Kazunori Koide

Oxidation-Resistant Fluorogenic Probe for Mercury Based on Alkyne Oxymercuration

Mercury continues to be a major safety hazard to the general public. Therefore, a fluorescence method that has potential for on-site use can be very useful. Most fluorescent probes for mercury are based on the interaction between mercury ions and sulfur atoms, which may not be compatible with mercury samples prepared by oxidation processes. Herein we report a fluorogenic probe for mercury based on the unique ability of Hg2+ to convert an alkyne to a ketone. By this methodology, the probe, prepared in two steps in 80% overall yield, is converted to a very bright green fluorescent molecule by Hg2+ in pH 7 buffer. This fluorescence method is sensitive, showing a signal-to-background ratio of 3 at 8 ppb mercury level, and highly specific for this metal ion. Since this detection method does not rely on mercury−heteroatom interactions, the probe is resistant to oxidants and thus can be used against mercury samples prepared by oxidative procedures. We demonstrate that our method can be applied to fish and dental...

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Development and Applications of Fluorogenic Probes for Mercury(II) Based on Vinyl Ether Oxymercuration

Mercury is a major threat to the environment and to human health. It is highly desirable to develop a user-friendly kit for on-site mercury detection. Such a method must be able to detect mercury below the threshold levels for drinking water, 1-2 ppb. We developed a fluorescence method based on the oxymercuration of vinyl ethers to detect mercury in dental and environmental samples. Chloride ions interfered with the oxymercuration reaction, but the addition of AgNO(3) solved this problem. Fine electronic and structural tuning led to the development of a more responsive probe that was less sensitive to chloride ion interference. This second-generation probe could detect 1 ppb mercury ions in water.

Enhancement of a catalysis-based fluorometric detection method for palladium through rational fine-tuning of the palladium species.

Metal analyses in chemistry, materials science, and environmental science are currently performed using techniques such as inductively coupled plasma mass spectrometry and X-ray fluorescence, which require expensive instrumentation and are not high-throughput. Although fluorescent probes are known for their sensitivity and specificity and are amenable to high-throughput analyses, the robustness of such analyses are typically limited due to their binding-based nature. Herein we report an improvement of our previously reported catalysis-based fluorescent probe for palladium by rationally fine-tuning the redox and coordination chemistries of the palladium species involved in the O-deallylation reaction. This method now rivals current analytical methods with respect to sensitivity. We demonstrate palladium detection in various active pharmaceutical ingredients, spent catalytic converter materials, and a metal scavenger resin. Thus, fluorescent methods may have the potential for substituting the current instrument-intensive techniques.

Oxidation State-Specific Fluorescent Method for Palladium(II) and Platinum(IV) Based on the Catalyzed Aromatic Claisen Rearrangement

Contamination of active pharmaceutical ingredients and healthcare products with oxidized forms of palladium and platinum are important problems. We report a convenient fluorogenic probe based on the catalyzed aromatic Claisen rearrangement capable of detecting PdII and PtIV. Using this probe, we can fluorescently detect PdII in functionalized organic compounds and PtIV in Pt0-water without sample pretreatment. This method may find broad applications in the pharmaceutical industry, the environment, and Pd/Pt quality control.

Crystal Structure of the Potent Natural Product Inhibitor Balanol in Complex with the Catalytic Subunit of cAMP-Dependent Protein Kinase †

Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar Ki values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanols specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 A resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.

Meayamycin inhibits pre–messenger RNA splicing and exhibits picomolar activity against multidrug-resistant cells

FR901464 is a potent antitumor natural product that binds to the splicing factor 3b complex and inhibits pre-mRNA splicing. Its analogue, meayamycin, is two orders of magnitude more potent as an antiproliferative agent against human breast cancer MCF-7 cells. Here, we report the picomolar antiproliferative activity of meayamycin against various cancer cell lines and multidrug-resistant cells. Time-dependence studies implied that meayamycin may form a covalent bond with its target protein(s). Meayamycin inhibited pre-mRNA splicing in HEK-293 cells but not alternative splicing in a neuronal system. Meayamycin exhibited specificity toward human lung cancer cells compared with nontumorigenic human lung fibroblasts and retained picomolar growth-inhibitory activity against multidrug-resistant cells. These data suggest that meayamycin is a useful chemical probe to study pre-mRNA splicing in live cells and is a promising lead as an anticancer agent. [Mol Cancer Ther 2009;8(8):2308–18]

Specific fluorogenic probes for ozone in biological and atmospheric samples

Ozone exposure is a growing global health problem, especially in urban areas. While ozone in the stratosphere protects the earth from harmful ultraviolet light, tropospheric or ground-level ozone is toxic and can damage the respiratory tract. It has recently been shown that ozone may be produced endogenously in inflammation and antibacterial responses of the immune system; however, these results have sparked controversy owing to the use of a non-specific colorimetric probe. Here we report the synthesis of fluorescent molecular probes able to unambiguously detect ozone in both biological and atmospheric samples. Unlike other ozone-detection methods, in which interference from different reactive oxygen species is often a problem, these probes are ozone specific. Such probes will prove useful for the study of ozone in environmental science and biology, and so possibly provide some insight into the role of ozone in cells.

Gallium reagents in organic synthesis: Dimethylgallium chloride and triflate as activators in glycosidation using glycopyranosyl fluorides

Abstract Gallium compounds were utilized in organic synthesis for the first time. Dimethylgallium chloride and dimethylgallium triflate was found to efficiently promote the glycosidation using several glycopyranosyl fluorides.

Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol

BACKGROUND The protein kinase C (PKC) family of serine/threonine-specific protein kinases is involved in many cellular processes, and the unregulated activation of PKC has been implicated in carcinogenesis. PKC inhibitors thus have significant potential as chemotherapeutic agents. Recently, the fungal metabolite balanol was shown to be an exceptionally potent inhibitor of PKC. We previously developed a practical and efficient total synthesis of balanol. We set out to use this synthetic molecule, and several synthetic analogs, to probe the mechanism of PKC inhibition and to determine the effect of balanol on the activity of other protein kinases. RESULTS As well as inhibiting PKC, balanol is a potent inhibitor of cyclic AMP-dependent protein kinase (PKA), another protein serine/threonine kinase. Balanol does not, however, inhibit the Src or epidermal growth factor receptor protein tyrosine kinases. The inhibition of both PKC and PKA by balanol can be overcome by high concentrations of ATP, and molecular modeling studies suggest that balanol may function as an ATP structural analog. Although balanol discriminates rather poorly between PKC and PKA, only minor modifications to its molecular structure are required to furnish compounds that are highly specific inhibitors of PKA. CONCLUSIONS A number of balanol analogs have been designed and synthesized that, unlike balanol itself, exhibit dramatic selectivity between PKA and PKC. Thus, despite the substantial homology between the catalytic domains of PKA and PKC, there is enough difference to allow for the development of potent and selective inhibitors acting in this region. These inhibitors should be useful tools for analyzing signal transduction pathways and may also aid in the development of drugs with significant therapeutic potential.