γ-AApeptides: Design, synthesis and evaluation

Article abstract of DOI:10.1039/c0nj00943a

A new class of peptide mimics termed "γ-AApeptides" has been described. The design and synthesis of γ-AApeptides, and potential bioactivities towards p53/MDM2 interaction were demonstrated. γ-AApeptides were also found to be highly resistant to proteolysis. The development of sequence-specific γ-AApeptides may lead to a family of peptidomimetics with a new framework for drug discovery or peptide/protein mimicry.

Full text of DOI:10.1039/c0nj00943a

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LETTER
c-AApeptides: design, synthesis and evaluationw
Youhong Niu,a Yaogang Hu,a Xiaolong Li,b Jiandong Chenb and Jianfeng Cai*a
Received (in Montpellier, France) 30th November 2010, Accepted 12th January 2011
DOI: 10.1039/c0nj00943a
A new class of peptide mimics termed ‘‘c-AApeptides’’ has been
described. The design and synthesis of c-AApeptides, and potential
bioactivities towards p53/MDM2 interaction were demonstrated.
c-AApeptides were also found to be highly resistant to proteolysis.
The development of sequence-specific c-AApeptides may lead
to a family of peptidomimetics with a new framework for drug
discovery or peptide/protein mimicry.
Fig. 1 Structures of an a-peptide and the corresponding g-AApeptide.
Sequence-specific peptidomimetic oligomers with a variety of
frameworks, structures and functions are increasingly impor-
tant in chemical biology and biomedical sciences.1,2 In the last
decade there have been extensive efforts to develop biomimetic
oligomers, such as b-peptides,3–5 peptoids,6 a-aminoxy-peptides,7
a/b-peptides,8,9 azapeptides,10,11 and phenylene ethynylenes,12
etc. These peptidomimetics have many advantages over con-
ventional peptides including resistance to enzymatic hydrolysis
and improved bioavailability, due to the presence of their
unnatural backbones.13 The area of research has led to some
important functions and applications of peptidomimetics ranging
from drug discovery to protein surface mimicry.14,15 Nonetheless,
the development of peptidomimetics is far less straightforward
than expected, limited by the availability of backbones. A
variety of backbones are needed to generate different classes of
peptide mimics with discrete structures and functions.2 Novel
peptide mimics with new frameworks are crucial in order to
further understand protein folding and functional properties,
to generate a new chemically diverse library so as to identify
novel therapeutic agents, and to further facilitate the applica-
tion of peptidomimetics in chemical biology.2,9
chosen because of their superior resistance to proteolysis and their
potential for peptide mimicry.
The repeating unit of g-AApeptide is comparable to a
di-a-peptide residue. In such a unit, there are two side chains;
one is from an a-amino acid side chain, while the other comes
from a tertiary amide. In g-AApeptides, nitrogen atoms on the
backbone are either forming secondary or tertiary amide bonds.
g-AApeptide side chains on the g position of the backbone
are derived from a-amino acids. Compared with conventional
a-peptides, g-AApeptides are able to project the same number
of side functional groups on the backbone of same length.
Therefore, g-AApeptides were presumably able to mimic
peptide structures and reproduce their functions, and generate
a novel chemically diverse library for drug discovery. One
should be aware that although the primary structures of
g-AApeptides can in principal mimic those of a-peptides
(side chains of g-AApeptides can perfectly overlap with those
of a-peptides), direct inter-conversion of sequences between
g-AApeptides and a-peptides may not present same functions
or bioactivities. This is because g-AApeptide and peptide back-
bones are different despite some similarities, therefore their
hydrogen bonding properties and conformational flexibilities
are not identical (g-AApeptides tend to be more flexible). Such
distinctions will result in different conformational structures,
which lead to possibly different functional behavior.
In an effort to search for new class of peptide mimics to
facilitate drug discovery and protein surface mimicry, we herein
describe a new family of sequence-specific peptide mimics derived
from g-chiral PNAs.16–18 They are termed ‘‘g-AApeptides’’ be-
cause they are comprised of g-substituted-N-acylated-N-amino-
ethyl amino acids (Fig. 1). Such g-chiral PNA backbones were
Bioactivity is one of the most important functions expected
for peptidomimetics. As the proof of concept, initially we chose
p53/MDM2 interaction as the modeling system to investigate
if certain g-AApeptides can disrupt this protein–protein inter-
action using an ELISA assay. This protein–protein interaction
was chosen because it has been a testing basis for the design
strategy of sequence-specific peptidomimetics.19 Peptidomimetics
with different scaffolds such as b-peptides,20 peptoids,21,22 and
N-acylpolyamine23 have been reported to disrupt p53/MDM2
interactions. Hence, any activity shown for g-AApeptides to
inhibit this interaction would be important for evaluating their
a Department of Chemistry, University of South Florida,
4202 E. Fowler Ave, Tampa, FL 33620, USA.
E-mail: jianfengcai@usf.edu; Fax: +1 813-974-3203;
Tel: +1 813-974-9506
b Department of Molecular Oncology, H. Lee Moffitt Cancer Center
& Research Institute, 12902 Magnolia Drive, Tampa, FL 33612,
USA
w Electronic supplementary information (ESI) available: Additional
experimental procedures and physical data for new compounds. See
DOI: 10.1039/c0nj00943a
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Fig. 4 Synthesis of g-AApeptide building block
with most carboxylic acids, while the use of other activation
agents such as PyBOP, HBTU/HOBt, DIC/HOBt provided
the desired product 3 when 2 was coupled with a few acids, but
in most cases the yields were poor. Solid phase synthesis of
g-AApeptides by assembling g-AApeptide building blocks 4
was problem-free and highly efficient. These sequences were
prepared over 80% yield in crude and eluted by HPLC with
more than 95% purity (Fig. 5). Three g-AApeptides were
described here for demonstration. Due to the freedom of
using almost countless carboxylic acids, the derivatization of
g-AApeptides is virtually unlimited, which allows the genera-
tion of a g-AApeptide library relatively simply and with much
more diversity than regular peptides. Such features will greatly
expand their potential applications in drug discovery in the
future.
Fig. 2 g-AApeptide sequences designed for p53/MDM2 disruption.
capability to mimic peptides and to demonstrate their potential
application in chemical biology. Therefore, we designed several
g-AApeptide sequences (Fig. 2) bearing side functional groups
of phenylalanine, tryptophan, and leucine, which are critical for
disruption of p53/MDM2 interaction.19
We initially attempted to directly synthesize g-AApeptides
on a solid phase through a submonomer strategy by building
the sequence step by step (Fig. 3). Unfortunately, overalkyla-
tion was constantly observed during the reductive amination
step. A few coupling cycles yielded a mixture of unidentifiable
products on HPLC after cleavage from the solid phase.
We then carried out the synthesis using a ‘‘monomer building
block’’ strategy (Fig. 4), the same protocol adopted from the
standard solid phase synthesis of conventional peptides.
In this route, g-AApeptide building blocks were prepared
and used for solid phase synthesis. Briefly, Fmoc-amino
aldehydes18,24 reacted with glycine benzyl ester through reductive
amination; the products were then acylated with carboxylic
acids. Subsequent hydrogenation provided desired g-AApeptide
building blocks, which were assembled on a Knorr resin using
Fmoc chemistry to produce the g-AApeptides. Preparation of
3 under different conditions was investigated. Oxohydroxy-
benzotriazole/DIC was found to be efficient for the coupling
The above sequences were then investigated for their
capability to disrupt p53/MDM2 protein–protein interactions
by the ELISA assay (Table 1). A previously reported p53-derived
peptide25 is included for comparison.
ELISA results show that g-AA3, with an IC50 of 50 mM, is
an effective inhibitor of p53/MDM2 interaction. It is only a
few fold less potent than a-peptides, and comparable to some
peptoids or b-peptides reported previously.21,26 This result
successfully demonstrated the functions of g-AApeptide as
novel peptide mimics. The selectivity of g-AApeptides is also
excellent. Different selected sequences are giving different activities
for p53/MDM2 inhibition. g-AA1 has the least activity, while
g-AA3 is the best inhibitor among three sequences. This
suggests that g-AApeptides do not interact with protein
Fig. 3 Initial unsuccessful attempt to synthesize g-AApeptides.
Fig. 5 HPLC traces of g-AApeptides after purification.
c
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Table 1 ELISA results of g-AApeptides for the disruption of
p53/MDM2
by introducing new frameworks and structures. Obtaining
structural requirements for g-AApeptides to form predicable
structures using Circular Dichroism (CD), 1D/2D NMR,
and X-ray crystallography, and design of novel g-AApeptide
sequences as better inhibitors/effectors for p53/MDM2 and
other proteins/nucleic acids/membrane are currently ongoing.
g-AApeptides
IC50/mM
g-AA1
g-AA2
>400
120 Æ 15
50 Æ 8
8.725
g-AA3
p53-derived peptide (Ac-QETFSDLWKLLP)
Experimental
General experimental methods
All the reagents and solvents were used as received without
further purification. g-AApeptide sequences were prepared on a
Knorr resin in peptide synthesis vessels, analyzed and purified
on an analytical and a preparative HPLC, respectively, and then
lyophilized. Molecular weights of g-AApeptides were identified
on a MALDI-TOF mass spectrometer.
Typical synthesis of 2
Fig. 6 Energy minimized structure (MM2) of g-AApeptide 3 is
superimposed with the p53 17-29 helical domain (blue colored).
g-AApeptide 3 is shown as the wire frame presentation. Three critical
residues (Phe19, Trp23, and Leu 26) in p53 responsible for binding to
MDM2 are red colored.
A glycine benzyl ester hydrochloride in 15 ml methanol in a
100 ml round bottom flask was added to 1.1 equiv. of triethyl-
amine and stirred at 0 1C for 15 min. A stoichiometric amount
of a Fmoc protected amino acid aldehyde18,24 was added
and the solution mixture was stirred for another 30 min.
A catalytic amount of acetic acid was then added followed
by 2 equiv. of NaBH3CN. The solution was allowed to stir at
0 1C for 1 h and continued at room temperature overnight.
The solvent was evaporated and 100 ml ethyl acetate and
100 ml saturated sodium bicarbonate solution were added to
the residue. The organic layer was separated and washed with
100 ml brine, dried over anhydrous sodium sulfate, and removed
in vacuo. Flash chromatography using ethyl acetate/hexane 1 : 1
gave 2 (Rf = 0.2) as a colorless oil.
randomly. Computer modeling (Fig. 6) indeed shows that the
side groups of an energy-minimized structure of g-AA3 are
able to overlap with Phe, Trp, and Leu of the p53 helical
domain that interacts with MDM2. Although a detailed
structure–activity relationship (SAR) will require the test of
more derivatives, and it is beyond the scope of this report,
much more potent g-AApeptide inhibitors are expected with
the introduction of halogen atoms onto the indole group of the
Trp residue21,27 and/or bulky and charged groups,19 and with
the assistance of in silico computer modeling and screening.
Another important feature of sequence-specific peptidomimetics
is their superior resistance to enzymatic hydrolysis, owing to
their artificial backbones, which is a key advantage over
regular peptides. To determine the stability of g-AApeptides
against enzyme degradation, the typical sequence 3 was incubated,
respectively, with chymotrypsin, thermolysin, and pronase
(0.1 mg mlÀ1) in a 100 mM pH 7.8 ammonium bicarbonate
buffer for 24 h. HPLC was used to analyze the reaction
mixtures by comparing their retention time, integration and
molecular weight to those of the starting material. The results
(see ESIw) show that AApeptides are potentially highly resis-
tant to proteolysis. None of the above proteinases is able to
hydrolyze 3 after 24 h incubation.
Typical synthesis of 3
Compound 2, 1.2 equiv. of DIC, oxohydroxybenzotriazole,
and R2COOH were stirred in 20 ml DMF overnight. The
solution was then partitioned in 100 ml ethyl acetate and 100 ml
water. The organic layer was separated and washed with water
(3 Â 100 ml) and brine (2 Â 100 ml), dried over anhydrous
sodium sulfate, and then concentrated in vacuo. Flash chromato-
graphy using ethyl acetate/hexane 1 : 3 gave 3 (Rf = 0.1) as a
colorless oil.
Typical synthesis of 4
3 in 20 ml ethyl acetate was added to 10% Pd/C and hydro-
genated at atmospheric pressure and room temperature over-
night. The solution was evaporated and the residue was
purified by flash chromatography, 5–7% MeOH/CH2Cl2, to
give 4 (Rf = 0.2 in 7% MeOH/CH2Cl2) as a white foam solid.
In conclusion, we have designed a new family of peptide
mimics—g-AApeptides—and demonstrated their efficient
synthesis on the solid phase using a monomer approach based
on g-substituted N-acylated-N-Fmocamino ethyl amino acid
building blocks. Diversifying g-AApeptides by introducing a
wide variety of side chains onto g-AApeptide backbones is
straightforward and easy considering the availability of numerous
carboxylic acids. Furthermore, g-AApeptides are found to be
highly resistant to enzymatic degradation. Further charac-
terization and development of sequence-specific g-AApeptides
will enrich the current peptidomimetic family and expand their
potential applications in drug discovery and chemical biology
Synthesis of c-AApeptides
g-AApeptides were prepared on the Knorr resin in peptide
synthesis vessels on a shaker following the standard Fmoc
chemistry of the solid phase peptide synthesis protocol.
Each coupling cycle included an Fmoc deprotection using
20% piperidine in DMF, and 4 h coupling of 1.5 equiv. of
g-AApeptide building blocks onto the resin in the presence
of
2 equiv. of DIC/oxohydroxybenzotriazole in DMF.
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After desired sequences were assembled, they were transferred
into a 4 ml vial and cleaved from the solid support in 74 : 24 : 2
TFA/CH2Cl2/triisopropylsilane for 2 h. Then the solvent was
evaporated and the residues were purified by HPLC. The
desired fractions were collected and lyophilized.
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GST-MDM2-1-150 and full-length His6-p53 were expressed in
E. coli and affinity purified under non-denaturing conditions.
ELISA plates were incubated with 2.5 mg mlÀ1 His6-p53 in
phosphate buffered saline (PBS) for 16 h. After washing with
PBS + 0.1% Tween 20 (PBST), the plates were blocked
with PBS + 5% non-fat dry milk + 0.1% Tween 20 (PBSMT)
for 0.5 h. GST-HDM2 was mixed with g-AApeptides in
PBSMT + 10% glycerol + 10 mM DTT and added to the
wells. The plates were washed with PBST after incubating for
1 h at room temperature, incubated with MDM2 antibody
4B2 in PBSMT for 1 h, followed by washing and incubation
with HRP–rabbit-anti-mouse Ig antibody for 1 h. The
plates were developed by incubation with a TMB peroxidase
substrate (KPL) and measured by absorbance at 450 nm.
Acknowledgements
This work was supported by the start-up fund from University
of South Florida.
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