Highly regioselective synthesis of a 3-O-sulfonated arabino Lewisa asparagine building block suitable for glycopeptide synthesis

Article abstract of DOI:10.1016/j.carres.2006.03.003

Using the stannylene method, the trisaccharide 2-acetamido-3-O-[6-O-benzyl-β-d-galactopyranosyl]-4-O-[2,3,4-tri-O-benzyl-β-d-arabinopyranosyl]-6-O-benzyl-2-deoxy-β-d-glucopyranosyl azide was regioselectively sulfonated and, after reduction of the anomeric

Full text of DOI:10.1016/j.carres.2006.03.003

Carbohydrate Research 341 (2006) 1597–1608
Highly regioselective synthesis of a 3-O-sulfonated arabino
Lewisa asparagine building block suitable for glycopeptide synthesis
Alexander Ro¨sch and Horst Kunz*
Institut fu¨r Organische Chemie, Johannes Gutenberg-Universita¨t Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany
Received 19 January 2006; received in revised form 17 February 2006; accepted 1 March 2006
Available online 11 April 2006
Abstract—Using the stannylene method, the trisaccharide 2-acetamido-3-O-[6-O-benzyl-b-D-galactopyranosyl]-4-O-[2,3,4-tri-O-
benzyl-b-D-arabinopyranosyl]-6-O-benzyl-2-deoxy-b-D-glucopyranosyl azide was regioselectively sulfonated and, after reduction
of the anomeric azide, coupled to Fmoc a-allyl aspartate. After Pd(0)-catalyzed deallylation, the sulfatyl Lewisa asparagine building
block was obtained, suitable for solid-phase glycopeptide synthesis applying the fluoride labile PTMSEL linker system.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: E-Selectin ligand; Sulfatyl Lewisa; Regioselective sulfonation; Glycopeptides
1. Introduction
compounds for binding to P- and E-selectin,2 although
the binding affinity for sLea/x is rather low (IC50
1 mM) (Fig. 1).
ꢀ
Communication between different types of cells is of
tremendous importance for human life, for example, in
immuno differentiation and in inflammatory processes.
At the beginning of inflammation, Ca2+-dependent
cell-surface receptors, so-called selectins, are expressed
on the inner surface of blood vessels and on leukocytes.1
Through recognition of specific carbohydrate epitopes
of their ligands, the selectins mediate the rolling of
leukocytes on endothelial cells. This is the first step of
leukocyte adhesion on the endothelium which finally
results in the leukocyte invasion into inflamed tissue.
On the other hand, overexpression of selectins leads to
pathological recruitment of endogenous leukocytes and
can cause acute diseases such as reperfusion syndrome
or rheumatoid arthritis. To prevent these pathological
processes, soluble selectin ligands could be applied as
antagonists binding competitively to the selectins and
thus suppressing the adhesion cascade from the begin-
ning. The tetrasaccharide sialyl-Lewisa (sLea) 1 and the
isomeric sialyl-Lewisx (sLex) have been identified as lead
In 1992, a mixture of sulfated Lex and Lea pentasac-
charides was isolated from an ovarian cystoadenoma
glycoprotein,3 which turned out to be an even more
potent inhibitor than the sialylated Lewis antigens.4 In
addition, previous studies have shown a distinct influ-
ence of the peptide backbone of sLex glycopeptides upon
receptor binding to P-selectin5 and E-selectin,6 respec-
tively. An artificial glycoconjugate consisting of the
highly conserved partial amino acid sequence 672–681
of the E-selectin-ligand 1 (ESL-1)7 and arabino sLex sig-
nificantly amplified the binding to E-selectin compared
to the tetrasaccharide 1 by a factor of 10.8
In this context, it appeared interesting to combine
both aspects and to elucidate the potential of a sul-
fated glycopeptide containing the partial structure
I670V G N L T E L E S E D I682 2 of ESL-1, in which
the sulfated arabino Lewisa is incorporated into the pep-
tide via a N-glycosidic linkage to asparagine. Although
the chemical syntheses of sialylated glycopeptides
succeeded in a number of cases, the assembly of defined
sulfated Lewis antigen glycopeptide analogues still is
challenging due to the extreme sensitivity of their fuco-
side moiety to acidic conditions. Hence, instead of
*
Corresponding author. Tel.: +49 6131 39 22334; fax: +49 6131 39
24786; e-mail addresses: hokunz@uni-mainz.de; hokunz@mail.
uni-mainz.de
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.003

1598
A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
OH
OH
OH
O
OH
O
OH
OH
OH
OH
OH
OH
OH
OH
H
O
O
HO2C
O
O
O
O
O
OH
NHAc
N
O
O
HO3SO
O
Peptide
OH
HO
NHAc
OH
OH
HO
AcHN
1
2
HO
Figure 1. Natural ligand sLea 1 and mimetic sulfonyl arabino Lewisa 2.
acid-labile protecting groups and linkers commonly
used for solid-phase glycopeptide synthesis, a new ap-
proach was chosen: exclusively hydrogenolytically cleav-
able benzyl ester9 and ether10 groups were utilized in
combination with the fluoride labile PTMSEL11 linker.
Fewer side reactions occur and, thus, purification was
considerably simplified. Under these conditions, the 6-
O-benzyl ether 7 was regioselectively formed in a yield
of 76%. The per-O-benzylated ethylthio-arabinoside 5
was activated with copper(II)bromide25 and tetrabutyl-
ammonium bromide26 furnishing the desired b-stereo-
chemistry of the arabino Lewisa trisaccharide 8 formed
as a single isomer in a yield of 84%. Treatment of 8 with
sodium methoxide in methanol yielded 9 (93%) suitable
for subsequent sulfonation. The sulfonate was regio-
selectively introduced taking advantage of the stannyl-
ene procedure and sulfur trioxide–trimethylamine27
complex in dimethylformamide at room temperature
with a reproducible yield of 91%. For the exclusive for-
mation of the 3-O-monosulfonated product, previous
activation by dibutyltin oxide28 is essential (Scheme 1).
Without pre-activation as a stannylene intermediate,
mixtures of mono- and disulfonated products had been
obtained.29 The regioselectivity of the reaction was pro-
ven by NMR chemical shift differences for the signals of
protons and carbon atoms in 20-, 30- and 40-positions of
the galactose residue in compounds 10, 9 and 11 (Table
1).
The sulfate group in position 30 of 10 caused down-
field shifts of the carbon signal of 6 ppm and for the pro-
ton signal of 0.52 ppm, whereas the signals for carbon
atoms carrying the remaining free hydroxyl groups at
positions 20 and 40 occurred at almost unchanged values.
After O-acetylation at these positions, downfield shifts
of 0.14 and 2.94 ppm (13C) and 1.26 and 1.55 ppm
(1H) gave evidence of the proposed structure 11.
To find the optimal conditions for the reduction of the
anomeric azido group and the subsequent conjugation
with the Fmoc a-allyl aspartate 13, the nonsulfated
trisaccharide Lea 9 and the sulfated analogue 10 were
investigated. Using commercially available Raney-nickel
at pH 7,30 both glycosyl azides were reduced to give
the corresponding amines in nearly quantitative yield.
Due to the increased solubility caused by the sulfate
group, 10 reacted even faster and with complete reten-
tion of sulfate and benzyl protecting groups. The build-
ing blocks 12a/b were coupled with N-Fmoc a-allyl
aspartate12,31 13 using O-(7-aza-benzotriazol-1-yl)-1,1,
3,3-tetramethyluroniumhexafluorophosphate/1-hydr-
oxy-7-aza-benzotriazole32 (HATU/HOAt) and diisopro-
pylethylamine (i-Pr2NEt, Huenigs base) to furnish the
2. Results and discussion
For the preparation of a sulfated arabino Lewisa ana-
logue conjugated to asparagine, the strategy efficient
for the synthesis of sialylated analogues was appropri-
ately modified.12a,b The key building blocks for the
construction of the sulfatyl Lewisa mimic are N-acetyl-
glucosaminyl azide 3,13 galactosyl bromide 414 and the
1-thio-D-arabinoside 5.15 The latter was chosen instead
of the natural L-fucose because of its assumed increased
stability against enzymatic degradation16 while it pre-
sents the three hydroxyl groups in a sterically identical
arrangement.17 The anomeric azido group of the N-acetyl-
glucosamine receptor 3 acts as a temporary protecting
group18 throughout the synthesis and can finally be re-
duced to give the corresponding glycosylamine suitable
for the conjugation with amino acids and peptides
(Fig. 2).19
The glycosylation of acceptor 3 with 4 obtained
from 1,2,3,4-tetra-O-acetyl-6-O-benzyl-galactopyranose
by treatment with TMSBr/BiBr320 was carried out using
Hg(CN)2 as the promoter,21 because the corresponding
trichloroacetimidate,22 usually an excellent galactosyl
donor, gave only moderate yields. By changing the
solvent from dichloromethane/nitromethane (3:1) to
dichloromethane/dichloroethane (1:1), the yield of
b-galactoside 6 was increased from 63% to 97%. The
regioselective opening of the benzylidene acetal using
triethylsilane/trifluoromethanesulfonicacid23indichloro-
methane at ꢁ78 ꢁC is superior to the usually applied
sodium cyano-borohydride/HCl24 in THF at 0 ꢁC.
OAc
OBn
OBn
OBn
O
Ph
O
O
O
OBn
SEt
O
N3
HO
AcO
NHAc
AcO
Br
5
3
4
Figure 2. Carbohydrate building blocks.

A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
1599
OBn
O
N3
NHAc
O
Ph
OAc
OBn
O
N3
NHAc
OAc
OBn
O
b
a
O
HO
O
3
+ 4
O
O
AcO
AcO
OAc
OAc
6
7
c
OBn
OBn
OBn
O
OBn
O
OBn
OBn
OBn
OBn
O
OAc
OH
O
O
OBn
OBn
d
e
O
O
N3
O
N3
NHAc
O
O
NHAc
HO
AcO
OH
OAc
8
9
OBn
OBn
OBn
OBn
OBn
OBn
O
O
OBn
OBn
OH
OAc
O
O
OBn
OBn
O
O
f
O
N3
NHAc
N3
O
O
O
NHAc
HO3SO
HO3SO
OH
OAc
11
10
Scheme 1. Reagents and conditions: (a) 3 (1.0 equiv), 4 (2.8 equiv), CH2Cl2/C2H4Cl2 (1:1), Hg(CN)2 (2.8 equiv), 20 ꢁC, 6 d, 97%; (b) Et3SiH, TfOH,
CH2Cl2, ꢁ78 ꢁC, 3 h, 64%; (c) 5 (3 equiv), CuBr2, Bu4NBr, CH2Cl2, 20 ꢁC, 6 d, 84%; (d) NaOCH3, CH3OH, 20 ꢁC, 3 h, 93%; (e) (1) Bu2SnO
(1.1 equiv), CH3OH, 65 ꢁC, 4 h; (2) SO3/N(CH3)3 (1.1 equiv), DMF, 20 ꢁC, 14 h, 91% (two steps); (f) pyridine/Ac2O, 20 ꢁC, 15 h, quant.
Table 1. NMR spectroscopy of trisaccharides 9, 10 and 11: chemical shift of protons and carbon atoms in positions 20, 30 and 40 of the galactose
residue
Position
9
10
11
13C (ppm)
1H (ppm)
13C (ppm)
1H (ppm)
13C (ppm)
1H (ppm)
20
30
40
70.34
73.20
68.01
3.35
3.28
3.61
69.09
79.20
66.29
3.51
3.80
3.85
69.23
73.70
69.23
4.77
4.19
5.40
arabino Lewisa asparagine conjugate 14 and its sulfated
analogue 15 (Scheme 2).
sulfatyl arabino Lewisa-asparagine analogue 17 both
suitable for solid-phase peptide synthesis.
Deprotection of the carboxylic acid by palladium(0)
catalyzed33 allyl transfer to p-toluenesulfinic acid34 or
N-methylaniline35 as the allyl scavenger yielded the
arabino Lewisa asparagine building block 15 and the
The solid-phase synthesis of the target sulfatyl
arabino-Lewisa glycopeptide was performed in a peptide
synthesizer according to the Fmoc strategy.36 Amino-
functionalized TentaGelꢂ-resin was loaded with the
O
OBn
OBn
H
N
OBn
Fmoc
OBn
OBn
O
O
OBn
O
OBn
O
OBn
13
CO2H
a
OH
OH
O
9, 10
H
OBn
OBn
O
O
b
N
NH2
NHAc
O
CO2R'
O
O
O
NHAc
RO
RO
O
NHFmoc
OH
OH
14 R = H, R' = All
15 R = R' = H
12a R = H
12b R = SO3H
c
d
16 R = SO3H, R' = All
17 R = SO3H, R' = H
Scheme 2. Reagents and conditions: (a) Raney-nickel, H2, 2-propanol/water, 5 h (12a), 3.5 h (12b), 20 ꢁC, quant.; (b) Fmoc-Asp-OAll12 (13), HATU/
HOAt, DMF, 20 ꢁC, 14 h, 82% (14), 66% (16); (c) Pd(PPh3)4, N-methylaniline, THF, 20 ꢁC, 74%; (d) Pd(PPh3)4, N-methylaniline, THF, 20 ꢁC, 5 h,
92%.

1600
A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
novel fluoride-labile PTMSEL-linker11 18, carrying the
C-terminal amino acid Fmoc isoleucine to give 19. The
subsequent eight Fmoc amino acids were coupled
according to a standard Fmoc protocol. After removal
of the Fmoc-protecting group from 19 with 30% of
piperidine in N-methylpyrrolidone (NMP), the peptide
chain was extended by iterative coupling using 10 equiv
of each Fmoc amino acid activated by O-(1-hydroxy-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (HBTU)31 and 1-hydroxybenzotriazole
(HOBt) as additive in N-methylpyrrolidone (NMP)
(Scheme 3).
To avoid deletion sequences, unreacted amino groups
were capped after each coupling step with Ac2O/
i-Pr2NEt/HOBt (4:1:0.1) in NMP. The sulfated arabino
Lewisa building block 17 was dissolved in NMP. For its
coupling the reaction time was extended to 3 h, and the
more reactive HATU/HOAt system32 was used for
activation. A large excess of the activating reagent was
applied to complete the carboxy activation and to
accelerate the coupling reaction of the sterically
demanding building block. The final three amino acids
were coupled according to the standard protocol. After
exchanging the terminal Fmoc for an acetyl group to
PTMSEL
O
O
O
O
O
OH
O
N
H
H
H
O
N
N
a
O
Fmoc
O
O
Si(CH3)3
Si(CH3)3
18
19
b
8x
SPGS
Ac-Ile-Val-Gly-Asn-Leu-Thr(OBn)-Glu(OBn)-Leu-Glu(OBn)-Ser(OBn)-Glu(OBn)-Asp(OBn)-Ile-PTMSEL
sulfatyl arabino-Lea
20
c
CO2Bn
N
CO2Bn
N
CO2Bn
N
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
OH
N
H
N
N
N
H
H
H
O
H
O
H
O
H
O
O
O
O
OBn
CO2Bn
OBn
O
OBn
OBn
O
21
OBn
OBn
O
OH
OBn
O
NH
NHAc
O
O
d
HO3SO
OH
CO2H
CO2H
N
CO2H
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
OH
N
H
N
N
N
N
N
H
H
H
H
OH
H
O
H
OH
O
O
O
O
O
O
CO2H
O
OH
OH
O
OH
OH
O
OH
22
OH
O
NH
NHAc
O
O
HO3SO
OH
Scheme 3. Reagents and conditions: (a) CH2Cl2/DMF, 20 ꢁC, NovaSyn TGꢂ HL, HBTU/HOBt/NMP, 98%; (b) solid-phase glycopeptide synthesis
(SPGS): (1) Fmoc cleavage: piperidine/NMP (30%), (2) coupling: 2.-9., 11.-13.: Fmoc-amino acid (10 equiv), HBTU/HOBt/i-Pr2NEt, DMF; 10.: 17
(1.6 equiv), HATU/HOAt/NMP, DMF, 3 h, (3) capping: Ac2O/i-Pr2NEt/HOBt (4:1:0.1); (c) (1) TBAFÆ3H2O (2 · 1 equiv), CH2Cl2, 20 min, (2)
Amberliteꢂ CG120 (Na+), 15% (based on 19); (d) H2, Pd(OH)2, 3 d, CH3OH/dioxane (1:1), quant.

A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
1601
give 20, the protected glycopeptide was detached from
the resin by a twofold treatment with 1 equiv TBAFÆ
3H2O in dichloromethane for 20 min. These conditions
are almost neutral. The formation of isoaspartate struc-
tures by rearrangement via aspartimides was not ob-
served. The sulfate group was kept intact as well. The
glycoconjugate 21 was furnished in a yield of 15% based
on the loaded resin 19. The incomplete couplings of the
glycosylated amino acid and of the N-terminal isoleu-
cine as well as the required purification by a repeated
chromatography are the major reasons for the low yield
of isolated 21. The cleavage of the anchor by TBAFÆ
3H2O resulted in the formation of tetrabutylammonium
salts of the negatively charged sulfonate and carboxylate
moieties. Unfortunately, the ionic bonds between O-sul-
fonate and the tetrabutylammonium cations were too
favoured to be completely exchanged for Na+ by ion
exchange chromatography and hence a precise analysis
of 21 by NMR spectroscopy was not possible. There-
fore, the use of the PTMSEL linker was also demon-
strated in the synthesis of the glycopeptide 24
containing the nonsulfonated arabino Lewisa asparagine
15. After fluoride-induced cleavage of the linker, the
fully protected arabino Lewisa glycopeptide 24 was iso-
lated in a yield of 43%, demonstrating the efficiency of
this method. NMR analysis and mass spectroscopy of
24 proved its correct structure (Scheme 4).
by Pd(OH)2 in a mixture of methanol and 1,4-dioxane
without previous purification to give 22. The target com-
pound was purified by preparative RP-thin-layer chro-
matography and identified by MALDI-TOF mass
spectroscopy in negative ion mode. Its acid-rich nature
is responsible for it retaining a mixture of ammonium
and sodium ions, leading to broad signals in the NMR
spectra.
3. Experimental
3.1. General methods
NMR-Spectra were recorded on a Bruker AC-300,
Bruker AM-400 or Bruker DRX-600 spectrometer.
The following abbreviations were used to explain multi-
plicities: s (singlet), d (doublet), t (triplet), m (multiplet).
Mass spectra were recorded on a ESI Navigator-1
(ThermoQuest) or a Tofspec E instrument (Micromass,
2,5-dihydroxy-benzoic acid (dhb) as the matrix). Optical
rotations were recorded using a Perkin–Elmer 241
polarimeter. Elemental analyses were performed by the
microanalytical laboratory of the Institut fur Organische
¨
Chemie, Universitaet Mainz.
All reactions were monitored by thin-layer chromato-
graphy (TLC) carried out on 0.25 mm silica gel plates
(60F254/RP-C18254 E. Merck, Darmstadt, Germany)
using UV light, KMnO4 or p-anisaldehyde solutions
and heat for developing. Silica gel 60 (particle size 0.04–
0.0063, E. Merck, Darmstadt, Germany) was used for
flash chromatography. All reactions were carried out
Because of the poor solubility of the protected sulfatyl
glycopeptide 21 in solvents applicable to RP-HPLC, the
benzyl ether and -ester protecting groups of the carbo-
hydrate moiety and of the peptide side chains were
simultaneously removed by hydrogenolysis catalyzed
Fmoc-Ile-PTMSEL
19
9x
a
SPGS
Fmoc-Asn-Leu-Thr(OBn)-Glu(OBn)-Leu-Glu(OBn)-Ser(OBn)-Glu(OBn)-Asp(OBn)-Ile-PTMSEL
arabino-Lea
23
b
CO2Bn
N
CO2Bn
N
CO2Bn
N
O
O
O
O
O
H
N
H
H
H
H
N
N
N
N
OH
Fmoc
N
N
H
H
O
H
O
H
O
H
O
O
OBn
CO2Bn
OBn
O
OBn
OBn
O
24
OBn
OBn
O
OH
OBn
O
NH
O
O
NHAc
HO
OH
Scheme 4. Reagents and conditions: (a) solid-phase glycopeptide synthesis (SPGS): (1) Fmoc cleavage: piperidine/NMP (30%), (2) coupling: 2.-9.:
Fmoc amino acid (10 equiv), HBTU/HOBt/i-Pr2NEt, DMF; 10.: 15 (1.65 equiv), HATU/HOAt/NMP, DMF, 3 h; (3) capping: Ac2O/i-Pr2NEt/
HOBt (4:1:0.1); (b) TBAFÆ3H2O, CH2Cl2, 20 min, 43%.

1602
A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
under argon atmosphere with dried solvents. Yields refer
to chromatographically and spectroscopically (1H NMR)
homogeneous materials. Analytical RP-HPLC was per-
formed on a Phenomenex Jupiter 300 A C18 5 lm col-
umn, 250 · 4.6 mm, flow 1 mL/min using a Knauer
HPLC equipment (Maxistar K1000, DAD 2026 detec-
tor). Solvent: (CH3CN/H2O + 0.1% TFA); Gradient:
(A) (20:80)!(20:80) [5 min]!(50:50) [20 min]!(80:20)
[40 min]!(100:0) [45 min]!100:0 [60 min]; (B) (50:
50)!(90:10) [5 min]!(0:100) [15 min]!(0:100) [25 min].
1.5 equiv of each reagent was added. For neutralization,
1.6 mL NEt3 was added, the solution subsequently fil-
tered through Hyflo-Supercelꢂ and the filter washed
with CHCl3 (150 mL). The organic layer was washed
with satd NaHCO3 (3 · 150 mL), dried (MgSO4), con-
centrated in vacuo and purified by flash chromatogra-
phy (cyclohexane/EtOAc) on silica gel (50 g). Yield:
22
0.76 g (1.03 mmol, 76%; 78%12a); amorphous; ½aꢂD
ꢁ11.4 (c 1, CHCl3); ꢁ11.9 (c 1, CHCl312b); Rf = 0.54
(EtOAc); 1H NMR (CDCl3, 300 MHz): d 7.32–7.26
(m, 10H, Harom.), 5.80 (d, 1H, JNH,2 = 7.7 Hz NH),
5.34 (d, 1H, J4 ,3 = 3.0 Hz, H-40), 5.16 (dd, 1H,
0
0
3.2. 2-Acetamido-3-O-(2,3,4-tri-O-acetyl-6-O-benzyl-b-
D-galactopyranosyl)-4,6-O-benzylidene-2-deoxy-b-D-
glucopyranosyl azide (6)
J2 ,3 = 10.3 Hz, J2 ,1 = 8.1 Hz, H-20), 4.96 (m, 2H, H-
30, H-1), 4.62–4.54 (m, 1H, H-10), 4.56 (s, 1H, OH),
4.49 (d, 2H, Jgem = 11.8 Hz, CH2Ph), 4.38 (d, 2H,
Jgem = 12.1 Hz, CH2Ph), 4.10 (m, 2H, H-6a/b), 3.91
(dd, 1H, Jvic = 12.1 Hz, H-50), 3.80 (d, 1H,
J4,3 = 8.6 Hz, H-4), 3.66 (dd, 1H, Jgem = 11.0 Hz,
J5,4 = 4.8 Hz, H-5), 3.48 (m, 3H, H-3, H-60a/b), 3.21
(dd, 1H, H-2), 2.05, 2.04, 1.97, 1.94 (4s, 12H, CH3OAc,
CH3NAc); 13C NMR (CDCl3, 75.6 MHz): d 170.59,
170.12, 169.96, 169.30 (CO (OAc, NHAc)), 138.21,
137.02 (Cipso-aryl), 128.49, 128.32, 128.02, 127.96,
127.56, 127.53 (Carom.), 101.03 (C-10), 87.17 (C-1),
83.03 (C-4), 77.58 (C-5), 76.58 (C-50), 72.41 (C-30),
69.30 (C-3), 73.69, 73.57 (CH2Ph), 70.93 (C-30), 69.27,
67.34 (C-6, C-60), 67.34 (C-40), 56.31 (C-2), 23.59
(CH3(NHAc)), 20.82, 20.58, 20.50 (CH3(OAc)); ESIMS
calcd: 714.35. Found: 737.3 [M+Na]+. Anal Calcd for
C34H42N4O13Æ3H2O: C, 53,12; H, 6.29; N, 7.29. Found:
C, 53.47; H, 5.87; N, 7.21.
0
0
0
0
To a suspension of 8.5 g (25.5 mmol) of glucosaminyl
azide 3, 21.5 g (85.1 mmol) of Hg(CN)2 and 15 g of
˚
dried molecular sieves (3 A) in CH2Cl2 (300 mL) was
added 48.9 g (111.5 mmol) of galactosyl bromide14 4 in
1,2-dichloroethane (350 mL). The mixture was stirred
for 6 d, filtered through Hyflo-Supercelꢂ and washed
with CH2Cl2. The combined organic layers were washed
with 30% NaI in water and with water, then dried
(MgSO4), concentrated in vacuo and purified by flash
chromatography (cyclohexane/EtOAc). Yield: 17.63 g
22
(24.6 mmol, 97%; 63%12a); amorphous; ½aꢂD ꢁ8.7 (c 1,
CHCl3); ꢁ16.2 (c 1, CHCl312b); Rf = 0.67 (EtOAc); H
1
NMR (CDCl3, 300 MHz): d 7.42–7.16 (m, 10H, Harom.),
5.78 (d,1H, JNH,2 = 7.0 Hz, NH), 5.40 (s, H, CH-aryl),
5.31–5.26 (m, 2H, J1,2 = 9.2 Hz, H-1, H-40), 5.09 (dd,
1H, J2 ,3 = 10.3 Hz, J2 ,1 = 8.1 Hz, H-20), 4.89 (d, 1H,
H-30), 4.70 (d, 1H, H-10), 4.53–4.20 (m, 4H, H-6a/b,
CH2Ph), 3.71–3.50 (m, 4H, Jvic = 6.2 Hz, Jvic = 6.6, H-
3, H-4, H-5, H-50), 3.40–3.29 (m, 2H, H-60a/b), 3.19
(dd, 1H, H-2), 2.05 (s, 3H, CH3(NHAc)); 1.97, 1.95,
1.92 (3s, 9H, CH3(OAc)); 13C NMR (CDCl3,
75.6 MHz): d 170.89, 170.14, 170.04, 169.59 (CO (OAc,
NHAc)), 137.40, 137.05 (Cipso-aryl), 129.30, 128.55,
128.47, 128.32, 127.99 (Carom.), 126.14 (Cipso-aryl),
99.84 (C-10), 87.32 (C-1), 80.29 (C-4), 76.07 (C-5),
71.11 (C-30), 69.67 (C-3), 68.38 (C-6), 68.14 (C-20),
67.60 (C-60), 67.42(C-40), 57.63 (C-2), 23.55
(CH3(NHAc)), 20.78, 20.63, 20.57 (CH3(OAc)); ESIMS
calcd: 712.33. Found: 735.61 [M+Na]+. Anal Calcd for
C34H40N4O13Æ2H2O: C, 54.53; H, 5.65; N, 7.48. Found:
C, 54.07; H, 5.69; N, 7.33. The lyophilized compound is
hygroscopic.
0
0
0
0
3.4. 2-Acetamido-3-O-[2,3,4-tri-O-acetyl-6-O-benzyl-b-D-
galactopyranosyl]-4-O-[2,3,4-tri-O-benzyl-b-D-arabino-
pyranosyl]-6-O-benzyl-2-deoxy-b-D-glucopyranosyl azide
(8)
Disaccharide 7 (0.7 g,1.0 mmol) and 1.47 g (3.1 mmol)
of ethyl 2,3,4-tri-O-acetyl-1-thio-a,b-D-arabinopyran-
oside 5 were dissolved in CH2Cl2 (50 mL) and stirred with
˚
molecular sieve (3 A) for 30 min. After addition of 1.3 g
(4 mmol) tetrabutylammonium bromide and 0.9 g
(4 mmol) CuBr2, the mixture was stirred under exclusion
of light at rt for 6 d. The suspension was filtered through
a thin layer of silica gel. The organic phase was washed
with satd NaHCO3 until the blue colour disappeared.
After washing with brine, the solution was dried
(MgSO4), concentrated in vacuo and the residue purified
by flash-chromatography (2:1, cyclohexane/EtOAc).
3.3. 2-Acetamido-3-O-(2,3,4-tri-O-acetyl-6-O-benzyl-b-
D-galactopyranosyl)-6-O-benzyl-2-deoxy-b-D-gluco-
pyranosyl azide (7)
22
Yield: 935 mg (0.84 mmol, 84%); amorphous; ½aꢂD
ꢁ90.9 (c 1, CH2Cl2); Rf = 0.72 (EtOAc); 1H NMR
(CDCl3, 400 MHz ): d 7.37–7.18 (m, 25H, Harom.), 6.68
0
0
To a solution of 1 g (1.36 mmol) 6 and 3 g molecular
(d, 1H, JNH,2 = 9.4 Hz, NH), 5.41 (d, 1H, J4 ,3
=
2.8 Hz, H-40), 5.20 (d, 1H, J1 ,2 = 3.5 Hz, H-100), 5.14
˚
00 00
sieves (4 A) in CH2Cl2 (75 mL) at ꢁ78 ꢁC was added
(dd, 1H, J2 ,1 = 7.8 Hz, J2 ,3 = 10.6 Hz, H-20), 4.99–
0
0
0
0
175 lL (2 mmol, 1.5 equiv) TfOH and 320 lL (2 mmol,
1.5 equiv) Et3SiH. After 2 h of stirring, an additional
4.95 (m, 3H, H3, H-1, H-30), 4.71–4.31 (m, 11H, H-10,

A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
1603
CH2Ph), 4.09 (m, 1H, H-5), 4.04–3.97 (m, 4H, H-40, H-
200, H-6a, H-3), 3.92 (m, 2H, H-2, H-400), 3.82 (dd, 1H,
Jvic = 6.3 Hz, Jvic = 6.7 Hz, H-50), 3.69–3.63 (m, 6H,
H-500a/b, H6b, H-5, H-4, H-300), 3.48 (dd, 1H,
Jgem = 17.6 Hz, H-60a), 3.41 (dd, 1H, Jvic = 6.7, H-
60b), 2.08 (s, 3H, CH3NHAc), 1.95, 1.92, 1.53 (3s, 9H,
CH3(OAc)); 13C NMR (CDCl3, 100.9 MHz): d 169.99,
169.92, 169.78, 169.62 (CO (OAc, NHAc)), 138.27,
138.15, 138.07, 137.65, 137.41 (Cipso-aryl), 128.45,
128.43, 128.40, 128.30, 128.11, 127.90, 127.84, 127.69,
127.61, 127.57, 127.39, 127.30 (Carom.), 99.23 (C-10),
94.12 (C-100), 87.84 (C-1), 77.22 (C-300), 76.05 (C-200),
73.92, 73.37, 73.31, 72.38, 71.46 (CH2Ph), 73.84 (C-400),
73.14 (C-4), 72.93 (C-5); 72.17 (C-50), 70.88 (C-30),
69.82 (C-3), 69.06 (C-6), 68.38 (C-20), 67.40 (C-60),
67.46 (C-40), 60.58 (C-500), 50.68 (C-2), 22.63
(CH3(NHAc)), 21.02, 20.75, 20.53 (CH3(OAc)); ESIMS
calcd: 1117.19. Found: 1140.2 [M+Na]+. Anal Calcd for
C60H68N4O17: C, 64.50; H, 6.12; N, 5.01. Found: C,
63.65; H, 5.98; N, 5.05. The compound retains inorganic
impurities.
Found: 1013.2 [M+Na]+; 985.2 [MꢁN2+Na]+. Anal
Calcd for C54H62N4O14: C, 65.44; H, 6.31; N, 5.65.
Found: C, 65.40; H, 6.30; N, 5.49.
3.6. 2-Acetamido-3-O-[6-O-benzyl-3-O-sulfatyl-b-D-
galactopyranosyl]-4-O-[2,3,4-tri-O-benzyl-b-D-arabino-
pyranosyl]-6-O-benzyl-2-deoxy-b-D-glucopyranosyl
azide (10)
A mixture of 156 mg (0.158 mmol) 9 and 43 mg
(0.173 mmol, 1.1 equiv) of dibutyltin oxide was dissolved
in CH3OH (10 mL) and heated under reflux. After
20 min, the suspension became clear and was kept at this
temperature for additional 3.5 h. After concentration in
vacuo and co-distillation twice with toluene (10 mL),
the colourless residue was dried until constant weight
was achieved. After dissolution of the residue in DMF
(15 mL), 24 mg (0.173 mmol, 1.1 equiv) SO3/NMe3 com-
plex was added and the mixture was stirred at rt for 14 h.
The solvent was removed under high vacuum, and the
residue was co-distilled twice with toluene (10 mL). The
resulting colourless oil was purified by flash-chromato-
graphy (15:1, CHCl3/CH3OH) on silica gel (15 g). Yield:
3.5. 2-Acetamido-3-O-[6-O-benzyl-b-D-galactopyran-
osyl]-4-O-[2,3,4-tri-O-benzyl-b-D-arabinopyranosyl]-
6-O-benzyl-2-deoxy-b-D-glucopyranosyl azide (9)
22
0.144 mg (0.14 mmol, 91%); amorphous; ½aꢂD ꢁ83.5 (c 1,
CH3CN); Rf = 0.17 (10:1, CHCl3/CH3OH); tR =
1
38.3 min (A); H NMR (DMSO-d6, 400 MHz): d 8.01
Lewisa derivative 8 (7.41 g, 6.64 mmol) was dissolved in
CH3OH (150 mL) and 200 mg Na was added. After 20 h
of stirring at rt, 7.3 g of ion exchange resin Amberlystꢂ
15 was added to neutralize the reaction mixture. The sol-
vent was decanted and the resin washed three times with
CH3OH (25 mL). The combined organic layers were
dried (MgSO4), concentrated in vacuo and the residue
purified on silica gel (200 g) by flash-chromatography
(20:1, CH2Cl2/CH3OH). Yield: 5.83 g (5.88 mmol,
(d, JNH,H2 = 9.1 Hz, NH), 7.47–7.14 (m, 25 H, Harom.),
4.97 (d, 1H, J1 ,2 = 3.5 Hz, H-100), 4.78 (d, 1H,
00 00
J2 -OH,H-2 = 2.5 Hz, 20-OH), 4.70–4.59 (m, 4H, 40-OH,
CH2Ph, H-500a), 4.56–4.33 (m, 10H, CH2Ph, H-10,
H-1), 3.97–3.73 (m, 8H, H-400, H-40, H-30, H-300, H-200,
H-60a, H-3, H-2), 3.67–3.58 (m, 3H, H-6a, H-4, H-60b),
3.54–3.47 (m, 5H, H-50, H-500e, H-5, H-6b, H-20),
1.81 (s, 3H, CH3(NHAc)); 13C NMR (DMSO-d6,
100.6 MHz): d 169.59 (CO(NHAc)), 139.03, 138.95,
138.71, 138.54, 138.27 (Cipso-aryl), 128.38, 128.32,
128.23, 128.17, 127.77, 127.50, 127.44, 127.38, 127.30,
127.18, 127.08 (Carom.), 102.92 (C-10), 97.43 (C-100),
88.01 (C-1), 79.12 (C-30), 77.50 (C-300), 76.82 (C-5),
75.82 (C-3), 75.44 (C-200), 74.51 (C-400), 73.45, 72.33,
72.22, 70.74, 70.34 (CH2Ph), 72.78 (C-50), 72.43 (C-4),
69.05 (C-20), 68.63 (C-6), 67.44 (C-60), 66.26 (C-40),
60.23 (C-500), 54.68(C-2), 22.95 (CH3(NHAc)); ESIMS
calcd: 1070.38. Found: 1115.35 [MꢁH+2Na]+. Anal
Calcd for C54H62N4O17SÆ2H2O: C, 58.58; H, 6.01; N,
5.06; S, 2.90. Found: C, 58.46; H, 6.38; N, 4.94; S, 3.24.
0
0
22
88%); ½aꢂD ꢁ60.9 (c 1, CH2Cl2); Rf = 0.21 (15:1,
CH2Cl2/CH3OH); 1H NMR (DMSO-d6, 600 MHz):
d 8.06 (d, JNH,H2 = 9.1 Hz, NH), 7.36–7.18 (m, 25H,
H
arom.), 4.99 (d, 1H, J1 ,2 = 3.6 Hz, H-100), 4.87 (d,
00 00
0
0
0
1H, J3 -OH,H-3 = 5.3 Hz, 3 -OH), 4.70–4.67 (m, 2H, H-
500a, CH2Ph), 4.62–4.52 (m, 5H, CH2Ph, H-1), 4.46–
4.35 (m, 6H, CH2Ph, H-10), 3.99 (br s, 1H, H-400), 3.94
(d, 1H, J3 ,2 = 10.4 Hz, H-300), 3.87–3.78 (m, 4H,
00 00
H-600a, H-3, H-200, H-2), 3.68–3.59 (m, 4H, H-6a, H-40,
H-6 b, H-4), 3.55–3.50 (m, 4H, H-5, H-6b, H-50,
H-500e), 3.35–3.34 (m, 2H, H-20, 20-OH), 3.29–3.27
(m, 1H, H-30), 1.83 (s, 3H, CH3(NHAc)); 13C NMR
(DMSO-d6, 150.9 MHz): d 171.31 (CO(NHAc)), 138.31,
138.22, 138.05, 173.26 (Cipso-Ar), 128.41, 128.38,
128.18, 127.98, 127.89, 127.76, 127.69, 127.62, 127.53
(Carom.), 103.05 (C-10), 97.42 (C-100), 87.88 (C-1), 77.55
(C-300), 76.72 (C-5), 75.76 (C-3), 75.72 (C-200), 74.69
(C-400), 73.46, 72.88, 72.16, 70.82, 70.48 (CH2Ph), 73.20
(C-30), 73.06 (C-50), 72.42 (C-4), 70.36 (C-20), 68.93
(C-6), 68.01 (C-40), 67.41 (C-60), 60.67 (C-500), 54.74
(C-2), 23.23 (CH3(NHAc)); ESIMS calcd: 990.43.
3.7. Na-Fluorenylmethoxycarbonyl-Nx-(2-acetamido-3-
O-[6-O-benzyl-b-D-galactopyranosyl]-4-O-[2,3,4-tri-O-
benzyl-b-D-arabinopyranosyl]-6-O-benzyl-2-deoxy-b-D-
glucopyranosyl)-L-asparagine allyl ester (14)
To 280 mg (0.26 mmol) 9 in 75 mL of i-PrOH/water
(9:1) was added a catalytic amount (10 mg) of freshly
washed (pH 7) Raney-nickel. Hydrogen was bubbled
through the stirred solution via a cannula. After

1604
A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
completion of the reaction (5 h, TLC), the catalyst
was filtered off and washed thoroughly with i-PrOH
and CH3OH. The combined organic solutions were
concentrated in vacuo, and the amine 12a was used
in the subsequent reaction without purification and
characterization. Yield: 252 mg (quant.); amorphous;
Rf = 0.10 (5:1, CHCl3/CH3OH); MS calcd for
C54H64N2O14: 964.43.
CH2-Asn), 22.79 (CH3(NHAc)); ESIMS calcd for
C76H83O19N3: 1341.56. Found: 1364.97 [M+Na]+.
3.8. Na-Fluorenylmethoxycarbonyl-Nx-(2-acetamido-3-
O-[6-O-benzyl-b-D-galactopyranosyl]-4-O-[2,3,4-tri-O-
benzyl-b-D-arabinopyranosyl]-6-O-benzyl-2-deoxy-b-D-
glucopyranosyl)-L-asparagine (15)
To a mixture of 147 mg (0.37 mmol, 1.3 equiv) Fmoc-
Asp-OAll12 13 with 51 mg (0.37 mmol, 1.3 equiv)
HOAT in DMF (5 mL) were added 0.13 mL
To 390 mg (0.29 mmol) 14 in freshly degassed THF
(30 mL) were added N-methylaniline (0.06 mL,
0.56 mmol) and a catalytic amount (about 10 mg) of
Pd(PPh3)4. After 24 h, another 10 mg Pd(PPh3)4 and
0.2 mL (1.8 mmol) N-methylaniline were added, but
total deallylation was not achieved. The solvents were
removed in vacuo, the residue was dissolved in EtOAc
(100 mL) and washed with 0.1 N HCl (50 mL). After
drying (MgSO4), the solvent was removed in vacuo,
yielding a yellow oil of which the product was extracted
with CH3OH (5 mL) and filtered through a C18
cartridge. The solvent was removed in vacuo and the
resulting colourless oil was purified by semi-preparative
HPLC (Knauer Eurosphere C18). Yield: 279 mg
(0.74 mmol,
2.6 equiv)
i-Pr2NEt
and
140 mg
(0.37 mmol, 1.3 equiv) HATU. After keeping the solu-
tion at rt for 10 min, glycosylamine 12a in DMF
(5 mL) was added, and the solution was stirred for
18 h. After removal of the solvents in high vacuum,
the residue was dissolved in CH2Cl2 (100 mL) and
washed with satd NaHCO3 (50 mL) and brine
(50 mL). The organic layer was dried (MgSO4), concen-
trated in vacuo and the residue purified by flash-chro-
matography on silica gel (30 g) with CH2Cl2/CH3OH
(15:1). Lyophilization from benzene yielded a colourless
amorphous solid. Yield: 313 mg (0.30 mmol, 82%);
22
(0.21 mmol, 74%); ½aꢂD ꢁ58.3 (c 1, CH3CN); Rf = 0.32
22
½aꢂD ꢁ69.8 (c 1, CH3CN); Rf = 0.32 (15:1, CHCl3/
(15:1, CHCl3/CH3OH); tR = 12.7 min (B); 1H NMR
(DMSO-d6, 400 MHz): d 12.61 (br s, 1H, acid); 8.41
(d, 1H, JNH,H1 = 9.2 Hz, xNH-Asn), 7.96 (d, 1H,
JNH,H2 = 8.9 Hz, NH-Glc), 7.87 (d, 2H, J = 7.5 Hz,
H4-, H5-Fmoc), 7.70 (d, 2H, J = 7.5 Hz, H1-,
H8-Fmoc), 7.48 (d, 1H, JNH,aCH = 8.4 Hz, NH-
urethane), 7.40 (m, 3H, H3-, H6-Fmoc), 7.33–7.21 (m,
CH3OH); tR = 46.3 min (A); 1H NMR (DMSO-d6,
400 MHz): d 8.49 (d, 1H, JNH,H1 = 9.3 Hz, xNH-Asn),
7.96 (d, 1H, JNH,H2 = 8.9 Hz, NH), 7.88 (d, 2H,
JH4,H3 = H5H6 = 7.9 Hz, H4-, H5-Fmoc), 7.69 (d, 3H,
JH1,H2 = H8,H7 = 7.9 Hz, H1-, H8-Fmoc), 7.40 (t, 2H,
JH3,H2/4 = H6,H5/7 = 7.9 Hz, H3-, H6-Fmoc), 7.36–7.14
(m, 27H, H2-, H7-Fmoc, Harom.), 5.89–5.79 (m, 1H,
25H, Harom.), 4.98 (d, 1H, J1 ,2 = 3.44 Hz, H-100), 4.92
00 00
3
3Jtrans = 17.2 Hz, Jcis = 10.6 Hz, All-H2), 5.26 (dd,
(pt, 1H, JH1,NH = 9.3 Hz, JH1,H2 = 9.4 Hz, H-1), 4.86
1H, 3Jtrans = 17.3 Hz, All-H3a), 5.14 (dd, 1H,
(d, 1H, J3 -OH,H3 = 5.2 Hz, 30-OH), 4.68 (m, 2H, H-
500a, CH2Ph), 4.62–4.19 (m, 14H, CH2Ph, H-10, a-CH-
Asn, CH2-Fmoc, H9-Fmoc), 4.02–3.97 (m, 2H, H-300,
H-400), 3.86–3.77 (m, 4H, H-60a, H-3, H-200, H-2), 3.69–
3.49 (m, 7H, H-6a, H-40, H-4, H-6b, H-60b, H-50,
H-500e), 3.34 (m, 3H, H-20, 20-OH, H-5), 3.25–3.20 (m,
0
0
3
00 00
Jcis = 10.4 Hz, All-H3b), 4.98 (d, 1H, J1 ,2 = 3.5 Hz,
H-100), 4.91 (t, 1H, H-1), 4.70–4.40 (m, 15H, H-500a,
CH2Ph, a-CH-Asn), 4.31–4.17 (m, 5H, CH2Ph, Fmoc-
CH2, H9-Fmoc), 4.00–3.97 (m, 2H, H-300, H-400), 3.86–
3.78 (m, 4H, H-3, H-60a, H-200, H-2), 3.69–3.48 (m,
8H, H-60, H-40, H-4, H6b, H-60b, H-500e, H-50), 3.36–
3.29 (m, 3H, H-20, 20-OH, H-5), 3.23 (dd, 1H,
1H, JH-3 ,3 -OH = 5.0 Hz, H-30), 2.62 (m, 1H, b-CH2-
Asn), 2.45–2.41 (m, 1H, b-CH2-Asn), 1.74 (s, 3H,
CH3(NHAc)); 13C NMR (DMSO-d6, 100.6 MHz): d
173.10 (CO-acid), 170.27, 169.61 (CO-amide, -NHAc),
155.89 (CO-urethane), 143.91, 143.87 (C1a-, C8a-Fmoc),
140.76 (C4a-, C5a-Fmoc), 139.08, 138.97, 138.79, 138.75,
138.28 (Cipso-aryl), 128.43, 128.32, 128.23, 127.74,
127.57, 127.44, 127.27 (Carom.), 127.30 (C3-,C6-Fmoc),
127.15 (C2-,C7-Fmoc), 125.36 (C1-, C8-Fmoc), 120.18
(C4-, C5-Fmoc), 102.94 (C-10), 97.41 (C-100), 78.41
(C-1), 77.59 (C-300), 76.69 (C-3), 76.63 (C-5), 75.81
(C-200), 74.81 (C-400), 73.45, 72.23, 70.92, 70.53 (CH2Ph),
73.45 (C-30), 73.14 (C-50), 72.62 (C-4), 70.46 (C-20), 69.12
(C-6), 69.07 (C-6), 68.18 (C-40), 67.69 (C-60), 65.79
(CH2-Fmoc), 60.33 (C-500), 54.64 (C-2), 50.23 (a-CH-
Asn), 46.79 (C9-Fmoc), 37.07 (b-CH2-Asn), 22.89
(CH3(NHAc)). ESIMS calcd for C73H80N3O19:
1302.5386 [M+H]+. Found: 1302.5345 [M+H]+.
0
0
J3 ,2 = 9.4 Hz, H-30), 2.65 (m, 1H, b-CH2-Asn), 2.45
(m, 1H, b-CH2-Asn), 1.73 (s, 3H, CH3(NHAc)); 13C
NMR (DMSO-d6, 100.6): d 171.08 (CO-ester), 171.00,
169.23 (CO-NHAc, -amide), 155.79 (CO-urethane),
143.74 (C1a-, C8a-Fmoc), 140.69 (C4a-, C5a-Fmoc),
138.99, 138.87, 138.71, 138.66, 138.14 (Cipso-aryl),
132.30 (All-C2), 128.21, 128.12; 127.46; 127.22, 128.02,
127.16, 127.05, 126.98 (Carom.), 127.63 (C3-, C6-Fmoc),
127.33 (C2-, C7-Fmoc), 125.22 (C1-, C8-Fmoc), 120.11
(C4-, C5-Fmoc), 117.41 (All-C3), 102.85 (C-10), 97.30
(C-100), 78.31 (C-1), 77.49 (C-300), 76.58 (C-3), 76.54 (C-
5), 75.78 (C-200), 74.67 (C-400), 73.31 (CH2Ph, C-30),
72.11, 70.77 (CH2Ph), 70.38 (CH2Ph, C-20), 73.03 (C-
50), 72.49 (C-4), 68.97 (C-6), 68.08 (C-40), 67.62 (C-60),
65.75 (CH2-Fmoc), 64.89 (All-C1), 60.23 (C-500), 54.45
(C-2), 50.21 (a-CH-Asn), 46.54 (C9-Fmoc), 36.82 (b-
0
0

A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
1605
3.9. Na-Fluorenylmethoxycarbonyl-Nx-(2-acetamido-3-
O-[6-O-benzyl-3-O-sulfatyl-b-D-galactopyranosyl]-4-O-
[2,3,4-tri-O-benzyl-b-D-arabinopyranosyl]-6-O-benzyl-(2-
deoxy-b-D-glucopyranosyl)-L-asparagine a-allyl ester (16)
Fmoc), 120.12 (C4-, C5-Fmoc), 117.43 (All-C3),
102.76 (C-10), 97.29 (C-100), 79.24 (C-30), 78.25 (C-1),
77.45 (C-300), 76.68 (C-5), 75.96 (C-3), 75.79 (C-200),
74.49 (C-400), 73.33, 72.18, 72.11, 70.68, 70.29 (CH2Ph),
72.72 (C-50), 72.51 (C-4), 68.95 (C-20), 68.64 (C-6),
67.58 (C-60), 66.31 (C-40), 65.77 (CH2-Fmoc), 64.90
(All-C1), 60.12 (C-500), 54.56 (C-2), 50.19 (a-CH-Asn),
To 196 mg (0.18 mmol) 10 in 47 mL of i-PrOH/water
(9:1) was added a catalytic amount (about 5 mg) of
freshly washed (pH 7) Raney-nickel. Hydrogen was
bubbled through the stirred solution via a cannula.
After completion of the reaction (3.5 h, TLC), the cata-
lyst was filtered off and washed thoroughly with i-PrOH
and CH3OH. The combined organic solutions were con-
centrated in vacuo and the amine 12b was used in the
subsequent reaction without purification and characteri-
zation. Yield: 187 mg (quant); amorphous; Rf = 0.09
(5:1, CHCl3/CH3OH); MS calcd for C54H64N2O17S:
1044.40.
46.55
(C9-Fmoc),
36.82
(b-CH2-Asn),
22.76
(CH3(NHAc)); ESIMS calcd for C76H83N3O22S:
1421.51. Found: 1466.41 [MꢁH+2Na]+.
3.10. Na-Fluorenylmethoxycarbonyl-Nx-(2-acetamido-3-
O-[6-O-benzyl-3-O-sulfatyl-b-D-galactopyranosyl]-4-O-
[2,3,4-tri-O-benzyl-b-D-arabinopyranosyl]-6-O-benzyl-(2-
deoxy-b-D-glucopyranosyl)-L-asparagine (17)
To 165 mg (0.116 mmol) 16 in freshly degassed THF
(15 mL) were added two drops of N-methylaniline and
catalytic amounts (about 3 mg) of Pd(PPh3)4. After
6 h complete consumption of the starting material was
detected by TLC. Purification was achieved by prepara-
tive RP-TLC in CH2Cl2/CH3OH (2:1) as the eluent.
To a solution of 90 mg (0.23 mmol, 1.3 equiv) of
Fmoc-Asp-OAll12 13 and 31 mg (0.194 mmol, 1.3 equiv)
HOAt in DMF (10 mL) were added 0.08 mL (2.6 equiv)
i-Pr2NEt and 90 mg (0.194 mmol, 1.3 equiv) HATU.
After keeping the mixture at rt for 10 min, glycosyl-
amine 12b in DMF (5 mL) was added and the solution
was stirred for 17 h. After removal of the solvents in
high vacuum, the residue was dissolved in CH2Cl2
(100 mL) and washed with satd NaHCO3 (50 mL) and
brine (50 mL). The organic layer was dried (MgSO4)
and concentrated in vacuo. Lyophilization from benzene
yielded a colourless amorphous solid. Yield: 172 mg
22
Yield: 142 mg (0.103 mmol, 92%); ½aꢂD ꢁ51.9 (c 1,
DMSO); Rf = 0.78 (RP-C18; 2:1, CH3CN/H2O);
1
tR = 41.7 min (A); H NMR (DMSO-d6, 400 MHz): d
8.57 (br s, 1H, xNH-Asn), 7.91 (d, 1H, JNH,H2 = 9.0 Hz,
NH), 7.88 (d, 2H, JH4,H3 = H5,H6 = 7.5 Hz, H4-,
H5-Fmoc), 7.69 (d, 3H, JH1,H2 = H8,H7 = 7.768 Hz,
H1-, H8-Fmoc), 7.40 (t, 2H, JH3,H2/4 = H6,H5/7
=
22
(0.12 mmol, 66%); ½aꢂD ꢁ46.1 (c 1, CH3CN); Rf = 0.49
7.4 Hz, H3-,H6-Fmoc), 7.36–7.14 (m, 27H, H2-, H7-
(5:1, CHCl3/CH3OH); tR = 45.5 min (A); 1H NMR
DMSO-d6, 600 MHz): d 8.42 (d, 1H, JNH,H1 = 9.3 Hz,
xNH-Asn), 7.91 (d, 1H, JNH,H2 = 9.8 Hz, NH), 7.88
(d, 2H, JH4,H3 = H5,H6 = 7.8 Hz, H4-, H5-Fmoc), 7.69
(d, 3H, JH1,H2 = H8,H7 = 7.8 Hz, H1-, H8-Fmoc), 7.40
(t, 2H, JH3,H2/4 = H6,H5/7 = 7.5 Hz, H3-, H6-Fmoc),
7.36–7.14 (m, 27H, H2-, H7-Fmoc, Harom.), 5.84 (m,
Fmoc, Harom.), 4.98 (d, 1H, J1 ,2 = 3.3 Hz, H-100), 4.89
00 00
(t, 1H, J1,2 = 9.0 Hz, J1,NH = 9.1 Hz, H-1), 4.69–4.60
(m, 5H, CH2Ph, H-500a, a-CH-Asn), 4.54–4.42 (m, 8H,
H-10, CH2Ph), 4.33–4.20 (m, 4H, CH2Ph, Fmoc-CH2,
H9-Fmoc), 4.01–3.72 (m, 8H, H-300, H-400, H-40, H-3,
H-30, H-60a, H-200, H-2), 3.68–3.47 (m, 7H, H-6a, H-4,
H-6b, H-60b, H-50, H-500e, H-20), 3.28 (m, 1H, H-5),
2.56 (d, 1H, Jgem 15.7 Hz, b-CH2-Asn), 2.42 (d, 1H,
Jgem = 15.4 Hz, b-CH2-Asn), 1.73 (s, 3H, CH3(NHAc));
13C NMR (DMSO-d6, 100.6 MHz): d 171.11 (CO-ester),
169.60, 169.11 (CO-amide, -NHAc), 155.82 (CO-
urethane), 143.75 (C1a-, C8a-Fmoc), 140.71 (C4a-,
C5a-Fmoc), 138.98, 138.91, 138.65, 138.62, 138.18
(Cipso-aryl), 132.83 (All-C2), 128.23, 128.14, 128.11,
127.66, 127.57, 127.05, 127.44, 127.08, 127.04 (Carom.),
127.59 (C3-, C6-Fmoc), 127.21 (C2-, C7-Fmoc),
125.31 (C1-, C8-Fmoc), 120.10 (C4, C5-Fmoc), 102.54
(C-10), 97.09 (C-100), 79.14 (C-30), 78.34 (C-1), 77.47
(C-300), 76.32 (C-5), 75.97 (C-3), 75.58 (C-200), 74.37
(C-400), 73.18, 72.03, 70.78, 70.26, 70.15 (CH2Ph),
72.62 (C-50), 72.21 (C-4), 68.86 (C-20), 68.44 (C-6),
67.40 (C-60), 66.35 (C-40), 65.38 (CH2-Fmoc), 59.88
(C-500), 54.56 (C-2), 50.19 (a-CH-Asn), 46.87 (C9-
Fmoc), 36.82 (b-CH2-Asn), 22.81 (CH3(NHAc));
ESIMS calcd for C73H79N3O22S: 1382.4949 [M+H]+.
Found: 1383.4910 [M+H]+.
3
1H, All-H2), 5.26 (dd, 1H, Jtrans = 17.2 Hz, All-H3a),
3
5.14 (dd, 1H, Jcis = 10.5 Hz, All-H3b), 4.98 (d, 1H,
J1 ,2 = 3.40 Hz, H-100), 4.91 (t, 1H, J1,NH = 9.3 Hz, H-
00 00
1), 4.68–4.42 (m, 14H, CH2Ph, H-500a, AllH1a/b, a-
CH-Asn), 4.31–4.18 (m, 4H, Fmoc-CH2, H9-Fmoc),
3.97–3.84 (m, 6H, H-300, H-400, H-40, H-3, H-30,H-60a),
3.79 (dd, 1H, J2 ,3 = 9. 6 Hz, H-200), 3.75 (d, 1H,
00 00
J2,1 = 9.6 Hz, H-2), 3.65 (dd, 1H, Jgem = 14.2 Hz, H-
60b), 3.58 (d, 1H, J4,3 = 9.2 Hz, H-4), 3.55–3.47 (m,
7H, H-4, H-6b, H-60b, H-50, H-500e, H-2), 3.30 (d, 1H,
J5,4 = 9.6 Hz, H-5), 2.64 (m, 1H, b-CH2-Asn), 2.46 (m,
1H, b-CH2-Asn), 1.73 (s, 3H, CH3(NHAc)); 13C NMR
(DMSO-d6, 150.9 MHz): d 171.11 (CO (ester)), 169.60,
169.11 (CO (NHAc), (amide)), 155.82 (CO-urethane),
143.75 (C1a-, C8a-Fmoc), 140.71 (C4a-, C5a-Fmoc),
138.98, 138.91, 138.65, 138.62, 138.18 (Cipso-aryl),
132.83 (All-C2), 128.23, 128.14, 128.11, 127.66, 127.57,
127.05, 127.44, 127.08, 127.04 (Carom.), 127.33 (C3-,
C6-Fmoc), 127.23 (C2-, C7-Fmoc), 125.25 (C1-, C8-

1606
A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
a
3.11. Loading of NovaSyn TGꢂ Amino Resin (HL) with
4-[2-(N-fluorenylmethoxycarbonyl-L-isoleucyloxy)-1-
(trimethylsilyl)-ethyl]-phenoxy-acetic acid (19)
3.13. N-Acetyl-L-isoleucinyl-L-valinyl-L-glycyl-(2-acet-
amido-3-O-[3-sulfatyl-b-D-galactopyranosyl]-4-O-[b-D-
arabinopyranosyl]-[2-deoxy-b-D-glucopyranosyl]-L-aspa-
raginyl-L-leucyl-L-threonyl-L-glutamyl-L-leucyl-L-glut-
amyl-L-seryl-L-glutamyl-L-aspariginyl-L-isoleucine (22)
NovaSyn Tentagel Amino Resin HLꢂ (2.7 g) was placed
in a Merrifield solid-phase reactor and swollen in CH2Cl2
(20 mL) for 1 h. In a separate flask, 4-[2-(N-fluorenyl-
methoxycarbonyl-L-isoleucyloxy)-1-(trimethylsilyl)-ethyl]-
phenoxyacetic acid 18 (561 mg, 0.93 mmol) was dis-
solved in CH2Cl2 (40 mL) and DMF (5 mL) and
127 mg of HOAT, 297 mg of HBTU and 0.21 mL N-
methylmorpholine (NMM) were added. After 20 min,
the solution was added to the resin via a cannula and sha-
ken for 14 h. The solution was filtered off, the loaded
polymer washed with DMF (5 · 10 mL) and CH2Cl2
(5 · 10 mL) and then dried in high vacuum. Yield:
3.07 g. Photometric determination of resin loading gave
c = 0.3 mmol/g (this corresponds to a yield of 87%).
To 70 mg (0.023 mmol) of glycotridecapeptide 21 in
CH3OH/dioxane (10 mL, 1:1), 20 mg Pd(OH)2 on char-
coal was added. Hydrogen was bubbled through the
solution until complete consumption of starting material
(3 d) was detected by TLC. The solution was filtered
through Hyflo-Supercelꢂ and washed with CH3OH
(50 mL) and i-PrOH (50 mL). After evaporation of sol-
vents, the product was purified by preparative RP-TLC
with CH3CN/CH3OH (2:1). Yield: 48 mg (0.023 mmol,
22
quant.); ½aꢂD ꢁ17.2 (c 0.5, DMSO); amorphous;
Rf = 0.20–0.35 (RP-C18; 2:1, CH3CN/CH3OH); MAL-
DI-TOF-MS (dhb, negative ion mode): calcd for
C82H133N15O43S: 2049.85. Found: 2087.98 [Mꢁ2H+
K]ꢁ. The glycopeptide containing six acidic functions
retains tetrabutylammonium cations obviously as a mix-
ture of ammonium, sodium salts and free acids. Its com-
plex NMR spectrum therefore shows broad signals.
a
3.12. N-Acetyl-L-isoleucinyl-L-valinyl-L-glycyl-Nx-
(2-acetamido-3-O-[6-O-benzyl-3-sulfatyl-b-D-galactopyr-
anosyl]-4-O-[2,3,4-tri-O-benzyl-b-D-arabinopyranosyl]-6-
O-benzyl-(2-deoxy-b-D-glucopyranosyl)-L-asparaginyl-L-
leucyl-(3-O-benzyl)-L-threonyl-(5-O-benzyl)-L-glutamyl-
L-leucyl-(5-O-benzyl)-L-glutamyl-(3-O-benzyl)-L-seryl-
(5-O-benzyl)-L-glutamyl-(4-O-benzyl)-L-aspariginyl-L-
isoleucine (21)
3.14. N-Fluorenylmethoxycarbonyl-Nx-(2-acetamido-3-
O-[6-O-benzyl-b-D-galactopyranosyl]-4-O-[2,3,4-tri-O-
benzyl-b-D-arabinopyranosyl]-6-O-benzyl-(2-deoxy-b-D-
glucopyranosyl)-L-asparaginyl-L-leucyl-(3-O-benzyl)-L-
threonyl-(5-O-benzyl)-L-glutamyl-L-leucyl-(5-O-benzyl)-
L-glutamyl-(3-O-benzyl)-L-seryl-(5-O-benzyl)-L-glutam-
yl-(4-O-benzyl)-L-aspariginyl-L-isoleucine (24)
Resin 19 loaded with Fmoc isoleucine (0.5 g, 0.15 mM)
was placed in a solid-phase reactor. The Fmoc group
was removed with 30% piperidine in NMP. For sequen-
tial coupling reactions, Fmoc amino acids were used in
an excess of 10 equiv and activated with HBTU/HOBt/
i-Pr2NEt in DMF/NMP 1:1 (4 mL) for 20 min. After
each coupling, the resin was treated with 0.5 M Ac2O,
0.125 M i-Pr2NEt and 0.015 M HOBt for capping. The
sulfated glycosyl amino acid 17 was coupled in an excess
of 1.65 equiv and HATU/HOAt (10 equiv) as the cou-
pling reagent. The coupling time was extended to 3 h.
The final three Fmoc amino acids were coupled according
to the standard protocol, and the glycopeptide was acetyl-
ated at the N-terminus to give 20. Cleavage from the
polymer support was achieved by shaking 20 with
40 mg (0.15 mmol, 1 equiv) of TBAFÆ3H2O in CH2Cl2
(20 mL) for 20 min at rt. This procedure was repeated
once, and the collected organic solvents were washed
twice with 50 mL of H2O, dried with MgSO4, concen-
trated in vacuo and the residue purified by preparative
TLC (5:1, CHCl3/CH3OH) and ion exchange chromato-
graphy on Amberliteꢂ CG120 (Na+-form) (25 g; 5:1,
CHCl3/CH3OH). Yield: 70 mg (0.023 mmol, 15%); amor-
phous: Rf = 0.30 (5:1, CHCl3/CH3OH). MALDI-TOF-
MS (dhb, negative ion mode): calcd for C154H201N15O43S:
3042.43. Found: 3099.80 [Mꢁ3H+Na+K]ꢁ. The com-
pound retains tetrabutylammonium cations even after
ion exchange chromatography.
Resin 19 loaded with Fmoc isoleucine (0.165 g,
0.05 mM) was placed in a solid-phase reactor. The Fmoc
group was removed with 30% piperidine in NMP. For
sequential coupling reactions, Fmoc amino acids were
used in an excess of 10 equiv and activated with HBTU
(10 equiv)/HOBt (10 equiv)/i-Pr2NEt (20 equiv) in
DMF/NMP 1:1 (4 mL) for 20 min. After each coupling,
the resin was treated with 0.5 M Ac2O, 0.125 M
i-Pr2NEt and 0.015 M HOBt for capping. The glycosyl
amino acid 15 was coupled in an excess of 1.65 equiv
and HATU/HOAt (10 equiv) as the coupling reagent.
The coupling time was extended to 3 h to give 23. Cleav-
age from the polymer support was achieved by shaking
of 23 with 38 mg (0.12 mmol, 2.4 equiv) of TBAFÆ3H2O
in CH2Cl2 (20 mL) for 20 min at rt. This procedure was
repeated once with 15 mg (0.9 equiv) TBAFÆ3H2O. The
polymer was washed with CH2Cl2 (3 · 10 mL), and the
collected organic solvents were washed twice with H2O
(50 mL), dried with MgSO4, concentrated in vacuo
and the residue was purified by preparative TLC (15:1,
CHCl3/CH3OH). Yield: 62 mg (0.022 mmol, 43%);
amorphous: Rf = 0.43 (5:1, CHCl3/CH3OH). 15:1);
22
½aꢂD ꢁ74.9 (c 1, DMSO); 1H NMR (DMSO-d6,
400 MHz): d 8.37–8.25 (m, 2H, NH-urethane, xNH),

A. Ro¨sch, H. Kunz / Carbohydrate Research 341 (2006) 1597–1608
1607
1990, 250, 1130–1132; (b) Walz, G.; Aruffo, A.; Kolanus,
W.; Becilacqua, M. P.; Seed, B. Science 1990, 250, 1132–
1135; (c) Lowe, J. B.; Stoolman, L. M.; Nair, R. P.;
Larsen, R. D.; Behrend, T. L.; Marks, R. M. Cell 1990, 63,
475–482.
8.13–7.95
(m,
8H,
NH),
7.86
(d,
2H,
JH4,H3 = H5,H6 = 7.4 Hz, H4-, H5-Fmoc), 7.68 (m, 2H,
H1, H8-Fmoc), 7.40–7.15 (m, 59H, Harom., H3-, H6-,
H2-, H7-Fmoc), 5.03–5.00 (m, 2H, H-100, H-1), 4.71–
4.66 (m, 3H, CH2Ph, a-CH-Asn, H-500e), 4.65–4.25 (m,
16H, CH2Ph, CH2-Fmoc, a-CH-(Glu, Ser, Thr, Asn,
Leu)), 4.20–4.12 (m, 3H, H9-Fmoc, a-CH-Ile, CH2-
Fmoc), 4.09–3.73 (m, 7H, H-300, H-400, H-60a, H-3,
b-CH-Thr, H-200, H-2), 3.61–3.39 (m, 9H, H-4, H-50,
H-40, H6a/b, H-60b, H-500a, b-CH2-Ser), 2.81–2.75 (m,
1H, b-CH2-Asn), 2.59–2.52 (m, 1H, b-CH2-Asn), 2.39–
2.31 (m, 8H, c-CH2-Glu, b-CH2-Asp), 1.96–1.74 (m,
10H, b-CH2-Glu, b-CH-Ile, CH3(NHAc)), 1.01–0.71
(m, 27H, c-CH3-Thr, d-CH3-Leu, c-CH3-Ile, d-CH3-
Ile); 13C NMR (DMSO-d6, 100.6 MHz): d 173.08 (CO-
acid), 172.19 (br s, CO-amide), 170.70, 169.97, 169.78,
169.12, 156.36 (CO-urethane), 143.75 (C1a-, C8a-
Fmoc), 140.64 (C4a-, C5a-Fmoc), 138.99, 138.87,
138.61, 138.46, 138.09, 137.99 (Cipso-aryl), 128.34,
128.10, 128.01, 127.87, 127.83, 127.60, 127.52, 127.46,
127.40, 127.30, 127.21, 127.13, 127.06, 126.98 (C3-,
C6-, C2-, C7-Fmoc, Carom.), 125.10 (C1-, C8-Fmoc),
120.06 (C4-, C5-Fmoc), 101.84 (C-10), 97.33 (C-100),
79.01 (C-1), 73.29 (C-300), 76.47 (C-3, C-5), 75.82 (C-
200), 74.85 (b-CH2-Thr), 74.61 (C-400), 73.32 (C-30),
73.32, 72.13, 70.44, 70.73 (CH2Ph), 72.84 (C-50), 72.35
(C-4), 70.50 (C-20), 68.80 (C-6), 68.08 (C-40), 67.59 (C-
60), 67.82 (CH2-Fmoc), 60.57 (C-500), 57.82 (b-CH2-
Ser), 56.28 (a-CH-Ile), 54.60 (C-2), 52.87 (a-CH-Glu),
51.66 (a-CH-Asn), 51.22 (a-CH-Thr), 50.94 (a-CH-
Leu), 49.23 (a-CH-Asp), 46.59 (C9-Fmoc), 40.63 (b-
CH2-Leu), 37.99 (b-CH2-Asp), 36.20 (b-CH-Ile), 35.25
(b-CH2-Asn), 30.12 (d-CH2-Glu), 27.78 (b-CH2-Glu),
24.81 (c-CH2-Ile), 23.04 (c-CH-Leu), 23.01, 21.35 (d-
CH3-Leu), 16.33 (c-CH3-Thr), 15.92 (c-CH3-Ile), 11.11
(d-CH3-Ile); MALDI-TOF-MS (dhb, positive ion mode):
calcd for C154H186N12O38: 2873.23. Found: 2896.11
[M+Na]+.
3. Yuen, C.-T.; Lawson, A. M.; Chai, W.; Larkin, M.; Stoll,
M. S.; Stuart, A.; Sullivan, F. X.; Ahern, T. J.; Feizi, T.
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9. Hartung, W. H.; Simonoff, R. Org. React. 1953, VII, 263–
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