Synthetic Biomimetic Polymethacrylates: Promising Platform for the Design of Anti-Cyanobacterial and Anti-Algal Agents

Extensive, uncontrolled growth of algae and cyanobacteria is an environmental, public health, economic, and technical issue in managing natural and engineered water systems. Synthetic biomimetic polymers have been almost exclusively considered antimicrobial alternatives to conventional antibiotics to treat human bacterial infections. Very little is known about their applicability in an aquatic environment. Here, we introduce synthetic biomimetic polymethacrylates (SBPs) as a cost-effective and chemically facile, flexible platform for designing a new type of agent suitable for controlling and mitigating photosynthetic microorganisms. Since SBPs are cationic and membranolytic in heterotrophic bacteria, we hypothesized they could also interact with negatively charged cyanobacterial or algal cell walls and membranes. We demonstrated that SBPs inhibited the growth of aquatic photosynthetic organisms of concern, i.e., cyanobacteria (Microcystis aeruginosa and Synechococcus elongatus) and green algae (Chlamydomonas reinhardtii and Desmodesmus quadricauda), with 50% effective growth-inhibiting concentrations ranging between 95 nM and 6.5 μM. Additionally, SBPs exhibited algicidal effects on C. reinhardtii and cyanocidal effects on picocyanobacterium S. elongatus and microcystin-producing cyanobacterium M. aeruginosa. SBP copolymers, particularly those with moderate hydrophobic content, induced more potent cyanostatic and cyanocidal effects than homopolymers. Thus, biomimetic polymers are a promising platform for the design of anti-cyanobacterial and anti-algal agents for water treatment.


SUPPLEMENTARY FIGURES Figure S1
Illustrative images of studied cyanobacterial and algal species and a 96-well plate at the end of assays S10

Figure S2
The cytocidal activity of homopolymer HP2 S11 Figure S3 The cytocidal activity of copolymer CP1 S12 Figure S4 The cytocidal activity of copolymer CP3 S13 Figure S5 The cytocidal activity of copolymer CP5 S14 4-((tert-butoxycarbonyl)amino)ethyl methacrylate (4 mmol, 4 ml of 1 M solution in acetonitrile (MeCN)), 2cycanopropyl-2-yl-dithiobenzoate (0.6 mmol, 132.6 mg), and AIBN (0.06 mmol, 9.8 mg) were mixed in a vial with magnetic stirrer. The oxygen from the mixture was removed by bubbling with nitrogen for 2 min. The reaction mixture was then stirred at 70 °C. After 16 h, the reaction was stopped by cooling the vial in a dry ice-acetone bath. The polymer product was isolated by evaporating the MeCN solvent under reduced pressure, and then the residue was dissolved in dichloromethane and precipitated in excess hexanes twice to give Boc-HP1. The mol percentage of EMA (MPethyl) was determined by comparing integrated peaks of butylene groups of Boc-ABMA and ethylene groups of EMA in the 1 H NMR spectrum. The degree of polymerization (DP) was calculated by comparing integrated peaks of the phenyl group of chain transfer agent at the polymer ω-end and side chains. The number average molecular weight (Mn) was calculated using the DP, MPethyl, and the molecular weights of monomers and RAFT agent. GPC: Mn = 2400 g/mol, Mw = 2731 g/mol, dispersity (Đ) = 1.15. 1 H NMR (solvent deuterochloroform CDCl3, with tetramethylsilane TMS as stabilizer , 0 ppm): DP = 10.9, MPethyl = 0 mol %, Mn = 2700 g/mol.
Boc-HP1 (200 mg) was mixed with MMP (100 µL), followed by the addition of TFA (8 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol and then deprotected HP1 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate was dissolved in distilled water and lyophilized to yield a light pink fluffy product (200.7 mg).
Boc-HP2 (123mg) was mixed with MMP (60 µL), followed by the addition of TFA (4.5 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol and then deprotected HP2 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate was dissolved in distilled water and lyophilized to yield a light, fluffy product (105.9 mg, 81%).

Boc-HP3 HP3
4-((tert-butoxycarbonyl)amino)ethyl methacrylate (5 mmol, 1.15 g), RAFT B (0.5 mmol, 211 mg), and Aza B (0.05 mmol, 28 mg) were mixed in MeCN (5 mL). The oxygen from the mixture was removed by bubbling with nitrogen for 2 min. The reaction mixture was then stirred at 70 °C. After 16 h, the reaction was stopped by cooling the vial in a dry ice-acetone bath. The polymer product was isolated by evaporating the MeCN solvent under reduced pressure, and then the residue was dissolved in dichloromethane and precipitated in excess hexanes twice to give Boc-HP3. The DP was calculated by comparing integrated peaks of phenyl group of chain transfer agent at the polymer ω-end and side chains. The number average molecular weight (Mn) was calculated using the DP, MPethyl, and the molecular weights of monomers and RAFT agent. GPC: Mn = 2400 g/mol, Mw = 2600 g/mol, Đ = 1.11. 1 H NMR (CDCl3): DP = 14.0, Mn = 3600 g/mol (including TFA) or 2370 g/mol (excluding TFA).
Boc-HP3 (100 mg) was mixed with MMP (50 µL), followed by the addition of TFA (1 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol and then deprotected HP3 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate S5 was dissolved in distilled water and lyophilized to yield a light pink fluffy product (84.1 mg). 1 H NMR (CD3OD): DP = 15.7, Mn = 3,600 g/mol (including TFA) or 2370 g/mol (excluding TFA).

Boc-CP1-3 CP1-3 D-1. Synthesis of copolymer CP1
4-((tert-butoxycarbonyl)amino)butyl methacrylate (5 mmol, 1.29g), EMA (5 mmol, 0.63 mL), 2cycanopropyl-2-yl-dithiobenzoate (1.5 mmol, 331.86 mg), and AIBN (0.10 mmol, 16.4 mg) were mixed in MeCN (5 mL) with magnetic stirrer. The oxygen from the mixture was removed by bubbling with nitrogen for 2 min. The reaction mixture was then stirred at 70 °C. After 16 h, the reaction was stopped by cooling the vial in a dry ice-acetone bath. The polymer product was isolated by evaporating the MeCN solvent under reduced pressure, and then the residue was dissolved in dichloromethane and precipitated in excess hexanes twice to give Boc-CP1. The mol percentage of EMA (MPethyl) was determined by comparing integrated peaks of butylene groups of Boc-ABMA and ethylene groups of EMA in the 1 H NMR spectrum. The DP was calculated by comparing integrated peaks of phenyl group of chain transfer agent at the polymer ω-end and side chains. The number average molecular weight (Mn) was calculated using the DP, MPethyl, and the molecular weights of monomers and RAFT agent. GPC: Mn = 2100 g/mol, Mw = 2350 /mol, Đ = 1.12. 1 H NMR (CDCl3): DP = 13.7, MPethyl = 48.7 mol %, Mn = 2800 g/mol.
Boc-CP1 (400 mg) was mixed with MMP (200 µL) in DCM (0.5 mL), followed by the addition of TFA (8 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol, and then deprotected CP1 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate was dissolved in distilled water and lyophilized to yield a light pink fluffy product (329 mg). 1 H NMR (CD3OD): DP = 16.8, MPethyl = 47.5 mol %, Mn = 3500 g/mol (including TFA) or 2530 g/mol (excluding TFA).
Boc-CP2 (200 mg) was mixed with MMP (100 µL), followed by the addition of TFA (4 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol, and then deprotected CP2 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate was dissolved in distilled water and lyophilized to yield a light pink fluffy product (179 mg).
Boc-CP3 (100 mg) was mixed with MMP (50 µL), followed by the addition of TFA (1 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol, and then deprotected CP3 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate was dissolved in distilled water and lyophilized to yield a light pink fluffy product ( Boc-CP4 (100 mg) was mixed with MMP (50 µL), followed by the addition of TFA (1 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol, and then deprotected CP4 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate (CD3OD): DP = 10.5, MPethyl = 27.6 mol %, Mn = 2900 g/mol (including TFA) or 1860 g/mol (excluding TFA).
Boc-CP5 (170 mg) was mixed with MMP (85 µL), followed by the addition of TFA (1.7 mL). After stirring for 30 min, the TFA was removed by blowing with nitrogen gas. The residue was dissolved in methanol, and then deprotected CP5 was isolated by precipitating in excess diethyl ether. Subsequently, the precipitate was dissolved in distilled water and lyophilized to yield a light, fluffy product (159.7 mg). 1 H NMR (CD3OD): MPethyl = 26.8 mol %.