Staphylococcus aureus entanglement in self-assembling β-peptide nanofibres decorated with vancomycin

The increasing resistance of pathogenic microbes to antimicrobials and the shortage of antibiotic drug discovery programs threaten the clinical use of antibiotics. This threat calls for the development of new methods for control of drug-resistant microbial pathogens. We have designed, synthesised and characterised an antimicrobial material formed via the self-assembly of a population of two distinct β-peptide monomers, a lipidated tri-β-peptide (β3-peptide) and a novel β3-peptide conjugated to a glycopeptide antibiotic, vancomycin. The combination of these two building blocks resulted in fibrous assemblies with distinctive structures determined by atomic force microscopy and electron microscopy. These fibres inhibited the growth of methicillin resistant Staphylococcus aureus (MRSA) and associated directly with the bacteria, acting as a peptide nanonet with fibre nucleation sites on the bacteria observed by electron microscopy and confocal microscopy. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity and establish an innovative strategy to develop self-assembled antimicrobial materials for future biomedical application.

The reaction was stirred overnight at room temperature. The reaction mixture was then concentrated under vacuum and the residue was dissolved in 30% aq. CH 3 CN (60 mL). The crude product was purified by injecting the sample (~7.5 mL) onto a reverse-phase preparative column, eluted over a 55 min gradient from 0 to 50 % solvent B, (solvent A: 0.1 % TFA/H2O; solvent B: 0.1 % TFA/CH 3 CN) with a flow rate of 6 mL/min. The fractions were collected and analysed for purity by injecting the samples onto a reverse-phase analytical column, eluted over a 45 min gradient from 0 to 45 % solvent B, with a flow rate of 1 mL/min. The pure fractions were pooled to provide the propargyl amide vancomycin trifluoroacetate 1 (180 mg, 19 % yield) after the recovery of starting material (100 mg).

Synthesis of tri β-peptide AcβSerAzAlaOH
The peptide was synthesised on a 0.25 mmol scale using standard Fmoc chemistry on Wang resin. The resin was swollen in DMF (2 mL) and then soaked in Fmoc-protected β amino acid (2.1 eq. to resin loading), dissolved in DMF (2 mL) along with HBTU (2 eq. to resin loading),

S3
HOBt (2 eq. to resin loading), DMAP (10 mol %) and DIPEA (4 eq. to resin loading), overnight with gentle agitation. The resin was thoroughly washed with DMF (3 x 4 mL) and the Fmoc protecting group on the amino acid was removed by soaking the resin twice in 20 % piperidine, with 0.1 M HOBt, in DMF (4 mL) for 15 minutes each. The resin was washed with DMF (3 x 4 mL), soaked in Fmoc-protected amino acid (2.1 eq. to resin loading), dissolved in DMF (4 mL) along with HBTU (2 eq. to resin loading), HOBt (2 eq. to resin loading) and DIPEA (4 eq. to resin loading), for 2 hours. β Peptide elongation cycle was then repeated until the sequence was complete. After removing the terminal Fmoc protecting group on the peptide, the resin was treated with a solution of 10 % v/v acetic anhydride and 2.5 % v/v DIPEA in DMF (4 mL) for 30 minutes to afford an acetyl-capped N-terminus. The resin was washed with DMF (2 x 4 mL), CH 2 Cl 2 (2 x 4 mL), Et 2 O (2 x 4 mL) and air dried. Cleavage was performed on the resin (0.25 mmol), by treating the resin with a cleavage solution (10 mL) comprising of  H 2 O (7.5 mL) and CH 3 CN (0.5 mL) was degassed with argon for 5 min. To this was added NaHCO 3 (32 mg) ascorbic acid (26 mg), propargyl vancomycin trifluoroacetate 1 (24 mg, S4 15 µmol) and AcβSerAzAlaOH 2 (7 mg, 19 µmol). CuSO 4 .5H 2 O (16 mg, 64 µmol) was then added to the reaction solution and vortexed until completely dissolved. The reaction was gently agitated for 60 min. The crude product was purified by injecting it onto a reverse-phase preparative column, eluted over a 55 min gradient from 0 to 55 % solvent B, (solvent A: 0.1 % TFA/H 2 O; solvent B: 0.1 % TFA/CH 3 CN) with a flow rate of 6 mL/min. The fractions were collected and analysed for purity by reverse-phase analytical HPLC, eluted over a 45 min gradient from 0 to 45 % solvent B, with a flow rate of 1 mL/min. Pure fractions were pooled to provide AcβSerineTriazole(Vancomycin)βAlanine trifluoroacetate 3 (21 mg, 71 % yield). Scheme S1: Synthesis of lipidated tri β-peptide conjugated to fluorophore AcβAlaC14Cy5βLysOH (4)

Synthesis of functional lipidated tri-β-peptides
The peptide was synthesised on a 0.1 mmol scale using standard Fmoc chemistry on chlorotrityl resin (0.3 mmol/g loading). The resin was swollen in DCM (2 mL) and then soaked in Fmocprotected β amino acid (2 eq. to resin loading), dissolved in DCM (2 mL) along with DIPEA (3 eq. to resin loading), overnight with gentle agitation. The resin was thoroughly washed with DMF (3 x 3 mL) and the Fmoc protecting group on the amino acid was removed by soaking the resin twice in 20 % piperidine, with 0.1 M HOBt, in DMF (3 mL) for 15 minutes each. The resin was washed with DMF (3 x 3 mL), soaked in Fmoc-protected amino acid (3 eq. to resin loading), dissolved in DMF (3 mL) along with HBTU (3 eq. to resin loading), HOBt (3 eq. to resin loading) and DIPEA (4 eq. to resin loading), for 2 hours. β Peptide elongation cycle was S5 then repeated until the sequence was complete. After removing the terminal Fmoc protecting group on the peptide, the resin was treated with a solution of 10 % v/v acetic anhydride and 2.5 % v/v DIPEA in DMF (4 mL) for 30 minutes to afford an acetyl-capped N-terminus. The resin was washed with DMF (2 x 3 mL), CH 2 Cl 2 (2 x 3 mL), Et 2 O (2 x 4 mL), air dried for 10 minutes, and transferred to a 50 mL vial for further manipulation.
Derivatisation of the N-acetyl β 3 -tripeptide, on solid support, was preceded by the reduction of the azido-alanine residue on the β 3 -peptide. The resin (0.1 mmol) was swollen in THF (2.5 mL) and then soaked in a solution of PPh 3 (4 eq. to resin loading), in THF (2 mL) and H 2 O (50 μL), overnight with gentle agitation. The resin was filtered through a sintered glass funnel and washed with THF (2 x 3 mL) and DMF (2 x 3 mL). The resin was soaked in myristic acid (3 eq. to resin loading), dissolved in DMF (4 mL) along with HBTU (3 eq. to resin loading), HOBt (3 eq. to resin loading) and DIPEA (4 eq. to resin loading), for 1 h. The resin was subsequently washed with DMF (2 x 3 mL), CH 2 Cl 2 (2 x 3 mL), Et 2 O (2 x 4 mL), air dried for 10 min and continued with further chemical manipulation to afford AcβAlaC14Cy5βLysOH (4) or cleaved to afford AcβAlaC14βLysβAlaOH (5).
To a 50 mL vial was added CHCl 3 (10 mL) which was rigorously degassed by bubbling a stream of argon. A portion of the degassed CHCl 3 (~2 mL) was then used to swell the resin (0.1 mmol), contained in a separate 15 mL vial pre-purged of air with argon. PhSiH 3 (250 µL) was added to the remaining CHCl 3 (~4 mL) whilst still bubbling with a stream of argon. Pd(PPh 3 ) 4 (250 mg) was then added and the mixture was shaken until a homogeneous solution was achieved. The resin was then soaked in the Pd(PPh 3 ) 4 solution for 2 hours, with gentle agitation, filtered through a sintered glass funnel, and washed with CH 2 Cl 2 (3 x 3 mL) and DMF (3 x 3 mL) to remove the catalyst. The resin was then soaked in Quasar ® 670 carboxylic acid (1 eq. to resin loading), dissolved in DMF (2 mL) along with HBTU (1 eq. to resin loading), HOBt (1 eq. to resin loading), DMAP (0.1 eq. to resin loading) and DIPEA (2.5 eq. to resin loading), for 2 hours with occasional stirring and excluded from light. The resin was subsequently washed with DMF (2 x 3 mL), CH 2 Cl 2 (2 x 3 mL), Et 2 O (2 x 4 mL), air dried and subsequently cleaved from the resin.
Cleavage was performed on the resin (0.1 mmol), by treating the resin with a cleavage solution (10 mL) comprising of H 2 O (2.5 % v/v), triisopropylsilane (2.5 % v/v) in CF 3 COOH, for S6 by addition of Et 2 O (50 mL). The precipitate was filtered and dissolved in 50 % aqueous CH 3 CN for lyophilisation. The peptide was redissolved in 60-70 % aqueous CH 3 CN (5 mL) and purified by injecting the sample onto a reverse-phase preparative column, eluted over a The peptide was synthesised on a 0.10 mmol scale using standard Fmoc chemistry on Wang resin as described above. The peptides were then cleaved and purified by injecting the them onto a reverse-phase preparative column, eluted over a 55 min gradient from 0 to 20 % solvent