A practical strategy to create near-infrared luminescent probes: conversion from fluorescein-based sensors

Luminescent lanthanide complexes incorporating Yb(3+) and Nd(3+) are attracting much attention as imaging agents, but there have been few practical methods to make responsive sensors with these complexes. Here, we introduce a general strategy to synthesize near-infrared luminescent probes by conjugating a Yb(3+) chelate to established fluorescein-based probes. As the first demonstration, we present a complex, based on the green-emitting probe DAF-4, that responds to nitric oxide (NO) in aqueous solution with a significant increase in luminescence intensity at 980 nm.     

A long-lived luminescent probe to sensitively detect arylamine N-acetyltransferase (NAT) activity of cells

Arylamine N-acetyltransferase (NAT) is an important phase II metabolizing enzyme that influences drug efficacy and adverse effects. Here, we report a long-lived luminescent lanthanide complex as a probe for NAT, employing an intraligand photoinduced electron transfer strategy. The probe shows approximately 100-fold increase of luminescence upon N-acetylation catalyzed by NAT, with relatively high specificity for NAT2 over NAT1. It is the first NAT probe that is suitable for sensitive, homogeneous, and rapid detection of NAT activity of recombinant enzyme or cell lysate, and is expected to be useful for drug discovery and clinical diagnosis.     

Possibility of scanning electron microscope observation and energy dispersive X-ray analysis in microscale region of insulating samples using diluted ionic liquid

The possibility of scanning electron microscope (SEM) observation and energy dispersive X-ray (EDX) spectrometry analysis in microscale regions of insulating samples using diluted ionic liquid was investigated. It is possible to obtain clear secondary electron images of insulating samples such as a rock and mineral at 5,000 times magnification by dropping 10 μL of 1 wt% of 1-ethyl-3-methylimidazolium acetate (EMI-CH?COO) diluted with ethanol onto the samples. We also obtained EDX spectra of the samples in microscale regions (~5 μm2) without overlapping EDX spectra of other minerals with different composition. It might be possible to perform quantitative analysis of the samples if a method that does not need standard samples is applied or an X-ray detector sensitive for light elements was attached. The method of dropping 1 wt% EMI-CH?COO diluted with ethanol onto insulating samples is useful for SEM observation, EDX analysis in microscale regions, and the preservation of scarce rock and mineral samples because ionic liquid can be easily removed with acetone.     

Rhodium-catalyzed asymmetric coupling reaction of allylic ethers with arylboronic acids

An asymmetric allylic substitution of simple allylic ethers with arylboronic acids in the presence of a rhodium(I)/(R)-DTBM-SEGPHOS catalyst has been developed. The reactions proceeded smoothly at room temperature to give the corresponding branch products with excellent regioselectivities and good to excellent enantioselectivities.     

Stereoselective formation of trisubstituted (z)-chloroalkenes adjacent to a tertiary carbon stereogenic center by organocuprate-mediated reduction/alkylation

A robust and efficient method for the synthesis of trisubstituted (Z)-chloroalkenes is described. A one-pot reaction of γ,γ-dichloro-α,β-enoyl sultams involving organocuprate-mediated reduction/asymmetric alkylation affords α-chiral (Z)-chloroalkene derivatives in moderate to high yields with excellent diastereoselectivity, and allylic alkylation of internal allylic gem-dichlorides is also demonstrated. This study provides the first examples of the use of allylic gem-dichlorides adjacent to the chiral center for novel 1,4-asymmetric induction.     

π-Fused bis-BODIPY as a candidate for NIR dyes

Benzene-fused bis-(borondipyrromethene)s (bis-BODIPYs) were synthesized by retro-Diels-Alder reaction of the corresponding bicyclo[2.2.2]octadiene-fused (BCOD-fused) bis-BODIPYs, which were, in turn, prepared from 4,8-ethano-4,8-dihydropyrrolo[3,4-f]isoindole derivatives. The π-fused bis-BODIPY chromophores were designed to show intensive absorption and strong fluorescence in the near-infrared region and not to have any strong absorption in the visible region. A 6,10-dibora-5a,6a,9a,10a-tetraaza-s-indaceno[2,3-b:6,5-b']difluorene derivative (syn-bis-benzoBODIPY) obtained by a thermal retro-Diels-Alder reaction of the corresponding BCOD-fused BODIPY dimer has strong absorption and emission bands at 775 and 781 nm, respectively. The absolute quantum yield is 0.36. The absorption is more than 5.0 times stronger than other absorptions observed in the visible region. In the case of 6,15-dibora-5a,6a,14a,15a-tetraaza-s-indaceno[2,3-b:6,7-b']difluorene derivatives (anti-bis-benzoBODIPY), the absorption and emission maxima exceed 840 nm.     

Design and Synthesis of a Highly Sensitive Off–On Fluorescent Chemosensor for Zinc Ions Utilizing Internal Charge Transfer

Fluorescence imaging is a powerful tool for the visualization of biological molecules in living cells, tissue slices, and whole bodies, and is important for elucidating biological phenomena. Furthermore, zinc (Zn²⁺) is the second most abundant heavy metal ion in the human body after iron, and detection of chelatable Zn²⁺ in biological studies has attracted much attention. Herein, we present a novel, highly sensitive off–on fluorescent chemosensor for Zn²⁺ by using the internal charge transfer (ICT) mechanism. The rationale of our approach to highly sensitive sensor molecules is as follows. If fluorescence can be completely quenched in the absence of Zn²⁺, chemosensors would offer a better signal‐to‐noise ratio. However, it is difficult to quench the fluorescence completely before Zn²⁺ binding, and most sensor molecules still show very weak fluorescence in the absence of Zn²⁺. But even though the sensor shows a weak fluorescence in the absence of Zn²⁺, this fluorescence can be further suppressed by selecting an excitation wavelength that is barely absorbed by the Zn²⁺‐free sensor molecule. Focusing on careful control of ICT within the 4‐amino‐1,8‐naphthalimide dye platform, we designed and synthesized a new chemosensor ( 1 ) that shows a pronounced fluorescence enhancement with a blueshift in the absorption spectrum upon addition of Zn²⁺. The usefulness of 1 for monitoring Zn²⁺ changes was confirmed in living HeLa cells. There have been several reports on 4‐amino‐1,8‐naphthalimide‐based fluorescent sensor molecules. However, 1 is the first Zn²⁺‐sensitive off–on fluorescent sensor molecule that employs the ICT mechanism; most off–on sensor molecules for Zn²⁺ employ the photoinduced electron transfer (PeT) mechanism.     

Hg(II) ion specifically binds with T:T mismatched base pair in duplex DNA

Metal-mediated base pair formation, resulting from the interaction between metal ions and artificial bases in oligonucleotides, has been developed for its potential application in nanotechnology. We have recently found that the T:T mismatched base pair binds with Hg(II) ions to generate a novel metal-mediated base pair in duplex DNA. The thermal stability of the duplex with the T-Hg-T base pair was comparable to that of the corresponding T:A or A:T. The novel T-Hg-T base pair involving the natural base thymine is more convenient than the metal-mediated base pairs involving artificial bases due to the lack of time-consuming synthesis. Here, we examine the specificity and thermodynamic properties of the binding between Hg(II) ions and the T:T mismatched base pair. Only the melting temperature of the duplex with T:T and not of the perfectly matched or other mismatched base pairs was found to specifically increase in the presence of Hg(II) ions. Hg(II) specifically bound with the T:T mismatched base pair at a molar ratio of 1:1 with a binding constant of 10(6) M(-1), which is significantly higher than that for nonspecific metal ion-DNA interactions. Furthermore, the higher-order structure of the duplex was not significantly distorted by the Hg(II) ion binding. Our results support the idea that the T-Hg-T base pair could eventually lead to progress in potential applications of metal-mediated base pairs in nanotechnology.     

A time-resolved fluorescence probe for dipeptidyl peptidase 4 and its application in inhibitor screening

The prevalence of type 2 diabetes is increasing dramatically throughout the world. Recently, dipeptidyl peptidase 4 (DPP4) was identified as a potential antidiabetes target. Many DPP4 inhibitors, such as sitagliptin and vildagliptin, have been developed and marketed, but superior therapeutic agents are still required. Therefore, we have developed new methodology for screening of DPP4 inhibitors. Absorption-based measurements with para-nitroaniline or fluorescence-based measurements with the coumarin derivative 7-amino-4-methylcoumarin are often used for the screening of protease inhibitors, including DPP4 inhibitors, but these strategies are not sufficiently sensitive because of interfering background absorption and fluorescence, thus giving rise to many false-positive and false-negative results. Therefore, we have designed and synthesised a novel DPP4 probe (Gly-Pro-BCD-Tb; Gly=glycine, Pro=proline, andBCD defines the backbone of the probe comprising an aniline derivative as on/off switch, a 7-amino-4-methyl-2(1H)-quinolinone (cs-124) as antenna moiety, and a diethylenetriamine-N,N,N',N',N'-pentaacetic acid (DTPA) as chelator moiety, Tb=terbium) for time-resolved fluorescence (TRF) measurements. TRF measurements with Gly-Pro-BCD-Tb showed high sensitivity and reliability in the inhibitory assay relative to Gly-Pro-MCA (MCA=4-methylcoumarin-7-amide), a conventional fluorescence probe for DPP4. Further, we employed our probe for high-throughput DPP4 inhibitor screening with 3841 randomly selected compounds and found that epibestatin, an epimer of bestatin (a well-known anticancer drug and general aminopeptidase inhibitor), showed dose-dependent DPP4 inhibitory activity. Interestingly, bestatin did not exhibit DPP4 inhibitory activity. We believe that this screening system will be useful for the discovery of DPP4 inhibitors with novel structural scaffolds.