Crystallization of Amorphous Nifedipine Under Isothermal Conditions: Inter-laboratory Reproducibility and Investigation of the Factors Affecting Reproducibility.

High inter-laboratory reproducibility is required for conducting collaborative experiments among several laboratories. The primary aim of our evaluation of the physical stability of amorphous drugs, conducted in co-operation with eight laboratories, was to establish a protocol for isothermal storage tests to obtain data of the same quality from all the participating laboratories. Sharing a protocol that contained the same level of detail as the experimental section of general papers was insufficient for high inter-laboratory reproducibility. We investigated the causes of variations in the data from the various laboratories and restricted the protocol step-by-step to achieve high inter-laboratory reproducibility. The various experimentalists had very different levels of awareness regarding how to control the temperature of a sample as the samples were transferred into and out of thermostatic chambers. Specific instructions on how to conduct this operation, such as regarding the time required for the transfer and thermal protection of the container during the transfer, helped to reduce variation. Improved inter-laboratory reproducibility revealed that the physical stabilities of amorphous drugs differed when samples were prepared in differently shaped aluminum pans designed for various differential scanning calorimeters.

Turn-on fluorescent probe with visible light excitation for labeling of hexahistidine tagged protein.

We report here the development of a novel fluorescein-based probe which shows selective fluorescence enhancement on binding to a hexahistidine-tagged protein. No fluorescence change was observed with untagged protein. This probe is excitable with visible light and is considered to be suitable for use in biological applications.

Novel probes showing specific fluorescence enhancement on binding to a hexahistidine tag.

The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel-nitrilotriacetic acid (Ni2+-NTA) complex. To develop efficient "turn-on" fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal-NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal-NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni2+ and Co2+ were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni2+ and Co2+ complexes of NTACs in pH 7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)6-Tyr-NH2; Tyr was included to allow convenient concentration measurement) in pH 7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co2+ complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide.

[Selective recognition and detection of biomacromolecules utilizing chemical property of amino Acid or Peptide].

Recently, the fluorometric detection of biomacromolecules has attracted much attention. In this paper, we report the development of two new techniques utilizing the chemical properties of amino acids or peptides: 1) a fluorescence assay for serine/threonine kinase activity; and 2) "turn-on" fluorescent probes for protein labeling, which could be useful for bioimaging. To develop the novel kinase assay, we utilized the chemical reactivity of phosphorylated serine or threonine. Phosphorylated peptide on resin was successfully labeled fluorescently via base-mediated beta-elimination, followed by Michael addition with novel coumarin derivatives. Protein kinase A and casein kinase I activities were detectable with our method. Also, this method was confirmed to be applicable for kinase inhibitor screening. For the development of the novel protein labeling technique, the selective interaction between "His-tag (His(6))" and "metal ion nitrilotriacetic acid (NTA) complex" was utilized. This interaction is useful for protein purification and immobilization. We designed fluorescent probes composed of a fluorophore and Ni2+ or Co2+-NTA complex. These probes were found to be weakly fluorescent as expected. When His-tag peptide was added, these probes became brightly fluorescent. On the other hand, these probes remained non fluorescent with the addition of angiotensin I (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH). These probes will be powerful tools for the bioimaging of target proteins.