Supporting Information for: Daniel Knappe, Stefania Piantavigna, Anne Hansen, Adam Mechler, Annegret Binas,

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1 Supporting Information for: Oncocin (VDKPPYLPRPRPPRRIYNR-NH 2 ): a novel antibacterial peptide optimized against Gram-negative human pathogens Daniel Knappe, Stefania Piantavigna, Anne Hansen, Adam Mechler, Annegret Binas, Oliver Nolte, Lisandra L. Martin and Ralf Hoffmann Contents: Table S1 lists all reagents used in this study including their purities and the suppliers. Table S2 lists the media used to culture the bacteria and eukaryotic cells Page S2: Statement on the purity of the synthesized peptides Figures S1 to S12: Analytical reversed-phase HPLC chromatograms and MALDI mass spectra for purified peptides 1 to 12. S1

2 Table S1: Reagents used in this study including their purities and the suppliers. Reagent Purity Supplier Fmoc-amino acid derivatives >99% (HPLC) Orpegen Pharma, Heidelberg, Germany 5,6-Carboxyfluorescein For fluorescence >95% (HPLC) Fluka Chemie GmbH; Buchs (Switzerland) 5,6-Carboxytetramethylrhodamine For fluorescence Merck, Darmstadt (Germany) >85% HPCE Acetonitrile HPLC-S, >99.95% (GC) Biosolve; Valkenswaard (Netherlands) Dimethyl sulfoxide Puriss., absolute over molecular Biosolve sieve, >99.5% (GC) Ethane dithiole Purum >98% (GC) Fluka Chemie GmbH Hydroxybenzotriazole Puriss >99% Fluka Chemie GmbH; m-cresole p.a. >99% (GC) Fluka Chemie GmbH; N,N'-Diisopropylcarbodiimide Purum >98% (GC) Fluka Chemie GmbH N,N- Peptide synthesis grade Biosolve Dimethylformamide >99.8% (GC) Sodium chloride For microbiology SIGMA-Aldrich-Laborchemikalien; Taufkirchen (Germany) Thioanisole Purum 99% (GC) Fluka Chemie GmbH Trans-retinoic acid >95% by assay Calbiochem, Darmstadt (Germany) Trifluoroacetic acid for Purum >98% (T) Fluka Chemie GmbH peptide synthesis Trifluoroacetic acid for Spectroscopy grade >99% Fluka Chemie GmbH HPLC Diethyl ether >99% (GC) Biosolve Sodium hydroxide >99% Carl Roth GmbH & Co. KG S2

3 Table S2: Media used in this study to culture the bacteria and eukaryotic cells and the suppliers Medium Purity Composition Supplier Nutrient solution for bacteria For microbiology Meat extract (3 g/l) Peptone from gelatine (5 g/l) Carl Roth GmbH & Co. KG; ph 6.8 ±.2 Tryptic soy broth For microbiology Casein peptone (pancreatic, 17 g/l) Dipotassium phosphate (2.5 g/l) Glucose (2.5 g/l) Sodium chloride (5 g/l) Soy peptone (3 g/l) ph 7.3 ±.2 Fluka Chemie GmbH Statement about the peptide purity: General procedures for all peptide syntheses, purifications, and analyses were performed as described previously (Singer, D.; Lehmann, J.; Hanisch, K.; Hartig, W.; Hoffmann, R. Biochemical and Biophysical Research Communications 6, 346, ). The purified peptides were analyzed by RP-HPLC on a Jupiter C 18 -column (4.6 mm internal diameter, 15 mm length, 5 µm particle size, 3 nm pore size; Phenomenex) using a linear gradient from 5 to 33.6% aqueous acetonitrile for 17 min in the presence of trifluoroacetic acid as ion pair reagent. The purities were above 95% based on the peak areas obtained after background correction. The molecular weight of the peptides were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS; 47 proteomic analyzer; Applied Biosystems GmbH, Darmstadt, Germany) using α-cyano-4-hydroxycinnamic acid (4 mg/ml in 6% aqueous acetonitrile containing.1% TFA) as matrix (see Supporting information). S3

4 Figure S1: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 1. 1 A25 A4 C6 ( )2:1_UV2_214nm min E S4

5 Figure S2: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 2. 2 A25 A5 G3:1_UV2_214nm min E S5

6 Figure S3: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 3. 3 A28 B2 G4:1_UV2_214nm min E S6

7 Figure S4: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 4. 4 A29 B1 C5:1_UV2_214nm min E S7

8 Figure S5: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 5. 5 A25 A6 A16:1_UV2_214nm min S8

9 Figure S6: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 6. 6 A29 A2 D77:1_UV2_214nm min E S9

10 Figure S7: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 7. 7 A29 A6 B8:1_UV2_214nm min E S1

11 Figure S8: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 8. 8 A35 A2 E9:1_UV2_214nm min E S11

12 Figure S9: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 9. 9 A35 A4 B91:1_UV2_214nm min S12

13 Figure S1: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide 1. 1 A66 B3 B5B611:1_UV2_214nm min E S13

14 Figure S11: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide A53 F2 D112:1_UV2_214nm min S14

15 Figure S12: Analytical reversed-phase HPLC chromatogram (top) and MALDI mass spectrum (bottom) for purified peptide A51 A6 A1113:1_UV2_214nm min S15