NMR spectra were acquired by a Bruker Advance III HD spectrometer at the frequency of 400 MHz for 1H (100 MHz for 13C). High-resolution mass spectrometry (HR-MS) was performed on an Agilent accurate-mass 6520B Q-TOF mass spectrometer. A Perkin Elmer Spectrum Two FTIR spectrometer equipped with a universal diamond ATR unit was used for the analysis of dissolved cellulose. Spectra of samples were recorded between range of 450 cm− 1 and 4500 cm− 1, at a resolution of 4 cm− 1. The spectra shown in Fig. 4 represent the accumulation of 4 scans/sample with baseline correction applied. All purchased chemicals were used without purification.
Procedure for the synthesis of ILs 2, 4, 9 and 10.
Tetrabutylammonium acetate (2) was prepared by dissolving tetrabutylammonium bromide (3) (322 mg, 1 mmol, 1 eq) of in 20 mL of acetonitrile. Silver acetate (167 mg, 1 mmol, 1 eq) was then added to the mixture. The reaction was allowed to stir at RT for 1 hr. After the reaction, the solution was centrifuged at 4000 x G for 10 min, and the supernatant was collected and dried under vacuum (0.3 Torr, 1 hr) at 50oC. The product was obtained as a light yellow oil in 89% yield. 1H NMR (acetone -d6): 0.97 (12H, t), 1.41 (8H, m), 1.68 (3H, s), 1.78 (8H, m), 3.48 (8H, t). 13C NMR: 13.93, 20.34, 24.50, 25.81, 59.14, 59.16, 174.47. HR-MS: [C16H36N]+, found 242.2845, calcd. 242.2842 (1.2 ppm). The 1H and 13C NMR of compound 2 are shown in Figure S1-S2.
Benzyltrimethylammonium acetate (4) was prepared by mixing 418 mg (1 mmol, 1 eq) of benzyltrimethylammonium hydroxide (5, 40 wt% aqueous solution) with 60 mg glacial acetic acid (1 mmol, 1 eq). The reaction was allowed to stir at RT for 1 hr. After the reaction, the solution was dried under vacuum vacuum (0.3 Torr, 1hr) at 50oC. The product was obtained as a clear liquid in quantitative yield. 1H NMR (D2O): 1.86 (3H, s), 3.06 (9H, s), 4.44 (2H, s), 7.52 (5H, m). 13C NMR: 23.42, 52.36, 52.40, 52.44, 69.63, 127.43, 129.25, 130.90, 132.86, 181.29. HR-MS: [C10H16N]+, found 150.1278, calcd. 150.1277 (0.7 ppm). The 1H and 13C NMR of compound 4 are shown in Figure S3-S4.
N-(3,4-dimethoxybenzyl)-N-ethyl-N-methylethanaminium acetate (9) was prepared by dissolving 209 mg (1 mmol, 1 eq) of 4-((diethylamino)methyl)-2-methoxyphenol in 2 mL of acetone. Dry K2CO3 (138 mg, 1 mmol, 1 eq) was added, followed by methyl iodide (568 mg, 4 mmol, 4 eq). The reaction was kept at 70 °C for 24 hr. After the reaction, the solution was filtered and then dried under vacuum (0.3 Torr, 1 hr) at 50oC. The product was then re-dissolved in 20 mL of acetonitrile and mixed with silver acetate (167 mg, 1 mmol, 1 eq). After 1 hr of reaction at RT, the solution was centrifuged at 4000 x G for 10 min, and the supernatant was collected and dried under vacuum (0.3 Torr, 1 hr) at 50oC. The final product was obtained as a dark brown oil in 85% overall yield. 1H NMR (DMSO-d6 with 1 µL acetone as calibration standard): 1.19 (6H, t), 1.73 (3H, s), 2.96 (6H, s), 3.29 (4H, q), 3.71 (3H, m), 4.91 (2H, m), 6.86 (3H, m). 13C NMR: 7.42, 24.29, 48.52, 55.17, 56.66, 57.54, 73.71, 112.17, 115.04, 120.40, 127.22, 147,41, 147.53, 173.10. HR-MS: [C14H24NO2]+, found 238.1803, calcd. 238.1802 (0.4 ppm). The 1H and 13C NMR of compound 9 are shown in Figure S5-S6.
N-(3,4-dimethoxybenzyl)-N-propylpropan-1-amine was prepared by dissolving 3,4-dimethoxybenzaldehyde (10.0 g, 60 mmol, 1 eq) into 200 mL dry acetonitrile. While stirring, dipropylamine (7.92 g, 78 mmol, 1.3 eq) was added followed by sodium triacetoxyborobydride (17.85 g, 84.2 mmol, 1.4 eq). The reaction was allowed to stir at RT overnight and worked up as reported elsewhere . After vacuum drying (0.3 Torr, 1 hr) at 50oC, the product (14.1 g, 55.8 mmol) was obtained as a pale yellow oil in 93% yield. 1H NMR (CDCl3): 0.86 (6H, t), 1.49 (4H, m), 2.40 (4H, t), 3.53 (2H, s), 3.86 (3H, s), 3.88 (3H, s), 6.79 (2H, m), 6.99 (1H, s). 13C NMR: 11.99, 19.97, 55.61, 55.94, 55.98, 58.40, 110.76, 112.09, 120.92, 148.01, 148.95. HRMS: [C15H26NO2]+, found 252.1955, calcd. for 252.1958 (1.2 ppm). The 1H and 13C NMR of N-(3,4-dimethoxybenzyl)-N-propylpropan-1-amine are shown in Figure S7-S8.
N-(3,4-dimethoxybenzyl)-N-methyl-N-propylpropan-1-aminium acetate (10) was prepared by dissolving N-(3,4-dimethoxybenzyl)-N-propylpropan-1-amine (325 mg, 1 mmol) in 2 mL of acetone. Methyl iodide (284 mg, 2 mmol, 2 eq) was then added to the mixture. The reaction was kept at 70 °C for 24 hr. After the reaction, the solution was filtered and dried under vacuum (0.3 Torr, 1 hr) at 50oC, and the product was then re-dissolved in 20 mL of acetonitrile and mixed with silver acetate (167 mg, 1 mmol, 1 eq). After 1 hr of reaction at RT, the solution was centrifuged at 4000 x G for 10 min, and the supernatant was collected and dried under vacuum (0.3 Torr, 1 hr) at 50oC. The product was obtained as a dark brown oil in 77% overall yield. 1H NMR: 0.89 (6H, t), 1.60 (3H, s), 1.75 (4H, m), 2.92 (3H, s), 3.14 (4H, m), 3.78 (6H, s), 4.52(2H, s), 7.09 (3H, m). 13C NMR (DMSO-d6): 10.58, 15.28, 25.58, 46.82, 48.43, 55.51, 55.67, 61.31, 64.44, 111.54, 116.34, 120.18, 125.90, 148.54, 150.12, 173.18. HR-MS: [C16H28NO2]+, found 266.2118, calcd. 266.2115 (1.1 ppm). The 1H and 13C NMR of compound 10 are shown in Figure S9-S10.
Avicel PH 101 microcrystalline cellulose obtained from Sigma Aldrich was used in the dissolution experiments. To test cellulose solubility, 500 mg of selected ILs were collected in a 7 mL transparent glass tube and heated in a sand bath at 100 °C for 1 hr (compound 8 was heated to 120 °C for 1 hr for due to its higher melting point). Cellulose (10 mg, 2 wt%) was added slowly into the tube, and the solution was stirred with a spatula. After 20 min of incubation, a drop of the solution was taken and observed under a light microscope (10x magnification). The procedure was repeated with 20 mg (4%) of total cellulose addition.
FTIR spectroscopy of dissolved cellulose
The IL solutions with 2 wt% cellulose concentration were used for FTIR analysis. Methanol (5 mL) was added into the tube to dissolve the IL and precipitate the dissolved cellulose. The solution was then transferred to a 50 mL centrifuge tube and centrifuged at 4000 x G for 10 min. After removal of the supernatant, the cellulose at bottom was dried in an oven at 70 °C for 1 h before FTIR analysis.
E. coli strain 1A1  was obtained from the microbial strain repository at the Joint Bioenergy Institute at Lawrence Berkeley National Laboratory. A 10 mL culture in Luria Broth (LB) media was grown for 18 hr at 37 °C with shaking at 200 rpm. The culture was then diluted into LB media to an absorbance of 0.05 at 600 nm, and added into the wells of a 96-well plate (first row = 190 µL, all other wells = 100 µL/well). Approximately 700 mg of IL was dissolved into 1 mL of methanol, and 10 µL of each IL solution was added to first row wells of the plate. A 2-fold serial dilution was performed down the plate by mixing and transferring 100 µL using a multi-channel pipette. To maintain isochoric conditions, 100 µL was discarded from bottommost wells. The plate was then incubated at 37° C with shaking at 150 rpm. After 24 hr incubation, E. coli growth was quantified at 600 nm using an Epoch Microplate Spectrophotometer (Gen5 software). Cells grown with 10 µL of methanol were used as negative controls, and resulted in zero growth inhibition. All experiments were repeated 3–7 times and error bars in Fig. 5 represent standard deviation. Statistical F-test was performed to determine the appropriate t-test (i.e., equal or unequal variance) to analyze significant difference between IC50 data.
Daphnia magna cultivation
Procedures for cultivating and performing acute toxicity assays with D. magna generally followed the US EPA protocol . Daphnia magna was cultured from stocks supplied by Aquatic Bio Systems Inc. (Fort Collins, CO). The organisms were cultured in 900 mL jars filled with moderately hard water from the recipe: 0.473 g CaSO4, 0.959 g NaHCO3, 1.223 g MgSO4 • 7H2O, and 0.039 g KCl per 10L of deionized water. 2 mL of algae (Selenastrum capriconutum) containing 3.0 × 107 cells/mL were fed to D. magna on both Tuesdays and Thursdays with water changes as well as feedings on Saturdays. All glass jars were placed in a Thermo Scientific Precision incubator (Fischer Scientific, Hampton, NH) at a holding temperature of 20°C and on a 12 hr: 12 hr light: dark cycle. This procedure was conducted approximately a month prior to the beginning of the project. To prepare D. magna for use in acute experiments, three hundred adult daphnids were separated into five 1L beakers with 60 daphnids each. These daphnids were used a week later in the experiment. All daphnid cultures followed the same protocol for feeding and water changes as mentioned previously.
Acute Toxicity Study of ILs on D. magna
A 48-hour acute toxicity study was carried out to examine the toxic effects of 11 and 12 on D. magna, Glass jars were filled with moderately hard water and spiked with the appropriate concentration of IL from stock solution to a total volume of 50 mL. For each IL, 5 different concentrations were studied. Control jars were set up with only 50 mL of moderately hard water. Four replicates were used for each concentration and each replicate had four daphnids. The daphnids were placed into glass jars for each concentration of ILs. All glass jars were placed in a Thermo Scientific Precision incubator (Fischer Scientific, Hampton, NH) at a holding temperature of 20°C and on a 12 hr: 12 hr light: dark cycle. Mortality in each jar was recorded every 24-hour.