Unveiling the Antifouling Potential of Stabilized Poly(phosphorus ylides)

Zwitterionic polymers have emerged as highly attractive building blocks for antifouling coatings in biomedical applications. Notably, these polymers offer effective alternatives to the widely used poly(ethylene glycol) (PEG), which has raised concerns regarding its immunotoxicity and the development of PEG-specific antibodies. Polymeric ylides, a largely overlooked class of zwitterionic polymers, have been reported as effective antifouling scaffolds. However, the reported subclasses, poly(sulfur ylides) and N-oxides, lack structural diversity and chemical variability. In this study, we present the synthesis and characterization of polymeric phosphorus ylides as an unexplored class of poly(ylides) with significantly increased structural diversity, which is of high value when designing future ylide-based antifouling materials. Our findings demonstrate that, owing to their low dipole moments and hydration layers, these polymeric phosphorus ylides significantly reduce bacterial attachment. Furthermore, we observe selective toxicity toward bacteria rather than mammalian cells. The bactericidal nature of poly(phosphorus ylides), coupled with their expanded chemical space, provides a distinct advantage over existing materials, including zwitterionic polymers from betaine scaffolds. We anticipate that these unexplored structures will broaden the scope of antifouling applications for poly(ylides).


1.
Materials Reagents were obtained from Sigma Aldrich/Merck (Zwijndrecht, The Netherlands), Fluorochem BV (Amsterdam, The Netherlands) and TCI Europe (Zwijndrecht, Belgium) and were used without purification unless otherwise stated.Vinyl benzoic acid was obtained from Carbosynth.Solvents were obtained from VWR, Fisher, Acros Organic and Sigma Aldrich/Merck.Solvents were dried by passing over activated alumina columns in a MBraun MB SPS800 under a nitrogen atmosphere and stored under argon.Reactions were carried under air unless stated otherwise.Typically, such air-sensitive reactions were carried out under atmosphere of nitrogen using Schlenk technique.Ultrapure Milli-Q water was obtained from QPOD Milli-Q system.Reactions and fractions from flash column chromatography were monitored by thin layer chromatography using glass TLC plates (Merck, TLC Silica gel 60 F254) and if necessary visualized by staining with KMnO4 solution.Column chromatography was performed on VWR SiO2 Type (40-63 mesh) using a forced flow of air at 0.5-1.0bar.

Instrumentation
Nuclear Magnetic resonance (NMR) characterization was carried out on a Bruker AVANCE HD nanobay console with a 9.4 T Ascend magnet (400 MHz) and a Bruker AVANCE III console with a 11.7 T UltraShield Plus magnet (500 MHz) equipped with a Bruker Prodigy cryoprobe, in chloroform (CDCl3) or DMSO-d6.NMR spectra were recorded at 298 K unless otherwise specified.Chemical shifts are given in parts per million (ppm) with respect to tetramethylsilane (TMS, δ 0.00 ppm) as internal standard for 1 H NMR. Coupling constants are reported as J values in Hz.Peak assignment is based on 2D COSY, 1 H− 13 C HSQC, and 1 H− 13 C HMBC spectra.The splitting patterns are indicated as follows: s, singlet; br.s, broad singlet; d, doublet; t, triplet; m, multiplet.Gel permeation chromatography (GPC) equipped with PL gel 5 μm mixed D column calibrated for polystyrene (580− 377400 g/mol) was carried out on a Shimadzu instrument with NMP or DMA as eluent using differential refractive index and UV absorbance (254 nm).

Dipole calculations
The static dipole moments of model compounds displaying N-oxide, sulfur ylide and phosphorus ylide have been computed with the Gaussian16 code. 1 The B3LYP 2,3 functional, augmented with the Grimme D3 dispersion term, 4 and the 6-311++G** basis set have been chosen.The geometry has been fully optimized before computing the dipole (Figure 1).Synthetic route to access triphenyl phosphorus ylide 2 from triphenyl phosphine.

Stability
In order to assess the stability of phosphorus ylide, small molecule PY S3 was incubated in varying conditions and the stability was investigated using 31 P-NMR after 24 h.PY S3 was incubated at room temperature and left on the bench exposed to daylight.Only upon treatment with 4M NaOH, degradation was observed, namely formation of triphenyl oxide.

Solubility
Solubility of polymeric phosphorus ylides was tested in various solvents at room temperature by dissolving 1 mg of the polymer in 1 mL of solvent.The mixtures were vortexed for a while and the solubility was determined by visual appearance.If the polymer did not dissolve, then it was sonicated for 10 min with heating and then the solubility was again checked.

Polymer
Water

Surface-Attachment of polymeric ylides
For surface attachment, we used amine-coated well plates that were obtained from biomat (MCB02F-AM1).Well-plates were modified with polymers that bear carboxylic acids using the following protocol.In brief, carboxylic acid containing polymers were dissolved in THF/H2O (1:3, v/v, 10 mM) and EDC (3 equiv) with NHS (5 equiv) were added.The solution was stirred for 10 min and added to the well plates.After 4 hours, the solution was removed and the wells were thoroughly washed with water, EtOH, water/THF, water and finally with EtOH again.Finally, the well-plates were air-dried overnight.Because of the autofluorescence of polymeric phosphorous ylides, it was possible to confirm the modification by measuring fluorescence (λex = 380 nm, λem = 450 nm, bandwidth 20 nm).

Stability of Phosphorous Ylide residue in BHI Media
For determining stability of phosphorous ylide residue, compound 2 was incubated in BHI media at 37 o C for 24 hours.Stability was determined with 31 P-NMR at different time points (Figure S5).Fluorescence intensity was measured at λex = 485 nm, λem = 535 nm and λex = 300 nm, λem = 632 nm, respectively, with a bandwidth of 20 nm, 30 flashes and an Integration time of 40 µs using a Tecan Spark M10 plate reader.

Cytotoxicity assays
HEK293T, Chinese Hamster Ovarian (CHO) and NIH 3T3 cells were cultured in DMEM medium supplemented with 10% FBS.After cells reached a confluence of around 50 %, they were rinsed with 1x PBS three times and detached with 4 ml Trypsin for 3 minutes.
Trypsin was quenched by adding 8 ml of DMEM medium.The cells were transferred to a 15 ml falcon and spun down 5 minutes at 0.

Bacterial toxicity assays
P. aeruginosa cultures were diluted to an OD of 0.005 in BHI supplemented with Tyloxapol 0.04%.The bacterial solutions were incubated with varying concentrations of P(TMPY) 7 (1.0, 0.1, 0.01, 0.001 mg/mL) and added to clear wellplates.Absorbance (600 nm) was immediately measured to get a baseline, before returning the plate to the incubator for 24 hours at 37 °C.Finally, the absorbance was measured at 600 nm and data evaluated by subtracting the final OD from the baseline to retrieve an OD increase after 24 hours.

Triphenyl phosphonium salt S1
The triphenyl phosphonium salt S1 was synthesized according to a literature protocol. 5,6iphenylphosphine (5.0 g, 19.1 mmol, 1.0 eq) was dissolved in dry toluene (30 mL) in a flame dried and Argon flushed Schlenk tube.Then chloacetonitrile (2.4 mL, 38.1 mmol, 2 eq) was added to the colorless solution and the reaction mixture was bubbled with Argon for 30min.The reaction mixture was stirred overnight at 60 o C under Argon atmosphere and a white precipitated was formed.The suspension was cooled to room temperature and the precipitate was collected by filtration under vacuum and washed with cold Et2O.

Trimethyl phosphonium salt S2
The trimethyl phosphonium salt was synthesized according to a literature protocol. 7imethyl phosphine solution (15.0 mL, 15.0 mmol, 1.0 eq) was added to a flame dried and Ar flashed Schlenk tube.Chloroacetonitrile (1.42 mL, 22.5 mmol, 1.5 eq) was added to the solution of PMe3 at 0 °C.After the reaction mixture was stirred at room temperature for 1 h, the precipitate was collected by filtration and washed with cold THF to obtain the product as a white solid (yield 86%). 1

Benzoic acid triphenyl phosphorus ylide S3
Benzoyl chloride (0.036 mL, 0.306 mmol, 1.0 eq) was dissolved in CH2Cl2 (1.6 mL) and the mixture was stirred.Triethylamine (0.130 mL, 0.918 mmol, 3.0 eq) was added and the mixture was stirred for a while and then the phosphonium salt S1 (0.145 g, 0.429 mmol, 1.4 eq) was added.The reaction mixture was stirred at room temperature overnight.It was then diluted with CH2Cl2 and it was washed with sat.NaHCO3 (x1), H2O (x1) and brine (x1).The crude product was dried over Na2SO4 and purified via column chromatography on silica gel eluting with CH2Cl2/MeOH mixtures (gradient 95:5 to 9:1 by volume) to obtain the product as a yellow solid (yield 84%). 1
Et3N (1.1 mL, 7.77 mmol, 3.0 eq) was added to the solution, followed by T3P (3 mL, 3.37 mmol, 1.4 eq).The reaction mixture was saturated with nitrogen for 15 min and then phosphonium salt S2 (551 mg, 3.63 mmol, 1.4 eq) was added.The resulting mixture was allowed to stir overnight at room temperature.The mixture was diluted with CH2Cl2 and washed with sat.NaHCO3 (x1), H2O (x1) and brine (x1), dried over Na2SO4 and concentrated under reduced pressure.The residue was purified via column chromatography on silica gel eluting with CH2Cl2/MeOH mixtures (gradient 100:0 to 20:1 by volume) to obtain the product as a pink solid (5 %). 1

Polymerizations
General Protocol: A flame dried Schlenk tube was purged with Argon and charged with the RAFT agent (2-[[(2-Carboxyethyl)sulfanylthiocarbonyl]-sulfanyl]propanoic acid) and dissolved in anhydrous DMF (unless stated otherwise).The monomers were added under an Argon atmosphere and the solution was stirred.Subsequently, AIBN was added to the solution (in a stock solution), followed by trioxane as internal standard.The solution was degassed for 20 min and a NMR sample for was taken (t = 0 h).The solution was heated to 80 o C (unless stated otherwise) and the reaction was monitored by 31 P and 1 H-NMR.
After the desired conversion was indicated, the reaction solution was allowed to reach to room temperature and exposed to air.The product was precipitated dropwise in either cold Et2O or MeOH, re-dissolved in CH2Cl2 and subsequently precipitated dropwise in either cold Et2O or MeOH again.The product was dried under high vacuum overnight.
The molecular weight Mn(NMR) was determined via 1 H-NMR using the alpha-protons adjacent to the trithiocarbonate residue for polymers 3 and 4. For polymers 5, 6 and 7 the acidic protons of the carboxylic acid were used (example in Figure S6a and b).
The product was precipitated dropwise in cold Et2O, re-dissolved in CH2Cl2 and subsequently precipitated dropwise in cold Et2O again.The product was isolated as a yellow solid (146 mg).
Figure S10. 31P NMR at tfinal (9 h) of the polymerization of the P(TMPY) 7. According to NMR, the percentage of monomer oxidation was found to be overall 3.0%.

Surface energy -Contact angle
The contact angle and surface energy were measured according to a literature protocol. 9e contact angle of the polymers was measured by spreading the polymers on a glass surface.Glass surfaces are polar and therefore it is easier for the polymers to spread.
Before using them, the glass surfaces were cleaned using 2% Hellmanex solution and sonicated for 10 min with heating.Then they were thoroughly rinsed with demi water and dried well.
The following polymer solutions were prepared: 10 μL of each solution were spread on different glass surfaces, using a pipette and performing a circular movement in order to create a thin layer of evenly spread solution.
Then, solutions 1 and 2 were put in the oven at 65 ⁰C for 5 min while the rest for 15 min For the contact angle measurement, MilliQ water, glycerol or diidomethane (5 μL) was placed on the glass coated polymer surface and then snapshots were taken using optical microscopy.The images were then analyzed using ImageJ program to determine the contact angles.Each measurement was performed twice.
The surface energy was calculated using the acid-base Van Oss method. 10For this study, only the homopolymer P(TMPY) was used.) 0.  To find γS LW , γS + and γS -, equation 1 was solved using the contact angles measured and the constants of table 1.Then γS was calculated using equation 2.

Figure S5 .
Figure S5.31P-NMR spectra of incubation of 2 in BHI media at 37 o C. Signal at 0 ppm corresponds to residual PO4 3-from BHI buffer.

2. 8 . 2
Live/Dead Assay 96 well plates with various modified surfaces were inoculated with 100 µl of bacterial solution in BHI broth (OD 0.005) and incubated for 4 hours at 37 °C to allow for adhesion and biofilm formation.After 4 hours, all wells were gently washed three times with 1x PBS buffer (pH 7.4) to remove planktonic cells.BacLight stain (Molecular Probles) containing Syto9 and Propidium Iodide was used to create a suitable working solution: for Syto9 a final concentration of c = 11.1 nM and for Propidium Iodide a final concentration of c = 66.6 nM in PBS (150 mM NaCl, 100 mM NaPO4 mM, pH 7.4).Wells were stained for 10 minutes and washed three times with PBS (150 mM NaCl, 100 mM NaHPO4, pH 7.4).
3 rcf.The supernatant was discarded, and cells seeded with DMEM complete medium in a 96 well plate at a density of 4.0 to 4.5 x 10 4 cells/ml and incubated for 24 hours at 37 °C with 5 % CO2.Afterwards, varying concentrations of polymer in DMEM complete medium were added to the wells and left to further incubate at 37 °C and 5 % CO2 for 72 hours.10 µl of CCK8 (Sigma Aldrich) was added to the wells, incubated for 3 hours and the absorbance measured at 450 nm.

Figure S7 .
Figure S7.Growth factors of planktonic bacteria in the presence of 1 to 0.001 mg/ml over 24 hours.Samples were conducted in replicates of 10.P value ≤ 0.01.

Figure S8 .
Figure S8.Reference protons used for the calculation of the Mn and 1 H NMR of the polymers, a) The protons used as reference for the triphenyl phosphorus ylide polymers 3 and 4, b) The protons used as reference for the trimethyl phosphorus ylide polymers 5, 6 and 7.

1 H
NMR spectrum of the triphenyl phosphonium salt S1.

1 H
NMR spectrum of the trimethyl phosphonium salt S2.

Table S1 .
Overview of stability assays performed using small molecule PY S3.

3-from BHI buffer. 2.8 Bioassays 2.8.1 Crystal Violet Stain
water (v/v) and transferred to a new clear bottom well plate.Absorbance was measured at 590 nm in a Tecan Spark M10 plate reader.