· crosslinking (0.3 mmol/g) from Advanced ChemTech Inc. NMR spectra were obtained on a Joel 400 MHz and Varian 600 MHz spectrometer. …

Supporting Information

Expedient on-resin modification of a peptide C-terminus through a benzotriazole linker

Anand Selvaraj [a], Hui-Ting Chen[b,c] Adela Ya-Ting Huang[a] and Chai-Lin Kao*[a, c, d]

[a] Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan

[b] Department of Fragrance and Cosmetic Science, Kaohsiung Medical University. Kaohsiung 807, Taiwan

[c] Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan

[d] Department of Chemistry, National Sun Yat-sen University, Kaohsiung 80424, Taiwan

Content Materials and Methods ........................................................................................................................................................... 1

General procedure for solid phase peptide synthesis ............................................................................................................ 2

General procedure for nucleophilic substitution on benzotriazole resin ............................................................................... 4

General procedure for nucleophilic acyl substitution on peptide .......................................................................................... 7

Preparation of peptide decorated dendrimer ...................................................................................................................... 11

Preparation of cyclic peptide ................................................................................................................................................ 15

General procedure for preparation of elastin peptide ......................................................................................................... 18

NMR Spectra ......................................................................................................................................................................... 22

Chiral HPLC chromatogram ................................................................................................................................................... 38

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2017

S1

Materials and Methods

All commercial materials were used without further purification. All solvents were reagent grade or HPLC grade from

Fisher. Diethyl ether, dichloromethane, ethanol, methanol, DMF, and DMSO were obtained from a dry solvent system and

used without further purification. Trifluoroacetic acid (TFA) and isoamyl nitrite were purchased from Acros .

Poly(amidoamine) (PAMAM) dendrimers were purchased from Dendritech. Inc. Triisopropylsilane (TIS), N, N-

diisopropylethylamine (DIPEA), N-methy morpholin (NMM), 4-amino-3-nitrobenzoic acid (ANB), piperidine, 3-

mercaptopropionic acid (MPA) and Tin(II) chloride were purchased from Alfa Aesar. o-(1-Benzotriazol-1-yl)-1,1,3,3-

tetramethyluronium hexafluorophosphate (HBTU) and amino acids were purchased from AK Scientific, Inc. Analytical

thin-layer chromatography was pre-coated and detected at 254 nm by fluorescence detector. Water was purified using a

Millipore MilliQ water purification system. Peptides were synthesized on a Rink amide resin(LS) 100-200 mesh 1% DVB

crosslinking (0.3 mmol/g) from Advanced ChemTech Inc.

NMR spectra were obtained on a Joel 400 MHz and Varian 600 MHz spectrometer. ESI Mass spectra were taken on Bruker

APEII. HRMS (FAB+) were recorded with a JEOL JMS SX/SX 102A four sector mass spectrometer. MALDI-Mass spectra

were recorded from Autoflex III MALDI-TOF system (Bruker Daltonics).

HPLC

Analytical HPLC was carried out in Agilent Model 1100 equipped and performed using a Pyramid C18 column (Nucleodex,

5.0 micron, ɸ4.6 x 250 mm) at a flow rate of 0.5 mL/min with a gradient from 0 to 80% of B in 40 min (eluent A: 0.1%

TFA in H2O, B: 0.1% TFA in CH3CN), 25oC. Analytical injections were monitored at 220 nm.

Gel permeation chromatography (GPC) experiments were carried out in Agilent Model 1100 HPLC system (1100 and

performed using TSK G2000sw column (ɸ7.5 x 300 mm, 10 m size) at a flow rate of 1 mL/min with an isocratic in 20

min (eluent A: 0.1% TFA in H2O), 25oC. Analytical injections were monitored at 214 nm.

Chiral experiments were performed using CHIRACEL OD-H (Chiral Technologies Europe, ɸ4.6 x 250 mm) columns at a

flow rate of 1 mL/min with an isocratic in 30 min (eluent: iPrOH/n-hexane), 25oC. Analytical injections were monitored at

220 nm.

S2

Scheme S1: Synthesis of on resin diaminobenzoic acid linker (Dbz)

The suspension of rink amide resin (0.3 mmol/g loading; 0.1 mmol) in DMF and swelled for 30 min, then the resin was

treated with 20% piperidine stand for 5 min twice and then washed with DMF and coupled to 4-amino-3-nitrobenzoic acid

(ANB) (4 equiv.), HBTU (4 equiv.) and NMM (8 equiv.) in DMF (3 mL). The reaction mixture was allowed to stirred for

2 h. The resin was washed with DCM (3 mL x 3) and DMF (3 mL x 3). The unreacted resin was capped by acetic anhydride.

The supernatant was removed and the resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3). The resulting resin-

bound ANB was bubbled with N2 for 10 min and then carried out with the dihydrate of tin (II) chloride (2.5 M) in the

presence of DBU (2 mmol) in DMF during 6 h at room temperature. The resulting resin-bound o-aminoanilide was filtered

off and then washed DMF (3 mL x 3) and DCM (3 mL x 3). The resin was dried under vacuo and treated with TFA: H2O

(95:5) to afford 95% yield. 1H NMR (400 MHz, DMSO) : δ 7.10 (s, 1H), 7.04 (d, J = 7.2 Hz, 1H), 6.47 (d, J = 8.1 Hz, 1H).

HRMS calcd for C7H10N3O (M+H)+ 152.0824, found 152.0821.

Loading Estimation of the First Amino Acid

The resin was treated with piperidine/DMF (1:4, v/v, 3 mL, 5 min × 3) and the combined deprotection solution (20%

piperidine in DMF) made up to 10 mL with DMF. The solution was diluted 200-fold with DMF and the UV absorbance of

the piperidine-fulvene adduct measured (λ = 301 nm, ε = 7800 M−1cm−1) to estimate the amount of amino acid loaded onto

the resin.

General procedure for solid phase peptide synthesis

The peptide synthesis was carried out using Fmoc-SPPS on rink amide resin (0.3 mmol/g, 0.05 mmol). The resin was treated

with 20% piperidine (5 min x 2) washed with DMF and coupled with 4-amino-3-nitrobenzoic acid (4 equiv.) using HBTU

(4 equiv.), NMM (8 equiv.) in DMF for 1 h. After the resin bound ANB was reduced with tin(II) chloride (2.5 M) in the

presence of DBU (2 mmol) in DMF. The remaining peptide was synthesized on an automated PS3 peptide synthesizer using

Fmoc-Xaa-OH (2 equiv.), NMM (4 equiv.) and HBTU (2 equiv.). The coupling was performed for 60 min and Fmoc-

deprotection was achieved using 20% piperidine (10 min x 2). Coupling reactions were checked by Kaiser test.

S3

General procedure for cyclization and cleavage of the benzotriazole linker

The resin-bound Dbz peptide was washed with DMF (3 mL x 3) and DCM (3 mL x 3), then treated with isoamyl nitrite (10

equiv.) in DMF (4 mL) for 90 min at room temperature. Unreacted isoamylnitrite was washed with DCM. The nucleophilic

(4 equiv.) and DIPEA (8 equiv.) in DMF was bubbled with N2 for 20 min and then immediately added into the activated

peptidyl-resin. The reaction was allowed to shake for 2 h at room temperature. After completion of the reaction, the

supernatant was removed and concentrate under vacuo. The resulting peptide derivative was deprotected with 20%

piperidine in DMF and then TFA cocktail (TFA/TIS/H2O (95:2.5:2.5) or TFA/phenol/water/thioanisole/EDT

(82.5:5:5:5:2.5)). The peptide was precipitated in cold ether and collected by centrifugation. The supernatant was removed

and the residue was dissolved in 30% acetonitrile/H2O and lyophilized to afford pure product without further purification.

Table S1: Optimization of reaction conditions for synthesis of resin bounded benzotriazole (17)

Entry Reagent (equiv.) Acid Solvent Time(h) Conversiona(%)

1 NaNO2(10 equiv.) AcOH DMF 24 40

2 NaNO2(10 equiv.) Acetic acid/Con. HCl DMF 24 15

3 NaNO2(10 equiv.) Con. HCl DMF 24 30

4

5

6

7

Isoamyl nitrite (10 equiv.)




-

-

-

-

DMF

DMF

NMP

DCM

1.5

1.5

3

1.5

99

85

99

99

Reaction conditions: Peptide (25 mg, 0.30 mmol/g) on solid support at room temperature. aConversion to 17

was calculated from the absorbance at 220 nm using HPLC. The entry in bold represents the optimized reaction

conditions for cyclization. Cleavage condition: TFA: H2O (95:5) for 1.5 h at RT.

Synthesis of benzotriazole (17, Bt)

S4

The resin-bound Dbz linker 3 (0.05 mmol) in DMF (4 mL) was added isoamyl nitrite (10 equiv.) at room temperature. The

reaction mixture was allowed to shaken for 90 min at room temperature. The resin was filtered and then washed DCM (3

mL x 3). The benzotriazole was cleaved from resin using TFA: H2O (95:5) to afford the compound 17. The crude mixture

was applied directly to flash chromatography (50-75% EtOAc/ hexanes) to yield 8 mg (92%) as a yellow solid. 1H NMR

(400 MHz, DMSO) : δ 8.44 (s, 1H), 7.90 (dd, J = 16.2, 6.8 Hz, 2H). HRMS calcd for C7H7N4O (M+H)+ 163.0614, found

163.0612.

General procedure for nucleophilic substitution on benzotriazole resin

The resin-bound Dbz 2 (0.05 mmol, 157mg) was coupled with amino acid (4 equiv.) in the presence of HBTU (2 equiv.)

and NMM (4 equiv.) in DMF for 1 h. The resin was washed with DMF (3 mL x 3) and DCM (3 mL x 3). After that resin

bound Dbz amino acid was treated with a solution of isoamylnitrite (10 equiv.) in 4 mL of DMF for 90 min at room

temperature. After washing with DCM (3 mL x 3), the resulting resin was shaken with a mixture of ethanol (10 equiv.) and

DIPEA (8 equiv.) in DCM for 2 h at room temperature. The resin was washed with DCM (3 mL x 3), then the supernatant

was concentrated under vacuo and washed through silica gel pad (ɸmm x 12 mm) to afford pure product.

Fmoc-L-Leu-OEt (6a): Rf = 0.4 (Hexane: Ethyl acetate, 8:2). White solid (18 mg, 91%). 1H NMR (400 MHz, CDCl3) : δ

7.76 (d, J = 7.5 Hz, 2H), 7.59 (dd, J = 6.8, 5.2 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.4

Hz, 2H), 5.20 (d, J = 8.5 Hz, 1H), 4.40-4.38 (m, 3H), 4.24-4.16 (m, 3H), 1.71-1.60 (m, 2H), 1.56

-1.51 (m, 1H), 1.27 (t, J = 7.1 Hz, 3H), 0.95 (dd, J = 6.1, 3.4 Hz, 6H); 13C NMR (100 MHz, CDCl3):

δ 173.3, 156.1, 144.1, 143.9, 141.4, 127.8, 127.1, 125.2, 120.1, 120.1, 67.0, 61.5, 52.6, 47.3, 42.0,

24.8, 23.0, 22.0, 14.3. HRMS (FAB) calcd for C23H28NO4 (M+H)+ 382.2018, found 382.2016.

Fmoc-L-Phe-OEt (6b): Rf = 0.2 (Hexane: Ethyl acetate, 8:2). White solid (19 mg, 90%). 1H NMR (400 MHz, CDCl3): δ

7.78 (d, J = 7.5 Hz, 2H), 7.58-7.55 (m, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.33-7.25 (m, 5H), 7.12 (d,

J = 6.9 Hz, 2H), 5.30 (d, J = 7.4 Hz, 1H), 4.67-4.64 (m, 1H), 4.46-4.42 (m, 1H), 4.36-4.32 (m,

1H), 4.23-4.15 (m, 3H), 3.12 (t, J = 5.6 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz,

CDCl3): δ 171.6, 155.6, 144.0, 143.9, 141.4, 135.9, 129.5, 128.7, 127.8, 127.2, 127.2, 125.2,

125.2, 120.1, 120.1, 67.0, 61.7, 54.9, 47.3, 38.4, 14.2. HRMS (FAB) calcd for C26H26NO4

(M+H)+ 416.1862, found 416.1859.

S5

Fmoc-L-Asp(OtBu)-OEt (6c): Rf = 0.5 (Hexane: Ethyl acetate, 8:2). White solid (19 mg, 87%). 1H NMR (400 MHz,

CDCl3): δ 7.76 (d, J = 7.5 Hz, 2H), 7.61-7.59 (m, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.4

Hz, 2H), 5.84 (d, J = 8 Hz, 1H), 4.60-4.58 (m, 1H), 4.44-4.40 (m, 1H), 4.36-4.32 (m, 1H), 4.26-

4.21 (m, 3H), 2.97-2.92 (dd, J= 16.8, 4.7 Hz, 1H), 2.80-2.75 (dd, J= 16.8, 4.5 Hz, 1H), 1.45 (s,

9H), 1.27 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 171.0, 170.1, 156.1, 144.0, 143.9,

141.4, 127.8, 127.2, 125.3, 125.2, 120.1, 81.9, 67.3, 61.9, 50.7, 47.2, 37.9, 28.1, 14.2. HRMS

(FAB) calcd for C25H30NO6 (M+H)+ 440.2073, found 440.2074.

Fmoc-L-Gln(Trt)-OEt (6d): Rf = 0.4 (Hexane: Ethyl acetate, 6:4). White solid (27 mg, 86%). 1H NMR (400 MHz, CDCl3):

δ 7.77 (d, J = 7.5 Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.43-7.38 (m, 2H), 7.34-7.23 (m, 17H), 5.66

(d, J = 8 Hz, 1H), 4.48-4.449 (m, 1H), 4.40-4.33 (m, 2H), 4.25-4.16 (m, 3H), 2.39-2.34 (m, 2H),

2.25-2.21 (m, 1H), 1.96-1.90 (m, 1H), 1.27 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ

172.2, 171.0, 156.5, 144.8, 144.0, 143.8, 141.5, 128.8, 128.1, 127.9, 127.2, 127.2, 125.3, 120.1,

70.8, 67.1, 61.8, 53.8, 47.3, 31.7, 22.8, 14.3. HRMS (FAB) calcd for C41H39N2O5 (M+H)+

639.2859, found 639.2861.

Fmoc-L-His(Trt)-OEt (6e): Rf = 0.3 (Hexane: Ethyl acetate, 6:4). White solid (26 mg, 82%). 1H NMR (400 MHz, CDCl3):

δ 7.75 (d, J = 7.5 Hz, 2H), 7.65-7.61 (m, 2H), 7.43-7.33 (m, 3H), 7.32-7.30 (m, 9H), 7.28-7.27

(m, 2H), 7.13-7.12 (m, 6H), 6.59 (m, 2H), 4.63-4.60 (m,1H), 4.39-4.25 (m, 3H), 4.13-4.06 (m,

2H), 3.10 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 171.7, 156.3, 144.2,

144.1, 142.4, 142.4, 141.3, 141.3, 139.0, 136.5, 129.9, 128.2, 127.2, 125.5, 125.4, 120.0, 119.7,

75.4, 67.3, 61.2, 54.4, 47.3, 29.8, 14.3. HRMS (FAB) calcd for C42H38N3O4 (M+H)+ 648.2862,

found 648.2859.

Fmoc- L-Cys(Trt)-OEt (6f): Rf = 0.5 (Hexane: Ethyl acetate, 8:2). White solid (26 mg, 84%). 1H NMR (400 MHz, CDCl3):

δ 7.77 (d, J = 7.5 Hz, 2H), 7.63 (m, 2H), 7.42-7.38 (m, 8H), 7.33-7.19 (m, 11H), 5.32-5.28 (m,

1H), 4.39-4.26 (m, 3H), 4.21-4.17 (m, 3H), 2.66 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H); 13C NMR

(100 MHz, CDCl3): δ 170.6, 155.7, 144.4, 144.0, 143.9, 141.4, 141.4, 129.6, 128.1, 127.9, 127.8,

127.2, 127.0, 125.3, 125.3, 120.1, 67.2, 61.9, 53.1, 47.2, 29.8, 14.2. HRMS (FAB) calcd for

C39H36NO4S (M+H)+ 614.2365, found 614.2366.

S6

Fmoc-L-Lys(Boc)-OEt (6g): Rf = 0.4 (Hexane: Ethyl acetate, 8:2). White solid (22 mg, 88%). 1H NMR (400 MHz, CDCl3):

δ 7.76 (d, J = 7.4 Hz, 2H), 7.59 (dd, J = 7.1, 2.4 Hz, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.30 (t, J =

7.5 Hz, 2H), 5.45 (d, J = 7.6 Hz , 1H), 4.59 (m, 1H), 4.40-4.33 (m, 3H), 4.23-4.17 (m, 3H), 3.10

(m, 2H), 1.85-1.82 (m, 1H), 1.71-1.66 (m, 1H), 1.49-1.47 (m, 1H), 1.42 (s, 9H), 1.37-1.35 (m,

2H), 1.26 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 172.6, 156.2, 144.0, 143.9, 141.4,

127.8, 127.2, 125.2, 120.1, 79.2, 67.1, 61.6, 53.9, 47.3, 40.2, 32.3, 29.7, 28.5, 22.4, 14.3. HRMS

(FAB) calcd for C26H26NO4 (M+H)+ 497.2652, found 497.2654

Fmoc-L-Trp(Boc)-OEt (6h): Rf = 0.4 (Hexane: Ethyl acetate, 8:2). White solid (24 mg, 86%). 1H NMR (400 MHz, CDCl3):

δ 8.13 (m, 1H), 7.76 (d, J = 7.5 Hz, 2H), 7.58-7.53 (m, 3H), 7.44-7.37 (m, 4H), 7.33-7.22 (m,

4H), 5.49 (d, J = 8.2 Hz,1H), 4.77-4.75 (m, 1H), 4.41-4.37 (m, 2H), 4.22-4.15 (m, 3H), 3.28 (m,

1H), 1.66 (m, 9H), 1.23 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 171.7, 155.8, 143.9,

141.4, 127.8, 127.2, 127.2, 125.3, 125.2, 124.7, 124.3, 122.8, 120.1, 119.0, 115.4, 115.1, 83.8,

67.3, 61.8, 54.3, 47.2, 28.3, 14.2. HRMS (FAB) calcd for C33H35N6O2 (M+H)+ 555.2495, found

555.2498.

Fmoc-L-Arg(Pbf)-OEt (6i): Rf = 0.4 (Hexane: Ethyl acetate, 5:1). White solid (26 mg, 78%). 1H NMR (400 MHz, CDCl3):

δ 7.72 (d, J = 7.5 Hz, 2H), 7.54 (d, J = 7.0 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H), 7.26-7.23 (m, 2H),

5.73 (d, J = 4.8 Hz, 1H), 4.34-4.24 (m, 2H), 4.16-4.11 (m, 3H), 3.21 (m, 2H), 2.88 (s, 2H), 2.56

(s, 3H), 2.48 (s, 3H), 2.05 (s, 3H), 1.88-1.80 (m, 2H), 1.68-1.64 (m, 1H), 1.57-1.56 (m, 2H),

1.40 (s, 6H), 1.21 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 172.3, 158.8, 156.3, 143.9,

143.7, 141.3, 138.4, 132.3, 127.8, 127.2, 125.2, 124.7, 120.1, 117.6, 86.5, 67.2, 61.7, 47.2, 43.3,

38.7, 28.6, 22.7, 19.4, 18.0, 14.2, 12.5. HRMS (FAB) calcd for C36H45N4O7S (M+H)+ 677.3009,

found 677.3007.

Fmoc-L-Met-OEt (6j) Rf = 0.4 (Hexane: Ethyl acetate, 5:1). White solid (18 mg, 90%). 1H NMR (400 MHz, CDCl3): δ

7.77 (d, J = 7.5 Hz, 2H), 7.60 (dd, J = 7.3, 2.6 Hz, 2H), 7.41 (t, J = 7.3 Hz, 2H), 7.32 (tt, J = 7.4,

1.3 Hz, 2H), 5.44 (d, J = 8.2 Hz, 1H), 4.51-4.46 (m, 1H), 4.43-4.41 (d, J = 7.0 Hz, 2H), 4.25-

4.20 (m, 2H), 2.55-2.51 (m, 2H), 2.18-2.13 (m, 1H), 2.10 (s, 3H), 2.02-1.93 (m, 1H), 1.29 (t, J

= 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 172.0, 155.9, 143.8, 143.7, 141.3, 127.70, 127.0,

125.0, 120.0, 119.9, 67.0, 61.7, 53.2, 47.1, 32.0, 29.9, 15.5, 14.2. HRMS (FAB) calcd for C22H26NO4S (M+H)+ 400.1583,

found 400.1581.

HN

NH

NH

SO

O

O

FmocHN OEt

O

S7

General procedure for nucleophilic acyl substitution on peptide

Peptide was prepared according to Fmoc-strategy SPPS on rink amide resin (0.05 mmol), as outlined in the general

procedures. The resin-bound o-aminoanilide peptide was washed with DMF, then treated with isoamyl nitrite (10 equiv.) in

DMF at room temperature for 90 min. After the resin was filtered, washed sequentially with DCM (3 mL x 3). The

nucleophiles (4 equiv.) and DIPEA (8 equiv.) in DMF was bubbled with N2 for 20 min and then immediately added into the

activated peptidyl-resin. After 2 h, the resin was washed with DMF (3 mL x 3) and the supernatant was concentrate under

vacuo. Then, the resulting product was deprotected by using 20% piperidine in DMF for 30 min and TFA cocktail for 1.5

h. The peptide was precipitated in cold ether and collected by centrifugation. The supernatant was removed and the residue

was dissolved in 30% acetonitrile/H2O and lyophilized to afford pure product without further purification.

HSSKLQL (8a)

H2N

N NH

NH

OOH

HN

OOH

NH

O

NH2

HN

O

NH

O

H2N O

HN

O

O

OH

8a

Peptide 8a was prepared according to Fmoc-strategy SPPS, as outlined in the general procedures. After the peptide-resin

was activated and cleavage with ACN/H2O (1:1) in the presence of DIPEA (8 equiv.) in DMF (4 mL) for 2 h at room

temperature. The Fmoc was deprotected with 20% piperidine in DMF and global deprotection was effected using a solution

of TFA/TIS/H2O (95:2.5:2.5, 4 mL) affording pure peptide 8a as a white solid after lyophilization (34 mg, 82% yield). 1H

NMR (400 MHz, D2O): δ 8.77 (s, 1H), 7.51 (s, 1H), 4.66-4.63 (m, 1H), 4.60-4.58 (m, 1H), 4.46-4.44 (m, 3H), 4.39-4.31

(m, 2H), 3.97-3.92 (m, 4H), 3.50-3.48 (m, 2H), 3.06-3.02 (m, 2H), 2.55-2.52 (t, 1H), 2.18-2.14 (m, 1H), 2.06-2.02 (m, 1H),

1.90-1.87 (m, 1H), 1.75-1.72 (m, 9H), 1.50-1.47 (m, 2H), 0.99-0.93 (m, 12H). ESI-FTMS calcd for C35H61N11O11 (M+H)+

812.4630, found 812.4633.

Figure S1. HPLC chromatogram of compound 8a (linear gradient, 10 to 90% B over 40 min, 0.1% TFA, flow rate: 0.4 ml/min, λ = 220 nm)

S8

HSSKLQL- propargyl amine (8b)

H2N

N NH

NH

OOH

HN

OOH

NH

O

NH2

HN

O

NH

O

H2N O

HN

O

O

NH

8b

Peptide 8b was prepared according to Fmoc-strategy SPPS, as outlined in the general procedures. After the peptide-resin

was activated and cleavage with propargyl amine (4 equiv.) in the presence of DIPEA (8 equiv.) in DMF(4 mL) for 2 h at

room temperature. Then Fmoc was deprotected with 20% piperidine in DMF and global deprotection was effected using a

solution of TFA/TIS/H2O (95:2.5:2.5, 4 mL) affording pure peptide 8b as a white solid after lyophilization (33 mg, 78%

yield). 1H NMR (400 MHz, D2O): δ 8.77 (s, 1H), 7.52 (s, 1H), 4.66 (t, J = 5.8 Hz, 1H), 4.60 (t, J = 5.4 Hz, 1H), 4.46-4.37

(m, 5H), 4.04-3.93 (m, 6H), 3.51-3.48 (m, 2H), 3.05 (t, J = 7.5 Hz, 2H), 2.66-2.64 (m, 1H), 2.66-2.64 (m 2H), 2.16-2.13 (m,

1H), 2.07-2.03 (m, 1H), 1.93-1.46 (m, 10H), 1.52-1.46 (m, 2H), 0.98 (d, J = 5.4 Hz, 6H), 0.93 (d, J = 5.7 Hz, 6H). MS(ESI):

calcd for C38H64N12O10 (M+H)+ 849.4941, found 849.4944.

Figure S2. HPLC chromatogram of compound 8b (linear gradient of 10 to 90% B over 40 min, 0.1% TFA, flow rate: 0.4 ml/min, λ = 220 nm)

S9

LMYKA-aniline (8c)

H2N NH

O

S

HN

O

OH

NH

O

NH2

HN

O

NH

O

8c

Peptide 8c was prepared according to Fmoc-strategy SPPS, as outlined in the general procedures. After the peptide-resin

was activated and cleavage with aniline (4 equiv.) in the presence of DIPEA (8 equiv.) in DMF (4 mL) for 5 h at room

temperature. Then Fmoc was deprotected with 20% piperidine in DMF and global deprotection was effected using a solution

of TFA/phenol/water/thioanisole/EDT (82.5:5:5:5:2.5, 4 mL) for 3 h affording pure peptide 8c as a white solid after

lyophilization (22 mg, 66% yield). 1H NMR (400 MHz, CD3OD): δ 7.54 (d, J = 8 Hz, 2H), 7.28 (t, J = 7.0 Hz, 2H), 7.09-

7.02 (m, 3H), 6.68-6.65 (m, 2H), 4.52-4.42 (m, 3H), 4.35-4.33 (m, 2H), 3.92-3.90 (m, 1H), 3.12-3.10 (m, 1H), 3.00-2.83

(m, 6H), 2.65-2.62 (m, 1H), 2.51-2.45 (m, 2H), 2.04 (s, 3H), 1.65-1.63 (m, 8H), 1.45-1.38 (m, 6H), 0.95-0.94 (m, 6H).

MS(ESI): calcd for C38H64N12O10 (M+H)+ 700.3850, found 700.3851.

Figure S3. HPLC chromatogram of compound 8c (linear gradient of 10 to 90% B over 40 min, 0.1% TFA flow rate: 0.4 ml/min, λ = 220 nm)

S10

Ac-AYRGA-mercaptopropionic acid (8d)

NH

OHN

O

OH

NH

O

HN

HN NH2

HN

O

NH

O

S

O

OH

O

8d

Peptide 8d was prepared according to Fmoc-strategy SPPS, as outlined in the general procedures. The Fmoc was deprotected

with 20% piperidine in DMF peptide-resin and then added 2 equvi. of acetic anhydride in DMF for 10 min. Thereafter, the

peptide-resin was activated and cleavage with 3-mercaptopropionic acid (4 equiv.) in the presence of DIPEA (8 equiv.) in

DMF (4 mL) for 2 h at room temperature. The global deprotection was effected using a solution of TFA/TIS/H2O (95:2.5:2.5,

4 mL) affording pure peptide 8d as a white solid after lyophilization (27 mg, 80% yield). 1H NMR (400 MHz, CD3OD): δ

7.03 (d, J = 8.5 Hz, 2H), 6.68 (d, J = 8.5 Hz, 2H), 4.49-4.44 (m, 2H), 4.30-4.27 (m, 1H), 4.20-4.1 (m, 1H), 3.87 (s, 2H),

3.29-3.28 (m, 1H), 3.25-3.21 (m, 2H), 3.04-3.01 (m, 3H), 2.91-2.84 (m, 1H), 2.54 (t, J = 7.0 Hz, 2H), 1.93 (s, 3H), 1.88-

1.84 (m, 1H), 1.59-1.55 (m, 2H), 1.34 (d, J = 7.3 Hz, 3H), 1.23 (d, J = 7.2 Hz, 3H). MS(ESI): calcd for C38H64N12O10

(M+H)+ 667.2868, found 667.2865.

Figure S4. HPLC chromatogram of compound 8d (linear gradient of 10 to 90% B over 40 min, 0.1% TFA, flow rate: 0.4 ml/min, λ = 220 nm).

S11

Preparation of peptide decorated dendrimer

Scheme S2:

Peptide was prepared according to Fmoc-strategy SPPS on rink amide resin (0.2 mmol), as outlined in the general procedures.

At the end of the synthesis, the resin-bound o-aminoanilide-peptide was washed with DMF and DCM, then treated with

isoamyl nitrite (10 equiv.) at room temperature for 90 min. The resin was filtered, washed with DCM (3 mL x 3). The

solvent DMF (4 ml) with DIPEA (8 equiv.) was bubbled with N2 for 20 min. Then, it was treated with peptidyl resin and

PAMAM dendrimer. After 2 h, the resin was washed with DMF and the solution was concentrate in vacuo. The resulting

product was deprotected by using 20% piperidine in DMF for 30 min and then precipitated with cold ether (25 mL x 2) and

collected by centrifugation. After that, the product was concentrate under vacuo and then treated with TFA/TIS/H2O

(95:2.5:2.5 v/v/v, 4 mL) was used to remove the protecting group at 25 °C for 1.5 h. Finally, the resulting peptide decorated

dendrimer was allowed to precipitated with cold ether (25 mL x 2) and collected by centrifugation. The supernatant was

removed and the residue was dissolved in 30% ACN/H2O and lyophilized to afford pure product without further purification.

PDI value was obtained from Gel Permeation chromatogram(GPC) instrumentation. Conditions: preparation of

compound 11a-f (1 mg/ml in H2O); TSK G2000SW column; elute: 0.1 % TFA in H2O; follow rate: 1 ml/min;

detector: UV detector @ 214nm. A typical combined SEC of a reference G0-G7 PAMAM dendrimer having MW

from 517 to 116,000 dalton.

S12

(G:5)-dendric- PAMAM- (Leu)60 (8e)

H2N

O

NHG5

PAMAM

(NH2)

60

60

8e

White solid (11 mg, 79% yield). 1H NMR (400 MHz, D2O): δ 3.95-3.91 (m, 60H), 3.64-3.30 (m, 905H), 2.82-2.76 (m,

312H), 1.70-1.59 (m, 195H), 0.91-0.90 (m, 362H). PDI = 1.07.

Figure S5. GPC chromatogram of peptide decorated dendrimer 8e (isocratic of 0.1% TFA in DIW over 30 min, flow rate: 1.0 ml/min, λ = 214 nm)

min2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

250

300

350

DAD1 A, Sig=214,8 Ref=360,100 (D:\DATA\ANAND\DENDRIMER\G5-LEU.D)

NH

NH

NH

NH

NH

NH

NH

NH

NH

S13

(G:5)-dendric-PAMAM-( LQLKSSH)32 (8f)

N

ONH

O

HN

N

O NH

O

HN

NH

NH2

N O

O NH

N

O NH

O

HN

NH2

NH2

N

ONH

O

HN

NH2

NH2

NO

ONH

N

ONH

O

HN

NH2NH

N

O

HN

N

O NH

O

NH

N

N

O NH

O

NH

NHO

HN

O

N

ONH

O

HN

N

O

HN

O

HN

NH2

NH2

NO

NH

ONH

N

O NH

O

HN

NH2

NHN

O NH

O

HN

NH2

NH2

N O

O NH

N

O NH

O

HN

HNNH2

N

O

HN

O

NH

N

O

HN

O

NH

NH

NH2

N

O

NH

O

HN

N

O

HN

O

NH

NH2

NH2

N

O

HN

O

HN

NH2

NH2

NO

NH

ONH

N

O NH

O

HN

NH2

NH

N

O

HN

O

NH

N O

HN

ONH NH

NH2

N

ONH

O

HN N

O

HN

ONH

NH2

NH2

NO

HN

ONH NH

NH2

N

ONH

O

HN

N

O

HN

O

NH NH2

NH2

N O

HN

ONH

NO

HNO NH

NH2

NH

N

O NH

O

HN

N O

HN

O NHNH2

NH2

N O

HN

ONH NH2

NH

N

ONH

O

HN N

O

HN

ONH

NH2

NH2

OHN

N O

NHONH

N

N

O

HN

ONHH

N

O

HNO

N

O

HNONH

N

O

HNONH

NH2NH

N

O NH

O

HN

NO

HNO NH

NH2NH2

NO

HNO NH

NH2

NH

N

O NH

O

HN

N O

HNO NH

NH2

NH2

N

O

HN

O

NH

N

O

HN

O

NH

NH2NH

N

O O

HN

N

O

HNO

NH

NH2H2N

N

O

HNONH

NH2HN

N

O NH

O

HN

NO

HNO NH

NH2NH2

N

O

HN

O

NH

N

OHN

O

NH

H2NNH

NO

OHN

N

OHN

O

NH

H2NH2N

N

OHN

O

NH

NHH2N

N

OO

HN

N

OHN

ONH

NH2H2N

NH2

NHN

HN

OHO

NH

OHO

HN

O

H2N

NH

OHN

O

NH2O

NH

O

O

NH2

NHN

HN

OHO

NH

OHO

HN

O

NH2

NH

OHN

O

NH2O

NH

O

O

NH2

NHN

HN

OHO

NH

O

HOHN

O

H2N

NH

OHN

O

NH2O

NH

O

O

NH2

NHN

HN

O

HO

NH

OHO

HN

O

NH2

NH

O

HN

O

NH2O

NH

O

O

NH2

NHN

HN

O

HO

NH

OHO

HN

O

NH2

NH

O

HN

O

NH2O

NH

O

O

NH2

N

HN

HNO

HONH

O

HO

HNO

NH2NH

O

HNO

NH2

ONH

O

O

NH2N

HN HN O

OHNH

O

HO

HN ONH2

NH

O

HN ONH2

ONH

O

O

NH2

N

NH

HN

O

OH

NHO

HOHN

ONH2

NHO

HN

ONH2

O

NHO

O

NH2

NHN

NH

O

HO

HNO

HONH

O

H2N

HNO

NH

O

NH2

O

HNOO

NH2

NHN

NH

O

HO

HNO

HONH

O

H2N

HNO

NH

O

NH2

O

HNOO

NH2

NHN

HN

OHO

NH

OHOHN

O

NH2

NH

OHN

O

NH2O

NH

O

O

NH2

N

HN

HNO

HONH

O

HO

HNO

NH2

NH

O

HNO

NH2

O

NH

O

O

NH2N

HN HN O

OHNH

O

HO

HN ONH2

NH

O

HN ONH2

ONH

O

O

NH2

N

NH

HN

O

OH

NHO

HO HN

O

NH2

NHO

HN

ONH2

O

NHO

O

NH2

N

NH

HN

O

OH

NHO

HO HN

ONH2

NHO

HN

ONH2

O

NHO

O

N

OHN

O

NH

N

OHN

O

NH

NH

H2N

NO

OHN

N

OHN

O

NH

H2NH2N

N

OHN

O

NH

H2NH2N

NO

OHN

N

OHN

O

NH

H2NHN

N

ONH

N

OHN

O

HN

N

N

OHN

O

HN

HN O

NH

O

N

OHN

O

NH

N

ONH

O

HN

H2N

H2N

N

O

NH

OHN

N

OHN

O

NH

NH

H2N N

OHN

O

NH

H2N

H2N

NO

OHN

N

OHN

O

NH

NHH2N

N

O

NH

O

HN

N

ONH

O

HN

NH

H2N

N

O

NH

O

NH

N

ONH

O

HN

H2N

H2N

N

ONH

O

HN

H2N

H2N

N

O

NH

OHN

N

OHN

O

NH

H2N

NH

N

O

NH

O

HN

NO

NH

OHNNH

H2N

N

O

NH

O

NHN

O

NH

O

HNH2N

H2N

NO

NH

O

HN

NH

H2N

N

O

NH

O

NH

N

ONH

O

HN

H2N

H2N

NO

NH

OHN

NO

NHOHN

H2N NH

N

O

NH

O

NH

NO

NH

OHNH2N

H2N

NO

NH

OHNH2N

NH

N

O

NH

O

NHN

O

NH

O

HNH2N

H2N

ONH

NO

HN OHN

N

N

O

NH

OHN

NH

O

NHO

N

O

NH OHN

N

O

NH OHN

H2N NH

N

ONH

O

NH

N

O

NH OHN

H2NH2N

NO

NH OHN

H2N NH

N

O

NH

O

NH

NO

NHOHN

H2NH2N

N

O

NH

O

HN

N

ONH

O

HN

H2N HN

N

OO

NH

N

O

NHO

HN

H2N NH2

N

O

NH OHN

H2N NH

N

ONH

O

NH

NO

NH OHN

H2NH2N

N

O

NH

O

HN

N

O NH

O

HN

NH2

NH

NO

O NH

N

ONH

O

HN

NH2

NH2

N

ONH

O

HN

HNNH2

N

OO

NH

N

ONH

OHN

NH2NH2

H2N

N NH

NH

O OH

HN

OOH

NH

O

NH2

HN

O NH

O

H2N O

HN

O

O

H2N

N NH

NH

OOH

HN

OOH

NH

O

NH2

HN

O

NH

O

H2N O

HN

O

O

H2N

N NH

NH

O OH

HN

O

OH

NH

O

NH2

HN

O NH

O

H2N O

HN

O

O

H2N

NNH

NH

O

OHHN

O OH

NH

O

H2N

HN

O

NH

O

H2NO

HN

O

O

H2N

NNH

NHO

OHHN

OOH

NHO

H2N

HN

O

NHO

H2NO

HN

O

O

H2NN

NH

NHO

OHHN

O

OH

NHO

H2N HN

O

NHO

H2N

OHN

O

O

H2NN

NHNHO

HOHN

O

OH

NHO

H2N

HN

O

NHO

H2N

OHN

O

O

H2N

N

HN

NH

O

HO

HNO

OHNH

OH2N

HNO

NH

OH2N

O

HNO

O

H2N

NNH

HN

O

OH

NHO

OHHN

O

NH2

NHO

HN

O

H2N

O

NHO O

H2N

NNH

HN

O

OH

NHO

OHHN

O

NH2

NHO

HN

O

H2N

O

NHO O

H2N

N NH

NH

OOH

HN

O OH

NH

O

H2N

HN

O

NH

O

H2N O

HN

O

O

H2N

N

NH

NHO

OHHN

O

OH

NHO

H2NHN

O

NHO

H2N

O

HN

O

O

H2NN

NHNHO

HOHN

O

OH

NHO

H2N

HN

O

NHO

H2N

OHN

O

O

H2N

N

HN

NH

O

HO

HN O

OHNH

O

H2N

HN O

NH

OH2N

O

HN O

O

H2N

N

HN

NH

O

HO

HN O

OHNH

O

H2N

HN O

NH

OH2N

O

HN O

O

H2N

N NH

NH

O OH

HN

O

OH

NH

O

NH2

HN

O NH

O

H2N O

HN

O

O

NH2

NHN

HN

OHO

NH

O

HOHN

O

H2N

NH

OHN

O

NH2O

NH

O

O

C2382H4416N858O572

Exact Mass: 54198.2840

White solid (12 mg, 70 %). 1H NMR (400 MHz, D2O): δ 8.77 (s, 32H), 7.52 (s, 32H), 4.38-4.37 (m, 212H), 3.94 (m, 210H),

3.72-3.22 (m, 1739H), 3.05 (m,130H), 2.88-2.70 (m, 529H), 2.41(m, 103H), 1.82-1.37 (m, 410H), 0.98-0.93 (m, 388H).

PDI = 1.10.

Figure S6. GPC chromatogram of peptide decorated dendrimer 8f (isocratic of 0.1% TFA in DIW over 30 min, flow rate: 1.0 mL/min, λ = 214 nm)

min0 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

VWD1 A, Wavelength=214 nm (D:\DATA\ANAND\DENDRIMER\G5-PSA..D)

S14

Gel Permeation Chromatography

The experiments were conducted at 25oC at a flow rate of 1.0 mL/min with 0.1% TFA in DIW as eluent and detected at 214 nm. The results of filtration were followed with the figures that (8a) (9.323 min), ((G:5)-dendri-PAMAM -(NH2)128) (6.774 min), (8e) (6.016 min) and (8f) (5.395 min) are discovered.

Figure S7. GPC chromatogram of compound 8a, (G:5)-dendri-PAMAM -(NH2)128, 8e and 8f.

Figure S8. 1H NMR Spectrum of Compound 8a, 8f, 8e and (G:5)-dendri-PAMAM -(NH2)128

S15

Preparation of cyclic peptide

HN

N

HN

HNNH

N

N

NH

NH

NH

NH

HN

HN

HN

O

O

O

O O

O

O

O

O

O

OO

O

O

OH

NH2

HO

NH

NH2

HN

HO

O

S

S

Asp

Pro

Phe

Cys

IlePro

Pro

Ile

Ser

Lys

CysArg

GlyThr

11

The linear SFTI-1 peptide was prepared according to Fmoc-strategy SPPS on rink amide resin (0.05 mmol), as outlined in

the general procedures. At the end of the synthesis, the Fmoc protected peptide-resin was deprotected with 20% piperidine

in DMF and washed with DMF (3 mL x 3). The free N-terminal amine resin-bound o-aminoanilide-peptide is activated with

isoamyl nitrite (10 equiv.) at room temperature for 90 min. The resin was filtered, washed with DCM (3 mL x 3). The on

resin N-terminal amine is reacted with C-terminal acid and cleaved from resin in the presence of DIPEA (4 equiv.) in DMF

(3 mL) for 5 h at room temperature. Then global deprotection was effected using a solution of TFA/TIS/H2O (95:2.5:2.5, 4

mL) affording the oxidized SFTI-1 peptide was acquired 11 as a white solid after lyophilization (12 mg, 42% yield). 1H

NMR (600 MHz, D2O): δ 8.23 (s, 1H), 7.51-7.15 (m, 5H), 4.58-4.24 (m, 6H), 4.23-4.05 (m, 2H), 4.06-3.57 (m, 8H), 3.56-

3.27 (m, 4H), 3.17 (m, 3H), 2.94 (m, 5H), 2.24 (m, 4H), 2.17-1.78 (m, 10H), 1.75 (m, 4H), 1.57-1.34 (m, 4H), 1.29-1.04

(m, 4H), 1.02-0.66 (m, 8H). Mass (MALDI, m/z) calcd for C67H105N18O18S2 (M+H)+ 1513.7, found 1513.6.

Figure S9. HPLC chromatogram of compound 11 (linear gradient of 10 to 90% B over 30 min, 0.1% TFA, flow rate: 0.5 ml/min, λ = 220 nm)

S16

Figure S10. MALDI-TOF MS spectrum of cyclic peptide 11

Figure S11. MALDI-TOF MS spectrum of linear peptide SFTI-1.

S17

Synthesis of VPGVG (13)

Peptide was prepared according to the Fmoc-strategy SPPS on rink amide resin (0.1 mmol/g loading; 0.78 mmol). The

peptide was synthesized on an automated PS3 peptide synthesizer using Fmoc-Xaa-OH (2 equiv.), NMM (4 equiv.) and

HBTU (2 equiv.). The coupling was performed for 60 min and Fmoc-deprotection was achieved using 20% piperidine (10

min x 2). After washing the resin, the cleavage was carried out by treating the resin with cleavage cocktail TFA: H2O (95:5)

for 1.5 h. Then the solution was precipitation with cold Et2O and centrifugation the crude peptides can be obtained. After

lyophilisation, the desired peptide was afford in 86 % yield. 1H NMR (400 MHz, D2O): δ 4.39- 4.35 (m, 1H), 4.06-4.02 (m,

2H), 3.85-3.77 (m, 4H), 3.68-3.50 (m, 2H), 2.21-1.81 (m, 6H), 0.98 (d, J = 7.0 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H), 0.82 (dd,

J = 6.8, 5.3 Hz, 6H). ESI MS calcd for C19H35N6O5 (M+H)+ 427.2, found 427.2.

NH2

OHN

O

NH

OHN

O

N

13O

H2N


Figure S13. ESI-MS spectrum of compound 13

min5 10 15 20 25

Norm.

0

200

400

600

800

DAD1 C, Sig=220,8 Ref=360,100 (D:\DATA\ANAND\20170427\023-0301.D)

12

.83

2

S18

General procedure for preparation of elastin peptide

Scheme S3:

Peptide was prepared according to Fmoc-strategy SPPS on rink amide resin, as outlined in the general procedures. The

resin-bound o-aminoanilide peptide was washed with DMF, then treated with isoamyl nitrite (10 equiv.) in DMF at room

temperature for 90 min. After the resin was filtered, washed sequentially with DCM (3 mL x 3). The peptide nucleophile

and DIPEA (4 equiv.) in DMF was bubbled with N2 for 20 min and then immediately added into the activated peptidyl-

resin. After 2 h, the resin was washed with DMF (3 mL x 3) and the supernatant was concentrate. The resulting product was

deprotected by using 20% piperidine in DMF for 30 min, thereafter completely removing the DMF. The peptide was

precipitated in cold ether and collected by centrifugation. The supernatant was removed and the residue was dissolved in

30% acetonitrile/H2O and lyophilized to afford pure product without further purification.

S19

(VPGVG)2 (14)

Peptide 7g (0.05 mmol) was prepared according to Fmoc-strategy SPPS, as outlined in the general procedures. After the

peptide-resin was activated and cleavage with compound 13 (17 mg, 0.04 mmol) in the presence of DIPEA (4 equiv.) in

DMF for 2 h at room temperature. Then Fmoc was deprotected with 20% piperidine in DMF affording pure peptide 14 as a

white solid after lyophilization (31 mg, 92% yield). 1H NMR (400 MHz, D2O): δ 4.38-4.27 (m, 3H), 4.06-4.01 (m, 3H),

3.85-3.65 (m, 9H), 3.78-3.50 (m, 3H), 2.26-1.77 (m, 12H), 0.99 (d, J = 7.0 Hz, 3H). 0.92-0.78 (m, 21H). Mass (MALDI,

m/z) calcd for C38H66N11O10 (M+H)+ 836.4, found 836.5.

NH2

OHN

O

NH

OHN

O

N

O

NH

14 2

H


Figure S15. MALDI-TOF MS spectrum of compound 14

S20

(VPGVG)3 (15)



DMF for 2 h at room temperature. Then Fmoc was deprotected with 20% piperidine in DMF affording pure peptide 15 as

a white solid after lyophilization (35 mg, 93% yield). 1H NMR (400 MHz, D2O): δ 4.38-4.27 (m, 4H), 4.06-400 (m, 4H),

3.84-3.77 (m, 12H), 3.66-3.50 (m, 4H), 2.21-1.78 (m, 18H), 0.99 (d, J = 7.0 Hz, 3H). 0.92-0.79 (m, 33H). Mass (MALDI,

m/z) calcd for C57H96N16NaO15 (M+Na)+ 1267.7, found 1267.5.

NH2

OHN

O

NH

OHN

O

N

O

NH

15 3

H



S21

(VPGVG)4 (16)



DMF for 2 h at room temperature. Then Fmoc was deprotected with 20% piperidine in DMF affording pure peptide 16 as a

white solid after lyophilization (30 mg, 90% yield). 1H NMR (400 MHz, D2O): δ 4.36-4.28 (m, 6H), 4.06-4.01 (m, 5H),

3.84-3.72 (m, 20H), 3.65-3.50 (m, 5H), 2.24-1.78 (m, 24H), 0.99 (d, J = 7.0 Hz, 3H), 0.91-0.71 (m, 45H). Mass (MALDI,

m/z) calcd for C76H127N21NaO20 (M+Na)+ 1676.9, found 1676.6.

NH2

OHN

O

NH

OHN

O

N

O

NH

16 4

H

Figure S18. HPLC chromatogram of compound 16 (linear gradient of 10 to 90% B over 30 min, 0.1% TFA, flow rate: 0.5 ml/min, λ = 220 nm).


S22

NMR Spectra

Figure S20. 1H and 13C NMR spectrum of compound 6a

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

14.2

6

21.9

622

.95

24.8

4

41.9

547

.29

52.6

1

61.4

6

67.0

2

120.

0512

0.07

125.

1812

7.14

127.

78

141.

4014

3.86

144.

05

156.

07

173.

29

S23

Figure S21. 1H and 13C NMR spectrum of compound 6b

3.20

1.95

3.47

1.11

1.28

0.97

0.86

1.91

5.92

2.30

2.37

2.37

1.22

51.

242

1.26

0

3.10

83.

122

3.13

64.

153

4.17

14.

189

4.20

84.

227

4.34

44.

362

4.41

94.

437

4.44

54.

463

5.28

05.

298

7.10

27.

119

7.24

87.

268

7.28

87.

309

7.32

87.

383

7.40

17.

420

7.55

17.

567

7.58

47.

757

7.77

6

14.2

2

38.4

0

47.2

7

54.8

9

61.6

5

67.0

3

120.

0712

0.09

125.

1612

5.23

127.

1512

7.21

127.

8112

8.65

129.

4813

5.89

141.

4114

3.86

143.

97

155.

64

171.

58

S24

Figure S22. 1H and 13C NMR spectrum of compound 6c

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

3.11

8.80

0.95

0.85

3.00

0.93

1.00

0.87

0.85

1.97

2.02

1.90

2.00

1.25

61.

274

1.29

21.

454

2.74

82.

759

2.79

02.

801

2.91

92.

931

2.96

22.

973

4.21

24.

229

4.23

34.

245

4.25

14.

262

4.31

54.

342

4.36

04.

400

4.41

84.

426

4.58

14.

602

5.82

45.

844

7.28

57.

304

7.32

37.

373

7.39

17.

410

7.58

77.

602

7.61

87.

745

7.76

4

14.2

3

28.1

3

37.9

2

47.2

050

.70

61.8

9

67.3

3

81.8

6

120.

0812

5.23

125.

2812

7.16

127.

81

141.

3914

3.84

144.

02

156.

08

170.

1117

0.99

S25

Figure S23. 1H and 13C NMR spectrum of compound 6d

2.86

0.95

1.02

1.61

2.87

1.67

1.03

0.81

17.0

82.

021.

842.

00

1.25

31.

271

1.28

81.

900

1.91

81.

936

1.95

82.

206

2.21

62.

234

2.33

62.

353

2.37

52.

395

4.18

24.

188

4.19

94.

206

4.21

64.

232

4.25

04.

358

4.37

64.

384

4.40

34.

437

4.45

54.

464

5.63

55.

655

7.22

77.

230

7.24

67.

259

7.26

27.

279

7.29

27.

297

7.30

27.

311

7.31

57.

320

7.32

37.

338

7.37

57.

394

7.41

17.

425

7.59

77.

615

7.75

47.

773

0102030405060708090100110120130140150160170180190200f1 (ppm)

S26

Figure S24. 1H and 13C NMR spectrum of compound 6e

3.04

1.66

2.09

2.89

0.83

1.70

5.91

14.1

61.

792.

00

1.14

61.

164

1.18

2

3.08

83.

098

4.06

94.

080

4.08

64.

104

4.11

14.

113

4.12

04.

129

4.24

74.

266

4.27

44.

299

4.31

84.

349

4.36

74.

374

4.39

24.

614

4.62

24.

634

6.56

46.

593

7.11

17.

118

7.12

47.

132

7.24

77.

249

7.26

77.

268

7.28

27.

301

7.31

27.

318

7.32

67.

358

7.37

77.

395

7.42

67.

612

7.63

17.

649

7.74

07.

758

0102030405060708090100110120130140150160170180190200f1 (ppm)

S27

Figure S25. 1H and 13C NMR spectrum of compound 6f

3.12

1.89

3.09

2.82

0.94

11.4

88.

151.

782.

00

1.24

21.

260

1.27

7

2.66

2

4.16

54.

177

4.19

54.

211

4.22

64.

244

4.26

14.

363

4.37

64.

392

5.27

95.

298

5.31

6

7.19

47.

197

7.21

57.

230

7.23

37.

248

7.26

07.

279

7.29

87.

309

7.32

87.

383

7.40

37.

424

7.61

57.

626

7.76

17.

779

0102030405060708090100110120130140150160170180190f1 (ppm)

14.2

4

29.8

1

47.2

0

53.0

7

61.9

2

67.2

4

120.

0912

5.25

125.

2812

7.01

127.

2012

7.83

127.

8412

8.13

129.

6314

1.39

141.

4014

3.86

144.

0014

4.39

155.

70

170.

60

S28

Figure S26. 1H and 13C NMR spectrum of compound 6g

3.84

12.1

40.

890.

99

1.85

2.92

2.80

0.76

0.77

1.93

1.99

1.92

2.00

1.24

81.

266

1.28

41.

350

1.37

11.

420

1.47

01.

487

1.66

21.

676

1.69

61.

708

1.81

81.

830

1.84

21.

854

3.09

13.

106

4.16

64.

184

4.19

44.

202

4.21

14.

220

4.22

84.

329

4.33

64.

340

4.36

14.

384

4.40

2

5.43

75.

456

7.28

07.

298

7.31

77.

365

7.38

47.

402

7.58

17.

588

7.59

97.

605

7.73

87.

757

14.2

8

22.4

428

.50

29.6

932

.29

40.1

8

47.2

6

53.8

5

61.5

7

67.0

7

79.2

4

120.

0712

5.20

127.

1512

7.79

141.

3914

3.85

144.

02

156.

16

172.

56

S29

Figure S27. 1H and 13C NMR spectrum of compound 6h

3.64

9.23

1.70

3.02

2.11

0.85

0.82

4.24

3.11

2.92

2.00

0.97

1.22

11.

238

1.25

6

1.65

7

3.27

74.

169

4.18

64.

203

4.22

04.

368

4.38

74.

409

4.75

04.

763

4.76

9

5.47

05.

490

7.23

47.

248

7.25

37.

287

7.30

57.

321

7.33

17.

374

7.39

27.

410

7.44

57.

534

7.55

47.

750

7.76

8

8.13

5

14.2

1

28.2

9

47.2

3

54.3

2

61.8

1

67.2

9

83.8

0

115.

0511

5.42

118.

9712

0.09

122.

7712

4.29

124.

6912

5.24

125.

2712

7.17

127.

1912

7.81

141.

3814

3.87

155.

83

171.

71

S30

Figure S28. 1H and 13C NMR spectrum of compound 6i

3.02

6.00

1.67

1.05

1.81

2.93

2.96

2.91

0.63

1.86

1.88

2.89

2.83

0.93

2.31

2.23

2.03

1.91

2.00

1.19

81.

216

1.23

41.

402

1.54

01.

557

1.57

31.

644

1.65

81.

677

1.69

51.

798

1.81

11.

884

2.05

12.

484

2.56

02.

777

2.85

32.

854

2.88

12.

927

3.19

23.

206

4.11

34.

127

4.14

34.

160

4.24

34.

256

4.27

74.

306

4.32

44.

342

5.71

95.

731

6.22

3

7.22

77.

248

7.26

47.

332

7.35

07.

369

7.53

27.

550

7.70

87.

727

0102030405060708090100110120130140150160170180190200f1 (ppm)

12.5

414

.22

18.0

319

.37

22.7

428

.64

38.7

143

.26

47.1

6

61.7

4

67.2

1

86.4

7

117.

5912

0.06

124.

7212

5.19

127.

1712

7.82

132.

3213

8.41

141.

3414

3.73

143.

86

156.

3415

8.83

172.

33

S31

Figure S29. 1H and 13C NMR spectrum of compound 6j

3.40

1.16

2.76

0.98

1.91

3.15

1.86

1.04

0.94

2.07

2.06

2.04

2.00

1.27

61.

294

1.31

21.

967

1.98

62.

003

2.10

42.

164

2.51

22.

531

2.54

42.

551

4.19

94.

216

4.23

34.

249

4.40

84.

425

4.45

94.

478

4.49

14.

510

5.42

45.

444

7.29

87.

301

7.30

57.

317

7.32

07.

323

7.33

57.

338

7.34

27.

388

7.40

67.

424

7.58

97.

595

7.60

77.

614

7.75

97.

760

7.77

87.

779

14.1

515

.47

29.8

532

.03

47.1

4

53.1

7

61.7

0

66.9

6

119.

9511

9.97

125.

0312

7.03

127.

70

141.

2814

3.65

143.

83

155.

84

171.

95

S32

Figure S30. 1H NMR spectrum of compound 8a

Figure S31. 1H NMR spectrum of compound 8b

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

6.17

5.93

2.35

10.7

31.

061.

032.

040.

80

1.97

1.92

6.19

5.34

1.02

0.99

0.99

1.00

0.92

0.94

0.98

0.99

1.64

1.65

1.71

1.72

1.74

1.76

2.39

2.41

2.64

2.65

3.03

3.05

3.50

3.51

3.95

3.96

4.01

4.37

4.38

4.41

4.43

4.44

4.46

4.59

4.60

4.62

4.64

4.66

4.67

7.52

8.77

S33

Figure S32. 1H NMR spectrum of compound 8c

Figure S33. 1H NMR spectrum of compound 8d

6.54

6.92

8.53

2.99

3.04

6.62

1.15

1.27

1.90

3.37

2.65

3.19

1.82

1.87

0.92

0.93

0.94

0.94

0.95

0.95

0.96

0.97

1.38

1.39

1.40

1.43

1.45

1.61

1.63

1.64

1.65

1.66

1.67

1.77

2.04

2.05

2.49

2.62

2.63

2.64

2.65

2.77

2.83

2.88

2.89

2.90

2.90

2.91

2.96

3.11

3.27

3.28

3.28

3.29

3.29

3.32

4.33

4.34

4.35

4.49

4.85

6.65

6.65

6.65

6.66

6.67

6.67

6.68

7.02

7.02

7.03

7.04

7.04

7.05

7.06

7.07

7.26

7.28

7.30

7.53

7.53

7.55

7.55

3.00

2.99

2.02

1.02

0.98

2.90

1.84

1.45

2.94

2.28

1.40

1.76

1.00

0.90

1.82

2.04

2.00

1.22

1.24

1.33

1.35

1.55

1.57

1.59

1.69

1.70

1.72

1.73

1.84

1.86

1.88

1.93

2.54

2.55

3.01

3.03

3.04

3.28

3.28

3.29

3.87

4.16

4.17

4.19

4.20

4.27

4.28

4.29

4.30

4.44

4.46

4.46

4.47

4.48

4.49

6.67

6.69

7.02

7.04

S34

Figure S34. 1H NMR spectrum of compound 8e

Figure S35. 1H NMR spectrum of compound 8f

362.

09

195.

28

312.

32

905.

84

60.5

0

0.80

0.81

1.49

1.51

1.51

1.53

1.56

1.58

1.60

2.66

2.71

2.72

2.72

2.72

3.20

3.21

3.22

3.39

3.52

3.52

3.52

3.52

3.52

3.52

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.53

3.54

3.54

3.54

3.81

3.83

3.85

388.

60

410.

74

103.

30

529.

00

130.

20

1739

.50

210.

56

212.

42

31.8

4

32.9

6

0.92

0.98

1.65

1.66

1.72

1.74

1.74

1.74

2.41

2.41

2.41

2.91

3.45

3.45

3.45

3.59

3.59

3.59

3.71

3.72

4.36

4.36

4.36

4.36

4.37

4.37

4.37

4.38

7.52

8.77

S35

Figure S36. 1H NMR spectrum of compound 14

Figure S37. 1H NMR spectrum of compound 8g

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

S36

Figure S38. 1H NMR spectrum of compound 8h

Figure S39. 1H NMR spectrum of compound 8i

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

S37

Figure S40. 1H NMR (600 MHz) spectrum of cyclic peptide 11

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

HN

N

HN

HNNH

N

N

NH

NH

NH

NH

HN

HN

HN

O

O

O

O O

O

O

O

O

O

OO

O

O

OH

NH2

HO

NH

NH2

HN

HO

O

S

S

Asp

Pro

Phe

Cys

IlePro

Pro

Ile

Ser

Lys

CysArg

GlyThr

11

S38

Chiral HPLC chromatogram

Figure S41. Chiral HPLC chromatogram of Fmoc-D-Ala-OEt (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm). Synthesis of Fmoc-D-Ala-OEt using general procedure for nucleophilic acyl substitution on benzotriazole resin.

Figure S42. Chiral HPLC chromatogram of Fmoc-L-Ala-OEt (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm). Synthesis of Fmoc-L-Ala-OEt using general procedure for nucleophilic acyl substitution on benzotriazole resin.

Figure S43. Chiral HPLC chromatogram of compound 6a (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm).

Fmoc-D-Ala-OEt

Fmoc-L-Ala-OEt

Fmoc-L-Leu-OEt (6a)

S39

Figure S44. Chiral HPLC chromatogram of compound 6b (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

Figure S45. Chiral HPLC chromatogram of compound 6c (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

Figure S46. Chiral HPLC chromatogram of compound 6d (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

Fmoc-L-Phe-OEt

6b

Fmoc-L-Asp(OtBu)-OEt

6c

Fmoc-L-Gln(Trt)-OEt

6d

S40

Figure S47. Chiral HPLC chromatogram of compound 6e (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

Figure S48. Chiral HPLC chromatogram of compound 6f (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

Figure S49. Chiral HPLC chromatogram of compound 6g (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

Fmoc-L-His(Trt)-OEt

6e

Fmoc-L-Cys(Trt)-OEt

6f

Fmoc-L-Lys(Boc)-OEt

6g

S41

Figure S50. Chiral HPLC chromatogram of compound 6h (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

Figure S51. Chiral HPLC chromatogram of compound 6i (isocratic of 40:60 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

Figure S52. Chiral HPLC chromatogram of compound 6j (isocratic of 60:40 n- hexane/iPrOH; flow rate: 1.0 mL/min, λ = 220 nm)

References

[1] S. Krupkova, P. Funk, M. Soural, J. Hlavac, ACS Combinatorial Science 2013, 15, 20-28.

[2] A. R. Katritzky, S. A. Belyakov, D. O. Tymoshenko, Journal of Combinatorial Chemistry 1999, 1, 173-176.

Fmoc-L-Trp(Boc)-OEt

6h

Fmoc-L-Arg(pbf)-OEt

6i

Fmoc-L-Met-OEt

6j

Documents

· crosslinking (0.3 mmol/g) from Advanced ChemTech Inc. NMR spectra were obtained on a Joel 400 MHz and Varian 600 MHz spectrometer. …