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Jiang et al. Hepatoma Res 2019;5:5 I http://dx.doi.org/10.20517/2394-5079.2018.97 Page 3 of 11

[21]
[20]
reinforced structures. These include disulfide bond formation , lactam formation , ruthenium-catalyzed
[15]
[22]
ring closing metathesis , and copper-catalyzed azide-acetylene cycloaddition . While these reactions have
enabled the synthesis of stapled peptide helices, the development of additional stapling reactions with high
yields and predictable structural effect is still highly desirable. Herein, we report the first synthesis of stapled
PMI helices using 1,2(1,3 or 1,4)-dimethylbromobenzene reacting with the sulfydryl of cycstine and the
subsequent structural, protein chemistry and in vitro anticancer activity studies of the stapled PMI.

METHODS
Patient data
The data of p53, MDM2 and MDMX expression at mRNA level in HCC patients were collected and obtained
from The Cancer Genome Atlas (TCGA) project [23,24] via the data portal on 03/24/2018.

General remarks
All synthetic peptide sources were obtained from CS Bio (Shanghai) Ltd. All other chemicals used in this
study were purchased from Sigma-Aldrich unless otherwise specified. Acetonitrile and water (HPLC grade)
were purchased from Fisher Scientific Ltd. All products were used as received without further purification.

Synthesis of peptides
All peptides were synthesized on appropriate resins on an CS bio 336X automated peptide synthesizer using
the optimized HBTU activation/DIEA in situ neutralization protocol developed by an HBTU/HOBt protocol
for Fmoc-chemistry SPPS.2 After cleavage and deprotection in a reagent cocktail containing 88% TFA, 5%
phenol, 5% H O and 2% TIPS, crude products were precipitated with cold ether and purified to homogeneity
2
by preparative C18 reversed-phase HPLC. The molecular masses were ascertained by electrospray ionization
mass spectrometry (ESI-MS).

Reversed phase analytical and preparative HPLC
Analytical HPLC was run on a Waters instrument using an analytical C18 column purchased from Waters
at a flow rate of 1.0 mL/min. Solution A was ultrapure water containing 0.1% trifluoroacetic acid (TFA),
and solution B was acetonitrile containing 0.1% TFA. The gradient is liner from 5% B to 65% B in 30 min.
Preparative HPLC was run on a Preparative Waters instrument using an analytical C4 column purchased
from Waters at a flow rate of 15.0 mL/min. Eluent A and B were same as the solution used in analytical
HPLC. The gradient is liner from 25% B to 50% B in 60 min.

Preparation of stapled PMI
To prepare stapled PMI, the peptide was firstly dissolved in reaction buffer [80% 10 mmol/L PBS (pH 7.4)
and 20% acetonitrile] at a concentration of 100 μmol/L, meanwhile 1,2(1,3 or 1,4)-dimethylbromobenzene
were dissolved in DMSO at a concentration of 10 mmol/L. After the preparation of reaction fluid, 10
mL peptide buffer were magnetic stirred in a beaker at room temperature, and then 50 μL 1,2 (1,3 or
1,4)-dimethylbromobenzene buffers were mixed into the buffer in four times every 10 min. After the
reaction, pure stapled PMI can be collected by preparative HPLC.

CD spectroscopy
CD spectra of variants at a concentration of 20 μmol/L in 10 mmol/L phosphate buffer (pH 7.4) were
obtained at room temperature on a J-810 spectropolorimeter (Jasco, Easton, MD) using a 1-mm quartz
cuvette as previous reports [25-27] . Scanned area was from 250 nm to 190 nm, and the scanning speed was 50
nm/min. Every curve was the average of three independent detections.

Fluorescence polarization-based competitive binding assay
As for fluorescence polarization assay, Fluorescein (FITC) was conjugated to 15-29 p53 via its N-terminal
amino group in DMF, and the resultant product 15-29 p53-FITC were HPLC-purified and lyophilized. The