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Mg2+ plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg2+ regulation and the Mg2+ concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg2+ in mitochondria in intact cells. Here, we have developed a novel Mg2+–selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg2+ concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg2+, KMG-104, enabled us to compare the dynamics of Mg2+ in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)–induced Mg2+ mobilization from mitochondria to the cytosol was visualized. Although a FCCP–induced decrease in the Mg2+ concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg2+ and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP+). A gradual decrease in the Mg2+ concentration in mitochondria was observed in response to MPP+ in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg2+ dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).

Highly sterically demanding 2,4,6‐tri‐t‐butylphenyllithium (1) reacts with carbon disulfide to give 1,2‐dibutylthio‐1,2‐bis(2,4,6‐tri‐t‐butylphenylthio) ethene (3), l‐butylthio‐1‐(2,4,6‐tri‐t‐butylphenylthio)pent‐l‐ene (4), and 2‐butylthio‐3‐(2,4,6‐tri‐t‐butylphenylthio)‐1,3‐dithiole‐2‐thione (5) when 1 is prepared by the reaction of 1‐bromo‐2,4,6‐tri‐t‐butylbenzene with n‐butyllithium. When the reaction is carried out using 1 prepared from 2 and t‐butyllithium and then quenched with l‐iodoethane, 2‐ethylthio‐3‐(2,4,6‐tri‐butylphenylthio)‐1,3‐dithiole‐2‐thione and 1,2‐diethylthio‐1,2‐bis(2,4,6–tri‐t‐butylphenylthio) ethene are produced. The formation of all these products can be explained in terms of initial thiophilic attack of 1 on the sulfur of carbon disulfide and an intermediate with a dual property of carbanion and carbene (i.e., ArSC(Li)(S) ⇌ ArSC̈SLi) is suggested. The reaction of 1 with thiophosgene affords 1,2‐dichloro‐1,2‐bis(2,4,6‐tri‐t‐butylphenylthio) ethene, bis(2,4,6‐tri‐t‐butylphenylthio)acetylene, and 1‐chloro 2,4,6‐tri‐t‐butylbenzene, the first two of which are explained to be formed by thiophilic attack of 1 with thiophosgene.

We report a novel near-infrared fluorescent calcium probe (KFCA), which has good optical properties such as intense NIR fluorescence emission (670 nm, QY: 0.24), excellent ON/OFF ratio (120-fold), and good wavelength-compatibility with visible-light-emissive fluorophores (Fluo-4, DsRed2), and which is applicable for real-time dual-colour intracellular Ca(2+) imaging.

Although the magnesium ion (Mg(2+)) is one of the most abundant divalent cations in cells and is known to play critical roles in many physiological processes, its mobilization and underlying mechanisms are still unknown. Here, we describe a novel fluorescent Mg(2+) probe, "KMG-104-AsH", composed of a highly selective fluorescent Mg(2+) probe, "KMG-104", and a fluorescence-recoverable probe, "FlAsH", bound specifically to a tetracysteine peptide tag (TCtag), which can be genetically incorporated into any protein. This probe was developed for molecular imaging of local changes in intracellular Mg(2+) concentration. KMG-104-AsH was synthesized, and its optical properties were investigated in solution. The fluorescence intensity of KMG-104-AsH (at λ(em/max) = 540 nm) increases by more than 10-fold by binding to both the TCtag peptide and Mg(2+), and the probe is highly selective for Mg(2+) (K(d/Mg) = 1.7 mM, K(d/Ca) ≫ 100 mM). Application of the probe for imaging of Mg(2+) in HeLa cells showed that this FlAsH-type Mg(2+) sensing probe is membrane-permeable and binds specifically to tagged proteins, such as TCtag-actin and mKeima-TCtag targeted to the cytoplasm and the mitochondrial intermembrane space. KMG-104-AsH bound to TCtag responded to an increase in intracellular Mg(2+) concentration caused by the release of Mg(2+) from mitochondria induced by FCCP, a protonophore that eliminates the inner membrane potential of mitochondria. This probe is expected to be a strong tool for elucidating the dynamics and mechanisms of intracellular localization of Mg(2+).

It is difficult to completely remove carcinomas in unguided ablative surgery because they cannot be distinguished with the unaided human eye. Therefore, in order to precisely visualize tiny tumors and the borders between cancerous lesions and normal tissues, we have been developing fluorescence probes activatable only in cancer cells. We previously reported the hydroxymethylrhodamine green (HMRG)-based fluorescence probe gGlu-HMRG for γ-glutamyltransferase (GGT), which is overexpressed in a variety of cancer cells, and we showed that it enables in vivo rapid detection of human ovarian cancer SHIN-3 nodules with a high tumor-to-background (T/B) fluorescence ratio in model mice. However, cancer cell lines with low GGT expression could hardly be detected with gGlu-HMRG. Here we developed two new HMRG-based fluorescence probes for the cathepsin family of cysteine proteases, including cathepsin B (CatB) and cathepsin L (CatL), which show increased expression and/or activity, secretion, and altered localization in many kinds of cancer cells. The developed probes, Z-Phe-Arg-HMRG and Z-Arg-Arg-HMRG, are colorless and nonfluorescent at the physiological pH of 7.4, but are hydrolyzed to HMRG upon reaction with purified cathepsins, resulting in a more than 200-fold fluorescence increase. These probes could visualize human ovarian cancer cell lines SHIN-3, SK-OV-3, and OVCAR-3, of which the latter two were hardly detectable with gGlu-HMRG. Z-Phe-Arg-HMRG showed higher applicability than Z-Arg-Arg-HMRG for in vivo imaging, and we confirmed that 0.5-mm-sized SK-OV-3 tumor nodules disseminated on the mesentery in a mouse model could be rapidly visualized by Z-Phe-Arg-HMRG, with a T/B fluorescence ratio of 4.2. Further, intraperitoneally disseminated tumor could be visualized in real time in vivo by fluorescence endoscopy after spraying Z-Phe-Arg-HMRG, with a T/B ratio of 3. In conclusion, our HMRG-based activatable probes targeted to cathepsins have expanded the detectable range of cancers, and appear to be suitable for clinical application.

Alzheimers disease (AD), [1] the most common cause of age-related dementia which affects approximately 20 million people worldwide, is characterized by neuronal loss and the presence of large numbers of senile plaques, consisting of fibrillar aggregates of 40-and 42-residue amyloid b (Ab) peptides, in the brain. [2] In recent studies, it was suggested that soluble oligomers of the Ab 42 peptide are responsible for synaptic dysfunction in the brains of patients with AD and are the key intermediate neurotoxic species in the pathology of AD. [3] One way of suppressing Ab 42 assembly in the brain is through small molecules with a high affinity for, [4] or an ability to degrade, [5] Ab 42 . A major effort should be directed toward inhibiting amyloid formation at very early stages of the disease. In this context, we recently reported that a fullerene derivative could degrade Ab peptides upon photoirradiation in the absence of any additives and under neutral conditions. [6] Herein, in a significant application of this fundamental result, we report that a newly designed and synthesized fullerene derivative with high water solubility effectively inhibited Ab 42 peptide aggregation and degraded Ab 42 peptide monomer and oligomers under photoirradiation conditions. In addition, it is noteworthy that the fullerene derivative effectively inhibited cytotoxicity induced by Ab 42 in neuron-like PC12 cells [7] upon photoirradiation. To the best of our knowledge, this is the first example of the inhibi-tion of Ab-mediated cytotoxicity against neuron-like PC12 cells by light-switching degradation of Ab under neutral conditions.In our previous study, we designed and synthesized fullerene-sugar hybrid 1, which was found to inhibit Ab 42 peptide aggregation and cause degradation of its monomer and oligomers upon photoirradiation, in the absence of any additives and under neutral conditions. [6] However, it has been revealed that 1 could not be applied to further biological studies using cells, mainly owing to its low solubility in aqueous media. Spurred on by this negative result, herein, we designed the fullerene derivatives 2 and 3, each of which consist of a fullerene attached to a sulfo or an amino group (Scheme 1). The design was based on an expectation that the hydrophobic and electrophilic fullerene moiety of the hybrids would exhibit a high affinity for the hydrophobic and electrophilic 16-20 amino-acid residue KLVFF segment in the central part of the Ab 42 peptide. [8] We also expected that the hydrophilicity and the ionic state of the sulfo or amino groups of the hybrids under neutral conditions and neutral pH would enhance not only the water solubility, but also the interaction with Ab 42 , owing to the formation of hydrogen bond(s) and/or ionic interactions with the termini of the Ab 42 peptide.The synthesis of 2 and 3 is outlined in Scheme 2. The known fullerene derivative 4, [9] which was prepared from the commercially available fullerene C 60 , was subjected to deprotection by removal of the trityl (Tr) group using trif...

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