Supramolecular Assemblies of Melamine-2-Thiobarbiturate and Melamine-Barbiturate-2-Thiobarbiturate: Experimental and Theoretical Studies

: In this work, we considered the formation of supramolecular assemblies of melamine-thiobarbiturate and melamine-barbiturate-thiobarbiturate. It is known that thiobarbituric acid can form many tautomers, as well as different motifs due to the change of C2=O to C2=S hydrogen bonds. We formed the crystal. The resulting crystals were studied with scanning electron microscopy (SEM), optical ﬂuorescence microscopy, single crystal and powder (PXRD) X-ray diffraction analyses, and solid state nuclear magnetic resonance (ss NMR). These systems were theoretically studied using density functional theory (DFT) and classical molecular dynamics (MD) simulations. Interestingly, just as in the case of melamine barbiturate, during the crystallization process, hydrogen from the C5 moiety of thiobarbituric acid migrates to the melamine molecule. In addition, the resulting melamine thiobarbiturate crystals exhibit ﬂuorescence behavior in the red region (~565–605 nm), while the melamine barbiturate crystals are ﬂuorescent in the green region (512–542 nm)


Introduction
An important role in supramolecular chemistry is played by hydrogen bonds between hydrogen atoms and electronegative elements and groups. This type of bond contributes to the formation of ordered molecular structures [1]. The formation of hydrogen bonds can be described as the transfer of hydrogen between a donor and an acceptor [2]. Oxygen or nitrogen often act as a hydrogen acceptor. On the other hand, it is of interest to study the formation of hydrogen bonds between other elements, such as sulfur or selenium [3]. Studies show that the formation of hydrogen between sulfur and hydrogen leads to the formation of many structural motifs [4,5].
Thiobarbituric acid (TBA), barbituric acid (BA), and melamine (M) formulas are shown in Figure 1. TBA is well known as one of the reagents used for determining lipid peroxidation [6]. The presence of an acidic proton in the C5 position in its structure makes it a convenient substrate for various modifications, including amination [7] and Another important property of TBA is the presence of many tautomeric forms [12,20,21]. Five tautomeric forms were experimentally observed out of ten theoretically possible [12]. In addition, it is known that the most thermodynamically favorable structures of TBA can form cocrystals [21] and deposit on the surface of gold nanodots [22,23].
BA and cyanuric acids have a similar structural motif to TBA. For these compounds, supramolecular assemblies with M are known [24][25][26][27][28][29][30][31][32]. Despite their similar spatial shape, these acids form different co-crystals with M. M cyanurate forms monoclinic crystals with the space group I2/m, composed of flat two-dimensional layers with a threefold rotation axis [33], while for M-BA the symmetry space group was defined as Ccc2 [29]. The crystal is composed of endless flat ribbons with alternating M and BA molecules arranged one above the other. Electrostatic bonds play an important role in the stabilization of this crystal [29]. Surprisingly, self-assemblies of melamine and TBA have not been reported, and this work intended to fill the corresponding gap in the supramolecular chemistry of TBA.

Sample Preparation
Samples of crystalline M-BA containing TBA were prepared by mixing 20 mM solutions of either M or BA containing similar concentrations of TBA.
M-TBA crystals were obtained by mixing 20 mM solutions of M and TBA. Next, about 80% of the liquid was evaporated and left to crystallize until pink crystals precipitated.
Then, the supernatant liquid of the powder precipitate was decanted, and the remaining powder was centrifuged and washed three times with water. Finally, the powder was dried.
The diffusion-controlled samples were prepared as follows. Agar-agar gel was laid out in a Petri dish, while a third of the volume was occupied by a gel prepared based on a 20 mM solution of M, and a gel prepared based on a 20 mM solution of BA was in the other half. A 20 mM TBA solution was added to the central space not occupied by the gel. Three days later, the resulting precipitate was filtered. Another important property of TBA is the presence of many tautomeric forms [12,20,21]. Five tautomeric forms were experimentally observed out of ten theoretically possible [12]. In addition, it is known that the most thermodynamically favorable structures of TBA can form cocrystals [21] and deposit on the surface of gold nanodots [22,23].
BA and cyanuric acids have a similar structural motif to TBA. For these compounds, supramolecular assemblies with M are known [24][25][26][27][28][29][30][31][32]. Despite their similar spatial shape, these acids form different co-crystals with M. M cyanurate forms monoclinic crystals with the space group I2/m, composed of flat two-dimensional layers with a threefold rotation axis [33], while for M-BA the symmetry space group was defined as Ccc2 [29]. The crystal is composed of endless flat ribbons with alternating M and BA molecules arranged one above the other. Electrostatic bonds play an important role in the stabilization of this crystal [29]. Surprisingly, self-assemblies of melamine and TBA have not been reported, and this work intended to fill the corresponding gap in the supramolecular chemistry of TBA.

Sample Preparation
Samples of crystalline M-BA containing TBA were prepared by mixing 20 mM solutions of either M or BA containing similar concentrations of TBA.
M-TBA crystals were obtained by mixing 20 mM solutions of M and TBA. Next, about 80% of the liquid was evaporated and left to crystallize until pink crystals precipitated.
Then, the supernatant liquid of the powder precipitate was decanted, and the remaining powder was centrifuged and washed three times with water. Finally, the powder was dried.
The diffusion-controlled samples were prepared as follows. Agar-agar gel was laid out in a Petri dish, while a third of the volume was occupied by a gel prepared based on a 20 mM solution of M, and a gel prepared based on a 20 mM solution of BA was in the other half. A 20 mM TBA solution was added to the central space not occupied by the gel. Three days later, the resulting precipitate was filtered.

Microluminescence Spectra
Microluminescence spectra of solid-state M-TBA crystals were acquired using a Zeiss Axio Imager, A2m, equipped with an Ocean Optics QE Pro spectrometer (St. Petersburg, Russia) (excitation by HAL 100 and HBO 100).

Microscopy
Scanning electron microscopy (SEM) and electron dispersion X-ray (EDX) studies were performed with a Hitachi S-3400N scanning electron microscope (Tokyo, Japan) and Oxford X-Max 20 EDX spectrometer (Abingdon, UK). EDX analysis was performed at 5 kV accelerating voltage and 0.2 nA beam current, to increase the locality of the method.

Powder X-ray Diffraction
Powder X-ray diffraction (PXRD) measurements of M-BA-TBA powder were performed on a Bruker D2 (Karlsruhe, Germany) with Cu Kα radiation from diffractionless silicon as a special substrate for the sample, with a speed of 1 degree per minute. All calculations for power XRD were made using regular Topas software (version 6.0).

Single Crystal X-ray Diffraction
Single crystal X-ray diffraction (XRD) studies were performed at 100 K using an XtaLAB Synergy-S diffractometer (Rigaku Oxford Diffraction, Oxford, UK) equipped with a hybrid photon counting HyPix-6000 detector that was operated with monochromated, microfocused CuKα radiation (λ[CuKα] = 1.54184 Å) at 50 kV and 1.0 mA. The data obtained were processed and normalized for polarization, Lorentz, and background effects. The CrysAlisPro [34] program was utilized to perform an empirical absorption correction based on spherical harmonics implemented in the SCALE3 ABSPACK algorithm. The unit-cell parameters were obtained via refining using the least-squares algorithms. The SHELX program [35,36] incorporated in the OLEX2 program package [37] was applied to refine the structure previously solved using a dual-space algorithm. The finalized model incorporated coordinates and anisotropic displacement parameters for all non-hydrogen atoms. The coordinates of carbon-bound hydrogen atoms were found from different Fourier maps and were included in the refinement without any restraints with individual isotropic displacement parameters (see Supplementary Materials).

Solid-State Nuclear Magnetic Resonance
The cross polarization/magic-angle spinning (CP-MAS) and cross polarization polarization inversion (CPPI) nuclear magnetic resonance spectra (NMR) were recorded (at 292 ± 1 K) on a Bruker AMX-400 (Rheinstetten, Germany) instrument operating at 7.05 T. The MAS rate was 8 kHz. Acquisition was performed with a standard CP pulse sequence using a 3.5 µs proton 90 • pulse, a 800 µs contact pulse, and a 2.5 s delay between repetitions.

Quantum Chemical Computations
The full geometry optimization for all model structures was carried out at the B3LYP-D3/def2-SVP level of theory with the help of the Orca 5.0.3 program package [38]. The RIJCOSX approximation [39,40] utilizing the def2-SVP/C auxiliary basis set and spin restricted approximation were employed. The convergence tolerances for the geometry optimization were energy change = 5.0 × 10 −6 Eh, maximal gradient = 3.0 × 10 −4 Eh/Bohr, RMS gradient = 1.0 × 10 −4 Eh/Bohr, maximal displacement = 4.0 × 10 −3 Bohr, and RMS displacement = 2.0 × 10 −3 Bohr. The couple perturbed self-consistent field (CPSCF) equations were solved using the conjugated gradient (CG) method, with convergence tolerance on a residual of 1.0 × 10 −6 Eh. The solvent effects were taken into account by the SMD model [41]. The Hessian matrices were calculated for all optimized model structures, to determine the location of the correct stationary points on the potential energy surfaces (no imaginary frequencies were found in all cases) and to estimate the thermodynamic properties (Gibbs free energy) for all model systems at 298.15 K and 1 atm. Cartesian atomic coordinates of all model structures are presented in the Supplementary Materials.

Molecular Dynamics Simulation Details
Molecular dynamics simulations of M-TBA and M-BA-TBA were performed utilizing the GROMACS program package version 2021.4 [42]. Interatomic interactions were described by the OPLS-AA/CM1A force field [43] with topology and charges generated by LigParGen [43][44][45]. For the water model, a rigid non-polarizable TIP3P [46] model was used. The cut-off for non-bonded and long-range interactions was 1.2 nm. For the calculation of long-range Coulomb interactions, the Particle-Mesh Ewald was used [47]. Energy minimization was performed with the steepest descent algorithm. Simulations were performed in the NPT ensemble at T = 300 K and P = 1 atm in the cubic unit cell (approximate size 85 × 85 × 85 A 3 ). The integration timestep for the trajectory calculations was 2 fs, and for relaxation 0.5 fs. Spatial distribution functions and dimer existence autocorrelation functions were calculated from the resulting trajectories using the TRAVIS program package version 21 May 2021 [48].

Results and Discussion
Since TBA can form different tautomers [12,15,49], we evaluated through quantum chemical calculations the Gibbs free energies of all possible tautomeric forms in the gas phase and aqueous solution (Figure 2a,c). Solvation reduces the relative Gibbs energy of formation. The difference between the most thermodynamically favorable (viz. TBA1 and TBA10) forms of TBA is 27.5 kJ/mol, thus it is clear that TBA1 prevails in the aqueous solution. However, one should not exclude the possibility of ketone-enol isomerization during the formation of crystals [21]. The calculation of TBA-M dimers ( Figure 2b) showed that the formation of the M-TBA7 dimer (∆G = −47.66 kJ/mol) is most beneficial, which is much more profitable than the formation of the BA-M dimer (∆G = −40.9 kJ/mol) [29]. However, the geometry of this dimer does not fit into the structure determined by single crystal XRD. In addition, all TBA(9-10) tautomers are unsymmetrical, which reduces the likelihood of symmetrical patterns being formed. [41]. The Hessian matrices were calculated for all optimized model structures, to determine the location of the correct stationary points on the potential energy surfaces (no imaginary frequencies were found in all cases) and to estimate the thermodynamic properties (Gibbs free energy) for all model systems at 298.15 K and 1 atm. Cartesian atomic coordinates of all model structures are presented in the Supplementary Materials.

Molecular Dynamics Simulation Details
Molecular dynamics simulations of M-TBA and M-BA-TBA were performed utilizing the GROMACS program package version 2021.4 [42]. Interatomic interactions were described by the OPLS-AA/CM1A force field [43] with topology and charges generated by LigParGen [43][44][45]. For the water model, a rigid non-polarizable TIP3P [46] model was used. The cut-off for non-bonded and long-range interactions was 1.2 nm. For the calculation of long-range Coulomb interactions, the Particle-Mesh Ewald was used [47]. Energy minimization was performed with the steepest descent algorithm. Simulations were performed in the NPT ensemble at T = 300 K and P = 1 atm in the cubic unit cell (approximate size 85 × 85 × 85 A 3 ). The integration timestep for the trajectory calculations was 2 fs, and for relaxation 0.5 fs. Spatial distribution functions and dimer existence autocorrelation functions were calculated from the resulting trajectories using the TRAVIS program package version 21 May 2021 [48].

Results and Discussion
Since TBA can form different tautomers [12,15,49], we evaluated through quantum chemical calculations the Gibbs free energies of all possible tautomeric forms in the gas phase and aqueous solution (Figure 2a,c). Solvation reduces the relative Gibbs energy of formation. The difference between the most thermodynamically favorable (viz. TBA1 and TBA10) forms of TBA is 27.5 kJ/mol, thus it is clear that TBA1 prevails in the aqueous solution. However, one should not exclude the possibility of ketone-enol isomerization during the formation of crystals [21]. The calculation of TBA-M dimers (Figure 2b) showed that the formation of the M-TBA7 dimer (∆G = −47.66 kJ/mol) is most beneficial, which is much more profitable than the formation of the BA-M dimer (∆G = −40.9 kJ/mol) [29]. However, the geometry of this dimer does not fit into the structure determined by single crystal XRD. In addition, all TBA(9-10) tautomers are unsymmetrical, which reduces the likelihood of symmetrical patterns being formed.  The calculation of the energy of the formation of dimers and trimers M-(BA)-TBA1 is shown in Figure 3. The least thermodynamically favorable is the formation of a dimer (compound 1, Figure 2a). In this case, the Gibbs energy of dimer formation (1) practically coincides with the Gibbs energy of the formation of a similar M-BA dimer, which indicates the absence of a noticeable effect of sulfur on the formation of hydrogen bonds between carboxyl groups and -NH 2 of melamine. Compared to the BA-M dimer [29], the formation of M-TBA1 (compound 2) is less favorable (−27.00 kJ/mol), while the distance between the sulfur atoms in TBA and the hydrogen atom in melamine is 2.382 Å rather than 1.877 Å for a similar bond in the case of M-BA, and the length of the hydrogen bond between the NH 2 of melamine and the carboxyl group of TBA1 is reduced to 1.839 Å (in the case of BA-M, the bond is 1.874 Å). Thus, the M and TBA molecules in this dimer are not in the same plane. The formation of trimer (5) turned out to be slightly more thermodynamically favorable than trimers (3) and (4). The trimer consisting of M-BA-TBA1 molecules (6) has a calculated Gibbs energy of formation equal to −65.97 kJ/mol, which indicates the possible incorporation of TBA into the M-BA structure. The decrease in Gibbs free energy during the formation of M-TBA1 aggregates, as compared to M-BA, can be attributed to the lower energy negativity of sulfur relative to oxygen [50,51].
The calculation of the energy of the formation of dimers and trimers M-(BA)-TBA1 is shown in Figure 3. The least thermodynamically favorable is the formation of a dimer (compound 1, Figure 2a). In this case, the Gibbs energy of dimer formation (1) practically coincides with the Gibbs energy of the formation of a similar M-BA dimer, which indicates the absence of a noticeable effect of sulfur on the formation of hydrogen bonds between carboxyl groups and -NH2 of melamine. Compared to the BA-M dimer [29], the formation of M-TBA1 (compound 2) is less favorable (−27.00 kJ/mol), while the distance between the sulfur atoms in TBA and the hydrogen atom in melamine is 2.382 Å rather than 1.877 Å for a similar bond in the case of M-BA, and the length of the hydrogen bond between the NH2 of melamine and the carboxyl group of TBA1 is reduced to 1.839 Å (in the case of BA-M, the bond is 1.874 Å). Thus, the M and TBA molecules in this dimer are not in the same plane. The formation of trimer (5) turned out to be slightly more thermodynamically favorable than trimers (3) and (4). The trimer consisting of M-BA-TBA1 molecules (6) has a calculated Gibbs energy of formation equal to −65.97 kJ/mol, which indicates the possible incorporation of TBA into the M-BA structure. The decrease in Gibbs free energy during the formation of M-TBA1 aggregates, as compared to M-BA, can be attributed to the lower energy negativity of sulfur relative to oxygen [50,51].   Figure 4a depicts RDF for the intermolecular bonds between the H(N) atom of TBA and N atom of M. We can conclude that the most intense peak on the graph is at approximately 1.9 Å, which is in correspondence with the average length of the hydrogen bond between TBA and M. Alternatively, Figure 4b shows the RDF for intermolecular bond between S atom of TBA and H(N) atom of M. There are no peaks at the distance corresponding to hydrogen bonds. Therefore, all hydrogen bonds between M and TBA molecules are represented by H(N)-TBA and N-BA hydrogen bonds. Figure 4c shows the DACF for system with and without   Figure 4a depicts RDF for the intermolecular bonds between the H(N) atom of TBA and N atom of M. We can conclude that the most intense peak on the graph is at approximately 1.9 Å, which is in correspondence with the average length of the hydrogen bond between TBA and M. Alternatively, Figure 4b shows the RDF for intermolecular bond between S atom of TBA and H(N) atom of M. There are no peaks at the distance corresponding to hydrogen bonds. Therefore, all hydrogen bonds between M and TBA molecules are represented by H(N)-TBA and N-BA hydrogen bonds. Figure 4c shows the DACF for system with and without BA molecules-in other terms the lifetime of the M-TBA dimers. The inclusion of BA molecules in the system slightly decreases the lifetime of M-TBA dimes, due to the competitive process of M-BA dimer formation. Figure 4d,e depicts the neighbor count of M-TBA for the M-BA-TBA system and M-TBA system, respectively. There are almost no changes in the neighbor count for systems with and without inclusion of BA.
BA molecules-in other terms the lifetime of the M-TBA dimers. The inclusion of BA molecules in the system slightly decreases the lifetime of M-TBA dimes, due to the competitive process of M-BA dimer formation. Figure 4d,e depicts the neighbor count of M-TBA for the M-BA-TBA system and M-TBA system, respectively. There are almost no changes in the neighbor count for systems with and without inclusion of BA.  Figure 5a,b,d,e represent the spatial distribution function between M and TBA molecules with and without the inclusion of BA molecules. For both types of systems one can conclude that the M-TBA dimer forms both by π-π stacking and hydrogen bonds. In correspondence with the RDF data, the hydrogen bonds only form between the H(N) atom of TBA and N atom of M.

Data in
The most significant change between the M-BA-TBA and M-BA systems is represented in Figure 5c (without the inclusion of BA) and Figure 5f (with the inclusion of BA). The time-dependent radial distribution function between M and TBA depicted in Figure  5c,f shows the dynamics of early-stage nucleation. The wider the area on the horizontal (distance) axis, the bigger the nuclei; the larger the area on the vertical (time) axis, the greater the lifetime of a nuclei. This shows that the inclusion of BA molecules in the system suppresses the formation of M-TBA nuclei.  Figure 5a,b,d,e represent the spatial distribution function between M and TBA molecules with and without the inclusion of BA molecules. For both types of systems one can conclude that the M-TBA dimer forms both by π-π stacking and hydrogen bonds. In correspondence with the RDF data, the hydrogen bonds only form between the H(N) atom of TBA and N atom of M.

Data in
The most significant change between the M-BA-TBA and M-BA systems is represented in Figure 5c (without the inclusion of BA) and Figure 5f (with the inclusion of BA). The timedependent radial distribution function between M and TBA depicted in Figure 5c,f shows the dynamics of early-stage nucleation. The wider the area on the horizontal (distance) axis, the bigger the nuclei; the larger the area on the vertical (time) axis, the greater the lifetime of a nuclei. This shows that the inclusion of BA molecules in the system suppresses the formation of M-TBA nuclei. The data from the theoretical calculations were also confirmed experimentally: at room temperature, the self-assembly of thiobarbiturate and melamine did not form spontaneously from saturated solutions of TBA and M. To obtain M-TBA crystals, a solution containing 10 mmol of TBA and 10 mmol of M was boiled, evaporating 80% of the water. At the same time, no visible signs of crystal precipitation were observed; on the second day, rhombic crystals precipitated, which could be identified as melamine crystals. A few days later, pink crystals precipitated, having a lamellar structure (Figure 6a); less often "hedgehogs" and fluorescing in the DAPI and RHOD ranges (Figure 4a). At the same time, M-BA fluoresced in the DAPI and FITC ranges [30]. From the SEM images (Figure 6b), M-TBA had a layered structure, and the EDX analysis showed the sulfur content in the sample (Figure 6c). Recrystallization of M-TBA crystals from ethanol formed rose-type structures, the dimensions of which were much smaller than those of the M-TBA crystals formed in water. As well as the crystals obtained from an aqueous solution, these agglomerates had fluorescence in the DAPI and RHOD ranges. The data from the theoretical calculations were also confirmed experimentally: at room temperature, the self-assembly of thiobarbiturate and melamine did not form spontaneously from saturated solutions of TBA and M. To obtain M-TBA crystals, a solution containing 10 mmol of TBA and 10 mmol of M was boiled, evaporating 80% of the water. At the same time, no visible signs of crystal precipitation were observed; on the second day, rhombic crystals precipitated, which could be identified as melamine crystals. A few days later, pink crystals precipitated, having a lamellar structure (Figure 6a); less often "hedgehogs" and fluorescing in the DAPI and RHOD ranges (Figure 4a). At the same time, M-BA fluoresced in the DAPI and FITC ranges [30]. From the SEM images (Figure 6b), M-TBA had a layered structure, and the EDX analysis showed the sulfur content in the sample (Figure 6c). Recrystallization of M-TBA crystals from ethanol formed rose-type structures, the dimensions of which were much smaller than those of the M-TBA crystals formed in water. As well as the crystals obtained from an aqueous solution, these agglomerates had fluorescence in the DAPI and RHOD ranges. Of the starting compounds, only M exhibited noticeable fluorescence. Thus, it can be assumed that fluorescence in this system can arise for four reasons: (1) fluorescence due to the transition to another tautomeric form in the excited state [52,53]; (2) hydrogen atom transfer from TBA to M; (3) fluorescence of radical species; and (4) fluorescence on crystal defects [54].
We succeeded in isolating and characterizing a single M-TBA crystal. The parameters and volume of the crystal cell are shown in Table 1. Only one tautomeric form of TBA was found in the studied crystal. Apparently, this is because the TBA1 tautomer predominates in the aqueous phase.  Of the starting compounds, only M exhibited noticeable fluorescence. Thus, it can be assumed that fluorescence in this system can arise for four reasons: (1) fluorescence due to the transition to another tautomeric form in the excited state [52,53]; (2) hydrogen atom transfer from TBA to M; (3) fluorescence of radical species; and (4) fluorescence on crystal defects [54].
We succeeded in isolating and characterizing a single M-TBA crystal. The parameters and volume of the crystal cell are shown in Table 1. Only one tautomeric form of TBA was found in the studied crystal. Apparently, this is because the TBA1 tautomer predominates in the aqueous phase.  (Figure 7b). Thus, the cracks in the SEM images (Figure 6b) can be associated with the release of water from the structure during the vacuumization of the sample. be associated with the release of water from the structure during the vacuumization of the sample.  As in the case of M-TBA, hydrogen connected with C5 passes from the CH2 group of TBA to the M molecule. However, a more accurate confirmation of the transition of hydrogen would be the application of such methods as X-ray photoelectron spectroscopy (XPS) and solid-state nuclear magnetic resonance [55,56]. The Supplementary Materials show ( Figure S3) the spectra of CP-MAS and CPPI. No negative signals are observed in the CPPI spectra, which indicates the absence of CH2 groups in the crystals. As in the case of M-TBA, hydrogen connected with C5 passes from the CH 2 group of TBA to the M molecule. However, a more accurate confirmation of the transition of hydrogen would be the application of such methods as X-ray photoelectron spectroscopy (XPS) and solid-state nuclear magnetic resonance [55,56]. The Supplementary Materials show ( Figure S3) the spectra of CP-MAS and CPPI. No negative signals are observed in the CPPI spectra, which indicates the absence of CH 2 groups in the crystals.
Using the PXRD method, it was established (Figure 8) that the structure of these supramolecular assemblies coincided with the initial M-BA. At the same time, with an increase in the concentration of TBA, the crystallinity parameter decreased. Samples obtained by diffusion control had the highest crystallinity. However, the lattice parameters differed slightly in M-BA, and the volume of the crystal lattice increased from 971.9 Å 2 to 1006.7 Å 2 . Apparently, this was due to an increase in the distance, due to the complexity of the formation of a hydrogen bond between the TBA sulfur and the hydrogens of the melamine amino groups. Thus, TBA changes the morphology and shape of M-BA crystals.
Crystals 2023, 13, x FOR PEER REVIEW 10 of 14 Using the PXRD method, it was established ( Figure 8) that the structure of these supramolecular assemblies coincided with the initial M-BA. At the same time, with an increase in the concentration of TBA, the crystallinity parameter decreased. Samples obtained by diffusion control had the highest crystallinity. However, the lattice parameters differed slightly in M-BA, and the volume of the crystal lattice increased from 971.9 Å 2 to 1006.7 Å 2 . Apparently, this was due to an increase in the distance, due to the complexity of the formation of a hydrogen bond between the TBA sulfur and the hydrogens of the melamine amino groups. Thus, TBA changes the morphology and shape of M-BA crystals. As we noted earlier, it follows from quantum chemical calculations and molecular dynamics modeling that TBA will be incorporated into the structure of the M-BA crystal ribbon. M-BA-TBA crystals obtained by adding low concentrations of TBA have the form of melamine barbiturate crystals with defective twin growth points, at high concentrations the shape of "hedgehogs", the "needles" of which have the shape of a hexagonal prism topped with a triangular pyramid head. Upon receipt of melamine-barbiturate-thiobarbiturate under diffusion control, hexagonal crystals grow (Figure 9). As we noted earlier, it follows from quantum chemical calculations and molecular dynamics modeling that TBA will be incorporated into the structure of the M-BA crystal ribbon. M-BA-TBA crystals obtained by adding low concentrations of TBA have the form of melamine barbiturate crystals with defective twin growth points, at high concentrations the shape of "hedgehogs", the "needles" of which have the shape of a hexagonal prism topped with a triangular pyramid head. Upon receipt of melamine-barbiturate-thiobarbiturate under diffusion control, hexagonal crystals grow ( Figure 9).
All crystals glow in fluorescent channels (RHOD, FITC, and DAPI). The EDX spectrum shows the presence of sulfur in the sample (Supplementary Materials). Crystals 2023, 13, x FOR PEER REVIEW 11 of 14

Conclusions
In conclusion, in this work, the supramolecular assemblies of M and TBA were studied theoretically (DFT and MD) and experimentally (single crystal XRD, ss-NMR, SEM, EDX), and the possibility of embedding TBA into M-BA was shown. The obtained crystal structure of M-TBA does not have tautomeric forms in its structure, except for TBA1. The crystalline cell is layered, while the layers are interconnected using hydrogen bonds that are part of the structure of water molecules. Sheets consist of ribbons alternating M and TBA, as well as water. At the same time, the motif of the structure was in good agreement with the theoretically calculated TBA-M-TBA trimer (compound 5, Figure 3). From a molecular dynamics point of view, M-TBA is predominantly formed through π-π stacking and hydrogen bonds between H(N) atom of TBA and N atom of M. Moreover, the inclusion of BA molecules in the system led to slower formation of M-TBA nuclei.
It is interesting that, as in the case of M-BA, M-TBA crystals have a fluorescence shifting towards RHOD. We hypothesized the following structure-based causes of fluorescence: (1) fluorescence due to the transition to another tautomeric form in the excited state; (2) proton transfer from TBA to M; (3) fluorescence due to radicals contained in the crystal structure; and (4) fluorescence on crystal defects. The incorporation of TBA into M-BA crystals leads to a decrease in crystallinity, apparently since the TBA molecule becomes a source of a defect, resulting in structure twinning. In this case, the crystal cell remains the same. The resulting M-BA-TBA crystals have the shape of "hedgehogs", and the crystal cell repeats the M-BA cell, but at the same time has somewhat larger parameters.

Conclusions
In conclusion, in this work, the supramolecular assemblies of M and TBA were studied theoretically (DFT and MD) and experimentally (single crystal XRD, ss-NMR, SEM, EDX), and the possibility of embedding TBA into M-BA was shown. The obtained crystal structure of M-TBA does not have tautomeric forms in its structure, except for TBA1. The crystalline cell is layered, while the layers are interconnected using hydrogen bonds that are part of the structure of water molecules. Sheets consist of ribbons alternating M and TBA, as well as water. At the same time, the motif of the structure was in good agreement with the theoretically calculated TBA-M-TBA trimer (compound 5, Figure 3). From a molecular dynamics point of view, M-TBA is predominantly formed through π-π stacking and hydrogen bonds between H(N) atom of TBA and N atom of M. Moreover, the inclusion of BA molecules in the system led to slower formation of M-TBA nuclei.
It is interesting that, as in the case of M-BA, M-TBA crystals have a fluorescence shifting towards RHOD. We hypothesized the following structure-based causes of fluorescence: (1) fluorescence due to the transition to another tautomeric form in the excited state; (2) proton transfer from TBA to M; (3) fluorescence due to radicals contained in the crystal structure; and (4) fluorescence on crystal defects. The incorporation of TBA into M-BA crystals leads to a decrease in crystallinity, apparently since the TBA molecule becomes a source of a defect, resulting in structure twinning. In this case, the crystal cell remains the same. The resulting M-BA-TBA crystals have the shape of "hedgehogs", and the crystal cell repeats the M-BA cell, but at the same time has somewhat larger parameters.