Synthesis of Peptide and Glycopeptide Partial Structures of the Homophilic Recognition Domain of Epithelial Cadherin

Article abstract of DOI:10.1055/s-2003-42429

Peptide and glycopeptide sequences of the homophilic recognition site of epithelial cadherin (E-cadherin) were synthesized by solid-phase technique based on acid-sensitive Wang anchor according to Fmoc strategy. Fmoc serine building blocks with T_N

Full text of DOI:10.1055/s-2003-42429

PAPER
2487
Synthesis of Peptide and Glycopeptide Partial Structures of the Homophilic
Recognition Domain of Epithelial Cadherin
Glycopeptides
o
a
f
the Hom
n
ophilic Reco
j
gnition
a
Site of E-Cadher
i
R
n
eipen, Horst Kunz*
Institut für Organische Chemie der Johannes Gutenberg-Universität Mainz, Duesbergweg 10-14, 55128 Mainz, Germany
Fax +49(6131)3924786; E-mail: hokunz@uni-mainz.de
Received 15 July 2003; revised 27 August 2003
Dedicated to Professor Gunter Fischer on the occasion of his 60th birthday.
Its extracellular portion consists of five domains of which
Abstract: Peptide and glycopeptide sequences of the homophilic
the terminal E-CAD1 is considered responsible for the ho-
recognition site of epithelial cadherin (E-cadherin) were synthe-
sized by solid-phase technique based on acid-sensitive Wang an-
chor according to Fmoc strategy. Fmoc serine building blocks with
motypic and homophilic cell adhesion activity.11,12 The
recognition between E-cadherin molecules proceeds via
TN-, T-, (2-6)-sialyl T-antigen and -N-acetylglucosamine side formation of dimers.12,13 The proposed specific binding
chains were prepared for the construction of E-cadherin glycopep-
site is embedded into a surface formed by three antiparal-
tides. The T- and (2-6)-sialylT-serine derivatives have been ob-
tained by chemical glycosylations of the TN-antigen serine
derivative carrying Fmoc/tert-butyl ester protecting group combi-
nation. According to NOESY and ROESY NMR experiments, E-
cadherin(glyco)peptides not acylated at the N-terminus prefer turn-
type conformations in water.
lel -sheets including the cell adhesion motif His-Ala-Val
presented in
a
well-defined steric arrangement
(Figure 1).11 Two of these antiparallel -sheets are linked
by a -turn-type structure (Ser83- Gly85) (Figure 2). As re-
cently reported, biological evaluation showed that syn-
thetic (glyco)peptides of this region induce differentiation
in transformed HaCaT keratinocytes.14
Key words: peptides, glycopeptides, solid-phase synthesis, glyco-
sylation, oligosaccharides
Carbohydrate
Introduction
S83
S V A H S78
E A V E89
H
N
Cadherins constitute a class of recently discovered calci-
um-dependent cell adhesion molecules which play crucial
roles in cell adhesion processes.1 Their interactions result
in homophilic cell adhesion in tissues between cells ex-
pressing the same type of cadherin on their surfaces. Cad-
herins are also involved in other important cellular
processes including differentiation,2 growth regulation
and morphogenesis.3–5 A down-regulation of cadherins
can cause invasiveness of cancer cells,6–10 while CD44-
mediated adhesion is increased in tumours.6,7A member of
the classical cadherin family is epithelial cadherin (E-cad-
herin).
G
OH
Figure 2 Partial stucture from the -turn region of the homophilic
recognition site of E-CAD1
In this case the differentiation marker proteins involucrin
and contactinhibin receptor were up-regulated after treat-
ment of the transformed cells with a synthetic glycopep-
tide from the proposed homophilic binding site whereas
the expression of the tumour-associated receptor CD44
was down-regulated at the same time. However, to date it
has not been possible to exert influence on the cell adhe-
βC
Thr
Lys
Ser
Val Phe35 Tyr Ser Ile Thr Gly
Gln
βF
Ala
Ser
Val
Ala80 His Ser Tyr Leu Ile Tyr Lys
Asn
Gly85 Glu Ala Val
βG
Met Glu Ile Val95 Ile Thr Val
Thr
Glu
Asp Pro
homophilic recognition domain
Figure 1 Homophilic recognition domain of E-cadherin in E-CAD111
SYNTHESIS 2003, No. 16, pp 2487–2502
x
x.
x
x
.
2
0
0
3
Advanced online publication: 14.10.2003
DOI: 10.1055/s-2003-42429; Art ID: T06903SS
© Georg Thieme Verlag Stuttgart · New York

2488
T. Reipen, H. Kunz
PAPER
sion processes. Thus, our goal was to synthesize a variety amino acid building blocks were synthesized in multi-step
of partial structures from the -turn region of the ho- reactions and manually coupled to the resin-linked pep-
mophilic recognition site of E-CAD1 (Figure 2) in order tide.
to investigate structural details of E-cadherin-mediated
cell adhesion. Results of such experiments are interesting
because of the importance of E-cadherin in processes like
Peptide Syntheses
morphogenesis, tumour development and self-recogni- The first synthesis of the dodecapeptide Ser78-Glu89 suf-
tion. Glycosylation of the recognition sequence on serine fered from low coupling yields from the eighth coupling
83 with carbohydrate residues of different sterical demand step onward (Val81) obviously as a result of backfolding
should induce or support the formation of -turn-type phenomena.14 It was possible to overcome these difficul-
structures and, thus, influence the cell biological effects.14 ties by utilizing double coupling steps and extended cou-
pling times during the automated synthesis.
For the synthesis of the resin-linked dodecapeptide Ser78-
Results and discussion
Glu89 (2), the standard protocol was modified (Scheme 1).
In order to avoid the formation of diketopiperazines, the
second and third amino acids (Ala-Val) were introduced
as a dipeptide building block in a double coupling. UV-
monitoring of the fluorenylmethylpiperidine indicated
difficulties in the Fmoc-removal from the ninth amino
acid onward. Due to these difficulties, the valine residue
81 was also introduced in a double coupling step. The de-
sired peptide 3 was obtained in an overall yield of 52% af-
ter treatment of the resin-linked form 2 with a mixture of
trifluoroacetic acid, triisopropylsilane and water
(15:0.9:0.9) and subsequent purification by preparative
RP-HPLC. For further investigations of the adhesion
The peptide and glycopeptide sequences of the ho-
mophilic recognition site of E-cadherin were synthesized
by automated solid-phase methods. As the solid support
either Tentagel® or polystyrene equipped with the acid-la-
bile Wang anchor and loaded with the Fmoc-protected
starting amino acid were used. The syntheses were carried
out in a peptide synthesizer. The Fmoc-protected amino
acids were used in an excess of 10 equivalents, HBTU/
HOBt served as coupling reagent (coupling time 20–30
min). Fmoc groups were removed with piperidine in 1-
methyl-2-pyrrolidinone (NMP) (20%). The glycosylated
Scheme 1 Wang resin: p-hydroxybenzyl (PHB) ester; HBTU: O-(1H-benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium hexafluorophosphate;
HOBt: 1-hydroxybenzotriazole; DIPE: diisopropylethylamine (Hünig’s base)
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

PAPER
Glycopeptides of the Homophilic Recognition Site of E-Cadherin
2489
properties of the synthetic structures, a peptide Ser78- fication (pH 8.5).21 An accurate adjustment of the pH is
Glu89 4 containing a fluorescent label was synthesized mandatory to avoid simultaneous cleavage of the base-la-
(Figure 3). The dansyl-lysine conjugate15 was coupled to bile Fmoc-group. The crude product was then reacted
the N-terminus of the solid-phase-bound dodecapeptide. with p-anisaldehyde dimethylacetal to yield compound
The condensation gave a N-terminally labelled structure 11. Several glycosylation methods had to be examined for
still carrying a free N-terminus considered essential for the generation of the -linked T-antigen disaccharide.
the biological activity of the synthetic peptides.14 The syn- Koenigs–Knorr glycosylation22 and reactions with
thesis was carried out as described for 3. The -dansyl- trichloroacetimidate23 resulted in the formation of com-
lysine was reacted with immobilized dodecapeptide in a plex mixtures and undesired ortho ester, respectively. The
threefold excess using O-(7-azabenzotriazol-1-yl)- Helferich glycosylation24 with glycosyl bromide 12 and
N,N,N ,N -tetramethyluronium hexafluorophosphate/1- mercury cyanide as the activator in a mixture of dichlo-
hydroxy-7-azabenzotriazole (HATU/HOAt) as the acti- romethane and nitromethane proved most effective. The
vating reagent.16 After acidolytic release from the resin, disaccharide 13 was isolated in a yield of 80%. Subse-
the peptide 4 was isolated by preparative RP-HPLC in an quently, the p-methoxybenzylidene acetal was removed
overall yield of 75%. In addition, the non-labelled tride- by treatment with ceric ammonium nitrate25 in acetonitrile
capeptide Lys-Ser78-Glu89 5 was also synthesized (yield: to furnish the disaccharide 14 with deprotected 4- and 6-
84%) in order to assure that potential activity in biological position. Acetylation with acetic anhydride and pyridine
assays is independent of the fluorescent dye.
furnished the completely protected T-antigen unit 15. C-
Terminal deprotection was carried out in a mixture of tri-
fluoroacetic acid and anisole to afford the T-antigen build-
ing block 16 suitable for solid-phase synthesis.
Synthesis of the Glycosylated Building Blocks
The synthesis of the glycosylated amino acids started
from Fmoc serine tert-butyl ester [Fmoc-Ser(OH)-O-t-
Bu] 6 obtained from Fmoc serine in one step
(Scheme 2).17 Glycosylation of 6 was achieved using the
2-azidogalactosyl bromide (7) of Paulsen and Hölck.18
The lacking neighboring group-participation favors the
formation of the desired -glycoside 8. Subsequently, the
azide 8 was reduced and N-acetylated with activated zinc
in a mixture of THF, acetic anhydride and acetic acid
(3:2:1),19 to give 9. The synthesis of the monosaccharide
TN-antigen serine building block 10 was completed by the
acidolysis of the tert-butyl ester.
The disaccharide serine conjugate 14 was also used for the
synthesis of the demanding 2,6-sialyl-T-antigen building
block (Scheme 3). In contrast to another strategy,26 the
complex sialic acid was introduced at a late stage of the
synthesis. Sialylation reactions still are demanding as they
often suffer from low yields and low stereoselectivity. Us-
ing the xanthate 17 of sialic benzyl ester as donor27 at low
temperature in the presence of acetonitrile proved suc-
cessful.
The xanthate 17 was applied in an excess of 2.5 equiva-
lents and activated by methylsulfenyl triflate28 formed in
situ from methylsulfenyl bromide29 and silver triflate at
–62 °C. Due to the sterically demanding structure of the
xanthate and the enhanced reactivity of the primary hy-
droxy group the sialylation proceeded regioselectively at
OH-6. In addition to the desired -glycoside 18, the -
In order to furnish a suitably protected acceptor for the
synthesis of the Thomsen–Friedenreich (T)-antigen disac-
charide,20 the acetyl groups in positions 3, 4, and 6 of 9
were selectively removed by careful Zemplén transesteri-
HO
O
O
OH
OH
O
NH2
O
O
O
O
O
O
O
H
H
N
H
N
H
H
N
H
N
H2N
N
N
N
H
N
N
H
N
N
N
H
OH
O
H
H
H
O
O
O
O
O
OH
N
75 %
4
HO
N
H
NH
O S O
N
HO
O
O
O
OH
OH
O
NH2
O
O
O
O
O
O
O
H
H
N
H
N
H
H
N
H
N
H2N
N
N
N
H
N
N
H
N
N
N
H
OH
O
H
H
H
O
O
O
O
OH
N
84 %
5
HO
N
H
NH2
Figure 3 Structures of peptides 4 and 5
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2490
T. Reipen, H. Kunz
PAPER
Scheme 2 a) Fmoc-Ser-t-BuO, Ag2CO3, AgClO4, toluene, CH2Cl2; 54%; b) Zn, THF–Ac2O, AcOH (3:2:1); 92%; c) TFA–anisole (10:1);
89%; d) 1. NaOMe, MeOH, pH 8.5; 2. p-anisaldehyde dimethyl acetal, p-TosOH, DMF; 52%; e) Hg(CN)2, nitromethane, CH2Cl2; 80%; f)
Ce(NH4)2(NO3)6, MeCN–H2O (9:1); 76%; g) Ac2O–pyridine (1:3); 88%; h) TFA–anisole (10:1); 84%
Scheme 3 a) AgOTf, MeSBr, CH2Cl2–MeCN (1:2), 28%; b-anomer: 7%; b) TFA–anisole (10:1), 94%
OAc
anomer 19 was also formed in the ratio of 1:4. Purification
OAc
O
of the trisaccharide turned out difficult. The sialic glycal
also formed as a by-product could only be removed by
careful column chromatography. The - and -sialyl-T
antigens were separated by preparative RP-HPLC yield-
ing the pure -glycoside 18 in a yield of 28%. Trisaccha-
ride threonine conjugate 18 was deprotected with
trifluoroacetic acid and anisole to furnish the building
block 20 in a yield of 94%.
O
AcO
AcO
a)
AcO
AcO
SEt
O
TrocNH
21
TrocNH
22
FmocHN
COOtBu
b)
OAc
OAc
O
O
AcO
AcO
c)
AcO
AcO
O
O
As a further variation, a -linked glucosamine serine con-
jugate was incorporated into the peptide chain
(Scheme 4). To this end, glucosamine hydrochloride was
transformed into the -thioglycoside 2130 which was re-
acted with Fmoc-Ser(OH)-O-t-Bu17 6 under activation
AcNH
AcNH
24
23
FmocHN
COOH
FmocHN
COOtBu
Scheme 4 a) Fmoc-Ser-t-BuO, NIS, TfOH, CH2Cl2, 80%; b) 1. Zn,
AcOH; 2. pyridine, Ac2O; 86%; c) TFA, anisole; 87%
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

PAPER
Glycopeptides of the Homophilic Recognition Site of E-Cadherin
2491
with N-iodosuccinimide (NIS) and trifluoromethane-
sulfonic acid31,32 to furnish the -glycoside 22 in a yield of
80%. The Troc group was cleaved with zinc in acetic acid,
and the resulting glucosamine subsequently acetylated
with acetic anhydride to give 23. Finally, acidolysis of the
tert-butyl ester was accomplished to give 24 in a yield of
87%.
Glycopeptide Synthesis
The preformed glycosylated amino acid building blocks
10, 16, 20 and 24 were incorporated into dodecapeptide
sequences derived from the homophilic recognition do-
main of epithelial cadherin. The first glycopeptide synthe-
sized for testing in biological assays was the glycosylated
dodecapeptide 27 (Ser78-Glu89) (Scheme 5).14
Scheme 5
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

2492
T. Reipen, H. Kunz
PAPER
The synthesis was carried out in analogy to the procedure into the dodecapeptide sequence according to the proce-
described for the non-glycosylated peptides. The glycosy- dure described above (Scheme 6). After release from the
lated amino acid 10 was manually coupled to the resin- solid support, the partially protected glycopeptide 30 was
linked peptide 25 after N-terminal deprotection. The TN- obtained which showed low solubility in common sol-
antigen building-block 10 was used in an excess of 2.5 vents making purification by preparative HPLC impossi-
equivalents and activated with HATU/HOAt16 within 5 ble. Thus, the benzyl ester protecting group was directly
hours. The detachment from the resin-linked form 26 was cleaved by hydrogenolysis using palladium on charcoal.
carried out by treatment with trifluoroacetic acid, water, After three days the reaction was terminated. The crude
thioanisole and 1,2-ethanedithiol in order to furnish a se- product 31 could hence be purified by preparative RP-
lectively deprotected glycopeptide. Due to its insolubility, HPLC. After lyophylization, the desired compound 31
the crude product was further deprotected under Zemplén was isolated in a yield of 33%. In the subsequent depro-
conditions (pH 9.5) to yield the water-soluble glycodode- tection, the O-acetyl groups were removed by transesteri-
capeptide 27 in a yield of 65% after purification by pre- fication (pH 9.5). The solution was stirred for 18 hours,
parative RP-HPLC.
carefully monitored by analytical RP-HPLC. The target
molecule 32 was obtained in a yield of 61% (overall yield:
20%) after purification by preparative RP-HPLC.
For the synthesis of the glycopeptide containing the
-
linked serine-glucosamine conjugate, the building block
24 was applied in an excess of two equivalents to couple
with N-deprotected 25. After acidolytic release from the
resin, the crude glycopeptide was deacetylated (pH 9.5) to
yield the glycopeptide 28 in an overall yield of 51%
(Figure 4).
Synthesis of a Dimeric Peptide
To impose influence on cell adhesion phenomena in cells
carrying E-cadherin molecules on their surfaces, a dimeric
structure of the recognition peptide sequence simulating
the native dimerization process was synthesized. To this
end, two monomeric nonapeptide units were connected
via a disulfide bridge (Scheme 7). The octapeptide Ser78-
Gly85 was synthesized by solid-phase peptide synthesis as
described above and subsequently N-terminally acylated
with a cysteine residue.
The disaccharide T-antigen serine conjugate 16 (1.3
equiv) was applied to the synthesis of glycopeptide 29
carried out in analogy to the other glycopeptide syntheses.
Coupling time for the reaction with the N-deprotected im-
mobilized peptide 25 was extended to 4 hours. The subse-
quent Fmoc amino acids were coupled according to the
standard protocol, but also for an extended reaction time.
Due to the low solubility of the resulting glycopeptide, it
was deacetylated without further purification. After stir-
ring with a catalytic amount of NaOMe in methanol for 48
hours (monitoring by analytical RP-HPLC), the pure
product 29 (Figure 4) was isolated by preparative RP-
HPLC in an overall yield of 32%.
Polystyrene resin preloaded with Fmoc-Gly-O-PHB was
used as solid support. Since cysteine residues are known
to be sensitive,33 the coupling of Fmoc-Cys(Trt)-OH was
carried out manually with O-(1H-benzotriazol-1-yl)-
N,N,N ,N -tetramethyluronium tetrafluoroborate (TBTU)
and HOBt without preactivation in dichloromethane. sym-
Collidine was used as the base. Subsequently, the peptide
was released from the solid support with a mixture of tri-
fluoroacetic acid, triisopropylsilane and water. According
In order to further modify the glycan, the sterically de-
manding 2,6-sialyl-T-antigen serine 20 was incorporated
HO
O
O
OH
OH
O
NH2
O
O
O
O
O
O
H
H
N
H
H
H
N
H
N
N
N
N
H2N
N
H
N
H
N
H
N
N
OH
O
H
H
O
O
O
O
O
N
NHAc
51 %
OH
OH
HO
O
N
H
O
28
HO
HO
O
O
OH
O
OH
NH2
O
O
O
O
O
O
O
H
H
N
H
H
H
N
H
N
N
N
N
H2N
N
H
N
H
N
H
N
N
H
OH
O
H
O
O
O
O
O
N
HO
O
NHAc
32 %
HO
N
H
OH
OH
O
29
O
O
H
HO
HO
Figure 4 Structures of peptide 28 and 29
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PAPER
Glycopeptides of the Homophilic Recognition Site of E-Cadherin
2493
Fmoc-Glu(OtBu)-
WANG
1
HO
O
O
O
OH
OH
O
NH2
O
O
O
O
O
O
H
N
N
H
N
H
H
H
N
H
N
N
N
H2N
N
H
N
H
N
N
H
OH
O
H
H
O
O
O
O
O
N
NHAc
O
30
AcO
HO
N
H
O
OAc
OAc
O
AcO
OAc
OH
O
NHAc
OAc
O
a)
BzlOOC
OAc
AcO
O
HO
O
O
OH
OH
O
NH2
H
N
O
O
O
O
H
N
N
H
N
H
H
N
H
N
N
H2N
N
H
N
H
N
N
H
OH
O
H
H
O
O
O
O
O
O
O
N
NHAc
O
31
AcO
HO
N
H
O
OAc
OAc
O
AcO
OH
O
OAc
NHAc
OAc
O
HOOC
OAc
AcO
b)
HO
O
O
OH
OH
O
NH2
H
N
O
O
O
O
O
H
N
N
H
H
H
N
H
N
N
N
H2N
N
N
N
N
OH
O
H
H
H
H
H
O
O
O
O
O
O
O
N
NHAc
O
HO
HO
N
H
O
OH
OH
32
O
HO
OH
OH
O
NHAc
OH
O
COOH
OH
HO
Scheme 6 a) H2, Pd-C, MeOH; overall yield: 33%; b) NaOMe, MeOH, pH 9.5; 61%
to the analytical RP-HPLC, the crude product 33 was of hours led to quantitative conversion.35 As the resulting
sufficient purity to be directly subjected to dimerization.
dimer 34 was completely insoluble in common solvents,
it was directly deprotected with DMF and piperidine (1:1)
to give the water-soluble dimer 35 in an overall yield of
68% after purification by gel permeation chromatogra-
phy.
To achieve this reaction, a number of conditions were ex-
amined. As the most common reagent, iodine was utilized
at first,34 but no conversion to the dimeric structure was
observed. Alternatively, compound 33 treated with a mix-
ture of DMSO and trifluoroacetic acid (10%) within 3
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

2494
T. Reipen, H. Kunz
PAPER
O
O
OH
OH
NH2
H
O
O
O
O
O
H
H
N
H
FmocHN
HS
N
N
N
N
H
N
N
H
N
OH
H
H
O
O
O
OH
N
33
N
H
a)
O
O
OH
OH
NH2
H
O
O
O
O
O
H
H
N
H
FmocHN
N
N
N
N
H
N
N
H
N
OH
H
N
H
H
O
O
O
S
S
OH
N
N
HO
N
H
O
O
O
O
O
34
H
H
N
H
H
N
HO
N
N
N
H
H2N
N
N
H
N
NHFmoc
H
H
O
O
O
O
O
HO
HO
b)
O
O
OH
OH
NH2
H
O
O
O
O
O
H
H
N
H
H2N
N
N
N
N
N
H
N
N
H
N
OH
H
N
H
H
O
O
O
S
S
OH
N
HO
35
N
H
O
O
O
O
O
H
H
N
H
H
HO
N
N
N
N
H
H2N
N
N
H
N
NH2
H
H
O
O
O
O
O
HO
HO
Scheme 7 a) DMSO–TFA (10%); b) DMF–piperidine (1:1); overall yield: 68%
Preliminary Evaluation of Structural Properties and
Biological Activity
ticular E-cadherin peptides 4 and 5 showed the strongest
anti-adhesive effects on cell cultures of E-cadherin ex-
pressing cells. Detailed description of conformational
analysis and biological evaluation of the synthetic E-cad-
herin (glyco)peptides will be reported elsewhere in due
course.
Preliminary results of conformational analysis by high-
resolution NOESY and ROESY experiments showed that
all synthesized E-cadherin peptides and glycopeptides
adopt preferred turn-type conformations in DMSO solu-
tion. Similar CH,NH- and NH,NH-contacts have been
found for protons within the turn sequence Ser-Asn-Gly
and in the C-terminal region as are shown in Figure 5 for
compounds 4 and 5. Arrows in Figure 5 indicate contacts
of protons in distances between 235 pm (strong, for exam-
ple, NH,NH of Ser-Asn and Asn-Gly in the turn and Glu-
Val in the C-terminal region) and 400 pm (weak, for ex-
ample, CH,CH of Hisimid-Val and Hisimid-Lys ). Glycosy-
lation might support, but is not essential for the formation
of these turn conformations, whereas no turn conforma-
tion is found for N-terminally protected peptides. In the
case of the E-cadherin peptides carrying an additional N-
terminal -dansyl-labelled 4 or free lysine residue 5, addi-
tional intensified contacts of protons in the N-terminal re-
gion have been observed (Figure 5), for example, NH,NH
of Ser-His and NH,CH between SerNH- Lys . This sug-
gests that the N-terminal region of these peptides contain-
ing the recognition motif His-Ala-Val is presented in a
preferred geometry. It is interesting to note that these par-
THF, Et2O, light petroleum (bp 50–70 °C), MeOH, CH2Cl2, ni-
tromethane, and MeCN were distilled and dried according to stan-
dard procedures.36 DMF (amine free, for peptide synthesis) was
purchased from Roth, Ac2O and pyridine in p.a. quality from Acros.
Reagents were purchased at highest commercially available quality
and used without further purification. Fmoc-protected amino acids
were purchased from Novabiochem. As the resins for solid-phase
synthesis preloaded Tentagel® resins (Rapp Polymers) or preloaded
polystyrene resins (Novabiochem) were used. Reactions were mon-
itored by TLC with aluminum-backed silica gel 60 F254 plates (Mer-
ck KGaA, Darmstadt). Flash column chromatography was
performed with silica gel (40–63 m) from Merck KGaA, Darms-
tadt. Optical rotations [ ]D were measured with a Perkin Elmer po-
larimeter 241. RP-HPLC analyses were carried out on a Knauer
HPLC system with a Eurospher C8 column (5 m, 250 × 4 mm) or
a Phenomenex Luna C18-2 (5 m, 250 × 4.6 mm) and a pump rate
of 1 mL/min. Preparative HPLC separations were carried out on a
Knauer HPLC system with a Eurospher C8 column (10 m,
250 × 20 mm) and a pump rate of 10 mL/min or with a Phenomenex
Luna C18-2 (10 m, 250 × 50 mm) at a pump rate of 20 mL/min.
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

PAPER
Glycopeptides of the Homophilic Recognition Site of E-Cadherin
2495
N
N
HN
HN
O
H
O
H2N
O
O
H
H
N
N
N
N
H2N
NH
N
N
H
H
N
HN
H
HN
O
H
O
O
O
HO
O
HO
HO
O
O
N
O
OH
HN
NH
HN
S
H2N
O
O
O
OH
O
OH
O
NH
NH
O
O
OH
OH
O
H2N
COOH
OH
O
H2N
COOH
O
HN
HN
O
O
HN
H
N
HN
O
O
H
N
N
N
N
N
H
H
O
O
H
H
OH
O
O
O
O
4
5
Figure 5 NH,NH, NH,CH and CH,CH contacts determined by 600 MHz NOESY and ROESY NMR spectroscopy
Mixtures of H2O and MeCN were used as the solvents, if required
0.1% TFA were added.
Rt 11.58 min (RP-HPLC, C8-Eurospher, 215 nm, 1% MeCN in
H2O 100% MeCN + 0.1% TFA in 42 min).
1H, 13C, and 2D-NMR spectra were recorded on Bruker AM-400,
ARX-400 or DRX-600 spectrometers. Proton chemical shifts are
1H NMR (400 MHz, D2O, 1H-COSY): = 8.52 (s, 1 H, Im2), 7.21 (s,
1 H, Im4), 4.70–4.60 (m, 2 H, N , H ), 4.42–4.23 (m, 6 H, 2 × A ,
given in ppm relative to CHCl3 ( = 7.24) or DMSO ( = 2.49). 13
C
2 × E , 2 × S ), 4.10–3.97 (m, 3 H, 2 × V , S ), 3.85–3.74 (m, 8 H,
a
chemical shifts are reported relative to CDCl3 ( = 77.0), DMSO
( = 39.5). Assignments of proton and carbon signals were achieved
by COSY, TOCSY and HMQC experiments when noted. MALDI-
TOF mass spectra were recorded on a Micromass Tofspec E spec-
trometer, ESI-mass spectra were recorded on ThemoQuest Naviga-
tor spectrometer.
G ,
3
×
S ), 3.29 (dd,
1
1
H, H , JH
b = 15.76 Hz,
a = 15.56 Hz,
a,H
b
JH
= 6.36 Hz), 3.14 (dd,
H, H , JH
a,H
b,H
JH
= 7.44 Hz), 2.73 (m, 2 H, N ), 2.35 (m, 4 H, E ), 1.98–1.88
b,H
(m, 6 H, 2 × E , 2 × V ), 1.25 (2 d, 6 H, 2 × A , JA
= 6.90 Hz),
,A
0.83 (d, 12 H, 2 × V , JV
= 6.74 Hz).
,V
13C NMR (100.6 MHz, D2O, HMQC): = 176.93, 174.63, 173.39,
173.29, 172.99, 172.86, 171.69, 170.79, 167.79, 163.02 (C = O),
133.70 (N=CN-Im), 128.14 (NC=CH-Im), 117.47 (HNCH=CR-
Im), 61.28, 61.01, 60.14 (3 × S ), 59.43 (2 × V ), 55.70, 55.23, 54.46
(3 × S ), 52.83, 52.54 (2 × E , H ), 50.62 (N ), 49.73 (2 × A ), 42.83
(G ), 36.09 (N ), 30.04, 30.23 (2 × V , 2 × E ), 26.51, 26.30, 25.86
(2 × E , H ), 18.47, 18.40, 17.70, 17.62, 16.65, 16.47 (2 × A , 2 ×
V ).
L-Seryl-L-histidyl-L-alanyl-L-valyl-L-seryl-L-seryl-L-asparagi-
nyl-glycyl-L-glutamyl-L-alanyl-L-valyl-L-glutamic Acid (3,
[H2N-SHAVSSNGEAVE-OH])
For the synthesis of the dodecapeptide 3, Tentagel® resin preloaded
with Fmoc-Glu(O-t-Bu)-O-PHB 1 (455 mg, loading: 0.22 mmol/g)
was used. Fmoc-groups were removed by treatment with 20% pip-
eridine in NMP (3–5 × 2.5 min). Peptide couplings were carried out
by reaction with Fmoc-protected amino acids (10 equiv), HBTU
(10 equiv), HOBt (10 equiv) and DIPEA (20 equiv) within 20–30
min. After cleavage of the first Fmoc group, the dipeptide Fmoc-
Ala-Val-OH was coupled to the resin in a double coupling (cou-
pling time: 2 × 20 min). Free amino groups were capped with Ac2O/
DIPEA/HOBt after every coupling step. The following amino acids,
Fmoc-Glu(O-t-Bu)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH and
2 × Ser(t-Bu)-OH, were reacted with the resin-linked peptide chain
according to the standard protocol. The ninth amino acid Fmoc-Val-
OH was coupled twice. The coupling times of the following amino
acids, Fmoc-Ala-OH, Fmoc-His(Trt)-OH und Fmoc-Ser(t-Bu)-OH,
were extended to 30 min. The resin was transferred into a solid-
phase reactor and thoroughly washed with CH2Cl2. The immobi-
lized peptide was released from the resin by treatment with trifluo-
roacetic acid (15 mL), triisopropylsilane (0.9 mL) and H2O
(0.9 mL) for 2 h. The acid-labile protecting groups were removed
simultaneously. Afterwards, the supernatant was filtered and the
solid support was washed with trifluoroacetic acid (3 × 10 mL). The
filtrates were combined and concentrated in vacuo. The liquid resi-
due was poured into cold Et2O (50 mL). The colorless precipitate
was centrifuged and washed with cold Et2O (2 ×). The crude prod-
uct was dissolved in H2O and purified by preparative RP-HPLC
(Luna C18, 1% MeCN in H2O 100% MeCN in H2O + 0.1% TFA
in 60 min). The completely deprotected peptide 3 was obtained as a
colorless lyophilisate (62 mg, 52%); [ ]D23 –40.9 (c = 0.97, H2O);
MALDI-TOF-MS (DHB): m/z calcd for C47H76N15O21: 1187.2;
found: 1187.8 (M + H+); m/z calcd for C47H75N15NaO21: 1209.2,
found: 1209.7 (M + Na+).
N -(5-Dimethylaminonaphthaline-1-sulfonyl)-L-lysyl-L-seryl-L-
histidyl-L-alanyl-L-valyl-L-seryl-L-seryl-L-asparaginyl-glycyl-
L-glutamyl-L-alanyl-L-valyl-L-glutamic Acid (4, [H2N-K(dan-
syl)SHAVSSNGEAVE-OH])
The synthesis of the fluorescent-labelled peptide was carried out ac-
cording to the preparation of the dodecapeptide 3 (see above). Resin
preloaded with Fmoc-Glu(O-t-Bu)-O- PHB 1 (500 mg, loading:
0.2 mmol Fmoc-Glu(O-t-Bu)/g) was utilized. After formation of the
resin-linked dodecapeptide 2 and Fmoc removal, the fluorescent-la-
belled building block15 was manually coupled. To this end, Fmoc-
Lys(dansyl)-OH (180 mg, 0.3 mmol, 3.0 equiv), HATU (114 mg,
0.3 mmol, 3.0 equiv), HOAt (40 mg, 0.3 mmol, 3.0 equiv) and
NMM (66 L, 0.6 mmol, 6.0 equiv) were dissolved in NMP (2 mL)
and added to the immobilized peptide and vortexed for 2 h. After re-
moval of the N-terminal Fmoc group, the polymer was treated with
trifluoroacetic acid to release the peptide 4 from the resin as de-
scribed above. The crude fluorescent-labelled peptide 4 was puri-
fied by preparative RP-HPLC (C8-Eurospher, 1% MeCN in H2O
100% MeCN in H2O + 0.1% TFA in 60 min). The completely
deprotected dansylpeptide 4 was obtained as a slightly yellow lyo-
philisate (115 mg, 75%); [ ]D23 –25.2 (c = 1.00, H2O); Rt 12.35 min
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

2496
T. Reipen, H. Kunz
PAPER
(RP-HPLC, Luna C18, 215 nm, 1% MeCN in H2O 100% MeCN
+ 0.1% TFA in 42 min).
MALDI-TOF-MS (DHB): m/z calcd for C53H88N17O22: 1315.4;
found: 1549.4 (M + H+); m/z calcd for C53H87N17NaO22: 1337.4;
found: 1337.9 (M + Na+).
1H NMR (400 MHz, D2O, 1H-COSY): = 8.73 (d, 1 H, CH-dansyl,
J = 9.00 Hz), 8.58 (s, 1 H, Im2), 8.42 (d, 1 H, CH-dansyl,
J = 8.60 Hz), 8.33 (d, 1 H, CH-dansyl, J = 7.44 Hz), 8.07 (d, 1 H,
CH-dansyl, J = 7.84 Hz), 7.19–7.83 (m, 2 H, CH-dansyl), 7.29 (s, 1
H, Im4), 4.70 (m, 2 H, N , H ), 4.50–4.44 (m, 3 H, 3 × S ), 4.41–4.31
(m, 4 H, 2 × A , 2 × E ), 4.17–4.06 (m, 2 H, 2 × V ), 4.01 (t, 1 H,
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-(2-acetamido-2-
deoxy-4,6-O-p-methoxybenzylidene- -D-galactopyranosyl)-L-
serine tert-Butyl Ester (11, [Fmoc-Ser( -4,6-O-Pmb-GalNAc)-
O-t-Bu])
To a solution of Fmoc-Ser( -Ac3GalNAc)-O-t-Bu (9;14,37 2.0 g,
2.8 mmol) in anhyd MeOH (50 mL) was added a 1% solution of
NaOMe in MeOH dropwise until a pH of 8.5 is reached. The mix-
ture was stirred for 7 h. After every 30 min, the pH was adjusted to
8.5. Finally, ion exchange resin Amberlyst IR 120 was added for
neutralization. After filtration, evaporation of the solvent in vacuo
and drying the remaining product in high vacuum, the product was
dissolved in DMF (100 mL). p-Methoxybenzylaldehyde dimethyl
acetal (0.96 mL, 5.6 mmol) and catalytic amounts of p-TsOH
(pH 4) were added. The mixture was kept at 50 °C and 25 mbar (ro-
tavapor) for 2 h. After neutralization with Hünig’s base, the solvent
was evaporated in vacuo, the remaining residue dissolved in EtOAc
(100 mL) and washed with aq sat. NaHCO3 solution (3 ×) and brine,
and dried (MgSO4). The solvent was evaporated, and the crude
product was purified by flash chromatography on silica gel (light
petroleum–EtOAc, 2:1) to give 11 as a colorless amorphous product
(1.03 g, 52%); [ ]D23 +60.1 (c = 1, CH2Cl2); Rf 0.20 (light petro-
leum–EtOAc).
1H NMR (400 MHz, CDCl3, 1H-COSY): = 7.84 (d, 2 H, H4-, H5-
Fmoc, JH3,H4 = JH5,H6 = 7.44 Hz), 7.67 (m, 2 H, H1-, H8-Fmoc),
7.49–7.37 (m, 6 H, H2-, H3-, H6-, H7-Fmoc, Hmeta-, Hpara-Pmb),
6.95 (d, 2 H, Hortho-Pmb, JHortho,Hmeta = 9.00 Hz), 6.21 (d, 1 H, NH-
Ac, JNH,H2 = 8.96), 6.08 (d, 1 H, NH-Fmoc, JNH,S = 8.24 Hz), 5.52
(s, 1 H, CH-Pmb), 4.97 (d, 1 H, H1-Gal, JH1,H2 = 1.96 Hz), 4.57–
4.40 (m, 4 H, H2-Gal, S , CH2-Fmoc), 4.31–4.16 (m, 3 H, H6a-,
H6b-Gal, H9-Fmoc), 4.03–3.89 (m, 4 H, H5-, H3-Gal, S ), 3.86 (s,
3 H, OCH3-Pmb), 2.08, (s, 3 H, NHCOCH3), 1.54 (s, 9 H, t-C4H9).
K , JK
= 6.24 Hz), 3.91–3.81 (m, 8 H, G , 3 × S ), 3.49 (s, 6 H,
,K
a
2 × CH3-dansyl), 3.30 (dd, 1 H, H , JH
b = 15.64 Hz,
a = 15.64 Hz,
a,H
b
JH
JH
= 5.88 Hz), 3.14 (dd, 1 H, H , JH
a,H
b,H
= 8.24 Hz), 2.91 (m, 2 H, K ), 2.81 (m, 2 H, N ), 2.43 (m, 4
b,H
H, 2 × E ), 2.17–1.78 (m, 6 H, 2 × E , 2 × V ), 1.79 (m, 2 H, K ),
1.43–1.31 (m, 10 H, K , K , 2 × A ), 0.91 (t, 12 H, 2 × V ).
13C NMR (100.6 MHz, D2O, HMQC): = 173.17, 172.85, 172.76,
172.68, 171.80, 171.71, 171.53, 171.30, 171.17, 171.03, 170.82,
169.69, 162.90, 162.55 (C=O), 138.40 (Cquart-dansyl), 135.20
(Cquart-dansyl), 133.22 (N=CN-Im), 130.01 (Ctert-dansyl), 128.52
(Cquart-dansyl), 128.09 (NC=CH-Im), 127.85, 126.84, 125.26 (Ctert
-
dansyl), 125.50 (Cquart-dansyl), 125.40, 119.82 (Ctert-dansyl),
117.21 (HNCH=CR-Im), 61.00, 60.82, 60.69 (3 × S ), 59.21 (2 ×
V ), 55.59, 55.45, 55.24, 55.01 (3 × S ), 52.63 (K ), 52.49 (E ),
52.19 (H ), 51.68 (E ), 50.37 (N ), 49.78, 49.50 (2 × A ), 46.57
(CH3-dansyl), 42.55 (G ), 41.94 (K ), 35.78 (N ), 30.03, 29.97,
29.87, 29.67, 28.20 (2 × V , 2 × E , K , K ), 26.15, 25.97, 25.91,
25.45, 25.35 (H , 2 × E ), 20.64 (K ), 18.23, 18.14, 17.55, 17.43,
17.40, 16.99, 16.43, 16.37, 16.17 (2 × A , 2 × V ).
MALDI-TOF-MS (DHB): m/z calcd for C65H99N18O24S: 1548.7;
found: 1549.4 (M + H+); m/z calcd for C65H98N18NaO24S: 1570.6;
found: 1571.5 (M + Na+).
L-Lysyl-L-seryl-L-histidyl-L-alanyl-L-valyl-L-seryl-L-seryl-L-
asparaginyl-glycyl-L-glutamyl-L-alanyl-L-valyl-L-glutamic
Acid (5, [H2N-KSHAVSSNGEAVE-OH])
The automated synthesis of the tridecapeptide was carried out in
analogy to the one of the labelled peptide 4. Tentagel® resin pre-
loaded with Fmoc-Glu(O-t-Bu)-O-PHB 1 (455 mg, loading:
0.22 mmol Fmoc-Glu(O-t-Bu)/g) served as the solid support, the
standard coupling time was 20 min. Fmoc-Lys-OH was finally cou-
pled to 2 after Fmoc removal. The peptide was detached from the
solid support and isolated as described above. The crude product
13C NMR (100.6 MHz, CDCl3): = 171.60, 169.36 (C=O), 160.15
(Cpara-Pmb), 155.98 (CO-urethan), 143.72, 143.64 (C4a-, C4b-
Fmoc), 141.30 (C8a-, C9a-Fmoc), 130.10 (Cipso-Pmb), 127.82,
127.71, 127.11 (C2-, C3-, C6-, C7-Fmoc, Ctert-Pmb), 125.05,
124.94 (C1-, C8-Fmoc), 120.9 (C4-, C5-Fmoc), 114.31, 113.56
(Ctert-Pmb), 101.11, 99.55 (CH-Pmb, C1-Gal), 83.00 [C(CH3)3],
75.80 (C3-Gal), 69.09 (S ), 68.73 (C4-Gal), 67.18 (CH2-Fmoc),
63.38 (C5-Gal), 63.30 (C6-Gal), 55.28 (OCH3-Pmb), 54.94 (S ),
50.15, 47.08 (C2-Gal, C9-Fmoc), 28.01 [C(CH3)3], 23.22
(NHCOCH3).
was purified by RP-HPLC (Luna C18, 5% MeCN in H2O
5%
MeCN in H2O in 15 min 20% MeCN in H2O in 50 min 100%
MeCN + 0.1% TFA in 60 min). The completely deprotected glyco-
peptide 5 was obtained as a colorless lyophilisate (110 mg, 84%);
[ ]D23 –47.9 (c = 1.00, H2O); Rt 20.71 min (RP-HPLC, Luna C18,
215 nm, 5% MeCN in H2O 5% MeCN in H2O in 10 min 60%
MeCN in H2O in 40 min 100% MeCN + 0.1% TFA in 60 min).
ESI-MS: m/z calcd for C38H45N2O11: 705.3, found: 705.3 (M + H+).
Anal. Calcd for C38H44N2O11 + H2O: C, 63.15; H, 41.41; N, 3.88.
Found: C, 62.81; H, 6.99; N, 4.12.
1
1H NMR (400 MHz, D2O, H-COSY): = 8.61 (d, 1 H, Im2,
J = 1.16 Hz), 7.29 (s, 1 H, Im4), 4.71 (m, 2 H, N , H ), 4.53–4.40
(m, 4 H, 1 × E , 3 × S ), 4.38–4.32 (m, 3 H, 2 × A , E ), 4.19–4.02
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-(2-acetamido-2-de-
oxy-4,6-O-p-methoxybenzylidene-3-O-[2,3,4,6-tetra-O-acetyl- -
D-galactopyranosyl]- -D-galactopyranosyl)-L-serine tert-Butyl
Ester (13, [Fmoc-Ser(Ac4Gal-( 1 3)- -4,6-O-Pmb-GalNAc)-
O-t-Bu])
To Fmoc-protected O-(methoxybenzylidene-N-acetylgalactosami-
nyl)threonine tert-butyl ester 11 (750 mg, 1.1 mmol, 1.0 equiv) and
molecular sieves (1.5 g, 4 Å) in CH2Cl2–nitromethane (20 mL, 3:1)
was added Hg(CN)2 (555 mg, 2.2 mmol, 2.0 equiv). The suspension
was stirred at r.t. for 1 h. After cooling to 0 °C, a solution of 2,3,4,6-
tetra-O-acetyl- -galactopyranosyl bromide (12; 1.36 g, 3.3 mmol,
3.0 equiv) in CH2Cl2–nitromethane (12 mL, 3:1) was added drop-
wise. Finally, an additional portion of Hg(CN)2 (555 mg, 2.2 mmol,
2.0 equiv) was added and the mixture was allowed to warm up to r.t.
The suspension was stirred for 15 h, diluted with CH2Cl2 (20 mL)
and filtered through Hyflo Supercel into a sat. aq solution of
NaHCO3 (30 mL). The phases were separated, and the organic layer
a
(m, 2 H, 2 × V ), 3.92–3.81 (m, 8 H, G , 3 × S ), 3.29 (dd, 1 H, H ,
b
JH
JH
JK
b = 15.24 Hz, JH
a = 15.24 Hz, JH
= 5.84 Hz), 3.14 (dd,
= 8.24 Hz), 2.91 (t,
1
2
H, H ,
H, K ,
a,H
b,H
,K
a,H
b,H
= 7.44 Hz), 2.82 (m, 2 H, N ), 2.44 (m, 4 H, 2 × E ), 2.08–1.88
(m, 8 H, 2 × E , K , 2 × V ), 1.69 (m, 2 H, K ), 1.45–1.33 (m, 8 H,
K , 2 × A ), 0.92 (d, 12 H, 2 × V , JV = 6.64 Hz).
,V
13C NMR (100.6 MHz, D2O, HMQC): = 133.27 (NC=CH-Im),
117.22 (HNCH=CR-Im), 60.99, 60.77 (3 × S ), 59.24, 59.09 (2 ×
V ), 55.51, 55.39, 55.27, 55.00 (3 × S ), 52.55 (K , E ), 52.18 (H ),
51.66 (E ), 50.36 (N ), 49.79, 49.45 (2 × A ), 42.48 (G ), 38.77
(K ), 35.72 (N ), 30.17 (K ), 29.97 (2 × V ), 29.72, 29.65 (2 × E ),
26.12, 25.96, 25.47, 25.36 (K , H , 2 × E ), 20.98 (K ), 18.22, 18.13,
17.55, 17.40, 17.01, 16.43, 16.38, 16.15 (2 × A , 2 × V ).
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

PAPER
Glycopeptides of the Homophilic Recognition Site of E-Cadherin
2497
was washed with aq NaHCO3 (2 × 30 mL), aq NaI (30 mL), and
with brine (30 mL). After drying (MgSO4), the solvents were re-
moved in vacuo. Purification was performed by flash chromatogra-
phy on silica gel (light petroleum–EtOAc, 1:2) to yield 13 as a
colorless amorphous solid (880 mg, 80%); [ ]D23 +53.4 (c = 1.00,
CH2Cl2); Rf 0.51 (light petroleum–EtOAc, 1:4).
H6 b-Gal), 3.67 (dd,
1
H, H3-Gal,
JH3,H2 = 10.56 Hz,
JH3,H4 = 2.76 Hz), 2.10 (s, 3 H, NHCOCH3), 2.01 (s, 3 H, COCH3),
1.96 (s, 3 H, COCH3), 1.93 (s, 3 H, COCH3), 1.89 (s, 3 H, COCH3),
1.43 (s, 9 H, t-C4H9).
13C NMR (100.6 MHz, CDCl3): = 170.39, 169.84, 169.51, 169.39
(4 × CO), 156.04 (CO-urethane), 143.68 (C4a-, C4b-Fmoc),
141.29, 141.24 (C8a-, C9a-Fmoc), 127.83 (C3-, C6-Fmoc), 127.10
(C2-, C7-Fmoc), 124.95 (C1-, C8-Fmoc), 120.10 (C4-, C5-Fmoc),
101.47 (C1 -Gal), 99.05 (C1-Gal), 83.07 [C(CH3)3], 77.94 (C3-
Gal), 70.84, 70.60, 70.13 (C3 -, C4-, C5-, C5 -Gal), 68.96 (S ),
68.58 (C2 -Gal), 67.02, 66.91 (CH2-Fmoc, C4 -Gal), 62.53 (C6-
Gal), 61.35 (C6 -Gal), 55.04 (S ), 47.65 (C2-Gal), 47.08 (C9-
Fmoc), 27.97 [C(CH3)3], 23.17 (NHCOCH3), 20.66, 20.60, 20.55,
20.52 (4 × COCH3).
1H NMR (400 MHz, CDCl3, 1H-COSY): = 7.76 (d, 2 H, H4-, H5-
Fmoc, JH3,H4 = JH5,H6 = 7.44 Hz), 7.61 (m, 2 H, H1-, H8-Fmoc,
J
H1,H2 = JH8,H7 = 9.26 Hz), 7.45–7.27 (m, 6 H, H2-, H3-, H6-, H7-
Fmoc, Hmeta-Pmb), 6.87 (2 d, H, Hortho-Pmb,
Hortho,Hmeta = 8.80 Hz), 5.77 (d, 1 H, NHAc, JNH,H2-Gal = 7.84 Hz),
2
J
5.79 (d, 1 H, NH-Fmoc, JNH,S = 8.64 Hz), 5.46 (s, 1 H, CH–Pmb),
5.34 (d, 1 H, H4 -Gal, JH4,H3 = 2.76 Hz), 5.12 (dd, 1 H, H2 -Gal,
JH2 ,H3 = 10.16 Hz, JH2 ,H1 = 7.80 Hz), 4.96–4.92 (m, 2 H, H1-, H3 -
Gal), 4.51–4.48 (m, 2 H, H1 -, H2-Gal, JH1 ,H2 = 7.80 Hz), 4.46–
4.33 (m, 3 H, S , CH2-Fmoc), 4.23–3.86 (m, 9 H, H5-, H5 -, H6a-,
H6b-, H6 a-, H6 b-Gal, S , H9-Fmoc), 3.79, 3.77 (2 s, 3 H, OCH3-
Pmb), 3.70–3.60 (m, 2 H, H3-, H4-Gal), 2.12 (s, 3 H, NHCOCH3),
2.04, 2.01, 1.98, 1.95, 1.92 (5 s, 12 H, 4 × COCH3), 1.46, 1.45 (2 s,
9 H, t-C4H9).
MALDI-TOF-MS (DHB): m/z calcd for C44H57N2O19: 917.9;
found: 916.3 (M + H+); m/z calcd for C44H56N2NaO19: 939.9; found:
939.4 (M + Na+).
Anal. Calcd for C44H56N2O19 (916.92): C, 57.64; H, 6.16; N, 3.06.
Found: C, 57.96; H, 6.07; N, 2.86.
13C NMR (100.6 MHz, CDCl3): = 170.28, 170.16, 169.64, 169.48
(C=O), 159.97 (Cpara-Pmb), 155.89 (CO-urethane), 143.62, 141.31
(C4a-, C4b-, C8a-, C9a-Fmoc), 130.12 (Cipso-Pmb), 127.89, 127.50,
127.35, 127.12, 124.86, 120.13 (C1-, C2-, C3-, C4-, C5-, C6-, C7-,
C8-Fmoc, Ctert-Pmb), 113.51 (Ctert-Pmb), 100.99, 100.64 (CH–
Pmb, C1 -Gal), 99.17 (C1-Gal), 83.08 [C(CH3)3], 77.80 (C3-Gal),
75.42, 75.36, 73.99, 70.96, 70.82, 69.12, 68.79 (C2 -, C3 -, C4-,
C5-, C5 -Gal), 69.03 (S ), 67.14 (CH2-Fmoc), 66.94 (C4 -Gal),
63.44, 61.36 (C6-, C6 -Gal), 55.28, 54.97 (OCH3-Pmb), 28.01
[C(CH3)3], 23.32 (NHCOCH3), 20.75, 20.58 (4 × COCH3); doubled
signals due to different conformers.
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-(2-acetamido-4,6-di-
O-acetyl-2-deoxy-3-O-[2,3,4,6-tetra-O-acetyl- -D-galactopyra-
nosyl]- -D-galactopyranosyl)-L-serine tert-Butyl Ester (15,
[Fmoc-Ser(Ac4Gal-( 1 3)- -Ac2GalNAc)-O-t-Bu])
A solution of partially deprotected disaccharide 14 (210 mg,
0.23 mmol) in pyridine–Ac2O (9 mL, 3:1) was stirred for 17 h at r.t.
The solution was poured onto ice and extracted with CH2Cl2 (3 ×
20 mL). The combined organic layers were washed with sat. aq
NaHCO3 solution (2 × 20 mL) and with brine (2 × 20 mL), dried
(MgSO4), and the solvents were evaporated in vacuo. The product
15 was isolated by flash chromatography on silica gel (EtOAc) as a
colorless amorphous solid (201 mg, 88%); [ ]D23 53.0 (c = 1.00,
CH2Cl2); Rf 0.52 (EtOAc).
MALDI-TOF-MS (DHB): m/z calcd for C52H62N2NaO20: 1058.0;
found: 1058.9 (M + Na+); m/z calcd for C52H62KN2O20: 1074.2;
found: 1074.9 (M + K+).
1H NMR (400 MHz, CDCl3, 1H-COSY): = 7.77 (d, 2 H, H4-, H5-
Fmoc,
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-(2-acetamido-2-de-
oxy-3-O-[2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl]- -D-ga-
lactopyranosyl)-L-serine tert-Butyl Ester (14, [Fmoc-
JH3,H4 = JH5,H6 = 7.84 Hz), 7.66 (d,
2
H, H1-, H8-Fmoc,
JH1,H2 = JH8,H7 = 7.04 Hz), 7.50–7.38 (m, 4 H, H2-, H3-, H6-, H7-
Fmoc), 5.78 (m, 2 H, NHAc, NHFmoc), 5.42 (m, 2 H, H4-, H4 -
Gal), 5.18 (dd, 1 H, H2 -Gal, JH2 ,H3 = 10.56 Hz, JH2 ,H1 = 8.25 Hz),
5.01 (dd, 1 H, H3 -Gal, JH3 ,H2 = 10.56 Hz, JH3 ,H4 = 3.16 Hz), 4.94
(br s, 1 H, H1-Gal), 4.63–4.45 (m, 5 H, H1 -, H2-Gal, CH2-Fmoc,
S ), 4.31 [t, 1 H, H9-Fmoc, J(H9,CH2) = 6.64 Hz), 4.22–4.17 (m, 3
H, H6a-, H6b-, H6 a-Gal), 4.12–4.10 (m, 1 H, H6 b-Gal), 4.03–4.00
(m, 2 H, S ), 3.93–3.90 (m, 2 H, H5-, H5 -Gal), 3.84 (dd, 1 H, H3-
Gal, JH3,H2 = 10.56 Hz, JH3,H4 = 2.72 Hz), 2.23 (s, 3 H, NHCOCH3),
2.19 (s, 3 H, COCH3), 2.13 (s, 3 H, COCH3), 2.09 (s, 6 H, 2
COCH3), 2.04 (s, 3 H, COCH3), 2.01 (s, 3 H, COCH3), 1.56 (s, 9 H,
t-C4H9).
Ser(Ac4Gal-( 1 3)- -GalNAc)-O-t-Bu])
To a solution of disaccharide 13 (494 mg, 0.48 mmol, 1.0 equiv) in
MeCN–H2O (20 mL, 9:1) was added Ce(NH4)2(NO2)6 (390 mg,
0.72 mmol, 1.5 equiv). The mixture was stirred for 45 min at r.t., di-
luted with H2O (20 mL) and neutralized by dropwise addition of sat.
aq NaHCO3. MeCN was removed in vacuo, and the remaining aque-
ous phase was extracted with EtOAc (3 × 20 mL). The combined or-
ganic layers were washed with brine (2 × 30 mL) and subsequently
dried (MgSO4). After removal of the solvents in vacuo, the crude
product was purified by flash chromatography (EtOAc) and subse-
quent preparative RP-HPLC (Luna C18, 45% MeCN in H2O) to
give 14 as a colorless amorphous solid (332 mg, 76%); [ ]D23 +50.6
(c = 1.00, CH2Cl2); Rf 0.17 (EtOAc); Rt 8.41 min (Luna C18, 45%
MeCN in H2O).
13C NMR (100.6 MHz, CDCl3):
= 170.48, 170.36, 170.33,
170,12, 169.72, 169.67, 168.96 (C=O), 155.80 (CO-urethane),
143.65 (C4a-, C4b-Fmoc), 141.32, 141.28 (C8a-, C9a-Fmoc),
127.85 (C3-, C6-Fmoc), 127.10 (C2-, C7-Fmoc), 124.86 (C1-, C8-
Fmoc), 120.11 (C4-, C5-Fmoc), 100.52 (C1 -Gal), 98.55 (C1-Gal),
83.10 [C(CH3)3], 77.38 (C3-Gal), 73.12, 70.87, 70.70 (C3 -, C4-,
C5-, C5 -Gal), 69.09 (S ), 68.65 (C2 -Gal), 67.14 (CH2-Fmoc),
66.71 (C4 -Gal), 62.65 (C6-Gal), 61.05 (C6 -Gal), 54.84 (S ), 48.66
(C2-Gal), 47.08 (C9-Fmoc), 28.00 [C(CH3)3], 23.24 (NHCOCH3),
20.70, 20.66, 20.63, 20.52 (6 × COCH3).
1H NMR (400 MHz, CDCl3, 1H-COSY): = 7.73 (d, 2 H, H4-, H5-
Fmoc, JH3,H4 = JH5,H6 = 7.84 Hz), 7.55 (d, 2 H, H1-, H8-Fmoc,
JH1,H2 = JH8,H7 = 7.04 Hz), 7.38–7.25 (m, 4 H, H2-, H3-, H6-, H7-
Fmoc), 5.93 (d, 1 H, NHAc, JNH,H2-Gal = 9.92 Hz), 5.85 (d, 1 H,
NHFmoc, JNH,S = 8.64 Hz), 5.31 (d,
1
H, H4 -Gal,
JH4,H3 = 2.72 Hz), 5.12 (t, H, H2 -Gal, JH2 ,H3
1
=
JH2 ,H1 = 10.16 Hz), 4.92 (dd, 1 H, H3 -Gal, JH3 ,H2 = 10.16 Hz,
JH3 ,H4 = 3.12 Hz), 4.84 (br s, 1 H, H1-Gal), 4.51–4.48 (m, 2 H, H1 -,
H2-Gal), 4.42–4.36 (m, 3 H, S , CH2-Fmoc), 4.18 (t, 1 H, H9-Fmoc,
J(H9, CH2) = 6.64 Hz), 4.12–4.00 (m, 4 H, H4-, H6a-Gal, S ),
3.93–3.91 (m, 1 H, H6b-Gal), 3.89–3.71 (m, 4 H, H5-, H5 -, H6 a-,
ESI-MS: m/z calcd for C48H61N2O21: 1001.4; found: 1001.3 (M +
H+); m/z calcd for C48H60N2NaO21: 1023.4; found: 1023.2 (M +
Na+).
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

2498
T. Reipen, H. Kunz
PAPER
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-(2-acetamido-4,6-di-
O-acetyl-2-deoxy-3-O-[2,3,4,6-tetra-O-acetyl- -D-galactopyra-
nosyl]- -D-galactopyranosyl)-L-serine (16,
in 80 min). The -anomer 18 (240 mg, 28%) and the -anomer 19
(61 mg, 7%) were obtained as colorless amorphous solids.
-Anomer 18
[Fmoc-Ser(Ac4Gal-( 1 3)- -Ac2GalNAc)-OH])
[ ]D23 +24.6 (c = 1.00, CH2Cl2); Rf 0.52 (EtOAc–MeOH, 10:1);
Disaccharide conjugate 15 (190 mg, 0.19 mmol, 1.0 equiv) was dis-
solved in trifluoroacetic acid (10 mL) and anisole (1 mL). After stir-
ring at r.t. for 1 h, the solvents were removed in vacuo. The residue
was repeatedly coevaporated with toluene. Subsequently, the crude
product was purified by flash chromatography on silica gel
(EtOAc–EtOH–AcOH, 10:1:0.1) to give 16 as a colorless amor-
phous solid (150 mg, 84%); [ ]D23 +102.9 (c = 1.00, CH2Cl2);
Rf 0.23 (light petroleum–EtOH–HOAc, 10:1:0.1).
Rt 26.57 min (Luna C18, 55% MeCN in H2O 55% MeCN in H2O
in 10 min
min).
70% MeCN in H2O in 60 min
100% MeCN in 80
1
1H NMR (400 MHz, CDCl3, H-COSY, TOCSY): = 7.73 (d, 2
H, H4-, H5-Fmoc, JH3,H4 = JH5,H6 = 7.44 Hz), 7.55 (m, 2 H, H1-,
H8-Fmoc, JH1,H2 = JH8,H7 = 7.44 Hz), 7.38–7.26 (m, 9 H, H2-,
H3-, H6-, H7-Fmoc, Ctert-benzyl), 5.83–5.76 (m, 2 H, NHFmoc,
NHAcGalNAc), 5.32–5.28 [m, 4 H, H4 -Gal (5.32), H7 -, H8 -
Neu5Ac, NHAcNeu5Ac], 5.17–5.11 [m, 3 H, H2 -Gal (5.13), CH2-
benzyl (5.16)], 4.91 (d, 1 H, H3 -Gal, JH3 ,H2 = 10.20 Hz), 4.77 (m,
1 H, H4 -Neu5Ac), 4.68 (br s, 1 H, H1-Gal), 4.51–4.43 [m, 3 H,
H1 - (4.53), H2-Gal (4.49), S (4.37)], 4.35 (m, 2 H, CH2-Fmoc),
1
1H NMR (400 MHz, CDCl3, H-COSY): = 7.73 (m, 2 H, H4-,
H5-Fmoc), 7.61 (m, 2 H, H1-, H8-Fmoc), 7.40–7.25 (m, 4 H, H2-,
H3-, H6-, H7-Fmoc), 6.14 (m, 1 H, NHAc), 5.90 (m, 1 H, NHF-
moc), 5.34 (br s, 1 H, H4-Gal), 5.31, 5.15 (2 d, 1 H, H4 -Gal,
JH4 ,H3 = 3.12 Hz), 5.05 (dd, 1 H, H2 -Gal, JH2 ,H1 = 7.8 Hz,
JH2 ,H3 = 9.80 Hz), 5.01 (d, 1 H, H1-Gal, JH1,H2 = 2.72 Hz), 5.02,
4.95 (2 dd, 1 H, H3 -Gal, JH3 ,H2 = 9.80 Hz, JH3 ,H4 = 3.12 Hz), 4.82,
4.66 (2 d, 1 H, H1 -Gal, JH1 ,H2 = 7.80 Hz), 4.49–4.35 (m, 3 H) and
4.26–3.74 (m, 11 H) (H2-, H3-, H5-, H-5 , H6-, H6 , H9-Fmoc,
CH2-Fmoc, S , S ), 2.11, 2.09, 2.07, 2.06, 2.04, 2.02, 2.00, 1.99,
1.98 (9 s, 21 H, 6 COCH3 and NHCOCH3).
4.26
(dd,
1
H,
H9 a-Neu5Ac,
JH6a,H5 = 2.36 Hz,
JH6a,H6b = 12.52 Hz), 4.19 [t, 1 H, H9-Fmoc, J(H9,CH2) = 6.64 Hz],
4.07–3.95 [m, 5 H, H6 -Gal (4.07, 3.99), H5 - (4.05), H6 - (4.06),
a
H9 b- Neu5Ac (4.05)], 3.89 (m, 1 H, S ), 3.84–3.66 [m, 6 H, H4-,
b
H5-Gal, H6a- (3.84), H5 -Gal, S (3.67)], 3.61 (m, 1 H, H3-Gal),
3.52 (m, 1 H, H6b-Gal), 2.62 (dd, 1 H, H3 eq-Neu5Ac,
JH3eq,H4 = 3.52 Hz, JH3eq,H3ax = 12.52 Hz), 2.10, 2.06, 2.05, 2.00,
13C NMR (100.6 MHz, CDCl3, HMQC):
= 170.07, 169.92,
1.96, 1.95, 1.93, 1.89, 1.81 (9 s, 30 H, 8 × COCH3, 2 × NHCOCH3),
1.88 (m, 1 H, H3 ax-Neu5Ac), 1.48 (s, 9 H, t-C4H9).
169.78, 169.31 (C=O), 155.92 (CO-urethane), 143.90, 143.58
(C4a-, C4b-Fmoc), 141.30, 141.21 (C8a-, C9a-Fmoc), 127.95,
127.85, 127.19, 127.15, 127.07 (C3-, C6-, C2-, C7-Fmoc), 125.36
(C1-, C8-Fmoc), 120.10 (C4-, C5-Fmoc), 100.94 (C1 -Gal), 97.74
(C1-Gal), 77.56 (C3-Gal), 72.72, 70.76, 70.64, 70.46 (C3 -, C4-,
C5-, C5 -Gal), 69.29 (S ), 68.91, 68.58, 68.35, 67.06 (CH2-Fmoc,
C2 -Gal), 66.74 (C4 -Gal), 62.88 (C6-Gal), 61.10 (C6 -Gal), 54.14
(S ), 49.21 (C2-Gal), 47.24, 47.07 (2 C9-Fmoc), 23.40, 22.76
(NHCOCH3), 20.73, 20.62, 20.56, 20.47, 20.40 (6 × COCH3), dou-
bled signals due to conformers.
13C NMR (100.6 MHz, CDCl3, HMQC):
= 170.78, 170.69,
170.25, 170.19, 170.170.16, 170.08, 169.51, 169.40 (C=O), 167.22
(C1 -Neu5Ac), 155.99 (CO-urethane), 143.69 (C4a-, C4b-Fmoc),
141.30, 141.24 (C8a-, C9a-Fmoc), 134.88 (Cquart-benzyl), 128.74,
128.62, 128.32, 127.84, 127.12 (C3-, C6-, C2-, C7-Fmoc, Ctert-ben-
zyl), 124.92 (C1-, C8-Fmoc), 120.08 (C4-, C5-Fmoc), 101.46 (C1 -
Gal), 98.94, 98.82 (C1-Gal, C2 -Neu5Ac), 82.99 [C(CH3)3], 77.28
(C3-Gal), 72.83, 70.58, 69.13, 68.76, 67.68 (C4-, C5-Gal, C5 -Gal,
C6 -, C7 -, C8 -Neu5Ac), 70.70 (C3 -Gal), 68.99 (H4 -Neu5Ac),
68.44 (C2 -Gal), 68.37 (S ), 67.76 (CH2-benzyl), 67.03 (CH2-
Fmoc), 66.74 (C4 -Gal), 63.24 (C6-Gal), 62.51 (C9 -Neu5Ac),
60.81 (C6 -Gal), 54.94 (S ), 49.09 (C5 -Neu5Ac), 47.63, 47.09
(C2-Gal, C9-Fmoc), 37.67 (C3 -Neu5Ac), 27.96 [C(CH3)3], 23.19,
23.11, 21.02, 20.78, 20.73, 20.62, 20.58, 20.53 (8 × COCH3, 2 ×
NHCOCH3).
ESI-MS: m/z calcd for C44H52N2NaO21: 967.3; found: 967.5 (M +
Na+); m/z calcd for C44H51N2Na2O21: 989.9; found: 989.4 (M + 2
Na+ – H+).
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-(2-acetamido-2-de-
oxy-3-O-[2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl]-6-O-
[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy- / -D-
glycero-D-galacto-2-nonulopyranosyl)onate]- -D-galactopyra-
nosyl)-L-serine tert-Butyl Ester (18/19, [Fmoc-Ser({Ac4Gal-
( 1 3)}[Ac4Neu5AcCOOBzl-( / 2 6)]- -Ac2GalNAc)-O-t-
Bu])
ESI-MS: m/z calcd for C70H88N3O31: 1466.5; found: 1466.6 (M +
H+); m/z calcd for C70H87N3NaO31: 1488.5; found: 1488.6 (M +
Na+).
-Anomer 19
[ ]D23 = 29.3 (c = 1.00, CH2Cl2); Rf 0.52 (EtOAc–MeOH 10:1);
Rt 34.54 min (Luna C18, 55% MeCN in H2O 55% MeCN in H2O
To a mixture of glycosyl acceptor 14 (530 mg, 0.58 mmol, 1.0
equiv) and xanthate donor 1727 (974 mg, 1.45 mmol, 2.5 equiv) dis-
solved in MeCN–CH2Cl2 (2:1) under argon were added molecular
sieves (1.50 g, 3 Å). After stirring for 1 h, anhyd AgOTf (373 mg,
1.45 mmol, 2.5 equiv) was added under the exclusion of light, and
the mixture was cooled to –62 °C. During a period of 15 min, me-
thylsulfenyl bromide [MSB, prepared by adding Br2 (410 L,
8.0 mmol, 1.0 equiv) to a solution of dimethyl disulfide (709 L,
8.0 mmol, 1.0 equiv) in 1,2-dichloroethane (10 mL) and stirring for
12 h under exclusion of oxygen and light; 0.91 mL, 1.6 M] cooled
to 0 °C, was added dropwise. After addition of MSB, the suspension
was stirred at –62 °C for 4 h. Diisopropylamine (0.2 mL) was add-
ed, and the suspension was stirred for 30 min. Subsequently, the
mixture was allowed to warm up to 10 °C, diluted with CH2Cl2
(50 mL) and filtered through Hyflo Supercel. After washing with
CH2Cl2 (20 mL), the solvents of the filtrate were evaporated in vac-
uo. The glycal formed as a by-product was removed by flash chro-
matography on silica gel (EtOAc). Separation of 18 from -anomer
19 and educt 14 was achieved by preparative RP-HPLC (Luna C18,
55% MeCN in H2O 55% MeCN in H2O in 15 min 70% MeCN
in H2O in 20 min 70% MeCN in H2O in 60 min 100% MeCN
in 10 min
min).
70% MeCN in H2O in 60 min
100% MeCN in 80
1H NMR (400 MHz, CDCl3, 1H-COSY): = 7.74 (d, 2 H, H4-, H5-
Fmoc, JH3,H4 = JH5,H6 = 7.44 Hz), 7.60 (m, 2 H, H1-, H8-Fmoc,
J
H1,H2 = JH8,H7 = 7.04 Hz), 7.40–7.26 (m, 9 H, H2-, H3-, H6-, H7-
Fmoc, Ctert-benzyl), 6.71 (d, 1 H, NHAcNeu5Ac, JNH,H5 = 9.76 Hz),
6.23 (d, 1 H, NHFmoc, JNH,S = 7.44 Hz), 5.53 (d, 1 H, NHAcGalNAc
NH,H2 = 8.60 Hz), 5.40–5.34 (m, 3 H, H4 -Gal, H4 -, H7 -
,
J
Neu5Ac), 5.25–5.11 (m, 4 H, H2 -Gal, H8 -Neu5Ac, CH2-benzyl),
4.96 (d, 1 H, H3 -Gal, JH3 ,H2 = 8.65 Hz), 4.80–4.73 (m, 2 H, H1-,
H9 a-Neu5Ac), 4.61–4.45 (m, 3 H, H1 -, H2-Gal, S ), 4.37 (m, 2 H,
CH2-Fmoc), 4.20 [t, 1 H, H9-Fmoc, J(H9,CH2) = 7.44 Hz], 4.13–
3.98 (m, 7 H, H4-, H5 -, H6 a, H6 b-Gal, H5 -, H6 -, H9 b-
a
Neu5Ac), 3.87 (m, 1 H, S ), 3.81–3.78 (m, 2 H, H5-, H6a-Gal),
b
3.68–3.62 (m, 3 H, S , H3-, H6b-Gal), 2.46 (dd, 1 H, H3 eq-
Neu5Ac, JH3eq,H4 = 4.72 Hz, JH3eq,H3ax = 12.96 Hz), 2.13, 2.12, 2.06,
1.96, 1.95, 1.94, 1.78 (7 s, 30 H, 8 × COCH3, 2 × NHCOCH3), 1.88
(m, 1 H, H3 ax-Neu5Ac), 1.48 (s, 9 H, t-C4H9).
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

PAPER
Glycopeptides of the Homophilic Recognition Site of E-Cadherin
2499
13C NMR (100.6 MHz, CDCl3, HMQC):
= 170.93, 170.63,
MALDI-TOF-MS: m/z calcd for C66H79N3NaO31: 1433.3; found:
1433.1 (M + Na+); m/z calcd for C66H79KN3O31: 1449.4; found:
1449.2 (M + K+).
170.31, 170.19, 170.12, 169.52 (C=O), 166.09 (C1 -Neu5Ac),
156.04 (CO-urethane), 143.62 (C4a-, C4b-Fmoc), 141.30, 141.24
(C8a-, C9a-Fmoc), 134.84 (Cquart-benzyl), 128.63, 128.56, 128.33,
127.86, 127.15, 127.11 (C3-, C6-, C2-, C7-Fmoc, Ctert-benzyl),
125.04 (C1-, C8-Fmoc), 120.07 (C4-, C5-Fmoc), 101.26 (C1 -Gal),
98.32, 98.26 (C1-Gal, C2 -Neu5Ac), 83.23 [C(CH3)3] 77.21 (C3-
Gal), 72.96, 70.61, 69.05, 68.76, 68.72, 67.56 (C4-, C5-, C2 -, C5 -
Gal, C4 -, C6 -, C7 -, C8 -Neu5Ac), 70.70 (C3 -Gal), 68.67 (S ),
67.62 (CH2-benzyl), 67.33 (CH2-Fmoc), 66.62 (C4 -Gal), 63.10
(C6-Gal), 62.44 (C9 -Neu5Ac), 60.62 (C6 -Gal), 54.58 (S ), 48.94
(C5 -Neu5Ac), 47.51, 47.00 (C2-Gal, C9-Fmoc), 37.25 (C3 -
Neu5Ac), 28.14 [C(CH3)3], 22.88, 20.98, 20.90, 20.76, 20.72,
20.56, 20.53 (CH3 – OAc, NHCOCH3).
L-Seryl-L-histidyl-L-alanyl-L-valyl-L-seryl-L-seryl-O-(2-aceta-
mido-2-deoxy- -D-galactopyranosyl)-L-asparaginyl-glycyl-L-
glutamyl-L-alanyl-L-valyl-L-glutamic Acid (27)
[H2N-SHAVSS( GalNAc)NGEAVE-OH])
The solid-phase glycopeptide synthesis was carried out according to
the standard protocol described above employing a Perkin Elmer
A433 peptide synthesizer. Starting from Rapp Tentagel® resin 1
preloaded with Fmoc-Glu(O-t-Bu)-O-PHB (500 mg, loading:
0.20 mmol/g), the protected fragment Fmoc-N(Trt)GE(O-t-
Bu)AVE(O-t-Bu)-PHB-TentaGel 25 was prepared by iterative cou-
pling of amino acids with coupling times of 30 min. In this course,
the dipeptide Fmoc-Ala-Val-OH was introduced by a double cou-
pling procedure. The glycosylated building block 10 was introduced
in a manual coupling step. To this end, a solution of Fmoc-Ser( -
Ac3GalNAc)-OH (10; 160 mg, 0.25 mmol, 2.5 equiv), HATU16
(106 mg, 0.28 mmol, 2.8 equiv), HOAt (38 mg, 0.28 mmol,
2.8 equiv) and N-methylmorpholine (NMM) (60 L, 0.56 mmol,
5.6 equiv) in DMF (2 mL) was added to the resin and vortexed for
5 h. The subsequent five amino acids were attached according to the
standard protocol. Among these, Fmoc-Val-OH was coupled twice.
The resin was washed with NMP and CH2Cl2. For the detachment,
the resin was transferred into a solid-phase reactor and treated with
a mixture of trifluoroacetic acid (13.13 mL), H2O (0.75 mL), thio-
anisole (0.75 mL) and 1,2-ethanedithiol (0.25 mL) for 90 min. The
crude glycopeptide was isolated as described above and subse-
quently dissolved in MeOH (50 mL). A 0.1 M solution of NaOMe
in MeOH was added until pH 9.5 was reached. After stirring for
18 h, the reaction mixture was neutralized by addition of AcOH.
The solvent was evaporated in vacuo, the residue dissolved in H2O
and purified by preparative RP-HPLC (Eurospher C8, 1% MeCN in
H2O 100% MeCN in H2O + 0.1% TFA in 60 min). The complete-
ly deprotected glycopeptide 27 was obtained as a colorless lyophi-
ESI-MS: m/z calcd for C70H88N3O31: 1466.5; found: 1466.6 (M +
H+); m/z calcd for C70H87N3NaO31: 1488.5; found: 1488.6 (M +
Na+).
N-(9H-Fluoren-9-yl)methoxycarbonyl-O-(2-acetamido-2-de-
oxy-3-O-[2,3,4,6-tetra-O-acetyl- -D-galactopyranosyl]-6-O-
[benzyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy- / -D-
glycero-D-galacto-2-nonulopyranosyl)onate]- -D-galactopyra-
nosyl)-L-serine (20, [Fmoc-Ser({Ac4Gal-
( 1 3)}[Ac4Neu5AcCOOBzl-( 2 6)]- -Ac2GalNAc)-OH])
A solution of sialyl-T conjugate 18 (220 mg, 0.15 mmol, 1.0 equiv)
in trifluoroacetic acid (5 mL) and anisole (0.5 mL) was stirred at r.t.
for 2 h. After evaporation of the solvents in vacuo, the residue was
repeatedly coevaporated with toluene. The crude product was puri-
fied by flash chromatography on silica gel (EtOAc
EtOAc–
EtOH–AcOH, 10:1:0.1) to yield an amorphous colorless solid 20
(200 mg, 94%); [ ]D23 +38.2 (c = 1.00, CH2Cl2); Rf 0.31 (EtOAc–
EtOH–AcOH, 10:1:0.1).
1H NMR (400 MHz, CDCl3, 1H-COSY, TOCSY): = 7.71 (m, 2 H,
H4-, H5-Fmoc), 7.57 (m, 2 H, H1-, H8-Fmoc), 7.38–7.22 (m, 9 H,
H2-, H3-, H6-, H7-Fmoc, Ctert-benzyl), 7.15 (m, 1 H, NHFmoc),
5.35 (m, 1 H, H8 -Neu5Ac), 5.40–5.09 [m, 5 H, H2 - (5.09), H4 -
Gal (5.26), H7 -Neu5Ac (5.28), CH2-benzyl (5.15)], 4.94–4.87 [m,
2 H, H1-Gal (4.93), H3 -Gal (4.88)], 4.80–4.69 [m, 2 H, H4 -
Neu5Ac (4.76), S (4.70)], 4.38–4.24 [m, 4 H, H1 -Gal (4.24),
H9 a-Neu5Ac (4.31), CH2-Fmoc], 4.10–3.95 [m, 6 H, H9-Fmoc
(4.06), H5 - (4.06), H-6 - (4.00), H9 b-Neu5Ac (4.03), S (4.06)],
3.89–3.67 [m, 6 H, H2- (3.83), H4- (3.80), H5- (3.69), H6a-Gal
(3.89), H6 -Gal (3.81)], 3.63 (m, 1 H, H3-Gal), 3.39–3.42 [m, 2 H,
H6b-Gal (3.41), H5 -Gal (3.39)], 2.58 (dd, 1 H, H3 eq-Neu5Ac,
JH3eq,H4 = 4.28 Hz, JH3eq,H3ax = 12.52 Hz), 2.09, 2.07, 2.02, 2.01,
1.98, 1.97, 1.95, 1.93, 1.83, 1.79 (10 s, 30 H, 8 × COCH3, 2 ×
NHCOCH3), 1.89 (m, 1 H, H3 ax-Neu5Ac).
24
lisate (90 mg, 65%); [ ]D23 –16.3 (c = 1.00, H2O) {Lit.38 [ ]D
–14.0 (c = 0.8, H2O)}; Rt 12.56 min (RP-HPLC, Eurospher C8,
215 nm, 1% MeCN in H2O
100% MeCN + 0.1% TFA in
42 min).
1
1H NMR (400 MHz, D2O, H-COSY, TOCSY): = 8.51 (s, 1 H,
Im2), 7.20 (s, 1 H, Im4), 4.88 (d, 1 H, H1-Gal, JH1,H2 = 3.82 Hz),
4.70–4.60 (m, 2 H, N , H ), 4.61 (m, 1 H, S ), 4.46 (m, 1 H, S ), 4.38
(m, 1 H, E ), 4.31–4.22 (m, 3 H, 2 × A , E ), 4.11–4.00 [m, 4 H, H2-
Gal (4.10), S (4.07), 2 × V (4.10, 4.04)], 3.90–3.86 [m, 13 H, H3-
(3.79), H4- (3.89), H5- (3.81), H6-Gal, G , 3 × S ], 3.22 (dd, 1 H,
a
b
H , JH
b = 15.55 Hz, JH
= 6.16 Hz), 3.17 (dd, 1 H, H ,
a,H
a,H
JH
b = 15.85 Hz, JH
= 7.62 Hz), 2.83–2.71 (m, 2 H, N ), 2.42
a,H
a,H
13C NMR (100.6 MHz, CDCl3, HMQC):
= 170.09, 170.02,
a
b
(m, 4 H, 2 × E ), 2.12 (m, 1 H, E ), 2.06–1.88 (m, 8 H, E , E , 2 ×
V , NHCOCH3), 1.30 (m, 6 H, 2 × A ), 0.86 (d, 12 H, 2 × V ,
169.97, 169.69, 167.35 (C=O), 167.35 (C1 -Neu5Ac), 156.01 (CO-
urethane), 143.92, 143.62 (C4a-, C4b-Fmoc), 141.20 (C8a-, C9a-
Fmoc), 134.87 (Cquart-benzyl), 128.76, 128.73, 128.59, 128.27,
127.94, 127.89, 127.27, 127.09 (C3-, C6-, C2-, C7-Fmoc, Ctert-ben-
zyl), 125.34 (C1-, C8-Fmoc), 120.07 (C4-, C5-Fmoc), 102.05,
101.26 (C1 -Gal), 98.72, 98.63, 98.03 (C1-Gal, C2 -Neu5Ac),
80.59 (C3-Gal), 72.89, 69.18, 69.07, 68.91, 68.82, 67.56, 67.49,
66.69 (C4-, C5-, C2 -, C5 -Gal, C4 -, C6 -, C7 -, C8 -Neu5Ac),
70.67 (C3 -Gal), 67.65 (CH2-benzyl), 67.38 (CH2-Fmoc), 66.97
(S ), 66.68 (C6 -Gal), 63.68 (C6-Gal), 62.56, 62.48 (C9 -Neu5Ac),
60.80 (C6 -Gal), 54.28 (S ), 49.13 (C2-Gal), 47.22, 47.00 (C5 -
Neu5Ac, C9-Fmoc), 37.67 (C3 -Neu5Ac), 23.13, 21.05, 20.80,
20.70, 20.62, 20.51, 20.40 (8 × COCH3, 2 × NHCOCH3); doubled
signals due to conformers.
JV
= 6.74 Hz).
,V
13C NMR (100.6 MHz, D2O, HMQC): = 176.91, 174.65, 173.32,
172.84, 172.78, 170.80, 170.79, 167.79, 163.02, 162.68 (C=O),
133.70 (N=CN-Im), 128.14 (NC=CH-Im), 117.47 (HNCH=CR-
Im), 97.97 (C1-Gal), 71.40 (C5-Gal), 68.56 (C4-Gal), 67.82 (C3-
Gal, S ), 61.27, 60.14 (C6-Gal, 2 × S ), 59.41 (2 × V ), 54.46, 52.95,
52.84 (3 × S ), 52.53, 51.92 (2 × E , H ), 50.65 (N ), 49.81, 49.71
(2 × A , C2-Gal), 42.69 (G ), 36.29 (N ), 30.34, 30.23, 30.00, 29.96
(2 × V , 2 × E ), 26.52 (H ), 25.79, 25.69 (2 × E ), 22.18
(NHCOCH3), 18.50, 18.40, 17.72, 17.63 (2 × V ), 16.67, 16.48 (2 ×
A ).
MALDI-TOF-MS (DHB): m/z calcd for C55H89N16O26: 1390.4;
found: 1391.0 (M + H+); m/z calcd for C55H88N16NaO26: 1412.4;
found: 1413.1 (M + Na+); m/z calcd for C55H87N16Na2O26: 1434.4;
found: 1435.0 (M + 2Na+ – H+).
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

2500
T. Reipen, H. Kunz
PAPER
L-Seryl-L-histidyl-L-alanyl-L-valyl-L-seryl-O-(2-acetamido-2-
deoxy- -D-glucopyranosyl)-L-seryl-L-asparaginyl-glycyl-L-glu-
tamyl-L-alanyl-L-valyl-L-glutamic Acid (28, [H2N-SHAVSS( -
GlcNAc)NGEAVE-OH])
isolated as described above. The acetyl groups were removed within
48 h, and the crude product was purified by preparative RP-RP-
HPLC (Luna C18, 5% MeCN in H2O
5% MeCN in H2O in 15
min 20% MeCN in H2O in 50 min 100% MeCN + 0.1% TFA
in 60 min). The completely deprotected glycopeptide 29 was ob-
tained as a slightly yellowish lyophilisate (50 mg, 32%); [ ]D23 –9.4
(c = 1.00, H2O); Rt 23.40 min (RP-HPLC, Luna C18, 215 nm, 5%
The synthesis of 28 was carried out according to the described stan-
dard procedure. Tentagel® resin preloaded with Fmoc-Glu(O-t-Bu)-
O-PHB (loading 500 mg, 0.20 mmol/g) 1 served as starting materi-
al. In order to react the glycosylated building block with the N-ter-
minally deblocked resin-linked peptide 25, Fmoc-Ser( -
Ac3GlcNAc)-OH (24; 130 mg, 0.2 mmol, 2.0 equiv), HATU
(83 mg, 0.22 mmol, 2.2 equiv), HOAt (30 mg, 0.22 mmol, 2.2
equiv) and NMM (47 L, 0.44 mmol, 4.4 equiv) were dissolved in
DMF (2 mL) and added to the resin. After 4 h, the reaction was ter-
minated by filtration. The subsequent amino acids were then cou-
pled according to the standard protocol. The acidolytic detachment
from the resin was carried out by treatment with trifluoroacetic acid
(15 mL), triisopropylsilane (0.9 mL) and H2O (0.9 mL) within
2.5 h. The solution of the crude product was poured into cold Et2O
resulting in precipitation of the crude glycopeptide. Subsequently,
the partially protected glycopeptide was deprotected with 0.1 M
NaOMe in MeOH as described above (reaction time: 15 h). The
crude deprotected glycopeptide 28 was dissolved in H2O and puri-
fied by preparative RP-HPLC (Luna C18, 1% MeCN in H2O
100% MeCN in H2O + 0.1% TFA in 60 min) to furnish the com-
pletely deprotected glycopeptide 28 as a colorless lyophilisate
(70 mg, 51%); [ ]D23 –55.6 (c = 2.50, H2O); Rt 11.27 min (RP-
MeCN in H2O
5% MeCN in H2O in 10 min
60% MeCN in
H2O in 40 min 100% MeCN + 0.1% TFA in 60 min).
1
1H NMR (400 MHz, D2O, H-COSY, TOCSY): = 8.62 (s, 1 H,
Im2), 7.32 (s, 1 H, Im4), 4.91 (1 H, H1-Gal), 4.78 (1 H, H ), 4.76 (1
H, N ), 4.67 (m, 1 H, S ), 4.54 (m, 1 H, S ), 4.48 (d, 1 H, H1 -Gal,
JH1 ,H2 = 7.44 Hz), 4.40–4.29 [m, 5 H, H2-Gal (4.36), 2 × A (4.38),
2 × E (4.33)], 4.25–4.08 [m, 4 H, H4-Gal (4.24), 2 × V (4.20,
4.15), S (4.17)], 4.05–3.85 [m, 11 H, H3- (4.02), H5-Gal (3.91),
H4 -Gal (3.92), G , 3 × S ], 3.74 (br s, 4 H, H6-Gal, H6 -Gal), 3.63–
3.59 [m, 2 H, H3 -Gal (3.62), H5 -Gal (3.65)], 3.51 (m, 1 H, H2 -
Gal), 3.21 (m, 2 H, H ), 2.83 (m, 2 H, N ), 2.32 (br s, 4 H, E ), 2.15–
1.90 (m, 9 H, 2 × V , 2 × E , NHCOCH3), 1.38 (m, 6 H, A ), 0.94
(br s, 12 H, V ).
13C NMR (100.6 MHz, D2O, HMQC): = 104.25 (C1 -Gal), 97.49
(C1-Gal), 76.83 (C3-Gal), 74.51 (C5 -Gal), 72.02 (C3 -Gal), 70.56
(C4 -Gal), 70.20 (C2 -Gal), 68.17 (C4-Gal), 68.06 (C5-Gal), 60.60
(C6-Gal, C6 -Gal), 66.46, 60.80, 59.71 (3 × S ), 59.01, 58.87 (2 ×
V ), 54.43, 53.43 (2 × S ), 54.00 (2 × E ), 53.13 (S ), 52.02 (H ),
50.19 (N ), 49.48 (2 × A ), 42.36 (C2-Gal), 42.14 (G ), 35.72 (N ),
31.86 (2 × E ), 29.79 (2 × V ), 27.02, 26.63 (2 × E ), 26.33 (H ),
21.62 (NHCOCH3), 18.10, 17.09 (2 × V ), 16.04 (2 × A ).
HPLC, Luna C18, 215 nm, 1% MeCN in H2O
100% MeCN +
0.1% TFA in 42 min).
1
1H NMR (400 MHz, D2O, H-COSY, TOCSY): = 8.47 (s, 1 H,
Im2), 7.17 (s, 1 H, Im4), 4.60–4.57 (m, 2 H, N , H ), 4.41–4.30 (m,
3 H, H1-Glc, 2 × S ), 4.25& ndash;4.12 (m, 3 H, 2 × A , E ), 4.11–
MALDI-TOF-MS (DHB): m/z calcd for C61H99N16O31: 1552.5;
found: 1552.3 (M + H+); m/z calcd for C61H98N16NaO31: 1574.5;
found: 1574.4 (M + Na+); m/z calcd for C61H98KN16O31: 1590.6;
found: 1590.3 (M + K+).
a
b
3.91 (m, 5 H, 2 × V , S , S , E ), 3.90–3.70 (m, 8 H, G , 2 × S , S ,
H6a-Glc), 3.61–3.52 (m, 2 H, H2-Glc, H6b-Glc), 3.39 (t, 1 H, H3-
Glc, JH3,H2 = JH3,H4 = 8.60 Hz), 3.29 (m, 2 H, H4-, H5-Glc), 3.13
a
L-Seryl-L-histidyl-L-alanyl-L-valyl-L-seryl-O-(2-acetamido-2-de-
oxy-3-O-[ -D-galactopyranosyl]-6-O-{(5-acetamido-3,5-dideoxy-
a-glycero-d-galacto-2-nonulopyranosyl)onate}-a-d-galacto-
pyranosyl)-L-seryl-L-asparaginyl-glycyl-L-glutamyl-L-alanyl-
L-valyl-L-glutamic Acid (32, [H2N-SHAVSS({Gal-
(dd, 1 H, H , JH
b = 15.68 Hz, JH
= 6.28 Hz), 3.04 (dd, 1 H,
a,H
a,H
b
H , JH
b = 15.64 Hz, JH
= 7.84 Hz), 2.74–2.65 (m, 2 H, N ),
a,H
a,H
2.15 (m, 4 H, 2 × E ), 2.14–1.77 (m, 9 H, 2 × E , 2 × V ,
NHCOCH3), 1.25 (m, 6 H, 2 × A ), 0.78 (m, 12 H, 2 × V ).
13C NMR (100.6 MHz, D2O, HMQC): = 174.12, 173.18, 172.81,
170.77, 167.66 (C=O), 133.52 (NC=CH-Im), 127.98 (NC=CH-Im),
117.32 (HNCH=CR-Im), 100.87 (C1-Glc), 73.85 (C3-Glc), 75.77,
69.75 (C4-, C5-Glc), 67.88 (S ), 60.59 (S ), 60.56 (C6-Glc), 60.01
(S ), 59.25, 59.12 (2 × V ), 55.22, 54.25 (2 × S , 2 × E ), 53.44 (S ),
52.38 (H ), 50.36 (N ), 49.50 (2 × A ), 42.63 (G ), 37.06 (N ), 32.69
(2 × E ), 30.10 (V ), 27.51 (2 × E ), 26.51 (H ), 22.18 (NHCOCH3),
18.43, 18.35, 17.56, 17.47, 17.38, 17.32, 16.70, 16.54, 16.44, 16.29
(2 × A , 2 × V ).
( 1 3)}[Neu5Ac-( 2 6)]- -GalNAc)NGEAVE-OH])
This glycopeptide was synthesized according to the procedure de-
scribed for 28. Tentagel® resin preloaded with Fmoc-Glu(O-t-Bu)-
O-PHB 1 (455 mg, loading 0.22 mmol/g) was used. The glycosylat-
ed building block was coupled manually by addition of a mixture of
18 (190 mg, 0.13 mmol, 1.3 equiv), HATU (148 mg, 0.39 mmol,
3.9 equiv), HOAt (53 mg, 0.39 mmol, 3.9 equiv) and NMM (86 L,
0.78 mmol, 7.8 equiv) in DMF (2 mL) to the resin (coupling time:
4 h). The crude product was isolated as described above.
MALDI-TOF-MS (DHB): m/z calcd for C55H89N16O26: 1389.6;
found: 1389.3 (M + H+); m/z calcd for C55H88N16NaO26: 1411.6;
found: 1411.5 (M + Na+); m/z calcd for C55H88KN16O26: 1427.6;
found: 1427.5 (M + K+).
MALDI-TOF-MS (DHB): m/z calcd for C95H138N17O47: 2270.2;
found: 2270.5 (M + H+); m/z calcd for C95H137N17NaO47: 2292.2;
found: 2292.2 (M + Na+).
The benzyl ester was subsequently removed by hydrogenation. For
this purpose, the partially protected glycopeptide 30 was dissolved
in MeOH (30 mL). After addition of a catalytic amount of 10% Pd/
C, the mixture was hydrogenated for 3 d. The hydrogenation was
terminated by flooding the flask with argon and filtration through
Hyflo Supercel. After removal of the solvents in vacuo, the crude
product 31 was dissolved in MeOH, and purified by preparative RP-
L-Seryl-L-histidyl-L-alanyl-L-valyl-L-seryl-O-(2-acetamido-2-
deoxy-3-O-[ -D-galactopyranosyl]- -D-galactopyranosyl)-L-
seryl-L-asparaginyl-glycyl-L-glutamyl-L-alanyl-L-valyl-L-glu-
tamic Acid (29, [H2N-SHAVSS(Gal-( 1 3)- -Gal-
NAc)NGEAVE-OH])
Glycopeptide 29 was synthesized according to the described proto-
col. As starting material Tentagel® resin preloaded with Fmoc-
Glu(O-t-Bu)-O-PHB 1 (455 mg, loading 0.22 mmol/g) was used.
The coupling of the glycosylated building block was carried out by
dissolving 16 (125 mg, 0.13 mmol, 1.3 equiv), HATU (152 mg,
0.4 mmol, 4.0 equiv), HOAt (54 mg, 0.4 mmol, 4.0 equiv) and
NMM (88 L, 0.8 mmol, 8.0 equiv) in DMF (2 mL). The mixture
was added to the resin and kept vortexing for 5 h. After coupling of
the following amino acids, the partially protected glycopeptide was
HPLC (Luna C18, 5% MeCN in H2O
5% MeCN in H2O in 15
min 20% MeCN in H2O in 50 min 100% MeCN + 0.1% TFA
in 60 min) and obtained as a colorless lyophilisate (71 mg, 33%).
31
[ ]D23 –1.7 (c = 1.00, MeOH); Rt 28.95 min (RP-HPLC, Luna C18,
215 nm, 5% MeCN in H2O 5% MeCN in H2O in 10 min 60%
MeCN in H2O in 40 min 100% MeCN + 0.1% TFA in 60 min).
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

PAPER
Glycopeptides of the Homophilic Recognition Site of E-Cadherin
2501
1H NMR (600 MHz, DMSO-d6, 1H-COSY, TOCSY): = 8.98 (s, 1
(1.94, 1.76; 1.87, 1.71)], 1.47 (m, 1 H, H3 ax-Neu5Ac,
H3 eq,H3 ax = JH3 eq,H4 = 11.32 Hz), 1.23 (d, H, A ,
JA = 6.68 Hz), 1.17 (d, 3 H, A , JA = 6.64 Hz), 0.83 (m, 12 H,
H, Im2), 8.69 (d, 1 H, HNH, JNH,H = 7.92 Hz), 8.31 (d, 1 H, ANH
,
J
3
JNH,A = 6.60 Hz), 8.23–8.13 (m, 5 H, ENH, GNH, 2 × SNH, NNH), 8.03
(d, 1 H, ANH, JNH,A = 6.60 Hz), 8.00–7.93 (m, 2 H, ENH, SNH), 7.82–
7.72 (m, 3 H, 2 × VNH, NHNeu5NAc), 7.46 (s, 1 H, NH2-N) 7.36 (s, 1
H, Im4), 7.28 (d, 1 H, NHGalNAc, JNH,H2 = 7.44 Hz), 6.93 (s, 1 H,
NH2-N), 5.22 (m, 2 H, H4 -Gal, H8 -Neu5Ac), 5.15 (d, 1 H, H7 -
,A
,A
2 × V ).
13C NMR (150.9 MHz, DMSO-d6, HMQC): = 104.54 (C1 -Gal),
98.04 (C1-Gal), 76.94 (C3-Gal), 75.04 (C5 -Gal), 73.02 (C3 -Gal),
71.21 (C4-Gal), 70.78 (C2 -Gal), 69.79 (C4 -Gal), 67.53 (C5-Gal),
73.04, 69.07, 68.51 (H6 -, H7 -, H8 -Neu5NAc), 66.24 (S ), 63.16
(C6-Gal), 62.88 (C9 -Neu5Ac), 61.58 (S ), 60.26 (C6 -Gal), 60.21
(S ), 57.57, 57.26 (2 × V ), 54.86, 53.97, 52.77 (3 × S ), 52.25
(C5 -Neu5Ac), 51.51 (H , E ), 50.90 (E ), 49.70 (N ), 48.29, 48.04
(A ), 47.70 (C2-Gal), 42.09 (G ), 41.13 (C3 -NeuNAc), 36.97 (N ),
30.72 (2 × V ), 29.98 (E ), 27.47 (H ), 27.39, 26.17 (E ), 22.84,
22.50 (NHCOCH3), 19.10, 17.88 (2 × V ), 17.64 (2 × A ).
Neu5Ac, JH7 ,H8 = 8.52 Hz), 5.01 (dd,
1
H, H3 -Gal,
JH3 ,H2 = 10.44 Hz), 4.95 (t, 1 H, H2 -Gal, JH2 ,H3 = 10.14 Hz), 4.81
(d, 1 H, H1 -Gal, JH1 ,H2 = 7.68 Hz), 4.72 (m, 1 H, H4 -Neu5Ac),
4.66 (m, 1 H, H ), 4.62 (d, 1 H, H1-Gal, JH1,H2 = 2.20 Hz), 4.53 (m,
1 H, N ), 4.46–4.27 [m, 7 H, 2 × A (4.36, 4.31), 2 × S (4.46, 4.43),
V (4.27), E (4.27), H2-Gal (4.24)], 4.18–4.09 [m, 5 H, H6a-Gal
(4.14), H5 - (4.11), H6 a-Gal (4.12), E (4.18), V (4.15)], 4.06–
3.93 [m, 3 H, H6b-Gal (4.03), 6 -Gal (3.98), H6 -Neu5Ac
a
MALDI-TOF-MS: m/z calcd for C61H98N16NaO31: 1574.5; found:
1576.0 (M – sialic acid + H+ + Na+); m/z calcd for C61H98KN16O31:
1590.6; found: 1590.3 (M – sialic acid + H+ + K+).
(4.02)], 3.90–3.76 [m, 4 H, S (3.87), G (3.80), H4-Gal (3.81),
H5 -Neu5Ac (3.83)], 3.74–3.53 [m, 10 H, H3- (3.62), H5-Gal
b
(3.69),
9
-Neu5Ac (3.69), G (3.62), 3 × S (3.72, 3.61; 3.70,
a
3.65; 3.66, 3.58), 3.55 (m,
9
-Neu5Ac)], 3.07 (dd, 1 H, H ,
b
L-Cystyl-di(L-seryl-L-histidyl-L-alanyl-L-valyl-L-seryl-L-seryl-
L-asparaginyl-glycine) (35)
JH
JH
b = 15.68 Hz, JH
b = 15.64 Hz, JH
= 6.28 Hz), 3.01 (dd,
= 7.84 Hz), 2.53–2.44 (m, 3 H, H3
1
H, H ,
a,H
a,H
a,H
-
a,H
eq
For automated peptide synthesis, polystyrene resin preloaded with
Fmoc-Gly-O-PHB (164 mg, loading 0.61 mmol/g) was used. The
subsequent seven amino acids were then coupled according to the
standard protocol. The last amino acid, Fmoc-Cys(Trt)-OH, was
coupled manually to the resin. For that purpose, a mixture of Fmoc-
Cys(Trt)-OH (293 mg, 0.5 mmol, 5 equiv), TBTU (161 mg,
0.5 mmol, 5 equiv), HOBt (77 mg, 0.5 mmol, 5 equiv) was dis-
solved in CH2Cl2 (2 mL) and rapidly added to the resin. Finally,
sym-collidine (67 L, 1.0 mmol, 10 equiv) was transferred into the
reaction vessel. After 20 min, the resin was washed thoroughly. The
crude peptide 33 was isolated by acidolysis with trifluoroacetic ac-
id, H2O, and triisopropylsilane as described above (90 mg). As the
compound showed sufficient purity according to RP-HPLC
Neu5Ac, N ), 2.24 (m, 4 H, E ), 2.09–1.74 (10 s, 30 H, 8 × COCH3,
2 × NHCOCH3), 1.99 (m, 2 H, V ), 1.95, 1.76 (m, 2 H, E ), 1.89,
1.73 (m, 2 H, E ), 1.61 (m, 1 H, H3 ax-Neu5Ac), 1.23 (m, 6 H, 2 ×
A ), 0.85, 0.81 (m, 12 H, 2 × V ).
13C NMR (150.9 MHz, DMSO-d6, HMQC): = 132.81 (N=CN-
Im), 117.43 (HNCH=CR-Im), 100.98 (C1 -Gal), 97.96 (C1-Gal),
77.10 (C3-Gal), 71.52 (C6 -Neu5Ac), 70.56 (C3 -Gal), 69.89 (C5 -
Gal), 69.41 (C4 -Neu5Ac), 69.13 (C5-Gal), 68.26 (C2 -Gal), 67.59,
67.20 (C4 -Gal, C8 -Neu5Ac), 67.30 (C4-Gal), 67.02 (C7 -
Neu5Ac), 66.25 (S ), 63.28 (C9 -Neu5Ac), 61.83 (S ), 61.84 (C6-
Gal), 60.94 (C6 -Gal), 60.48 (S ), 57.57, 57.41 (V ), 54.07 (H5 -
Neu5Ac), 54.34, 52.81 (S ), 51.73, 50.97 (E ), 51.43 (H ), 49.89
(N ), 48.41, 48.31 (2 × A ), 47.16 (C2-Gal), 42.18 (G ), 37.91 (C3 -
Neu5Ac), 37.33 (N ), 30.78 (V ), 29.83 (E ), 27.26 (H ), 27.35,
26.20 (E ), 22.83, 22.70, 22.64, 20.86, 20.67, 20.64, 20.42, 20.41 (8
CH3, 8 × COCH3, 2 × NHCOCH3), 19.08, 17.68 (2 × V ), 17.75 (2
× A ).
(Rt 14.56 min, Luna C18, 215 nm, 1% MeCN in H2O
100%
MeCN + 0.1% TFA in 42 min), a portion of the crude peptide
(73 mg) was directly oxidized by a mixture of trifluoroacetic acid
(5 mL) and DMSO (0.5 mL) at r.t. within 3 h to give 34 (RP-HPLC,
Rt 15.39 min, Luna C18, 215 nm, 1% MeCN in H2O
100%
Subsequently, removal of O-acetyl groups was carried out as de-
scribed above. Glycopeptide 31 (68 mg, 0.03 mmol) was treated
with 0.1 M NaOMe in MeOH for 18 h. The crude glycopeptide was
purified by preparative RP-HPLC (Luna C18, 5% MeCN in H2O
MeCN + 0.1% TFA in 42 min). Finally, the solvent was removed in
vacuo, and the residue was coevaporated efficiently with DMF and
toluene. The Fmoc-protected dimer 34 was treated with a mixture
of DMF and piperidine (1:1, 15 mL) for 2 h. After evaporation of
the solvents in vacuo and coevaporation with toluene, the dimeric
peptide 35 was dissolved in H2O and purified by gel permeation
chromatography (Sephadex G-25, 0.4 mL/min, fraction size:
15 min). Lyophilization yielded 35 as a slightly yellowish amor-
phous solid. (40 mg, 68% overall yield); [ ]D23 –31.4 (c = 1.00,
H2O).
5% MeCN in H2O in 15 min
20% MeCN in H2O in 50 min
100% MeCN + 0.1% TFA in 60 min) in order to obtain 32 as a col-
orless lyophilisate (34 mg, 61%).
32
[ ]D23 +13.9 (c = 1.0, H2O); Rt 22.83 min (RP-HPLC, Luna C18,
215 nm, 5% MeCN in H2O 5% MeCN in H2O in 10 min 60%
MeCN in H2O in 40 min 100% MeCN + 0.1% TFA in 60 min).
1H NMR (600 MHz, D2O, 1H-COSY): = 8.40 (m, 2 H, 2 × Im2),
7.24 (m, 2 H, 2 × Im4), 4.85 (m, 2 H, 2 × N ), 4.70 (m, 2 H, 2 × H ),
1H NMR (600 MHz, DMSO-d6, 1H-COSY, TOCSY, NOESY,
4.54–4.46 (m, 6 H, 6 × S ), 4.34 (q, 2 H, 2 × A , JA
= 7.04 Hz),
,A
ROESY):
= 8.83 (s, 1 H, Im2), 8.67 (d, 1 H, HNH
,
4.15 (dd, 2 H, 2 × V , JV a = 7.04 Hz, JV b = 2.76 Hz), 3.92–
,V ,V
JNH,H = 7.44 Hz), 8.36 (d, 1 H, ANH, JNH,A = 5.48 Hz), 8.37–7.91
(m, 9 H, 2 × ENH, NNH, ANH, 2 × SNH, VNH, GNH, NHNeu5NAc), 7.92 (d,
1 H, ENH, JNH,E = 7.44 Hz), 7.77 (d, 1 H, VNH, JNH,V = 8.60 Hz),
7.47 (s, 1 H, NH2-N), 7.28 (m, 2 H, NHGalNAc, Im4), 6.93 (s, 1 H,
NH2-N), 4.66 (m, 2 H, H1-Gal, H ), 4.52 (m, 1 H, N ), 4.46–4.25
[m, 6 H, 2 × A (4.30, 4.33), 2 × S (4.45, 4.40), V (4.22), E
(4.26)], 4.18–4.09 [m, 3 H, H2-Gal (4.18), E (4.17), V (4.15)],
3.90–3.87 (m, 2 H, H5-Gal, S ), 3.76–3.25 [m, 24 H, G (3.76,
3.61), 3 × S (3.70, 3.64; 3.73, 3.57; 3.65, 3.58), H3- (3.67), H4-
(3.61), H6-Gal (3.67, 3.55), H2 - (3.32), H3 - (3.25), H4 - (3.61),
H6 -Gal (3.48), H4 - (3.54), H5 - (3.44), H6 -, H7 -, H8 -, H9 a-
Neu5Ac (3.61), H9 b-Neu5Ac (3.37))], 3.10–2.96 (m, 2 H, H ),
2.57–2.44 (m, 3 H, H3 eq-Neu5Ac, N ), 2.27 (m, 4 H, 2 × E ), 1.96–
1.73 [m, 12 H, 2 × NHCOCH3 (1.86, 1.81), 2 × V (1.94), 2 × E
3.81 (m, 12 H, 6 × S ), 3.75 (m, 4 H, 2 × G ), 3.30–3.09 (m, 4 H, 2
a
× H ), 2.86 (dd, 2 H, 2 × N , JN
b = 15.64 Hz, JN
= 5.48 Hz),
a,N
a,N
b
2.71 (dd, 2 H, 2 × N , JN
a = 15.64 Hz, JN
= 8.60 Hz), 2.07
b,N
b,N
(m, 2 H, 2 × V ), 1.35 (m, 6 H, 2 × A ), 0.94 (d, 12 H, 2 × V ,
JV = 7.04 Hz); not visible: C , C .
,V
13C NMR (150.9 MHz, DMSO-d6, HMQC): = 177.31, 177.22,
175.97, 174.35, 174.22, 173.97, 173.81 (C=O), 119.8
(NCH=CRIm), 63.72, 63.47 (S ), 62.01 (V ), 58.19, 57.71 (S ),
55.06 (H ), 53.04 (N ), 52.21 (A ), 45.92 (G ), 38.99 (N ), 32.70
(V ), 29.45 (H ), 20.93, 20.17 (V ), 19.01 (A ); not visible: C , C .
MALDI-TOF-MS: m/z calcd for C64H103N24O28S2: 1720.8; found:
1720.7 (M + H+); m/z calcd for C64H102N24NaO28S2: 1742.8; found:
Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York

2502
T. Reipen, H. Kunz
PAPER
(17) (a) Kunz, H. In Preparative Carbohydrate Chemistry;
1742.6 (M + Na+); m/z calcd for C64H102KN24O28S2: 1758.9; found:
1758.8 (M + K+).
Hanessian, S., Ed.; Marcel Decker: New York, 1997, 265.
(b) Braum, G. Ph.D. Dissertation; University of Mainz:
Germany, 1991.
Acknowledgment
(18) Paulsen, H.; Hölck, J.-P. Carbohydr. Res. 1982, 109, 89.
(19) Meinjohanns, E.; Meldal, M.; Schleyer, A.; Paulsen, H.;
Bock, K. J. Chem. Soc., Perkin Trans. 1 1996, 985.
(20) Springer, G. F. J. Mol. Med. 1997, 75, 594.
(21) Zemplén, G.; Kunz, A. Ber. Dtsch. Chem. Ges. 1923, 56,
1705.
This work was supported by the Volkswagen-Stiftung and by the
Fonds der Chemischen Industrie.
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Synthesis 2003, No. 16, 2487–2502 © Thieme Stuttgart · New York