Teixobactin analogues reveal enduracididine to be non-essential for highly potent antibacterial activity and lipid II binding

Leu10-teixobactin and Ile10-teixobactin have shown comparable activity to natural teixobactin.


II. Equipment used for the analysis and purification of compounds
All peptides were analysed on a Thermo Scientific Dionex Ultimate 3000 RP-HPLC equipped with a Phenomenex Gemini NX C18 110 Å (150 x 4.6 mm) column using the following buffer systems: A: 0.1% HCOOH in milliQ water. B: ACN using a flow rate of 1 ml/min. The column was flushed with 95% A for 5 min prior to an injection and was flushed for 5 min with 95% B and 5% A after the run was finished.
Peptides were dissolved in 0.1% HCOOH buffer in water and in ACN (10-30% ACN) and purified using the same gradient as mentioned above, on a Thermo Scientific Dionex Ultimate 3000 RP-HPLC with a flow rate of 5 mL/min using a Phenomenex Gemini NX C18 110 Å (150 x 10 mm) semi-prep column.
LC-MS data were collected on an Agilent 1100 Series instrument with a Phenomenex Kinetex C18 100Å column (150 x 4.6 mm, 5 μm at 35 °C) connected to an ESMSD type VL mass detector with a flow rate of 1.5 ml/min was used with the following solvent systems: (A): 0.1% HCOOH in H2O and (B) MeCN. The column was flushed with 100% A for 2 min, then a gradient from 0 to 100% B over 6 min was used, followed by 2 min of flushing with 100% B.
NMR spectra were recorded on a Bruker AV 500 NMR. HRMS spectra were recorded on a Thermo Scientific Q Exactive Plus Orbitrap Mass Spectrometer in the positive ion mode.

III. Syntheses of teixobactin analogues
Fig. S1: Synthesis of Leu10-teixobactin (step a) Commercially available 2-Chlorotrityl chloride resin (manufacturer's loading = 1.2 mmol/g, 170 mg resin) was swelled in DCM in a reactor. To this resin was added 4 eq. Fmoc-Ala-OH/8 eq. DIPEA in DCM and the reactor was shaken for 3h. The loading determined by UV absorption of the piperidine-dibenzofulvene adduct was calculated to be 0.6 mmol/g, (170mg resin, 0.102 mmol). Any unreacted resin was capped with MeOH:DIPEA:DCM = 1:2:7 by shaking for 1h. (step b) The Fmoc protecting group was deprotected using 20% piperdine in DMF by shaking for 3 min, followed by draining and shaking again with 20% piperidine in DMF for 10 min. AllocHN-D-Thr-OH was then coupled to the resin by adding 3 eq. of the AA, 3 eq. HATU and 6 eq. DIPEA in DMF and shaking for 1.5h at room temperature. (step c) Esterification was performed using 10 eq. of Fmoc-Ile-OH, 10 eq. DIC and 5 mol% DMAP in DCM and shaking the reaction for 2h. This was followed by capping the unreacted alcohol using 10% Ac2O/DIPEA in DMF shaking for 30 min and Fmoc was removed using protocol described earlier in step (b). (step d) Fmoc-Leu-OH was coupled using 4 eq. of AA, 4 eq. HATU and 8 eq. DIPEA in DMF and shaking for 1h followed by Fmoc deprotection using 20% piperidine in DMF as described earlier.
(step e) The N terminus of Leu was protected using 10 eq. Trt-Cl and 15% Et3N in DCM and shaking for 1h. The protection was verified by the Ninhydrin colour test.
(step f) The Alloc protecting group of D-Thr was removed using 0.2 eq. [Pd(PPh 3 )] 0 and 24 eq. PhSiH3 in dry DCM under argon for 20 min. This procedure was repeated again increasing the time to 45 min and the resin was washed thoroughly with DCM and DMF to remove any Pd stuck to the resin. (step g) All amino acids were coupled using 4 eq. Amino Acid, 4 eq. DIC/Oxyma using a microwave peptide synthesizer. Coupling time was 10 min. Deprotection cycles were performed as described earlier.
(step h) The peptide was cleaved from the resin without cleaving off the protecting groups of the amino acid side chains using TFA:TIS:DCM = 2:5:93 and shaking for 1h. (step i) The solvent was evaporated and the peptide was redissolved in DMF to which 1 eq. HATU and 10 eq. DIPEA were added and the reaction was stirred for 30 min to perform the cyclization. (step j) The side-chain protecting groups were then cleaved off using TFA:TIS:H2O = 95:2.5:2.5 by stirring for 1h. The peptide was precipitated using cold Et2O (-20°C) and centrifuging at 7000 rpm to obtain a white solid. This solid was further purified using RP-HPLC using the protocols described previously 1 .
All other teixobactin analogues were synthesised according to the above procedure.

Sr
No. 12 Table S1: Compound number, code, exact mass, chemical formula, mass found and overall yields for compounds 1-15.

Compound
* Gly10-teixobactin afforded a yield of 2% when synthesised for the first time possibly due to deletion sequences. Since the yield was unusually low, therefore, the synthesis was repeated a second time thereby affording a yield of 18%.

V. NMR Analysis
All NMR was carried out in DMSO-d6 at 27°C on a Bruker Avance III HD 500 MHz spectrometer equipped with a room-temperature broadband probe. The following spectra were utilised in the assignment of 1 mM solutions of the  Table S2. Proton chemical shifts obtained from the mutants used in this study. The residue replaced by alanine is shown with a grey background, with the introduced methyl group shown in bold.

VI. MIC testing (screening)
For MIC assays all peptides were dissolved in DMSO containing 0.002% polysorbate 80 7 . All bacteria were grown in Mueller Hinton broth (Oxoid) in triplicate. All incubations were at 37°C. Dilutions were carried out in triplicate. 100 µl of autoclaved Mueller Hinton broth was added to wells 2-12 in a 96well plate. 200 µl of the peptide was added to well one at a concentration of 256 µg/mL. 100µl of peptide in well one was taken up and pipetted into well two. The mixture was then mixed via pipetting before 100µl was taken up and pipetted into well three. This process was repeated up to well 11. Once peptide was added to well 11 100 µl was taken up and then discarded ensuring the well 12 had no peptide present. Thus, the concentrations (in µg/mL) were: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and no peptide present. Each well was then inoculated with 100µl of bacteria that had been diluted to an OD600nm of 0.1. This was repeated three times. The 96-well plates were then incubated at 37°C for 24 hours. The MIC was determined to be the lowest concentration at which there was no growth visible.
For all the compounds in which the MIC lower than 1 µg/ml for the initial test, the above procedure was repeated at an altered initial concentration of 64 µg/ml. To determine the MIC of M. smegmatis ATCC 607 an inoculum was shaken at 140rpm, 37°C in 5ml Middlebrook 7H9 broth (SIGMA) supplemented to 5% Middlebrook ADC (SIGMA) growth supplement for 3-4 days and harvested mid-late exponential phase (OD ~0.6). The harvested cells were washed once in fresh media and diluted 10-fold from the original volume. Then plated out in a 96-well plate as previously described, incubated at 37°C 140RPM with MIC readings taken after 72 hours.
In order to determine the effect of serum on antibacterial activity, the MIC of compounds 12 and 13 were measured in presence of 10% human serum using the above protocols. Both the compounds were pre-treated with 10% human serum (Sigma, H4522) for 30 mins and 2 hours. These pre-treated samples were used for MIC determination using Mueller Hinton Broth supplemented with 10% human serum.

VII. Antagonization assay
An antagonization assay was performed using Leu10-teixobactin (13) and Lipid II as reported in literature. 7 MIC was tested using the protocols described in section VI.

IX. Time-dependent killing of bacteria by teixobactin analogues 12 and 13
Time-kill kinetics against MRSA DM21455 strains (clinical isolates from patients) was carried out in MHB. Cultures were grown overnight in MHA plates and adjusted to a final inoculum of 10 5 -10 6 CFU/ml in MHB (containing 0.002% v/v, polysorbate 80) with teixobactin analogues 12 and 13 maintained at a final concentration of 0.5 µg/ml. For vancomycin, the concentration was varied from 0.5 -16 µg/ml without polysorbate 80. The tubes were then incubated at 37 °C. 100 µl of cell suspension was withdrawn at various time points (0, 2, 4, 8, 24 h), serially diluted (10 1 -10 5 fold dilutions) and plated onto a MHA plates and incubated for 24 h at 37 °C. Colonies were then enumerated using a haemocytometer. Colony counting too numerous to count (>300 colonies) was taken as 10 10 CFU. Average values from two independent experiments are reported.

X. Complex formation of teixobactin with lipid II and geranyl pyrophosphate
Complex formation of teixobactin analogues 8 (Ala10-teixobactin) and 16 (Arg10-teixobactin) with lipid II and geranyl pyrophosphate was performed using TLC as described previously. 7 Binding of teixobactin to lipid II and geranyl pyrophosphate was analysed by incubating 30 µL of 2 nmol of each precursor with 2 or 4 nmoles of teixobactin in 50 mM Tris/HCl, pH 7.5, for 30 min at room temperature. Complex formation was analysed by extracting unbound precursors from the reaction mixture with 30 µL n-butanol/6M pyridine acetate (pH 4.2) (2:1; vol/vol) followed by TLC analysis of the organic layer using chloroform/methanol/water/ammonia (88:48:10:1, v/v/v/v) as the solvent and detection of lipid/phosphate containing precursors by phosphomolybdic acid staining. The TLC figures represent the results obtained through three independent experiments. Figure S76: Binding of teixobactin analogues 8 (Ala10-teixobactin) and 16 (Arg10-teixobactin) with lipid II using the protocols described in literature. 7 Partial binding is observed when the ratio of lipid II to the analogue is 1:1 (indicated by lighter spots on the TLC) and complete binding is observed when the ratio of lipid II to the analogue is 1:2 in case of analogue 16 and 1:4 in case of analogue 8 (indicated by no spots on TLC).  Figure S77: Binding of teixobactin analogues 8 (Ala10-teixobactin) and 16 (Arg10-teixobactin) with geranyl pyrophosphate using the protocols described in literature. 7 complete binding is observed when the ratio of the phosphate to the analogue is 1:2 (indicated by no spots on the TLC). Figure S78: Binding of teixobactin analogue 13 (Leu10-teixobactin) with lipid II using the protocols described in literature. 7 Partial binding is observed when the ratio of lipid II to the analogue is 1:1 (indicated by lighter spots on the TLC) and complete binding is observed when the ratio of lipid II to the analogue is 1:2 (indicated by no spots on TLC).

Cytotoxicity assay by Formazan bioreduction
HeLa cells were seeded in a 96-well plate at 10 4 cells/cm 2 density in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% serum. The cells were repeatedly rinsed with Hank's Balanced Salt Solution (HBSS) prior to be exposed to different peptides in the range of 0.5 -100 µM in HBSS 24 hrs post-seeding. Following 6 hrs of exposure to the teixobactin analogue, CellTiter 96 AQueous Nonradioactive Cell Proliferation Assay (Promega) was used according to the manufacturer's instructions. 8 Not ingested teixobactin analogue was removed by repeated washings with fresh medium. 20 μL of the combined MTS/PMS solution was added to 100 μL fresh medium in each well and plates were incubated for 3 hrs at 37°C. Absorbance was measured at 490 nm on Tecan Infinite M200 PRO plate reader with i-control 1.10 software (Molecular Devices).

XII. Haemolytic Assay Protocol
This assay was done at Singapore Eye Research Institute, Singapore. Hemolytic assay was performed on rabbit red blood cells (RBCs) immediately after collecting the blood samples from adult rabbits. All procedures for isolating blood from rabbits were approved by IACUC Singhealth and performed according to the standards of the Association for the Research in Vision and Ophthalmology.
Haemolytic activity of peptides was determined for rabbit red blood cells (rRBC), as reported before. 9 Rabbit erythrocytes were isolated from freshly collected blood samples and washed twice with sterile PBS. Two-fold serial dilutions of peptides (0.195 -250 µg/ml) was mixed with rRBC (final concentration 4% v/v), incubated at 37 o C for 1h and centrifuged at 3000 rpm for 5 minutes. The release 8 11 13