Supporting Information

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1 Supporting Information Chitosan-based Peptidopolysaccharides as Cationic Antimicrobial Agents and Antibacterial Coatings Dicky Pranantyo 1, Li Qun Xu 1, En-Tang Kang 1 *, Mary B. Chan-Park 2 * 1 Department of Chemical & Biomolecular Engineering National University of Singapore 4 Engineering Drive 4, Kent Ridge Singapore Centre of Antimicrobial Bioengineering School of Chemical and Biomedical Engineering Nanyang Technological University Singapore * Corresponding Authors cheket@nus.edu.sg (E.T.K) mbechan@ntu.edu.sg (M.B.C.P) * ORCID En-Tang Kang: Mary B. Chan-Park: S1

2 Table S1. Minimum bactericidal concentration (MBC) values of the polysaccharides and peptidopolysaccharide conjugates. MBC, µg/ml a Sample E. coli P. aeruginosa S. aureus S. epidermidis CS > 2048 b > 2048 b > 2048 b > 2048 b CSOMI > 2048 b > 2048 b > 2048 b > 2048 b CysHHC CSNHHC 256 (116) 256 (116) 256 (116) 32 (14) CSOHHC 128 (61) 128 (61) 64 (30) 16 (8) a Values in bracket express the mass of peptide only in the conjugate, calculated based on the peptide mass fraction. b The MBC values were not observed up to the highest concentration of compound tested (2048 µg/ml). S2

3 Figure S1. The 1 H 13 C heteronuclear multiple bond correlation (HMBC) spectrum of CSOMI. S3

4 Figure S2. The 1 H 13 C heteronuclear multiple bond correlation (HMBC) spectrum of CSNMI. S4

5 Figure S3. Size-exclusion GPC chromatograms of the CS, CSNMI, CSOMI, CSNHHC, and CSOHHC compounds. S5

6 (a) SS-CSNHHC 10 C 1s (b) SS-CSNHHC 10 N 1s (c) SS-CSNHHC 10 S 2p C H C N C O O C=O >N >N + < S 2p 3/2 S 2p 1/2 sulfonate (d) SS-CSNHHC 20 C 1s (e) SS-CSNHHC 20 N 1s (f) SS-CSNHHC 20 S 2p Intensity (a.u.) (g) SS-CSOHHC 10 C 1s (h) SS-CSOHHC 10 N 1s (i) SS-CSOHHC 10 S 2p (j) SS-CSOHHC 20 C 1s (k) SS-CSOHHC 20 N 1s (l) SS-CSOHHC 20 S 2p S 2p 1/2 sulfonate S 2p 3/2 Binding Energy (ev) Figure S4. XPS (a,d,g,j) C 1s, (b,e,h,k) N 1s, and (c,f,i,l) S 2p core-level spectra of the (a c) SS-CSNHHC 10, (d f) SS-CSNHHC 20, (g i) SS-CSOHHC 10, and (a c) SS- CSOHHC 20 surfaces. S6

7 Figure S5. Surface elemental stoichiometries of the SS-TA, SS-CSNHHC, and SS- CSNHHC substrates derived from the respective peak-area ratios of the XPS spectra. S7

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15 S15

16 The parameters for NMR characterization: Number of scans (for proton) = 64 Number of scans (for HMBC) = 144 Line broadening = 0.30 Hz Pulse program (for proton) = zg30 Pulse program (for HMBC) = hmbcgplpndqf Reference used = H 2 O 4.79 ppm, DMSO 2.50 ppm, and CDCl ppm Water suppression (zgpr) was used when characterizing the CSOMI sample. Samples were dissolved in the corresponding solvents using vortex mixer at a concentration of 15 mg/ml. The parameters for size-exclusion GPC characterization: Poly(ethylene glycol)/poly(ethylene oxide) (Agilent) with M n 12600, 20260, 30310, 49640, and g/mol were used as standards. Samples were dissolved using vortex mixer at a concentration of 2 mg/ml in aqueous sodium sulfate 0.05 M as eluent. Determination of the degree of substitution (DS) Degree of substitution (DS) was determined from the NMR peak area integrations. The theoretical CSOMI structure (page S11) has two proton d and one proton 2, so: = =.. hence DS = 0.27 The theoretical CSNMI structure (page S12) has two proton d and one proton 2, so: = =.. hence DS = 0.19 The theoretical CSOHHC structure (page S14) has six proton * and one proton 2, so: = = (.. ). hence DS = 0.23 The theoretical CSNHHC structure (page S15) has six proton * and one proton 2, so: = = (.. ). hence DS = 0.18 S16

17 Determination of theoretical yields and experimental yields The CSOMI has DS 0.27, with starting material of CS 645 mg (4 mmol saccharide unit) and MIHCl 459 mg, so the theoretical CSOMI mass obtained is: = + =645+( )= mg From experiment, the experimental CSOMI mass obtained is: = ( + )=0.76( )= mg The CSNMI has DS 0.19, with starting material of CS 1289 mg (8 mmol saccharide unit) and MIHAc 845 mg, so the theoretical CSNMI mass obtained is: = + =1289+( )= mg From experiment, the experimental CSOMI mass obtained is: = ( + )=0.65( )= mg The CSOHHC has DS 0.23, with starting material of CSOMI 103 mg (0.5 mmol saccharide unit) and peptide 309 mg, so the theoretical CSOMI mass obtained is: = + =103+( )=274.6 mg From experiment, the experimental CSOMI mass obtained is: = + =0.57( )=235.1 mg The CSNMI has DS 0.18, with starting material of CSNMI 52 mg (0.3 mmol saccharide unit) and MIHAc 845 mg, so the theoretical CSNMI mass obtained is: = + =52+( )=125 mg From experiment, the experimental CSOMI mass obtained is: = + =0.55(52+155)=114 mg S17

18 Determination of the surface deposition density of the polymers and peptide With ρ is the density of tannic acid and chitosan derivatives (2.12 g/ml), h is the coating thickness, M n is number average molecular weight of tannic acid and chitosan derivatives (1112 g/mol), and N A is Avogadro s number, the deposition density (σ) of the bilayer coatings is estimated as: = h For the SS-CSOHHC 10 surface, the density of the deposited polymers is: = =459 unit nm From the XPS elemental analysis, the N/C mol ratio (8.84 / 65.91) is used as the mol ratio of peptide over the total polymers. So for the the SS-CSOHHC 10 surface, the density of the deposited peptide is: = 8.84 = =62 unit nm For the SS-CSNHHC 10 surface, the density of the deposited polymers is: = =310 unit nm From the XPS elemental analysis, the N/C mol ratio (6.83 / 66.19) is used as the mol ratio of peptide over the total polymers. So for the the SS-CSOHHC 10 surface, the density of the deposited peptide is: = 6.83 = =32 unit nm S18