# Exploring Naproxen Cocrystals Through Solid-State Vibrational Circular Dichroism

Adam Sklenář, Anne Zehnacker-Rentien, Jakub Kaminský, Jan Rohlíček, Petr Bouř

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Adam Sklenář, Anne Zehnacker-Rentien, Jakub Kaminský, Jan Rohlíček, Petr Bouř. Exploring Naproxen Cocrystals Through Solid-State Vibrational Circular Dichroism. Chirality, 2025, 37 (3), pp.e70027. 10.1002/chir.70027. hal-04966062

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Exploring Naproxen Cocrystals through Solid-State Vibrational Circular Dichroism

Adam Sklenář,¹,² Anne Zehnacker-Rentien,³ Jakub Kaminský,¹ Jan Rohlíček,⁴ and Petr Bour¹,²*

¹ Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo nám. 2, 166
10 Prague, Czech Republic

² Department of Analytical Chemistry, University of Chemistry and Technology, Technická 5, 16628
Prague 6, Czech Republic

³ Institut des Sciences Moléculaires d'Orsay, CNRS, Université Paris-Saclay, 91405 Orsay, France

⁴ Institute of Physics, Academy of Sciences, Na Slovance 2, Prague, Czech Republic

* E-mail: bour@uochb.cas.cz
Abstract

Vibrational circular dichroism (VCD) spectroscopy appears as a useful method for characterizing optically active substances in the solid state. This is particularly important for active pharmaceutical ingredients. However, measurement and interpretation of the spectra bring about many difficulties. To assess the experimental and computational methodologies, we explore an anti-inflammatory drug, naproxen. Infrared (IR) and VCD spectra of the pure compound and its cocrystals with alanine and proline were recorded, and the data interpreted by quantum chemical simulations based on a cluster model and density functional theory. Although unpolarized IR spectroscopy can already distinguish pure ingredients from cocrystals or a mixture, the VCD technique is much more sensitive. For example, the naproxen carboxyl group strongly interacts with the zwitterionic alanine in the cocrystal via two strong hydrogen bonds, which results in a rather rigid structure crystallizing in the chiral P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub> Sohncke group and its VCD is relatively strong. In contrast, the D-proline and (S)-naproxen cocrystal (P2<sub>1</sub> group) involve a single hydrogen bond between the subunits, which together with a limited motion of the proline ring gives a weaker signal. Solid-state VCD spectroscopy thus appears useful for exploring composite crystal structures and interactions within them, including studies of pharmaceutical compounds.
Introduction

Solid state is a very convenient form of drug delivery.¹ It can circumvent the low solubility of active pharmaceutical ingredients (API), improve their bioavailability or stability.² For example, from class II of the Biopharmaceutics classification system containing low-solubility drugs, 30% of all marketed products are in the solid state.³ Structural characterization of such compounds is essential to ensure their proper activity, safety, and regulatory approval.

For the crystal structure determination, diffraction methods including powder diffraction remain the gold standard.⁴,⁵ Solid-state nuclear magnetic resonance (NMR) spectroscopy does not provide atomic resolution, but offers insight into the chemical composition and molecular packing as well.⁶ The construction of the crystal structure from such data is sometimes referred to as NMR crystallography.⁷ One can also use cryo-electron⁸ or transmission electron microscopies.⁹

Compared to these techniques, infrared absorption (IR) and vibrational circular dichroism (VCD) offer a relatively simple, fast, and inexpensive experiment, still sometimes surprisingly sensitive to the structure and composition. The (in principle) non-destructive nature of IR techniques enables the treatment of expensive samples, or of those available in small quantities. Unlike for spectroscopies using visible light, almost all compounds provide absorption bands useful for solid-state IR studies.¹⁰⁻¹² The chiral extension of IR, VCD, is in addition sensitive to chirality, detecting differential absorption of the left and right circularly polarized light.¹³ Since more than 50% of all drugs are optically active, this technique can be used for a large segment of APIs.¹⁴

In general, VCD is also more sensitive to the structure than IR.¹⁵,¹⁶ The price to pay is the weakness of the differential signal. The typical ratio of VCD to IR is about 10⁻⁴, which makes the experiment prone to artifacts. Solid-state VCD (ssVCD) experimental procedures thus involve additional steps minimizing the false signals in the spectra. For example, it is very convenient when both enantiomers of investigated systems are available for the measurement.¹⁷ It has been shown that ssVCD can distinguish crystal polymorphs,¹⁸ hydrates from dried API,¹⁹ different solvates²⁰ or pure compound from a cocrystal.¹⁷,²¹

For interpretation of ssVCD spectra, methods fully exploring the crystal periodicity are often based on first principle molecular dynamics simulations and the nuclear velocity perturbation theory in a plane waves approach.²²⁻²⁴ These are often restricted to general gradient approximation functionals.²⁵ A much simpler way consists in extracting a cluster of limited size from the crystal structure and calculating its spectroscopic properties. However, this approach does not fully take into account the environmental effects in the whole crystal.²⁶,²⁷ Even when the periodic boundary conditions are used for the optimization of a subunit, edge effects appear in the spectra and complicate their interpretation. In this work, we use an extended cluster approach that allows us to
treat bigger systems using more precise (hybrid) functionals in the frame of the magnetic field perturbation theory.²⁸,²⁹ Vibrational properties are calculated for smaller parts of the crystal and transferred back using the Cartesian coordinate tensor transfer (CCT) technique.³⁰,³¹ In particular, the Hessian (force constant matrix) and the phonon modes can be obtained in this way.

The CCT-based approach appeared extremely flexible and was previously applied to systems of thousands of atoms,³² crystal assemblies¹⁷,³³ or liquids.²⁹ For crystal, the periodicity is fully considered by calculating the dynamic force field matrix of the elementary cell for the zero phonon mode determining the optical spectra. Longer-range forces could be in principle included in the model, for example by adding larger fragments calculated at a lower approximation level. Compared to ad hoc cluster approaches²⁰,²¹,³⁴,³⁵ the fragmentation is automatized and flexible, based on pre-defined interaction distances and desired cluster sizes. Although probably not suitable for crystals with long-range electron conjugation, for the molecular systems studied here this computational methodology provided basic understanding of the spectral features and a solid basis for spectral band assignment.

Cocrystals are convenient systems to explore how the VCD methodology captures various structural differences. They can be universally found in the realm of APIs. A cocrystal is a crystalline material composed of two or more compounds within a single crystal lattice. In what follows, we focus on the cocrystal of the anti-inflammatory drug naproxen with alanine and proline as a cocrystal formers (coformers) (Figure 1). We investigate whether ssVCD is able to distinguish the cocrystals from a pure APIs, and a mixture of pure solid components.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Studied compounds.</figcaption></figure>
## Methods

**Sample Preparation.** Commercially available (R) and (S)-naproxen (Merck, pharmaceutical impurity standard) and L or D-alanine/proline (Fluka) were used without further purification. The cocrystals were prepared using a liquid-assisted grinding (LAG) method, consisting of grinding the solid components with a small amount of solvent³⁶ following refs. ³⁷ and ³⁸. Cocrystals of (S)-naproxen and L-alanine (and enantiomeric form), and (S)-naproxen and D-alanine, (S)-naproxen and D-proline (and enantiomers), and (S)-naproxen and L-proline were prepared. To explore the computational analysis, the (S)-naproxen/L-alanine and (S)-naproxen/D-proline combinations were analyzed in detail. The chemicals were ground together at ambient conditions for 60 minutes at a 1:1 stoichiometric ratio with 10 µl of methanol added to 100 mg of the mixture. We found that the cocrystal formation does not depend strongly on the amount of methanol. For a test measurement, partially cocrystallized sample of (S)-naproxen and L-alanine (in 1:1 mass ratio, i.e. with molar excess of alanine) and its enantiomer were prepared as well, which gave spectra close to a sum of the cocrystal and excessive alanine (Figure S1). Retsch mixer mill MM 200 with three stainless steel balls of 0.5 cm diameter was used. Finally, pellets were made by mixing the analyte with the KBr matrix in a ratio of about 1:100, adjusted to optimize the maximal absorbance within 0.8-1.0, and pressing.

**Measurement of Vibrational Spectra.** IR and VCD spectra were recorded using two spectrometers, according to availability or for a verification: a ChiralIR-2XTM spectrometer (BioTools, Inc., USA) equipped with a dual-PEM™ system using two ZnSe photoelastic modulators, and a Bruker Vertex70 spectrometer with VCD extension (PMA 50 Bruker), consisting of a photoelastic modulator (PEM) and a fast-acquisition dual channel. A previously described procedure was used to minimize instrumental artifacts, which is documented in ref. ¹⁷ and its supporting material. As reported in the literature most artifacts can be eliminated by averaging the orientations of the sample (flipping) combined with its continuous rotation around the beam axis.³⁹,⁴⁰ In our case, we found that the effect of flipping was rather minor.¹⁷ Control measurements were conducted for a suspension in nujol oil. A 4 cm⁻¹ resolution, the Blackman-Harris apodization function, and PEM calibration at 1400 cm⁻¹ were used. The resulting spectra are averages of 6 recorded blocks, with each one consisting of 1560 scans. The experiment took approximately 3 hours for each sample.

**X-ray powder diffraction.** At the Université Paris-Saclay powder X-ray diffraction (PXRD) patterns were recorded at the MORPHEUS platform of the Laboratoire de Physique des Solides at room temperature on a Bruker D8 Advance diffractometer equipped with a LYNXEYE position-sensitive detector, using Cu Kα radiation (λ = 0.15418 nm). Other experiments were performed at
the Institute of Physics of Prague, where the sample was ground and placed in a 0.5 mm borosilicate-glass capillary. Powder diffraction data were collected using the Debye-Scherrer transmission configuration on a powder diffractometer Empyrean (PANalytical, λ<sub>Cu,Kα</sub> = 0.15418 nm), equipped with a focusing mirror, capillary holder, and a PIXcel<sup>3D</sup> detector. The measurement range was 3 – 80° 2θ with a 0.013° step size and 150 s per step.

**Modeling and Calculations.** Fragmentation and tensor transfer approaches<sup>17,30,31</sup> were used, where the basic crystal model consisted of 27 elementary cells. It was obtained by propagating the elementary cell 3-times in each crystallographic direction *a*, *b*, *c*. X-ray geometries downloaded from the Cambridge Crystallographic Data Centre (CCDC) were used for alanine, proline, naproxen and the cocrystals (Table S1). Using our scripts<sup>29</sup> smaller fragments were created comprising interactions of all molecules in the central elementary cell with the closest ones. The fragment geometries were partially optimized in the vibrational normal mode coordinates<sup>41,42</sup> and harmonic force constants, atomic polar and axial tensors were calculated at the B3LYP<sup>43</sup>/6-311++G**<sup>44</sup> level comprising the GD3BJ<sup>45</sup> dispersion correction. The Gaussian 16 Rev.A.03 program package<sup>46</sup> was used for the quantum chemistry calculations.

Similarly as in previous studies,<sup>35,47</sup> we used the CPCM<sup>48</sup>(H<sub>2</sub>O) solvent model to mimic the crystal environment, in particular the effect of the polar groups. Note that within the CPCM methodology the main parameter is the dielectric constant and the result are not particularly sensitive to it for large relative permittivities. For our systems the CPCM environment is particularly important to stabilize zwitterionic forms of the amino acids.

The vibrational parameters were transferred back to the 27-cell system, and the force constants matrix and zero-phonon spectra calculated.<sup>49</sup> For the naproxen/proline cocrystal where the x-ray data<sup>50</sup> indicated two geometries of the proline ring the spectra were simulated for both possibilities and averaged (cf. Table S2, Figure S2). In addition, the effect of longer-range interactions was explored using the transition dipole coupling model.<sup>51,52</sup> From the dipole and rotational strengths IR and VCD spectra were generated by convolution with a Lorentzian function, using full width at half height of 10 cm<sup>-1</sup>.

**Results and discussion**

**Crystal and Cocrystals Structure.** X-ray geometries of naproxen and its cocrystals with alanine and proline (CCDC identifiers COYRUD, RODSEK, and FEVZOX, respectively) are displayed in **Figure 2**. Clearly, the packing patterns are different. In pure naproxen (P2<sub>1</sub> symmetry) the carboxyl groups are linked by H-bonds and the naphthyl subunits are in a parallel-displaced geometry, allowing for a limited π-π interaction. In both cocrystals, the amino acids are present as
zwitterions, and the naproxen molecules are not directly connected by H-bonds. In the naproxen/alanine system (P2₁2₁2₁ Sohncke symmetry group), the NH₃⁺ residue is bonded to the O=C group of naproxen, and the aromatic rings adopt a parallel-displaced positions, allowing for a more favorable overlap between the π clouds than in pure naproxene. For naproxen/proline (P2₁ Sohncke symmetry group), the proline NH₂⁺ group is H-bonded to other prolines only whereas the naproxen interacts with the carboxylate group of the amino acid. The naproxen-naproxen packing seems to be looser here, with the naphthyl planes tilted more to each other than in the naproxen/alanine cocrystal.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Crystal packing in naproxen and the two cocrystals, in two projections.</figcaption></figure>

**Powder Diffraction.** The formation of the cocrystals was confirmed by powder X-ray diffraction (PXRD) measurements. The observed diffraction patterns were compared with those calculated from previous studies,³⁸ providing an evidence of the cocrystal formation (Figure 3). The patterns are almost identical, although in the naproxen/alanine cocrystal data additional weak peaks indicate a small contamination of the sample by crystalline alanine. Such impurities may have resulted from the different solubilities of naproxen and alanine in methanol. The data analyzed below (Figure 4), however, suggest that they have no detectable influence on the vibrational spectra.
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Experimental PXRD intensities of polycrystalline LAG *S*-naproxen/*L*-alanine, peaks due to contamination with crystalline alanine marked by asterisks (*), and (*S*)-naproxen/*D*-proline cocrystals, and calculated patterns based on the crystal structure.<sup>38</sup></figcaption></figure>

**Solid State Vibrational Circular Dichroism Experiments.** IR and VCD spectra of the pure components, naproxen, alanine, and proline, are plotted in **Figure 4**. The “mirror imaging” of the S/R and D/L enantiomers confirms reliability of the experimental protocol.<sup>17</sup> Most visible distortions of naproxen VCD spectra appear in the regions around 1200 cm<sup>-1</sup> and 900 cm<sup>-1</sup>. Alanine and proline give straighter VCD baseline and fewer artifacts. Overall, VCD signal of a reasonable quality is recorded, comparable with previous studies.<sup>17,21,25,53</sup>

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Experimental VCD (ΔA) and IR (A) spectra of naproxen, alanine and proline enantiomers.</figcaption></figure>

IR absorption and VCD spectra of (*S*)-naproxen/*L*-Ala and (*S*)-naproxen/*D*-Pro cocrystals are plotted in **Figure 5**, the band assignment is given in **Table 1**. Experimental noise is indicated by grey lines. Clearly, the two cocrystals give different IR and VCD spectra, and the spectra differ
from those of individual components. In the naproxen/alanine system (left in the figure), for example, the carbonyl bands converge and overlap. Band 4, corresponding to the symmetric bending (umbrella) vibration of the NH₃ group, is blue-shifted by 4 cm⁻¹ relative to the bare L-alanine and changes VCD sign. The strong signals (13) come from the naphthyl ring, the solitary bands 14 and 15 correspond to out of plane vibrations of the carboxylate group and C-C bond stretching in naproxen. Band 1 of the C=O stretching of naproxen appears as a strong negative peak in the VCD spectrum at 1667 cm⁻¹. The strong difference compared to pure naproxen probably arises from the different nature of the hydrogen bond, neutral OH...O in naproxen vs. ionic NH⁺...O in the cocrystal. Indeed, in the naproxen/proline cocrystal where this type of H-bonding does not exist the C=O naproxen stretching frequency is observed much higher, at 1710 cm⁻¹.

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Experimental VCD and absorption spectra of the (S)-naproxen/L-Ala and (S)-naproxen/D-Pro cocrystals, cf. **Table I** for the band assignment. The grey lines represent the experimental noise.</figcaption></figure>

VCD intensities of the naproxen/alanine are on average stronger than those of the naproxen/proline cocrystal (Figure 5, right). In addition, the IR absorption is much broader for the latter. Several hypotheses could explain these differences: first, the two co-crystals crystallize in different space groups (P2₁ for proline and P2₁2₁2₁ for alanine), which can affect the VCD intensity.⁵⁴ For example, non-local effects enabled by the crystal packing can enhance it.⁵⁵,⁵⁶ Second, the conformational mobility of proline is larger than that of alanine, as both puckering
isomers can exist in the crystal. This might result in a blurring of the VCD spectrum due to the contribution of the two isomers. For naproxen/alanine the interaction between the two components involves two strong ionic hydrogen bonds, which makes the system more conformationally stable and rigid than for naproxen/proline involving one hydrogen bond only.

These VCD and IR spectra are compared with those of the complementary combinations, (S)-naproxen/D-Ala and (S)-naproxen/L-Pro, in **Figure S3**. One can see that while IR spectra of the diastereoisomers are rather similar, VCD spectra exhibit profound differences.

<table><caption>**TABLE I** Experimental and calculated vibrational frequencies (cm⁻¹) of the (S)-naproxen/L-alanine and (S)-naproxen/D-proline cocrystals.</caption><thead><tr><th>Band<sup>a</sup></th><th>ω<sub>Exp(VCD)</sub></th><th>ω<sub>Calc</sub></th><th>VCD sign</th><th>Assignment</th></tr></thead><tbody><tr><td colspan="5">(S)-naproxen/L-alanine</td></tr><tr><td>1</td><td>1667</td><td>1731</td><td>-</td><td>v(C=O) naproxen</td></tr><tr><td>2</td><td>1642</td><td>1662</td><td>+</td><td>Asym. δ(NH<sub>3</sub>) alanine</td></tr><tr><td>3</td><td>1633</td><td>1646</td><td>-</td><td>Naphthalene core naproxen, v(C=C)</td></tr><tr><td>4</td><td>1612</td><td>1627</td><td>-</td><td>Sym. δ(NH<sub>3</sub>) alanine</td></tr><tr><td>5</td><td>1498</td><td>1514</td><td>+</td><td>δ, C-H, CH<sub>3</sub> naproxen</td></tr><tr><td>6</td><td>1455</td><td>1462</td><td>+</td><td>δ(O-H) naproxen</td></tr><tr><td>7</td><td>1427</td><td>1419</td><td>-</td><td>Sym. δ(CH<sub>3</sub>) alanine; δ(C-H), v(C-C) naproxen</td></tr><tr><td>8</td><td>1372</td><td>1396</td><td>+</td><td>δ(CH<sub>3</sub>)</td></tr><tr><td>9</td><td>1354</td><td>1357</td><td>-</td><td>δ, C-H, O-H naproxen</td></tr><tr><td>10</td><td>1307</td><td>1320</td><td>-</td><td>δ(C-H) alanine</td></tr><tr><td>11</td><td>1265</td><td>1291</td><td>-</td><td>δ(C-H), naphthalene ring</td></tr><tr><td>13</td><td>1215</td><td>1213</td><td>-</td><td>v(C-C), δ(C-H) naproxen</td></tr><tr><td>13</td><td>1207</td><td>1213</td><td>+</td><td>δ(CH<sub>3</sub>). oop(COOH) naproxen</td></tr><tr><td>14</td><td>1033</td><td>1028</td><td>-</td><td>Delocalized, naproxen</td></tr><tr><td>15</td><td>885</td><td>908</td><td>+</td><td>Naproxen methyl group def.</td></tr><tr><td colspan="5">(S)-naproxen/D-proline</td></tr><tr><td>1</td><td>1710</td><td>1761</td><td>+</td><td>v(C=O) naproxen</td></tr><tr><td>2</td><td>1626</td><td>1634</td><td>+</td><td>Asym. v(C=O), Pro</td></tr><tr><td>2</td><td>1609</td><td>1615</td><td>-</td><td>δ(N-H), Pro</td></tr><tr><td>3</td><td>1504</td><td>1540</td><td>+</td><td>δ(C-H), naproxen, arom.</td></tr><tr><td>4</td><td>1485</td><td>1515</td><td>-</td><td>δ(C-H), Pro</td></tr><tr><td>5</td><td>1454</td><td>1496</td><td>-</td><td>δ(C-H), naproxen, methyl scissor</td></tr><tr><td>6</td><td>1393</td><td>1397</td><td>-</td><td>δ(C-H), Pro, naproxen</td></tr><tr><td>7</td><td>1264</td><td>1246</td><td>+</td><td>v(C-C), δ(C-H) naproxen</td></tr><tr><td>8</td><td>1209</td><td>1194</td><td>-</td><td>δ(C-H), naproxen, arom</td></tr><tr><td>8</td><td>1197</td><td>1199</td><td>+</td><td>δ(C-H), naproxen, arom</td></tr><tr><td>9</td><td>1164</td><td>1172</td><td>-</td><td>δ(C-H), naproxen, arom</td></tr><tr><td>10</td><td>1031</td><td>1038</td><td>-</td><td>v(C-C), δ(C-H) naproxen</td></tr><tr><td>11</td><td>858</td><td>843</td><td>-</td><td>δ(CH<sub>3</sub>). oop(COOH) naproxen</td></tr></tbody></table>

<footnote><sup>a</sup>Figure 5. v - stretching, δ - bending, oop - out of plane</footnote>
<figure><img src="image_7.png" /><figcaption>**Figure 6.** Experimental absorption and VCD of (S)-naproxen/L-alanine cocrystal (black), (S)-naproxen/L-alanine mixture in the same 1:1 molar proportion (red), sum of L-alanine and (S)-naproxen spectra (green), L-alanine (pink) and (S)-naproxen (blue).</figcaption></figure>

The cocrystal IR and VCD spectra are also different from those obtained as mere sums of the two components. For naproxen/alanine this is shown in **Figure 6**, where we see that whereas the spectra of a 1:1 molar ratio mixture can be obtained as the sum, the cocrystal behaves as an autonomous species.

**Spectra Simulations.** To explore the variations coming from parameters used in the fragmentation methodology, we simulated IR and VCD spectra of alanine. Second energy derivatives, atomic polar and axial tensors were calculated including molecular pairs, trimers, and a cluster of one molecule with 14 neighboring ones in the crystal (“15-mer”). After the tensors were transferred back to the crystal, the dynamic force field matrix was calculated, and resultant spectra plotted in **Figure S4**. For all models, a problematic part of the spectrum is the region of 1720-1560 cm⁻¹, including N-H bending (band 1) and C=O stretching (band 2). Perhaps by accident, the trimer gives the best result here. Other calculated IR and VCD bands reproduce the experimental ones better, and their intensities are not so much dependent on the details of the fragmentation approach.
<figure><img src="image_8.png" /><figcaption>**Figure 7.** Calculated and experimental IR absorption and VCD spectra of the (S)-naproxen/L-alanine and (S)-naproxen/D-proline cocrystals.</figcaption></figure>

For the cocrystals, with our computational resources we were limited to the pair-wise
fragmentation scheme, which gave the spectra plotted in **Figure 7**. Given the approximations, we
consider the agreement with the naproxen/alanine experiment to be very good, providing reliable
assignment for most of the strongest IR and VCD spectral features (**Table I**). The frequency of the
C=O stretching band (#1) is calculated too high, but with reasonable intensities and correct VCD
sign. Around 1300 cm⁻¹ the band assignment based on IR would be problematic; however, VCD
with the sign information is quite helpful in this respect. The 1030 cm⁻¹ -/+ VCD couplet in the
experiment is reproduced with a lower intensity only.
For naproxen/proline, the agreement is not so good. The computation reproduced the trends in the C=O stretching frequencies, which are significantly lower (exp./cal. 1667/1731 cm⁻¹) in naproxen/alanine than in naproxen/proline (1710/1761 cm⁻¹). This is due to the much stronger hydrogen bonding of the carboxyl to the NH₃⁺ group in the former. The overall absorption profile is also reasonable, given the unresolved background-like signal in the experiment.

Regarding VCD, the intensities computed for naproxen/proline (a typical VCD/absorption ratio of ~5×10⁻⁵) are much smaller than for naproxen/alanine (~1.5×10⁻⁴), which corresponds to the experiment. The w-shape of the aromatic naproxen signals around 1200 cm⁻¹ is also reproduced correctly, whereas the calculated couplet around 1035 cm⁻¹ has much lower intensity experimentally. The predicted 1634/1615 cm⁻¹ “-/+” VCD band pair is seen only in nujol, whereas a positive signal was measured in the KBr pellet, indicating possible artifacts in the ssVCD measurement. For naproxen/proline the computations predict more than 4-times lower intensity of the C=O stretching mode than for naproxen/alanine. This agrees with the experimental trend, although the observed signal is even weaker (cf. also **Figure 5**, band 1). Further weakening may occur due to a limited motion of the naproxen carboxylate groups, not included in the calculations. The dynamical factors may also explain the splitting of both C=O stretching IR bands (1 and 2), hardly discernable in the simulation. Around 1450 cm⁻¹ a positive VCD is predicted, not seen in experiment; again, this can be explained by a limited motion of the five-membered proline ring in the crystal, averaging out the CH bending VCD signals, and similar anharmonic effects.⁵⁷

IR and VCD spectra simulated for the two proline ring conformations are plotted in **Figure S5**. Indeed, the averaging leads to partial cancelation of the VCD signal, in particular around 1300 cm⁻¹, although it cannot explain all observed features. In **Figure S6**, we compare the spectra of the naproxen/proline cocrystal simulated with and without the transition dipole coupling correction. While the spectra suggest that the longer-range interactions in the crystal simulated by the correction may be important for fine VCD pattern of the C=O stretching, they do not principally change the overall shape. Note also, because of the weakness of VCD, that we cannot rule out experimental artifacts for naproxen/proline, especially those competing with weak VCD signals.

Overall, however, we find the IR/VCD methodology viable to study the cocrystals capturing very sensitively the structural details, such as giving clearly different spectra between mere mixtures and true cocrystals. In spite of the drawbacks, the computational methodology appears indispensable to interpret, understand, and verify the experimental solid state VCD data for the naproxen cocrystals. The advantages of combining the polarized and unpolarized spectroscopy can be documented, for example, for the region of 1200 cm⁻¹ where absorption of the naproxen/alanine cocrystal does not significantly deviate from that of the solid mixture, whereas VCD spectrum is very different.
## Conclusions

In our study of the naproxen-alanine and naproxen-proline cocrystals, we aimed to evaluate the solid-state vibrational circular dichroism (VCD) methodology for structural identification and confirmation. Additionally, we sought to assess the feasibility of computational approaches for interpreting the spectra. The results were promising, as the experimental protocol yielded spectra with minimal artifacts, although these were still present in the naproxen/proline cocrystal providing very weak VCD signal. In spite of that the cocrystals could be clearly distinguished from the pure components or their mere mixtures. Due to the relatively large cocrystal size, the fragmentation computational model had to be restricted to the closest pair-wise interactions. However, it proved to be flexible and adaptable with respect to the computational sources available. For the molecular crystals studied the model was able to reproduce the main experimental features and trends. More generally, the higher sensitivity of VCD to crystal morphology, combined with advances in experimental methods, makes this approach an interesting tool for distinguishing polymorphic forms, probing intermolecular interactions, crystal dynamics, and studying structural chirality, particularly in the pharmaceutical context.

## Acknowledgements

This work was supported by the Czech Science Foundation (24-10558S), Ministry of Education, Youth and Sports of the Czech Republic (e-INFRA CZ 90254), University of Chemistry and Technology (A2_FCHI_2024_028), and the French National Research Agency (ANR) Dichrosol project (Grant ANR-23-CE29-0006).

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