- Supporting Information - for

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1 - 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 Frankfurt am Main, Germany. 2 Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D Frankfurt am Main, Germany. 3 Swiss Tropical and Public Health Institute, Parasite Chemotherapy, Socinstrasse 57, CH 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 Frankfurt am Main, Germany. - Content - General Procedures... 2 Synthesis of Amines S1-S Design of Experiment (DOE)... 4 Supplementary Tables... 6 Supplementary Figures NMR Spectra References S1

2 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), (m, 1H), (m, 1H), (m, 1H), 6.90 (s, 1H), 3.75 (s, 3H), (m, 2H), (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, S2

3 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), (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), (m, 1H), 7.43 (s, 1H), (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), (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

4 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

5 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 cm² V D = volume in the donor well (cm 3 ) V A = volume in the acceptor well (cm 3 ) t = incubation time (s) S5

6 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 extra none none extra extra none none extra extra none none extra extra none none extra extra none none extra extra none none extra extra none none extra extra none none S6

7 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 extra none Mean n Median STDEV Max Min graph a 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 Mean n Median STDEV Max Min graph a 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

8 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 % 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 ml-ml-ml-tra ml-ml-l-ml-tra ml-ml-ml-ml-tra* ml-ml-l-ml-ml-tra ml-ml-ml-ml-ml-tra ml-ml-l-ml-ml-ml-tra ml-ml-ml-ml-ml-ml-tra 6.3 MTRA ml-ml-ml-mtra ml-ml-l-ml-mtra ml-ml-ml-ml-mtra* ml-ml-l-ml-ml-mtra ml-ml-ml-ml-ml-mtra ml-ml-ml-ml-ml-ml-mtra 2.9 1Nphth ml-ml-ml-1nphth ml-ml-l-ml-1nphth ml-l-ml-ml-1nphth ml-ml-ml-ml-1nphth* ml-ml-l-ml-ml-1nphth ml-ml-ml-ml-ml-1nphth ml-ml-ml-ml-ml-ml-1nphth 2.6 2Nphth ml-ml-ml-2nphth ml-ml-l-ml-2nphth ml-l-ml-ml-2nphth ml-ml-ml-ml-2nphth* ml-ml-l-ml-ml-2nphth ml-ml-ml-ml-ml-2nphth ml-ml-ml-ml-ml-ml-2nphth 5.4 S8

9 Substrate 4FPEA 4BrPEA MW (g/mol) RXP m/z R t (min) MS 2 -fragmentation relative amount ml-ml-ml-4fpea ml-ml-l-ml-4fpea ml-l-ml-ml-4fpea ml-ml-ml-ml-4fpea* ml-ml-l-ml-ml-4fpea ml-ml-ml-ml-ml-4fpea ml-ml-l-ml-ml-ml-4fpea ml-ml-ml-ml-ml-ml-4fpea ml-ml-l-ml-ml-ml-ml-4fpea ml-ml-ml-ml-ml-ml-ml-4fpea ml-ml-4brpea ml-l-ml-4brpea ml-ml-ml-4brpea ml-ml-l-ml-4brpea ml-l-ml-ml-4brpea ml-ml-ml-ml-4brpea* ml-ml-l-ml-ml-4brpea ml-ml-ml-ml-ml-4brpea ml-ml-l-ml-ml-ml-4brpea ml-ml-ml-ml-ml-ml-4brpea ml-ml-ml-ml-ml-ml-ml-4brpea 0.3 S9

10 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 n.d. I-I-I-I n.d. mi-mi-mi-mi 7 V-V-V-V-PEA mv-mv-mv-mv-pea V-V-V-V-V-PEA S10

11 ID structure and sequence MS 2 -fragmentation crude BPC mv-mv-mv-mv-mv-pea EIC V-V-V-V-V-V-PEA mv-mv-mv-mv-mv-mv-pea EIC V-V-V-V-V-V-V-PEA mv-mv-mv-mv-mv-mv-mv-pea EIC L-L-L-L-PEA S11

12 ID structure and sequence MS 2 -fragmentation crude BPC ml-ml-ml-ml-pea L-L-L-L-L-PEA ml-ml-ml-ml-ml-pea L-L-L-L-L-L-PEA ml-ml-ml-ml-ml-ml-pea L-L-L-L-L-L-L-PEA S12

13 ID structure and sequence MS 2 -fragmentation crude BPC ml-ml-ml-ml-ml-ml-ml-pea EIC F-F-F-F-PEA mf-mf-mf-mf-pea F-F-F-F-F-PEA mf-mf-mf-mf-mf-pea S13

14 ID structure and sequence MS 2 -fragmentation crude BPC F-F-F-F-F-F-PEA mf-mf-mf-mf-mf-mf-pea EIC V-V-V-L-PEA mv-mv-mv-ml-pea V-L-V-V-PEA S14

15 ID structure and sequence MS 2 -fragmentation crude BPC mv-ml-mv-mv-pea F-F-V-V-PEA mf-mf-mv-mv-pea F-V-L-V-PEA mf-mv-ml-mv-pea S15

16 ID structure and sequence MS 2 -fragmentation crude BPC V-V-V-F-PEA mv-mv-mv-mf-pea EIC F-V-L-V-V-PEA mf-mv-ml-mv-mv-pea F-V-V-V-F-PEA S16

17 ID structure and sequence MS 2 -fragmentation crude BPC mf-mv-mv-mv-mf-pea EIC V-L-V-V-V-PEA mv-ml-mv-mv-mv-pea EIC L-L-V-V-V-PEA ml-ml-mv-mv-mv-pea EIC S17

18 ID structure and sequence MS 2 -fragmentation crude BPC V-V-V-V-V-L-PEA mv-mv-mv-mv-mv-ml-pea V-V-V-V-V-F-PEA mv-mv-mv-mv-mv-mf-pea F-V-V-V-V-F-PEA S18

19 ID structure and sequence MS 2 -fragmentation crude BPC mf-mv-mv-mv-mv-mf-pea EIC V-L-V-V-V-V-PEA mv-ml-mv-mv-mv-mv-pea 55 V-V-V-V-TRA n.d. 56 mv-mv-mv-mv-tra n.d. S19

20 ID structure and sequence MS 2 -fragmentation crude BPC n.d. V-V-V-L-TRA n.d. mv-mv-mv-ml-tra n.d. V-V-V-F-TRA n.d. mv-mv-mv-mf-tra n.d. V-L-V-V-TRA n.d. mv-ml-mv-mv-tra S20

21 ID structure and sequence MS 2 -fragmentation crude BPC n.d. L-L-L-A-PEA ml-ml-ml-ma-pea EIC n.d. L-L-A-L-PEA n.d. ml-ml-ma-ml-pea EIC n.d. L-A-L-L-PEA EIC S21

22 ID structure and sequence MS 2 -fragmentation crude BPC n.d. ml-ma-ml-ml-pea EIC n.d. A-L-L-L-PEA n.d. ma-ml-ml-ml-pea EIC n.d. L-L-L-l-PEA n.d. ml-ml-ml-ml-pea S22

23 ID structure and sequence MS 2 -fragmentation crude BPC n.d. L-L-l-L-PEA n.d. ml-ml-ml-ml-pea n.d. L-l-L-L-PEA n.d. ml-ml-ml-ml-pea n.d. l-l-l-l-pea n.d. ml-ml-ml-ml-pea S23

24 ID structure and sequence MS 2 -fragmentation crude BPC n.d. l-l-l-l-pea n.d. ml-ml-ml-ml-pea 81 Abu-Abu-Abu-Abu-PEA n.d. 82 mabu-mabu-mabu-mabu-pea n.d. Nva-Nva-Nva-Nva-PEA S24

25 ID structure and sequence MS 2 -fragmentation crude BPC mnva-mnva-mnva-mnva-pea n.d. I-I-I-I-PEA mi-mi-mi-mi-pea n.d. V-V-V-A-PEA mv-mv-mv-ma-pea EIC S25

26 ID structure and sequence MS 2 -fragmentation crude BPC n.d. V-V-V-C-PEA mv-mv-mv-mc-pea n.d. V-V-V-D-PEA mv-mv-mv-md-pea EIC n.d. V-V-V-E-PEA S26

27 ID structure and sequence MS 2 -fragmentation crude BPC mv-mv-mv-me-pea n.d. V-V-V-M-PEA EIC mv-mv-mv-mm-pea n.d. V-V-V-S-PEA mv-mv-mv-ms-pea EIC S27

28 ID structure and sequence MS 2 -fragmentation crude BPC n.d. V-V-V-T-PEA mv-mv-mv-mt-pea EIC n.d. V-V-V-W-PEA mv-mv-mv-mw-pea n.d. V-V-V-Y-PEA S28

29 ID structure and sequence MS 2 -fragmentation crude BPC mv-mv-mv-my-pea EIC n.d. L-L-L-L-TYA n.d. ml-ml-ml-ml-tya n.d. L-L-L-L-TRA ml-ml-ml-ml-tra S29

30 ID structure and sequence MS 2 -fragmentation crude BPC n.d. L-L-L-L-MTRA ml-ml-ml-ml-mtra n.d. L-L-L-L-2Npth ml-ml-ml-ml-2npth n.d. L-L-L-L-1Npth S30

31 ID structure and sequence MS 2 -fragmentation crude BPC ml-ml-ml-ml-1npth n.d. L-L-L-L-4FPEA ml-ml-ml-ml-4fpea n.d. L-L-L-L-4BrPEA ml-ml-ml-ml-4brpea S31

32 ID structure and sequence MS 2 -fragmentation crude BPC n.d. L-L-L-L-Bz ml-ml-ml-ml-bz n.d. L-L-L-L-PPA ml-ml-ml-ml-ppa n.d. L-L-L-L-PBA S32

33 ID structure and sequence MS 2 -fragmentation crude BPC ml-ml-ml-ml-pba n.d. L-L-L-L-cHex ml-ml-ml-ml-chex n.d. L-L-L-L-Me ml-ml-ml-ml-me S33

34 ID structure and sequence MS 2 -fragmentation crude BPC n.d. L-L-L-L-Et ml-ml-ml-ml-et n.d. L-L-L-L-Pr ml-ml-ml-ml-pr n.d. L-L-L-L-Bu S34

35 ID structure and sequence MS 2 -fragmentation crude BPC ml-ml-ml-ml-bu n.d. L-L-L-L-Amyl ml-ml-ml-ml-amyl n.d. L-L-L-L-iPn ml-ml-ml-ml-ipn S35

36 ID structure and sequence MS 2 -fragmentation crude BPC n.d. L-L-L-C-PEA n.d. ml-ml-ml-mc-pea n.d. L-L-L-C-1Npth n.d. ml-ml-ml-mc-1npth S36

37 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 ± ± 3.7 >100 >100 >50 >50 >100 >100 8 mv-mv-mv-mv-pea 16.9 ± ± 1.8 >100 > V-V-V-V-V-PEA 78 ± ± 19 >100 >100 >50 >50 >100 > mv-mv-mv-mv-mv-pea 22.3 ± ± 10.0 >100 > >100 > V-V-V-V-V-V-PEA 63 ± ± 33 >100 >100 >50 >50 >100 > mv-mv-mv-mv-mv-mv-pea 13.5 ± ± 7.3 >100 > ± ± V-V-V-V-V-V-V-PEA 62.6 ± ± 5.3 >100 >100 >50 >50 >100 > mv-mv-mv-mv-mv-mv-mv-pea 4.56 ± ± ± ± ± ± L-L-L-L-PEA 13.2 ± ± 1.1 >100 > ml-ml-ml-ml-pea 1.41 ± ± ± ± ± ± L-L-L-L-L-PEA 54.8 ± ± 7.1 >100 >100 >50 >50 >100 > ml-ml-ml-ml-ml-pea 3.0 ± ± ± ± ± ± L-L-L-L-L-L-PEA 39 ± ± 17 >100 > >100 > ml-ml-ml-ml-ml-ml-pea 2.09 ± ± ± ± ± ± L-L-L-L-L-L-L-PEA >100 >100 >50 >50 >100 > ml-ml-ml-ml-ml-ml-ml-pea 2.3 ± ± ± ± ± ± F-F-F-F-PEA 5.7 ± ± 0.2 >100 > ± ± mf-mf-mf-mf-pea 4.1 ± ± ± ± ± ± F-F-F-F-F-PEA 10 ± ± >100 >100 >50 >50 >100 > mf-mf-mf-mf-mf-pea 2.6 ± ± ± ± ± ± 1.7 S37

38 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 >100 >100 >50 >50 >100 > mf-mf-mf-mf-mf-mf-pea 4.1 ± ± ± ± ± ± V-V-V-L-PEA 14.0 ± ± 2.4 >100 >100 >50 >50 >100 > mv-mv-mv-ml-pea 11.7 ± ± 0.9 >100 > ± ± V-L-V-V-PEA 43.2 ± ± 11 >100 >100 >50 >50 >100 > mv-ml-mv-mv-pea ± ± 0.09 >100 > ± ± F-F-V-V-PEA 38.2 ± >100 >100 >50 >50 >100 > mf-mf-mv-mv-pea 4.5 ± ± ± ± ± ± F-V-L-V-PEA 42.6 ± ± 1.6 >100 >100 >50 >50 >100 > mf-mv-ml-mv-pea 4.5 ± ± ± ± ± ± V-V-V-F-PEA 12.3 ± ± 3.4 >100 >100 >50 > > mv-mv-mv-mf-pea 4.50 ± ± ± ± ± ± F-V-L-V-V-PEA 47.4 ± ± 10.0 >100 >100 >50 >50 >100 > mf-mv-ml-mv-mv-pea 3.9 ± ± ± ± 3.8 >50 > ± ± F-V-V-V-F-PEA 34.1 ± ± 9.9 >100 >100 >50 >50 >100 > mf-mv-mv-mv-mf-pea 4.0 ± ± > ± ± V-L-V-V-V-PEA 52.0 ± ± ± ± 6.7 >50 >50 >100 > mv-ml-mv-mv-mv-pea 10.8 ± ± ± ± ± ± L-L-V-V-V-PEA 53.1 ± ± 15 >100 >100 >50 >50 >100 > ml-ml-mv-mv-mv-pea 3.4 ± ± 0.8 >100 > ± ± V-V-V-V-V-L-PEA >100 >50 >50 >100 > mv-mv-mv-mv-mv-ml-pea 0.59 ± ± 0.14 >100 > ± ± 2.0 S38

39 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 >100 >100 >50 >50 >100 > mv-mv-mv-mv-mv-mf-pea 0.9 ± ± ± ± ± ± F-V-V-V-V-F-PEA >100 >100 >50 >50 >100 > mf-mv-mv-mv-mv-mf-pea 3.9 ± ± ± ± ± ± V-L-V-V-V-V-PEA 54.9 ± ± 07 > ± 5.8 >50 >50 >100 > mv-ml-mv-mv-mv-mv-pea 3.3 ± ± ± ± ± mv-mv-mv-mv-tra 2.8 ± ± ± ± ± ± ± ± mv-mv-mv-ml-tra 1.7 ± ± ± ± ± ± ± ± mv-mv-mv-mf-tra 1.8 ± ± > ± ± ± ± mv-ml-mv-mv-tra 2.14 ± ± ± ± ± ± ± 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 ± ± ± ± 1.5 >100 > L-L-A-L-PEA 15.1 ± ± 0.4 >100 >100 >50 >50 >100 > ml-ml-ma-ml-pea 15.2 ± ± 1.0 >100 > ± ± 1.3 >100 > L-A-L-L-PEA 14.5 ± ± 0.3 >100 >100 >50 >50 >100 > ml-ma-ml-ml-pea 5.47 ± ± 0.01 >100 > ± ± ± ± A-L-L-L-PEA 14.4 ± ± 0.3 >100 > ± ± ± ± ma-ml-ml-ml-pea 1.85 ± ± ± 3.1 > ± ± ± ± ml-ml-ml-ml-pea 1.87 ± ± ± ± ± ± ± ± ml-ml-ml-ml-pea 17.5 ± ± 2.6 >100 > ± ± ± ± ml-ml-ml-ml-pea 2.2 ± ± ± ± ± ± ± ± ml-ml-ml-ml-pea 2.1 ± ± ± ± ± ± 1.3 S39

40 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 ± ± ± ± ± ± ± ± mabu-mabu-mabu-mabu-pea 70 ± ± 29 >100 >100 >50 >50 >100 > mnva-mnva-mnva-mnva-pea 48.7 ± ± 1.0 >100 > ± ± 4.1 >100 > mi-mi-mi-mi-pea 5.4 ± ± ± ± ± ± ± ± mv-mv-mv-ma-pea 51.9 ± ± 2.4 >100 > >100 > mv-mv-mv-mc-pea 3.0 ± ± ± ± ± ± ± ± mv-mv-mv-md-pea >100 > mv-mv-mv-me-pea 67 ± ± ± ± 3.6 >100 > mv-mv-mv-mm-pea 33.2 ± ± ± >100 > mv-mv-mv-ms-pea 47.6 ± ± 0.7 >100 >100 >50 >50 >100 > mv-mv-mv-mt-pea 39.8 ± ± ± ± 13 >100 > mv-mv-mv-mw-pea 6.07 ± ± ± ± mv-mv-mv-my-pea 15.6 ± ± 3.9 >100 > ± ± 1.4 >100 > ml-ml-ml-ml-tya 0.94 ± ± 0.01 >100 > ± ± ± ± ml-ml-ml-ml-tra 1.59 ± ± ± ± ± ± ± ± ml-ml-ml-ml-mtra 4.0 ± ± ± ± ± ± ml-ml-ml-ml-2npth 1.53 ± ± ± ± ± ± ± ± ml-ml-ml-ml-1npth 1.0 ± ± ± ± ± ± ± ± ml-ml-ml-ml-4fpea 1.8 ± ± ± ± ± ± ml-ml-ml-ml-4brpea 1.88 ± ± ± ± ± ± ± ± ml-ml-ml-ml-bn 4.1 ± ± ± ± ± ± ± ± ml-ml-ml-ml-ppa 4.3 ± ± ± ± ± ± 2.0 S40

41 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 ± ± ± ± ± ± ml-ml-ml-ml-chex 5.0 ± ± ± ± ± ± ml-ml-ml-ml-me 35.5 ± ± 6.0 >100 > >100 > ml-ml-ml-ml-et 13.3 ± ± 1.4 >100 > ± ± ± ± ml-ml-ml-ml-pr 12.4 ± ± ± ± ± ± ± ± ml-ml-ml-ml-bu 4.9 ± ± ± ± ± ± ± ± ml-ml-ml-ml-amyl 5.8 ± ± ± ± ± ± ± ml-ml-ml-ml-ipn 5.73 ± ± ± ± ± ± ml-ml-ml-mc-pea 5.6 ± ± ± ± ± ± ml-ml-ml-mc-1npth 7.5 ± ± ± ± ± ± 2.6 Reference* ± ± 0.12 ± ± ± ± ± ± S41

42 Supplementary Figures Figure S2. Exemplarily comparison of heterologous produced natural and synthetic RXP ml-ml-ml-ml-4brpea (118) (m/z ) 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

43 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

44 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 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

45 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

46 Figure S7. Metabolic stability of nonmethylated (7) and permethylated (8) RXP using liver microsomes. S46

47 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 for 7 and m/z 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 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

48 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 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

49 Figure S10. a) MS 2 -fragmentation pattern and structure of starting material (8, m/z 574.4) and b/c) metabolic products (m/z at different retention times) using liver microsomes and possible structures derived from observed fragmentation pattern. S49

50 Figure S11. Comparison of MS 2 -fragmentation and retention time of synthetic tyramine derivative and the metabolic tyramine product. S50

51 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

52 Figure S14: 1 H-NMR spectra (400 MHz, CDCl3) of S3. FigureS15: 1 H-NMR spectra (400 MHz, CDCl 3) of S4. S52

53 References (1) Lygin, A. V.; Meijere, A. de. Synthesis of 1-substituted benzimidazoles from o- bromophenyl isocyanide and amines. Eur. J. Org. Chem. 2009, 2009, (2) Uno, T.; Beausoleil, E.; Goldsmith, R. A.; Levine, B. H.; Zuckermann, R. N. New submonomers for poly N-substituted glycines (peptoids). Tetrahedron Lett. 1999, 40, (3) Cai, X.; Nowak, S.; Wesche, F.; Bischoff, I.; Kaiser, M.; Fürst, R.; Bode, H. B. Entomopathogenic bacteria use multiple mechanisms for bioactive peptide library design. Nat. Chem 2016, 9, (4) Shinkyo, R.; Sakaki, T.; Kamakura, M.; Ohta, M.; Inouye, K. Metabolism of vitamin D by human microsomal CYP2R1. Biochem. Biophys. Res. Commun. 2004, 324, (5) Feyereisen, R. Insect CYP genes and P450 enzymes. Insect Molecular Biology and Biochemistry (edited by L. I. Gilbert), Academic Press, London 2012, S53