Peptoid Efficacy against Polymicrobial Biofilms Determined by Using Propidium Monoazide‐Modified Quantitative PCR

Abstract Biofilms containing Candida albicans are responsible for a wide variety of clinical infections. The protective effects of the biofilm matrix, the low metabolic activity of microorganisms within a biofilm and their high mutation rate, significantly enhance the resistance of biofilms to conventional antimicrobial treatments. Peptoids are peptide‐mimics that share many features of host defence antimicrobial peptides but have increased resistance to proteases and therefore have better stability in vivo. The activity of a library of peptoids was tested against monospecies and polymicrobial bacterial/fungal biofilms. Selected peptoids showed significant bactericidal and fungicidal activity against the polymicrobial biofilms. This coupled with low cytotoxicity suggests that peptoids could offer a new option for the treatment of clinically relevant polymicrobial infections.


Peptoid Synthesis
Synthesis as previously described and shown in Figure S1 (G. A. Eggimann, H. L. Bolt, P. W. Denny, S. L. Cobb, ChemMedChem, 2015, 10, 214-214). Fmoc-protected Rink Amide resin (normally 100 mg, 0.1 mmol, typical loading between 0.6-0.8 mmol g -1 ) was swollen in DMF (at least 1 hour at room temperature, overnight preferred) in a 20 mL polypropylene syringe fitted with two polyethylene frits (Crawford Scientific). The resin was deprotected with piperidine (20% in DMF v/v, 2 x 20 min) and washed with DMF (3 x 2mL). The resin was treated with bromoacetic acid (8 eq. with respect to the resin, 2M in DMF) and DIC (8 eq., 2M in DMF) for 15 minutes at 50 ˚C on a heated shaker at 400 rpm. The resin was washed with DMF (3 x 2 mL), before the desired amine sub-monomer was added (4 eq., 1M in DMF) and allowed to react for 15 minutes at 50 ˚C 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 sub-monomer had been added and the desired peptoid sequence had been obtained. The resin was shrunk in diethyl ether (3 ml) and final cleavage from resin was achieved using a TFA cleavage cocktail (4 ml, TFA:TIPS:H 2 O, 95:2.5:2.5) on the shaker at 400 rpm for 60 minutes. The resin was removed by filtration and the cleavage cocktail removed in vacuuo. The crude product was precipitated in diethyl ether (30 mL) and the precipitate retrieved by centrifuge for 15 min at 5,000 rpm. The ether phase was decanted and the crude product dissolved in a mixture of acidified H 2 O and MeCN and lyophilised before purification. Preparative RP-HPLC was performed with a semi-preparative Perkin Elmer Series 200 lc pump fitted with a 785A UV/Vis detector using a SB-Analytical ODH-S optimal column (250 × 10 mm, 5 µm); flow rate 2 ml min −1 ; λ = 250 nm, typical linear gradient elution 0-50% of solvent B over 60 min (A = 0.1% TFA in 95% H 2 O and 5% MeCN, B = 0.1% TFA in 5% H 2 O and 95% MeCN). Analytical RP-HPLC was performed with a Perkin Elmer Series 200 lc pump fitted with a 785A UV/Vis detector using a SB-Analytical ODH-S optimal column (100 × 1.6 mm, 3.5 µm); flow rate 1 ml min −1 ; λ = 220 nm, linear gradient elution 0-100% of solvent B over 30 min (A = 0.05% TFA, 95% H 2 O, 5% MeCN, B = 0.03% TFA, 5% H 2 O, 95% MeCN).
Peptoids were characterised by accurate LC-MS (QToF mass spectrometer and an Acquity UPLC from Waters Ltd.) using an Acquity UPLC BEH C8 1.7μm (2.1mm × 50mm) column with a flow rate of 0.6 ml min -1 and a linear gradient of 5-95% of solvent B over 3.8 min (A = 0.1% formic acid in H 2 O, B = 0.1% formic acid in MeCN). Peptide identities were also confirmed by MALDI-TOF mass spectra analysis (Autoflex II ToF/ToF mass spectrometer Bruker Daltonik GmBH) operating in positive ion mode using an α-cyano-4-hydroxycinnamic acid (CHCA) matrix. Data processing was done with MestReNova Version 8.1. Figure S1. The submonomer method of peptoid synthesis on solid phase; [i] swelling and deprotection of resin; [ii] acylation using bromoacetic acid and DIC in DMF; [iii] displacement step (primary amine in DMF); [iv/v] successive cycles of acylation and displacement; [vi] acidic TFA cleavage of product from resin.

Characterisation of building blocks and peptoids used in this study
Table S1: The abbreviations used for the peptoid monomers synthesised in this study, and the amines that they were derived from.

Monomer
Chemical Structure Amine Sub-monomer    Figure S3. LC-MS spectra for the peptoid library; for each peptoid the mass spectrum (top) and UV chromatogram at λ = 250 nm (bottom) are shown.

qPCR reaction mixes and conditions
Supplementary

Proteolytic Stability Study
Although it is generally acknowledged that peptoid backbone structures should be inherently resistant proteolysis, we compared the tryptic digestion profile of one selected peptoid, peptoid 7, against the naturally occurring alpha helical peptide LL-37. Peptoid 7 showed no degradation following treatment with trypsin for 24 hours whereas LL-37 was completely degraded to peptide fragments during the same time period.