Aurora J. Cruz Cabeza

HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid) is a widely used buffering agent in biological and biomedical research. Here, we reveal an exceptional solid-form landscape for HEPES. In addition to the two previously known polymorphs, we discover and characterize three new polymorphs and three new hydrates. These solid forms differ not only in crystal packing but also in conformation and protonation states, giving rise to a unique case of rich conformational multi-zwitterionic polymorphism. All forms are characterized using a comprehensive suite of experimental techniques, their phase transformations are examined, and their structures are further investigated through computational modeling. Energetic analyses uncover an unusual interplay between conformation, ionization state, and intermolecular interactions in determining the thermodynamically stable form. Together, the experimental and computational insights reveal the complex solid-state behavior of zwitterionic molecules and the resulting challenges for modeling, crystallization control, and overall early solid form and process development in the pharmaceutical and other industries.

The control of crystallization processes in polymorphic systems remains a long-term goal at both laboratory and industrial scales. The rational design of processes that yield specific, desired crystal structures with suitable physical properties requires not only phase equilibrium data but also insights into the relative nucleation and growth rates of available crystal forms. For polymorphic systems that crystallize concomitantly, not only is such data largely unavailable, but also it is difficult to measure. This study determines the nucleation and growth kinetics of tolfenamic acid forms I and II from induction time distributions obtained from their crystallization in isopropanol in small volumes. The significant influence of crystal growth on both the form and analysis of induction time distributions has been examined, and methods to appropriately account for this effect are discussed. An analytical solution is developed, describing the induction time distribution in a dimorphic system in terms of the nucleation and growth kinetics of both forms. Despite the relevance of such data, this appears to be the first occasion in which such kinetic data have been obtained in a concomitantly crystallizing system.

Mapping crystallization outcomes in ternary solvent mixtures for systems able to solvate remains experimentally intensive due to the significant compositional phase space that needs to be explored to derive complete phase diagrams. Here, we show that these phase diagrams can be generated readily by combining rapid mechanochemical determination of critical solvent activities using binary solvent mixtures with thermodynamic activity mapping such as NRTL modeling. This approach enables quantitative prediction of ternary phase diagrams without direct experimental mapping of the full compositional space. Using nitrofurantoin as a model system, which forms an anhydrous phase, a monohydrate, and a dmf solvate, we determine the critical solvent activities from binary mixtures and use these together with predicted activity maps to generate ternary phase diagrams. The resulting framework accurately predicts phase boundaries and solid-form outcomes across two ternary solvent systems. Comparison with experimental data across 108 controlled solvent activity liquid assisted grinding (CSA-LAG) outcomes shows excellent agreement, with predictive accuracy exceeding 90% and correct reproduction of phase diagram topology. This strategy reduces the required experimental effort by more than 70% while providing mechanistic insight into competing solvation equilibria through solvent activity and activity-ratio landscapes. Since multicomponent solvate phase behavior is governed by solvent activity rather than composition, determination of critical activities in low dimensional space with activities modeling allow for a shift from empirical screening to thermodynamically guided prediction in crystallization design.

Hydrates are common solid forms that can significantly affect a compound’s stability, physicochemical properties, and commercial viability. Despite their importance in pharmaceutical development, hydrate structures, their relationship to material properties, and their propensity for formation remain poorly understood. In 2003, Gillon et al. introduced a simple yet powerful framework for the structural classification of hydrates based on the hydrogen-bonding environment of crystalline water molecules. This framework categorizes water molecules according to the number of hydrogen-bond donor (D) and acceptor (A) interactions in which they participate, giving rise to eight distinct environments. In the original study, the DDA environment, where water donates two hydrogen bonds and accepts one, was reported as the most common. The statistical distribution of these environments in the Cambridge Structural Database (CSD) has since provided a useful benchmark for the qualitative assessment of hydrate structures. Here, we revisit Gillon’s water environment classification using a substantially expanded data set comprising 13,881 hydrate entries from the CSD and provide updated statistics on hydrate structures. Our analysis confirms that the DDA environment remains the most prevalent, followed by DDAA and DD, while the remaining environments occur less frequently. Extending beyond the original work, we quantify the energetics associated with each environment and demonstrate that the four-hydrogen-bond DDAA environment is energetically the most favorable, followed by environments involving three, two, and one hydrogen bonds, respectively. We further show that water-mediated intermolecular interactions contribute up to 40% of the total lattice interaction energy in the hydrate structures, highlighting the surprisingly large influence of this small solvent molecule on crystal stability. Despite its superior energetic stability, the DDAA environment is not the most frequently observed experimentally. This apparent discrepancy arises because the hydrogen-bonding capability and topology of the main component in the hydrate constrains the maximum hydrogen-bonding environment that water can achieve. Overall, this work provides a comprehensive analysis of hydrate structures and energetics across the CSD. The resulting insights offer a valuable framework for the qualitative assessment of newly discovered hydrate forms and for evaluating whether their structures conform to established crystallographic trends.

Surface properties of molecular crystals play a central role in determining their nucleation, growth, and overall crystallisation behaviour. In particular, surface rugosity has been suggested as a meaningful descriptor of how readily a crystal may nucleate and grow, but the existing rugosity metrics have been limited by incomplete descriptions of surface topology. In this study, we present a new workflow for the computation of surface descriptors across crystal facets. Here the surface rugosity description for each facet is based on the Surface Area Ratio (SAR) definition proposed in the Cambridge Structural Database (CSD) tools. Given a crystal structure, our algorithm computes an average overall particle rugosity with three offset selection criteria that account for different crystallisation conditions. We then calculate particle rugosities and analyse data for a range of systems, including: polymorphic families, datasets from the CSD, and crystal structure prediction (CSP) landscapes. Our analysis shows that, in most cases, polymorphs with lower rugosities tend to nucleate and grow more readily, suggesting that this metric can distinguish experimentally accessible forms from more elusive ones. These findings demonstrate that particle rugosities can serve as a complementary tool for predicting and classifying the experimental feasibility of polymorphs generated computationally.

Predicting when and why molecular salts form hydrates has remained a significant problem in solid-state chemistry, despite its impact on the stability and performance of industrially relevant materials. Here, we identify counterion charge localization as a key determinant of hydrate formation in molecular salts, providing a framework for predicting and controlling solid-state hydration. By analyzing over 31,000 crystal structures and performing electron density calculations, we show that delocalized counterion charge suppresses hydrate formation, while localized charge promotes it. This principle makes hydration propensity a more tuneable property (from ~80% to K _a rule, we experimentally demonstrate rational control of hydration in salts of 4-aminoacetanilide. Our results establish a direct link between molecular electrostatics and crystallization outcomes, affording predictive design of molecular salts with tailored hydration behavior.

The ability to understand crystallization and predict the resulting solid form of a system is not always easily achieved, but it is critical, particularly in the field of materials science. Intriguing (and previously unreported) crystallization behavior is observed with terephthalic dihydrazide (TeDi) as it rapidly forms two concomitant crystalline polymorphs upon cooling in solution. The crystal morphology of Form I (FI) has not been seen before in organic systems and involves impressive, accordion-like stacks, composed of numerous twin domains and remains stable in solution for years. Form II (FII) exists as large needles that disappear in solution after 20 h. All experimental methods employed reveal that FI is the most stable polymorph. Conversely, all computational methods utilized (conformational analyses, lattice energy calculations, and crystal structure prediction) suggest that FII is the most stable polymorph. Isolation of FII was achieved by the crystallization of TeDi powder with a supramolecular mimetic gelator, as the gel fibers act as a template for the preferential crystallization of FII, due to the comparable crystal packing of FII and the gelator. This work highlights the impact of crystallization behavior in a real laboratory and the defects, disorder, and twinning that lead to remarkable crystal morphologies that may not be accounted for with idealized calculations, and also explores approaches for controlling and directing crystallization outcomes.

Crystalline solvates, including hydrates, hold untapped potential in pharmaceutical development, yet their exploitation remains minimal due to the difficult and laborious task of unequivocally establishing their physical stabilities. We introduce Controlled Solvent-Activity Liquid-Assisted Grinding (CSA-LAG), a mechanochemical protocol that offers solvate phase boundary elucidation by varying the activity of a chosen solvent in defined binary/ternary mixtures and analysing the equilibrated resulting solid form. Using small API amounts, CSA-LAG reaches equilibrium within minutes and yields critical solvent activities that delimit neat, hydrated, solvated and competing-solvate domains. The method uses mixtures of known thermodynamic activities, requires far less material and time than traditional slurries and affords high reproducibility. Applied to four pharmaceutical compounds, CSA-LAG reproduces slurry boundaries and quantifies activity thresholds for single, stepwise and competitive solvations. Defining these boundaries enables rational form selection and process design either by avoiding or targeting solvates, whilst turning a month-scale empirical screening into a rapid, thermodynamically guided workflow.

Professor Angelo Gavezzotti’s scientific works have shaped the field of structural chemistry and inspired crystallographers and molecular modellers alike. To honour his legacy in this contribution, the Durham Molecular Crystals and Crystallisation (DuMoC ² ) group (and friends) have reviewed his work with a touch of humour and enormous gratitude. After presenting a concise review of his scientific contributions to crystallography and molecular modelling, the Gavezzotti Structural Database (GSD) was compiled and compared to the Cambridge Structural Database (CSD). We then used Angelo’s PIXEL method to analyse some of the GSD crystal structures. Together, through the writing of this paper, we celebrate Angelo’s career.

Flexible molecules change their conformations to optimize intermolecular interactions in the solid-state ─a process that comes at an intramolecular energy cost. The interplay between these opposing forces leads to beautifully balanced and elegant crystal structures. Yet, the relationship between intramolecular energy penalties and intermolecular stabilization has not been fully explored, largely because it requires models capable of accurately capturing components with distinct physical origins. In this work, we employ state-of-the-art hybrid DFT models to probe these interactions and quantify their energetic contributions to crystal lattice energies. After benchmarking 18 computational methods, we identify PBE-MBD/B2PLYPD as the method best reproducing experimental polymorph stabilities with mean absolute deviations of just 2.3 kJ·mol^-1 across 17 polymorphic pairs. Using this model, we compute and analyze lattice energy partitions for 125 crystal structures spanning a diverse set of flexible compounds. Our analysis reveals a striking empirical trend─the "40% limit"─which shows that up to 40% of the intermolecular stabilization energy can compensate for intramolecular energy penalties associated with conformational variations. Furthermore, the probability of a higher-energy conformation being observed in the solid-state decreases as a function of the intra-to-intermolecular energy ratio and becomes negligible at the 40% mark. These findings are significant: they define the energetic limits of conformational flexibility in the solid-state and the intra-to-intermolecular energy ratios provide a quantitative tool to predict structures. They can be used to guide the conformational phase space sampling for crystal structure prediction, to rank hypothetical structures according to their crystallizability, or to anticipate difficulties of nucleation and crystal growth in crystal forms of flexible compounds.