- Supporting Information - for Combined Approach of Backbone Amide Linking and On-Resin N-Methylation for the Synthesis of Bioactive and Metabolically Stable Peptides Frank Wesche 1, Hélène Adihou 1, Astrid Kaiser 2, Mario Wurglics 2, Manfred Schubert- Zsilavecz 2, Marcel Kaiser 3,4, Helge B. Bode 1,5 1 Merck Stiftungsprofessur für Molekulare Biotechnologie, Goethe University Frankfurt, Maxvon-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany. 2 Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany. 3 Swiss Tropical and Public Health Institute, Parasite Chemotherapy, Socinstrasse 57, CH- 4051 Basel, Switzerland. 4 University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland. 5 Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, Maxvon-Laue-Strasse 15, D-60438 Frankfurt am Main, Germany. - Content - General Procedures... 2 Synthesis of Amines S1-S4... 2 Design of Experiment (DOE)... 4 Supplementary Tables... 6 Supplementary Figures... 42 NMR Spectra... 51 References... 53 S1
General Procedures All reactions were carried out under an atmosphere of nitrogen and with dry solvents, unless otherwise stated. Dry solvents and chemicals were purchased from Sigma-Aldrich (Germany) or Acros Organics (Belgium) in highest commercial available purity and were used without further purification. Protected amino acids and resins were supplied from Carbolution (Germany), Bachem (Switzerland), Iris Biotech (Germany) and Merck Millipore (Germany). NMR The 1 H and 13 C spectra were recorded on an AV-400 or AV-500-spectrometer from Bruker. All measurements were done at room temperature and chemical shifts are declared in ppm (parts per million) compared to tetramethylsilane [ = 0 ppm], referring on second internal standard. To explain multiplicities the following abbreviations were used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Synthesis of Amines S1-S4 2-(1-Methyl-1H-indol-3-yl)ethanamine (S1) Title compound was synthesized according to Lygin et al. 1 Therefore, to a mixture of sodium hydride (60% in mineral oil, 0.88 g, 22 mmol, 1.1 equiv.) in 60 ml dry N,N-dimethylformamide (DMF) was slowly added a solution of tryptamine (3.32 g, 20 mmol, 1.0 equiv.) in 40 ml dry DMF. The mixture was stirred for 30 min at RT, cooled to 0 C and methyl iodide (1.37 ml, 22 mmol) was added dropwise and stirred overnight. Afterwards DMF was evaporated and the resulting residue was dissolved in 300 ml water and extracted three times with EtOAc. The combined organic layers were dried over MgSO 4 and the solvents were removed under reduced pressure. The crude product was purified by column chromatography on silica gel (DCM/MeOH/Et 3N 85:10:5) giving 2.35 g (68%) of S1 as a yellow oil. 1 H NMR (500 MHz, CDCl 3) δ 7.61 (d, J = 7.9 Hz, 1H), 7.32 7.28 (m, 1H), 7.26 7.21 (m, 1H), 7.14 7.09 (m, 1H), 6.90 (s, 1H), 3.75 (s, 3H), 3.04 2.99 (m, 2H), 2.93 2.88 (m, 2H), 1.63 (s, 2H). 13 C NMR (126 MHz, CDCl 3) δ 137.2, 128.0, 127.0, 121.7, 119.1, 118.8, 112.3, 109.3, 42.6, 32.7, 29.4. S2
Tert-butyl 3-(2-aminoethyl)-1H-indole-1-carboxylate (N-Boc-tryptamine) (S4) Title compound was synthesized according to Uno et al. 2 N-(2-(1H-Indol-3-yl)ethyl)-2,2,2-trifluoroacetamide (S2) Tryptamine (3.20 g, 20 mmol, 1.0 eq.) was dissolved in 150 ml dry DCM and pyridine (17.95 ml) and cooled to 0 C. Subsequently trifluoroacetic anhydride (3.10 ml, 22 mol, 1.1 eq.) was added dropwise, and the mixture was stirred for 5 min at 0 C. The ice bath was removed and the mixture was stirred for another 2 h at RT. After addition of 150 ml saturated aqueous NaHCO 3, the phases were separated and the organic layer was washed with saturated aqueous NH 4Cl and water and dried over MgSO 4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography (Flash 40+M KP-Sil, 0% 50% EtOAc in n-hexane) to give 4.82 (94%) of S2 as an off-white solid. 1 H NMR (400 MHz, CDCl 3) δ 8.14 (s, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.26 7.21 (m, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 2.0 Hz, 1H), 6.40 (s, 1H), 3.70 (q, J = 6.5 Hz, 2H), 3.06 (t, J = 6.7 Hz, 2H). Tert-butyl 3-(2-(2,2,2-trifluoroacetamido)ethyl)-1H-indole-1-carboxylate (S3) To a mixture of S2 (4.80 g, 18.7 mmol, 1.0 eq.) and 4-N,N-dimethylaminopyridine (114 mg, 0.94 mmol, 0.05 eq.) in 190 ml THF was slowly added a solution of di-tert-butyl dicarbonate (4.90 g, 22.4 mmol, 1.2 eq.) in 20 ml THF and stirred for 1 h at 40 C. The reaction mixture was diluted with DCM (100 ml) and washed once with water. The phases were separated and the organic layer dried over MgSO 4 and purified by flash chromatography (Flash 40+M KP- Sil, 0% 50% EtOAc in n-hexane) to give 5.00 g (75%) of S3 as an off-white solid. 1 H NMR (250 MHz, CDCl 3) δ 8.15 (d, J = 8.1 Hz, 1H), 7.56 7.48 (m, 1H), 7.43 (s, 1H), 7.39 7.22 (m, 2H), 6.51 (s, 1H), 3.69 (q, J = 6.7 Hz, 2H), 2.99 (t, J = 6.9 Hz, 2H), 1.67 (s, 9H). Tert-butyl 3-(2-aminoethyl)-1H-indole-1-carboxylate (N-Boc-tryptamine) (S4) A mixture of S3 (4.8 g, 13.5 mmol, 1.0 eq.) and K 2CO 3 (6.53 g, 47.2 mmol, 3.5 eq.) was stirred in 135 ml MeOH/water (70:30) (v/v) for 48 h. The reaction mixture was diluted with 100 ml water and extracted three times with 100 ml DCM. The combined organic layers were dried over MgSO 4. The solvent was removed under reduced pressure to give 3.14 g (89%) of title compound as a slightly yellow oil. 1 H NMR (250 MHz, CDCl 3) δ 8.13 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 7.1 Hz, 1H), 7.43 (s, 1H), 7.37 7.27 (m, 1H), 7.22 (dd, J = 7.4, 1.0 Hz, 1H), 3.06 (t, J = 6.7 Hz, 2H), 2.86 (t, J = 6.7 Hz, 2H), 1.71 (s, 2H), 1.67 (s, 9H). S3
Design of Experiment (DOE) A linear sequence of Boc-Ile-Ile-Ile-Ile-2CTC-PS was synthesized in parallel four times on a preloaded resin on a 100 µm scale with a Syro Wave peptide synthesizer (Biotage, Uppsala, Sweden) by using standard Fmoc/t-Bu chemistry. Therefore, the resin was placed in a plastic reactor vessels with a Teflon frit and an amount of 6 eq. of amino acid derivatives (c = 0.2 M) was activated in situ at room temperature with 6 eq. of HCTU (c = 0.6 M) in DMF in the presence of 12 eq. DIPEA (c = 2.4 M) in NMP for 50 min. Fmoc-protecting groups were removed with a solution of 40% piperidine in NMP (v/v%) for 5 min and the deprotection step was repeated for another 10 min with 20% piperidine in NMP (v/v%). After each coupling and deprotection step, the resin was washed with NMP (4 ). After the addition of the final residue, Boc-Ile-OH, the resin was washed with NMP (5 ), DMF (5 ) and DCM (5 ). The synthesis was confirmed with a test-cleavage. Therefore, a few beads of the resin were treated with cleavage cocktail TFA/TIS/water (95:2.5:2.5) for 1 h. The solution was evaporated and the residue was dissolved in MeOH and analysed by HPLC-MS. Subsequently the four resins (in total 400 µmol) were mixed and split into 10 µmol portions. Conditions for on-resin permethylation of peptide backbone were performed with synthesised peptidyl resin ((Boc-Ile-Ile-Ile-Ile-2CTC-PS, 10 µmol) from above. Therefore, the resin was placed in a plastic reactor vessel equipped with a Teflon frit and a Teflon valve. The reactor vessel was flushed three times with nitrogen and the resins were washed three times with dry THF. Additionally, under an atmosphere of nitrogen in a separate round bottom flask, a mixture of 0.25 or 1 M LiOtBu (1.0 ml, 5 or 20 eq./amide bond), respectively, in THF and when indicated dry DMSO (0.4 ml, 100 µl/amide bond) was prepared and added to the resin. When no DMSO was added, 400 µl of dry THF were added. The deprotonation took place for 5 or 60 min at room temperature. Afterwards the LiOtBu containing solution was poured into a round bottom flask under nitrogen and MeI (100 µl of 12.5% or 49.8% MeI (v/v%) in THF, 5 or 20 eq./amide bond), respectively, was added to it. The mixture was added again to the resin and agitated for another 5 or 60 min at room temperature. Afterwards the resin was washed with MeOH (5 ), DCM (5 ) and dried. The achieved conversion was determined with a testcleavage as previously described. For statistical analysis of the DOE, the trial version of the program DEVELVE was used with a full factorial array with five factors. Number of observations = 32 (2 5 for complete factorial design). Factor 1: amount of LiOtBu (two levels: 5 eq. and 20 eq.) Factor 2: time for deprotonation (two levels: 5 and 60 min) Factor 3: amount of MeI (two levels: 5 eq. and 20 eq.) Factor 4: time for methylation (two levels: 5 and 60 min) Factor 5: DMSO (two levels: added and not added) Design of the matrix and measured conversions are shown in Table S1. S4
Formula for Parallel Artificial Membrane Permeability Assay (PAMPA) Log P e was calculated with formula below. ln (1 c ) equilibrium log P e (cm/s) = log [ S ( 1 V + 1 ] D V ) t A c A c A = final drug concentration in the acceptor well (μm) c equilibrium = theoretical equilibrium concentration = [c D V D + c A V A ]/[V D + V A ] where: c D = final drug concentration in the donor well (μm) V D = volume in the donor well (cm 3 ) c A = final drug concentration in the acceptor well (μm) V A = volume in the acceptor well (cm 3 ) S = surface area (cm²), typically 0.268 cm² V D = volume in the donor well (cm 3 ) V A = volume in the acceptor well (cm 3 ) t = incubation time (s) S5
Supplementary Tables Table S1. Reaction conditions for on-resin permethylation of Boc-Ile-Ile-Ile-Ile-2CTC-PS (1); respresenting the design matrix of the DOE with measured conversion of permethlyated peptide (4 x Me). a percentages are estimated from the areas of HPLC-ESI/MS (extracted ion chromatograms) of the corresponding masses of the appropriate cleaved peptides; b equivalents of LiOtBu and MeI refer to one amide bond. Conditions Number of Methylgroups/ % a # equiv. LiOtBu b DMSO Deprotonation time/ min equiv. MeI b Methylation time/ min 0 x Me 1 x Me 2 x Me 3 x Me 4 x Me (permethylated product) 1 5 extra 5 5 5 56.2 13.9 17.9 3.20 8.8 2 20 extra 5 5 5 18.1 6.2 3.6 11.9 60.2 3 5 none 5 5 5 98.7 1.1 0.2 0.0 0.0 4 20 none 5 5 5 92.7 7.1 0.2 0.0 0.0 5 5 extra 60 5 5 48.3 20.6 6.4 13.7 11.0 6 20 extra 60 5 5 22.7 7.8 38.1 3.6 27.8 7 5 none 60 5 5 100.0 0.0 0.0 0.0 0.0 8 20 none 60 5 5 100.0 0.0 0.0 0.0 0.0 9 5 extra 5 5 60 98.4 1.6 0.0 0.0 0.0 10 20 extra 5 5 60 23.3 12.7 22.5 7.4 34.1 11 5 none 5 5 60 100.0 0.0 0.0 0.0 0.0 12 20 none 5 5 60 78.9 20.3 0.7 0.1 0.0 13 5 extra 60 5 60 68.5 23.5 3.1 4.0 0.9 14 20 extra 60 5 60 9.2 4.0 45.2 2.4 39.2 15 5 none 60 5 60 100.0 0.0 0.0 0.0 0.0 16 20 none 60 5 60 92.6 6.6 0.8 0.0 0.0 17 5 extra 5 20 5 82.1 12.6 1.5 2.3 1.5 18 20 extra 5 20 5 18.3 1.1 4.8 2.1 73.7 19 5 none 5 20 5 99.2 0.8 0.0 0.0 0.0 20 20 none 5 20 5 98.0 1.7 0.3 0.0 0.0 21 5 extra 60 20 5 65.0 22.1 4.2 6.4 2.3 22 20 extra 60 20 5 3.5 1.2 10.2 0.8 84.3 23 5 none 60 20 5 96.3 3.4 0.0 0.0 0.3 24 20 none 60 20 5 90.2 9.2 0.5 0.0 0.1 25 5 extra 5 20 60 88.7 9.0 0.9 0.8 0.6 26 20 extra 5 20 60 9.7 1.2 2.9 3.7 82.5 27 5 none 5 20 60 100.0 0.0 0.0 0.0 0.0 28 20 none 5 20 60 84.0 15.1 0.6 0.3 0.0 29 5 extra 60 20 60 79.3 18.4 1.7 0.5 0.1 30 20 extra 60 20 60 0.0 0.7 5.5 1.0 92.8 31 5 none 60 20 60 95.5 4.4 0.1 0.0 0.0 32 20 none 60 20 60 83.9 12.4 3.2 0.4 0.1 S6
Table S2. Result table of all five factors showing calculated mean, size of data set (n), median and standard deviation (STDEV) as well as min./max. values of each factor. equiv. LiOtBu equiv. MeI Deprotonation time/ min Methylation time/ min DMSO all 5 20 5 20 5 60 5 60 extra none Mean 16.2 1.6 11.4 11.4 21.1 16.9 16.2 16.9 15.6 32.5 0.02 n 32 16 16 16 16 16 16 16 16 16 16 Median 0.1 0.1 0.0 0.0 0.2 0.9 0.2 0.9 0.04 19.4 0.0 STDEV 29.4 3.4 18.7 18.7 37.2 29.0 30.5 29.0 30.8 35.0 0.1 Max 92.8 11.1 92.8 60.3 92.8 82.5 92.8 84.3 92.8 92.8 0.3 Min 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 67.5 graph a -16.1 a Response graph of performed DOE with all five factors (eq. LiOtBu. eq. MeI, deprotonation time, methylation time, DMSO). Black line is the mean value of the factor, and the two gray lines are the mean plus and minus STDEV. Visualizing a great influence of the amount of LiOtBu and added DMSO. Table S3. Result table of three factors showing calculated mean, size of data set (n), median and STDEV as well as min./max. values of each factor. equiv. MeI Deprotonation time/ min Methylation time/ min all 5 20 5 60 5 60 Mean 61.9 40.5 83.3 62.8 61.0 61.5 62.3 n 8 4 4 4 4 4 4 Median 66.7 36.9 83.4 67.0 61.7 67.0 60.8 STDEV 25.2 14.0 7.8 20.8 32.3 24.6 29.6 Max 92.8 60.3 92.8 82.5 92.8 84.3 92.8 Min 27.7 27.7 73.7 34.7 27.7 27.7 34.7 graph a 93.3 26.5 a Response graph of performed DOE with three factors (equiv. MeI, deprotonation time, methylation time). Whereas the amount of LiOtBu is set to 20 equiv. and DMSO was added. Black line is the mean value of the factor, and the two gray lines are the mean plus and minus STDEV. Emphasising the influence of the amount of added MeI. S7
Table S4: Heterologous expression of kj12b and kj12c from Xenorhabdus KJ12.1 with A-MT in Kj12B replaced by A-MT from vietb in E. coli DH10 MtaA + 0.1 % arabinose fed with different amines. 3 * RXPs were synthesized by approach of backbone amide linking and on-resin methylation and tested for their bioactivity. Substrate TRA MW (g/mol) RXP m/z R t (min) MS 2 -fragmentation relative amount - 542.34 7.9 ml-ml-ml-tra 16.4-655.63 8.5 ml-ml-l-ml-tra 10.2 108 669.89 8.6 ml-ml-ml-ml-tra* 100 160.10-782.78 9.2 ml-ml-l-ml-ml-tra 8.8-796.57 9.3 ml-ml-ml-ml-ml-tra 31.4-909.98 9.8 ml-ml-l-ml-ml-ml-tra 3.3-923.64 10.0 ml-ml-ml-ml-ml-ml-tra 6.3 MTRA - 556.38 9.1 ml-ml-ml-mtra 16.9-669.55 9.6 ml-ml-l-ml-mtra 6.6 174.11 110 683.49 9.9 ml-ml-ml-ml-mtra* 100-796.54 10.4 ml-ml-l-ml-ml-mtra 6.0-810.58 10.6 ml-ml-ml-ml-ml-mtra 24.2-937.67 11.2 ml-ml-ml-ml-ml-ml-mtra 2.9 1Nphth - 553.42 8.8 ml-ml-ml-1nphth 65.1-666.53 9.2 ml-ml-l-ml-1nphth 4.4-666.53 9.4 ml-l-ml-ml-1nphth 7.8 171.10 114 680.53 9.5 ml-ml-ml-ml-1nphth* 100-793.52 9.9 ml-ml-l-ml-ml-1nphth 5.4-807.61 10.1 ml-ml-ml-ml-ml-1nphth 20.8-934.68 10.8 ml-ml-ml-ml-ml-ml-1nphth 2.6 2Nphth - 553.44 8.7 ml-ml-ml-2nphth 22.2-666.50 9.2 ml-ml-l-ml-2nphth 2.9-666.50 9.4 ml-l-ml-ml-2nphth 6.2 171.10 112 680.51 9.5 ml-ml-ml-ml-2nphth* 100-793.54 9.9 ml-ml-l-ml-ml-2nphth 9.5-807.61 10.1 ml-ml-ml-ml-ml-2nphth 28.9-934.68 10.8 ml-ml-ml-ml-ml-ml-2nphth 5.4 S8
Substrate 4FPEA 4BrPEA MW (g/mol) 139.08 199.00 RXP m/z R t (min) MS 2 -fragmentation relative amount - 521.40 8.1 ml-ml-ml-4fpea 10.3-634.39 8.6 ml-ml-l-ml-4fpea 2.8-634.39 8.7 ml-l-ml-ml-4fpea 5.9 116 648.48 8.9 ml-ml-ml-ml-4fpea* 100-761.53 9.4 ml-ml-l-ml-ml-4fpea 12.0-775.58 9.6 ml-ml-ml-ml-ml-4fpea 47.7-888.49 10.0 ml-ml-l-ml-ml-ml-4fpea 4.8-902.67 10.3 ml-ml-ml-ml-ml-ml-4fpea 10.4-1015.58 10.6 ml-ml-l-ml-ml-ml-ml-4fpea 0.9-1029.73 10.9 ml-ml-ml-ml-ml-ml-ml-4fpea 1.1-454.24 7.8 ml-ml-4brpea 1.6-567.29 8.3 ml-l-ml-4brpea 2.7-581.36 8.6 ml-ml-ml-4brpea 51.1-694.43 9.1 ml-ml-l-ml-4brpea 4.9-694.43 9.2 ml-l-ml-ml-4brpea 9.1 118 708.44 9.3 ml-ml-ml-ml-4brpea* 100-821.19 9.8 ml-ml-l-ml-ml-4brpea 6.9-835.49 10.0 ml-ml-ml-ml-ml-4brpea 24.2-948.51 10.5 ml-ml-l-ml-ml-ml-4brpea 1.0-962.60 10.7 ml-ml-ml-ml-ml-ml-4brpea 3.2-1089.64 11.3 ml-ml-ml-ml-ml-ml-ml-4brpea 0.3 S9
Table S5. HPLC-ESI/MS analysis of synthesized RXPs showing sequence, MS 2 -fragmentation, base peak chromatogram (BPC) of crude peptide and and calculated masses. ID structure and sequence MS 2 -fragmentation crude BPC 1 471.27 471.35 n.d. I-I-I-I 2 527.31 527.42 n.d. mi-mi-mi-mi 7 V-V-V-V-PEA 518.3698 518.3701 0.6 8 mv-mv-mv-mv-pea 574.4321 574.4327 1.0 9 V-V-V-V-V-PEA 617.4385 617.4372 2.1 S10
ID structure and sequence MS 2 -fragmentation crude BPC 10 687.5167 687.5171 0.6 mv-mv-mv-mv-mv-pea EIC 11 716.5069 716.5044 3.5 V-V-V-V-V-V-PEA 12 800.6008 800.6009 0.1 mv-mv-mv-mv-mv-mv-pea EIC 13 815.5753 815.5712 5.0 V-V-V-V-V-V-V-PEA 14 913.6849 913.6825 2.6 mv-mv-mv-mv-mv-mv-mv-pea EIC 15 574.4327 574.4327 0.0 L-L-L-L-PEA S11
ID structure and sequence MS 2 -fragmentation crude BPC 16 630.4943 630.4953 1.6 ml-ml-ml-ml-pea 17 687.5167 687.5148 2.8 L-L-L-L-L-PEA 18 757.5950 757.5948 0.3 ml-ml-ml-ml-ml-pea 19 800.6008 800.5985 2.9 L-L-L-L-L-L-PEA 20 884.6947 884.693 1.9 ml-ml-ml-ml-ml-ml-pea 21 913.6849 913.6812 4.0 L-L-L-L-L-L-L-PEA S12
ID structure and sequence MS 2 -fragmentation crude BPC 22 1011.7944 1011.7913 3.1 ml-ml-ml-ml-ml-ml-ml-pea EIC 23 710.3689 710.3701 1.7 F-F-F-F-PEA 24 766.4303 766.4327 3.1 mf-mf-mf-mf-pea 25 857.4385 857.4376 1.0 F-F-F-F-F-PEA 26 927.5167 927.5145 2.4 mf-mf-mf-mf-mf-pea S13
ID structure and sequence MS 2 -fragmentation crude BPC 27 1004.5069 1004.5071 0.2 F-F-F-F-F-F-PEA 28 1088.6008 1088.5968 3.7 mf-mf-mf-mf-mf-mf-pea EIC 29 532.3858 532.3857 0.2 V-V-V-L-PEA 30 588.4478 588.4483 0.8 mv-mv-mv-ml-pea 31 532.3855 532.3857 0.4 V-L-V-V-PEA S14
ID structure and sequence MS 2 -fragmentation crude BPC 32 588.4477 588.4483 1.0 mv-ml-mv-mv-pea 33 614.3698 614.3701 0.5 F-F-V-V-PEA 34 670.4319 670.4327 1.2 mf-mf-mv-mv-pea 35 580.3855 580.3857 0.3 F-V-L-V-PEA 36 636.4477 636.4483 0.9 mf-mv-ml-mv-pea S15
ID structure and sequence MS 2 -fragmentation crude BPC 37 566.3712 566.3701 1.9 V-V-V-F-PEA 38 622.431 622.4327 2.7 mv-mv-mv-mf-pea EIC 39 679.4541 679.4515 3.8 F-V-L-V-V-PEA 40 749.5324 749.5335 1.5 mf-mv-ml-mv-mv-pea 41 713.4385 713.4403 2.5 F-V-V-V-F-PEA S16
ID structure and sequence MS 2 -fragmentation crude BPC 42 783.5167 783.5147 2.6 mf-mv-mv-mv-mf-pea EIC 43 631.4541 631.4556 2.4 V-L-V-V-V-PEA 44 701.5324 701.5302 3.1 mv-ml-mv-mv-mv-pea EIC 45 645.4698 645.4716 2.8 L-L-V-V-V-PEA 46 715.5480 715.5475 0.7 ml-ml-mv-mv-mv-pea EIC S17
ID structure and sequence MS 2 -fragmentation crude BPC 47 730.5226 730.5209 2.3 V-V-V-V-V-L-PEA 48 814.6165 814.6145 2.5 mv-mv-mv-mv-mv-ml-pea 49 764.5069 764.5082 1.7 V-V-V-V-V-F-PEA 50 848.6008 848.5983 2.9 mv-mv-mv-mv-mv-mf-pea 51 812.5069 812.5096 3.3 F-V-V-V-V-F-PEA S18
ID structure and sequence MS 2 -fragmentation crude BPC 52 896.6008 896.5979 3.2 mf-mv-mv-mv-mv-mf-pea EIC 53 730.5226 730.5239 1.8 V-L-V-V-V-V-PEA 54 814.6165 814.6139 3.2 mv-ml-mv-mv-mv-mv-pea 55 V-V-V-V-TRA 557.42 557.38 n.d. 56 mv-mv-mv-mv-tra 613.43 613.44 n.d. S19
ID structure and sequence MS 2 -fragmentation crude BPC 57 571.43 571.40 n.d. V-V-V-L-TRA 58 627.43 627.46 n.d. mv-mv-mv-ml-tra 59 605.42 605.38 n.d. V-V-V-F-TRA 60 661.44 661.44 n.d. mv-mv-mv-mf-tra 61 571.44 571.40 n.d. V-L-V-V-TRA 62 627.45 627.46 n.d. mv-ml-mv-mv-tra S20
ID structure and sequence MS 2 -fragmentation crude BPC 63 532.33 532.39 n.d. L-L-L-A-PEA 64 588.4483 588.4482 0.2 ml-ml-ml-ma-pea EIC 65 532.36 532.39 n.d. L-L-A-L-PEA 66 588.32 588.45 n.d. ml-ml-ma-ml-pea EIC 67 532.33 532.39 n.d. L-A-L-L-PEA EIC S21
ID structure and sequence MS 2 -fragmentation crude BPC 68 588.33 588.45 n.d. ml-ma-ml-ml-pea EIC 69 532.36 532.39 n.d. A-L-L-L-PEA 70 588.34 588.45 n.d. ma-ml-ml-ml-pea EIC 71 574.45 574.43 n.d. L-L-L-l-PEA 72 630.48 630.50 n.d. ml-ml-ml-ml-pea S22
ID structure and sequence MS 2 -fragmentation crude BPC 73 574.45 574.43 n.d. L-L-l-L-PEA 74 630.53 630.50 n.d. ml-ml-ml-ml-pea 75 574.45 574.43 n.d. L-l-L-L-PEA 76 630.52 630.50 n.d. ml-ml-ml-ml-pea 77 574.44 574.43 n.d. l-l-l-l-pea 78 630.50 630.50 n.d. ml-ml-ml-ml-pea S23
ID structure and sequence MS 2 -fragmentation crude BPC 79 574.44 574.43 n.d. l-l-l-l-pea 80 630.46 630.50 n.d. ml-ml-ml-ml-pea 81 Abu-Abu-Abu-Abu-PEA 462.32 462.31 n.d. 82 mabu-mabu-mabu-mabu-pea 518.3701 518.3695 1.2 83 518.33 518.37 n.d. Nva-Nva-Nva-Nva-PEA S24
ID structure and sequence MS 2 -fragmentation crude BPC 84 574.4327 574.4323 0.7 mnva-mnva-mnva-mnva-pea 85 574.46 574.43 n.d. I-I-I-I-PEA 86 630.4953 630.4952 0.2 mi-mi-mi-mi-pea 87 490.28 490.34 n.d. V-V-V-A-PEA 88 546.4014 546.4010 0.7 mv-mv-mv-ma-pea EIC S25
ID structure and sequence MS 2 -fragmentation crude BPC 89 522.26 522.31 n.d. V-V-V-C-PEA 90 578.3735 578.3733 0.3 mv-mv-mv-mc-pea 91 534.29 534.33 n.d. V-V-V-D-PEA 92 590.3912 590.3905 1.2 mv-mv-mv-md-pea EIC 93 548.29 548.34 n.d. V-V-V-E-PEA S26
ID structure and sequence MS 2 -fragmentation crude BPC 94 604.4069 604.4071 0.3 mv-mv-mv-me-pea 95 550.28 550.34 n.d. V-V-V-M-PEA EIC 96 606.4048 606.4049 0.2 mv-mv-mv-mm-pea 97 506.28 506.33 n.d. V-V-V-S-PEA 98 562.3963 562.3958 0.9 mv-mv-mv-ms-pea EIC S27
ID structure and sequence MS 2 -fragmentation crude BPC 99 520.30 520.35 n.d. V-V-V-T-PEA 100 576.4119 576.4114 0.9 mv-mv-mv-mt-pea EIC 101 605.33 605.38 n.d. V-V-V-W-PEA 102 661.4436 661.4432 0.6 mv-mv-mv-mw-pea 103 582.31 582.37 n.d. V-V-V-Y-PEA S28
ID structure and sequence MS 2 -fragmentation crude BPC 104 638.4276 638.4269 1.1 mv-mv-mv-my-pea EIC 105 590.43 590.44 n.d. L-L-L-L-TYA 106 646.49 646.45 n.d. ml-ml-ml-ml-tya 107 613.49 613.44 n.d. L-L-L-L-TRA 108 669.5062 669.5068 0.9 ml-ml-ml-ml-tra S29
ID structure and sequence MS 2 -fragmentation crude BPC 109 627.51 627.46 n.d. L-L-L-L-MTRA 110 683.5218 683.5215 0.4 ml-ml-ml-ml-mtra 111 624.49 624.44 n.d. L-L-L-L-2Npth 112 680.5109 680.5103 0.9 ml-ml-ml-ml-2npth 113 624.40 624.44 n.d. L-L-L-L-1Npth S30
ID structure and sequence MS 2 -fragmentation crude BPC 114 680.5109 680.5105 0.6 ml-ml-ml-ml-1npth 115 592.45 592.42 n.d. L-L-L-L-4FPEA 116 648.4859 648.4861 0.3 ml-ml-ml-ml-4fpea 117 652.43 652.34 n.d. L-L-L-L-4BrPEA 118 708.4058 708.4054 0.6 ml-ml-ml-ml-4brpea S31
ID structure and sequence MS 2 -fragmentation crude BPC 119 560.37 560.42 n.d. L-L-L-L-Bz 120 616.4796 616.4771 4.1 ml-ml-ml-ml-bz 121 588.39 588.45 n.d. L-L-L-L-PPA 122 644.5109 644.5085 3.7 ml-ml-ml-ml-ppa 123 602.40 602.46 n.d. L-L-L-L-PBA S32
ID structure and sequence MS 2 -fragmentation crude BPC 124 658.5266 658.5243 3.5 ml-ml-ml-ml-pba 125 580.42 580.48 n.d. L-L-L-L-cHex 126 636.5422 636.5391 4.9 ml-ml-ml-ml-chex 127 484.32 484.39 n.d. L-L-L-L-Me 128 540.4483 540.4475 1.5 ml-ml-ml-ml-me S33
ID structure and sequence MS 2 -fragmentation crude BPC 129 498.34 498.40 n.d. L-L-L-L-Et 130 554.4640 554.4622 3.2 ml-ml-ml-ml-et 131 512.36 512.42 n.d. L-L-L-L-Pr 132 568.4796 568.4780 2.8 ml-ml-ml-ml-pr 133 526.39 526.43 n.d. L-L-L-L-Bu S34
ID structure and sequence MS 2 -fragmentation crude BPC 134 582.4953 582.4932 3.6 ml-ml-ml-ml-bu 135 540.39 540.45 n.d. L-L-L-L-Amyl 136 596.5109 596.5095 2.3 ml-ml-ml-ml-amyl 137 540.38 540.45 n.d. L-L-L-L-iPn 138 596.5109 596.5089 3.4 ml-ml-ml-ml-ipn S35
ID structure and sequence MS 2 -fragmentation crude BPC 139 564.29 564.36 n.d. L-L-L-C-PEA 140 620.30 620.42 n.d. ml-ml-ml-mc-pea 141 614.32 614.37 n.d. L-L-L-C-1Npth 142 670.34 670.44 n.d. ml-ml-ml-mc-1npth S36
Table S6. Bioactivity of synthesized RXPs (IC 50 [μg/ml]; [µm]) against different protozoa and L6; *Activity of reference compounds: Trypanosoma brucei rhodesiense (melarsoprol), Leishmania donovani (miltefosine), Plasmodium falciparum (chloroquine), L6 cell line (podophyllotoxin). RXP sequence T. b. rhodesiense L. donovani P. falciparum L6 cell µg/ml µm µg/ml µm µg/ml µm µg/ml µm 7 V-V-V-V-PEA 42.0 ± 2.7 81.1 ± 3.7 >100 >100 >50 >50 >100 >100 8 mv-mv-mv-mv-pea 16.9 ± 1.5 29.37 ± 1.8 >100 >100 24.9 43.4 97.2 169 9 V-V-V-V-V-PEA 78 ± 17 125 ± 19 >100 >100 >50 >50 >100 >100 10 mv-mv-mv-mv-mv-pea 22.3 ± 9.8 32.4 ± 10.0 >100 >100 23.4 34.1 >100 >100 11 V-V-V-V-V-V-PEA 63 ± 33 88 ± 33 >100 >100 >50 >50 >100 >100 12 mv-mv-mv-mv-mv-mv-pea 13.5 ± 8.3 16.8 ± 7.3 >100 >100 31.6 39.5 45.2 ± 6.2 56.4 ± 7.7 13 V-V-V-V-V-V-V-PEA 62.6 ± 6.1 76.8 ± 5.3 >100 >100 >50 >50 >100 >100 14 mv-mv-mv-mv-mv-mv-mv-pea 4.56 ± 0.11 4.99 ± 0.09 27.5 ± 3.6 34.0 ± 3.9 6.5 7.1 29 ± 14 31 ± 15 15 L-L-L-L-PEA 13.2 ± 0.9 22.9 ± 1.1 >100 >100 25.0 43.6 68.6 119.6 16 ml-ml-ml-ml-pea 1.41 ± 0.03 2.24 ± 0.03 16.4 ± 1.8 26.0 ± 2.8 2.4 3.7 16.7 ± 3.5 26.5 ± 4.9 17 L-L-L-L-L-PEA 54.8 ± 6.9 79.8 ± 7.1 >100 >100 >50 >50 >100 >100 18 ml-ml-ml-ml-ml-pea 3.0 ± 1.5 3.9 ± 1.4 27.2 ± 1.5 35.9 ± 2.0 1.2 1.6 15.6 ± 1.7 20.6 ± 2.2 19 L-L-L-L-L-L-PEA 39 ± 20 49 ± 17 >100 >100 21.0 26.2 >100 >100 20 ml-ml-ml-ml-ml-ml-pea 2.09 ± 0.14 2.37 ± 0.11 11.5 ± 1.0 13.0 ± 1.1 0.6 0.7 8.5 ± 3.6 9.7 ± 4.0 21 L-L-L-L-L-L-L-PEA 57.1 62.5 >100 >100 >50 >50 >100 >100 22 ml-ml-ml-ml-ml-ml-ml-pea 2.3 ± 1.2 2.3 ± 0.8 10.2 ± 3.7 10.1 ± 3.6 0.7 0.7 15.1 ± 1.8 14.9 ± 1.7 23 F-F-F-F-PEA 5.7 ± 0.2 8.1 ± 0.2 >100 >100 10.0 14.1 50.0 ± 0.1 70.44 ± 0.14 24 mf-mf-mf-mf-pea 4.1 ± 0.2 5.4 ± 0.2 9.0 ± 0.9 11.8 ± 1.1 2.0 2.6 7.6 ± 1.9 10.0 ± 2.5 25 F-F-F-F-F-PEA 10 ± 7 11.3 ± >100 >100 >50 >50 >100 >100 26 mf-mf-mf-mf-mf-pea 2.6 ± 0.2 2.74 ± 0.15 11.6 ± 2.6 12.5 ± 2.8 0.2 0.25 5.9 ± 1.6 6.3 ± 1.7 S37
RXP sequence T. b. rhodesiense L. donovani P. falciparum L6 cell µg/ml µm µg/ml µm µg/ml µm µg/ml µm 27 F-F-F-F-F-F-PEA 55.9 55.6 >100 >100 >50 >50 >100 >100 28 mf-mf-mf-mf-mf-mf-pea 4.1 ± 0.2 3.79 ± 0.12 10.5 ± 0.1 9.60 ± 0.05 0.3 0.3 3.0 ± 1.4 2.7 ± 1.3 29 V-V-V-L-PEA 14.0 ± 1.8 26.2 ± 2.4 >100 >100 >50 >50 >100 >100 30 mv-mv-mv-ml-pea 11.7 ± 0.8 19.8 ± 0.9 >100 >100 19.3 32.8 78 ± 17 132 ± 29 31 V-L-V-V-PEA 43.2 ± 8.0 81 ± 11 >100 >100 >50 >50 >100 >100 32 mv-ml-mv-mv-pea 13.15 ± 0.05 22.37 ± 0.09 >100 >100 15.4 26.2 45.3 ± 0.8 77.0 ± 1.3 33 F-F-V-V-PEA 38.2 ± 0.9 62.15 >100 >100 >50 >50 >100 >100 34 mf-mf-mv-mv-pea 4.5 ± 0.3 6.7 ± 0.3 24.40 ± 0.00 36.42 ± 0.00 2.5 3.8 14.0 ± 1.6 20.9 ± 2.4 35 F-V-L-V-PEA 42.6 ± 1.3 73.4 ± 1.6 >100 >100 >50 >50 >100 >100 36 mf-mv-ml-mv-pea 4.5 ± 0.1 7.01 ± 0.13 48.8 ± 4.8 76.7 ± 7.6 0.1 0.16 32.1 ± 8.3 50 ± 13 37 V-V-V-F-PEA 12.3 ± 2.7 21.7 ± 3.4 >100 >100 >50 >50 62.5 >100 38 mv-mv-mv-mf-pea 4.50 ± 0.11 7.23 ± 0.12 69.3 ± 2.3 111.4 ± 3.6 12.6 20.3 45.1 ± 7.0 72 ± 11 39 F-V-L-V-V-PEA 47.4 ± 9.1 69.8 ± 10.0 >100 >100 >50 >50 >100 >100 40 mf-mv-ml-mv-mv-pea 3.9 ± 0.9 5.2 ± 0.8 38.2 ± 2.9 50.9 ± 3.8 >50 >50 31.3 ± 10.7 42 ± 14 41 F-V-V-V-F-PEA 34.1 ± 9.9 47.8 ± 9.9 >100 >100 >50 >50 >100 >100 42 mf-mv-mv-mv-mf-pea 4.0 ± 0.3 5.2 ± 0.2 74.7 >100 2.1 2.7 28.4 ± 10.1 36 ± 13 43 V-L-V-V-V-PEA 52.0 ± 0.7 82.4 ± 0.8 55.9 ± 4.3 88.5 ± 6.7 >50 >50 >100 >100 44 mv-ml-mv-mv-mv-pea 10.8 ± 2.9 15.4 ± 3.0 10.4 ± 0.3 14.8 ± 0.4 2.5 3.6 42 ± 16 59 ± 22 45 L-L-V-V-V-PEA 53.1 ± 13.4 82 ± 15 >100 >100 >50 >50 >100 >100 46 ml-ml-mv-mv-mv-pea 3.4 ± 0.8 4.7 ± 0.8 >100 >100 2.3 3.2 25 ± 12 35 ± 16 47 V-V-V-V-V-L-PEA 58.8 80.6 0.3 >100 >50 >50 >100 >100 48 mv-mv-mv-mv-mv-ml-pea 0.59 ± 0.17 0.73 ± 0.14 >100 >100 1.2 1.5 3.6 ± 1.7 4.4 ± 2.0 S38
RXP sequence T. b. rhodesiense L. donovani P. falciparum L6 cell µg/ml µm µg/ml µm µg/ml µm µg/ml µm 49 V-V-V-V-V-F-PEA 60.1 78.7 >100 >100 >50 >50 >100 >100 50 mv-mv-mv-mv-mv-mf-pea 0.9 ± 0.3 1.1 ± 0.3 67 ± 17 79 ± 19 1.1 1.3 15.3 ± 2.1 18.0 ± 2.5 51 F-V-V-V-V-F-PEA 83.0 102.3 >100 >100 >50 >50 >100 >100 52 mf-mv-mv-mv-mv-mf-pea 3.9 ± 0.7 4.4 ± 0.5 29.1 ± 6.3 32.4 ± 6.9 0.14 0.16 14.6 ± 4.1 16.2 ± 4.5 53 V-L-V-V-V-V-PEA 54.9 ± 8.0 71.2 ± 07 >100 76.5 ± 5.8 >50 >50 >100 >100 54 mv-ml-mv-mv-mv-mv-pea 3.3 ± 1.6 13.3 ± 2.6 58.9 12.7 ± 0.3 1.9 3.1 15.6 ± 0.3 19.1 ± 0.3 56 mv-mv-mv-mv-tra 2.8 ± 0.6 4.5 ± 1.0 39 ± 22 64 ± 36 0.76 ± 0.11 1.20 ± 0.17 44.1 ± 7.5 72 ± 12 58 mv-mv-mv-ml-tra 1.7 ± 0.8 3.6 ± 0.2 73 ± 26 116 ± 40 0.54 ± 0.08 0.86 ± 0.12 40.9 ± 10.0 65 ± 16 60 mv-mv-mv-mf-tra 1.8 ± 0.7 3.40 ± 0.02 38.2 >100 0.65 ± 0.15 1.0 ± 0.2 37 ± 11 56 ± 17 62 mv-ml-mv-mv-tra 2.14 ± 0.13 3.4 ± 0.2 43 ± 15 68 ± 24 0.49 ± 0.06 0.78 ± 0.10 44.7 ± 1.3 71.3 2.1 63 L-L-L-A-PEA n.d. n.d n.d. n.d. n.d. n.d. n.d. n.d. 64 ml-ml-ml-ma-pea 31 ± 12 52 ± 20 63.7 108.4 25.4 ± 0.9 43.2 ± 1.5 >100 >100 65 L-L-A-L-PEA 15.1 ± 0.2 28.4 ± 0.4 >100 >100 >50 >50 >100 >100 66 ml-ml-ma-ml-pea 15.2 ± 0.6 25.8 ± 1.0 >100 >100 11.9 ± 0.8 20.2 ± 1.3 >100 >100 67 L-A-L-L-PEA 14.5 ± 0.2 27.3 ± 0.3 >100 >100 >50 >50 >100 >100 68 ml-ma-ml-ml-pea 5.47 ± 0.01 9.30 ± 0.01 >100 >100 9.4 ± 0.3 16.0 ± 0.5 56.0 ± 3.7 76 ± 25 69 A-L-L-L-PEA 14.4 ± 0.10 27.1 ± 0.3 >100 >100 35.5 ± 3.5 66.7 ± 6.5 64.1 ± 2.9 87 ± 27 70 ma-ml-ml-ml-pea 1.85 ± 0.01 3.15 ± 0.02 55.8 ± 3.1 >100 3.45 ± 0.07 5.87 ± 0.12 39.5 ± 3.0 47 ± 15 72 ml-ml-ml-ml-pea 1.87 ± 0.01 2.97 ± 0.02 17.0 ± 0.9 26.9 ± 1.4 1.15 ± 0.05 1.82 ± 0.07 14.6 ± 0.7 23.1 ± 1.0 74 ml-ml-ml-ml-pea 17.5 ± 2.3 27.7 ± 2.6 >100 >100 7.80 ± 0.07 12.38 ± 0.11 51.9 ± 4.6 82.3 ± 7.2 76 ml-ml-ml-ml-pea 2.2 ± 0.3 3.4 ± 0.3 49.4 ± 3.8 78.4 ± 6.0 1.10 ± 0.01 1.74 ± 0.01 6.9 ± 0.4 10.9 ± 0.7 78 ml-ml-ml-ml-pea 2.1 ± 03. 3.3 ± 0.3 99.8 158 1.31 ± 0.14 2.1 ± 0.2 17.7 ± 0.9 28.0 ± 1.3 S39
RXP sequence T. b. rhodesiense L. donovani P. falciparum L6 cell µg/ml µm µg/ml µm µg/ml µm µg/ml µm 80 ml-ml-ml-ml-pea 1.96 ± 0.01 3.10 ± 0.01 65.5 ± 9.2 103 ± 14 1.69 ± 0.01 2.67 ± 0.01 15.1 ± 0.6 24.0 ± 1.0 82 mabu-mabu-mabu-mabu-pea 70 ± 15 135 ± 29 >100 >100 >50 >50 >100 >100 84 mnva-mnva-mnva-mnva-pea 48.7 ± 0.6 84.8 ± 1.0 >100 >100 33.6 ± 2.4 58.5 ± 4.1 >100 >100 86 mi-mi-mi-mi-pea 5.4 ± 0.3 8.6 ± 0.4 24.3 ± 13 39 ± 20 2.17 ± 0.09 3.44 ± 0.14 17.6 ± 4.1 27.9 ± 6.6 88 mv-mv-mv-ma-pea 51.9 ± 1.3 95.1 ± 2.4 >100 >100 42.4 77.7 >100 >100 90 mv-mv-mv-mc-pea 3.0 ± 2.8 7.2 ± 3.7 28 ± 14 49 ± 23 4.3 ± 1.8 7.40 ± 0.12 30.1 ± 7.4 52 ± 13 92 mv-mv-mv-md-pea 50.8 88.1 58.8 99.7 36.5 61.9 >100 >100 94 mv-mv-mv-me-pea 67 ± 26 110 ± 43 64.9 107.5 33.7 ± 2.2 55.8 ± 3.6 >100 >100 96 mv-mv-mv-mm-pea 33.2 ± 15.8 55 ± 26 60.5 99.9 24.5 ± 0.1 40.44 0.17 >100 >100 98 mv-mv-mv-ms-pea 47.6 ± 0.4 84.7 ± 0.7 >100 >100 >50 >50 >100 >100 100 mv-mv-mv-mt-pea 39.8 ± 2.2 69.1 ± 3.8 40.5 70.3 41.8 ± 7.6 73 ± 13 >100 >100 102 mv-mv-mv-mw-pea 6.07 ± 0.17 9.2 ± 0.3 31.8 48.1 3.5 ± 0.5 5.3 ± 0.8 54.3 82.2 104 mv-mv-mv-my-pea 15.6 ± 2.5 24.5 ± 3.9 >100 >100 5.6 ± 0.9 8.8 ± 1.4 >100 >100 106 ml-ml-ml-ml-tya 0.94 ± 0.01 1.46 ± 0.01 >100 >100 1.4 ± 0.3 2.2 ± 0.5 30.6 ± 3.4 47.4 ± 5.3 108 ml-ml-ml-ml-tra 1.59 ± 0.13 2.37 ± 0.19 20.7 ± 0.9 30.9 ± 1.3 0.60 ± 0.03 0.89 ± 0.05 16.8 ± 3.1 25.1 ± 4.7 110 ml-ml-ml-ml-mtra 4.0 ± 1.2 5.9 ± 1.7 34.6 50.7 1.45 ± 0.13 2.1 ± 0.1 15.7 ± 5.5 23.0 ± 8.0 112 ml-ml-ml-ml-2npth 1.53 ± 0.08 2.25 ± 0.12 35 ± 13 50.7 ± 18. 0.84 ± 0.09 1.20 ± 0.14 6.8 ± 2.4 10.0 ± 3.5 114 ml-ml-ml-ml-1npth 1.0 ± 0.3 1.5 ± 0.5 29 ± 15 42.8 ± 22. 0.26 ± 0.09 0.30 ± 0.13 9.1 ± 3.9 13.4 ± 5.7 116 ml-ml-ml-ml-4fpea 1.8 ± 0.3 2.8 ± 0.5 44.4 68.5 0.80 ± 0.06 1.23 ± 0.09 7.4 ± 0.7 11.5 ± 1.0 118 ml-ml-ml-ml-4brpea 1.88 ± 0.01 2.65 ± 0.02 33 ± 13 46.8 ± 18. 0.52 ± 0.04 0.70 ± 0.06 8.3 ± 0.3 11.7 ± 0.4 120 ml-ml-ml-ml-bn 4.1 ± 0.3 6.7 ± 0.5 36.7 ± 0.9 59.5 ± 1.4 2.5 ± 0.5 4.1 ± 0.8 32.6 ± 0.3 52.9 ± 0.5 122 ml-ml-ml-ml-ppa 4.3 ± 0.3 6.7 ± 0.4 22.9 ± 4.6 35.5 ± 7.1 2.3 3.6 12.6 ± 1.3 19.6 ± 2.0 S40
RXP sequence T. b. rhodesiense L. donovani P. falciparum L6 cell µg/ml µm µg/ml µm µg/ml µm µg/ml µm 124 ml-ml-ml-ml-pba 3.5 ± 0.7 5.3 ± 1.0 15.0 ± 1.8 22.7 ± 2.6 1.7 2.6 6.9 ± 0.9 10.5±1.4 126 ml-ml-ml-ml-chex 5.0 ± 0.2 7.8 ± 0.4 11.0 ± 2.9 17.3 ± 4.5 1.4 2.2 8.7 ± 0.8 13.6 ± 1.3 128 ml-ml-ml-ml-me 35.5 ± 3.2 65.7 ± 6.0 >100 >100 14.4 26.7 >100 >100 130 ml-ml-ml-ml-et 13.3 ± 0.8 24.0 ± 1.4 >100 >100 10.2 ± 0.8 18.4 ± 1.5 90.9 ± 5.3 164 ± 10 132 ml-ml-ml-ml-pr 12.4 ± 0.7 21.8 ± 1.1 25.1 ± 3.0 44.1 ± 5.2 6.8 ± 1.0 11.9 ± 1.8 71.5 ± 4.5 125 ± 8 134 ml-ml-ml-ml-bu 4.9 ± 0.3 8.3 ± 0.5 10.6 ± 0.6 18.2 ± 1.0 2.3 ± 0.5 3.9 ± 0.8 39.6 ± 0.9 68.0 ± 7.8 136 ml-ml-ml-ml-amyl 5.8 ± 0.3 9.7 ± 0.6 34.8 2.2 58.4 ± 3.6 3.9 ± 1.1 6.6 ± 1.8 42.1 ± 0.2 70.6 ± 1.5 138 ml-ml-ml-ml-ipn 5.73 ± 0.17 9.6 ± 0.3 13.6 ± 4.7 22.7 ± 7.9 2.9 4.9 43.2 ± 0.7 72.6 ± 1.1 140 ml-ml-ml-mc-pea 5.6 ± 0.5 9.0 ± 0.8 49.3 ± 9.8 79 ± 15 2.1 3.3 32.1 ± 8.3 51.8 ± 13.4 142 ml-ml-ml-mc-1npth 7.5 ± 1.5 11.2 ± 1.7 39.4 ± 2.5 58.7 ± 3.8 1.3 1.9 17.3 ± 1.8 25.8 ± 2.6 Reference* 0.005 ± 0.001 0.013 ± 0.12 ± 0.03 0.29 ± 0.07 0.002 ± 0.001 0.006 ± 0.003 0.007 ± 0.001 0.017 ± 0.002 S41
Supplementary Figures Figure S2. Exemplarily comparison of heterologous produced natural and synthetic RXP ml-ml-ml-ml-4brpea (118) (m/z 708.4 ) for structure confirmation. Structures of RXP and proposed MS/MS fragments (a) in addition to HPLC-ESI/MS analysis (BPC) of natural (b) and synthetic RXP (d). MS 2 fragmentation shows the actual measured fragmentation pattern in good agreement with proposed fragments (c and e). Both peptides have identical retention times (R t = 9.3 min) and MS 2 fragmentation. Figure S2. Structure activity study of permethylated RXPs with single and double substituted methyl valine and different chain length from four to six amino acids with C-terminal PEA against protozoa T. b. rhodesiense, P. falciparum and mammalian L6 cells (* against P. falciparum an IC 50 >50 µg/ml was assumed to show inactivity). S42
Figure S3. Structure activity study of permethylated RXPs with different aliphatic side chains (top) and single substituted methyl valine (bottom) and C-terminal PEA against protozoa T. b. rhodesiense, P. falciparum and mammalian L6 cells (* against P. falciparum an IC 50 >50 µg/ml was assumed to show inactivity) (Abu = aminobutanoic acid, Nva = norvaline). Bioactivity increased by increasing the size of the amino acid side chains in a permethylated RXP could be observed in T. b. rhodesisense, P. falciparum, and L6 cells. Figure S4. Structure activity study of permethylated RXPs with different C-terminal amines against protozoa T. b. rhodesiense, P. falciparum and mammalian L6 cells (* against P. falciparum an IC 50 >50 µg/ml was assumed to show inactivity) (MTRA = 1-methyltryptamine, 2Npth = 2-(2-Naphthyl) ethylamine, 1Npth = 2-(1-Naphthyl) ethylamine, 4FPEA = 4- fluorophenylethylamine, 4BrPEA = 4-bromophenylethylamine, Bz = benzyl, PPA = 3-phenyl- 1-propylamine, PBA = 4-phenylbutlyamine, chex = 2-cyclohexylethylamine). S43
Figure S5. HPLC-MS after permethylation of six stereoisomers containing four leucine with C-terminal PEA. Extracted ion chromatograms (EIC of m/z 630.5) of RXPs (16, 72-80) are shown. RXPs 72-78 contain one N-methyl-D-leucine (mi) at different positions, whereas 80 consist of only D-leucine and is the enantiomer of 16. These results demonstrate that epimerization is minimal during deprotonation and methylation step. S44
Figure S6. HPLC-MS analysis (BPC) of nonmethylated (29, upper two chromatograms) vs. permethylated (30, lower two chromatograms) RXP in insect hemolymph plasma of G. mellonella after 0 and 240 min depicting biostability. Spectra are illustrated in same intensity for comparison. The position of asterisk (*) in the chromatogram highlight the position of proteolytic digested starting material to V-PEA and double asterisk (**) highlight the position of proteolytic digested starting material to V-V-PEA, whereas no V-V-V-PEA and any other proteolytic/hydrolytic product could be observed during assay. Any proteolytic/hydrolytic products of 30 could not be observed either. S45
Figure S7. Metabolic stability of nonmethylated (7) and permethylated (8) RXP using liver microsomes. S46
Figure S8. a) HPLC-MS analysis (BPC) of metabolic stability assay using liver microsomes of nonmethylated RXP (7, m/z 518.4) and b) and permethylated RXP (8, m/z 574.4) at different time points. Highlighted by arrows are the starting materials as well as new appearing masses (m/z 534.4 for 7 and m/z 590.4 for 8) in the chromatograms. c) and d) are EICs of the corresponding starting material illustrating the disappearance of starting material through metabolic degradation. e) and f) are the EICs of m/z 534.4 and m/z 590.4, respectively showing products of the metabolic degradation. By HR-HPLC-MS analysis the products are confirmed as single oxidized products of starting material at different positions (oxidized valine or phenyl ring). Multiple oxidized products could not be observed or are below detection limit. Structures of oxidized products and their MS 2 -fragmentation pattern are shown in Fig. S5 and S6. S47
Figure S9. a) MS 2 -fragmentation pattern and structure of starting material (7, m/z 518.4) and b-d) MS 2 -fragmentation of metabolic products (m/z 534.4 at different retention times) using liver microsomes and possible structures derived from observed fragmentation pattern. Shown structures are hypothetically and are inspired form the metabolism of vitamin D by CYP2R1 4 and for example deltamethrin 5. S48
Figure S10. a) MS 2 -fragmentation pattern and structure of starting material (8, m/z 574.4) and b/c) metabolic products (m/z 590.4 at different retention times) using liver microsomes and possible structures derived from observed fragmentation pattern. S49
Figure S11. Comparison of MS 2 -fragmentation and retention time of synthetic tyramine derivative and the metabolic tyramine product. S50
NMR Spectra Figure S12: 1 H-NMR spectra (500 MHz, CDCl 3) of S1. Figure S13: 1 H-NMR spectra (400 MHz, CDCl3) S2. S51
Figure S14: 1 H-NMR spectra (400 MHz, CDCl3) of S3. FigureS15: 1 H-NMR spectra (400 MHz, CDCl 3) of S4. S52
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