Fully Oxygen-Tolerant Visible-Light-Induced ATRP of Acrylates in Water: Toward Synthesis of Protein-Polymer Hybrids

Over the last decade, photoinduced ATRP techniques have been developed to harness the energy of light to generate radicals. Most of these methods require the use of UV light to initiate polymerization. However, UV light has several disadvantages: it can degrade proteins, damage DNA, cause undesirable side reactions, and has low penetration depth in reaction media. Recently, we demonstrated green-light-induced ATRP with dual catalysis, where eosin Y (EYH2) was used as an organic photoredox catalyst in conjunction with a copper complex. This dual catalysis proved to be highly efficient, allowing rapid and well-controlled aqueous polymerization of oligo(ethylene oxide) methyl ether methacrylate without the need for deoxygenation. Herein, we expanded this system to synthesize polyacrylates under biologically relevant conditions using CuII/Me6TREN (Me6TREN = tris[2-(dimethylamino)ethyl]amine) and EYH2 at ppm levels. Water-soluble oligo(ethylene oxide) methyl ether acrylate (average Mn = 480, OEOA480) was polymerized in open reaction vessels under green light irradiation (520 nm). Despite continuous oxygen diffusion, high monomer conversions were achieved within 40 min, yielding polymers with narrow molecular weight distributions (1.17 ≤ D̵ ≤ 1.23) for a wide targeted DP range (50–800). In situ chain extension and block copolymerization confirmed the preserved chain end functionality. In addition, polymerization was triggered/halted by turning on/off a green light, showing temporal control. The optimized conditions also enabled controlled polymerization of various hydrophilic acrylate monomers, such as 2-hydroxyethyl acrylate, 2-(methylsulfinyl)ethyl acrylate), and zwitterionic carboxy betaine acrylate. Notably, the method allowed the synthesis of well-defined acrylate-based protein-polymer hybrids using a straightforward reaction setup without rigorous deoxygenation.


Instrumentation Nuclear Magnetic Resonance (NMR)
1 H NMR spectra were recorded on Bruker Avance III 500 MHz spectrometers with D2O or DMSO-d6 as the solvent.

Size Exclusion Chromatography
SEC measurements of polymers were performed using an Agilent GPC equipped with a RI detector and PSS columns (Styrogel 10 5 , 10 3 , 10 2 Å) with DMF as an eluent at 50 °C and the flow rate of 1 mL/min. Linear poly(methyl methacrylate) standards were used for calibration.

Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)
SEC-MALS measurements of zwitterionic polyacrylate and bioconjugates were performed using Agilent SEC system (Agilent, 1260 Infinity II) coupled with MALS, DLS, UV, Viscometer and RI detectors (Wyatt Technology, USA). Measurements were performed using Waters Ultra hydrogel Linear column with 1X DPBS as an eluent at room temperature and the flow rate of 0.5 mL/min.

dn/dc value of poly(OEOA480)
The dn/dc value of poly(OEOA480) in DMF with 0.05 M LiBr at 50 °C was determined using the refractive index (RI) detector Waters 2414 (Milford, MA). Five samples of poly(OEOA480) (Mn,app = 51 500, Đ = 1.16) purified by dialysis were prepared using the standard dilution method (Table   S1). Using syringe pump Chemyx, Fusion 4000 (Stafford, TX), the DMF was injected into the RI detector at a flow rate of 0.1 mL/min until it stabilized. Then 1 mL of polymer sample was injected at a constant flow rate, and RI values was measured (Table S1). Each sample was repeated three times, and the averaged RI values were used to obtain a calibration plot, the slope of which gives the dn/dc value of poly(OEOA480) as 0.047 mL/g.

Kinetics of EY/Cu-catalyzed ATRP (Figure 3)
The ATRP cocktail (5 mL Samples were drawn at regular intervals and monitored by 1 H NMR in D2O ( Figure S4) and by SEC ( Figure 2).

EY/Cu-catalyzed ATRP of OEOA480 with varying degrees of polymerization (Table 2)
The target degrees of polymerization (DP) were varied by adjusting the HO-EBiB concentration Expanding the scope to other hydrophilic acrylates (Table 3) The polymerization reaction mixtures were prepared according to the general procedure. The

Synthesis of linear poly(OEOA480) (DPT = 100)
The ATRP cocktail (5 mL 25.0 mW/cm 2 ). The resultant copolymers were analyzed by 1 H NMR and SEC ( Figure S7). The block copolymers of desired molecular weight could be prepared, although they showed a slightly broad distribution. Further optimizations may be needed to achieve better control over the block copolymers comprising of different monomers.

Synthesis and characterization of CT macroinitiators with 7 and 12 ATRP initiators
The synthesis of chymotrypsin macroinitiator was achieved by reaction of NHS-functionalized ATRP initiator (NHS-Br) with purified CT. NHS-Br (146 mg, 437 µmol) was dissolved in DMSO (0.5 mL), divided into three portions and added to CT (260 mg, 10.4 µmol) solution (20 mL, 100 mM sodium phosphate buffer (pH = 8.0)) every 30 minutes. After stirring the mixture in a refrigerator (4 °C) for 3 hours, the functionalized CT was purified by dialysis using a 15 KDa molecular weight cut-off dialysis tube against 25 mM sodium phosphate (pH = 8.0) and deionized water in a refrigerator for 24 hours. The final product was isolated by lyophilization. The number of functionalized sites on CT was determined by fluorescamine assay as reported in our previous publication. [2] Increased functionalization of CT was achieved by stopping the reaction at a longer time.
General procedure for the synthesis of CT-poly(OEOA480) biohybrids (Table 4) The ATRP cocktail in a 2 mL volumetric flask was prepared according to the general procedure.
The final concentrations were: OEOA480 (100 mM), CT-7 or -12 (0.285-0.071 mM), EYH2 (10 µM Then 2 mL of the ATRP cocktail was transferred to a 2 mL HPLC vial and placed in the Lumidox® photoreactor equipped with green light LEDs (527 nm, 125 mW/cm 2 ) and a cooling system. The polymerization was stopped after 3 h by turning the light off. For SEC analysis, the polymerization solution was purified by dialysis using a 15 KDa molecular weight cut-off dialysis tube in deionized water.