Supporting Information:

SI1

Supporting Information:

Synthesis, Antimicrobial Activity, and Membrane Permeabilizing Properties of C-Terminally Modified Nisin Conjugates Accessed by CuAAC

Jack C. Slootweg,† Steffen van der Wal,† H (Linda). C. Quarles van Ufford,† Eefjan Breukink,§ Rob M. J. Liskamp,†,|| and Dirk T. S. Rijkers*,†

†

Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands

§

Membrane Biochemistry & Biophysics, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands

||

Chemical Biology and Medicinal Chemistry, School of Chemistry, University of Glasgow, Glasgow, G12 8QQ, United Kingdom

*

Corresponding author. Tel.: +31 (0)6 2026 0572 / +31 (0)30 253 7307; fax: +31 (0)30 253 6655. E-mail: [email protected].

Contents

1. Copies of 1H and 13C NMR spectra of compounds 5 – 9 SI2–SI11

2. Copies of ESI-MS spectra of compounds 8 and 9 SI12–SI13

3. Copies of MALDI-TOF spectra and HPLC chromatograms

of nisin-conjugates 2, 13 – 15 SI14–SI21

SI2

1.

Copies of

H and

C NMR Spectra of Compounds 5 – 9.

Figure SI 1: 1H NMR Spectrum of compound 5 (300 MHz, CDCl3, T = 298 K).

-0 .5 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5 5 .0 5 .5 6 .0 6 .5 7. 0 7. 5 8 .0 8 .5 9 .0 9 .5 1 0 .0 1 0 .5 1 1 .0 f1 ( p p m ) -5 0 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 8.94 1.94 2.07 2.15 2.14 12.18 O O O N H B o c N3 0.81

SI3

Figure SI 2: 13C NMR Spectrum of compound 5 (75.5 MHz, CDCl3, T = 298 K).

-1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 f1 ( p p m ) -2 0 0 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0 2 2 0 0 2 4 0 0 2 6 0 0 2 8 0 0 O O O N H B o c N3

SI4

Figure SI 3: 1H NMR Spectrum of compound 6 (300 MHz, CDCl3, T = 298 K).

-0 .5 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5 5 .0 5 .5 6 .0 6 .5 7. 0 7. 5 8 .0 8 .5 9 .0 9 .5 1 0 .0 1 0 .5 1 1 .0 f1 ( p p m ) -1 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 1 7 0 0 1 8 0 0 1 9 0 0 2.0 1 1.9 6 1.9 3 2.4 0 12 .0 6 2.6 4 O O O N H3 N3 C l

SI5

Figure SI 4: 13C NMR Spectrum of compound 6 (75.5 MHz, CDCl3, T = 298 K).

-3 0 -2 0 -1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 f1 ( p p m ) 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 O O O N H3 N3 C l

SI6

Figure SI 5: 1H NMR Spectrum of compound 7 (300 MHz, CDCl3, T = 298 K).

-0 .5 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5 5 .0 5 .5 6 .0 6 .5 7. 0 7. 5 8 .0 8 .5 9 .0 9 .5 1 0 .0 1 0 .5 1 1 .0 f1 ( p p m ) -1 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 4.0 0 4.0 6 12 .0 3 O O O N3 N3

SI7

Figure SI 6: 13C NMR Spectrum of compound 7 (75.5 MHz, CDCl3, T = 298 K).

-1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 f1 ( p p m ) -2 0 0 -1 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 1 7 0 0 1 8 0 0 1 9 0 0 2 0 0 0 2 1 0 0 O O O N3 N3

SI8

Figure SI 7: 1H NMR Spectrum of compound 8 (300 MHz, CDCl3, T = 298 K).

-0 .5 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5 5 .0 5 .5 6 .0 6 .5 7. 0 7. 5 8 .0 8 .5 9 .0 9 .5 1 0 .0 1 0 .5 1 1 .0 f1 ( p p m ) -2 0 0 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 2 0 0 0 2 2 0 0 2 4 0 0 2 6 0 0 2 8 0 0 2.2 8 1.9 6 6.0 0 2.0 4 7.7 6 5.6 6 1.0 2 1.3 0 1.9 9 1.8 5 0.9 3 S N H O O O O O N3 N

SI9

Figure SI 8: 13C NMR APT Spectrum of compound 8 (75.5 MHz, CDCl3, T = 298 K).

-1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 10 1 20 1 30 1 40 1 50 1 60 1 70 1 80 1 90 2 00 2 10 2 20 f1 ( p p m ) -3 0 0 0 -2 5 0 0 -2 0 0 0 -1 5 0 0 -1 0 0 0 -5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 S N H O O O O O N3 N

SI10

Figure SI 9: 1H NMR Spectrum of compound 9 (300 MHz, CDCl3, T = 298 K).

-0 .5 0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .0 3 .5 4 .0 4 .5 5 .0 5 .5 6 .0 6 .5 7. 0 7. 5 8 .0 8 .5 9 .0 9 .5 1 0 .0 1 0 .5 1 1 .0 f1 ( p p m ) -5 0 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0 2.50 1.00 1.96 7.86 4.49 1.88 5.26 0.47 0.38 0.27 0.42 0.73 0.45 O H O O H O O N H O O O O N3 0.42

SI11

Figure SI 10: 13C NMR Spectrum of compound 9 (75.5 MHz, CDCl3, T = 298 K).

-1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 f1 ( p p m ) -5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0 4 0 0 0 4 5 0 0 5 0 0 0 O H O O H O O N H O O O O N 3

SI12

2.

Copies of ESI-MS Spectra of Compounds 8 and 9.

mass calcd. for C22H33N5O5S: 479.22 Da

found: m/z [M + H]+ 480.30; [M + Na]+ 502.20

Figure SI 11: ESI-MS Spectrum of compound 8.

[M+H]

[M+Na]

SI13 mass calcd. for C31H32N4O9: 604.22

found: m/z [M + H]+ 604.95; [M + Na]+ 627.40

Figure SI 12: ESI-MS Spectrum of compound 9.

[M+H]

[M+Na]

SI14

3.

Copies of MALDI-TOF Spectra and HPLC Chromatograms of

Nisin-Conjugates 2, 13 – 15.

SI15 mass calcd. for C146H233N43O36S7: 3388.58

found: m/z [M + H]+ 3390.11

Figure SI 14: MALDI-TOF MS Spectrum and HPLC chromatogram of nisin alkyne 2.

[M+H]+ [M+Na]+ [M+K]+ -20 0 20 40 60 80 100 -0.5 1.5 3.5 5.5 7.5 9.5 0 5 10 15 20 25 30 35 40 % b u ff e r B A b s o r b a n c e ( A U) Time (min)

SI16

SI17 mass calcd. for C168H266N48O41S8: 3867.80

found: m/z [M + H]+ 3868.53

Figure SI 16: MALDI-TOF MS Spectrum and HPLC chromatogram of nisin-dansyl conjugate 13.

[M+H]+ [M+Na]+ [M+K]+ -20 0 20 40 60 80 100 -0.5 1.5 3.5 5.5 7.5 9.5 0 5 10 15 20 25 30 35 40 % b u ff e r B A b s o r b a n c e ( A U) Time (min)

SI18

SI19 mass calcd. for C177H265N47O45S7: 3992.79

found: m/z [M + H]+ 3993.50; [M + Na]+ 4014.82

Figure SI 18: MALDI-TOF MS Spectrum and HPLC chromatogram of nisin-CF conjugate 14.

[M+H]+ [M+Na]+ [M+K]+ -20 0 20 40 60 80 100 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 5 10 15 20 25 30 35 40 % b u ff e r B A b s o r b a n c e ( A U) Time (min)

SI20

SI21 mass calcd. for C302H486N92O75S14: 7054.60 (average mass)

found: m/z [M + H]+ 7055.5; [M + Na]+ 7077.3; [M + K]+ 7093.0

Figure SI 20: MALDI-TOF MS Spectrum and HPLC chromatogram of nisin-dimer 15.

[M+H]+ [M+Na]+ [M+K]+ -20 0 20 40 60 80 100 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 0 5 10 15 20 25 30 35 40 % b u ff e r B A b s o r b a n c e ( A U) Time (min)

SI22

4.

MIC determination data

MIC in Bacillus subtilis

Nisin N alkyne N dansyl N CF N dimer conc in µM

A 0.054 0.444 0.364 0.043 0.042 0.04 0.052 0.053 0.036 0.047 0.048 0.065 0.566 0.103 0.116 50 B 0.04 0.044 0.038 0.042 0.042 0.039 0.049 0.047 0.039 0.051 0.051 0.045 0.048 0.076 0.063 16.7 C 0.04 0.04 0.038 0.039 0.04 0.04 0.042 0.041 0.045 0.043 0.044 0.046 0.046 0.045 0.045 5.56 D 0.04 0.043 0.04 0.041 0.037 0.041 0.044 0.044 0.046 0.195 0.282 0.047 0.043 0.043 0.04 1.85 E 0.049 0.076 0.042 0.072 0.067 0.041 0.049 0.046 0.047 0.427 0.402 0.055 0.578 0.293 0.04 0.62 F 0.424 0.46 0.047 0.357 0.32 0.051 0.106 0.091 0.045 0.43 0.416 0.047 0.723 0.507 0.042 0.21 G 0.504 0.408 0.042 0.512 0.577 0.046 0.326 0.229 0.048 0.33 0.331 0.043 0.532 0.478 0.041 0.07 H 0.551 0.441 0.049 0.569 0.598 0.05 0.31 0.393 0.045 0.32 0.43 0.045 0.544 0.453 0.04 0 MIC in SA

Nisin N alkyne N dansyl N CF N dimer conc in µM

A 0.052 0.047 0.041 0.043 0.043 0.039 0.058 0.057 0.037 0.118 0.125 0.05 0.114 0.108 0.041 50 B 0.045 0.044 0.038 0.045 0.045 0.039 0.05 0.049 0.042 0.429 0.458 0.049 0.053 0.052 0.04 16.7 C 0.046 0.048 0.039 0.242 0.043 0.039 0.215 0.174 0.053 0.463 0.471 0.049 0.219 0.228 0.048 5.56 D 0.213 0.272 0.041 0.362 0.266 0.043 0.372 0.368 0.051 0.471 0.493 0.065 0.445 0.421 0.066 1.85 E 0.427 0.424 0.043 0.38 0.404 0.042 0.461 0.451 0.051 0.454 0.484 0.059 0.525 0.507 0.042 0.62 F 0.475 0.498 0.046 0.494 0.488 0.042 0.433 0.439 0.045 0.454 0.468 0.052 0.513 0.549 0.054 0.21 G 0.51 0.515 0.044 0.497 0.507 0.039 0.397 0.414 0.055 0.406 0.442 0.064 0.577 0.566 0.046 0.07 H 0.618 0.541 0.043 0.486 0.527 0.047 0.492 0.495 0.055 0.412 0.429 0.085 0.636 0.624 0.049 0

Note: Row H: maximal bacterial growth since the concentration of the peptide = 0 µM. The first two columns of a

nisin-derivative of interest indicate a duplo measurement: bacteria/medium/peptide-sample (0.07-50 µM); the third column indicates a measurement without bacteria: medium/peptide-sample (0.07-50 µM). The values represent the optical density of the bacterial suspensions, values in bold correspond to the MIC value of the respective peptide sample.