Spacer-separated sialyl LewisX cyclopeptide conjugates as potential E-selectin ligands

Article abstract of DOI:10.1016/j.carres.2006.09.012

Completely protected sialyl LewisX azide was synthesized from a neolactosamine azide precursor carrying a 3-O-allyloxycarbonyl group as the temporary protecting group. After its Pd(0)-catalyzed deprotection and stereoselective α-fucosylation, the obtained

Full text of DOI:10.1016/j.carres.2006.09.012

Carbohydrate Research 342 (2007) 541–557
Spacer-separated sialyl LewisX cyclopeptide conjugates as
potential E-selectin ligands
Holger Herzner and Horst Kunz*
Institut fu¨r Organische Chemie, Johannes Gutenberg-Universita¨t Mainz, Duesbergweg 10-14, D-55128 Mainz, Germany
Received 1 September 2006; accepted 15 September 2006
Available online 9 October 2006
Dedicated to the memory of Professor Nikolay K. Kochetkov
Abstract—Completely protected sialyl LewisX azide was synthesized from a neolactosamine azide precursor carrying a 3-O-allyloxy-
carbonyl group as the temporary protecting group. After its Pd(0)-catalyzed deprotection and stereoselective a-fucosylation, the
obtained LewisX azide was subjected to O-deacetylation in the galactose unit and subsequent regio- and stereoselective sialylation.
Reduction of the anomeric azido group afforded the sialyl LewisX amine building block. Two molecules of this tetrasaccharide
ligand were conjugated to a preformed cyclooctapeptide containing two equidistant L-asparagine units equipped with carboxy-ter-
minated tetraethyleneglycol side chains to give, after deprotection, the target glycopeptide conjugate. Preliminary biological evalu-
ation of the synthesized bivalent sialyl LewisX cyclopeptide conjugate showed only slightly enhanced inhibition of E-selectin binding
in spite of the given flexibility of the two linked saccharide determinants.
ꢀ 2006 Published by Elsevier Ltd.
Keywords: Glycopeptides; Sialyl LewisX; E-Selectin ligand; Oligoethylene glycol spacer; Glycosyl azides
1. Introduction
liposomes.9 An interesting concept of multivalent
presentation of sialyl LewisX is based on simultaneous
enzymatic formation of seven sialyl LewisX units linked
to b-cyclodextrin as a cyclic scaffold.10 In the course of
our investigations of sialyl LewisX glycopeptides it
was found that a trivalent sialyl LewisX cycloheptapep-
tide is a selective selectin inhibitor.11 It showed binding
to E-selectin, but not to P-selectin. Influence of the pep-
tide structure on the inhibitory effect of sialyl LewisX
towards selectins was also observed for other synthetic
glycopeptides.12 Since the binding affinity of oligovalent
sialyl LewisX has been found dependent on the distance
between the sialyl LewisX structures,1,13 it appeared
interesting to find out the effect a spacer between the sia-
lyl LewisX saccharide and the cyclopeptide backbone
has on the binding affinity. Because information about
the optimal distance between two sialyl LewisX saccha-
rides in the natural ligands of E-selectin is not available,
the chosen spacer should be flexible enough in order to
ascertain an adjustable fit in the binding sites of the
selectin. Furthermore, the two sialyl LewisX structures
Biological recognition of cell membrane glycoconjugates
plays an important role in regulatory processes within
mammalian organisms. However, carbohydrate ligands
only weakly bind to their receptors in most cases. Multi-
valent presentation of the saccharide ligands often
strongly amplifies the binding efficiency and selectivity.1
For example, multivalency obviously is important for
the binding of carbohydrate ligands like sialyl LewisX
to selectins.2 Synthetic oligosaccharide selectin ligands
can function as inhibitors of the inflammatory cascade3,4
and of tumour cell metastasis.5 As a consequence, a
number of multivalent sialyl LewisX derivatives have
been synthesized by chemical and chemoenzymatic
methods including dendrimers terminating with sialyl
LewisX,6 polymer-linked sialyl LewisX,7 polylysine-
bound sialyl LewisX8 or sialyl LewisX presented on
*
Corresponding author. Tel.: +49 6131 39 22334; fax: +49 6131 39
24786; e-mail: hokunz@mail.uni-mainz.de
0008-6215/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.carres.2006.09.012

542
H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
O
O
O
AcHN
O
Aloc-Cl, pyridine
O
O
N3
N3
O
HO
O
-35 °C, 45 min, 85%
AcHN
2
O
1
AcO OBn
O
AcO OBn
O
O
CCl3
BnO
AcO
OAc
TMS-OTf, CH2Cl2
-50 °C 20 °C, 14 h, 62%
O
4
O
NaCNBH3, HCl
ether, THF
NH
O
O
AcO
N3
O
O
OAc
HO
O
AcHN
N3
O
O
2 h, 0 °C, 73%
AcHN
O
5
3
AcO OBn
O
BnO
O
Morpholine, Pd(PPh3)4, THF
2 h, room temp., 74%
O
AcO
N3
HO
OAc
AcHN
6
SEt
O
AcO OBn
O
AcO
BnO
OBn
O
OBn
O
O
7
BnO
N3
OAc
AcHN
OBn
Bu4NBr, CuBr2, DMF, CH2Cl2
O
18 h, room temp., 69%
OBn
8
BnO
Scheme 1. Synthesis of a selectively deprotectable LewisX azide 8.
should be equivalent. As a model compound, which
meets these requirements a symmetrical cyclooctapep-
tide containing two equidistant spacer-separated sialyl
LewisX side chains was synthesized.
affecting the O-acetyl groups. Fucosylation using donor
722 gave the selectively deprotectable LewisX azide 8.
After O-deacetylation of 8 with methanol/sodium
methoxide to furnish 9,12a and sialylationꢀ using ethyl-
thio sialoside 1023 under conditions described by Hase-
gawa et al.,24 sialyl LewisX azide 11 was obtained with
satisfying regio- and stereoselectivity (Scheme 2). The
remaining free hydroxy groups were acetylated to afford
12. The reduction of the azido group of 12 to furnish the
glycosylamine 13 is best carried out immediately prior to
further conversion. The best results were obtained by
hydrogenolysis catalyzed by Raney-nickel that was
washed to neutral conditions (pH 7.5).17a,25 In this
way benzyl ether groups are not affected, and anomer-
ization of the glycosylamines is prevented.
The spacer amino acid required for the construction
of spacer-separated sialyl LewisX cyclopeptide conju-
gates was synthesized from tetraethylene glycol and its
base catalyzed conjugate addition to tert-butyl acrylate
to yield 14.
After mesylation, 15 was subjected to nucleophilic
introduction of the azido group. Finally, 16 was hydro-
genolyzed to furnish spacer amino acid ester 1726
(Scheme 3).
2. Results and discussion
Apart from efficient enzymatic syntheses of the sialyl
LewisX tetrasaccharide14 most strategies for chemical
syntheses start with fucosylation of a glucosamine
derivative.4b,11,12,15 As the stereoselective galactosyla-
tion of the fucosyl-glucosamine structure suffered from
only moderate yields in many cases, a variation of the
alternative fucosylation of an appropriately preformed
neolactosamine structure16 was pursued in this work.
The 4,6-benzylidene glucosamine derivative 1 served as
the starting material.12a It contained the azido group
as anomeric protecting group and precursor of the gly-
cosylamine required for later coupling reactions.17 After
introduction of the O-allyl carbonate18 to give 2 and reg-
ioselective reductive opening of the benzylidene-acetal,19
galactosylation of 3 with trichloroacetimidate 420 fur-
nished the fully protected type 2 lactosamine azide 5
(Scheme 1).
Removal of the allyloxycarbonyl (Aloc) group by
Pd(0)-catalyzed allyltransfer to morpholine as a weakly
basic allyl-trapping nucleophile21 proceeded without
ꢀ The sialylation of 9 was not optimized, higher, albeit varying yields of
sialylation reactions with O-glycosides of LewisX acceptor molecules
have been reported in the literature.

H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
543
OAc
AcO
COOCH3
SEt
OBn
O
HO
HO
AcO
AcHN
O
BnO
O
O
O
AcO
10
N3
NaOMe, MeOH
OH
8
NIS, TfOH, H3CCN
AcHN
5 h, room temp., 97%
O
OBn
-28 °C, 14 h, 59%
OBn
9
BnO
OAc
OBn
O
RO
O
AcO
MeOOC
O
BnO
O
11 X = N3, R = H
AcO
AcHN
O
O
X
OR
AcHN
OBn
AcO
12 X = N3, R = Ac
13 X = NH2, R = Ac
O
OBn
BnO
Scheme 2. Synthesis of sialyl LewisX amine 13.
a) Na
b)
O
O
O
O
O
O
O
O
HO
O
OH
HO
O
OtBu
24 h,room temp.,
78%
14
Mes-Cl, Et3N, CH2Cl2
0 °C, 10 h, quant.
O
O
O
O
NaN3, DMF
O
O
O
O
N3
O
O
S
OtBu
O
O
O
OtBu
H3C
2d, room temp.,
68%
15
16
Raney-nickel pH8, H2, isopropanol
12 h, 93%
O
O
O
O
O
OtBu
H2N
17
Scheme 3. Formation of spacer amino acid derived from tetraethylene glycol.
In order to explore the conjugation technique Fmoc
protected b-tert-butyl aspartate was converted to the
a-allyl ester 18. Acidolysis of the tert-butyl ester gave
the desired a-allyl Fmoc aspartate 19 (Scheme 4).
Condensation of 19 with the glycol spacer 17 pro-
moted by isobutyl 2-isobutoxy-1,2-dihydroquinoline-1-
carboxylate (IIDQ)27 afforded spacer-linked asparagine
20 carrying three orthogonally stable protecting
groups. Selective removal of the allyl ester protection
using Pd(0)-catalysis and N-methylaniline as the allyl
scavenger28 furnished Fmoc asparagine building block
21.
Alternative selective acidolysis of the side chain tert-
butyl ester of 20 formed 22, which served as a model
compound for coupling reactions with sialyl LewisX
amine 13. Among the condensing reagents tested O-ben-
zotriazolyl tripyrrolidinophoshonium hexafluorophos-
phate (PyBOP)29 afforded the highest yield of the sialyl
LewisX-spacer asparagine conjugate 23 (Scheme 5).
Due to the long reaction time partial anomerization of
the glycosylamine occurred. Its condensation gave the a-
anomer of 23 as a side product (25%).
The use of the benzyl ester-containing sialyl LewisX
analogue of 13 and O-pentafluorophenyl-bis-tetrameth-

544
H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
O
FmocHN
COOH
O
FmocHN
O
O
Br,
i
Pr2EtN
FmocHN
O
O
O
TFA, CH2Cl2, 5% H2O
reflux, 30 min, 85%
O
O
or
30 min, room temp., quant.
Cs2CO3, MeOH, 30 min
Br, DMF
HO
18
IIDQ
19
2 h, room temp.,95%
N
O
O
iBu
17
O
Oi
Bu
14 h, room temp.,
85%
cat. Pd(PPh3)4,
-methylaniline, THF
O
HO
O
O
N
O
O
O
O
O
O
O
OtBu
FmocNH
N
FmocNH
OtBu
N
H
5 h, room temp.
70%
4
H
4
21
20
Scheme 4. Synthesis of the protected spacer-equipped asparagines 21.
TFA/CH2Cl2 1:1
O
O
O
O
O
20
O
4
OH
FmocNH
N
H
22
O
O
FmocNH
O
13
PyBOP, HOBt
NH
OAc
OBn
O
AcO
O
AcO
MeOOC
O
BnO
O
O
H
N
O
AcO
AcHN
O
O
O
O
OAc
AcHN
OBn
O
AcO
O
OBn
BnO
23: 72%
Scheme 5. Model condensation to form the spacer-separated sialyl LewisX asparagines 23.
ylene-uronium-hexafluorophosphate (PfPyU)30 or (7-
aza-benzotriazol-1-yl)-tetramethyluronium-hexafluoro-
phosphate (HATU)31 as the condensing agents resulted
in lower yields.
For the solid-phase synthesis of an octapeptide
containing two spacer-equipped aspartate residues
polystyrene-based resin having the allylic HYCRON
anchor26b 24 was applied according to Scheme 6.
Benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium-tetra-
fluoroborate (TBTU)32 in combination with 1-hydro-
benzotriazol (HOBt)33 served as the condensing
reagent.
Palladium(0)-catalyzed detachment of the octapeptide
25 using the very weak base N-methyl aniline as the allyl
scavenger28,30b did not affect the Fmoc group.
After purification of peptide 25 and removal of the
Fmoc group cyclization was achieved using HATU
and 1-hydroxy-7-aza-benzotriazole (HOAt).31 The sym-
metric cyclooctapeptide 26 was subjected to acidolysis of
the side chain tert-butyl esters. Subsequently the two
spacer carboxylic groups were condensed simulta-
neously with sialyl LewisX amine 13 (Scheme 7).
Again the long reaction time resulted in some anomer-
ization of 13. The cyclic sialyl LewisX glycopeptide 27bb

H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
545
H
H
O
O
N
N
O
O
O
FmocHN
24
O
O
O
1. morpholine/ DMF or TFCH2Cl2
2. -proteced aminoacid, TBTU, HOBt, NMM
3. Ac2O, pyridine
solid-phase synthesis
N
Fmoc-Asn(glycol4-COOtBu)-Val-Pro-Gly-Asn(glycol4-COOtBu)-Val-Pro-Gly-HYCRON-βAla-PS
cat. [Pd(PPh3)4], DMF/Me2SO,
-methylaniline
N
Fmoc-Asn(glycol4-COOtBu)-Val-Pro-Gly-Asn(glycol4-COOtBu)-Val-Pro-Gly-OH
25
Scheme 6. Solid-phase synthesis of spacer-containing octapeptide 25.
25
1. DMF, morpholine; quant.
2. HATU, HOAt;
CH2Cl2, DMF
tBuOOC
COO
t
Bu
O
O
O
O
71%
O
O
O
N
O
O
N
H
N
H
HN
N
O
O
O
H
CH3
CH3
H3C
N
H
N
H
O
O
CH3
O
O
H
N
H
N
26
NHAc
AcO
N
O
AcO
AcO
OAc
O
NHAc
AcO
O
OAc
AcO
O
O
O
AcO
MeO
OAc
O
1. TFA/CH2Cl2 1:1
O
O
OAc
OBn
O
AcO
O
OBn
O
MeO
OBn
O
2. 13, PyBOP, HOBt
O
O
OBn
O
AcO
O
OBn
OBn
AcHN
NH
NH
O
BnO
NHAc
OBn
O
O
OBn
BnO
O
O
O
O
O
O
O
N
O
O
N
H
H
HN
N
O
O
H
O
CH3
N
27ββ: 38.5%
27αβ: 18.5%
N
O
O
CH3
H3C
O
CH3O
H
N
H
N
H
H
N
N
O
O
Scheme 7. Peptide cyclization and condensation with sialyl LewisX amine.

546
H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
27ββ
NHAc
HO
HO
HO
1. Pd(OH)2, MeOH, AcOH, H2O
NHAc
OH
2. 0.1 N NaOMe, MeOH
HO
O
OH
O
HO
HO
3. NaOH (aq.), pH 10.5
O
O
OH
O
HO
O
O
OH
OH
O
HO
O
OH
O
HO
OH
O
O
O
O
O
HO
OH
OH
NH
NH
OH
HO
AcHN
O
NHAc
OH
O
O
OH
HO
O
O
O
O
O
O
O
O
O
N
N
H
H
N
28ββ: 89%
HN
N
O
O
H
O
CH3
CH3
H3C
N
N
H
O
O
O
CH3
analogously
O
H
28αβ: 92%
N
H
H
N
N
O
O
Scheme 8. Deprotection of the spacer-separated sialyl LewisX cyclopeptide.
containing two b-glycosyl amide bonds was isolated in a
yield of 39%. The analogue having one a- and one b-gly-
cosyl amide linkage was obtained as a by-product (18%).
The NMR spectra give evidence of the structure of com-
pounds 27. While the spectrum of 27bb shows only one
methyl ester signal, that of 27ab shows two (d = 3.833
and 3.823 ppm). The separated signals of H-1a of 27ab
at d = 5.74 ppm has only half of the intensity compared
to that of H-400 of Gal at d = 5.03 ppm in the same range
of the spectrum. Further characterization was carried
out for the deblocked compounds.
proceeds in a statistical fashion and shows no exponen-
tial enhancement. It should be noticed that in the same
assay the monomeric sialyl LewisX asparagine showed
an IC50 ꢀ 2.4 mM suggesting that in this series the ami-
no acid decreases the binding affinity to E-selectin com-
pared to the tetrasaccharide itself.
3. Experimental
3.1. General methods
Deprotection was achieved by Pd-catalyzed hydroge-
nation in methanol/acetic acid/water (30:3:0.2), subse-
NMR spectra were recorded on a Bruker AC-200 or a
Bruker AM-400 spectrometer. The following abbrevia-
tions were used to explain multiplicities: s (singlet), d
(doublet), t (triplet), m (multiplet). Indication of 1H
and 13C NMR signals to monosaccharide un0i0t0s: no index
´
quent Zemplen transesterification in methanol/sodium
methoxide at pH 10 and saponification of methyl ester
groups in aqueous NaOH at pH 10.5. Neutralization
was performed with solid carbondioxide (Scheme 8).
The resulting crude products containing salts were
purified by gel permeation chromatography (Sephadex
LH15) in water giving the sialyl LewisX-spacer-cyclo-
peptide conjugates 28bb and 28ab in a pure form and
high yield.
0
00
GlcNAc, index Fuc, index Gal, index NeuNAc.
Mass spectra were recorded on a ESI Navigator-1 (Ther-
moQuest), a Tofspec E instrument (Micromass, 2,5-
dihydroxy-benzoic acid (dhb) or a-cyanocinnamic acid
(cca) as the matrix) or a Finnigan MAT 95 (FD and
FAB) instrument. Optical rotations were recorded using
a Perkin Elmer 241 polarimeter. Elemental analyses were
performed by the microanalytical laboratory of the Insti-
tut fuer Organische Chemie, Universitaet Mainz.
Preliminary evaluation of inhibitory effects showed an
IC50 ꢀ 0.8 mM in an assay of adhesion of 32Dc3 cells
(murine neutrophiles) to
a
E-selectin-IgG con-
struct.12b,26a From this result the bivalent 28bb is just
twice as active as sLeX itself. Obviously, the inserted
spacer is too flexible. The binding of 28bb to E-selectin
All reactions were monitored by thin-layer chroma-
tography (TLC) carried out on 0.25 mm silica gel plates

H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
547
(60F254/RP-C18254 E. Merck, Darmstadt, Germany)
using UV light, KMnO4 or p-anisaldehyde solns and
heat for visualization. Silica gel 60 (particle size 0.04–
0.0063, E. Merck, Darmstadt, Germany) was used for
flash chromatography, silica particle size 0.63–0.2 mm
(J. T. Baker, Gross-Gerau, Germany) for atmospheric
pressure chromatography. Gel permeation chromato-
graphy was carried out on Sephadex LH 15 (Pharma-
cia). All reactions were carried out under an argon
atmosphere with dried solvents. Yields refer to chro-
matographically and spectroscopically (1H NMR)
homogeneous materials. Analytical RP-HPLC was per-
formed on a Phenomenex LUNA C18 (5 lm column,
FDMS calcd: 418.2. Found: 419.2 (100%) [M+H]+.
Anal. Calcd for C19H22N4O7: C, 54.54; H, 5.30; N,
13.39. Found: C, 54.48; H, 5.57; N, 13.21.
3.1.2. 2-Acetamido-3-O-allyloxycarbonyl-6-O-benzyl-2-
deoxy-b-D-glucopyranosyl azide 3 (3-Aloc-6-BzlGlcNAc-
N3). To a soln of benzylidene acetal 2 (11.30 g,
27.0 mmol) in dry tetrahydrofuran (130 mL), 10 g of
˚
pulverized molecular sieves (3 A) and 17.0 g (270 mmol)
of sodium cyanoborohydride were added. After cooling
to 0 ꢁC saturated (at 0 ꢁC) HCl in diethyl ether was
added dropwise within 1 h until the evolution of gas
deceased (about 35 mL). Under careful monitoring by
TLC, additional small amounts of NaCNBH3 and
HCl in ether (5 mL) were added twice. Compound 2
had then been completely consumed. After neutraliza-
tion by addition of solid NaHCO3, the solid materials
were separated by centrifugation. The soln was diluted
with CH2Cl2 (700 mL), and the organic layer was
washed with satd aq NaHCO3 and water. After drying
with MgSO4 and evaporation of the solvents, colourless
crystals of 3 remained. The product was purified by flash
chromatography in 60:1 CH2Cl2–MeOH. Yield: 8.32 g
250 4.6 mm), flow 1 mL/min using a Knauer HPLC
*
equipment (MaxiStar K1000, DAD 2026 detector). Sol-
vent: (MeCN-water + 0.1% TFA); For semi-preparative
(flow 10 mL/min) and preparative HPLC (flow 20 mL/
min) a Knauer HPLC equipment (two MaxiStar K500,
DAD 2026 detector) was used. Columns are specified
for the different compounds.
3.1.1. 2-Acetamido-3-O-allyloxycarbonyl-4,6-O-benzyli-
dene-2-deoxy-b-D-glucopyranosyl azide 2 (3-Aloc-4,6-
BzdGlcNAc-N3). In dry CH2Cl2 (200 mL) and pyridine
(300 mL), 10.75 g (32.15 mmol) glycosyl azide17b 1 was
dissolved and cooled to ꢁ35 ꢁC. Allyl chloroformate
(12.0 mL, 113.1 mmol) was added within 30 min, and
the soln was stirred for 15 min. The precipitating pyrid-
inium hydrochloride was filtered off. The soln was
washed with 100 mL of satd aq NaHCO3 and 100 mL
of water. After drying with magnesium sulfate and evap-
oration of the solvents under diminished pressure, the
residue was co-distilled three times with toluene. The
solid residue was dissolved in boiling ethyl acetate
(300 mL) and crystallized by cooling and addition of
light petroleum. Concentration of the mother liquor
and further addition of light petroleum afforded addi-
tional amounts of product 2. Yield: 11.47 g (85%),
23
(73%), colourless crystals; mp: 164 ꢁC; ½aꢂD ꢁ63.5 (c 1,
CHCl3); Rf = 0.42 (2:3 light petroleum–ethyl acetate).
1H NMR (Me2SO-d6, 400 MHz): d 1.77 (s, 3H,
CH3CO–); 3.40–3.47 (m, 1H, H-2); 3.58–3.64 (m, 2H,
H-3, H-4); 3.67–3.75 (m, 2H, H-1, H-5); 4.53 (s, 2H,
–CH2Bn); 4.53 (s, 2H, –CH2Bn); 4.55 (d, 1H, Jgem
16.7 Hz, Jvic 5.3 Hz, CH2@CH–CH2–O–); 4.62 (d, 1H,
Jgem 16.7 Hz, Jvic 4.7 Hz, CH2@CH–CH2–O–); 4.66–
4.74 (m, 2H, H-6a, H-6b); 5.22 (d, 1H, Jcis 10.6 Hz,
CH2@CH–CH2–O–); 5.28 (d, 1H, Jtrans 17.3 Hz,
CH2@CH–CH2–O–); 5.72 (d, 1H, J4,OH 6.5 Hz, 4-OH);
5.90 (mc, 1H, CH2@CH–CH2–O–); 7.24–7.37 (m, 5H,
Ar–H); 8.06 (d, 1H, JNH,2 9.4 Hz, –NHAc). 13C NMR
(Me2SO-d6, 50.3 MHz) DEPT: d 22.51 (CH3CO–);
52.77 (C-2); 67.55 (C-3); 67.61, 68.88 (C-6, CH2@CH–
CH2–O–); 72.30 (–CH2–Bn); 77.25, 79.42 (C-4, C-5);
87.43 (C-1); 117.65 (CH2@CH–CH2–O–); 127.22,
127.25, 128.06 (C–Ar); 132.03 (CH2@CH–CH2–O–);
138.29 (Cipso); 154.16 (O–CO–O–); 169.17 (CH3CO–).
Anal. Calcd for C19H24N4O7: C, 54.28; H, 5.75; N,
13.33; Found: C, 53.82; H, 5.75; N, 13.31.
22
colourless crystals; mp: 168 ꢁC; ½aꢂD ꢁ82.2 (c 1,
CHCl3); Rf = 0.28 (1:1 light petroleum–ethyl acetate);
1H NMR (CDCl3, 200 MHz): d 1.97 (s, 3H, CH3CO–);
3.54–3.72 (m, 3H, H-2, H-5, H-6a); 4.00 (dd, 1H, Jgem
19.5 Hz, Jvic 9.3 Hz, –O–CH2a–CH@CH2); 4.30 (dd,
1H, J6a,6b 10.3 Hz, J6a,5 4.4 Hz, H-6a); 4.56–4.66 (m,
3H, H-1, H-4, –O–CH2b–CH@CH2); 5.16 (t, 1H,
J3,2 ꢃ J3,4 9.8 Hz, H-3); 5.23 (dd, 1H, Jcis 11.2 Hz, Jgem
1.0 Hz, –CH@CH2cis); 5.30 (dd, 1H, Jtrans 17.1 Hz, Jgem
1.0 Hz, –CH@CH2trans); 5.49 (s, 1H, @CH–Bzd); 5.76–
5.97 (m, 1H, –CH@CH2); 6.16 (d, 1H, JNH,2 9.3 Hz,
–NH–C2); 7.31–7.48 (m, 5H, H-benzylidene).
3.1.3. 2-Acetamido-3-O-allyloxycarbonyl-4-O-(600-O-ben-
zyl-200,300,400-tetra-O-acetyl-b-D-galactopyranosyl)-6-O-
benzyl-2-deoxy-b-D-glucopyranosyl azide 5 (b-3-Aloc-4-
(b-600-Bn-Ac3Gal)-6-BnGlcNAc-N3). Under
argon
atmosphere a soln of 3 (250 mg, 0.462 mmol) and
of galactosyl trichloroacetimidate20
(352 mg,
13C NMR (Me2SO-d6, 50.3 MHz): d 22.57 (CH3CO–);
52.92 (C-2); 67.25, 67.57, 67.88 (C-3, C-6, –CH2–
CH@CH2); 75.38, 77.55 (C-4, C-5); 88.11 (C-1); 100.26
(@CH–Bzd); 117.77 (CH2@CH–CH2–); 126.29, 128.08
(C–Arortho, C–Armeta); 128.92 (C–Arpara); 137.06 (C–
Aripso); 153.96 (–O–CO–OAll); 169.52 (CH3CO–).
4
0.837 mmol) in dry CH2Cl2 (30 mL) was stirred with
˚
0.5 g of pulverized molecular sieves 4 A at room temper-
ature for 1 h. After cooling to ꢁ50 ꢁC, a soln of tri-
methylsilyl trifluoromethanesulfonate (triflate) (20 lL,
0.111 mmol) in CH2Cl2 (3 mL) was added via a syringe

548
H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
within 1 h. The cooling was removed after 15 min and
the soln stirred at room temperature for 14 h. The soln
was neutralized with solid NaHCO3, filtered through
Hyflo, which was washed with CH2Cl2, and the com-
bined organic layers were washed with satd aq NaHCO3
and water, dried with MgSO4, and the solvents were
evaporated under diminished pressure. The obtained
brownish-yellow solid (730 mg) was purified by flash
chromatography with light petroleum/ethyl acetate.
portion (about 170 mL) of THF was distilled off, the
remaining suspension was diluted with ethyl acetate
(500 mL) and extracted with water. After drying with
MgSO4, the solvent was evaporated under diminished
pressure. The remaining viscous oil (4.90 g) was purified
by flash chromatography in light petroleum/ethyl ace-
tate. Yield: 3.24 g (74%), colourless amorphous solid;
21
½aꢂD ꢁ17.9 (c 1, CHCl3); Rf = 0.24 (1:2 light petro-
1
1
leum–ethyl acetate). H NMR (CDCl3, 400 MHz), H,
1H-COSY: d 1.96 (s, 6H, CH3CO–); 1.97 (s, 3H,
CH3CO–); 2.03 (s, 3H, CH3CO–); 3.29–3.37 (m, 2H,
24
Yield: 229 mg (62%), colourless amorphous solid; ½aꢂD
ꢁ50.0 (c 1, CHCl3); Rf = 0.32 (2:3 light petroleum–ethyl
acetate). 1H NMR (CDCl3, 400 MHz), 1H, 1H-COSY: d
1.91 (s, 3H, CH3CO–); 2 · 1.94 (s, 6H, CH3CO–); 1.98
H-6b, H-600b); 3.47 (t, 1H, J6 a;6 b ꢃ J6 a;5 8:4 Hz, H-
600a); 3.55–3.68 (m, 4H, H-2, H-4, H-6a, H-500); 3.79–
3.83 (m, 1H, H-5); 4.05 (dd, 1H, J3,4 10.3 Hz, J3,2
8.2 Hz, H-3); 4.33 (d, Jgem 12.0 Hz, –CH2Bn); 4.48 (d,
12.0 Hz, –CH2Bn); 4.51 (d, Jgem 12.9 Hz, –CH2Bn);
00
00
00
00
00
00
00
00
(s, 3H, CH3CO–); 3.33 (t, J6 b;6 a ꢃ J6 b;5 8:8 Hz, H-
600b); 3.42 (dd, J6 a;6 b 9:0 Hz, J6 a;5 5:3 Hz, H-600a);
3.55 (dt, 1H, J5,4 9.4 Hz, J5,6a J5,6b 2.5 Hz, H-5); 3.67
00
00
00
00
(dd, 1H, J5 ;6 a 6:1 Hz, J5 ;6 b 8:4 Hz, H-500); 3.68–3.71
(m, 2H, H-6a, H-6b); 3.89 (q, 1H, J2,1 ꢃ J2,NH ꢃ J2,3
9.6 Hz, H-2); 3.95 (t, 1H, J4,3 ꢃ J4,5 9.1 Hz, H-4); 4.34
(d, 1H, Jgem 11.9 Hz, –CH2Bn); 4.37 (ddt, 4J 1.4 Hz, Jgem
13.1 Hz, Jvic 5.3 Hz, CH2@CH–CH2–O–); 4.48 (d, 1H,
4.53 (d, 1H, J1 ;2 9:1 Hz, H-100); 4.94 (dd, J3 ;4 3:2 Hz,
00 00
00 00
00 00
00 00
00 00
J3 ;2 10:3 Hz, H-300); 5.00 (d, 1H, J1,2 9.1 Hz, H-1);
5.11 (dd, J2 ;3 10:3 Hz, J2 ;1 7:9 Hz, H-200); 5.17 (d,
00 00
00 00
1H, J4 ;3 2:9 Hz, H-400); 5.96 (d, 1H, JNH,2 7.6 Hz,
00 00
–NHAc); 7.23–7.35 (m, 10H, H–Ar). 13C NMR (CDCl3,
100.6 MHz): d 20.49, 20.64, 23.44 (CH3CO–); 56.85 (C-
2); 67.33, 67.65, 67.81 (C-6, C-400, C-600); 69.12 (C-200);
70.89 (C-300); 71.44, 72.36 (C-4, C-500); 73.67 (2 ·
–CH2Bn); 76.17 (C-5); 80.79 (C-3); 87.70 (C-1); 101.15
(C-100); 127.75, 127.85, 128.02, 128.52 (H–Ar); 137.18,
137.92 (Hipso); 169.19, 169.98, 170.68 (CH3CO–). Anal.
Calcd for C34H42N4O13 (714.72): C, 57.14; H, 5.92; N,
7.84. Found: C, 56.94; H, 6.03; N, 7.85.
J1 ;2 7:9 Hz, H-100); 4.50 (d, 1H, Jgem 11.7 Hz, –CH2Bn);
00 00
4.51 (d, 1H, Jgem 12.3 Hz, –CH2Bn); 4.55 (ddt, 4J 1.4 Hz,
Jgem 13.1 Hz, Jvic 7.4 Hz, CH2@CH–CH2–O–); 4.59 (d,
1H, J1,2 9.1 Hz, H-1); 4.69 (d, 1H, Jgem 12.0 Hz,
00 00
00 00
–CH2Bn); 4.86 (dd, 1H, J3 ;4 3:2 Hz, J3 ;2 10:3 Hz,
H-300); 4.92 (dd, 1H, J3,2 10.3 Hz, J3,4 9.1 Hz, H-3);
5.00 (dd, 1H, J2 ;3 10:3 Hz, J2 ;1 7:9 Hz, H-200); 5.18
(dd, 1H, Jgem 1.2 Hz, Jcis 10.6 Hz, CH2@CH–CH2–
O–); 5.27 (dd, 1H, Jgem 1.2 Hz, Jtrans 17.0 Hz,
00 00
00 00
CH2@CH–CH2–O–); 5.41 (d, 1H, J4 ;3 3:2 Hz, H-400);
3.1.5. 2-Acetamido-3-O-(20,30,40-tri-O-benzyl-a-L-fuco-
pyranosyl)-6-O-benzyl-2-deoxy-4-O-(200,300,400-tri-O-ace-
tyl-600-O-benzyl-b-D-galactopyranosyl)-b-D-glucopyrano-
00 00
5.79 (mc, 1H, CH2@CH–CH2–O–); 5.82 (d, 1H, JNH,2
8.5 Hz, –NHAc); 7.22–7.37 (m, 10H, H–Ar). 13C
1
NMR (Me2SO-d6, 100.6 MHz), DEPT, H, 13C-COSY:
syl azide
GlcNAc-N3). To
(3.10 g, 4.34 mmol) and thiofucoside22
8 (b-3-(a-Bn3Fuc)-4-(b-Ac3-6-BnGal)-6-Bn-
d 20.44, 20.48, 20.63, 23.13 (CH3CO–); 53.86 (C-2);
66.65 (C-600); 67.20 (C-400); 67.36 (C-6); 68.56
(CH2@CH–CH2–O); 69.32 (C-200); 71.06 (C-300); 73.53
(–CH2Bn); 73.81 (–CH2Bn); 74.81 (C-4); 71.92 (C-500);
76.72 (C-5); 76.75 (C-3); 88.25 (C-1); 100.64 (C-100);
118.76 (CH2@CH–CH2–O); 127.82, 127.90, 128.37,
128.47 (C–Ar); 131.17 (CH2@CH–CH2–O); 137.51,
137.68 (Cipso); 154.71 (–O–CO–O–); 169.17, 169.76,
169.79, 170.08 (CH3CO–). Anal. Calcd for C38H46-
N4O15: C, 57.14; H, 5.80; N, 7.01. Found: C, 57.19; H,
5.78; N, 6.91.
a
soln of lactosamine azide
6
7
(2.91 g,
6.08 mmol) in dry DMF (70 mL) and dry CH2Cl2
˚
(70 mL), 7 g of pulverized molecular sieve (4 A) were
added. The mixture was stirred for 1 h at room temper-
ature. After addition of tetrabutylammonium bromide
(2.38 g, 7.38 mmol) and copper(II)bromide (1.45 g,
6.51 mmol), the stirring was continued for 18 h at room
temperature. After filtration through Hyflo, which was
washed with CH2Cl2 (400 mL), the organic soln was
extracted with brine and six times with approx. 80 mL
of satd aq NaHCO3. The aqueous extracts were re-
extracted with 40 mL of CH2Cl2. The combined organic
solns were dried over MgSO4, and the solvent was evap-
orated under diminished pressure to give 9.3 g of a
brown, viscous oil, which was purified by flash chroma-
tography (4:1 light petroleum–ethyl acetate). Yield:
3.1.4. 2-Acetamido-4-O-(600-O-benzyl-200,300,400-tetra-O-
acetyl-b-D-galactopyranosyl)-6-O-benzyl-2-deoxy-b-D-
glucopyranosyl azide 6 (b-4-(b-600-Bn-Ac3Gal)-6-BnGlc-
NAc-N3). To a soln of allyloxycarbonyl protected
disaccharide 5 (4.90 g, 6.13 mmol) in dry and oxygen
free tetrahydrofuran (200 mL) under argon atmosphere,
morpholine (3.20 mL, 36.81 mmol) was added trough a
syringe. Under an argon counter stream a catalytic
amount of (PPh3)4Pd(0) (30 mg) was added, and the
soln was stirred at room temperature for 2 h. The major
21
3.40 g (69%), colourless amorphous solid; ½aꢂD ꢁ66.3
(c 1, CHCl3); Rf = 0.37 (1:1 light petroleum–ethyl ace-
1
1
1
tate). H NMR (CDCl3, 400 MHz), H, H-COSY: d
1.10 (d, 3H, J6 ;5 6:5 Hz, H-60); 1.72 (s, 3H, CH3CO–);
1.95 (s, 3H, CH3CO–); 1.98 (s, 3H, CH3CO–); 3.34 (t,
0
0

H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
549
J5 ;6 a ꢃ J5 ;6 b 8:4 Hz, H-500); 3.42 (q, 1H, J2,1
ꢃ
J4 ;3 2:7 Hz, H-400); 3.90 (dd, 1H, J3 ;2 10:2 Hz,
00 00
00 00
00 00
0
0
J3 ;4 2:3 Hz, H-30); 3.95 (dd, 1H, J6a,6b 11.7 Hz, J6b,5
0
0
J2,3 ꢃ J2,NH 7.3 Hz, H-2); 3.46–3.58 (m, 4H, H-5,
H-40, H-600a, H-600b); 3.76–3.82 (m, 2H, H-6a, H-
3.5 Hz, H-6b); 4.02–4.09 (m, 2H, H-4, H-20); 4.10 (t,
6b); 3.83 (dd, 1H, J3 ;2 10:3 Hz, J3 ;4 2:5 Hz, H-30);
3.93 (t, 1H, J4,3 ꢃ J4,5 7.3 Hz, H-4); 4.08 (dd, 1H,
1H, J2,3 ꢃ J3,4 8.6 Hz, H-3); 4.30 (q, 1H, J5 ;6 6:3 Hz,
0
0
0
0
0
0
H-50); 4.46 (s, 2H, –CH2Bn); 4.48 (d, 1H, J1 ;2 7:8 Hz,
00 00
J2 ;1 3:8 Hz, J2 ;3 10:3 Hz, H-20); 4.10 (t, 1H, J3,2 ꢃ J3,4
8.2 Hz, H-3); 4.26 (m, 1H, H-50); 4.28 (d, 1H, Jgem
12.3 Hz, –CH2–Bn); 4.43 (d, 1H, Jgem 12.0 Hz, –CH2–
Bn); 4.45 (d, 1H, Jgem 12.0 Hz, –CH2–Bn); 4.53 (d, 1H,
H-100); 4.51 (d, 1H, Jgem 12.1 Hz, –CH2Bn); 4.55 (d,
1H, Jgem 11.4 Hz, –CH2Bn); 4.60 (d, 1H, Jgem 9.8 Hz,
–CH2Bn); 4.63 (d, 2H, Jgem 11.7 Hz, –CH2Bn); 4.72 (d,
1H, Jgem 11.4 Hz, –CH2Bn); 4.81 (d, 1H, J1,2 9.0 Hz,
H-1); 4.89 (d, 1H, Jgem 11.4 Hz, –CH2Bn); 4.90 (d, 1H,
0
0
0
0
J1 ;2 8:2 Hz, H-100); 4.62 (d, 1H, Jgem 12.0 Hz, –CH2–
00 00
0
0
Bn); 4.63 (d, 1H, Jgem 11.5 Hz, –CH2–Bn); 4.65 (d, 1H,
Jgem 12.0 Hz, –CH2–Bn); 4.66 (d, 1H, Jgem 12.3 Hz,
–CH2–Bn); 4.71 (d, 1H, Jgem 11.7 Hz, –CH2–Bn); 4.84
Jgem 11.3 Hz, –CH2Bn); 5.13 (d, 1H, J1 ;2 3:5 Hz, H-
10); 6.16 (d, 1H, JNH,2 5.9 Hz, –NH–); 7.21–7.36 (m,
25H, H–Ar). 13C NMR (CDCl3, 100.6 MHz), DEPT,
13
(dd, J3 ;2 10:6 Hz, J3 ;4 3:7 Hz, H-300); 4.85 (d, 1H, Jgem
11.5 Hz, –CH2–Bn); 4.94 (d, 1H, Jgem = 12.0 Hz, –CH2–
1H, C-COSY: d 16.85 (C-60); 23.00 (CH3CO–); 56.59
00 00
00 00
(C-2); 67.26 (C-50); 68.16 (C-6); 68.25 (C-400); 68.69
(C-600); 72.03 (–CH2Bn); 72.13 (C-200); 72.89 (C-500);
73.30 (–CH2Bn); 73.45 (–CH2Bn, C-300); 74.13 (C-4);
74.47, 74.97 (–CH2Bn); 76.07 (C-3); 76.77 (C-5); 76.94
(C-20); 77.30 (C-40); 79.70 (C-30); 87.81 (C-1); 98.16 (C-
10); 100.98 (C-100); 127.12, 127.47, 127.56, 127.64,
127.70, 127.73, 127.94, 127.99, 128.17, 128.35, 128.41,
128.67 (C–Ar); 137.68, 137.71, 138.10, 138.28, 138.50
(Cipso); 170.91 (CH3CO–). Anal. Calcd for C55H64N4-
O14: C, 65.72; H, 6.42; N, 5.57. Found: C, 65.45; H,
6.60; N, 5.41.
Bn); 4.99 (dd, 1H, J2 ;1 8:2 Hz, J2 ;3 10:6 Hz, H-200);
00 00
00 00
5.02 (d, 1H, J1 ;2 3:8 Hz, H-10); 5.12 (d, 1H, J1,2
0
0
00 00
00 00
7.3 Hz, H-1); 5.39 (d, 1H, J4 ;3 3:2 Hz, J4 ;5 ꢄ 1 Hz,
H-400); 5.90 (d, 1H, J–NH–,2 5.9 Hz, –NH–COCH3);
7.14–7.38 (m, 25H, Ar–H). 13C NMR (CDCl3, 100.6
MHz), DEPT, 1H, C-COSY: d 16.61 (C-60); 20.42,
13
20.50, 20.63, 22.98 (4 · CH3CO–); 54.78 (C-2); 66.69
(C-600); 66.70 (C-500); 67.32 (C-400); 68.09 (C-6); 69.18
(C-200); 70.77 (C-300); 71.80 (C-5); 72.75 (–CH2–Bn);
73.33 (–CH2–Bn); 73.46 (C-3); 73.52 (–CH2–Bn); 73.53
(C-50); 73.67 (C-4); 74.46 (–CH2–Bn); 76.71 (C-20);
77.14 (C-40); 79.76 (C-30); 87.44 (C-1); 97.29 (C-10);
99.47 (C-100); 127.15, 127.51, 127.54, 127.58, 127.65,
127.71, 127.79, 127.95, 128.14, 128.23, 128.34, 128.40,
128.46, 128.49 (C–Ar); 170.23, 169.77, 169.68, 169.31
(4 · CH3CO–). Anal. Calcd for C61H70N4O17: C, 64.77;
H, 6.24; N, 4.95. Found: C, 64.41; H, 6.20; N, 4.76.
3.1.7. 2-Acetamido-3-O-(20,30,40-tri-O-benzyl-a-L-fuco-
pyranosyl)-6-O-benzyl-2-deoxy-4-O-(600-O-benzyl-300-O-
[methyl 5000-acetamido-4000,7000,8000,9000-tetra-O-acetyl-3000,5000-
dideoxy-D-galacto-2000-nonulopyranosylate]-b-D-galacto-
pyranosyl)-b-D-glucopyranosyl azide 11 (b-Ac4-Bn5sLex-
COOMe-N3). To a soln of LewisX azide 9 (1.023 g,
1.018 mmol) and ethyl thiosialoside23,24 10 (1.090 mg,
2.035 mmol) in dry acetonitrile (80 mL) under an argon
3.1.6. 2-Acetamido-3-O-(20,30,40-tri-O-benzyl-a-L-fuco-
pyranosyl)-6-O-benzyl-2-deoxy-4-O-(600-O-benzyl-b-D-
galactopyranosyl)-b-D-glucopyranosyl azide 9 (b-3-(a-
Bn3Fuc)-4-(b-600-BnGal)-6-BnGlcNAc-N3). To a soln
of LewisX derivative 8 (3.13 g,2.77 mmol) in dry MeOH
(100 mL), 3.3 mL of freshly prepared 0.1 M sodium
methoxide in MeOH was added. The soln was stirred
for 5 h at room temperature. After neutralization with
acidic ion-exchange resin (Amberlite IR-120) and filtra-
tion, the solvent was evaporated under diminished pres-
sure. The remaining colourless amorphous solid was
purified by flash chromatography with 40:1 CH2Cl2–
MeOH. Yield: 2.67 g (97%), colourless amorphous solid;
˚
atmosphere, molecular sieves (3 A, 4 g) were added.
After stirring for 40 min and subsequent cooling to
ꢁ40 ꢁC, a soln of N-iodosuccinimide (0.962 g, 4.276
mmol) in 20 mL of acetonitrile was added. Within
40 min, 1.30 mL of a 0.4 N soln of trifluoromethanesulf-
onic acid in acetonitrile was added dropwise, and the
soln was stirred for 15 min at ꢁ40 ꢁC. After keeping
the soln overnight at ꢁ28 ꢁC, the mixture was neutral-
ized with solid NaHCO3. The suspension was filtered
through Hyflo, and the filtrate was diluted with CH2Cl2
(500 mL) and washed twice with satd aq NaHCO3. The
brownish soln became wine red. After decolourizing by
washing with 80 mL of 20% aq sodium thiosulfate and
water, the organic layer was dried with MgSO4 and con-
centrated under diminished pressure to give 2.03 g of a
yellow amorphous solid. Column chromatography in
6:1 light petroleum–ethyl acetate and a second chroma-
tography in 60:1 CH2Cl2–MeOH gave the sialyl LewisX
11 in a pure form.
22
½aꢂD ꢁ63.1 (c 1, CH2Cl2); Rf = 0.33 (18:1 CH2Cl2–
1
1
1
MeOH). H NMR (CDCl3, 400 MHz), H, H-COSY:
d 1.12 (d, 3H, J6 ;5 6:3 Hz, H-60); 1.62 (s, 3H,
CH3CO–); 3.37 (q, 1H, J2,1 ꢃ J2,3 ꢃ J2,NH 9.0 Hz, H-
0
0
2); 3.41 (dd, 1H, J3 ;4 3:1 Hz, J3 ;2 9:4 Hz, H-300); 3.47
00 00
00 00
(t, 1H, J5 ;6 a ꢃ J5 ;6 b 5:9 Hz, H-500); 3.52 (dd, 1H,
00 00
00 00
J2 ;1 7:8 Hz, J2 ;3 9:0 Hz, H-200); 3.58–3.61 (m, 2H,
00 00
00 00
26
H-5, H-600a); 3.70 (d, 1H, J4 ;3 2:3 Hz, H-40); 3.71 (dd,
Yield: 882 mg (59%), colourless amorphous solid; ½aꢂD
0
0
1H, J6 b;6 a 9:8 Hz, J6 b;5 6:7 Hz, H-600b); 3.76 (dd, 1H,
J6a,6b 11.7 Hz, J6a,5 1.6 Hz, H-6a); 3.89 (d, 1H,
ꢁ45.3 (c 1, CHCl3); Rf = 0.32 (1:1 toluene–acetone);
1H NMR (CDCl3, 400 MHz), 1H, 1H-COSY: d 1.05
00
00
00
00

550
H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
(d, 3H, J6 ;5 6:5 Hz, H-60); 1.63 (s, 3H, –CO–CH3); 1.87
(s, 3H, –CO–CH3); 1.96 (s, 3H, –CO–CH3); 2.02 (s,
3H, –CO–CH3); 2.03 (m, 1H, H-3000ax); 2.07 (s, 3H,
–CO–CH3); 2.08 (s, 3H, –CO–CH3); 2.68 (dd, 1H,
–COOCH3, H-6a, H-6b, H-40); 3.88–3.98 (m, 5H, H-3,
0
0
H-5, H-50, H-5000, H-9000b); 4.01 (dd, 1H, J2 ;1 3:5 Hz,
0
0
J2 ;3 10:3 Hz, H-20); 4.25 (d, 1H, Jgem 12.0 Hz,
10:8 Hz, H-
0
0
000a;9000
b
000a;8000
–CH2Bn); 4.26 (t, 1H, J9
ꢃ J9
000eq;3000ax
J3
12:9 Hz, J3
4:5 Hz, H-3000eq); 3.48–3.73
000eq;4000
9000a); 4.32 (d, 1H, Jgem 12.0 Hz, –CH2Bn); 4.36 (d, 1H,
Jgem 11.7 Hz, –CH2Bn); 4.50 (d, 1H, Jgem 12.0 Hz,
–CH2Bn); 4.52–4.59 (m, 3H, 2 · –CH2Bn, H-300); 4.67
(d, 2H, Jgem 12.0 Hz, 2 · –CH2Bn); 4.68 (d, 1H,
(m, 7H, H-5, H-40, H-400, H-500, H-600a, H-600b, H-6000);
3.74 (s, 3H, –COOCH3); 3.76–4.19 (m, 10H, H-2, H-4,
H-3, H-6a, H-6b, H-20, H-30, H-50, H-300, H-5000, H-
9a000); 4.20–4.32 (m, 2H, –CH2Bn, H-9000b); 4.43 (s, 2H,
–CH2Bn); 4.52–4.77 (m, 7H, H-1, H-100, 5 · –CH2Bn);
4.84–4.92 (m, 3H, 2 · –CH2Bn, H-4000); 5.18 (d, 1H,
J1 ;2 7:0 H, H-100); 4.72 (d, 1H, Jgem 11.7 Hz, –CH2Bn);
00 00
4.81–4.88 (m, 3H, H-200, H-4000, –CH2Bn); 4.91 (d, 1H,
J1,2 5.6 Hz, H-1); 4.99 (d, 1H, J4 ;3 2:6 Hz, H-400); 5.02
00 00
J1 ;2 3:3 Hz, H-10); 5.24–5.33 (m, 2H, H-200, H-7000);
5.43 (mc, 1H, H-8000); 6.11 (d, 1H, Jvic 7.2 Hz, –NH–
C2); 7.21–7.36 (m, 25H, H–Ar). 13C NMR (CDCl3,
100.6 MHz): d 16.81 (C-60); 20.61, 20.69, 20.77, 21.09,
23.08, 23.12 (–CO–CH3); 37.49 (C-3000); 49.61 (C-5000);
53.05 (–COOCH3); 55.98 (C-2); 62.31 (C-9000); 67.07,
67.23, 67.67, 68.24 (C-5, C-400, C-7000, C-8000); 68.29,
68.50 (C-6, C-600); 68.63, 70.01 (C-200, C-4000); 72.14
(–CH2Bn); 72.53, 72.70, 73.48 (C-3, C-300, C-500, C-6000);
73.01, 73.28, 74.38, 75.03 (–CH2–Bn); 76.13, 77.02,
77.29, 77.72 (C-4, C-20, C-40, C-50); 79.93 (C-30); 88.23
(C-1); 97.95 (C-2000); 98.17 (C-10); 101.14 (C-100); 127.15,
127.34, 127.42, 127.48, 127.52, 127.65, 128.04, 128.07,
128.16, 128.23, 128.37, 128.64 (C–Ar); 138.05, 138.29,
138.42, 138.54, 138.75 (C-ipso); 168.16, 169.78, 169.91,
170.28, 170.42, 170.47, 170.76 (–CO–CH3, –COOMe).
ESI-MS calcd for C75H91N5O26: 1477.8. Found: 1501.1
(100%), [M+Na]+; 1517.1 (5%), [M+K]+.
(d, 1H, JNH;5 10:3 Hz, –NH–C5000); 5.06 (d, 1H,
0
0
000
0
0
0
000;6000
J1 ;2 3:2 Hz, H-1 ); 5.30 (dd, 1H, J7
2:1 Hz,
11:8 Hz,
9:2 Hz, H-7000); 5.53 (ddd, 1H, J8
000;8000
000;9000
a
J7
J8
6:1 Hz, H-8000); 6.14 (d, 1H, JNH,2
000;7000
000;9000
b
3:2 Hz, J8
7.9 Hz, –NH–C2); 7.14–7.32 (m, 25H, H–Ar). 13C
NMR (CDCl3, 100.6 MHz), DEPT, H, 13C-COSY: d
1
16.64 (C-60); 20.60, 20.74, 2 · 20.78; 20.92; 21.39;
23.03; 23.12 (CH3CO–); 37.54 (C-3000); 49.03 (C-5000);
52.13 (C-2); 53.19 (–O–CH3); 62.47 (C-9000); 66.96
(C-5)00; 67.22 (C-7000); 67.46 (C-600); 67.64 (C-8000); 67.87
(C-4 ); 68.84 (C-6); 69.37 (C-4000); 70.27 (C-200); 71.22
(C-300); 71.81 (C-500); 71.97 (C-6000); 72.78 (C-3); 72.86
(–CH2Bn); 72.92 (–CH2Bn); 73.09 (C-50); 2 · 73.27
(2 · –CH2Bn); 74.53 (–CH2Bn); 75.60 (C-40); 76.50
(C-20); 77.31 (C-4); 79.35 (C-30); 87.63 (C-1); 96.59 (C-
10); 96.88 (C-2000); 99.05 (C-100); 127.21, 127.24, 127.43,
127.53, 127.59, 127.64, 127.70, 127.79, 128.20, 128.23,
128.32, 128.38, 128.43 (C–Ar); 137.69, 138.28, 138.57,
138.62, 138.75 (Cipso); 167.83, 169.68, 169.89, 170.30,
170.33, 170.42, 2 · 170.51, 170.82 (8 · –COCH3, C-1000).
MALDI-MS (dhb) calcd for C79H95N5O28: 1562.6.
Found 1585.7 [M+Na]+; 1601.5 [M+K]+. Anal. Calcd
for C79H95N5O28ÆH2O: C, 60.00; H, 6.19; N, 4.43.
Found: C, 59.84, H, 6.32; N, 4.43.
3.1.8. 2-Acetamido-3-O-(20,30,40-tri-O-benzyl-a-L-fuco-
pyranosyl)-6-O-benzyl-2-deoxy-4-O-(200,400-di-O-acetyl-
600-O-benzyl-300-O-[methyl 5000-acetamido-4000,7000,8000,9000-tetra-
O-acetyl-3000,5000-dideoxy-D-galacto-2000-nonulopyranosylate]-
b-D-galactopyranosyl)-b-D-glucopyranosyl azide 12 (b-
Ac6-Bn5sLexCOOMe-N3). The sLeX derivative 11
(773.5 mg, 0.523 mmol) was dissolved in pyridine
(15 mL) and Ac2O (15 mL). After addition of 50 mg of
4-dimethylaminopyridine (DMAP), the soln was stirred
for 3 h at room temperature. After concentration in high
vacuum the residue was co-distilled three times with
30 mL of toluene and purified by flash chromatography
in 6:1 light petroleum–ethyl acetate. Yield: 725 mg
3.1.9. 2-Acetamido-3-O-(20,30,40-tri-O-benzyl-a-L-fuco-
pyranosyl)-6-O-benzyl-2-deoxy-4-O-(600-O-benzyl-300-O-
[methyl 5000-acetamido-4000,7000,8000,9000-tetra-O-acetyl-3000,5000-
dideoxy-D-galacto-2000-nonulopyranosylate]-b-D-galacto-
pyranosyl)-b-D-glucopyranosylamine 13 (b-Ac6-Bn5sLex-
COOMe-NH2). To a soln of sLex azide 12 (444.0 mg,
0.284 mmol) in isopropanol (12 mL) and 2 mL of water
a catalytic amount of neutrally washed Raney nickel was
added. After exchange of the air for argon and then for
hydrogen, hydrogenation was carried out under vigorous
stirring for 1–2 h at atmospheric pressure. After filtration
through Hyflo and washing of the catalyst with isopropa-
nol, evaporation of the solvents under diminished pressure
at 35 ꢁC and drying in high vacuum quantitatively gave
unstable sLex amine 13 (Rf = 0.74, 8:1 CH2Cl2–MeOH),
which was immediately subjected to further conversion.
22
(89%), colourless amorphous solid; ½aꢂD ꢁ59.9 (c 1,
1
CHCl3); Rf = 0.35 (12:1 CH2Cl2 – MeOH). H NMR
(CDCl3, 600 MHz), 1H, 1H-COSY: d 1.00 (d, 1H,
J6 ;5 6:2 Hz, H-60); 1.65 (t, 1H, J3
ꢃ J3
12:3
0
0
000a;3000
e
000a;4000
Hz, H-3000a); 1.79 (s, 3H, CH3CO–); 1.80 (s, 3H,
CH3CO–); 1.88 (s, 3H, CH3CO–); 1.89 (s, 3H, CH3-
CO–); 1.94 (s, 3H, CH3CO–); 2.01 (s, 3H, CH3CO–);
2.04 (s, 3H, CH3CO–); 2.14 (s, 3H, CH3CO–); 2.50
000
(dd, 1H, J3
000 12:4 Hz, J3
4:3 Hz, H-3000e); 3.28
000e;4000
(t, 1H, J6 a;6e;b3aꢃ J6 a;5 8:5 Hz, H-600a); 3.39 (dd, 1H,
00
00
00
J6 b;6 a 9:4 Hz, J6 b;5 5:6 Hz, H-600b); 3.42 (s, breit, H-
3.1.10. tert-Butyl 15-hydroxy-4,7,10,13-tetraoxapentade-
canoate 14 (Glycol4-(CH2)2COOt-Bu). To dry tetra-
ethylene glycol (172.6 mL, 1.00 mol) in 400 mL of dry
00
00
00
00
000;7000
4); 3.56 (dd, 1H, J6
2:1 Hz, J6
10:7 Hz, H-6000);
000;5000
3.66–3.73 (m, 3H, H-2, H-30, H-500); 3.78–3.81 (m, 6H,

H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
551
tetrahydrofuran sodium (0.2 g, 8.7 mmol) was added.
After 2 h, the sodium had been dissolved and tert-butyl
acrylate (43.5 mL, 0.3 mol) was added. The soln was
stirred under exclusion of moisture for 24 h. After
neutralization with 8 mL of 1 M HCl, the solvent was
evaporated under diminished pressure. The residue
was dissolved in 400 mL of brine and extracted three
times with 250 mL of ethyl acetate. The combined
organic layers were washed with water (100 mL) and
dried with MgSO4. The solvent was evaporated under
diminished pressure, and the remaining 14 was dried
in high vacuum. Yield: 43.15 g (78%), clear, pale yellow
liquid, n2D0 ¼ 1:448; Rf = 0.52 (4:1 EtOAc–EtOH). 1H
NMR (CDCl3, 200 MHz): d 1.41 (s, 9H, –C(CH3)3);
2.47 (t, 2H, Jvic 6.6 Hz, –CH2–COOt-Bu); 2.76 (s, 1H,
–OH); 3.53–3.75 (m, 18H, –CH2–O–). 13C NMR
(CDCl3, 50.3 MHz), BB, d [ppm]: 28.03 (–C(CH3)3);
36.20 (–CH2–COOt-Bu); 61.59 (–CH2OH); 66.81
(–CH2CH2COOt-Bu); 70.49, 70.43, 70.29 (–CH2–O–);
72.49 (HO-CH2–CH2–O–); 80.39 (–C(CH3)3); 170.83
(–COOt-Bu). FDMS calcd for C15H30O7: 322.4. Found
345.7 (23%) [M+Na]+, 646.1 (9%) [2M+H]+ 668.1
(9%) [2M+Na]+.
5.08 Hz, –CH2N3); 3.56–3.64 (m, 14H, –O–CH2–); 3.67
(t, 2H, Jvic 6.7 Hz, –CH2–CH2–COOt-Bu). 13C NMR
(CDCl3, 50.3 MHz), d 28.03 (–C(CH3)3); 36.23 (–CH2–
COOt-Bu); 50.62 (–CH2N3); 66.84 (–CH2CH2COOt-
Bu); 69.97, 70.32, 70.45, 70.62 (–CH2–O–); 80.36
(–C(CH3)3); 170.79 (–COOt-Bu). FDMS calcd for
C15H29N3O6: 347.4, gef.: 322.8 (100%) [MꢁN2+2H]+,
348.8 (36%) [M+H]+, 668.2 (64%) [2MꢁN2+H]+. Anal.
Calcd for C15H29N3O6Æ1/2H2O: C, 50.55; H, 8.48, N,
11.79. Found: C, 50.44, H; 8.37; N, 11.93.
3.1.13. tert-Butyl 15-amino-4,7,10,13-tetraoxapentade-
canoate 17 (H2N-Glycol4-(CH2)2COOt-Bu). To a soln
of azide 16 (10.0 g, 28.78 mmol) in 120 mL of isopropa-
nol freshly prepared Raney nickel (7 g, pH 9) was given.
Air was exchanged for argon and then for hydrogen.
After hydrogenation for 12 h, the catalyst was filtered
off through Hyflo and washed with 50 mL of isopropa-
nol. The solvent was evaporated and the remaining prod-
uct 17 filtered through a syringe filter in order to remove
traces of Raney nickel. Yield: 8.57 g (93%), colourless
liquid; Rf = 0.05 (ethyl acetate); 1H NMR (CDCl3,
200 MHz): 1.40 (s, 9H, –C(CH3)3); 1.56 (s, br, 2H,
–NH2); 2.46 (t, 2H, Jvic 6.6 Hz, –CH2–COOt-Bu); 2.81
(t, 2H, Jvic 5.4 Hz, H2N–CH2–); 3.46 (t, Jvic 5.4 Hz, –
CH2–CH2–NH2); 3.54–3.62 (m, 12H, –O–CH2–); 3.66
(t, 2H, Jvic 6.6 Hz, –CH2–CH2–COOt-Bu). 13C NMR
(Me2SO-d6, 50.3 MHz): d 28.10 (–C(CH3)3); 36.27
(–CH2–COOt-Bu); 41.84 (–CH2–NH2); 66.89 (–CH2–
CH2–COOt-Bu); 70.31, 70.37, 70.51, 70.58, 70.61
(–CH2–O–); 73.52 (–CH2–CH2–NH2); 80.49 (–C(CH3)3);
170.90 (–COOt-Bu). FDMS: calcd for C15H31N1O6:
321.4. Found: 322.1 (2%) [M+H]+, 643.6 (100%)
[2M+H]+. Anal. Calcd for C15H31N1O6 (321.41): C,
56.05; H, 9.72; N, 4.36. Found: C, 56.10; H, 9.62; N, 4.37.
3.1.11. tert-Butyl 15-methylsulfonyloxy-4,7,10,13-tetra-
oxapentadecanoate 15 (MesO-Glycol4-(CH2)2COOt-
Bu). To a soln of 14 (40.0 g, 124 mmol) in 300 mL of
dry CH2Cl2 and 42.4 mL (300 mmol) of Et3N at 0 ꢁC,
20.3 mL (260 mmol) of methanesulfonyl chloride were
added dropwise within 15 min. After stirring for 15 h
at 0 ꢁC, filtration through Hyflo and washing with water
and brine, the CH2Cl2 soln was dried with MgSO4 and
evaporated to dryness under diminished pressure. Yield:
49.69 g (124 mmol, quant.); dark brown oil; Rf = 0.66
(ethyl acetate). The product was used for further conver-
sion without purification.
3.1.14. a-Allyl b-tert-butyl N-fluorenylmethoxycarbonyl-
L-aspartate 18 (Fmoc-Asp(Ot-Bu)-OAll)
3.1.12. tert-Butyl 15-azido-4,7,10,13-tetraoxapentadecan-
oate 16 (N3-Glycol4-(CH2)2COOt-Bu). Sodium azide
(16.3 g, 250 mmol) was added to soln of mesylate 15
(49.69 g, 124 mmol) in DMF (300 mL). The mixture
was stirred under exclusion of moisture for 2 d at room
temperature. After centrifugation and extraction of the
precipitate with DMF (twice 50 mL), the combined
organic layers were evaporated in high vacuum. The
residue was dissolved in diethyl ether (400 mL) and
washed with 200 mL of satd aq NaHCO3. The aq soln
was re-extracted three times with 80 mL of ether. The
combined ether solns were dried with MgSO4 and evap-
orated under diminished pressure to give 40.02 g of a
brown oil, which was purified by flash chromatography
in light petroleum/ethyl acetate. Yield: 28.47 g (66%),
pale yellow oil; n2D3 ¼ 1:449; Rf = 0.39 (1:2 light petro-
3.1.14.1. Method (a). Fmoc-Asp(Ot-Bu)-OH (3.0 g,
7.51 mmol) and diisopropylethylamine (2.57 mL,
15.02 mmol) in allylbromide (15 mL) were heated under
reflux for 30 min. The resulting clear slightly brownish
soln, after cooling, was poured into ethyl acetate
(200 mL). Precipitating diisopropylethylammonium
hydrobromide was filtered off, and the filtrate was rap-
idly washed with 0.1 N HCl, NaHCO3/Na2CO3 buffer
(pH 9.5) and brine. After drying over MgSO4, the sol-
vent was evaporated under diminished pressure and
resulting brown oil was lyophilized from benzene. Yield:
3.39 g (quant.), beige-coloured amorphous solid.
3.1.14.2. Method (b). To Fmoc-Asp(Ot-Bu)-OH
(2.50 g, 6.08 mmol) in 120 mL of dry MeOH, dry
Cs2CO3 (0.99 g, 3.04 mmol) was added. The soln was
stirred for 30 min at room temperature, the MeOH was
evaporated under diminished pressure, and the salt dried
1
leum–ethyl acetate). FT-IR: 2104 cmꢁ1 (N3); H NMR
(CDCl3, 400 MHz): d 1.41 (s, 9H, –C(CH3)3); 2.47
(t, 2H, Jvic 6.7 Hz, –CH2–COOt-Bu); 3.36 (t, 2H, Jvic

552
H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
in high vacuum. The thus obtained caesium salt was dis-
solved in dry dimethylformamide (90 mL). At 0 ꢁC a soln
of allylbromide (1.10 mL, 13.00 mmol) in dry dimethyl-
formamide (10 mL) was added within 30 min. After stir-
ring for 2 h at room temperature, the precipitating CsBr
was filtered off and DMF evaporated in high vacuum.
The residue was dissolved in 300 mL of CH2Cl2 and
washed twice with 50 mL of water. Drying with MgSO4
afforded a yellow viscous oil, which was lyophilized from
benzene. Yield: 2.75 g (quant.), colourless amorphous
solid; Rf = 0.78 (2:1 light petroleum–ethyl acetate).
added and stirring was continued for additional 14 h.
After dilution with CH2Cl2 (100 mL) and washing with
brine (50 mL) and water (50 mL), the organic soln was
dried with MgSO4 and the solvent was evaporated under
diminished pressure. The oily yellow crude product was
purified by column chromatography in 60:1 CH2Cl2–
MeOH. Yield: 2.85 g (85%), colourless oil, which solid-
22
ifies to a waxy mass within some days. ½aꢂD 15.3
(c 1, CHCl3); Rf = 0.44 (100:1 ethyl acetate–acetic acid).
1H NMR (CDCl3, 400 MHz):
d
1.41 (s, 9H,
–C(CH3)3); 2.47 (t, 2H, Jvic 6.6 Hz, –CH2–COOt-Bu);
2.72 (dd, 1H, Jgem 15.9 Hz, Jvic 4.3 Hz, (Asn-b-H)a);
2.96 (dd, 1H, Jgem 15.9 Hz, Jvic 4.7 Hz, (Asn-b-H)b);
3.38–3.45 (m, 2H, –O–CH2–CH2–NH–); 3.47–3.64 (m,
12H, –CH2–O–); 3.67 (t, 2H, Jvic 6.5 Hz, –CH2–CH2–
COOt-Bu); 4.21 (t, 1H, Jgem 7.2 Hz, (H-9)Fmoc); 4.27
(dd, 1H, Jgem 10.3 Hz, Jvic 7.6 Hz, –CH2–Fmoc); 4.41
(dd, 1H, Jgem 10.3 Hz, Jvic 7.0 Hz, –CH2–Fmoc); 4.59–
4.65 (m, 3H, Asp-a-H, –O–CH2–CH@CH2); 5.20 (d, Jcis
10.6 Hz, –O–CH2–CH@CH2cis); 5.31 (d, Jtrans 17.3 Hz,
–O–CH2–CH@CH2trans); 5.92–5.84 (mc, 1H, –O–CH2–
CH@CH2); 6.22 (d, 1H, JNH,Ha 8.8 Hz, Asn-a-NH);
6.65 (t, Jvic 1.6 Hz, –NH–CH2–); 7.24–7.39 (m, 2H, H–
ArFmoc); 7.55–7.61 (m, 2H, H–ArFmoc); 7.74 (d, 2H, Jvic
7.3 Hz, (H-4)Fmoc, (H-5)Fmoc). 13C NMR (CDCl3,
3.1.15. a-Allyl N-fluorenylmethoxycarbonyl-L-aspartate
19 (Fmoc-Asp-OAll). A soln of Fmoc-Asp(t-Bu)-OAll
18 (2.74 g, 6.07 mmol) in a mixture of TFA (25 mL),
CH2Cl2 (25 mL) and water (1.8 mL) was stirred for
40 min at room temperature. CH2Cl2 and TFA were
evaporated under diminished pressure, and the residue
was co-distilled three times with toluene (30 mL). Crude
19 was purified by flash chromatography in 20:10:1 light
petroleum–ethyl acetate–acetic acid. Yield: 2.29 g (95%),
colourless crystals; mp: 93 ꢁC, Rf = 0.66 (100:10:1
23
CH2Cl2–MeOH–acetic acid), ½aꢂD 22.6 (c 1, CHCl3).
1H NMR (CDCl3, 400 MHz): 2.94 (dd, 1H, Jgem
17.6 Hz, Jb,a 4.4 Hz, (Asp-b-H)a); 3.11 (dd, 1H, Jgem
17.6 Hz, Jb,a 4.7 Hz, (Asp-b-H)b); 4.21 (t, 1H, Jvic
7.0 Hz, (H-9)Fmoc); 4.35 (dd, 1H, Jvic 7.3 Hz, Jgem
10.6 Hz, –CH2–Fmoc); 4.42 (dd, 1H, Jvic 7.3 Hz, Jgem
10.6 Hz, –CH2–Fmoc); 4.65–4.70 (m, 3H, Asp-a-H,
–O–CH2–CH@CH2); 5.24 (d, 1H, Jvic 10.3 Hz, –CH2–
CH@CH2cis); 5.31 (d, 1H, Jvic 17.0 Hz, –O–CH2–
CH@CH2trans); 5.84 (d, 1H, JNH,a-Asp 8.8 Hz, Asp–
NH); 5.84–5.92 (m, 1H, –O–CH2–CH@CH2); 7.29 (t,
2H, Jgem 7.3 Hz, (H-2)Fmoc, (H-7)Fmoc); 7.38 (t, 2H, Jgem
7.3 Hz, (H-3)Fmoc, (H-6)Fmoc); 7.58 (d, 2H, Jgem 7.3 Hz,
(H-1)Fmoc, (H-8)Fmoc); 7.74 (d, 2H, Jgem 7.3 Hz, (H-
4)Fmoc, (H-5)Fmoc). 13C NMR (CDCl3, 50.3 MHz): d
36.42 (C-b Asp); 47.10 (C-9 Fmoc); 50.28 (C-a Asp);
66.62 (CH2@CH–CH2–O–); 67.43 (–CH2–Fmoc);
119.10 (CH2@CH–CH2–O–); 120.04, 125.13, 127.14,
127.79 (C–Ar); 131.31 (CH2@CH–CH2–O–); 141.33,
143.67, 143.79 (Cipso); 156.11 (C-c Asp); 170.3
(–COOAll); 175.93 (–COOH). Anal. Calcd for
C22H21NO6: C, 66.83; H, 5.35; N, 3.54. Found: C,
66.79; H, 5.32; N, 3.48.
50.3 MHz), GASPE:
d
28.09 (–C(CH3)3); 36.18
(–CH2–COOt-Bu); 37.45 (C-b Asn); 39.34 (–CH2–
NH–); 47.12 (C-9 Fmoc); 51.00 (C-a Asn); 66.17
(–CH2–CH2–NH–); 66.88 (–CH2–CH2–COOt-Bu);
67.15 (–CH2–Fmoc); 69.70 (CH2@CH–CH2–O–); 70.19,
70.32, 70.37 (–CH2–O–); 80.62 (–C(CH3)3); 118.44
(CH2@CH–CH2–O–); 119.96, 125.21, 125.29, 127.11,
127.70 (C–Ar Fmoc); 131.77 (CH2@CH–CH2–O–);
141.28, 143.83, 143.96 (C–Ar ipso); 156.27 (Fmoc–CO–
NH–); 169.90, 170.89, 171.06 (–COOt-Bu, C-c Asn,
C-1 Asn). Anal. Calcd for C37H30N2O11: C, 63.59; H,
7.21; N, 4.01. Found: C, 63.65; H, 7.28; N, 4.14.
3.1.17. N2-Fluorenylmethoxycarbonyl-N4-{14-tert-butyl-
oxycarbonyl-3,6,9,12-tetraoxatetradecyl}-L-asparagine
21 (Fmoc-Asn(glycol4-(CH2)2COOt-Bu)-OH). To
a
soln of asparagine-spacer-conjugate 20 (2.04 g, 2.92
mmol) in dry tetrahydrofuran (60 mL) under an argon
atmosphere, 476 lL of N-methylaniline and 40 mg of
(Ph3P)4Pd0 were added. After stirring for 5 h at room
temperature under exclusion of light, THF was distilled
off. The residue was dissolved in 150 mL of CH2Cl2 and
the soln extracted with 50 mL of each 0.25 N HCl and
water. After drying with MgSO4, the solvent was evap-
orated under diminished pressure and the residue
purified by column chromatography (200:5:1 CH2Cl2–
MeOH–AcOH). Yield: 1.353 g (70%), yellow oil, which
3.1.16. Allyl N2-fluorenylmethoxycarbonyl-N4-{14-tert-
butyloxycarbonyl-3,6,9,12-tetraoxatetradecyl}-L-aspar-
aginate
20
(Fmoc-Asn(glycol4-(CH2)2COOt-Bu)-
OAll). To a soln of Fmoc-Asp-OAll 19 (2.28 g,
5.77 mmol) in 50 mL of dry CH2Cl2, isobutyl (2-iso-
butoxy)-1,2-dihydroquinoline-1-carboxylate27 (IIDQ,
2.63 g, 8.66 mmol) was added, and the mixture was stir-
red for 15 min at room temperature. Subsequently,
amino-spacer component 17 (1.55 g, 4.81 mmol) was
24
solidifies to a waxy mass within some days. ½aꢂD 40.5
(c 1, CHCl3); Rf = 0.35 (10:1 CH2Cl2–MeOH).
1H NMR (CDCl3, 400 MHz):
d
1.41 (s, 9H,
–C(CH3)3); 2.47 (t, 2H, Jvic 6.6 Hz, –CH2COOt-Bu);

H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
553
2.71 (dd, 1H, Jgem 15.3 Hz, Jvic 7.6 Hz, Asn-Hba); 2.92
(dd, 1H, Jgem 15.3 Hz, Jvic 3.2 Hz, Asn-Hbb); 3.30–3.34
(m, 1H, –O–CH2–CH2–NH–); 3.52–3.64 (m, 17H, –O–
CH2–); 3.65 (t, 2H, Jvic 6.5 Hz, –CH2–CH2–COOt-Bu);
4.20 (t, 1H, Jvic 7.2 Hz, H-9 Fmoc); 4.30 (dd, 1H,
Jgem 10.6 Hz, Jvic 7.3 Hz, –CH2–Fmoc); 4.37 (dd, 1H,
Jgem 10.3 Hz, Jvic 7.3 Hz, –CH2–Fmoc); 4.55 (td,
1H, JAsn-Ha,NH ꢃ JAsn-Ha,Asn-Hba 7.0 Hz, JAsn-Ha,Asn-Hbb
3.2 Hz, Asn-Ha); 6.20 (d, 1H, JNH,Asn-Ha 7.0 Hz,
Asn-NHa); 7.06 (m, 1H, Asn-NHb); 7.27–7.31 (m, 2H,
H–ArFmoc); 7.35–7.39 (m, 2H, H–ArFmoc); 7.57–7.61
(m, 2H, H–ArFmoc); 7.73–7.74 (d, 2H, Jvic 7.3,
(H-4)Fmoc, (H-5)Fmoc).
ꢁ34.2 (c 1, CHCl3); Anal. HPLC: tR = 31.5 min (Chro-
masil C-18, Knauer, gradient: 50% H2O/50%
H3CCN ! 100% CH3CN in 40 min). 1H NMR, 1H,
1H-COSY (CDCl3, 400 MHz):
d
0.94 (d, 1H,
J3 12:3 Hz,
J6 ;5 6:2 Hz, H-60); 1.70 (t, 1H, J3
0
0
000a;3000
e
000a;4000
H-3000a); 1.82, 2 · 1.90, 1.96, 1.98, 2.04, 2.05,
2.20 (8 · s, 3H, –CH3 Acetyl); 2.17 (m, 2H, –CH2–
000e;4000
000e;3000
a
12:6 Hz,
CO–); 2.55 (dd, 1H, J3
4:4 Hz, J3
H-3000e); 2.69 (dd, 1H, Jgem 15.8 Hz, Jvic 4.1 Hz, H-
ba-Asn); 2.93 (dd, 1H, Jgem 15.8 Hz, Jvic 4.4 Hz, H-
bb-Asn); 3.31 (m, H-600a); 3.37 (m, –NH–CH2–); 3.40
(m, –CH2–CH2–NH–); 3.42 (m, H-600b); 3.43–3.57 (m,
16H, –O–CH2-Glycol); 3.48 (m, H-40); 3.58 (m, H-6a);
3.60 (m, H-6000); 3.63 (m, H-6b); 3.68 (m, H-30); 3.71
(m, H-500); 3.80 (m, H-5); 3.81 (s, 3H, –OCH3); 3.88
(m, H-50); 3.92 (m, H-3); 3.94 (m, H-9000a); 4.01 (m, H-
4, H-5000); 4.02 (m, H-20); 4.13 (mc, H-2); 4.20 (t, 1H, Jgem
6.9 Hz, H-9 Fmoc); 4.24 (m, –CH2a–Fmoc); 4.28 (m,
–CH2Bn); 4.32 (m, H-9000b); 4.38 (m, –CH2Bn); 4.40
(m, –CH2b–Fmoc); 4.50–4.78 (m, 7 · –CH2–Bn); 4.60
(m, H-300, H-a Asn); 4.62 (m, –O–CH2–CH@CH2);
4.70 (m, 1H, H-100); 4.83–4.98 (m, H-200, H-4000, –CH2–
Bn); 5.02–5.14 (m, 5H, H-1, H-10, H-400, C5000-NH);
5.19 (d, 1H, Jvic 10.6 Hz, –CH@CH2cis); 5.29 (d, 1H, Jvic
13C NMR (CDCl3, 50.3 MHz): d 28.09 (–C(CH3)3);
36.15 (–CH2–COOt-Bu); 37.88 (C-b Asn); 39.78
(–CH2–NH–); 47.09 (C-9 Fmoc); 50.76 (C-a Asn);
66.84 (–CH2–CH2–COOt-Bu); 67.26 (–CH2–Fmoc);
69.22, 70.05, 70.27, 70.34, 70.43, 70.67 (–CH2–O–);
80.74 (–C(CH3)3); 119.97, 125.27, 127.14, 127.73
(C–ArFmoc); 141.26, 143.93 (C–Ar ipso); 155.96
(Fmoc–CO–NH–); 170.98, 171.94 (–COOt-Bu, C-c
Asn). Anal. Calcd for C34H46N2O11: C, 61.99; H, 7.04;
N, 4.25. Found: C, 61.76; H, 7.17; N, 4.27.
000;6000
17.0 Hz, –CH@CH2trans); 5.35 (dd, 1H, J7
J7
2:6 Hz,
3.1.18. Allyl [N4-{14-N-(2-acetamido-3-O-(20,30,40-tri-O-
benzyl-a-L-fucopyranosyl)-2-deoxy-4-O-(200,400-di-O-ace-
tyl-600-O-benzyl-300-O-[methyl 5000-acetamido-4000,7000,8000,9000-
tetra-O-acetyl-3000,5000-dideoxy-D-galacto-2000-nonulopyranosyl-
ate]-b-D-galactopyranosyl)-b-D-glucopyranosyl)-carbam-
oyl-3,6,9,12-tetraoxatetradecyl}-L-asparaginate] 23 (Fmoc-
Asn(glycol4-(CH2)2CO-b-Ac6Bn5sLex-COOMe)-OAll).
Fmoc-Asn(glycol4-(CH2)2COOt-Bu)-OAll 20 (56 mg,
80.5 lmol) was reacted with 10 mL of 1:1 CH2Cl2–
TFA for 2 h at room temperature. The solvents were
evaporated under diminished pressure and the residue
co-distilled three times with toluene (10 mL). The quan-
titatively obtained Fmoc-Asn(Glycol4-(CH2)2COOH)-
OAll 22 (54.0 mg, 80 lmol) was dissolved in dimeth-
ylformamide (8 mL). PyBOP29 (83.2 mg, 160 lmol),
diisopropylethylamine (68.5 lL, 393 lmol), HOBt33
(12.3 mg, 80.3 lmol) and sLex amine 13 (156.3 mg,
100 lmol) were added, and the soln was stirred at room
temperature for 15 h. DMF was evaporated in high vac-
uum and the residue co-distilled three times with 5 mL
of toluene, then dissolved in 100 mL of CH2Cl2 and
washed with 15 mL of each satd aq NaHCO3 and water.
After evaporation of CH2Cl2 under diminished pressure
the remaining residue was dissolved in 0.4 mL of aceto-
nitrile. Insoluble material was removed by filtration
through a syringe and the remaining clear yellow soln
was purified by preparative RP-HPLC (column: Euro-
spher C-18, Knauer, Berlin, Germany; gradient: 70%
H2O/30% H3CCN ! 100% CH3CN in 140 min;
tR = 63.4 min). Yield 125.0 mg (72%), colourless amor-
8:8 Hz, H-7000); 5.52 (mc, 1H, H-8000); 5.87 (mc,
000;8000
1H, –CH@CH2); 6.23 (d, 1H, a-NH Asn); 6.57 (s, 1H,
–NH–CH2–); 6.80 (d, 1H, J2,NH 8.5 Hz, C2–NH);
7.11–7.42 (m, 29H, H–Ar); 7.52–7.64 (m, 3H, C1–NH,
H-1 Fmoc, H-8 Fmoc); 7.73 (d, 2H, Jvic 7.6 Hz, H-4
Fmoc, H-5 Fmoc). 13C NMR (CDCl3, 100.6 MHz-),
DEPT: d 16.49 (C-60); 20.52, 20.71, 20.95, 21.24, 22.88,
23.08 (–CH3Ac); 36.74 (–CH2–CH2–CO–); 37.52 (C-b
Asn); 37.63 (C-3000); 39.36 (–CH2–NH–); 47.17 (C-9
Fmoc); 49.19 (C-5000); 49.60 (C-2); 51.05 (C-a Asn);
53.08 (–OCH3); 62.35 (C-9000); 66.11 (–CH2–CH@CH2);
66.80 (C-6); 67.13 (–CH2–Fmoc); 67.30 (C-7000); 67.39
(C-5); 67.59 (C-600); 67.94 (C-400); 68.07 (C-8000); 69.41,
69.61, 69.69, 70.21, 70.26, 70.34, 70.39, 70.74 (C-200, C-
300, C-4000, –O–CH2-Glycol); 71.26, 71.88, 72.16, 72.40,
72.75, 72.86, 73.25 (C-4, C-50, C-500, C-6000, 3 · –CH2Bn);
74.73, 74.61, 73.99 (C-3, 2 · –CH2–Bn); 76.48 (C-20);
77.28 (C-40); 78.01 (C-1); 79.48 (C-30); 96.97 (C-2000);
97.81 (C-10); 99.05 (C-100); 118.36 (–O–CH2–CH@CH2);
119.89 (C-4 Fmoc, C-5 Fmoc); 125.15, 125.22 (C-1
Fmoc, C-8 Fmoc); 127.01, 127.06, 127.17, 127.37,
127.59, 127.63, 128.20, 128.24, 128.46, 128.65 (C–Ar);
131.77 (–O–CH2–CH@CH2); 137.44, 137.82, 138.41,
138.50, 138.57 (C-ipso); 141.27 (C-4a Fmoc, C-4b
Fmoc); 143.97, 143.85 (C-8a Fmoc, C-9a Fmoc); 156.20
(–NH–CO–Fmoc); 167.87, 169.65, 169.80, 169.94,
170.17, 2 · 170.33, 170.52, 170.75, 170.85, 170,94,
171.18 (–C=O). MALDIMS: calcd for C112H137N5O38:
2161.3. Found: 2185.0 [M+Na]+, 2200.8 [M+K]+. Anal.
Calcd for C112H137N5O38Æ3H2O: C, 60.72; H, 6.56; N,
3.18. Found: C, 60.72; H, 6.71; N, 3.09.
22
phous solid; Rf = 0.46 (6:1 CH2Cl2–MeOH); ½aꢂD

554
H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
In addition, 43.0 mg (25%) of the a-configured ano-
3.78 mmol), TBTU (607 mg, 1.9 mmol) in DMF
(10 mL), 17 h; (d) Fmoc-Gly-OH (944 mg, 3.18 mmol),
HOBt (488 mg, 3.18 mmol), N-methylmorpholine
(0.7 mL, 6.35 mmol), TBTU (1.02 g, 3.18 mmol) in
DMF (10 mL), 18 h; (e) Fmoc-Pro-OH (1.072 g,
3.18 mmol), HOBt (488 mg, 3.18 mmol), N-methylmorph-
oline (0.7 mL, 6.35 mmol), TBTU (1.02 g, 3.18 mmol) in
DMF (10 mL), 18 h; (f) Fmoc-Val-OH (950 mg,
2.80 mmol), HOBt (430 mg, 2.39 mmol), N-methylmorph-
oline (616 lL, 4.78 mmol), TBTU (0.90 mg, 2.80 mmol) in
DMF (10 mL), 16 h; (g) Fmoc-Asn(spacer)-OH 21
(1.57 g, 2.39 mmol), HOBt (367 mg, 3.18 mmol), N-meth-
ylmorpholine (526 lL, 4.78 mmol), TBTU (767 mg,
2.39 mmol) in DMF (10 mL), 18 h.
mer of 23 was isolated. Anal. HPLC: tR = 31.5 min
(Chromasil C-18, Knauer, gradient: 50% H2O/50%
H3CCN ! 100% CH3CN in 40 min).
1H NMR (CDCl3, 400 MHz): characteristic signals:
d 0.81 (d, 1H, J6 ;5 6:5 Hz, H-60); 1.71 (t, 1H,
0
0
000a;3000
e
000a;4000
J3
J3
12:3 Hz, H-3000a); 1.83, 1.90, 1.98, 2.03,
2.06, 2.10, 2.12, 2.19 (8 · s, 3H, –CH3Ac); 2.58 (dd,
1H, J3 12:5 Hz, J3
4:2 Hz, H-3000e); 2.71 (dd,
000e;3000
a
000e;4000
1H, JH-ba,H-bb 15.6 Hz, JHba,H-a 3.9 Hz, H-ba Asn);
2.96 (dd, 1H, JH-bb,H-ba 15.8 Hz, JHbb,H-a 4.4 Hz, H-bb
00 00
Asn); 3.84 (s, 3H, –OCH3); 5.04 (d, 1H, J4 ;3 2:9 Hz,
H-400); 5.10 (d, 1H, J5
10:6 Hz, C5000–NH); 5.19 (d,
000;NH
1H, Jvic 10.6 Hz, H2C@CH–CH2–O–cis); 5.29 (d, 1H, Jvic
17.3 Hz, H2C@CH–CH2–O–trans); 5.35 (dd, 1H,
000;6000
J7
2:3 Hz, J7
8:5 Hz, H-7000); 5.51 (mc, 1H, H-
000;8000
3.1.19.1. Detachment of linear peptide 25 from
resin. After the final coupling reaction and consecutive
washings, the resin was suspended in 1:1 DMF–Me2SO
(30 mL). N-Methylaniline (1.4 mL) was added, and air
was exchanged for an argon atmosphere. [Ph3P]4Pd(0)
(40 mg) was added and the mixture shaken for 15 h
under an argon atmosphere and exclusion of light. The
resin was filtered off and washed with DMF
(6 · 20 mL). The combined filtrate and washing solns
were concentrated under diminished pressure at 30 ꢁC.
The residue was co-distilled under diminished pressure
with toluene (3 · 50 mL). The yellow residue was dis-
solved in CH2Cl2 (250 mL) and the soln washed with
0.05 N HCl (50 mL) and brine (2 · 40 mL). After drying
with MgSO4, the soln was concentrated under dimin-
ished pressure to a volume of 5mL and kept in a refrig-
erator at 4 ꢁC for 16 h. The soln was separated from
precipitating amounts of the palladium catalyst, which
were washed with cold CH2Cl2 (3 mL). The combined
solns were evaporated under diminished pressure, dried
in high vacuum, dissolved in 8:1 CH2Cl2–MeOH and fil-
tered through a short column (3 cm) of silica gel. After
evaporation of the solvent, the oily residue was purified
by preparative RP-HPLC (Eurosphere C-18, Knauer,
gradient: 30% CH3CN/70% H2O ! 75% CH3CN/25%
H2O in 90 min). The fractions containing product 25
(anal. HPLC) were combined and evaporated under
diminished pressure. The remaining peptide was lyophi-
lized. Yield: 892 mg (61%); colourless amorphous solid;
8000); 5.73 (d, 1H, J1,NH 8.5 Hz, J1,2 ꢄ 1 Hz, H-1); 5.87
(mc, 1H, H2C@CH–); 6.21 (d, 1H, JH-a,NH 8.8 Hz, a–
NH Asn); 6.63 (s, 1H, –NH–CH2–); 6.71 (d, 1H, J2,NH
9.7 Hz, C2–NH); 7.03 (d, 1H, J1,NH 8.5 Hz, C1–NH);
7.53–7.62 (m, 2H, H-1 Fmoc, H-8 Fmoc); 7.73 (d,
2H, H-4 Fmoc, H-5 Fmoc). MALDIMS calcd for C112
137N5O38:2161.3, Found: 2184.7 [M+Na]+, 2200.8
[M+K]+.
-
H
3.1.19. N2-Fluorenylmethoxycarbonyl-N4-{14-tert-butyl-
oxycarbonyl-3,6,9,12-tetraoxatetradecyl}-L-asparaginyl-
L-valyl-L-prolyl-L-glycyl-N4-{14-tert-butyloxycarbonyl-
3,6,9,12-tetraoxatetradecyl}-L-valyl-L-prolyl-L-glycine 25
(Fmoc-Asn(Glycol4-(CH2)2COOt-Bu)-Val-Pro-Gly-Asn-
(Glycol4-(CH2)2COOt-Bu)-Val-Pro-Gly-OH). The solid-
phase synthesis of the octapeptide containing two aspara-
gine units equipped with a spacer-acid side chain was
carried out using an aminopolystyrene resin loaded with
Fmoc glycine through an allyl HYCRON anchor 24
according to a procedure described earlier.26,34 Loading
of 24 was ca. 0.6 mmol/g. The reactions on Fmoc-Gly-
HYCRON resin 24 (1.41 g, 0.84 mmol) were performed
in a solid-phase reactor. Fmoc removal: 1:1 DMF–mor-
pholine, 80 mL, 30–45 min, washing DMF (3 · 20 mL);
Boc removal: 1:1 CH2Cl2–TFA, 30 mL, washing CH2Cl2
(6 · 20 mL); neutralization with 10:1 CH2Cl2–diisopropyl-
ethylamine, 10 min, then washing CH2Cl2 (6 · 20 mL)
and DMF (20 mL). Capping was carried out after each
coupling reaction: 3:1 pyridine–Ac2O, 24 mL, 15 min,
washing DMF (6 · 20 mL). Coupling reactions: Suspen-
sion of the resin in DMF (20 mL), after each coupling
washing with DMF (6 · 20 mL); applied amounts (a)
Boc-Pro-OH (0.85 g, 3.95 mmol), HOBt (0.61 g, 3.95
mmol), N-methylmorpholine (0.87 mL, 7.40 mmol),
TBTU32 (1.27 g, 3.95 mmol) in DMF (10 mL, 15 h); (b)
Fmoc-Val-OH (1.46 g, 4.31 mmol), HOBt (0.66 g, 4.31
mmol), N-methylmorpholine (0.95 mL, 8.61 mmol),
TBTU (1.38 g, 4.31 mmol) in DMF (10 mL), 17 h; (c)
Fmoc-Asn(spacer)-OH 21 (1.24 g, 1.9 mmol), HOBt
(286 mg, 1.9 mmol), N-methylmorpholine (0.45 mL,
22
½aꢂD ꢁ47.3 (c 1, MeOH); Rf = 0.67 (5:1 CH2Cl2–
MeOH); anal. HPLC (Eurosphere C-18, Knauer): gradi-
ent 25% H3CCN/75% H2O ! 75% H3CCN/25% H2O,
tR = 32.2 min. 1H NMR (Me2SO-d6, 400 MHz), 1H,
1H-COSY: d 0.81 (d, 6H, JH-b,H-ca 4.7 Hz, 2 · H-ca
Val); 0.87 (d, 6H, JH-cb,H-b 5.1 Hz, 2 · H-cb Val); 1.39
(s, 18H, 2 · –C(CH3)3); 1.68–2.11 (m, 10H, 2 · H-b
Val (1.99, 2.00), 4 · H-b Pro (1.82), 4 · H-c Pro
(2.00)); 2.38 (t, 2H, Jvic 6.3 Hz, –O–CH2CH2–COOt-
Bu); 2.39 (t, 2H, Jvic 6.3 Hz, –O–CH2CH2–COOt-Bu);
2.35–2.44 (m, 3H, H-ba Fmoc-Asn, H-bb Fmoc-Asn,
H-ba –Asn–); 2.51 (m, 1H, H-bb –Asn–); 3.17 (t, 4H, Jvic

H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
555
5.1 Hz, 2 · –O–CH2CH2–NH–); 3.36 (t, 4H, Jvic 5.9 Hz,
2 · –O–CH2CH2–NH–); 3.55 (t, 2H, Jvic 6.3 Hz,
–CH2CH2–COOt-Bu); 3.56 (t, 2H, Jvic 6.3 Hz,
–CH2CH2–COOt-Bu); 3.59 (m, 2H, 2 · H-aa Gly); 3.77
(m, 2H, 2 · H-ab Gly); 4.14–4.26 (m, 3H, H-9 Fmoc,
–CH2–Fmoc); 4.26–4.37 (m, 4H, 2 · H-a Val, 2 · H-a
Pro); 4.38 (q, 1H, JH-a,H-ba ꢃ JH-a,H-bb ꢃ H-a,–NH– 5.5 Hz,
residue was co-distilled with toluene (5 mL), dissolved
in acetonitrile (5 mL) and filtered through a C18 RP-
silica gel cartridge, which was washed with 20 mL of
acetonitrile. The combined solns were concentrated to
a volume of 2 mL, and cyclopeptide 26 was purified
by preparative RP-HPLC (column Eurospher C18;
Knauer, gradient: 30% H3CCN/70% H2O ! 100%
H3CCN in 90 min, tR = 51.8 min). Yield: 176 mg
(71%), colourless amorphous solid; Rf = 0.29 (RP
C-18), CH3CN/H2O = 2:3. 1H NMR (CD3OD, 400
MHz): characteristic signals: d 0.83 (d, 3H, JH-c,H-b
6.2 Hz, H-c Val); 0.99–1.08 (m, 6H, H-c Val); 1.14
(d; 3H, JH-c,H-b 5.9 Hz, H-c Val); 1.49 (s, 18H, 2 ·
–C(CH3)3); 2.52 (t, 4H, Jgem 6.2 Hz, 2 · –CH2–COOt-
Bu); 3.74 (t, 4H, Jgem 6.2 Hz, 2 · –CH2–CH2–COOt-
Bu). In addition in Me2SO-d6 the following NH-signals
are visible: d 7.24–7.42 (m, 2H, 2 · –NHc–), 7.82–7.93
(m, 1H, –NHa–); 7.97–8.07 (m, 1H, 1H, –NHa–); 8.08–
8.17 (m, 1H, –NHa–); 8.19–8.30 (m, 1H, –NHa–);
9.02–9.15 (m, 1H, –NHa–). 13C NMR (CD3OD,
100.3 MHz): d 17.77, 19.64, 20.51, 21.66 (2 · C-b
Val, 2 · C-c Val); 26.71, 29.50, 29.87, 32.84 (2 · C-b
Pro, 2 · C-c Pro); 37.27 (–CH2COOt-Bu); 28.38
(–C(CH3)3); 40.26, 40.44 (2 · –CH2–NH–); 43.49, 43.69
(2 · C-a Gly); 47.91 (C-d Pro); 50.38, 51.38, 52.78,
56.60 (2 · C-a Val, 2 · C-a Asn); 63.03, 64.56 (2 · C-a
Pro); 67.88 (–CH2–CH2–COOt-Bu); 70.42, 71.27,
71.37, 71.47, 71.53 (–CH2–O–); 171.39, 171.52, 173.36,
174.72, 175.29 (–CO–NH–); 172.74 (–COOt-Bu).
MALDIMS (dhb) calcd for C62H106N10O22: 1344.6.
Found: 1366.9 [M+Na]+, 1382.7 [M+K]+.
H-a Fmoc-Asn); 4.57 (q, 1H, JH-a,H-ba ꢃ JH-a,H-bb
ꢃ
6.6 Hz, H-a –Asn–); 7.31 (t, 2H, Jvic 7.4 Hz,
H-a,–NH–
H-2 Fmoc, H-7 Fmoc); 7.40 (t, 2H, Jvic 7.4 Hz, H-3,
H-6 Fmoc); 7.61 (d, 1H, JNH,H-a 8.2 Hz, –NH–a
Fmoc-Asn); 7.67 (m, 1H, –NH– Val); 7.68 (d, 2H, Jvic
7.1 Hz, H-1 Fmoc, H-8 Fmoc); 7.78 (d, 1H, JNH,H-a
8.2 Hz, –NH– Val); 7.87 (d, 2H, Jvic 7.4 Hz, H-4 Fmoc,
H-5 Fmoc); 7.91 (t, 1H, Jvic 5.1 Hz, –O–CH2–CH2–NH–);
7.95 (t, 1H, Jvic 5.1 Hz, –O–CH2–CH2–NH–); 8.04
(d, 1H, JNH,H-a 7.4 Hz, –NH– Val); 8.14 (t, 2H, JNH,H-a
5.0 Hz, 2 · –NH– Gly). 13C NMR (MeOD,
100.6 MHz), DEPT: d 18.66, 19.89, 19.93 (C-c Val);
25.92, 26.14 (C-c Pro); 28.39 (–C(CH3)3); 30.35 (C-b
Pro); 31.78, 31.68 (C-b Val); 37.27 (–CH2–COOt-Bu);
38.48, 38.60 (C-b Asn); 40.50 (–O–CH2CH2–NH–);
43.72 (C-a Gly); 48.33 (C-9 Fmoc); 49.60 (C-d Pro);
51.81 (C-a Asn); 53.34 (C-a Val); 61.78, 58.06 (C-a
Pro); 67.86, 68.15 (–CH2–Fmoc, –CH2CH2–COOt-Bu);
81.68 (–C(CH3)3); 120.92, 126.17, 128.17, 128.77 (C–
Ar Fmoc); 142.50, 145.16 (C-ipso Fmoc); 158.02
(–NH–CO–O); 171.36, 172.07, 172.29, 172.65, 173.21,
173.96, 174.64 (–CO–). MALDIMS (dhb) calcd for
C77H118N10O25: 1583.9. Found: 1606.9 [M+Na]+,
1622.9 [M+K]+, 1628.9 [M+2NaꢁH]+; Anal. Calcd
for C77H118N10O25Æ3H2O: C, 56.47; H, 7.63; N, 8.55.
Found: C, 56.35; H, 7.55; N, 8.44.
3.1.21. cyclo-[N4-{14-N-(2-Acetamido-3-O-(20,30,40-tri-
O-benzyl-a-L-fucopyranosyl)-2-deoxy-4-O-(200,400-di-O-
acetyl-600-O-benzyl-300-O-[methyl 5000-acetamido-4000,7000,8000,
9000-tetra-O-acetyl-3000,5000-dideoxy-D-galacto-2000-nonulopyr-
anosylate]-b-D-galactopyranosyl)-b-D-glucopyranosyl)-car-
bamoyl-3,6,9,12-tetraoxatetradecyl}-L-asparaginyl-L-valyl-L-
prolyl-L-glycinyl-N4-{14-N-(2-acetamido-3-O-(20,30,40-tri-
O-benzyl-a-L-fucopyranosyl)-2-deoxy-4-O-(200,400-di-O-ace-
tyl-600-O-benzyl-300-O-[methyl 5000-acetamido-4000,7000,8000,9000-
tetra-O-acetyl-3000,5000-dideoxy-D-galacto-2000-nonulopyranosy-
late]-b-D-galactopyranosyl)-b-D-glucopyranosyl)-carbam-
oyl-3,6,9,12-tetraoxatetradecyl}-L-asparaginyl-L-valyl-L-
prolyl-L-glycine] 27 (cyclo-[Asn(Glycol4-(CH2)2CO-a/b-
Ac6Bn5sLexCOOMe)-Val-Pro-Gly-Asn(Glycol4-(CH2)2-
CO-b-Ac6Bn5sLexCOOMe)-Val-Pro-Gly-])
3.1.20. cyclo-[N4-{14-tert-Butyloxycarbonyl-3,6,9,12-tet-
raoxatetradecyl}-L-asparaginyl-L-valyl-L-prolyl-L-glycyl-
N4-{14-tert-butyloxycarbonyl-3,6,9,12-tetraoxatetrade-
cyl}-L-asparaginyl-L-valyl-L-prolyl-L-glycine] 26 (cyclo-
[Asn(Glycol4-(CH2)2COOt-Bu)-Val-Pro-Gly-Asn(Glycol4-
(CH2)2COOt-Bu)-Val-Pro-Gly-])
3.1.20.1. Fmoc-removal. A soln of octapeptide 25
(300 mg, 0.19 mmol) in 8:1 DMF–morpholine (14 mL)
was stirred at room temperature for 1 h. The solvents
were evaporated in high vacuum, the residue co-distilled
with toluene (10 mL) and the pale yellow residue puri-
fied by column chromatography in 5:2 acetonitrile–
water on 50 g of reversed phase C18 silica gel.
3.1.21.1. Side chain deprotection. The side chain-
protected cyclo-octapeptide 26 (174 mg, 0.13 mmol)
was stirred in a mixture of TFA (5 mL), CH2Cl2
(5 mL), ethylmethylsulfide (0.4 mL) and ethanedithiole
(0.1 mL) at room temperature for 2 h. After evaporation
of the solvents, the remaining residue was co-distilled
with toluene (4 · 10 mL), dried in high vacuum and
purified by preparative RP-HPLC (column Eurospher,
100-C18, Knauer, gradient: 99% H2O ! 35% H2O/
3.1.20.2. Cyclization. The deprotected peptide ob-
tained (250 mg, 0.184 mmol, Rf = 0.29, reversed phase
TLC (C18), 3:2 acetonitrile–water was dissolved in
200 mL of 1:1 CH2Cl2–DMF. To this soln HOAt31
(50.3 mg, 0.367 mmol), diisopropylethylamine (94.3 lL,
0.551 mmol) and HATU31 (104.6 mg, 0.275 mmol) were
added, and the soln was stirred at room temperature for
2 h. The solvents were evaporated in high vacuum, the

556
H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
65% H3CN in 60 min, tR = 41.3 min). Yield: 156 mg
Pearlman’s catalyst (10 mg, Pd(OH)2 20% on charcoal)
was added. After hydrogenation for 25 h at atmospheric
pressure, the catalyst was filtered off through Hyflo and
washed with MeOH (150 mL). The combined filtrate
solns were evaporated under diminished pressure, the
residue was co-distilled with toluene (10 mL) and dried
in high vacuum. The debenzylated product was dis-
solved in MeOH (30 mL). Freshly prepared 0.1 N
sodium methoxide in MeOH was added until a drop
of the soln on a moist pH-paper showed pH 10 (about
2.4 mL). After stirring for 20 h at room temperature,
solid CO2 was added for neutralization, and the solvent
was evaporated under diminished pressure. The remain-
ing solid was dissolved in aq NaOH of pH 10–10.5 and
stirred at room temperature for 20 h. After neutraliza-
tion with solid CO2, the water was evaporated under
diminished pressure and the residue purified by gelper-
meation chromatography on Sephadex LH 15 in water
(column: 80 · 2 cm; eluent: water).
26
(0.127 mmol, 97%); colourless amorphous solid; ½aꢂD
5.9 (c 1, CH3CN); anal. RP-HPLC (column Eurospher
100 C18 Knauer): gradient: 1% CH3CN/99% H2O !
100% CH3CN in 50 min, tR = 19.7 min; MALDIMS
calcd for C54H90N10O22: 1231.4. Found: 1233.0 (100%)
[M+H]+, 1254.6 (18%) [M+Na]+, 1270.7 (7%) [M+K]+.
3.1.21.2. Conjugation with sLeX amine 17. To a soln
of the deprotected cyclooctapeptide (67 mg, 0.054
mmol) in DMF (5 mL), HOBt (16.9 mg, 0.11 mmol),
diisopropylethylamine (92 lL, 0.54 mmol) and PyBOP29
(114.5 mg, 0.22 mmol) were added. sLeX amine 17
(221 mg, 0.144 mmol) was then added and the soln stir-
red at room temperature for 22 h. The solvent was dis-
tilled off under diminished pressure, and the residue
was co-distilled with toluene (3 · 5 mL). The crude
product was dissolved in CH2Cl2 (120 mL), washed with
water (80 mL), satd aq NaHCO3 (80 mL) and water
again. After drying with MgSO4 and evaporation under
diminished pressure, the remaining brown amorphous
solid (362 mg) was purified by preparative RP-HPLC
to give the two diastereomers 27bb and 27ab: (column
LUNA, C18, Phenomenex): gradient 75% CH3CN/
25% H2O ! 100% CH3CN in 90 min, tR = 59.0 min
(27bb) and 68.4 min (27ab).
23
Compound 28bb: Yield: 45.0 mg (89%); ½aꢂD ꢁ66.6
1
(c 1, H2O); H NMR (D2O, 400 MHz): characteristic
signals: d 0.94 (d, 12H, JHc,Hb 6.5 Hz, H-c Val); 1.15
(d, 6H, J6 ,5 6.5 Hz, H-60); 1.77 (t, 2H, J3
ꢃ
0
0
000a;3000
e
11:9 Hz, H-3000a); 1.96 (s, 6H, –NHAc); 2.00 (s,
000a;4000
J3
6H, –NHAc); 2.57 (t, 4H, Jvic 5.9 Hz, –CH2–CO–);
2.73 (d, 2H, J3
10:7 Hz, H-3000e); 3.32–3.42 (m,
000e;3000
a
00 00
00 00
Compound 27bb: Yield: 89.4 g (38.5%); colourless
amorphous solid; Anal. RP-HPLC (column Eurospher
100, C8, Knauer): tR = 22.6 min, gradient: 50% CH3-
CN/50% H2O ! 100% CH3CN/0% H2O in 50 min.
MALDIMS calcd for C212H280N16O76: 4268.6. Found:
4292.8 [M+Na]+, 4308.7 [M+Ka]+.
Compound 27ab: Yield: 43.0 mg (18.5%); colourless
amorphous solid; Anal. RP-HPLC (column Eurospher
100, C8, Knauer): tR = 27.4 min, gradient: 50% CH3-
CN/50% H2O ! 100% CH3CN in 50 min. MALDIMS
calcd for C212H280N16O76: 4268.6 Found: 4292.3 [M+
Na]+, 4308.2 [M+Ka]+.
4H, –NH–CH2–); 3.56 (t, 2H, J2 ;1 ꢃ J2 ;3 8:5 Hz, H-
200); 4.11 (d, 2H, J3 ;2 8:8 Hz, H-300); 4.52 (d, 2H,
00 00
J1 ;2 7:6 Hz, H-100); 4.88 (mc, 2H, H-50); 5.08 (d, 2H,
00 00
J1 ;2 3:5 Hz, H-10); 5.11 (d, 2H, J1,2 9.7 Hz, H-1). 13C
NMR (D2O, 100.6 MHz): d 15.44 (C-60); 18.85 (C-c
Val); 22.24 (–CH3Ac); 22.33 (–CH3Ac); 25.36; 28.71;
36.27 (–CO–CH2–); 39.06 (–CH2–NH–); 40.15 (C-3000);
42.57; 47.87; 51.98 (C-5000); 54.86 (C-2); 59.84 (C-600);
61.65; 62.92 (C-9000); 66.54; 66.85 (C-50); 67.55; 67.94;
68.39; 68.91; 69.46; 69.60; 69.66; 69.75; 72.12; 73.13;
73.29; 75.11; 75.44; 75.94; 77.35; 78.33 (C-1); 98.66
(C-10); 101.87 (C-100, C-2000); 170.97, 171.53, 172.57,
174.30, 174.80, 175.28 (–CO–NH–). ESI-MS calcd for
(C116H192N16O64): 2834.9. Found: 1448.3 (27%)
[(M+Na+K)/2]+, 1451.1 (18%) [(MꢁH+3Na)/2]+,
1456.1 (45%) [(M+2K)/2]+, 1459.3 (100%) [(MꢁH+
2Na+K)/2]+, 1462.1 (53%) [(Mꢁ2H+4Na)/2]+, 1467.3
(28%) [(MꢁH+Na+2K)/2]+, 1478.8 (16%) [(Mꢁ2H+
2Na+2K)/2]+.
0
0
3.1.22. cyclo-[N4-{14-N-(2-Acetamido-3-O-(20,30,40-tri-
O-benzyl-a-L-fucopyranosyl)-2-deoxy-4-O-(200,400-di-O-
acetyl-600-O-benzyl-300-O-[methyl 5000-acetamido-4000,7000,8000,
9000-tetra-O-acetyl-3000,5000-dideoxy-D-galacto-2000-nonulopyr-
anosylate]-b-D-galactopyranosyl)-b-D-glucopyranosyl)-car-
bamoyl-3,6,9,12-tetraoxatetradecyl}-L-asparaginyl-L-valyl-
L-prolyl-L-glycinyl-N4-{14-N-(2-acetamido-3-O-(20,30,40-
tri-O-benzyl-a-L-fucopyranosyl)-2-deoxy-4-O-(200,400-di-O-
acetyl-600-O-benzyl-300-O-[methyl 5000-acetamido-4000,7000,8000,
9000-tetra-O-acetyl-3000,5000-dideoxy-D-galacto-2000-nonulo-
pyranosylate]-b-D-galactopyranosyl)-b-D-glucopyranosyl)-
carbamoyl-3,6,9,12-tetraoxatetradecyl}-L-asparaginyl-L-
In an analogous preparation, 27ab (37.1 mg, 0.0087
mmol) was converted to 28ab: Yield: 22.7 mg (92%);
23
1
colourless amorphous solid. ½aꢂD ꢁ12.6 (c 1, H2O); H
NMR (D2O, 400 MHz): characteristic signals: d 0.94
(d, 12H, JHc,Hb 6.4 Hz, H-c Val); 1.13 (d, 3H,
J5 ;6 5:6 Hz, H-60); 1.15 (d, 3H, J5 ;6 5:6 Hz, H-60);
0
0
0
0
11:7 Hz, H-3000a); 1.95 (s, 3H,
000a;3000
e
valyl-L-prolyl-L-glycine]
28bb
(cyclo-[Asn(Glycol4-
1.76 (t, 2H, J3
(CH2)2CO-a/b-sLexCOOH)-Val-Pro-Gly-Asn(Glycol4-
(CH2)2CO-sLexCOOH)-Val-Pro-Gly-]). To a soln of
the cyclic glycopeptide 27bb (76 mg, 0.0178 mmol) in
MeOH (30 mL), water (0.2 mL) and acetic acid (3 mL)
–NHAc); 1.97 (s, 3H, –NHAc); 1.99 (s, 6H, –NHAc);
2.04–2.13 (m, 2H, H-c Pro); 2.13–2.23 (m, 2H, H-b
Val); 2.24–2.38 (m, 2H, H-c Pro); 2.52 (t, 2H, Jgem
13.9 Hz, –CH2–CO–); 2.58–2.67 (m, 2H, –CH2–CO–);

H. Herzner, H. Kunz / Carbohydrate Research 342 (2007) 541–557
557
11:2 Hz, H-3000e); 3.30–3.42 (m, 4H,
a
Vestweber, D.; Kunz, H. Angew. Chem., Int. Ed. 2001, 40,
3836.
13. Wittmann, V.; Takayama, S.; Gong, K. W.; Weitz-
Schmidt, G.; Wong, C.-H. J. Am. Chem. Soc. 1998, 63,
5137.
14. For a recent example: Huang, K.-T.; Wu, B.-C.; Lin, C.-
C.; Luo, S.-C.; Chen, C.; Wong, C.-H. Carbohydr. Res.
2006, 341, 2152.
15. (a) Kamayama, A.; Ishida, H.; Kiso, M.; Hasegawa, A.
Carbohydr. Res. 1990, 100, 143; (b) Eisele, T.; Toepfer, A.;
Kretzschmar, G.; Schmidt, R. R. Tetrahedron Lett. 1996,
37, 1389; (c) Danishefsky, S. J.; Gervay, J.; Peterson, J.
M.; McDonald, F. E.; Koseki, K.; Griffith, D. A.;
Oriyama, T.; Marsden, S. P. J. Am. Chem. Soc. 1995,
117, 1940.
000e;3000
2.73 (d, 2H, J3
–CH2–NH–); 4.51 (d, 2H, J1 ;2 7:6 Hz, H-100); 5.06 (d,
00 00
1H, J1 ;2 4:1 Hz, H-10); 5.07 (d, 1H, J1 ;2 4:1 Hz, H-10);
5.11 (d, 1H, J1b,2 9.7 Hz, H-1b); 5.58 (d, 1H, J1a,2
4.4 Hz, H-1a). MALDIMS calcd for (C116H192N16-
O64): 2834.9. Found: 2564.8 [MꢁNeuNAc+Na]+,
2900.7 [Mꢁ2H+3Na]+, 2894.9 [MꢁH+Na+K]+, 2878.7
[MꢁH+2Na]+, 2872.8 [M+K]+, 2856.9 [M+Na]+.
0
0
0
0
References
1. Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321.
2. Nicholson, M. W.; Barclay, A. N.; Singer, M. S.; Rosen, S.
D.; van der Merwe, P. A. J. Biol. Chem. 1998, 273, 763.
3. (a) Bevilaqua, M. P.; Stengelein, S.; Gimbrone, M. A., Jr.;
Seed, B. Science 1989, 243, 1160; (b) Philips, M. L.;
Nudelman, E.; Gaeta, F. C. A.; Peretz, M.; Singhal, A. K.;
Hakomori, S. I.; Paulsen, J. C. Science 1990, 250, 1130.
4. For reviews see, for example: (a) Simanek, E. E.; McGar-
vey, G. J.; Jablonski, J. A. K.; Wong, C.-H. Chem. Rev.
1998, 98, 833; (b) Unger, F. M. Adv. Carbohydr. Chem.
Biochem. 2001, 57, 207; (c) Ehrhardt, C.; Kneuer, C.;
Bakowsky, U. Adv. Drug. Delivery Rev. 2004, 56, 527.
5. See, for example: (a) Brown, J. R.; Mark, M.; Li, R.;
Varki, N.; Glass, C. A.; Esko, J. D. Clin. Cancer Res.
2006, 12, 2894; recent review: (b) Kannagi, R. Glycocon-
jugate J. 2004, 20, 353.
16. See, for example: (a) Nicolaou, K. C.; Hummel, C. W.;
Bochkovic, N. J.; Wong, C.-H. J. Chem. Soc., Chem.
Commun. 1991, 870; (b) Nunomura, S.; Iida, M.; Numata,
M.; Sugimoto, M.; Ogawa, T. Carbohydr. Res. 1994, 263,
C1.
17. (a) Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl.
1988, 27, 1697; (b) Unverzagt, C.; Kunz, H. J. Prakt.
Chem. 1992, 334, 570.
´
18. Zhang, H. X.; Guibe, F.; Balvoine, G. Tetrahedron Lett.
1981, 22, 623.
19. Garegg, P. J.; Hultberg, H.; Wallin, S. Carbohydr. Res.
1982, 108, 97.
20. Jung, K.-H.; Hoch, M.; Schmidt, R. R. Liebigs Ann.
Chem. 1989, 1099.
21. (a) Kunz, H.; Waldmann, H. Angew. Chem., Int. Ed. Engl.
1984, 23, 71; (b) Kunz, H.; Unverzagt, C. Angew. Chem.,
Int. Ed. Engl. 1984, 23, 436.
6. Palcic, M. M.; Li, H.; Zanini, D.; Bhella, R. S.; Roy, R.
Carbohydr. Res. 1997, 305, 433.
22. Sato, S.; Ito, Y.; Ogawa, T. Carbobydr. Res. 1986, 155, C6.
7. (a) Bovin, N. V.; Korchagina, E. Y.; Zemlyanukhina, T.
V.; Byramova, N. E.; Galamina, O. E.; Zemlyakov, A. E.;
Ivanov, A. E.; Zubov, V. P.; Mochalova, L. V. Glycocon-
jugate J. 1993, 10, 142; (b) Bovin, N. V.; Gabius, H.-J.
Chem. Soc. Rev. 1995, 24, 413; (c) Thoma, G.; Ernst, B.;
Schwarzenbach, F.; Duthaler, R. O. Bioorg. Med. Chem.
Lett. 1997, 7, 1705; (d) Sallas, F.; Nishimura, S.-I. J.
Chem. Soc., Perkin Trans. 1 2005, 2091.
23. Marra, A.; Sinay, P. Carbohydr. Res. 1989, 187, 35.
¨
24. (a) Murase, T.; Ishida, H.; Kiso, M.; Hasegawa, A.
Carbohydr. Res. 1988, 156, C1; (b) Nifant’ev, N. E.;
Tsvetkov, A. E.; Shashkov, A. S.; Kononov, L. O.;
Menshov, V. M.; Tuzikov, A. B.; Bovin, N. J. Carbohydr.
Chem. 1996, 15, 939; (c) Makimura, Y.; Ishida, H.; Kiso,
M.; Hasegawa, A. Carbohydr. Res. 1996, 15, 1097; For a
review on O-sialylation reactions: (d) Boons, G.-J.;
Demschenko, A. V. Chem. Rev. 2000, 100, 4539.
25. Kunz, H.; Sager, W.; Schanzenbach, D.; Decker, M.
Liebigs Ann. Chem. 1991, 649.
8. Thoma, G.; Potton, J. T.; Magnani, J. L.; Ernst, B.;
Oehrlein, R.; Duthaler, R. O. J. Am. Chem. Soc. 1999,
121, 5919.
9. (a) Lin, C.-C.; Kimura, T.; Wu, S.-H.; Weitz-Schmidt, G.;
Wong, C.-H. Bioorg. Med. Chem. Lett. 1996, 6, 2755; (b)
Bruehl, R. E.; Dasgupta, F.; Katsumoto, T. R.; Tan, J. H.;
Bertozzi, C. R.; Spevak, W.; Ahn, D. J.; Rosen, S. D.;
Nagy, J. O. Biochemistry 2001, 40, 5964; (c) Zeisig, R.;
Stahn, R.; Wenzel, K.; Behrens, D.; Fichtner, I. Biochim.
Biophys. Acta, Biomembranes 2004, 1660, 31; (d) Vod-
ovozova, E. L.; Nazarova, A. I.; Feofanova, A. V.;
Kholodenkov, R. V.; Pazyina, G. V.; Gaenko, G. P.;
Bovin, N. V.; Molotkovsky, J. G. Biol. Membr. 2004, 21,
53; (e) Schumacher, G.; Bakowsky, U.; Gege, C.; Schmidt,
R. R.; Rothe, U.; Bendas, G. Biochemistry 2006, 45, 2894.
10. Furnike, T.; Sadamoto, R.; Niikura, R.; Monde, K.;
Sakairi, N.; Nishimura, S.-I. Tetrahedron 2005, 61, 1737.
11. Sprengard, U.; Schudok, M.; Kretzschmar, G.; Kunz, H.
Angew. Chem., Int. Ed. Engl. 1996, 35, 321.
26. (a) Herzner, H. Dissertation, Universitaet Mainz, 2001;
for analogous triethyleneglycol spacers: (b) Seitz, O.;
Kunz, H. Angew. Chem., Int. Ed. Engl. 1995, 34, 803.
27. Kiso, Y.; Kai, Y.; Yajima, H. J. Am. Chem. Soc. 1972, 86,
4557.
28. Ciommer, M.; Kunz, H. Synlett 1991, 593.
29. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett.
1990, 31, 205.
30. (a) Habermann, J.; Kunz, H. J. Prakt. Chem. 1998, 340,
233; (b) Habermann, J.; Kunz, H. Tetrahedron Lett. 1998,
39, 265.
31. Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.;
Carpino, L. A.; Bienert, M. Tetrahedron Lett. 1993, 34,
4681.
32. Knorr, R.; Treciak, A.; Baumwarth, W.; Gillessen, D.
Tetrahedron Lett. 1989, 30, 1927.
33. Ko¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 788.
34. Seitz, O.; Kunz, H. J. Org. Chem. 1997, 62, 813.
12. (a) Sprengard, U.; Kretzschmar, G.; Bartnik, E.; Huls, C.;
¨
Kunz, H. Angew. Chem., Int. Ed. Engl. 1995, 34, 990;
(b) Ro¨sch, M.; Herzner, H.; Dippold, W.; Wild, M.;