Convergent synthesis of a fluorescence-quenched glycopeptide as a potential substrate for peptide: N-glycosidases

Article abstract of DOI:10.1016/S0008-6215(98)00011-1

The fluorescence-quenched glycopeptide N(v)-[2-acetamido-6-(2'- amino)benzamido-2,6-dideoxy-β-D-glucopyranosyl]-N(α)-benzoyl-Asn-Tyr(NO₂)- OMe, having anthranilamide/3-nitrotyrosine as the donor/acceptor pair, was synthesized via a convergent route involving the EEDQ-mediated coupling of 2- acetamido-2,3-di-O-acetyl-6-(2'-tert-butoxycarbonylamino)benzamido-2,6- dideoxy-β-D-glucopyranosylamine with N-benzoyl-Asp-Tyr(NO₂)-OMe, followed by deprotection.

Full text of DOI:10.1016/S0008-6215(98)00011-1

Carbohydrate Research 306 (1998) 531±538
Convergent synthesis of a ¯uorescence-quenched
glycopeptide as a potential substrate for peptide:
N-glycosidases
Vito Ferro, Larry Weiler, Stephen G. Withers *
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC,
Canada, V6T 1Z1
Received 3 November 1997; accepted 2 January 1998
Abstract
The ¯uorescence-quenched glycopeptide Nꢀ-[2-acetamido-6-(20-amino)benzamido-2,6-dideoxy-
ꢁ-d-glucopyranosyl]-Nꢂ-benzoyl-Asn-Tyr(NO2)-OMe, having anthranilamide/3-nitrotyrosine as
the donor/acceptor pair, was synthesized via a convergent route involving the EEDQ-mediated
coupling of 2-acetamido-2,3-di-O-acetyl-6-(20-tert-butoxycarbonylamino)benzamido-2,6-dideoxy-
ꢁ-d-glucopyranosylamine with N-benzoyl-Asp-Tyr(NO2)-OMe, followed by deprotection. # 1998
Elsevier Science Ltd. All rights reserved
Keywords: PNGase assay; ¯uorimeter; peptide; nitrotyrosine
1. Introduction
properties of certain target proteins involved in
embryonal development [5]. The enzyme hydro-
lyzes all types of asparagine-linked oligosacchar-
ides, provided that both the amino and carboxyl
groups of the asparagine are involved in amide
bonds. The enzyme has a broad substrate speci®-
city [2], liberating all N-linked oligosaccharides,
regardless of their structure, size or charge, from
glycoproteins of vertebrate origin. However, the
binding of the enzyme to the inner di-N-acetyl-
chitobiose core of the carbohydrate chain is more
speci®c [6]. For example, N-glycans with a fucose
linked ꢂ-(1!6) to the asparagine-proximal N-acet-
ylglucosamine are good substrates, but if the fucose
is in an ꢂ-(1!3) linkage as found in glycoproteins
from plants and insects, the enzyme is inactive [7].
PNGase F is probably the most widely used
agent for the deglycosylation of glycoproteins [8]
Peptide-N4-(N-acetyl-ꢁ-d-glucosaminyl)asparagine
amidase F [1] (peptide: N-glycosidase F, glyco-
peptidase F or PNGase F) from Flavobacterium
meningosepticum is an amidase/amidohydrolase
that cleaves the ꢁ-aspartylglucosylamine bond of
N-linked glycoproteins, converting the asparagine
residue to an aspartic acid [2]. The released 1-ami-
nooligosaccharide spontaneously degrades to
ammonia and the free oligosaccharide [3,4].
PNGases are also found in plants, and recently
they have been found to be widely distributed in
animals [5]. It is speculated that they may play an
important role in the non-lysosomal regulation of
various physiological and/or physicochemical
* Corresponding author.
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00011-1

532
V. Ferro et al./Carbohydrate Research 306 (1998) 531±538
and is thus a valuable biochemical tool for investi-
gating oligosaccharide structure and the biosyn-
thesis of N-linked glycoproteins [9]. Despite the
importance of these enzymes, and the fact that the
three-dimensional X-ray crystallographic structure
of PNGase F was recently determined [10,11], very
few kinetic studies have been performed beyond
recent kinetic comparisons of PNGase F and
PNGase A (from almond) which revealed several
di€erences in substrate speci®city [12].
The currently available assays for PNGases are
quite tedious and time consuming, involving the
extensive use of HPLC [8]. In addition, the expen-
sive glycopeptide substrates required for the assay
must ®rst be derivatized with a ¯uorescent label. A
more sensitive and convenient assay would therefore
be valuable. This paper describes the design and
synthesis of a ¯uorescence-quenched glycopeptide
substrate as a ®rst step towards the development of
a more convenient assay for PNGase activity.
Glycopeptide 4 possesses an N-acetylglucosamine
derivative N-linked via asparagine to a dipeptide.
The donor/acceptor pair chosen to provide the
¯uorescence quenching via resonance energy
transfer was anthranilamide/nitrotyrosine [13], a
highly ecient pair that has found utility in ¯uor-
escence-quenched assays of proteolytic enzymes
[14,15]. This donor/acceptor pair is also the only
one currently available that is amenable to solid-
phase synthesis techniques [13], an important con-
sideration for ultimately producing substrates of
varying peptide length. The anthranilamide donor
was placed at C-6 of the N-acetylglucosamine resi-
due for ease of synthesis and because PNGases are
known to accept substrates with substitution at C-6
[7]. Glycopeptide 4 was prepared by a convergent
approach [16±19] involving syntheses of the glyco-
sylamine 1 and the dipeptide 2 and then coupling
as outlined in Scheme 1.
The dipeptide 2 was prepared by conventional
solution synthesis techniques as shown in Scheme
2. Commercially available ꢁ-tert-butyl-l-aspartate
(7) was N-benzoylated in 94% yield with benzoyl
chloride and sodium carbonate in aqueous dioxane
and then coupled with 3-nitro-l-tyrosine methyl
ester hydrochloride 6 [20] under standard peptide
coupling conditions (DCC/DhbtOH) to give the
protected dipeptide 9 in 91% yield. The terminal
2. Results and discussion
A substrate was desired having both the mini-
mum requirements for recognition by the enzyme
PNGase. The glycopeptide 4 was thus designed as
the minimal structure to meet these requirements.
Scheme 1.

V. Ferro et al./Carbohydrate Research 306 (1998) 531±538
533
¯ash chromatography. The benzyloxycarbonyl
group of compound 15 was quantitatively removed
by hydrogenolysis with palladium-on-charcoal as
catalyst to give the glycosylamine 1, in readiness
for coupling with the dipeptide (2). Compound 1,
isolated as a foam, was pure by TLC and 1H NMR
spectroscopy and was used immediately in the
coupling step without further puri®cation.
Scheme 2.
methyl ester of the dipeptide proved to be very
stable, and all attempts to convert it into an amide
by treatment with ammonia in methanol led to
recovery of starting material. The tert-butyl group
of 9 was then quantitatively removed by brief
exposure to neat tri¯uoroacetic acid (TFA) at
room temperature to give 2.
The coupling of glucosylamine 1 with dipeptide
2 in DMF at room temperature via the agency of
2-ethoxy-l-N-ethoxycarbonyl-1,2-dihydroquinoline
EEDQ [24] a€orded the fully protected glycopep-
tide 3 in 43% yield after ¯ash chromatography.
The coupling could also be performed in similar
yield using DCC in the presence of DhbtOH. As a
consequence of its poor solubility properties (solu-
ble only in Me2NCHO, Me2SO or MeOH±CH2Cl2
mixtures), compound 3 proved to be dicult to
purify, and repeated chromatography was
required, which partly accounted for the moderate
The synthesis of the glycosylamine 1 began from
the readily available glycosyl azide 10 [21]. Com-
pound 10 was hydrogenated in ethanol with platinum
oxide catalyst for 5 h. The resulting glycosylamine
is known to be unstable, giving rise to dimerization
products even when stored at room temperature
after recrystallization [21]. The crude amine was
thus treated immediately with benzyl chloroformate
and pyridine in dichloromethane at 0 ꢀC to give the
carbamate 11 in 61% overall yield, after recrys-
tallization. Compound 11 was then deacetylated
with ammonia in methanol to give the triol 12.
Crude 12 was selectively tosylated in 82% overall yield
by treatment with 1.2 equivalentsꢀ of p-toluene-
sulfonyl chloride in pyridine at 0 C to give the
tosylate 13. The tosylate 13 was smoothly converted
into the azide 14 in good yield by heating with
sodium azide in DMF. The ¯uorescent anthranila-
mide group was then readily introduced in an e-
cient, one-pot procedure. The azido group was ®rst
reduced by heating with triphenylphosphine in 7:1
THF±water [22] and was then coupled with Boc-
ABz-ODhbt [23]. The crude reaction mixture was
acetylated (excess acetic anhydride±pyridine), and
the desired product 15 was obtained as a crystalline
solid in 91% overall yield following puri®cation by
1
yield. Compound 3 was fully characterized by H
and 13C NMR spectroscopy and mass spectro-
metry. Deprotection to give the target compound 4
was achieved by deacetylation with ammonia in
methanol followed by brief treatment with aqueous
TFA. The crude product, obtained as the TFA salt
in essentially quantitative yield, was approximately
1
92% pure as estimated by H NMR spectroscopy,
analytical reversed-phase HPLC and ESIMS (M,
780.5). Final puri®cation was performed by semi-
preparative reversed-phase HPLC to give pure
glycopeptide 4 as a yellow, amorphous solid.
3. Experimental
General.ÐMelting points (mp) were determined
on a Laboratory Devices Mel-Temp II melting

534
V. Ferro et al./Carbohydrate Research 306 (1998) 531±538
point apparatus and are uncorrected. Optical rota-
ꢀ
C-ꢂ, Me), 121.2±139.0 (5 C, Ar), 153.6 (ArOH),
170.4 (C=O).
tions were determined at 25 C on a Perkin±Elmer
241 MC polarimeter. NMR spectra were recorded
on Bruker AC 200, Bruker WH 400 or Varian XL
300 instruments and are referenced to the solvent
peak unless otherwise stated. Where required, the
interpretations were supported by COSY or APT
NMR experiments. Mass spectra were obtained by
desorption chemical-ionization (DCI) on a Delsi
Nermag R10-10C mass spectrometer with ammo-
nia as the reactive gas, by the liquid secondary-ion
(LSIMS) technique on a Kratos Concept mass
spectrometer using thioglycerol±water as the
matrix, or by electrospray-ionization (ESIMS) on a
PE/Sciex API 300 mass spectrometer. Semi-
preparative and analytical reversed-phase HPLC
were performed on a Waters 600E HPLC system
using a Waters 10 ꢃm RCM C18 (8 mmÂ10 cm)
N-Benzoyl-l-aspartic acid ꢁ-tert-butyl ester
(8).ÐH-Asp(OtBu)-OH (7) (378 mg, 2.00 mmol)
was dissolved in aq Na2CO3 (4.8 mL of 1 M,
4.8 mmol) and dioxane (4 mL). A solution of ben-
zoyl chloride (338 mg, 278 ꢃL, 2.40 mmol) in diox-
ane (2 mL) was added, followed by water (5 mL).
After stirring for 20 min at room temperature, a
clear, homogeneous solution resulted. The solution
was stirred for a further 16 h at room temperature
and then was extracted with ether. The aqueous
phase was acidi®ed to pH 2 with M HCl and
extracted with EtOAc. The EtOAc extracts were
washed successively with water and brine, dried
(MgSO4), ®ltered and concentrated. The residue
was recrystallized from diisopropyl ether±petro-
leum ether to give title compound 8 as a white
microcrystalline solid (550 mg, 94%): mp 162±163 ꢀC;
[ꢂ]d 2 ꢀ (c 1.5, MeOH); 1H NMR (200 MHz,
Me2SO-d6): ꢄ 1.36 (s, Me3), 2.66, 2.84 (2 dd, AB
part of an ABX system, JAB 15.9, JAX 8.6, JBX
5.9 Hz, ꢁ-H), 4.78 (m, ꢂ-H), 7.40±7.60, 7.78±7.89 (2
m, 5 H, Ph), 8.74 (d, J 8.3 Hz, NH); 13C NMR
(50 MHz, Me2SO-d6): ꢄ 27.5 (Me3), 37.0 (C-ꢁ), 49.5
(C-ꢂ), 80.3 (CMe3), 127.4±134.0 (Ph), 166.2, 169.5,
172.5 (C=O); DCI: m/z 294 (M+H+, 60.3%), 311
(M+NH4+, 58.8%). Anal. Calcd for C15H19NO5:
C, 61.42; H, 6.53; N, 4.78. Found: C, 61.57; H,
6.64; N, 4.73.
1
column with a ¯ow rate of 2 mL min and detec-
tion at 254 nm using a Waters 486 tunable absor-
bance detector. Solvent system A: 0.05% TFA in
49:1 water±acetonitrile; B: 0.05% TFA in 1:4
water±acetonitrile. Microanalyses were performed
by Mr. Peter Borda in the Microanalytical
Laboratory of the University of British Columbia.
Solvents and reagents used were either reagent
grade, certi®ed, or spectral grade and where neces-
sary were dried by standard procedures. Petroleum
ꢀ
ether had a boiling range of 35±60 C. Reactions
were monitored by thin-layer chromatography
(TLC) using E. Merck Kieselgel 60F254 aluminium-
backed sheets. Compounds were detected (where
possible) under UV light or by charring with 10%
sulfuric acid in MeOH or with 10% ammonium
molybdate in 2 M sulfuric acid. Flash chromato-
graphy was performed on short columns (10±
15 cm) of E. Merck Kieselgel 60 (230±400 mesh)
under a positive pressure with speci®ed eluants.
3-Nitro-l-tyrosine and H-Asp(OtBu)-OH were
from Aldrich Chemical Company and Bachem,
respectively.
3-Nitro-l-tyrosine methyl ester hydrochloride
(6).ÐThe title compound was prepared according
to the method of Hanson and Law [20] in 80%
yield as ®ne yellow needles: mp 195±197 ꢀC (EtOH/
EtOAc; lit [20]. 195±197 ꢀC); [ꢂ]d+8.6ꢀ (c 0.8,
MeOH); lit [20].+8.9 ꢀ; 1H NMR (200 MHz, D2O,
ext. DSS): ꢄ 3.12±3.34 (m, 2 H, AB part of an ABX
system, JAB 14.7, JAXꢁJBX=6.7 Hz, ꢁ-H), 3.79 (s,
Me), 4.40 (t, J 6.7 Hz, ꢂ-H), 7.09 (d, J5,6 8.5 Hz, H-
5), 7.47 (dd, J2,6 2.2 Hz, H-6), 7.92 (d, H-2); 13C
NMR (50 MHz, D2O): ꢄ 35.0 (C-ꢁ), 54.4 (2 C,
N-Benzoyl-Asp(ꢁ-OtBu)-Tyr(NO2)-OMe (9).Ð
A solution of 8 (300 mg, 1.02 mmol), DhbtOH
(167 mg, 1.02 mmol) and 6 (304ꢀmg, 1.10 mmol) in
DMF (10 mL) was cooled to 0 C. DCC (210 mg,
1.02 mmol) and N-ethyldiisopropylamine (142 mg,
192 ꢃL, 1.10 mmol) wꢀere then added, and the mix-
ture was stirred at 0 C for 1 h and then at room
temperature for 16 h. The mixture was ®ltered
(Celite), and the ®lter cake was washed well with
EtOAc. The combined ®ltrate and washings were
washed with successively with M HCl, water and
brine, dried (MgSO4), ®ltered and concentrated.
Flash chromatography (3:7 EtOAc±petroleum
ether) gave the dipeptꢀide 9 (480 mg, 91%) as yellow
needles: mp 102±105 C (EtOAc±petroleum ether);
[ꢂ]d +22 ꢀ (c 0.5, CHCl3); H NMR (400 MHz,
1
CDCl3): ꢄ 1.44 (s, Me3), 2.56, 2.94 (2 dd, AB part
of an ABX system, JAB 16.9, JAX 6.2, JBX 4.0 Hz,
Asp ꢁ-H), 2.97, 3.15 (2 dd, AB part of an ABX
system, JAB 14.2, JAX 6.9, JBX 5.2 Hz, Tyr ꢁ-H),
3.73 (s, OMe), 4.81 (ddd, JH,NH 12.8 Hz, Tyr ꢂ-H),
4.92 (ddd, JH,NH 10.2 Hz, Asp ꢂ-H), 6.87±7.83 (10

V. Ferro et al./Carbohydrate Research 306 (1998) 531±538
535
H, ArH, NH), 10.36 (s, OH); 13C NMR (75 MHz,
CDCl3): ꢄ 27.9 (Me3), 35.8, 36.5 (C-ꢁ), 49.6 (C-ꢂ
Asp), 52.6, 53.0 (C-ꢂ Tyr, OMe), 82.1 (CMe3),
120.0±138.5 (11 C, Ar), 154.0 (ArOH), 167.2,
170.4, 170.9, 172.0 (C=O); DCI: m/z 516 (M+H+,
58.4%), 533 (M+NH4+, 100%). Anal. Calcd for
C25H29N3O9: C, 58.24; H, 5.67; N, 8.15. Found: C,
58.27; H, 5.72; N, 8.14.
1.88, 2.01, 2.03, 2.06 (4 s, Me), 3.71 (m, H-5), 4.06±
4.15 (m, 2 H, H-2, 6A), 4.27 (dd, B part of an ABX
system, J6A,6B 12.5, J5,6B 4.0 Hz, H-6B), 4.84 (dd,
J3 ,4&J =9.2 Hz, H-4), 4.99±5.12 (m, 4 H, H-l, 3,
4,5
PhCH2), 5.88 (d, J 8.2 Hz, NH), 6.20 (d, J 8.6 Hz,
NH), 7.27±7.39 (m, Ph); 13C NMR (50 MHz,
CDCl3): ꢄ 20.5, 20.7, 23.0 (4ÂCOMe), 52.9 (C-2),
61.9 (C-6), 67.1 (PhCH2), 68.0 (C-4), 73.1, 73.2 (C-
3, 5), 82.5 (C-1), 127.9±135.9 (Ph), 156.0 (NCO2),
169.3±171.6 (C=O). Anal. Calcd for C22H28N2O10:
C, 54.99; H, 5.87; N, 5.83. Found: C, 54.95; H,
5.84; N, 5.87.
N-Benzoyl-Asp-Tyr(NO2)-OMe (2).ÐThe dipep-
tide 9 (270 mg, 0.52 mmol) was dissolved in neat
tri¯uoroacetic acid (1.5 mL) and stirred at room
temperature for 1 h. The mixture was then diluted
with toluene and concentrated. The residue was
treated with toluene and concentrated (Â2) to give
title compound 2 (240 mg, 100%) as a yellow solid:
2 - Acetamido - 2 - deoxy- 6 - O - p - toluenesulfonyl-
N-(benzyloxycarbonyl) - ꢁ - d - glucopyranosylamine
(13).ÐThe carbamate 11 (2.13 g, 4.44 mmꢀol) was
suspended in MeOH (50 mL), cooled to 0 C, and
the mixture was saturated with ammonia. The
ꢀ
mp 204±205 C (MeOH); [ꢂ]d 33 ꢀ (c 1.1, DMF);
1H NMR (200 MHz, Me2SO-d6): ꢄ 2.60, 2.71(2 dd,
AB part of an ABX system, JAB 16-6, JAX 8.5, JBX
5.4 Hz, Asp ꢁ-H) 2.92, 3.02 (2 dd, AB part of an
ABX system, JAB 14.2, JAX 8.6, JBX 5.6 Hz Tyr ꢁ-
H) 3.60 (s, OMe), 4.39±4.50, 4.73±4.84 (2 m, ꢂ-H),
6.90±8.58 (10 H, ArH, NH), 10.81, 12.24 (2 bs, OH,
CO2H); 13C NMR (75 MHz, Me2SO-d6): ꢄ 34.9,
35.8 (C-ꢁ), 49.9 (C-ꢂ Asp), 52.0, 53.5 (C-ꢂ Tyr,
OMe), 119.0±136.2 (11 C, Ar), 150.9 (ArOH), 166.2
(CO2H), 171.0, 171.6, 171.8 (C=O); DCI: m/z 460
(M+H+, 100%). Anal. Calcd for C21H21N3O9: C,
54.90; H, 4.61; N, 9.15. Found: C, 54.70; H, 4.70;
N, 9.02.
ꢀ
mixture was stirred at 0 C for 6 h and then con-
centrated. The residue was dissolved in pyridine
(20 mL) and cooled to 0 ꢀC, p-toluenesulfonyl
chloride (1.02 g, 5.35 mmol) was added, and the
ꢀ
mixture was stirred at 0 C for 1 h and then at
room temperature for 16 h. Water (0.5 mL) was
added, and the mixture was stirred for 20 min and
then concentrated. Flash chromatography (1:9
MeOH±EtOAc) then gave the p-toluenesulfonate
ꢀ
13 (1.84 g, 82%) as needles: mp 116±118 C (dec.;
EtOH±diisopropyl ether); [ꢂ]d 5.5 ꢀ (c 0.2, CHCl3);
1H NMR (200 MHz, CDCl3): ꢄ 1.88 (s, Me), 2.36
(s, ArMe), 3.40±3.65 (m, H-3, 4, 5), 3.80 (m, H-2),
4.14, 4.39 (2 bd, AB part of an ABX system, J6A,6B
10.1 Hz, H-6A, 6B), 4.78 (dd, J1,2 9.3 J1,NH 8.9 Hz,
H-1), 5.06 (s, PhCH2), 5.10±5.27 (bm, 2 H, OH),
6.56 (d, NHZ), 6.85 (d, J2,NH 7.6 Hz, NHAc), 7.12±
7.80 (m, 9 H, Ar); 13C NMR (50 MHz, CDCl3): ꢄ
21.6 (ArMe), 23.0 (Me), 54.4 (C-2), 66.9, 69.1 (C-6,
PhCH2), 69.8 (C-4), 74.6, 74.8 (C-3, 5), 81.9 (C-l),
127.9±145.0 (Ar), 156.2 (NCO2), 173.3 (C=O).
Anal. Calcd for C23H28N2O9S: C, 54.32; H, 5.55;
N, 5.51. Found: C, 54.24; H, 5.52; N, 5.38.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-N-(benzyl-
(11).ÐA
oxycarbonyl)-ꢁ-d-glucopyranosylamine
solution of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
ꢁ-d-glucopyranosyl azide (10) [21] (3.00 g,
8.06 mmol) in EtOH (80 mL) was hydrogenated at
room temperature and atmospheric pressure in the
presence of platinum oxide (250 mg) for 5 h. The
mixture was then ®ltered (Celite) and concentrated.
The residue was dissolved in a mixture of CH2Cl2
ꢀ
(25 mL) and pyridine (5 mL) and cooled to 0 C.
Benzyl chloroformate (2.17 g of 95%, 1.82 mL,
12.1 mmol) was then added dropwise, and the
2-Acetamido-6-azido-2,6-dideoxy-N-(benzyloxy-
carbonyl)-ꢁ-d-glucopyranosylamine (14).ÐA mix-
ture of the p-toluenesulfonate 13 (2.08 g, 4.09 mmol)
and sodium azide (1.33 g, 20.5 mmol) in DMF
ꢀ
mixture was stirred at 0 C for 1 h and then at
room temperature for 16 h. The mixture was then
diluted with CH2Cl2 and poured into water. The
organic phase was separated, and the aqueous
phase was extracted with CH2Cl2. The combined
organic phases were washed successively with M
HCl, water and brine, dried (MgSO4), ®ltered and
concentrated to a solid. Recrystallization from
MeOH±diethyl ether then gave the carbamate 11
(2.36 g, 61%) as needles: mp 221±222 ꢀC; [ꢂ]d 32 ꢀ
ꢀ
(40 mL) was heated at 80 C for 16 h. The mixture
was then poured into water and extracted with
EtOAc. The combined extracts were washed suc-
cessively with water and brine, dried (MgSO4),
®ltered and concentrated to give a white solid.
Recrystallization from MeOH±diethyl ether then
gaveꢀthe azide 14 (1.16 g, 75%) as needles: mp 203±
(c 0.3, CHCl3); H NMR (400 MHz, CDCl3): ꢄ
205 C (dec.); [ꢂ]d 33 ꢀ (c 1.7, Me2NCHO); H
1
1

536
V. Ferro et al./Carbohydrate Research 306 (1998) 531±538
NMR (200 MHz, Me2SO-d6): ꢄ 1.78 (s, Me), 3.06
(bddd, Jl,2 9.3, J2,3 8.3, J2,NH 8.0 Hz, H-2), 3.19±
3.60 (m, 5 H, H-3, 4, 5, 6A, 6B), 4.70 (bdd, J1,NH
9.0 Hz, H-l), 5.04 (s, PhCH2), 5.08 (d, 1 H, J
5.4 Hz, OH), 5.26 (d, 1 H, J 5.9 Hz, OH), 7.09±7.23
(m, Ph), 7.64 (d, NHZ), 7.88 (d, NHAc); 13C NMR
(50 MHz, Me2SO-d6): ꢄ 23.0 (Me), 51.3 (C-6), 54.6
(C-2), 65.6 (PhCH2), 71.3 (C-4), 74.2 (C-5), 76.5
(C-3), 81.8 (C-l), 127.6±137.0 (Ph), 155.8 (NCO2),
170.4 (C=O). Anal. Calcd for C16H21N5O6: C,
50.65; H, 5.58; N, 18.46. Found: C, 50.29; H, 5.70;
N, 18.29.
Nꢀ -[2-Acetamido-2,3-di-O-acetyl-6-(20 -tert-
butoxycarbonylamino)benzamido-2,6-dideoxy-ꢁ-d-
glucopyranosyll-Nꢂ-benzoyl-Asn-Tyr(NO2)-OMe
(3).ÐA solution of the carbamate 15 (344 mg,
0.52 mmol) in EtOH (10 mL) was hydrogenated at
room temperature and atmospheric pressure in the
presence of 10% palladium-on-charcoal (30 mg)
for 1 h. The mixture was then ®ltered (Celite) and
concentrated to give the amine, 2-acetamido-2,3-
di-O-acetyl-6-(20 -tert-butoxycarbonylamino)benz-
amido-2,6-dideoxy-ꢁ-d-glucopyranosylamine (1)
(275 mg, 100%) as a white foam that was used
2-Acetamido-2,3-di-O-acetyl-6-(20 -tert-butoxy-
carbonylamino)benzamido-2,6-dideoxy-N-(benzyl-
oxycarbonyl)-ꢁ-d-glucopyranosylamine (15).ÐA
solution of the azide 14 (540 mg, 1.42 mmol) and
triphenylphosphine (560 mg, 2.13 mmol) in 7:1
THF±water (16 mL) was heated at re¯ux for 3 h.
The mixture was cooled to room temperature and
Boc-ABz-ODhbt [23] (655 mg, 1.71 mmol) was
added. The mixture was stirred for 3 h and then
was concentrated. The residue was dissolved in
CH2Cl2 (20 mL) and treated with acetic anhydride
(5 mL) and pyridine (5 mL) for 6 h at room tem-
perature. MeOH (5 mL) was added, and the mix-
ture was stirred for 20 min and then concentrated.
The residue was dissolved in EtOAc and was
washed successively with M HCl, water, saturated
aqueous sodium bicarbonate and brine, dried
(MgSO4), ®ltered and concentrated. The residue
was puri®ed by ¯ash chromatography (7:3 EtOAc±
petroleum ether) to give the title compound 15
without further puri®cation. H NMR (200 MHz,
1
CDCl3): ꢄ 1.50 (s, Me3), 1.8±2.10 (bs, NH2), 1.95,
2.03, 2.09 (3 s, Ac), 3.36 (ddd, A part of an ABX
system, J6A,6B 14.5, J5,6A&J6A,NH=5.8 Hz, H-6A),
3.59 (ddd, J5,6B 2.5, J4,5 9.0 Hz, H-S), 3.85 (ddd, B
part of an ABX system, J6B,NH 7.0 Hz, H-6B), 3.97
(ddd, J1,2 9.5 J2,3 J2,NH=9.0 Hz, H-2), 4-13 (d, H-
1), 4.95 (dd, J3,4 9.0 Hz, H-4), 5.05 (dd, H-3), 5.68
(d, NHAc), 6.66 (bdd, NHCOAr), 6.98±8.40 (4 H,
Ar), 10.02 (s, NHBoc).
(a) Using DCC. The dipeptide Bz-Asp-
Tyr(NO2)-OMe (2, 74 mg, 0.16 mmol), DCC
(33 mg, 0.16 mmol) and DhbtOH (26 mg,
0.16 mmol) were dissolved in DMF (0.5 mL) and
stirred at room temperature for 20 min. The amine
1 (84 mg, 0.16 mmol) in DMF (0.5 mL) was then
added, and the mixture stirred at room tempera-
ture overnight. The mixture was ®ltered, diluted
with ethyl acetate, washed with water and brine,
dried over sodium sulfate, and the solvent was
removed in vacuo. Repeated ¯ash chromatography
(1:9 MeOH±CH2Cl2) gave the protected glycopep-
tide 3 (65 mg, 42%) as a yellow amorphous solid.
(b) Using EEDQ. EEDQ (99 mg, 0.40 mmol)
was added to a solution of the amine 1 (155 mg,
0.29 mmol) and Bz-Asp-Tyr(NO2)-OMe 2 (138 mg,
0.30 mmol) in DMF (5 mL). The mixture was stir-
red at room temperature under nitrogen for 4 days.
The mixture was concentrated, and the residue was
subjected to repeated ¯ash chromatography (1:9
MeOH±CH2Cl2) to give the protected glycopeptide
3 (122 mg, 43%) as a yellow amorphous solid: [ꢂ]d
ꢀ
(850 mg, 91%) as needles: mp 216±218 C (EtOH±
diisopropyl ether); [ꢂ]d +17 ꢀ (c 1.5, CHC13); H
1
NMR (400 MHz, CDCl3): ꢄ 1.50 (s, Me3), 1.90,
2.04, 2.08 (3 s, Ac), 3.45 (ddd, A part of an ABMX
system, J6A,6B 14.2, J5,6A&J6A,NH=6.0 Hz, H-6A)
3.64±3.72 (m, H-5), 3.76 (ddd, B part of an ABMX
system, J5,6b 2.7, J6b,NH 6.2 Hz, H-6B) 4.10 (ddd,
J1,2 9.0, J2,3 10.1, J2,NH 8.1 Hz, H-2), 4.86 (dd,
J1,NH 8.1 Hz, H-l), 4.97 (dd, J3,4&J4,5=9.5 Hz, H-
4), 5.04 (dd, H-3), 5.02±5.14 (m, 2 H, PhCH2), 6.06
(d, NHAc), 6.38 (d, NHZ), 6.61 (bdd, NHCOAr),
6.29±8.36 (9 H, Ar), 10.02 (s, NHBoc); 13C NMR
(50 MHz, CDCl3): 20.60, 20.65, 23.0 (4ÂCOMe),
28.3 (CMe3), 39.5 (C-6), 52.9 (C-2), 67.3 (PhCH2),
69.2 (C-4), 73.2, 73.7 (C-3, 5), 80.1 (CMe3), 82.2
(C-l), 119.6±140.2 (Ar), 153.0, 156.3 (NCO2),
168.9±171.7 (C=O). DCI: m/z 657 (M+H+,
6.57%), 674 (M+NH4+, 1.77%). Anal. Calcd for
C32H40N4O11: C, 58.53; H, 6.14; N, 8.53. Found:
C, 58.58; H, 6.27; N, 8.51.
+4.8 ꢀ (c 1.3, Me2NCHO); H NMR (400 MHz,
1
Me2SO-d6): ꢄ 1.47 (s, Me3), 1.62, 1.87, 1.95 (3 s,
Ac), 2.57, 2.62 (2 dd, AB part of an ABX system,
JAB 16.2, JAX 8.5, JBX 5.1 Hz, Asn ꢁ-H), 2.91, 2.99
(2 dd, AB part of an ABX system, JAB 14.0, JAX
8.7, JBX 5.4 Hz, Tyr ꢁ-H), 3.35 (obscured by H2O,
H-6A), 3.51 (ddd, B part of an ABMX system,
J6A,6B 14.2, J5,6B 5.5, J6B,NH 5.6 Hz, H-6B), 3.60 (s,

V. Ferro et al./Carbohydrate Research 306 (1998) 531±538
OMe), 3.77 (ddd, J5,6a 3.4, J4,5 9.7 Hz H-5) 3.92 Acknowledgements
537
(ddd J2,3 9.5, J2,3 9.8, J2,NH 9.2 Hz, H-2), 4.44 (ddd,
JH,NH 7.7 Hz, Tyr ꢂ-H), 4.71 (dd, J3,4 9.8 Hz, H-4),
4.80 (ddd, JH,NH 8.0 Hz, Asn ꢂ-H) 5.03 (dd, H-3),
5.14 (dd, J1,NH 9.1 Hz, H-l), 6.92±8.18 (Ar), 7.84
(d, NHAc), 8.25 (d, NHTyr), 8.45 (d, NHBz), 8.49
(d, Asn NꢀH), 8.65 (bdd, J6,ANH 5.6 Hz, C-6 NH)
10-44 (s, NHBoc); 13C NMR (75 MHz, Me2SO-d6):
ꢄ 20.4, 20.6, 22.5 (COMe), 27.9 (CMe3), 35.0, 36.7
(C-ꢁ), 39.5 (C-6), 49.8, 52.0, 53.4 (4 C, C-ꢂ, C-2,
OMe), 70.0 (C-4), 73.0, 73.8 (C-3, 5), 78.2 (C-l),
79.7 (CMe3), 118.4±139.5 (17 C, Ar), 151.1, 152.1
(ArOH, NCO2), 166.1±171.5 (8 C, C=O). LSIMS:
m/z 964 (M+H+, 0.5%); HRMS calcd for
C45H54N7O17: 964.3576; found: 964.3592. Anal.
The authors wish to thank Herman Ziltener for
valuable discussions, and Todd Schindler and
Edward Koepf for technical assistance. Funding
for this research was provided by the Natural Sci-
ences and Engineering Research Council of
Canada.
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Calcd for C45H53N7O17 H2O: C, 55.04; H, 5.64; N,
9.98. Found: C, 55.17; H, 5.58; N, 9.85.
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ꢀ
in MeOH (5 mL), the mixture was cooled to 0 C,
then saturated with ammonia and stirred overnight
ꢀ
at 0 C. The resulting orange solution was con-
centrated, and the residue was dissolved in 19:1
tri¯uoroacetic acid±water (2 mL) and stirred at
room temperature for 10 min. Toluene (2Â5 mL)
was then added, and the mixture was concentrated
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1
mately 92% pure as estimated by H NMR and
analytical reversed-phase HPLC/ESIMS. Final
puri®cation was achieved by semipreparative
reversed-phase HPLC using a linear gradient of
30±80% solvent B in 20 min. [ꢂ]d 37ꢀ (c 0.2
1
DMF); H NMR (400 MHz, Me2SO-d6+D2O): ꢄ
1.64 (s, Ac), 2.52, 2.58 (2 dd, AB part of an ABX
system, JAB 17.1, JAX 8.4, JBX 5.1 Hz, Asn ꢁ-H),
2.87, 2.98 (2 dd, AB part of an ABX system, JAB
13.9, JAX 8.6, JBX 5.4 Hz, Tyr ꢁ-H), 3.01 (dd,
J3,4&J4,5=9.0 Hz, H-4), 3.24±3.39 (m, 3H, H-3, 5,
6A), 3.52 (dd, J1,2 9.8, J2,3 9.9 Hz, H-2) 3.56 (s,
OMe), 3.55±3.62 (m, H-6B), 4.43 (dd, Tyr ꢂ-H),
4.75 (dd, Asn ꢂ-H), 4.81 (d, H-l), 6.62±7.76 (Ar);
13C NMR (75 MHz, Me2SO-d6): ꢄ 22.7 (COMe),
35.0, 36.8 (C-ꢁ), 39.5 (obscured by Me2SO, C-6),
49.8 (C-ꢂ Asn), 52.0, 53.4, 54.5 (C-ꢂ Tyr, C-2,
OMe), 72.3, 73.9, 76.2 (C-3, 4, 5), 79.1 (C-l), 115.3±
150.9 (Ar), 158.2 (q, JCF 33 Hz, F3CCO2), 166.1,
169.2, 169.8, 170.0, 171.4, 171.6 (C=O). LSIMS:
m/z 780 (M, 4.7%) 802 (M+Na+, 5.5%); HRMS
Calcd for C36H42N7O13: 780.2841; found 780.2853.

538
V. Ferro et al./Carbohydrate Research 306 (1998) 531±538
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