C-Terminal Functionalization of Nylon-3 Polymers: Effects of C-Terminal Groups on Antibacterial and Hemolytic Activities Supporting Information Part 2 MALDI-TF, GPC, 1 H NMR, Bioassay and CAC data Jihua Zhang, Matthew J. Markiewicz, Brendan P. Mowery, Bernard Weisblum, Shannon S. Stahl * and Samuel H. Gellman * Department of Chemistry and Department of Pharmacology, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706 S2-1
1 MALDI-TF data samples generated during efforts to achieve C-terminal functionalization of poly(c) MALDI-TF spectrum of the product from Table S1, entry 1. M refers to poly(c) containing 15 units with an acetyl N-terminus and an imide C-terminus. S2-2
MALDI-TF spectrum of the product from Table S1, entry 2. M refers to poly(c) containing 10 subunits with an acetyl N-terminus and an imide C-terminus. Nu = benzylamine. S2-3
MALDI-TF spectrum of the product from Table S1, entry 3. M refers to poly(c) containing 12 subunits with an acetyl N-terminus and an imide C-terminus. Nu = benzylamine. S2-4
MALDI-TF spectrum of the product from Table S1, entry 4. M refers to poly(c) containing 19 subunits with an acetyl N-terminus and an imide C-terminus. Nu = benzylamine, Nu = benzyl mercaptan. S2-5
MALDI-TF spectrum of the product from Table S1, entry 5. M refers to poly(c) containing 9 subunits with an acetyl N-terminus and an imide C-terminus. Nu = benzylamine. S2-6
MALDI-TF spectrum of the product from Table S1, entry 6. The labels 1, 2 and 3 indicate [M+Nu+Li] + (2165), [M+Nu+Na] + (2181) and [M+Nu+K] + (2197) peaks, respectively, where M refers to poly(c) containing 13 subunits with an acetyl N-terminus and an imide C-terminus. Nu = benzyl mercaptan. S2-7
MALDI-TF spectrum of the product from Table S1, entry 7. M refers to poly(c) containing 23 subunits with an acetyl N-terminus and an imide C-terminus. S2-8
MALDI-TF spectrum of the product from Table S1, entry 8. M refers to poly(c) containing 19 subunits with an acetyl N-terminus and an imide C-terminus. S2-9
MALDI-TF spectrum of the product from Table S1, entry 10. M refers to poly(c) containing 19 subunits with an acetyl N-terminus and an imide C-terminus. Nu = MeH. S2-10
MALDI-TF spectrum of the product from Table S1, entry 11. M refers to poly(c) containing 8 subunits with an acetyl N-terminus and an imide C-terminus. Nu = EtH. S2-11
MALDI-TF spectrum of the product from Table S1, entry 12. M refers to poly(c) containing 9 subunits with an acetyl N-terminus and an imide C-terminus. Nu = BnH. S2-12
MALDI-TF spectrum of the product from Table S1, entry 13. M refers to poly(c) containing 8 subunits with an acetyl N-terminus and an imide C-terminus. Nu = HCH 2 CH 2 2. S2-13
MALDI-TF spectrum of the product from Table S1, entry 14. M refers to poly(c) containing 31 subunits with an acetyl N-terminus and an imide C-terminus. S2-14
MALDI-TF spectrum of the product from Table S1, entry 15. M refers to poly(c) containing 10 subunits with an acetyl N-terminus and an imide C-terminus. S2-15
MALDI-TF spectrum of the product from Table S1, entry 16. M refers to poly(c) containing 29 subunits with an acetyl N-terminus and an imide C-terminus. S2-16
2 GPC, 1 H NMR and MALDI-TF characterization data for C-terminal imide and C- terminal functionalized 37:63 CH:MM nylon-3 random copolymers (R = side chain of either CH or MM) Solvents GPC trace (RI detection) of Boc-protected 1 in THF. M n = 4627, PDI = 1.10, dn/dc = 0.1. 23.22 0.81 41.02 6.82 3.00 113.21 4.806 4.328 4.173 4.163 3.797 3.775 3.343 3.148 3.012 2.936 2.600 2.033 2.016 1.977 1.700 1.586 1.389 1.260 1.243 1.222 1.193 8 7 6 5 4 3 2 1 PPM 1 H NMR of 1 in D 2 at 10 mg/ml. S2-17
MALDI-TF spectrum of 1. S2-18
Solvents GPC trace (RI detection) of Boc-protected 2 in THF. M n = 4749, PDI = 1.13, dn/dc = 0.1. 4.00 18.89 1.89 40.75 95.35 7.777 7.749 7.724 7.629 4.315 4.119 3.803 3.777 3.647 3.337 3.135 3.007 2.586 1.685 1.548 1.332 1.246 1.180 8 7 6 5 4 3 2 1 PPM 1 H NMR of 2 in D 2 at 10 mg/ml. S2-19
MALDI-TF spectrum of 2. S2-20
Solvents GPC trace (RI detection) of Boc-protected 3 in THF. M n = 5845, PDI = 1.15, dn/dc = 0.1. 21.12 1.88 40.30 6.68 1.68 3.00 121.16 4.329 4.159 3.806 3.782 3.725 3.675 3.654 3.352 3.153 3.123 3.008 2.611 2.282 2.262 1.700 1.608 1.407 1.394 1.361 1.264 0.865 0.842 0.37 N 0.63 n R' + H 3 N 8 7 6 5 4 3 2 1 PPM 1 H NMR of 3 in D 2 at 10 mg/ml. S2-21
MALDI-TF spectrum of 3. S2-22
Solvents TrtS 0.37 0.63 N n R' BocHN GPC trace (RI detection) of Boc-protected 4 (with trityl-proteced thiol) in THF. M n = 5337, PDI = 1.07, dn/dc = 0.1. 0.82 31.86 1.00 10.94 0.32 62.75 170.28 7.936 7.641 4.320 4.164 3.814 3.782 3.725 3.656 3.347 3.147 3.018 2.863 2.609 2.095 1.698 1.390 1.256 1.184 HS 0.37 0.63 N n R' + H 3 N 8 7 6 5 4 3 2 1 PPM 1 H NMR of 4 in D 2 at 10 mg/ml. S2-23
HS 0.37 0.63 N n R' + H 3 N MALDI-TF spectrum of 4. S2-24
N 0.37 0.63 n HN BocHN Solvents GPC trace (RI detection) of Boc-protected 1B-a in THF. M n = 4890, PDI = 1.13, dn/dc = 0.1. 1.55 31.79 57.02 9.25 3.00 5.43 157.85 7.902 7.680 4.821 4.741 4.325 3.768 3.358 3.150 3.017 2.985 2.621 2.019 1.950 1.702 1.598 1.456 1.418 1.395 1.367 1.339 1.334 1.266 1.254 1.227 1.202 8 7 6 5 4 3 2 1 PPM 1 H NMR of 1B-a in D 2 at 10 mg/ml. S2-25
MALDI-TF spectrum of 1B-a. S2-26
N 0.37 0.63 n HN BocHN Solvents GPC trace (RI detection) of Boc-protected 1B-b in THF. M n = 5975, PDI = 1.07, dn/dc = 0.1. 1.38 22.41 7.28 3.00 6.24 47.29 117.01 7.924 7.683 4.820 4.818 4.804 4.801 4.787 4.784 4.783 4.781 4.780 4.328 4.169 3.347 3.131 3.007 2.605 2.226 2.224 2.020 1.939 1.699 1.679 1.260 1.239 1.217 1.192 8 7 6 5 4 3 2 1 PPM 1 H NMR of 1B-b in D 2 at 10 mg/ml. S2-27
MALDI-TF spectrum of 1B-b. S2-28
N 0.37 0.63 n HN BocHN Solvents GPC trace (RI detection) of Boc-protected 2B-a in THF. M n = 3948, PDI = 1.13, dn/dc = 0.1. 4.00 21.13 0.16 0.35 39.40 6.52 4.19 122.33 7.774 7.744 7.738 7.712 7.619 4.808 4.726 4.295 4.251 3.800 3.754 3.666 3.593 3.565 3.542 3.506 3.346 3.332 3.322 3.119 3.104 3.070 3.059 2.991 2.585 1.927 1.588 1.569 1.673 1.551 1.527 1.441 1.419 1.379 1.315 1.242 1.232 1.191 1.168 1.144 N 0.37 0.63 n HN + H 3 N 8 7 6 5 4 3 2 1 PPM 1 H NMR of 2B-a in D 2 at 10 mg/ml. S2-29
N 0.37 0.63 n HN + H 3 N MALDI-TF spectrum of 2B-a. S2-30
N 0.37 0.63 n HN BocHN GPC trace (RI detection) of Boc-protected 2B-b in THF. M n = 4837, PDI = 1.12, dn/dc = 0.1. 1.71 10.00 3.53 3.00 21.01 50.49 7.788 7.756 7.728 7.638 4.809 4.321 4.283 3.378 3.353 3.342 3.324 3.308 3.159 3.127 3.121 3.080 3.020 3.003 2.864 2.232 1.610 1.583 1.957 1.947 1.568 1.699 1.559 1.542 1.533 1.529 1.523 1.511 1.482 1.454 1.413 1.391 1.339 1.266 1.252 N 0.37 0.63 n HN + H 3 N 8 7 6 5 4 3 2 1 PPM 1 H NMR of 2B-b in D 2 at 10 mg/ml. Polymer 2B-b does not produce a MALDI-TF spectrum. S2-31
Solvents GPC trace (RI detection) of Boc-protected 3B in THF. M n = 4112, PDI = 1.12, dn/dc = 0.1. 16.51 5.79 2.53 3.28 3.00 34.53 111.88 7.996 7.958 7.716 7.665 7.619 7.536 7.490 4.810 4.336 4.312 4.305 3.372 3.364 3.341 3.169 3.146 3.037 3.028 2.625 2.300 2.278 2.249 1.738 1.970 1.725 1.720 1.704 1.682 1.643 1.619 1.577 1.499 1.485 1.458 1.427 1.405 1.392 1.379 1.286 0.883 8 7 6 5 4 3 2 1 PPM 1 H NMR of 3B in D 2 at 10 mg/ml. S2-32
MALDI-TF spectrum of 3B. S2-33
Solvents N 0.37 0.63 n HN BocHN GPC trace (RI detection) of Boc-protected 1C-a in THF. M n = 5072, PDI = 1.09, dn/dc = 0.1. 9.78 3.00 106.21 214.12 579.10 7.697 7.592 4.832 4.811 4.810 4.786 4.782 4.781 4.780 4.776 4.743 4.741 4.312 4.258 3.352 3.128 3.095 3.013 2.610 2.230 2.019 1.696 1.679 1.588 1.455 1.433 1.418 1.395 1.382 1.362 1.321 1.259 1.241 1.221 1.196 8 7 6 5 4 3 2 1 PPM 1 H NMR of 1C-a in D 2 at 10 mg/ml. S2-34
MALDI-TF spectrum of 1C-a. S2-35
N 0.37 0.63 n HN BocHN GPC trace (RI detection) of Boc-protected 1C-b in THF. M n = 6135, PDI = 1.12, dn/dc = 0.1. 3.44 27.20 50.86 5.73 3.00 145.45 7.686 7.623 4.817 4.332 4.288 3.353 3.326 3.146 3.135 3.107 3.069 3.063 3.052 3.017 3.006 2.985 2.229 1.717 1.699 1.690 1.656 1.639 1.614 1.601 1.588 1.579 1.563 1.536 1.514 1.464 1.449 1.326 1.261 1.197 8 7 6 5 4 3 2 1 PPM 1 H NMR of 1C-b in D 2 at 10 mg/ml. S2-36
MALDI-TF spectrum of 1C-b. S2-37
N 0.37 0.63 n HN BocHN Solvents GPC trace (RI detection) of Boc-protected 2D in THF. M n = 5753, PDI = 1.11, dn/dc = 0.1. 57.86 2.82 104.76 18.16 8.57 3.00 304.29 4.840 4.811 4.781 4.318 4.237 3.580 3.556 3.350 3.147 3.138 3.128 3.093 3.006 2.963 2.952 2.589 2.030 2.013 1.693 1.605 1.587 1.377 1.258 1.196 1.183 1.159 0.861 8 7 6 5 4 3 2 1 PPM 1 H NMR of 1E in D 2 at 10 mg/ml. S2-38
MALDI-TF spectrum of 1E. S2-39
Solvents GPC trace (RI detection) of Boc-protected 1D-a in THF. M n = 5676, PDI = 1.06, dn/dc = 0.1. 213.74 10.93 387.00 80.15 32.93 3.00 1004.34 4.835 4.812 4.788 4.739 4.317 4.274 4.191 4.179 4.136 3.604 3.580 3.556 3.540 3.347 3.143 3.088 3.010 2.897 2.599 2.029 2.012 1.699 1.684 1.594 1.576 1.395 1.363 1.257 1.247 1.182 1.159 0.860 8 7 6 5 4 3 2 1 PPM 1 H NMR of 1D-a in D 2 at 10 mg/ml. S2-40
MALDI-TF spectrum of 1D-a. S2-41
N 0.37 0.63 n HN BocHN GPC trace (RI detection) of Boc-protected 1D-b in THF. M n = 5544, PDI = 1.09, dn/dc = 0.1. 21.07 7.67 3.92 3.00 0.43 40.61 100.28 4.831 4.810 4.783 4.773 4.324 3.347 3.153 3.141 3.134 3.119 3.111 3.022 3.010 1.703 2.233 1.687 1.676 1.617 1.608 1.594 1.574 1.549 1.545 1.510 1.485 1.474 1.451 1.414 1.393 1.364 1.342 1.284 1.264 8 7 6 5 4 3 2 1 PPM 1 H NMR of 1D-b in D 2 at 10 mg/ml. S2-42
MALDI-TF spectrum of 1D-b. S2-43
N 0.37 0.63 n HN STrt BocHN Solvents GPC trace (RI detection) of Boc-protected 1F (with trityl-proteced thiol) in THF. M n = 5449, PDI = 1.11, dn/dc = 0.1. 0.06 0.02 0.04 1.50 1.00 1.36 1.46 7.350 7.298 7.267 5.536 5.301 4.234 3.352 3.129 2.557 1.997 1.900 1.609 11.99 1.421 1.175 N 0.37 0.63 n HN STrt BocHN 8 7 6 5 4 3 2 1 PPM 1 H NMR of Boc-protected 1F (with trityl-proteced thiol) in CDCl 3 at 10 mg/ml. S2-44
Solvents N 0.37 0.63 n HN STrt BocHN GPC trace (RI detection) of Boc-protected 2F (with trityl-proteced thiol) in THF. M n = 4917, PDI = 1.12, dn/dc = 0.1. 2.00 18.65 4.16 12.76 16.37 19.12 7.801 7.777 7.449 7.423 7.352 7.322 7.299 7.263 5.536 5.296 4.464 4.234 3.378 3.208 2.543 140.44 2.300 1.867 1.622 1.421 1.322 1.174 N 0.37 0.63 n HN STrt BocHN 8 7 6 5 4 3 2 1 PPM 1 H NMR of Boc-protected 2F (with trityl-proteced thiol) in CDCl 3 at 10 mg/ml. S2-45
4.00 1.29 19.10 5.63 37.19 111.79 7.789 7.757 7.734 7.648 7.640 7.358 4.335 3.346 3.160 3.111 3.010 2.623 1.693 1.339 1.258 N 0.37 0.63 n HN SH + H 3 N 8 7 6 5 4 3 2 1 PPM 1 H NMR of 2F D 2 at 10 mg/ml. N 0.37 0.63 n HN SH + H 3 N MALDI-TF spectrum of 2F. S2-46
Solvents TrtS N 0.37 0.63 n HN BocHN GPC trace (RI detection) of Boc-protected 4C (with trityl-proteced thiol) in THF. M n = 4573, PDI = 1.13, dn/dc = 0.1. 0.87 0.86 0.29 1.00 0.06 1.36 0.94 0.88 7.428 7.403 7.298 7.291 7.288 5.318 4.251 3.771 3.753 3.378 2.990 2.561 2.216 1.879 10.68 1.649 1.445 1.370 1.354 1.196 TrtS N 0.37 0.63 n HN BocHN 8 7 6 5 4 3 2 1 PPM 1 H NMR of Boc-protected 4C (with trityl-proteced thiol) in CDCl 3 at 10 mg/ml. S2-47
4.00 3.86 28.99 11.00 0.68 58.58 155.88 7.936 7.693 7.665 7.616 7.367 4.313 4.154 3.348 3.157 3.019 2.863 2.611 1.690 1.324 1.251 1.184 8 7 6 5 4 3 2 1 PPM 1 H NMR of 4C D 2 at 10 mg/ml. MALDI-TF spectrum of 4C. S2-48
3 Antibacterial and hemolytic assays Dose-response curves of antibacterial activity of polymer 1. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. Dose-response curves of antibacterial activity of polymer 2. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. S2-49
Dose-response curves of antibacterial activity of polymer 3. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. Dose-response curves of antibacterial activity of polymer 1B-a. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. S2-50
Dose-response curves of antibacterial activity of polymer 1B-b. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. Dose-response curves of antibacterial activity of polymer 2B-a. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. S2-51
Dose-response curves of antibacterial activity of polymer 2B-b. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. Dose-response curves of antibacterial activity of polymer 3B. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. S2-52
Dose-response curves of antibacterial activity of 1C-a. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. Dose-response curves of antibacterial activity of 1C-b. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. The polymer is not fully soluble in BHI > 100 µg/ml. S2-53
Dose-response curves of antibacterial activity of 1D-a. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. Dose-response curves of antibacterial activity of 1D-b. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. S2-54
Dose-response curves of antibacterial activity of 1E. Numbers in the parentheses are the MIC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. The polymer is not fully soluble in BHI > 200 µg/ml. S2-55
Dose-response curves of hemolytic activity. Numbers in the parentheses are the MHC values (µg/ml) deduced from the corresponding curves. The lines simply connect the data points. S2-56
Dose-response curves of hemolytic activity. Numbers in the parentheses are the MHC (µg/ml) values deduced from the corresponding curves. The lines simply connect the data points. S2-57
3 CAC measurements in Tris-buffered saline DPH fluorescence plot for the non-aggregating polymers DPH fluorescence plot for 1C-a. CAC = 220 µg/ml. S2-58
DPH fluorescence plot for 1C-b. CAC = 70 µg/ml. DPH fluorescence plot for 1E. CAC = 360 µg/ml. S2-59
DPH fluorescence plot for DDM. CAC = 0.19 mm (lit. 0.17mM). S2-60