- Determination of the structure of co-crystals
Example: Characterisation of pharmaceutical co-crystals
- Examination of the insects using phase-contrast computer tomography (CT)
Example: Investigation of morphological changes in the irradiated malaria transmitting mosquito specimens.
- Structure determination of short DNA oligomers
Example: Recognition of Z-DNA by dinuclear transition metal complexes.
Pharmacy and Biology – Examples of services
Co-crystals may open the door to new patents and for product tailoring of pharmaceutical products. As there is no clear definition of the term evidence is difficult. Starting from this situation, pharmaceutical ingredients were co-crystallized by mechanical and supercritical methods at Fraunhofer ICT and the products have been characterized by X-ray diffraction, performed at the Diff-beamline at ANKA, Karlsruhe. The results give evidence to structural interaction of ingredients beyond pure physical blend, which may help define and identify co-crystals.
Co-crystals are discussed within the particle engineering community, particularly, in context of pharmaceutical applications. The interest originates from investigations showing that co-crystallized ingredients may reveal a disproportional enhancement of properties not only additional. The term »co-crystal« is not clearly defined but most commonly used in order to describe a crystal containing two or more components together. Thus, co-crystals may encompass molecular compounds, molecular complexes, solvates, inclusion compounds, channel compounds, clathrates and other types of multi-component systems in a crystalline state. An additional requirement may be that ingredients show structural interaction one another beyond a pure physical blend. One should be aware that on that base a co-crystallization not necessarily yields co-crystals.
In literature mechanical processes for co-crystals are described, where crystalline particles of different ingredients are brought together tightly by means of a ball mill. By adding a few solvent droplets to the mixture, particles may be glued together to co-crystals, as the solved surface molecules may chain the ingredients. Another approach is the fast crystallization of mixtures, so that ingredients have no time to follow their affinity to separate (what is used in chemistry to increase purity by slow recrystallization processes).
Investigations were started, testing co-crystallization of 1:1 mixtures of the pharmaceutical ingredients paracetamol, cholesterol, caffeine, ibuprofen, lactose and poly lactic acid using the supercritical fluid technologies RESS and PCA and a mini ball mill process.
SEM-pictures of paracetamol,caffeine and
as yielded by PCA-process.
Hence, the crystal structures of ingredients and mixtures have been investigated by means of X-ray powder diffraction. A differentiation of co-crystals from physical blends is obvious when crystal structures change completely. More difficult is a differentiation on the basis of slighly disturbed crystal lattices, particularly, of low absorbing materials]. Therefore the high flux and resolution of the synchrotron radiation at ANKA, Karlsruhe, was used.
Fig. 2 depicts selected diffraction pattern of the ingredients paracetamol and cholesterol, and a physical and a ball milled blend. Here emerging new peaks at about 10.18 and 10.93 °2Theta indicate changes of the crystal structures of the ball milled compared to the physical blend. Further changes are depicted in Fig. 3 where shifting of selected caffeine peaks indicate an anisotropic residual strain of the caffeine-lattice, when co-crystallized with cholesterol.
|Fig. 2: Diffraction patterns of paracetamol, cholesterol, a physical and a ball milled mixture of both (from up to down). New peaks indicate changes of the crystal structures.||
|Fig. 3: Two peaks of separately and co-crystallized caffeine (β-phase). The peak shift indicates anisotropic residual strain of the caffeine lattice due to co-crystallization with cholesterole by RESS-process.||
The International Atomic Energy Agency (IAEA) in Vienna is carrying out an assessment of sterile insect technology (SIT) for control of malaria transmitting mosquitoes. The male mosquitoes under study have been irradiated to render them sterile, a treatment with the goal to reduce the transmission of malaria. Dariusz Wegrzynek (IAEA) and co-workers from IAEA and the Atomic Institute of the Austrian Universities (ATI) have, together with Timm Weitkamp from ANKA, examined the insects using phase-contrast computer tomography (CT) to find out whether the male mosquitoes undergo morphological changes with irradiation, or in other words whether they stay otherwise healthy, in order to compete with non-treated males during the mating process.
White beam topography is a non-destructive imaging technique for the analysis of e. g. dislocations in real single crystals where usually a complete Laue pattern is collected during one exposure on a X-ray film. Every reflection contains one topograph which is magnified and digitized with a light microscope to analyse number and type of dislocations. In experiments by Andreas Danilewsky (University Freiburg) and Alexander Rack (ANKA) , for the first time a digital camera was used with a white beam, providing better resolution and higher dynamic range than high resolution x-ray films. The 111 topograph from a highly sulphur doped InP shows that all the details are visible: strong dopant inhomogeneities in the center of the crystal and V-shaped straight lines which are 60 ° dislocations. Even single curved dislocations at the border of the crystal with very high dislocation density are resolved.
|Figure 3: Two individual projections of the abdomens
of irradiated mosquito pupae (a) and adult specimens (b) are shown.
The collected data demonstrated the usefulness of X-ray phase-contrast tomography for investigating morphological changes in the irradiated mosquito specimens. In the next experiment a cooled CCD camera with specially designed optics will be used. The data acquired with cooled CCD camera will have much better signal to noise ratio as compared to the data collected during the first experiment with the thermally stabilized CCD. In combination with phase retrieval algorithms, e.g. , this should allow for image segmentation and visualization of the individual morphological details of mosquito specimens.
Z-DNA is the left-handed conformation of DNA. Its presence has been linked to gene regulation and transcription in particular.(1) Therefore it is an attractive new target for cancer therapy. It is the goal of this project to design and study small molecules that are able to recognize Z-DNA by strong and selective binding to this structural motive. Since Z-DNA is distinguished from the other conformations of DNA by its own very characteristic geometry, the recognition must be mainly based on geometrical features and to a lesser extent on the sequence of the nucleobases. As small molecules we have chosen dinuclear metal complexes, which are expected to coordinate to N7 of neighboring guanosine nucleobases. Although there are several structures of Z-DNA known, no three dimensional structure of a specially designed Z-DNA recognition unit together with its target can be found in the literature. In order to optimize the recognition of Z-DNA by these small molecules, the knowledge of their three dimensional structure in complex with Z-DNA at the atomic level is essential. We have co-crystallized several short DNA oligomers, some of them bromine substituted, with either just polyamines or metal complexes. The halogen substitution should facilitate structure solution by the multiwavelength anomalous dispersion (MAD) method.
First an X-ray fluorescence scan was taken from a brominated crystal. The most intensive signal at 11,9 keV was assigned to arsenic, being present in the crystallization buffer in the form of cacodylate. A signal from the K edge of bromine at 13.5 keV could also be detected. With the help of the program CHOOCH, the needed wavelengths for the bromine MAD measurements could be calculated (Figure 4). This presents the first fluorescence scan on the new APEX detector at the protein beamline of ANKA.
Have been collected complete data sets from two crystals with different cells and space groups contained brominated nucleotides. In both cases, a two wavelength MAD experiment with a dataset at the inflection point and one in the high energy remote region was done. However when trying to solve the structures with the help of MAD or SAD, the remote data set was always having a higher correlation coefficient. In case of km190106, the structure could be solved by molecular replacement, however during the refinement the R-values remained at high values. Further data processing is ongoing in order to find a reason for this behaviour.
|Figure 4: From left to right, the structures of A, B and Z-DNA||