High-Throughput Tertiary Amine Deoxygenated Photopolymerizations for Synthesizing Polymer Libraries

The huge chemical space potential of synthetic polymers means that in many studies only a small parameter range can be realistically synthesized in a short time and hence the “best” formulations may not be optimum. Throughput is traditionally limited by the need for deoxygenation in radical polymerizations, but advances in photopolymerization now provide opportunities for “in-air” polymerizations. Here, we have developed a protocol using liquid handling robots (or multichannel pipettes) with blue light photolysis of reversible addition fragmentation chain transfer agents and tertiary amine deoxygenation to enable the synthesis of polymer libraries in industry-standard 96-well plates with no specialized infrastructure and no degassing step. The roles of solvents and amine deoxygenators are explored to optimize the polymerization, particularly to look at alternatives to dimethyl sulfoxide (DMSO) for hydrophobic monomer (co)polymerization. DMSO is shown to aid the degassing process but is not easy to remove and hence prevents isolation of pure polymers. In contrast, using dioxane in-plate evaporation or precipitation of the tertiary amine allowed isolation of polymers in-plate. This removed all reaction components yielding pure polymers, which is not easily achieved with systems using metal catalysts and secondary reductants, such as ascorbic acid. As an example of the throughput, in just under 40 h, 392 polymers were synthesized and subsequently analyzed direct from plates by a 96-well plate sampling size exclusion chromatography system to demonstrate reproducibility. Due to less efficient degassing in dioxane (compared to DMSO), the molecular weight and dispersity control were limited in some cases (with acrylates giving the lowest dispersities), but the key aim of this system is to access hundreds of polymers quickly and in a format needed to enable testing. This method enables easy exploration of chemical space and development of screening libraries to identify hits for further study using precision polymerization methods.

Plus with a differential refractive index (DRI) detector. The system was equipped with either PL Rapide M (7.5 x 150 mm) and PL Rapide F (10 x 100 mm) columns, or 2x PL Rapide M (7.5 x 150 mm) columns. The eluent is DMF with 1% LiBr as the additive. Samples were run at 50 °C at either 2 mL.min -1 or 1 mL.min -1 based on column set respectively. Poly(methyl methacrylate) standards (Agilent EasiVials) were used for calibration to create a third order calibration between 500 -1,000,000 Da. Respectively, experimental molar mass (Mn SEC ) and dispersity (Đ) values of synthesized polymers were determined by conventional calibration using Agilent SEC software.
Polymers were prepared using a Gilson Pipette Max 268, with 200 μL and 20 μL pipette heads.
Pipette Error -As solutions were pipetted using a system designed for water, the ability of a pipette to accurately measure 1 mL dioxane was tested. 1 mL was aspirated 5 times and each time weighed into a clean vial. Given the density of dioxane, a mass of 1.033 g is expected. Values recorded: 1.021 g, 1.028 g, 1.024 g, 1.030 g, 1.024 g. These values give and average of 1.025 g, and a standard deviation of 0.0032 g. This suggests that the values are likely close to the desired volume, and that the values measured are consistent. However, we would recommend testing solutions before use to ensure consistent pipetting volumes.

Synthetic Section
Determination of the effect of headspace -Into a 20 mL vial was added N-hydroxyethyl acrylamide (2.34 g), triethanolamine (3.02 g), dioxane (5.15 mL) and 2-cyano-2-propyl dodecyltrithiocarbonate (39 μL). This solution was vortexed until homogenous, and then 0.5 mL, 1 mL and 1.5mL pipetted into separate wells of a 2.2 mL deep 96 well plate. The plate was then covered with a TiterTop and wrapped in blue LED lights. The reaction was allowed to proceed for S4 dioxane. 1 mL DMF was added to each of the three wells and the plate shaken for 6 h. These samples were then filtered, diluted to 2 mg.mL -1 and analysed by SEC in DMF. was then placed back into the liquid handling robot and 1 mL DMF was added to each well. This plate was then agitated for 6 h, followed by the addition of 200 μL of this solution to each well of an empty polypropylene 96 well plate. These samples were then analysed by high throughput SEC and the Mp of each peak picked out and plotted in figure 3. lights. The reaction was allowed to proceed for 24 h after which the lights were removed and the plate dried under vacuum for 24 h to remove dioxane. The plate was then placed back into the liquid handling robot and 1 mL DMF was added to each well. This plate was then agitated for 6 h, followed by the addition of 200 μL of this solution to each well of an empty polypropylene 96 well plate. These samples were then analysed by high throughout SEC and the Mp of each peak picked out and plotted in Figure 3B.  was pipetted into wells C2:D7, followed by 406 μL of column 5 into C2:C7, and 406 μL of column 6 into D2:D7. 500 μL of column 7 was pipetted into wells E2:E7, 500 μL of column 8 was pipetted into wells F2:F7, 500 μL of column 9 was pipetted into wells G2:G7 and finally 500 μL of column 10 was pipetted into wells H2:H7. The plate was then covered with a TiterTop and wrapped in blue LED lights. The reaction was allowed to proceed for 24 h after which the lights were removed and the plate dried under vacuum for 24 h to remove dioxane. The plate was then placed back into the liquid handling robot and 1 mL DMF was added to each well. This plate was then agitated for 6 hours, followed by the addition of 200 μL of this solution to the same well of an empty polypropylene 96 well plate. These samples were then analysed by high throughput SEC, and the DP of the polymers plotted in Figure 3.   (3.52 mL) and CTA (5 μL) (col 9). 216 μL from column 1 was pipetted into wells A2:B7 of a deep well 96 well plate, followed by 284 μL of column 2 into A2:A7, and 284 μL of column 3 into B2:B7. 111 μL from column 1 was pipetted into wells C2:D7, followed by 389 μL of column 4 into C2:C7, and 389 μL of column 5 into D2:D7. 55 μL from column 1 was pipetted into wells E2:F7, followed by 445 μL of column 6 into E2:E7, and 445 μL of column 7 into F2:F7. 111 μL from column 1 was pipetted into wells G2:H7, followed by 472 μL of column 8 into G2:G7, and 472 μL of column 9 into H2:H7. The plate was then covered with a TiterTop and wrapped in blue LED lights. The reaction was allowed to proceed for 24 h after which the lights were removed and the plate dried under vacuum for 24 h to remove dioxane. The plate was then placed back into the liquid handling robot and 1 mL DMF was added to each well. This plate was then agitated for 6 h, followed by the addition of 200 μL of this solution to the same well of an empty polypropylene 96 well plate. These samples were then analysed by high throughput SEC, and the Mp of the polymers plotted in Figure 4A. added to wells B2:B7, followed by 397 μL from column 3 to wells C2:C7, and finally 296 μL from column 4 to wells D2:D7. The plate was then covered with a TiterTop and wrapped in blue LED lights. The reaction was allowed to proceed for 24 hours after which the lights were removed and the plate dried under vacuum for 24 hours to remove dioxane. The plate was then placed back into the liquid handling robot and 1mL DMF was added to each well. This plate was then agitated for 6 hours, followed by the addition of 200 μL of this solution to the same well of an empty polypropylene 96 well plate. These samples were then analyzed by high throughput SEC, and the DP of the polymers plotted in Figure 4B. A small sample of this stock solution was analyzed by 1 H NMR spectroscopy as a t0 sample. The plate was then covered with a TiterTop and wrapped in blue LED lights. At various timepoints, the plate was uncovered and the contents of one well was removed, followed by analysis by 1 H NMR spectroscopy and SEC. After 28 h the reaction was stopped. Conversion was determined by integration of the vinyl peaks against the DMF solvent standard. Conversion was plotted against molecular weight, and reaction time against the Ln[Mo/Mn] in order to investigate the kinetics of the reaction. This is shown in Figure 5.

Molecular weight variable polymerizations -
Amine Free Control Polymerization -To a 10 mL vial was added methyl methacrylate (0.5 g), dioxane (1.95 mL) and CTA (10 μL Degassed Control Experiment -To a 10 mL vial was added methyl methacrylate (0.5 g), triethanolamine (0.74 g), dioxane (1.28 mL) and CTA (10 μL). This solution was stirred and then 0.5 mL pipetted into a vial, the vial was sparged with nitrogen for 5 min and a sample removed for 1 H NMR spectroscopy, followed by irradiation for 24 hours. After this time, another 1 H NMR sample was removed and the sample was diluted with more dioxane and transferred to a 2 mL Eppendorf, 0.1 mL HCl was added and the sample centrifuged to remove the amine. The resulting solution was dried and the sample dissolved in DMF followed by SEC analysis. SEC DMF: Mn 13,500 Da, Mw/Mn = 1.50. Conversion = 94 %.
Standard Control Experiment -To a 10 mL vial was added methyl methacrylate (0.5 g), triethanolamine (0.74 g), dioxane (1.28 mL) and CTA (10 μL). This solution was stirred and then 0.5 mL pipetted into a vial, the vial was left open to air and irradiated for 24 h. After this time, the sample was diluted with more dioxane and transferred to a 2 mL Eppendorf, 0.1 mL HCl was added and the sample centrifuged to remove the amine. The resulting solution was dried and the S12 sample dissolved in DMF followed by SEC analysis. SEC DMF: Mn 12,400 Da, Mw/Mn = 2.12.