No abstract

The crystallization mechanisms of organic molecules in solution are not well-understood. The mechanistic scenarios where crystalline order evolves directly from the molecularly dissolved state (“classical”) and from initially formed amorphous intermediates (“nonclassical”) are suggested and debated. Here, we studied crystallization mechanisms of two widely used analgesics, ibuprofen (IbuH) and etoricoxib (ETO), using direct cryogenic transmission electron microscopy (cryo-TEM) imaging. In the IbuH case, parallel crystallization pathways involved diverse phases of high and low density, in which the instantaneous formation of final crystalline order was observed. ETO crystallization started from well-defined round-shaped amorphous intermediates that gradually evolved into crystals. This mechanistic diversity is rationalized by introducing a continuum crystallization paradigm: order evolution depends on ordering in the initially formed intermediates and efficiency of molecular rearrangements within them, and there is a continuum of states related to the initial order and rearrangement rates. This model provides a unified view of crystallization mechanisms, encompassing classical and nonclassical pictures.

Structural analysis of beam‐sensitive materials by transmission electron microscopy (TEM) represents a significant challenge, as high‐resolution TEM (HRTEM) requires high electron doses that limit its applicability to stable inorganic materials. Beam‐sensitive materials, e.g., organic crystals, must be imaged under low dose conditions, leading to problematic contrast interpretation and loss of fine structural details. Here, HRTEM imaging of organic crystalline materials with near‐atomic resolution of up to 1.6 Å is described, which enables real‐space studies of crystal structures, as well as observation of co‐existing polymorphs, crystal defects, and atoms. This is made possible by a low‐dose focal‐series reconstruction methodology, which provides HRTEM images where contrast reflects true object structure and can be performed on contemporary cryo‐EM instruments available to many research institutions. Copper phthalocyanine (CuPc), a perchlorinated analogue of CuPc, and indigo crystalline films are imaged. In the case of indigo crystals, co‐existing polymorphs and individual atoms (carbonyl oxygen) can be observed. In the case of CuPc, several polymorphs are observed, including a new one, for which the crystal structure is found based on direct in‐focus imaging, accomplishing real‐space crystal structure elucidation. Such direct analysis can be transformative for structure studies of organic materials.

Organic crystal nucleation and growth are complex processes that often do not fit into the framework of the existing crystallization theories. We investigated a crystal growth mechanism of an organic dye, perylene diimide, using high-resolution cryogenic transmission electron microscopy and optical spectroscopy. The elucidated mechanism involves classical (monomer attachments) and nonclassical pathways, exhibiting a self-assembly sequence where all steps are interconnected. It starts from the assembly of molecular π-stacks that are initially disordered. They gradually optimize their structure, rigidify, and interact to form crystalline domains. The latter further evolve via the addition of individual molecules, and crystal fusion (via oriented attachment). All the observed supramolecular transformations are connected and follow a clear hierarchy starting from the molecular-scale interactions. The elucidation of the complex pathway of organic crystallization as a series of coordinated supramolecular transformations at multiple scales conceptually advances the understanding of order evolution in organic matter.

Detoxification of heme in Plasmodium and other blood-borne parasites results in crystallization of hemozoin. This crystallization is a major target of antimalarial drugs. The structure of hemozoin has been studied primarily by X-ray powder diffraction, which showed that the unit cell contains a centrosymmetric cyclic hematin dimer. The pro-chiral nature of heme would support formation of two centrosymmetric and two enantiomeric chiral dimer structures. Here we apply emerging methods of cryo-electron microscopy, tomography, and diffraction to obtain a definitive structure of biogenic hemozoin. Individual crystals typically take a polar shape with morphology distinct from that of the more commonly-studied synthetic form. Diffraction analysis and density functional theory indicate a compositional mixture of one centrosymmetric and one chiral dimer, whose absolute configuration has been determined on the basis of crystal morphology and interaction with the aqueous medium. Structural modeling of the heme detoxification protein suggests a mechanism for such enantiomeric selection in dimer production. The refined structure of native hemozoin should serve as a guide to new drug development.