Novel Fmoc-polyamino acids for solid-phase synthesis of defined polyamidoamines

Article abstract of DOI:10.1021/ol200381z

A versatile solid-phase approach to sequence-defined polyamidoamines was developed. Four different Fmoc-polyamino acid building blocks were synthesized by selective protection of symmetrical oligoethylenimine precursors followed by introduction of a carboxylic acid handle using cyclic anhydrides and subsequent Fmoc-protection. The novel Fmoc-polyamino acids were used to construct polyamidoamines demonstrating complete compatibility to standard Fmoc reaction conditions. The straightforward synthesis of the building blocks and the high efficiency of the solid-phase coupling reactions allow the versatile synthesis of defined polycations.

Full text of DOI:10.1021/ol200381z

ORGANIC
LETTERS
2011
Vol. 13, No. 7
1586–1589
Novel Fmoc-Polyamino Acids for Solid-
Phase Synthesis of Defined
Polyamidoamines
David Schaffert, Naresh Badgujar, and Ernst Wagner*
Pharmaceutical Biotechnology, Department of Pharmacy, Center for Drug Research,
€
and Center of NanoScience, Ludwig-Maximilians-Universitat Munich, Butenandtstr.
5-13, D-81377 Munich, Germany
ernst.wagner@cup.uni-muenchen.de
Received December 1, 2010
ABSTRACT
A versatile solid-phase approach to sequence-defined polyamidoamines was developed. Four different Fmoc-polyamino acid building blocks
were synthesized by selective protection of symmetrical oligoethylenimine precursors followed by introduction of a carboxylic acid handle using
cyclic anhydrides and subsequent Fmoc-protection. The novel Fmoc-polyamino acids were used to construct polyamidoamines demonstrating
complete compatibility to standard Fmoc reaction conditions. The straightforward synthesis of the building blocks and the high efficiency of the
solid-phase coupling reactions allow the versatile synthesis of defined polycations.
Polyamines are involved in a number of biological
functions such as neuromodulation,1 cell proliferation,2
and DNA condensation,3 constituting important tem-
plates in drug design4 and synthetic scaffolds in alkaloid
and heterocycle synthesis.5 Furthermore they are key
components in the syntheses of gene delivery systems such
as PAMAM dendrimers,6 cationic lipids,7 or linear poly-
(amidoamines).8 Poly(amidoamines) have received a lot of
scientific attention because of their favorable properties
regarding DNA compaction,9 solubility, and biocompat-
ibility as well as their favorable properties regard-
ing hemolysis and cytotoxicity10 compared to other
commonly used polymers such as poly(L-lysine)11 and
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Natl. Acad. Sci. U.S.A. 1987, 84, 7413. (b) Wang, X. L.; Jensen, R.; Lu,
Z. R. J. Controlled Release 2007, 120, 250.
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Lewandowski, S.; Begin, A.; Lewandowski, R.; Talbot, B. G.; ElAzhary,
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r
10.1021/ol200381z
Published on Web 03/04/2011
2011 American Chemical Society

Scheme 1. Building Block Synthesis
polyethylenimine.12 Poly(amidoamines) are classically
synthesized by solution-phase polyaddition reactions13
resulting in a limited design space and products with a
high degree of polydispersity.
carboxylic acidfunctionata terminalamino groupbyring-
opening acylation with cyclic anhydrides (Scheme 1). To
prevent side reactions during building block synthesis and
SPPS, the secondary amines of the oligoethylenimines had
to be protected. The primary and secondary amines of the
polyamine scaffold were differentiated by selective acyla-
tion of the primary amines16 using ethyl trifluoroacetate.
Secondary amines were protected as carbamates using di-
tert-butyl dicarbonate, allowing deprotection during glo-
bal deprotection and cleavage from the resin. Alkaline
hydrolysis of 3 and 4 yielded the corresponding Boc-
protected diamines (5 and 6). The conversion of the
symmetrical Boc-protected diamines into functionalized
polyamino acids required a monoacylation in the first step,
thereby differentiating the two amino functions. Desym-
metrization reactions are by their nature low yielding and
in the case of polyamines often involve laborious chroma-
tographic purification procedures. We optimized a one-
pot strategy allowing introduction of the carboxylic acid
function and subsequent fmoc protection of the remaining
amino function. The yield limiting step was the desymme-
trization reaction, so different reaction conditions were
tested for an effective monoacylation. Acylation at -70 °C
using cyclic anhydrides followed by conversion into the
A literature survey reveals that reports of the application
of solid-phase chemistry to the synthesis of polyamido-
amines are scarce and mostly deal with rather short target
structures.14 The only examples for the synthesis of longer,
linear polyamidoamines by a solid-phase methodology
were reported by Hartmann et al.15 They employed a
two-step condensation synthesis strategy characterized
by an alternating assembly of the diacid succinic acid
and diamines, which nicely demonstrated the feasibility
of the approach. Our efforts to apply their strategy to
larger oligoamines failed because of unexpected cross-
links which block further polymer extension (see Sup-
porting Information). In this letter we report a versatile
synthetic route to fmoc-polyamino acid building blocks
and their use in solid-phase poly(amidoamine) synth-
esis. Use of these building blocks greatly improves the
versatility and efficiency of solid-phase poly(amidoamine)
synthesis.
In designing oligoethylenimine based building blocks
compatible with Fmoc-SPPS protocols we introduced the
(12) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.;
Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 7297.
(13) Ferruti, P.; Ranucci, E.; Di Silvestro, G.; Yuan, C.-M.; Caranzolo,
N.; Donetti, R.; Marchisio, M. A. Chim. Ind. 1999, 81, 739–748.
(14) (a) Wells, N. J.; Basso, A.; Bradley, M. Peptide Science 1998, 47,
381. (b) Manku, S.; Laplante, C.; Kopac, D.; Chan, T.; Hall, D. G. J.
Org. Chem. 2001, 66, 874. (c) Jørgensen, M. R.; Jaroszewski, J. W.; Witt,
M.; Franzyk, H. Synthesis 2005, 2687.
(16) (a) Xu, D.; Prasad, K.; Repic, O.; Blacklock, T. J. Tetrahedron
Lett. 1995, 36, 7357. (b) Koscova; S.; Budesinsky; M.; Hodacova; J. G.;
Czechoslovak Academy of Sciences, Institute of Organic Chemistry and
Biochemistry: Praha, Tcheque Republique, 2003; Vol. 68, p 7.
€
(17) Bernatowicz, M. S.; Daniels, S. B.; Koster, H. Tetrahedron Lett.
1989, 30, 4645–4648.
(18) Chan, W. C.; White, P. D. Fmoc solid phase peptide synthesis: a
practical approach; Oxford University Press: New York, 2000.
(19) Tam, J. P. Proc. Natl. Acad. Sci. USA. 1988, 85, 5409–5413.
(20) Li, Z.; Zhao, R.; Wu, X.; Sun, Y.; Yao, M.; Li, J.; Xu, Y.; Gu, J.
Faseb J 2005, 19, 1978–1985.
(15) (a) Hartmann, L.; Krause, E.; Antonietti, M.; Borner, H. G.
€
Biomacromolecules 2006, 7, 1239. (b) Hartmann, L.; Hafele, S.; Peschka-
€
€
Suss, R.; Antonietti, M.; Borner, H. G. Macromolecules 2007, 40, 7771.
(c) Hartmann, L.; Hafele, S.; Peschka-Suss, R.; Antonietti, M.; Borner,
H. G. Chemistry 2008, 14, 2025.
(21) Schaffert, D., Dohmen, C.Wagner, E. Polymers for delivery of
nucleic acids. European patent application 2010, EP10165502.
Org. Lett., Vol. 13, No. 7, 2011
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Table 1. Synthesized PAAs
no.
sequence
puritya
98
MALDI calcd
1561.02
MALDI found
11
12
13
14
15
16
H2N-Stp-Stp-Stp-Stp-Stp-Trp-CONH2
1561.9 [MH]
1583.2 [MHþNa]
1416.4 [MH]
H2N-Gtt-Gtt-Gtt-Gtt-Gtt-Trp-CONH2
97
97
94
91
97
1415.82
1631.15
1801.24
2443.21
2083.4
1438.4 [MHþNa]
1631.9 [MH]
H2N-Gtp-Gtp-Gtp-Gtp-Gtp-Trp-CONH2
1653.6 [MHþNa]
1802.2 [MH]
H2N-Ptp- Ptp- Ptp- Ptp-Ptp-Trp-CONH2
1823.9 [MHþNa]
2443.4 [MH]
(H2N-Lys-Gtt-Stp-Gtp-Lys) 2-Lys-Trp-CONH2
H2N-Stp-Stp-Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile-COOH
2465.3 [MHþNa]
2084.9 [MH]
a IEX-HPLC purity at 220 nm of crude peptide. Elution by linear gradient from 0 to 100% B (A: 20 mM NaCl in 10 mM HCl pH 2.2; B: 3 M NaCl in
10 mM HCl pH 2.2). Stp, Gtt, Gtp, and Ptp are the deprotected forms of compounds 7, 8, 9, and 10, respectively.
Fmoc-derivative by Fmoc-OSu proved to be the most
effective route to fully protected polyamino acids (7, 8,
9). This synthetic route allowed the isolation of the poly-
amino acids at very convenient yields above 40% of
purified polyamines substituted with aliphatic anhydrides.
Other strategies (complexation using 9-BBN, desym-
metrization using Fmoc-OSu or Fmoc-Cl) did not
further improve the yield of desymmetrization. Despite
modest yields, these syntheses were performed on
10-30 g scales. Use of phthalic anhydride resulted in
a lower product yield (10), most likely because of the
acid lability of the product.
was above 90% demonstrating the feasibility of the syn-
thetic protocols.
After successful homosequencesynthesisthe three build-
ing blocks containing aliphatic spacers (7, 8, 9) were used
to construct a mixed sequence with a branching point (15).
The two-arm MAP-like19 structure was synthesized by
incorporation of N,N0-bis(fmoc)lysine as a second amino
acid. The increased sterical demands enforced by the
branching point and the combination of the different
building blocks had no apparent influence on product
quality.
To evaluate the use of the polyamino acid building block
in a more complex protection group scheme as routinely
applied in standard solid-phase peptide synthesis, we chose
the moderately hydrophobic, EGFR targeting peptide
GE1120 and modified the sequence with two N-terminal
Stp units. The peptide precursor was synthesized in a
completely automatic fashion, checked for purity using
RP-HPLC (see Supporting Information), and modified
with two N-terminal Stp units. IEX-HPLC of 16 showed a
purity >95% similar to the result of the RP-HPLC run of
the unmodified peptide. These results demonstrate the
compatibility of the building block with automated pep-
tide synthesis and with the typically used protection
schemes in Fmoc SPPS.
Polyamidoamines (PAAs) 11-15 (Table 1) were synthe-
sized as carboxamides using Rink MBHA resin,17 pre-
loaded with tryptophane as the first amino acid for an
increased UV absorption of the products (Scheme 2). 16
was synthesized on a peptide synthesizer using a standard
Wang linker, with manual introduction of the Stp units. A
series of homopolymers (five repeating units) was synthe-
sized to evaluate the coupling efficiency and overall per-
formance of the different polyamino acid building blocks.
The PyBOP/HOBt coupling and deprotection protocols
used for the synthesis were described previously.18 Char-
acterization of the PAAs was done by high performance
1
cation-exchange chromatography, H NMR, and MAL-
In summary, a versatile and straightforward route to
oligoethylenimine-based Fmoc-polyamino acids was de-
veloped, allowing a multigram synthesis of orthogonally
protected, cationic building blocks for Fmoc-SPPS. Use of
these building blocks in solid-phase synthesis showed full
compatibility with standard fmoc SPPS protocols and the
excellent purity of the resulting poly(amidoamines). Linear
and branched sequences can be synthesized, optionally
fused with natural peptide sequences. The convenience of
the presented strategy has recently been proven in the
synthesis of a library of >300 defined polycationic carriers
for nucleic acid delivery21 including very efficient plasmid
DNA and siRNA carriers (Schaffert et al., manuscript in
preparation). The novel building blocks may also find use
in the synthesis of peptides with increased positive charge
density.
DI-TOF MS.
The 1H and 13C NMR spectra of all building blocks and
intermediates show different extents of line broadening of
most of their signals and an increased measurement tem-
perature (50 °C) leading to coalescence and sharpening of
signals.16b Building blocks containing aliphatic diacids as a
spacer (7, 8, 9) displayed excellent coupling efficiencies and
resulted in products (11, 12, 13) with a crude purity of
>95% (exemplary HPLC traces are shown in Figure 1)
after deprotection and cleavage from the resin. Building
block 10, containing the phthalic acid spacer, demon-
strated slightly positive Kaiser tests from time to time
during the synthesis of its homosequence. This was ob-
served independently from coupling time and used solvent.
Despite this problem the purity of the crude product (14)
1588
Org. Lett., Vol. 13, No. 7, 2011

Scheme 2. Solid-Phase Synthesis of PAAs 11-15 Using Tryptophane-Preloaded Rink-Amide MBHA Resin Support, Followed by
Deprotection and Cleavage from the Resin As Described in the Supporting Information
Figure 1. IEX-HPLC traces of crude (a) 11, (b) 15, and (c) 16 after cleavage from resin. The PAAs were eluted (2.5 mL/min) using a
Resource S 1 mL column at rt by a linear gradient from 0 to 100% B (A: 20 mM NaCl in 10 mM HCl; B: 3 M NaCl in 10 mM HCl).
€
Acknowledgment. We thank Olga Bruck and Miriam
Supporting Information Available. Experimental syn-
thetic procedures and characterization data (NMR
spectra, MS spectra, HPLC traces). This material is
available free of charge via the Internet at http://pubs.
acs.org.
Sindelar (LMU Munich) for skillful assistance. This work
was supported by the DFG excellencecluster Nanosystems
Initiative Munich (NIM), a grant by Roche Kulmbach,
and the Munich Biotech Cluster m4 T12.
Org. Lett., Vol. 13, No. 7, 2011
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