Dendrimers of vaccines consisting of tumor-associated glycopeptide antigens and T cell epitope peptides

Article abstract of DOI:10.1055/s-0028-1088033

According to the principle of multiple antigen presentation (MAP), a di-lysyl lysine core was used for the synthesis of tetramers of a glycododecapeptide antigen from the tandem-repeat sequence of the tumor-associated mucin, MUC1, which carries a sialyl T_N-antigen saccharide side chain. The methodology was extended to the analogous construction of a tetrameric vaccine consisting of a T cell epitope from tetanus toxoid and the sialyl T_N-glycododecapeptide of MUC1. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0028-1088033

PAPER
1355
Dendrimers of Vaccines Consisting of Tumor-Associated Glycopeptide
Antigens and T Cell Epitope Peptides
Dendrimeric
G
t
lycope
e
ptide
A
ntig
f
ens anie Keil, Anton Kaiser, Fauziya Syed, Horst Kunz*
Institut für Organische Chemie, Johannes Gutenberg-Universität Mainz, Duesbergweg 10-14, 55128 Mainz, Germany
Fax +49(6131)3924786; E-mail: hokunz@uni-mainz.de
Received 17 October 2008; revised 15 December 2008
Dedicated to Professor Bernt Krebs on the occasion of his 70th birthday
sponse (T8 cells)9 and, therefore, should enhance the
chance of eliciting a cytotoxic immune reaction. In con-
sideration of these observations, we have synthesized den-
drimeric glycopeptide antigen structures of the tandem
Abstract: According to the principle of multiple antigen presenta-
tion (MAP), a di-lysyl lysine core was used for the synthesis of tet-
ramers of a glycododecapeptide antigen from the tandem-repeat
sequence of the tumor-associated mucin, MUC1, which carries a
sialyl TN-antigen saccharide side chain. The methodology was ex- repeat region of tumor-associated mucin MUC1 and den-
tended to the analogous construction of a tetrameric vaccine con-
drimers of conjugates of these antigens with T cell epitope
sisting of a T cell epitope from tetanus toxoid and the sialyl TN-
peptides.
glycododecapeptide of MUC1.
As a core structure for a dendrimeric presentation of the
Key words: tumor-associated antigens, glycopeptides, fragment
condensation, MUC1, multiple antigen presentation
glycopeptides, the di-lysyl lysine structure was used.7 It
should be mentioned that dendrimers of carbohydrate an-
tigens have been successfully synthesized on other core
structures.10 In order to prevent undesired interactions be-
The O-glycan pattern of the epithelial mucin MUC1 in tu-
tween the glycopeptides, as well as of the glycopeptides
mor cells is characteristically altered compared to MUC1
with the lysine core, a tri(ethylene glycol)-derived spacer
in normal cells of the same tissues.1 As MUC1 is highly
amino acid8 was inserted in order to separate the core from
over-expressed in tumor cells2 and the altered O-glycan
the glycopeptides linked to it. According to this concept,
side chains are located in the extracellular region of
the C-terminal amino acids Fmoc-proline and Fmoc-va-
MUC1, which consists of numerous tandem repeat pep-
line were coupled to the spacer amino acid tert-butyl ester
tide sequences, partial structures of this tandem repeat re-
1,8 which was obtained from the corresponding w-hy-
gion containing tumor-associated saccharide antigens are
droxy-spacer acid.11 Acidolysis of the tert-butyl ester of
considered attractive target structures for the development
the Fmoc-amino acid spacer conjugates 2 and nucleo-
of anti-tumor vaccines.3 As a rule, the immunogenicity of
philic coupling of the obtained Fmoc-amino-acyl spacer
such glycopeptide structures from natural glycoproteins is
carboxylic acid 3 (as the sodium salts) to the allylic
low.4 Therefore, synthetic glycopeptide antigens have
HYCRON linker 4, gave the linker conjugates 5. These
been conjugated with carrier proteins in order to enhance
were immobilized on aminomethyl-polystyrene resin
their immunogenicity.5 However, peptide epitopes con-
(AMPS) in the presence of tetrabutylammonium bromide
tained in the carrier protein can overwrite the immune re-
as the phase-transfer catalyst, to afford the loaded resins 7
sponse directed towards the glycopeptide antigen. One
(Scheme 1).
way to prevent this problem consists of the conjugation of
Because of the sensitivity of the allylic anchor towards
strongly basic primary and secondary amines, the removal
of the Fmoc group from the Fmoc proline loaded resin 7a
was performed with a mixture of morpholine (pKa 8.33)
and N,N-dimethylformamide (1:1) within 90 minutes.
Coupling reactions with Fmoc amino acids were carried
out with O-(benzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluro-
nium tetrafluoroborate (TBTU)12 and 1-hydroxybenzotri-
azole (HOBt)13 in the presence of N-methylmorpholine
(NMM) in dimethylformamide (DMF). However, the
Fmoc-protected sialyl-TN-threonine was coupled with O-
(7-azabenzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluronium
tetrafluoroborate (HATU)14 and 1-hydroxy-7-azabenzo-
triazole (HOAt) in DMF in a Merrifield reactor (typically
a flask with sinter-filter and valve at the bottom). After
completion of the solid-phase synthesis (Scheme 2) the
Fmoc group was removed, and the N-terminal amino
group was acetylated. Palladium(0)-catalyzed cleavage11
the synthetic glycopeptide antigens with a T cell epitope
peptide within a fully synthetic vaccine.6 An alternative to
circumvent this problem was introduced by J. P. Tam7 in
the form of a multiple antigen presentation (MAP) of pep-
tide epitopes. We had shown that conjugates of tumor-
associated glycopeptide antigens from MUC1 with T cell
epitope peptides from tetanus toxoid (TTX) were able to
induce proliferation of CD8-positive T cells (cytotoxic T8
cells).8 The combination of this B cell-/T cell epitope prin-
ciple with the multiple antigen presentation should pro-
vide favorable aspects for the development of an anti-
tumor vaccine. In addition, peptide antigens administered
according to the multiple antigen presentation (MAP)
were shown to induce a MHCI restricted immune re-
SYNTHESIS 2009, No. 8, pp 1355–1369
x
x
.
x
x
.2
0
0
9
Advanced online publication: 25.03.2009
DOI: 10.1055/s-0028-1088033; Art ID: T18508SS
© Georg Thieme Verlag Stuttgart · New York

1356
S. Keil et al.
PAPER
1. MeSO2Cl
Et3N, CH2Cl2
H2, Raney-Ni
i-PrOH
O
O
N3
CO2t-Bu
HO
CO2t-Bu
3
3
2. NaN3, DMF
Fmoc-Pro-OH
Fmoc-Val-OH
O
or
O
O
H2N
CO2t-Bu
Fmoc-Xaa
N
H
CO2R
3
3
EEDQ
1
2a R = t-Bu, Xaa = Pro 74%
2b R = t-Bu, Xaa = Val 70%
TFA
3a R = H, Xaa = Pro 82%
3b R = H, Xaa = Val quant.
Br
O
O
CO2t-Bu
O
3
4
O
O
O
3
Fmoc-Xaa
N
O
CO2R
H
3
NaHCO3, CH2Cl2
3
O
Bu4N+Br–, 20 °C, 12 h
5a R = t-Bu, Xaa = Pro 50%
5b R = t-Bu, Xaa = Val 75%
H2
C
TFA
PS
6a R = H, Xaa = Pro 82%
6b R = H, Xaa = Val 95%
H2N
7a Xaa = Pro 94%
O
loading 0.58 mmol/g
O
O
O
O
Fmoc-Xaa
N
O
AMPS
H
3
7b Xaa = Val 74%
loading 0.58 mmol/g
3
O
Scheme 1
of the allylic HYCRON linker using morpholine as the al- Fmoc removal using morpholine/DMF and coupling of
lyl trapping nucleophile, gave the conjugate 8 of the fully Fmoc amino acids using TBTU/HOBt and completion by
protected MUC1 glycopeptide antigen with the hydro- coupling of benzyloxycarbonyl (Z)-protected O-tert-butyl
philic spacer amino acid in an overall yield of 36% (after tyrosine under analogous conditions, the conjugate 9 of
25 steps and preparative HPLC).
the fully protected tetanus toxoid T cell epitope peptide
and spacer amino acid was obtained by palladium(0)-
catalyzed cleavage of the allylic linker.
In an analogous procedure, the spacer-conjugated peptide
sequence 9 of a T cell epitope of tetanus toxoid16 was as-
sembled starting with AMPS resin 7b loaded with Fmoc For the multiple antigen presentation7 (MAP), the di-lysyl
valine (Scheme 3). The first coupling was performed with lysine core was prepared according to Scheme 4. Lysine
the Fmoc seryl proline dipeptide. Through alternating tert-butyl ester16 (10) was acylated with di-benzyloxycar-
7a
a
b
Fmoc-G-V-T(α-NeuAc4NAcCO2Bn-(2→6)-α-GalAc2NAc)-S(t-Bu)-A-P-D(t-Bu)-T(t-Bu)-R(Pmc)-P-A-P-NH-(CH2CH2O)3CH2CH2CO2HYCRON-AMPSm
OAc
OAc
AcO
AcHN
Bn
CO2
O
c
O
AcO
d
AcO
AcO
O
AcHN
O
O
O
O
O
O
O
O
O
O
H
H
H
H
H
O
O
N
N
N
N
N
N
N
O
AcHN
N
N
N
N
N
H
H
H
H
O
O
O
O
O
O
O
NH
PmcHN
NH
O
HO
8
Scheme 2 Reagents and conditions: (a) morpholine–DMF (1:1); (b) [repeat 10 times]: (i) Couplings 1–8, 10 and 11: Fmoc-Xaa-OH, TBTU,
HOBT, NMM, DMF; Coupling 9: Fmoc sialyl-Tn antigen threonine,8,15 HOAt, HATU, NMM, DMF, 24 h, 76%; (ii) Capping: Ac2O/py (1:3);
(iii) Fmoc-removal: morpholine–DMF (1:1); (c) Ac2O/py (1:3); (d) [Pd(PPh3)4], morpholine, DMF–DMSO (1:1), 36% with respect to 7a.
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

PAPER
Dendrimeric Glycopeptide Antigens
1357
7b
a
b
H-S(t-Bu)-Y(t-Bu)-F-P-S(t-Bu)-V-CONH(CH2CH2O)3CH2CH2COO-HYCRON-AMPS
c
Z-Y(t-Bu)-S(t-Bu)-Y(t-Bu)-F-P-S(t-Bu)-V-CONH(CH2CH2O)3CH2CH2COO-HYCRON-AMPS
d
O
O
O
O
O
O
O
O
H
H
H
N
N
O
N
O
OH
N
N
H
N
N
O
N
O
H
H
H
O
O
O
O
O
9
Scheme 3 Reagents and conditions: (a) morpholine–DMF (1:1); (b) [repeat 5 times]: (i) Coupling: Fmoc-Xaa-OH, TBTU, HOBT, NMM,
DMF; (ii) Capping: Ac2O/py (1:3); (iii) Fmoc removal: morpholine–DMF (1:1); (c) (i) Coupling for 6: Z-Tyr(t-Bu)-OH, TBTU, HOBT, NMM,
DMF; (ii) Capping: Ac2O/py (1:3); (d) [Pd(PPh3)4], morpholine, DMF–DMSO (1:1), 42% with respect to 7b.
bonyl-lysine (11) to give the Z-protected di-lysyl lysine alytical HPLC of the product showed two compounds:
ester core 12.
one was isolated in pure form (27 mg, 30%) which, ac-
cording to both its ESI mass spectrum (m/z = 8018.5) and
its NMR spectrum, was the target tetrameric glycopeptide
antigen 16 as a sodium salt.
Hydrogenolytic removal of the benzyloxycarbonyl groups
furnished the tetravalent di-lysyl lysine ester 13.
Fragment condensation using eight equivalents of the si-
alyl TN dodecapeptide-spacer conjugate 8 per molecule of
core 13 with HATU/HOAt, and N-methylmorpholine for
carboxylic activation, resulted in the formation of the ful-
ly protected dendrimer 14 exposing four glycopeptide an-
tigens (Scheme 5). The pure compound was separated
from excess of 8, its benzotriazole ester (active ester) and
a few minor by-products by preparative reverse-phase
HPLC. Removal of the benzyl ester protection from the
The second fraction isolated (32 mg, 35%) also contained
16 and, in addition, a compound with a mass 56 g/mol
higher than that of 16. This compound was probably a tet-
ramethyl ester of 16 formed either during the dissolution
of the hydrotrifluoroacetate of partly deblocked 15 in
methanol prior to the transesterification, or during the
chromatographic purification in the presence of 1% tri-
fluoroacetic acid.
sialic acid residues was achieved by hydrogenation in The synthesis of an even more complex dendrimer, pre-
methanol. Subsequent acidolysis with trifluoroacetic acid senting four spacer-linked glycopeptide antigen-T cell
in dichloromethane in the presence of cation scavengers epitope conjugates, was accomplished by sequential frag-
gave dendrimer 15, which still carried the O-acetyl groups ment condensations on the tetravalent lysine core. The
in the disaccharide portion. Analytical RP-HPLC revealed benzyloxycarbonyl-protected TTX-T cell epitope peptide
a small shoulder on the product peak; however, no impu- spacer conjugate 9 (8 equivalents) was coupled with the
rity was observed in the 1H NMR spectrum. It is notewor- lysine core 13 using HATU/HOAt for carboxylic activa-
1
thy and characteristic that the H NMR spectra of the tion. The dendrimer 17, presenting four protected T cell
glycopeptide dendrimers of 14 and 15 showed only one epitope peptides, was isolated after separation from ex-
set of signals for the glycopeptide portions. This gives ev- cess of 9 and its hydroxy-azabenzotriazole ester by pre-
1
idence that the glycopeptide molecules separated from the parative HPLC in a yield of 49% (Scheme 6). The H
core behave independently from each other and are not in- NMR spectrum of 17 was almost identical to that of the T
fluenced by the core. For removal of the O-acetyl groups, cell peptide spacer conjugate 9, except for additional sig-
15 was subjected to controlled transesterification in meth- nals from the lysine core, e.g. between d = 1.68 and 1.33
anol catalyzed by sodium methanolate at pH 8.5. The an- ppm (Kb, Kg, Kd) and at d = 1.35 ppm (t-Bu). The signal
O
O
NHZ
O
NH2
O
H
H
N
N
ZHN
O
H2N
O
H2N
O
O
a
b
ZHN
NH2
NH
NH
ZHN
H2N
NH2
10
12
13
O
O
Scheme 4 Reagents and conditions: (a) Z-Lys(Z)-OH (11), TBTU, HOBt, NMM, DMF, 20 °C, 2 d, 36%; (b) Pd/C (10%), H2, MeOH, 20 °C,
2 h, 93%.
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

1358
S. Keil et al.
PAPER
AcO
AcHN
OAc
OAc
CO2
Bn
O
AcO
8 + 13
AcO
O
AcO
a
NHPmc
O
HN
HN
O
O
O
O
O
O
AcHN
O
O
H
O
H
N
O
NHAc
H
N
O
H
N
O
O
N
H
O
N
N
N
N
N
H
H
O
O
NH
N
N
H
AcHN
N
O
H
O
N
H
H
N
H
O
O
O
N
O
H
O
N
O
N
O
O
O
O
N
H
NHAc
N
H
O
O
O
N
N
H
N
N
O
O
O
O
O
H
O
OAc
OAc
O
O
O
O
NH
NH
O
O
PmcHN
NH
C
BnO2
O
OAc
HN
O
AcO
AcO
NHAc
OAc
O
NH
OAc
OAc
O
AcO
AcHN
O
O
H
O
N
O
AcO
Bn
O
CO2
HN NHPmc
HN
N
H
O
O
AcO
AcO
O
O
O
O
O
H
O
O
O
H
O
O
H
O
H
HN
N
O
N
N
O
O
N
N
O
N
N
NHAc
N
O
N
N
N
N
H
H
O
H
O
O
H
O
O
H
O
AcHN
O
O
O
O
NHAc
H
H
O
O
H
N
O
H
O
N
AcHN
N
N
N
HN
N
N
O
N
14
N
O
H
O
N
OAc
OAc
H
N
O
O
H
O
O
H
O
O
O
O
C
O
NH
OAc
NHAc
OAc
b
O
BnO2
PmcHN
NH
AcO
AcO
[Ac-G-V-T(α-NeuAc4NAcCO2H-(2→6)-α-GalAc2NAc)-S-A-P-D-T-R-P-A-P-NH-(CH2CH2O)3-CH2CH2CONH]2-Lys2Lys-OH
15
16
c
[Ac-G-V-T(α-NeuNAcCO2H-(2→6)-α-GalNAc)-S-A-P-D-T-R-P-A-P-NH-(CH2CH2O)3-CH2CH2CONH]2-Lys2Lys-OH
Scheme 5 Reagents and conditions: (a) HATU, HOAt, NMM, DMF, 20 °C, 2 d, 65%; (b) (i) Pd/C (10%), H2, MeOH, 20 °C, 3 h; (ii) CH2Cl2/
TFA/thioanisole/ethanedithiol (10:10:1:1), 20 °C, 4 h, 66%; (c) NaOMe, MeOH, pH 8.5, 20 °C, 18 h, preparative HPLC: P1 contained 16
(30%), P2 contained impure 16 (35%).
of the C-terminal CH2 group of the spacer was shifted The second peak (P2; 35 mg, 23%) was identified as com-
slightly upfield due to the amide formation (from d = 2.42 pound 19 according to its ESI mass spectrum (m/z =
ppm to d = 2.23–2.19 ppm). After hydrogenolytic remov- 13216.33; calcd: 13215.96 [8 × 13C]). This compound was
al of the four benzyloxycarbonyl groups from 17, an ex- subjected to the removal of the O-acetyl groups by con-
cess of the MUC1 glycopeptide antigen-spacer compound trolled transesterification in methanol in the presence of
8 (10 equivalents) was activated with HATU/HOAt and catalytic sodium methanolate at pH 8.5. After 14 hours at
simultaneously coupled to the four terminal amino func- room temperature, methanolic acetic acid was added. Pu-
tions. After separation from the excess of 8 and its 1-hy- rification by semi-preparative RP-HPLC gave two frac-
droxy-azabenzotriazole ester by preparative HPLC, the tions with similar characteristics, both containing the
complex dendrimer 18 presenting four tumor-associated target dendrimer 20 according to their ESI mass spectra
MUC1 glycopeptide antigen-TTX T cell epitopes8 was (m/z = 12207.02, calcd: 12206.88). The slightly more hy-
isolated in a yield of 73%. The 1H and 13C NMR spectra, drophilic fraction consisted of the target dendrimer 20 and
although complex, showed mainly one set of signals and, minor components which still contained one, two and
thus, suggested that the antigen-T cell epitope combina- three O-acetyl groups. The second, slightly more hydro-
tions do not influence each other in DMSO solution. In the phobic mixture also contained the completely deprotected
ESI mass spectrum, molecules 18 (molecular weight target dendrimer 20 together with its mono-, di- and tri-O-
16212.89 g/mol) carrying 7,8,9,10,11 and 12 positive acetylated derivatives and, in addition, components
charges (protons) (average m/z = 16211.92 g/mol) were formed by elimination of the glycal of N-acetyl-neuramin-
1
detected and confirm the structure.
ic acid. The H and 13C NMR spectra of both fractions
were in agreement with structure 20, but showed small
impurities in the range of the O-acetyl signals (d = 2.0
ppm, d = 20 ppm). As for the other dendrimers, only one
set of signals was found for the four antigen-T cell epitope
conjugates except for the carbohydrate carbons carrying
the N-acetyl groups (C2 and C5¢).
Deprotection of the complex dendrimer 18 was performed
by hydrogenolytic cleavage of the NeuNAc benzyl ester
and subsequent treatment with TFA/CH2Cl2 in the pres-
ence of thiols.
Preparative RP-HPLC of the crude product afforded two
fractions: fraction P1 (86 mg, 57%) was a mixture. Ac-
cording to its ESI mass spectrum it contained the partly
deblocked dendrimer 19, however, the major compound
showed a molecular mass of M – 56 (13159.21 g/mol). Its
structure remained unclear.
The obtained results show that complex glycopeptide an-
tigen structures can be presented in a multimeric form on
a di-lysyl lysine core. Fragment condensation reactions
using protected glycopeptides synthesized on solid-phase
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

PAPER
Dendrimeric Glycopeptide Antigens
1359
are efficient in the synthesis of these complex structures. in aqueous solution. Thus, complex conjugates such as 16,
Due to the separation of the glycopeptide from the lysine 18 and 20 show only one set of signals in their NMR spec-
core by oligo(ethylene glycol)-derived spacer amino tra. Since the obtained dendrimeric glycopeptide antigens
acids, the glycopeptide chains do not influence each other 16 and 20 are molecules as large as small proteins (molar
9 + 13
O
O
a
O
H
O
O
O
O
O
O
O
H
O
O
H
N
H
O
N
H
H
N
O
O
H
N
N
O
N
N
N
N
N
N
H
N
N
N
H
N
H
O
H
O
H
O
O
O
O
O
O
NH
H
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
HN
O
O
O
O
HN
O
O
O
O
O
O
H
N
H
N
HN
H
N
O
O
O
O
H
O
O
N
H
N
O
N
N
H
O
H
N
H
O
O
N
N
H
H
N
H
N
N
O
O
O
N
O
O
N
N
O
O
NH
O
H
O
H
O
H
O
O
O
O
17
O
O
b
OAc
OAc
OAc
OAc
AcO
AcHN
AcO
AcO
AcHN
O
O
AcO
O
AcO
AcO
O
AcO
AcO
O
AcHN
O
O
AcHN
O
O
O
N
H
O
H
O
H
N
O
H
H
N
N
N
O
N
N
N
NHAc
AcHN
O
N
H
O
N
O
N
H
N
H
N
O
O
H
O
H
H
N
H
O
O
O
H
O
N
H
O
O
N
N
O
O
N
N
N
H
N
H
N
H
N
N
O
O
O
O
O
H
O
O
O
O
HN
O
O
O
O
NHPmc
HN
O
NH
O
PmcHN
NH
O
O
O
O
O
O
H
N
O
O
H
O
O
O
O
H
N
O
H
N
H
N
H
N
N
O
H
N
H
N
N
N
N
N
N
H
N
H
N
O
H
O
O
H
O
NH
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
HN
O
O
O
O
HN
O
O
O
O
O
O
H
H
N
HN
H
N
O
O
N
O
O
O
H
N
O
O
N
H
N
H
N
H
N
H
O
H
N
O
H
N
N
H
N
N
O
O
O
N
H
O
O
N
H
N
O
O
NH
O
O
H
O
O
O
O
O
O
O
O
O
NH
PmcHN
O
HN
HN
NHPmc
O
NH
O
O
O
O
O
O
O
N
O
H
O
H
N
O
O
O
H
H
N
O
O
O
O
N
N
N
O
O
AcHN
N
O
N
N
H
O
O
N
H
O
N
O
O
N
H
N
H
H
O
N
H
O
N
N
H
O
H
O
N
O
H
N
NHAc
N
N
N
N
H
O
N
N
H
O
O
H
O
H
O
O
H
NHAc
OAc
OAc
NHAc
O
O
OAc
OAc
O
OAc
NHAc
OAc
O
18
O
OAc
NHAc
OAc
O
AcO
AcO
AcO
c
AcO
[Ac-G-V-T(α-NeuAc4NAcCO2H-(2→6)-α-GalAc2NAc)-S-A-P-D-T-R-P-A-P-NH-(CH2CH2O)3-CH2CH2CONH-Y-S-Y-F-P-S-V-CONH(CH2CH2O)3-CH2CH2CONH]2-Lys2-Lys-OH
19
20
d
[Ac-G-V-T(α-NeuNAcCO2H-(2→6)-α-GalNAc)-S-A-P-D-T-R-P-A-P-NH-(CH2CH2O)3-CH2CH2-CONH-Y-S-Y-F-P-S-V-CONH(CH2CH2O)3-CH2CH2CONH]2-Lys2-Lys-OH
Scheme 6 Reagents and conditions: (a) HATU, HOAt, NMM, DMF, 20 °C, 2 d, 49%; (b) (i) Pd/C (10%), H2, MeOH, 20 °C, 3 h; (ii) 8,
HATU, HOAt, NMM, DMF, 20 °C, 2 d, 73%; (c) (i) Pd/C (10%), H2, MeOH, 20 °C, 4 h; (ii) CH2Cl2/TFA/thioanisole/ethanedithiol (20:20:1:1),
20 °C, 3 h; 19: 23%, by-product: 57%; (d) NaOMe, MeOH, pH 8.5, 20 °C, 18 h; after HPLC: 20.
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

1360
S. Keil et al.
PAPER
1H NMR (200 MHz, CDCl3):
CH2O(CH2CH2O)2CH2], 3.30 (t, J = 5.1 Hz, 2 H, N3CH2), 2.42 (t,
J = 6.6 Hz, 2 H, CH2COOt-Bu), 1.37 [s, 9 H, C(CH3)3].
13C NMR (50 MHz, CDCl3): d = 170.83 (C=O), 80.40 [C(CH3)3],
70.65, 70.60, 70.55, 70.33 [O(CH2CH2O)2CH2], 69.99
(CH2CH2COOt-Bu), 66.85 (N3CH2CH2), 50.62 (N3CH2CH2), 36.23
(CH2COOt-Bu), 28.04 [C(CH3)3].
d
=
3.66–3.49 [m, 12 H,
weight of about 12000), they should be useful for the in-
duction of immune responses without being bound to car-
rier proteins.
Solvents for moisture-sensitive reactions (MeOH, CH2Cl2, MeCN
and MeNO2) were distilled and dried according to standard proce-
dures.17 DMF for peptide synthesis was purchased from Roth, Ac2O
and pyridine (p.a. quality) from Acros. Reagents were of the highest
available commercial quality and used without further purification.
Fmoc-protected amino acids were purchased from Orpegen Phar-
ma, Heidelberg, Germany. Petroleum ether (PE), where used, had a
boiling range of 40–60 °C. For solid-phase syntheses, preloaded
Tentagel S resins (Rapp Polymere, Tübingen, Germany) were em-
ployed. Reactions were monitored by TLC with precoated silica gel
60 F254 aluminium plates (Merck). Flash column chromatography
was performed with silica gel (32–63 mm) from Merck. RP-HPLC
analyses were carried out on a Knauer HPLC system at a flow rate
of 1 mL/min using Knauer Eurospher 100 C-8 and C-18, Kromasil
100 C-18, Vydag Protein and Peptide C-18 and Vydag Protein C-4
columns. Semi-preparative RP-HPLC separations were performed
on a Knauer HPLC system at a flow rate of 10 mL/min. Preparative
RP-HPLC separations were carried out on a Knauer HPLC system
at a flow rate of 20 mL/min. Mixtures of H2O and MeCN were used
as solvents, if required 0.1% TFA was added. 1H, 13C, and 2D-NMR
spectra were recorded with either a Bruker WT-200 or a Bruker
AM-400 spectrometer. The chemical shifts (d) are reported in ppm
relative to the signal of the deuterated solvent. Multiplicities are
given as: s (singlet), br s (broad singlet), d (doublet), t (triplet), q
(quartet) or m (multiplet). Assignment of proton and carbon signals
was achieved by additional COSY and HMQC experiments when
noted. In complex saccharides, the signals of N-acetyl-D-galac-
tosamine derivatives are named as usual, the signals of N-acetyl-D-
neuraminic acid derivatives are marked with one apostrophe. With-
in the glycopeptide spectra, the glycosylated amino acid threonine
is characterized by a star. FAB- and FD-mass spectra were recorded
on a Finnigan MAT-95 instrument. For FAB spectra, either 3-ni-
trobenzylalcohol (nba) or glycerol (gly) were used as the matrix.
MALDI-TOF mass spectra were recorded on a Micromass Tofspec
E spectrometer (Micromass) using either 2,3-dihydroxybenzoic
acid (dhb) or cyanocinnamic acid (cca) as the matrix. ESI-mass
spectra were measured on ThemoQuest Navigator or a Micromass-
Q-TOF-Ultima-3 instruments. Optical rotations [a]D were mea-
sured with a Perkin–Elmer polarimeter 241.
tert-Butyl 12-Amino-4,7,10-trioxadodecanoate
[NH2(CH2CH2O)3CH2CH2COOt-Bu, 1]
Raney-nickel alloy (15 g) was suspended in H2O (400 mL) and solid
NaOH was added until the foaming stopped. After 10 min at r.t., the
mixture was heated to 70 °C for 30 min. Subsequently, the basic
soln was decanted from the Raney-nickel, which was then
washed with H2O until pH 8 was reached. The catalyst was
washed with i-PrOH (3 × 150 mL), and the azido compound
[N3(CH2CH2O)3CH2CH2COOt-Bu] prepared as described above
(20.0 g, 66.0 mmol) dissolved in i-PrOH (150 mL), was added. The
mixture was degassed in vacuo, flooded with Ar and then with H2.
After stirring under a H2 atmosphere for 14 h, the catalyst was fil-
tered off through Celite. After removal of the solvent, a grey-green-
ish liquid [15.0 g; Rf = 0.12 (PE–EtOAc, 2:1 + 1% Et3N)] was
obtained, which was used for further transformations without puri-
fication.
tert-Butyl (12-N-Fluorenylmethoxycarbonyl-L-prolylamido)-
4,7,10-trioxadodecanoate [Fmoc-Pro-
NH(CH2CH2O)3CH2CH2COOt-Bu, 2a]
Fmoc-Proline (15.0 g, 44.4 mmol) and ethyl 2-ethoxy-1,2-dihydro-
quinoline-1-carboxylate (EEDQ;17 12.1 g, 48.8 mmol) dissolved in
CH2Cl2 (200 mL) were stirred at r.t. for 15 min. The spacer amino
acid 1 (15.0 g, 54.1 mmol) dissolved in anhydrous CH2Cl2 (200 mL)
was added dropwise and the mixture was stirred at r.t. for 14 h. After
dilution with CH2Cl2 (200 mL) and separation, the organic layer
was washed with 0.1 M HCl (3 × 200 mL), sat. NaHCO3 (3 × 200
mL) and brine (3 × 200 mL). After drying (MgSO4) and evaporation
of the solvent in vacuo, the crude product was purified by flash
chromatography (300 g silica gel; PE–EtOAc, 1:4→EtOAc).
Yield: 19.5 g (74%); colorless viscous oil; Rf = 0.45 (EtOAc + 1%
AcOH); [a]D22 –29.0 (c 1.0, CHCl3).
1H NMR (200 MHz, CDCl3): d = 7.74 (d, J = 7.3 Hz, 2 H, H4-, H5-
Fmoc), 7.63–7.51 (m, 2 H, H1-, H8-Fmoc), 7.41–7.24 (m, 4 H,
H2-, H3-, H6-, H7-Fmoc), 6.83 (br s, 0.5 H, NH), 6.42 (br s, 0.5 H,
NH), 4.35 (d, J = 7.3 Hz, 2 H, CH2-Fmoc), 4.31–4.13 (m, 2 H, Pa,
H9-Fmoc), 3.64 (t, J = 6.6 Hz, 2 H, CONHCH2CH2O), 3.59–3.32
[m, 14 H, CH2O(CH2CH2O)2CH2, Pd], 2.45 (t, J = 6.4 Hz, 2 H,
CH2COOt-Bu), 2.32–2.12 (m, 1 H, Pba), 2.06–1.81 (m, 3 H, Pbb, Pg),
1.41 [s, 9 H, C(CH3)3].
13C NMR (50 MHz, CDCl3 [DEPT]): d = 171.83, 170.83 (C=O),
156.4 (C=O urethane), 143.87 (C4a-, C4b-Fmoc), 141.27 (C8a-,
C9a-Fmoc), 127.75 (C3-, C6-Fmoc), 127.10 (C2-, C7-Fmoc),
125.10, 125.04 (C1-, C8-Fmoc), 119.98 (C4-, C5-Fmoc), 80.45 [-
C(CH3)3], 70.48, 70.43, 70.32, 70.29 [CH2O(CH2CH2O)2], 69.05
(CH2CH2COOt-Bu), 67.64 (OCH2-Fmoc), 66.85 (CONHCH2CH2),
60.76 (Pa), 47.23 (C9-Fmoc, Pd), 39.26 (CONHCH2CH2), 36.25
(CH2COOt-Bu), 28.90 (Pb), 28.10 [C(CH3)3], 24.55 (Pg).
tert-Butyl 12-Azido-4,7,10-trioxadodecanoate
[N3(CH2CH2O)3CH2CH2COOt-Bu]
To
tert-butyl
12-hydroxy-4,7,10-trioxadodecanoate11
[HO(CH2CH2O)3CH2CH2COOt-Bu; 40.0 g, 0.14 mole] dissolved in
anhydrous CH2Cl2 (40 mL), Et3N (47.9 mL, 0.34 mole) was added.
Under cooling in an ice-bath, MsCl (22.3 mL, 0.29 mole) was added
dropwise. After 3 h, precipitated Et3N·HCl was filtered off through
Celite. The filtrate was washed with ice-water (2 × 20 mL) and
brine (2 × 20 mL), then dried (MgSO4), and the solvent was evapo-
rated in vacuo. The remaining residue was dissolved in DMF (200
mL). NaN3 (56.1 g, 0.86 mmol) was added and stirring was contin-
ued for 14 h at 60 °C. The solvent was evaporated in vacuo and the
remainder dissolved in H2O (100 mL). The aqueous soln was ex-
tracted with Et2O (5 × 100 mL), and the combined organic solns
were dried (MgSO4). After removal of the solvent in vacuo, the
crude product was purified by flash chromatography (500 g silica
gel; PE–EtOAc, 6:1→2:1).
FD-MS (5 kV, 10 mA/min): m/z = 596.5 [M+; calcd: 596.31], 374.4
[M – Fmoc + H+], 375.4 [M(1 × 13C) – Fmoc + H+], 376.4
[M(2 × 13C) – Fmoc + H+], 178.1 [fulvene + H+].
tert-Butyl (12-N-Fluorenylmethoxycarbonyl-L-valylamido)-
4,7,10-trioxadodecanoate [Fmoc-Val-
NH(CH2CH2O)3CH2CH2COOt-Bu, 2b]
Yield: 32.0 g (73%); colorless oil; Rf = 0.47 (mesylate; EtOAc),
0.36 (azide; PE–EtOAc, 2:1).
IR: 2100 cm–1(azide).
Fmoc-Val-OH (16.0 g, 47.1 mmol) and ethyl 2-ethoxy-1,2-dihydro-
quinoline-1-carboxylate (EEDQ;18 14.0 g, 56.5 mmol) were dis-
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

PAPER
Dendrimeric Glycopeptide Antigens
1361
solved in anhydrous CH2Cl2 (200 mL) and stirred at r.t. for 15 min.
Amino compound 1 (19.6 g, 70.7 mmol) dissolved in anhydrous
CH2Cl2 (200 mL) was slowly added. The mixture was stirred at r.t.
for 14 h, diluted with CH2Cl2 (200 mL) and washed with 0.1 M HCl
(3 × 200 mL), sat. NaHCO3 (3 × 200 mL) and brine (3 × 200 mL).
After drying (MgSO4) and removal of the solvent in vacuo, the
crude product 2b was purified by flash chromatography (400 g sili-
ca; PE–EtOAc, 2:1→1:1).
tert-Butyl (E)-17-[12-(N-Fluorenylmethoxycarbonyl-L-valyl-
amido)-4,7,10-trioxadodecanoyloxy]-4,7,10,13-tetraoxa-15-
heptadecenoate [Fmoc-Val-NH(CH2CH2O)3CH2CH2CO-
HYCRON-Ot-Bu, 5b]
Compound 2b (20.0 g, 33.4 mmol) in TFA (20 mL) were stirred at
r.t. for 2 h. After dilution with toluene (200 mL) the solvents were
evaporated in vacuo. Toluene (80 mL) was co-distilled from the re-
mainder to give the carboxylic acid 3b (18.1 g, quant.). Acid 3b,
tert-butyl (E)-17-bromo-4,7,10,13-tetraoxa-15-heptadecenoate11
(4; 13.7 g, 33.4 mmol) and TBAB (21.6 g, 66.8 mmol) in a mixture
of CH2Cl2 (340 mL) and sat. aq NaHCO3 (310 mL) were vigorously
stirred for 14 h. After phase separation, the aqueous layer was ex-
tracted with CH2Cl2 (3 × 150 mL). The combined organic solns
were dried (MgSO4) and, after evaporation of the solvent in vacuo,
the crude product was purified by flash chromatography (600 g sil-
ica; PE–EtOAc, 1:1→EtOAc).
22
Yield: 20.0 g (70%); colorless waxy solid; Rf = 0.42 (EtOAc); [a]D
–3.9 (c 1.0, CHCl3).
1H NMR (200 MHz, CDCl3): d = 7.74 (d, J = 7.3 Hz, 2 H, H4-, H5-
Fmoc), 7.58 (d, J = 6.8 Hz, 2 H, H1-, H8-Fmoc), 7.41–7.24 (m, 4 H,
H2-, H3-, H6-, H7-Fmoc), 6.77 (br s, 1 H, NH), 5.58 (d, J = 8.8 Hz,
1 H, NH), 4.44–4.30 (m, 1 H), 4.25–4.14 (m, 1 H), 4.01–3.93 (m,
1 H, Va, H9-Fmoc, CH2-Fmoc), 3.67 (t, J = 6.6 Hz, 2 H,
CONHCH2), 3.56–3.44 [m, 12 H, CH2O(CH2CH2O)2CH2], 2.47 (t,
J = 6.6 Hz, 2 H, CH2COOt-Bu), 2.13–2.09 (m, 1 H, Vb), 1.42 [s,
9 H, C(CH3)3], 0.92 (dd, J = 6.8, 12.7 Hz, 6 H, Vg).
22
Yield: 22.0 g (75%); colorless waxy solid; Rf = 0.21 (EtOAc); [a]D
–3.1 (c 1.0, CHCl3).
1H NMR (200 MHz, CDCl3): d = 7.74 (d, J = 6.8 Hz, 2 H, H4-, H5-
Fmoc), 7.54 (d, J = 7.3 Hz, 2 H, H1-, H8-Fmoc), 7.41–7.24 (m, 4 H,
H2-, H3-, H6-, H7-Fmoc), 6.56 (br s, 1 H, NH), 5.87–5.78 (m, 2 H,
CH=CH), 5.60 (d, J = 8.8 Hz, 1 H, NH), 4.51 (d, J = 4.4 Hz, 2 H,
COOCH2CH=CHCH2), 4.40–3.99 (m, 4 H, CH2-Fmoc, Va, H9-
Fmoc), 3.99 (d, J = 3.9 Hz, 2 H, COOCH2CH=CHCH2), 3.70–3.64
(m, 2 H, CONHCH2), 3.58–3.33 [m, 26 H, CH2O(CH2CH2)2OCH2,
CH2O(CH2CH2O)2CH2], 2.53 (t, J = 6.6 Hz, 2 H), 2.43 (t, J = 6.6
Hz, 2 H, CH2COOt-Bu, CH2COOR), 2.06–1.98 (m, 1 H, Vb), 1.38
[s, 9 H, C(CH3)3], 0.92 (br s, 6 H, Vg).
13C NMR (50 MHz, CDCl3): d = 171.20, 170.85 (C=O), 156.31
(C=O urethane), 143.89, 143.83 (C4a-, C4b-Fmoc), 141.27 (C8a-,
C9a-Fmoc), 130.83 (COOCH2CH=CHCH2), 127.68 (C3-, C6-
Fmoc), 127.05 (C2-, C7-Fmoc), 126.41 (COOCH2CH=CHCH2),
125.10, 125.06 (C1- C8-Fmoc), 119.93 (C4-, C5-Fmoc), 80.42
[C(CH3)3], 71.12, 70.82, 70.56, 70.48, 70.37, 70.34, 70.26
13C NMR (50 MHz, CDCl3 [DEPT]): d = 171.22, 170.89 (C=O),
156.33 (C=O urethane), 143.90, 143.84 (C4a-, C4b-Fmoc), 141.27
(C8a-, C9a-Fmoc), 127.69 (C3-, C6-Fmoc), 127.07 (C2-, C7-
Fmoc), 125.07 (C1-, C8-Fmoc), 119.93 (C4-, C5-Fmoc), 80.51
[C(CH3)3], 70.51, 70.43, 70.33, 70.29 [CH2O(CH2CH2O)2], 69.61
(CH2CH2COOt-Bu), 66.93 (OCH2-Fmoc), 66.87 (CONHCH2CH2),
60.40 (Va), 47.19 (C9-Fmoc, Pd), 39.28 (CONHCH2CH2), 36.25
(CH2COOt-Bu), 31.41 (Vb), 28.10 [C(CH3)3], 19.19, 17.94 (Pg).
FD-MS (5 kV, 10 mA/min): m/z = 598.8 [M+; calcd: 598.33], 599.8
[M(1 × 13C)+], 600.8 [M(2 × 13C)].
Anal. Calcd for C33H46N2O8: C, 66.20; H, 7.74; N, 4.68. Found: C,
65.94; H, 7.99; N, 4.58.
(12-N-Fluorenylmethoxycarbonyl-L-prolylamido)-4,7,10-triox-
adodecanoic Acid [Fmoc-Pro-NH(CH2CH2O)3CH2CH2COOH,
3a]
[CH2OCH2CH2O,
CH2O(CH2CH2O)2CH2],
69.65,
69.45
Compound 2a (20.0 g, 33.5 mmol) in TFA (20 mL) were stirred at
r.t. for 2 h. After dilution with toluene (200 mL), the solvents were
evaporated in vacuo and toluene (80 mL) was twice co-distilled
from the remainder. Purification by flash chromatography (400 g
silica; EtOAc) afforded the carboxylic acid 3a.
(CH2COOR, CH2COOt-Bu, COOCH2CH2O), 66.87 (OCH2-Fmoc),
66.50, 64.31 (COOCH2CH=CHCH2, COOCH2CH2O), 60.32 (Va),
47.20 (C9-Fmoc), 39.26 (CONHCH2CH2), 36.26, 34.96
(CH2COOR, CH2COOt-Bu), 19.15, 17.88 (Vg).
FD-MS (5 kV, 10 mA/min): m/z = 895.5 [M + Na+], 896.5
22
[M(1 × 13C) + Na+], 872.5 [M+; calcd: 872.47], 873.5 [M(1 × 13C)+].
Yield: 14.8 g (82%); colorless viscous oil; [a]D –22.7 (c 1.0,
CHCl3).
1H NMR (200 MHz, CDCl3): d = 9.69 (br s, 1 H, COOH), 7.74 (d,
J = 7.3 Hz, 2 H, H4-, H5-Fmoc), 7.63–7.51 (m, 2 H, H1-, H8-
Fmoc), 7.41–7.24 (m, 4 H, H2-, H3-, H6-, H7-Fmoc), 7.19 (br s,
0.5 H, NH), 7.01 (br s, 0.5 H, NH), 4.45–4.34 (m, 3 H, CH2-Fmoc,
Pa), 4.30–4.12 (m, 1 H, H9-Fmoc), 3.70 (t, J = 5.9 Hz, 2 H,
CONHCH2CH2O), 3.61–3.32 [m, 14 H, CH2O(CH2CH2O)2CH2,
Pd], 2.55 (t, J = 5.9 Hz, 2 H, CH2COOH), 2.32–2.12 (m, 1 H, Pba),
2.06–1.81 (m, 3 H, Pbb, Pg).
13C NMR (50 MHz, CDCl3 [DEPT]): d = 174.75, 172.74 (C=O),
155.80 (C=O urethane), 143.79 (C4a-, C4b-Fmoc), 141.29 (C8a-,
C9a-Fmoc), 127.75 (C3-, C6-Fmoc), 127.15 (C2-, C7-Fmoc),
125.11, 125.06 (C1- C8-Fmoc), 120.01 (C4-, C5-Fmoc), 70.49,
70.30, 70.21 [CH2O(CH2CH2O)2], 69.66 (CH2CH2COOH), 67.89
(OCH2-Fmoc), 66.49 (CONHCH2CH2), 60.76 (Pa), 47.18 (C9-
Fmoc, Pd), 39.46 (CONHCH2CH2), 34.84 (CH2COOH), 28.90 (Pb),
24.55 (Pg).
(E)-17-[12-(N-Fluorenylmethoxycarbonyl-L-prolylamido)-
4,7,10-trioxadodecanoyloxy]-4,7,10,13-tetraoxa-15-hepta-
decenoic Acid [Fmoc-Pro-NH(CH2CH2O)3CH2CH2CO-
HYCRON-OH, 6a]
Compound 3a (14.8 g, 27.4 mmol), tert-butyl (E)-17-bromo-
4,7,10,13-tetraoxa-15-heptadecenoate11 4 (11.3 g, 27.4 mmol) and
TBAB (17.7 g, 54.8 mmol) in a mixture of CH2Cl2 (150 mL) and
sat. aq NaHCO3 (120 mL) were vigorously stirred for 14 h. After
phase separation, the aqueous layer was extracted with CH2Cl2
(3 × 80 mL). The combined organic solns were dried (MgSO4) and
the solvent was removed in vacuo. The crude product was purified
by flash chromatography (300 g silica; EtOAc→EtOAc–MeOH,
10:1) to give tert-butyl ester 5a (12.0 g, 50%), which was isolated
as a colorless viscous oil [Rf = 0.46 (EtOAc–MeOH, 10:1)]. This oil
was dissolved in TFA (20 mL) and stirred at r.t. for 2 h. The soln
was then diluted with toluene (200 mL), and the solvents were evap-
orated in vacuo. Toluene (80 mL) was co-distilled twice from the re-
mainder, which was subsequently purified by flash chromatography
(350 g silica; EtOAc–MeOH, 20:1).
FD-MS (5 kV, 10 mA/min): m/z = 563.5 [M + Na+], 564.5
[M(1 × 13C) + Na+], 565.4 [M(2 × 13C) + Na+], 541.5 [M + H+; cal-
cd: 541.31], 542.5 [M(1 × 13C) + H+], 319.3 [M – Fmoc + H+].
22
Yield: 9.58 g (82%); colorless viscous oil; [a]D –20.4 (c 1.0,
Anal. Calcd for C33H44N2O8·2H2O: C, 60.40; H, 6.99; N, 4.86.
Found: C, 60.52; H, 7.12; N, 4.63.
CHCl3).
1H NMR (200 MHz, CDCl3): d = 7.74 (d, J = 6.8 Hz, 2 H, H4-, H5-
Fmoc), 7.63–7.51 (m, 2 H, H1-, H8-Fmoc), 7.41–7.24 (m, 4 H,
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

1362
S. Keil et al.
PAPER
(E)-17-[12-(N-Fluorenylmethoxycarbonyl-L-valylamido)-
4,7,10-trioxadodecanoyloxy]-4,7,10,13-tetraoxa-15-hepta-
decenoyl-b-alanyl-aminomethyl Polystyrene [Fmoc-Val-
NH(CH2CH2O)3CH2CH2CO-HYCRON-b-Ala-AMPS, 7b]
The coupling of compound 6b to the resin was carried out according
to a previously described procedure,11 using tert-butyloxycarbonyl-
b-alanyl-aminomethylpolystyrene (4.20 g, 0.97 mmol/g) and com-
pound 6b (7.99 g, 9.78 mmol).
H2-, H3-, H6-, H7-Fmoc), 6.92, 6.60, 5.89 (br s, 2–3 H, NH), 5.87–
5.78 (m, 2 H, CH=CH), 4.54 (d, J = 4.4 Hz, 2 H,
COOCH2CH=CHCH2), 4.39 (d, J = 7.3 Hz, 2 H, CH2-Fmoc), 4.29–
4.04 (m, 2 H, Pa, H9-Fmoc), 3.99 (d, J = 3.9 Hz, 2 H,
COOCH2CH=CHCH2), 3.77–3.64 (m, 2 H, CONHCH2), 3.58–3.33
[m, 26 H, CH2O(CH2CH2)2OCH2, CH2O(CH2CH2O)2CH2, Pd],
2.61–2.44 (m, 2 H, CH2COOH, CH2COOR), 2.35–2.12 (m, 1 H,
Pba), 2.06–1.91 (m, 3 H, Pbb, Pg).
13C NMR (50 MHz, CDCl3 [DEPT]): d = 174.22, 171.26 (C=O),
143.87 (C4a-, C4b-Fmoc), 141.27 (C8a-, C9a-Fmoc), 130.83
(COOCH2CH=CHCH2), 127.77 (C3-, C6-Fmoc), 127.11 (C2-, C7-
Fmoc), 126.61 (COOCH2CH=CHCH2), 125.14, 125.06 (C1-, C8-
Fmoc), 119.99 (C4-, C5-Fmoc), 70.81, 70.51, 70.44, 70.39, 70.33,
70.30, 70.25 [CH2OCH2CH2O, CH2O(CH2CH2O)2CH2], 69.69,
69.63 (CH2COOR, CH2COOH, COOCH2CH2O), 66.44 (OCH2-
Fmoc), 64.32 (COOCH2CH=CHCH2, COOCH2CH2O), 60.08 (Pa),
47.22 (C9-Fmoc, Pd), 39.26 (CONHCH2CH2), 34.95, 34.84
(CH2COOR, CH2COOH), 28.93 (Pb), 24.24 (Pg).
Yield: 6.57 g (74%, loading 0.58 mmol/g).
Solid-Phase Synthesis of Peptides and Glycopeptides; General
Procedure
The reactions on solid-phase were carried out in a Merrifield reac-
tor (flask equipped with a frit and a valve at the bottom). The
amounts of solvent refer to overall loading of the resin of 0.5–1.5
mmol.
Cycle for chain extension: (A) Fmoc-removal: morpholine–DMF
(1:1, 20 mL) for 90 min and subsequent washing with DMF (6 × 10
mL). (B) Coupling reactions: Fmoc amino acid (10 equiv), TBTU
(10 equiv), HOBt (10 equiv) and NMM (10 equiv) in DMF (10 mL)
were added to the filtered resin and shaken for 20 h. After filtration,
the resin was washed with DMF (6 × 20 mL). For glycopeptide syn-
theses, Fmoc amino acids (20 equiv) and the corresponding
amounts of TBTU/HOBt and NMM were used. The amounts of gly-
cosylamino acid are given for the particular syntheses. (C) Capping:
The resin was shaken with pyridine (18 mL) and Ac2O (6 mL) for
30 min and subsequently washed with DMF (6 × 20 mL).
FAB-MS (gly, neg): m/z = 813.7 [M – H+; calcd: 813.90], 591.4.
(E)-17-[12-(N-Fluorenylmethoxycarbonyl-L-valylamido)-
4,7,10-trioxadodecanoyloxy)-4,7,10,13-tetraoxa-15-hepta-
decenoic Acid [Fmoc-Val-NH(CH2CH2O)3CH2CH2CO-
HYCRON-OH, 6b]
Compound 5b (22.0 g, 25.2 mmol) was dissolved in TFA (20 mL)
and stirred at r.t. for 2 h. The soln was diluted with toluene (200
mL), and the solvents were evaporated in vacuo. Toluene (80 mL)
was co-distilled twice from the remainder, which was subsequently
purified by flash chromatography (300 g silica; EtOAc→EtOAc–
AcOH, 10:1).
Detachment from resin: The resin was suspended in a mixture of
DMF (10 mL), DMSO (10 mL) and morpholine (1 mL). For careful
exclusion of oxygen, air was exchanged for argon. Pd(PPh3)4 (20
mg) was added and the mixture shaken under exclusion of light for
18 h. The filtered soln was collected and the resin was washed with
DMF (8 × 25 mL) and CH2Cl2 (6 × 20 mL). The solvents were
evaporated from the combined filtrates to give the crude products.
Yield: 19.5 g (95%); colorless viscous oil; Rf = 0.23 (EtOAc–
AcOH, 5:1); [a]D22 –3.42 (c 1.0, CHCl3).
1H NMR (200 MHz, CDCl3): d = 7.74 (d, J = 6.8 Hz, 2 H, H4-, H5-
Fmoc), 7.54 (d, J = 7.3 Hz, 2 H, H1-, H8-Fmoc), 7.41–7.24 (m, 4 H,
H2-, H3-, H6-, H7-Fmoc), 6.75 (br s, 1 H, NH), 5.95 (br s, 1 H,
NH), 5.80–5.74 (m, 2 H, CH=CH), 4.51 (d, J = 4.4 Hz, 2 H,
COOCH2CH=CHCH2), 4.41–4.28 (m, 2 H), 4.23–3.92 (m, 1 H),
4.40 (m, 3 H, CH2-Fmoc, Va, H9-Fmoc, COOCH2CH=CHCH2),
N-Acetyl-glycyl-L-valyl-O-{2-acetamido-3,4-di-O-acetyl-2-
deoxy-6-O-[benzyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-a-glycero-D-galacto-2-nonulopyranosyl)onat]-a-D-ga-
lactopyranosyl}-L-threonyl-O-tert-butyl-L-seryl-L-alanyl-L-
prolyl-4-O-tert-butyl-L-aspartyl-O-tert-butyl-L-threonyl-N-
(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginyl-L-prolyl-
L-alanyl-L-prolylamido-4,7,10-trioxadodecanoic Acid {Ac-G-
V-T[a-NeuAc4NAcCOOBn-(2→6)-a-GalAc2NAc]-S(t-Bu)-A-P-
D(t-Bu)-T(t-Bu)-R(Pmc)-P-A-P-
3.70–3.35
[m,
28 H,
CONHCH2CH2O(CH2CH2)2OCH2,
CH2O(CH2CH2O)2CH2], 2.62–2.49 (m, 4 H, CH2COOH,
CH2COOR), 2.06–1.98 (m, 1 H, Vb), 0.92 (d, J = 6.8 Hz, 6 H, Vg).
13C NMR (50 MHz, CDCl3): d = 174.72, 172.08, 171.33 (C=O),
156.57 (C=O urethane), 143.84, (C10-, C11-Fmoc), 141.27 (C12-,
C13-Fmoc), 130.83 (COOCH2CH=CHCH2), 127.68 (C3-, C6-
Fmoc), 127.07 (C2-, C7-Fmoc), 126.51 (COOCH2CH=CHCH2),
125.14 (C1-, C8-Fmoc), 119.91 (C4-, C5-Fmoc), 70.77, 70.48,
NH(CH2CH2O)3CH2CH2COOH, 8}
Compound 7a (2.0 g, 0.58 mmol/g) was used according to the gen-
eral procedure, and Fmoc amino acids (10.3 equiv, 11.6 mmol)
were applied. The first eight coupling reactions, capping reactions,
and Fmoc removal reactions were carried out as described in the
general procedure. In the ninth coupling after photometric determi-
nation of the loading (0.823 mmol according to Fmoc cleavage) the
glycosyl amino acid N-Fmoc-O-[benzyl-(5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-a-glycero-D-galacto-2-nonulopyrano-
70.45,
70.40,
70.31,
70.16
[CH2OCH2CH2O,
CH2O(CH2CH2O)2CH2], 69.52, 67.00, 66.56 (CH2COOR,
CH2COOH, COOCH2CH2O), 66.49 (OCH2-Fmoc), 64.32
(COOCH2CH=CHCH2, COOCH2CH2O), 60.58 (Va), 47.11 (C9-
Fmoc), 39.33 (CONHCH2CH2), 34.91, 34.82 (CH2COOR,
CH2COOH), 19.15, 18.13 (Vg).
syl)onat]-a-D-galactopyranosyl-L-threonine,8,15
Fmoc-Thr[a-
FD-MS (5 kV, 10 mA/min): m/z = 595.6 [M – Fmoc + H+; calcd:
NeuAc4NAcCOOBn-(2→6)-a-GalAc2NAc]-OH (4.00 g, 3.40
mmol, 4 equiv), was applied together with HOAt (462 mg, 3.40
mmol), HATU14 (1.29 g, 3.40 mmol) and NMM (747 mL, 6.80
mmol) in DMF (8 mL). In this coupling, the reaction time was ex-
tended to 24 h.
595.34].
(E)-17-[12-(N-Fluorenylmethoxycarbonyl-L-prolylamido)-
4,7,10-trioxadodecanoyloxy]-4,7,10,13-tetraoxa-15-hepta-
decenoyl-aminomethyl Polystyrene [Fmoc-Pro-
NH(CH2CH2O)3CH2CH2CO-HYCRON-AMPS, 7a]
The coupling of compound 6a to the resin was carried out according
to a previously described procedure,11 using tert-butyloxycarbonyl-
b-alanyl-aminomethylpolystyrene (H-AMPS; 4.30 g, 1.10 mmol/g)
and compound 6a (9.25 g, 11.35 mmol).
After completion of the solid-phase synthesis and N-terminal Fmoc
removal, acetylation, detachment reaction and work up, the crude
glycopeptide 8 was purified by flash chromatography (100 g silica,
CH2Cl2–MeOH, 20:1→10:1→5:1→1:1→MeOH) and subsequent
preparative HPLC on a Eurospher C18 column (H2O–MeCN,
60→30% over 2 h) and isolated by lyophilization.
Yield: 8.11 g (94%, loading 0.58 mmol/g).
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

PAPER
Dendrimeric Glycopeptide Antigens
1363
Yield: 1.15 g (36% with respect to 7a); colorless amorphous solid;
Rf = 0.44 (CH2Cl2–MeOH, 6:1); Rt = 23.9 min (Eurospher C18; 220
nm; H2O–MeCN, 60→30 over 40 min); [a]D22 –2.5 (c 1.0, CHCl3).
from the resin and work-up was carried out according to the general
procedure. Purification of the crude peptide 9 was achieved by pre-
parative HPLC on a Eurospher C8 column (H2O–MeCN, 60→0%
over 1 h).
1H NMR (400 MHz, DMSO-d6 [1H-1H-COSY]): d = 8.27–8.15 (m,
1 H), 8.13–7.84 (m, 8 H), 7.82–7.67 (m, 2 H), 7.25–7.11 (m, 2 H,
13 × NH), 7.42–7.31 (m, 5 H, HAr-Bn), 6.65, 6.40 (br s, 2 × RNH),
5.27–5.12 (m, 5 H, OCH2-Bn, H-4, H-7¢, H-8¢), 4.92 (d, J = 12.4
Hz, 1 H, H-3), 4.87 (br s, 1 H, H-1), 4.75–4.65 (m, 1 H, H-4¢), 4.61–
4.39 (m, 5 H, Ra, Da, Va, 2 × Aa), 4.34–4.09 (m, 9 H, 3 × Pa, Sa, 2 ×
Ta, 1 × Tb, H-2, H-9¢a), 4.08–4.00 (m, 2 H, H-5, H-6¢), 3.98–3.91
(m, 1 H, H-9¢b), 3.90–3.83 (m, 2 H, H-5¢, 1 × Tb), 3.77–3.69 (m,
Yield: 888 mg (42%); colorless lyophilizate; Rt = 23.4 min (Euro-
spher C8; 220 nm; H2O–MeCN, 60→0% over 40 min); [a]D22 –8.9
(c 1.0, CHCl3).
1H NMR (400 MHz, DMSO-d6): d = 8.38 (d, J = 7.6 Hz, 1 H), 8.03–
7.99 (m, 2 H), 7.94 (d, J = 7.6 Hz, 1 H), 7.75 (d, J = 7.9 Hz, 1 H),
7.53 (d, J = 8.8 Hz, 1 H), 7.49 (d, J = 8.8 Hz, 1 H, 7 × NH), 7.32–
7.21 (m, 10 H, HAr-F, HAr-Z), 7.16 (d, J = 7.9 Hz, 2 H), 7.03 [d,
J = 8.2 Hz, 2 H, 2 × (H2-, H6-Y)], 6.82 (d, J = 8.2 Hz, 2 H), 6.77 [d,
J = 8.2 Hz, 2 H, 2 × (H3-, H5-Y)], 4.91 (s, 2 H, CH2-Z), 4.68–4.59
(m, 1 H), 4.56–4.50 (m, 1 H), 4.49–4.44 (m, 1 H, 2 × Ya, Fa), 4.34–
4.26 (m, 3 H), 4.16 (t, J = 7.6 Hz, 1 H, 2 × Sa, Pa, Va), 3.58 (t,
J = 6.5 Hz, 2 H, CONHCH2), 3.55–3.34 [m, 16 H, 2 × Sb,
CH2(OCH2CH2)2OCH2], 3.26–3.10 (m, 2 H, Pd), 3.02–2.94 (m, 1 H,
Fba), 2.91–2.87 (m, 2 H), 2.86–2.74 (m, 2 H), 2.72–2.63 (m, 1 H,
Fbb, 2 × Yb), 2.42 (t, J = 6.3 Hz, 2 H, CH2COOH), 2.04–1.68 (m,
5 H, Pb, Pg, Vb), 1.24, 1.20, 1.11, 1.06 [s, 36 H, 4 × C(CH3)3], 0.83
(d, J = 3.5 Hz, 3 H, Vga), 0.81 (d, J = 3.2 Hz, 3 H, Vgb).
3 H, Ga, H-6a), 3.63–2.94 [m, 25 H, Sb,
3
×
Pd,
NH(CH2CH2O)3CH2CH2COOH, H-6b, Rd], 2.75–2.67 (m, 1 H,
Dba), 2.62–2.53 (m, 2 H, CH2-Pmc), 2.51–2.38 (m, 10 H, H-3¢e,
Dbb, o,o¢-CH3-Pmc, CH2COOH), 2.10–1.58 (m, 15 H, 3 × Pb,
3 × Pg, Vb, CH2-Pmc), 2.03, 2.01, 1.98, 1.93, 1.92, 1.85, 1.82, 1.75,
1.66 (s, 30 H, m-CH3-Pmc, 3 × NHAc, 6 × OAc), 1.50–1.00 (m,
14 H, Rg, H-3¢a, 1 × Tg, 2 × Ab, Rb), 1.34, 1.09, 1.06 [s, 24 H, 3 ×
C(CH3)3], 1.24 [s, 6 H, C(CH3)2-Pmc], 0.94 (d, J = 5.5 Hz, 3 H, Tg),
0.87 (d, J = 6.5 Hz, 3 H, Vga), 0.81 (d, J = 6.5 Hz, 3 H, Vgb).
13C NMR (100 MHz, DMSO-d6 [DEPT]): d = 171.31, 171.19,
170.71, 170.32, 169.82, 169.71, 169.61, 169.54, 169.48, 169.41,
169.28, 169.19, 169.15, 168.96, 168.83, 168.67, 168.57 (C=O),
166.68 (C-1¢), 155.85 (Cq-guanidine), 152.22, 134.88, 134.88,
133.91, 128.38, 128.27, 127.83, 122.52, 117.58 (Pmc), 98.42 (C-1),
97.99 (C-2¢), 80.12, 73.60, 73.33, 72.85 [C(CH3)2-Pmc, C(CH3)3],
13C NMR (100 MHz, CDCl3 [DEPT]): d = 171.17, 170.80, 170.73,
170.00 (C=O), 154.62, 154.40, (C=O urethane), 136.41, 136.29,
129.86, 129.76, 129.68, 128.62, 128.51, 128.25, 127.97, 126.99,
124.21, 123.98 (CAr-F, CAr-Y, CAr-Z), 78.40, 78.33, 74.50, 74.43
[C(CH3)3], 70.47, 70.31, 70.18, 69.81, 66.92, 66.58
[CONHCH2CH2(OCH2CH2)2OCH2, CH2-Z], 61.27, 60.92 (2 × Sb),
75.58,
69.58,
69.55,
69.46,
68.84,
67.20
[Tß,
NH(CH2CH2O)3CH2CH2COOH, C-3, C-4, C-5, C-4¢, C-6¢, C-7¢, C-
8¢], 66.13 (OCH2-Bn), 62.69, 61.77, 61.61 (C-6, C-9¢, Sb), 59.37,
59.04, 58.84 (Sa, 2 × Ta), 57.02, 56.90, 56.76 (3 × Pa), 55.99 (Va),
52.97, 49.60, 49.11, 47.53 (Ra, Da, C-2, C-5¢), 46.67, 46.41 (3 × Pd,
C-2, C-5¢), 46.22, 46.03 (2 × Aa), 41.87 (Ga), 38.33, 37.41, 36.60
(C-3¢, Db, Rd), 34.67 (CH2COOH), 32.03 (CH2-Pmc), 30.67 (Vb),
29.09, 28.92 (Rb, 3 × Pb), 27.75, 27.49, 27.00, 26.30 [C(CH3)2-Pmc,
C(CH3)3], 25.54, 24.52, 24.29, 24.09 (Rg, 3 × Pg, CH2-Pmc), 22.66,
22.43, 22.20 (NHAc), 20.65, 20.49, 20.41, 20.31, 20.08 (OAc),
19.02, 18.80, 17.91, 16.93 (Tg, Vg), 18.10, 18.00 (o,o¢-CH3-Pmc),
16.75, 16.61 (2 × Ab), 11.77 (m-CH3-Pmc).
a
a
59.23 (Pa), 57.76 (S1 ), 55.26 (Va), 53.32, 52.91, 52.31, 51.41 (S2 ,
2 × Ya, Fa), 46.67 (Pd), 39.40, 38.99 (2 × Yb, Fb), 35.16
(CH2COOH), 31.19 (Vb), 28.94 (Pb), 29.01, 28.96, 27.50, 27.39
[C(CH3)3], 25.00 (Pg), 19.30 (Vga), 18.23 (Vgb).
HRMS: m/z calcd for C76H110N8O18: 1422.79; found: 1423.7.
MALDI-MS (CCA): m/z = 1447.0 [M + Na+], 1468.9 [M – H+ + (2
× Na+)].
N2,N7-Di-(N2,N7-dibenzyloxycarbonyl-L-lysyl)-L-lysine tert-
Butyl Ester {[Z-Lys(Z)]2-Lys-Ot-Bu, 12}
Lysine tert-butyl ester16 (10; 2.00 g, 9.89 mmol) was co-distilled
with DMF (3 × 30 mL) under high vacuum. A soln of Z-Lys(Z)-OH
(11; 16.4 g, 39.6 mmol), TBTU (12.7 g, 39.6 mmol), HOBt (6.00 g,
39.6 mmol) and NMM (8.7 mL, 79.1 mmol) in DMF (50 mL) was
added, and the mixture was stirred at r.t. for 2 d. After evaporation
of the solvent under high vacuum, the remainder was dissolved in
CH2Cl2 (100 mL) and the soln was washed with 0.5 M HCl (50 mL),
sat. NaHCO3 (50 mL) and sat. NaCl (50 mL). The combined aque-
ous solns were extracted with CH2Cl2 (50 mL) and the combined or-
ganic layers were dried (MgSO4). After evaporation of the solvent,
the remaining residue was purified by flash chromatography (150 g
silica; PE–EtOAc, 1:1).
HRMS: m/z calcd for C124H190N18O45S: 2683.29; found: 2685.0.
MALDI-MS (CCA): m/z = 2709.0 [M + Na+], 2725.4 [M + K+],
2652.9 [M – t-Bu + H+ + Na+], 2658.7 [M – t-Bu + H+ + Na+ + Li+],
2674.9 [M – t-Bu + H+ + 2 × Na+].
ESI-MS (MeCN–H2O): m/z = 1387.7 {[M(2 × 13C) – (2 × H+) + (4
× Na+)]/2}, 1376.7 {[M(2 × 13C) – H+ + (3 × Na+)]/2}, 1365.7
{[M(2 × 13C) + (2 × Na+)]/2}.
N-Benzyloxycarbonyl-O-tert-butyl-L-tyrosyl-O-tert-butyl-L-
seryl-O-tert-butyl-L-tyrosyl-L-phenylalanyl-L-prolyl-O-tert-
butyl-L-seryl-L-valylamido-4,7,10-trioxadodecanoic Acid
[Z-Y(t-Bu)-S(t-Bu)-Y(t-Bu)-F-P-S(t-Bu)-V-
Yield: 3.50 g (36%); colorless amorphous solid; Rf = 0.26 (PE–
EtOAc, 1:2); [a]D22 –35.1 (c 1.0, CHCl3).
NH(CH2CH2O)3CH2CH2COOH, 9]
The synthesis was performed according to the general procedure
starting with resin 7b (2.59 g, 0.58 mmol/g). However, only 4 equiv
of Fmoc amino acids (6.00 mmol), HOBt (948 mg, 6.00 mmol),
TBTU (1.93 g, 6.00 mmol) and NMM (1.32 mL, 12.0 mmol) in
DMF (20 mL) were applied in the couplings 1–5. The sixth cou-
pling was carried out with Z-Tyr(t-Bu)-OH*DCHA (Novabiochem;
5 g, 9 mmol), which was dissolved in EtOAc (50 mL) and extracted
with 2M H2SO4 (10.8 mL, 10.8 mmol). The organic layer was sep-
arated and the aqueous soln was diluted with ice-water (10 mL) and
extracted with EtOAc (15 mL). The combined organic solns were
dried (MgSO4) and the solvent was evaporated in vacuo. The ob-
tained Z-Tyr(t-Bu)-OH (3.3 g, 8.89 mmol) was coupled with TBTU
(2.85 g, 8.89 mmol), HOBt (1.40 g, 8.89 mmol), and NMM (2.10
mL, 17.8 mmol) in DMF (20 mL). Pd(0)-catalyzed detachment
1H NMR (200 MHz, CDCl3): d = 7.48–7.43 (m, 1 H, NH), 7.26 (s,
20 H, 4 × HAr-Bn), 6.19–6.13 (m, 2 H, NH), 5.35–5.29 (m, 1 H),
5.28–5.12 (m, 1 H, 2 × NH), 5.11–5.83 (m, 8 H, 4 × CH2-Bn), 4.48–
4.32 (m, 1 H), 4.31–4.16 (m, 2 H, 3 × Ka), 3.52–3.28 (m, 1 H),
3.22–3.00 (m, 4 H), 2.99–2.79 (m, 1 H, 3 × Ke), 1.89–1.02 (m,
18 H, 3 × Kb, 3 × Kg, 3 × Kd), 1.39 [s, 9 H, C(CH3)3].
13C NMR (50 MHz, CDCl3): d = 172.80, 172.53, 171.16 (C=O),
156.83, 156.59 (C=O urethane), 136.75, 136.69, 136.19, 128.47,
128.42, 128.07, 128.02, 127.94 (CAr-Bn), 81.76 [C(CH3)3], 67.01,
66.55, 66.49 (4 × CH2-Bn), 54.73, 54.34, 52.87 (3 × Ka), 40.41,
38.64, 38.38 (3 × Ke), 32.42, 32.02, 31.25, 29.19, 28.57, 27.98,
22.51, 22.30 [3 × (Kb, Kg, Kd)], 27.98 [C(CH3)3].
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

1364
S. Keil et al.
PAPER
FD-MS (5 kV, 10 mA/min): m/z = 995.2 [M + H+], 96.2 [M(1 × 13C)
106 H, 3 × Ke, 4 × [Sb, 3 × Pd, NH(CH2CH2O)3CH2CH2CONH, H-
6b, Rd]}, 2.75–2.67 (m, 4 H, 4 × Dba), 2.62–2.53 (m, 8 H, 4 × CH2-
Pmc), 2.51–2.22 [m, 10 H, 4 × (H-3¢e, Dbb, o,o¢-CH3-Pmc,
CH2CONH)], 2.10–1.60 [m, 60 H, 4 × (3 × Pb, 3 × Pg, Vb, CH2-
Pmc)], 2.03, 2.01, 1.99, 1.93, 1.92, 1.85, 1.82, 1.75, 1.66 [s, 90 H, 4
× (m-CH3-Pmc, 3 × NHAc, 6 × OAc)], 1.50–1.00 [m, 74 H, 3 × (Kb,
Kg, Kd), 4 × (Rg, H-3¢a, 1 × Tg, 2 × Ab, Rb)], 1.35, 1.24, 1.19, 1.18,
1.16, 1.09, 1.06 [s, 144 H, 13 × C(CH3)3, 4 × C(CH3)2-Pmc], 0.94
(d, J = 5.3 Hz, 12 H, Tg), 0.87 (d, J = 6.8 Hz, 12 H, Vga), 0.81 (d,
J = 6.5 Hz, 12 H, Vgb).
13C NMR (100 MHz, DMSO-d6 [DEPT]): d = 171.52, 171.26,
170.38, 169.78, 169.67, 169.48, 169.22, 169.06, 168.90, 168.64
(C=O), 166.75 (C-1¢), 155.92 (Cq-guanidine), 152.28, 134.93,
134.44, 133.96, 128.43, 128.33, 127.88, 122.57, 117.61 (CAr-Bn,
CAr-Pmc), 98.39 (C-1), 98.02 (C-2¢), 80.12, 73.59, 73.32, 72.85
[C(CH3)2-Pmc, C(CH3)3], 75.58, 71.81, 69.61, 69.50, 69.41, 68.92,
67.25, 66.95 [C-3, C-4, C-5, C-4¢, C-6¢, C-7¢, C-8¢,
NH(CH2CH2O)3CH2CH2COOH, Tb], 66.80 (OCH2-Bn), 62.75,
61.82 (C-6, C-9¢, Sb), 59.42, 59.09, 58.89 (Sa, Ta), 56.98, 56.06 (Pa),
53.05, 49.63, 49.16, 47.56 (Ka, Va, Ra, Da, C-2, C-5¢), 46.74, 46.47
(C-2, C-5¢, Pd), 46.25, 46.08 (Aa), 41.92 (Ga), 38.35, 37.45, 36.64,
36.04, 35.9, 35.86 (C-3¢, Ke, Db, Rd, CH2CONH), 32.07 (CH2-Pmc),
30.74 (Vb), 30.52, 29.19, 29.18, 29.09, 29.01, 28.89, 28.80 (Kb, Kd,
Rb, 3 × Pb), 27.79, 27.53, 27.05, 26.35 [C(CH3)2-Pmc, C(CH3)3],
24.57, 24.32, 24.29, 24.12 (Kg, Rg, 3 × Pg, CH2-Pmc), 22.72, 22.47,
22.24 (NHAc), 20.70, 20.55, 20.47, 20.37, 20.15 (OAc), 19.07,
18.86, 17.91, 16.93 (Tg, Vg), 18.10, 18.00 (o,o¢-CH3-Pmc), 16.75,
16.61 (2 × Ab), 11.77 (m-CH3-Pmc).
+ H+].
Anal. Calcd for C54H70N6O12: C, 65.17; H, 7.09; N, 8.44. Found: C,
64.95; H, 7.04; N, 8.44.
N2,N7-Di-l-lysyl-L-lysine tert-Butyl Ester {[H-Lys(H)]2-Lys-
Ot-Bu, 13}
Protected di-lysyl lysine [Z-Lys(Z)]2-Lys-Ot-Bu (12; 3.50 g, 3.52
mmol) was dissolved in MeOH (40 mL) and hydrogenated over Pd/
C (10%, 50 mg) for 2 h. The catalyst was filtered off through Celite,
MeOH was evaporated under vacuo and the product was dried un-
der high vacuum.
Yield: 1.50 g (93%); colorless oil; [a]D22 +0.42 (c 1.0, MeOH).
1H NMR (200 MHz, CD3OD): d = 4.34–4.23 (m, 1 H, K1 ), 3.36–
a
a
a
e
3.32 (m, 2 H, K2 , K3 ), 3.29–3.17 (m, 2 H, K1 ), 2.75–2.63 (m, 4 H,
K2 , K3 ), 1.97–1.32 [m, 18 H, 3 × (Kb, Kg, Kd)], 1.51 [s, 9 H,
e
e
C(CH3)3].
13C NMR (50 MHz, CD3OD [DEPT]): d = 177.67, 172.86 (C=O),
82.78 [C(CH3)3], 56.18, 55.78, 54.25 (3 × Ka), 42.23, 42.20, 39.94
(3 × Ke), 36.38, 36.34, 33.36, 32.31, 29.99, 24.20, 24.10, 23.87 [3 ×
(Kb, Kg, Kd)], 28.29 [C(CH3)3].
FD-MS (5 kV, 10 mA/min): m/z = 459.7 [M + H+].
N2,N6-Di{N2,N6-di(N-acetyl-glycyl-L-valyl-O-{2-acetamido-3,4-
di-O-acetyl-2-deoxy-6-O-[benzyl(5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-a-glycero-D-galacto-2-nonulopyrano-
syl)onat]-a-D-galactopyranosyl}-L-threonyl-O-tert-butyl-L-ser-
yl-L-alanyl-L-prolyl-4-O-tert-butyl-L-aspartyl-O-tert-butyl-L-
threonyl-N-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-argi-
nyl-L-prolyl-L-alanyl-L-prolylamido-4,7,10-trioxadodecanoyl)-
L-lysyl}-L-lysine tert-Butyl Ester ({Ac-G-V-T[a-
HRMS: m/z calcd for C518H798N78O180S4: 11119.46; found: 11126.6.
MALDI-MS (THF/dithranol): m/z = 11149.5 [M + Na+].
Active ester of 8 with HOAt:
NeuAc4NAcCOOBn-(2→6)-a-GalAc2NAc]-S(t-Bu)-A-P-D(t-
Bu)-T(t-Bu)-R(Pmc)-P-A-P-NH(CH2CH2O)3CH2CH2CO}4-
Lys2-Lys-Ot-Bu, 14)
1H NMR (400 MHz, DMSO-d6): d = 8.74 (d, J = 4.1 Hz, 1 H, HAr-
HOAT), 8.51 (d, J = 8.5 Hz, 1 H, HAr-HOAt), 8.23–8.18 (m, 1 H),
8.11–7.84 (m, 8 H), 7.82–7.72 (m, 2 H), 7.25–7.13 (m, 2 H, 13 ×
NH), 7.49 (dd, J = 4.4, 8.5 Hz, 1 H, HAr-HOAT), 7.40–7.31 (m,
5 H, HAr-Bn), 6.65, 6.40 (br s, 2 × RNH), 5.27–5.12 (m, 5 H, OCH2-
Bn, H-4, H-7¢, H-8¢), 4.92 (d, J = 12.4 Hz, 1 H, H-3), 4.87 (br s, 1 H,
H-1), 4.75–4.65 (m, 1 H, H-4¢), 4.61–4.39 (m, 5 H, Ra, Da, Va, 2 ×
Aa), 4.34–4.09 (m, 9 H, 3 × Pa, Sa, 2 × Ta, 1 × Tb, H-2, H-9¢a), 4.08–
4.00 (m, 2 H, H-5, H-6¢), 3.98–3.91 (m, 1 H, H-9¢b), 3.90–3.83 (m,
2 H, H-5¢, 1 × Tb), 3.77–3.69 (m, 3 H, Ga, H-6a), 3.63–2.94 [m,
25 H, Sb, 3 × Pd, NH(CH2CH2O)3CH2CH2COOH, H-6b, Rd], 2.75–
2.67 (m, 1 H, Dba), 2.62–2.53 (m, 2 H, CH2-Pmc), 2.51–2.38 (m,
10 H, H-3¢e, Dbb, o,o¢-CH3-Pmc, CH2COOH), 2.10–1.58 (m, 15 H,
3 × Pb, 3 × Pg, Vb, CH2-Pmc), 2.03, 2.01, 1.98, 1.93, 1.92, 1.85, 1.82,
1.75, 1.66 (s, 30 H, m-CH3-Pmc, 3 × NHAc, 6 × OAc), 1.50–1.00
(m, 14 H, Rg, H-3¢a, 1 × Tg, 2 × Ab, Rb), 1.34, 1.09, 1.06 [s, 24 H, 3
× C(CH3)3], 1.24 [s, 6 H, C(CH3)2-Pmc], 0.94 (d, J = 5.5 Hz, 3 H,
Tg), 0.87 (d, J = 6.5 Hz, 3 H, Vga), 0.81 (d, J = 6.5 Hz, 3 H, Vgb).
13C NMR (100 MHz, DMSO-d6): d = 169.70, 169.63, 169.50,
169.26, 169.07, 168.90, 168.76 (C=O), 166.75 (C-1¢), 155.92 (Cq-
guanidine), 152.28, 134.93, 134.44, 133.96, 128.43, 128.34,
127.88, 122.57, 117.63 (Pmc), 150.89, 139.57, 134.54, 128.66,
120.55 (HOAT), 98.48 (C-1), 98.03 (C-2¢), 80.12, 73.59, 73.33,
72.85 [C(CH3)2-Pmc, C(CH3)3], 75.58, 71.80, 69.63, 68.92, 68.58,
67.58, 67.25, 66.94 [Tß, NH(CH2CH2O)3CH2CH2COOH, C-3, C-4,
C-5, C-4¢, C-6¢, C-7¢, C-8¢], 66.17 (OCH2-Bn), 62.69, 61.86, 61.66
(C-6, C-9¢, Sb), 59.46, 59.16, 58.97 (Sa, 2 × Ta), 57.04, 56.91, 56.14
(3 × Pa), 55.99 (La, Va), 49.60, 49.25, 47.69 (Ra, Da, C-2, C-5¢),
46.72, 46.46, 46.13 (C-2, C-5¢, 3 × Pd, 2 × Aa), 42.05 (Ga), 38.43,
37.47, 36.70 (C-3¢, Db, Rd), 34.69 (CH2COOH), 32.12 (CH2-Pmc),
30.66 (Vb), 29.09, 28.90 (Rb, 3 × Pb), 27.83, 27.56, 27.05, 26.33
[C(CH3)2-Pmc, C(CH3)3], 25.55, 24.32, 24.29, 24.09 [Rg, 3 × Pg,
CH2-Pmc], 22.67, 22.45, 22.23 (NHAc), 20.65, 20.51, 20.42, 20.32,
DMF (3 × 25 mL) was co-distilled from di-lysyl lysine ester [H-
Lys(H)]2-Lys-Ot-Bu (13; 17.0 mg, 37.1 mmol) under high vacuum.
A soln of spacer-linked glycopeptide 8 (800 mg, 0.30 mmol),
HATU14 (113 mg, 0.30 mmol), HOAt (40.6 mg, 0.30 mmol) and
NMM (65.6 mL, 0.60 mmol) in DMF (2 mL) was added. The mix-
ture was stirred at r.t. for 2 d then DMF was evaporated under high
vacuum, and DMF (20 mL) was co-destilled from the remainder un-
der high vacuum. The remaining residue was dissolved in CH2Cl2
(50 mL) and washed with 0.5 M HCl (2 × 10 mL) and H2O (10 mL).
The combined aqueous layers were extracted with CH2Cl2 (2 × 15
mL), and the combined organic solns were washed with sat.
NaHCO3 (10 mL) and dried (MgSO4). After evaporation of the sol-
vent under vacuo, the crude dendrimeric glycopeptide was purified
by preparative HPLC on a Eurospher C8 column (220 nm; H2O–
MeCN, 60→25% over 90 min). During this chromatography, ex-
cess of glycopeptide-spacer conjugate 8 (129 mg; colorless lyo-
philizate; Rf = 0.44; Rt = 18.7 min) and its 1-hydroxy-azobenzotria-
zolyl ester {99.0 mg; colorless lyophilizate; Rf = 0.58; Rt = 23.7 min;
[a]D22 –13.7 (c 1.0, CHCl3)} were separated.
Yield: 267 mg (65%); colorless amorphous solid; Rf = 0.39
(CH2Cl2–MeOH, 8:1); Rt = 36.0 min (Eurospher C8; 220 nm; H2O–
MeCN, 60→25% over 45 min).
1H NMR (400 MHz, DMSO-d6 [1H-1H-COSY]): d = 8.27–8.15 (m,
4 H), 8.13–7.67 (m, 46 H), 7.25–7.11 (m, 8 H, NH), 7.42–7.31 (m,
20 H, HAr-Bn), 6.61, 6.38 (br s, 8 × RNH), 5.27–5.12 [m, 20 H, 4 ×
(OCH2-Bn, H-4, H-7¢, H-8¢)], 4.92 (d, J = 12.4 Hz, 4 H, 4 × H-3),
4.87 (br s, 4 H, 4 × H-1), 4.75–4.65 (m, 4 H, 4 × H-4¢), 4.61–4.39
[m, 23 H, 3 × Ka, 4 × (Ra, Da, Va, 2 × Aa)], 4.34–4.09 [m, 36 H, 4 ×
(3 × Pa, Sa, 2 × Ta, 1 × Tb, H-2, H-9¢a)], 4.08–4.00 [m, 8 H, 4 × (H-
5, H-6¢)], 3.98–3.91 (m, 4 H, 4 × H-9¢b), 3.90–3.83 [m, 8 H, 4 × (H-
5¢, 1 × Tb)], 3.77–3.69 [m, 12 H, 4 × (Ga, H-6a)], 3.63–2.94 {m,
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

PAPER
Dendrimeric Glycopeptide Antigens
1365
20.12 (OAc), 19.07, 18.86, 17.91, 16.93 (Tg, Vg), 18.10, 18.00 (o,o¢-
CH3-Pmc), 16.75, 16.61 (2 × Ab), 11.77 (m-CH3-Pmc).
N2,N6-Di{N2,N6-di(N-acetyl-glycyl-L-valyl-O-{2-acetamido-2-
deoxy-6-O-[(5-acetamido-3,5-dideoxy-a-glycero-D-galacto-2-
nonulopyranosyl)onat]-a-D-galactopyranosyl}-L-threonyl-L-
seryl-L-alanyl-L-prolyl-L-aspartyl-L-threonyl-L-arginyl-L-pro-
lyl-L-alanyl-L-prolylamido-4,7,10-trioxadodecanyl)-L-lysyl}-L-
lysine ({Ac-G-V-T[a-NeuNAcCOOH-(2→6)-a-GalNAc]-S-A-P-
D-T-R-P-A-P-NH(CH2CH2O)3CH2CH2CO}4-Lys2-Lys-OH, 16)
Dendrimeric glycopeptide 15 (100 mg, 11.1 mmol) was dissolved in
MeOH (20 mL). A soln of 1% NaOMe was added dropwise until a
pH of 8.5–9 (wet pH paper) was achieved. The soln was stirred at
r.t. for 14 h, then methanolic AcOH was added until pH 6 was
reached. After evaporation of the MeOH, the remainder was dis-
solved in H2O (25 mL), the soln was filtered and lyophilized. After
semi-preparative HPLC of the lyophilizate (Vydag C18 column;
H2O–MeCN, 90→80 + 0.1% TFA over 2 h) two substances P1 and
P2 were isolated.
HRMS: m/z calcd for C129H192N22O45S: 2801.31; found: 2803.1.
ESI-MS (MeCN–H2O): m/z = 1400.1 [(M2+)/2].
N2,N6-Di{N2,N6-di(N-acetyl-glycyl-L-valyl-O-{2-acetamido-3,4-
di-O-acetyl-2-deoxy-6-O-[(5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-a-glycero-D-galacto-2-nonulopyranosyl)onat]-a-D-
galactopyranosyl}-L-threonyl-L-seryl-L-alanyl-L-prolyl-L-as-
partyl-L-threonyl-L-arginyl-L-prolyl-L-alanyl-L-prolylamido-
4,7,10-trioxadodecanyl)-L-lysyl}-L-lysine ({Ac-G-V-T[a-
NeuAc4NAcCOOH-(2→6)-a-GalAc2NAc]-S-A-P-D-T-R-P-A-
P-NH(CH2CH2O)3CH2CH2CO}4-Lys2-Lys-OH, 15)
Dendrimer 14 (222 mg, 20.0 mmol) was dissolved in MeOH (6 mL)
and hydrogenated over Pd/C (10%, 50 mg) for 3 h. The catalyst was
filtered off through Celite, MeOH was evaporated under vacuo and
the product was dried under high vacuum to give a colorless amor-
phous solid (188 mg), which was analyzed by HPLC (Eurospher
C18 column; 220 nm; H2O–MeCN, 60→20 over 45 min; Rt = 36.0
min) and found to be sufficiently pure for the subsequent conver-
sion. The solid was dissolved in a mixture of CH2Cl2–TFA–
ethanedithiol–thioanisole (10:10:1:1, 5 mL) and stirred at r.t. for 4
h (reaction monitored by TLC). After evaporation of the solvents,
toluene was co-distilled from the remainder (3 × 25 mL). The crude
product was dissolved in H2O (20 mL) and lyophylized. The lyo-
philizate was dissolved in a little MeOH and purified by preparative
HPLC (Eurospher C18 column; 215 nm; H2O–MeCN, 90→60
+0.1% TFA over 2 h).
P1 which contained 16:
Yield: 27.0 mg (30%); colorless lyophilizate; Rt = 30.7 min (Vydag
C18; 215 nm; H2O–MeCN, 90→80 + 0.1% TFA over 40 min);
[a]D22 –88.7 (c 0.58, H2O).
1H NMR (400 MHz, D2O): d = 4.91 (d, J = 3.8 Hz, 4 H, H-1), 4.70
a
(t, J = 6.5 Hz, 4 H, 4 × Da), 4.67–4.63 [m, 8 H, 4 × (Ra, A1 )], 4.62–
a
4.53 (m, 4 H, 4 × A2 ), 4.52–4.43 (m, 8 H), 4.39–4.24 (m, 27 H),
4.22–4.14 [m, 4 H, 3 × Ka, 4 × (Va, 3 × Pa, Sa, 2 × Ta, 2 × Tb)], 4.12–
4.04 (m, 8 H, H-2, H-9¢a), 3.98–3.52 [m, 140 H, 4 × (H-3, H-4, H-
5, H-6, H-4¢, H-5¢, H-6¢, H-7¢, H8¢, H-9¢b, Ga, Sb, 3 × Pd,
NH(CH2CH2O)3CH2CH2CONH)], 3.48–3.39 (m, 4 H, H-6a), 3.37–
3.29 (m, 1 H, H-6b), 3.22–3.12 (m, 14 H, 3 × Ke, 4 × Rd), 2.96 (dd,
Jb,b¢ = 17.0 Hz, Ja,b = 6.2 Hz, 4 H, 4 × Dba), 2.88 (dd, 4 H, 4 × Dbb,
Jb,b¢ = 17.0 Hz, Ja,b = 6.1 Hz), 2.66 (dd, J = 12.6, 4.4 Hz, 4 H, 4 × H-
3¢e), 2.59–2.51 (m, 4 H), 2.48 (t, J = 6.0 Hz, 4 H, 4 × CH2CONH),
2.36–2.20 (m, 12 H, 12 × Pba), 2.13–1.93 [76 H, 4 × (3 × Pbb, 3 × Pg,
Vb, 3 × NHAc{s, 2.03, 2.00})], 1.92–1.81 (m, 10 H, 3 × Kb, 4 × Rba),
1.78–1.63 [m, 18 H, 3 × Kg, 4 × (H-3¢a, Rg, Rbb)], 1.58–1.42 (m, 6 H,
3 × Kd), 1.40–1.35 (m, 24 H, 8 × Ab), 1.29 (d, J = 6.1 Hz, 12 H, 4 ×
Yield: 120 mg (66%); colorless lyophilizate; Rt = 33.6 min (Euro-
spher C18; 220 nm; H2O–MeCN, 90→60 over 40 min); [a]D22 –84.4
(c 1.0, H2O).
1H NMR (400 MHz, DMSO-d6): d = 8.27–8.15 (m, 4 H), 8.13–7.67
(m, 32 H), 7.50 (m, 4 H), 7.42–6.72 (m, 10 H, NH, RNH), 5.22 [m,
8 H, 4 × (H-4, H-8¢)], 5.17–5.12 (m, 4 H, 4 × H-7¢), 4.98–4.87 [m,
8 H, 4 × (H-3, H-1)], 4.75–4.65 (m, 4 H, 4 × H-4¢), 4.57–3.89 [m,
75 H, 3 × Ka, 4 × (Ra, Da, Va, 2 × Aa, 3 × Pa, Sa, 2 × Ta, 2 × Tb, H-
2, H-5, H-6¢, H-9¢)], 3.83 (q, J = 10.0 Hz, H-5¢), 3.76–3.68 [m,
12 H, 4 × (Ga, H-6a)], 3.66–3.38 {m, 88 H, 4 × [Sb, 3 × Pd,
NH(CH2CH2O)3CH2CH2CONH]}, 3.26–2.91 [m, 18 H, 3 × Ke, 4 ×
(H-6b, Rd)], 2.79–2.67 (m, 4 H, 4 × Dba), 2.56–2.32 [m, 8 H, 4 × (H-
3¢e, Dbb)], 2.31–2.23 (m, 8 H, CH2CONH), 2.13–1.41 [m, 82 H, 4 ×
(H-3¢a, 3 × Pb, 3 × Pg, Rg, Vb), 3 × (Kb, Kg, Kd)], 2.05, 2.00, 1.93,
1.89, 1.83, 1.81, 1.73, 1.64 [s, 90 H, 4 × (3 × NHAc, 6 × OAc)],
1.40–1.08 [m, 44 H, 4 × (Tg, 2 × Ab, Rb)], 0.96 (d, J = 5.0 Hz, 12 H,
Tg), 0.87 (d, J = 5.9 Hz, 12 H, Vga), 0.81 (d, J = 5.9 Hz, 12 H, Vgb).
g
g
T1 ), 1.18 (d, J = 6.2 Hz, 12H, 4 × T2 ), 0.97 (d, J = 4.4 Hz, 12 H, 4
× Vga), 0.96 (d, J = 4.1 Hz, 12 H, 4 × Vgb).
13C NMR (100 MHz, D2O [DEPT]): d = 175.04, 174.74, 174.17,
174.07, 173.99, 173.83, 173.44, 173.14, 172.47, 171.56, 171.24,
170.75 (C=O), 156.81 (Cq-guanidine), 99.43 (C-1), 99.21 (C-2¢),
77.24, 72.62, 71.08, 69.78, 68.52, 68.20, 67.60, 66.93 (C-3, C-4, C-
5, C-4¢, C-6¢, C-7¢, C-8¢, Tb), 69.78, 69.55, 69.37, 69.29, 68.68,
66.70, 66.56 [NH(CH2CH2O)3CH2CH2CONH], 63.84, 62.73 (C-6,
C-9¢), 61.36 (Sb), 60.56, 60.00, 59.20, 58.81, 56.97 (2 × Ta, Pa, Sa),
54.86 (Va), 51.02, 50.08, 49.46 (Ka, Ra, Da, C-2, C-5¢), 47.79,
47.62, 47.45 (Pd), 47.61, 47.42 (Aa), 42.33 (Ga), 40.67, 39.46,
39.10, 38.96 (C-3¢, Rd, Ke), 35.95, 35.59, 34.81 (CH2CONH, Db,
Kb), 30.01 (Vb), 29.52, 29.38, 29.31, 27.97, 27.88, 27.65 (Rb, Pb,
Kg), 24.66, 24.09 (Rg, Pg, Kd), 22.32, 22.14, 21.74 (NHAc), 18.92,
18.52, 18.30, 17.64 (Tg, Vg), 15.50, 15.44 (Ab).
13C NMR (100 MHz, DMSO-d6 [DEPT]): d = 173.36, 171.85,
171.76, 171.63, 171.33, 171.20, 170.98, 170.44, 170.29, 169.99,
169.90, 169.71, 169.62, 169.56, 169.39, 169.30, 169.17, 169.10,
169.01, 168.83, 168.54 (C=O), 158.63, 158.27, 157.91, 156.82
(Cq-guanidine), 98.53 (C-1), 98.17 (C-2¢), 75.91, 71.43, 69.31,
68.65, 67.20, 66.94, 66.87 (Tb, C-3, C-4, C-5, C-4¢, C-6¢, C-7¢, C-
HRMS: m/z calcd for C338H558N78O144: 8013.87; found: 8018.5.
8¢),
69.74,
69.61,
69.51,
69.01,
66.87
ESI-MS (MeCN–H2O): m/z = 1361.2 {[M(4 × 13C) + (6 × Na+)]/6},
1169.2 {[M(4 × 13C) + (7 × Na+)]/7}, 1025.3 {[M(4 × 13C) + (8 ×
Na+)]/8}, 1308.7, 1125.3.
[NH(CH2CH2O)3CH2CH2CONH], 62.61, 62.00, 61.79 (C-6, C-9¢,
Sb), 59.58, 59.11, 57.83, 57.27, 56.18 (Sa, Ta, Pa), 54.82 (Va), 52.46,
52.05 (Ka), 50.07, 49.54 (Ra, Da), 46.81, 46.45, 45.87 (C-2, C-5¢),
47.85, 46.45, 46.23 (C-2, C-5¢, Pd, Aa), 42.07 (Ga), 40.57, 38.49,
37.78, 36.15, 35.93, 35.54 (C-3¢, Ke, Db, Rd, CH2CONH), 30.70
(Vb), 29.32, 28.94, 28.31 (Kb, Kd, Rb, 3 × Pb), 25.94, 25.87, 24.46
(Kg, Rg, 3 × Pg), 22.79, 22.53, 22.31 (NHAc), 20.82, 20.68, 20.55,
20.48, 20.39 (OAc), 19.54, 19.15 (Vg), 18.41, 18.11 (Tg), 16.76,
16.48 (Ab).
ESI-MS (MeCN–H2O, 40:60 + 1% HOAc, after deconvolution of
the measured values including all charged forms): m/z = 7999.89
[M(4 × 13C) – H2O; calcd: 7999.88], 7708.78 {[M(4 × 13C) – H2O –
Neu2enNAcCOOH; calcd: 7708.79}.
P2 which contained the tetramethyl ester of 16:
Yield: 31.7 mg (35%); colorless lyophilizate; Rt = 31.9 min (Vydag
C18; 215 nm; H2O–MeCN, 90→80 + 0.1% TFA over 40 min);
[a]D22 –92.8 (c 0.58, H2O).
HRMS: m/z calcd for C386H606N78O168: 9022.13; found: 9027.4.
ESI-MS (MeCN–H2O): m/z = 1525.6 {[M(4 × 13C) + (6 × Na+)]/6},
1313.4 {[M(4 × 13C) + (7 × Na+)]/7}, 1152.0 {[M(4 × 13C) + (8 ×
Na+)]/8}, 1023.9 {[M(4 × 13C) + (9 × Na+)]/9}.
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

1366
S. Keil et al.
PAPER
1H NMR (400 MHz, D2O) corresponds to the specrum of 16, but
shows additional integration between 3.98–3.50 (m, >140 H, + 1–4
× OMe).
C(CH3)3], 0.83 (d, J = 3.1 Hz, 12 H, Vga), 0.81 (d, J = 3.5 Hz, 12 H,
Vgb).
13C NMR (100 MHz, DMSO-d6 [DEPT]): d = 171.66, 171.40,
171.30, 171.09, 170.95, 170.49, 170.00, 169.87, 169.66, 169.55,
169.27, 168.93 (C=O), 155.74, 153.42, 153.34 (C=O urethane),
137.33, 136.86, 132.54, 131.62, 129.63, 129.55, 129.08, 128.12,
128.01, 127.52, 127.26, 126.22, 123.01, 122.93 (CAr-F, CAr-Y, CAr-
Z), 80.25, 77.41, 77.36, 72.85, 72.78 [C(CH3)3], 69.61, 69.47,
69.37, 68.83, 66.78, 65.14 [CONHCH2CH2(OCH2CH2)2OCH2,
13C NMR (100 MHz, D2O [DEPT]): d = 175.04, 174.72, 174.17,
174.04, 173.78, 173.42, 173.12, 172.92, 172.46, 171.56, 171.34,
171.23, 170.75 (C=O), 156.81 (Cq-guanidine), 99.36 (C-1), 99.21
(C-2¢), 77.24, 72.88, 71.22, 69.97, 68.72, 68.48, 67.65, 67.12 (C-3,
C-4, C-5, C-4¢, C-6¢, C-7¢, C-8¢, Tb), 69.71, 69.55, 69.49, 68.85,
66.87, 66.73 [NH(CH2CH2O)3CH2CH2CONH], 63.93, 62.97 (C-6,
C-9¢), 61.53 (Sb), 60.75, 60.23, 59.38, 58.97, 57.20 (2 × Ta, Pa, Sa),
55.06 (Va), 51.88, 51.14, 50.23, 49.69 (La, Ra, Da, C-2, C-5¢, OMe),
47.79, 47.62 (Pd), 47.46 (Aa), 42.58 (Ga), 40.44, 39.30, 38.94, 38.77
(C-3¢, Rd, Le), 35.95, 35.58, 34.81 (CH2CONH, Db, Lb), 30.01 (Vb),
29.39, 29.19, 27.70, 27.41 (Rb, Pb, Lg), 24.54, 23.92 (Rg, Pg, Ld),
22.16, 21.96, 21.54 (NHAc), 18.76, 18.37, 18.20, 17.52 (Tg, Vg),
15.50, 15.44 (Ab).
a
CH2-Z], 61.43 (Sb), 59.29 (Pa), 57.41 (S1 ), 55.89 (Va), 53.39,
a
53.24, 53.11, 52.59, 51.88 (S2 , Ya, Fa, Ka), 46.65 (Pd), 38.40, 38.30,
36.97, 36.58, 36.09, 35.87 (Yb, Fb, Ke, CH2CONH), 31.92 (Kb),
30.92 (Vb), 28.70 (Pb, Kd), 28.45, 27.51, 27.04 [C(CH3)3], 24.18
(Pg), 22.57 (Kg), 18.95 (Vga), 17.89 (Vgb).
HRMS: m/z calcd for C326H478N38O72: 6077.49; found: 6081.5.
ESI-MS (MeCN–H2O): m/z = 1542.3 {[M(3 × 13C) + (4 × Na+)]/4},
1237.5 {[M(3 × 13C) + (5 × Na+)]/5}, 1031.7 {[M(3 × 13C) + H+ +
(5 × Na+)]/6}.
ESI-MS (MeCN–H2O): m/z = 1370.6 {[M(4 × 13C) + (4 × OMe) +
(6 × Na+)]/6}, 1175.2 {[M(4 × 13C) + (4 × OMe) + (7 × Na+)]/7},
1031.4 {[M(4 × 13C) + (4 × OMe) + (8 × Na+)]/8}, 1308.8, 1125.1.
ESI-MS (MeCN–H2O, 40:60 + 1% HOAc, after deconvolution of
the measured values including all charged species): m/z = 8100.67,
8059.67 [M(4 × 13C) + (3 × methyl ester); calcd: 8059.93], 8045.64
[M(4 × 13C) + (2 × methyl ester); calcd: 8045.92], 8032.66 [M(4 ×
13C) + methyl ester; calcd: 8031.90].
N2,N6-Di{N2,N6-di(N-acetyl-glycyl-L-valyl-O-{2-acetamido-3,4-
di-O-acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-a-glycero-D-galacto-2-nonulopyrano-
syl)onat]-a-D-galactopyranosyl}-L-threonyl-O-tert-butyl-L-ser-
yl-L-alanyl-L-prolyl-4-O-tert-butyl-L-aspartyl-O-tert-butyl-L-
threonyl-N-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-argi-
nyl-L-prolyl-L-alanyl-L-prolylamido-4,7,10-trioxadodecanoyl-
amido-O-tert-butyl-L-tyrosyl-O-tert-butyl-L-seryl-O-tert-butyl-
L-tyrosyl-L-phenylalanyl-L-prolyl-O-tert-butyl-L-seryl-L-valyl-
amido-4,7,10-trioxadodecanoyl)-L-lysyl}-L-lysine tert-Butyl Es-
ter ({Ac-G-V-T[a-NeuAc4NAcCOOBn-(2→6)-a-GalAc2NAc]-
S(t-Bu)-A-P-D(t-Bu)-T(t-Bu)-R(Pmc)-P-A-P-
N2,N6-Di{N2,N6-di(N-benzyloxycarbonyl-O-tert-butyl-L-ty-
rosyl-O-tert-butyl-L-seryl-O-tert-butyl-L-tyrosyl-L-phenylala-
nyl-L-prolyl-O-tert-butyl-L-seryl-L-valylamido-4,7,10-
trioxadodecanoyl)-L-lysyl}-l-lysine tert-Butyl Ester {[Z-Y(t-
Bu)-S(t-Bu)-Y(t-Bu)-F-P-S(t-Bu)-V-
NH(CH2CH2O)3CH2CH2CO]4-Lys2-Lys-Ot-Bu, 17}
DMF (3 × 30 mL) was co-distilled from di-lysyl lysine ester 13
(35.5 mg, 77.4 mmol) under high vacuum. Subsequently, a soln of
peptide-spacer conjugate 9 (880 mg, 0.62 mmol), HATU (235 mg,
0.62 mmol), HOAt (84.1 mg, 0.62 mmol) and NMM (136 mL, 1.24
mmol) in DMF (5 mL) was added. After stirring at r.t. for 2 d, DMF
was evaporated under high vacuum. DMF (3 × 20 mL) was co-dis-
tilled from the remainder under high vacuum. The remaining resi-
due was dissolved in CH2Cl2 (50 mL) and the soln was washed with
0.5 M HCl (2 × 10 mL), sat. NaHCO3 (10 mL) and sat. NaCl (10
mL). The aqueous solns were extracted with CH2Cl2 (15 mL) and
the combined organic layers were dried (MgSO4). After evaporation
of the solvent in vacuo, the crude product 17 was separated from the
excess of educt 9 {30 mg; colorless lyophilizate; Rt = 18.1 min; ESI-
MS (MeCN–H2O); m/z = 1446.7 [M + Na+]} and its hydroxy-
azobenzotriazolyl ester (50 mg; colorless lyophilizate; Rt = 21.9
min; ESI-MS (MeCN–H2O): m/z = 1565.9.1 [M + Na+]} by prepar-
ative HPLC (Eurospher C8 column; 220 nm; H2O–MeCN, 50→0
over 1 h).
NH(CH2CH2O)3CH2CH2CO-Y(t-Bu)-S(t-Bu)-Y(t-Bu)-F-P-S(t-
Bu)-V-NH(CH2CH2O)3CH2CH2CO}4-Lys2-Lys-Ot-Bu, 18)
The dendrimeric peptide 17 (180 mg, 29.6 mmol) was dissolved in
MeOH (8 mL) and hydrogenated over Pd/C (10%, 50 mg) for 3 h.
The catalyst was filtered off through Celite, MeOH was evaporated
under vacuo and the remainder was dried under high vacuum to give
the N-terminally deblocked dendrimeric peptide (100 mg, 61%) as
a colorless viscous oil. DMF (5 × 25 mL) was co-distilled from this
substance under high vacuum. Then, a soln of glycopeptide-spacer
conjugate 8 (775 mg, 0.29 mmol), HATU (110 mg, 0.29 mmol),
HOAt (39.3 mg, 0.29 mmol) and NMM (63.5 mL, 0.58 mmol) in
DMF (3 mL) was added. After stirring at r.t. for 2 d, DMF was evap-
orated under high vacuum. DMF (50 mL) was co-distilled from the
residue, which was then dissolved in CH2Cl2 (30 mL) and washed
with 0.5 M HCl (3 × 10 mL). The combined aqueous solns were ex-
tracted with CH2Cl2 (10 mL) and the combined organic layers were
washed with brine (2 × 15 mL) and sat. NaHCO3 (10 mL). The
aqueous solns were separately extracted with CH2Cl2 (10 mL) and
the combined organic solns were dried (MgSO4). After evaporation
of the solvent, the product was separated from excess of glycopep-
tide 8 (323 mg; colorless lyophilizate; Rt = 7.8 min) and its hydroxy-
azobenzotriazolyl ester (100 mg; colorless lyophilizate; Rt = 10.8
min) by preparative HPLC (Eurospher C8; 220 nm; H2O–MeCN,
60→0% over 90 min).
Yield: 230 mg (49%); colorless lyophilizate; Rt = 37.7 min (Euro-
22
spher C8; 220 nm; H2O–MeCN, 50→0 over 40 min); [a]D –12.5
(c 1.0, CHCl3).
1H NMR (400 MHz, DMSO-d6): d = 8.38 (d, J = 7.1 Hz, 4 H), 8.03–
7.72 (m, 22 H), 7.54–7.48 (m, 8 H, 34 × NH), 7.32–7.15 [m, 48 H,
HAr-F, HAr-Z, 4 × (H2-, H6-Y)], 7.02 [d, J = 8.2 Hz, 8 H, 4 × (H2-,
H6-Y)], 6.82 (d, J = 8.2 Hz, 8 H), 6.77 [d, J = 8.2 Hz, 8 H, 8 × (H3-,
H5-Y)], 4.91 (s, 8 H, 4 × CH2-Z), 4.68–4.59 (m, 4 H), 4.56–4.50 (m,
4 H), 4.49–4.44 [m, 4 H, 4 × (2 × Ya, Fa)], 4.34–4.26 (m, 14 H), 4.16
[t, J = 7.6 Hz, 5 H, 4 × (2 × Sa, Pa, Va), 3 × Ka], 3.58–3.34 [m, 76 H,
8 × Sb, 4 × Pda, 4 × CONHCH2CH2(OCH2CH2)2OCH2], 3.26–3.10
(m, 4 H, 4 × Pdb), 3.02–2.59 (m, 30 H, 4 × Fb, 8 × Yb, 3 × Ke), 2.41–
2.30 (m, 4 H), 2.29–2.23 (m, 4 H, 4 × CH2CONH), 2.04–1.69 [m,
20 H, 4 × (Pb, Pg, Vb)], 1.68–1.33 [m, 27 H, 3 × (Kb, Kg, Kd), Lys-
OC(CH3)3 {s, 1.35}], 1.24, 1.19, 1.10, 1.05 [s, 144 H, 16 ×
Yield: 212 mg (73%); colorless lyophilisate; Rf = 0.42 (CH2Cl2–
MeOH, 8:1); Rt = 24.7 min (Eurospher C8; 220 nm; H2O–MeCN,
50→0 over 40 min); [a]D22 –39.3 (c 1.0, MeOH).
1H NMR (400 MHz, DMSO-d6): d (characteristic signals) = 8.44–
8.32 (m, 4 H), 8.27–8.14 (m, 4 H), 8.12–7.58 (m, 70 H), 7.57–7.48
(m, 4 H, 82 × NH), 7.42–7.31 (m, 20 H, 4 × HAr-Bn), 7.29–6.94 [m,
36 H, 4 × HAr-F, 8 × (H2-, H6-Y)], 6.83–6.72 [m, 16 H, 8 × (H3-,
H5-Y)], 6.61, 6.38 (br s, RNH), 5.27–5.09 [m, 20 H, 4 × (OCH2-Bn,
H-4, H-7¢, H-8¢)], 4.96–4.80 [m, 8 H, 4 × (H-3, H-1)], 4.75–3.80 [m,
111 H, 4 × (Ra, Da, 2 × Va, 2 × Aa, 4 × Pa, 2 × Sa, 2 × Ta, 2 × Tb, 2
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

PAPER
Dendrimeric Glycopeptide Antigens
P2 (containing 19):
1367
× Ya, Fa, H-2, H-5, H-4¢, H-5¢, H-6¢, H-9¢), 3 × Ka], 3.77–3.69 [m,
12 H, 4 × (Ga, H-6a)], 3.68–2.18 {m, 262 H, 3 × Ke, 4 × [2 × Sb, 4
× Pd, 2 × NH(CH2CH2O)3CH2CH2CONH, H-6b, Rd (3.01), 2 × Yb,
Fb, Db, H-3¢e, CH2-Pmc (2.56), o,o¢-CH3-Pmc (2.49), 2 ×
CH2CONH (2.28)]}, 2.10–1.58 [m, 200 H, 4 × (4 × Pb, 4 × Pg, 2 ×
Vb, CH2-Pmc), 4 × (m-CH3-Pmc, 3 × NHAc, 6 × OAc) (2.03, 2.01,
1.99, 1.93, 1.92, 1.85, 1.82, 1.75, 1.66)], 1.53–1.00 [m, 347 H, 3 ×
(Kb, Kg, Kd), 4 × (Rg, H-3¢a, 2 × Ab, Rb), 29 × C(CH3)3, 4 × C(CH3)2-
Pmc], (1.35, 1.24, 1.23, 1.20, 1.11, 1.09, 1.07, 1.06, 1.05)], 0.99–
0.74 [m, 72 H, 8 × (Tg, Vg)].
13C NMR (100 MHz, DMSO-d6 [DEPT]): d = 170.36, 169.77,
169.47, 169.25, 169.02, 168.88, 168.72 (C=O), 166.72 (C-1¢),
153.26 (Cq-guanidine), 134.43, 133.95, 129.63, 129.55, 129.07,
128.42, 128.00, 127.87, 126.21, 123.02, 122.92 (CAr-F, CAr-Y, CAr-
Bn, CAr-Pmc), 98.42 (C-1), 98.02 (C-2¢), 80.11, 77.38, 73.30, 72.84
[C(CH3)2-Pmc, C(CH3)3], 75.57, 71.69, 69.56, 69.49, 69.43, 69.32,
69.26, 68.78, 68.23, 67.35, 67.22, 67.16, 67.03, 66.89, 66.73, 66.60
Yield: 34.8 mg (23%); colorless lyophilizate; Rt = 58.8 min (Euro-
spher C18; 215 nm; H2O–MeCN, 75→55 + 0.1% TFA over 90
min).
1H NMR (400 MHz, DMSO-d6): d (characteristic signals) = 8.35–
8.33 (m, 4 H), 8.24–8.21 (m, 4 H), 8.13–7.74 (m, 58 H), 7.57–7.51
(m, 8 H), 7.34–7.32 (m, 4 H, 78 × NH), 7.25–7.16 (m, 24 H, 4 ×
NH, 4 × HAr-F), 6.99 [d, J = 8.5 Hz, 16 H, 4 × (H2-, H6-Y)], 6.90
[d, J = 8.2 Hz, 16 H, 4 × (H2-, H6-Y)], 6.58 [dd, J = 7.9, 4.4 Hz,
16 H, 8 × (H3-, H5-Y)], 5.25–5.12 [m, 12 H, 4 × (H-4, H-7¢, H-8¢)],
4.98–4.89 [m, 8 H, 4 × (H-3, H-1)], 4.70–3.81 [m, 151 H, 4 × (Ra,
Da, 2 × Va, 2 × Aa, 4 × Pa, 3 × Sa, 2 × Ta, 2 × Tb, 2 × Ya, Fa, 2 × SOH
,
TOH, 2 × YOH, FOH, H-2, H-5, H-4¢, H-5¢, H-6¢, H-9¢, 2 ×
NHCH2CH2), 3 × Ka], 3.80–3.18 {m, 167 H, 4 × [Ga, H-6a, 3 × Sb,
4 × Pd, 2 × NHCH2CH2(CH2CH2O)2CH2CH2CONH]}, 3.16–2.95
(m, 34 H), 2.85–2.49 (m, 20 H), 2.47–2.25 [m, 16 H, 3 × Ke, 4 × (H-
6b, Rd, 2 × Yb, Fb, Db, H-3¢e, 2 × CH2CONH {2.28})], 2.07–1.64
[m, 180 H, 4 × (4 × Pb, 4 × Pg, 2 × Vb, {3 × NHAc, 6 × OAc} {2.06,
2.00, 1.96, 1.93, 1.90, 1.84, 1.83, 1.81, 1.75, 1.65})], 1.51–1.08 [m,
[C-3,
C-4,
C-5,
C-4¢,
C-6¢,
C-7¢,
C-8¢,
NH(CH2CH2O)3CH2CH2COOH, Tb, OCH2-Bn], 61.78, 61.63,
61.42 (C-6, C-9¢, Sb), 59.37, 59.03, 58.83, 57.30, 56.92, 56.75,
56.00 (Sa, Ta, Pa), 53.26, 52.97, 52.65, 52.26, 51.83, 49.59, 49.12
(Ka, Va, Ra, Da, Ya, Fa, C-2, C-5¢), 47.53, 46.63, 46.44, 46.19, 46.01
(C-2, C-5¢, Pd, Aa), 41.87 (Ga), 38.31, 36.82, 36.70, 35.99, 35.82 (C-
3¢, Yb, Fb, Ke, Db, Rd, CH2CONH), 32.02 (CH2-Pmc), 31.65 (Kb),
30.95, 30.70 (Vb), 29.11, 28.96, 28.76 (Kb, Kd, Rb, Pb), 28.46, 27.82,
27.55, 27.04, 26.33 [C(CH3)2-Pmc, C(CH3)3], 24.54, 24.38, 24.30,
24.20 (Lg, Rg, Pg, CH2-Pmc), 22.66, 22.43, 22.22 (NHAc), 20.64,
20.42, 20.31, 20.11 (OAc), 19.05, 18.95, 18.74, 18.07, 17.98, 17.89
(Tg, Vg, o,o¢-CH3-Pmc), 16.92, 16.68 (Ab), 11.76 (m-CH3-Pmc).
74 H, 3 × (Kb, Kg, Kd), 4 × (Rg, Rb, H-3¢a, 2 × Ab, T1 )], 0.99 (d,
g
g
J = 5.9 Hz, 12 H, 4 × T2 ), 0.89–0.79 (m, 48 H, 8 × Vg).
13C NMR (100 MHz, DMSO-d6 [DEPT]): d = 171.70, 171.27,
170.53, 170.17, 169.77, 169.61, 169.48, 169.33, 169.16, 168.94,
168.86 (C=O), 156.60, 156.44, 155.63, 155.53 (Cq-guanidine),
129.92, 129.05, 128.00, 127.83, 114.63 (CAr-F, CAr-Y), 98.07 (C-
2¢), 75.73, 71.32, 69.53, 69.47, 69.43, 69.29, 69.20, 68.90, 68.77,
67.08, 66.76, 66.59 [C-3, C-4, C-5, C-4¢, C-6¢, C-7¢, C-8¢,
NH(CH2CH2O)3CH2CH2CONH, Tb], 62.50, 61.89, 61.66, 61.54,
61.44 (C-6, C-9¢, Sb), 59.44, 58.97, 57.66, 57.46, 57.11, 56.06 (Sa,
Ta, Pa), 54.81, 54.65, 53.85, 53.64, 51.88, 49.90, 49.38 (Ka, Va, Ra,
Da, Ya, Fa, C-2, C-5¢, OMe), 47.71, 46.68, 46.45, 46.28, 46.08 (C-
2, C-5¢, Pd, Aa), 41.93 (Ga), 40.46, 38.38, 37.65, 36.73, 36.44, 36.01,
35.82, 35.42 (C-3¢, Yb, Fb, Ke, Db, Rd, CH2CONH), 31.85 (Kb),
30.62 (Vb), 29.21, 28.93, 28.70, 28.58, 28.25, 27.57 (Kd, Rb, Pb),
24.35, 24.19, 24.02 (Kg, Rg, Pg), 22.71, 22.46, 22.24 (NHAc), 20.74,
20.58, 20.47, 20.41, 20.29, 20.02 (OAc), 19.44, 19.07, 18.98, 18.31,
18.03, 17.75 (Tg, Vg), 16.70, 16.36 (Ab).
HRMS: m/z calcd for C790H1206N110O240S4: 16202.44; found:
16212.9.
ESI-MS (diluted 1:100 in MeCN–H2O, 30:70 + 1% AcOH): m/z =
2316.71 {[M + (7 × H+)]/7}, 2027.32 {[M + (8 × H+)]/8}, 1802.31
{[M + (9 × H+)]/9}, 1622.27 {[M + (10 × H+)]/10}, 1474.9 {[(M +
(11 × H+)]/11}, 1352.00 {[M + (12 × H+)]/12}.
N2,N6-Di[N2,N6-di(N-acetyl-glycyl-L-valyl-O-{2-acetamido-3,4-
di-O-acetyl-2-deoxy-6-O-[(5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-a-glycero-D-galacto-2-nonulopyranosyl)onat]-a-D-
galactopyranosyl}-L-threonyl-L-seryl-L-alanyl-L-prolyl-L-as-
partyl-L-threonyl-L-arginyl-L-prolyl-L-alanyl-L-prolylamido-
4,7,10-trioxa-dodecanoylamido-L-tyrosyl-L-seryl-L-tyrosyl-L-
phenylalanyl-L-prolyl-L-seryl-L-valylamido-4,7,10-trioxado-
decanoyl)-l-lysyl]-L-lysine ({Ac-G-V-T[a-NeuAc4NAcCOOH-
(2→6)-a-GalAc2NAc]-S-A-P-D-T-R-P-A-P-
ESI-MS (diluted 1:20 in MeCN–H2O, 70:30 + 1% AcOH, after de-
convolution of measured values including all charged species):
m/z = 13173.18, 13216.23 {major component [M(8 × 13C)], calcd:
13215.96}, 13271.29, 13313.35.
P1 (major component according to mass spectrum: 19 – 56 g/mol).
Yield: 85.7 mg (57%); colorless lyophilizate; Rt = 56.4 min (Euro-
spher C18; 215 nm; H2O–MeCN, 75→55% + 0.1% TFA over 90
min); [a]D22 –39.3 (c 1.00, MeOH).
1H NMR (400 MHz, DMSO-d6) corresponds to the spectrum of 19,
but shows somewhat lower integral between 3.80–3.18 ppm (m,
164 H).
NH(CH2CH2O)3CH2CH2CO-Y-S-Y-F-P-S-V-
NH(CH2CH2O)3CH2CH2CO}4-Lys2-Lys-OH, 19)
For cleavage of the sialic benzyl esters, dendrimer 18 (185 mg, 11.4
mmol) was dissolved in MeOH (6 mL) and hydrogenated over Pd/C
(10%, 50–60 mg) for 4 h. The catalyst was filtered off through
Celite, MeOH was evaporated in vacuo and the remainder was dried
under high vacuum. The obtained colorless amorphous solid (158
mg) was sufficiently pure according to its analytical HPLC (Euro-
spher C8; 220 nm; H2O–MeCN, 100→0% over 40 min; Rt = 32.2
min). The solid was dissolved in a mixture of CH2Cl2–TFA–
ethanedithiol–thioanisole (20:20:1:1, 5 mL) and stirred at r.t. for 3
h. The progress of the reaction was monitored by analytical HPLC.
After addition of toluene (20 mL), the solvents were evaporated in
vacuo. Toluene (3 × 25 mL) was co-distilled from the remainder,
which was then dissolved in H2O (20 mL) and isolated by lyo-
philization. The lyophilizate was dissolved in a mixture of MeOH–
MeCN (1:2, 2 mL) and purified by preparative HPLC (Eurospher
C18; 215 nm; H2O–MeCN, 75→55 + 0.1% TFA over 5 h). Two
substances were isolated P1 and P2 (19).
13C NMR (100 MHz, DMSO-d6 [DEPT]): d = 171.78, 171.56,
171.37, 171.26, 171.13, 170.62, 170.39, 170.26, 169.94, 169.84,
169.75, 169.68, 169.64, 169.57, 169.51, 169.42, 169.34, 169.26,
169.19, 169.05, 168.95, 168.47 (C=O), 156.74, 155.70, 155.61 (Cq-
guanidine), 137.49, 129.98, 129.11, 128.06, 127.87, 127.26,
126.21, 114.70 (CAr-F, CAr-Y), 98.46 (C-1), 98.11 (C-2¢), 75.79,
71.32, 70.48, 69.59, 69.52, 69.47, 69.43, 69.37, 69.29, 69.18, 69.08,
68.88, 68.75, 68.53, 67.08, 66.82, 66.76, 66.59 [C-3, C-4, C-5, C-
4¢, C-6¢, C-7¢, C-8¢, NH(CH2CH2O)3CH2CH2COOH, Tb], 62.47,
61.89, 61.66, 61.53, 61.43 (C-6, C-9¢, Sb), 59.44, 58.99, 57.70,
57.50, 57.12, 56.06 (Sa, Ta, Pa), 54.94, 54.83, 54.68, 54.42, 53.89,
53.69, 52.31, 51.91, 51.72, 49.93, 49.41 (Ka, Va, Ra, Da, Ya, Fa, C-
2, C-5¢), 47.72, 46.68, 46.47, 46.31, 46.10 (C-2, C-5¢, Pd, Aa), 42.09
(Ga), 40.02, 38.39, 37.65, 36.72, 36.41, 36.15, 36.01, 35.82, 35.57,
35.42 (C-3¢, Yb, Fb, Ke, Db, Rd, CH2CONH), 31.78 (Lb), 30.87,
30.55 (Vb), 29.19, 28.95, 28.84, 28.56, 28.19 (Kb, Kd, Rb, Pb), 24.35,
HRMS: m/z calcd for C594H886N110O228: 13208.11; found: 13216.2.
Synthesis 2009, No. 8, 1355–1369 © Thieme Stuttgart · New York

1368
S. Keil et al.
PAPER
24.22 (Kg, Rg, Pg), 22.69, 22.43, 22.21 (NHAc), 20.71, 20.57, 20.45,
20.38, 20.28 (OAc), 19.44, 19.05, 18.96, 18.30, 18.01, 17.74 (Tg,
Vg), 16.67, 16.36 (Ab).
ESI-MS (diluted 1:10 in MeCN–H2O, 1:1 + 1% HOAc): m/z =
1355.82 {[M + (9 × H+)]/9}, 1525.15 {[M + (8× H+)]/8}, 1742.96
{[M + (7 × H+)]/7}. After deconvolution of the measured values in-
cluding all charged species: m/z = 12207.02 [M(7 × 13C), calcd.:
12206.88], 12249.10 [M(7 × 13C) + (1 × Acetyl)], 12292.21 [M(7 ×
13C) + (2 × Acetyl)].
ESI-MS (diluted 1:20 in MeCN–H2O, 70:30 + 1% HOAc, after de-
convolution of the measured values including all charged species):
m/z = 13117.19, 13159.17 (major component), 13215.28 [M(8 ×
13C); calcd: 13215.96], 13257.34.
P2 (containing 20 and also components formed by elimination).
Yield: 10.3 mg; colorless lyophilizate; Rt = 27.5 min (Vydag C18;
215 nm; H2O–MeCN, 90→50% + 0.1% TFA over 40 min).
N2,N6-Di{N2,N6-di(N-acetyl-glycyl-L-valyl-O-{2-acetamido-2-
deoxy-6-O-[(5-acetamido-3,5-dideoxy-a-glycero-D-galacto-2-
nonulopyranosyl)onat]-a-D-galactopyranosyl}-L-threonyl-L-
seryl-L-alanyl-L-prolyl-L-aspartyl-L-threonyl-L-arginyl-L-pro-
lyl-L-alanyl-L-prolylamido-4,7,10-trioxa-dodecanyl-amido-L-
tyrosyl-L-seryl-L-tyrosyl-L-phenylalanyl-L-prolyl-L-seryl-L-va-
lylamido-4,7,10-trioxadodecanyl)-L-lysyl}-L-lysine (Ac-G-V-
T[a-NeuNAcCOOH-(2→6)-a-GalNAc]-S-A-P-D-T-R-P-A-P-
NH(CH2CH2O)3CH2CH2CO-Y-S-Y-F-P-S-V-
1H NMR (400 MHz, D2O, [1H-1H-COSY]) was very similar to that
of 20.
13C NMR (100 MHz, D2O): d = 174.98, 173.96, 173.80, 173.67,
173.47, 173.12, 173.06, 172.85, 172.62, 172.56, 172.40, 171.73,
171.52, 171.30, 171.20, 170.69, 170.62 (C=O), 156.77, 154.67 (Cq-
guanidine), 136.43, 130.51, 129.40, 128.65, 128.03, 127.06,
115.46, 115.40 (CAr-F, CAr-Y), 72.90, 69.97, 69.73, 69.68, 69.55,
69.46, 68.84, 68.71, 68.64, 68.48, 68.36, 68.25, 68.13, 67.67, 67.15,
66.87, 66.72, 66.61 [C-3, C-4, C-5, C-4¢, C-6¢, C-7¢, C-8¢,
NH(CH2CH2O)3CH2CH2CONH, Tb], 64.03, 63.87, 62.99, 61.54,
61.28, 61.14 (C-6, C-9¢, Sb), 60.71, 60.22, 59.48, 59.38, 58.96,
57.18, 57.02 (Sa, Ta, Pa), 55.63, 55.49, 55.19, 55.06, 54.94, 52.62,
51.88, 51.13, 50.28, 49.66, 49.59 (Ka, Va, Ra, Da, Ya, Fa, C-2, C-5¢,
XMe), 47.90, 47.84, 47.72, 47.55 (C-2, C-5¢, Pd, Aa), 42.59 (Ga),
40.67, 39.09, 38.98, 36.57, 36.20, 36.05, 35.85, 35.02, 33.67 (C-3¢,
Yb, Fb, Ke, Db, Rd, CH2CONH), 30.33, 30.19 (Vb), 29.49, 29.38,
29.31, 28.04, 27.73 (Kb, Kd, Rb, Pb), 24.64, 24.09 (Kg, Rg, Pg), 22.33,
22.14, 21.75 (NHAc), 18.94, 18.53, 18.29, 17.71, 17.65 (Tg, Vg),
15.57, 15.46 (Ab).
NH(CH2CH2O)3CH2CH2CO}4-Lys2-Lys-OH, 20)
Glycopeptide dendrimer 19 (33.7 mg) was dissolved in MeOH (15
mL). A soln of 1% NaOMe in MeOH was added dropwise until pH
8.5–9 (wet pH paper) was reached. The soln was stirred at r.t. for 14
h, then AcOH in MeOH was added until pH 6 was reached. After
evaporation of the MeOH, the remainder was dissolved in H2O (25
mL). The soln was filtered and the product was isolated by lyo-
philization. Purification by semi-preparative HPLC of the lyo-
philizate (Vydag C18; H2O–MeCN, 90→50% + 0.1% TFA over 2
h) afforded two substances P1 und P2.
HRMS: m/z calcd for C546H838N110O204: 12199.86; found: 12207.1.
P1 (consisting of 20 and minor amounts of mono- and di-O-acety-
lated derivatives)
ESI-MS (diluted 1:10 in MeCN–H2O, 1:1): m/z = 1355.82 {[M + (4
× Na+) + (5 × H+)]/9}, 1537.42 {[M + (4 × Na+) + (4 × H+)]/8},
1742.96 {[M + (4 × Na+) + (3 × H+)]/7}.
Yield: 5.00 mg; colorless lyophilizate; Rt = 25.5 min (Vydag C18;
215 nm; H2O–MeCN, 90→50% + 0.1% TFA over 40 min).
ESI-MS (diluted 1:10 in MeCN–H2O, 1:1 + 1% HOAc, after decon-
volution of the measured values including all charged species):
m/z = 11917.18 [M(7 × 13C) – Neu2enNAcCOOH], 11958.14 [M(7
1H NMR (400 MHz, D2O, [1H-1H-COSY]): d (characteristic sig-
nals) = 7.30–7.17 (m, 20 H, 4 × HAr-F), 7.07–6.91 [m, 16 H, 8 ×
(H2-, H6-Y)], 6.74–6.67 [m, 16 H, 8 × (H3-, H5-Y)], 4.85 (m, 1 H,
H-1), 4.75–4.73 (m, 4 H, 4 × Da), 4.66–4.22 [m, 71 H, 4 × (Fa, Ra,
×
13C) + (1 × Acetyl) – Neu2enNAcCOOH], 12000.15 [M(7 × 13C)
+ (2 × Acetyl) – Neu2enNAcCOOH], 12042.04 [M(7 × 13C) + (3 ×
Acetyl) – Neu2enNAcCOOH], 12082.94 [M(7 × 13C) + (4 × Acetyl)
– Neu2enNAcCOOH], 12098.00, 12207.05 [M(7 × 13C), calcd:
12206.88], 12250.16 [M(7 × 13C) + (1 × Acetyl)], 12291.32 [M(7 ×
13C) + (2 × Acetyl)], 12333.38 [M(7 × 13C) + (3 × Acetyl)],
12375.38 [M(7 × 13C) + (4 × Acetyl)], 12417.38 [M(7 × 13C) + (5 ×
Acetyl)].
a
b
2 × Aa, 3 × Sa, 2 × Ya, 4 × Pa, 2 × Ta, V1 , T1 ), 3 × Ka], 4.21–4.18
b
a
b
(m, 4 H, 4 × T2 ), 4.11–4.02 [m, 12 H, 4 × (H-2, V2 , S1 )], 3.99–
3.23 {m, 282 H, 4 × [H-3, H-4, H-5, H-6, H-4¢, H-5¢, H-6¢, H-7¢, H-
8¢, H-9¢, 2 × NH(CH2CH2O)3CH2CH2CONH, 2 × Sb, Ga, 4 × Pd,
H2O]}, 3.20–2.62 [m, 50 H, 3 × Ke, 4 × (Rd, Fb, 2 × Yb, Db, H-3¢e
{2.77})], 2.57–2.38 (m, 16 H, 2 × CH2CONH-G-V), 2.35–1.58 [m,
140 H, 4 × (2 × Vb {2.08}, 4 × Pb, 4 × Pg, Rb, Rg, 3 × NHAc {2.02,
2.00}, H-3¢a {1.68}), 3 × (Kb, Kg)], 1.48–1.22 {m, 41 H, 3 × Kd, 8
g
× Ab [1.36 (d, J = 6.8 Hz), 1.32 (d, J = 6.8 Hz)], 4 × T1 [1.27 (d,
Acknowledgment
g
J = 5.6 Hz)]}, 1.15 (d, J = 6.2 Hz, 12 H, T2 ), 1.00–0.85 (m, 48 H,
8 × Vg).
This work was supported by the Deutsche Forschungsgemeinschaft
and by the Fonds der Chemischen Industrie.
13C NMR (100 MHz, D2O [DEPT]): d = 174.98, 174.59, 173.97,
173.68, 173.55, 173.39, 173.26, 173.05, 172.76, 172.40, 171.65,
171.45, 171.24, 171.11, 170.64, 170.55 (C=O), 156.70, 154.66 (Cq-
guanidine), 136.38, 130.44, 129.36, 129.21, 128.87, 128.59,
127.97, 127.74, 126.99, 115.40 (CAr-F, CAr-Y), 99.14 (C-1), 99.05
(C-2¢), 77.07, 72.64, 71.60, 69.95, 69.79, 69.68, 69.56, 68.78,
68.64, 68.38, 68.20, 68.06, 67.07, 66.82, 66.72, 66.56 [C-3, C-4, C-
5, C-4¢, C-6¢, C-7¢, C-8¢, NH(CH2CH2O)3CH2CH2CONH, Tb],
63.74, 62.76, 61.47, 61.27, 61.11 (C-6, C-9¢, Sb), 60.65, 60.36,
60.16, 59.41, 59.28, 58.89, 57.94, 57.18, 57.01 (Sa, Ta, Pa), 55.45,
55.12, 55.02, 54.90, 52.53, 51.93, 51.07, 50.29, 49.66 (Ka, Va, Ra,
Da, Ya, Fa, C-2, C-5¢), 47.78, 47.49 (C-2, C-5¢, Pd, Aa), 42.64 (Ga),
40.63, 39.99, 38.91, 38.18, 37.96, 37.76, 36.46, 35.81 (C-3¢, Yb, Fb,
Ke, Db, Rd, CH2CONH), 30.25, 30.13 (Vb), 29.43, 29.25, 28.04,
27.67 (Kb, Kd, Rb, Pb), 24.60, 24.03 (Kg, Rg, Pg), 22.29, 22.09, 21.71
(NHAc), 18.89, 18.50, 18.29, 17.67, 17.59 (Tg, Vg), 15.54, 15.44
(Ab).
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