SUPPORTING INFORMATION

Directional assembly of stapled α‑helical peptideKuan Hu,a,† Feng Yin,a,† Ziyuan Zhou,a,b,† Chenshan Lian,a Yinghuan Liu,a Chengjie Sun,a Wenjun Li,a Jianing Zhang,a Zigang Lia,*

aState Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School, Peking University, Shenzhen 518055, China

ContentMethods and materials.....................................................................................................................2

Chemicals and reagents ............................................................................................................2Unnatural amino acids synthesis ..............................................................................................2The brief procedures for linear peptide synthesis and purification .........................................2Oxidation of the linear peptide and characterization...............................................................2Scanning electron microscopy ..................................................................................................3Atomic force microscopy ..........................................................................................................3Optical characterization............................................................................................................3Circular dichroism (CD) spectroscopy .......................................................................................4Gel retardation assay to access the quantity of siRNA .............................................................4Tumor cell line and culture .......................................................................................................4siRNA transfection and flow cytometry ....................................................................................4Cell Imaging ..............................................................................................................................5Cell viability test........................................................................................................................5RNA isolation and quantitative RT-PCR ....................................................................................5Western blotting.......................................................................................................................5

Supplementary Figures: ....................................................................................................................6Supplementary MS data .................................................................................................................10

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019

Methods and materials

Chemicals and reagentsAll reagents, including amino acids and resins, were purchased from GL Biochem (Shanghai), Shanghai Hanhong Chemical Co., J&K Scientific or Energy Chemical and were used without further purification. Unnatural amino acids were synthesized followed by reported procedures. NMP was purchased from Shenzhen Tenglong Logistics Co. and used without purification. All other solvents were purchased from Cantotech Chemicals, Ltd. Anhydrous solvents were purchased from J&K Scientific.

Unnatural amino acids synthesis The detailed unnatural amino acids and peptide synthesis procedures can be found in our previous reported articles.1-6

KOH, DMFNi N

NH

O

ON

OH

(R) Ni NNH

O

ON

O

H(R)

Minor product

3 M HCl/MeOH, refluxO

HO(R)

HH3N

ClClPh Ph

Ph

Fmoc-OSu, NaHCO3

O

HO(R)

HFmocHN Ph

Ni NNH

O

ON

O Cl

R

(R) I

Major product

HNOH

O Cl

NOH

O

1. KOH/iPrOH, 40¡æ2. HCl, pH 5-6 PCl5 , DCM

O NH2

NNH

O

O Gly(Ala), Ni(NO3)2 6H2O, KOH

MeOH, reflux

Ni NNH

O

ON

O

1

2

Cl

Cl

ClCl

R

A

3

B

Scheme S1. Synthesis procedures of unnatural amino acid S5(Ph).

The brief procedures for linear peptide synthesis and purificationPeptides were synthesized on MBHA resin (loading capacity: 0.37 mmol/g) by standard Fmoc-based SPPS. At first, the resin was swelled in NMP for 30 min. Then 50% (vol/vol) morpholine in NMP was used to deprotect the Fmoc group on amine group for 30min × 2. Next the resin was washed with DCM and NMP alternatively for three time(3*1min). In coupling process, for natural amino acids, the Fmoc-protected amino acids (5.0 equiv), HCTU (4.9 equiv), DIPEA (10.0 equiv) were dissolved in NMP and mixed with resin for 2h, followed by washing with DCM and NMP for three times(3*1 min). For unnatural amino acids, Fmoc-protected acids (2.5 equiv), HCTU (2.4 equiv) and DIEA (5.0 equiv) were dissolved in NMP and mixed with resin for 4h, followed by washing with DCM and NMP for several times.

Oxidation of the linear peptide and characterizationThe monomer peptide was dissolved in deionized water/acetonitrile (v/v=1:1) and its concentration was 1 mM. After the solutions in 2 mL eppendorf tubes rendered uniformly by sonicate for 2 min, they were placed in a heat block at 80°C for air oxidation. After incubation for

hours, the disulfide bonding is formed from sulfhydryl residues of cysteine in monomer peptides. For HPLC analysis of the oxidation state of peptides at different time points, an aliquot of peptides was taken out from the system and injected into LC/MS. The peptide fractions were analyzed by ESI mass spectra. To further validate the cyclomer, a MALDI-TOF mass spectra was used and performed in Shenzhen University.

1.HCTU,DIEA,AAs2,acetic anhydride,DIEA H

NNH

HN

NH

O

O

O

Arg Glu

Cys

NH

OONH

Rink amide MBHA resin

95:2.5:2.5TFA:H2O:TIS

Ph

FmocHNHN

O

Ph

repeated SPPS

FmocNH

HN

NHO

O

Arg

Arg CysHN

O

O

Ph

Glu

HN

NH

HN

NH

O

O

O

Arg Glu

NH2

OONH

Ph

FmocNH

HN

NHO

O

Arg

Arg HN

O

O

Ph

Glu

SH HS

NH

HNN

H

HN

O

O

O

ArgGlu

H2N

O O HN

Ph

FmocHNN

H

HN

O

O

Arg

ArgNH

O

OPh

Glu

SS

HN N

H

HN N

H

O

O

O

Arg Glu

NH2

OONH

Ph

Fmoc NH

HN N

HO

O

Arg

Arg HN

O

OPh

Glu

S S

HN N

H

HN N

H

O

O

O

Arg Glu

NH2

OONH

Ph

Fmoc NH

HN N

HO

O

Arg

Arg HN

O

OPh

Glu

S S

´Oxidation

No

Yes

Scheme S2. Procedures for solid phase peptide synthesis (SPPS). Peptide (RCES5)2 was taken as an example.

Scanning electron microscopySamples of peptide nanospheres and linear peptides were placed on silicon slides and were left to dry at room temperature. Samples were then viewed using a scanning electron microscope (ZEISS Supra 55, Oxford X-Max 20, 10 kV, 10 μA).

Atomic force microscopyAFM measurements were performed using a Nanoscope V multimode atomic force microscope (Veeco Instruments, USA). Samples for AFM images were diluted with deionized H2O to yield a final concentration of 1×10-4 M. The sample (20 μL) was then dispersed on freshly cleaved muscovite mica, and then dried at room temperature. Tapping mode was used to acquire the images under ambient conditions.

Optical characterization Raman data were measured by Horiba Jobin-Yvon LabRam Aramis Raman spectrometer. A 785 nm Ar-ion laser was used as the excitation source, and the substrate plate was stainless steel which had no Raman signal and guaranteed a free fluorescence background. Wave numbers ranging from 200 to 3000 cm−1 were scanned, and each spectrum was accumulated for 60 s. The

analysis was executed more than three points for each sample for reproducibility and reliability. To get the data of peptide sheets, it was transferred to the substrate, and just a single transfer of film provided enough signals for interpreting the Raman peaks. The UV-VIS absorption spectra were determined using a Shimadzu UV-2450 spectrophotometer. The hydrodynamic size distribution profiles and the zeta potentials of the nanocomplexes were measured using a particle size analyzer system (90 Plus, Brookhaven Instruments). The peptide cyclo-(RCES5)2 nanosphere was characterized with a Bruker IFS 66 v/s infrared spectrometer (Bruker, Karlsruhe, Germany) by Fourier transform infrared (FT-IR) spectroscopy in the range 400-4000 cm-1 with a resolution of 4 cm-1. The sample was dropped into a clean silicon substrate and formed a uniform film for recording in the FTIR instrument.

Circular dichroism (CD) spectroscopyThe peptides were dissolved at a concentration of 25 μM in water/acetonitrile (v/v=1:1). CD spectra were obtained on an applied photophysics chirascan CD Spectrometer at 20 °C using the following standard parameters: wavelength, 185-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path 3 length, 1mm. Every sample was scanned twice, and the final CD spectrum was averaged and smoothed. Thermal disruption curves were acquired by monitoring the signal at 215 nm while increasing the temperature stepwise by 5°C, with a 10min equilibration between steps. The data were fit to a two-state folding model using Origin Pro 9.0.

Gel retardation assay to access the quantity of siRNAThe siRNA binding ability of the cyclo-(RCES5)2 nanocarriers was studied using agarose gel electrophoresis. The cyclo-(RCES5)2 (1 mg mL-1) was mixed with siRNA (0.4 mg mL-1) at five different mass ratios (10 mg:1 mg, 20 mg:1 mg, 40 mg: 1 mg, 80 mg:1 mg, 100 mg: 1 mg and 150 mg: 1 mg). Electrophoresis was carried out on a 1% agarose gel with a voltage of 100 V for 15 min in a TAE buffer solution (40mM Tris-HCl, 1 v/v% acetic acid, and 1mM EDTA). The retardation of the complexes was visualized by staining with ethidium bromide and then analyzed on a UV illuminator to show the position of the complex siRNA band relative to that of naked siRNA.

Tumor cell line and culture Human ovarian teratocarcinoma cells, PA-1 (CRL-1572, American Type Culture Collection), were maintained in Eagle’s Minimum Essential Medium (EMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone) and penicillin/streptomycin (100 mg mL1, Gibco). Cells were cultured at 37°C in a humidified atmosphere with 5% CO2.

siRNA transfection and flow cytometryThe day before transfection, PA-1 cells were seeded onto 6-well plates in EMEM medium with 10% FBS to give 30% − 50% confluence at the time of transfection. cyclo-(RCES5)2 dispersion (1 mg/mL, 20μL) was mixed with LSD1 siRNACy3 (0.4mg/mL, 3.25μL) with gentle vortex and left undisturbed for 30 min. The cyclo-(RCEF)2 peptide, lipofectamine and oligo2000 used as controls at the same dosage. Before transfection, the culture medium was replaced with OPTI-MEM (950μL, Invitrogen), the above-mentioned cyclo-(RCES5)2@iRNACy3 and other complexes mixture was then added to the medium and the cells were continuously cultured. Free siRNACy3 or cyclo-

(RCES5)2 peptides were also used in another parallel experiment at the same dosage level. Four hours later, EMEM medium (500μL) with 30% FBS was added to the medium. The gene expression was monitored at 48 hour post-transfection. For transfection efficiency examination, fluorescent imaging and flow cytometry assays were performed at 4 hour post-treatment.

Cell ImagingPA-1 cells were cultured in EMEM with 10% FBS (v/v) in imaging dishes (50,000 cells/well) at 37°C and 5% CO2 for one day until they were about 80% confluent. The peptide@siRNACy3 complexes were added to the dishes at indicated concentration. The cells were incubated with nanocomplexes for 4 hours at 37°C, washed 3 times with PBS and then fixed with 4% formaldehyde (Alfa Aesar, MA) in PBS for 10 minutes. They were then washed 3 times with PBS and stained with 1µg/ml 4', 6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA) in PBS for 5 minutes. Images of peptide localization in cells were taken on a Perkin Elmer confocal microscope. Image processing was done with the Volocity software package (Zeiss Imaging).

Cell viability testCell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) assay. Cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated with different peptide@siRNA nanocomplexes for indicated time (4, 24, 48 hours). MTT (5 mg/ml, 20 mL) in PBS was added and the cells were incubated for 4 hours at 37°C with 5% CO2. DMSO (dimethylsulfoxide, 150 mL, Sigma) was then added to solubilize the precipitate with 5 min gentle shaking. Absorbance was measured with a microplate reader (Bio-Rad) at a wavelength of 490 nm. The cell viability was obtained by normalizing the absorbance of the sample well against that from the control well and expressed as a percentage, assigning the viability of non-treated cells as 100%.

RNA isolation and quantitative RT-PCR48 hours after transfection, total RNA was extracted from PA-1 cells using TRIzol reagent (Invitrogen) and the amount of RNA was quantitated using a spectrophotometer (Nano-Drop ND-2000). Total RNA (2 mg) was reverse transcribed to cDNA using the reverse transcriptase kit from Takara according to the manufacturer’s instructions. The mRNA levels of the target genes were quantified by real time PCR using SYBR green (Takara) in a Bio-Rad CFX Connect real-time PCR system.

Western blottingFor western blot analysis, cells were seeded in 6-well plates and treated for 44 hours with different peptide@siRNA nanocomplexes as described for the RT-PCR assay. To isolate protein, cells were washed with PBS and harvested using the lysis buffer (50 mM TrisCl pH = 6.8, 2% SDS, 6% glycerol, 1% β-mercaptoethanol, 0.004% bromophenol blue). Total cellular protein concentrations were determined using a spectrophotometer (Nano-Drop ND-2000). 20 mg of denatured cellular extracts were resolved using 10% SDS-PAGE gels. Protein bands in the gel were then transferred to nitrocellulose blotting membranes and incubated with appropriate primary antibodies. The antibody dilutions were as follows: 1:1000 for LSD1 and 1: 3000 for histone H3. Membranes were incubated overnight at 4℃ and washed the next day with buffer (1

PBS, 0.05% Tween 20). Goat antirabbit secondary antibodies were used for secondary incubation for 1 hour at room temperature. Proteins were then visualized using chemilluminescent substrates.

Supplementary Figures:

Figure S1 MALDI-TOF mass data for cyclo-(RCES5)

2, the M/Z clearly indicated the linear peptide

oxidized as cyclomer rather than dimer.

Sequence Stapling efficiencya Selectivityb

Fmoc-RRCES5RCES5 95% 98%Fmoc-RRCEFRCEF 96% 98%Fmoc-RRCEARCEA 98% 99%Fmoc-RRCEGRCEG 99% 99%Fmoc-RRCELRCEL 98% 100%

Figure S2. The peptide sequences used for oxidation stapling and the corresponding stapling efficiency and selectivity. a, the efficiency was calculated from the integrating area in the HPLC curve. b, the selectivity indicates the percentage of intramolecular oxidation relative to the intermolecular oxidation.

Figure S3 HPLC curves at indicated time points to study the oxidation kinetics of the linear peptide. Only a small fraction of peptide oxidized to dimer after 24 hours.

Figure S4 FTIR spectrum of cyclo-(RCES5)2 nanospheres.

Figure S5. (A) CD spectra of S-(RCES5)2 and L-(RCES5)2 in water, 25℃, 10-100 µM. CD spectra of peptides (B) S-(RCEA)2 and L-(RCEA)2, (C) S-(RCEA)2 and L-(RCEA)2, and (D) S-(RCEA)2 and L-(RCEA)2 in 20% TFE solution, 25℃, 10-100 µM.

Figure S6 (A-D) SEM images of the peptide S-(RCES5)2 assemblies.

Figure S7 Gel retardation analysis of interaction between siRNA and different peptides. The

results suggested the S-(RCES5)2 peptide has higher loading efficiency than all the other unstapled peptides.

PA-1 cell

Blank 2)5

S-(RCES

lipo20

00

oligofec

tamine

siRNA

@siRNA

2

S-(RCEF) @siR

NA

2)5

S-(RCES

oligofec

tamine@

siRNA

lipo20

00@siR

NA0

5

10

15

**

**

** **

Aver

age

Fluo

resc

ence

Inte

nsity

(Cy3

)

Figure S8 Mean fluorescence intensity of cells treated with different reagents in flow cytometry analysis. The values were derived from the Flowjo software. Values are means ± SD, n = 3, **, P < 0.01.

Figure S9 Fluorescent images of PA-1 cells treated with L-(RCES5)2@siRNACy3. The cell nuclei were stained with DAPI (blue) and the signals from Cy3 are marked in red.

Figure S10 Protein relative expression levels detected by Western Blotting. H3 was used as the protein loading control for samples.

Supplementary MS data

Entry Peptide Calc. mass Obs. mass

1 Linear Fmoc-RRCES5RCES5 1573.73 787.88

2 Stapled Fmoc-RRCES5RCES5 1571.72 786.88

3 Linear Fmoc-RRCEFRCEF 1465.64 489.85, 734.05

4 Stapled Fmoc-RRCEFRCEF 1463.62 489.35, 733.20

5 Linear Fmoc-RRCEARCEA 1313.58 438.85, 657.90, 1315.05

6 Stapled Fmoc-RRCEARCEA 1311.56 438.15, 656.85

7 Linear Fmoc-RRCEGRCEG 1285.54 429.50, 643.75, 1287.00

8 Stapled Fmoc-RRCEGRCEG 1283.53 428.85, 642.80

8 Linear Fmoc-RRCELRCEL 1397.67 466.90, 699.85, 1399.15

10 Stapled Fmoc-RRCELRCEL 1395.65 466,25, 698.95, 1398.15

11 Fmoc-RRAES5RAES5 1509.78 504.25, 756.10

12 Fmoc-RRGES5RGES5 1481.76 494.95, 742.00

13 Fmoc-RRMEFRCEF 1493.67 499.20, 748.05

14 Fmoc-RRMEFRCEF dimer 2987.34 499.05, 747.90

Fmoc-RRCES5RCES5 or linear (RCES5)2

stapled-(RCES5)2

Linear Fmoc-RRCEFRCEF

Stapled Fmoc-RRCEFRCEF

Fmoc-RRMEFRCEF

Fmoc-RRMEFRCEF dimer

Fmoc-RRAES5RAES5

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z0.00

0.25

0.50

0.75

1.00Inten.(x10,000,000)

504.25

756.10

件 件 件 件 件 :23S5-A2.lcd件 件 件 :1

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0

100

200mV

Fmoc-RRGES5RGES5

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z0.0

0.5

1.0

1.5

2.0Inten.(x10,000,000)

494.95

742.00

415.05

件 件 件 件 件 :23S5-G2.lcd件 件 件 :1

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0

100

200mV

Linear Fmoc-RRCEARCEA

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z0.0

0.5

1.0

1.5

2.0

2.5

Inten.(x10,000,000)

657.90

438.85

1315.05

Stapled Fmoc-RRCEARCEA件 件 件 件 件 :22Li-AA2.lcd件 件 件 :1

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0

500

1000mV

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

Inten.(x10,000,000)

438.15

656.85

Linear Fmoc-RRCEGRCEG

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z0.0

0.5

1.0

1.5

2.0

2.5Inten.(x10,000,000)

643.75429.50

588.25 1287.00

Stapled Fmoc-RRCEGRCEG件 件 件 件 件 :22Li-GG2.lcd件 件 件 :1

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0

500

1000

mV

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

Inten.(x10,000,000)

428.85

642.80

588.25

Linear Fmoc-RRCELRCEL

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z0.00

0.25

0.50

0.75

1.00

1.25

Inten.(x10,000,000)

699.85

466.90

480.501399.15

Stapled Fmoc-RRCELRCEL

件 件 件 件 件 :22Li-LL2.lcd件 件 件 :1

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0

1000

mV

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00Inten.(x10,000,000)

698.95

466.25

1398.15

Reference:

1. K. Hu, H. Geng, Q. Zhang, Q. Liu, M. Xie, C. Sun, W. Li, H. Lin, F. Jiang, T. Wang, Y.-D. Wu and Z. Li, Angew. Chem. Int. Ed., 2016, 55, 8013-8017.

2. K. Hu, W. Li, M. Yu, C. Sun and Z. Li, Bioconjugate Chem., 2016, 27, 2824-2827.3. K. Hu, C. Sun and Z. Li, Bioconjugate Chem., 2017, 28, 2001-2007.4. K. Hu, C. Sun, D. Yang, Y. Wu, C. Shi, L. Chen, T. Liao, J. Guo, Y. Liu and Z. Li, Chem. Commun., 2017,

53, 6728-6731.5. K. Hu, C. Sun, M. Yu, W. Li, H. Lin, J. Guo, Y. Jiang, C. Lei and Z. Li, Bioconjugate Chem., 2017, 28,

1537-1543.6. Y. Jiang, K. Hu, X. Shi, Q. Tang, Z. Wang, X. Ye and Z. Li, Org. Bio. Chem., 2017, 15, 541-544.