Greasy Cations Bind to Neutral Macromolecules in Aqueous Solution

Ions influence the solution properties of macromolecules. Although much is known about anions, cationic effects are considered mostly in terms of weak interactions or exclusion from neutral interfaces. Herein, we have systematically studied the effect of quaternary tetraalkylammonium cations (NH4+, NMe4+, NEt4+, NPr4+, NBu4+) on the phase transition of poly(N-isopropylacrylamide) (PNIPAM) in aqueous solution. Solubility measurements were coupled to 1H NMR and ATR-FTIR spectroscopic measurements. The solubility and NMR measurements revealed a direct binding between the greasiest cations and the isopropyl group of the macromolecule, evidenced from the nonlinear, Langmuir-type chemical shift response only at the isopropyl NMR signals with increasing salt concentrations. The ATR-FTIR measurements focusing on the amide oxygen showed that it is not the main direct-binding site. Additionally, the salting-out effects of the greasier cations correlate with their hydration entropies. These results demonstrate that the most weakly hydrated cations can bind to macromolecules as strongly as the weakly hydrated Hofmeister anions.

The remaining salts were used as is without further purification.Poly(N-isopropylacrylamide) (PNIPAM) was purchased from Polymer Source Inc., with reported number average molecular mass of 118,500 Da and a PDI of 2.16.

Lyophilization of polymer samples:
Stock solutions of the polymer were prepared and refrigerated to ensure complete dissolution.Aliquots of appropriate volume were transferred into 2 mL Eppendorf tubes/microtubes and frozen in liquid nitrogen.The samples were then dried under vacuum overnight and stored in a refrigerator until usage.To prepare a particular sample for measurement, the freeze-dried polymer samples were re-dissolved in the relevant solutions.

Measurements
LCST Measurements: Lower critical solution temperature (LCST) measurements were made at minimum in triplicate, using capillary samples in OptiMelt instrument (Stanford Research Systems).LCST points were taken as the onset of the rise in detected scattering, approximated as the intersection points of the linearized curves before and after the onset (as shown below in Figure S1, left).LCST-cosolute concentration curves were fit using OriginLab 2016 nonlinear function fitting, with Levenburg-Marquadt iteration algorithm.
ATR-FTIR Measurements: ATR-FTIR measurements were conducted using a Bruker Alpha FTIR spectrometer, equipped with a custom water-circulator/copper chamber head set-up to control the temperature of the spectrometer plate, ATR crystal and sample.The circulator head was fixed over the plate during measurement, without contact with the crystal or sample after delivery.The circulator was run in this position for at least 2 hours prior to measurement to ensure the instrument temperature was stabilized at the desired value.ATR-FTIR spectra were obtained from 32 scans with frequent background spectrum measurement.precision 7″ NMR tubes, fitted with Wilmad® coaxial inserts for external referencing (2,2dimethyl-2-silapentane sulfonate sodium salt (DSS) as chemical shift reference, purchased from Sigma-Aldrich, 97% purity).The reference solution was always contained in the inner insert tube, and the measurement sample in the outer tube.The two solutions were thus measured together without any physical or chemical contact.NMR spectra were obtained from 32 scans, and corrected for phase and baseline.Exact peak points of polymer bands were determined by taking the midpoint of two points on the curve at an equal height between above the half-maximum (shown below in Figure S1, right).NMR titration curves were fit to an empirical form (Equation 2 in the main text) similar to the one used for the LCST measurements.Alternatively, mathematical fitting of polymer bands to Gaussian functions produced peak positions that agreed with the midpoint method within ~0.0001 ppm (i.e., ~1% of typical differences between samples).For the tetraalkylammonium chloride salts, although the LCST measurements were carried out in the largest range of salt concentration possible, it was observed that at the highest salt concentrations tested, the LCST profiles exhibited excessive downward curvature.2][3][4] Because this behavior is beyond the scope of our study and also incongruous with the mathematical form of Equation 1, the highest concentration data points (between one and two points) were purposefully omitted from the analysis.The range of concentrations employed for the fitting are given above in the second column of Table S1.Mathematically, it was necessary to impose a lower bound to the c parameter, which would otherwise tend to unbounded negative values in an attempt to optimize the fit.This was done by taking the tangential slope of the data between the two highest concentration points (e.g., for NBu4Cl, the slope between 0.8 -1.0 M), where the nonlinear term should have saturated and the slope should give the best approximation for the c parameter.A consequence of this treatment is that several of the c values shown in Table S1 have vanishingly small apparent errors; such errors are not statistically meaningful and thus were not included.

Figure S1 :
Figure S1: Left: Representative scattering intensity vs. temperature plot for PNIPAM in neat water, with tangent lines to the curve before and after the LCST onset drawn.The point of intersection (red dot) is taken as the LCST point of onset.Right: A snapshot representative of polymer band processing and peak determination.The 32-scan spectrum (black) is first smoothed (red) and then intersected with a horizontal line (green) close to the apex.The best estimate for the peak position is then taken as the average of the two intersection points between the line and the smoothed band curve.

Figure S2 :
Figure S2: Normalized amide I ATR-FTIR bands of PNIPAM in various D2O solutions: (a) PNIPAM in pure D2O as reference, PNIPAM in 1.0 M solution of NMe4Cl in D2O and residual (difference between curves); (b) reference spectrum, PNIPAM in 1.0 M NEt4Cl solution and residual; (c) reference spectrum, PNIPAM in 1.0 M NPr4Cl solution and residual; (d) reference spectrum, PNIPAM in 1.0 M NBu4Cl solution, and residual.

Figure S3 :
Figure S3: LCST and 1 H-NMR chemical shift titration curves for PNIPAM and NaCl, NaSCN salts.Structure of PNIPAM with color annotations of groups on the polymer is given at the top.(a) LCST of PNIPAM and NaCl, NaSCN salts up to 0.8 M. (b) -(e): 1 chemical shift curves of the PNIPAM signals, as color-coded according to the top structure, with NaCl and NaSCN salts.The legend for (a) -(e) is shared, and shown at the bottom.

Figure S4 :
Figure S4: The 1 H-NMR chemical shifts for the four PNIPAM signals (legend and colorannotated structure of PNIPAM are included at the top) as a function of concentration of (a) NMe4Cl, (b) NEt4Cl, (c) NPr4Cl, and (d) NBu4Cl.Note that the backbone -CH2-signal is absent in (c) and (d), because the signal coincides with a large salt peak and is unobservable in these cases.

Figure S5 :
Figure S5: Plots of c values obtained by fitting the PNIPAM LCST's for the series of salts to Equation 2, as shown in TableS1, as correlated against: (a) the entropy of hydration (R 2 ≈ 0.89), (b) polarizability (R 2 ≈ 0.97), and (c) ionic radius (R 2 ≈ 0.92), for the respective cations.The source for the cation-specific values is reference 5, which partitions experimental values between cation and anion pairs through explicit assumptions, some extra-thermodynamic.The ionic radii given for the tetraalkylammonium cations are their van der Waals radii.The red lines in each panel are meant as guides to the eye.

Table S1 :
Table of Fitted Parameters for PNIPAM LCST Curves Table S1 above shows the fit values for the parameters c, KD and Bmax according to Equation 1 in the main text, for LCST values of PNIPAM in salt solutions and for the range of concentrations indicated in the second column.Values in parentheses are errors.For the first two salts, the fit is purely linear and thus there are no nonlinear terms (KD, Bmax).