A highly K(+)-selective two-photon fluorescent probe for the in vitro monitoring of physiological K(+) levels in the range of 1-100 mM is reported. The two-photon excited fluorescence (TPEF) probe shows a fluorescence enhancement (FE) by a factor of about three in the presence of 160 mM K(+), independently of one-photon (OP, 430 nm) or two-photon (TP, 860 nm) excitation and comparable K(+)-induced FEs in the presence of competitive Na(+) ions. The estimated dissociation constant (Kd ) values in Na(+)-free solutions (Kd (OP) =(28±5) mM and Kd (TP)=(36±6) mM) and in combined K(+)/Na(+) solutions (Kd (OP) =(38±8) mM and Kd (TP)=(46±25) mM) reflecting the high K(+)/Na(+) selectivity of the fluorescent probe. The TP absorption cross-section (σ2PA ) of the TPEF probe+160 mM K(+) is 26 GM at 860 nm. Therefore, the TPEF probe is a suitable tool for the in vitro determination of K(+).

A desirable goal is to synthesize easily accessible and highly K(+) /Na(+) -selective fluoroionophores to monitor physiological K(+) levels in vitro and in vivo. Therefore, highly K(+) /Na(+) -selective ionophores have to be developed. Herein, we obtained in a sequence of only four synthetic steps a set of K(+) -responsive fluorescent probes 4, 5 and 6. In a systematic study, we investigated the influence of the alkoxy substitution in ortho position of the aniline moiety in π-conjugated aniline-1,2,3-triazole-coumarin-fluoroionophores 4, 5 and 6 [R=MeO (4), EtO (5) and iPrO (6)] towards the K(+) -complex stability and K(+) /Na(+) selectivity. The highest K(+) -complex stability showed fluoroionophore 4 with a dissociation constant Kd of 19 mm, but the Kd value increases to 31 mm in combined K(+) /Na(+) solutions, indicating a poor K(+) /Na(+) selectivity. By contrast, 6 showed even in the presence of competitive Na(+) ions equal Kd values (Kd (K+) =45 mm and Kd (K+/Na+) =45 mm) and equal K(+) -induced fluorescence enhancement factors (FEFs=2.3). Thus, the fluorescent probe 6 showed an outstanding K(+) /Na(+) selectivity and is a suitable fluorescent tool to measure physiological K(+) levels in the range of 10-80 mm in vitro. Further, the isopropoxy-substituted N-phenylaza[18]crown-6 ionophore in 6 is a highly K(+) -selective building block with a feasible synthetic route.

There is a tremendous demand for highly Na -selective fluoroionophores to monitor the top analyte Na in life science. Here, we report a systematic route to develop highly Na /K selective fluorescent probes. Thus, we synthesized a set of fluoroionophores 1, 3, 4, 5, 8 and 9 (see Scheme ) to investigate the Na /K selectivity and Na - complex stability in CH CN and H O. These Na -probes bear different 15-crown-5 moieties to bind Na stronger than K . In the set of the diethylaminocoumarin-substituted fluoroionophores 1-5, the following trend of fluorescence quenching 1>3>2>4>5 in CH CN was observed. Therefore, the flexibility of the aza-15-crown-5 moieties in 1-4 determines the conjugation of the nitrogen lone pair with the aromatic ring. As a consequence, 1 showed in CH CN the highest Na -induced fluorescence enhancement (FE) by a factor of 46.5 and a weaker K induced FE of 3.7. The Na -complex stability of 1-4 in CH CN is enhanced in the following order of 2>4>3>1, assuming that the O-atom of the methoxy group in the ortho-position, as shown in 2, strengthened the Na -complex formation. Furthermore, we found for the N-(o-methoxyphenyl)aza-15-crown-5 substituted fluoroionophores 2, 8 and 9 in H O, an enhanced Na -complex stability in the following order 8>2>9 and an increased Na /K selectivity in the reverse order 9>2>8. Notably, the Na -induced FE of 8 (FEF=10.9), 2 (FEF=5.0) and 9 (FEF=2.0) showed a similar trend associated with a decreased K -induced FE [8 (FEF=2.7)>2 (FEF=1.5)>9 (FEF=1.1)]. Here, the Na -complex stability and Na /K selectivity is also influenced by the fluorophore moiety. Thus, fluorescent probe 8 (K =48 mm) allows high-contrast, sensitive, and selective Na measurements over extracellular K levels. A higher Na /K selectivity showed fluorescent probe 9, but also a higher K value of 223 mm. Therefore, 9 is a suitable tool to measure Na concentrations up to 300 mm at a fluorescence emission of 614 nm.

This work presents two molecular fluorescent probes 1 and 2 for the selective determination of physiologically relevant K levels in water based on a highly K /Na selective building block, the o-(2-methoxyethoxy)phenylaza-18-crown-6 lariat ether unit. Fluorescent probe 1 showed a high K -induced fluorescence enhancement (FE) by a factor of 7.7 of the anthracenic emission and a dissociation constant (K ) value of 38 mm in water. Further, for 2+K , we observed a dual emission behavior at 405 and 505 nm. K increases the fluorescence intensity of 2 at 405 nm by a factor of approximately 4.6 and K decreases the fluorescence intensity at 505 nm by a factor of about 4.8. Fluorescent probe 2+K exhibited a K value of approximately 8 mm in Na -free solutions and in combined K /Na solution a similar K value of about 9 mm was found, reflecting the high K /Na selectivity of 2 in water. Therefore, 2 is a promising fluorescent tool to measure ratiometrically and selectively physiologically relevant K levels.

The new K -selective fluorescent probes 1 and 2 were obtained by Cu -catalyzed 1,3-dipolar azide alkyne cycloaddition (CuAAC) reactions of an alkyne-substituted [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ester fluorophore with azido-functionalized N-phenylaza-18-crown-6 ether and N-(o-isopropoxy) phenylaza-18-crown-6 ether, respectively. Probes 1 and 2 allow the detection of K in the presence of Na in water by fluorescence enhancement (2.2 for 1 at 2000 mm K and 2.5 for 2 at 160 mm K ). Fluorescence lifetime measurements in the absence and presence of K revealed bi-exponential decay kinetics with similar lifetimes, however with different proportions changing the averaged fluorescence decay times (τ ). For 1 a decrease of τ from 12.4 to 9.3 ns and for 2 an increase from 17.8 to 21.8 ns was observed. Variation of the substituent in ortho position of the aniline unit of the N-phenylaza-18-crown-6 host permits the modulation of the K value for a certain K concentration. For example, substitution of H in 1 by the isopropoxy group (2) decreased the K value from >300 mm to 10 mm. 2 was chosen for studying the efflux of K from human red blood cells (RBC). Upon addition of the Ca ionophor ionomycin to a RBC suspension in a buffer containing Ca , the fluorescence of 2 slightly rose within 10 min, however, after 120 min a significant increase was observed.