Synthesis And Antimicrobial Activity Of Stilbene-based Peptoid Mimics

Citrus canker,which is caused by the Gram-negative bacterial Xanthomonas citri pv.citri(Xcc),is a severe disease that results in leaf lesions and fruit drop.As a consequence,the bacterial disease leads to significant economic losses to citrus industry.Copper biocides are currently the primary citrus canker protection product.However,the aggressive use of copper biocides for citrus protection has resulted in an increased risk of bacterial copper tolerance and ecological environment pollution.Therefore,the search for copper biocides alternatives with high bioactivity and low propensity to develop bacterial resistance are desirable for the treatment of citrus pathogenic bacteria.Small peptoid mimics reproduce the critical AMPs amphiphilic structure,which aim to retain membrane-active mechanism of AMPs.Therefore,peptoid mimics have received increasing attention as a new type of potential antibacterial agent due to its strong bactericidal activity.Natural stilbene compounds are widely distributed in plants and function as phytoalexin.Stilbenes have some pharmacological properties and play a biological role in defending against pathogens,making it a lead skeleton for drugs design and development.Considering the amphiphilic topology of peptoid mimics and the bioactivity of stilbene,a class of stilbene-based peptoid mimics was designed and synthesized.Bioassays in vitro of stilbene-based peptoid mimics against Xcc was tested,and the compounds with obviously antibacterial activity were selected to control the citrus canker in vivo,inhibit the biofilm formation.To understand the antibacterial mode of action of stilbene-based peptoid mimic,acridine orange/ethidium bromide(AO/EB)staining,outer membrane permeability assay,260 nm nucleic acid leakage assay and the experiment of scanning electron microscopy(SEM)for bacterial morphology were performed.The main conclusions were drawn as follows:(1)The hydrophobic core(E)-4-(4-(trifluoromethyl)styryl)benzoic acid 3 was successfully prepared using Arbuzov reaction and Wittig-Horner reaction from benzyl bromide and methyl p-formylbenzoate as starting materials.In the synthesis of stilbene-based peptoid mimics,coupling agent(EDCI)and activating agent(HOBt)were used for amide bond formation by liquid method.Totally 21 new stilbene-based peptoid mimics were synthesized,8 of which are stilbene-capped single amino acids,9 of which are stilbene-capped dipeptides,4 of which are stilbene-capped tripeptides.The structures of the target compounds were confirmed by NMR and HRMS techniques.(2)The minimum inhibitory concentrations(MICs)of the target compounds against Xcc were determined using the microdilution assay.The stilbene-capped single amino acids 5aand 6 showed high activity against Xcc with the MIC values of 25 ?M.The stilbene-capped tripeptides 11 c and 11 d with two positive charges displayed the highest activity,and showed the same MIC value of 25 ?M.The all stilbene-capped dipeptides with two hydrophobic amino acid residues were inactive against Xcc.Although the stilbene-capped dipeptides with one positive charge showed antibacterial activity,the MIC values were lower than compound5 a and 6.On the bases of bioassay in vitro,the in vivo antibacterial activity of compounds 5a,6,11 c and 11 d were tested on detached citrus leaves.These results showed that compound 11 c and 11 d were effective in suppressing the lesion development,with very little lesion on leaves.Moreover,compound 11 c and 11 d also effectively inhibited biofilm formation with the inhibition ratio of 77.8% and 86.3% at a concentration of 50 ?M.(3)AO/EB staining assay confirmed that on treatment with 5e and 9b at 2 × MIC,Xcc could be killed quickly and bacterial cell membranes were indeed compromised.To further investigate the membrane-active mechanism of stilbene-based peptoid mimics,the effects of compounds 6 and 11 d on the outer membrane permeability,leakage of nucleic acid at 260 nm,and morphological change of Xcc.The results showed that tested compounds could destroy bacterial membranes,and compound 11 d leads more damage to the membrane of Xcc compared to compound 6.The stilbene-based cationic peptoid mimics killed Xcc by binding to the cell membrane,then inducing outer membrane rupture,affecting permeation of membrane and causing the leakage of cytoplasm,a mechanism analogous to that of antimicrobial peptides.