Synthesis of Antibacterial Nisin–Peptoid Hybrids Using Click Methodology

Antimicrobial peptides and structurally related peptoids offer potential for the development of new antibiotics. However, progress has been hindered by challenges presented by poor in vivo stability (peptides) or lack of selectivity (peptoids). Herein, we have developed a process to prepare novel hybrid antibacterial agents that combine both linear peptoids (increased in vivo stability compared to peptides) and a nisin fragment (lipid II targeting domain). The hybrid nisin–peptoids prepared were shown to have low micromolar activity (comparable to natural nisin) against methicillin-resistant Staphylococcus aureus.


Materials and methods
All reagents used in this project were purchased from commercial sources and used without further purification unless otherwise specified.m Amines used in submonomer peptoid synthesis were obtained either from Sigma Aldrich (Gillingham, UK) or TCI Europe (Zwijndrecht, Belgium). These chemicals were used without further purification and stored under appropriate conditions, as detailed in the manufacturer's instructions. Bond Elut solid phase extraction cartridges (20 mL, polypropylene with two polypropylene frits) were purchased from Crawford Scientific and used as reaction vessels for solid phase synthesis.
Solvents were removed under reduced pressure using a Büchi Rotavapor R11. A Radleys Discovery Technology shaker was also used to mix solutions where indicated and aqueous solutions were lyophilised using a Christ Alpha 1-2 LD Plus freeze-drier.

Quadrupole time of flight accurate mass spectrometry
Accurate mass measurements were performed using a QToF Premier mass spectrometer with an Acquity ultra-performance liquid chromatography system (Waters Ltd, UK). Samples were injected to the Acquity UPLC BEH C18 column (1.7 µm, 2.1 mm x 100 mm) with a flow rate of 0.6 mL min -1 and a linear gradient of 0-99 % of solvent B over 6 min (A = 0.1 % formic acid in H2O, B = 0.1 % formic acid in acetonitrile). The solvent flow from the UPLC was injected into a 0.2 mL/min flow of acetonitrile which was introduced into the electrospray ion source.
Modified protocol following the submonomer synthesis of peptoids [H.L. Bolt, S.L. Cobb, Org. Biomol. Chem., 2016, 14, 1211. Fmoc-protected Rink Amide resin (0.1 mmol, loading 0.82 mmol g -1 ) was swollen in DMF (at least 1 hour, overnight preferred, at room temperature) in a 20 mL polypropylene syringe fitted with two polyethylene frits. The resin was deprotected with piperidine (20 % in DMF v/v, 2 x 20 min) and washed with DMF (3 x 2 mL). The resin was treated with bromoacetic acid (1 mL, 0.6 M in DMF) and DIC (0.20 mL, 50 % v/v in DMF) for 20 minutes at room temperature at 400 rpm. The resin was washed with DMF (3 x 2 mL), before the desired amine sub-monomer was added (1 mL, 0.8-2.0 M in DMF) and allowed to react for 60 minutes at room temperature on the shaker. The resin was again washed with DMF (3 x 2 mL) and the bromoacetylation and amine displacement steps were repeated until the final submonomer had been added and the desired peptoid sequence had been obtained.
The resin was shrunk in diethyl ether to remove DMF in preparation for cleavage. Final cleavage from resin was achieved using TFA (95 %), H2O (2.5 %) and TIPS (2.5 %). For test cleaves approximately 1 mL of the cleavage cocktail was used and for cleavage from 100 mg resin, approximately 4 mL of the cleavage cocktail was added. The resin was then placed on the shaker at 400 rpm for 45 minutes and the resin removed by filtration. The cleavage cocktail was removed in vacuo, the crude product precipitated in diethyl ether (45 mL) and the precipitate retrieved by centrifuge for 15 min at 5,000 rpm. The ether phase was decanted, the crude product dissolved in a mixture of acidified H2O and MeCN and lyophilised. Crude peptoid sequences were purified using RP-HPLC prior to ligation with nisin A/B .    Nisin (600 mg, 0.18 mmol) was dissolved in 250 mL Tris buffer (25 mmol, NaOAc, 5 mmol Tris acetate, 5 mmol CaCl2, pH 7.0) and the solution cooled on ice for 15 minutes. Trypsin (50 mg) was added and stirred at room temperature for 15 minutes. The mixture was then heated to 30 °C for 16 hours, then another 50 mg of trypsin was added and after an additional 24 hours the reaction was complete by HPLC. The reaction was acidified with HCl (1 M) to pH 4.0 and solvents removed in vacuo.

Sequence
The nisin fragment was isolated by preparative HPLC and product fractions lyophilised to obtain a white powder (80 mg, 39 %).

Time /min
Cytotoxicity analyses were performed in 96-well plates (Costar, Fisher Scientific) using alamarBlue® (Invitrogen) for cell viability detection using a modified protocol as previously described. 9 The HepG2 or HaCaT cells were grown at 37 °C, 5 % CO2 in DMEM10 high glucose supplemented with heat-inactivated foetal bovine sera (FBS, 10 %; Biosera Ltd) and penicillin/streptomycin (P/S, 1 %). Cells were counted using a Neubauer Improved Haemocytometer. Cells were seeded 1 day prior to treatment in 96 well plates at a concentration of 2 x 10 5 cells mL -1 in 100 µL of medium (2 x 10 4 cells/well). Empty wells were filled with 100 µL PBS. After 24 hours, cells were incubated with the compounds in a dilution series in triplicate from 2-100 µM (5 mM stock solutions in DMSO; untreated cells with DMSO as a negative control) in 50 µL of the media for 1 hour. Afterwards, 40 µL of medium was removed from each well before the addition of 90 µL of the media, followed by incubation for 24 hours at 37 °C, 5 % CO2. Then, 10 µL of alamarBlue® (Invitrogen) was added to each well before a 2 hour incubation prior to assessing cell viability using a fluorescent plate reader (Biotek; λex 560 nm, λem 600 nm).