An approach to ratiometric fluorescence detection of quenching metal ions was devised by copolymerizing N-isopropylacrylamide with small percentages of bipyridine and amine monomers. The copolymer was divided into two portions. The amine group on one portion was functionalized with AlexaFluor555 (donor fluorophore) and the other with AlexaFluor647 (acceptor fluorophore). The indicator consists of a mixture of these two portions. Aggregation above the lower critical solution temperature (LCST) of this copolymer brings about a large increase in fluorescence resonance energy transfer (FRET). Addition of Cu(II) and other complexing metal ions to the aggregated copolymer introduces charge onto the backbone, causing the copolymer to redissolve with a resulting decrease in FRET. The ratio of acceptor to donor fluorescence varies with Cu(II) concentration. A plot of intensity ratio vs. pCu is sigmoidal with a log K(f) of 6.1 for the Cu(II)-bipyridine complex. The data are consistent with the formation of a 1 : 1 complex. The copolymer responds to higher concentrations of other transition metal ions. The selectivity for Cu(II) is consistent with the literature values for 1 : 1 formation constants for bipyridine with metal ions.

The fluorescence emission of poly(N-isopropylacrylamide) (PNIPAM) covalently tagged with a 5-(dimethylamino)naphthalene-1-sulfonyl (dansyl) fluorophore and an iminodiacetic acid (IDA) chelator changes with temperature and with Cu(II) complexation. Increasing the temperature above the lower critical solution temperature (LCST) causes the polymer to collapse from a coil to a compact globule. This changes the environment experienced by the fluorophore causing a shift in maximum emission wavelength from 546 to 508 nm and an increase in the ratio of emission intensity at 508 nm to emission intensity at 546 nm from 0.70 to almost 1.40. Metal ions can be sensed by working at a temperature where the uncomplexed polymer is in an expanded state due to the charges on the ligand. Complexation with a metal ion such as Cu(II) neutralizes the charges on the ligand causing the polymer to collapse. At 35 °C, the emission intensity maximum shifted from 535 to 510 nm as Cu(II) concentration was increased and the intensity ratio increased from 0.84 to 1.28. By decoupling complexation from fluorescence, we have prepared a ratiometric fluorescent indicator for a metal ion that normally quenches fluorescence. The affinity for Cu(II) was found to be thermally tunable. The log apparent formation constants for the indicator-Cu(II) complex were estimated as the half way point in the intensity ratio vs. pCu curve. The values were determined to be 4.3 at 35 °C and 3.2 at 34 °C respectively.

Visualizing disease heterogeneity remains a challenging task since most imaging agents are targeted to a single receptor.

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a b s t r a c tHerein we report an efficient eight-step synthesis of N 1 -(3-(methylthio)propyl)-2-((3-(methylthio)propyl)thio)benzene-1,4-diamine (1) from 6-nitrobenzo [d]thiazole. Incorporation of a nitro rather than a hydroxyl functionality off the aromatic ring serves to enhance conversion during thia-and aza-conjugate additions. Ultimate reduction of the nitro substituent affords a bifunctional Cu(I) chelate in gram-scale quantities for subsequent sensing applications.Ó 2015 Elsevier Ltd. All rights reserved.As the third most abundant metal in biology, copper is intimately involved in the transport and activation of molecular oxygen. 1 This role is due in large part to its facile redox nature. However, the ability to access multiple oxidation states also renders this metal potentially toxic, with an increase in oxidative and nitrosative stress being linked with cancer 2 and neurodegeneration. 3 Monitoring this metal in a biological context is therefore necessary to elucidate the relationship between disruptions in metal-ion homeostasis and pathophysiology. The most attractive method for doing so is through the design and development of ligands that result in a measurable change in fluorescence upon metal-ion binding. 4 Fahrni and co-workers have developed a series of fluorescent pyrazolines exhibiting increased fluorescence in the presence of Cu(I) through inhibition of photoinduced electron transfer (PET) (Fig. 1). 5 Chelation of Cu(I) serves to lower the ligand's HOMO, thereby deactivating the PET process and restoring fluorescence. The authors quickly accessed these probes by first synthesizing a bifunctional N 1 S 3 ligand (2) that then underwent an aldol condensation with 4-acetylbenzonitrile. Subsequent condensation with one of five fluoro-substituted phenylhydrazine derivatives afforded the corresponding N 1 S 3 -functionalized pyrazolines (3). This ligand is extremely selective for Cu(I) over other interfering metal ions given the soft sulfur donors 6 and the propensity of this ligand to form a tetrahedral coordination environment, thus stabilizing Cu(I) relative to Cu(II). 5 Despite their elegant synthesis, these probes suffered from poor solubility in aqueous media in addition to high-energy excitation (346-391 nm) and emission profiles (432-486 nm) unsuitable for biological imaging.To improve tissue penetration, Chang and coworkers recently reported a near-infrared fluorescent sensor for monitoring biological Cu(I) also using PET. 7 They achieved this by developing a similar bifunctional N 1 S 3 ligand (4) that was then covalently attached to cyanine 7 to afford 5 (Fig. 1). Although this account demonstrated in vivo visualization of labile Cu(I), these 'turn-on' approaches toward metal-ion monitoring are incapable of providing quantitative measurements. Thus, sensors capable of modulating the ratio of multiple emission bands (ratiometric) are preferred in that they generate quantitative information. 8 However, few ratiometric sensors for copper exist, while most suffer from poor water solubility 9...