Serial crystallography has made remarkable progress since its origin. The advantages of serial crystallography cover the aspects of room-temperature structure determination of the biomacromolecules, micron or sub-micron sized crystal structure determination, as well as time-resolved research. Sample delivery system is one of the key parts of serial crystallography. It is the main limiting factor affecting the application of serial crystallography. In the existing sample delivery technologies, the samples are usually delivered in linear motion. Here we show that the samples can be also delivered using circular motion, which is a novel motion mode never tested before. We report a microfluidic rotating-target sample delivery device, which is characterized by the circular motion of the samples, and verify the performance of the device on the synchrotron radiation facility [1]. The microfluidic rotating-target sample delivery device consists of two parts: a microfluidic sample plate and a motion control system. Sample delivery is realized by rotating the microfluidic sample plate containing in-situ grown crystals as shown in Fig. 1. This device offers significant advantages, including a very wide adjustable range of delivery speed, low background noise, and low sample consumption. Using the microfluidic rotating-target device, we carried out in-situ serial crystallography experiments with lysozyme and proteinase K as model samples on the Shanghai Synchrotron Radiation Facility, and performed the structural determination based on the serial crystallographic data. The results showed that the designed device is fully compatible with the synchrotron radiation facility, and the structure determination of proteins is successful using the serial crystallographic data obtained with the device.

New "dynamic theory of protein crystallization (DTPC)" considers protein crystallization as a competitive process between different “protein-protein adhesion modes (PPAM)” mutually incompatible in molecular stacking into the crystal lattice. Large surface of protein molecules offers variety of different adhesion modes and only some of them are compatible in a single crystal form. The DTPC searches for the crystallization conditions supporting spontaneous preference of a dominant protein-protein adhesion mode and suppression of all adverse adhesion modes in the newly formed solid phase. The DTPC is based on the concept of PPAM giving us tools for manipulation with protein adhesion and allowing the intuitive design of experiment to increase the number of crystallizable proteins, to choose rationally the desired polymorph form and to increase the resolution of protein structures. The necessary condition for formation of single crystal is its growth according to the „principle of the dominant adhesion mode (PDAM)". It provides non-conflicting explanation for all available experimental observations regarding the protein crystallization and also for catalysis of crystal growth on heterogeneous substrates [1]. In a very simplified form, the necessary condition for the successful crystal growth can be written as a high difference of free energies of the competitive processes ΔF_cryst ~ F_{dominant AM} - Σ F_{incompatible AM}

This offers the experimenter a rational way to grow diffraction quality crystals and also to select the required crystalline form. The new approach changes the situation significantly and leads to enhancement of efficiency and accuracy of all standing crystallization methods. DTPC with PDAM are general and should be strictly respected by any method of protein crystallization.

Heterogeneous crystallization. Historically, there were different explanations why some heterogeneous materials initiate protein crystallization. Here, we propose the universal explanation why some materials are suitable for crystal initiation and other not. The DTPC explains efficiency of crystallization catalyzers (e.g. bioglass, coarsely wrinkled foils, nano-carbon materials, imprinted polymers, porous Si, hoarse hairs, properly coated nanotubes and nanostructured carbon black [1]) as follows. The specific adhesion in depressions in the substrate surface leads to identical orientation of protein molecules. Thus, it restricts an access to their adhesive surfaces responsible for incompatible PPAM. It enforces a unique PPAM in the growing crystal nuclei. They are more stable because of lower number of stacking faults. Therefore, they do not dissolve and can continue to grow even after being released into the seemingly under-saturated bulk solution. Contrary to a number former mutually contradicting explanation, this explains all available experiments on a unique common basis. By active control of crystallization process in slightly under-saturated conditions, one can rationally limit crystal formation to cavities only. The new insight promises better design of natural and artificially prepared crystallization catalyzers promising an increase in a number of crystallizable proteins and higher resolution in structure determination.

Homogeneous crystallization. Also here, one should look for the systems providing the highest difference between free energies of mutually exclusive protein-protein adhesion modes. The temporary molecular clusters formed in the overcrowded crystallization solution play here an important role. The adhesion properties of the protein molecule in these complexes may differ radically from the adhesion properties of the original molecule. If the crystallographer knows the rules for the formation of these temporary complexes, he can control preferences of the adhesion modes active in the emerging crystal. He can decide which of the mutually exclusive protein-protein adhesion modes succeeds and becomes dominant by using his knowledge of the adhesion modes between the target protein and the „protein-surface-active molecules (PSAM)“. Very reach and natural source of the adhesion mode examples is the PDB offering a deep inshight how the „protein surface shielding agents“ work in practice and how the „crystal structure forming elements“ help in finding the best crystal architecture [2]. If the crystallographer fails in suppressing the mutually incompatible adhesion modes, the result cannot be quality crystal. Thus even very good precipitation agent can be a bad crystallization agent, if it does not differentiate among adhesion modes of the target protein.

Classical theories of protein crystallization and majority of papers on protein crystallography concentrated in last decades to an efficient and rapid precipitation. Low attention was given to reasons why the growing solid phase is regular and to the fact that protein crystallization is a competitive process between different adhesion modes. The abstract notion “adhesion mode” introduced by the DTPC gives the experimenter new tools controlling the crystallization and increasing predictability in protein crystallization methods.

[1] Yau S.T. et al, Nature 406, 494 (2000); Chayen, N.E., et al, J. Mol. Biol. 312, 591 (2001); Redecke L. et al. Nature Methods 9, 259 (2012); Khurshid S., et al, Nature Protocols 9, 1621 (2014); Ghatak A.S. et al, Crystal Growth & Design 16, 5323 (2016); Krishnan V. et al J. Advanced Pharmaceutical Technology & Research 4, 78 (2016); Govada, L. et al, Sci. Rep. 6, 1,(2016); Nanev, C.N., et al, Scientific Reports, 7, 35821 (2017); Pechkova E., et al, Nature Protocols 12, 2570 (2017); Nanev C., et al, IUCrJ 8, 270 (2021).[2] Hašek, J. Zeitschrift fur Kristallogr. 23, 613 (2006); Hašek, J. J. Synchrotron Radiation 18, 50 (2011)

Supported by projects MEYS ERDF fund (CZ02.1.01/0.0/0.0/16_013/0001776) and the Czech Academy of Sciences no. 86652036.

Protein crystallization is an ideal theoretical model for studying nucleation and growth of crystals due to features like slow kinetics. In the research field of crystal nucleation, there are a number of important theories describing the nucleation process. Among the theoreis, nucleation from dense liquid droplets after a process called liquid-liquid phase separation (LLPS) is widely accepted. According to this theory, the number and distribution of the phase separated droplets can determine the number, size and distribution of the final crystals in the solution. Here in this report, we will present our effort to obtain a single, suspended crystal in the crystallization solution through manipulation of the phase separated droplets. It is known that gradient magnetic field can exert magnetic force on the objects in the field so that gradient magnetic field can be fully utilized in protein crystallization [1-3]. By using a large gradient magnetic field we can merge the phase separated droplets in the solution non-contactly. The merged dense liquid droplet will be the location of protein crystallization and a single suspended crystal can be thus obtained. Figure 1 shows an example of suspened lysozyme crystal grown in a superconducting magnet. The results show that crystallization can be controlled via manipulation of the phase-separated droplets. Further, this study can also provide a strong support for the two-step nucleation theory.

The protein crystal has a wide range of potential applications (catalytic transformation, cell imaging and drug delivery) due to its highly ordered morphology. The hemoglobin, as the earliest discovered protein that can be crystallization, is the most commonly used protein crystal due to its mature crystallization process, lower cost, extensive source and can be mass produced. The protein crystal are generally prepared by vapor diffusion crystallization which already realize the size-controllable protein crystal in millimeter level. However, the preparation of protein crystal with micro-nano scale remains a challenge, which limits the application in the field of material science. In this study, the hemoglobin crystals in micron scale were prepared by stirring to make dense liquid phase well dispersive in the process of vapor diffusion crystallization, and successfully obtain the hemoglobin crystals around the size of 20 µm. Furthermore, nano-crystals were obtained by the water-in-oil micro-emulsion method. This work will boost the application of hemoglobin crystals in functional materials. Our next research will focus on the water-in-oil micro-emulsion process’ optimization to realize size-controllable and fine reproducibility.

The applications and potential advantages of serial crystallography, at both synchrotron and XFEL light sources, are growing. Despite advances in delivery methods, the sample volumes of micro-crystals required for serial crystallography, particularly time-resolved experiments, are still demanding. Batch crystallisation methods are the primary means in crystallographer's toolbox to create these samples. However, the process to convert single crystals grown by vapour diffusion to large volumes (> 100 µL) of micro-crystalline slurry can be exceptionally challenging. Here we present a strategy to perform this translation and it is divided into three stages: (1) optimising crystal morphology, (2) transitioning to batch, and (3) scaling. Given the variation of protein crystallisation, we hope that this protocol can act as a useful framework when attempting the conversion from vapour diffusion to batch. Tips and tricks will also be presented that may also be useful. Ultimately, we hope that this methodology improves the samples used for serial crystallographic studies and that this also improves the quality of acquired data.

Phosphine ligands are considered by many as one of the most significant class of ligands in organometallic chemistry. The search of new phosphine chelators, as well as the subsequent functionalization thereof, is a continuing process in order to induce appropriate properties for highly effective catalyst and to a lesser extend in medicinal purposes. Of particular interest is the search for water-soluble and highly stable ligands that can preserve their aquatic solubility even after metal coordination.

In this study, we aim to improve the efficiency of middle/late transition metal homogeneous catalysts (i.e. Re, Rh, Pd and Pt) and fac-[M(CO)₃] (M = Re and Tc) radiopharmaceutical synthons by selectively introducing monodentate and bidentate phosphine ligands consisting of various electronic and steric properties. The use of systematically altered bidentate phosphine ligands such as diphosphinoamine ligands has already been reported to show high selectivity improvements in catalytic reactions such as ethylene tri- and tetramerization [1].

A series of diphosphinoamine ligands was synthesized using methods described in literature [2, 3]. These ligands were then coordinated to various metal (i.e. Re(I), Tc(I), Pt(II) and Pd(II)). Results obtained from the biological analysis and catalytic evaluations have opened up a new window of opportunities for such compounds.

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Drugs in a crystalline state are preferably used due to their physicochemical stability [1]. Around 70% of the drug candidates to be newly available drugs have low solubility, which can compromise bioavailability and, consequently, product development [2]. Therefore, amorphous phases have become more attractive to the industry field as a promising strategy to improve solubility. The amorphous structure can be defined as a long-range tridimensional molecular packing loss. Compared to its crystalline counterpart, the amorphous can have higher inner energy that can increase the drug solubility, dissolution rate, and extension, improving the final formulation bioavailability. Several processes can be used to reach the drug’s amorphous structure, such as solvent evaporation, melting-cooling, lyophilization, and ball-milling. However, the amorphous drug metastability can induce structure recrystallization, which can be a problem [3]. Furthermore, different amorphization processes can promote local molecular packing variations yielding changes in the properties.

Herein, we combined the pair distribution function (PDF) and Reverse Monte Carlo (RMC) methods with data from a high-resolution diffractometer (model STADI-P, STOE^®) equipped with a MoKa₁ source, available at the “Laboratory of Crystallography and Structural Characterization of Materials” (LCCEM) at the Federal University of ABC, Santo André-SP, Brazil to analyze crystalline drugs under amorphization process. By the results, we could identify the amorphization mechanisms when using ball-mill and solvent evaporation process for different drugs, which can be induced by displacing O-O and O-H correlations (molecule distortions) as well as variations in hydrogen bonds. In both cases, the molecule was preserved after the amorphization process.

[1] D.A. Snider, W. Addicks, W. Owens, Polymorphism in generic drug product development. (2004). Adv. Drug Deliv. Rev. 56 391–395.

[2] B.Y. Shekunov, P. York, Crystallization processes in pharmaceutical technology and drug delivery design. (2000). J. Cryst. Growth. 211, 122–136.

[3] S.L. Raghavan, A. Trividic, A.F. Davis, J. Hadgraft, Crystallization of hydrocortisone acetate: influence of polymers. (2001). Int. J. Pharm. 212, 213–221.

The authors thank the financial support of The São Paulo Research Foundation (FAPESP) grant #2018/11990-5 and the National Council for Scientific and Technological Development (CNPq) grant #305661/2019-9.

Catalysis plays a vital role in numerous stages of petroleum refinement and fuel production, with one of the major energy sources globally being crude oil for fuels and further production of a variety of chemicals [1]. However, development of highly selective catalysts still poses a significant challenge in many of these processes [2]. Methanol carbonylation is one of the major homogeneously catalysed process for production of the acetic acid from methanol. With the oxidative addition of methyl iodide being the rate determining step in this catalytic process; the selectivity of the catalyst to favour the formation of the desired product may be achieved by carefully varying the ligand system of said catalyst as well as the reaction conditions[3]. The selectivity for acetic acid production with rhodium-based catalysts in a homogeneous medium is roughly 99% [4]. Catalytic rhodium system’s activity and selectivity are vastly improved by phosphine ligands leading to favourable results under milder conditions [5]. Our functionalised halogenated rhodium(I) complexes, [Rh(N,O)(CO)(PR₃)](R= Ph, Cy), coordinated to N,O bidentate Schiff-base ligands and select phosphine ligands are hereby reported. The extensive structural characterization of the complexes followed by the kinetic mechanistic study using UV/Vis, infrared and nuclear magnetic resonance spectroscopy will also be reported. The influence of halogens (F, Cl, Br) on the para-position of the Schiff base ligand on the methyl iodide oxidative addition to the rhodium(I) monocarbonyl specie will also be described .

[1] Lemonidou, A. A., Lappas, A. A. & Vasalos, L. A., 2011. Catalysis and Refinery. Thessaloniki: Encyclopedia of Life Support Systems

[2] Kumar, M, Chaudhari, R.V; Subramaniam, B; Jackson, T.A. Organometallics., 2014, 33, 4183−4191.

[3] Van Leeuwen P.W.N.M; Homogeneous Catalysis: Understanding the Art, 2004, Dordrecht: Kluwer Academic Publishers.

[4] Schurell, M., 1977. Rhodium catalysts for methanol carbonylation. Platinum Metals Reviews, 21(3), pp. 92-96.

[5] Osborn, J.A; Wilkinson, G; Young, J.F. Chem. Commun., 1965, 17.

The development of sterically encumbered ligands that contain anionic nitrogen donor sites (NR^2-) has played a pivotal role in advancing our overall knowledge of fundamental chemical reactivity throughout the Periodic Table. Recently, we are introducing a methyl-pyridine side arm in the β-diketiminato framework leads to a ligand that is tridentate in its nacnac imino-pyridine state (2,6-iPr₂-C₆H₃NC(Me)CHC(Me)NH(CH₂py))¹. Such ligands have not been used for compounds with low valent p-block elements. We presumed that additional donation from the nitrogen atom of the pyridine moiety may provide sufficient electronic stabilization that would compensate for the decrease in the sterics. Here we have successfully synthesized and characterized methylpyridinato β-diketiminate ligand stabilized chlorogermylene 1 which undergoes unusual smooth ring contraction in presence of Lewis acid (GeCl₂.dioxane) via C–N bond cleavage (2), facile dehydrocoupling and six membered Al-heterocycle formation (5), which are not observed for the nacnac based systems (Scheme 1). But, in presence of another Lewis acid containing group 13 element like AlCl₃, leads to the formation of dicholoaluminim complex 4 via the transmetallation process (Scheme 1). Single crystal X-ray study reveals that the pyridine moiety coordinates to the aluminum center in 4, possibly due to the radius of the aluminum atom is apparently too small compared to the ligand's bite angle, which leads to the asymmetric coordination. The work is another testimony to the fact that small variations can yield unprecedented outcomes.²