Reversible cross-linking of hyperbranched polymers: A strategy for the combinatorial decoration of multivalent scaffolds

Article abstract of DOI:10.1002/anie.200460559

All decked out: Peptide-decorated dendritic polymers were synthesized on macroscopic polymer resins and then released by cleaving the reversible cross-linkers (see scheme). The released multivalent structures entered cells efficiently at low concentrations by endocytosis.

Full text of DOI:10.1002/anie.200460559

Communications
zation, transfection, adhesion, and drug delivery.[1] Multi-
valent scaffolds such as linear or branched polymers including
dendrimers have been especially useful in these applica-
tions.[2] They have served for the multiple presentation of
single effective ligands in order to amplify low-affinity
binding.[3] In addition, dendritic structures have been found
to be significantly more stable towards proteolysis in vivo
than the respective monovalent ligands.[4]
Whereas the plain, nondecorated polymers are easily
accessible,[5] the generation of chemically modified polymers
with specific biological activity is much more demanding.
Synthesis on soluble polymers has been studied extensively[6]
and has been been inefficient in most cases due to tedious
workup procedures and low yields. The one practical method
to date is the conjugation of the polymer with preformed
small molecules. Conjugation, however, is restricted to few
coupling reactions and does not allow a flexible variation of
the polymer decoration and loading. For structure–activity
studies for example, during the biological optimization of
polymer drugs, the preparation and variation of decorated
multivalent scaffolds with increased throughput by parallel or
combinatorial methods will be necessary.
Herein, we present a strategy that eliminates the problems
of polymer modification and facilitates flexible access to
complex decorated scaffolds (Scheme 1). Branched polymers
Dendritic Polymers
Reversible Cross-Linking of Hyperbranched
Polymers: A Strategy for the Combinatorial
Decoration of Multivalent Scaffolds**
Michael Barth, Rainer Fischer, Roland Brock, and
Jꢀrg Rademann*
Functional polymers in the nanometer range have been
reported to control biomedical processes including immuni-
[*] Prof. Dr. J. Rademann
Medizinische Chemie
Forschungsinstitut fꢀr Molecular Pharmakologie
Robert-Rꢁssle-Strasse 10, 13125 Berlin (Germany)
Fax: (+49)30-9479-3280
E-mail: rademann@fmp-berlin.de
and
Institut fꢀr Chemie
Freie Universitꢂt Berlin
Takustrasse 3, 14195 Berlin (Germany)
Scheme 1. Reversibly cross-linked hyperbranched polymers facilitate
the multivalent decoration macromolecular structures. a) Highly
branched PEI is cross-linked to yield a swellable resin that serves as a
robust support in polymer-supported synthesis. b) Complex molecules
can be constructed by multistep solid-phase synthesis. c) Finally, disin-
tegration of the resin is effected by cleaving the reversible cross-link-
ing.
Dipl.-Chem. M. Barth
Institut fꢀr Organische Chemie
Eberhard Karls Universitꢂt Tꢀbingen
Auf der Morgenstelle 18, 72076 Tꢀbingen (Germany)
were cross-linked to yield a swellable resin which served as a
robust support for subsequent solid-phase synthesis
(Scheme 1, top). Making use of easy-to-perform polymer-
supported protocols for the multistep synthesis, we could
assemble multiple copies of a target molecule on each
macromolecule. Finally, the cross-linking units of the polymer
support were cleaved, yielding the decorated scaffolds
(Scheme 1, bottom).
Dipl.-Biochem. R. Fischer, Dr. R. Brock
Interfakultꢂres Institut fꢀr Zellbiologie
Eberhard Karls Universitꢂt Tꢀbingen
Auf der Morgenstelle 15, 72076 Tꢀbingen (Germany)
[**] This research was supported by the DFG (Graduiertenkolleg
“Chemie in Interphasen, Projekt 6”, fellowship for M.B.). We thank
Dr. Volker Braig, BASF AG, Ludwigshafen for conducting the gel
permeation chromatography.
Highly branched polyethylene imine (PEI) was selected
as the ideal starting material for the preparation of a
reversibly cross-linked resin.[16,17] In several biological appli-
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200460559
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Angewandte
Chemie
cations including in vivo transfection,[7] PEI served as an
efficient hyperbranched structure. Recently, it was demon-
strated that various PEIs are well suited for the construction
of ultrahigh-loaded polymer supports (“Ultraresins”) useful
in organic synthesis and for polymer reagents.[8–10]
To obtain a robust solid support constructed of reversibly
cross-linked hyperbranched polymers, the cross-linker must
be cleaved orthogonally. Dialdehyde 2, which contains a
dialkoxysilane tether, was chosen for this purpose. Reversibly
cross-linked resins were obtained by polycondensation of
highly branched PEI 3 (Mn = 10000; Mw = 25000, polydis-
persity = 2.5) with 2 (Scheme 2). For homogeneous cross-
Figure 1. GPC of a) starting PEI 3 (Mn =10000, Mw =25000, continu-
ous line), b) PEI obtained by decomposition of reversibly cross-linked
resin 4 (dotted line), and c) peptide-decorated PEI polymer 8
(Mn =24000, Mw =75000, dashed line). V=elution volume,
I=intensity of the detector signal.
Scheme 2. Cross-linker 2 was constructed from 1 and employed for
construction of polymer support 4. a) Diisopropyldichlorosilane,
pyridine, 1 h, 608C; b) 2, THF, 4 h, RT; c) Sodium borohydride
THF/MeOH 2:1, 16 h, RT.
Scheme 3. Reversibly cross-linked resin 4 was employed in the synthe-
sis of decorated multivalent scaffolds. a) Fmoc-AA, TBTU, HOBT,
DIPEA, DMF; b) di-tert-butyl dicarbonate, DIPEA, DMF, 2ꢃ2 h, RT;
c) peptide synthesis following the Fmoc strategy; d) 4-[(acetyloxy)me-
thyl]benzoic acid, TBTU, HOBT, DIPEA, DMF, 4 h, RT; e) NaOMe
0.1m in MeOH, 0.5 h, RT; f) 95% trifluoroacetic acid, 2.5% triisopro-
pylsilane, 2.5% water, 4 h, RT. R=unprotected peptide, Boc=tert-
butyloxycarbonyl, Fmoc=9-fluorenylmethoxycarbonyl.
linking in high yields, the concentrations of PEI and of the
cross-linker 2 were critical. Resin 4 was characterized by H
1
MAS NMR spectroscopy, FT-ATR-IR spectroscopy, and
elemental analysis (MAS = magic angle spinning, ATR =
attenuated total reflection). Disintegration of resin 4 could
be effected by cleaving the silicon–oxygen bonds in the cross-
linker with acid (50% trifluoroacetic acid in dichlorome-
thane, 2 h) or fluoride (1m tetrabutylammonium fluoride in
THF, 2 h). Completion of the disintegration of 4 was proven
by gel-permeation chromatography (GPC) yielding a PEI
product with an Mn value identical to that of the starting
polymer (Figure 1).
on the secondary amines of resin 4 to yield resin 5.
Alternatively, the peptide sequences were assembled on the
4-hydroxymethyl benzoic acid linker (HMBA) (!resin 6).
This base-labile linker can be used such that orthogonal
cleavage of the protected peptides from resin 6 does not affect
resin integrity. In addition, the linker enables the cleavage of
deprotected peptides from the released multivalently deco-
rated scaffolds 8–16 as required for analytical purposes or as
desirable for specific in vivo applications. As a third option,
Resin
4
was employed in solid-phase synthesis
(Scheme 3). Peptide synthesis could be conducted directly
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only a small fraction of free amines were coupled with the
HMBA linker in order to allow for the analytic monitoring of
peptide synthesis by partial cleavage (!resin 7).
To adjust the peptide content of the resulting multivalent
decorated scaffolds, a substoichiometric amount of the linker
or the first amino acid was coupled on resin 4. The remaining
secondary amines of the polymer backbone were capped by
reaction with Boc-anhydride. Benzotriazolyltetramethyl-
uranium tetrafluoroborate (TBTU) was used to activate the
Fmoc-amino acids. The success of the synthesis could be
monitored by employing the Kaiser test or by cleavage and
deprotection to give the final peptide product.
To demonstrate the feasibility of the concept, a selection
of potentially bioactive decorated multivalent scaffolds was
prepared (Table 1). The selected peptides include sequences
for intracellular targeting (decorated scaffolds 8 and 9),[11]
B
and T cell epitopes for vaccination (10–14), and a peptide
described to disrupt molecular interactions involved in the
regulation of apoptosis inside the cytoplasm (15).[12] This
selection represents a broad spectrum of biological and
biomedical applications.
Resins 5, 6 and 7 were decomposed under acidic
conditions (95% trifluoroacetic acid, 2.5% triisopropylsilane,
2.5% water, 4 h), and the standard side-chain protection
groups of amino acids were also removed in the same step. For
workup of the decorated polymers, protocols routinely used
in peptide synthesis could be employed. Repeated precipita-
tion of the peptide-functionalized polymers in cold diethyl
ether furnished pure decorated polymer scaffolds and
removed the nonvolatile residues from the trityl and the
Figure 2. HeLa cells were incubated with serum-free medium contain-
ing fluorescein-labeled dendrimer (400 nm) and AlexaFluor 647-dextran
(10 mm) for 2 h, washed, and analyzed by multichannel confocal laser
scanning microscopy. A) fluorescein fluorescence, B) AlexaFluor 647-
dextran fluorescence, C) superposition of both fluorescence channels,
D) transmission picture.
(10000 Da) dextrans, which are internalized by fluid-phase
endocytosis.[13] The observed subcellular distribution is indi-
cative of uptake by endocytosis and subsequent escape from
endocytic compartments. The cytoplasmic and nuclear local-
ization could be inhibited by incubation with bafilomycin A1,
a highly potent and selective inhibitor of vacuolar-type H+-
ATPases (see the Supplementary Information).[14] The homo-
geneity of the uptake on the level of a population of cells and
the cellular toxicity of the nanoscale constructs was studied by
flow cytometry. A low concentration of 8 (50 nm) was
sufficient for homogeneous loading of the cell population.[15]
In addition, no toxicity was observed at concentrations up to
1 mm (data are given in the Supplementary Information).
Reversibly cross-linked resins prepared from hyper-
branched polymers are powerful new tools for the generation
of decorated multivalent scaffolds. By creating a transition
from solution to solid-phase methods, the concept combines
the advantages of polymer-supported syn-
2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl
(Pbf)
protecting groups as determined by NMR spectroscopy (see
the Supplementary Information).
To evaluate the biological applicability of the peptide-
decorated polymer scaffolds obtained by the novel strategy,
cellular uptake was investigated by confocal microscopy of
living cells. The decorated scaffold 8 bearing the fluorescein-
labeled nuclear localization sequence Fluo-PKKKRKV was
selected for this purpose. HeLa cells were incubated with the
scaffold at a concentration of 400 nm for 2 h at 378C. Both a
distinct vesicular staining and a homogeneous cytoplasmic
and nuclear localization were observed (Figure 2). The
vesicular staining colocalized with high-molecular-weight
thesis with the ease of conventional reaction
Table 1: Synthesis of decorated multivalent scaffods with potentially bioactive peptide sequences.
monitoring, including on-bead and off-bead
analysis. With these resins, the repertoire of
combinatorial methods including parallel
synthesis, automation, and split-and-mix
operations is applicable to the decoration
of macromolecular structures. Furthermore,
the multivalent scaffolds we obtained are
suitable for cellular applications. The entire
cell population was affected homogene-
ously by scaffold 8. In future studies the
concept will be used for the development
and optimization of biologically active
polymers targeting the cytoplasm or specific
organelles of eukaryotic cells.
Decorated Sequence
scaffold
Resin Loading
[mmolgꢀ1
MP
Purity
Mn
Copies
per PEI
molecule
]
[gmolꢀ1 [a]
]
(214 nm) [%] [gmolꢀ1 [b]
]
8
9
Fluo-PKKKRKV
ANWTGPKKKRKV
EQPRKFG
5
5
5
6
6
6
7
7
7
1.6
1.6
3.0
1.8
1.8
1.8
2.2
2.2
2.2
–
–
–
–
–
–
30000
32000
35000
32000
41000
38500
48000
38000
38000
16
16
40
18
18
18
22
22
22
10
11
12
13
14
15
16
MAYPRISVNNG
1234.6
1745.8
1595.9
1745.8
1088.7[c]
1088.7[c]
82.5
84.3
82.5
88.3
88.3[c]
86.6[c]
QSQPQPPHPTPYWIG
KVSTLPAITLKLGGKG
QSQPQPPHPTPYWIG
AVPIAQKK(Fluo)G
KQAIPVAK(Fluo)G
[a] Mass of the peptide methyl ester. [b] Mass of the product. [c] Mass and purity of AVPIAQKK(Dde)G-
OMe and KQAIPVAK(Dde)G-OMe. Fluo=5(6)-carboxyfluorescein.
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Chemie
b) E. Bayer, A. Geckeler, Justus Liebigs Ann. Chem. 1974,
1671 – 1674; c) R. Haag, A. Sunder, A. Hebel, S. Roller, J. Comb.
Chem. 2002, 4, 112 – 119.
Experimental Section
Synthesis of 4: Polyethylene imine (Mn = 10000, Mw = 25000, 1.25 g)
was dissolved in THF (6.1 mL), and a solution of 2 (0.495 g,
1.29 mmol) in THF (4.6 mL) was added rapidly. After one minute
the stirring bar ceased rotating. After 4 h the polymer was crushed,
washed with THF, and suspended in THF/MeOH (2:1, 24 mL).
Sodium borohydride (0.097 g, 2.56 mmol) was added, and the
suspension was shaken for 16 h at RT. The polymer was washed
with THF and MeOH, pressed through a sieve (400 mm pores),
washed again with MeOH and CH2Cl2, and dried in vacuo to give
[7] K. Aoki, S. Furuhata, K. Hatanaka, M. Maeda, J.-S. Remy, J.-P.
Behr, M. Terada, T. Yoshida, Gene Ther. 2001, 8, 508 – 514.
[8] M. Barth, J. Rademann, J. Comb. Chem. 2004, 6, 340 – 349.
[9] J. Rademann, M. Barth, Angew. Chem. 2002, 114, 3087 – 3090;
Angew. Chem. Int. Ed. 2002, 41, 2975 – 2978.
[10] M. Barth, S. T. Ali Shah, J. Rademann, Tetrahedron 2004, 60,
8703 – 8709.
[11] D. A. Jans, C.-Y. Xiao, M. H. C. Lam, Bioessays 2000, 22, 532 –
544.
[12] C. R. Arnt, M. V. Chiorean, M. P. Heldebrant, G. J. Gores, S. H.
Kaufmann, J. Biol. Chem. 2002, 277, 44236 – 44243.
[13] C. Plank, B. Oberhauser, K. Mechtler, C. Koch, E. Wagner, J.
Biol. Chem. 1994, 269, 12918 – 12924.
1
resin 4 (1.5 g, 89%). Elemental analysis: C 55.6, H 9.7, N 21.0; H
MAS NMR (400 MHz, MeOD, rotation frequency 4500 Hz): d = 0.9–
1.2 (m, isopropyl, rel. integration 16.7), 2.2–3.0 ppm (m, PEI-CH2,
100), 3.72 (br.s, sec-N-CH2-aryl, 2.52), 4.56 (br.s, tert-N-CH2-aryl,
0.53) 7.2–7.5 ppm (br.s, aryl-H, 6.51); FT-ATR-IR: n˜ = 815, 1063,
1090, 1461, 1572, 2815, 2932, 3277 cmꢀ1
.
[14] a) E. J. Bowman, A. Siebers, K. Altendorf, Proc. Natl. Acad. Sci.
USA 1988, 85, 7972 – 7976; b) T. Merdan, K. Kunath, D. Fischer,
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[15] For comparison, in a recent publication 50 mm of a fluorophore-
labeled trimeric peptide was required for detectable incorpo-
ration in HeLa cells: J. Fernandez-Carneado, M. J. Kogan, S.
Castel, E. Giralt, Angew. Chem. 2004, 116, 1847 – 1850; Angew.
Chem. Int. Ed. 2004, 43, 1811 – 1814.
[16] The fragmentation of a macroscopic polymer to give a modified,
multivalent structure as described in this article must be clearly
distinguished from the numerous works on enzymatically or
chemically cleavable dendrimers. Cleavable dendrimers are
fragmented into monomeric building blocks, and thereby the
multivalent character is lost. Two recent examples on triggered
dendrimer cleavage are: a) F. M. H. de Groot, C. Albrecht, R.
Koekkoek, P. H. Beusker, H. W. Scheeren, Angew. Chem. 2003,
115, 4628 – 4632; Angew. Chem. Int. Ed. 2003, 42, 4490 – 4494;
b) J. R. Amir, N. Pessah, M. Shamis, D. Shabat, Angew. Chem.
2003, 115, 4632 – 4636; Angew. Chem. Int. Ed. 2003, 42, 4494 –
4499.
General procedure for the synthesis of peptide-decorated poly-
mer scaffolds: Fmoc-glycine (446 mg, 1.5 mmol) was coupled with
TBTU (482 mg, 1.5 mmol) N-hydroxybenzotriazole (HOBt; 230 mg,
1.5 mmol) and N,N-diisopropylethyl amine (DIPEA; 257 mL,
1.5 mmol) in DMF to resin 4 (100 mg). After 4 h the resin was
washed with DMF and CH2Cl2 and dried in vacuo. The resin was
capped using di-tert-butyl dicarbonate (1.1 g, 5 mmol) and DIPEA
(1.7 mL, 10 mmol) in DMF (2 ꢀ 2 h) at RT. The absence of primary
and secondary amines was indicated by the Kaiser test and the
chloranil test, respectively. The loading of the resin was determined
photospectrometrically by cleaving the Fmoc group from the resin.
Peptides were synthesized by the Fmoc strategy using four
equivalents of amino acid (based on the loading with first amino acid),
TBTU, HOBT, and DIPEA in DMF for 90 min. The Fmoc group was
cleaved by treatment with 20% piperidine in DMF (2 ꢀ 8 min).
Completion of the acylation was determined by the Kaiser test.
Decomposition together with removal of the amino acid side-
chain protection of the peptide-decorated resin was performed by
using 95% trifluoroacetic acid (TFA), 2.5% H2O, and 2.5%
triisopropylsilane for 4 h at RT. The solution was filtered, and the
filter was washed with TFA. Collected solvents were evaporated.
After precipitation with cold diethyl ether (4 ꢀ ) the soluble peptide-
decorated polymer scaffold was lyophilized (tert-butanol/water 4:1).
[17] Polyethylene imine was selected as the most inexpensive
commercially available starting polymer. In principle, the
protocol should be applicable for other branched polyamines
including amine dendrimers.
Received: May 5, 2004
Revised: August 23, 2004
Published online: February 2, 2005
Keywords: combinatorial chemistry · drug delivery · peptides ·
.
polymers · solid-phase synthesis
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Angew. Chem. Int. Ed. 2005, 44, 1560 –1563
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