Solid-phase synthesis of γ-AApeptides using a submonomeric approach

Article abstract of DOI:10.1021/ol301406a

The solid-phase synthesis of γ-AApeptides using a novel submonomeric approach that utilizes an allyl protection is reported. The strategy successfully circumvents the necessity of preparing γ-AApeptide building blocks in order to prepare γ-AApeptide sequences. This method will maximize the potential of developing chemically diverse γ-AApeptide libraries and thereby facilitate the biological applications of γ-AApeptides in the future.

Full text of DOI:10.1021/ol301406a

ORGANIC
LETTERS
2012
Vol. 14, No. 13
3446–3449
Solid-Phase Synthesis of γ-AApeptides
Using a Submonomeric Approach
Haifan Wu,† Mohamad Nassir Amin,† Youhong Niu, Qiao Qiao, Nassier Harfouch,
Abdelfattah Nimer, and Jianfeng Cai*
Department of Chemistry, University of South Florida, Tampa, Florida 33620,
United States
jianfengcai@usf.edu
Received May 21, 2012
ABSTRACT
The solid-phase synthesis of γ-AApeptides using a novel submonomeric approach that utilizes an allyl protection is reported. The strategy successfully
circumvents the necessity of preparing γ-AApeptide building blocks in order to prepare γ-AApeptide sequences. This method will maximize the potential
of developing chemically diverse γ-AApeptide libraries and thereby facilitate the biological applications of γ-AApeptides in the future.
Unnatural peptidomimetics have been investigated for
more than a decade and are of increasing importance in
chemical biology and drug discovery.1,2 Besides being resis-
tant to protease degradation and straightforward derivatiza-
tion, many classes of peptidomimetics, such as β-peptides,3À5
peptoids,6 R/β-peptides,7,8 oligoureas,9,10 azapeptides,11À13
and oligocarbamates,14 have shown versatile biological ap-
plications by mimicking structures and functions of bioactive
peptides. One of the most important applications is to
generate short peptide-like oligomeric ligands that specifi-
cally target proteins of interest, so as to facilitate the discovery
of potential drug candidates14 or identification of protein-
binding molecules.15,16 Such research efforts, with the devel-
opment of proteomics, lead to an unprecedented need for
the rapid generation of a chemically diverse combina-
torial library.17 A very elegant and successful example is
the development of peptoid combinatorial libraries by
Kodadek’s group to identify short peptoid ligands that
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Hynan, L.; German, D.; Kodadek, T. Cell 2011, 144, 132–142.
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r
10.1021/ol301406a
Published on Web 06/25/2012
2012 American Chemical Society

bind to a range of proteins with excellent specificity and
affinity.15,16,18À22 Nonetheless, there are urgent needs to
develop novel combinatorial libraries with new scaffolds
and functional groups in order to discover new classes of
ligands with enhanced specificity, affinity, and other biologi-
cal properties. This is because, with the presence of new side
chains and 3D structures, ligands with distinct functional
properites are expected to be identified.23
We have recently developed a new class of peptidomi-
metics termed “γ-AApeptides”, as they comprise N-acy-
lated-N-aminoethyl amino acid building blocks (Figure 1),
and chiral side chains are linked to the γ-carbon in the
building blocks.24 The other half of the side chains are
introduced onto the γ-AApeptide scaffold through acyla-
tion of the center N in each building block using a wide
variety of commercially available carboxylic acids, which
endow γ-AApeptides with a limitless potential for the
generation of chemically diverse libraries. In contrast to
R-peptides, each γ-AApeptide unit is comparable to a
dipeptide, and γ-AApeptides and R-peptides of the same
lengths project the same number of side chains. As such,
there is a strong potential to identify γ-AApeptides that
can mimic the structures and functions of R-peptides.
γ-AApeptides are potential antibiotic agents to combat
drug resistance by mimicking the mechanism of action of
natural antimicrobial peptides.27À29 Thus, it is envisioned
that there is great potential to identify γ-AApeptide-based
ligands from a combinatorial library to bind to proteins of
interest with high specificity and affinity.
However, the previous approach of solid-phase synth-
esis of γ-AApeptides (Figure 2)24À29 is not suitable for the
development of combinatorial libraries. In this method, a
γ-AApeptide sequence is prepared by assembling γ-AA-
peptide building blocks on solid phase. Each building
block requires a three-step synthesis (reductive amination,
acylation, and deprotection) starting from the correspond-
ing Fmoc-amino aldehyde. For instance, to prepare a
random library of short γ-AApeptides containing three
building blocks (6 side chains, comparable to 6-mer
peptides), with the availability of 10 Fmoc-amino alde-
hydes (R = 10) and 10 carboxylic acids (R = 10), 100
different building blocks have to be generated, which is
almost impossible to achieve.
Figure 1. Representative structure of a native R-peptide and a
γ-AApeptide.
Figure 2. Previous method for the synthesis of γ-AApeptides.
Submonomer approach has been used by many groups
to synthesize different classes of oliogmeric peptido-
mimetics.30À33 To rapidly develop γ-AApeptide libraries,
so as to maximize their biological potential, herein we
report the development of a novel submonomeric ap-
proach for the solid-phase synthesis of short γ-AApeptides
by utilizing an allyl protection. This method circumvents
the necessity of γ-AApeptide building block preparation,
thereby it is expected to greatly facilitate the application of
γ-AApeptides in biomedical sciences in the future.
Indeed, similar to other classes of peptidomimetics,
γ-AApeptides have been shown to be highly resistant to
protease degradation.24 More importantly, they are able to
disrupt proteinÀprotein interactions24 and mimic the Tat
peptide by binding to HIV-1 RNA25 and facilitating
membrane translocation26 with comparable affinity and
efficiency. More recently, we have also demonstrated that
(21) Zuckermann, R. N.; Kodadek, T. Curr. Opin. Mol. Ther. 2009,
11, 299–307.
The new route for the solid-phase synthesis of γ-AApep-
tides using the submonomeric approach is shown in Figure 3.
The first two steps have been used in the microwave-assisted
(22) Astle, J. M.; Simpson, L. S.; Huang, Y.; Reddy, M. M.; Wilson,
R.; Connell, S.; Wilson, J.; Kodadek, T. Chem. Biol. 2010, 17, 38–45.
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B.; Cao, C.; Cai, J. Chem. Sci. 2012, 10.1039/C2SC20428B.
(30) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H.
J. Am. Chem. Soc. 1992, 114, 10646–10647.
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J. Org. Biomol. Chem. 2011, 9, 6832–6843.
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1995, 5, 1159–1162.
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Chem. 2011, 9, 6604–6609.
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Buzzeo, R.; Cao, C.; Cai, J. Mol. Pharm. 2012, 9, 1529–1534.
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Org. Lett., Vol. 14, No. 13, 2012
3447

preparation of peptoids17 and have proven to be highly
efficient. In brief, 1 is obtained through the microwave-
assisted coupling of bromoacetic acid with the amino group
on the Rink amide resin using DIC as the activation agent.17
With the assistance of a 1000 W commercial microwave, the
reaction is accomplished in 4 min (8 Â 30 s). Then excess allyl
amine is added as the nucleophilic agent to form a secondary
amine on the solid phase to give 2, which again is assisted by
microwave and finished in 4 min (8 Â 30 s). We reason the
introduction of the allyl protecting group is critical since it
completely avoids the constant overalkylation occurring in
the reductive amination of Fmoc-amino aldehyde with the
primary amino group on the solid phase.34,35 Although over
alkylation can be potentially alleviated by draining out excess
aldehyde remaining in the solution during the imine forma-
tion step, it does not solve the problem;34 on the contrary,
incomplete imine formation is seen when the draining meth-
od is used since the formation of the imine is not efficient.34
As such, successful preparation of sequences employing
repetitive reductive amination reactions on the solid phase
is rare due to such complexity of overalkylation and incom-
plete reaction.
4. Although this method has only been used to convert
allyl-protected secondary amines to primary amines,36 we
found that deprotection of tertiary amines 3 under the
same condition is also extremely efficient. Followed by
double coupling of carboxylic acids and deprotection of
Fmoc, the first building block 5 is accomplished, which
ends the first synthetic cycle on the solid phase. The desired
γ-AApeptides therefore can be generated by repeating the
synthetic cycles.
To demonstrate the efficiency of this approach, Fmoc-
Phe-CHO was first used for the reductive amination of 2a,
and CH3COOH was used to acylate 4a. Both 4a and 5a
were cleaved by 95% TFA/H2O and analyzed by HPLC.
As shown in Figure 4, every step in the synthetic cycle,
including reductive amination, allyl deprotection, and
acylation, is highly efficient, as crude 4a and 5a showed
more than 95% purity.
Figure 4. HPLC traces of crude 4a and 5a that were monitored at
215 nm.
To further prove the practical application of this ap-
proach in the future development of a γ-AApeptide com-
binatorial library, three random γ-AApeptide sequences
6a, 6b, and 6c (Figure 5a) were synthesized from a pool of
Fmoc-amino aldehydes34,37 and carboxylic acids (Figure 5b)
that contain a variety of charged and hydrophobic groups.
As shown in Figure 5a, 6a and 6b are γ-AApeptides contain-
ing three building blocks, which are comparable to 6-mer
peptides in length, whereas 6c is a γ-AApeptide having five
building blocks and thereby a 10-mer peptide mimic. We
believe these γ-AApeptide sequences are sufficiently long
enough to compose combinatorial libraries in the future for
the identification of potential drug candidates or protein-
binding ligands. If the satisfactory yield of these γ-AApep-
tides can be achieved, this submonomeric approach will
definitely be able to be used to generate γ-AApeptide
libraries much more rapidly than the current building block
strategy.
Figure 3. New route for the synthesis of γ-AApeptides by
submonomeric approach. DIC = Diisopropylcarbodiimide,
PMHS = polymethylhydrosiloxane, DhBtOH = 3-hydroxy-
4-oxo-3,4-dihydro-1,2,3-benzotriazine.
With the protection of the allyl group, 3 can be obtained
free of side reactions, as the reductive amination step can
be repeated in order to achieve quantitative conversion.
The allyl protecting group is selectively removed by using
PMHS-ZnCl2/Pd(PPh3)4 in THF for 4 h (twice) to provide
(34) Iera, J. A.; Jenkins, L. M. M.; Kajiyama, H.; Kopp, J. B.;
Appella, D. H. Bioorg. Med. Chem. Lett. 2010, 20, 6500–6503.
(35) Szardenings, A. K.; Burkoth, T. S.; Look, G. C.; Campbell,
D. A. J. Org. Chem. 1996, 61, 6720–6722.
(36) Chandrasekhar, S.; Reddy, C. R.; Rao, R. J. Tetrahedron 2001,
57, 3435–3438.
(37) Debaene, F.; Mejias, L.; Harris, J. L.; Winssinger, N. Tetrahe-
dron 2004, 60, 8677–8690.
3448
Org. Lett., Vol. 14, No. 13, 2012

using the previous building block strategy, which is much
more tedious and time-consuming. Thus, this new sub-
monomeric approach is a real breakthrough.
Figure 5. (a) Random γ-AApeptide sequences 6a, 6b, and 6c.
(b) Fmoc-amino aldehydes and carboxylic acids used to prepare
6a, 6b, and 6c.
Figure 6. HPLC profiles of γ-AApeptide 6a and 6c. (a) Top,
HPLC trace of crude 6a; bottom, HPLC trace of purified 6a.
(b) Top, HPLC trace of crude 6c; bottom, HPLC trace of purified 6c.
Table 1. Product Characteristics Based on the Monomeric
Approach
γ-AApeptide
6a
6b
6c
In summary, we have reported a novel submonomeric
method to prepare short γ-AApeptides. This strategy cir-
cumvents the needs to prepare γ-AApeptide building blocks
and therefore greatly facilitates the rapid preparation of
chemically diverse γ-AApeptide libraries. The application
of this approach will unprecedentedly enhance the biologi-
cal potential of γ-AApeptides. The preparation of the γ-
AApeptide combinatorial library for specific protein target-
ing is currently under investigation.
puritya
yieldb
68%
31%
60%
27%
56%
22%
a Calculated based on crude HPLC. b Calculated based on the
purified product after lyophilization.
Surprisingly, the submonomeric method led to the pro-
duction of these three γ-AApeptide sequences with good
yield and purity (Table 1, Figure 6, and Supporting
Information Figure S1). Although there are some impu-
rities seen in crude HPLC traces, which may come from the
long-time exposure of resin to air and to water moisture in
the solvents during solid-phase synthesis, the quality of
these crude γ-AApeptides is consistent and considerably
high. They can be easily purified (Figure 6) and provided
with the excellent overall yields. However, as seenin Figure
S2, to prepare same three γ-AApeptide sequences 6a, 6b,
and 6c, seven building blocks would have to be prepared
Acknowledgment. The work was supported by USF
Start-up fund.
Supporting Information Available. Experimental details,
characterization of compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
† These authors contributed equally to this work.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 13, 2012
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