Beyond Stereoisomeric Effects: Exploring the Importance of Intermolecular Electron Spin Interactions in Biorecognition

This work shows that electron spin polarization and stereoisomeric effects make comparable contributions to the enantioselective binding of amino acids. Magneto-electrochemical quartz crystal microbalance methods are used to study the adsorption of chiral amino acids onto a monolayer film of chiral molecules that are spin polarized by an underlying ferromagnetic substrate. The direction of the electron spin polarization affects both the kinetics and thermodynamics of the enantiospecific adsorption of the amino acids. Comparison of these data with the circular dichroism (CD) spectra of the amino acid adsorbates shows that the CD spectrum of the interacting group provides a good figure-of-merit for predicting the contributions of electron spin to the intermolecular interaction. These findings demonstrate the importance of electron spin in enantioselective intermolecular interactions between chiral amino acids and represent a paradigm shift for how selectivity should be viewed in biorecognition.

The mEQCM experiments were performed using a 7.9995 MHz quartz crystal in an EQCM cell attachment and a 430A potentiostat (CH Instruments). The surface area of the crystal is 0.205 cm 2 and is coated with 100 nm of nickel and 10 nm polycrystalline gold as the working electrode area (CH Instruments). The counter electrode was a Pt wire and the reference electrode was a saturated Ag/AgCl electrode. Magnetic field studies placed a permanent magnet, 0.54 T (K&J Magnetics) underneath the working electrode during the experiment.

Self-assembled Monolayer Formation and Stability measurement.
The quartz crystal working electrodes were incubated in a 50 mM solution of L-NAC (or D-NAC) in 0.1 M phosphate buffer at pH 9 overnight. Then the electrode was rinsed three times with phosphate buffer and water to remove any loosely, physisorbed material. The electrodes were then blown dry with a stream of Argon. Linear sweep voltammetry (LSV) and QCM measurements were used to study the stability of the L-and D-NAC SAMs. Here, the SAM coated quartz crystal working electrode was incubated in a 0.1 M phosphate buffer (pH 9) electrolyte solution for 30 min, to allow the QCM frequency to stabilize. Next the electrode was scanned from 0 V to -0.8 V versus saturated Ag|AgCl at a scan rate = 25 mV/s and the change in mass was monitored.
Adsorption study with mEQCM.
The SAM coated quartz crystal was first incubated in a 0.1 M phosphate buffer (pH 9) electrolyte solution containing a 150 µM of an amino acid and allowed to equilibrate for 30 minutes. Next, linear sweep voltammetry and QCM measurements were performed to determine the desorption and re-adsorption of the amino acid onto the NAC SAM coated film; the data were collected by scanning from -0 V to -0.8 V versus saturated Ag|AgCl at a scan rate = 25 mV/s. Upon determination of the potentials required for adsorption and desorption, the SAM coated quartz crystal was incubated for an additional 30 min to allow the system to equilibrate and then chronoamperometry experiments were performed. Take for example L-LeuME; An initial potential of -0.4 V corresponding to reductive adsorption of L-LeuME was applied and then a more positive potential, 0 V, corresponding to oxidative desorption. The applied voltage was jumped between these two values and the QCM response was collected as a function of time after the potential jump. A 5 s pulse width was applied during the experiment and >150 cycles were performed to calculate the average mass change for thermodynamic analysis and to build up the statistics for histogram plots to perform a kinetic analysis of the adsorption and desorption process. It is important to stress that the measurements made using North and South magnetizations occur on the same NAC film and therefore any imperfections in the quality of the SAM should not influence the observed spin effects.

Optical Measurements.
Absorbance spectroscopy was performed using an Agilent model 8453 spectrometer and Circular dichroism (CD) spectra were measured using a JASCO J-810 CD spectrometer with a scan rate of 20 nm/min and a bandwidth of 1 nm. For the CD measurements each sample was scanned three times and the average of the three curves is reported. The absorbance and circular dichroism data were collected in a 2 mm quartz cuvette containing 2 mM phosphate buffer (pH 9) and 0.5mM concentration of amino acid.

Section 2 -Stability of NAC assemblies
Linear sweep voltammetry measurements were performed to determine the potential required for desorption and re-adsorption of LeuME enantiomers from the NAC coated Ni/Au films. First, the stability of L-NAC and D-NAC coated Ni/Au film was studied. When scanning negatively from 0 V to -0.8V, a cathodic desorption peak occurs at around -0.65 V for both L-NAC and D-NAC ( Figure S1A), and the mass begins to decrease from -0.45 V ( Figure S1B). These results indicate that the L-NAC and D-NAC are stable if the applied potential is more positive than -0.4 V.

Section 3 Determination of Adsorption and Desorption Behavior of LeuME on NAC
To determine the adsorption of LeuME enantiomers onto NAC coated Ni/Au films the potential was scanned from 0 V to -0.8V and the change in mass studied. As the potential was swept, a mass increase process followed by a mass decrease process was observed ( Figure S2B & E). This behavior is attributed to cathodic adsorption of LeuME enantiomers onto the L-NAC SAM surface (0 V to around -0.4V) that is followed by a complete cathodic desorption of L-NAC with LeuME (-0.4V to -0.8V). Interestingly, more L-LeuME adsorbs onto the surface under a North magnetic field (red) than a South magnetic field (blue); See Figure S2 C. Conversely the opposite behavior manifests for D-LeuME ( Figure S2F). Also, the desorption peak of L-NAC with LeuME exhibits a dependence on the enantiomeric form of LeuME and the magnetic field direction; L-LeuME on L-NAC shows a more negative desorption peak under a North magnetic field, indicating a stronger binding onto Ni/Au film, whereas D-LeuME on L-NAC shows the opposite dependence on magnetic field. These results indicate that the charge delocalization across the NAC-LeuME composite is spin-dependent and enantiospecific. Fig. S3 shows complimentary measurements on D-NAC SAMs, and the result is opposite to that on L-NAC SAMs.  Figure S4A shows representative data for the chronoamperometry measurements; the top panel shows the potential sequence and the bottom panel shows the corresponding frequency response.

Section 4 Chronoamperometry Measurements and Data Analysis
To extrapolate the rate constant data from the frequency response, the following calculations were performed. First, the adsorption process was assumed to follow a simple Langmuir model and thus,the adsorption rate can be written as Eq. (1) Eq. (1) where is the concentration of LeuME in solution, θ is the concentration of adsorbed molecule on the surface, is the adsorption rate constant and is the effective adsorption ′ rate constant. To quantify the adsorption rates, the time responses of the QCM frequency were fit to an exponential decay equation for adsorption ( Figure S4B top panel), where = • -/ 1 + 0 A, , and were adjusted for a best fit to the data. Then the effective adsorption rate constant 0 1 S7 was calculated as . A histogram with >150 , determined through this method was ′ = 1 | 1 | ′ then built and fit to a Gaussian distribution to obtain the average and standard deviation of the mean for the adsorption rate constant ( Figure S4C top panel).
The desorption rate can be written as Eq. (2) Eq. ( where θ is the concentration of adsorbed molecule on the surface and is the desorption rate constant. The time responses of the QCM frequency were fit to an exponential growth equation for desorption (Fig, S4B bottom panel), where A, , and were adjusted for a = • / 2 + 0 0 2 best fit to the data. Then, the desorption rate constant was calculated as . A histogram = 1 | 2 | with >150 , determined through this method was then built and again fit to a Gaussian distribution to obtain the average and standard deviation of the mean for the desorption rate constant ( Figure S4C bottom panel). (bottom) processes under a north magnetic field (red) and a south magnetic field (blue). A best fit of the data using a Gaussian distribution is shown as a solid curve.
To quantify the mass change during the adsorption (desorption) process, eighty QCM frequency responses were selected at random and the average change in frequency during the 5s adsorption (desorption) process was quantified. A frequency shift of -1.0 Hz corresponds to a mass change of 1.4 ng based on the Sauerbrey equation and characteristics of our EQCM set-up. To determine the mass change, the data were fit to an exponential growth equation, (Fig. S4), in = • / 1 + 0 which A, , and τ 1 and were adjusted for a best fit to the data, allowing the coverage after 5s to be 0 calculated. Fig. S5 (A) -(C) replots the average mass change, reported in Fig. 3 (A)-(C) in the main text, for L-LeuME adsorption on L-NAC, MPA, and D-NAC, and the complimentary measurements on the same substrates for D-LeuME are plotted in Figure S5(D)-(F).  (red) and South (blue) magnetic field. In all three SAM configurations the adsorption rate constant for D-LeuME is faster when the magnetic field is oriented South rather than North. The change in mass of D-LeuME at 5s, after the kinetically controlled adsorption process is shown in Figure  S6D. Values for the mass change in Figure S6D and Figure 3 in the main text are reported in Table  S1. Homochiral ensembles exhibited the largest average mass (L-LeuME on L-NAC, and D-LeuME on D-NAC). Heterochiral ensembles exhibited the smallest average mass (L-LeuME on D-NAC, and D-LeuME on L-NAC). Achiral SAMs were intermediate. Analogous adsorption rate constant measurements and mass changes occurring during the desorption process are shown in Figure S7 and S8. for L-LeuME and D-LeuME, respectively, on L-NAC, MPA, and D-NAC SAMs. A summary of the polarization in adsorption rate constant for LeuME desorption is shown in Figure S9A and complimentary measurements on phenylalanine, Phe, for adsorption is shown in Figure S9B.   Ni/Au films under North magnetic field (red) and South magnetic field (blue). A best fit of the data using a Gaussian distribution is shown as a solid line.

Section 6 Control Experiments on Gold
Control experiments were performed for the adsorption of L-LeuME and L-Phe onto L-NAC SAM coated Au films, which are not ferromagnetic, under North and South magnetic fields; See Fig.  S11. The polarization in adsorption rate constant for L-LeuME is 0.94 ± 0.64% and for L-Phe is 1.09 ± 0.58%, indicating the adsorption behavior of a chiral molecule onto a nonmagnetic substrate is the same under the North and South magnetic field. Thus, the enantiospecific adsorption is not arising from the magnetic field per se, but instead from the CISS-mediated spin dependent exchange interactions at the ferromagnetic surface.