Flexible Molecules Research Articles

Quantum chemical investigation of predominant conformation of the antibiotic azithromycin in water and DMSO solutions: thermodynamic and NMR analysis.

Azithromycin (AZM) is a macrolide-type antibiotic used to prevent and treat serious infections (mycobacteria or MAC) that significantly inhibit bacterial growth. Knowledge of the predominant conformation in solution is of fundamental importance for advancing our understanding of the intermolecular interactions of AZM with biological targets. We report an extensive density functional theory (DFT) study of plausible AZM structures in solution considering implicit and explicit solvent effects. The best match between the experimental and theoretical nuclear magnetic resonance (NMR) profiles was used to assign the preferred conformer in solution, which was supported by the thermodynamic analysis. Among the 15 distinct AZM structures, conformer M14, having a short intramolecular C6-OH … N H-bond, is predicted to be dominant in water and dimethyl sulfoxide (DMSO) solutions. The results indicated that the X-ray structure backbone is mostly conserved in solution, showing that large flexible molecules with several possible conformations may assume a preferential spatial orientation in solution, which is the molecular structure that ultimately interacts with biological targets.

Dimeric Calix[4]arenes, Synthesis and their Application: A Review

Abstract: Calix[n]arene is an attractive host for molecular recognition due to its accessibility through the hollow cavity and shallow bowl shape and has been used as a receptor over the last 30 years. Calix[n]arene has a small cavity, so designing a flexible molecule to recognize nano to large biomolecules is a challenging goal in host-guest chemistry. Dimeric calix[n]arene is formed by linking two calix[n]arene sub-units to each other. Their considerable structural features and relative diversity of modifying the upper or lower rim represent outstanding and greatly adaptive structures for designing bulky and complex building blocks adequate for selfassembly and molecular recognition. Their ability to form supramolecular structures for a wide range of applications, including the recognition of nano-molecules and large biological molecules, has been extensively studied. This review details the progress of the host-guest chemistry of dimeric calix[n]arenes, emphasizing the synthetic pathways employed for their production and their self-assembly properties. Dimerization of calix[n]arene occurs in two ways (1-through non-covalent bonding such as H-bonding or self-assembly, and 2-through covalent bond formation such as amide bond formation, multi-component reactions and Sonogashira cross-coupling reaction and metathesis reactions). In this work, we focused on dimerization through covalent bond formation, due to having more applications and diverse synthetic applications.

Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

Monoclonal antibodies (mAbs) make up a major class of biotherapeutics with a wide range of clinical applications. Their physical stability can be affected by various environmental factors. For instance, an acidic pH can be encountered during different stages of the mAb manufacturing process, including purification and storage. Therefore, understanding the behavior of flexible mAb molecules in acidic solution environments will benefit the development of stable mAb products. This study used small-angle X-ray scattering (SAXS) and complementary biophysical characterization techniques to investigate the conformational flexibility and protein-protein interactions (PPI) of a model mAb molecule under near-neutral and acidic conditions. The study also characterized the interactions between Fab and Fc fragments under the same buffer conditions to identify domain-domain interactions. The results suggest that solution pH significantly influences mAb flexibility and thus could help mAbs remain physically stable by maximizing local electrostatic repulsions when mAbs become crowded in solution. Under acidic buffer conditions, both Fab and Fc contribute to the repulsive PPI observed among the full mAb at a low ionic strength. However, as ionic strength increases, hydrophobic interactions lead to the self-association of Fc fragments and, subsequently, could affect the aggregation state of the mAb.

Cryogenic Ion Vibrational Predissociation (CIVP) Spectroscopy of Aryl Cobinamides in the Gas Phase: How Good Are the Calculations for Vitamin B12 Derivatives?

Aryl corrins represent a novel class of designed B12 derivatives with biological properties of "antivitamins B12". In our previous study, we experimentally determined bond strength in a series of aryl-corrins by the threshold collision-induced dissociation experiments (T-CID) and compared the measured bond dissociation energies (BDEs) with those calculated with density functional theory (DFT). We found that the BDEs are modulated by the side chains around the periphery of the corrin unit. Given that aryl cobinamides have many side chains that increase their conformational space and that the question of a specific structure, measured in the gas phase, was important for further evaluation of our T-CID experiment, we proceeded to analyze structural properties of aryl cobinamides using cryogenic ion vibrational predissociation (CIVP) spectroscopy, static DFT, and Born-Oppenheimer molecular dynamic (BOMD) simulations. We found that none of the examined DFT models could reproduce the CIVP spectra convincingly; both "static" DFT calculations and "dynamic" BOMD simulations provide a surprisingly poor representation of the vibrational spectra, specifically of the number, position, and intensity of bands assigned to hydrogen-bonded versus non-hydrogen-bonded NH and OH moieties. We conclude that, for a flexible molecule with ca. 150 atoms, more accurate approaches are needed before definitive conclusions about computed properties, specifically the structure of the ground-state conformer, may be made.

First-Principles Study of the Dispersion Effects in the Structures and Keto-Enol Tautomerization of Curcumin.

Noncovalent interactions, such as dispersion, play a significant role in the stability of flexible molecules, such as curcumin. This study revealed the importance of dispersion correction in the structure and keto-enol tautomerization of curcumin, which has rarely been addressed in computational studies. We rigorously constructed all possible unique curcumin conformers in the enol and keto forms within the first-principles framework. Regardless of the different environments, we carefully explained the agreement between the computational geometry (in the gas phase) and the experimental measurement (in the polymorph) by using dispersion correction. The calculation results for the aqueous solution of conformational abundance, thermochemistry, and reaction kinetics support the experimental observations after considering the dispersion correction. The study also suggests a water-catalyzed mechanism for keto-enol tautomerization, where dispersion correction plays a role in decreasing the energy barrier and making the keto form thermochemically and kinetically favorable. Our results could be helpful in future computational studies to find a method for increasing the aqueous solubility of curcumin; hence, the potential of curcumin as a multifunctional medicine can be fully achieved.

Dynamozones are the most obvious sign of the evolution of conformational dynamics in HIV-1 protease

Proteins are not static but are flexible molecules that can adopt many different conformations. The HIV-1 protease is an important target for the development of therapies to treat AIDS, due to its critical role in the viral life cycle. We investigated several dynamics studies on the HIV-1 protease families to illustrate the significance of examining the dynamic behaviors and molecular motions for an entire understanding of their dynamics-structure–function relationships. Using computer simulations and principal component analysis approaches, the dynamics data obtained revealed that: (i) The flap regions are the most obvious sign of the evolution of conformational dynamics in HIV-1 protease; (ii) There are dynamic structural regions in some proteins that contribute to the biological function and allostery of proteins via appropriate flexibility. These regions are a clear sign of the evolution of conformational dynamics of proteins, which we call dynamozones. The flap regions are one of the most important dynamozones members that are critical for HIV-1 protease function. Due to the existence of other members of dynamozones in different proteins, we propose to consider dynamozones as a footprint of the evolution of the conformational dynamics of proteins.

Mechanistically transparent models for predicting aqueous solubility of rigid, slightly flexible, and very flexible drugs (MW

Yalkowsky's General Solubility Equation (GSE), with its three fixed constants, is popular and easy to apply, but is not very accurate for polar, zwitterionic, or flexible molecules. This review examines the findings of a series of studies, where we have sought to come up with a better prediction model, by comparing the performances of the GSE to Abraham's Solvation Equation (ABSOLV), and Random Forest regression (RFR) machine-learning (ML) method. Large, well-curated aqueous intrinsic solubility databases are available. However, drugs may be sparsely distributed in chemical space, concentrated in clusters. Even a large database might overlook some regions. Test compounds from under-represented portions of space may be poorly predicted, as might be the case with the 'loose' set of 32 drugs in the Second Solubility Challenge (2020). There appears to be still a need for better coverage of drug space. Increasingly, current trends in predictions of solubility use calculated input descriptors, which may be an advantage for exploring properties of molecules yet to be synthesized. The risk may be that overall prediction approaches might be based on accumulated uncertainty. The increasing use of ML/AI methods can lead to accurate predictions, but such predictions may not readily suggest the strategies to pursue in selecting yet-to-be-synthesized compounds. Based on our latest findings, we recommend predictions based on both 'grouped' ABSOLV(GRP) and 'Flexible Acceptor' GSE(Φ,B) models with the provided best-fit parameters, where Φ is the Kier molecular flexibility index and B is the Abraham H-bond acceptor strength. For molecules with Φ < 11, the prudent choice is to pick the Consensus Model, the average of ABSOLV(GRP) and GSE(Φ,B). For more flexible molecules, GSE(Φ,B) is recommended.

Structure and Conformation of Hydroxypropylmethyl Cellulose with a Wide Range of Molar Masses in Aqueous Solution─Effects of Hydroxypropyl Group Addition.

The structure of hydroxypropylmethyl cellulose (HpMC) samples with a wide range of weight average molar masses (Mw) from 23 to 5000 kg mol-1, a controlled degree of substitution (DS) of 1.9 by methyl groups, and a molar substitution number (MS) of 0.25 by hydroxypropyl groups dissolved in aqueous solution was examined using static light scattering (SLS), dynamic light scattering (DLS), small-to-wide angle neutron scattering (S-WANS) techniques, and intrinsic viscosity ([η]) measurements. The determined Mw and the radius of gyration (Rg) showed the relationships Rg ∝ Mw1.0 and [η] ∝ Mw1.7 in a range of Mw < 100 kg mol-1, similar to rigid rod molecules in solution. However, exponents in the relationships decreased gradually with increasing Mw and reached ∼0.5 in a high Mw region, which is a typical value of flexible chain molecules for both Rg and [η]. These observations suggest that the HpMC samples behave as semiflexible rods with a certain persistence length (lp). The ratios of the hydrodynamic radius via DLS measurements to Rg also supported semiflexible rod behavior. Particle form factors and the average lengths (L) resulting from SLS and S-WANS experiments are well described with rigid rod particles in the range of Mw < 100 kg mol-1 and semiflexible rods with lp ∼ 100 nm in Mw > 100 kg mol-1. Because the average contour length (lc) calculated from Mw is approximately twice as long as L in the Mw range < 100 kg mol-1, the formed HpMC particles possess a folded hairpin-like elongated rigid rod structure. However, the lc/L value increases gradually in the range Mw > 200 kg mol-1, where the formed HpMC particles behave as semiflexible rods. The formed particle structure was substantially different from that found in methyl cellulose samples with a similar DS value, which showed rod-like behavior over a wide Mw range. The addition of hydroxypropyl groups only at MS = 0.25 effectively changed the formed particle structure.

Melamine–Urea–Formaldehyde Resin Adhesive Modified with Recycling Lignin: Preparation, Structures and Properties

The wettability, bonding strength and flexibility of modified melamine–urea–formaldehyde resin adhesive with hydroxymethyl lignin (LMUF) were investigated. Moreover, the curing performance, thermal properties and chemical structure of LMUF were analyzed by differential scanning calorimetry (DSC), thermogravimetry (TG), scanning electronmicroscopy (SEM), X–ray diffraction (XRD), Fourier–transform infrared spectroscopy (FTIR) and 13C nuclear magnetic resonance (13C–NMR). The synthesis mechanism of LMUF was also discussed. The results demonstrated that: (1) LMUF resin is characterized by low free formaldehyde and a short pot life. (2) With the increase in hydroxymethyl lignin, the bonding strength and flexibility of the LMUF increased first and then decreased. They reached their maximum when the hydroxymethyl lignin content was 6%–8%, which represented increases of 36% and 102%, respectively. (3) The DSC and TG analyses showed that the LMUF resin had a high hot-pressing temperature, but its thermostability was increased. (4) The XRD and SEM analyses proved that the LMUF resin was characterized by flexibility failure, especially at 6% of hydroxymethyl lignin. (5) Hydroxymethyl lignin is a type of flexible long-chain molecule, which was introduced into spaces between the rigid triazine rings through adhesion and penetration, thus improving the regular single structural form of MUF resin and increasing the compactness of the system. As a result, the resin had stronger deformability and cohesion in the molecules. The bonding strength and flexibility were improved to different degrees.

Theory of phase transitions in the assembly of biaxial molecules interacting via dispersion forces II: on phase transitions of flexible molecules

Previously, I investigated phase transitions in an assembly of biaxial molecules interacting via dispersion forces. Consequently, a uniaxial and two biaxial nematic phases, as well as a disco-nematic phase, were reported to appear in such an assembly of biaxial molecules. However, the investigation was performed at limited points in the space of anisotropy parameters ɛx and ɛy. In the present study, phase transitions at all points in the space of anisotropy parameters are investigated because understanding phase transitions in a system of flexible molecules, whose shape and/or anisotropy of molecular interaction change with temperature or density, is valuable and important.

Deciphering the Influence of Ground-State Distributions on the Calculation of Photolysis Observables.

Nonadiabatic molecular dynamics offers a powerful tool for studying the photochemistry of molecular systems. Key to any nonadiabatic molecular dynamics simulation is the definition of its initial conditions (ICs), ideally representing the initial molecular quantum state of the system of interest. In this work, we provide a detailed analysis of how ICs may influence the calculation of experimental observables by focusing on the photochemistry of methylhydroperoxide (MHP), the simplest and most abundant organic peroxide in our atmosphere. We investigate the outcome of trajectory surface hopping simulations for distinct sets of ICs sampled from different approximate quantum distributions, namely harmonic Wigner functions and ab initio molecular dynamics using a quantum thermostat (QT). Calculating photoabsorption cross-sections, quantum yields, and translational kinetic energy maps from the results of these simulations reveals the significant effect of the ICs, in particular when low-frequency (∼ a few hundred cm-1) normal modes are connected to the photophysics of the molecule. Overall, our results indicate that sampling ICs from ab initio molecular dynamics using a QT is preferable for flexible molecules with photoactive low-frequency modes. From a photochemical perspective, our nonadiabatic dynamics simulations offer an explanation for a low-energy tail observed at high excitation energy in the translational kinetic energy map of MHP.

Equilibrium structures of selenium compounds: The torsionally flexible molecule of selenophenol.

The equilibrium structure of selenophenol has been investigated using rotational spectroscopy and high-level quantum mechanical calculations, offering electronic and structural insight into the scarcely studied selenium compounds. The jet-cooled broadband microwave spectrum was measured in the 2-8GHz cm-wave region using broadband (chirped-pulse) fast-passage techniques. Additional measurements up to 18GHz used narrow-band impulse excitation. Spectral signatures were obtained for six isotopic species of selenium (80Se, 78Se, 76Se, 82Se, 77Se, and 74Se), together with different monosubstituted 13C species. The (unsplit) rotational transitions associated with the non-inverting μa-dipole selection rules could be partially reproduced with a semirigid rotor model. However, the internal rotation barrier of the selenol group splits the vibrational ground state into two subtorsional levels, doubling the dipole-inverting μb transitions. The simulation of the double-minimum internal rotation gives a very low barrier height (B3PW91: 42cm-1), much smaller than for thiophenol (277cm-1). A monodimensional Hamiltonian then predicts a huge vibrational separation of 72.2GHz, justifying the non-observation of μb transitions in our frequency range. The experimental rotational parameters were compared with different MP2 and density functional theory calculations. The equilibrium structure was determined using several high-level abinitio calculations. A final Born-Oppenheimer (reBO) structure was obtained at the coupled-cluster CCSD(T)_ae/cc-wCVTZ level of theory, including small corrections for the wCVTZ → wCVQZ basis set enlargement calculated at the MP2 level. The mass-dependent method with predicates was used to produce an alternative rm(2) structure. The comparison between the two methods confirms the high accuracy of the reBO structure and offers information on other chalcogen-containing molecules.

Coupling of nematic in-plane orientational ordering and equilibrium shapes of closed flexible nematic shells

The impact of the intrinsic curvature of in-plane orientationally ordered curved flexible nematic molecules attached to closed 3D flexible shells was studied numerically. A Helfrich-Landau-de Gennes-type mesoscopic approach was adopted where the flexible shell’s curvature field and in-plane nematic field are coupled and concomitantly determined in the process of free energy minimisation. We demonstrate that this coupling has the potential to generate a rich diversity of qualitatively new shapes of closed 3D nematic shells and the corresponding specific in-plane orientational ordering textures, which strongly depend on the shell’s volume-to-surface area ratio, so far not predicted in mesoscopic-type numerical studies of 3D shapes of closed flexible nematic shells.

Flexible Colloidal Molecules with Directional Bonds and Controlled Flexibility.

Colloidal molecules are ideal model systems for mimicking real molecules and can serve as versatile building blocks for the bottom-up self-assembly of flexible and smart materials. While most colloidal molecules are rigid objects, the development of colloidal joints has made it possible to endow them with conformational flexibility. However, their unrestricted range of motion does not capture the limited movement and bond directionality that is instead typical of real molecules. In this work, we create flexible colloidal molecules with an in situ controllable motion range and bond directionality by assembling spherical particles onto cubes functionalized with complementary surface-mobile DNA. By varying the sphere-to-cube size ratio, we obtain colloidal molecules with different coordination numbers and find that they feature a constrained range of motion above a critical size ratio. Using theory and simulations, we show that the particle shape together with the multivalent bonds creates an effective free-energy landscape for the motion of the sphere on the surface of the cube. We quantify the confinement of the spheres on the surface of the cube and the probability to change facet. We find that temperature can be used as an extra control parameter to switch in situ between full and constrained flexibility. These flexible colloidal molecules with a temperature switching motion range can be used to investigate the effect of directional yet flexible bonds in determining their self-assembly and phase behavior, and may be employed as constructional units in microrobotics and smart materials.

Ultrafast Electronic Coupling Estimators: Neural Networks versus Physics-Based Approaches.

Fast and accurate estimation of electronic coupling matrix elements between molecules is essential for the simulation of charge transfer phenomena in chemistry, materials science, and biology. Here we investigate neural-network-based coupling estimators combined with different protocols for sampling reference data (random, farthest point, and query by committee) and compare their performance to the physics-based analytic overlap method (AOM), introduced previously. We find that neural network approaches can give smaller errors than AOM, in particular smaller maximum errors, while they require an order of magnitude more reference data than AOM, typically one hundred to several hundred training points, down from several thousand required in previous ML works. A Δ-ML approach taking AOM as a baseline is found to give the best overall performance at a relatively small computational overhead of about a factor of 2. Highly flexible π-conjugated organic molecules like non-fullerene acceptors are found to be a particularly challenging case for ML because of the varying (de)localization of the frontier orbitals for different intramolecular geometries sampled along molecular dynamics trajectories. Here the local symmetry functions used in ML are insufficient, and long-range descriptors are expected to give improved performance.

Accuracy Meets Feasibility for the Structures and Rotational Constants of the Molecular Bricks of Life: A Joint Venture of DFT and Wave-Function Methods.

A fully unsupervised computational protocol is proposed with the aim of obtaining reliable structural properties for molecular bricks of life in the gas phase. The results of the new composite scheme approach spectroscopic accuracy at a moderate cost without any empirical parameter in addition to those of the underlying electronic structure method. The whole workflow is fully automated and provides optimized geometries and equilibrium rotational constants. Direct comparison with experimental ground state rotational constants can be performed thanks to the effective computation of vibrational corrections in the framework of second-order vibrational perturbation theory. The results for all the nucleic acid bases and several flexible molecules of biological or medicinal interest show that the accuracy of the new tool is close to that delivered by state-of-the-art composite wave function methods for small semirigid molecules.

HF trimer: 12D fully coupled quantum calculations of HF-stretch excited intramolecular and intermolecular vibrational states using contracted bases of intramolecular and intermolecular eigenstates.

We present the computational methodology, which for the first time allows rigorous twelve-dimensional (12D) quantum calculations of the coupled intramolecular and intermolecular vibrational states of hydrogen-bonded trimers of flexible diatomic molecules. Its starting point is the approach that we introduced recently for fully coupled 9D quantum calculations of the intermolecular vibrational states of noncovalently bound trimers comprised of diatomics treated as rigid. In this paper, it is extended to include the intramolecular stretching coordinates of the three diatomic monomers. The cornerstone of our 12D methodology is the partitioning of the full vibrational Hamiltonian of the trimer into two reduced-dimension Hamiltonians, one in 9D for the intermolecular degrees of freedom (DOFs) and another in 3D for the intramolecular vibrations of the trimer, and a remainder term. These two Hamiltonians are diagonalized separately, and a fraction of their respective 9D and 3D eigenstates is included in the 12D product contracted basis for both the intra- and intermolecular DOFs, in which the matrix of the full 12D vibrational Hamiltonian of the trimer is diagonalized. This methodology is implemented in the 12D quantum calculations of the coupled intra- and intermolecular vibrational states of the hydrogen-bonded HF trimer on an abinitio calculated potential energy surface (PES). The calculations encompass the one- and two-quanta intramolecular HF-stretch excited vibrational states of the trimer and low-energy intermolecular vibrational states in the intramolecular vibrational manifolds of interest. They reveal several interesting manifestations of significant coupling between the intra- and intermolecular vibrational modes of (HF)3. The 12D calculations also show that the frequencies of the v = 1, 2 HF stretching states of the HF trimer are strongly redshifted in comparison to those of the isolated HF monomer. Moreover, the magnitudes of these trimer redshifts are much larger than that of the redshift for the stretching fundamental of the donor-HF moiety in (HF)2, most likely due to the cooperative hydrogen bonding in (HF)3. The agreement between the 12D results and the limited spectroscopic data for the HF trimer, while satisfactory, leaves room for improvement and points to the need for a more accurate PES.

Ab Initio Thermochemistry of Highly Flexible Molecules for Thermal Decomposition Analysis.

Pyrolysis is a promising technology for chemical recycling of waste plastics, since it enables the generation of high-value chemicals with low capital and operating cost. The calculation of thermodynamic equilibrium composition using the Gibbs free energy minimization approach can determine pyrolysis operating conditions that produce desired products. However, the availability of thermochemical data can limit the application of equilibrium calculations. While density functional theory (DFT) calculations have been commonly used to produce accurate thermochemical data (e.g., enthalpies of formation) of small molecules, the accuracy and computational cost of these calculations are both challenging to handle for large, flexible molecules, exhibiting multiple conformations at elevated (i.e., pyrolysis) temperatures. In this work, we develop a computational framework to calculate accurate, temperature-dependent thermochemistry of large and flexible molecules by combining force field based conformational search, DFT calculations, thermochemical corrections, and Boltzmann statistics. Our framework produces accurately calculated thermochemistry that is used to predict equilibrium thermal decomposition profiles of octadecane, a model compound of polyethylene. Our thermochemistry results are compared against literature data demonstrating a great agreement, and the predicted decomposition profiles rationalize a series of pyrolysis experimental observations. Our work systematically addresses entropic contributions of large molecules and suggests paths for accurate and yet computationally feasible calculations of Gibbs free energies. The first-principles-based thermodynamic equilibrium analysis proposed in this work can be a significant step toward predicting temperature-dependent product distributions from plastic pyrolysis and guide experimentation on chemical plastic recycling.

Spectroscopic and Theoretical Studies on Conformational Stability of Benzyl Methyl Ether.

Conformer-selected electronic and vibrational spectra of benzyl methyl ether and its terminal methyl group-substituted derivative in a supersonic jet have been measured using ultraviolet (UV)-UV hole burning and fluorescence-detected infrared spectroscopy to investigate the conformational stability of flexible molecules. Various quantum chemical calculations as well as experimental observations reveal the coexistence of three conformers with different CCOC dihedral angles and side-chain orientations relative to the benzene ring plane. Vibrational analysis in the excited state with time-dependent density functional theory and IR simulations containing anharmonic coupling sufficiently reproduce the experimental results, suggesting that these three conformers can be distinguished into one gauche-conformer and two trans-ones with respect to the CCOC dihedral angle. We also observe that the gauche conformer exhibits higher-frequency CH2 modes. The natural bond orbital analysis indicates that this phenomenon is attributed to the electron delocalization from the non-bonding orbitals and the C-O orbitals associated with the neighboring oxygen atom, which leads to a conformer dependence of the methylene C-H bond strength.

How Accurate Can Crystal Structure Predictions Be for High-Energy Molecular Crystals?

Molecular crystals have shallow potential energy landscapes, with multiple local minima separated by very small differences in total energy. Predicting molecular packing and molecular conformation in the crystal generally requires ab initio methods of high accuracy, especially when polymorphs are involved. We used dispersion-corrected density functional theory (DFT-D) to assess the capabilities of an evolutionary algorithm (EA) for the crystal structure prediction (CSP) of well-known but challenging high-energy molecular crystals (HMX, RDX, CL-20, and FOX-7). While providing the EA with the experimental conformation of the molecule quickly re-discovers the experimental packing, it is more realistic to start instead from a naïve, flat, or neutral initial conformation, which reflects the limited experimental knowledge we generally have in the computational design of molecular crystals. By doing so, and using fully flexible molecules in fully variable unit cells, we show that the experimental structures can be predicted in fewer than 20 generations. Nonetheless, one must be aware that some molecular crystals have naturally hindered evolutions, requiring as many attempts as there are space groups of interest to predict their structures, and some may require the accuracy of all-electron calculations to discriminate between closely ranked structures. To save resources in this computationally demanding process, we showed that a hybrid xTB/DFT-D approach could be considered in a subsequent study to push the limits of CSP beyond 200+ atoms and for cocrystals.