Rational Design of Right-Handed Heterogeneous Peptidomimetics as Inhibitors of Protein-Protein Interactions

Article abstract of DOI:10.1021/acs.jmedchem.0c01638

Peptidomimetics have gained great attention for their function as protein-protein interaction (PPI) inhibitors. Herein, we report the design and investigation of a series of right-handed helical heterogeneous 1:1 α/Sulfono-I-AA peptides as unprecedented inhibitors for p53-MDM2 and p53-MDMX. The most potent helical heterogeneous 1:1 α/Sulfono-I-AA peptides were shown to bind tightly to MDM2 and MDMX, with Kd of 19.3 and 66.8 nM, respectively. Circular dichroism spectra, 2D-NMR spectroscopy, and the computational simulations suggested that these helical sulfono-I-AA peptides could mimic the critical side chains of p53 and disrupt p53/MDM2 PPI effectively. It was noted that these 1:1 α/Sulfono-I-AA peptides were completely resistant to proteolytic degradation, boosting their potential for biomedical applications. Furthermore, effective cellular activity is achieved by the stapled 1:1 α/Sulfono-I-AA peptides, evidenced by significantly enhanced p53 transcriptional activity and much more induced level of MDM2 and p21. The 1:1 α/Sulfono-I-AA peptides could be an alternative strategy to antagonize a myriad of PPIs.

Full text of DOI:10.1021/acs.jmedchem.0c01638

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Article
Rational Design of Right-Handed Heterogeneous Peptidomimetics
as Inhibitors of Protein−Protein Interactions
Yan Shi,# Peng Sang,# Junhao Lu,# Pirada Higbee,# Lihong Chen, Leixiang Yang, Timothy Odom,
Gary Daughdrill,* Jiandong Chen,* and Jianfeng Cai*
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ABSTRACT: Peptidomimetics have gained great attention for their
function as protein−protein interaction (PPI) inhibitors. Herein, we
report the design and investigation of a series of right-handed helical
heterogeneous 1:1 α/Sulfono-γ-AA peptides as unprecedented inhib-
itors for p53-MDM2 and p53-MDMX. The most potent helical
heterogeneous 1:1 α/Sulfono-γ-AA peptides were shown to bind tightly
to MDM2 and MDMX, with Kd of 19.3 and 66.8 nM, respectively.
Circular dichroism spectra, 2D-NMR spectroscopy, and the computa-
tional simulations suggested that these helical sulfono-γ-AA peptides
could mimic the critical side chains of p53 and disrupt p53/MDM2 PPI
effectively. It was noted that these 1:1 α/Sulfono-γ-AA peptides were
completely resistant to proteolytic degradation, boosting their potential for biomedical applications. Furthermore, effective cellular
activity is achieved by the stapled 1:1 α/Sulfono-γ-AA peptides, evidenced by significantly enhanced p53 transcriptional activity and
much more induced level of MDM2 and p21. The 1:1 α/Sulfono-γ-AA peptides could be an alternative strategy to antagonize a
myriad of PPIs.
an urgent task to exploit new peptidomimetic foldamers
bearing new molecular scaffolds and frameworks due to current
limited availability and application of peptidomimetics.
INTRODUCTION
■
In the endeavor of searching and designing molecules that bind
to important biological targets such as proteins with high
affinity and specificity, peptides predominately serve as the
leads.1,2 This is particularly true for the identification of
molecules that modulate protein−protein interactions (PPIs)
in which large, flat, and noncontiguous interfaces are involved.
However, natural peptides are deemed not suitable for the
therapeutic development due to their intrinsic drawbacks such
as susceptibility to proteolytic degradation.3 To alleviate the
problem, non-natural sequence-specific peptidomimetics have
emerged to be a viable alternative strategy.4−14 Compared to
their natural counterparts, the peptidomimetics could not only
retain or mimic the folding domain of the peptides such as
helices but also display distinct structures and functions.
Additionally, the chemodiversity is enhanced due to the ease of
introducing diverse and unnatural functional side chains.3,15 Of
course, thanks to their unnatural backbones, they are generally
resistant to enzymatic hydrolysis, which enhances bioavail-
ability.16 Furthermore, peptidomimetic foldamers could mimic
the primary, secondary, and even tertiary structures of peptides
and proteins, and their potential application in biomedical
science is enhanced through the structure-based design.17,18 In
the past two decades, significant efforts have been made to
develop new classes of peptidomimetics and successfully
employ them into the study of biomolecular recognition and
protein−protein interactions (PPIs).19−23 However, it remains
To advance the field of peptidomimetics, we have recently
developed a new class of foldamer, γ-AA peptides (oligomers
of N-acylated-N-aminoethyl amino acids), which were created
based on the molecular scaffold γ-chiral PNA but with an
enlarged chemodiversity to mimic peptides rather than nucleic
acids.3,17 γ-AA peptides are highly resistant to enzymatic
hydrolysis and have shown excellent cellular translocation
capability.15,16,25−27 The modular synthesis of γ-AA peptides
and their enormous chemodiversity make them excellent
candidates for identification of valuable molecular probes or
drug candidates through combinatorial library screening.15,28,29
More importantly, as a subclass of γ-AA peptides, sulfono-γ-AA
peptides have been found to adopt a series of unprecedented
helical secondary structures, stabilized by both intramolecular
hydrogen bonding and the curved nature of sulfonamido
moieties in the molecular framework,24,30−32 endowing them
with superior folding propensity to the α-helix in solution. As
Received: September 18, 2020
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Figure 1. (A) Chemical structure of 1:1 α/Sulfono-γ-AA peptides. a and b denote the chiral side chain and the sulfonamido side chain from the
building block, respectively. (B) The crystal structure of a 1:1 α/Sulfono-γ-AA peptide.24 (C) Top view of panel (D,E). The schematic
representation of the distribution of side chains from 1:1 α/Sulfono-γ-AA peptides. (D) Side view. (E) Top view.
Figure 2. (A) Superimposition of the p53 α-helix (purple) with a designed 1:1 α/Sulfono-γ-AA peptide (blue) (a PDB file is shown in the
Supporting Information). (B) The structure overlay of p53 (purple) binding and MDM2 (green) (PDB: 1YCR). (C) Overlay of the p53 helix
(purple) with a 1:1 α/Sulfono-γ-AA peptide (blue) on the binding of MDM2 (green) (a PDB file is shown in the Supporting Information).
such, sulfono-γ-AA peptides could be ideal candidates that
could be rationally designed to mimic helical domains of
proteins and disrupt medicinally related PPIs. Indeed, the
homogeneous sulfono-γ-AA peptides, which form the left-
handed 414 helical structure,31 have been shown to mimic the
critical residues of the helical domain of BCL9 and disrupt the
β-catenin/B cell lymphoma 9 protein−protein interaction
(PPI)16 and to mimic GLP-1 to recognize GLP1-R and
decrease blood glucose.33 Recently, we have also demonstrated
that they could be designed to mimic those important residues
of the p53 helix and antagonize p53/MDM2 PPI.27 However,
even if the results are highly promising, the mimicry of a right-
handed α-helix by a left-handed helix may not be ideal,
particularly when residues on the multiface of the helix are
involved in interactions at the protein−protein interface.
Previous structural studies show that 1:1 heterogeneous α/
Sulfono-γ-AA peptides adopt a robust right-handed 413
windmill-shaped helices with a helical pitch of 5.34 Å (Figure
B
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Scheme 1. Structures of the p53 and 1:1 α/Sulfono-γ-AA peptide 1−11
1A−E),30,34 implying their close similarity to the α-helix
(pitch: 5.34 Å). Inspired by this desired structural information,
we speculated that 1:1 heterogeneous α/Sulfono-γ-AA
peptides could be a new class of helix mimetics as potential
PPI inhibitors. In this paper, as a proof of concept, for the first
time, we explored the heterogeneous 1:1 α/Sulfono-γ-AA
peptides for the mimicry of the α-peptide helix. It is known
that p53 is a tumor suppressor that plays a prominent role in
the oncogenic transformation process and protects higher
organisms from cancer. MDM2 and MDMX share consid-
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erable structural homology, binding to the N-terminus of p53
and leading to its transcriptional activity inhibition and
degradation.35 Recent findings suggest that overexpression of
MDM2 and MDMX is a key factor that results in multiple
human cancers, making them the promising target for
antitumor drug development.22,36−40 Hence, p53−MDM2
and p53−MDMX protein−protein interactions were selected
as the model applications to evaluate the feasibility and
practicality of our new developed foldamer.
the MDM2 binding pocket. As shown in Figure 1D, since the
1:1 α/Sulfono-γ-AA peptide helical scaffold has four faces, we
postulated that residues on the other faces, such as 2b-5-8a and
3-6a-8b, could also be used for the mimicry of three hot-spot
side chains F19, W23, and L26 in p53. As such, we synthesized
compound 10 (Scheme 1) in which the side chains on the
positions of 2b, 5, and 8a were designed to mimic F19, W23,
and L26 in p53. To our disappointment, the binding affinity
decreased sharply compared to that of 1 (Scheme 1, Kd = 653
nM). Also, if the side chains at the positions of 3, 6a, and 8b
are used to mimic those three crucial residues in p53, although
the activity is slightly improved (compound 11, Scheme 1, Kd
= 344 nm) and comparable to that of the p53 peptide, it is still
much less potent than compound 1. These findings suggest
that the precise arrangement of side chains on each helical face
of 1:1 α/Sulfono-γ-AA peptides is not identical. In the
meantime, the binding affinity is also highly affected by the
neighboring side chains that are not directly involved in the
binding to the target protein. The IC50 values of these
compounds are shown in Figure S4.
As MDMX is also involved in p53 binding and affecting the
p53 signaling pathway, the binding affinities of these helical
mimetics to MDMX are obtained. As shown in Scheme 1 and
Figure S3, most of the 1:1 α/Sulfono-γ-AA peptides have
shown good Kd values to MDMX compared to p53,
demonstrating that these peptides are potential dual-inhibitors
to both MDM2 and MDMX.
CD (Circular Dichroism) Spectra. In order to evaluate the
helical propensity of these peptides in solution, the circular
dichroism (CD) spectra are next recorded in PBS (100 μM) in
the range of 195−260 nm. As shown in Figure 3, most of our
RESULTS AND DISCUSSION
■
Peptide Design and Activity Test. p53/MDM2 PPI has
been a testing base for validation of helical peptidomimetics for
the mimicry of the α-helix. As shown in Figure 2, three crucial
residues of p53, Phe19, Trp23, and Leu26, on one face of the
p53 helix, make multiple hydrophobic contacts at the deep
pocket of the MDM2 cleft. It is demonstrated that molecules
that can reproduce the binding modes of these three residues
are expected to bind MDM2 tightly.41−45 As shown in Figure
1D, the pitch of 1:1 right-handed α/Sulfono-γ-AA peptides is
5.34 Å, essentially the same as that for the α-helix. Due to the
characteristic of four side chains per turn (Figure 1E), we
could select any face of the helix to mimic p53 (Figure 2).
As an initial attempt, we randomly selected 2a, 4b, and 7 of
1:1 α/sulfono-γ-AA peptides to mimic F19, W23, and L26 in
p53. 2a comes from the chiral side chain of the sulfono-γ-AA
building block (Scheme 1, compounds 1−3) to mimic F19, 7
is the leucine amino acid residue to mimic L26, and 4b
possesses sulfonyl side chains such as sulfonyl-2-naphthalene
to mimic the W23 (Scheme 1, compounds 1−3). To our
delight, the first peptide 1, which contains the sulfonyl-2-
naphthalene group, shows a Kd of 19.3 nM, revealing an almost
18-fold higher binding affinity toward MDM2 than the p53
(16−29) peptide (Kd, 360 nM, Scheme 1; Figure S2). It
strongly suggested that the approach of 1:1 α/Sulfono-γ-AA
peptides for the mimicry of the α-helix is viable. We next
probed the effect of the side chains on positions 2a and 7.
When the Trp group is used to mimic Phe on the 2a position
(compound 2, Scheme 1), Kd = 82.7 nM, suggesting that
although both indole and phenyl groups are hydrophobic, a
phenyl group (compound 1) is more preferable to mimic F19
in p53. Interestingly, replacing the Leu 7 in compound 1 with
the cyclobutylmethyl group residue (Scheme 1, compound 3),
the activity is still slightly weaker than that of 1 (Scheme 1, Kd
= 72.6 nM), implying that Leu is the optimized residue.
Subsequently, we explored the importance of the side chains at
the position 4b. It is noted that compound 4 bearing the
sulfonyl-1-naphthalene side chain induces a dramatic drop in
the binding affinity (Scheme 1, Kd = 482 nM), possibly due to
the short group that could not make hydrophobic contact with
the p53 binding domain of MDM2. The hypothesis is
supported by the result that when changing this position to
sulfonyl-TPE groups, the binding affinity of compound 5
becomes 6-fold potent as p53 (Scheme 1, Kd = 62 nM).
Instead of the naphthalene moiety, the effect of the phenyl
group, as well as the substituent effect on its aromatic ring, was
also investigated at the 4b position. Interestingly, although
their binding affinities are all less than that of 1, they bind
more tightly to MDM2 than the p53 peptide.
Figure 3. CD spectra of the p53 and compounds 1−11.
compounds revealed a pronounced maximum at 198−202 nm
and a minimum at 212 nm, suggesting that these peptides (1−
11) adopt similar right-handed helical conformations.34
Interestingly, a minimum of less than 200 nm was observed
for p53 (16−29), signifying a random coil with almost no α-
helix population. The data may explain why 1:1 α/Sulfono-γ-
AA peptides are better MDM2 binders than p53.
HSQC NMR of Lead Peptide 9 in Complex with
MDM2. As both 1 and 9 have comparable binding activities
whereas 9 has slightly better solubility than 1, we chose 9 for
NMR study to further elucidate its binding to MDM2. Nuclear
magnetic resonance (NMR) spectroscopy was used to
determine if the compound 9 binding site on MDM217−125
was similar to the binding site for the p53 transactivation
It is intriguing that the binding affinity reveals the
relationship of 4-CF3 > 4-Cl > 4-Br > 4-OCH3 (compounds
9, 7, 8, and 6; Scheme 1). We speculated that the stronger
electron-withdrawing group may facilitate the interaction with
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1
Figure 4. (A) Overlay of H−15N HSQC spectra for before (blue) and after (red) the addition of increasing concentrations of 9. (B) Average
chemical shift changes in ppm (parts per million) of the MDM2 p53 binding domain (residues 17−125) when bound to 9. The 2° structure of
MDM217−125 is shown below the graph. (C) Ribbon structure of MDM217−125 (orange) and p53TAD (green, residues 15−29) (PDB: 1YCR).
Residues colored red have chemical shifts above the average of 0.048 ppm when bound to 9. (D) Ribbon structure of MDM217−125 (orange) and
p53TAD15−29 (green) (PDB: 1YCR). Residues colored red have chemical shifts above the average of 0.048 ppm when bound to p53TAD1−73
.
domain (TAD). Figure 4A demonstrates the overlay of
1H−15N HSQC spectra before (blue) and after (red) the
addition of increasing concentrations of 9. We measured the
amide proton and nitrogen chemical shift changes of a
uniformly 15N-labeled sample of MDM217−125 after a
stoichiometric excess of 9 was added. Figure 4B shows the
average chemical shift changes in ppm for the amide proton
and nitrogen resonances in MDM217−125. The average
chemical shift change for all the detectable MDM217−125
resonances when bound to 9 was 0.048 ppm. Figure 4C,D
shows the structure of MDM217−125 in orange bound to
p53TAD15−29 in green.46 Figure 4C shows MDM217−125
residues with chemical shift changes greater than 0.048 ppm
when bound to 9 colored red; 33 residues had chemical shift
changes above the average. Figure 4D shows MDM217−125
residues with chemical shift changes greater than 0.048 ppm
when bound to p53TAD1−73 colored red; 52 residues had
chemical shift changes above the average.
luciferase activities (1.5−2-fold) were observed in the cells
treated with stapled sequences. U2OS cells expressing
endogenous wild-type p53 under negative regulation by both
MDM2 and MDMX were treated with the stapled peptides.
Treatment with all four stapled compounds led to increased
p53 levels to various degrees. Modest induction of p53 target
genes p21 and MDM2 were also detected, suggesting increased
activities of p53 due to these compounds (Figure 5D). The
modest effect of the compounds in activating p53 in cell
culture suggests that further optimization is needed to improve
cell permeability and fully exploit their MDM2 binding ability.
Overall, the results demonstrated the inhibitory function of
these compounds for MDM2 in vitro, as well as their potential
to be further developed into cell-permeable activators of p53
for cancer treatment.
Enzymatic Stability Study. One distinctive characteristic
of peptidomimetics is the remarkable resistance to enzymatic
degradation, which has been proven in our previous study.16
Herein, the stability of the 1:1 α/Sulfono-γ-AA peptide was
evaluated by using p53 (16−29) as the control peptide. 0.1
mg/mL of compound 1 and p53 were incubated with 0.1 mg/
mL pronase in 100 mM ammonium bicarbonate buffer (pH
7.8) at 37 °C for 24 h. After being monitored by HPLC-MS
(Figures S6−S7), the peptide p53 was found to be completely
degraded by pronase as multiple unidentified peaks with no
intact peptide remaining were detected. Meanwhile, the
peptide 1 showed negligible degradation, indicating that
these 1:1 α/Sulfono-γ-AA peptides were of great stability
against proteolysis.
MDM217−125 β1 and β2 had shifts in the presence of 9 and
p53TAD1−73, as well as β3’. α1, β1’, β2’, and α2’ had more
residues with shifts above the 0.048 ppm threshold in the
presence of p53TAD1−73 than 9 and β3 had shifts only in the
presence of 9 while α1’ had shifts only in the presence of
p53TAD1−73. α2 had shifts in the presence of both 9 and
p53TAD1−73. For 9, most of the large chemical shift changes
were localized to the N-terminal half of α2. For p53TAD1−73
,
most of the large chemical shift changes were localized to the
C-terminal half of α2. Based on these results, it appears that the
binding site for 9 is similar to p53TAD1−73 but not identical.
We are currently using this data to optimize peptide design.
Stapled 1:1 α/Sulfono-γ-AA Peptide Induces Activa-
tion of p53 in Cells. To investigate the abilities of the
compounds in activating p53 in cells, stapled 1:1 lactam-
bridged α/Sulfono-γ-AA peptides 12−15 were synthesized via
a side chain cross-linking strategy based on the most potent
sequences 1 and 2 (Figure 5A−C) in the hope of enhancing
cell permeability.47 The IC50s of compounds 12−15 are 11.7,
9.4, 4.9, and 9.0 μM, respectively (Figure S4), which was better
than those of corresponding linear compounds 1 and 2. The
luciferase reporter assay was employed to check the efficiency
of wild-type p53 in activating the BP-100 MDM2 promoter,
which constructed into the promoting sequence of luciferase
(Figure S9). Unlike the linear compounds 1 and 2, enhanced
CONCLUSIONS
■
In this paper, we have designed and synthesized a series of
unprecedented right-handed helical 1:1 α/Sulfono-γ-AA
peptide inhibitors based on the single crystal structure. The
feasibility and practicality of the new helical peptidomimetic
scaffolds were evaluated by mimicking the p53 helix for
targeting MDM2 and MDMX. The activities of these peptides
were evaluated by Kd. Besides, through the 2D-NMR
experiment, the lead compound 9 was proven to interact
with the p53-binding pocket of MDM2. The induction of p53
accumulation and its transcriptional targets p21 and MDM2
were also detected among the stapled 1:1 α/Sulfono-γ-AA
peptide, suggesting that these peptides have the potential to be
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Figure 5. (A) Design and synthesis of the stapled sequences (a PDB file is shown in the Supporting Information). (B) The structures of the stapled
1:1 α/Sulfono-γ-AA peptides 12−15. (C) Overlay of the stapled 1:1 α/Sulfono-γ-AA peptide (blue) on the binding of MDM2 (a PDB file is shown
in the Supporting Information). (D) Activation of cellular p53 by compounds 1 and 2 and stapled p53 helical mimetics. U2OS osteosarcoma cells
expressing endogenous wild-type p53 were treated with indicated compounds at 30 μM for 16 h. The cells were analyzed by Western blot for the
level of p53 pathway markers.
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6.75 (d, J = 8 Hz, 2H), 6.62 (d, J = 8 Hz, 1H), 4.14−4.18 (m, 1H),
4.08−4.11 (m, 2H), 4.01−4.05 (m, 2H), 3.85−3.92 (m, 2H), 3.28−
3.32 (m, 1H), 3.03−3.09 (m, 1H), 2.67−2.75 (m, 1H), 1.14 (s, 9H).
13C NMR (100 MHz, DMSO-d6): δ 170.6, 155.9, 153.6, 148.1, 144.2,
143.0, 142.8, 141.1, 139.4, 137.4, 131.7, 131.1, 130.9, 130.1, 128.4,
128.3, 128.0, 127.4, 127.3, 126.9, 125.6, 123.8, 120.5, 77.9, 65.8, 51.7,
49.5, 47.1, 37.5, 31.7, 28.9 HRMS (ESI) ([M + H]+) Calcd. for
C56H53N2O7S: 897.3573, found: 897.3582.
further developed into in vivo p53 activators. Furthermore,
these peptidomimetics have shown great resistance toward the
enzymatic degradation, which also make them to be promising
therapeutic agents. This work provided a streamlined approach
to discover potent peptidomimetic inhibitors of a myriad of
protein−protein interactions.
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-phenyl-
propyl)-N-((4-chlorophenyl)sulfonyl)glycine (BB4). 1H NMR (400
MHz, DMSO-d6): δ 12.82 (s, 1H), 7.83 (d, J = 8 Hz, 2H), 7.73 (d, J =
8 Hz, 2H), 7.47 (t, J = 4 Hz, 4H), 7.33−7.38 (m, 2H), 7.19−7.24 (m,
7H), 7.13 (d, J = 4 Hz, 1H), 4.13 (s, 1H), 4.07−4.09 (m, 2H), 3.97−
4.02 (m, 1H), 3.84 (s, 1H), 3.35 (q, J = 12 Hz, 2H), 3.17 (q, J = 12
Hz, 1H), 2.83−2.87 (m, 1H), 2.55−2.61 (m, 1H). 13C NMR (100
MHz, DMSO-d6): δ 170.5, 155.9, 144.2, 141.1, 139.0, 138.8, 129.7,
129.5, 129.3, 128.5, 128.0, 127.4, 126.5, 125.6, 120.5, 65.7, 52.2, 51.7,
49.3, 47.1, 37.9 HRMS (ESI) ([M + H]+) Calcd. for C32H30ClN2O6S:
605.1513, found: 605.1520.
EXPERIMENTAL SECTION
■
General Information. Fmoc-protected amino acids were
purchased from Chem-impex (Wood Dale, IL). Rink-Amide-MBHA
resin (0.64 mmol/g) was purchased from GL Biochem (Shanghai,
China). 1-Hydroxybenzotriazole wetted with no less than 20 wt %
water (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC), and N,N-diisopropylethylamine (DIPEA) were purchased
from Oakwood Chemical (Estill, SC). Tetraphenylethylene was
purchased from Alfa Aesar. Chlorosulfonic acid was purchased from
Sigma Aldrich. Solid phase synthesis was conducted in peptide
synthesis vessels on a Burrell Wrist-Action shaker. γ-AA peptides were
analyzed and purified on a Waters Breeze 2 HPLC system installed
with both the analytic module (1 mL/min) and preparative module
(16 mL/min), by employing a method using a 5−100% linear
gradient of solvent B (0.1% TFA in acetonitrile) in solvent A (0.1%
TFA in water) over 40 min followed by 100% solvent B over 10 min.
Then the pure peak was collected and lyophilized on a Labcono
lyophilizer. The purity of the compounds was determined to be >95%
by analytical HPLC. High-resolution mass spectra were obtained on
an Agilent 6220 using electrospray ionization time-of-flight (ESI-
TOF). 1H NMR spectra were recorded at 400 MHz using TMS as the
internal standard. 13C NMR spectra were recorded at 100 MHz using
TMS as the internal standard. The multiplicities are reported as
follows: singlet (s), doublet (d), doublet of doublets (dd), triplet (t),
quartet (q), multiplet (m). Coupling constants are reported in hertz
(Hz).
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-phenyl-
1
propyl)-N-((4-bromophenyl)sulfonyl)glycine (BB5). H NMR (400
MHz, DMSO-d6): δ 12.83 (s, 1H), 7.83 (d, J = 8 Hz, 2H), 7.72 (d, J =
8 Hz, 2H), 7.65 (d, J = 4 Hz, 2H), 7.54−7.56 (m, 2H), 7.36 (q, J = 12
Hz, 2H), 7.19−7.29 (m, 8H), 7.13 (d, J = 4 Hz, 1H), 4.13(s, 1H),
4.07−4.08 (m, 2H), 3.97−4.01 (m, 1H), 3.84 (s, 1H), 3.34 (q, J = 12
Hz, 1H), 3.18 (q, J = 12 Hz, 1H), 2.84 (q, J = 12 Hz, 1H), 2.55−2.61
(m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 170.5, 155.9, 144.2,
141.1, 139.2, 139.0, 129.5, 129.4, 128.5, 128.0, 127.4, 126.5, 125.6,
120.5, 65.7, 52.2, 51.7, 49.3, 47.1, 37.9. HRMS (ESI) ([M + H]+)
Calcd. for C32H30BrN2O6S: 649.1008, found: 649.1001.
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-phenyl-
propyl)-N-((4-(trifluoromethyl)phenyl)sulfonyl)glycine (BB6). 1H
NMR (400 MHz, DMSO-d6): δ 12.86 (s, 1H), 7.95 (d, J = 8 Hz,
2H), 7.88 (d, J = 8 Hz, 2H), 7.83 (d, J = 8 Hz, 2H), 7.55 (d, J = 8 Hz,
2H), 7.36 (q, J = 12 Hz, 2H), 7.19−7.29 (m, 7H), 7.13 (d, J = 4 Hz,
1H), 4.13(s, 1H), 4.07−4.08 (m, 2H), 3.96−4.01 (m, 1H), 3.86 (s,
1H), 3.39 (q, J = 12 Hz, 2H), 3.23 (q, J = 12 Hz, 1H), 2.85 (q, J = 12
Hz, 1H), 2.57−2.62 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ
170.4, 155.9, 144.2, 144.1, 143.8, 141.1, 138.9, 129.5, 128.5, 128.4,
127.9, 127.4, 126.7, 126.5, 125.6, 125.3, 122.7, 120.5, 65.7, 52.2, 51.7,
49.3, 47.1, 37.9. HRMS (ESI) ([M + H]+) Calcd. for
C33H30F3N2O6S: 639.1777, found: 639.1780.
Preparation of Sulfono-γ-AA Building Blocks. The sulfono-γ-
AA building blocks were synthesized based on a previous report.16 As
shown in Scheme S1, the initial starting materials were Fmoc-
protected amino acids. Sulfono-γ-AA building blocks 4−10 and 15
were synthesized based on route 1, while sulfono-γ-AA building
blocks 1−3 and 11−14 were synthesized based on route 2.
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-
(tert-butoxy)phenyl)propyl)-n-(naphthalen-2-ylsulfonyl)glycine
1
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-phenyl-
propyl)-N-(methylsulfonyl)glycine (BB7). 1H NMR (400 MHz,
DMSO-d6): δ 7.80 (d, J = 8 Hz, 2H), 7.53 (d, J = 8 Hz, 2H), 7.33
(t, J = 8 Hz, 2H), 7.23 (q, J = 12 Hz, 3H), 7.16 (d, J = 4 Hz, 4H),
7.08−7.11 (m, 1H), 4.12 (d, J = 8 Hz, 2H), 4.05−4.06 (m, 1H),
4.00−4.03 (m, 2H), 3.87 (s, 1H), 3.31 (q, J = 12 Hz, 1H), 3.14(q, J =
16 Hz, 1H), 2.88 (s, 3H), 2.79−2.853 (m, 1H), 2.49−2.55 (m, 1H).
13C NMR (100 MHz, DMSO-d6): δ 171.3, 156.1, 144.2, 144.1, 139.1,
(BB1). H NMR (400 MHz, DMSO-d6): δ 8.38 (d, J = 12 Hz, 1H),
8.05−8.13 (m, 4H), 7.82 (s, 2H), 7.63−7.65 (m, 3H), 7.23−7.35 (m,
6H), 7.04 (d, J = 8 Hz, 1H), 6.94 (d, J = 8 Hz, 1H), 6.71 (d, J = 8 Hz,
1H), 6.69 (d, J = 8 Hz, 1H), 4.12−4.18 (m, 2H), 3.8 (s, 2H), 3.52−
3.57 (m, 2H), 3.36−3.43 (m, 1H), 3.14−3.23 (m, 1H), 2.71−2.84
(m, 1H), 1.11 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 170.8,
155.9, 153.6, 144.2, 141.1, 132.1, 130.4, 130.0, 129.8, 129.7, 129.2,
128.3, 128.1, 127.9, 127.8, 127.4, 125.6, 123.7, 123.0, 120.5, 115.3,
77.9, 65.6, 52.1, 51.9, 49.4, 47.0, 31.7, 28.9, 16.6. HRMS (ESI) ([M +
H]+) Calcd. for C40H41N2O7S: 693.2634, found: 693.2650.
129.5, 128.5, 127.9, 127.4, 126.4, 125.5, 120.5, 65.7, 51.9, 51.6, 49.0,
47.1, 37.9. HRMS (ESI) ([M + H]+) Calcd. for C27H29N2O6S:
509.1746, found: 509.1750.
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-meth-
ylpentyl)-N-(methylsulfonyl)glycine (BB8). 1H NMR (400 MHz,
DMSO-d6): δ 7.80 (d, J = 8 Hz, 2H), 7.60 (d, J = 4 Hz, 2H), 7.34 (t, J
= 4 Hz, 2H), 7.24−7.27 (m, 2H), 7.06 (d, J = 12 Hz, 1H), 4.27 (d, J =
8 Hz, 2H), 4.13−4.15 (m, 1H), 3.89 (s, 2H), 3.64 (s, 2H), 3.16 (q, J
= 16 Hz, 1H), 3.01 (q, J = 12 Hz, 1H), 2.85 (s, 3H), 1.46−1.47 (m,
1H), 1.13−1.20 (m, 1H), 0.76 (q, J = 12 Hz, 6H). 13C NMR (100
MHz, DMSO-d6): δ 171.2, 156.3144.3, 141.1, 127.9, 127.4, 125.5,
120.5, 65.5, 51.9, 48.8, 48.1, 47.2, 41.2, 24.6, 23.6, 21.9. HRMS (ESI)
([M + H]+) Calcd. for C24H31N2O6S: 475.1903, found: 475.1910.
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-meth-
ylpentyl)-N-(isobutylsulfonyl)glycine (BB9). 1H NMR (400 MHz,
DMSO-d6): δ 7.81 (d, J = 8 Hz, 2H), 7.61 (q, J = 8 Hz, 2H), 7.34 (t, J
= 8 Hz, 2H), 7.23−7.27 (m, 2H), 7.08 (d, J = 12 Hz, 1H), 4.28−4.31
(m, 1H), 4.19−4.24 (m, 1H), 4.12−4.15 (m, 1H), 3.90 (s, 2H),
3.62−3.65 (m, 1H), 3.19 (q, J = 12 Hz, 1H), 3.05 (q, J = 16 Hz, 1H),
2.89 (t, J = 8 Hz, 2H), 1.96−2.04 (m, 1H), 1.46−1.48 (m, 1H),
(tert-butoxy)phenyl)propyl)-N-(naphthalen-1-ylsulfonyl)glycine
1
(BB2). H NMR (400 MHz, DMSO-d6): δ 8.52 (d, J = 8 Hz, 1H),
8.18 (d, J = 4 Hz, 1H), 8.02−8.09 (m, 2H), 7.82 (s, 2H), 7.56−7.66
(m, 5H), 7.27−7.36 (m, 5H), 6.97 (d, J = 8 Hz, 1H), 6.85 (d, J = 8
Hz, 1H), 6.71 (d, J = 8 Hz, 2H), 6.58 (d, J = 4 Hz, 1H), 4.08−4.26
(m, 2H), 4.02−4.06 (m, 1H), 3.72 (s, 2H), 3.33−3.49 (m, 2H),
2.58−2.72 (m, 1H), 2.37−2.46 (m, 2H), 1.11 (s, 9H). 13C NMR (100
MHz, DMSO-d6): δ 170.6, 155.9, 153.6, 144.2, 141.1, 135.4, 134.5,
130.3, 129.9, 129.4, 129.1, 128.5, 128.4, 128.0, 127.4, 127.2, 125.6,
124.9, 123.7, 120.5, 115.3, 77.9, 67.4, 65.7, 51.78, 49.2, 47.1, 37.4,
37.1, 31.7, 28.9, 27.7 HRMS (ESI) ([M + H]+) Calcd. for
C40H41N2O7S: 693.2634, found: 693.2650.
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-
(tert-butoxy)phenyl)propyl)-N-((4-(1,2,2-triphenylvinyl)phenyl)-
sulfonyl)glycine (BB3). 1H NMR (400 MHz, DMSO-d6): δ 7.85 (d, J
= 8 Hz, 2H), 7.60 (d, J = 4 Hz, 2H), 7.47 (d, J = 4 Hz, 2H), 7.37 (s,
2H), 7.27−7.31 (m, 4H), 7.05−7.08 (m, 12H), 6.88−6.96 (m, 5H),
G
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Article
1.14−1.21 (m, 2H), 0.9 (q, J = 8 Hz, 6H), 0.77 (q, J = 8 Hz, 6H). 13
C
Preparation of the α/Sulfono-γ-AA Peptide. The synthesis of
the α/sulfono-γ-AA peptides was carried out on 150 mg of Rink
Amide-MBHA resin (0.64 mmol/g) at room temperature (Scheme
S2). The resin was soaked in DMF for 5 min, and then the Fmoc
protecting group was deprotected in 20% piperidine in DMF solution
(2 mL, 10 min × 2). The resin was thoroughly washed with DCM (3
mL, 10 min × 3) and DMF (3 mL, 10 min × 3). After that, Fmoc-
deprotected amino acid (2 equiv.), HOBt (4 equiv.), and DIC (4
equiv.) were added and shaken in 2 mL of DMF. After 3 h, the resin
was washed with DCM and DMF and then treated with 20%
piperidine/DMF solution (10 min × 2). Next, a solution of the α/
Sulfono-γ-AA peptide building block (2 equiv.), HOBt (4 equiv.), and
DIC (4 equiv.) in 2 mL of DMF was added and reacted for 4 h, and
then the Fmoc protecting group was removed as stated above. The
reaction cycles were repeated until the desired α/Sulfono-γ-AA
peptides were synthesized. The N-terminus of the sequence was
capped with acetic anhydride (1 mL) in pyridine (2 mL,15 min)
followed by treatment with TFA/DCM (4 mL, 1:1, v/v) for 2.5 h.
The cleavage solution was collected, and the resin was washed with
DCM (3 mL × 2). The solution was combined and evaporated under
air flow to give the crude product, which was analyzed and purified
using a Waters HPLC system. The gradient eluting method of 5 to
100% of solvent B (0.1% TFA in acetonitrile) in A (0.1% TFA in
water) over 40 min was performed. All the α/Sulfono-γ-AA peptides
were obtained with moderate yields after prep-HPLC purification.
For the FITC-labeled α/sulfono-γ-AA peptide synthesis, after
attaching the last α-amino acid, the Fmoc protecting group was then
removed. FITC (1.2 equiv.) in 2 mL of DMF and DIPEA (6 equiv.)
were added to the resin and shaken overnight to complete the
reaction. After washing with DMF (×3) and DCM (×3), the resin
was cleaved using TFA/DCM (6 mL, 1:1, v/v) for 3 h. Then, the pure
FITC-labeled α/Sulfono-γ-AA peptides were obtained using the same
abovementioned method.
NMR (100 MHz, DMSO-d6): δ 171.2, 156.4, 144.3, 144.1, 141.1,
127.9, 127.4, 125.4, 120.5, 65.5, 59.3, 51.8, 48.6, 47.9, 47.2, 24.6, 23.6,
22.5, 21.9. HRMS (ESI) ([M + H]+) Calcd. for C27H37N2O6S:
517.2372, found: 517.2386.
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-
butoxycarbonyl)amino)hexyl)-N-(isobutylsulfonyl)glycine (10BB).
1H NMR (400 MHz, DMSO-d6): δ 7.81 (d, J = 8 Hz, 2H), 7.63
(q, J = 4 Hz, 2H), 7.34 (t, J = 8 Hz, 2H), 7.25−7.28 (m, 2H), 7.10 (d,
J = 8 Hz, 1H), 6.66 (s, 1H), 4.19−4.26 (m, 2H), 4.14−4.16 (m, 1H),
3.92 (s, 2H), 3.55 (s, 2H), 3.20−3.24 (m, 1H), 3.05−3.11 (m, 1H),
2.90−2.95 (m, 2H), 2.83−2.88 (m, 2H), 1.98−2.05 (m, 1H), 1.29 (s,
9H), 1.18−1.24 (m, 5H), 0.9 (q, J = 8 Hz, 6H). 13C NMR (100 MHz,
DMSO-d6): δ 171.2, 156.4, 155.9, 144.2, 141.1, 128.0, 127.4, 125.6,
120.5, 77.8, 65.6, 59.4, 51.5, 49.9, 48.6, 48.5, 47.2, 31.9, 29.7, 28.6,
24.6, 23.1, 22.6. HRMS (ESI) ([M + H]+) Calcd. for C32H46N3O8S:
632.3006, found: 632.3009.
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-
(tert-butoxycarbonyl)-1H-indol-3-yl)propyl)-N-(methylsulfonyl)-
glycine (BB11). δ 7.96 (d, J = 8 Hz, 1H), 7.78 (q, J = 4 Hz, 2H), 7.62
(d, J = 8 Hz, 1H), 7.49 (d, J = 4 Hz, 3H), 7.30−7.33 (m, 3H), 7.23−
7.26 (m, 1H), 7.17 (q, J = 16 Hz, 3H), 4.12−4.15 (m, 2H), 4.08−
4.09 (m, 1H), 3.90 (s, 2H), 3.42 (q, J = 12 Hz, 1H), 3.23 (q, J = 12
Hz, 1H), 2.91 (s, 3H), 2.87 (s, 1H), 2.66−2.72 (m, 1H), 1.5 (s, 1H),
1.47 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 171.3, 171.2, 158.9,
158.6, 156.3, 149.4, 144.1, 144.0, 141.1, 135.1, 130.9, 127.9, 127.8,
127.4, 125.5, 120.4, 117.8, 83.8, 65.9, 52.1, 50.1, 49.2, 47.1, 27.9.
HRMS (ESI) ([M + H]+) Calcd. for C34H38N3O8S: 648.2380, found:
648.2371.
(R)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-
butoxy)propyl)-N-((4-methoxyphenyl)sulfonyl)glycine (BB12). 1H
NMR (400 MHz, DMSO-d6): δ 7.77 (d, J = 4 Hz, 6H), 7.33 (t, J
= 4 Hz, 2H), 7.24−7.27 (m, 2H), 7.06 (d, J = 8 Hz, 1H), 6.68 (d, J =
8 Hz, 2H), 4.23−4.25 (m, 2H), 4.16 (d, J = 8 Hz 1H), 3.96−4.10 (m,
2H), 3.74 (s, 1H), 3.69 (s, 3H), 3.42 (d, J = 12 Hz 1H), 3.20−3.22
(m, 3H), 1.02 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 170.7,
162.8, 156.2, 144.2, 144.1, 131.5, 129.7, 128.0, 127.4, 125.6, 120.4,
114.6, 72.9, 65.9, 61.8, 55.9, 55.8, 51.1, 49.4, 47.1, 27.5. HRMS (ESI)
([M + H]+) Calcd. for C31H37N2O8S: 597.2271, found: 597.2268.
(R)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-
butoxy)propyl)-N-(methylsulfonyl)glycine (BB13). 1H NMR (400
MHz, DMSO-d6): δ 7.82 (d, J = 8 Hz, 2H), 7.64 (d, J = 8 Hz, 2H),
7.36 (t, J = 4 Hz, 2H), 7.27 (t, J = 8 Hz, 2H), 7.11 (d, J = 8 Hz, 1H),
4.25−4.26 (m, 2H), 4.16−4.18 (m, 1H), 3.9−4.2 (m, 2H), 3.70 (s,
1H), 3.33−3.38 (m, 1H), 3.13−3.24 (m, 3H), 2.9 (s, 3H), 1.05 (s,
9H). 13C NMR (100 MHz, DMSO-d6): δ 171.2, 156.2, 144.2, 128.0,
127.4, 125.6, 120.5, 73.0, 65.8, 61.2, 50.9, 49.1, 48.9, 47.1, 27.6.
HRMS (ESI) ([M + H]+) Calcd. for C25H33N2O7S: 505.2008, found:
505.2012.
N-((2R,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-
(tert-butoxy)butyl)-N-(benzylsulfonyl)glycine (BB14). 1H NMR (400
MHz, DMSO-d6): δ 7.81 (d, J = 8 Hz, 2H), 7.65 (d, J = 8 Hz, 2H),
7.34−7.35 (m, 5H), 7.28−7.29 (m, 3H), 7.17−7.24 (m, 2H), 4.27−
4.38 (m, 4H), 4.17 (t, J = 8 Hz, 1H), 3.92−3.87 (m, 1H), 3.77 (s,
1H), 3.52−3.55 (m, 3H), 3.9 (t, J = 12 Hz, 1H), 1.07 (s, 9H), 0.88
(d, J = 8 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 171.2, 156.5,
144.3, 141.1, 131.3, 130.1, 128.7, 128.5, 128.0, 127.4, 125.6, 125.5,
120.5, 120.4, 73.8, 67.2, 65.8, 58.5, 54.8, 49.2, 47.5, 47.3, 28.5, 17.9.
HRMS (ESI) ([M + H]+) Calcd. for C32H39N2O7S: 595.2478, found:
595.2468.
Fluorescence Polarization Competition Assays. The binding
affinities (Kd) of the AA peptides were investigated by fluorescence
polarization (FP). GST-MDM2-1-150 containing human MDM2 was
expressed in E. coli as previously described by us. FP was carried out
by incubating the 50 nM FITC-labeled AA peptide with MDM2/
MDMX (0 to 1 μM) in 1× PBS with 0.1% Pluronic F-68. Dissociation
constants (Kd) were determined by plotting the fluorescence
anisotropy values as a function of protein concentration, and the
plots were fitted to the following equation, where Lst is the
concentration of the peptide, and x stands for the concentration of
the protein. The experiments were performed in triplicate and
repeated three times.
Y = FPmin + (FPmax − FPmin)
(Kd + Lst + x) − (Kd + Lst + x)2 − 4Lstx
2Lst
Circular Dichroism. Circular dichroism (CD) spectra were
measured on an Aviv 215 circular dichroism spectrometer using a 1
mm path length quartz cuvette, and compound solutions in PBS
buffer (or in trifluoroethanol) were prepared using dry weight of the
lyophilized solid followed by dilution to give the desired
concentration (100 uM) and solvent combination. 10 scans were
averaged for each sample, and three independent experiments were
conducted, and the spectra were averaged. The final spectra were
normalized by subtracting the average blank spectra. Molar ellipticity
[θ] (deg·cm2·dmol−1) was calculated using the equation
(S)-N-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)propyl)-N-
((2-((tert-butoxycarbonyl)amino)ethyl)sulfonyl)glycine (BB15). 1H
NMR (400 MHz, DMSO-d6): δ 7.81 (d, J = 8 Hz, 2H), 7.62 (s, 2H),
7.34 (t, J = 8 Hz, 2H), 7.26−7.28 (m, 2H), 7.16−7.18 (m, 1H), 6.83
(s, 1H), 4.23 (t, J = 8 Hz, 2H), 4.14 (s, 1H), 3.68 (s, 2H), 3.25 (s,
2H), 3.17 (s, 3H), 1.29 (s, 9H), 1.00 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ 171.2, 156.0, 155.7, 144.3, 141.1, 128.0, 127.4, 125.6,
120.5, 78.5, 65.7, 52.4, 51.5, 48.8, 47.2, 45.7, 35.2, 28.5, 18.6. HRMS
(ESI) ([M + H]+) Calcd. for C27H36N3O8S: 562.2223, found:
562.2216.
[θ] = θobs/(n × l × c × 10)
where θobs is the measured ellipticity in millidegree, n is the number of
side groups, l is the path length in centimeter (0.1 cm), and c is the
concentration of the sulfono-γ-AA peptide in molar unit.
Luciferase Reporter Assay. U2OS cells (p53 wild-type
osteosarcoma) with stably integrated BP100-luc (p53-responsive
luciferase reporter) and CMV-lacZ (internal control for cell mass and
toxicity) were treated with compounds for 18 h. Luciferase and lacZ
H
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Article
activities were determined, and the ratio of luc/lacZ indicates p53
transcriptional activity. Positive control Nutlin was used at 4 and 8
μM, respectively.
Leixiang Yang − Department of Molecular Oncology, H. Lee
Moffitt Cancer Center and Research Institute, Tampa, Florida
33612, United States
Timothy Odom − Department of Chemistry, University of
South Florida, Tampa, Florida 33620, United States
Western Blotting. To detect proteins by Western blotting, cells
were lysed in lysis buffer (50 mM Tris−HCl [pH 8.0], 150 mM NaCl,
0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1×
protease inhibitor cocktail) and centrifuged at 4 °C for 10 min at
14,000g. The supernatant was boiled in Laemmli sample buffer for 5
min and subjected to SDS-PAGE and Western blotting to detect the
expression of proteins indicated in the figures using the corresponding
antibodies.
Enzymatic Stability Study. Lead compounds and peptide
control p53 (0.1 mg/mL) were incubated with 0.1 mg/mL pronase
in 100 mM ammonium bicarbonate buffer (pH 7.8) at 37 °C for 24 h.
Then, the reaction mixtures were concentrated in a speed vacuum at
medium temperature to remove water and ammonium bicarbonate.
The resulting residues were redissolved in H2O/CH3CN and analyzed
on a Waters analytical HPLC system with a 1 mL/min flow rate and 5
to 100% linear gradient of solvent B (0.1% TFA in acetonitrile) in A
(0.1% TFA in water) over the duration of 50 min. The UV detector
was set to 215 nm.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01638
Author Contributions
#Y.S., P.S., J.L., and P.H. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported by NIH 9R01AI152416 and NIH
5R01AG056569.
ABBREVIATIONS USED
■
DMF, dimethylformamide; DCM, dichloromethane; DIPEA,
N,N-diisopropylethylamine; EDC, ethyl-3-(3-
dimethylaminopropyl)carbodiimide; FITC, fluorescein isothio-
cyanate; HOBt, hydroxybenzotriazole; IC50, half maximal
inhibitory concentration; Kd, dissociation constant; MDM2,
mouse double minute 2; MDMX, also known as MDM4; TFA,
trifluoroacetic acid
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01638.
■
sı
Preparation of sulfono-γ-AA building blocks; preparation
of α/Sulfono-γ-AA peptides; fluorescence polarization
competition assays; circular dichroism; enzymatic
1
stability study; H−15N HSQC NMR of lead peptide 9
REFERENCES
■
1
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AUTHOR INFORMATION
Corresponding Authors
■
Gary Daughdrill − Department of Cell Biology, Microbiology
and Molecular Biology, University of South Florida, Tampa,
Florida 33620, United States; Email: gdaughdrill@usf.edu
Jiandong Chen − Department of Molecular Oncology, H. Lee
Moffitt Cancer Center and Research Institute, Tampa, Florida
33612, United States; Email: jiandong.chen@moffitt.org
Jianfeng Cai − Department of Chemistry, University of South
Florida, Tampa, Florida 33620, United States; orcid.org/
0000-0003-3106-3306; Email: jianfengcai@usf.edu
́
́
(8) Berlicki, Ł.; Pilsl, L.; Weber, E.; Mandity, I. M.; Cabrele, C.;
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Martinek, T. A.; Fulop, F.; Reiser, O. Unique α,β- and α,α,β,β-Peptide
Authors
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