Synthesis of fully O-benzylated N-linked core pentasaccharide glycosyl azide

Article abstract of DOI:10.1080/07328303.2011.642433

The fully O-benzylated pentasaccharide glycosyl azide representing the common core structure of N-glycans was synthesized. The β-mannosidic linkage was created by C-2 epimerization of the initially introduced β-d-gluco-unit via DMSO/Ac₂O oxidat

Full text of DOI:10.1080/07328303.2011.642433

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Synthesis of Fully O-Benzylated N-Linked
Core Pentasaccharide Glycosyl Azide
László Kalmár a , Károly Ágoston a , Zoltán Szurmai b , Boglárka
Döncző c & János Kerékgyártó c
a Glycom Hungary, MTA-KKKI, H-1025, Budapest, Hungary
b University of Debrecen, Faculty of Sciences and Technology,
Department of Applied Ecology, Bio-organic Laboratory, H-4010,
Debrecen, Hungary
c University of Debrecen, Faculty of Sciences and Technology,
Department of Botany, Bio-organic Laboratory, H-4010, Debrecen,
Hungary
To cite this article: László Kalmár, Károly Ágoston, Zoltán Szurmai, Boglárka Döncző & János
Kerékgyártó (2012): Synthesis of Fully O-Benzylated N-Linked Core Pentasaccharide Glycosyl Azide,
Journal of Carbohydrate Chemistry, 31:3, 203-219
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Journal of Carbohydrate Chemistry, 31:203–219, 2012
ꢀ
C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2011.642433
Synthesis of Fully
O-Benzylated N-Linked
Core Pentasaccharide
Glycosyl Azide
1
1
2
´
La´ szlo´ Kalma´ r, Ka´ roly Agoston, Zolta´ n Szurmai,
Bogla´ rka Do¨nczo˝,3 and Ja´ nos Kere´kgya´ rto´3
1Glycom Hungary, MTA-KKKI, H-1025 Budapest, Hungary
2University of Debrecen, Faculty of Sciences and Technology, Department of Applied
Ecology, Bio-organic Laboratory, H-4010, Debrecen, Hungary
3University of Debrecen, Faculty of Sciences and Technology, Department of Botany,
Bio-organic Laboratory, H-4010, Debrecen, Hungary
The fully O-benzylated pentasaccharide glycosyl azide representing the common core
structure of N-glycans was synthesized. The β-mannosidic linkage was created by C-2
epimerization of the initially introduced β-D-gluco-unit via DMSO/Ac2O oxidation fol-
lowed by stereoselective reduction with tetrabutylammonium borohydride.
Keywords Glycosyl azides; N-glycopeptides; Core-pentasaccharide; ß-D-Manno-
pyranoside; Thioglycosides
INTRODUCTION
N-Glycoproteins are ubiquitous on eukaryotic cell surfaces and in body flu-
ids. Their glycans play critical roles both in normal biological and patho-
logical recognition events, such as chronic inflammation, viral and bacterial
infections, and tumorigenesis and metastasis, and in cellular adhesion pro-
cesses, immunology, and cell growth regulation.[1] Protein glycosylation is not
under direct genetic control and, therefore, results in the formation of glyco-
forms that possess the same peptide backbone but differ in the oligosaccharide
Received June 7, 2011; accepted November 16, 2011.
´
Address correspondence to Ka´roly Agoston, Glycom Hungary, MTA-KKKI, H-1025
Budapest, Pusztaszeri ut 59-67, Hungary. E-mail: karoly.agoston@glycom.com; Ja´nos
Kere´kgya´rto´, University of Debrecen, Faculty of Sciences and Technology, Department
of Botany, Bio-organic Laboratory, H-4010, Debrecen, Egyetem ter 1, Hungary. E-mail:
kerek@puma.unideb.hu
203

L. Kalma´ r et al.
204
component and the site of glycosylation.[2] Due to this type of microhetero-
geneity, glycopeptides, the characteristic partial structures of glycoproteins,
are not obtained easily from biological sources or recombinant gene technol-
ogy. Therefore, chemical synthesis provides a valuable means to produce ho-
mogeneous glycopeptides, which serve as model compounds to gain insight into
N-glycoprotein structure and function, as tools to study biomolecular interac-
tions, and as effectors to evoke biological responses.[3]
A number of excellent reviews have been published on the synthetic meth-
ods for constructing glycopeptides.[4] In general, three major strategies have
been used to generate N-glycopeptides. The chemo-enzymatic synthesis of
N-glycopeptides has the advantage of a considerable reduction of protecting
groups, but it requires the availability of glycosyltransferases and nucleoside-
activated sugar building blocks. The stepwise method or linear assembly in-
volves the incorporation of preformed glycosyl amino acids as building blocks in
conventional solid-phase or solution-phase peptide synthesis. The third strat-
egy is the direct coupling between an oligosaccharide glycosylamine and a
previously synthesized, selectively protected polypeptide containing a free as-
partyl side chain (convergent assembly). For both strategies of the latter, it is
critical to have access to oligosaccharides with an azido or free amino group
linked to their reducing end.
Our synthesis of N-glycopeptides was based on the third strategy. First,
fully O-benzylated mono-, di-, and trisaccharide glycosyl azides represent-
ing the reducing terminal of the core structure of N-glycans were pre-
pared.[5] Chemoselective reduction of their azido functionalities resulted in
fully O-benzylated glycosyl amines. These electrophilic glycosyl amines were
incorporated into a peptide sequence, such as Leu-Lys-Asn∗-Gly-Gly-Pro,
which is a partial structure of the Trp-cage mini-protein, by either conver-
gent or linear assembly to form glycopeptides.[6] Here, we report the synthesis
of the fully O-benzylated glycosyl azido derivative of the core pentasaccha-
ride of N-linked glycoprotein glycans, which is useful for the preparation of
N-glycopeptides.
RESULTS AND DISCUSSION
Our target molecule contains a β-D-Man-(1→4)-β-D-GlcNAc linkage, which is
one of the most difficult anomeric linkages to construct because of the con-
comitant occurrence of both the α-directing anomeric effect of mannose and
the repulsion between mannose axial C-2 oxygen and the approaching nucle-
ophiles. Furthermore, the 4-OH group in D-GlcNAc derivatives has relatively
4
low reactivity, especially in compounds with the C1 conformation. The direct
chemical synthesis of β-mannosides was achieved via glycosyl triflates.[7] The
method provided β-mannopyranosides in high yields (76–95%) and stereose-
lectivity for a wide variety of primary and secondary glycosyl acceptors except

Synthesis of Glycosyl Azide
205
Figure 1: Retrosynthetic plan for the N-glycan core pentasaccharide.
for D-GlcNAc derivatives with the 4-OH (the corresponding β-mannoside was
obtained in a yield of 31%, with the anomeric ratio of β:α = 3.8:1). Later, this
method was improved to reach 60% to 70% total yield but still remained tech-
nically difficult to handle.[8] The development of diverse and innovative strate-
gies for the synthesis of β-D-mannopyranosides has been well reviewed.[9]
The synthesis of the core pentasaccharide glycosyl azides with protecting
groups different from our synthetic target, which was fully O-benzylated, was
carried out by many research groups.[10] In those syntheses, the “insoluble pro-
moter strategy,”[10a] “oxidation-reduction method,”[10b] and SN2 displacement of
a triflate leaving group[10c,d] were selected for the construction of the β-D-Man-
(1→4)-β-D-GlcNAc linkage. Our synthesis was based on a different strategy
with a retrosynthetic pathway shown in Figure 1. The key steps of our ap-
proach included the synthesis of glycosyl azide via thioalkyl glycoside and the
preparation of a suitably protected disaccharide-thioglycoside containing a β-
mannosidic linkage.[5]
For the synthesis of the key suitably protected disaccharide-thioglycoside,
first, the glycosyl donor bearing O-allyl ether temporary protecting groups
at the subsequently α-mannosylated positions was prepared as shown in
Scheme 1. Initially, we tried to regioselectively introduce an allyl group to
the 6-OH of 3-O-allyl-1,2-O-isopropylidene-α-D-glucofuranose (1)[11] under the
phase-transfer-catalyzed conditions, which failed in our hand, as a ∼1:1 mix-
ture of the desired 3,6-di-O-allyl-derivative 2 and the corresponding 3,5-di-O-
allyl derivative was obtained. Treatment of compound 1 with allyl bromide
in the presence of Bu2SnO and Bu4NBr in toluene resulted in 3,6-di-O-allyl-
derivative 2 in a yield of 65% (Sch. 1). Removal of the isopropylidene group
with Amberlite IR 120 (H+)/H2O followed by conventional acetylation gave two
anomers of 3.
Compound 3 was treated with HBr/AcOH in dichloromethane at 0◦C
for 20 min to afford the glycosyl bromide 4, which was converted into the

L. Kalma´ r et al.
206
RO
OAll
HO
O
R4O
OAll
R1
OAll
O
R3O
R2
O
O
R
H
R1
R2
R3
Ac
Ac
R4
1
3
4
5
6
7
OAc, H
Ac
Ac
b
a
c
d
e
f
2
All
H
H
H
H
Br
OCH(CH3)OEt Ac
OCH(CH3)OEt Bn
Br
Ac
Bn
Scheme 1: a: AllBr, Bu2SnO, TBABr, in toluene, 65%; b: Amberlite IR120, water, 50◦C, followed
by Ac2O, pyridine, 63% (over two steps); c: HBr/AcOH, DCM, 0◦C; d: EtOH, TBABr, s-collidine,
MeCN, 59% (over two steps); e: BnBr, KOH, THF, 0◦C, 80%; f: HBr/AcOH, DCM, 0◦C.
corresponding orthoester 5 with Bu4NBr/EtOH/collidine. The acetyl function-
ality of 5 was changed to the benzyl group in one pot using benzyl bro-
mide and KOH, yielding orthoester 6 (Sch. 1). Treatment of compound 6
with HBr/AcOH in dichloromethane at 0◦C gave glycosyl bromide 7 that was
used as a glycosyl donor without any purification. Condensation of 7 with
acceptor 8[12] in dichloromethane-toluene in the presence of silver triflate
afforded crystalline disaccharide 9 in a yield of 82% (Sch. 2). The stereochem-
istry of the newly formed glycosidic linkage of compound 9 was determined by
NMR analysis. Coupling constants of H-1ꢁ/H-2ꢁ (J1 ,2 = 8.2 Hz) and C-1ꢁ/H-1ꢁ
ꢁ
ꢁ
Scheme 2: a: AgOTf, toluene-DCM, −50◦C, 82% (over two steps); b: K2CO3, THF, MeOH, 76%;
c: DMSO, Ac2O; d: TBABH4, THF, 90% (over two steps); e: BnBr, Ag2O, KI, DMF, 0◦C, 87%; f:
(PPh3)3PhCl, EtOH, acetone, HCl (1N), 63%; g: pyridine, Ac2O, 90%.

Synthesis of Glycosyl Azide
207
ꢁ
ꢁ
(JC1 -H1 = 161 Hz) confirmed the β-gluco-configuration, since for the corre-
ꢁ
ꢁ
ꢁ
ꢁ
sponding α-anomer J1 ,2 and JC1 -H1 values were expected to be ∼2 Hz and
∼170 Hz, respectively.
Removal of the 2ꢁ-O-acetyl group of compound 9 with K2CO3 in methanol
and tetrahydrofuran (9 → 10) followed by oxidation of the 2ꢁ-OH group with
DMSO and acetic anhydride afforded crude ulose 11. During the reduction
the highest stereoselectivity was obtained when crude ulose 11 was reduced
with Bu4NBH4 in tetrahydrofuran, furnishing disaccharide 12 bearing β-
mannosidic linkage (90% isolated yield).[5,13] The C-2ꢁ epimerization would not
effect the configuration of the glycosidic linkage, and the coupling constant
between H-1ꢁand H-2ꢁ (J1 ,2 ) of β-mannopyranoside was <1 Hz. Only a faint
spot of the gluco derivative 10 was detectable by TLC. The hydroxyl func-
tion of compound 12 was benzylated using silver oxide as a base affording
disaccharide glycosyl donor 13. Condensation of thioglycoside 13 and glyco-
syl azide 14[5,14] in the presence of methyl triflate resulted in trisaccharide 15
(Sch. 3; 15 was also prepared along another route[10a]). For the preparation of
our target structure the removal of allyl groups in the presence of an anomeric
azido functionality was required. However, under the tested conditions with
commercially available catalysts, including palladium(II) chloride–copper(I)
chloride, tris(triphenylphosphine)rhodium(I) chloride in ethanol, sodium
borohydride–iodine, and palladium on carbon–acetic acid, complex reaction
mixtures were obtained and the desired products were isolated in low yields.
To overcome this difficulty, our preliminary synthetic strategy to utilize 13 as
ꢁ
ꢁ
OR
OBn
OBn
OBn
OBn
O
OBn
O
OBn
O
O
a
BnO
RO
HO
BnO
13 or 17 +
O
BnO
N3
O
BnO
BnO
O
BnO
N3
NPhth
NPhth
NPhth
SEt
R
All
20
14
15
18 Ac
b
19
H
OBn
BnO
BnO
c
OBn
O
OBn
OBn
O
O
BnO
BnO
OBn
OBn
O
OBn
BnO
O
O
O
O
O
BnO
BnO
N3
NR
NR
R
21 Phth
22 H,Ac
d
Scheme 3: a: to 15; MeOTf, DCM, 0◦C, 69%, to 18 NIS/AgOTf, MeCN-DCM, −25◦C, 51%; b:
K2CO3, THF, MeOH, 66%; c: MeOTf, DCM, 0◦C, 71%; d: ethylenediamine, n-ButOH, 90◦C, then
pyridine, Ac2O, 76% (over two steps).

L. Kalma´ r et al.
208
a key disaccharide donor had to be modified. Allyl groups of disaccharide 13
were eventually removed with tris(triphenylphosphine)rhodium(I) chloride in
ethanol (→ 16), and the 3ꢁ-OH and 6ꢁ-OH groups were acetylated to give dis-
accharide 17 (Sch. 2). Condensation of glycosyl donor 17 with acceptor 14 in
the presence of NIS/AgOTf afforded fully protected trisaccharide 18 (Sch. 3).
The coupling constant between H-1ꢁ and H-2ꢁ (J1 ,2 = 8.1 Hz) confirmed the
β-configuration of the newly formed glycosidic linkage.
ꢁ
ꢁ
Removal of 3ꢁꢁ- and 6ꢁꢁ-O-acetyl groups from compound 18 furnished
trisaccharide glycosyl acceptor 19. Glycosylation of 19 with ethyl 2,3,4,6-
tetra-O-benzyl-1-thio-α-D-mannopyranoside (20)[15] in the presence of methyl
triflate yielded fully protected pentasaccharide 21. The coupling constants of
C-1ꢁꢁꢁ/H-1ꢁꢁꢁ and C-1ꢁꢁꢁꢁ/H-1ꢁꢁꢁꢁ (both ∼170 Hz) confirmed the α-configuration of
the newly formed glycosidic linkages. The phthalimido functionalities of the
fully protected pentasaccharide 21 were removed by treatment with ethylene-
diamine and the resulting product was N-acetylated to furnish our target
compound 22.
SUMMARY
A fully O-benzylated pentasaccharide glycosyl azide representing the com-
mon core structure of N-glycans was synthesized for the first time. The
β-mannosidic linkage was created by C-2 epimerization of the initially
introduced β-D-gluco-unit via oxidation followed by stereoselective reduction
with tetrabutylammonium borohydride. Our practical synthetic route to the
core pentasaccharide glycosyl azide does not involve very special or difficult-
to-handle reaction conditions. Very similar structures were synthesized before
with comparable yields, but delicate separations of the anomeric mixtures were
necessary in those cases. The investigation of the coupling reactions between
selectively protected oligopeptides and fully O-benzylated (electron-donating)
glycosyl amine formed after the chemoselective reduction (PtO2/H2) of the
azido functionality of the core pentasaccharide glycosyl azide 22 is in progress.
EXPERIMENTAL
General
Commercially available starting materials were used without further pu-
rification. Solvents were dried according to standard methods. Melting points
(uncorrected) were determined on a Kofler hot-stage apparatus. Optical rota-
tions were measured with a Perkin-Elmer 241 polarimeter. IR spectra were
recorded on a Perkin-Elmer 700 instrument. NMR spectra were recorded on
a DRX-500 (125.83 MHz for 13C) spectrometer in CDCl3 as a solvent. All
chemical shifts are quoted in ppm downfield from the characteristic signals

Synthesis of Glycosyl Azide
209
(1H: 0.00 ppm [TMS], 13C: 77.00 ppm [solvent signal]). Kieselgel 60 (E. Merck,
Darmstadt, Germany) was used for column chromatography. MALDI-TOF
measurements were carried out on a Bruker Biflex III mass spectrometer. 2,5-
Dihydroxybenzoic acid was used as matrix and 100 to 200 laser shots were
applied for each spectrum. Elemental analyses were measured in the Analyti-
cal Department of the University of Debrecen.
3,6-Di-O-allyl-1,2-O-isopropylidene-α-D-glucofuranose (2)
3-O-Allyl-1,2-O-isopropylidene-α-D-glucofuranose[11] (1) (21.0 g, 80.68
mmol) and Bu2SnO (20.1 g, 80.75 mmol) was suspended in toluene (100 mL)
and concentrated to its one-fifth volume, then diluted with toluene (80 mL).
The dilution-concentration procedure was repeated 15 times. Then the residue
˚
was dissolved in dry toluene (200 mL) and molecular sieves were added (4 A,
10 g) and stirred for 20 min. Bu4NBr (29.8 g, 92.44 mmol) and AllBr (18 mL,
212 mmol) was added to the mixture. When TLC showed the complete conver-
sion the mixture was diluted with CH2Cl2 (300 mL), filtered through Celite,
washed with water (3 × 60 mL), dried (MgSO4), filtered, and concentrated.
Column chromatography (hexane:EtOAc 7:3, Rf 0.37) of the residue gave 2
(15.77 g, 65%) isolated as a syrup; [α]2D5 −33 (c 0.22, CHCl3); 1H NMR (CDCl3):
δ 5.95–5.87 (m, 2H, 2 CH ), 5.90 (d, 1H, J1,2 = 4.0 Hz, H-1), 5.33–5.18 (m,
4H, 2 CH2), 4.55 (d, 1H, J2,3 = 3.8 Hz, H-2), 4.19–4.04 (m, 7H, 2 –CH2–CH ,
H-3, H-4 and H-5), 3.72–3.55 (m, 2H, H-6), 2.80 (m, 1H, 5-OH), 1.48 and 1.32
(2s, each 3H, C(CH3)2); 13C NMR (CDCl3) δ 134.45 and 133.92 (2 –CH ), 117.62
and 117.10 (2 CH2), 111.60 (C(CH3)2), 105.03 (C-1), 82.33 (C-2), 81.82 (C-3),
79.34 (C-4), 72.23 and 71.24 (2 –CH2–CH ), 71.90 (C-6), 67.81 (C-5), 26.71 and
26.21 (C(CH3)2). Anal. Cald for C15H24O6: C, 59.97; H, 8.06. Found: C, 60.92;
H, 8.26.
1,2,4-Tri-O-acetyl-3,6-di-O-allyl-α,β-D-glucopyranose (3)
2 (10.20 g, 33.96 mmol) and Amberlite IR 120 resin (H+) (6 g) were
stirred in water (60 mL) at 50◦C for 2 h. The resin was filtered, and the fil-
trate was concentrated and dissolved in the mixture of pyridine and acetic-
anhydride (1:1, 20 mL) and stirred overnight. The mixture was concentrated
and co-concentrated with toluene (3 × 10 mL). Column chromatography (hex-
ane:EtOAc 7:3) of the crude product gave 3 (8.30 g, 63%) isolated as a syrup
[α]2D5 +39 (c 0.43, CHCl3); H NMR (CDCl3): δ 6.30 (d, 1H, J1,2 = 3.5 Hz, H-
1
1α), 5.90–5.74 (m, 4H, 2 CH ), 5.65 (d, 1H, J1,2 = 8.5 Hz, H-1β), 5.31–5.05
(m, 11H, H-2β, H-4α, H-4β and 2 × 2 CH2 ), 4.98 (dd, 1H, J2,3 = 9.9 Hz,
H-2α), 4.10–3.96 (m, 8H, 2 × 2 CH CH2 ), 3.85 (dd, 1H, J3,4 = 10.3 Hz,
H-3α), 3.71–3.45 (m, 7H, H-3β, H-5α and H-5β, H-6α and H-6β), 2.15–2.05 (m,
18H, 2 × 3 OCOCH3); 13C NMR (CDCl3): δ 169.46, 169.23, 169.09, 168.96 and

L. Kalma´ r et al.
210
168.74 (2 × 3 C O), 134.37, 134.20, 134.17 and 134.07 (2 × 2 CH ), 117.42,
117.35, 116.96 and 116.53 (2 × 2 CH2 ), 91.96 (C-1β), 89.43 (C-1α), 79.68 and
74.16 (C-5α and C-5β), 76.50 and 71.53 (C-3α and C-3β), 73.35, 72.83, 72.50
and 72.42 (2 × 2 CH CH2 ), 71.46 and 71.33 (C-2α and C-2β), 70.21 and
70.08 (C-4α and C-4β), 68.91 and 68.74 (C-6α and C-6β), 20.88, 20.81, 20.77,
20.68 and 20.55 (2 × 3 OCOCH3). Anal. Cald for C18H26O9: C, 55.94; H, 6.79.
Found: C, 56.01; H, 6.77.
4-O-Acetyl-3,6-di-O-allyl-1,2-O-(1-ethoxyetylidene)-α-D-
glucopyranose (5)
HBr/acetic acid (11 mL) in dry CH2Cl2 (15 mL) was added to a mixture
of 1,2,4-tri-O-acetyl-3,6-di-O-allyl-α,β-D-glucopyranose (3, 4.39 g, 11.36 mmol)
◦
˚
and 4 A molecular sieves (2 g) in dry CH2Cl2 (15 mL) over 10 min at 0 C.
The mixture was diluted with cooled CH2Cl2 (150 mL), washed with aq 5%
NaHCO3 (2 × 30 mL) and water (2 × 30 mL), dried (CaCl2, MgSO4), filtered,
and concentrated. The residue (4) was dried under high vacuum and then dis-
˚
solved in dry CH3CN (9 mL), 4 A molecular sieves (500 mg) were added, and
the mixture was stirred under argon. Sym collidine (3.2 mL, 24.14 mmol), dry
EtOH (9 mL, 156.26 mmol), and Bu4NBr (1.19 g, 3.69 mmol) were added to
the mixture and stirred overnight. The mixture was diluted with CH2Cl2 (200
mL) and filtered through Celite, and the filtrate was washed with aq 1 M HCl
(20 mL), aq 5% NaHCO3 (2 × 20 mL), and water (2 × 20 mL); dried (MgSO4);
filtered; and concentrated. Column chromatography (hexane:EtOAc 75:25 +
5% TEA, Rf 0.57) of the residue gave 5 (2.50 g, 59%) isolated as a syrup; [α]D25
1
+22 (c 0.38, CHCl3); H NMR (CDCl3): δ 5.93–5.83 (m, 2H, 2 CH ), 5.72 (d,
ꢁ
ꢁ
1H, J1,2 = 5.0 Hz, JC1 -H1 = 180 Hz, H-1), 5.30–5.16 (m, 4H, CH2), 4.96 (dd,
1H, J4,5 = 9.0 Hz, H-4), 4.38 (dd, 1H, J2,3 = 4.2 Hz, H-2), 4.28–4.00 (m, 4H,
CH2 CH ), 3.88 (m, 1H, H-5), 3.73 (dd, 1H, J3,4 = 3.2 Hz, H-3), 3.57–3.52
(m, 4H, H-6 and CH2CH3), 2.08 (s, 3H, OCOCH3), 1.71 (s, 3H, CCH3), 1.21
(t, 3 H, CH2 CH3); 13C NMR (CDCl3): δ 169.94 (C O), 134.46 and 133.98 (2
CH ), 121.18 (C(CH3)OEt), 117.69 and 117.15 (2 H2C ), 97.18 (C-1), 75.19
(C-3), 73.95 (C-2), 72.41 and 70.78 (2 CH2 CH ), 69.92 (C-6), 68.52 (C-5),
68.30 (C-4), 58.79 (OCH2CH3), 20.98 (C CH3), 15.26 (CH2CH3). Anal. Calcd
for C18H28O8: C, 58.04; H, 7.58. Found: C, 57.93; H, 7.59.
3,6-Di-O-allyl-4-O-benzyl-1,2-O-(1-ethoxyethylidene)-α-D-
glucopyranose (6)
KOH (1.68 g, 29.94 mmol) and BzlBr (1.00 mL, 8.41 mmol) were added
to the solution of 5 (2.50 g, 6.71 mmol) in THF (12 mL) at 0◦C. The reac-
tion mixture was stirred overnight at rt, then diluted with CH2Cl2 (150 mL),
washed with water (3 × 30 mL), dried (MgSO4), filtered, and concentrated. Col-
umn chromatography (hexane:EtOAc 95:5 + 5% TEA → hexane:EtOAc 8:2 +

Synthesis of Glycosyl Azide
211
5% TEA, Rf 0.58) of the residue afforded 6 (2.25 g, 80%) isolated as a syrup;
[α]2D5 +52 (c 0.33, CHCl3); H NMR (CDCl3): δ 7.38–7.27 (m, 5H, aromatics),
1
5.98–5.82 (m, 2H, 2 CH ), 5.73 (d, 1 H, J1,2 = 5.0 Hz, H-1), 4.77 and 4.58 (q,
each 2H, PhCH2O), 4.36 (dd, 1H, J2,3 = 4.0 Hz, H-2), 1.52 (s, 3H, CCH3), 1.21
(t, 3H, CH2 CH3); 13C NMR (CDCl3): δ 134.67 and 134.38 (2 CH ), 117.47
and 117.16 (2 H2C ), 97.77 (C-1), 58.61 (OCH2CH3), 21.95 and 15.32 (CH3 and
CH2CH3). Anal. Calcd for C23H32O7: C, 65.68; H, 7.67. Found: C, 65.43; H, 7.61.
Ethyl-(2-O-acetyl-3,6-di-O-allyl-4-O-benzyl-β-D-glucopyranosyl)-
(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-
glucopyranoside (9)
˚
A mixture of 6 (2.07 g, 4.92 mmol), 4 A molecular sieves (600 mg), and
dry CH2Cl2 (11 mL) was stirred for 30 min under argon, then cooled to 0◦C.
HBr/acetic acid (4.1 mL) in CH2Cl2 (11 mL) was added to the mixture over 10
min. The reaction mixture was diluted with cooled CH2Cl2 (150 mL), washed
with water (2 × 30 mL) and aq 5% NaHCO3 (2 × 30 mL), dried (CaCl2 and
MgSO4), filtered, and concentrated.
˚
A mixture of the residue (7), 8 (789 mg, 1.48 mmol), 4 A molecular sieves
(2 g), and dry CH2Cl2 (20 mL) was stirred for 20 min under argon in the dark.
AgOTf (975 mg, 3.79 mmol) in toluene (16 mL) was added to the mixture over
30 min at −50◦C. Pyridine (2 mL) was added to the mixture and diluted with
CH2Cl2 (200 mL) and filtered through Celite, and the filtrate was washed
with aq 10% Na2S2O3 (2 × 40 mL), aq 5% NaHCO3 (2 × 40 mL), and water
(2 × 40 mL); dried (MgSO4); filtered; and concentrated. The residue was
crystallized from EtOH (5 mL) and the mother liquor was column chro-
matographed (hexane:EtOAc 6:4 Rf 0.31) to give 9 (1.10 g, 82%); [α]2D5 +35
1
(c 0.20, CHCl3); H NMR (CDCl3): δ 7.75–6.77 (m, 19H, aromatics), 5.90–5.72
(m, 2H, 2 CH ), 5.26–5.04 (m, 4H, 2 CH2 ), 5.02 (d, 1H, J1,2 = 10.0 Hz,
JC1 H1 = 157 Hz, H-1), 4.95 (dd, 1H, J2 ,3 = 9.7 Hz, H-2ꢁ), 4.87–4.45 (m, 6H,
ꢁ
ꢁ
3 PhCH2O), 4.56 (d, 1H, J1 ,2 = 8.2 Hz, JC1 H1 = 161 Hz, H-1ꢁ), 4.28–4.08
ꢁ
ꢁ
ꢁ
ꢁ
(m, 2H, CH2 O3ꢁ), 4.25 (dd, 1H, J3,4 = 9.8 Hz, H-3), 4.22 (dd, 1H, J2,3
=
10.4 Hz, H-2), 4.06 (dd, 1H, J4,5 = 8.4 Hz, H-4), 3.92–3.90 (m, 2H, CH2 O6ꢁ),
3.83–3.77 (m, 2H, H-6), 3.66 (dd, 1H, J4 ,5 = 9.2 Hz, H-4ꢁ), 3.69–3.56 (m, 2H,
ꢁ
ꢁ
H-6ꢁ), 3.54 (m, 1H, H-5), 3.40 (dd, 1H, J3 ,4 = 9.3 Hz, H-3ꢁ), 3.29 (m, 1H, H-5ꢁ),
2.69–2.54 (m, 2H, SCH2 CH3), 1.94 (s, 3H, OCOCH3), 1.16 (t, 3H, SCH2CH3);
13C NMR (CDCl3): δ 169.07, 167.62 and 167.35 (3 C O), 138.60, 138.06 and
137.92 (3 benzyl quaterner), 134.60 and 133.55 (2 CH ), 116.52 (2 H2C ),
100.18 (C-1ꢁ), 82.63 (C-3ꢁ), 80.86 (C-1), 79.21 (C-5), 77.86 (C-3), 77.69 (C-4),
77.56 (C-4ꢁ), 77.48 (C-5ꢁ), 74.72 and 74.65 (2 CH2 CH ), 73.66, 73.46 and
72.16 (3 PhCH2O), 73.53 (C-2ꢁ), 68.36 (C-6ꢁ), 67.83 (C-6), 54.62 (C-2), 23.65
(SCH2CH3), 20.92 (OCOCH3), 14.80 (SCH2CH3). Anal. Calcd for C51H57NO12S:
C, 67.45; H, 6.33. Found: C, 67.55; H, 6.29.
ꢁ
ꢁ

L. Kalma´ r et al.
212
Ethyl-(3,6-di-O-allyl-4-O-benzyl-β-D-glucopyranosyl)-(1→4)-3,6-
di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-
glucopyranoside (10)
K2CO3 (174 mg, 1.26 mmol) was added to the solution of 9 (572 mg, 0.63
mmol) in dry THF/MeOH (1:1, 8 mL). The reaction mixture was neutralized
with Amberlite IR 120 (H+), the resin was filtered, and the filtrate was concen-
trated. Column chromatography (CH2Cl2:EtOAc 95:5, Rf 0.25) of the residue
afforded 10 (412 mg, 76%) isolated as a syrup; [α]2D5 +62 (c 0.79, CHCl3). H
1
NMR (CDCl3): δ 7.73–6.77 (m, 19H, aromatics), 6.03–5.70 (m, 2H, 2 CH ),
5.33–5.01 (m, 4H, 2 CH2 ), 5.25 (d, 1H, J1,2 = 10.3 Hz, JC1 H1 = 156 Hz,
ꢁ
ꢁ
ꢁ
ꢁ
H-1), 4.96–4.46 (m, 6H, 3 PhCH2O), 4.63 (d, 1H, J1 ,2 = 8.2 Hz, JC1 H1
=
ꢁ
161 Hz, H-1ꢁ), 4.43–4.32 (m, 2H, CH2 O3 ), 4.41 (dd, 1H, J3,4 = 8.8 Hz, H-3),
4.28 (dd, 1H, J2,3 = 10.3 Hz, H-2), 4.16 (dd, 1H, J4,5 = 9.5 Hz, H-4), 4.07–3.85
ꢁ
(m, 2H, H-6), 3.90–3.88 (m, 2H, CH2 O6 ), 3.69 (m, 1H, H-5), 3.65–3.57 (m,
3H, H-4ꢁ and H-6ꢁ), 3.52 (ddd, 1H, J2 ,3 = 9.5 Hz and J2 -OH = 2.8 Hz, H-
ꢁ
ꢁ
ꢁ
2ꢁ), 3.39 (dd, 1H, J3 ,4 = 9.1 Hz, H-3ꢁ), 3.32 (m, 1H, H-5ꢁ), 2.71–2.55 (m, 2H,
SCH2 CH3), 1.16 (t, 3H, SCH2CH3); 13C NMR (CDCl3): δ 167.61 and 167.06
(2 C O), 138.35, 138.05 and 137.49 (3 benzyl quaterner), 135.05 and 134.36
(2 CH ), 116.36 and 116.19 (2 H2C ), 102.77 (C-1ꢁ), 84.05 (C-3ꢁ), 80.72 (C-
1), 79.03 (C-3), 78.79 (C-5), 78.05 (C-4), 77.05 (C-4ꢁ), 74.85 (C-2ꢁ), 74.71 (C-5ꢁ),
74.52 and 74.42 (2 CH2 CH ), 73.65, 73.12 and 71.88 (3 PhCH2O), 68.36 (C-
6ꢁ), 68.15 (C-6), 54.49 (C-2), 23.29 (SCH2CH3), 14.62 (SCH2CH3). Anal. Calcd
for C49H55NO11S: C, 67.95; H, 6.41. Found: C, 67.83; H, 6.45.
ꢁ
ꢁ
Ethyl-(3,6-di-O-allyl-4-O-benzyl-β-D-mannopyranosyl)-(1→4)-3,6-
di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-
glucopyranoside (12)
10 (968 mg, 1.12 mmol) was dissolved in the mixture of DMSO and acetic
anhydride (2:1 15 mL) and kept for overnight at rt. The reaction mixture was
concentrated and co-concentrated with toluene (4 × 40 mL). Bu4NBH4 (576
mg, 2.24 mmol) was added to the mixture of the residue (11) and dry THF (5
mL) at 0◦C. The mixture was diluted with CH2Cl2 (100 mL), washed with water
(3 × 20 mL), dried (MgSO4), filtered, and concentrated. Column chromatogra-
phy (hexane:EtOAc 6:4 Rf 0.24), of the residue gave 12 (875 mg, 90%) isolated
as a syrup; [α]2D5 +49 (c 0.34, CHCl3). H NMR (CDCl3): δ 7.77–6.78 (m, 19H,
1
aromatics), 5.94–5.70 and 5.30–5.03 (2m, 2H, 2 CH ), 5.24 (d, 1H, J1,2
=
10.2 Hz, JC1 H1 = 156 Hz, H-1), 4.82–4.50 (m, 6H, 3 PhCH2O), 4.70 (bs, 1H,
J1 ,2 <1 Hz, JC1 -H1 = 161 Hz, H-1ꢁ), 4.43 (dd, 1H, J3,4 = 8.8 Hz, H-3), 4.27
(dd, 1H, J2,3 = 10.2 Hz, H-2), 4.11 (dd, 1H, J4,5 = 10 Hz, H-4), 4.03–3.97 (m,
ꢁ
ꢁ
ꢁ
ꢁ
2H, CH2 O3ꢁ) 4.02 (dd, 1H, J2 ,3 = 3.0 Hz, H-2ꢁ), 3.91 (m, 2H, CH2 O6ꢁ),
ꢁ
ꢁ
3.89–3.79 (m, 2H, H-6), 3.69 (m, 1H, H-5), 3.66–3.55 (m, 2H, H-6ꢁ), 3.32 (dd, 1H,

Synthesis of Glycosyl Azide
213
J3 ,4 = 8.8 Hz, H-3ꢁ), 3.29 (m, 1H, H-5ꢁ), 2.71–2.56 (m, 2H, SCH2 CH3), 1.17
(t, 3H, SCH2CH3); 13C NMR (CDCl3): δ 167.74 and 167.32 (2 C O), 138.27,
138.23 and 137.76 (3 benzyl quaterner), 134.51 and 134.41 (2 CH ), 117.07
and 116.65 (2 H2C ), 100.31 (C-1ꢁ), 81.43 (C-3ꢁ), 80.92 (C-1), 79.00 (C-3), 78.70
(C-5), 77.91 (C-4), 75.23 (C-5ꢁ), 74.89 and 74.70 (2 CH2 CH ), 73.72 (C-2ꢁ),
73.35, 72.13 and 70.32 (3 PhCH2O), 68.78 (C-6), 68.69 (C-6ꢁ), 67.96 (C-4ꢁ), 54.61
(C-2), 23.57 (SCH2CH3), 14.77 (SCH2CH3). Anal. Calcd for C49H55 NO11S: C,
67.95; H, 6.41. Found: C, 68.07; H, 6.37.
ꢁ
ꢁ
Ethyl-(3,6-di-O-allyl-2,4-di-O-benzyl-β-D-mannopyranosyl)-
(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-
glucopyranoside (13)
Silver(I) oxide (740 mg, 3.19 mmol), benzyl bromide (0.50 mL, 4.21 mmol),
and potassium iodide (199 mg, 1.20 mmol) were added to the mixture of 12
˚
(690 mg, 0.80 mmol) and 4 A molecular sieves (600 mg) in DMF (8 mL) at
0◦C. The reaction mixture was stirred for 2 h at 0◦C; then diluted with CH2Cl2
(100 mL); filtered through Celite; washed with aq 10% Na2S2O3 (2 × 20 mL),
aq 5% NaHCO3 (2 × 20 mL), and water (2 × 20 mL); dried (MgSO4); fil-
tered; and concentrated. Column chromatography (CH2Cl2 → CH2Cl2:EtOAc
9:1 Rf 0.59) of the residue gave 13 (665 mg, 87%) isolated as a syrup; [α]2D5 +20
1
(c 0.30, CHCl3); H NMR (CDCl3): δ 7.77–6.76 (m, 24H, aromatics), 5.92–5.73
and 5.31–5.01 (2m, each 1H, 2 CH ), 5.23 (d, 1H, J1,2 = 10 Hz, JC1 H1 =
ꢁ
ꢁ
ꢁ
ꢁ
156 Hz, H-1), 5.00–4.48 (m, 8H, 4 PhCH2O), 4.60 (bs, 1H, J1 ,2 <1 Hz, JC1 H1
= 158 Hz, H-1ꢁ), 4.30 (dd, 1H, J3,4 = 8.4 Hz, H-3), 4.26 (dd, 1H, J2,3 = 10.1 Hz,
H-2), 4.05 (dd, 1H, J4,5 = 10 Hz, H-4), 3.96–3.93 (m, 4H, CH2 CH ) 3.83
(dd, 1H, J4 ,5 = 9.5 Hz, H-4ꢁ), 3.76 (dd, 1H, J2 ,3 = 2.9 Hz, H-2ꢁ), 3.73–3.63 (m,
ꢁ
ꢁ
ꢁ
ꢁ
3H, H-5 and H-6), 3.60–3.56 (m, 2H, H-6ꢁ), 3.32 (m, 1H, H-5ꢁ), 3.26 (dd, 1H,
J3 ,4 = 9.5 Hz, H-3ꢁ), 2.72–2.56 (m, 2H, SCH2 CH3), 1.17 (t, 3H, SCH2CH3);
13C NMR (CDCl3): δ 167.83 and 167.32 (2 C O), 138.85, 138.79, 138.50 and
137.81 (4 benzyl quaterner), 134.94 and 134.69 (2 CH ), 116.43 and 116.16
(2 H2C ), 101.37 (C-1ꢁ), 82.45 (C-3ꢁ), 80.87 (C-1), 79.05 (C-5), 78.90 (C-4), 78.14
(C-3), 75.86 (C-5ꢁ), 74.89 and 74.63 (2 CH2 CH ), 74.79 (C-2ꢁ), 74.60 (C-4ꢁ),
73.88, 73.38, 72.21 and 70.60 (4 PhCH2O), 69.31 (C-6ꢁ), 68.79 (C-6), 54.61 (C-2),
23.48 (SCH2CH3), 14.81 (SCH2CH3). Anal. Calcd for C56H61NO11S: C, 70.34;
H, 6.43. Found: C, 69.58; H, 6.49.
ꢁ
ꢁ
3,6-Di-O-allyl-2,4-di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-
O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→4)-
3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-
glucopyranosyl Azide (15)
MeOTf (134 µL, 1.18 mmol) was added to the mixture of 14 (92 mg,
˚
0.18 mmol), 13 (216 mg, 0.23 mmol), dry CH2Cl2 (2 mL), and 4 A molecular

L. Kalma´ r et al.
214
sieves (50 mg) at 0◦C. The reaction mixture was stirred overnight and then
pyridine (0.2 mL) was added and diluted with CH2Cl2 (100 mL). The or-
ganic solution was washed with aq 5% NaHCO3 (2 × 20 mL) and water (2 ×
20 mL), dried (MgSO4), filtered, and concentrated. Column chromatography
(hexane:CH2Cl2:EtOAc 5:4:1 Rf 0.35) of the residue gave 15 (174 mg, 69%) iso-
1
lated as a syrup; [α]D +2◦ (c 0.26, CHCl3); H NMR (CDCl3): δ 7.58–6.72 (m,
ꢁ
ꢁ
38 H, aromatic), 5.98–5.62 (m, 2 H, 2 CH2 CH CH2O), 5.29 (d, 1 H, J1 ,2
=
8.0 Hz, H-1ꢁ), 5.16 (d, 1 H, J1,2 = 9.0 Hz, H-1), 4.56 (bs, 1 H, J1 ,2 <1 Hz, H-
1ꢁꢁ); 13C δ 116.53 and 116.22 (2 H2C ), 101.46 (C-1ꢁꢁꢁ), 96.99 (C-1ꢁꢁ), 85.49 (C-1),
56.51 and 55.14 (C-2 and C-2ꢁ). IR(KBr) νmax 2160 cm−1 (N3); Anal. Calcd for
C82H81N5O17: C, 69.91; H, 5.80. Found: C, 70.01; H, 5.77.
ꢁꢁ ꢁꢁ
Ethyl-(2,4-di-O-benzyl-β-D-mannopyranosyl)-(1→4)-3,6-di-O-
benzyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside
(16)
A solution of 13 (100 mg, 0.10 mmol) and (PPh3)3RhCl (133 mg, 0.14 mmol)
in EtOH (8 mL) was boiled under reflux for 2.5 h, then cooled and concen-
trated. A solution of the residue in acetone/1M HCl (9:1 10 mL) was boiled for
10 min. Then, the mixture was diluted with toluene (50 mL), washed with
aq 5% NaHCO3 (2 × 10 mL) and water (2 × 10 mL), dried (MgSO4), filtered,
and concentrated. Column chromatography (hexane:EtOAc 6:4 Rf 0.46) of the
residue gave 16 (58 mg, 63%) isolated as a syrup; [α]2D5 +16 (c 0.66, CHCl3);
1H NMR (CDCl3): δ 7.80–6.88 (m, 24H, aromatics), 5.25 (d, 1H, J1,2 = 10 Hz,
ꢁ
ꢁ
JC1 H1 = 156 Hz, H-1), 5.03–4.42 (m, 8H, 4 PhCH2O), 4.61 (bs, 1H, J1 ,2 <1
ꢁ
ꢁ
ꢁ
Hz, JC1 H1 = 158 Hz, H-1 ), 4.30–4.28 (m, 2H, H-2 and H-3), 4.05 (dd, 1H, H-
4), 3.78–3.63 (m, 5H, H-6, H-6ꢁ and H-2ꢁ), 3.51 (m, 1H, H-5), 3.47–3.42 (m, 2H,
H-3ꢁ and H-4ꢁ), 3.16 (m, 1H, H-5ꢁ), 2.74–2.58 (m, 2H, SCH2 CH3), 1.17 (t, 3H,
SCH2CH3); 13C NMR (CDCl3): δ 168.00 and 167.33 (2 C O), 138.19, 138.14,
138.05 and 137.48 (4 benzyl quaterner), 101.13 (C-1ꢁ), 80.89 (C-1), 79.02 (C-4ꢁ),
78.72 (C-4), 78.21 (C-2ꢁ), 78.05 (C-3), 76.44 (C-3ꢁ), 75.32 (C-5ꢁ), 73.88, 73.38,
72.21 and 70.60 (4 PhCH2O), 74.08 (C-5), 68.37 (C-6), 62.21 (C-6ꢁ), 54.54 (C-2),
23.54 (SCH2CH3), 14.81 (SCH2CH3). Anal. Calcd for C50H53NO11: C, 68.55; H,
6.10. Found: C, 68.59; H, 6.04.
Ethyl-(3,6-di-O-acetyl-2,4-di-O-benzyl-β-D-mannopyranosyl)-
(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-
glucopyranoside (17)
Compound 16 (200 mg, 0.23 mmol) was treated with pyridine/acetic anhy-
dride (1:1 2 mL) for 2 h. The mixture was concentrated and co-concentrated
with toluene (3 × 5 mL). Column chromatography (hexane:EtOAc 6:4 Rf 0.57)
of the residue gave 17 (196 mg, 90%) isolated as a syrup; [α]2D5 +4 (c 0.40,

Synthesis of Glycosyl Azide
215
CHCl3); 1H NMR (CDCl3): δ 7.76–6.75 (m, 24H, aromatics), 5.22 (d, 1H, J1,2
10 Hz, JC1 H1 = 156 Hz, H-1), 4.89–4.45 (m, 8H, 4 PhCH2O), 4.77 (dd, 1H,
=
J3 ,4 = 9.7 Hz, H-3ꢁ) 4.72 (bs, 1H, J1 ,2 <1 Hz, JC1 H1 = 158 Hz, H-1ꢁ),
4.27–4.23 (m, 4H, H-2, H-3 and H-6ꢁ), 4.08 (dd, 1H, J4,5 = 9.9 Hz, H-4), 3.94
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
(dd, 1H, J2 ,3 = 3.0 Hz, H-2ꢁ), 3.87 (dd, 1H, J4 ,5 = 10 Hz, H-4ꢁ), 3.78–3.67 (m,
2H, H-6), 3.57 (m, 1H, H-5), 3.39 (m, 1H, H-5ꢁ), 2.71–2.55 (m, 2H, SCH2 CH3),
1.93 and 1.91 (2s, 6H 2 OCOCH3), 1.17 (t, 3H, SCH2CH3); 13C NMR (CDCl3): δ
170.73, 170.03, 167.82 and 167.31 (4 C O), 138.77, 138.36, 137.74 and 137.71
(4 benzyl quaterner), 100.81 (C-1ꢁ), 80.89 (C-1), 79.05 (C-4), 79.00 (C-5), 77.96
(C-2ꢁ), 75.91 (C-3ꢁ), 75.77 (C-2ꢁ), 74.70, 74.50, 74.39 and 73.41 (4 PhCH2O),
73.09 (C-5 and C-5ꢁ), 68.57 (C-6), 63.09 (C-6ꢁ), 54.58 (C-2), 23.59 (SCH2CH3),
20.87 and 20.63 (2 OCOCH3), 14.83 (SCH2CH3). Anal. Calcd for C54H57NO13S:
C, 67.55; H, 5.99. Found: C, 67.62; H, 6.02.
ꢁ
ꢁ
ꢁ
ꢁ
3,6-Di-O-acetyl-2,4-di-O-Benzyl-β-D-mannopyranosyl-(1→4)-3,6-
di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-
(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-
glucopyranosyl azide (18)
A mixture of 17 (298 mg, 0.31 mmol), 14 (133 mg, 0.26 mm◦ol), dry CH2Cl2
˚
(3 mL), and 4 A molecular sieves (300 mg) was cooled to –40 C under Ar. A
solution of NIS (204 mg, 0.91 mmol) and AgOTf (24 mg, 0.09 mmol) in ace-
tonitrile (2 mL) was added to the mixture and stirred at –25◦C. After the ad-
dition of pyridine (0.5 mL) and CH2Cl2 (100 mL), the mixture was filtered
through Celite; the filtrate was washed with aq 10% Na2S2O3 (2 × 20 mL),
aq 5% NaHCO3 (2 × 20 mL), and water (2 × 20 mL); dried (MgSO4); filtered;
and concentrated. Column chromatography (hexane:EtOAc 6:4 Rf 0.38) of the
residue afforded 18 (188 mg, 51%) isolated as a syrup; [α]2D5 –10 (c 0.16, CHCl3);
1H NMR (CDCl3) δ 7.83–6.78 (m, 38 H, aromatic), 5.28 (d, 1H, J1 ,2 = 8.1 Hz,
ꢁ
ꢁ
JC1 H1 = 166 Hz, H-1ꢁ), 5.16 (d, 1H, J1,2 = 9.5 Hz, JC1 H1 = 166 Hz, H-1),
ꢁ
ꢁ
4.93–4.41 (m, 12H, 6 PhCH2O), 4.77 (dd, 1H, J3 ,4 = 9.9 Hz, H-3ꢁꢁ), 4.71 (bs,
ꢁꢁ ꢁꢁ
1H, J1 ,2 <1 Hz, JC1 H1 = 156 Hz, H-1ꢁꢁ), 4.27–4.21 (m, 4H, H-3ꢁ, H-4ꢁ and
ꢁꢁ ꢁꢁ
ꢁꢁ
ꢁ
H-6ꢁꢁ), 4.19–4.14 (m, 2H, H-3 and H-2ꢁ), 4.10–4.04 (m, 2H, H-2 and H-4), 3.93
ꢁꢁ
ꢁꢁ
ꢁꢁ ꢁꢁ
ꢁꢁ ꢁꢁ
(dd, 1H, J2 ,3 = 2.7 Hz, H-2 ), 3.86 (dd, 1H, J4 ,5 = 9.6 Hz, H-4 ), 3.69–3.49 (m,
2H, H-6ꢁ), 3.57–3.41 (m, 2H, H-6), 3.38 (m, 1H, H-5ꢁ), 3.38 (dd, 1H, H-5ꢁ), 3.36
(m, 1H, H-5ꢁꢁ), 3.28 (m, 1H, H-5), 1.93 and 1.85 (2 OCOCH3); 13C NMR (CDCl3)
δ 170.66 and 170.04 (C O), 138.96, 138.46, 138.38, 138.17, 137.79 and 137.77
(6 benzyl quaterner), 100.91 (C-1ꢁ), 96.89 (C-1ꢁ), 85.50 (C-1), 79.15 (C-4), 77.13
(C-4ꢁ), 76.78 (C-5ꢁ), 76.52 (C-3), 76.02 (C-3ꢁꢁ), 75.97 (C-2ꢁꢁ), 75.19 (C-5), 74.72,
74.60, 74.54, 74.47, 73.21 and 72.71 (6 PhCH2O), 74.66 (C-3ꢁ), 73.13 (C-5ꢁꢁ),
73.04 (C-4ꢁꢁ), 67.82 and 67.71 (C-6 and C-6ꢁ), 63.11 (C-6ꢁꢁ), 56.46 (C-2ꢁ), 55.17

L. Kalma´ r et al.
216
(C-2), 20.87 and 20.58 (2 OCOCH3). IR(KBr) νmax 2100 cm−1 (N3); Anal. Calcd
for C80H77N5O19: C, 68.01; H, 5.50. Found: C, 68.14; H, 5.47.
2,4-Di-O-benzyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl-(1→4)-3,6-di-O-
benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl
azide (19)
K2CO3 (12 mg, 0.09 mmol) was added to the solution of 18 (60 mg,
0.04 mmol) in dry THF/MeOH (1:1, 2 mL). The reaction mixture was neutral-
ized with Amberlite IR 120 (H+), the resin was filtered off, and the filtrate
was concentrated. Column chromatography (hexane:EtOAc 6:4 Rf 0.22) of the
residue gave 19 (37 mg, 66%); [α]2D5 +6 (c 0.31, CHCl3); H NMR (CDCl3) δ
1
ꢁ
ꢁ
ꢁ
ꢁ
7.89–6.76 (m, 38 H, aromatic), 5.30 (d, 1H, J1 ,2 = 7.5 Hz, JC1 H1 = 160 Hz,
H-1ꢁ), 5.18 (d, 1H, J1,2 = 9.4 Hz, JC1 H1 = 158 Hz, H-1), 5.04–4.39 (m, 12H,
6 PhCH2O), 4.61 (bs, 1H, J1 ,2 <1 Hz, JC H1 = 158 Hz, H-1ꢁꢁ), 4.28–4.22
(m, 2H, H-2ꢁ and H-4), 4.17 (dd, 1H, J3,4 = 18.5 Hz, H-3), 4.16–4.06 (m, 2H, H-
ꢁꢁ ꢁꢁ
ꢁ
ꢁ
2 and H-4ꢁ), 4.11 (dd, 1H, J3 ,4 = 7.1 Hz, H-3ꢁ), 3.71–3.66 (m, 2H, H-6ꢁꢁ), 3.64
ꢁ
ꢁ
(dd, 1H, J2 ,3 = 3.5 Hz, H-2ꢁꢁ), 3.59–3.52 (m, 2H, H-6ꢁ), 3.49 (dd, 1H, J3 ,4
=
ꢁꢁ ꢁꢁ
ꢁꢁ ꢁꢁ
9.1 Hz, H-3ꢁꢁ), 3.45–3.39 (m, 4H, H-5, H-6 and H-4ꢁꢁ), 3.26 (m, 1H, H-5ꢁ), 3.12
(dd, 1H, H-5ꢁꢁ); 13C NMR (CDCl3) δ 168.48 and 167.48 (C O), 138.41, 138.33,
138.18, 138.13, 138.09 and 137.53 (6 benzyl quaterner), 101.21 (C-1ꢁ), 96.96
(C-1ꢁ), 85.50 (C-1), 78.81 (C-4ꢁ), 78.28 (C-2ꢁꢁ), 76.93 (C-4), 76.63 (C-3ꢁꢁ), 76.45 (C-
3ꢁ), 76.35 (C-4ꢁꢁ), 75.22 (C-3), 75.18, 74.66, 74.60, 74.47 and 73.31 (6 PhCH2O),
75.11 (C-5ꢁꢁ), 74.55 (C-5ꢁ), 74.11 (C-5), 67.64 and 67.54 (C-6 and C-6ꢁ), 62.20
(C-6ꢁꢁ), 56.34 (C-2ꢁ), 55.11 (C-2); IR(KBr) νmax 2100 cm−1 (N3). MALDI-TOF:
C76H73O17N5 (1328.44): m/z 1350.79 [M+Na]+, 1366.46 [M+K]+.
2,3,4,6-Tetra-O-benzyl-α-D-mannopyranosyl-(1→6)-[2,3,4,6-tetra-
O-benzyl-α-D-mannopyranosyl-(1→3)]-2,4-di-O-benzyl-β-D-
mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl
azide (21)
MeOTf (65 µL 0.59 mmol) was added to a mixture of 19 (44 mg, 33 µmol),
˚
20 (60 mg, 0.103 mmol), and 4 A molecular sieves (50 mg) in dry CH2Cl2 (2 mL)
at 0◦C. After stirring overnight, pyridine (0.1 mL) was added and the mixture
was diluted with CH2Cl2 (50 mL), washed with aq 5% NaHCO3 (2 × 10 mL)
and water (2 × 10 mL), dried (MgSO4), filtered, and concentrated. Column
chromatography (hexane:EtOAc 6:4 Rf 0.51) of the residue gave 21 (55 mg,
71%) isolated as a syrup; [α]D25 +9 (c 0.20, CHCl3); H NMR (CDCl3) δ 5.26 (d,
1

Synthesis of Glycosyl Azide
217
1H, J1 ,2 = 7.9 Hz, JC1 H1 = 167 Hz, H-1ꢁ), 5.22 (d, 1H, J1
= 1.8 Hz,
ꢁ
ꢁ
ꢁ
ꢁ
ꢁꢁꢁ,2ꢁꢁꢁ
JC1 H1 = 170 Hz, H-1ꢁꢁꢁ), 5.17 (d, 1H, J1,2 = 9.4 Hz, JC1 H1 = 164 Hz,
ꢁꢁꢁ
ꢁꢁꢁ
H1 = 170 Hz, H-1ꢁꢁꢁꢁ), 4.67 (bs,
ꢁꢁꢁꢁ,2ꢁꢁꢁꢁ
ꢁꢁꢁꢁ
ꢁꢁ
ꢁꢁꢁꢁ
H-1), 4.99 (d, 1H, J1
= 1.2 Hz, JC1
1H, J1 ,2 <1 Hz, JC1 H1 = 157 Hz, H-1 ), 13C NMR (CDCl3) δ 102.34 (C-1ꢁꢁ),
100.57 (C-1ꢁꢁꢁ), 98.52 (C-1ꢁꢁꢁꢁ), 97.29 (C-1ꢁ), 85.80 (C-1); IR(KBr) νmax 2100 cm−1
(N3). MALDI-TOF: C144H141O27N5 (2373.72): m/z 2395.81 [M+Na]+, 2411.71
[M+K]+
ꢁꢁ ꢁꢁ
ꢁꢁ
ꢁꢁ
2,3,4,6-Tetra-O-benzyl-α-D-mannopyranosyl-(1→6)-]2,3,4,6-Tetra-
O-benzyl-α-D-mannopyranosyl-(1→3)]-2,4-di-O-benzyl-β-D-
mannopyranosyl-(1→4)-2-acetamido-3,6-di-O-benzyl-2-
deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-3,6-di-O-
benzyl-2-deoxy-β-D-glucopyranosyl
azide (22)
Ethylenediamine (10 µL, 0.15 mmol) was added to a solution of 21 (17 mg,
7 µmol) in n-butanol (2 mL). The mixture was stirred under argon for 18 h
at 90◦C, then co-concentrated with toluene and dried under high vacuum. The
residue was treated with pyridine/acetic anhydride (1:1 2 mL), and then the
reaction mixture was concentrated and co-concentrated with toluene (3 × 10
mL). The residue was purified by column chromatography (CH2Cl2:EtOAc 6:4
Rf 0.41), affording 22 (11.9 mg, 76%) isolated as a syrup; [α]2D5 –15 (c 0.15,
1
ꢁꢁꢁ,2ꢁꢁꢁ
CHCl3); H NMR (CDCl3) δ 5.22 (bs, 1H, J1
<3 Hz, H-1ꢁꢁꢁ), 5.01 (bs, 1H,
ꢁꢁ ꢁꢁ
ꢁꢁꢁꢁ,2ꢁꢁꢁꢁ
J1
<3 Hz, H-1ꢁꢁꢁꢁ), 4.77 (d, 1H, J1,2 = 6.0 Hz, H-1), 4.57 (bs, 1H, J1 ,2 <3
Hz, H-1ꢁꢁ), 4.31 (d, 1H, J1 ,2 = 8.1 Hz, H-1ꢁ), 13C NMR (CDCl3) δ 101.65 (C-1ꢁꢁ),
100.43 (C-1ꢁꢁꢁ), 100.05 (C-1ꢁ), 98.50 (C-1ꢁꢁꢁꢁ), 88.46 (C-1); IR(KBr) νmax 2100 cm−1
(N3). MALDI-TOF: C132H141O25N5 (2197.59): m/z 2220.26 [M+Na]+, 2236.58
[M+K]+.
ꢁ
ꢁ
ACKNOWLEDGMENT
We are grateful for the grant from the Hungarian National Science Foundation
´
(OTKA-71753) and TAMOP-4.2.1/B-09/1/KONV-2010-0007.
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