Amyloidogenesis highlighted by designed peptides forming supramolecular self-assemblies

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1 Electronic Supplementry nformtion Amyloidogenesis highlighted y designed peptides forming suprmoleculr self-ssemlies M. ueri,. T. Dolphin, P. Dumy, nd J. rci* Déprtement de Chimie Moléculire, UM-CNS 5250, CM F 2607, Université Joseph Fourier, BP53, renole Cedex 9, Frnce E-mil: julin.grci@ujf-grenole.fr Tle of Contents: Synthesis nd Experimentl Section HPLC profiles nd SM spectr inetics dt

2 Figure S1. Chemicl structures of peptide edifices nd corresponding precursors. Synthesis of cyclic decpeptide 3 (Figure S2). The liner peptide (Pmc)-(Dde)-(Pmc)- P--(Pmc)-(Dde)-(Pmc)-P- ws first uilt up utomticlly (Advnce Chem Tech 348 Ω peptide synthesizer) on Fmoc-ly-Ssrin resin (500 mg, 0.69 mmol.g -1 ) nd cyclized (415 2

3 mg, mol) in DMF (0.5 mmol.l -1 ) under high dilution using PyBP (1.2 eq.) nd DPEA. The white solid powder otined fter precipittion nd wshing in diethyl ether ws soluilized in solution of 2% of hydrzine in DMF to remove Dde protecting groups. The cyclopeptidic intermedite 1 (272 mg, mol) ws then otined fter precipittion nd wshing in diethyl ether (76% yield from the liner peptide). To solution of compound 1 (272 mg, mol) in DMF (0.01 mol.l -1 ), were dded Boc-Ser(tBu)-H (3 eq), PyBP (3 eq.) nd DPEA. The mixture ws stirred for 2 h t r.t. Then Boc, tbu nd Pmc protecting groups were removed using solution of TFA/TS/H 2 (95:2.5:2.5) (0.01 mol.l -1 ). After 2 h, the solvent ws evported nd the crude compound 2 (145 mg, mol) ws otined y precipittion with diethyl ether s white solid powder with 59% yield. HPLC t = 4.9 min. ES-MS clc , found To solution of compound 2 (5 mg, mol) in H 2 /CH 3 CN (1:1) (0.01 mol.l -1 ) ws dded N 4 (20 eq.). The rection ws stirred for 20 min t r.t. nd immeditely purified y P-HPLC (C18 Nucleosil column, 5-100% B in 30 min). This procedure ws relized 8 times to fford compound 3 (21 mg, mol) s white powder with 61% yield. HPLC t = 5.0 min. ES-MS clc , found Fmoc SPPS c,d,e P P Pmc Pmc Pmc Pmc f H H NH 2 H 2 N P P 1 2 g H H P P 3 Figure S2. Synthesis of the cyclodecpeptide 3. () Piperidine/DMF (1:4); () Fmoc-X-H (2 eq.), PyBP (2 eq.), DPEA (3-4 eq.), DMF; (c) TFA/CH 2 Cl 2 (1:99), 10 min (three times); (d) PyBP (1.2 eq.), DPEA (3-4 eq.), DMF ( M), 1 h; (e) Hydrzine/DMF (2:98), 2 h, 76% from the liner form of 1; (f) i) Boc-Ser(tBu)-H (3 eq.), PyBP (3 eq.), DPEA, DMF (10-2 M), 2 h; ii) TFA/TS/H 2 (95:2.5:2.5), 2 h, 59% from 1 (2 steps); (g) N 4 (20 eq.), H 2 /CH 3 CN (1:1), 61%. Synthesis of peptide 4 (Figure S3). The CAβ Y C peptide sequence ws synthesized utomticlly (Applied Biosystems) y solid phse synthesis on NovSyn T Sieer resin (300 mg, 0.19 mmol.g -1 ). Peptide on resin ( mol) ws then solvted in 10 ml of DMF nd the ph ws djusted with DEPA to ph (1- ethoxyethylideneminooxy)cetic cid (2 eq.), nd PyBP (2 eq.) were dded to the resin 3

4 solution. The mixture ws stirred for 1 h t r.t. Peptide ws then solvted in DMF nd iodine (20 eq.) ws dded. The peptide ws relesed from the resin using 10 ml clevge solution of TFA/H 2 /TS (95:2.5:2.5). The mixture ws stirred for 2 h t r.t then 10 eq. of NH 4 ws dded nd the mixture ws stirred for nother 30 min. The crude free peptide 4 ws otined s white powder (142 mg, mol) nd then purified y P-HPLC (C18 Nucleosil column, 5-100% B in 30 min) ffording pure peptide 4 (15.1 mg, mol) s white powder with 8% overll yield from the resin. HPLC t = 8.3 min. ES-MS clc , found HN PE SPPS Trt tbu Boc Trt H C L V F Y A D X N A 1 L M V C Boc Trt Boc Boc Trt c N Trt tbu Boc Trt N A C L V F Y A D X 1 L M V C Boc Trt Boc Boc Trt d Boc Trt N A L M V C S X 1 D A Y F V L S C Boc Boc Trt Boc tbu N e N A S D L M V C NH 2 S A Y F V L S C 4 NH 2 X 1 = -S(ψ Me,Me pro) Figure S3. Synthesis of the peptide 4. () Piperidine/NMP (1:4); () Fmoc-X-H (10 eq.), HBTU (10 eq.), DPEA (20 eq.), NMP; (c) 2-(1-ethoxyethylideneminooxy)cetic cid (2 eq.), PyBP (2 eq.), DPEA (3-4 eq.), DMF; (d) 2 (20 eq.), DMF; (e) TFA/H 2 /TS (95:2.5:2.5), NH 4 (10 eq.), 8% from the resin. The cyclic decpeptide 5 nd the peptide 6 (Figure S1) were synthesized s previously descried. 1 Synthetic Aβ 1-40 ws prepred s previously descried. 2 4

5 t = 8.7 min [M+5H] 5+ [M+6H] 6+ [M+7H] 7+ [M+8H] 8+ [M+9H] 9+ [M+4H] 4+ Figure S4. Chrcteriztion of compound 2Lin y chromtogrphy nd mss spectrometry. P-HPLC profile (); ES-MS nlysis (). 5

6 t = 9.0 min [M+4H] 4+ [M+5H] 5+ [M+8H] 8+ [M+7H] 7+ [M+6H] 6+ Figure S5. Chrcteriztion of compound 2Loop y chromtogrphy nd mss spectrometry. P-HPLC profile (); ES-MS nlysis (). 6

7 t = 10.2 min [M+11H] 11+ [M+12H] 12+ [M+10H] 10+ [M+13H] 13+ [M+9H] 9+ [M+14H] 14+ [M+8H] 8+ Figure S6. Chrcteriztion of compound 4Loop y chromtogrphy nd mss spectrometry. P-HPLC profile (); ES-MS nlysis (). 7

8 inetics studies. A concentrtion 25 µm of 4Loop, 2Loop nd 2Lin nd 6 µm of 4Lin were used for kinetic studies t 20 C. Firil formtion ws monitored y the inding of Thioflvin T (10 µm), studying the fluorescence t 480 nm with excittion t 440 nm. The kinetic constnts k 1 nd k 2 were otined using the Finke-Wtzky (F-W) two-step mechnism of nucletion followed y utoctlytic surfce growth. Using this mechnism (where A is the initil monomer nd B (ctlytic) ggregted form of peptide edifices pst the criticl nucleus size) we cn extrct from experimentl dt, two constnts, k 1, which represents the nucletion process nd k 2, which represents the extension of the fire (Tle S1). This model cn e mthemticlly trnslted y the following equtions: or 4Lin 4Loop 2Loop 2Lin k 1 (min -1 ) ± ± ± ± k 2 (µm -1.min -1 ) ± ± ± ± t 1/2 (min) Tle S1. te constnts nd hlf-life time vlues from fitting kinetic dt with the F-W twostep mechnism. 1.. T. Dolphin, P. Dumy nd J. rci, Angew. Chem.-nt. Edit., 2006, 45, T. Dolphin, M. ueri, P. Dumy nd J. rci, ChemMedChem, 2007, 2,