Enantioselective hyperporous molecularly imprinted thin film polymers

Significant enantioselective recognition has been achieved through the introduction of long range ordered and highly interconnected 300 nm diameter pores in molecularly imprinted polymer matrices.


Table of Contents
Table S1 S18 Table S2 S19

Chemicals used for synthesizing the transition state analogues
L-and D-phenylalaninamide, pyridoxal hydrochloride (≥99%), methanol and acetonitrile (HPLC grade) and d-chloroform (>99.8 atom % D) and d6-DMSO (>99.9 atom % D) were purchased from Chemtronica or Sigma-Aldrich.The L-TSA and D-TSA were both prepared according to a previously reported procedure 1 . 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy, and liquid chromatography mass spectrometry (LC-MS) were used to characterize all isolated compounds.The characterization data correspond with the previously reported one 1 .NMR spectra were recorded on a Varian 500 MHz instrument.All 1 H NMR experiments are reported in the unit parts per million (ppm), and were measured relative to the signal for residual solvent, CDCl3 (7.26 ppm).
All 13 C NMR spectra are reported in ppm relative to solvent, DMSO-d6 (39.52 ppm), unless otherwise stated, and all were obtained with 1 H decoupling.The coupling constants are reported in Hertz (Hz).

Procedures for preparing the QCM chips
Silicon dioxide (SiO2) coated Au-quartz QCM chips (obtained from Attana AB) were sonicated for 10 min in 99.5% ethanol (1 mL), and subsequently in dry acetone (1 mL).Next, the QCM chips were cleaned with "Piranha" solution (H2SO4:H2O2 3:1) for 1 min.(Caution!"Piranha" solution must be handled with extreme care since it is a hazardous oxidizing agent and reacts violently with most organic materials!)The QCM chips were sonicated for 10 min in a 0.1 M solution of NaOH in H2O (1 mL), rinsed for 10 min in the following solvents, respectively: deionized water (1 mL), dry acetone (1 mL), dry tetrahydrofuran (1 mL), and dry toluene (1 mL).
Next, the QCM chips were immersed for 16 h in a freshly prepared solution containing 3-(trimethoxysilyl)propyl methacrylate (14.4 µL), triethylamine (1.44 µL), and dry toluene (720 µL).To clean away excessive silane, the QCM chips were rinsed with a series of 1 mL volumes of solvents for 10 min each (dry toluene, dry tetrahydrofuran, dry acetone) and were subsequently dried with nitrogen gas.An O-ring (ID 7 mm), corresponding to the diameter of the silanized surface of the QCM chip, was positioned to fence the area for silanization on the surface of the QCM chip.For the polymer systems with polystyrene, polystyrene suspension (30 µL; 0.3 µm bead diameter, 10% w/w aqueous suspension) was deposited by drop coating on the silanized area of the QCM chip (i.e.within the O-ring).After that, the drop-coated surfaces were placed in a desiccator to evaporate the solvent, leaving dry polystyrene beads on the silanized surfaces.
Prepolymerization mixtures were prepared with MAA, EGDMA and AIBN as given in Table S1.Briefly, L-TSA (1.30 mg, 3.97 µmol), MAA (4.04 µL, 47.6 µmol) and EGDMA (41.6 µL, 0.221 mmol) were thoroughly mixed in n-heptane (74 µL) [The components not included in all systems are underlined].Then, AIBN (1.30 mg, 7.92 µmol) was added to this mixture and purged with N2 for 10 min.N2-purged pre-polymerization mixture (1 µL) was deposited on the polystyrene beads coated silanized QCM chip, and a cover glass was placed on top of the pre-polymerization mixture.Immediately, the QCM chip with the pre-polymerization mixture was placed under UV irradiation (50 W UV lamp, 365 nm from Labino AB) for 2 h.After that, the cover glass was removed, and the chips were rinsed for 10 min in dry toluene (1 mL), a 5 mM solution of NaOH in H2O (1 mL), and deionized water (1 mL).

Procedure for preparing the borosilicate and silicon substrates
Borosilicate glass and silicon substrates (Sigma-Aldrich) were prepared for deposition of polymer samples for Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) studies, respectively, according to the following protocol.Borosilicate glass slides and silicon wafers were put in a beaker, and immersed in the following solvents: 15 mL 30% H2O2 + 15 mL HCl (37%, fuming) + 90 mL deionized water, 80 °C, 5 min 20 mL a 25% solution of NH3 in H2O + 100 mL deionized water, 80 °C, 5 min 20 mL 30% H2O2 + 100 mL deionized water, 80 °C, 5 min 120 mL deionized water, room temperature (RT), 5 min 120 mL deionized water, RT, 5 min 120 mL dry acetone, RT, 5 min 120 mL dry acetone, RT, 5 min S9 120 mL dry tetrahydrofuran, RT, 5 min 120 mL dry tetrahydrofuran, RT, 5 min 120 mL dry toluene, RT, 5 min 120 mL dry toluene, RT, 5 min After the washing steps described above, the substrates were silanized as described above, in section S2.2.Subsequently, the systems containing polystyrene were drop-coated with polystyrene suspension (note: here two layers, i.e. 2×30 µL, of polystyrene was sequentially deposited (the second layer was deposited after the first layer had dried)), and pre-polymerization mixture was deposited on them, as described in section S2.2.The borosilicate and silicon substrates were additionally washed as described in section S2.2, with the exception that the borosilicate substrates which were drop coated with polystyrene prior to deposition of prepolymerization mixture were washed in 99.5% ethanol, subjected to FTIR analysis, and subsequently washed in the solvents listed for washing the QCM chips after polymerization (section S2.2) before recording another FTIR spectrum.The borosilicate substrates prepared without any polystyrene and the silicon substrates were only washed in the solvents described (section S2.2) for washing the QCM chips after polymerization.
The thickness of the polymer films was estimated using the following approach: For the film systems (no polystyrene included), only 1 uL pre-polymerization mixture was deposited within the O-ring.This gave, according to the formula for calculating the volume of a cylinder: V=πr 2 h the layer thickness: h= V/πr 2 The ID of the O-ring used to fence the area for deposition of polystyrene and pre-polymerization mixture on the QCM chip was 7 mm, giving the r = 3.5 mm.à h of the polymer film = 0.025984 mm ≈ 26 µm For the systems with PS beads, we have to account for the packing coefficient of spheres (the shape of the PS beads) in a cylinder (the volume fenced off with the O-ring).The packing coefficient of spheres in a cylinder is 0.74048.This gives that, when depositing 1 uL of prepolymerization mixture on a drop coated area, this will fill the remaining 0.25952 volume, which is not packed by the PS beads.This gives that the height resulting from the PS drop coated systems is the height of the thin film systems/0.25952.Thus, 0.25952 -> 0.025984/0.25952= 0.1001 mm ≈ 100 µm.
The ID of the O-ring used to fence the area for deposition of polystyrene and pre-polymerization mixture on the QCM chip was 7 mm.Herein 30 uL of polystyrene suspension in water was deposited.The liquid of the suspension was subsequently evaporated.Latex beads: 10% solids -> in 30 uL suspension, 3 uL PS beads.Thus 3 uL is the resulting evaporated volume.The height of the PS beads after evaporation of water, before accounting for the packing coefficient: h= V/πr 2 à h= 0.077853 mm.Then, the packing coefficient of cylinders is: 0.74048.Accounting for this gives the height: 0.077853/0.74048= 0.10527 mm.
The height of the evaporated PS beads, if optimal packing is assumed, is slightly higher than the height that results of the pre-polymerization mixture if it fills the space between the PS beads.Thus, we may assume that there should be some "uncovered" PS beads, which may come in contact with toluene so that they can be extracted out and form pores in the polymer.The height of the pre-polymerization mixture is thus what limits the height of the layer, i.e. ≈ 100 µm (after toluene wash).

QCM experiments set-up
The analytical performance of the polymer covered QCM chips was investigated under flow injection analysis (FIA) conditions, by placing the chips in QCM holders (provided by Attana AB), using the Attana Cell 200 instrument (Attana AB).All QCM experiments were done at 20 °C, using a 1:1 mixture of methanol and a 0.1 M solution of sodium acetate in H2O (pH 7) as the carrier solvent.This was infused by a peristaltic pump integrated in the QCM instrument with the flow rate 50 µL/min.Before doing any analyte injections, the frequency of the QCM chips in the carrier solvent was allowed to stabilize, as defined by a resonant frequency change of ≤0.5 Hz over 300 s.The analyte to be studied (150 µL) in a 1:1 mixture of methanol and a 0.1 M solution of sodium acetate in H2O (pH 7) was injected with a 250 µL gas tight syringe (ID 10.9 mm, Kloehn) using the injection valve of the QCM instrument.At least three injections were done for each analyte on each polymer system and the average value is used for FIA calibration plots.The obtained data was collected with the software Attester (version 1.5.3,Attana AB), and analyzed with the software Attana Evaluation (version 3.3.4,Attana AB).The sensitivity was obtained from the slope of the calibration plots obtained by plotting the resonant frequency changes detected when injecting the respective analytes on the polymer films, as a function of the analyte concentration.

Scanning electron microscopy
The polymer films, deposited on silicon substrates (preparation of silicon substrates with polymer films is described in sections S2.2 and S2.3) were investigated with scanning electron microscopy (SEM) using a LEO Ultra 55 instrument (Carl Zeiss AG, Oberkochen, Germany) equipped with a field emission electron gun.Initially, polymer films were sputter coated with a thin layer of palladium S11 using an LEICA EM SCD 500 sputtering unit and placed on black carbon tape attached to alumina stubs before being inserted in the SEM instrument.A 3 kV potential was applied to the electron gun to generate the electron beam used to scan the polymer particles.

Fourier-transform infrared spectroscopy
FTIR analyses were performed using an Agilent Cary 630 FTIR Spectrometer (Agilent Technologies) equipped with KBr optics and complementary diamond attenuated total reflectance (ATR) sampling accessory.The polymer coated substrates were placed on a type IIa diamond crystal, and measured with ATR.This system is configured with the Agilent MicroLab FTIR Software (Agilent Technologies) for collecting a background spectrum and a sample spectrum.The samples were recorded within 400-4000 cm -1 range with 32 scans and 4 cm -1 resolutions.

Figure S2 .
Figure S2.Scanning electron micrographs obtained for the polymer system imprinted with L-TSA, prepared in n-heptane (P4), drop-coated with two layers of 30 µL polystyrene suspension with A) lower and B), higher magnifications.

Figure S5 .
Figure S5.Histogram of sensitivity of polymer systems (Table1, TableS1 and Table S2) obtained from slope of the FIA calibration plots of various analytes on different polymer systems

Table 1 ,
TableS1 and Table S2) obtained from slope of the FIA calibration plots of various analytes on different polymer systems

Table S1 .
Composition of the prepared polymer systems.

Table S2 .
The sensitivity of the studied polymer systems (obtained from the slope of the calibration plots) for all investigated analytes.Value in parenthesis is the correlation co-efficient of the sensitivity.