SUPPORTING INFORMATION. Reversible and versatile on-tether modification of chiral. content

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1 UPPRTIG IFRMATI Reversible and versatile on-tether modification of chiral center-induced helical peptides Kuan u,,# Chengjie un,,# Zigang Li #,* # chool of Chemical Biology and Biotechnology, Peking University henzhen Graduate chool, henzhen, (China) lizg@pkusz.edu.cn [ ] These authors contributed equally to this work content General information for unnatural amino acid and peptide synthesis upplementary Figures... 4 upplementary Tables... 8 Appendix: selected Peptide s PLC and LC/M data

2 General procedures for unnatural amino acid and peptide synthesis. The detailed unnatural amino acids and peptide synthesis procedures have been covered elsewhere in our previous reported articles, for example (1. u K et al., Angew Chem Int Ed., 2016, 55, ; 2. u K. et al., 2. Lin et al., Chem Commun., 2016, 52, ; 3. Lin et al., rg. Bio. Chem., 2016, 14, ; 4. Bioconjugate Chem., 2016, 27, ; 5. Zhang Q et al., ci. Rep., 6, ; 6. u K. et al., Bioconjugate Chem., 2017, AAP, DI: /acs.bioconjchem.7b00171). A () Bn Cl n-buli () Bn 1. LDA -78ºC Br (R) LiB 4 (R) pph3, I 2 (R) I () Bn Imidazole 1 2 Cl Cl 1. K/iPr, Cl, p 5-6 Cl 2 2 PCl 5, DCM Cl Gly(), i( 3) 2 62, K Me, reflux R Cl i 3 4 R Cl i K, DMF (R) I Cl Cl (R) i (R) i Major product Minor product 3 M Cl/Me, reflux 3 (R) Fmoc-u, a 2 C 3 Fmoc (R) cheme 1. (A) The flow chart for non-natural amino acid 5 (R) synthesis. Fmoc 1.CTU,DIEA,AAs 2,acetic anhydride, DIEA MAP,MMP 365nm 2R 95:2.5:2.5 TFA: 2 :TI 2 1.2eq FG-X, X=Cl, Br, I C FG 2 Rink amide MBA resin 2R-(FG)-A/B cheme 2. Presentation of the solid phase peptide synthesis procedure. Take peptide 2R-(a-e)-A/B as an example. Peptides (1-7)-R-(a-e)-A/B were all synthesized by this procedure.

3 Fmoc 2 (R) 2 Fmoc thiol-ene click reaction MMP, MAP 365nm, hv DMF C 2 C (R) 2 Fmoc (R) Amidation PyBP, ATU,MM DMF PP deprotection, coupling... acetylation Fmoc (R) 2 (R) eq FG-X, X=Cl, Br, I 2. C 95:2.5:2.5 TFA: 2 :TI FG (R) 2 2 cheme 3. Presentation of the solid phase peptide synthesis procedure. Peptides PDI-R-(a-e) were synthesized by this procedure.

4 upplementary Figures Epimer A Epimer B 2R 3R 4R 5R 6R Figure 1. CD spectra of sulfonium functionalized peptide epimers of (2-6)-R-(a-e)-A/B. CD spectra were recorded at a peptide concentration of 25μM in PB (50mM, p 7.4) at 20.

5 Figure 2. A-E) Temperature dependence of mean residue ellipticity of peptides 2R-(a-e)-B from 5 to 65. CD spectra were recorded at a peptide concentration of 25μM in PB buffer (50mM, p 7.4).

TAT- 3 CC C 2 C 2 C 2 CC C 2 C 2 C 2 C 2 CC C 2 C 2 C 2 C 2 CC C 2 C 2 C 2 C 2 2 Cu 4, odium ascorbate 2 :ipr 1 hour CC CC CC CC

6 Figure 3. PLC detection of the isomerization of peptide 2R-(c)-B after heating to 70. A C 7R-(c)-B C CC C 2 C 2 3 C 2 C 2 CC C 2 C 2 C 2 C 2 2 CC C 2 C 2 C 2 C 2 2 CC C 2 C 2 C 2 C 2 CC C 2 C 2 C 2 C 2 TAT- 3 CC C 2 C 2 C 2 CC C 2 C 2 C 2 C 2 CC C 2 C 2 C 2 C 2 CC C 2 C 2 C 2 C 2 2 Cu 4, odium ascorbate 2 :ipr 1 hour CC CC CC CC CC CC CC CC CC 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C + C 2 C 2 C C 2 C 2 C 2 2 C 2 C 2 C C 2 C 2 C 2 2 C 2 C 2 C 2 8R C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 B

A TAT-analogue peptide TAT- 3 was conjugated to peptide 7R-(c)-B.

products of peptides 7R-(c)-B and 8R. Figure 5.

(green), TAT conjugated peptide 8R (blue) 5 µm at 37 C for 2h.

7 Figure 4. (A) chematic presentation of the Cu(I) catalyzed click reaction for peptide conjugation. A TAT-analogue peptide TAT- 3 was conjugated to peptide 7R-(c)-B. (B) The PLC chromatogram of the reaction mixture after CUAAC reaction and the pure products of peptides 7R-(c)-B and 8R. Figure 5. (A) Flow cytometry analysis of ela cells treated with peptide 7R (range), 7R-(c)-B (green), TAT conjugated peptide 8R (blue) 5 µm at 37 C for 2h. (B) The fluorescence confocal images of ela cells treated with peptide 7R, 7R-(c)-B, and TAT conjugated peptide 8R 5 µm at 37 C for 2h, FITC: green. Figure 6. Dealkylation of peptide 2R-(e)-B over time in the presence of Py in vitro detected via LC-M at different time points.

8 7R 7R-(c)-B Figure 7. The de-alkylation of peptide 7R-(c)-B in cellular circumstances monitored by RM. Figure 8. Comparison of the biophysical properties of sulfonium peptides 2R-(a-e)-B with D-CI peptide in our previous article. (A) Molar ellipticity at 215 nm of 2R, 2R-(a-e)-B and 1R-c (the peptide number 1R-c was used in the same with previous article) at different concentration of guanidine hydrochloride. (B) Molar ellipticity at 215 nm of 2R, 2R-(a-e)-B and 1R-c at different temperatures from 5 to 65. (C) In vitro serum digestion assay of peptides 2R, 2R-(a-e)-B and 1R-c. The percentage of intact peptide was recorded from PLC integration. (D) istogram represents the intracellular fluorescence signal of peptides measured by flow cytometry. Values represent the average of three independent experiments. *: peptide 1R-c and pep-16 were cited from ref. 13. upplementary Tables Table 1: The conversion efficiency of alkylation reaction using Bn-Br as the alkylating reagents under different conditions.

9 -cyclo[c(fg)aai 5 (R )]- 2 FG=Bn Entry olvent Catalyst Reaction time Conversion(%) b 1 DMF TFA MeC AgBF 4-4h DMF, DIPEA M C - > 98 a Reaction conditions: sulfide (1.0 equiv), FG-X (1.2 equiv) in 0.2M C at r.t., 4 h. b The conversion were calculated based on integration of peak areas in PLC

10 Table 2: Peptides Characterization. Calculated and Found m/z are presented as [M+1] + / [M/2+] + / [M/3+] + / [M/4+] +.. Peptide FG Calculated mass m/z bserved mass (A/B) m/z 1R -cyclo[caai 5 (-R )]- 2 2R -cyclo[caaa 5 (-R )]- 2 3R -cyclo[caqa 5 (-R )]- 2 4R -cyclo[caa 5 (-R )]- 2 5R 6R 7R -cyclo[cara 5 (-R )]- 2 -cyclo[crrr 5 (-R )]- 2 FITC-βA-cyclo[CRRR 5 (-R )]- 2 PDI-R -βa-cyclo-ltf[cyw 5 (-R)]QLT- 2 Bn / Me / C / Allyl / Propinyl / Bn / Me (B) C (B) Allyl / Propinyl / Bn / Me / C / Allyl / Propinyl / Bn / Me / C (B) Allyl / Propinyl / Bn / Me / C , / , Allyl / Propinyl / Bn / Me / C (B) Allyl / Propinyl / Bn , Me , C , Allyl , Propinyl , Bn Me C Allyl Propinyl R TAT( 3 -RKKRRQRRR)-7R (c)-b ,921.80

11 Table 3:A library of sulfonium functionalized pentapeptides 1-6R-(a-e) with different sequences and their molar ellipticities, alkylation conversion efficiency, and percentage of helical contents, product ratio of epimers A: epimer B. ote: 1R -cyclo[caai 5 (R )]- 2 FG conversion ratio [θ] 215 [θ] 207 [θ] 190 [θ] 215 /[θ] 207 Relative helicity a Me : : : C : : R -cyclo[caaa 5 (R )]- 2 FG conversion ratio [θ] 215 [θ] 207 [θ] 190 [θ] 215 /[θ] 207 Relative helicity a Me 0.58 only product : : C 0.76 only product : A 3R -cyclo[caqa 5 (R )]- 2 FG conversion ratio [θ] 215 [θ] 207 [θ] 190 [θ] 215 /[θ] 207 Relative helicity a Me : : : C : : R -cyclo[caa 5 (R )]- 2 FG conversion ratio [θ] 215 [θ] 207 [θ] 190 [θ] 215 /[θ] 207 Relative helicity a Me : : : C 0.67 only product :

12 5R -cyclo[cara 5 (R )]- 2 FG conversion ratio [θ] 215 [θ] 207 [θ] 190 [θ] 215 /[θ] 207 Relative helicity a Me : : : C : ; R -cyclo[crrr 5 (R )]- 2 FG conversion ratio [θ] 215 [θ] 207 [θ] 190 [θ] 215 /[θ] 207 Relative helicity a Me : /A /A : /A /A : /A /A C 0.65 only product : /A /A

13 Appendix: selected Peptide s PLC and LC/M data. LC/M spectra of peptide 1-7R-(a-e)-A/B Me Peptide 1R-(a)-A/B Ile 2 Me Peptide 1R-(a)-A Ile 2 Me Peptide 1R-(a)-B Ile 2

14 Peptide 1R-(b)-A/B Ile 2 Peptide 1R-(b)-A Ile 2

15 Peptide 1R-(b)-B Ile 2 Peptide 1R-(c)-A/B Ile 2 Peptide 1R-(c)-A Ile 2

16 Peptide 1R-(c)-B Ile 2 Peptide 1R-(d)-A/B Ile 2 C

17 Peptide 1R-(d)-A Ile 2 C Peptide 1R-(d)-B Ile 2 C Peptide 1R-(e)-A/B Ile 2

18 Peptide 1R-(e)-A Ile 2 Peptide 1R-(e)-B Ile 2

19 Peptide 2R-(a)-B 2 Me Peptide 2R-(b)-A/B 2

20 Peptide 2R-(b)-A 2 Peptide 2R-(b)-B 2 Peptide 2R-(c)-A/B 2

21 Peptide 2R-(c)-A 2 Peptide 2R-(c)-B 2

22 Peptide 2R-(d)-B 2 C Peptide 2R-(e)-A/B 2

23 Peptide 2R-(e)-A 2 Peptide 2R-(e)-B 2

24 Me Peptide 3R-(a)-A/B Gln 2 Me Gln 2 Peptide 3R-(a)-A

25 Peptide 3R-(a)-B Gln 2 Me Peptide 3R-(b)-A/B Gln 2 Peptide 3R-(b)-A Gln 2

26 Peptide 3R-(b)-B Gln 2 Peptide 3R-(c)-A/B Gln 2

27 Peptide 3R-(c)-A Gln 2 Peptide 3R-(c)-B Gln 2 Peptide 3R-(d)-A/B Gln 2 C

28 Peptide 3R-(d)-A Gln 2 C Peptide 3R-(d)-B Gln 2 C

29 Peptide 3R-(e)-A/B Gln 2 Peptide 3R-(e)-A Gln 2

30 Peptide 3R-(e)-B Gln 2 Me Peptide 4R-(a)-A/B er 2 Me er 2 Peptide 4R-(a)-A

31 Peptide 4R-(a)-B er 2 Me Peptide 4R-(b)-A/B er 2 Peptide 4R-(b)-A er 2

32 Peptide 4R-(b)-B er 2 Peptide 4R-(c)-A/B er 2 Peptide 4R-(c)-A er 2

33 Peptide 4R-(c)-B er 2 C Peptide 4R-(d)-B er 2

34 Peptide 4R-(e)-A/B er 2 Peptide 4R-(e)-A er 2

35 Peptide 4R-(e)-B er 2 Me Peptide 5R-(a)-A/B 2 Me Peptide 5R-(a)-A 2

36 Peptide 5R-(a)-B 2 Me Peptide 5R-(b)-A/B 2 Peptide 5R-(b)-A 2

37 Peptide 5R-(b)-B 2 Peptide 5R-(c)-A/B 2

38 Peptide 5R-(c)-A 2 Peptide 5R-(c)-B 2 Peptide 5R-(d)-A/B 2 C

39 Peptide 5R-(d)-A 2 C Peptide 5R-(d)-B 2 C Peptide 5R-(e)-A/B 2

40 Peptide 5R-(e)-A 2 Peptide 5R-(e)-B 2

41 Me Peptide 6R-(a)-A/B 2 Peptide 6R-(b)-A/B 2 2 Peptide 6R-(b)-A

42 Peptide 6R-(b)-B 2 Peptide 6R-(c)-A/B 2

43 Peptide 6R-(c)-A 2 Peptide 6R-(c)-B 2 C 2 Peptide 6R-(d)-B

44 Peptide 6R-(e)-A/B 2

45 Peptide 6R-(e)-A 2 2 Peptide 6R-(e)-B Me Peptide 7R-(a)-B FITC 2

46 Peptide 7R-(b)-B FITC 2

47 Peptide 7R-(c)-B 2 FITC Peptide 7R-(d)-B 2 C FITC

48 Peptide 7R-(e)-B FITC 2 Peptide PDI-R-(a) Me Leu e Tyr Leu er 2 Thr is Trp Gln Thr

49 Peptide PDI-R-(b) Leu e Tyr Leu er 2 Thr is Trp Gln Thr

50 Peptide PDI-R-(c) Leu e Tyr Leu er 2 Thr is Trp Gln Thr Peptide PDI-R-(d) C Leu e Tyr Leu er 2 Thr is Trp Gln Thr

51 Peptide PDI-R-(e) Leu e Tyr Leu er 2 Thr is Trp Gln Thr uv(x1,000,000) min

52 3 C CC C 2 CC C 2 CC C 2 CC C 2 CC C 2 CC C 2 CC C 2 CC C 2 CC C 2 2 TAT-3 C 2 C 2 C 2 C 2 C 2 C 2 2 C 2 C 2 C 2 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 CC CC CC CC CC CC CC CC CC 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C + C 2 C 2 C 2 2 C 2 C 2 C 2 2 C 2 C 2 C 2 2 C 2 C 2 C 2 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 Peptide 8R 2 2

54 Reference: (1) Y.. Belokon, V. I. Tararov, V. I. Maleev, T. F. avel eva, M. G. Ryzhov. (1998) Improved procedures for the synthesis of ()-2-[ - ( -benzylprolyl) amino] benzophenone (BPB) and i (II) complexes of chiff's bases derived from BPB and amino acids. Tetrahedron: Asymmetry, 9, (2) X. Tang, V. A. oloshonok, V.J. ruby. (2000) Convenient, asymmetric synthesis of enantiomerically pure 2,6 -dimethyltyrosine (DMT) via alkylation of chiral equivalent of nucleophilic glycine. Tetrahedron: Asymmetry, 11, (3) V. A. oloshonok, X. Tang, V. J. ruby. (2001) Large-scale asymmetric synthesis of novel sterically constrained 2,6 -dimethyl- and α,2,6 -trimethyltyrosine and -phenylalanine derivatives via alkylation of chiral equivalents of nucleophilic glycine and alanine. Tetrahedron: Asymmetry, 57, (4) B. Aillard,.. Robertson, A. R. Baldwin,. Robins, A. G. Jamieson. (2014) Robust asymmetric synthesis of unnatural alkenyl amino acids for conformationally constrained α-helix peptides. rg. Biomol. Chem., 12, (5) K. u,. Geng, Q. Zhang, Q. Liu, M. Xie, C. un, W. Li,. Lin, F. Jiang, T. Wang, et al. (2016) An In-tether Chiral Center Modulates the elicity, Cell Permeability and Target Binding Affinity of a Peptide. Angew. Chem. Int. Ed. 55, (6) D. A. Evans, M. D. Ennis and D. J. Mathre. (1982) Asymmetric alkylation reactions of chiral imide enolates. A practical approach to the enantioselective synthesis of.alpha.-substituted carboxylic acid derivatives. J. Am. Chem. oc., 104, (7) U. chollkopf. (1983) Enantioselective synthesis of non-proteinogenic amino acids via metallated bis-lactim ethers of 2,5-diketopiperazines. Tetrahedron. 39, (8) K. u,. Geng, Q. Zhang, Q. Liu, M. Xie, C. un, W. Li,. Lin, F. Jiang, T. Wang, et al. (2016) An In-tether Chiral Center Modulates the elicity, Cell Permeability and Target Binding Affinity of a Peptide. Angew. Chem. Int. Ed. 55, (9) J. R. Kramer, T. J. Deming. (2013) Reversible chemoselective tagging and functionalization of methionine containing peptides. Chem. Commun., 49, (10) J. R. Kramer, T. J. Deming. (2012) Enzyme-Triggered Cargo Release from Methionine ulfoxide Containing Copolypeptide Vesicles. Biomacromolecules, 13, (11). E. hepherd,.. oang, G. Abbenante, D. P. Fairlie, (2005) J. Am. Chem. oc., 127, (12) hepherd,. E.; oang,..; Abbenante, G.; Fairlie, D. P. (2005) ingle turn peptide alpha helices with exceptional stability in water. J. Am. Chem. oc., 127, (13) u, K.; un, C.; Yu, M.; Li, W.; Lin,.; Guo, J.; Jiang, Y.; Lei, C.; Li, Z. (2017) Dual in-tether chiral centers modulate peptide helicity. Bioconjugate Chem. 28,

SUPPORTING INFORMATION

SUPPORTING INFORMATION SUPPRTIG IFRMATI Salicylaldehyde Ester-Induced Chemoselective Peptide Ligations Enabling Generation of atural Peptidic Linkages at the Serine/Threonine Sites Xuechen Li,* iu Yung Lam, Yinfeng Zhang, Chun