DOI: 10.1002/chem.201501473
Communication
& Fluorescent Probes
A Highly K + -Selective Two-Photon Fluorescent Probe Thomas Schwarze,[a] Janine Riemer,[a] Sascha Eidner,[b] and Hans-Jìrgen Holdt*[a] Abstract: 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 twophoton 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 (KdOP = (28 5) mm and KdTP = (36 6) mm) and in combined K + /Na + solutions (KdOP = (38 8) mm and KdTP = (46 25) mm) reflecting the high K + /Na + selectivity of the fluorescent probe. The TP absorption cross-section (s2PA) 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 + .
Two-photon (TP) active cation-responsive fluorescent probes are suitable tools for monitoring cations in living cells.[1a–e] Using two-photon excited fluorescence (TPEF) probes in TP excitation microscopy, essential benefits can be obtained compared to one-photon (OP) excitation, for example, increased depth penetration, reduced phototoxicity, and intrinsic 3D capabilities. The design of effective TPEF probes is still a challenge and there is a demand for tailoring TPEF probes for cations. An important quality of TPEF probes is the brightness (fFs2PA), that is, the product of the TP absorption cross-section (s2PA) and the fluorescence quantum yield (fF). Desirable features of cation-responsive TPEF probes for biological applications are their stability and fast response, TP excitation in the range of 700–1000 nm, emission maxima (> 500 nm), high brightness, no interference by changes in pH value, and their high selectivity. Nowadays, highly selective TPEF probes for several biologically important cations like Na + ,[2a,b] Ca2 + ,[2c–f] Mg2 + ,[2g] and Zn2 + ,[2h,i] based on the two-photon excitable chromophore 2acetyl-6-(dimethylamino)-naphthalene (acedan) were devel[a] Dr. T. Schwarze, J. Riemer, Prof. H.-J. Holdt Institut fìr Chemie, Anorganische Chemie, Universitt Potsdam Karl-Liebknecht-Str. 24–25, 14476 Golm (Germany) E-mail: [email protected] [b] Dr. S. Eidner Institut fìr Chemie, Physikalische Chemie, Universitt Potsdam Karl-Liebknecht-Str. 24–25, 14476 Golm (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501473. Chem. Eur. J. 2015, 21, 11306 – 11310
oped. However, to the best of our knowledge, a selective TPEF probe for K + ions has not been studied. In recent years, several fluorescent probes for K + ions were synthesized. Minta and Tsien designed the first fluorescent indicator PBFI (potassium-binding benzofuranisophthalate) to measure intracellular K + ion concentration, which incorporates a diaza-18-crown-6 as an ionophore.[3a] The commercially available PBFI[3b] is the most popular K + indicator,[3c] but it lacks K + /Na + selectivity. To overcome the poor K + /Na + selectivity, calix[4]bisazacrown-5[4] or cryptand-based[5a–l] K + -fluoroionophores were developed. Most of all, the [2.2.3]-triazacryptand (TAC) is a highly K + -selective ionophore established by He and co-workers.[5f] This K + receptor is an essential feature of numerous fluoroionophores with emission wavelengths > 550 nm[5c–e] as a part of a blood sensor device[5f,g] or as components of dextran conjugates for studies in vivo.[5h,i] A drawback of the TAC-based fluoroionophores is their multistep synthesis. Recently, we synthesized easily accessible fluoroionophores for K + ions[6a,b] or Na + ions[6c] by using CuAAC (CuI-catalyzed 1,3-dipolar azide alkyne cycloaddition) reactions. Furthermore, we found that in these fluoroionophores, the signal-transduction chain, that is, p-conjugated aniline(donor)-1,4-triazole-coumarin (acceptor), works nicely under simulated physiological conditions.[6a–c] Firstly, we incorporated a N-phenylaza-18crown-6 ionophore for sensing of K.[6a] The estimated dissociation constant Kd value of 260 mm was not suitable for monitoring physiological K + levels. In the next step, we designed a new highly selective K + /Na + ionophore, a N-(2-methoxyethoxyphenyl)aza-18-crown-6 unit.[6b] The attachment of a 2-methoxyethoxy lariat group at the ortho position of the anilino moiety reduces the Kd value to a physiologically relevant concentration range of K + . Afterwards, we synthesized two isomeric 1,2,3-triazol-1,4-diyl-fluoroionophores. In these probes the regioisomeric 1,4-triazole linkage influences the K + /Na + selectivity and the fF.[6b] A higher fF value and a high K + /Na + selectivity were reached when the aniline donor was connected to position 1 of the triazole and the coumarin acceptor to position 4, as shown in fluorescent probe 2 (Scheme 1). Probe 2 showed a high K + /Na + selectivity and a fluorescence quantum yield of fF = 0.18 in the presence of 160 mm of K + .[6b] To study physiological relevant K + levels by a highly K + /Na + selective TPEF probe, we selected as an ionophore the N-(2methoxyethoxyphenyl)aza-18-crown-6 unit with the same triazole arrangement between the ionophore and the coumarin fluorophore as in compound 2. Furthermore, to enhance the fF value of the K + complexed fluorescent probe for designing an effective TPEF probe, we selected the 2,3,6,7-tetrahydro1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one fluoro-
11306
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication Table 1. Photophysical properties of 1, 2, and 3 in Tris-buffer.[a]
1 1 + K+ 1 + Na + 2 2 + K+ 3 3 + K+ 3 + Na +
Chem. Eur. J. 2015, 21, 11306 – 11310
www.chemeurj.org
lf [nm]
fF[b]
FEF[c,d]
s2PA[e] [GM]
s2PAfF[f] [GM]
434 434 434 430 430 365 365 365
511 511 511 493 493 501 501 501
0.12 0.36 0.13 0.06 0.18 0.03 0.04 0.03
– 2.9 (2.7) 1.1 (1.1) – 2.5 – 1.1 0.9[g]
26 26 26 95 89 n.d.[h] n.d. n.d.
3.1 9.4 3.4 5.7 16.1 n.d. n.d. n.d.
[a] All data were measured in a buffered H2O/DMSO mixture (99:1, v/v; 10 mm Tris; pH 7.2) in the absence and presence of K + or Na + ions (160 mm). The ionic strength was maintained at 160 mm with choline chloride. [b] Fluorescence quantum yield, ( 15) %. [c] Fluorescence enhancement factor, [FEF = I/I0], ( 0.1), measured by a one-photon (OP) process. [d] Values in parentheses measured by two-photon (TP) process (lexTP = 860 nm). [e] TP cross section in 10¢50 cm4 s photon¢1 (GM) for 1 at 860 nm and for 2 at 840 nm. [f] Action cross section in 10¢50 cm4 s photon¢1 (GM). [g] For 3 + Na + , we observed a fluorescence quenching; [h] n.d. = not determined.
Scheme 1. Structures of K + -responsive fluorescent probes 1–3 and of the Na + -selective TPEF probe 4.[2a]
phore (cf. Scheme 1, compound 1). This coumarin-derived fluorophore exhibits a high fF > 0.70,[7a,b] a moderate s2PA value > 150 GM,[7c] and a fluorescence emission lF > 500 nm[7a,b] in polar solvents. Moreover, we synthesized fluorescent probe 3 as an analogue to the Na + -responsive TPEF probe 4, which consists of a 1,7-diaza-15-crown-5 ionophore. The fluoroionophore 3 is also derived from the well-known TP acedan fluorophore and for K + -binding studies the N-(2-methoxyethoxyphenyl)aza-18-crown-6 ionophore was introduced. In this paper, we show that probe 1 (Scheme 1) is a highly K + /Na + -selective TPEF probe for the in vitro determination of K + concentration levels in the range of 1–100 mm. 1 shows a K + -induced fluorescence enhancement factor (FEF) of 2.7 0.1 in Na + -free solutions and a FEF of 2.4 0.1 in combined K + /Na + solutions by a TP excitation at 860 nm. We found for 1, after addition of 160 mm K + , a fluorescence quantum yield of fF = 0.36 and a s2PA of 26 GM. The synthesis of the 1,2,3-triazol-fluoroionophore 1 was realized by a CuAAC of the azido-functionalized o-(2-methoxyethoxy)-phenylaza-18-crown-6[6b] ionophore and the 3-ethynylfuctionalized coumarin.[8] The UV/Vis absorption spectrum of 1 under simulated physiological conditions (10 mm Tris buffer, pH 7.2) showed two charge-transfer (CT) absorption bands (Figure S1 in the Supporting Information). The long-wavelength coumarin CT band is centered at 434 nm in 1. The second CT band at approximately 300 nm is typical for p-conjugated 1,2,3-triazol-1,4-diylfluoroionophores.[6a] In addition, we investigated the influence of K + and Na + in the OP absorption behavior of 1. The coumarin absorption band around 434 nm is essentially unchanged by K + but the complexation of K + ions can be seen from the short wavelength CT absorption at approximately 300 nm (Figure S1 in the Supporting Information). K + reduces the electron donor ability of the anilino unit in 1, which leads to a decrease of the absorption of the CT band at approximately 300 nm (Figure S1). The UV/Vis absorption spectrum of 1 is almost unaffected by Na + ions (Figure S1), demonstrating the high K + /Na + selectivity of probe 1. The fluorescence spectrum of 1 (OP, lex = 430 nm) shows, compared to 2[6b] (lF = 493 nm, fF = 0.06), a red shifted emission centered at 511 nm and a higher fF value of 0.12 under si-
labs [nm]
mulated physiological conditions (Table 1). In both compounds, 1 and 2, the low fluorescence quantum yield is caused by a photoinduced electron transfer (PET)[9a–c] across a virtual spacer, as already observed for 1,2,3-triazol-fluoroionophores for K + [6a,b] and Na + .[6c] The TPEF spectrum of 1 (TP, lex = 860 nm) shows also an emission centered at 511 nm. The highest s2PA value of 26 GM for 1 was found by a TP excitation at 860 nm (Figure S9 in the Supporting Information). We investigated the influence of K + and Na + on the fluorescence of 1 in the physiologically relevant concentration range of 1–160 mm K + and Na + . Figure 1 a shows the fluorescence enhancement of 1 (OP, lex = 430 nm) in a buffered H2O/DMSO mixture (99:1, v/v; 10 mm Tris; pH 7.2) in the presence of 1– 160 mm K + at a constant ionic strength of 160 mm adjusted with choline chloride. We observed K + -induced fluorescence enhancements of 1 (FEF = 2.9 0.2, Figure 1 a) and 2 (FEF = 2.5[6b]). The maximum signal change was observed at 100 mm K + , with fF values for 1 of 0.36 and for 2 of 0.18. Thus, the fF of 1 + 160 mm K + is two times higher than 2 + 160 mm K + . Furthermore, the TPEF intensity of 1 (TP, lex = 860 nm) is increased by K + ions under simulated physiological conditions (Figure 2 a). We observed a FEF of 2.7 0.1 (1 + 160 mm K + ), when 1 was excited at 860 nm (Figure 2 a). The FEFs of 1 + 160 mm K + by OP- and TP-excitation were similar to each other (Figure 2 a). The run of the titration curves of 1 + K + were independent of the excitation process (OP or TP, cf. Figure 2 a). We found for 1 + 160 mm K + a s2PA value of 26 GM at 860 nm (Table 1). The s2PA values of 1 (26 GM) and 1 + K + (26 GM) were the same, concluding that the TP excitation process at 860 nm is not influenced by K + ions. For probe 2 we found a higher s2PA value of 95 GM at 840 nm and an almost identical s2PA value of 89 GM at 840 nm for 2 + 160 mm K + (see Table 1). To verify the K + /Na + selectivity of probe 1, we measured the fluorescence intensities at various concentrations of Na + ions (1–160 mm) under simulated physiological conditions ex-
11307
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
Figure 1. Fluorescence response of probe 1 (10¢5 m, lex = 430 nm) to K + in the presence of various concentrations of ions under simulated physiological conditions (10 mm Tris-buffer, pH 7.2); the ionic strength was maintained at 160 mm with: a) choline chloride and b) various concentrations of Na + ions in combined K + /Na + solutions.
cited through an OP and TP process, respectively. As shown in Figure 2 a, the fluorescence of probe 1 at 511 nm is slightly affected by Na + ions (fF = 0.13 at 160 mm Na + ). Furthermore, this high K + /Na + selectivity of probe 1 was validated by using solutions that contained both K + and Na + ions to ensure a total metal ion concentration of 160 mm (Figure 1 b, lex = 430 nm). The fluorescence enhancement by a factor of 2.4 0.2 at 160 mm K + was only slightly lower than that in the experiment without Na + ions (FEF = 2.9 0.2). Continuing, we observed a marginal change of the FEF of 1 + K + from 2.7 0.1 (without Na + , see Figure 2 a) to 2.4 0.1 (in the presence of Na + ) by an excitation at 860 nm (Figure S10 in the Supporting Information). In addition, the K + selectivity of probe 1 was substantiated by titration experiments with other metal ions such as Mg2 + and Ca2 + (at 160 mm), and Mn2 + , Fe3 + , Co2 + , Ni2 + , Cu2 + , and Zn2 + (at 100 mm) under simulated physiological conditions. The fluorescence intensity of 1 showed only negligible changes in the presence of these cations (Figure S3c in the Supporting Information). Importantly, probe 1 recognized K + even in the co-presence of these cations (Figure S3c). Moreover, probe 1 showed high sensitivity towards K + ions with a detection limit of 0.2 mm.[8] Chem. Eur. J. 2015, 21, 11306 – 11310
www.chemeurj.org
Figure 2. a) Fluorescence-enhancement factors (If(max)/If0(max)) for probe 1 (10¢5 m, lex = 430 nm (OP) and lex = 860 nm (TP)) under simulated physiological conditions (10 mm Tris buffer, pH 7.2) in the presence of various concentrations of K + or Na + ions; the ionic strength was maintained at 160 mm with choline chloride. b) Two-photon action spectra of 1 (10¢5 m) in the presence of 160 mm K + and 160 mm Na + ions.
Dissociation constants (Kd) were calculated from plots of fluorescence intensities versus K + ion concentration.[8] The Kd values of probe 1 in Na + -free solution were KdOP = (28 5) mm and KdTP = (36 6) mm for the OP and TP process, respectively. These values increased to KdOP = (38 8) mm and KdTP = (46 25) mm for the combined K + /Na + solutions. A similar result was found for 2 + K + in Na + -free and combined Na + /K + solutions (KdOP values increases from 26 to 29 mm).[6b] Thus, these close Kd values demonstrate the high K + /Na + selectivity of probe 1. Hence, Na + ions have a negligible effect on the performance of probe 1 under simulated physiological conditions. Changes in pH value showed only a minor effect towards the sensitivity of 1 at various concentrations of K + ions within the pH range 6.8–8.8 (see Figure S8 in the Supporting Information). Overall, the fluorescence signal of 1 is mainly increased by K + ions and marginal influenced by Na + ions independently of the excitation process (one or two photon) (Figure 2 a). This demonstrates the high K + /Na + selectivity of the TPEF probe 1. Figure 2 b displays the fFs2PA values of 1, 1 + K + , and 1 + Na + in the range of 780–900 nm. The brightness of 1 + K + at 860 nm is about 10 GM and that of 2 + K + at 840 nm is about
11308
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication 16 GM (Table 1). Herein, 1 + K + and 2 + K + showed a lower brightness than the acedan-based TPEF probes for Na + (95 GM[2a] and 83 GM[2b]), but 1 + K + and 2 + K + are moderately excitable by a TP process and should be applicable in TP excitation microscopy. Therefore, 1 and 2 are suitable TPEF probes for the highly selective sensing of K + in the range of 1– 100 mm under simulated physiological conditions. Herein, to the best of our knowledge, we report for the first time on highly K + -selective TPEF probes. To investigate the K + binding of the N-(2-methoxyethoxyphenyl)aza-18-crown-6 ionophore in combination with the bright TP acedan fluorophore, we synthesized fluoroionophore 3. Compound 3 was prepared by the coupling reaction between 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]-naphthalene[2g] and N-(4-amino-2-methoxyethoxyphenyl)aza-18-crown-6 ether[6b] .[8] For 3, we found a broad OP absorption band centered at 365 nm, which is not influenced by K + or Na + ions (Table 1). This is typical for acedan-based TPEF probes for cations.[2a–i] Probe 3 showed a fluorescence emission centered around 501 nm (OP, lex = 365 nm) with a fluorescence quantum yield of 0.03 under simulated physiological conditions (Table 1), comparable to reported TPEF probes[2] based on a PET-quenching mechanism of the acedan fluorophore. The K + /Na + selectivity of the fluorescent probe 3 is not as high as that observed for probe 1. Probe 3 had a FEF of only 1.1 in the presence of 160 mm K + (fF = 0.04) under simulated physiological conditions (Table 1, Figures S4a and S5b in the Supporting Information). However, the fluorescence of probe 3 is slightly quenched in the presence of 160 mm Na + (fF = 0.03, Table 1, Figure S5a and S5b). This contrary result was further substantiated by using solutions that contained both Na + and K + ions. We observed a higher FEF of 1.15 for 3 + K + in the presence of Na + (Figure S4b) caused by the lower initial fluorescence intensity of 3 + 160 mm Na + . The KdOP values of 3 + K + in Na + -free solutions were 49 and 45 mm in combined Na + /K + solutions, forming a weaker K + complex as 1. Thus, the combination of a N-(2-methoxyethoxyphenyl)aza18-crown-6 ionophore and an acedan fluorophore bridged by an amide is more unsuitable to determine K + ions than the 1,2,3-triazole-fluoroionophores 1 and 2. We assume, that the triazole unit and their arrangement, as found in 1 and 2, is substantial for the high K + /Na + selectivity. Furthermore, for the design of highly selective TPEF probes for K + based on the o-(2-methoxyethoxy)-phenylaza-18-crown-6 ionophore, an integration of the triazole unit should be considered. In summary, we have synthesized the fluorescent probes 1 and 3 based on the N-(2-methoxyethoxyphenyl)aza-18crown-6 ionophore. Both consisting of different TP chromophores, 1 contains a coumarin unit and 3 an acedan moiety. K + induces fluorescence intensity enhancements of 1 and 3 under simulated physiological conditions, but 3 shows a FE by only a factor of 1.09 in the presence of 160 mm K + . On the other hand, 1 shows a FEF of 2.9 0.2 and 2.7 0.1 by OP and TP excitation, respectively. The fluorescence quantum yield is fF = 0.36 and the two-photon excitation cross section is s2PA = 26 GM in the presence of 160 mm K + . A similar FEF of 2.4 0.2 Chem. Eur. J. 2015, 21, 11306 – 11310
www.chemeurj.org
for 1 was observed in combined K + /Na + solutions. This high K + /Na + selectivity is substantiated by the close Kd values for Na + -free (KdOP = (28 5) mm and KdTP = (36 6) mm) and combined K + /Na + solutions (KdOP = (38 8) mm and KdTP = (46 25) mm) by OP or TP excitation. We have shown that 1 is a highly K + -selective TPEF probe with a suitable Kd value to determine physiologically relevant K + levels. Currently in our laboratories, we generate TPEF probes for K + , Na + , Ca2 + , Mg2 + , and Zn2 + based on the signal-transduction chain aniline-triazole-coumarin with a high brightness.
Experimental Section General methods and reagents All commercially available chemicals were used without further purification. Solvents were distilled prior to use. 1H and 13C NMR spectra were recorded on 300 and 600 MHz instruments, respectively. Data are reported as follows: chemical shifts in ppm (d), multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet), integration, coupling constant (Hz). ESI spectra were recorded using a Micromass Q-TOF micro mass spectrometer in a positive electrospray mode. Column chromatography was performed with silica gel (Merck; silica gel 60 (0.04–0.063 mesh)). 10-{1-[4-(1,4,7,10,13-Pentaoxa-16-azacyclooctadecan-16-yl)-3-(2methoxyethoxy)phenyl]-1H-1,2,3-triazol-4-yl}-2,3,6,7-tetrahydro1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (1): A mixture of N-(4-azido-2-methoxyethoxyphenyl)aza-[18]crown-6 ether[6b] (190 mg, 0.415 mmol), 10-ethynyl-2,3,6,7-tetrahydro-1H,5H,11H[1]benzopyrano[6,7,8-ij]quinolizin-11-one[8] (110 mg, 0.415 mmol), CuSO4·5H2O (5.3 mg) and sodium ascorbate (8.2 mg) in 9 mL THF/ H2O (v/v, 2:1) was stirred at 60 8C for 48 h. After that, 5 mL H2O were added to the mixture and extracted with CHCl3 (30 mL). The organic layer was dried with MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica using CHCl3/CH3OH (v/v, 9:1) as an eluent mixture to afford 1 as a dark yellow solid (70 mg, 24 %). M.p.: 79 8C (decomp.); 1H NMR (CDCl3, 600 MHz): d = 8.64 (s, 1 H), 8.57 (s, 1 H), 7.36–7.33 (m, 1 H), 7.27–7.25 (m, 1 H), 7.17 (s, 1 H), 7.00 (s, 1 H), 4.25–4.17 (m, 2 H), 3.78–3.76 (m, 2 H), 3.75–3.49 (m, 24 H), 3.43 (s, 3 H), 3.29 (dd, J = 5.3, 11.2 Hz, 4 H), 2.93 (t, J = 6.5 Hz, 2 H), 2.79 (t, J = 6.2 Hz, 2 H), 2.01– 1.95 ppm (m, 4 H); 13C NMR (CDCl3, 75 MHz): d = 160.96, 151.14, 147.90, 146.21, 142.53, 138.94, 136.17, 125.63, 121.29, 120.34, 119.01, 113.02, 109.59, 108.50, 106.43, 106.29, 102.63, 70.85, 70.74, 70.62, 70.57, 70.29, 69.79, 68.02, 58.96, 52.87, 50.09, 49.70, 27.53, 21.44, 20.53, 20.31 ppm; HRMS (ESI + ): m/z calcd for C38H50N5O9 : 720.3609; found: 720.3620. N-(4-(1,4,7,10,13-Pentaoxa-16-azacyclooctadecan-16-yl)-3-(2-methoxyethoxy)phenyl)-2-((6-acetylnaphthalen-2-yl)(methyl)amino)acetamide (3): A mixture of N-(4-amino-2-methoxyethoxyphenyl)aza-[18]crown-6 ether[6b] (127 mg, 0.49 mmol), 1-hydroxybenzotriazole (49 mg, 0.33 mmol), 1,3-dicyclohexyl carbodiimide (102 mg, 0.49 mmol) in dry dichloromethane (20 mL) was stirred for 30 min. To this solution, 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene[2g] (85 mg, 0.33 mmol) was added and stirred for 12 h under argon. The mixture was extracted with CH2Cl2 (60 mL) and washed with 1 N NaOH. The organic layer was dried over MgSO4, and removed in vacuo. The product was purified by column chromatography (silica gel) using chloroform/methanol (v/v, 10:1 to 5:1) as the eluent. It was further purified by crystallization using
11309
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication chloroform/hexane to yield compound 3 as a light yellow solid (82 mg, 37 %). M.p.: 88 8C (decomp.); 1H NMR (CDCl3, 300 MHz): d = 11.10 (s, 1 H), 8.28 (s, 1 H), 7.89 (d, J = 8.7 Hz, 1 H), 7.78 (d, J = 8.8 Hz, 1 H), 7.64 (d, J = 8.8 Hz, 1 H), 7.52 (d, J = 8.6 Hz, 1 H), 7.35 (s, 1 H), 7.22 (d, J = 10.0 Hz, 1 H), 7.12 (dd, J = 8.7, 2.5 Hz, 1 H), 7.00 (s, 1 H), 4.22–4.18 (m, 2 H), 3.77–3.33 (m, 31 H), 3.24 (s, 3 H), 2.64 ppm (m, 3 H); 13C NMR (CDCl3, 75 MHz): d = 197.71, 166.99, 158.51, 149.37, 141.84, 131.73, 130.92, 130.24, 126.89, 126.50, 124.46, 123.91, 121.23, 118.94, 116.26, 111.67, 107.63, 105.64, 70.93, 70.57, 70.03, 69.88, 69.74, 69.45, 69.26, 68.99, 58.84, 58.33, 40.01, 26.41 ppm; HRMS (ESI + ): m/z calcd for C36H50N3O9 : 668.3547; found: 668.3537.
[4] [5]
Keywords: click chemistry · fluorescence · fluorescent probes · potassium · two-photon [1] a) M. Pawlicki, H. A. Collins, R. G. Denning, H. L. Anderson, Angew. Chem. Int. Ed. 2009, 48, 3244 – 3266; Angew. Chem. 2009, 121, 3292 – 3316; b) S. Yao, K. D. Belfield, Eur. J. Org. Chem. 2012, 2012, 3199 – 3217; c) S. Sumalekshmy, C. J. Fahrni, Chem. Mater. 2011, 23, 483 – 500; d) H. M. Kim, B. R. Cho, Acc. Chem. Res. 2009, 42, 863 – 872; e) A. R. Sarkar, D. E. Kang, H. M. Kim, B. R. Cho, Inorg. Chem. 2014, 53, 1794 – 1803. [2] a) M. K. Kim, C. S. Lim, J. T. Hong, J. H. Han, H.-Y. Jang, H. M. Kim, B. R. Cho, Angew. Chem. Int. Ed. 2010, 49, 364 – 367; Angew. Chem. 2010, 122, 374 – 377; b) A. R. Sarkar, C. H. Heo, M. Y. Park, H. W. Lee, H. M. Kim, Chem. Commun. 2014, 50, 1309 – 1312; c) H. M. Kim, B. R. Kim, J. H. Hong, J.-S. Park, K. J. Lee, B. R. Cho, Angew. Chem. Int. Ed., 2007, 46, 7445 – 7448; Angew. Chem. 2007, 119, 7589 – 7592; d) H. M. Kim, B. R. Kim, M. J. An, J. H. Hong, K. J. Lee, B. R. Cho, Chem. Eur. J. 2008, 14, 2075 – 2083; e) P. S. Mohan, C. S. Lim, Y. S. Tian, W. Y. Roh, J. H. Lee, B. R. Cho, Chem. Commun. 2009, 5365 – 5367; f) Y. N. Shin, C. S. Lim, Y. S. Tian, W. Y. Rho, B. R. Cho, Bull. Korean Chem. Soc. 2010, 31, 599 – 605; g) H. M. Kim, C. Jung, B. R. Kim, S.-Y. Jung, J. H. Hong, Y.-G. Ko, K. J. Lee, B. R. Cho, Angew. Chem. Int. Ed. 2007, 46, 3460 – 3463; Angew. Chem. 2007, 119, 3530 – 3533; h) H. M. Kim, M. S. Seo, M. J. An, J. H. Hong, Y. S. Tian, J. H. Choi, O. K. K. J. Lee, B. R. Cho, Angew. Chem. Int. Ed. 2008, 47, 5167 – 5170; Angew. Chem. 2008, 120, 5245 – 5248; i) I. A. Danish, C. S. Lim, Y. S. Tian, J. H. Han, M. Y. Kang, Bong R. Cho, Chem. Asian J. 2011, 6, 1234 – 1240. [3] a) A. Minta, R. Y. Tsien, J. Biol. Chem. 1989, 264, 19 449 – 19 457; b) Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, 11th ed. (Ed.: R. P. Haugland), Interchim, Molecular Probes, Eugene, 2010,
Chem. Eur. J. 2015, 21, 11306 – 11310
www.chemeurj.org
[6]
[7]
[8] [9]
Chapter 11; c) K. Meuwis, N. Boens, F. D. Schryver, J. Gallay, M. Vincent, Biophys. J. 1995, 68, 2469 – 2473. J.-P. Malval, I. Leray, B. Valeur, New J. Chem. 2005, 29, 1089 – 1094. a) K. Golchini, M. Mackovic-Basic, S. A. Gharib, D. Masilamani, M. E. Lucas, I. Kurtz, Am. J. Physiol. Renal Physiol. 1990, 258, F438 – F443; b) A. P. de Silva, H. Q. N. Gunaratne, K. R. A. S. Sandanayake, Tetrahedron Lett. 1990, 31, 5193 – 5196; c) P. Padmawar, X. Yao, O. Bloch, G. T. Manley, A. S. Verkman, Nat. Methods 2005, 2, 825 – 827; d) T. Hirata, T. Terai, T. Komatsu, K. Hanaoka, T. Nagano, Bioorg. Med. Chem. Lett. 2011, 21, 6090 – 6093; e) X. Zhou, F. Su, Y. Tian, C. Youngbull, R. H. Johnson, D. R. Meldrum, J. Am. Chem. Soc. 2011, 133, 18530 – 18533; f) H. He, M. A. Mortellaro, M. J. P. Leiner, R. J. Fraatz, J. K. Tusa, J. Am. Chem. Soc. 2003, 125, 1468 – 1469; g) J. K. Tusa, H. He, J. Mater. Chem. 2005, 15, 2640 – 2647; h) W. Namkung, P. Padmawar, A. D. Mills, A. S. Verkman, J. Am. Chem. Soc. 2008, 130, 7794 – 7795; i) W. Namkung, Y. Song, A. D. Mills, P. Padmawar, W. E. Finkbeiner, A. S. Verkman, J. Biol. Chem. 2009, 284, 15916 – 15926; j) M. Magzoub, P. Padmawar, J. A. Dix, A. S. Verkman, J. Phys. Chem. B 2006, 110, 21216 – 21221; k) R. D. Carpenter, A. S. Verkman, Org. Lett. 2010, 12, 1160 – 1163; l) R. D. Carpenter, A. S. Verkman, Eur. J. Org. Chem. 2011, 1242 – 1248. a) S. Ast, H. Mìller, R. Flehr, T. Klamroth, B. Walz, H.-J. Holdt, Chem. Commun. 2011, 47, 4685 – 4687; b) S. Ast, T. Schwarze, H. Mìller, A. Sukhanov, S. Michaelis, J. Wegener, O. S. Wolfbeis, T. Kçrzdçrfer, A. Dìrkop, H.-J. Holdt, Chem. Eur. J. 2013, 19, 14911 – 14917; c) T. Schwarze, H. Mìller, S. Ast, D. Steinbrìck, S. Eidner, F. Geißler, M. U. Kumke, H.-J. Holdt, Chem. Commun. 2014, 50, 14167 – 14170. a) G. Jones II, W. R. Jackson, A. M. Halpern, Chem. Phys. Lett. 1980, 72, 391 – 395; b) G. Jones II, W. R. Jackson, C.-Y. Choi, J. Phys. Chem. 1985, 89, 294 – 300; c) P.-H. Huang, J.-Y. Shen, S.-C. Pu, Y.-S. Wen, J. T. Lin, P.-T. Chou, M.-C. P. Yeh, J. Mater. Chem. 2006, 16, 850 – 857. Detailed experimental procedures and data can be found in the Supporting Information. a) R. A. Bissell, A. P. de Silva, H. Q. Nimal Gunarante, R. L. M. Lynch, G. E. M. Maguire, K. R. A. S. Sandanayake, Chem. Soc. Rev. 1992, 21, 187 – 195; b) J. F. Callan, A. P. de Silva, D. C. Magri, Tetrahedron 2005, 61, 8551 – 8588; c) B. Daly, J. Ling, A. P. de Silva, Chem. Soc. Rev. 2015, 44, 4203 – 4211.
Received: April 14, 2015 Published online on July 14, 2015
11310
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
& Fluorescent Probes
A Highly K + -Selective Two-Photon Fluorescent Probe Thomas Schwarze,[a] Janine Riemer,[a] Sascha Eidner,[b] and Hans-Jìrgen Holdt*[a] Abstract: 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 twophoton 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 (KdOP = (28 5) mm and KdTP = (36 6) mm) and in combined K + /Na + solutions (KdOP = (38 8) mm and KdTP = (46 25) mm) reflecting the high K + /Na + selectivity of the fluorescent probe. The TP absorption cross-section (s2PA) 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 + .
Two-photon (TP) active cation-responsive fluorescent probes are suitable tools for monitoring cations in living cells.[1a–e] Using two-photon excited fluorescence (TPEF) probes in TP excitation microscopy, essential benefits can be obtained compared to one-photon (OP) excitation, for example, increased depth penetration, reduced phototoxicity, and intrinsic 3D capabilities. The design of effective TPEF probes is still a challenge and there is a demand for tailoring TPEF probes for cations. An important quality of TPEF probes is the brightness (fFs2PA), that is, the product of the TP absorption cross-section (s2PA) and the fluorescence quantum yield (fF). Desirable features of cation-responsive TPEF probes for biological applications are their stability and fast response, TP excitation in the range of 700–1000 nm, emission maxima (> 500 nm), high brightness, no interference by changes in pH value, and their high selectivity. Nowadays, highly selective TPEF probes for several biologically important cations like Na + ,[2a,b] Ca2 + ,[2c–f] Mg2 + ,[2g] and Zn2 + ,[2h,i] based on the two-photon excitable chromophore 2acetyl-6-(dimethylamino)-naphthalene (acedan) were devel[a] Dr. T. Schwarze, J. Riemer, Prof. H.-J. Holdt Institut fìr Chemie, Anorganische Chemie, Universitt Potsdam Karl-Liebknecht-Str. 24–25, 14476 Golm (Germany) E-mail: [email protected] [b] Dr. S. Eidner Institut fìr Chemie, Physikalische Chemie, Universitt Potsdam Karl-Liebknecht-Str. 24–25, 14476 Golm (Germany) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501473. Chem. Eur. J. 2015, 21, 11306 – 11310
oped. However, to the best of our knowledge, a selective TPEF probe for K + ions has not been studied. In recent years, several fluorescent probes for K + ions were synthesized. Minta and Tsien designed the first fluorescent indicator PBFI (potassium-binding benzofuranisophthalate) to measure intracellular K + ion concentration, which incorporates a diaza-18-crown-6 as an ionophore.[3a] The commercially available PBFI[3b] is the most popular K + indicator,[3c] but it lacks K + /Na + selectivity. To overcome the poor K + /Na + selectivity, calix[4]bisazacrown-5[4] or cryptand-based[5a–l] K + -fluoroionophores were developed. Most of all, the [2.2.3]-triazacryptand (TAC) is a highly K + -selective ionophore established by He and co-workers.[5f] This K + receptor is an essential feature of numerous fluoroionophores with emission wavelengths > 550 nm[5c–e] as a part of a blood sensor device[5f,g] or as components of dextran conjugates for studies in vivo.[5h,i] A drawback of the TAC-based fluoroionophores is their multistep synthesis. Recently, we synthesized easily accessible fluoroionophores for K + ions[6a,b] or Na + ions[6c] by using CuAAC (CuI-catalyzed 1,3-dipolar azide alkyne cycloaddition) reactions. Furthermore, we found that in these fluoroionophores, the signal-transduction chain, that is, p-conjugated aniline(donor)-1,4-triazole-coumarin (acceptor), works nicely under simulated physiological conditions.[6a–c] Firstly, we incorporated a N-phenylaza-18crown-6 ionophore for sensing of K.[6a] The estimated dissociation constant Kd value of 260 mm was not suitable for monitoring physiological K + levels. In the next step, we designed a new highly selective K + /Na + ionophore, a N-(2-methoxyethoxyphenyl)aza-18-crown-6 unit.[6b] The attachment of a 2-methoxyethoxy lariat group at the ortho position of the anilino moiety reduces the Kd value to a physiologically relevant concentration range of K + . Afterwards, we synthesized two isomeric 1,2,3-triazol-1,4-diyl-fluoroionophores. In these probes the regioisomeric 1,4-triazole linkage influences the K + /Na + selectivity and the fF.[6b] A higher fF value and a high K + /Na + selectivity were reached when the aniline donor was connected to position 1 of the triazole and the coumarin acceptor to position 4, as shown in fluorescent probe 2 (Scheme 1). Probe 2 showed a high K + /Na + selectivity and a fluorescence quantum yield of fF = 0.18 in the presence of 160 mm of K + .[6b] To study physiological relevant K + levels by a highly K + /Na + selective TPEF probe, we selected as an ionophore the N-(2methoxyethoxyphenyl)aza-18-crown-6 unit with the same triazole arrangement between the ionophore and the coumarin fluorophore as in compound 2. Furthermore, to enhance the fF value of the K + complexed fluorescent probe for designing an effective TPEF probe, we selected the 2,3,6,7-tetrahydro1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one fluoro-
11306
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication Table 1. Photophysical properties of 1, 2, and 3 in Tris-buffer.[a]
1 1 + K+ 1 + Na + 2 2 + K+ 3 3 + K+ 3 + Na +
Chem. Eur. J. 2015, 21, 11306 – 11310
www.chemeurj.org
lf [nm]
fF[b]
FEF[c,d]
s2PA[e] [GM]
s2PAfF[f] [GM]
434 434 434 430 430 365 365 365
511 511 511 493 493 501 501 501
0.12 0.36 0.13 0.06 0.18 0.03 0.04 0.03
– 2.9 (2.7) 1.1 (1.1) – 2.5 – 1.1 0.9[g]
26 26 26 95 89 n.d.[h] n.d. n.d.
3.1 9.4 3.4 5.7 16.1 n.d. n.d. n.d.
[a] All data were measured in a buffered H2O/DMSO mixture (99:1, v/v; 10 mm Tris; pH 7.2) in the absence and presence of K + or Na + ions (160 mm). The ionic strength was maintained at 160 mm with choline chloride. [b] Fluorescence quantum yield, ( 15) %. [c] Fluorescence enhancement factor, [FEF = I/I0], ( 0.1), measured by a one-photon (OP) process. [d] Values in parentheses measured by two-photon (TP) process (lexTP = 860 nm). [e] TP cross section in 10¢50 cm4 s photon¢1 (GM) for 1 at 860 nm and for 2 at 840 nm. [f] Action cross section in 10¢50 cm4 s photon¢1 (GM). [g] For 3 + Na + , we observed a fluorescence quenching; [h] n.d. = not determined.
Scheme 1. Structures of K + -responsive fluorescent probes 1–3 and of the Na + -selective TPEF probe 4.[2a]
phore (cf. Scheme 1, compound 1). This coumarin-derived fluorophore exhibits a high fF > 0.70,[7a,b] a moderate s2PA value > 150 GM,[7c] and a fluorescence emission lF > 500 nm[7a,b] in polar solvents. Moreover, we synthesized fluorescent probe 3 as an analogue to the Na + -responsive TPEF probe 4, which consists of a 1,7-diaza-15-crown-5 ionophore. The fluoroionophore 3 is also derived from the well-known TP acedan fluorophore and for K + -binding studies the N-(2-methoxyethoxyphenyl)aza-18-crown-6 ionophore was introduced. In this paper, we show that probe 1 (Scheme 1) is a highly K + /Na + -selective TPEF probe for the in vitro determination of K + concentration levels in the range of 1–100 mm. 1 shows a K + -induced fluorescence enhancement factor (FEF) of 2.7 0.1 in Na + -free solutions and a FEF of 2.4 0.1 in combined K + /Na + solutions by a TP excitation at 860 nm. We found for 1, after addition of 160 mm K + , a fluorescence quantum yield of fF = 0.36 and a s2PA of 26 GM. The synthesis of the 1,2,3-triazol-fluoroionophore 1 was realized by a CuAAC of the azido-functionalized o-(2-methoxyethoxy)-phenylaza-18-crown-6[6b] ionophore and the 3-ethynylfuctionalized coumarin.[8] The UV/Vis absorption spectrum of 1 under simulated physiological conditions (10 mm Tris buffer, pH 7.2) showed two charge-transfer (CT) absorption bands (Figure S1 in the Supporting Information). The long-wavelength coumarin CT band is centered at 434 nm in 1. The second CT band at approximately 300 nm is typical for p-conjugated 1,2,3-triazol-1,4-diylfluoroionophores.[6a] In addition, we investigated the influence of K + and Na + in the OP absorption behavior of 1. The coumarin absorption band around 434 nm is essentially unchanged by K + but the complexation of K + ions can be seen from the short wavelength CT absorption at approximately 300 nm (Figure S1 in the Supporting Information). K + reduces the electron donor ability of the anilino unit in 1, which leads to a decrease of the absorption of the CT band at approximately 300 nm (Figure S1). The UV/Vis absorption spectrum of 1 is almost unaffected by Na + ions (Figure S1), demonstrating the high K + /Na + selectivity of probe 1. The fluorescence spectrum of 1 (OP, lex = 430 nm) shows, compared to 2[6b] (lF = 493 nm, fF = 0.06), a red shifted emission centered at 511 nm and a higher fF value of 0.12 under si-
labs [nm]
mulated physiological conditions (Table 1). In both compounds, 1 and 2, the low fluorescence quantum yield is caused by a photoinduced electron transfer (PET)[9a–c] across a virtual spacer, as already observed for 1,2,3-triazol-fluoroionophores for K + [6a,b] and Na + .[6c] The TPEF spectrum of 1 (TP, lex = 860 nm) shows also an emission centered at 511 nm. The highest s2PA value of 26 GM for 1 was found by a TP excitation at 860 nm (Figure S9 in the Supporting Information). We investigated the influence of K + and Na + on the fluorescence of 1 in the physiologically relevant concentration range of 1–160 mm K + and Na + . Figure 1 a shows the fluorescence enhancement of 1 (OP, lex = 430 nm) in a buffered H2O/DMSO mixture (99:1, v/v; 10 mm Tris; pH 7.2) in the presence of 1– 160 mm K + at a constant ionic strength of 160 mm adjusted with choline chloride. We observed K + -induced fluorescence enhancements of 1 (FEF = 2.9 0.2, Figure 1 a) and 2 (FEF = 2.5[6b]). The maximum signal change was observed at 100 mm K + , with fF values for 1 of 0.36 and for 2 of 0.18. Thus, the fF of 1 + 160 mm K + is two times higher than 2 + 160 mm K + . Furthermore, the TPEF intensity of 1 (TP, lex = 860 nm) is increased by K + ions under simulated physiological conditions (Figure 2 a). We observed a FEF of 2.7 0.1 (1 + 160 mm K + ), when 1 was excited at 860 nm (Figure 2 a). The FEFs of 1 + 160 mm K + by OP- and TP-excitation were similar to each other (Figure 2 a). The run of the titration curves of 1 + K + were independent of the excitation process (OP or TP, cf. Figure 2 a). We found for 1 + 160 mm K + a s2PA value of 26 GM at 860 nm (Table 1). The s2PA values of 1 (26 GM) and 1 + K + (26 GM) were the same, concluding that the TP excitation process at 860 nm is not influenced by K + ions. For probe 2 we found a higher s2PA value of 95 GM at 840 nm and an almost identical s2PA value of 89 GM at 840 nm for 2 + 160 mm K + (see Table 1). To verify the K + /Na + selectivity of probe 1, we measured the fluorescence intensities at various concentrations of Na + ions (1–160 mm) under simulated physiological conditions ex-
11307
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
Figure 1. Fluorescence response of probe 1 (10¢5 m, lex = 430 nm) to K + in the presence of various concentrations of ions under simulated physiological conditions (10 mm Tris-buffer, pH 7.2); the ionic strength was maintained at 160 mm with: a) choline chloride and b) various concentrations of Na + ions in combined K + /Na + solutions.
cited through an OP and TP process, respectively. As shown in Figure 2 a, the fluorescence of probe 1 at 511 nm is slightly affected by Na + ions (fF = 0.13 at 160 mm Na + ). Furthermore, this high K + /Na + selectivity of probe 1 was validated by using solutions that contained both K + and Na + ions to ensure a total metal ion concentration of 160 mm (Figure 1 b, lex = 430 nm). The fluorescence enhancement by a factor of 2.4 0.2 at 160 mm K + was only slightly lower than that in the experiment without Na + ions (FEF = 2.9 0.2). Continuing, we observed a marginal change of the FEF of 1 + K + from 2.7 0.1 (without Na + , see Figure 2 a) to 2.4 0.1 (in the presence of Na + ) by an excitation at 860 nm (Figure S10 in the Supporting Information). In addition, the K + selectivity of probe 1 was substantiated by titration experiments with other metal ions such as Mg2 + and Ca2 + (at 160 mm), and Mn2 + , Fe3 + , Co2 + , Ni2 + , Cu2 + , and Zn2 + (at 100 mm) under simulated physiological conditions. The fluorescence intensity of 1 showed only negligible changes in the presence of these cations (Figure S3c in the Supporting Information). Importantly, probe 1 recognized K + even in the co-presence of these cations (Figure S3c). Moreover, probe 1 showed high sensitivity towards K + ions with a detection limit of 0.2 mm.[8] Chem. Eur. J. 2015, 21, 11306 – 11310
www.chemeurj.org
Figure 2. a) Fluorescence-enhancement factors (If(max)/If0(max)) for probe 1 (10¢5 m, lex = 430 nm (OP) and lex = 860 nm (TP)) under simulated physiological conditions (10 mm Tris buffer, pH 7.2) in the presence of various concentrations of K + or Na + ions; the ionic strength was maintained at 160 mm with choline chloride. b) Two-photon action spectra of 1 (10¢5 m) in the presence of 160 mm K + and 160 mm Na + ions.
Dissociation constants (Kd) were calculated from plots of fluorescence intensities versus K + ion concentration.[8] The Kd values of probe 1 in Na + -free solution were KdOP = (28 5) mm and KdTP = (36 6) mm for the OP and TP process, respectively. These values increased to KdOP = (38 8) mm and KdTP = (46 25) mm for the combined K + /Na + solutions. A similar result was found for 2 + K + in Na + -free and combined Na + /K + solutions (KdOP values increases from 26 to 29 mm).[6b] Thus, these close Kd values demonstrate the high K + /Na + selectivity of probe 1. Hence, Na + ions have a negligible effect on the performance of probe 1 under simulated physiological conditions. Changes in pH value showed only a minor effect towards the sensitivity of 1 at various concentrations of K + ions within the pH range 6.8–8.8 (see Figure S8 in the Supporting Information). Overall, the fluorescence signal of 1 is mainly increased by K + ions and marginal influenced by Na + ions independently of the excitation process (one or two photon) (Figure 2 a). This demonstrates the high K + /Na + selectivity of the TPEF probe 1. Figure 2 b displays the fFs2PA values of 1, 1 + K + , and 1 + Na + in the range of 780–900 nm. The brightness of 1 + K + at 860 nm is about 10 GM and that of 2 + K + at 840 nm is about
11308
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication 16 GM (Table 1). Herein, 1 + K + and 2 + K + showed a lower brightness than the acedan-based TPEF probes for Na + (95 GM[2a] and 83 GM[2b]), but 1 + K + and 2 + K + are moderately excitable by a TP process and should be applicable in TP excitation microscopy. Therefore, 1 and 2 are suitable TPEF probes for the highly selective sensing of K + in the range of 1– 100 mm under simulated physiological conditions. Herein, to the best of our knowledge, we report for the first time on highly K + -selective TPEF probes. To investigate the K + binding of the N-(2-methoxyethoxyphenyl)aza-18-crown-6 ionophore in combination with the bright TP acedan fluorophore, we synthesized fluoroionophore 3. Compound 3 was prepared by the coupling reaction between 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]-naphthalene[2g] and N-(4-amino-2-methoxyethoxyphenyl)aza-18-crown-6 ether[6b] .[8] For 3, we found a broad OP absorption band centered at 365 nm, which is not influenced by K + or Na + ions (Table 1). This is typical for acedan-based TPEF probes for cations.[2a–i] Probe 3 showed a fluorescence emission centered around 501 nm (OP, lex = 365 nm) with a fluorescence quantum yield of 0.03 under simulated physiological conditions (Table 1), comparable to reported TPEF probes[2] based on a PET-quenching mechanism of the acedan fluorophore. The K + /Na + selectivity of the fluorescent probe 3 is not as high as that observed for probe 1. Probe 3 had a FEF of only 1.1 in the presence of 160 mm K + (fF = 0.04) under simulated physiological conditions (Table 1, Figures S4a and S5b in the Supporting Information). However, the fluorescence of probe 3 is slightly quenched in the presence of 160 mm Na + (fF = 0.03, Table 1, Figure S5a and S5b). This contrary result was further substantiated by using solutions that contained both Na + and K + ions. We observed a higher FEF of 1.15 for 3 + K + in the presence of Na + (Figure S4b) caused by the lower initial fluorescence intensity of 3 + 160 mm Na + . The KdOP values of 3 + K + in Na + -free solutions were 49 and 45 mm in combined Na + /K + solutions, forming a weaker K + complex as 1. Thus, the combination of a N-(2-methoxyethoxyphenyl)aza18-crown-6 ionophore and an acedan fluorophore bridged by an amide is more unsuitable to determine K + ions than the 1,2,3-triazole-fluoroionophores 1 and 2. We assume, that the triazole unit and their arrangement, as found in 1 and 2, is substantial for the high K + /Na + selectivity. Furthermore, for the design of highly selective TPEF probes for K + based on the o-(2-methoxyethoxy)-phenylaza-18-crown-6 ionophore, an integration of the triazole unit should be considered. In summary, we have synthesized the fluorescent probes 1 and 3 based on the N-(2-methoxyethoxyphenyl)aza-18crown-6 ionophore. Both consisting of different TP chromophores, 1 contains a coumarin unit and 3 an acedan moiety. K + induces fluorescence intensity enhancements of 1 and 3 under simulated physiological conditions, but 3 shows a FE by only a factor of 1.09 in the presence of 160 mm K + . On the other hand, 1 shows a FEF of 2.9 0.2 and 2.7 0.1 by OP and TP excitation, respectively. The fluorescence quantum yield is fF = 0.36 and the two-photon excitation cross section is s2PA = 26 GM in the presence of 160 mm K + . A similar FEF of 2.4 0.2 Chem. Eur. J. 2015, 21, 11306 – 11310
www.chemeurj.org
for 1 was observed in combined K + /Na + solutions. This high K + /Na + selectivity is substantiated by the close Kd values for Na + -free (KdOP = (28 5) mm and KdTP = (36 6) mm) and combined K + /Na + solutions (KdOP = (38 8) mm and KdTP = (46 25) mm) by OP or TP excitation. We have shown that 1 is a highly K + -selective TPEF probe with a suitable Kd value to determine physiologically relevant K + levels. Currently in our laboratories, we generate TPEF probes for K + , Na + , Ca2 + , Mg2 + , and Zn2 + based on the signal-transduction chain aniline-triazole-coumarin with a high brightness.
Experimental Section General methods and reagents All commercially available chemicals were used without further purification. Solvents were distilled prior to use. 1H and 13C NMR spectra were recorded on 300 and 600 MHz instruments, respectively. Data are reported as follows: chemical shifts in ppm (d), multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet), integration, coupling constant (Hz). ESI spectra were recorded using a Micromass Q-TOF micro mass spectrometer in a positive electrospray mode. Column chromatography was performed with silica gel (Merck; silica gel 60 (0.04–0.063 mesh)). 10-{1-[4-(1,4,7,10,13-Pentaoxa-16-azacyclooctadecan-16-yl)-3-(2methoxyethoxy)phenyl]-1H-1,2,3-triazol-4-yl}-2,3,6,7-tetrahydro1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (1): A mixture of N-(4-azido-2-methoxyethoxyphenyl)aza-[18]crown-6 ether[6b] (190 mg, 0.415 mmol), 10-ethynyl-2,3,6,7-tetrahydro-1H,5H,11H[1]benzopyrano[6,7,8-ij]quinolizin-11-one[8] (110 mg, 0.415 mmol), CuSO4·5H2O (5.3 mg) and sodium ascorbate (8.2 mg) in 9 mL THF/ H2O (v/v, 2:1) was stirred at 60 8C for 48 h. After that, 5 mL H2O were added to the mixture and extracted with CHCl3 (30 mL). The organic layer was dried with MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica using CHCl3/CH3OH (v/v, 9:1) as an eluent mixture to afford 1 as a dark yellow solid (70 mg, 24 %). M.p.: 79 8C (decomp.); 1H NMR (CDCl3, 600 MHz): d = 8.64 (s, 1 H), 8.57 (s, 1 H), 7.36–7.33 (m, 1 H), 7.27–7.25 (m, 1 H), 7.17 (s, 1 H), 7.00 (s, 1 H), 4.25–4.17 (m, 2 H), 3.78–3.76 (m, 2 H), 3.75–3.49 (m, 24 H), 3.43 (s, 3 H), 3.29 (dd, J = 5.3, 11.2 Hz, 4 H), 2.93 (t, J = 6.5 Hz, 2 H), 2.79 (t, J = 6.2 Hz, 2 H), 2.01– 1.95 ppm (m, 4 H); 13C NMR (CDCl3, 75 MHz): d = 160.96, 151.14, 147.90, 146.21, 142.53, 138.94, 136.17, 125.63, 121.29, 120.34, 119.01, 113.02, 109.59, 108.50, 106.43, 106.29, 102.63, 70.85, 70.74, 70.62, 70.57, 70.29, 69.79, 68.02, 58.96, 52.87, 50.09, 49.70, 27.53, 21.44, 20.53, 20.31 ppm; HRMS (ESI + ): m/z calcd for C38H50N5O9 : 720.3609; found: 720.3620. N-(4-(1,4,7,10,13-Pentaoxa-16-azacyclooctadecan-16-yl)-3-(2-methoxyethoxy)phenyl)-2-((6-acetylnaphthalen-2-yl)(methyl)amino)acetamide (3): A mixture of N-(4-amino-2-methoxyethoxyphenyl)aza-[18]crown-6 ether[6b] (127 mg, 0.49 mmol), 1-hydroxybenzotriazole (49 mg, 0.33 mmol), 1,3-dicyclohexyl carbodiimide (102 mg, 0.49 mmol) in dry dichloromethane (20 mL) was stirred for 30 min. To this solution, 6-acyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene[2g] (85 mg, 0.33 mmol) was added and stirred for 12 h under argon. The mixture was extracted with CH2Cl2 (60 mL) and washed with 1 N NaOH. The organic layer was dried over MgSO4, and removed in vacuo. The product was purified by column chromatography (silica gel) using chloroform/methanol (v/v, 10:1 to 5:1) as the eluent. It was further purified by crystallization using
11309
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication chloroform/hexane to yield compound 3 as a light yellow solid (82 mg, 37 %). M.p.: 88 8C (decomp.); 1H NMR (CDCl3, 300 MHz): d = 11.10 (s, 1 H), 8.28 (s, 1 H), 7.89 (d, J = 8.7 Hz, 1 H), 7.78 (d, J = 8.8 Hz, 1 H), 7.64 (d, J = 8.8 Hz, 1 H), 7.52 (d, J = 8.6 Hz, 1 H), 7.35 (s, 1 H), 7.22 (d, J = 10.0 Hz, 1 H), 7.12 (dd, J = 8.7, 2.5 Hz, 1 H), 7.00 (s, 1 H), 4.22–4.18 (m, 2 H), 3.77–3.33 (m, 31 H), 3.24 (s, 3 H), 2.64 ppm (m, 3 H); 13C NMR (CDCl3, 75 MHz): d = 197.71, 166.99, 158.51, 149.37, 141.84, 131.73, 130.92, 130.24, 126.89, 126.50, 124.46, 123.91, 121.23, 118.94, 116.26, 111.67, 107.63, 105.64, 70.93, 70.57, 70.03, 69.88, 69.74, 69.45, 69.26, 68.99, 58.84, 58.33, 40.01, 26.41 ppm; HRMS (ESI + ): m/z calcd for C36H50N3O9 : 668.3547; found: 668.3537.
[4] [5]
Keywords: click chemistry · fluorescence · fluorescent probes · potassium · two-photon [1] a) M. Pawlicki, H. A. Collins, R. G. Denning, H. L. Anderson, Angew. Chem. Int. Ed. 2009, 48, 3244 – 3266; Angew. Chem. 2009, 121, 3292 – 3316; b) S. Yao, K. D. Belfield, Eur. J. Org. Chem. 2012, 2012, 3199 – 3217; c) S. Sumalekshmy, C. J. Fahrni, Chem. Mater. 2011, 23, 483 – 500; d) H. M. Kim, B. R. Cho, Acc. Chem. Res. 2009, 42, 863 – 872; e) A. R. Sarkar, D. E. Kang, H. M. Kim, B. R. Cho, Inorg. Chem. 2014, 53, 1794 – 1803. [2] a) M. K. Kim, C. S. Lim, J. T. Hong, J. H. Han, H.-Y. Jang, H. M. Kim, B. R. Cho, Angew. Chem. Int. Ed. 2010, 49, 364 – 367; Angew. Chem. 2010, 122, 374 – 377; b) A. R. Sarkar, C. H. Heo, M. Y. Park, H. W. Lee, H. M. Kim, Chem. Commun. 2014, 50, 1309 – 1312; c) H. M. Kim, B. R. Kim, J. H. Hong, J.-S. Park, K. J. Lee, B. R. Cho, Angew. Chem. Int. Ed., 2007, 46, 7445 – 7448; Angew. Chem. 2007, 119, 7589 – 7592; d) H. M. Kim, B. R. Kim, M. J. An, J. H. Hong, K. J. Lee, B. R. Cho, Chem. Eur. J. 2008, 14, 2075 – 2083; e) P. S. Mohan, C. S. Lim, Y. S. Tian, W. Y. Roh, J. H. Lee, B. R. Cho, Chem. Commun. 2009, 5365 – 5367; f) Y. N. Shin, C. S. Lim, Y. S. Tian, W. Y. Rho, B. R. Cho, Bull. Korean Chem. Soc. 2010, 31, 599 – 605; g) H. M. Kim, C. Jung, B. R. Kim, S.-Y. Jung, J. H. Hong, Y.-G. Ko, K. J. Lee, B. R. Cho, Angew. Chem. Int. Ed. 2007, 46, 3460 – 3463; Angew. Chem. 2007, 119, 3530 – 3533; h) H. M. Kim, M. S. Seo, M. J. An, J. H. Hong, Y. S. Tian, J. H. Choi, O. K. K. J. Lee, B. R. Cho, Angew. Chem. Int. Ed. 2008, 47, 5167 – 5170; Angew. Chem. 2008, 120, 5245 – 5248; i) I. A. Danish, C. S. Lim, Y. S. Tian, J. H. Han, M. Y. Kang, Bong R. Cho, Chem. Asian J. 2011, 6, 1234 – 1240. [3] a) A. Minta, R. Y. Tsien, J. Biol. Chem. 1989, 264, 19 449 – 19 457; b) Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, 11th ed. (Ed.: R. P. Haugland), Interchim, Molecular Probes, Eugene, 2010,
Chem. Eur. J. 2015, 21, 11306 – 11310
www.chemeurj.org
[6]
[7]
[8] [9]
Chapter 11; c) K. Meuwis, N. Boens, F. D. Schryver, J. Gallay, M. Vincent, Biophys. J. 1995, 68, 2469 – 2473. J.-P. Malval, I. Leray, B. Valeur, New J. Chem. 2005, 29, 1089 – 1094. a) K. Golchini, M. Mackovic-Basic, S. A. Gharib, D. Masilamani, M. E. Lucas, I. Kurtz, Am. J. Physiol. Renal Physiol. 1990, 258, F438 – F443; b) A. P. de Silva, H. Q. N. Gunaratne, K. R. A. S. Sandanayake, Tetrahedron Lett. 1990, 31, 5193 – 5196; c) P. Padmawar, X. Yao, O. Bloch, G. T. Manley, A. S. Verkman, Nat. Methods 2005, 2, 825 – 827; d) T. Hirata, T. Terai, T. Komatsu, K. Hanaoka, T. Nagano, Bioorg. Med. Chem. Lett. 2011, 21, 6090 – 6093; e) X. Zhou, F. Su, Y. Tian, C. Youngbull, R. H. Johnson, D. R. Meldrum, J. Am. Chem. Soc. 2011, 133, 18530 – 18533; f) H. He, M. A. Mortellaro, M. J. P. Leiner, R. J. Fraatz, J. K. Tusa, J. Am. Chem. Soc. 2003, 125, 1468 – 1469; g) J. K. Tusa, H. He, J. Mater. Chem. 2005, 15, 2640 – 2647; h) W. Namkung, P. Padmawar, A. D. Mills, A. S. Verkman, J. Am. Chem. Soc. 2008, 130, 7794 – 7795; i) W. Namkung, Y. Song, A. D. Mills, P. Padmawar, W. E. Finkbeiner, A. S. Verkman, J. Biol. Chem. 2009, 284, 15916 – 15926; j) M. Magzoub, P. Padmawar, J. A. Dix, A. S. Verkman, J. Phys. Chem. B 2006, 110, 21216 – 21221; k) R. D. Carpenter, A. S. Verkman, Org. Lett. 2010, 12, 1160 – 1163; l) R. D. Carpenter, A. S. Verkman, Eur. J. Org. Chem. 2011, 1242 – 1248. a) S. Ast, H. Mìller, R. Flehr, T. Klamroth, B. Walz, H.-J. Holdt, Chem. Commun. 2011, 47, 4685 – 4687; b) S. Ast, T. Schwarze, H. Mìller, A. Sukhanov, S. Michaelis, J. Wegener, O. S. Wolfbeis, T. Kçrzdçrfer, A. Dìrkop, H.-J. Holdt, Chem. Eur. J. 2013, 19, 14911 – 14917; c) T. Schwarze, H. Mìller, S. Ast, D. Steinbrìck, S. Eidner, F. Geißler, M. U. Kumke, H.-J. Holdt, Chem. Commun. 2014, 50, 14167 – 14170. a) G. Jones II, W. R. Jackson, A. M. Halpern, Chem. Phys. Lett. 1980, 72, 391 – 395; b) G. Jones II, W. R. Jackson, C.-Y. Choi, J. Phys. Chem. 1985, 89, 294 – 300; c) P.-H. Huang, J.-Y. Shen, S.-C. Pu, Y.-S. Wen, J. T. Lin, P.-T. Chou, M.-C. P. Yeh, J. Mater. Chem. 2006, 16, 850 – 857. Detailed experimental procedures and data can be found in the Supporting Information. a) R. A. Bissell, A. P. de Silva, H. Q. Nimal Gunarante, R. L. M. Lynch, G. E. M. Maguire, K. R. A. S. Sandanayake, Chem. Soc. Rev. 1992, 21, 187 – 195; b) J. F. Callan, A. P. de Silva, D. C. Magri, Tetrahedron 2005, 61, 8551 – 8588; c) B. Daly, J. Ling, A. P. de Silva, Chem. Soc. Rev. 2015, 44, 4203 – 4211.
Received: April 14, 2015 Published online on July 14, 2015
11310
Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
