J ) 11.6 Hz, OCH2Ph), 4.92
(dd, 1H, J ) 0.9, 11.5 Hz, OCH2Ph), 4.93 (d, 1H, OCH2Ph),
7.28-7.39 (m, 15H, OCH2Ph); 13C NMR (CDCl3, 125 MHz) δ
69.72, 70.68, 70.74, 73.64, 74.16, 74.46 (d, J ) 2 Hz), 82.45 (d,
J ) 16 Hz), 92.86 (d, J ) 187 Hz), 99.10 (d, J ) 23 Hz), 127.67,
127.77, 127.88, 127.89, 127.99, 128.06, 128.43, 128.53, 136.78,
137.76, 137.91; 19F NMR (CDCl3, 282 MHz) δ -197.52 (dd, J
) 14, 49 Hz); [R]D -69.4° (c ) 1, CHCl3); MS (FAB) 453 (MH+);
HRMS m/z calcd for C27H30FNO5 453.207728, found 453.207200.
2-Azid o-3,4,6-tr i-O-ben zyl-2-d eoxy-â-D-glu cop yr a n osyl-
(1f4)-1,3,6-tr i-O-ben zyl-2-d eoxy-2-flu or o-â-D-glu cop yr a -
n ose 23. To a stirred solution of compound 9 (135.4 mg, 0.2992
mmol), azido imidate 8 (260.0 mg, 0.4194 mmol), and 4 Å
molecular sieves (500 mg) in CH2Cl2 (5 mL) at -78 °C was
added BF3‚Et2O (38 µL, 0.2992 mmol). The mixture was
allowed to warm to 5 °C over 5 h, followed by the addition of
Et3N (0.5 mL). The mixture was filtered through a pad of Celite
and washed with CHCl3. Concentration of the solvent followed
by column chromatography [n-hex, EtOAc (6:1 v/v) followed
by (4.5:1 v/v)] gave the desired disaccharide 23 (227.2 mg, 83%)
as an oil: IR (neat) cm-1 2913, 2862, 2108, 1496, 1453, 1362,
1056, 1026, 733, 692; 1H NMR (CDCl3, 500 MHz) δ 3.18 (ddd,
2-Aceta m id o-3,4,6-tr i-O-a cetyl-2-d eoxy-â-D-glu cop yr a -
n osyl-(1f4)-3,6-d i-O-a cetyl-2-d eoxy-2-flu or o-r-D-glu cop y-
r a n osyl Br om id e 26. To a stirred solution of anomeric
acetates 24 (120.0 mg, 0.1882 mmol) in CH2Cl2 at 0 °C was
added a solution of hydrogen bromide in acetic acid (30%, 3
mL). The mixture was allowed to warm to room temperature
and stirred for an extra 3.5 h. The solvent was then concen-
trated and coevaporated twice with toluene (4 mL). Flash
column chromatography [CHCl3, CH3CN (2:1 v/v)] of the crude
oil gave R-bromide 26 (105.0 mg, 85%) as a white solid, mp
210 ° C (decompose); IR (neat) cm-1 1746, 1666, 1531, 1436,
1372, 1227, 1113, 1047, 903; 1H NMR (CDCl3, 500 MHz) δ 1.94
(s, 3H, Ac), 2.01 (s, 3H, Ac), 2.02 (s, 3H, Ac), 2.10 (s, 3H, Ac),
2.12 (s, 6H, 2 × Ac), 3.71 (ddd, 1H, J ) 2.1, 5.0, 10.1 Hz, H-5′),
3.73 (dt, 1H, J ) 8.4, 10.5 Hz, H-2′), 3.82 (t, 1H, J ) 9.9 Hz,
H-4), 4.07 (dd, 1H, J ) 2.4, 12.4 Hz, H-6a′), 4.29 (dd, 1H, J )
4.3, 12.5 Hz, H-6a), 4.31 (dd, 1H, J ) 5.0, 12.5 Hz, H-6b′), 4.36
(ddd, 1H, J ) 2.0, 4.1, 10.4 Hz, H-5), 4.41 (dd, 1H, J ) 2.0,
12.2 Hz, H-6b), 4.46 (ddd, 1H, J ) 4.3, 9.2, 50 Hz, H-2), 4.88
(d, 1H, J ) 8.4 Hz, H-1′), 5.02 (dd, 1H, J ) 9.3, 10.1 Hz, H-3′),
5.33 (dd, 1H, J ) 9.2, 10.5 Hz, H-4′), 5.63 (dt, 1H, J ) 9.3,
11.6 Hz, H-3), 5.69 (d, 1H, J ) 8.7 Hz, NH), 6.48 (d, 1H, J )
4.9 Hz, H-1); 13C NMR (CDCl3, 125 MHz) δ 20.52, 20.54, 20.67,
20.74, 20.76, 23.15, 55.28, 61.35, 61.98, 68.38, 70.17 (d, J )
19 Hz), 71.97, 72.06, 73.12, 74.21 (d, J ) 6.4 Hz), 85.52 (d, J
) 25 Hz), 86.62 (d, J ) 198 Hz), 99.23, 169.33, 169.88, 170.34,
170.42, 170.47; [R]D +87.4° (c ) 0.6, CHCl3); MS (FAB) 680,
682 (MNa+); HRMS m/z calcd for C24H33BrFNO14Na 680.096612,
found 680.094500.

6226 J . Org. Chem., Vol. 66, No. 19, 2001
Imperiali
Diben zyl 2-Aceta m id o-3,4,6-tr i-O-a cetyl-2-d eoxy-â-D-
glu cop yr a n osyl-(1f4)-3,6-d i-O-a cetyl-2-d eoxy-2-flu or o-r-
D-glu cop yr a n osyl P h osp h a te 27a a n d Diben zyl 2-Aceta -
m ido-3,4,6-tr i-O-acetyl-2-deoxy-â-D-glu copyr an osyl-(1f4)-
3,6-di-O-acetyl-2-deoxy-2-flu or o-â-D-glu copyr an osyl P h os-
p h a te 27b. To a stirred solution of R-bromide 26 (28.5 mg,
0.04329 mmol) and dibenzyl phosphate 16 (24.1 mg, 0.08657
mmol) in THF (1.5 mL) at room temperature was added a
solution of AgOTf (12.2 mg) in THF (94 µL). After 30 min, Et3N
(4 drops) was added, and the silver salt was filtered through
a plug of glass wool. Concentration of the solvent followed by
flash chromatography [CHCl3, CH3CN (3:2 v/v) followed by (1:1
v/v)] gave first R-dibenzyl phosphate 27a (13.9 mg, 38%)
followed by â-dibenzyl phosphate 27b (8.5 mg, 23%), both as
oil. Da ta for r-d iben zyl p h osp h a te 27a : IR (neat) cm-1 1747,
1667, 1369, 1227, 1154, 1044, 959, 907, 743, 697; 1H NMR
(CDCl3, 500 MHz) δ 1.92 (s, 3H, Ac), 2.00 (s, 3H, Ac), 2.01 (s,
3H, Ac), 2.04(s, 3H, Ac), 2.07(s, 3H, Ac), 2.10(s, 3H, Ac), 3.63
(ddd, 1H, J ) 2.1, 4.0, 10 Hz, H-5′), 3.65 (dt, 1H, J ) 8.2, 10.4
Hz, H-2′), 3.70 (dd, 1H, J ) 9.5, 9.8 Hz, H-4), 4.00 (dd, 1H, J
) 2.1, 12.5 Hz, H-6a′), 4.03 (ddd, 1H, J ) 1.8, 4.0, 9.8 Hz, H-5),
4.11 (dd, 1H, J ) 1.8, 12.2 Hz, H-6a), 4.18 (dd, 1H, J ) 4.0,
12.2 Hz, H-6b), 4.38 (dd, 1H, J ) 4.0, 12.5 Hz, H-6b′), 4.49
(ddt, 1H, J ) 3.7, 9.8, 49 Hz, H-2), 4.70 (d, 1H, J ) 8.2 Hz,
H-1′), 5.03 (dd, 1H, J ) 9.3, 10 Hz, H-4′), 5.07-5.09 (m, 4H),
5.31 (dd, 1H, J ) 9.3, 10.4 Hz, H-3′), 5.52 (dt, 1H, J ) 9.5,
11.4 Hz, H-3), 5.78 (d, 1H, J ) 8.2 Hz, NH), 5.93 (dd, 1H, J )
3.6, 6.7 Hz, H-1), 7.33-7.38 (m, 10H, Ph); 13C NMR (CDCl3,
125 MHz) δ 20.59, 20.62, 20.66, 20.75, 20.83, 23.18, 55.30,
61.25, 61.66, 68.10, 69.50, 69.61 (d, J ) 5.8 Hz), 69.86 (d, J )
4.6 Hz), 70.24, 71.79, 72.01, 74.93 (d, J ) 6.9 Hz), 86.89 (dd,
J ) 8, 191 Hz), 93.50 (dd, J ) 4, 22 Hz), 100.17, 127.90, 128.15,
128.64, 128.71, 128.73, 135.20 (d, J ) 8 Hz), 135.32 (d, J ) 8
Hz), 169.39, 169.75, 170.41, 170.52, 170.57, 170.81; 19F NMR
(CDCl3, 282 MHz) δ -199.89 (dd, J ) 12.1, 49 Hz); 31P NMR
(CDCl3, 121 MHz) δ -2.08; [R]D +5.8° (c ) 1.0, CHCl3); MS
(FAB) 878 (MNa+); HRMS m/z calcd for C38H47FNO18PNa
878.241249, found 878.243300. Da ta for â-d iben zyl p h os-
p h a te 27b: IR (neat) cm-1 1748, 1672, 1556, 1456, 1370, 1229,
3.94 (ddd, 1H, J ) 2.0, 3.7, 10.1 Hz, H-5), 4.20 (dddd, 1H, J )
2.4, 3.7, 9.6, 50 Hz, H-2), 4.22 (dd, 1H, J ) 4.0, 12.4 Hz, H-6b′),
4.26 (dd, 1H, J ) 2.0, 12.5 Hz, H-6b), 4.55 (d, 1H, J ) 8.4 Hz,
H-1′), 4.78 (dd, 1H, J ) 9.3, 10.1 Hz, H-4′), 5.16 (dd, 1H, J )
9.3, 10.5 Hz, H-3′), 5.25 (dt, 1H, J ) 9.5, 11.9 Hz, H-3), 5.49
(dd, 1H, J ) 3.7, 6.9 Hz, H-1); 13C NMR [(CDCl3, CD3OD (2:1
v/v), 125 MHz] δ 7.90, 19.83, 19.91, 19.99, 20.12, 20.16, 21.93,
45.21, 54.81, 61.32, 61.48, 68.10, 68.30, 70.42 (d, J ) 18.5 Hz),
70.98, 71.63, 74.75 (d, J ) 6.3 Hz), 87.29 (dd, J ) 8, 195 Hz),
91.19 (dd, J ) 6, 22 Hz), 99.84, 169.60, 170.08, 170.32, 170.60,
171.00, 171.49; 19F NMR [(CDCl3, CD3OD (2:1 v/v) 282 MHz]
δ -200.11 (dd, J ) 12.1, 49 Hz); 31P NMR (CDCl3, 121 MHz)
δ -1.02; [R]D +39.8° (c ) 0.6, CHCl3); MS (-ve FAB) 674 (M
- H)-; HRMS m/z calcd for C24H34FNO18P 674.149755, found
674.148300.
P 1-[2-Acetam ido-3,4,6-tr i-O-acetyl-2-deoxy-â-D-glu copy-
r a n osyl-(1f4)-3,6-d i-O-a cet yl-2-d eoxy-2-flu or o-r-D-glu -
cop yr a n osyl] P 2-Dolich yl P yr op h osp h a te 29. 19F NMR
[CDCl3, CD3OD (5:1 v/v), 282 MHz] δ -201.5 (brd, J ) 50.8
Hz); 31P NMR [CDCl3, CD3OD (5:1 v/v), 202.5 MHz] -10.8,
-13.2; MS (-ve ESI) 922.7, 956.8, 990.8, 1024.9, 1058.9,
1092.9, 1127.0 (m/ 2 for n ) 12-18).
P 1-[2-Aceta m id o-2-d eoxy-â-D-glu cop yr a n osyl-(1f4)-2-
d eoxy-2-flu or o-r-D-glu cop yr a n osyl] P 2-Dolich yl P yr o-
p h osp h a te 1d . To a stirred solution of peracetylated Dol-PP-
2DFGlc-GlcNAc 29 (4.4 mg, 2.11 µmol) 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
50 × 8, pyridinium form, 200 mg) was added, and the mixture
was stirred for 10 min. The resin was then filtered and washed
with CHCl3/MeOH (2:1 v/v). Concentration of solvent followed
by column chromatography [CHCl3, MeOH, HO (72:21:2 v/v)
followed by (60:25:4 v/v)] gave Dol-PP-2DFGlc-GlcNAc 1d (3.1
mg, 73%): 31P NMR [CDCl3, CD3OD (2:1 v/v), 202.3 MHz] δ
-11.1, -13.0; MS (-ve ESI) 852.5, 885.8, 919.8, 953.8, 987.9
(m/ 2 for n ) 13-17).
2,3,4,6-Tet r a -O-a cet yl-â-D-glu cop yr a n osyl-(1f4)-2-a c-
eta m id o-3-O-a cetyl-1,6-d i-O-ben zyl-2-d eoxy-r-D-glu cop y-
r a n ose 30. To a solution of glycosyl acceptor 1121 (383.6 mg,
0.8649 mmol) and bromoacetyl glucose 10 (533.5 mg, 1.2974
mmol) in CH2Cl2/toluene (8 mL, 1:1 v/v) at -45 °C was added
AgOTf (366.7 mg, 1.4271 mmol) in one portion. The mixture
was allowed to warm to 0 °C over 4 h. Et3N (1 mL) and CH2-
Cl2 (40 mL) were then added. The organic phase was washed
with water (15 mL), a saturated aqueous solution of KI (15
mL), and brine (15 mL). The organic extracts were dried (Na2-
SO4) and filtered through a pad of silica gel topped with Celite.
Concentration of the filtrate followed by flash chromatography
[CHCl3, EtOAc (3:1 v/v) followed by (2:1 v/v)] gave disaccharide
30 (563.1 mg, 84%) as a white solid: mp 150-152 °C (EtOH);
IR (neat) cm-1 1756, 1676, 1540, 1456, 1348, 1230, 1038, 903,
1
1159, 1037, 892, 736, 692, 640; H NMR (CDCl3, 500 MHz) δ
1.93 (s, 3H, Ac), 2.01 (s, 3H, Ac), 2.02 (s, 3H, Ac), 2.03 (s, 3H,
Ac), 2.09 (s, 3H, Ac), 2.12 (s, 3H, Ac), 3.63 (dt, 1H, J ) 8.4,
10.5 Hz, H-2′), 3.68 (ddd, 1H, J ) 2.3, 4.6, 10.1 Hz, H-5′), 3.76
(dd, 1H, J ) 8.2, 10.1 Hz, H-4), 3.79 (ddd, 1H, J ) 1.5, 4.5,
10.1 Hz, H-5), 4.03 (dd, 1H, J ) 2.3, 12.6 Hz, H-6a′), 4.16 (dd,
1H, J ) 4.5, 12.2 Hz, H-6a), 4.36 (ddd, 1H, J ) 7.8, 9.2, 50.5
Hz, H-2), 4.36 (dd, 1H, J ) 4.6, 12.5 Hz, H-6b′), 4.41 (dd, 1H,
J ) 1.5, 12.2 Hz, H-6b), 4.82 (d, 1H, J ) 8.2, H-1′), 5.02 (dd,
1H, J ) 9.3, 10.1 Hz, H-4′), 5.05-5.12 (m, 4H, OCH2Ph), 5.33
(dt, 1H, J ) 9.0, 14.2 Hz, H-3), 5.36 (dd, 1H, J ) 9.3, 10.4 Hz,
H-3′), 5.36 (dt, 1H, J ) 2.7, 7.8 Hz, H-1), 5.70 (d, 1H, J ) 8.7
Hz, NH), 7.31-7.37 (m, 10H, OCH2Ph); 13C NMR (CDCl3, 125
MHz) δ 20.58, 20.60, 20.65, 20.69, 20.77, 23.17, 55.40, 61.64,
61.80, 68.23, 69.53 (d, J ) 5.8 Hz), 69.67 (d, J ) 5.8 Hz), 71.61
(d, J ) 20 Hz), 71.79, 71.83, 73.72, 75.12 (d, J ) 6.9 Hz), 89.47
(dd, J ) 9.2, 193 Hz), 95.75 (dd, J ) 4.6, 24.2 Hz), 99.66,
127.87, 128.54, 128.56, 128.60, 128.65, 135.18 (d, J ) 8 Hz),
135.31 (d, J ) 8 Hz), 169.40, 169.73, 170.45, 170.50 (2 ×),
170.60; 19F NMR (CDCl3, 282 MHz) δ -199.16 (dd, J ) 13.6,
50.3 Hz); 31P NMR (CDCl3, 121 MHz) δ -2.36; [R]D +46.9° (c
) 0.4, CHCl3); MS (FAB) 878 (MNa+); HRMS m/z calcd for
1
747, 699; H NMR (CDCl3, 500 MHz) δ 1.87 (s, 3H, Ac), 1.93
(s, 3H, Ac), 1.97 (s, 3H, Ac), 2.00 (s, 3H, Ac), 2.01 (s, 3H, Ac),
2.06 (s, 3H, Ac), 3.38 (ddd, 1H, J ) 2, 4.3, 10 Hz, H-5′), 3.55
(dd, 1H, J ) 1.3, 10.7 Hz, H-6a), 3.70 (brd, 1H, J ) 10.5 Hz,
H-5), 3.73 (dd, 1H, J ) 2.7, 10.7 Hz, H-6b), 3.94 (dd, 1H, J )
9.5, 9.9 Hz, H-4), 3.97 (dd, 1H, J ) 2, 12.4 Hz, H-6a′), 4.25
(ddd, 1H, J ) 3.7, 9.7, 10.8 Hz, H-2), 4.32 (dd, 1H, J ) 4.3,
12.4 Hz, H-6b′), 4.37 (d, 1H, J ) 8.1 Hz, H-1′), 4.44 (d, 1H, J
) 12 Hz, OCHHPh), 4.50 (d, 1H, J ) 12 Hz, OCHHPh), 4.69
(d, 1H, J ) 12 Hz, OCHHPh), 4.79 (d, 1H, J ) 12 Hz,
OCHHPh), 4.82 (dd, 1H, J ) 8, 10 Hz, H-2′), 4.91 (d, 1H, J )
3.7 Hz, H-1), 4.95-4.99 (m, 2H, H-3′, H-4′), 5.13 (dd, 1H, J )
9.4, 10.7 Hz, H-3), 5.65 (d, 1H, J ) 9.3 Hz, NH), 7.30-7.45
(m, 10H, OCH2Ph); 13C NMR (CDCl3, 125 MHz) δ 20.51, 20.53,
20.56, 20.59 (2 ×), 23.09, 51.97, 61.56, 67.05, 67.90, 70.10,
70.50, 71.12, 71.55, 71.60, 73.10, 73.67, 74.89, 96.75, 100.03,
128.07, 128.09, 128.16, 128.20, 128.55, 128.67, 136.80, 137.65,
168.79, 169.30, 169.90, 170.19, 170.48, 171.22; [R]D +55.3° (c
) 0.7, CHCl3); MS (FAB) 796 (MNa+); HRMS m/z calcd for
C
38H47FNO18PNa 878.241249, found 878.244200.
Mon o(tr ieth yla m m on iu m ) Sa lt of 2-Aceta m id o-3,4,6-
tr i-O-a cetyl-2-d eoxy-â-D-glu cop yr a n osyl-(1f4)-3,6-d i-O-
a cet yl-2-d eoxy-2-flu or o-r-D-glu cop yr a n osyl P h osp h a t e
28. Hydrogenolysis of R-dibenzyl phosphate 27a (25.2 mg,
0.0295 mmol) gave R-phosphate 28 (24.0 mg, quantitative) as
a colorless film; IR (neat) cm-1 1745, 1644, 1436, 1370, 1230,
1159, 1043, 959, 908, 841; 1H NMR [(CDCl3, CD3OD (2:1 v/v),
500 MHz] δ 1.05 (t, 9 H, J ) 7.3 Hz, CH2CH3), 1.68 (s, 3H,
Ac), 1.78 (s, 3H, Ac), 1.79 (s, 3H, Ac), 1.87 (s, 3H, Ac), 1.88 (s,
3H, Ac), 1.89 (s, 3H, Ac), 2.79 (q, 6H, J ) 7.3 Hz, CH2CH3),
3.36 (dd, 1H, J ) 8.4, 10.5 Hz, H-2′), 3.52 (ddd, 1H, J ) 2.4,
4.0, 10.2 Hz, H-5′), 3.56 (t, 1H, J ) 9.7 Hz, H-4), 3.82 (dd, 1H,
J ) 2.4, 12.5 Hz, H-6a′), 3.84 (dd, 1H, J ) 3.7, 12.5 Hz, H-6a),
C
33H47NO16Na 796.279255, found 796.280900.
2,3,4,6-Tet r a -O-a cet yl-â-D-glu cop yr a n osyl-(1f4)-2-a c-
eta m id o-1,3,6-tr i-O-a cetyl-2-d eoxy-r-D-glu cop yr a n ose 31.
Disaccharide 30 (171.1 mg, 0.2211 mmol) underwent hydro-
genolysis and acetylation to give octaacetate 31 (119.2 mg,

Oligosaccharyl Transferase Substrate Specificity
J . Org. Chem., Vol. 66, No. 19, 2001 6227
91%) as a white solid, mp 221-223 °C (MeOH/pentane); IR
1.93 (s, 3H, Ac), 2.90 [q, 6H, J ) 7.3 Hz, N(CH2CH3)3], 3.53
(ddd, 1H, J ) 2, 3.9, 10.3 Hz, H-5′), 3.65 (dd, 1H, J ) 9.5, 9.7
Hz, H-4), 3.85 (dd, 1H, J ) 1.8, 12.5 Hz, H-6a′), 3.89 (dd, 1H,
J ) 3.5, 12.1 Hz, H-6a), 3.93 (ddd, 1H, J ) 1.8, 3.5, 9.7, H-5),
4.04 (brdt, 1H, J ) 2.5, 10.7 Hz, H-2), 4.21 (dd, 1H, J ) 3.9,
12.6 Hz, H-6b′), 4.37 (dd, 1H, J ) 1.8, 11.6 Hz, H-6b), 4.38 (d,
1H, J ) 8 Hz, H-1′), 4.71 (dd, 1H, J ) 8, 9.3 Hz, H-2′), 4.87 (t,
1H, J ) 9.7 Hz, H-4′), 4.96 (t, 1H, J ) 9.4 Hz, H-3′), 5.04 (dd,
1H, J ) 9.5, 10.4 Hz, H-3), 5.22 (dd, 1H, J ) 3.3, 6.6 Hz, H-1);
13C NMR [CDCl3, CD3OD (2:1 v/v), 125 MHz] δ 7.92, 19.84(2
×), 19.95, 20.01, 20.17, 21.74, 29.19, 45.65, 51.60, 51.65, 61.13,
61.27, 67.61, 68.99, 70.92, 71.38, 72.80, 75.79, 93.38, 100.34,
169.12, 169.44, 170.15, 170.53, 170.62, 170.78, 171.60; 31P
NMR [CDCl3, CD3OD (2:1 v/v) 162 MHz] δ -0.08; [R]D +42.9°
(c ) 0.8, CHCl3); MS (-ve FAB) 714 (M-); HRMS m/z calcd
for C26H37NO20P 714.164657, found 714.164900.
(neat) cm-1 1746, 1674, 1540, 1433, 1370, 1226, 1130, 1036,
1
943, 908, 755; H NMR (CDCl3, 500 MHz) δ 1.93 (s, 3H, Ac),
1.99 (s, 3H, Ac), 2.02 (s, 3H, Ac), 2.05 (s, 3H, Ac), 2.07 (s, 3H,
Ac), 2.09 (s, 3H, Ac), 2.12 (s, 3H, Ac), 2.19 (s, 3H, Ac), 3.67
(ddd, 1H, J ) 2.2, 4.1, 9.8 Hz, H-5′), 3.84 (dd, 1H, J ) 8.4, 10
Hz, H-4), 3.88 (ddd, 1H, J ) 1.7, 3.8, 10.5 Hz, H-5), 4.04 (dd,
1H, J ) 2.2, 12.5 Hz, H-6a′), 4.11 (dd, 1H, J ) 3.8, 12.2 Hz,
H-6a), 4.35-4.40 (m, 2H, H-2, H-6b′), 4.42 (dd, 1H, J ) 1.7,
12.2 Hz, H-6b), 4.53 (d, 1H, J ) 7.9 Hz, H-1′), 4.94 (dd, 1H, J
) 7.9, 9.1 Hz, H-2′), 5.08 (t, 1H, J ) 9.5 Hz, H-4′), 5.15 (t, 1H,
J ) 9.3 Hz, H-3′), 5.20 (dd, 1H, J ) 8.5, 11.5 Hz, H-3), 5.54 (d,
1H, J ) 9.1 Hz, NH), 6.10 (d, 1H, J ) 3.6 Hz, H-1); 13C NMR
(CDCl3, 125 MHz) δ 20.49 (2 ×), 20.55 (2 ×), 20.61, 20.76,
20.93, 23.00, 51.08, 61.44, 61.59, 67.76, 70.45, 70.59, 71.61,
71.99, 72.88, 75.86, 90.49, 100.94, 168.71, 169.19, 169.29,
170.04, 170.09, 170.23, 170.40, 171.39; [R]D +47.7° (c ) 1.3,
P 1-[2,3,4,6-Tetr a -O-a cetyl-â-D-glu cop yr a n osyl-(1f4)-2-
a cet a m id o-3,6-d i-O-a cet yl-2-d eoxy-r-D-glu cop yr a n osyl]
P 2-Dolich yl P yr op h osp h a te 36. 31P NMR [CDCl3, CD3OD
(2:1 v/v), 162 MHz] δ -9.36, -11.50; MS (-ve ESI) 1954, 2022,
2090, 2158, 2227 (M- for n ) 13-17).
CHCl3); MS (FAB) 700 (MNa+); HRMS m/z calcd for C28H39
-
NO18Na 700.206484, found 700.206400.
2,3,4,6-Tet r a -O-a cet yl-â-D-glu cop yr a n osyl-(1f4)-2-a c-
eta m id o-3,6-d i-O-a cetyl-2-d eoxy-r,â-D-glu cop yr a n ose 32.
A solution of octaacetate 31 (61.9 mg, 0.09135 mmol) and
hydrazine monoacetate (42 mg, 0.4568) was stirred in DMF
(3 mL) for 4 days. The mixture was diluted with EtOAc (20
mL) and washed with brine (6 mL). The aqueous phase was
separated and extracted with EtOAc (2 × 6 mL). The combined
organic extracts were dried (Na2SO4) and filtered. Concentra-
tion of the filtrate followed by column chromatography [CHCl3,
MeOH (93:7 v/v)] gave compound 32 (48.9 mg, 84%) mainly
as the R-anomer: MS (FAB) 636 (MH+), 658 (MNa+); HRMS
m/z calcd for C26H38NO17 636.213974, found 636.211300.
Dib en zyl 2,3,4,6-Tet r a -O-a cet yl-â-D-glu cop yr a n osyl-
(1f4)-2-a ceta m id o-3,6-d i-O-a cetyl-2-d eoxy-r-D-glu cop y-
r a n osyl P h osp h a te 34. To a solution of compound 32 (48.9
mg, 0.0769 mmol) in THF (2.5 mL) at -65 °C was added a
solution of LiHMDS (1 M in hexanes, 100 µL, 0.1 mmol). The
mixture was stirred at this temperature for 10 min followed
by the slow addition of tetrabenzyl pyrophosphate 33 (53.9 mg,
0.1 mmol) in THF (0.8 mL). The mixture was allowed to warm
to 0 °C and stirred for an extra 30 min. The mixture was
diluted with Et2O (25 mL) and washed with a saturated
aqueous solution of NaHCO3 (10 mL) and brine (10 mL). The
organic phase was dried (Na2SO4), filtered, and concentrated.
Flash column chromatography [CHCl3, MeOH (97:3 v/v)] of the
crude product gave R-dibenzyl phosphate 34 (61.6 mg, 89%)
as a white film; IR (neat) cm-1 1747, 1678, 1544, 1369, 1228,
1166, 1133, 1036, 955, 749, 699; 1H NMR (CDCl3, 500 MHz) δ
1.69 (s, 3H, Ac), 1.98 (s, 3H, Ac), 2.00 (s, 3H, Ac), 2.01 (s, 3H,
Ac), 2.02 (s, 3H, Ac), 2.05 (s, 3H, Ac), 2.08 (s, 3H, Ac), 3.64
(ddd, 1H, J ) 2.2, 4.0, 9.7 Hz, H-5′), 3.79 (t, 1H, J ) 9.6 Hz,
H-4), 3.90 (ddd, 1H, 2, 3.8, 10.5 Hz, H-5), 3.98 (dd, 1H, J )
3.8, 12.3 Hz, H-6a), 4.02 (dd, 1H, J ) 2.0, 12.5 Hz, H-6a′), 4.26
(ddt, 1H, J ) 3, 9, 10 Hz, H-2), 4.31 (dd, 1H, J ) 2, 12.3 Hz,
H-6b), 4.37 (dd, 1H, J ) 4.2, 12.5 Hz, H-6b′), 4.49 (d, 1H, J )
7.9 Hz, H-1′), 4.92 (dd, 1H, J ) 8.3, 8.7 Hz, H-2′), 5.01-5.18
(m, 7H, OCH2Ph, H-3, H-3′, H-4′), 5.60 (d, 1H, J ) 9.3 Hz,
NH), 5.60 (dd, 1H, J ) 3.3, 6.0 Hz, H-1), 7.32-7.47 (m, 10H,
OCH2Ph); 13C NMR (CDCl3, 125 MHz) δ 20.47 (4 ×), 20.58,
20.68, 22.69, 51.77, 51.83, 61.09, 61.48, 67.68, 69.78, 69.82,
69.87, 69.90, 70.30, 71.55, 71.91, 72.90, 75.59, 96.01 (d, J ) 6
Hz, C-1), 100.80, 127.97, 128.04, 128.71, 128.78, 128.88, 128.89,
135.17 (d, J ) 6.5 Hz), 135.27 (d, J ) 6.5 Hz), 168.98, 169.23,
170.04, 170.13, 170.24, 170.41, 170.77; 31P NMR (CDCl3, 162
MHz) δ -0.75; [R]D 24.1° (c ) 1.3, CHCl3); MS (FAB) 918
(MNa+); HRMS m/z calcd for C40H50NO20NaP 918.256152,
found 918.254600.
P 1- [â-D-Glu cop yr a n osyl-(1f4)-2-a ceta m id o-2-d eoxy-r-
D-glu cop yr a n osyl] P 2-Dolich yl P yr op h osp h a te 1e. Similar
to the deprotection of 29, peracetylated Dol-PP-GlcNAc-Glc 36
(6.1 mg, 2.87 µmol) gave after column chromatography Dol-
PP-GlcNAc-Glc 1e (4.2 mg, 73%): 31P NMR [CDCl3, CD3OD
(2:1 v/v), 121 MHz] δ -11.4, -13.4; MS (-ve ESI) 850.8, 884.8,
918.8, 952.9, 987.0 (m/ 2 for n ) 13-17).
Bz-Asn -Leu -Th r -Lys-NH2‚TF A 42. Tetrapeptide Bz-Asn-
Leu-Thr-Lys-NH2‚TFA 42 was prepared by standard Fmoc-
based solid-phase peptide synthesis (SPPS) on PAL-PEG-PS
resins (0.175 mmol/g, 1.18 g). In situ activated esters were
generated from the free acid derivatives using (benzotriazol-
1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (Py-
BOP) chemistry. In general, coupling was carried out manually
with Fmoc free acid (1.5 equiv), PyBOP (1.5 equiv), and di-
iso-propylethylamine (1.5 equiv) in DMF (10 mL) inside a
reaction vessel with shaking (Fisher orbital shaker) for 1 h.
The resins were filtered through suction and washed succes-
sively with DMF (2 × 20 mL) and CH2Cl2 (20 mL). Fmoc
deprotection was performed with 20% piperidine/DMF (10 mL,
3 × 5 min) followed by washing with DMF (5 × 20 mL). The
peptides were deprotected and cleaved from the solid support
by treatment with trifluoroacetic acid (TFA)/CH2Cl2/triisopro-
pylsilane (TIPS)/water (47.5:47.5:2.5:2.5 v/v, 2 × 10 mL). The
resin was filtered and washed with TFA/CH2Cl2 (50:50 v/v, 2
× 10 mL). The combined filtrates were concentrated and
triturated with Et2O (3 × 10 mL). The white pellet was
dissolved in H2O (15 mL) and 0.1% TFA/CH3CN (100 µL) and
coevaporated with toluene to give the desired tetrapeptide 42
(101.4 mg, 71%); 1H NMR (d6-DMSO, 500 MHz) δ 0.81 (d, 3H,
J ) 6.6 Hz, Leu-Hδ), 0.85 (d, 3H, J ) 6.5 Hz, Leu-Hδ), 1.04 (d,
3H, J ) 6.4 Hz, Thr-Hγ), 1.24-1.34 (m, 2H, Lys-Hγ), 1.45-
1.75 (m, 9H), 2.58 (dd, 1H, J ) 8.4, 15.3 Hz, Asn-Hâ), 2.65
(dd, 1H, J ) 5.6, 15.3 Hz, Asn-Hâ), 2.75 (t, 2H, J ) 7.5 Hz,
Lys-Hꢀ), 4.01 (m, 1H, Thr-Hâ), 4.15 (dt, 1H, J ) 4.9, 8.7 Hz,
Lys-HR), 4.20 (dd, 1H, J ) 4.4, 8.1 Hz, Thr-HR), 4.33 (brq, 1H,
J ) 8.2 Hz, Leu-HR), 4.75 (dt, 1H, J ) 5.6, 7.9 Hz, Asn-HR),
5.00 (d, 1H, J ) 5.6 Hz, Thr-OH), 7.02 (brs, 1H, CONHH),
7.15 (brs, 1H, CONHH), 7.25 (brs, 1H, CONHH), 7.42 (brs,
1H, CONHH), 7.48 (t, 2H, J ) 7.8 Hz, Ph), 7.53-7.57 (m, 1H,
Ph), 7.61 (brs, 3H, NH3+), 7.75 (d, 1H, J ) 8.1 Hz, Leu-NH),
7.79 (d, 1H, J ) 8.1 Hz, Lys-NH), 7.84 (dd, 1H, J ) 1.5, 7.8
Hz, Ph), 8.14 (d, 1H, J ) 8.1 Hz, Thr-NH), 8.64 (d, 1H, J )
7.5 Hz, Asn-NH); MS (ESI) 579 (MH+).
Bz-Asn -Leu -Th r -Lys(Ac)-NH2 37. A solution of the tet-
rapeptide 42 (41.5 mg, 0.06 mmol), acetic anhydride (17.0 µL,
Mon o(tr ieth ylam m on iu m ) Salt of 2,3,4,6-Tetr a-O-acetyl-
â-D-glu cop yr a n osyl-(1f4)-2-a ceta m id o-3,6-d i-O-a cetyl-2-
d eoxy-r-D-glu cop yr a n osyl P h osp h a te 35. Hydrogenolysis
of R-dibenzyl phosphate 34 (25.8 mg, 0.0288 mmol) yielded
R-phosphate 35 (25.8 mg, quantitative); IR (neat) cm-1 3339,
2959, 1747, 1668, 1369, 1230, 1166, 1125, 1038, 962, 909, 753;
1H NMR [CDCl3, CD3OD (2:1 v/v), 500 MHz] δ 1.12 [t, 9H, J
) 7.3 Hz, N(CH2CH3)3], 1.74 (s, 3H, Ac), 1.79 (s, 3H, Ac), 1.82
(s, 3H, Ac), 1.83 (s, 3H, Ac), 1.84 (s, 3H, Ac), 1.89 (s, 3H, Ac),
i
0.18 mmol,) and Pr2NEt (62.7 µL, 0.36 mmol) was stirred in
DMF (4 mL) for 1.5 h. The solvent was blown down under N2
overnight (0.5 mL remaining) and triturated with Et2O/n-hex
(1:1 v/v, 4 × 5 mL). The white pellet was dried under high
vacuum to give a quantitative yield of the acetylated peptide
1
37 (42.5 mg); H NMR (d6-DMSO, 500 MHz) δ 0.81 (d, 3H, J
) 6.6 Hz, Leu-Hδ), 0.85 (d, 3H, J ) 6.6 Hz, Leu-Hδ), 1.03 (d,
3H, J ) 6.3 Hz, Thr-Hγ), 1.2-1.7 (m, 9H), 1.77 (s, 3H, Ac),

6228 J . Org. Chem., Vol. 66, No. 19, 2001
Imperiali
2.58 (dd, 1H, J ) 8.5, 15.3 Hz, Asn-Hâ), 2.65 (d, 1H, J ) 5.6,
15.3 Hz, Asn-Hâ), 2.97 (q, 2H, J ) 6.9 Hz, Lys-Hꢀ), 4.01 (m,
1H, Thr-Hâ), 4.12 (dt, 1H, J ) 4.8, 8.5 Hz, Lys-HR), 4.19 (dd,
1H, J ) 4.4, 8.2 Hz, Thr-HR), 4.32 (q, 1H, J ) 7.5 Hz, Leu-HR),
4.76 (dt, 1H, J ) 5.8, 8.1 Hz, Asn-HR), 4.95 (brd, 1H, Thr-OH),
7.02 (d, 1H, J ) 1.5 Hz, CONHH), 7.10 (d, 1H, J ) 1.5 Hz,
CONHH), 7.23 (d, 1H, J ) 1.7 Hz, CONHH), 7.41 (d, 1H, J )
1.7 Hz, CONHH), 7.48 (brt, 2H, J ) 7.7 Hz, Ph), 7.53-7.56
(m, 1H, Ph), 7.70 (d, 1H, J ) 8.0 Hz, Leu-NH), 7.79 (d, 1H, J
) 8.3 Hz, Lys-NH), 7.80-7.85 (m, 3H, NHAc, Ph), 8.15 (d, 1H,
J ) 7.9 Hz, Thr-NH), 8.61 (d, 1H, J ) 7.6 Hz, Asn-NH); MS
(ESI) 620 (MH+), 643 (MNa+).
MgCl2 (12:192:186:2.69 v/v), 3 mL}. The combined aqueous
phases were blown down under nitrogen for HPLC analysis.
For competition assay of unnatural analogues 1c and 1d , a
constant amount of Dol-PP-GlcNAc-GlcNAc 1b (22 µM) was
mixed with variable amounts of 1c or 1d .
HP LC An a lysis of Glycop ep tid e. HPLC analysis of assay
mixtures was carried out on a Beckman System Gold liquid
chromatography system equipped with a C18 column (micro-
sorb MV C18, 5 µm, 100 Å, 25 cm). Samples were resuspended
in 0.1% TFA in water (0.8 mL) and centrifuged before injection
onto the HPLC. Separation of glycopeptides and peptides were
accomplished by elution with a gradient of 15-30% acetonitrile
over 25 min. Fractions (0.5 mL) were collected for 15-30 min,
mixed with Ecolite (5.5 mL), and counted on a Beckman LS-
5000TD scintillation counter. The amount of glycopeptide
formation can be calculated on the basis of the specific activity
of radiolabeled peptide 37r (29.3 mCi/mmol).
Product formation was shown to be linear over 2 h in accord
with Coward’s results. The use of long assay times allows
enough radiolabeled glycopeptide to be detected. The detection
limit of this assay is ca. 3 pmol (which corresponds to 200 dpm)
and glycopeptide formation is generally within 10% of sub-
strate. Possible product inhibition has previously been ad-
dressed and shown to be lacking. Michaelis-Menten param-
eters were obtained by Lineweaver-Burk reciprocal plots. The
approximate Ki’s were calculated as described by Segel36 using
eq 1,
Bz-Asn -Leu -Th r -Lys(3H-Ac)-NH2 37r . To a solution of
i
the tetrapeptide 42 (41.5 mg, 0.06 mmol) and Pr2NEt (71.4
µL, 0.41 mmol) in DMF (1 mL) in a 15 mL Oakridge tube was
added tritium-labeled acetic anhydride [(3H)-Ac2O, 2.36 µL,
0.025 mmol, specific activity of 100 mCi/mmol from American
Radiolabeled Chemicals Inc., St. Louis, MO]. After 2 h, the
free amine was capped by treating with excess Ac2O (17 µL,
0.18 mmol) for another 2 h. The reaction was monitored by
TLC [EtOAc, nBuOH, AcOH, H2O (3:1:1:1 v/v)]; Rf of peptide
42 ) 0.26; Rf of peptide 37 ) 0.51. The product was triturated
with Et2O/n-hex (1:1 v/v, 8 × 4 mL) to give radiolabeled
tetrapeptide 37r (29.3 mg, 79%).
OT Assa y w it h R a d iola b eled P ep t id e 37r . Yeast (S.
cerevisiae) OT was purified to the solubilized membrane stage
according to the Pathak protocol.39 The final buffer solution
contains 140 mM sucrose, 50 mM HEPES, pH 7.5, 10 mM
MgCl2, 0.1 mM AEBSF, 0.5 µg/mL pepstatin, and 0.5 µg/mL
leupeptin. The assay buffer for OT assay consisted of 140 mM
sucrose, 50 mM HEPES, pH 7.5, 1.2% Triton X-100, 15 mM
MnCl2, and 0.5 mg/mL phosphatidylcholine. Different amounts
of dolichol-linked saccharides (1b-e, 3) were aliquoted from
chloroform/methanol stock solutions into eppendorf tubes, and
where i represents the fraction inhibition, [I] is the concentra-
tion of the inhibitor, and [S] is the concentration of in vitro
substrate 1b.
the solvents were evaporated under
a gentle stream of
nitrogen. DMSO (10 µL) and assay buffer (final volume of 200
µL) were added and vortexed vigorously. The reaction was
initiated by the addition of OT and the radiolabeled peptide
37r (0.42 mM, 10 µL in DMSO, 29.3 µCi/µmol, final concentra-
tion of 21 µM ≈ 5 Km). (Nonlabeled peptide 37 was used for
the mass spectroscopy analysis of glycopeptides.) The mixture
was shaken at 150 rpm for the specified time at room
temperature followed by quenching with 6 mL of chloroform/
methanol/4 mM MgCl2 (3:2:1 v/v). The upper aqueous phase
was removed and the lower phase reextracted twice with TUP
{theoretical upper phase, chloroform/methanol/water/0.25 M
Ack n ow led gm en t. This research was supported by
the National Institute of Health (GM39334). The au-
thors like to thank Prof. Daniel Kahne from Princeton
University for helpful discussions regarding the phos-
phorylation of bromide 26.
Su p p or tin g In for m a tion Ava ila ble: Graphs for kinetic
analysis of tetrapeptide 37 and dolichol-linked saccharides
1b-e and mass spectra of glycopeptides 38, 39, and 43. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(39) Pathak, R.; Parker, C. S.; Imperiali, B. FEBS Lett. 1995, 362,
229-234.
J O0100345