Chiroptical Enhancement of Chiral Dicarboxylic Acids from Confinement in a Stereodynamic Supramolecular Cage

The fundamental implications that chirality has in science and technology require continuous efforts for the development of fast, economic, and reliable quantitative methods for enantiopurity assessment. Among the different analytical approaches, chiroptical techniques in combination with supramolecular methodologies have shown promising results in terms of both costs and time analysis. In this article, a tris(2-pyridylmethyl)amines (TPMA)-based supramolecular cage is able to amplify the circular dichroism (CD) signal of a series of chiral dicarboxylic acids also in the presence of a complex mixture. This feature has been used to quantify tartaric acid in wines and to discriminate different matrixes using principal component analysis (PCA) of the raw CD data.


General Methods
NMR spectra were recorded at 301 K on Bruker 400 Avance III BBi-z grad 5 mm and Bruker Avance-III 500MHz instruments. All the 1 H-NMR spectra were referenced to residual isotopic impurity of DMSO-d 6 (2.50 ppm). The following abbreviations are used in reporting the multiplicity for NMR resonances; s=single, d=doublet, t= triplet, and m=multiplet. The NMR data were processed using Bruker Topspin 3.5 pl2 and MestReNova 12.0.0. High resolution electrospray ionization mass spectrometry HRMS (ESI-TOF) analyses were performed in positive mode with Waters Xevo G2-S QTof. The analysis was performed with Fast Flow Injection: 10 μL of sample injected ACN at 30 ul/min. Capillary: 3000V, Sample cone: 30V, Source temperature: 80 °C, Desolvation temperature: 250 °C. ECD spectra were recorded with a Jasco J-1500 spectrometer and processed with Spectra Manager Version 2.15.3.1 or OriginPro 2018 (64-bit) SR1 b9. 5.1.195. The computational searches of the most stable structures and the TD-DFT calculations were carried out Gaussian 16 package 1 Revision C.01 and processed with GaussView 6.0.16 2 or CYL view BETA 1.0. The experimental/theoretical comparison of the CD spectra were carried out with SpecDis Version 1.71 3 . Chemicals were purchased from Merck, TCI, or Apollo Scientific and used without further purification.

CD measurements
CD measurements were performed diluting with anhydrous DMSO the synthesized cage to obtain a final concentration equal to 1.0·10 -5 M (0.1 cm cuvette). The CD spectra were measured in millidegrees, normalized for the concentration of the cage and reported as [], following the formula: Where [] is the molar ellipticity,  is the CD value registered from the instrument (expressed in mdeg), C is the concentration of the sample, expressed in (mol/L) and l is the optical path (expressed in cm).

Conformational Analysis
Manual conformational search was run with Gaussian 16 package (DFT B3LYP/6-31G(d)), with default grids and loose convergence criteria 4 . The guests chosen for the computational investigation are the D-Tartaric acid and L-malic acid. Three stereogenic elements have been varied to generate eight different starting structures for the conformational search. In particular: i) the TPMA helix helicity sense ( Figure S3), ii) the dihedral angle between the pyridine ring and the imine ring ( Figure S4), iii) the dihedral angle of the imine bond relative to the aromatic ring ( Figure S5).

Figure S4
Dihedral angle formed between the pyridine-ring of TPMA and the phenyl-ring: example of positive angle on left (+) and negative angle on right (-). Hydrogen atoms are removed for clarity. Figure S5. Orientation of the C=N bond of the imine group respect the C-N highlighted on the TPMA pyridine ring: C=N forms an angle >|90°| (left); C=N forms an angle <|90°| (right). Hydrogen atoms are removed for clarity. Table S1. Classification of conformations assumed by cages stereoisomers: the propeller direction of TPMA, Λ (counterclockwise), Δ (clockwise); the sign of dihedral angle formed between the pyridine-ring of TPMA and the phenyl-ring (+ for a positive angle, -for a negative angle); the orientation of the C=N bond of the imine group respect the C-N bond on the TPMA pyridine ring (angle <|90°| or >|90°|). Table S3, relative energies for the eight different conformers clearly indicate a preference of the cage to adopt mainly two conformations. These two conformations, namely I and V are the lowest in energy for both the diacid under investigation. These two conformations were subsequently used to vary the conformations of the diacid within the cage. S11

Dicarboxylic Acid Coordination
The search for the more stable cage conformation was then followed by a conformational analysis over the dicarboxylic acids, trying to expand the number minima explored on the potential energy surface. Due to the large energy difference of the two conformations I and V among the others, this search was done only with these two conformations of the cage. In Figure S6 and Figure

Meso Structures
In the last part of the computational search structures with opposite helicity of the two TPMA zinc complexes have been considered (pseudo-meso). The dihedral angle between the pyridine ring and the phenyl ring ( Figure  S4) were also manually varied while the dihedral angle of the imine bond relative to the pyridine ring ( Figure  S5) was kept <|90°| since the most stable conformers calculated so far had this stereoconformation. The most stable dicarboxylic acid coordination was used for the calculation, i.e. c) for the D-Tartaric acid ( Figure S6) and e) for the L-Malic acid ( Figure S7). The classification of the different meso conformers is shown in Table  S2.

Table S2
Classification of the four meso conformers computed: the propeller direction of TPMA, Λ (counterclockwise), Δ (clockwise) and the sign of dihedral angle formed between the pyridine-ring of TPMA and the phenyl-ring (+ for a positive angle, -for a negative angle). The dihedral angle of the imine bond relative to the pyridine ring was kept <|90°|.
The conformational search over the four meso structures reveal the presence of other two good conformers, namely the IX and XI (Table S3). For both the diacid tested, they resulted close in energy to the best stable structures previously found. S13

Energy calculation of the most stable conformers
Sixteen different conformations were obtained by changing the three stereogenic elements of the cage and the diacid coordination. Relative energies are calculated by the difference from the most stable conformation energy. D-Tar@1 conformers summarized in Table S3 and L-Mal@1 summarized in Table S4. Highlighted in green the lowest energetical conformation and in yellow the second and third best. a) : two coordination of the malic acid are possible, the left one with the hydroxyl group pointing the Δ conformation of the zinc complex, the right one pointing the Λ. Table S3 and Table S4 summarize the conformational search conducted for the supramolecular cages D-Tar@1 and L-Mal@1. As it is possible to see, there are some conformations relatively close in energy and others considerably far from the best one. To consider that the energy difference is expressed in kcal/mol, and therefore a small value difference determines a relatively large difference in the Boltzmann population. As example, considering two best structures with an energy difference equal to 0.5 kcal/mol, their corresponding population will be 70% for the best conformation and 30% for the second one.
In the case of L-Mal@1, two structures resulted significantly close in energy (Entry 15 and 16). Noteworthy, these two conformations are characterized by an opposite TPMA helicity and thus opposite CD spectra. As a result, the contemporary presence of both the conformations in solution, could be the explanation of the low intensity of the experimental CD signal measured for this system.

TD-DFT Calculations
Once obtained the energy distribution of the conformers, TD-DFT B3LYP/6-31G(d) calculations were performed to simulate the CD spectrum. The calculations were carried out over 50 excited states for the three best conformational structures found for the supramolecular systems D-Tar@1 and L-Mal@1 and Boltzmannweighted ECD spectrum was then obtained with SpecDis software, 5 by using the population data shown in Table S5 and the bandwidth σ that gives the best superimposition of the spectra. To do this, the program maximize the similarity factor S, a parameter proposed by Bultinck 6 based on the cosine similarity equation.   The standard deviation has been calculated using the equation: The abscissa intercept is equal to -0.095 ± 0.020 mM, corresponding to a Tartaric acid content equal to 2.28 ± 0.13 g/L.            Due to the slow exchange regime, the determination of binding constant was possible thanks to the direct integration of the signals of the pyridine ring α proton of the filled cage and the empty cage in the region between 9.5 ppm and 8.5 ppm using equation:  Table S6. For the CD analysis, 200 μL of the mixture were diluted to 1 mL with dry DMSO and analyzed. As complex mixtures, 3 red wines, 3 white wines, 3 Apple juices, 3 Pear juices, 3 Blueberry juices, 1 orange juice, and 1 lemon juice have been employed. All the different mixtures were purchased at the local market in Padova.

ESI-MS spectrum L-Tar@1
The CD spectra were processed with Spectra Manager Version 2.15.3.1 and transformed into an xy coordinate sheet file. The non-vanishing portion of the spectra relative to the interval between 285 -335 ppm were selected and analyzed with OriginPro 2018 (64-bit) SR1 b9.5.1.195. The data set was represented by a matrix , where the rows represent the number of points acquired during the * measurement and the columns represented the experiments characterized by a different complex mixture. In detail, the matrix was composed by a =101 rows relative to the points of the spectra and =13 columns relative to the number of complex mixtures utilized. Once the matrix was created, the Principal Component Analysis (PCA) was performed through the PCA for Spectroscopy script implemented into Origin which performs principal component analysis for spectra (IR, Fluorescence, UV-Vis, Raman, etc.). This tool allows to easily obtain a loading score as a plot in function of the PC1 (or PC2) and the wavelength of the spectra. In the case of PCA obtained from spectra data this kind of loading score result much clear and informative since the high number of the data points make the usual loading score (PC1 vs PC2) too crowded and complicated to understand. The resulting PCA bidimensional plots were obtained from the score and coeff matrix from the pca function, considering the first and second principal components which account for the 99.6% of the total variance of the data set.  Figure S47 CD spectrum of cage R@1 formed using Lemon squeezed juice as complex mixture.

Quantification of Dicarboxylic acids Content in the Complex Mixtures for the PCA Analysis
In order to determine the concertation of Tartaric and Malic in the complex mixtures, the concentration of cages L-Tar@1 and L-Mal@1 has been determined using p-xylene as internal standard.