Peptide-functionalized polyphenylene dendrimers

Article abstract of DOI:10.1016/S0040-4020(03)00461-7

This contribution describes the synthesis of polyphenylene dendrimers that are functionalized with up to 16 lysine residues or substituted with short peptide sequences composed of 5 lysine or glutamic acid repeats and a C- or N-terminal cysteine residue. Polyphenylene dendrimers were prepared via a sequence of Diels-Alder cycloaddition and deprotection reactions from cyclopentadienone building blocks. Single amino acids could be introduced on the periphery of the dendrimers by using amino acid substituted cyclopentadienones in the last Diels-Alder addition reaction. Alternatively, peptide sequences were attached via a chemoselective reaction, which involved the addition of the sulfhydryl group of a cysteine residue of an oligopeptide to a maleimide moiety present on the surface of the dendrimer. These amino acid and peptide functionalized dendrimers may be of interest as model compounds to study DNA complexation and condensation or as building blocks for the preparation of novel supramolecular architectures via layer-by-layer self-assembly.

Full text of DOI:10.1016/S0040-4020(03)00461-7

Tetrahedron 59 (2003) 3925–3935
Peptide-functionalized polyphenylene dendrimers
Andreas Herrmann, Gueorgui Mihov, Guido W. M. Vandermeulen, Harm-Anton Klok
*
and Klaus Mu¨llen
Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany
Received 26 September 2002; revised 14 November 2002; accepted 10 January 2003
Abstract—This contribution describes the synthesis of polyphenylene dendrimers that are functionalized with up to 16 lysine residues
or substituted with short peptide sequences composed of 5 lysine or glutamic acid repeats and a C- or N-terminal cysteine residue.
Polyphenylene dendrimers were prepared via a sequence of Diels–Alder cycloaddition and deprotection reactions from cyclopentadienone
building blocks. Single amino acids could be introduced on the periphery of the dendrimers by using amino acid substituted
cyclopentadienones in the last Diels–Alder addition reaction. Alternatively, peptide sequences were attached via a chemoselective reaction,
which involved the addition of the sulfhydryl group of a cysteine residue of an oligopeptide to a maleimide moiety present on the surface of
the dendrimer. These amino acid and peptide functionalized dendrimers may be of interest as model compounds to study DNA complexation
and condensation or as building blocks for the preparation of novel supramolecular architectures via layer-by-layer self-assembly. q 2003
Elsevier Science Ltd. All rights reserved.
1. Introduction
are composed of five lysine or glutamic acid residues and
a C- or N-terminal cysteine moiety. Our interest in
polyphenylene–peptide dendrimers is twofold. First of
all, the cationic lysine functionalized dendrimers may
serve as model compounds to study DNA complexation
and condensation.11 Such model compounds may help to
further understand the pathway of gene transfection.
Second, both the cationic lysine and anionic glutamic acid
functionalized dendrimers are interesting as building
blocks for the electrostatic layer-by-layer self-assembly of
novel supramolecular architectures.12 Layer-by-layer self-
assembly may involve either alternating deposition of
oppositely charged dendrimers or of dendrimers and linear
polyelectrolytes of opposite charge. The shape persistence
of the polyphenylene dendrimer scaffold may allow the
preparation of supramolecular assemblies possessing
defined nanocavities.
In contrast to conventional polymers, dendrimers are highly
branched, monodisperse macromolecules with a high
number of surface functionalities.1–3 Dendrimers differ
from polymers with respect to their methods of prepa-
ration.4,5 They are synthesized either by a convergent or
divergent approach, both implicating the generation of a
core molecule and an iterative procedure for the creation of
the dendrons. The structural perfection of dendrimers is a
prerequisite for clinical applications. Recent advances in
this respect, concerning therapeutic and diagnostic
approaches, have been reviewed.6 For diagnostic purposes
we have recently created a well defined dendrimer,
containing three chromophores and one biotin moiety at
the periphery.7 The perfect structural and spatial definition
of functions results from the use of a shape persistent
dendritic polyphenylene scaffold, consisting of penta-
phenylbenzene units and a rigid tetrahedral core molecule.
In previous work we have demonstrated that, by applying
this dendrimer type, we cannot only place a certain number
of different functionalities into a defined volume element,
which also holds true for other dendrimer-types, but can
even control their orientation.8–10
2. Results and discussion
The divergent synthesis of polyphenylene dendrimers,
which will be described here, starts from multi ethyne-
substituted core molecules, the geometry of which
determines the overall shape of the resulting cascade
molecules.13 The generation of each dendritic layer is
based on an iterative process including a Diels–Alder
reaction and subsequent deprotection of ethynyl functions.
The branching agents that are employed are bis silylethyne
substituted tetracyclones, which can act both as diene and,
after removal of the silyl protective groups from the ethyne
groups, as dienophiles. In the [4þ2]-cycloaddition between
In this publication, we describe the synthesis of several new
polyphenylene dendrimers that contain a large number of
lysine residues on their periphery or which are functional-
ized with short peptide sequences. These peptide sequences
Keywords: dendrimers; lysine residues; hexapeptide.
*
Corresponding author. Tel.: þ49-6131-379-151; fax: þ49-6131-379-350;
e-mail: muellen@mpip-mainz.mpg.de
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00461-7

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A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
reagent, but act as terminating reagents bearing only
functional groups. These functional groups are then
converted into the desired functions in a polymer-analogous
reaction on the dendritic scaffold. A third approach to
introduce multiple functional groups involves the use of
cyclopentadienones that are already substituted with the
appropriate functional group.19,20 This approach requires
the possibility of incorporating the respective function into
the cyclopentadienone and sufficient stability of the
functional groups against the Diels–Alder conditions, i.e.
elevated temperatures. An advantage of this approach is that
it allows quantitative introduction of the functional groups
due to the high yield of the [4þ2]-cycloaddition. This paper
reports the use of the latter two approaches to introduce
lysine moieties and short amino acid sequences based on
lysine and glutamic acid onto the surface of polyphenylene
dendrimers.
Figure 1. Synthesis offunctionalized cyclopentadienones. (i) Benzophenone
imine, BINAP, Cs2CO3, Pd(dba)2, toluene.
the cyclopentadienone and a free ethyne group of the core
molecule or the preceding dendrimer generation, a tetra-
phenylbenzene unit is formed with the extrusion of carbon
monoxide.14–17
2.1. Synthesis of functionalized dendrimers via polymer-
analogous reactions
Three routes for the functionalization of polyphenylene
dendrimers have been reported.18 First, functional groups
can be introduced by electrophilic aromatic substitution.
This leads to a statistical distribution of functional groups on
the periphery of the dendrimer, which is not desired within
the context of this contribution. A second strategy, which
allows the controlled introduction of sensitive function-
alities like biological molecules, starts from dendrimers
which are already functionalized. The corresponding
functional groups on the rim of the dendrimer are generated
by a functionalized cyclopentadienone in a Diels–Alder
reaction at elevated temperatures. Such cyclopentadienones
do not carry any protected ethyne groups like the branching
As amino groups offer a wide range of possibilities for
further functionalization, we chose these as surface
functionalities. To incorporate amino groups at the rim of
the dendrimer the corresponding function must be intro-
duced into the cyclopentadienone. The synthesis of the
functionalized cyclopentadienones is outlined in Figure 1
and starts with monobromo- (1a) and dibromotetracyclo-
pentadienone (1b). In the first reaction step, the halogen
atoms are replaced in a palladium-catalyzed aryl-N-
coupling by benzophenone imine, which is an elegant way
for the introduction of an amino group as a synthetic
equivalent of ammonia.21–24 An additional advantage of
benzophenone imine, in contrast to a non-protected amine,
Figure 2. Abbreviations.

A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
3927
Figure 3. Synthesis of oligolysine-functionalized G1/G2-dendrimers. (i) 2a or 2b, o-xylene, 1458C (G1) or tetraethylene glycol/diphenyl ether, 1708C (G2).
(ii) HCl, rt, THF. (iii) 7, triethylamine, DMF, rt. (iv) 9, DMF, rt.
is its thermostability and therefore its suitability as a reagent
in the cycloaddition. The synthesis of both monofunctional
(2a)25 and difunctional (2b)26 cyclopentadienones, offers
the possibility to control the surface concentration of
functional groups.
five lysine residues, and 9b is composed of a N-terminal
cysteine and five glutamic acid residues. These peptides
were prepared via solid-phase peptide synthesis using Fmoc
protective group chemistry.29 For the coupling reaction with
For the sake of clarity, the terminology in Figure 2 will be
used to represent the central dendritic polyphenylene units
in the following paragraphs. Employing the terminating
reagents 2a,b in a Diels–Alder reaction with either
the tetrahedral core molecule 3 or the octaethynyl
G1-dendrimer 4 yielded the benzophenone imine-function-
alized dendrimers 5a,b and 11a,b (Fig. 3). The synthesis of
the starting materials 3 and 4 has been described
previously.27 For the generation of the G1-dendrimers
5a,b, o-xylene was applied as a solvent at 1458C. The
formation of the next higher generation dendrimers 11a,b
required temperatures of 1708C and a solvent mixture of
tetraethyleneglycol and biphenylether. Subsequently,
complete removal of the protective groups was achieved
with HCl in THF at rt. The resulting dendrimers 6a,b and
12a,b possess 4, 8 and 16 amino groups on their surface.
The attachment of a defined number of oligopeptide
sequences onto polyphenylene dendrimers involved the
addition of the sulfhydryl group of a terminal cysteine
residue to a maleimide function on the periphery of the
dendrimer. It was found that 4-maleimide-butyric acid
chloride (7), which was prepared according to a literature
procedure,28 reacted quantitatively with the amino functions
of the dendrimers 6a,b and 12a,b to yield the maleimide-
functionalized structures 8a,b and 13a,b (Fig. 3). In a
subsequent step, the thiol group of peptides 9a,b was reacted
with the maleimide functions of the dendritic scaffold.
Hexapeptide 9a is composed of a C-terminal cysteine and
Figure 4. HPLC-monitoring of the coupling of hexapeptide 9 to the
maleimide-functionalized dendrimer 8b to form dendrimer 10b.

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A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
10a which show the defect-free attachment of peptide 9a. It
is important to mention, that with the exception of the last
step of the reaction sequence, the work-up of all dendritic
intermediates is realized simply by precipitation from
methanol and filtration. Consequently, all reactions
described in this paragraph give yields higher than 80%,
thus illustrating the efficiency of this route of functionaliza-
tion. The obtained G1-dendrimers 10a,b contain 20 and 40
lysine residues on the surface, respectively. The next higher
generation dendrimer is decorated with 40 and 80 lysines
corresponding to 14a,b, respectively. For the G2-dendri-
mers 14a,b, there are therefore 48 and 96 primary amino
groups on the surface. The purity of 14a,b was also checked
with 1H NMR (spectra not shown) and RP-HPLC (Fig. 5). It
was not possible to obtain mass spectra.
2.2. Synthesis of lysine-functionalized dendrimers from
amino acid substituted cyclopentadienones
Figure 5. RP-HPLC chromatograms of all compounds. (a) Compounds 10a
(peak 1), 10b (peak 2), 14b (peak 3), and 14a (peak 4), prepared by
polymer-analogous reaction. (b) Compounds 21b (peak 5), 21a (peak 6),
and 19 (peak 7), prepared by functionalized cyclopentadienones.
The synthesis of the lysine-functionalized cyclopenta-
dienones started from the benzophenone imine tetraphenyl-
cyclopentadienone derivatives 2a,b, which were
transformed into the corresponding amino compounds
15a,b by the same method as applied for the dendritic
structures 6a,b and 12a,b (Fig. 7). For the next step, a
thermostable protective group for the amino acid must be
selected. Among commercially available bisamino-
protected derivatives of lysine, N a,N 1-di(benzyloxycarbo-
nyl)-L-lysine (16) is thermally very stable. Hence, 16 was
reacted with 15a,b, with EDC as a coupling reagent in
DMF, to obtain the Z-protected lysine-functionalized
cyclopentadienones 17a,b (Fig. 7). After column chroma-
tography, cyclopentadienones 17a,b were obtained in 75
and 45% yield, respectively. Both compounds show a
single peak in their mass spectrum with a mass of 796
and 1208 g/mol respectively, which agrees with the
calculated molecular weight. These compounds serve as
terminating reagents in the Diels–Alder reaction intro-
ducing one or two amino acid functions per free ethyne
group, respectively.
the dendrimer, 9a,b were applied in a three-fold excess with
respect to the dendrimers. The reaction was carried out
under mild conditions in DMF, which was removed in
the work-up by dialysis against water. The addition of
hexapeptide 9a to 8b was followed by reversed-phase
HPLC. Samples from the reaction mixture were analyzed at
6, 24 and 48 h after the start of the reaction (Fig. 4). The
total reaction time was 72 h. Two observations can be made.
First, there is a shift of the major peak from higher to lower
elution volumes during the process of reaction, i.e. to a
solvent mixture of higher water content. The compound
becomes more hydrophilic with increasing reaction time,
which can be explained by the attachment of an increasing
number of (hydrophilic) peptide chains. Second, the major
peak becomes sharper and more symmetrical at longer
reaction times, indicating the decrease of polydispersity. In
Figure 5, chromatograms of all compounds are shown to
give a single peak and document their purity and
monodispersity.
The cycloaddition of the cyclopentadienones 17a,b with
the tetrahedral core molecule 3 and the octaethyne-func-
tionalized G1-dendrimer 4 was carried out analogously to
the preparation of 11a,b, using the higher boiling solvent
mixture at 1708C (Fig. 8). The corresponding dendrimers 18
and 20a,b were obtained after precipitation in methanol
without any cleavage of the protective groups. Z-groups
were removed by applying a cleavage mixture consisting of
HBr in ice-acetic acid to yield the dendrimers 19 and
21a,b.30 Purification was achieved by precipitation from
diethylether. Comparison of the 1H NMR spectra of 18 and
19, which are shown in Figure 9, supports the complete
removal of all Z-groups. The 1H NMR spectrum of 18
exhibits the signals of the aromatic protons of the dendritic
scaffold and of the Z-groups at 6.6 to 7.6 ppm. The signals
of the lysines are detected at 4.12, 3.13, 1.88, 1.49 and
1.39 ppm, whereas the aliphatic benzylic protons of the
protective groups show up at 5.03 ppm. The 1H NMR
spectrum of 19 is nearly identical with the exception that all
benzylic protons at 7.35 and 5.03 ppm are absent.
Furthermore, the relative intensities of the aromatic and
aliphatic protons in both spectra perfectly match the
1
Figure 6 shows a comparison of the H NMR spectra of
dendrimer 8b and the peptide-decorated dendrimer 10b, the
latter being separated from the reaction mixture by dialysis
1
and purified by RP-HPLC. The H NMR spectrum of 8b
shows the aromatic signals of the dendritic scaffold in the
region of 6.4 to 7.4 ppm. The protons of the maleimide
linker appear as singlets at 1.71, 2.18 and 3.38 ppm as well
as a doublet at 6.82 ppm, which overlaps with the aromatic
protons of the polyphenylene signals. When the hexapeptide
9a is attached and dendrimer 10b is generated, the doublet
for the maleimide at low field disappears to form two
singlets. One appears at 3.82 ppm and the other coincides
with the water peak at 3.25 ppm and is therefore not
detectable. The remaining aliphatic signals of the maleimide
linker as well as all signals of the peptide are found in the 1H
NMR spectrum of 10b. For the exact assignment of the
peptide signals see Figure 6. The 1H NMR spectrum of 10b
supports the proposed structure and also proves the purity of
compound 10b. Similar spectra are obtained for dendrimer

A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
3929
Figure 6. 500 MHz 1H NMR-spectra (DMSO-d 6) of (a) 8b and (b) 10b.
Figure 7. Synthesis of lysine-functionalized cyclopentadienones. (i) HCl,
rt, THF; (ii) 16, EDC, DMF, rt.
Figure 8. Synthesis of lysine-substituted G1/G2-dendrimers. (i) 17a or 17b,
tetraethylene glycol/diphenyl ether, 1708C; (ii) HBr, HOAc, rt.

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A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
Figure 9. 300 MHz 1H NMR-spectra of (a) 18 (CD2Cl2) and (b) 19 (DMF d 6).
calculated values. The complete removal of Z-groups is also
shown in the MALDI-TOF mass spectra of 18 and 19 (Fig.
10). All yields for the cycloaddition and the deprotection are
higher than 90%. The G1-dendrimer 19 possesses four
lysine residues on the surface, whereas the G2-dendrimers
21a,b bear 8 and 16 lysine moieties on the surface,
respectively. Again expressed in terms of primary amine
groups this equals 16 and 32 for the respective structures.
structures 10a,b and 14a,b, the nature of the end groups
influences the properties of the whole dendrimer more
strongly. All polyphenylene dendrimers with free lysine
groups on the surface are nicely soluble in DMSO and DMF.
In contrast, when solubilization in water is desired,
structures 19 and 21a,b need to be predissolved in a very
small amount of DMF or DMSO, whereas structures 10a,b
and 14a,b are directly soluble in water. When no lysine
residues are attached to the surface the polyphenylene
dendrimers do not dissolve in water at all.
1
The purity of 21a,b was checked by H NMR (spectra not
shown) and RP-HPLC (Fig. 5). It was not possible to obtain
mass spectra for these compounds.
Summarizing, the two synthetic approaches give peptide-
functionalized dendrimers in high yields and purity. Both
routes provide oligolysine–dendrimers that behave in
accord with the surface density of lysine residues and
primary amine groups.
Further support for the attachment of the cationic moieties
to the surface comes from the change in solubility properties
of these materials. As the surface density of lysine moieties
increases when going from structures 19 and 21a,b to

A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
3931
Figure 11. Molecular simulation of (a) 10b, (b) 14a, (c) 14b and (d) 21b.
minimization of the entire hybrid structures. The peptide
building blocks have been optimized by applying the amber
force field, which proved to be suitable for such structures.
The diameters of all molecules can be determined from the
optimized three-dimensional structures, and are summar-
ized in Table 1. The smallest compound with a diameter of
3 nm is the G1-dendrimer 19 bearing four lysine on the
surface. The largest hybrid structures, exhibiting a diameter
of 6 nm, are the G2-dendrimers 14a,b, functionalized with
the hexapeptide 9. From Table 1, as well as from the
3D-structures in Figure 11, it can also be concluded that
G1-dendrimer 10b, containing a shell of the hexapeptide 9,
exhibits nearly the same size as the dendrimer 21b, which is
of the second generation, functionalized only with a shell of
one amino acid. According to the simulation (Fig. 11), the
lysine moieties are situated exclusively on the surface,
where their spatial arrangement is well-defined. Comparing
structures 14a,b, it is clearly visible that in the case of 14b, a
dense, and in the case of 14a, an open structure is formed.
Figure 10. MALDI-TOF mass spectra of (a) 18 (m/z¼3511 g/mol
(M(Naþ)), calcd MW 3488.26 g/mol) and (b) 19 (m/z¼2414 g/mol
(Mþ), calcd MW 2415.17 g/mol).
3. Visualization/simulation
The structures of the tetrahedral core and dendrons of the
first and second generation were optimized separately
using the MM2 (MMþ) force field, as implemented in
HyperChem 5.1 (Hypercube Inc.). Subsequently, optimi-
zation of the polyphenylene dendritic scaffold was
performed by combining four first-generation dendrons
and four second-generation dendrons with the core
molecule and minimizing both systems. The obtained
‘naked’ scaffolds resemble the structures obtained by a
more profound modeling approach of these structures very
closely.31 Only for the G1-dendritic framework was a global
minimum obtained. Due to the fairly flat hypersurface
obtained for the second-generation dendritic scaffold, the
three-dimensional structure represents one out of several
possible local minima. In the last step of our calculations to
obtain the structures 10a,b, 14a,b, 19 and 21a,b we attached
the minimized lysine residue as well as the hexapeptide
onto the G1- and the G2-dendrimer and performed a
4. Conclusions
We have presented two routes for the functionalization of
shape persistent polyphenylene dendrimers with a large
number of amino acids or oligopeptide sequences on the
surface. These routes are essentially complementary with
respect to the chemistry used for the preparation of the
biofunctional dendrimers, but result in structurally similar
molecules. The synthesis of the mono- and bis-lysine
functionalized cyclopentadienones 17a,b allows the con-
struction of G1- and G2-dendrimers with low surface
densities of amino acids. In the resulting structures the
lysine residues are directly attached to the polyphenyl-
ene scaffold. Functionalization via a polymer-analogous
Table 1. Diameters of the dendrimers as determined from molecular
modelling
Dendrimer
10a
10b
14a
14b
19
21a
21b
Diameter (nm)
4.0
4.0
6.0
6.0
3.0
4.5
4.5

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A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
reaction, in contrast, yields first- and second-generation
dendrimers with a high surface loading of lysine residues.
In these structures, coupling was achieved by the addition of
a terminal cysteine residue of a lysine or glutamic acid
based hexapeptide to a maleimide group on the rim of the
dendrimers. Important characteristics of the latter concept
are the use of unprotected peptides, mild reaction conditions
and the great flexibility for the attachment of different
sequences. Currently, we are exploring these shape-per-
sistent peptide functionalized dendrimers as model com-
pounds for DNA complexation and condensation and are
investigating their potential as building blocks for the
electrostatic layer-by-layer self-assembly of ultrathin
nanostructured supramolecular films.
(250 mL) was added. Subsequently, benzophenone imine
(10 mL, 0.055 mol) was added dropwise. The reaction
mixture was left at 808C for 4 days. Next, it was cooled
down to rt and the toluene was evaporated. The residue was
dissolved in tetrahydrofuran and precipitated in a mixture of
methanol/water (7/1) to give 2a as a brown powder. Yield:
1
4.5 g (82%). FD (Mþ): m/z¼563.7 g/mol; mp 2448C; H
NMR (250 MHz, CD2Cl2) d 7.75–7.25 (m, 10H), 7.23–
7.11 (m, 15H), 6.69 (d, J¼9 Hz, 2H), 6.48 (d, J¼9 Hz, 2H).
5.2.2. Compound 2b. 3,4-Bis(4-bromophenyl)-2,5-di-
phenylcyclopenta-2,4-dien-1-one (1b) (5.0 g, 9.22 mmol),
BINAP (0.631 g, 1.01 mmol), tris(dibenzylideneacetone)-
dipalladium(0) (0.337 g, 0.37 mmol) and CsCO3 (20 g,
0.084 mol) were mixed in a 500 mL flask. The system was
flushed with argon and freshly distilled toluene (250 mL)
was added. Subsequently, benzophenone imine (20 mL,
0.11 mol) was added dropwise. The reaction mixture was
left at 808C for 4 days. Next, it was cooled down to rt and the
toluene was evaporated. The residue was dissolved in
tetrahydrofuran and precipitated in a mixture of methanol/
water (7/1) to give 2b as a brown powder. Yield: 6.2 g
5. Experimental
5.1. General information
Unless stated, all solvents and reagents are commercially
available and were used as received. Compound 4 was
synthesized according to a published procedure.20 1H NMR
spectra were recorded on Bruker DRX 500 (500 MHz) and
Bruker AMX 250 (250 MHz) spectrometers. Spectra were
referenced to the residual proton signal of the deuterated
solvent. Molecular weights were determined with matrix-
assisted laser desorption/ionisation time-of-flight (MALDI-
TOF) or field desorption (FD) mass spectroscopy. MALDI-
TOF spectra were recorded on a Bruker MALDI-TOF mass
spectrometer. Peptide synthesis was carried out on an
Applied Biosystems peptide synthesizer Model 433A. The
amino acids were free acids and coupling was facilitated by
the use of O-benzotriazol-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HBTU) and N-hydroxybenzotriazole
(HOBt). Purity was checked using analytical reversed-phase
high pressure liquid chromatography (RP-HPLC, AKTA-
Purifier, Amersham-Pharmacia Biotech). The column used
was a reversed-phase C8 column (Sephasil peptide C8,
volume 4.155 mL, 5 mm particle size). The lysine-dendri-
mers were eluted with a linear AB gradient from A to B over
61 min, with A consisting of a mixture of 98% water/2%
acetonitrile (plus 0.065% TFA) and B of 100% acetonitrile
(plus 0.05% TFA) in the case of 10a,b and 14b,a. The flow
rate was 0.45 mL/min. Compounds 21a,b and 19 were
eluted with a linear AB gradient from A to B over 61 min,
with A consisting of a mixture of 98% water/2%
isopropanol (plus 0.1% TFA) and B of 100% isopropanol
(plus 0.1% TFA). The flow rate was 1.0 mL/min at a
temperature of 458C. For the glutamic acid-dendrimer 10c a
Resource RPC with a volume of 3 mL was used. Compound
10c was eluted with A consisting of a mixture of 98% water/
2% acetonitrile (plus 0.03% Et3N) and B of 100%
acetonitrile (plus 0.03% Et3N). The flow rate was 2 mL/min.
1
(83%). FD MS: (Mþ): m/z¼742.3 g/mol; mp 2058C; H
NMR (250 MHz, CD2Cl2) d 7.75–7.25 (m, 20H), 7.23–
7.11 (m, 10H), 6.69 (d, J¼9 Hz, 4H), 6.48 (d, J¼9 Hz, 4H).
5.2.3. Compound 5a. Tetra-(4-ethynylphen-1-yl)-methane
(0.1 g, 0.24 mmol) and 2a (0.810 g, 1.44 mmol) in o-xylene
(10 mL) were heated at 1608C for 24 h under argon
atmosphere. Next, the reaction mixture was concentrated
to 1/3 of its initial volume and the product was purified by
precipitation in a mixture of methanol and water (7/1) to
give a dark yellow powder. Yield: 0.520 g (85%). MALDI-
1
TOF MS: m/z¼2560.22 g/mol (Mþ); H NMR (250 MHz,
CD2Cl2) d 7.78–6.40 (m, 128H), 6.30–6.15 (m, 8H).
5.2.4. Compound 5b. Tetra-(4-ethynylphen-1-yl)-methane
(0.1 g, 0.24 mmol) and 2b (1.070 g, 1.44 mmol) in o-xylene
(15 mL) were heated at 1608C for 24 h under argon
atmosphere. The reaction mixture was concentrated to 1/4
of the initial volume and the product was purified by
precipitation in a mixture of methanol and water (7/1) to
give a dark yellow powder. Yield: 0.7 g (89%). MALDI-
TOF MS: m/z¼3277 g/mol (Mþ); 1H NMR (250 MHz,
CD2Cl2) d 7.78–6.40 (m, 156H), 6.38–6.15 (m, 16H).
5.2.5. Compound 6a. Compound 5a (0.100 g, 0.039 mmol)
was dissolved in THF (3 mL). Then, 2N HCl (6 mL) was
added. After 10 min the mixture was cooled with ice and
conc. HCl (3 mL) was added. The mixture was stirred for
20 min. The obtained precipitate was filtered and dried
under high vacuum. The final product is a brown powder.
Yield: 0.065 g (88%). 1H NMR (250 MHz, DMF-d 6) d 7.49
(s, 4H), 7.30–6.76 (m, 84H), 6.76–6.60 (m, 8H).
5.2.6. Compound 6b. This was deprotected analogously to
6a. From 5b (0.100 g, 0.030 mmol) was obtained 0.050 g
5.2. Procedures
1
(83%) 6b. H NMR (250 MHz, DMF-d 6) d 7.50 (s, 4H),
5.2.1. Compound 2a. 3-(4-Bromophenyl)-2,4,5-triphenyl-
cyclopenta-2,4-dien-1-one (1) (4.5 g, 9.71 mmol), BINAP
(0.720 g, 1.15 mmol), tris(dibenzylideneacetone)-dipalla-
dium(0) (0.356 g, 0.39 mmol), and CsCO3 (20.94 g,
0.088 mol) were mixed in a 500 mL flask. The system
was flushed with argon and freshly distilled toluene
7.30–7.11 (m, 48H), 7.09–6.80 (m, 24H), 6.78–6.60 (m
16H).
5.2.7. Compound 7. 4-Maleimide-butyric acid (1.0 g,
5.5 mmol) was dissolved in benzene (10 mL) and thionyl

A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
3933
chloride (1 mL) was added. The reaction mixture was
refluxed for 2 h at 708C. Subsequently, the benzene and
unreacted thionyl chloride was evaporated to give 7 as a
methanol to give 11a as a dark yellow powder. Yield: 0.17 g
(91%). MALDI-TOF MS: m/z¼6319 g/mol; 1H NMR
(250 MHz, CD2Cl2) d 7.78–6.40 (m, 316H), 6.30–6.15
(m, 16H).
1
white powder. Yield: 1.10 g (99%). H NMR (250 MHz,
CD2Cl2) d 6.73 (s, 2H), 3.58 (t, J¼6.7 Hz, 2H), 2.96 (t,
J¼7.22 Hz, 2H), 1.88 (p, J¼6.9 Hz, 2H).
5.2.12. Compound 11b. Compound 4 (0.060 g, 0.0295
mmol) and 2b (0.354 mmol) were dissolved in 9 mL of a
tetraethyleneglycol/biphenylether mixture (1/1). The result-
ing solution was heated for 24 h at 1708C under argon
atmosphere. The reaction mixture was precipitated in
methanol to give 11b as a dark yellow powder. Yield:
0.205 g (89%). MALDI-TOF MS: m/z¼7753 g/mol (Mþ);
1H NMR (250 MHz, CD2Cl2) d 7.78–6.40 (m, 372H),
6.37–6.15 (m, 32H).
5.2.8. Compound 8a. Compound 6a (0.065 g, 0.034 mmol)
and 7 (0.085 g, 0.41 mmol) were dissolved in a mixture of
DMF (5 mL) and triethylamine (0.5 mL). The solution was
stirred for 6 h at rt under argon atmosphere. Then the
product was purified by precipitation in methanol to give a
dark yellow powder. Yield: 0.080 g (92%). MALDI-TOF
MS: m/z¼2584 g/mol (M(Naþ)); 1H NMR (250 MHz,
CD2Cl2) d 7.52–6.40 (m, 104H, arom.), 3.53 (m, 8H),
2.19 (m, 8H), 1.90 (m, 8H).
5.2.13. Compound 12a,b. These were deprotected analo-
gously to 5a. From 11a (0.120 g, 0.019 mmol), 12a
(0.075 g, 80%) was obtained and from 11b (0.120 g,
0.015 mmol), 12b (0.065 g, 84%) was obtained.
5.2.9. Compound 8b. Compound 6b (0.050 g, 0.025 mmol)
and 7 (0.135 g, 0.61 mmol) were dissolved and stirred in a
mixture of DMF (5 mL) and triethylamine (0.5 mL) to give
8b after precipitation as a dark yellow powder. Yield:
0.072 g (88%). MALDI-TOF MS: m/z¼3393 g/mol
Compound 12a. 1H NMR (250 MHz, DMF-d 6) d 7.50–6.75
(m, 236H), 6.74–6.58 (m, 16H).
1
(M(Agþ)); H NMR (250 MHz, DMSO-d 6) d 7.40–6.40
(m, 108H, arom.), 3.44 (m, 16H), 2.17 (m, 16H), 1.78 (m,
16H).
Compound 12b. 1H NMR (250 MHz, DMF-d 6) d 7.50–6.76
(m, 212H), 6.75–6.58 (m, 32H).
5.2.10. Compound 9a,b. HOOC-Cys-(Lys)5-NH2 (9a) and
HOOC-(Glu)5-Cys-NH2 (9b) were prepared by solid-phase
peptide synthesis (SPPS) methods using standard Fmoc-
chemistry.29 The sulfhydryl group of cysteine was protected
with a trityl-group, the 1-amino function of lysine with a
tert(butoxycarbonyl) group (tBoc) and the g-carboxylic acid
function of glutamic acid with a tert(butyl) group (tBu).
A commercially available Wang resin was used, which
releases the peptide readily within 1 h after treatment with a
cleavage mixture consisting of 94.5% trifluoroacetic acid,
1% triisopropylsilane, 2.5% water and 2% ethanedithiol.
The protective groups are simultaneously removed during
this procedure and at the C-terminus of the peptide a
carboxylic group is generated. Subsequently, the free resin is
filtered off and washed with dichloromethane. The solvents
are evaporated leaving a small volume for easy precipitation
with cold diethylether. The peptide was dried overnight
under vacuum. No further purification step was required.
Typically, a 1.00 mmol SPPS resulted in 1.2 g (91%) of the
peptide as a white powder.
5.2.14. Compound 13a. This was prepared analogously to
8a. 12a (0.065 g, 0.013 mmol) and 7 (0.139 g, 0.63 mmol)
were dissolved and stirred in a mixture of DMF (5 mL) and
triethylamine (0.5 mL) to give 13a as a dark yellow powder.
Yield: 0.075 g (91%). MALDI-TOF MS: m/z¼6435 g/mol
1
M(Agþ)) (broad peak); H NMR (250 MHz, DMF-d 6) d
7.50–6.40 (m, 268H, arom.), 3.47 (m, 16H), 2.25 (m, 16H),
1.81 (m, 16H).
5.2.15. Compound 13b. This was prepared analogously to
8a. 12b (0.065 g, 0.013 mmol) and 11 (0.139 g, 0.63 mmol)
were dissolved and stirred in a mixture of DMF (5 mL) and
triethylamine (0.5 mL) to give 13b as a dark yellow powder.
Yield: 0.095 g (94%). MALDI-TOF MS: m/z¼7770 g/mol
1
(broad peak); H NMR (250 MHz, DMF-d 6) d 7.50–6.40
(m, 276H, arom.), 3.47 (m, 32H), 2.25 (m, 32H), 1.81 (m,
32H).
5.2.16. Compound 10a–c and 14b. 1 equiv. of the corre-
sponding maleimide-decorated dendrimer and 3 equiv. of
9a or 9b were dissolved in DMF (10 mL). The reaction
mixture was stirred for 72 h at rt. DMF was removed by
dialysis against distilled water for 48 h. Finally, the peptide-
decorated dendrimers were purified by RP-HPLC.
1
Compound 9a. H NMR (250 MHz, D2O) d 4.55 (t, 1H,
Ha-Cys, J¼5.5 Hz), 4.32–4.20 (m, 4H, Ha-Lys), 4.00–3.95
(t, 1H, N-terminal Ha-Lys, J¼6.5 Hz), 2.97–2.95 (10H,
H1-Lys and 2H, Hb-Cys), 1.88–1.58 (m, 20H, Hb- and
Hd-Lys), 1.50–1.38 (m, 10H, Hg-Lys).
1
Compound 10a. Yield: 80%. H NMR (500 MHz, DMSO-
d 6) d 7.55–6.40 (m, 96H, arom.), 4.49–4.20 (m, 20H), 4.01
(m, 4H), 3.79 (m, 4H), 3.49 (m, 8H), 3.25 (m, 8H), 3.05 (m,
8H), 2.80 (m, 40H), 2.22 (m, 8H) 1.89–1.60 (m, 88H),
1.51–1.38 (m, 40H); RP-HPLC: retention time 33 min.
Compound 9b. 1H NMR (250 MHz, D2O) d 4.47–4.27 (m,
5H, Ha-Glu), 4.21 (t, 1H, Ha-Cys, J¼5.5 Hz), 3.18–3.01
(2H, Hb-Cys), 2.60–2.32 (m, 10H, Hg-Glu), 2.30–1.83 (m,
10H, Hb-Glu).
1
Compound 10b. Yield: 65%. H NMR (500 MHz, DMSO-
5.2.11. Compound 11a. Compound 4 (0.060 g, 0.0295
mmol) and 2a (0.200 g, 0.354 mmol) were dissolved in
9 mL of a tetraethyleneglycol/biphenylether mixture (1/1).
The resulting solution was heated for 24 h at 1708C under
argon atmosphere. The reaction mixture was precipitated in
d 6) d 7.55–6.40 (m, 92H, arom.), 4.49–4.20 (m, 40H), 3.99
(m, 8H), 3.81 (m, 8H), 3.48 (m, 16H), 3.25 (m, 16H), 2.97
(m, 16H), 2.80 (m, 80H), 2.22 (m, 16H) 1.89–1.60 (m,
176H), 1.51–1.38 (m, 80H); RP-HPLC: retention time
47 min.

3934
A. Herrmann et al. / Tetrahedron 59 (2003) 3925–3935
1
Compound 10c. Yield: 62%. H NMR (500 MHz, DMSO-
d 6) d 7.55–6.40 (m, 92H, arom.), 4.22–4.10 (m, 40H), 4.01
(m, 8H), 3.79 (m, 8H), 3.49 (m, 16H), 3.25 (m, 16H), 3.05
(m, 16H), 2.40–2.15 (m, 96H), 1.89–1.70 (m, 96H);
RP-HPLC: retention time 10 min.
5.2.21. Compound 18. This was prepared analogously to
11a from tetra-(4-ethynylphen-1-yl)-methane (0.05 g,
0.12 mmol) and 17a (0.460 g, 0.58 mmol) to give 18 as a
yellow powder. Yield: 0.40 g (95%). MALDI-TOF MS:
m/z¼3511 g/mol (M(Naþ)); 1H NMR (250 MHz, CD2Cl2) d
7.55–6.40 (m, 136H, arom.), 5.05 (m, 16H), 4.06 (m, 4H),
3.08 (m, 8H), 1.89–1.60 (m, 16H), 1.51–1.38 (m, 8H).
Compound 14a. Yield: 39%. 1H NMR (500 MHz, DMF-d 6)
d 7.65–6.40 (m, 252H, arom.), 4.65–4.35 (m, 40H), 4.20–
4.00 (m, broad, 32H), 3.65–3.45 (m, 16H), 3.25 (m, 16H),
2.89–3.10 (m, broad, 96H), 2.38–2.50 (m, broad, 16H) 1.95
(m, 16H), 1.88–1.60 (m, 160H), 1.50–1.38 (m, 80H);
RP-HPLC: retention time 69 min.
5.2.22. Compound 20a. This was prepared analogously to
11a from 4 (0.060 g, 0.0295 mmol) and 17a (0.263 g,
0.354 mmol) to give 20a as a yellow powder. Yield: 0.21 g
1
(87%). MALDI-TOF MS: m/z¼8198 g/mol (M(Naþ)); H
NMR (250 MHz, CD2Cl2) d 7.55–6.40 (m, 332H, arom.),
5.05 (m, 32H), 4.06 (m, 8H), 3.08 (m, 16H), 1.89–1.60 (m,
32H), 1.51–1.38 (m, 16H).
Compound 14b. Yield: 45%. 1H NMR (500 MHz, DMF-d 6)
d 7.65–6.40 (m, 244H, arom.), 4.65–4.35 (m, 80H), 4.21–
4.00 (m, broad, 64H), 3.60–3.40 (m, 32H), 3.25 (m, 32H),
2.89–3.10 (m, broad, 192H), 2.38–2.50 (m, broad, 32H)
1.95 (m, 32H), 1.88–1.60 (m, 320H), 1.51–1.38 (m, 160H).
RP-HPLC: retention time 58 min.
5.2.23. Compound 20b. This was prepared analogously to
11a from 4 (0.060 g, 0.0295 mmol) and 17b (0.427 g,
0.354 mmol) to give 20b as a yellow powder. Yield: 0.30 g
(89%). 1H NMR (250 MHz, d 2-DCM): 7.55–6.40 (m,
404H, arom.), 5.05 (m, 64H), 4.06 (m, 16H), 3.08 (m, 32H),
1.89–1.60 (m, 64H), 1.51–1.38 (m, 32H).
5.2.17. Compound 15a. This was obtained analogously to
6a by deprotection of 2a (1.500 g, 2.66 mmol) to give 15a as
a red powder. Yield: 1.02 g (96%). FD (Mþ): m/z¼399.6 g/
mol; 1H NMR (250 MHz, DMF-d 6) d 7.30–7.16 (m, 15H),
6.90–6.79 (m, 4H).
5.2.24. Compound 19 and 21a,b. These were prepared by
deprotection of the 18 and 20a,b. To 0.100 g of each
dendrimer was dissolved 1 mL 10% solution of HBr in
ice-acetic acid. The mixture was stirred for 1 h at rt
and precipitated in diethylether to give deprotected
Lys-decorated dendrimers.
5.2.18. Compound 15b. This was obtained analogously to
6a by deprotection of 2b (1.800 g, 2.42 mmol) to give 15b as
a red powder. Yield: 0.95 g (94%). FD (Mþ): m/z¼414.5 g/
mol; 1H NMR (250 MHz, DMF-d 6) d 7.32–7.16 (m, 10H),
6.90–6.79 (m, 4H).
Compound 19. Yield: 0.065 g (95%). MALDI-TOF MS:
1
m/z¼2415 g/mol (Mþ); H NMR (250 MHz, DMF-d 6) d
5.2.19. Compound 17a. This was prepared from 15a
(0.799 g, 2 mmol), which was added to N a,N 1-Z-Lys
(2.484 g, 6 mmol), EDC (1.152 g, 6 mmol) and DMAP
(0.406 g, 3.33 mmol) in DMF (10 mL), and stirred for 72 h
under argon atmosphere. Subsequently, DCM (10 mL) was
added and the reaction mixture was washed twice with
water. The organic phase was isolated, dried on magnesium
sulfate and subsequently the solvent was evaporated. The
dried residue was purified by column chromatography with
DCM/ethylacetate (5/1) as eluent to give 17a as a red
powder. Yield: 1.25 g (79%). FD (Mþ): m/z¼796 g/mol; 1H
NMR (250 MHz, CD2Cl2) d 7.55–7.10 (m, 25H, arom.),
6.96 (d, 2H, J¼7.90 Hz), 6.85 (d, 2H, J¼7.90 Hz), 5.1 (s,
4H), 4.15 (t, 1H), 3.16 (m, 2H), 1.89–1.60 (m, 4H), 1.51–
1.38 (m, 2H).
7.40–6.40 (m, 96H, arom.), 4.38 (m, 4H), 2.94 (m, 8H),
1.95–1.60 (m, 16H), 1.51–1.38 (m, 8H).
1
Compound 21a. Yield: 0.07 g (96%). H NMR (250 MHz,
DMF-d 6) d 7.55–6.40 (m, 252H, arom.), 4.42 (m, 8H), 2.90
(m, 16H), 1.89–1.60 (m, 32H), 1.51–1.38 (m, 16H).
Compound 21b. Yield: 0.06 g (96%).1H NMR (250 MHz,
DMF-d 6) d 7.55–6.40 (m, 244H, arom.), 4.44 (m, 16H),
2.90 (m, 32H), 1.88–1.60 (m, 64H), 1.51–1.38 (m, 32H),
Acknowledgements
Financial support from the Deutsche Forschungsge-
meinschaft within the Sonderforschungsbereich 625 and
the Emmy Noether Program (KL 1049/2, H. -A. K.), the
Stiftung Stipendien-Fonds des Verbandes der Chemischen
Industrie and the Bundesministerium fu¨r Bildung und
Forschung (G. V.) is gratefully acknowledged.
5.2.20. Compound 17b. This was prepared from 15b
(0.829 g, 2 mmol), which was added to N a,N 1-Z-Lys
(4.968 g, 12 mmol), EDC (2.304 g, 12 mmol) and DMAP
(0.812 g, 6.66 mmol) in DMF (15 mL). The solution was
stirred for 72 h under argon atmosphere. Subsequently,
DCM (10 mL) was added and the reaction mixture was
washed twice with water. The organic phase was dried
on magnesium sulfate and the solvent evaporated. The
dried residue was purified by column chromatography
with DCM/ethylacetate (3/1) as eluent to give 17a as
red powder. Yield: 1.10 g (45%). FD (Mþ): m/z¼
1208 g/mol; 1H NMR (250 MHz, CD2Cl2) d 7.55–7.10
(m, 34H, arom.), 6.81 (d, 4H, J¼7.85 Hz), 5.06 (m, 8H),
4.18 (t, 2H), 3.14 (m, 4H), 1.89–1.60 (m, 8H), 1.51–1.38
(m, 4H).
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