Hydrophilic photolabelling of glycopeptides from the murine liver-intestine (LI) cadherin recognition domain

Article abstract of DOI:10.1016/j.bmc.2006.06.014

LI-Cadherin is a transmembrane glycoprotein involved in cell adhesion of epithelial cells. Its supposed recognition domain contains the peptide motif AAL and is distinctly hydrophobic. In order to obtain sufficiently soluble model compounds, glycan side c

Full text of DOI:10.1016/j.bmc.2006.06.014

Bioorganic & Medicinal Chemistry 14 (2006) 6149–6164
Hydrophilic photolabelling of glycopeptides from the murine
liver–intestine (LI) cadherin recognition domain
Sebastian Heiner, Heiner Detert, Axel Kuhn and Horst Kunz*
Institut fuer Organische Chemie, Universitaet Mainz, D-55099 Mainz, Germany
Received 24 March 2006; revised 2 June 2006; accepted 8 June 2006
Available online 7 July 2006
Abstract—LI-Cadherin is a transmembrane glycoprotein involved in cell adhesion of epithelial cells. Its supposed recognition
domain contains the peptide motif AAL and is distinctly hydrophobic. In order to obtain sufficiently soluble model compounds,
glycan side chains of T-antigen, (2,6)sialyl T-antigen and sialyl TN-antigen structure were linked to the serine located in the supposed
turn sequence of the LI-cadherin recognition domain. A quinic acid-glycine-7-amino-coumarine (Quiglac) chromophore was
constructed in order to enhance the solubility of labelled LI-cadherin (glyco)peptides in water.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
glycosylated at Ser83 and adopting a turn-type conforma-
tion induced cell differentiation8 in transformed basal
keratinocytes of the HaCat9 cell line. This effect turned
out to be dependent upon the conformation of these
(glyco)peptides.8
Cadherins constitute a family of transmembrane glyco-
proteins which exert decisive functions in cell adhesion
phenomena.1,2 In their extracellular portion they con-
tain consensus repeat units of about 110 amino acids.
According to the number of these repeats cadherins
are subdivided in different classes.3 Epithelial cadherin
(E-cadherin) is the most intensely investigated cadher-
in.4,5 It belongs to the classical cadherins containing five
extracellular consensus repeat (EC) units. It is strongly
expressed on polarized, differentiated epithelial cells,
however, down-regulated on tumour cells. Its Ca2+-de-
pendent interaction is homotypic and homophilic and
proceeds via a cis dimer (on the same cell) which binds
to a trans dimer on the neighbouring cell.6 The homo-
philic recognition site of E-cadherin has been identified
in consensus repeat 1 (EC 1).1–5 According to NOE
NMR spectroscopic analyses, the homophilic recognition
domain is formed by three b-sheets (bC, bF, and bG)
and includes the recognition motif His79-Ala80-Val81
in b-sheet F close to a turn motif Ser83-Asn84-Gly85
between b-sheets F and G.7 Synthetic glycopeptides
Liver intestine (LI) cadherin has been found as a mem-
ber of a new class of cadherins consisting of seven extra-
cellular EC domains.10 Instead of the tripeptide motif
HAV of E-cadherin, the tripeptide AAL in the further-
most consensus repeat EC1 was identified as the recog-
nition motif of LI-cadherin.11
Whereas the classical cadherins are involved in important
cellular processes such as differentiation12 or growth reg-
ulation and morphogenesis,2,4 there are indications that
murine LI-cadherin during differentiation influences tran-
scription factors CDx1 and CDx213 important for intes-
tine formation. In addition, a connection between
LI-cadherin expression and the differentiation of
pancreas carcinoma cells was found.14
Except for the amino acid sequence and the AAL recog-
nition motif10,11 not much is known about the detailed
structure of LI cadherin. A comparison of the amino
acid sequence around the AAL motif for human (A),
rat (B) and murine (C) LI cadherin with the related area
of E cadherin suggests that the recognition region of LI
cadherins also should preferably adopt a turn-type
conformation.
Keywords: Glycopeptides; LI-cadherin; Coumarine chromophore;
Sialyl-T antigen; Solid-phase synthesis.
*
Corresponding author. Tel.: +49 06131 39 22334; fax: +49 06131 39
24786; e-mail: hokunz@mail.uni-mainz.de
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.014

6150
S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
human
rat
mouse
LI-CAD
LI-CAD
LI-CAD
Q95
QL95
QL95
VAALD100
AALDS100
AALDS100
ANGII105
QGAIV105
HGAIV105
VEGPV
DGPV
DGPV
A
B
C
Conformational analysis of a glycodecapeptide contain-
ing the amino acid sequence L95-I104 of rat-LI-cadherin
and an a-O-galactosaminyl side chain at S100 by NOESY
NMR spectroscopy in DMSO-d6 showed that this mole-
cule actually prefers a turn-type conformation.15 The
interesting question is whether this result indicates a sim-
ilar homophilic recognition site conformation as was
found for E-cadherin.7 To answer this question, it
requires to investigate the compound and its biological
effects in water. However, the insufficient water-solubility
of this molecule opposed all these efforts. Therefore, we
turned our interest to the supposed recognition region
of murine LI-cadherin containing a histidine in the as-
sumed turn sequence. Introduction of sialic acid contain-
ing saccharides at serine100 should further enhance the
water-solubility. As localization ofthe binding site ofsuch
a LI-cadherin glycopeptide on a cell is of particular
interest, the labelling of the glycopeptides with a fluoro-
phore appeared highly desirable. Fluorescent chromoph-
ores usually are hydrophobic. Therefore, it was a first aim
to construct a fluorophore which promotes water-solubil-
ity of hydrophobic LI-cadherin glycopeptides.
coumarine chromophore, ethoxycarbonyl-protected
m-amino-phenol17 1 was subjected to a Pechmann con-
densation reaction18 with acetone 1,3-dicarboxylic acid.
After removal of the ethoxycarbonyl group, Brenner es-
ter formation19 with benzylic alcohol in SOCl2 at ꢀ5 ꢁC
gave 7-aminocoumarine derivative 2 in high yield. Its
amino function turned out to be a poor nucleophile.
Therefore, its acylation with Boc-glycine to give 3 was
promoted by the efficient reagent O-(7-azabenzotriazol-
1-yl-)-N,N,N0,N0,-tetramethyluronium-hexafluorophos-
phate (HATU)20 in combination with collidine. After
acidolytic removal of the Boc group, coupling of 4 with
tetra-O-acetyl quinic acid21 5 was achieved by activation
with O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluroni-
umtetrafluoroborate (TBTU)22/1-hydroxybenzotriazol
(HOBt) under microwave irradiation (50 W, 50 ꢁC).
The hydrogenolytic cleavage of the benzylic ester of 6 re-
quires selective conditions. Hydrogenation catalyzed by
Pd/C (10%) produced the coumarine carboxylic acid 7 in
a yield of only 22%, but the analogous compound satu-
rated in the pyrone ring as the major product. In con-
trast, hydrogenation over Pd/C (5%) and careful
monitoring of the reaction by TLC yielded the fluoro-
phoric carboxylic compound 7 almost quantitatively
(Scheme 1).
2. Construction of a novel chromophore promoting
solubility in water
During coupling reactions of the fluorescent amino acid
7, decarboxylation occurred as a notable side reaction.
The amount of the decarboxylated methyl derivative
depended upon the chosen reaction conditions. There-
Coumarine derivatives are fluorescent chromophores
frequently used for fluorescent resonance energy transfer
(FRET) spectroscopy.16 In order to synthesize a suitable
O
O
O
H2N
O
O
1.
HO
70%ige H2SO4 , 91 %
Boc-Gly-OH
H
N
OH
O
OH
HATU, sym-collidine
O
OBn
2
2.
NaOH/H2O
pH 14, 100 ˚C, 85 %
O
1
3.
PhCH2OH, SOCl2
-5 ˚C, 87 %
H
N
H
N
O
O
O
O
BocHN
AcO
H2N
O
TFA/TIS
O
OBn
AcO
OBn
O
quant.
3 , 74 %
4
O
O
AcO
AcO
AcO
O
H
N
AcO
AcO
OH
OAc
O
O
N
H
6, R = Bn, 75 %
7, R = H, 98 % %
H2
O
Pd-C (5 %)
MeOH
5
OR
TBTU/HOBt/EtiPr2N, DMF,
microwave 50W, 50 ˚C
O
Scheme 1.

S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
6151
fore, it appeared advisable to couple the N-terminal
amino acid of the glycopeptide to be synthesized prior
to the solid-phase synthesis. In coupling reactions with
leucine tert-butyl ester best results were obtained with
N,N0-dicyclohexylcarbodiimide (DCC) and HOBt
(Scheme 2). The chromophore-leucine ester conjugate
8 and 4-methyl-coumarine 9 derivative were formed in
a ratio of 2.8:1. The mixture of by-product and product
could only be separated by HPLC.
hydroxy groups in 3- and 4-position were acetylated to
give 16 which is sufficiently stable to acids. Cleavage
of its tert-butyl ester with trifluoroacetic acid and
anisole furnished the sialyl TN-serine building block 17
suitable for solid-phase glycopeptide synthesis. The
corresponding T-antigen (18) and 2,6-sialyl T-antigen
(19) serine conjugates obtained by a similar pathway
have already been described.25
However, after treating the mixture of 8 and 9 with
TFA/triisopropylsilane(TIS)/H2O (14:1:1), the free car-
boxylic acid 10 was separable by flash-chromatography
and isolated in an overall yield of 63% after the two
steps.
4. Solid-phase synthesis of LI-cadherin glycopeptides
The peptide and glycopeptide sequences of the supposed
homophilic recognition site of the murine LI-cadherin
were synthesized on solid-phase using a peptide synthe-
sizer. Tentagelꢂ resin30 preloaded with Fmoc valine via
the acid-labile Wang anchor31 20 was used. The synthe-
ses were carried out according to the Fmoc protocol.32
The Fmoc amino acids were used in an excess of
20 equiv. HBTU/HOBt22 served as coupling reagent
(coupling time 20–30 min). The Fmoc groups were re-
moved by treatment with piperidine (20%) in N-meth-
yl-pyrrolidinone (NMP). The glycosylated amino acid
building blocks were coupled to the resin-linked pep-
tides manually using HATU/HOAt20 as the coupling
reagent.
For fluorescence spectroscopy, the O-acetyl groups were
23
´
removed from 10 by Zemplen transesterification with
NaOMe in methanol to give 11.
3. Syntheses of O-glycosyl serine building blocks
The glycosylated serine building blocks were synthesized
from Fmoc serine tert-butyl ester24 as is shown for the
example of the sialyl TN derivative in Scheme 3.
Fmoc-protected 2-azido-galactosyl serine tert-butyl
ester25 12 was carefully O-deacetylated by Zemplen
The synthesis of the pentadecapeptide Leu95-Val109 of
murine LI-cadherin (see introduction) attempted at first
suffered from the insolubility of the product. It could
not be purified by chromatography and was only detect-
able by mass spectroscopy. In order to overcome the low
solubility rendering any purification and characteriza-
tion of the product impossible, a glycopeptide contain-
ing the shortened undecapeptide sequence Leu95-Val105
and carrying the disaccharide T-antigen side chain (from
18) was constructed (Scheme 4).
´
transesterification at pH 8.5.26
By these conditions, the base-labile Fmoc group re-
mained stable. Regioselective and stereoselective sialyla-
tion of 13 was achieved using the xanthate27 14 of sialic
acid benzyl ester promoted by methylsulfenyl trifluo-
romethylsulfonate28 in dichloromethane/acetonitrile at
ꢀ65 ꢁC29 to yield the desired sialyl TN-serine conjugate
15. After purification by flash-chromatography, the
AcO
O
AcO
H
N
O
O
AcO
AcO
N
H
O
HCl*H-Leu-OtBu/NEt3
7
9
DCC/HOBt, DMF
AcO
O
AcO
H
N
O
O
AcO
AcO
N
H
O
O
H
N
TFA/TIS/H2O
OtBu
8
O
RO
RO
RO
O
OR
H
N
O
O
N
H
O
10 R = Ac, 63 %
11 R = H, 52 %
O
H
N
OH
O
Scheme 2.

6152
S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
OR
RO
RO
AcO OAc
AcO
AcHN
AcO OAc
COOBn
O
COOBn
O
AcO
AcHN
O
O
S
S
OEt
AcHN
AcO
AcO
RO
O
14
O
OtBu
AgOTf, CH3SBr
CH3CN/CH2Cl2
-65 ˚C
RO
15 R = H, 58 %
FmocHN
AcHN
O
O
12 R = Ac
OtBu
13 R = H
Ac2O/pyridine
FmocHN
MeOH/NaOMe
pH = 8.5
16 R = Ac, 65 %
O
AcO OAc
AcO
AcHN
COOBn
O
O
TFA/anisole (10:1)
AcO
AcO
O
AcO
AcHN
O
OH
17, 92 %
FmocHN
AcO OAc
AcO
AcHN
O
COOBn
O
O
OAc
AcO
AcO
AcO
OAc
O
O
AcO
OAc
AcO
AcO
HO
O
AcO
AcHN
O
O
O
O
AcO
AcHN
OH
O
FmocHN
O
OH
O
FmocHN
19
18
Scheme 3.
´
Detachment from the resin 21 and simultaneous remov-
al of the amino acid side-chain-protecting groups were
achieved with TFA/triisopropylsilane(TIS)/water. Addi-
tion of diethyl ether to the reaction solution resulted in
the precipitation of crude glycopeptide 22. After remov-
catalyst and of the O-acetyl groups under Zemplen con-
ditions (pH 9) gave the water-soluble 2,6-sialyl T-glyco-
hexapeptide 26 which was isolated after purification by
preparative HPLC in an overall yield of 23% relative
to 20 (Scheme 7).
´
al of the O-acetyl groups from 22 by Zemplen reaction
at pH 9.5, a product of good solubility in water was ob-
tained (Scheme 5). Its purification by preparative HPLC
yielded the desired T antigen LI-cadherin glycopeptide
23 (45%) and two deletion sequences 23a (13%) and
23b (16%).
5. Synthetic LI-cadherin glycopeptide containing the
amino-coumarine chromophore
According to the good solubility of the LI-cadherin gly-
copeptide 26, a fluorescent-labelled partial sequence of
the LI-cadherin recognition domain was designed as a
pentadecapeptide containing the solubilizing quinic
acid–coumarine residue (Quiglac) 11 instead of the
N-terminal glutamine of 26 and the sialyl TN-antigen
saccharide considered necessary as the minimal
hydrophilizing saccharide side chain. The solid-phase
assembly was carried out in analogy to the protocol
described for 24.
Encouraged by the good solubility and successful purifi-
cation of 23 an extended partial sequence of the
supposed recognition domain of LI-cadherin, the
glycohexadecapeptide (Gln94-Val109) containing 2,6-sial-
yl-T-antigen 19 as a larger saccharide, was synthesized
according to an analogous solid-phase synthesis proto-
col (Scheme 6). Again, the glycosyl amino acid was cou-
pled manually to the resin-linked peptide using an excess
of 2.34 equiv and HATU/HOAt as the activating
reagent.
The 2,6-sialyl-TN-antigen 17 was coupled manually
using an excess of 1.02 equiv and activation by
HATU/HOAt. The coupling of the chromophore 10
was accomplished likewise by activation with HATU/
HOAt and an excess of 10 of 2 equiv (Scheme 8).
Release of the glycopeptide from the resin 27 was
achieved by treatment with trifluoroacetic acid,
triisopropylsilane and water. After purification by
Detachment of the product from the resin-linked form
24 using trifluoroacetic acid, triisopropylsilane and
water gave crude, partially protected glycopeptide 25.
It was sufficiently soluble for purification by preparative
HPLC to afford 25 in a yield of 42%. Removal of the
benzyl ester by hydrogenation using Pd/C (10%) as the

S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
6153
O
FmocHN
Linker
O
20
1.-4. Fmoc removal, Fmoc-Xaa-OH, HBTU/HOBt, capping
5. manual coupling: Fmoc removal,
Fmoc-Ser(β-Ac4Gal-(1 3)-α-Ac2GalNAc)-OH 18 (4 eq),
HATU/HO At/DIPEA, 5 h
6.-10. Fmoc removal, Fmoc-Xaa-OH, HBTU/HOBt, capping
11. Fmoc removal
OAc
AcO
OAc
AcO
O
O
AcO
O
AcO
AcHN
O
O
O
O
O
O
O
H
N
H
H
H
H
H2N
N
N
N
N
Linker
N
N
N
H
N
N
O
H
H
H
H
O
O
O
N
O
O
ButOOC
NTrt
21
TFA/TIS/H2O
OAc
OAc
AcO
AcO
AcO
O
O
O
AcHN
O
AcO
O
O
O
O
H
O
O
H
N
H
H
N
H
H2N
N
N
N
H
N
N
N
N
H
N
H
OH
H
H
O
O
O
O
O
HOOC
N
22
NH
Scheme 4.
preparative HPLC, fluorescent-labelled glycopeptide 28
was isolated in a yield of only 10% (Scheme 8).
found at 328 nm, whereas the fluorescence spectrum
has its maximum at kmax 404.5 nm. The flank which
can be seen in the UV absorption spectrum of com-
pound 11 is responsible for difficulties using the new
chromophore in a FRET experiment with tryptophane
as the donor (Fig. 1). Because of this flank, an overlap
of the absorption of tryptophane and compound 11 does
occur.
The main reasons for the poor yield are the two manual
couplings and only equimolar amounts of the sialyl TN
serine building block available for the coupling reaction.
Accordingly, the major product of the synthesis was
deletion sequence 29. Nevertheless, the structure 28
could be proven by NMR spectroscopy.
Accordingly, a sufficiently separated excitation of donor
and acceptor as would be necessary for FRET experi-
ments cannot reliably be achieved.
Benzyl ester and O-acetyl groups were removed from 28
by treatment with sodium methanolate in methanol at
pH 9.5 and, subsequently, with aqueous sodium hydrox-
ide at pH 10.5. After acidification with acetic acid, puri-
fication of the product was carried out by preparative
HPLC to yield the desired water-soluble glycopenta-
decapeptide 30 equipped with the fluorescent chromo-
phore Quiglac (11) in an overall yield of 4%. Due to
the small amount of 30, its characterization could only
be performed by mass spectroscopy.
7. Experimental
7.1. General instrumentation
Solvents were dried and distilled according to standard
procedures. Fmoc amino acids were purchased from
Novabiochem. The Tentagel-PHB resin preloaded with
valine was supplied by Rapp Polymere (Tubingen, Ger-
¨
6. Fluorescence and UV spectroscopic data
many). Reactions were monitored on TLC aluminium
plates coated with silica gel 60F254 (Merck, Darmstadt,
Germany). Purification by flash-chromatography was
performed with silica gel (32–63 lm) from Merck,
Darmstadt. For microwave heating a CEM Discover Sys-
tem was used. RP-HPLC analyses were performed by
application of a Knauer HPLC system on a Phenomenex
To analyze the spectroscopic behaviour of compound
11, the UV absorption and the fluorescence spectra were
measured on a Zeiss MCS 320/340 diode-array spec-
trometer and on a Perkin-Elmer LS 50B spectrometer
at an exciting wavelength of 330 nm. In UV, kmax was

6154
S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
22
NaOMe/MeOH
HO
HO
HO
O
OH
OH
O
O
HO
AcHN
O
O
O
O
O
O
O
H
H
H
H
H
H2N
N
N
N
N
N
N
N
N
H
N
N
OH
H
H
H
H
O
O
O
N
O
O
HOOC
NH
23
45%
HO OH
HO
HO
OH
O
O
+
O
HO
AcHN
O
O
O
O
O
H
H
H
N
N
N
N
H
N
H
N
OH
H
O
O
O
N
NH
HO
HO
O
13%
23a
OH
O
OH
O
HO
O
+
HO
AcHN
O
O
O
O
O
H
H
H
H
H
N
N
N
N
N
N
N
H
N
H
N
OH
H
H
O
O
O
N
O
O
HOOC
NH
16%
23b
Scheme 5.
20
1.-8. Fmoc removal, Fmoc-Xaa-OH, 20 eq., HBTU/HOBt, capping
9. manual coupling: Fmoc removal,
Fmoc-2 6-sialyl-T-antigen-OH 19 (2.34 eq.),
HATU/HOAt/DIPEA, 5 h
10.-15. Fmoc removal, Fmoc-Xaa-OH, HBTU/HOBt, capping
16. Fmoc removal
AcO OAc
AcO
COOBn
O
O
AcHN
AcO
OAc
AcO
HO
O
O
AcO
O
AcO
AcHN
O
COOtBu
O
O
O
O
O
O
H
N
H
H
H
H
H
O
N
N
N
N
N
H
N
N
H
N
N
N
HN
O
N
N
Linker
H
H
H
H
O
O
O
O
O
O
O
COOtBu
O
N
HN
O
N
24
Trt
O
H2N
NHBoc
Scheme 6.

S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
6155
24
AcO OAc
AcO
COOBn
O
TFA/TIS/H2O
O
AcHN
AcO
OAc
AcO
HO
O
O
AcO
O
AcO
AcHN
O
COOH
O
O
O
O
O
O
H
N
H
H
H
H
H
O
N
N
N
N
N
H
N
N
H
N
N
N
HN
O
N
N
H
H
H
H
OH
O
O
O
O
O
O
COOH
O
N
HN
O
N
25 42 %
H
O
H2N
NH2
1.H2, Pd/C(10%), MeOH
2.NaOMe/MeOH
HO OH
HO
COOH
O
O
O
AcHN
HO
OH
HO
HO
O
O
O
AcHN
HO
HO
O
COOH
O
O
O
O
O
H
H
H
H
H
H
O
N
N
N
N
N
N
H
N
N
H
N
N
N
HN
O
N
N
H
H
H
H
OH
O
O
O
O
O
O
COOH
O
N
HN
O
N
H
26 overall yield 23 %
O
H2N
NH2
Scheme 7.
Luna C18 column (5 lm, 250 · 4.6 mm) at a pump rate of
1 mL/min. Semi-preparative HPLC were carried out on a
Knauer HPLC system on a Phenomenex Luna C 18 col-
umn at a pump rate of 15 mL/min. Preparative RP-HPLC
purifications were carried out on a Knauer HPLC system
Gradients used for preparative HPLC: gradient h
(SH132): MeCN/H2O (+0.1% TFA) (25:75) ! MeCN/
H2O (+0.1% TFA) (100:0) in 120 min; gradient i
(SH133): MeCN/H2O (+0.1% TFA) (20:80) ! MeCN/
H2O (+0.1% TFA) (30:70) in 120 min; gradient j
(SH150): MeCN/H2O (+0.1% TFA) (40:60) ! MeCN/
H2O (+0.1% TFA) (90:10) in 90 min; gradient k
(SH151): MeCN/H2O (+0.1% TFA) (10:90) ! MeCN/
H2O (+0.1% TFA) (50:50) in 120 min.
on
a
Phenomenex Luna C18 column (10 lm,
250.0 · 50.0 mm) at a pump rate of 30 mL/min. MeCN
and H2O were used (+0.1% TFA) as the solvents.
Gradients used for analytical RP-HPLC: gradient a
(SH76): MeCN/H2O (+0.1% TFA) (30:70) ! MeCN/
H2O (+0.1% TFA) (80:20) in 40 min; gradient b
(SH80): MeCN/H2O (+0.1% TFA) (5:95) ! MeCN/
H2O (+0.1% TFA) (100:0) in 40 min; gradient c
(SH132): MeCN/H2O (+0.1% TFA) (3:97) ! MeCN/
H2O (+0.1% TFA) (100:0) in 40 min; gradient d
(SH133): MeCN/H2O (+0.1% TFA) (10:90) ! MeCN/
H2O (+0.1% TFA) (50:50) in 40 min; gradient e
(Sh150): MeCN/H2O (+0.1% TFA) (10:90) ! MeCN/
H2O (+0.1% TFA) (100:0) in 40 min; gradient f
(SH151): MeCN/H2O (+0.1% TFA) (10:90) ! MeCN/
H2O (+0.1% TFA) (50:50). Gradients used for semi-pre-
parative HPLC: gradient g (SH80) MeCN/H20 (+0.1%
TFA) (5:95) ! MeCN/H2O (+0.1% TFA) (100:0) in
60 min.
1H, 13C and 2 D NMR spectra were recorded on a
Bruker Ac-300 or a Bruker AM-400 spectrometer. The
chemical shifts are correlated to the signal of the deuter-
ated solvent. Multiplicities are given as s (singlet), sb
(broad singlet), d (doublet), t (triplet) or m (multiplet).
In case of complex structures the protons were assigned
by additional COSY and HMQC experiments. In the
synthesized complex carbohydrate structures the incor-
porated carbohydrate structures are named as follows:
N-acetyl-D-galactoseamine: normal, D-galactose: one
apostrophe and D-neuraminic acid: two apostrophes.
As abbreviations for quinic acid Qui and for the couma-
rine-core Cou were choosen. MALDI-TOF mass spectra
were recorded on a Micromass Tofpec E spectrometer,
ESI-mass spectra on a ThermoQuest Navigator spec-

6156
S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
20
1.-8. Fmoc removal, Fmoc-Xaa-OH, HBTU/HOBt, capping
9. manual coupling: Fmoc removal,
Fmoc-2 6-Sialyl-TN-serine-OH 17 (1.02 eq.),
HATU/HOAt/DIPEA, 5 h, capping
AcO OAc
AcO
AcHN
10.-13. Fmoc removal, Fmoc-Xaa-OH, HBTU/HOBt,
capping
14. manual coupling: Fmoc removal,
Ac4quinic-Gly-aminocoumarine-Leu-OH 10 (2 eq.),
HATU/HOAt/DIPEA, 5 h, capping
COOBn
O
O
AcO
AcO
O
O
H
AcO
OAc
AcO
AcO
N
O
O
AcO
N
H
AcHN
O
O
COOtBu
O
O
O
O
O
O
O
H
H
N
H
N
H
H
H
H
O
N
N
N
N
N
H
N
N
N
H
N
N
N
N
H
N
H
H
H
H
O
O
O
O
O
Linker
O
O
O
COOtBu
N
O
N
Trt
27
AcO OAc
AcO
AcHN
COOBn
O
TFA/TIS/H2O
O
AcO
AcO
AcO
O
O
H
N
AcO
OAc
AcO
AcO
O
O
N
H
AcHN
O
O
COOH
O
O
O
O
O
O
O
H
N
H
N
H
H
H
N
H
H
O
N
N
N
N
H
N
H
N
H
N
H
N
N
N
N
N
H
H
H
OH
O
O
O
O
O
O
O
COOH
N
O
N
H
+
28
1. NaOMe/MeOH, pH 9.5
2. NaOH/H2O, pH 10.5
COOtBu
O
O
O
O
H
H
N
H
N
H
N
O
N
H
N
N
H
N
H
N
H
N
HO OH
COOH
O
OH
O
O
O
O
N
O
HO
AcHN
O
O
N
Trt
HO
45%
HO
135
29
O
O
H
HO
HO
HO
N
O
O
HO
AcHN
N
H
O
O
COOH
O
O
O
O
O
O
H
N
H
N
H
H
H
H
N
H
O
OH
N
N
N
N
H
N
N
H
N
H
N
N
H
N
H
N
N
H
H
OH
O
O
O
O
O
O
O
COOH
N
O
N
H
30
Scheme 8.
trometer. Optical rotations [a]D were measured on a
Perkin Elmer polarimeter 241.
UV absorption trypthophane
UV absorption spectra new chromophore
1.0
7.2. Benzyl 2-(7-aminocoumarin-4-yl)-acetate (2)
To 10 mL of benzyl alcohol cooled to ꢀ5 ꢁC, thionyl
chloride (0.36 mL, 5.02 mmol) was added dropwise.
Subsequently 2-(7-aminocoumarin-4-yl)acetic acid17
(1.00 g, 4.56 mmol) was added. The mixture was stirred
at ꢀ5 ꢁC for 15 min and subsequently heated to 55 ꢁC
for 1 h. While cooling to room temperature a yellow sol-
id precipitated. It was washed with diethyl ether. Yield:
1.36 g (87%); mp 170 ꢁC (EtOH); 1H NMR (DMSO-d6),
d: 7.41 (d, 1H, H5-Cou, JH4,H5 = 8.82 Hz); 7.31 (sb, 5H,
Harom.-Bn); 6.74–6.68 (m, 2H, H6-, H8-Cou); 6.13 (s,
1H, H3-Cou); 5.12 (s, 2H, CH2-Bn); 3.93 (s, 2H, CH2-
Cou); 13C NMR (DMSO-d6), d: 169.27, 160.38 (CO);
155.20 (C4-Cou); 149.59 (C9-Cou); 149.30 (C7-Cou);
ption
0.5
absor
rel.
0.0
200
250
300
350
400
wavelength [nm]
Figure 1. UV absorption spectra of tryptophane and chromophore 11.

S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
6157
135.88 (Cipso-Bn); 128.51, 128.22, 128.01 (Carom.-Bn);
126.54 (C5-Cou); 113.35 (C6-Cou); 110.92 (C3-Cou);
110.44 (C10-Cou); 101.73 (C8-Cou); 66.37 (CH2-Bn);
36.88 (CH2-Cou); FD-MS, m/z: 309.5 (M)+, calcd:
309.1.
the residue codistilled with toluene (2·) and dried in vac-
uo. (1S,3R,4S,5R)-1,3,4,5-tetra-O-acetyl-quinic acid 5
(1.53 g, 4.24 mmol, 2 equiv), TBTU (1.36 g, 4.24 mmol,
2 equiv) and diisopropylethylamine (DIPEA, 741 lL)
were dissolved in 10 mL dry DMF and stirred for
15 min at room temperature. After adding DIPEA
(525 lL), the two solutions were combined and brought
into the microwave system for 20 min (50 ꢁC, 50 W).
DMF was removed, the residue diluted with EtOAc
(100 mL) and washed with aq citric acid solution (5%,
2·), satd aq NaHCO3 solution, satd aq NaCl solution,
dried over MgSO4 and evaporated in vacuo. The crude
product was purified by flash-chromatography on silica
gel (EtOAc/cyclohexane 4:1). Yield: 984 mg (75%); col-
ourless, amorphous solid; Rf = 0.20 (EtOAc/cyclohex-
7.3. Benzyl 2-(7-(N-tert-butyloxycarbonyl-glycyl)-amino-
coumarin-4-yl)-acetate (3)
In dry DMF (1.5 mL) Boc-Gly-OH (57 mg, 0.32 mmol,
2 equiv), HATU (123 mg, 0.32 mmol, 2 equiv) and
2,4,6-collidine (43.1 lL) were dissolved. After 5 min of
stirring, benzyl 2-(7-aminocoumarin-4-yl)-acetate
2
(50 mg, 0.16 mmol) was added and the mixture was stir-
red at room temperature for 42 h. After evaporation of
the solvent in vacuo, the residue was diluted by 20 mL
EtOAc, washed with 1 N HCl, satd aq NaHCO3 solu-
tion, satd aq NaCl solution, dried over MgSO4 and
evaporated in vacuo. Purification by flash-chromatogra-
phy on silica gel (EtOAc/cyclohexane 2:1) yielded a col-
ourless, amorphous solid. Yield: 56 mg (74%); Rf = 0.5
(cyclohexane/EtOAc 1:2); 1H NMR (DMSO-d6),
d: 10.40 (s, 1H, NH); 7.75 (d, 1H, H8-Cou,
22
1
ane 4.1); ½aꢁ ꢀ10.0 (c 1.00, DMSO); H NMR (1H,
D
COSY) (CD3OD), d: 7.79 (d, 1H, H8-Cou,
JH6,H8 = 1.96 Hz); 7.53–7.51 (m, 1H, H5-Cou); 7.41
(dd, 1H, H6-cou, JH6,H8 = 2.36 Hz, JH5,H6 = 9.00 Hz);
7.33 (s, 5H, Harom.-Bn); 6.33 (s, 1H, H3-Cou); 5.61 (m,
1H, H3-Qui); 5.50 (m, 1H, H5-Qui); 5.18 (s, 2H, CH2-
Bn); 5.12 (dd, 1H, H4-Qui, JH3,H4 = 3.92 Hz,
JH4,H5 = 10.56 Hz); 4.02 (s, 2H, CH2-Cou); 3.93 (d,
2H, Ga, JNH,Ga = 7.04 Hz); 2.76 (m, H2a-Qui); 2.68–
2.64 (m, 1H, H6a-Qui); 2.55 (1H, H2b-Qui,
JH2a,H3 = 3.52 Hz, JH2a,b = 16.04 Hz); 2.29, 2.15, 2.07,
2.02 (4· s, 12H, CH3-Ac); 2.07–2.04 (m, 1H, H6b-
Qui); 13C NMR (13C, HMQC) (CD3OD), d: 173.31,
172.67, 171.83, 171.70, 171.62, 170.47, 169.72 (CO);
162.65 (C2-Cou); 155.48 (C9-Cou); 150.63 (C4-Cou);
143.10 (C7-Cou); 137.04 (Cipso-Bn); 129.55, 129.36
(Carom.-Bn); 126.82 (C5-Cou); 116.79 (C6-Cou); 116.15
(C10-Cou); 115.68 (C3-Cou); 107.75 (C8-Cou); 82.27
(C1-Qui); 73.27 (C4-Qui); 69.46 (CH2-Bn); 68.20 (C3-
Qui); 67.78 (C5-Qui); 44.39 (Ga); 38.57, 38.32 (C2-Qui,
CH2-Cou); 31.97 (C6-Qui); 21.77, 20.82, 20.64, 20.58
(CH3-Ac); ESI-MS, m/z: 731.1 (M+Na)+, calcd: 731.2.
JH6,H8 = 1.83 Hz); 7.61 (d, 1H, H5-Cou, JH5,H6
=
8.82 Hz); 7.43–7.38 (m, 6H, H6-Cou, Harom-Bn); 7.12
(t, 1H, NHG, JNH,Ga = 5.88 Hz); 6.39 (s, 1H, H3-Cou);
5.14 (s, 2H, CH2–O); 4.04 (s, 2H, CH2-Cou); 3.27 (d,
2H, Ga, JGa,NH = 5.88 Hz); 1.39 (s, 9H, CH3-t-Bu);
13C NMR (DMSO-d6), d: 170.56, 169.26 (CO); 159.96
(C2-Cou); 156.08 (CO-Urethan); 153.97 (C9-Cou);
149.21 (C4-Cou); 142.45 (C7-Cou); 135.85 (Cipso-Bn);
128.52, 128.25, 128.08 (Carom.-Bn); 126.26 (C5-Cou);
115.24 (C6-Cou); 114.35 (C10-Cou); 114.22 (C3-Cou);
105.73 (C8-Cou); 78.27 (Cq-t-Bu); 66.45 (CH2-Bn);
44.04 (Ga); 36.71 (CH2-Cou); 28.31 (CH3-t-Bu); ESI-
MS, m/z: 499.3 (M+Na)+, calcd: 499.2.
7.4. (1S,3R,4S,5R)-1,3,4,5-Tetraacetoxycyclohexane
carboxylic acid (5)21
7.6. 2-(7-{N-[1S,3R,4S,5R]-1,3,4,5-Tetraacetoxycyclo-
hexanoyl-glycyl}-amino-coumarin-4-yl)-acetic acid (7)
Compound 5 was obtained from quinic acid by treat-
ment with acetic acid/aceticanhydride (2:1) and two
drops of 70% H2SO4. Yield 88%; 1H NMR (1H, COSY)
(CDCl3), d: 9.59 (sb, 1H, COOH); 5.52 (q, 1H, H3-Qui,
JH3,H4 = 3.66 Hz); 5.39 (m, 1H, H5-Qui); 4.99 (dd, 1H,
H4-Qui, JH3,H4 = 3.66 Hz, JH4,H5 = 9.93 Hz); 2.67–2.51
(m, 2H, H2a-, H6a-Qui); 2.33 (dd, 1H, H2b-Qui,
JH2b,H3 = 3.69 Hz, JH2a,b = 15.81 Hz); 2.10, 2.03, 2.00,
1.96 (4· s, 12H, CH3-Ac); 1.96–1.86 (m, 1H, H6b-
Qui); 13C NMR (13C, HMQC) (CDCl3), d: 175.32
(COOH); 170.13, 169.95, 169.86, 169.81 (CO); 78.26
(C1-Qui); 71.37 (C4-Qui); 67.51 (C3-Qui); 66.34 (C5-
Qui); 36.60 (C2-Qui); 31.58 (C6-Qui); 20.92, 20.88,
20.67, 20.62 (CH3-Ac).
In MeOH (15 mL) compound 6 (778 mg, 1.11 mmol)
was dissolved and repeatedly degassed. Subsequently
8 mg Pd/C (5%) was added and the argon atmosphere
was exchanged for hydrogen. After stirring for 45 min,
the mixture was filtered through Hyflo Supercel and
evaporated. Purification by flash-chromatography on
silica gel (EtOAc/EtOH/AcOH 6:1:0.1) gave a colour-
22
less, amorphous solid. Yield: 667 mg (98%); ½aꢁ ꢀ21.7
D
(c 1.00, DMSO); 1H NMR (DMSO-d6), d: 10.21 (s,
1H, NH); 8.44 (t, 1H, NHG, JNH,Ga = 5.52 Hz); 7.74
(m, 2H, H5-, H8-Cou); 7.44–7.39 (m, 1H, H6-Cou);
6.34 (s, 1H, H3-Cou); 5.40–5.28 (m, 2H, H3-, H5-Qui);
5.05 (dd, 1H, H4-Qui, JH3,H4 = 3.66 Hz, JH4,H5
J
=
NH,Ga = 5.52 Hz,
10.29 Hz); 3.92 (dd, 1H, Gaa,
7.5. Benzyl 2-(7-{N-[1S,3R,4S,5R]-1,3,4,5-tetraacetoxy-
cyclohexanoyl-glycyl}-amino-coumarin-4-yl)-acetate (6)
JGaa,b = 16.53 Hz); 3.83 (s, 2H, CH2-Cou); 3.75 (dd,
1H, Gab, JNH,Ga = 5.52 Hz, JGaa,b = 16.53 Hz); 2.63–
2.58 (m, 1H, H2a-Qui); 2.49–2.38 (m, 3H, H2b-, H6-
Qui); 2.05, 2.01, 1.93, 1.90 (4· s, 12H, CH3-Ac); 13C
NMR (DMSO-d6), d: 172.25, 170.43, 169.93, 179.87,
169.84, 169.66, 168.29 (CO); 160.09 (C2-Cou); 153.77
(C9-Cou); 153.20 (C4-Cou); 142.11 (C7-Cou); 126.17
(C5-Cou); 115.19 (C6-Cou); 115.08 (C10-Cou); 112.44
A solution of 2-(7-(N-tert-butyloxycarbonyl-glycyl)-
aminocoumarin-4-yl)-acetic acid benzyl ester3 (0.99 g,
2.12 mmol), 2.0 mL trifluoroacetic acid and 0.2 mL
H2O was stirred at room temperature for 1 h. Subse-
quently, the solution of 4 was concentrated in vacuo,

6158
S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
(C3-Cou); 105.56 (C8-Cou); 80.50 (C1-Qui); 71.36 (C4-
Qui); 68.05 (C3-Qui); 66.52 (C5-Qui); 44.39 (Ga); 39.12
(CH2-Cou); 37.37 (C2-Qui); 30.32 (C6-Qui); 21.76,
20.82, 20.58, 18.08 (CH3-Ac); ESI-MS, m/z: 641.2
(M+Na)+, calcd: 641.2.
5.05 (dd, 1H, H4-Qui, JH3,H4 = 3.66 Hz, JH4,H5
10.29 Hz); 4.14–4.09 (m, 1H, La); 3.93 (dd, 1H, Gaa,
NH,Ga = 5.97 Hz, JGaa,b = 16.65 Hz); 3.76 (dd, 1H,
=
J
Gab, JNH,Ga = 5.97 Hz, JGaa,b = 16.65 Hz); 2.62–2.57
(m, 1H, H2a-Qui); 2.52–2.46 (m, 2H, H2b-, H6a-Qui);
2.38 (s, 3H, CH3-Cou); 2.13, 2.05, 2.02, 1.94 (4· s, 13H,
CH3-Ac, H6b-Qui); 13C NMR (DMSO-d6) (13C,
HMQC), d: 170.42, 169.93, 169.87, 169.83, 169.65,
168.27 (CO); 160.09 (C2-Cou); 153.79 (C9-Cou); 153.19
(C4-Cou); 142.09 (C7-Cou); 126.17 (C5-Cou); 115.21
(C6-Cou); 115.10 (C10-Cou); 112.44 (C3-Cou); 105.58
(C8-Cou); 80.51 (C1-Qui); 71.37 (C4-Qui); 68.09 (C3-
Qui); 66.53 (C5-Qui); 43.00 (Ga); 37.37 (C6-Qui); 30.36
(C2-Qui); 21.76, 20.87, 20.82, 20.58 (CH3–CO); 18.08
(CH3-Cou); ESI-MS, m/z: 597.2 (M+Na)+.
7.7. 2-(7-{N-[1S,3R,4S,5R]-1,3,4,5-Tetraacetoxycyclohe-
xanoyl-glycyl}-amino-coumarin-4-yl)-acetyl-L-leucine
tert-butyl ester (8)
A mixture of compound 7 (541 mg, 0.88 mmol), DCC
(181 mg, 0.88 mmol) and HOBt (134 mg, 0.88 mmol)
was stirred in 5 mL of dry DMF at 0 ꢁC for 15 min.
Subsequently, H-Leu-O-t-Bu hydrochloride(196 mg,
0.88 mmol) and NEt3 (123 lL, 0.88 mmol) were added.
After 1.5 h at 0 ꢁC, the mixture was stirred at room tem-
perature for 17 h. The precipitated urea was filtered off
and the solvent evaporated in vacuo. The residue was dis-
solved in EtOAc and the precipitating urea filtered off.
The organic layer was washed with 1 N HCl, satd aq
NaHCO3 solution, satd aq NaCl solution and dried with
MgSO4. The solvent was removed in vacuo. An analytical
7.9. 2-(7-{N-[1S,3R,4S,5R]-1,3,4,5-Tetraacetoxycyclo-
hexanoyl-glycyl}-aminocumarin-4-yl)-acetyl-L-leucine
(10)
The crude product 8/9 (0.5 g) was dissolved in trifluoro-
acetic acid (7 mL), triisopropylsilane (0.5 mL), H2O
(0.5 mL) and stirred at room temperature for 1 h. The
solvent was evaporated in vacuo and the residue codis-
tilled with toluene. Purification by flash-chromatogra-
phy on silica gel (EtOAc/EtOH/AcOH 6:1:0.1) yielded
probe was isolated by HPLC [LUNA C 18, gradient (%
22
D
CH3CN): 30–80 (120 min)]: ½aꢁ ꢀ18.0 (c 0.50, DMSO);
tR = 27.12 min [LUNA C 18 gradient (% CH3CN): 30–
80 (40 min)]; 1H NMR (DMSO-d6) (1H, COSY), d:
10.19 (s, 1H, NH); 8.59 (d, 1H, NHL, JNH,La = 7.71 Hz);
8.43 (t, 1H, NHG, JNH,Ga = 5.88 Hz); 7.74 (d, 1H,
H8-Cou, JH6,H8 = 1.83 Hz); 7.71 (d, 1H, H5-Cou,
a colourless solid. Yield: 400 mg (63%, over two steps);
22
D
Rf = 0.28 (EtOAc/EtOH/AcOH 6:1:0.1); ½aꢁ ꢀ16.5 (c
1
0.5, DMSO); H NMR (DMSO-d6), d: 12.44 (sb, 1H,
JH5,H6 = 8.82 Hz);
7.40
(dd,
1H,
H6-Cou,
COOH); 10.23 (s, 1H, NH); 8.61 (d, 1H, NHL,
JH6,H8 = 1.83 Hz, JH5,H6 = 8.82 Hz); 6.31 (s, 1H, H3-
Cou); 5.39–5.27 (m, 2H, H3-, H5-Qui); 5.05 (dd, 1H,
H4-Qui, JH3,H4 = 3.66 Hz, JH4,H5 = 10.29 Hz); 4.14–
4.09 (m, 1H, La); 3.93 (dd, 1H, Gaa, JNH,Gaa = 5.97 Hz,
JGaa,b = 16.65 Hz); 3.80–3.73 (m, 3H, Gab, CH2-Cou);
2.62–2.57 (m, 1H, H2a-Qui); 2.52–2.46 (m, 2H, H2b-,
H6a-Qui); 2.12, 2.04, 2.01, 1.94 (4· s, 13H, CH3-Ac,
H6b-Qui); 1.70–1.41 (m, 3H, Lb, Lc); 1.35 (s, 9H, CH3-
t-Bu); 0.89 (d, 3H, Ld, JLc, Ld = 6.24 Hz); 0.80 (d, 3H,
Ld, JLc,Ld = 6.60 Hz); 13C NMR (DMSO-d6) (13C,
HMQC), d: 171.64, 170.41, 169.93, 169.86, 169.81,
169.63, 168.32, 167.85 (8C, CO); 159.99 (1C, C2-Cou);
153.92 (1C, C9-Cou); 150.82 (1C, C4-Cou); 142.09 (1C,
C7-Cou); 126.14 (1C, C5-Cou); 114.92 (1C, C6-Cou);
114.58 (1C, C10-Cou); 113.84 (1C, C3-Cou); 105.68
(1C, C8-Cou); 80.71 (1C, Cq-t-Bu); 80.52 (1C, C1-Qui);
71.36 (1C, C4-Qui); 68.09 (1C, C3-Qui); 66.53 (1C, C5-
Qui); 51.34 (1C, La); 43.02 (1C, Ga); 40.61 (1C, Lb);
38.45 (1C, CH2-Cou); 37.36 (1C, C6-Qui); 30.38 (1C,
C2-Qui); 27.66 (3C, CH3-t-Bu); 24.46 (1C, Lc); 21.78,
21.37, 20.87, 20.82, 20.58 (6C, CH3–CO, Ld); ESI-MS,
m/z: 810.4 (M+Na)+; HR-ESI-MS, m/z: 810.3078
(M+Na)+.
J
NH,La = 7.71 Hz); 8.44 (t, 1H, NHG, JNH,Ga = 5.49 Hz);
7.73–7.70 (m, 2H, H5-Cou, H8-Cou); 7.40 (d, 1H, H6-
Cou, JH5,H6 = 8.82 Hz); 6.32 (s, 1H, H3-Cou); 5.39–
5.27 (m, 2H, H3-, H5-Qui); 5.05 (dd, 1H, H4-Qui,
JH3,H4 = 3.69 Hz, JH4,H5 = 10.29 Hz); 4.14–4.09 (m,
1H, La); 3.93 (dd, 1H, Gaa,
JNH,Ga = 5.88 Hz,
JGaa,b = 16.53 Hz),; 3.80–3.73 (m, 3H, Gab, CH2-Cou);
2.62–2.57 (m, 1H, H2a-Qui); 2.52–2.46 (m, 2H, H2b-,
H6a-Qui); 2.12, 2.04, 2.01, 1.94 (4· s, 13H, CH3-Ac,
H6b-Qui); 1.70–1.41 (m, 3H, Lb, Lc); 0.88, 0.80 (2· d,
6H, Ld, JLc,Ld = 6.24 Hz).
13C NMR (DMSO-d6), d: 174.00, 170.41, 169.91, 169.83,
169.63, 168.32, 167.93 (CO); 160.02 (C2-Cou); 153.89
(C9-Cou); 150.91 (C4-Cou); 142.11 (C7-Cou); 126.17
(C5-Cou); 114.92 (C6-Cou); 114.58 (C10-Cou); 113.90
(C3-Cou); 105.65 (C8-Cou); 80.52 (C1-Qui); 71.37 (C4-
Qui); 68.09 (C3-Qui); 66.53 (C5-Qui); 50.58 (La); 43.02
(Ga); 40.61 (Lb); 38.50 (CH2-Cou); 37.36 (C2-Qui);
30.39 (C6-Qui); 24.48 (Lc); 21.77, 21.23, 21.17, 20.85,
20.82, 20.58 (CH3-Ac, Ld); ESI-MS, m/z: 732.3
(M+H)+; HR-ESI-MS, m/z: 732.2633 (M+H)+, calcd:
732.2616.
7.8. 7-{N-(1S,3R,4S,5R)-1,3,4,5-Tetraacetoxycyclohexa-
noyl-glycyl}-amino-4-methyl-coumarine (9)
7.10. 2-(7-{N-[1S,3R,4S,5R]-1,3,4,5-Tetrahydroxycyclo-
hexanoyl-glycyl}-aminocoumarin-4-yl)-acetyl-L-leucine
(11)
The compound was separated by analytical HPLC as an
analytical probe (see, 8): Colourless solid; tR = 17.0 min;
Compound 10 (20 mg, 0.027 mmol) was dissolved in
2 mL MeOH. NaOMe/MeOH (0.1 M) was added drop-
wise until a pH of 10.5 was reached. After 19 h stirring
at room temperature, the solution was neutralized by
addition of AcOH, and the sovent was evaporated in vac-
22
1
½aꢁ ꢀ16.7 (c 1.00 MeOH); H NMR (DMSO-d6), d:
D
10.18 (s, 1H, NH); 8.43 (t, 1H, NHG, JNH,Ga = 5.52 Hz);
7.72–7.69 (m, 2H, H5-, H8-Cou); 7.40 (m, 1H, H6-Cou);
6.26 (s, 1H, H3-Cou); 5.40–5.27 (m, 2H, H3-, H5-Qui);

S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
6159
uo. Purification was performed by semi-preparative
HPLC [LUNA C-18, gradient (% CH3CN+0.1% TFA):
5–100, 60 min]. Yield: 11 mg (73%); ½aꢁ ꢀ16.7 (c 1,
acid benzyl ester29 (a-NeuAc45NAcCOOBnXan) 14
(773 mg, 1.15 mmol) in 15 mL dry CH3CN/CH2Cl2
22
D
˚
(2:1), 1 g molecular sieve (4A) was added. The mixture
MeOH); tR = 14.80 min [LUNA C 18, gradient (%
CH3CN+0.1% TFA): 5–100, 40 min]; 1H NMR
(CD3OD), d: 7.81–7.75 (m, 2H, H5-Cou-, H8-Cou);
7.48 (d, 1H, H6-Cou, JH5,H6 = 8.43 Hz); 6.40 (s, 1H,
H3-Cou); 4.51–4.48 (m, 1H, La); 4.20–4.03 (m, 4H, H3-
Qui, Ga, H5-Qui); 3.85 (s, 2H, CH2-Cou); 3.48–3.43 (m,
1H, H4-Qui); 2.10–1.97 (m, 4H, H2-, H6-Qui); 1.73–
1.65 (m, 3H, Lb, Lc); 1.00 (d, 3H, Ld, JLc, Ld = 5.52 Hz);
0.95 (d, 3H, Ld, JLc,Ld = 5.61 Hz); 13C NMR (CD3OD),
d: 178.05, 170.91, 169.71 (4C, CO); 162.90 (1C, C2-
Cou); 155.62 (1C, C9-Cou); 151.92 (1C, C4-Cou);
143.47 (1C, C7-Cou); 126.94 (1C, C5-Cou); 116.86 (1C,
C6-Cou); 116.38 (1C, C10-Cou); 115.50 (1C, C3-Cou);
107.87 (1C, C8-Cou); 78.07 (1C, C4-Qui); 77.03 (1C,
C1-Qui); 72.21 (1C, C3-Qui); 68.19 (1C, C5-Qui); 42.44
(1C, Ga); 43.89 (1C, Lb) 41.47 (1C, C2-Qui); 39.74 (1C,
C6-Qui); 38.83 (1C, CH2-Cou); 26.16 (1C, Lc); 23.42
(1C, Ld); 21.67 (1C, Ld); ESI-MS, m/z: 564.3 (M+H)+;
HR-ESI-MS, m/z: 586.2040 (M+Na)+.
was stirred at room temperature for 1 h and subse-
quently cooled to ꢀ65 ꢁC. Anhydrous silver trifluo-
romethanesulfonate (AgOTf) (299 mg, 1.17 mmol)
was added under exclusion of light. Over a period
of 20 min, 719 lL methylsulfenyl bromide [MSB, pre-
pared by addition of Br2 (205 lL, 4 mmol) to a solu-
tion of dimethyl disulfide (355 lL, 4 mmol) in 1,2-
dichlorethane (5 mL) after stirring for 15 h under
exclusion of oxygen and light and cooled to 0 ꢁC]
was added dropwise.
After stirring for 4.5 h, N-ethyldiisopropylamine
(0.39 mL) was added. The stirring was continued at
ꢀ65 ꢁC for another 15 min. Then the mixture was
warmed to room temperature, diluted with CH2Cl2
(20 mL) and filtered through Hyflo Supercel. The filtrate
was evaporated in vacuo and the residue purified by
flash-chromatography on silica gel (EE ! EE/EtOH
50:1 ! EE/EtOH 40:1). Yield: 290 mg (58%); Rf = 0.15
22
(CH2Cl2/MeOH 20:1); ½aꢁ 4.0 (c 1.0, CH2Cl2); 1H
D
NMR (1H, COSY) (CDCl3), d: 7.72 (d, 2H, H4-, H5-
Fmoc, JH3,H4 = JH5,H6 = 7.44 Hz); 7.56 (d, 2H, H1-,
H8-Fmoc, JH1,H2 = JH7,H8 = 7.04 Hz); 7.40–7.28 (m,
9H, H2-, H3-, H6-, H7-Fmoc, Harom.-Bn); 6.51 (d,
1H, NHAc, JNH,H2 = 7.35 Hz); 5.83 (d, 1H, NH–Fmoc,
7.11. N-(9H-Fluoren-9-yl-methoxycarbonyl)-O-(2-acetami-
do-2-desoxy-a-D-galacto-pyranosyl)-L-serine tert-butyl ester
(13) [Fmoc-Ser(a-GalNAc)-O-t-Bu]
Fmoc-Ser(a-Ac3GalNAc)-O-t-Bu25
12
(500 mg,
J
NH,Sa = 9.45 Hz); 5.32–5.25 (m, 2H, H700 (5.31), H800
0.70 mmol) was dissolved in 15 mL dry MeOH. Fresh
NaOMe/MeOH solution (0.1 mol) was added until a
pH of 8.5 was reached. The reaction was terminated
after 3 h by addition of ion exchange resin Amberliteꢂ
IR-120. After filtration, evaporation of the solvent in
vacuo gave the crude product which was purified by
flash-chromatography on silica gel (EtOAc ! EtOH).
(5.28)); 5.22–5.14 (m, 2H, CH2–Bn); 4.83–4.72 (m, 2H,
H400 (4.80), H1 (4.73)); 4.41–4.20 (m, 6H, Sa (4.38),
CH2–Fmoc (4.34), H9a00 (4.31), H2 (4.23), H9-Fmoc
(4.21)); 4.07–4.00 (m, 3H, H500 (4.05), H600 (4.04),
H9b00 (4.02)); 3.92–3.50 (m, 7H, Sba (3.89), H6a (3.84),
Sbb (3.73), H4 (3.76), H5 (3.69), H3 (3.68), H6b
1
(3.51)); 2.58 (dd, 1H, H300 ; JH3 eq;H4 ¼ 4:72 Hz;
Yield: 275 mg (67%) Rf = 0.16 (toluene/EtOH 5:1). H
00
00
eq
NMR (CD3OD), d: 7.81 (d, 2H, H4-, H5-Fmoc,
JH3,H4 = JH5,H6 = 7.71 Hz); 7.69 (d, 2H, H1-, H8-Fmoc,
JH1,H2 = JH7,H8 = 6.63 Hz); 7.44–7.31 (m, 4H, H2-, H3-,
H6-, H7-Fmoc); 4.83 (d, 1H, H1, JH1,H2 = 3.69 Hz);
4.48–4.22 (m, 5H, CH2-Fmoc, Sba, H2, H9-Fmoc);
3.92–3.76 (m, 7H, Sbb, Sa, H3, H4, H5, H6); 1.99 (s,
3H, CH3–NHAc); 1.49 (s, 9H, CH3-t-Bu).
00
00
JH3 eq;H3 ax ¼ 12:92 Hz); 2.11, 2.03, 2.01, 1.98 (4· s,
18H, CH3-Ac); 1.91 (m, 1H, H30a0x); 1.44 (s, 9H, CH3-
t-Bu); 13C NMR (13C, HMQC) (CDCl3), d: 173.39,
170.95, 170.71, 170.25, 170.07, 169.59 (CO); 167.41
(C100); 155.90 (CO-urethane); 143.63 (C1a-, C8a-Fmoc);
141.23 (C4a-, C5a-Fmoc); 134.76 (Cipso-Bn); 128.72,
128.56, 128.42, 127.74, 127.06 (C3-, C6-, C2-, C7-Fmoc,
Carom.-Bn (5C)); 124.97 (C1-, C8-Fmoc); 119.98 (C4-,
C5-Fmoc); 98.90 (C1); 98.84 (C200); 82.98 (Cq-t-Bu);
72.83 (C600); 70.50 (C3); 69.30 (C5, C700); 68.90 (C400);
68.81 (Sb); 68.25 (C4); 67.87 (CH2–Bn); 67.44 (C800);
67.32 (CH2-Fmoc); 63.80 (C6); 62.65 (C900); 54.90 (Sa);
50.64 (C2); 49.16 (C500); 47.21 (C9-Fmoc); 37.50 (C300);
27.96 (CH3-t-Bu); 23.08, 22.85, 21.03, 20.75 (CH3-Ac,
CH3–NHAc); ESI-MS, m/z: 1158.6 (M+Na)+, 1174.6
(M+K)+, calcd: 1158.4, 1174.5.
13C NMR (CD3OD), d: 173.79, 171.06 (CO); 158.59
(CO-urethane); 145.24 (C1a-, C8a-Fmoc); 142.63 (C4a-,
C5a-Fmoc); 128.86 (C3-, C6-Fmoc); 128.23 (C2-, C7-
Fmoc); 126.16 (C1-, C8-Fmoc); 121.00 (C4-, C5-Fmoc);
100.03 (C1); 83.43 (Cq-t-Bu); 72.92 (Sb); 70.28, 69.73,
68.90 (C3, C4, C5); 67.99 (CH2-Fmoc); 62.79 (C6);
56.63 (Sa); 51.20 (C2); 47.25 (C9-Fmoc); 28.36 (CH3-t-
Bu); 23.03 (CH3-Ac); ESI-MS, m/z: 609.2 (M+Na)+,
calcd: 609.2.
7.13. N-(9H-Fluoren-9-ylmethoxycarbonyl)-O-(2-acetami-
do-3,4-di-O-acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,
7,8,9-tetra-O-acetyl-3,5-dideoxy-a-D-glycero-D-galacto-
2-nonulopyranosyl)onate]-a-D-galactopyranosyl)-L-serine
tert-butyl ester [Fmoc-Ser(a-NeuAc45NAcCOOBn-(2,6)-
a-Ac2GalNAc)-O-t-Bu] (16)
7.12. N-(9H-Fluoren-9-ylmethoxycarbonyl)-O-(2-acetami-
do-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-a-D-glycero-D-galacto-2-nonulopyr-
anosyl)onate]-a-D-galactopyranosyl)-L-serine tert-butyl ester
[Fmoc-Ser(a-NeuAc45NAcCOOBn-(2,6)-a-GalNAc)-O-
t-Bu] (15)
At 0 ꢁC Fmoc-Ser(a-NeuAc45NAcCOOBn-(2,6)-a-Gal-
NAc)- O-t-Bu 15 (290 mg, 0.26 mmol) was dissolved in
3.5 mL of pyridine. Ac2O (2 mL) was added dropwise.
To a solution of Fmoc-Ser(a-GalNAc)-O-t-Bu 13
(259 mg, 0.44 mmol) and xanthate of acetylated sialic

6160
S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
After stirring for 19 h, the mixture was diluted with
30 mL CH2Cl2. Ice was added, and the mixture was
extracted three times with 90 mL CH2Cl2. The combined
organic layers were washed with satd aq NaHCO3 solu-
tion (2·) and satd aq NaCl solution, dried (MgSO4) and
the solvent was evaporated in vacuo. The crude product
2.65 Hz, JH2,H3 = 11.51 Hz); 4.85 (d, 1H, H1, JH1,H2
H400,
=
¼
00
00
2.45 Hz);
4.68
0
(dt,
1H,
JH3 eq;H4
2:60 Hz; JH3 ax;H4 ¼ 11:58 Hz); 4.33 (d, 2H, Sb,
00
J
Sa,Sb = 6.50 Hz); 4.28–3.65 (m, 6H, CH2-Fmoc, H9-
Fmoc, H900, Sa); 4.20–4.15 (m, 1H, H2); 4.05–3.96 (m,
2H, H5, H600); 3.90–3.82 (m, 1H, H500); 3.82–3.68 (m,
1H, H6a); 3.10 (dd, 1H, H6b, JH5,H6b = 5.52 Hz,
JH6a,b = 8.70 Hz); 2.53 (dd, 1H, H3eq;JH3 eq;H4
was purified by flash-chromatography on silica gel
22
00
00
00
(EtOAc). Yield: 203 mg (65%); Rf = 0.15 (EtOAc); ½aꢁ
¼
D
23.2 (c 1, CHCl3); H NMR (1H, COSY) (CDCl3), d:
7.73 (d, 2H, H4-, H5-Fmoc, JH3,H4 = JH5,H6 = 7.44 Hz);
7.59 (d, 2H, H1-, H8-Fmoc, JH1,H2 = JH7,H8 = 7.04 Hz);
7.39-7.27 (m, 9H, H2-, H3-, H6-, H7-Fmoc, Harom.-Bn);
4:20 Hz; JH3 eq;H3 ax ¼ 12:30 Hz); 2.04, 2.01, 1.98, 1.95,
1
00
00
1.91, 184, 1.80 (7· s, 25H, CH3-Ac, H300 ); 13C NMR
ax
(13C, HMQC) (DMSO-d6), d: 170.20, 169.79, 169.53,
169.00 (CO, C100); 158.85 (CO-urethane); 143.80 (C1a-,
C8a-Fmoc); 141.00 (C4a-, C5a-Fmoc); 134.87 (Cipso-
5.79, 5.65 (2· d, 2H, NH-Fmoc, NHAc, JNH,Sa
=
9.76 Hz, JNH,H2 = 8.24 Hz); 5.33–5.24 (m, 2 H, H700
(5.30), H800 (5.26)); 5.18–5.11 (m, 3H, H4 (5.16), CH2-
Bn (5.12)); 5.00 (dd, 1H, H3, JH3,H4 = 2.58 Hz,
JH2,H3 = 11.40 Hz); 4.80–4.72 (m, 2H, H1 (4.78), H400
(4.77)); 4.59–4.49 (m, 1H, H2); 4.45–4.32 (m, 3H,
CH2a-Fmoc (4.41), Sa (4.38), CH2b-Fmoc (4.35));
4.25–4.20 (m, 2H, H9a00 (4.23), H9-Fmoc (4.22)); 4.05–
3.87 (m, 5H, H500 (4.03), H9b00 (4.03), H600 (4.01), Sba,
H5 (3.90)); 3.82–3.74 (m, 2H, Sbb (3.80), H6a (3.77));
Bn); 129.02, 128.63, 128.20, 127.53, 127.10 (C3-, C6-,
C2-, C7-Fmoc, Carom.-Bn (5C)); 125.89, 125.07 (C1-,
C8-Fmoc); 120.10 (C4-, C5-Fmoc); 98.57 (C1); 98.09
(C200); 72.53 (C600); 69.35 (C5); 68.00 (C700); 68.89 (Sb);
68.76 (C400); 68.52 (C3); 68.10 (CH2-Bn); 67.40 (CH2-
Fmoc); 67.20 (C4); 66.95 (C800); 63.00 (C6); 62.10
(C900); 58.54 (Sa); 49.00 (C500); 47.21 (C2); 47.10 (C9-
Fmoc); 38.40 (C300); 23.20, 23.14, 20.99, 20.75, 20.67,
20.62 (CH3-Ac, CH3–NHAc); ESI-MS, m/z: 1168.7
(M+Na)+, 1202.9 (M+K)+, calcd: 1186.4, 1202.4.
3.12 (dd, 1H, H6b, JH5,H6b = 5.48 Hz, JH6a,b
00
=
10.56 Hz); 2.55 (dd, 1H, H300 ; JH3 eq;H4 ¼ 4:68 Hz;
00
eq
00
JH3 eq;H3 ax ¼ 12:88 Hz); 2.06, 2.01, 1.98, 1.96, 1.91 (5·
7.15. General procedure for the automated solid-phase
glycopeptide synthesis
00
s, 24H, CH3-Ac); 1.88 (m, 1H, H30a0x.); 1.45 (s, 9H,
CH3-t-Bu); 13C NMR (13C, HMQC) (CDCl3), d:
170.90, 170.82, 170.65, 170.30, 170.12, 169.89, 169.70,
169.59 (CO); 167.30 (C100); 155.60 (CO-urethane);
143.71 (C1a-, C8a-Fmoc); 141.15 (C4a-, C5a-Fmoc);
134.56 (Cipso-Bn); 128.62, 128.42, 128.40, 127.84,
127.12 (C3-, C6-, C2-, C7-Fmoc, Carom.-Bn (5C));
125.07 (C1-, C8-Fmoc); 120.00 (C4-, C5-Fmoc); 98.88
(C1); 98.58 (C200); 82.87 (Cq-t-Bu); 72.70 (C600); 69.30
(C5); 68.10 (C700); 68.87 (Sb); 68.85 (C400); 68.70 (C3);
68.00 (CH2–Bn); 67.35 (CH2-Fmoc); 67.31 (C4); 67.10
(C800); 63.52 (C6); 62.43 (C900); 54.82 (Sa); 49.12 (C500);
47.31 (C2); 47.16 (C9-Fmoc); 37.60 (C300); 27.98 (CH3-
t-Bu); 23.20, 23.14, 20.99, 20.75, 20.67, 20.62 (CH3-Ac,
CH3–NHAc); ESI-MS, m/z: 1254.7 (M+Na)+, calcd:
1254.5.
The solid-phase syntheses were carried out in a Perkin
Elmer ABI 433A peptide synthesizer using Tentagelꢂ
resin (263 mg, 0.05 mmol) equipped with Wang linker31
and loaded with Fmoc valine 20 as the C-terminal ami-
no acid. Removal of the Fmoc group was performed
with piperidine (20%) in NMP in cycles of 4 min. As a
rule, this treatment was repeated. The coupling reactions
were carried out with Fmoc amino acids (1 mmol,
20 equiv) in a solution of 1 mmol of HBTU, 1 mmol
of HOBt and 2 mmol ethyl-diisopropylamine (DIPEA)
in NMP/DMF (1:1) within 15–17 min. For glycosylated
Fmoc amino acids less excess and extended reaction
times were applied. Unreacted amino groups were acet-
ylated using a solution of acetic anhydride (0.5 M), DI-
PEA (0.125 M) and HOBt (0.015 M) in NMP. After
each cycle, the resin was washed with NMP and
dichloromethane.
7.14. N-(9H-Fluoren-9-ylmethoxycarbonyl)-O-(2-acetami-
do-3,4-di-O-acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,
9-tetra-O-acetyl-3,5-didesoxy-a-D-glycero-D-galacto-2-non-
ulopyranosyl)onate]-a-D-galactopyranosyl)-L-serine [Fmoc-
Ser(a-NeuAc45NAcCOOBn-(2,6)-a- Ac2GalNAc)-OH] (17)
7.16. L-Leucyl-L-alanyl-L-alanyl-L-leucyl-L-aspartyl-L-ser-
yl-O-(2-acetamido-2-deoxy-3-O-[-b-D-galactopyranosyl]-
a-D-galactopyranosyl)-L-histidyl-L-glycyl-L-alanyl-L-iso-
leucyl-L-valine (23)
Compound 16 (135 mg, 0.11 mmol) was dissolved in
5 mL of dichloromethane, 3 mL of trifluoroacetic acid
and 0.5 mL of anisole and stirred at room temperature
for 6 h. Subsequently, the solvents were evaporated in
vacuo and the crude product codistilled with toluene
(3·). Purification was accomplished by flash-chromatog-
7.16.1. (H-Leu-Ala-Ala-Leu-Asp-Ser(b-Gal-(1,3)-a-Gal-
NAc)-His-Gly-Ala-Ile-Val-OH). The synthesis was car-
ried out according to the general protocol using
0.263 mg (0.05 mmol) of resin 20. After Fmoc-removal
from histidine, the resin was treated with a freshly
prepared solution of 95 mg (0.20 mmol, 4 equiv) of
Fmoc-Ser(b-Ac4Gal-(1 ! 3)-a- Ac2GalNAc)-OH 18,
30 mg HOAt (0.19 mmol), 82 mg HATU (0.21 mmol)
and 48 lL (0.29 mmol) DIPEA in NMP under vigor-
ous orbital shaking for 5 h. The coupling reactions
of the further Fmoc amino acids (20 equiv) again were
performed according to the standard protocol. After
coupling of the N-terminal leucine and Fmoc removal,
raphy on silica gel (EE ! EE/MeOH 2:1). Yield: 119 mg
22
(92%); Rf = 0.28 (EtOAc/MeOH 2:1); ½aꢁ 24.2 (c 1,
D
CHCl3); H NMR (1H, COSY) (DMSO-d6), d: : 8.11
(sb, 1 H, NH); 7.78 (d, 2H, H4-, H5-Fmoc,
JH3,H4 = JH5,H6 = 7.43 Hz); 7.75 (d, 1H, NHAc,
JH2,NH = 9.78 Hz); 7.60 (d, 2H, H1-, H8-Fmoc,
JH1,H2 = JH7,H8 = 7.40 Hz); 7.39–7.27 (m, 9H, H2-,
H3-, H6-, H7-Fmoc, Harom.-Bn); 5.30–5.11 (m, 5H,
1
H4, H700, H800, CH2–Bn); 4.98 (dd, 1H, H3, JH3,H4
=

S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
6161
no acetylation was performed. Release of the glyco-
7.17. N-Acetyl-L-seryl-O-(2-acetamido-2-deoxy-3-O-[b-
peptide from resin 21 was accomplished with 15 mL
TFA, 0.9 mL of H2O and 0.9 mL TIS and orbital
shaking for 2 h. After filtration and washing three
times with THF (10 mL each) the solvents were evap-
orated in vacuo. The residue was washed with cold
diethyl ether (30 mL) and the crude 22 lyophilized.
Yield: 81 mg.
D-galactopyranosyl]-a-D-galactopyranosyl)-L-histidyl-L-
glycyl-L-alanyl-L-isoleucyl-L-valine (23a)
7.17.1. (Ac-Ser(b-Gal-(1,3)-a-GalNAc)-His-Gly-Ala- Ile-
Val-OH). Yield 7 mg (13%, from 0.05 mmol resin 20).
MALDI-TOF-MS (positive, dhb), m/z: 1013.7
([M+Na]+, calcd: 1013.0).
ESI-MS (positive), m/z: 1707.0 ([M+Na]+, calcd:
1706.7).
1H NMR (400 MHz, D2O) (1H, COSY), d: 8.60 (s, 1H,
He1); 7.31 (s, 1H, Hd2); 4.86–4.70 (m, 2H, H1 (4.84), Ha
After mass-spectrometric investigation, the crude prod-
uct 23 was dissolved in 20 mL MeOH. Freshly prepared
NaOMe-solution was added until a pH of 9.5 was
reached. The solution was stirred for 68 h and then neu-
tralized by addition of a few drops of acetic acid. Evapo-
ration of the solvent yielded crude 23 (92 mg). Purification
by preparative HPLC [LUNA C-18, gradient (%
CH3CN+0.1% TFA): 3–20 (75 min)–20(140 min)] gave
pure 23: Yield: 32 mg (45%); colourless amorphous solid;
(4.75), under H2O-signal); 4.53 (t, 1H, Sa, JSa,Sb
=
6.28 Hz); 4.43 (d, 1H, H10, JH1 ;H2 ¼ 7:40 Hz); 4.33–
4.25 (m, 2H, Aa, H2); 4.23–4.17 (m, 3H, Va, Ia, H4);
3.97–3.82 (m, 5H, H3, H5, H40, Ga, Sba); 3.78–3.65
(m, 5H, C6, C60, Sbb); 3.64–3.57 (m, 2H, H30, H50);
3.32–3.28 (m, 1H, Hba); 3.22–3.15 (m, 1H, Hbb); 2.18–
2.10 (m, 1H, Vb); 2.02, 1.98 (2· s, 6H, CH3-Ac); 1.88–
1.80 (m, 1H, Ib); 1.53–1.45 (m, 1H, Ica); 1.36 (d, 3H,
Ab, JAb,Aa = 7.44 Hz); 1.22–1.14 (m, 1H, Icb); 0.92 (d,
0
0
tR = 38.55 min [LUNA C-18, gradient (% CH3CN+0.1%
6H, Vc, JVc,Vb = 7.04 Hz); 0.83 (t, 3H, Ic, JIc,Ib
=
7.44 Hz).
22
D
TFA): 3–21(40 min)–21(60 min)]; ½aꢁ ꢀ32.4 (c 1.4,
H2O).
13C NMR (100.6 MHz, D2O) (1H, HMQC), d: 104.94
(1C, C10); 98.41 (1C, C1); 77.33 (1C, C3); 75.22 (1C,
C50); 72.81 (1C, C30); 71.23 (1C, C40); 70.83 (1C, C20);
68.77 (2C, C4, C5); 67.20 (1C, Sb); 61.22 (2C, C6,
C60); 58.71 (1C, Va); 54.13 (1C, Sa); 52.62 (1C, Ha);
49.90 (1C, Aa); 48.51 (1C, C2); 42.38 (1C, Ga); 36.20
(1C, Ib); 30.24 (1C, Vb); 26.75 (1C, Hb); 24.60 (1C, Ic);
22.40, 21.95 (2C, CH3-Ac); 18.10 (2C, Vc); 16.72 (1C,
Ab); 14.80 (1C, Ic); 10.15 (1C, Id).
ESI-MS (positive), m/z: 1432.2 ([M+H]+, calcd:
1432.5); 1454.5 ([M+Na]+, calcd: 1454.5); 1470.3
([M+K]+, calcd: 1470.6); 1492.5 ([M+Na+K-H]+, calcd:
1492.6).
HR-ESI-MS (positive), m/z: 1431.7188 ([M+H+], calcd:
1431.7219).
1H NMR (400 MHz, D2O) (1H, COSY), d: 8.61 (s, 1H,
He1); 7.31 (s, 1H, Hd2); 4.85–4.64 (m, 3H, H1 (4.83), Da
(4.72), Ha (4.71), under H2O-signal); 4.54 (t, 1H, Sa,
7.18. N-Acetyl-L-alanyl-L-leucyl-L-aspartyl-L-seryl-O-(2-
acetamido-2-deoxy-3-O-[b-D-galactopyranosyl]-a-D-
galactopyranosyl)-L-histidyl-L-glycyl-L-alanyl-L-isoleu-
cyl-L-valine (23b)
0
0
J
Sa,Sb = 5.28 Hz); 4.44 (d, 1H, H10, JH1 ;H2 ¼ 7:44 Hz);
4.37–4.16 (m, 8H, 3· Aa (4.36–4.26), H2 (4.31), La
(4.29), Va (4.22), Ia (4.19), H4 (4.19)); 4.00–3.82 (m,
7H, H3 (3.98), Ga (3.99, 3.90), La (3.96), Sba (3.93),
H5 (3.89), H40 (3.85)); 3.80–3.66 (m, 5H, Sbb (3.78),
C6 (3.75–3.66), C60 (3.75–3.66)); 3.64–3.57 (m, 2H,
H50 (3.62), H30 (3.58)); 3.51–3.47 (m, 1H, H20); 3.31–
3.28 (m, 1H, Hba); 3.22–3.15 (m, 1H, Hbb); 2.96–
2.78 (m, 2H, Db); 2.17–2.10 (m, 1H, Vb); 1.99 (2· s,
6H, CH3-Ac); 1.87–1.80 (m, 1H, Ib); 1.66–1.40 (m,
7H, 2· Lc (1.71–1.58), 2· Lb (1.75–1.55), Ica (1.51));
1.36 (m, 9H, Ab); 1.23–1.13 (m, 1H, Icb); 0.92–0.80
(m, 18H, 4· Ld (0.94, 0.85), Ic (0.89), Vc (0.90), Id
(0.83)).
7.18.1. (Ac-Ala-Leu-Asp-Ser(b-Gal-(1,3)-a-GalNAc)-
His-Gly-Ala-Ile-Val-OH). Yield: 10 mg (16%, from
0.05 mmol resin).
MALDI-TOF (positive, dhb), m/z: 1289.7 ([M+H]+,
calcd: 1289.6); 1312.0 ([M+Na]+, calcd: 1311.6); 1334.0
([M+2Na–H]+, calcd: 1333.6).
1H NMR (400 MHz, D2O) (1H, COSY), d: 8.60 (s, 1H,
He1); 7.30 (s, 1H, Hd2); 4.85–4.64 (m, 3H, H1 (4.83), Ha
(4.71), Da (4.67), under H2O-signal); 4.54 (t, 1H, Sa,
0
0
J
Sa,Sb = 5.28 Hz); 4.44 (d, 1H, H10, JH1 ;H2 ¼ 7:84 Hz);
13C NMR (100.6 MHz, D2O) (13C, HMQC), d: 104.98
(1C, C10); 98.27 (1C, C1); 77.40 (1C, C3); 75.11 (1C,
C50); 72.71 (1C, C30); 71.21 (1C, C40); 70.80 (1C, C20);
68.81 (1C, C4); 68.80 (1C, C5); 67.27 (1C, Sb); 61.41
(2C, C6, C60); 58.60 (1C, Va); 58.42 (1C, Ia); 53.80
(1C, Sa); 52.74 (1C, Ha); 51.90 (1C, La); 50.09 (1C,
Da); 49.81 (2C, Aa); 49.70 (1C, La); 48.51 (1C, C2);
42.31 (1C, Ga); 39.85 (2C, Lb); 36.12 (1C, Ib); 35.60
(1C, Db); 30.11 (1C, Vb); 26.78 (1C, Hb); 24.23
(2C, Lc); 22.39 (2C, CH3-Ac); 21.67, 21.03 (4C, Ld);
18.37 (2C, Vc); 16.78 (3C, Ab); 14.80 (1C, Ic); 10.11
(1C, Id).
4.37–4.26 (m, 3H, Aa (4.31), La (4.31), H2 (4.29));
4.22–4.15 (m, 4H, Aa (4.19), H4 (4.19), Va (4.18), Ia
(4.17)); 3.97–3.82 (m, 6H, H3 (3.98), Ga (3.98, 3.90),
Sba (3.93), H5 (3.89), H40 (3.86)); 3.80–3.67 (m, 5H,
Sbb (3.79), C6 (3.71–3.67), C60 (3.71–3.67)); 3.64–3.57
(m, 2H, H50 (3.62), H30 (3.57)); 3.52–3.48 (m, 1H,
H20); 3.31–3.28 (m, 1H, Hba); 3.22–3.15 (m, 1H, Hbb);
2.91–2.78 (m, 2H, Db); 2.15–2.10 (m, 1H, Vb); 1.98 (2·
s, 6H, CH3-Ac); 1.87–1.80 (m, 1H, Ib); 1.66–1.43 (m,
4H, Lc (1.63), Lb (1.58), Ica (1.48)); 1.33 (m, 6H, Ab);
1.20–1.15 (m, 1H, Icb); 0.92–0.80 (m, 18H, 2· Ld
(0.92, 0.85), Ic(0.90), Vc (0.90), Id (0.84)).

6162
S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
13C NMR (100.6 MHz, D2O) (13C, HMQC), d/: 104.94
(1C, C10); 98.31 (1C, C1); 77.50 (1C, C3); 75.24 (1C,
C50); 72.80 (1C, C30); 71.25 (1C, C40); 70.84 (1C, C20);
68.80 (1C, C5); 68.75 (1C, C4); 67.20 (1C, Sb); 61.20
(2C, C6, C60); 58.80 (1C, Va); 58.71 (1C, Ia); 53.94
(1C, Sa); 52.81 (1C, Ha); 50.27 (1C, Da); 49.90 (2C,
Aa); 49.81 (1C, La); 48.50 (1C, C2); 42.29 (1C, Ga);
39.71 (1C, Lb); 36.10 (1C, Ib); 35.90 (1C, Db); 30.06
(1C, Vb); 26.81 (1C, Hb); 24.41 (1C, Lc); 22.41, 20.90
(2C, Ld); 21.80 (2C, CH3-Ac); 18.49 (2C, Vc); 16.78
(2C, Ab); 14.90 (1C, Ic); 10.21 (1C, Id).
7H, Da (4.66), Ha (4.66), Pa (4.57), Sa (4.53), Aa
(4.48), Aa (4.46), La (4.46)); 4.44–3.80 (m, 24H, H2
(4.43), Aa (4.34), Aa (4.33), La (4.33), CH2-Fmoc
(4.30), Va (4.29), Ia (4.29), H900 (4.27, 4.09), Va (4.24),
Ga (4.19, 4.14), H600 (4.12), H50 (4.11), Qa (4.00), H4
(4.00), Ga (3.99, 3.90), H9-Fmoc (3.98), H5 (3.87),
H6a (3.84), H3 (3.83)); 3.70–3.40 (m, 4H, Pd (3.62,
3.58), C6b (3.53), Hba (3.41)); 3.30–3.20 (m, 1H, Hbb
(3.21)); 3.05–2.75 (m, 2H, Db (2.93)); 2.57–2.50 (m,
00
1H, H3 ); 2.30–1.98 (m, 34H, Pb (2.19, 2.02), Vb
eq
00
(2.18), H3 (2.14), Qb (2.13), Vb (2.08), Pc (2.02),
ax
CH3-Ac); 1.90 (s, 3H, CH3-Ac); 1.84–1.58 (m, 7H, Ib
(1.84), 2· Lc (1.73), 2· Lb (1.63)); 1.58–1.38 (m, 10H,
Ica (1.57), 3· Ab); 1.30–1.15 (m, 1H, Icb); 1.10–0.88
(m, 30H, 4· Vc (0.99, 0.91), 4· Ld (0.95, 0.91, 0.97), Ic
(0.90), Id (0.87)).
7.19. L-Glutamyl-L-leucyl-L-alanyl-L-alanyl-L-leucyl-L-as
partyl-L-seryl-O-(2-acetamido-2-deoxy-3-O-[2,3,4,6-tetra-
O-acetyl-b-D-galactopyranosyl]-6-O-[benzyl-(5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-D-glycero-D-galacto-
2-nonulopyranosyl)onate]-a-D-galactopyranosyl)-L-histidyl-
L-glycyl-L-alanyl-L-isoleucyl-L-valyl-L-aspartyl-L-glycyl-
L-prolyl-L-valine (25)
13C NMR (100.6 MHz, CD3OD) (13C, HMQC), d:
102.68 (1C, C10); 99.21 (1C, C1); 78.19 (1C, C3); 73.12
(1C, C600); 72.00 (1C, C20); 71.52 (1C, C50); 70.22 (1C,
C30); 70.11 (2C, C700, C5); 69.59 (1C, C800); 68.91 (1C,
C4); 68.52 (1C, CH2-Bn); 68.18 (1C, C40); 64.05 (1C,
C6); 63.03 (1C, C900); 63.00 (1C, CH2-Fmoc); 60.93
(1C, Pa); 59.59 (1C, Va); 59.05 (1C, Va); 59.00 (1C, Ia);
53.82 (2C, 2· La); 53.51 (1C, Da); 53.42 (1C, Qa);
51.51 (1C, Ha); 50.96, 50.29, 49.04 (4C, Aa); 50.35
(1C, C2); 49.80 (1C, C9-Fmoc); 47.42 (1C, Pd); 43.00,
42.67 (1C, Ga); 41.41 (2C, 2· Lb); 37.44 (1C, Ib); 36.12
(1C, Db); 31.89, 31.46 (2C, 2· Vb); 31.31 (1C, C300);
30.25 (1C, Pb); 27.73 (1C, Qb); 27.52 (1C, Hb); 25.79
(1C, Ic); 25.52 (2C, 2· Lc); 25.31 (1C, Pc); 22.92–20.71
(9C, CH3-Ac); 23.09, 21.97, 21.69 (4C, Lc); 19.50,
18.30 (2C, 2· Vc); 17.65 (3C, Ab); 15.68 (1C, Ic); 10.90
(1C, Id).
7.19.1. H-Gln-Leu-Ala-Ala-Leu-Asp-Ser(b-Ac4Gal-(1,3)-
[a-NeuAc45NAcCOOBn-(2,6)]-a-GalNAc)-His-Gly-Ala-
Ile-Val-Asp-Gly-Pro-Val-OH. Using 227 mg (0.05 mmol)
of Fmoc-Val-Tentagelꢂ resin 20, the coupling reactions
of the initial eight Fmoc amino acids (20 equiv) were per-
formed according to the general protocol. For coupling
of sialyl T serine conjugate 19 the Fmoc histidine-terminat-
ed resin was transferred into separate reaction flask and
treated with a freshly prepared solution of 165 mg
(0.12 mmol, 2.34 equiv) of Fmoc-Ser(b-Ac4Gal-(1 ! 3)-
[a-NeuAc45NAcCOOBn-(2 ! 6)]-a-GalNAc)-OH
19,
18 mg (0.12 mmol) HOAt, 47 mg HATU (0.12 mmol)
and 25 lL (0.15 mmol) DIPEA in NMP. After orbital
shaking for 5 h, the resin was filtered off, washed with
NMP and subjected to the capping reaction. Coupling
reactions for the six N-terminal Fmoc amino acids were
carried in the automated synthesizer again. After removal
of the Fmoc group from the N-terminal glutamine, no
capping reaction was carried out.
7.20. L-Glutamyl-L-leucyl-L-alanyl-L-alanyl-L-leucyl-L-
aspartyl-L- seryl-O-{2-acetamido-2-deoxy-3-O-[-b-D-
galactopyranosyl]-6-O-[(5-acetamido-3,5-dideoxy-a-D-
glycero-D-galacto-2- nonulopyranosyl)onate]-a-D-galac-
topyranosyl}-L-histidyl-L-glycyl- L-alanyl-L-isoleucyl-L-
valyl-L-aspartyl-L-glycyl-L-prolyl-L-valine (26)
The detachment of the glycopeptide from resin 24 was
performed with 10 mL TFA, 0.9 mL H2O and 0.9 mL
TIS under orbital shaking within 2 h. The resin was
filtered off and washed twice with 10 mL TFA. After
concentration of the filtrate in vacuo, 30 mL of cold
diethyl ether was added, and the solvent was removed
by centrifugation. The remaining sialyl T glycopenta-
decapeptide 25 was lyophilized and purified by
preparative HPLC [LUNA C-18, gradient (% CH3CN+
0.1%TFA): 25–100 (120 min)].
7.20.1. H-Gln-Leu-Ala-Ala-Leu-Asp-Ser(b-Gal-(1,3)-[a-
Neu5NAc-(2,6)]-a-GalNAc)-His-Gly-Ala-Ile-Val-Asp-Gly-
Pro-Val-OH. Glycopeptide 25 was hydrogenated in
15 mL MeOH within 18 h using catalytic amounts of
Pd/C (10%) as the catalyst. After filtration, freshly pre-
pared solution of NaOMe in MeOH was added until a
pH of 9.0–9.5 was reached. The solution was stirred
for 72 h. A few drops of acetic acid were added for
neutralization, and the solvents were evaporated in
vacuo. Purification of the glycopeptides 26 was per-
formed by preparative HPLC [LUNA C-18, gradient
(% CH3CN+0.1% TFA): 20–30(120 min)].
Yield: 55 mg (42%); colourless amorphous solid;
tR = 20.20 min [LUNA C-18, gradient (% CH3CN+
0.1% TFA): 3–100 (40 min)].
ESI-MS (positive), m/z: 1323.7 ([M+2H]2+/2, calcd:
2647.8).
Yield: 26 mg (23%, from 20); colourless amorphous sol-
id; tR = 19.50 [(% CH3CN+0.1% TFA): 10–50 (40 min)];
½aꢁ ꢀ19.3 (c 1, H2O).
22
D
1H NMR (400 MHz, CD3OD) (1H, COSY), d: 8.86 (s,
1H, He1); 7.52–7.40 (m, 6H, Harom.-Bn, Hd2); 5.46–5.33
(m, 2H, H40 (5.35), H800 (5.34)); 5.30–5.10 (m, 4H,
CH2-Bn (5.21), H700 (5.14), H20 (5.12)); 4.87–4.71 (m,
3H, H10 (4.85), H30 (4.83), H1 (4.74)); 4.68–4.45 (m,
ESI-MS (positive), m/z: 2218.2 ([M]+, calcd: 2218.0).
1H NMR (600 MHz, D2O) (1H, COSY), d: 8.52 (d, 1H,
He1, JHe1,Hd2 = 7.04 Hz); 7.22 (d, 1H, Hd2,

S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
6163
JHe1,Hd2 = 7.04 Hz); 4.69–4.58 (m, 2· Da, H1); 4.47–4.42
(m, H10, Ha); 4.41–4.31 (m, 3· Aa, Pa, 2· La); 4.28–4.11
(m, Ga, Ia); 4.10–4.00 (m, Ga, Qa); 3.98–3.70 (m, H40
(3.88), H60 (3.73)); 3.68–3.54 (m, H50, H30, Pd); 3.53–
3.38 (m, H20); 3.35–3.11 (m, Hb); 3.11 (m, Hb); 2.91–
2.69 (m, 2· Db); 2.22–2.13 (m, Vb, Qb, Pb); 2.11–2.00
(m, Vb, Pc); 1.98–1.82 (m, NH-Ac, Pb); 1.78–1.69 (m,
1 H, Ib); 1.60–1.45 (m, 3H, Lb, Lc); 1.42–1.35 (m, 1H,
Ic); 1.30–1.22 (m, 9H, Ab); 1.17–1.12 (m, 1H, Ic); 0.88–
0.70 (m, 30H, 4· Vc, Ic, Id, 4· Ld).
tion and washing of the resin three times with 10 mL of
TFA, the filtered solutions were combined, the solvent
was evaporated, and diethyl ether (30 mL) was added
to the remaining residue. The solvent was removed by
centrifugation and the crude product containing the de-
sired glycopeptide 28 and the deletion peptide 29 was
lyophilized. Separation and purification was carried
out by preparative HPLC [LUNA C-18, gradient (%
MeOH+0.1TFA): 40–90 (90 min)].
Yield of 28: 15 mg (10%); colourless amorphous solid;
tR = 22.0 min [LUNA C-18, gradient (% CH3CN+0.1%
TFA) 10–100 (40 min)].
13C NMR (150.9 MHz, D2O) (HMQC), d: 104.58 (1C,
C10); 74.81 (1C, C50); 72.43 (1C, C30); 71.14 (1C, C40);
70.40 (1C, C20); 60.73 (1C, C60); 60.11 (1C, Pa); 59.05
(1C, Ia); 53.71 (1C, Ha); 52.50 (2C, Da); 52.37 (2C,
La); 52.02 (1C, Qa); 49.62, 49.58, 48.06 (3C, Aa); 46.81
(1C, Pd); 41.96, 41.68 (2C, Ga); 39.48 (2C, Lb); 35.66
(1C, Ib); 35.18 (2C, Db); 29.96, 29.71 (2C, Vb); 29.18
(1C, Pb); 26.38 (1C, Qb); 26.32 (1C, Hb); 24.47 (1C,
Ic); 24.23 (1C, Pc); 24.11 (2C, Lc); 21.48 (1C, CH3–
NHAc); 20.57, 21.94 (4C, Ld); 18.26, 17.83, 17.11 (4C,
Vc); 16.34 (3C, Ab); 14.51 (1C, Ic); 9.74 (1C, Id).
ESI-MS (positive), m/z: 1447.8 ([M+Na+H]2+/2, calcd:
1448.0).
1H NMR (400 MHz, CD3OD) (1H, COSY), d/ppm: 8.83
(m, 1H, He1); 7.89–7.78 (m, 1H, H8-Cou); 7.77–7.74 (m,
1H, H5-Cou); 7.63–7.58 (m, 1H, H6-Cou); 7.50–7.38 (m,
6H, Hd2, Harom.-Bn); 6.40 (s, 1H, H3-Cou); 5.63–5.61
(m, 1H, H3-Qui); 5.53–5.48 (m, 1H, H5-Qui); 5.44–
5.35 (m, 2H, H700 (5.41), H800 (5.37)); 5.33–5.27 (m, H4
(5.31), H900a (5.28)); 5.26–5.22 (m, 1H, H900b); 5.18–
5.09 (m, 2H, H3 (5.15), H4-Qui (5.11)); 4.90–4.80 (m,
H400 (4.83), Da 4.82); 4.72–4.60 (m, Da (4.65), Ha
(4.64), Pa (4.58)); 4.47–3.92 (m, Aa (4.42, 4.24, 4.18),
Va (4.31, 4.22), Ga (4.17, 4.01, 3.97)); 3.70–3.60 (m,
Pd); 3.50–3.40 (m, Hba); 3.31–3.20 (m, Hbb); 3.10–2.88
(m, Dba (2.99, 2.90, 2.80)); 2.84–2.75 (m, 2H, Dbb,
H2a-Qui); 2.59–2.55 (m, 1H, H2b-Qui); 2.29 (s, 3H,
CH3-Ac); 2.24–1.58 (m, CH3-Ac, Pb (2.22, 2.06), Vb
(2.20, 2.12), Pc (2.02), Ib (1.90), Ica (1.61)); 1.45–1.38
(m, 9H, Ab); 1.36–1.14 (m, Lc (1.30, 1.22, 1.16), Icb
(1.22)); 1.08–0.90 (m, 30H, Ld (1.04, 1.00, 0.94, 0.94),
Vc (1.01, 0.97, 0.95), Ic (0.94), Id (0.90)).
7.21. 2-(7-{N-[1S,3R,4S,5R]-1,3,4,5-Tetraacetoxycyclo-
hexanoyl-glycyl}-aminocumarin-4-yl)-acetyl-L-leucyl-L-
alanyl-L-alanyl-L-leucyl-L-aspartyl-L-seryl-O-(2-acetami-
do-3,4-di-O-acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-D-glycero-D-galac-
to-2-nonulopyranosyl)onate]-a-D-galactopyranosyl)-L-his-
tidyl-L-glycyl-L-alanyl-L-isoleucyl-L-valyl-L-aspartyl-L-
glycyl-L-prolyl-L-valine (28)
7.21.1.
Quiglac-Gly-Aca-Leu-Ala-Ala-Leu-Asp-Ser(a-
NeuAc45NAcCOOBn-(2,6)-a-Ac2GalNAc)-His-Gly-Ala-
Ile-Val-Asp-Gly-Pro-Val-OH. According to the general
protocol solid-phase synthesis was performed starting
with Fmoc-Val-loaded resin 20 (227 mg, 0.05 mmol).
Coupling reactions for the next 8 Fmoc amino acids
(20 equiv) were carried out as described above. For cou-
pling of the sialyl TN serine 17 the histidine-terminated
resin was transferred to a separate reaction flask. After
removal of the Fmoc group, a freshly prepared solution
of 60 mg (0.05 mmol, 1.02 equiv) of Fmoc-Ser(a-Neu-
Ac45NAcCOOBn-(2 ! 6)-a-Ac2GalNAc)-OH 17, 8 mg
(0.05 mmol) HOAt, 20 mg (0.05 mmol) HATU and
10.7 lL DIPEA in NMP was added. The mixture was
vigorously shaken for 6 h and filtered. After washing
with NMP, a capping reaction was carried out. The cou-
pling reactions for the further Fmoc amino acids were
performed according to the general protocol. For cou-
pling of the N-terminal quinic acid–coumarine chromo-
phore the resin-linked glycopeptide was placed in flask,
73 mg (0.10 mmol, 2 equiv) of Ac4Quiglac-Gly-Aca-
Leu-OH 10, 15 mg (0.10 mmol) HOAt, 38 mg
(0.10 mmol) HATU and 21 lL of DIPEA in 2 mL of
NMP were added, and the mixture was vigorously shak-
en for 6 h. After a final capping acetylation, the loaded
resin 27 was filtered off and washed with NMP and
dichloromethane.
13C NMR (100.6 MHz, CD3OD) (HMQC), d/ppm:
73.33 (1C, C4-Qui); 70.81 (1C, C400); 69.73 (1C, C3);
69.48 (1C, C3-Qui); 69.43 (1C, C700); 69.12 (1C, C900);
68.66 (1C, C4); 68.41 (1C, C800); 67.91 (1C, C5-Qui);
61.42 (1C, Pa); 60.38, 59.09 (2C, Va); 54.95, 54.83,
50.82 (3C, Aa); 59.73, 52.66 (3C, La); 59.41 (1C, Ia);
52.63 (1C, Ha); 52.58, 51.33 (2C, Da); 47.71 (1C, Pd);
44.41, 43.02 (2C, Ga); 40.84 (3C, Lb); 37.75 (1C, Ib);
36.71 (1C, Db); 36.69 (1C, C2-Qui); 36.22 (1C, Db);
31.96 (1C, C6-Qui); 31.87, 31.67 (2C, Vb); 30.63 (1C,
Pb); 27.71 (1C, Hb); 27.46 (3C, Lc); 26.23 (1C, Ic);
25.61 (1C, Pc); 23.37, 23.17, 22.30, 22.02 (4C, Ld);
19.91, 19.64, 18.81, 18.56 (4C, Vc); 18.26–17.04 (3C,
Ab); 16.01 (1C, Ic); 11.31 (1C, Id).
7.22. 2-(7-{N-[1S,3R,4S,5R]-1,3,4,5-Tetraacetoxycyclo-
hexanoyl-glycyl}-aminocumarin-4-yl)-acetyl- L-leucyl-L-
alanyl-L-alanyl-L-leucyl-L-aspartyl-L-seryl-O-(2-acetami-
do-2-deoxy-6-O-[(5-acetamido-3,5-dideoxy-a-D-glycero-
D-galacto-2-nonulopyranosyl)onate]-a-D-galactopyrano-
syl)-L-histidyl-L-glycyl-L-alanyl-L-isoleucyl-L-valyl-L-
aspartyl-L-glycyl-L-prolyl-L-valine (30)
Release of the labelled glycopeptide from resin 27 was
performed by treatment with 10 mL TFA, 0.9 mL H2O
and 0.9 mL TIS and orbital shaking for 2 h. After filtra-
7.22.1.
Quiglac-Gly-Aca-Leu-Ala-Ala-Leu-Asp-Ser(a-
Neu5NAcCOOH-(2,6)-a-GalNAc)-His-Gly-Ala-Ile-Val-
Asp-Gly-Pro-Val-OH. To a solution of glycopeptide 28

6164
S. Heiner et al. / Bioorg. Med. Chem. 14 (2006) 6149–6164
(15 mg, 5.23 · 10ꢀ6 mol) in 15 mL of dry MeOH was
added freshly prepared NaOMe solution until a pH of
9.5 is reached. The solution was stirred at room temper-
ature for 48 h, neutralized by addition of a drop of ace-
tic acid, and the solvent was removed in vacuo. The
crude product was dried in high vacuum, dissolved in
water (5 mL), and 0.1 N NaOH solution was added until
a pH of 10.0 was reached. The solution was stirred at
room temperature for 24 h, neutralized by addition of
a few drops of acetic acid and lyophilized. Purification
was performed by preparative HPLC [LUNA C 18 (%
CH3CN+0.1% TFA) 10–50 (120 min)].
11. Gessner, R.; Tauber, R. Ann. N.Y. Acad. Sci. 2000, 915,
136–143.
12. Saeki, Y.; Hazeki, K.; Matsumoto, M.; Toyoshima, K.;
Akedo, H.; Seya, T. Oncol. Rep. 2000, 7, 731–735.
13. Silberg, D. G.; Swain, G. P.; Suh, E. R.; Traber, P. G.
Gastroenterology 2000, 119, 961–971.
14. Takamura, M.; Sakamoto, M.; Ino, Y.; Shimamura,
T.; Ichida, T.; Hirohashi, S. Cancer Sci. 2003, 94,
425–430.
15. Kuhn, A. PhD thesis, Universityof Mainz 2005; Wagner,
M.; Kuhn, A.; Kunz, H., unpublished results.
16. (a) Aamir, E.; Haas, E. Biochemistry 1988, 27, 8889–8893;
(b) Kudlicki, W.; Odom, O. W.; Kramer, G.; Hardesty, B.
J. Biol. Chem. 1996, 271, 31160–31165.
17. Maly, D. J.; Leonetti, F.; Backes, B. J.; Daubner, D. S.;
Harris, J. L.; Craik, C. S.; Ellmann, J. A. J. Org. Chem.
2002, 67, 910–915.
18. von Pechmann, H.; Duisberg, C. Ber. Dtsch. Chem. Ges.
1883, 16, 2219–2228.
Yield of 30: 5 mg (4%, overall from 20); tR = 11.20 min
[LUNA C-18, (% CH3CN+0.1% TFA) 10–50 (40 min)];
½aꢁ ꢀ12.1 (c 0.3, H2O).
22
D
MALDI-TOF-MS (positive), m/z: 2363.6 ([M]+, calcd:
19. Brenner, M.; Huber, W. Helv. Chim. Acta 1953, 36, 1109–
1115.
2360.0); 2385.5 ([M+Na]+, calcd: 2383.0).
20. Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F.
J. Chem. Soc., Chem. Commun. 1994, 201–203.
21. Grandjean, C.; Rommens, C.; Gras-Masse, H.; Mel-
nyk, O. J. Chem. Soc., Perkin Trans. 1 1999, 20,
2967–2976.
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