Biosourced Functional Hydroxybenzoate- co-Lactide Polymers with Antimicrobial Activity

Abstract

Antimicrobial resistance is an urgent global health challenge, and compounds that address this issue have attracted significant attention. In particular, bioderived molecules that possess natural antimicrobial properties can be useful to prepare active macromolecules that are degradable. In this work, a 4-(methyl/allyl/benzyl)oxy-6-(H/alkyl)-2-oxy-benzoate-co-lactide-based polymer library was designed and studied for antimicrobial activity. The monomer precursors were heterologously produced and purified from an engineered fungal host, chemically modified with 4-(methyl/allyl/benzyl)oxy substituents, and ring-closed to form the 3-methyl-5H-benzo[e][1,4]dioxepine-2,5(3H)-diones. The polymers were synthesized by ring-opening polymerization using a 3-O urea/1-methyl-2,3,4,6,7,8-hexahydro-1H-pyrimido[1,2-a]pyrimidine catalytic system and 3-methyl-butan-1-ol as the initiator. Polymers at different degrees of polymerization were prepared by varying the [monomer]/[initiator] (M/I = 5-30) and tested for activity against the pathogen Staphylococcus aureus. Polymers were identified that were antimicrobial and disrupted biofilms while maintaining good in vitro biocompatibility. The degradability of the polymers was confirmed. Overall, these results demonstrate the power of utilizing a combination of synthetic biology and chemistry to produce functional and degradable polymers that are potent inhibitors of the Gram-positive bacterium S. aureus, with potential applications in medicine.

1. Polymeric Antimicrobial Agents against S. aureus Inspired by Depsides

1

Compounds tested for antimicrobial evaluation with different R₁ and R₂ groups on the aromatic ring.

2

Preparation of lactide monomers (compounds 11–15) from 4-(methyl/allyl/benzyl)­oxy-6-(H/alkyl)-2-hydroxybenzoates and structure determination via MicroED from small crystallites: (a) synthesis of monomers; (b) nanocrystals of compound 13 that (c) yield high-resolution diffraction by MicroED, (d) such that their structures can be readily determined; and the structure of the same molecule determined from (e) larger crystals by (f) X-ray diffraction SCXRD; and (g) superimposed structures from MicroED and SCXRD all-atom reported with root-mean-square deviation (RMSD) of 0.028 Å.

3

Preparation and characterization of polymers derived from 4-(methyl/allyl/benzyl)-oxy-6-alkyl-2-oxybenzoate lactide monomers by ring-opening polymerization. (a) Synthetic route to prepare polymers with different M/I. Variation of the [monomer]/[initiator] (M/I) during ROP of monomer 13: (b) refractive index (RI) trace SEC in THF and (c) plot of M _n (black) and dispersity (Đ, blue) versus M/I.

4

Biofilm inhibition and disruption of functional hydroxybenzoate-co-lactide polymers 22 (a, b) and 28 (c, d) on S. aureus. Antimicrobial properties were determined by evaluating the biofilm inhibition and disruption by staining with 1% crystal violet. The figure shows the 96-well plate picture accompanied by the UV–vis quantitation of the biofilm assays. Statistical significance was determined via a one-way ANOVA with multiple comparisons (p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)) (n = 3).

5

Representative examples of the in vitro biocompatibility evaluated in the NIH 3T3 fibroblast cell line by MTT assay for polymers 22 and 24 (a, b) having M/I = 6 and 30, respectively. Statistical significance was determined via a one-way ANOVA with multiple comparisons (p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)) (n = 3).

6

Degradation of polymers studied at 50 °C in 0.1 M NaOH (n = 3).

7

Key compounds with antimicrobial activity against S. aureus. MIC is the minimal inhibitory concentration against S. aureus, CC₈₀ is the concentration at which fibroblast cell viability ≥80%, and SI is the selectivity index. Polymers 22 and 28 inhibit and disrupt S. aureus biofilms.