A High-Resolution Magic Angle Spinning NMR Study of the Enantiodiscrimination of 3,4-Methylenedioxymethamphetamine (MDMA) by an Immobilized Polysaccharide-Based Chiral Phase

This paper reports the investigation of the chiral interaction between 3,4-methylenedioxy-methamphetamine (MDMA) enantiomers and an immobilized polysaccharide-based chiral phase. For that, suspended-state high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (1H HR-MAS NMR) was used. 1H HR-MAS longitudinal relaxation time and Saturation Transfer Difference (STD NMR) titration experiments were carried out yielding information at the molecular level of the transient diastereoisomeric complexes of MDMA enantiomers and the chiral stationary phase. The interaction of the enantiomers takes place through the aromatic moiety of MDMA and the aromatic group of the chiral selector by π-π stacking for both enantiomers; however, a stronger interaction was observed for the (R)-enantiomer, which is the second one to elute at the chromatographic conditions.


Introduction
In chromatography, separation mechanisms are based not only on the solvated structure of the analyte and of the stationary phase but also on their tridimensional structure; therefore, enantiorecognition is a result of the sum of all these different interactions. Pinpointing these interactions at the molecular level is, however, an analytical challenge. In this context, saturation transfer difference nuclear magnetic resonance (STD NMR) spectroscopy has been successfully employed as a key technique to differentiate interactions at the supramolecular level [1]. For STD experiments, two 1 H NMR spectra are acquired. The first spectrum corresponds to the 'saturation on-resonance' (STD on ), in which the chiral stationary phase (CSP) 1 H signals are selectively excited using a narrow NMR pulse that does not excite the enantiomer under analysis (the excitation is transferred throughout the CSP proton network by a mechanism called spin-diffusion, which is common for macromolecules). The second spectrum is a complementary control experiment and corresponds to the 'saturation off-resonance' (STD off ), with a selective excitation applied to a point far from these CSP 1 H signals. The difference spectrum (STD diff ) is simply a subtraction of both spectra, and all STD data are reported showing two spectra; STD diff and STD off [1].
This approach can easily differentiate the interactions of each enantiomer in the transient diastereoisomeric complexes formed by the analyte and the chiral selector. For that, suspended-state high-resolution magic angle spinning nuclear magnetic resonance spectroscopy ( 1 H HR-MAS NMR) can be used to mimic the chromatographic column conditions [2,3].
Among the wide variety of chiral stationary phases (CSPs), polysaccharide-based coated phases exhibit a broad range of applicability to a variety of classes of compounds; however, there is a limited number of solvents that can be used without dissolving or swelling the chiral selector, and thus damaging the columns [4]. These limitations can compromise the analytical and preparative efficiencies mainly for larger scale preparative separations in which good solubility of the analyte is required to achieve high productivity. In the last three decades, the immobilization of polysaccharide derivatives on chromatographic supports has emerged as a major improvement in the technology and can help to solve this type of problem, allowing for a greater range of solvents to be used as eluent [4]. These chiral phases, however, need further mechanistic studies to adequately understand their chiral recognition ability under liquid chromatographic (LC) conditions.
To probe their chiral separation ability by 1 H HR-MAS NMR, we selected the enantiomers of 3,4-methylenedioxy-methamphetamine (MDMA- Fig 1). Despite the large number of reported separations for chiral secondary amine derivatives, the enantioresolution of MDMA is a difficult task.
To date, derivatives of cyclodextrin have been the chiral selectors that yielded the best resolution of MDMA enantiomers by capillary electrophoresis and LC [5][6][7][8][9][10][11]. Moreover, MDMA is a chiral synthetic amphetamine used as a recreational racemic drug commonly known as "ecstasy" [12][13][14]. Toxicological studies have shown that the (S)-form is more active than the (R)-form in terms of "the degree and the disruptiveness of the induced intoxication" [9,12]. To proceed with toxicological studies, better analytical and multimilligram chiral separations are deemed necessary [11].
To meet this goal, the amylose tris(3-chlorophenylcarbamate) immobilized CSP yielded a high resolution at the analytical and semi-preparative LC scale, justifying the selection of MDMA as a probe for this CSP.
To throw light on the enantiorecognition mechanism of immobilized amylose based CSP towards the MDMA enantiomers, herein we fully discuss results obtained by 1 H HR-MAS NMR. Furthermore, HR-MAS STD NMR experiments were used to determine the dissociation constant (K D ) between the MDMA enantiomers and the used CSP. Competing Interests: Co-author Tiago C. Lourenço is employed by Apex Science, a commercial company. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Chromatographic conditions
The enantiomeric separation of MDMA was carried out by elution chromatography with stacked injection under mass overload condition using a mixture of acetonitrile and DEA (0.1%) as mobile phase at a flow rate of 5.0 mL.min -1 . The preparative conditions were obtained after scaling up from the analytical conditions. MDMA was dissolved in the mobile phase solution at a concentration of 10 mg.mL -1 . An injection volume of 0.5 mL of this solution was used, with a total of 27 injections in two cycles. The preparative HPLC system consisted of a Shimadzu LC-6AD pump, a Rheodyne 7725-018 injector fitted with a 500 μL loop, and a 10 AVvp variable wavelength UV-vis detector (λ = 270 nm) with a CBM SCL-10 Avp interface. Data acquisition was performed using the CLASS-VP software. The elution order was determined by optical rotation of the solutions (10 mg.mL -1 ) of the isolated enantiomers in either ethanol or water, (S)-(+)-and (R)-(-)-enantiomers. It is important to mention that the sign of the optical rotation of the enantiomers was inverted when determined in the chromatographic mobile phase (acetonitrile), compared to the ethanol and water solutions.

NMR conditions
All NMR experiments were carried out at 400 MHz on a Bruker Avance III spectrometer equipped with a 1 H HR-MAS probehead. The spectra were obtained using 1 mg of CSP-ID. All the stock and diluted solutions were prepared in ACNd 3 /DEA (100:0.1, V/V). The water signal at δ % 2.0 ppm was suppressed. Samples were prepared in 4 mm ZrO 2 rotors with a detection volume of 12 μL. The spectra were recorded at a spinning rate of 4kHz, at 298 K, under magic angle, properly adjusted by using anhydrous KBr. 1  were calculated by the following expression [15]: Where α STD is the maximum amplification factor. The K D and α STD values were obtained by linearization of the Eq 1, accordingly to a Lineweaver-Burk plot using QtiPlot software. For that, the yaxis corresponded to 1/A STD and x-axis to 1/[MDMA].

Results and Discussion
For 1 H HR-MAS NMR experiments, first it is necessary to obtain the enantiomers in a high degree of enantiomeric purity. The MDMA enantiomers (135 mg) were separated by a total of 27 continuous injections (2.64 h), providing a throughput of almost 577 mg.d -1 for the enantiomers. The first (S)-enantiomer (63.1 mg) and the second (R)-enantiomer (64.0 mg) were both obtained in recoveries higher than 93%. Enantiomeric purity higher than 99.9% was obtained for both enantiomers (Fig 2). The 1 H HR-MAS NMR spectrum of the CSP-ID at the chromatographic mobile phase used is illustrated in Fig 3. The signal broadening at 6 and 8 ppm is due to the limited molecular mobility causing a short T 2 Ã . As in the coated polysaccharide phases, hydrogen bonding, π-π, dipole-dipole stacking, and steric inclusion are the most likely interactions between the chiral selector and the enantiomers during the separation process.
The NMR spectra obtained for the (R)-enantiomer (A) and (S)-enantiomer (B) and the racemic mixture (C) in the presence of CSP-ID are illustrated in Fig 4. The spectrum of the racemic mixture (Fig 4C) shows the chemical shift for the protons located at H1 (δ = 2.55 ppm), H2 (δ = 1.14-1.13 ppm), H3 (δ = 2.65-2.80 ppm), H4 (δ = 2.91-3.31 ppm), H5 (δ = 6.60-6.71 ppm), H6 (δ = 6.79-6.71 ppm), H7 (δ = 6.75 ppm) and H8 (δ = 5.96 ppm), as assigned at the MDMA chemical structure (Fig 1). Changes in chemical shift can be perceived for the protons located at position H2, H3 and H4 when the spectra of Fig 4 are compared. These changes are better observed in the expanded spectra of both enantiomers shown in Fig 5. Major differences in chemical shift are observed for the (R)-enantiomers when compared to the other two spectra (B and C). This is an indicative that the (R)-enantiomer strongly interacts with CSP.
The T 1 ratio values [16] of the (S)-and (R)-enantiomers in homogeneous solution and in CSP-ID suspension are summarized on Table 1. The T 1 values show that the spin-lattice   relaxation times are shorter for the enantiomers in the diastereoisomeric complex, especially for the protons of the (R)-enantiomer, compared to analogous relaxation times for the dissolved species, indicating a binding affinity towards the CSP-ID. These results indicate that there is a difference in the dynamic behavior of the enantiomers when bound to CSP-ID, suggesting that the configuration of (R)-MDMA is the one that allows the closest interaction to the chiral selector, decreasing its correlation time. The ratio of T 1 values obtained at 4 mg of CSP-ID in comparison with the values obtained at 1 mg (Table 1) is significantly lower for all the protons, and somehow the values are closer to the (S/R) ratio in solution. This effect can be probably caused by a shift in the equilibrium of the diastereoisomeric complex favoring the less retained (S)-enantiomer, and losing enantioselectivity.
T 1 values have been already used to designate the most retained enantiomer [3,2,16].

STD Experiments
The 1 H STD HR-MAS NMR spectra essentially reveal the analyte's proton signals closer to the sorbent, since they are more sensitive to magnetization transfer. As a result, the proton with the strongest interaction with the stationary phase gives the most intense signals [17]. It was not possible to observe the STD signals at short saturation times (0.5 to 2.5 s). Thus, the value of 3.0 s was selected for the STD experiments. The comparison of the spectra of Fig 6A and 6B with the spectra at of Fig 6C reveals that the later has a new set of proton signals corresponding to H2 (-CH 3 ), H5-H6 (aromatic), and H8 (-CH 2 ). In the spectrum of (S)-MDMA-CSP-ID (Fig 6B), the aromatic proton signals are of lower intensity when compared to those of (R)-MDMA-CSP-ID (Fig 6A).
The STD signals were normalized with respect to the registered signal of highest intensity (ACNd 3 -solvent signal), which was assigned as 100%. The percentage signal intensities are given in Fig 7. The protons at the positions H8 (45.7%) and H6 (43.7%) of the (R)-MDMA enantiomer showed the highest interactions, corroborating the T 1 values as well as, the obtained chromatographic retention factors. Thus, it can be assumed that the aromatic ring is the most important moiety for the molecular enantiorecognition process.
Here it is worth noting that the chiral selector is based on 3-chlorophenyl derivatives that furnish a more effective π-π interactions.

Determination of K D
Chromatographic chiral recognition is a result of energy differences between the formations of the two transient diastereomeric complexes. Conceptually, it is similar to enantiomeric differentiation in biological process [18]. STD experiments have been commonly employed to determine K D values of the protein-ligand complexes by measuring the equilibrium concentration of the free and bound species [19]. The isotherms fitted from the STD experiments (Fig 8) for both enantiomers were used to calculate K D ; from these experiments, it was possible to estimate the equilibrium constants (K on /K off ) involved in the formation of the transient diastereoisomeric complex of (R)-MDMA and CSP-ID. It is important to note that the STD NMR titrations are strongly dependent of experimental conditions such as the, saturation time, relative concentration of the species, and monitored protons [19].
The results herein described extend the applicability of STD NMR spectroscopy for unrevealing the equilibrium constant for the weak interactions involved in enantioselective chromatography. As far as we could ascertain, this is the first time such experiments have been used to determine K D for a transient diastereoisomer, which is of particular relevance in designing new chiral selectors for liquid chromatography.
The difficulty to estimate K D for the first enantiomers ( Fig 8A) is due to the variability of the STD data at low concentrations. This behavior can be related to the poor integration of the proton signals in the spectra, even though at 256 scans. In addition, the MAS conditions and  the heterogeneity of the sample contribute to the poor estimation of K D especially for the (S)enantiomer, which is the one with the weakest interaction. The mean (n = 3) K D value for the (R)-enantiomer was of 1.52 ± 0.94 mmol.L -1 , calculated from the plot illustrated in Fig 8B. The low K D value obtained for the (R)-enantiomer implies a weak interaction [1] in the formation of the diastereoisomer, even though, it reflects the obtained elution order and corroborates the difficulty in measuring the K D value for the (S)-enantiomer.

Conclusions
The CHIRALPAK-ID stationary phase was fully able to separate MDMA enantiomers by stack injections under mass overload conditions with high throughput, yielding the enantiomers with high enantiomeric purity for the HR-MAS-NMR experiments. Amongst the MAS procedures, the STD NMR experiment was the one that provided more information about the molecular interactions of the MDMA enantiomers with the CSP-ID. As disclosed by these experiments, the interaction of the enantiomers takes place through the aromatic moiety of the MDMA and the aromatic group of the chiral selector (3-chlorophenyl group) by π-π stacking, among others. It is important to note that, although both enantiomers can be discriminated through this moiety, a strong interaction is observed for the (R)-enantiomer, thus justifying the elution order.