Desolvation Processes in Channel Solvates of Niclosamide.

The antiparasitic drug niclosamide (NCL) is notable for its ability to crystallize in multiple 1:1 channel solvate forms, none of which are isostructural. Here, using a combination of time-resolved synchrotron powder X-ray diffraction and thermogravimetry, the process-induced desolvation mechanisms of methanol and acetonitrile solvates are investigated. Structural changes in both solvates follow a complicated molecular-level trajectory characterized by a sudden shift in lattice parameters several degrees below the temperature where the desolvated phase first appears. Model fitting of kinetic data obtained under isothermal heating conditions suggests that the desolvation is rate-limited by the nucleation of the solvent-free product. The desolvation pathways identified in these systems stand in contrast to previous investigations of the NCL channel hydrate, where water loss by diffusion initially yields an anhydrous isomorph that converts to the thermodynamic polymorph at significantly higher temperatures. Taking the view that each solvate lattice is a unique "pre-organized" precursor, a comparison of the pathways from different starting topologies to the same final product provides the opportunity to reevaluate assumptions of how various factors (e.g., solvent binding strength, density) influence solid-state desolvation processes.

Enhancement of Hydrate Stability through Substitutional Defects.

Cytosine monohydrate (CM) and anhydrate crystal forms reversibly interconvert under high temperatures or high humidity conditions. Here, we demonstrate through defect engineering the ability to expand the thermal stability range of CM through the targeted creation of quantifiable defects in low-level concentrations. Twelve different molecular dyes with a variety of core structures and charges were screened as potential dopants in CM. CM-dye phases prepared with Congo red (CR), Evans blue (EB), and Azocarmine G (AG) exhibited the highest inclusion levels (up to 1.1 wt %). In these doped isomorphous materials, each dye is presumed to substitute for 4-7 cytosine molecules within the low-rugosity (102) planes of the CM matrixes, thereby creating a quantifiable substitutional defect and an impediment to the cooperative molecular motions which enable the transformation to the anhydrate. Dehydration of materials with these engineered defects requires significantly higher temperatures and proceeds with slower kinetics compared to pure CM. The CM-dye phases also exhibit a reduction in the thermal expansion along key crystallographic axes and yield dehydration products with altered particle morphologies.

Dehydration of Niclosamide Monohydrate Polymorphs: Different Mechanistic Pathways to the Same Product.

Many active pharmaceutical ingredients (APIs) can crystallize as hydrates or anhydrates, the relative stability of which depends on their internal structures as well as the external environment. Hydrates may dehydrate unexpectedly or intentionally, though the molecular-level mechanisms by which such transformations occur are difficult to predict a priori. Niclosamide is an anthelmintic drug on the World Health Organization's "List of Essential Medicines" that crystallizes in two monohydrate forms: HA and HB. Through complementary time-resolved synchrotron powder X-ray diffraction and thermogravimetric kinetic studies, we demonstrate that the two monohydrates dehydrate via distinctly different solid state pathways yet yield the same final anhydrate phase. Water loss from HA via diffusion yields an isomorphous desolvate intermediate which can rearrange to at least two different polymorphs, only one of which exhibits long-term stability. In contrast, dehydration of HB proceeds via a surface nucleation process where simultaneous water loss and product formation occur with no detectable crystalline intermediates. Comparative analysis of the two systems serves to highlight the complex relationship between lattice structure and solid state dehydration processes.

An Improved Model for Biogenic Ammonium Urate.

The pathological crystallization of ammonium urate inside the urinary tract is a well-documented medical condition; however, structural studies of the biogenic material have proven challenging owing to its propensity to precipitate as a powder and to exhibit diffraction patterns with widely varying intensities. Using block Rietveld refinement methods of powder diffraction data, here we identify ammonium urate hydrate (AUH) as a likely component in natural uroliths. AUH has a planar 2-D hydrogen-bonded organic framework of urate ions separated by ammonium ions with water molecules residing in bisecting channels. AUH is stable up to 150 °C for short time periods but begins to decompose with prolonged heating times and/or at higher temperatures. Changes in the solid-state structure and composition of synthetic material over a temperature range from 25 to 300 °C are elucidated through thermogravimetric and spectroscopic data, combustion analysis, and time-resolved synchrotron powder X-ray diffraction studies. We contend that biogenic ammonium urate is more accurately modeled as a mixture of AUH and anhydrous ammonium urate, in ratios that can vary depending on the growth environment. The similar but not identical diffraction patterns of these two forms likely account for much of the variability seen in natural ammonium urate samples.

Adhesion properties of uric acid crystal surfaces.

Two key steps in kidney stone formation--crystal aggregation and attachment to renal tissues--depend on the surface adhesion properties of the crystalline components. Anhydrous uric acid (UA) is the most common organic crystalline phase found in human kidney stones. Using chemical force microscopy, the adhesion force between various functional groups and the largest (100) surface of UA single crystals was measured in both aqueous solution and model urine. Adhesion trends in the two solutions were identical, but were consistently lower in the latter. Changes in the solution ionic strength and pH were also found to affect the magnitude of the adhesion. UA surfaces showed the strongest adhesion to cationic functionalities, which is consistent with ionization of some surface uric acid molecules to urate. Although hydrogen-bonding and van der Waals interactions are usually considered to be dominant forces in the association between neutral organic compounds, this work demonstrates that electrostatic interactions can be important, particularly when dealing with weak acids under certain solution conditions.

Evaluation of pyridoacridine alkaloids in a zebrafish phenotypic assay.

Three new minor components, the pyridoacridine alkaloids 1-hydroxy-deoxyamphimedine (1), 3-hydroxy-deoxyamphimedine (2), debromopetrosamine (3), and three known compounds, amphimedine (4), neoamphimedine (5) and deoxyamphimedine (6), have been isolated from the sponge Xestospongia cf. carbonaria, collected in Palau. Structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1-6 were evaluated in a zebrafish phenotype-based assay. Amphimedine (4) was the only compound that caused a phenotype in zebrafish embryos at 30 muM. No phenotype other than death was observed for compounds 1-3, 5, 6.

Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening.

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.

Crystal structure of a di-aryl carbonate: 1,3-phenyl-ene bis-(phenyl carbonate).

The whole mol-ecule of the title compound, C20H14O6, is generated by mirror symmetry, the mirror bis-ecting the central benzene ring. The carbonate groups adopt an s-cis-s-cis conformation, with torsion angles of 58.7 (2) and 116.32 (15)°. The crystal structure of 1,3-phenyl-ene bis-(phenyl carbonate) contains no strong hydrogen bonds, though weak C-H⋯O and offset π-π inter-actions are observed, forming layers parallel to the ac plane.

A new polymorph of 2,6-diaminopyridine.

2,6-Diaminopyridine (26-DAP, C5H7N3) is a common intermediate in the synthesis of aromatic azo chromophores, which are widespread in the dyes and pigments industry. Sublimation of commercial 26-DAP powder yielded a new polymorph, denoted Form II, which grew as colorless orthorhombic needles. Recrystallization from acetone or toluene also yielded Form II as the major phase. Thermal analysis shows that Form II is a less stable polymorph and it converts upon heating at 335 K to the previously reported Form I.

Control of Tungiasis in Absence of a Roadmap: Grassroots and Global Approaches.

Tungiasis is a tropical skin disease caused by the sand flea Tunga penetrans. It inflicts misery upon tens of millions of people, mostly children, across Central and South America and sub-Saharan Africa, and yet there is no globally accepted roadmap for its control. Here we review how research in the last 15 years has developed control methods and report on new grassroots and digital mapping approaches. Treatment is now possible with a two-component dimethicone, used for the treatment of headlice in Europe, Asia and Canada, but not yet available in most tungiasis-endemic areas. Prevention is possible through the daily use of repellents based on coconut oil. A Kenyan coastal community has successfully controlled tungiasis using a neem and coconut oil mix produced locally to treat cases, combined with spraying floors with neem solution and distributing closed shoes. Development of affordable hard floor technology is underway, although not yet widely available, but is a priority to control off-host stages in the floors of homes. A new web-based digital mapping application will enable researchers and health officials to collaborate, share data and map the prevalence of tungiasis. We conclude that tungiasis can be controlled through a multi-disciplinary, One Health approach.

Two tautomeric forms of 2-amino-5,6-dimethylpyrimidin-4-one.

Derivatives of 4-hydroxypyrimidine are an important class of biomolecules. These compounds can undergo keto-enol tautomerization in solution, though a search of the Cambridge Structural Database shows a strong bias toward the 3H-keto tautomer in the solid state. Recrystallization of 2-amino-5,6-dimethyl-4-hydroxypyrimidine, C6H9N3O, from aqueous solution yielded triclinic crystals of the 1H-keto tautomer, denoted form (I). Though not apparent in the X-ray data, the IR spectrum suggests that small amounts of the 4-hydroxy tautomer are also present in the crystal. Monoclinic crystals of form (II), comprised of a 1:1 ratio of both the 1H-keto and the 3H-keto tautomers, were obtained from aqueous solutions containing uric acid. Forms (I) and (II) exhibit one-dimensional and three-dimensional hydrogen-bonding motifs, respectively.

Concomitant polymorphs of 1,3-bis(3-fluorophenyl)urea.

Hydrogen bonding between urea functionalities is a common structural motif employed in crystal-engineering studies. Crystallization of 1,3-bis(3-fluorophenyl)urea, C13H10F2N2O, from many solvents yielded concomitant mixtures of at least two polymorphs. In the monoclinic form, one-dimensional chains of hydrogen-bonded urea molecules align in an antiparallel orientation, as is typical of many diphenylureas. In the orthorhombic form, one-dimensional chains of hydrogen-bonded urea molecules have a parallel orientation rarely observed in symmetrically substituted diphenylureas.

An in situ atomic force microscopy study of uric acid crystal growth.

Kidney stones are heterogeneous polycrystalline aggregates that can consist of several different building blocks. A significant number of human stones contain uric acid crystals as a crystalline component, though the molecular-level growth of this important biomaterial has not been previously well-characterized. In the present study, in situ atomic force microscopy (AFM) is used to investigate the real-time growth on the (100) surface of uric acid (UA) single crystals as a function of fundamental solution parameters. Layer-by-layer growth on UA (100) was found to be initiated at screw dislocation sites and to proceed via highly anisotropic rates which depend on the crystallographic direction. The smallest b-steps exhibited minimum heights corresponding to two molecular layers, while fast-moving c-steps more commonly showed monolayer heights. Growth kinetics measured under a range of flow rates, supersaturation levels, and pH values reveal linear trends in the growth kinetics, with faster growth attained in solutions with higher supersaturation and/or pH. The calculated kinetic parameters for UA growth derived from these experiments are in good agreement with the values reported for other crystal systems.

A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa.

BACKGROUND: Camelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits. RESULTS: Expressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50-57 %, while expression of full bypass increased seed yield by 57-73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions. CONCLUSIONS: The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops.

Selective growth of a less stable polymorph of 2-iodo-4-nitroaniline on a self-assembled monolayer template.

Orthorhombic and triclinic crystals of 2-iodo-4-nitroaniline (INA) grow concomitantly from supersaturated ethanol solutions, but the less stable orthorhombic phase can be selectively grown on 3'-X-4-mercaptobiphenyl (X = NO(2), I) self-assembled monolayer templates.

Retinoic acid is required for endodermal pouch morphogenesis and not for pharyngeal endoderm specification.

Because tissues from all three germ layers contribute to the pharyngeal arches, it is not surprising that all major signaling pathways are involved in their development. We focus on the role of retinoic acid (RA) signaling because it has been recognized for quite some time that alterations in this pathway lead to craniofacial malformations. Several studies exist that describe phenotypes observed upon RA perturbations in pharyngeal arch development; however, these studies did not address whether RA plays multiple roles at distinct time points during development. Here, we report the resulting phenotypes in the hindbrain, the neural crest-derived tissues, and the pharyngeal endoderm when RA synthesis is disrupted during zebrafish gastrulation and pharyngeal arch morphogenesis. Our results demonstrate that RA is required for the post-gastrulation morphogenesis and segmentation of endodermal pouches, and that loss of RA does not affect the length of the pharyngeal ectoderm or medial endoderm along the anterior-posterior axis. We also provide evidence that RA is not required for the specification of pharyngeal pouch endoderm and that the pharyngeal endoderm consists of at least two different cell populations, of which the pouch endoderm is sensitive to RA and the more medial pharyngeal endoderm is not. These results demonstrate that the developmental processes underlying pharyngeal arch defects differ depending on when RA signaling is disturbed during development.

Controlling molecular crystal polymorphism with self-assembled monolayer templates.

The control of crystal polymorphism is a long-standing issue in solid-state chemistry, which has many practical implications for a variety of commercial applications. At least four different crystalline forms of 1,3-bis(m-nitrophenyl) urea (MNPU), a classic molecular crystal system, are known to crystallize from solution in various concomitant combinations. Herein we demonstrate that the introduction of gold-thiol self-assembled monolayers (SAMs) of substituted 4'-X-mercaptobiphenyls (X = H, I, and Br) into the crystallization solution can serve as an effective means to selectively template the nucleation and growth of alpha-, beta-, and gamma-MNPU phases, respectively. Polymorph control in the presence of SAM surfaces persists under a variety of solution conditions and consistently results in crystalline materials with high phase purity. The observed selectivity is rationalized on the basis of long-range two-dimensional geometric lattice matching and local complementary chemical interactions at the SAM/crystal interfaces.

Selective growth and distribution of crystalline enantiomers in hydrogels.

The crystallization of sodium chlorate (NaClO3) is a classic example of spontaneous chirality, since it is achiral in solution but adopts a chiral form in the solid state. While crystal growth of NaClO3 from pure aqueous solutions yields a 50:50 statistical distribution of d- and l-crystals, large enantiomeric excesses of either d- and l-crystals can be achieved by crystal growth in agarose gel, a naturally occurring chiral polysaccharide. The influence of gel density (0.1-0.75 wt %), temperature, and the diffusion of cosolvents on crystal distribution was discerned from statistical data obtained from 752 gel-mediated crystallization experiments yielding 12,384 individual crystals. These studies demonstrate that the magnitude and direction of the bias can be selectively engineered toward either d- or l-forms by changing the gelation conditions. Aqueous agarose gels infused with 48 wt % NaClO3 at 6 degrees C, favored the growth of d-NaClO3 crystals, with ee's reaching 22% at the highest gel concentrations. Crystal growth under methanol diffusion favored deposition of the opposite enantiomorph, l-NaClO3. The bias in the crystal distribution is enhanced at higher temperatures. Aqueous gels at 24 degrees C infused with methanol cosolvent favored l-NaClO3, with ee's reaching 53%. The changing magnitude and direction of the enantiomorph bias can be ascribed to differences in the agarose conformation and intermolecular interactions between the gel and crystal surfaces that inhibit the formation of the two enantiomers to different extents.

Dyeing uric acid crystals with methylene blue.

The growth of anhydrous uric acid (UA) and uric acid dihydrate (UAD) crystals from supersaturated aqueous solutions containing methylene blue, a cationic organic dye, has been investigated. Low concentrations of dye molecules were found to be included in both types of crystal matrixes during the growth process. Incorporation of dye into UA crystals occurs with high specificity, affecting primarily [001] and [201] growth sectors, while UAD crystals grown from solutions of similar dye concentration show inclusion but little specificity. The orientation of the UA-trapped species was determined from polarization data obtained from visible light microspectrometry. To achieve charge neutrality, a second anionic species must also be included with the methylene blue into UA and UAD crystal matrices. Under high pH conditions, crystallization of 1:1 stoichiometric mixtures of methylene blue and urate yields methylene blue hexahydrate (MBU.6(H2O). The crystal structure of MBU.6(H2O) reveals continuous pi-pi stacks of planes of dye cations and urate anions mediated by water molecules. This structure provides an optimal geometry for methylene blue-urate pairs and additional support for the incorporation of these dimers in uric acid single-crystal matrices. The strikingly different inclusion patterns in UA and UAD demonstrate that subtle changes in the crystal surfaces and/or growth dynamics can greatly affect recognition events.

Epitaxial relationships between uric acid crystals and mineral surfaces: a factor in urinary stone formation.

Uric acid (C5H4N4O3) is one of the final products of purine metabolism. Its concentration balance is maintained in the kidneys, but compromised kidney function can result in its crystallization either in the renal tract or in the interstitial fluid of joints. In physiological deposits, crystalline uric acid is most frequently found either in a protonated state (anhydrous or dihydrate phases) or as a deprotonated urate ion (sodium or ammonium salts). Often these precipitates are found in association with a number of mineral phases (e.g., calcium oxalates, calcium phosphates, and magnesium phosphates). Their frequent and common coexistence suggests that synergistic relationships between these crystalline phases may exist. A comprehensive list of different heterogeneous uric acid/uric acid and uric acid/mineral interfaces that are epitaxially matched was generated with the lattice-matching program EpiCalc. Two hundred twenty-five coincident epitaxial matches and four commensurate epitaxial matches were identified using this screening procedure.