Red- to NIR-Emitting, BODIPY-Based, K+-Selective Fluoroionophores and Sensing Materials

Red- to NIR-Emitting, BODIPY-Based, K⁺-Selective

Fluoroionophores and Sensing Materials

Bernhard J. Müller, Sergey M. Borisov,* and Ingo Klimant

Extracellular potassium (whole blood,

serum) is the key analyte in clinical diag-

nostics as elevated K⁺concentration

(hyperkalemia) is an indication for car-

diac arrhythmia which can lead to sudden

heart failure.^[8]The crucial requirement

and greatest challenge for potassium sen-

sors for clinical diagnostics is the selec-

tive detection of the low extracellular K⁺

(5 × 10⁻³m) over a high Na⁺concentration

(150 × 10⁻³m).^[9]Apart from good selec-

tivity, inertness to variations of pH in the

relevant range is of extreme importance.

Optical sensing materials for the selective measurement of potassium ions

(K⁺) in water are presented. The indicator dyes are based on an aza-crown

ether as a receptor and borondipyrromethenes (BODIPY) dyes as ﬂuoro-

phores. Fluorescence enhancement is caused by the reduction of photoin-

duced electron transfer (PET) upon complexation with K⁺ions. The family of

new indicators possesses tuneable optical properties (green to red excitation,

red to NIR emission) and PET efﬁciencies. They exhibit high brightness with

quantum yields between 0.20 and 0.47 in the “on” state and a molar absorp-

tion coefﬁcient between 30 000 and 290 000 ^m⁻¹cm⁻¹. The new indicator

dyes are immobilized in biocompatible hydrogel matrices to obtain stable

nonleaching and fast responding (t₉₀≈ 10 s) sensing materials for continuous

measurements of potassium. They are realized in various formats such as

planar optodes, ﬁber-optic sensors, and water-dispersible polymer-based

nanoparticles. Apart from ﬂuorescence intensity measurements, self-

referenced read-out of ﬂuorescence decay time is demonstrated. All sensor

materials display a high K⁺/Na⁺selectivity and are not inﬂuenced by pH within

the physiologically relevant range. Practical applicability of the materials is

emphasized by application of a ﬁber-optic sensor to quantiﬁcation of K⁺in

serum, which shows excellent correlation with the reference measurements.

Fluorescence-based

measurements

offer several advantages compared to other

analytical techniques (e.g., electrochemical

measurements with ion selective elec-

trodes) as they are free of electromagnetic

interferences and are noninvasive. Fluo-

rescent sensors are available in various

formats including planar sensors and

spots, ﬁber-optic sensors and (nano)par-

ticles. Nanoscale sensing materials can

be incorporated into small objects such

as cells to gain information in real time

or enable high-resolution imaging.^[10]Conventional ﬂuores-

cence and laser-scanning microscopes can be used for this

purpose.^[11,12]

A ﬂuorescent indicator dye typically consists of a ﬂuorophore

linked to a recognition unit (receptor/ionophore) leading to a

ﬂuoroionophore. Fluoroionophores based on intramolecular

quenching due to photoinduced electron transfer (PET) have

been applied successfully for sensing cations in the last years.^[13]

Typically, the receptor unit bears a tertiary amine group which

is responsible for the emission enhancement in presence of

ions, due to the reduction of the PET effect.

PBFI (potassium binding benzofuran isophthalate) con-

sisting of a diaza-18-crown-6 ether as a receptor and a benzo-

furan derivative as a ﬂuorophore is the most popular indicator

dye for molecular biology studies and the only commercially

available ﬂuoroionophore.^[14]However, this PBFI indicator

suffers from a poor K⁺/Na⁺selectivity and is expensive. A tri-

azacryptand (TAC) receptor designed by He et al.^[15]shows

excellent K⁺selectivity and sensitivity, but its preparation is very

tedious due to extensive multistep synthesis. Hence, there is a

high demand for simple, sensitive, and selective receptors.

Recently, Ast et al. introduced a phenylaza-[18]crown-6

with ortho-substituted 2-methoxyethoxy group as receptor

which has a good K⁺/Na⁺selectivity and is simpler to prepare

than the TAC receptor.^[16]Combining this receptor with a

1. Introduction

Potassium (K⁺) plays a central role in the human body and

is necessary for the function of all living cells. Inside the

cell K⁺is the main ion with an approximate concentration

of 150 × 10⁻³m while extracellular concentrations are about

5 × 10⁻³m.^[1,2]The difference in concentration causes a disparity

in electric potential between the interior and exterior of cells,

known as the membrane potential.^[3]It takes part in substantive

processes and functions such as the regulation of cell growth,

acid-base equilibrium, and maintaining the normal blood pres-

sure.^[4–6]Cells can control this potential by opening or blocking

K⁺channel transmembrane proteins.^[7]This regulation of intra-

and extracellular K⁺concentration plays a key role in metabolic

processes and is of high interest for pharmacological research.

However, the molecular mechanism of potassium physiology

and pathology are still insufﬁciently understood, partly due to

the lack of tools for measuring K⁺.

B. J. Müller, Dr. S. M. Borisov, Prof. I. Klimant

Institute of Analytical Chemistry and Food Chemistry

Graz University of Technology

Stremayrgasse 9, 8010 Graz, Austria

E-mail: sergey.borisov@tugraz.at

DOI: 10.1002/adfm.201603822

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

coumarin ﬂuorophore enabled measurement of K⁺in the range

of 2 × 10⁻³–100 × 10⁻³m with a negligible cross-sensitivity to

Na⁺under physiological conditions and a 2.5-fold ﬂuorescence

enhancement at 160 × 10⁻³m K⁺at 493 nm. The ﬂuorophores

used for optical K⁺sensing included derivates of coumarins,^[16]

xanthene dyes,^[17–19]naphthalimides,^[15,20]borondipyrrome-

thenes (BODIPYs),^[21–26]and other dyes.^[27]Most of them are

excitable below 600 nm (Table S1, Supporting Information).

However, indicator dyes with longer wavelength of absorption

and emission (>600 nm) are of particular interest as they allow

measurements in highly scattering and absorbing media (e.g.,

tissues) as well as in autoﬂuorescent media (e.g., biological

samples).

The ﬂuoroionophores for intracellular imaging of K⁺

reported by the groups of Verkman and Meldrum were modi-

ﬁed with charged groups in order to facilitate the solubility in

water and to enable cell uptake. Application of such probes is

limited to assays in small volumes (such as cells) since they

have to be added to the analyzed media. Thus, immobilization

of the ﬂuoroionophores into polymeric matrices is necessary

to design sensors for continuous monitoring of the analytes

(e.g., in diagnostics). Despite seemingly straightforward, this

can be a challenging task since (i) it should be ensured that the

material possesses good permeability for the analyte, i.e., is suf-

ﬁciently hydrophilic; (ii) the indicator should be compatible to

the matrix to prevent aggregation; (iii) it should not leach out

of the matrix which is not unlikely for the dyes bearing hydro-

philic receptors. Last but not least, not all indicators which

work properly in solution show a response when immobilized

in a hydrogel, as the environmental polarity plays an impor-

tant role in the PET effect. In fact, very few sensors based on

immobilized indicators have been reported so far.^[15,16,20]They

employed ﬂuoroionophores which show excitation and emis-

sion at shorter wavelength and only moderate brightness. Thus,

preparation of novel high performance sensing materials for K⁺

remains of utmost importance.

Figure 1. Operating principle of a ﬂuorescent K⁺probe. Fluorescence

enhancement is caused by complexation of K⁺and a reduction of the

photoinduced electron transfer (PET).

Supporting Information). Importantly, this convenient proce-

dure relies on readily available reagents and allows prepara-

tion of multigram quantities of the receptor. All the BODIPY

indicators are prepared via condensation of a pyrrole and the

aromatic aldehyde from the receptor, subsequent oxidation

with DDQ and complexation using a base and BF₃–etherate.

We prepared differently substituted pyrroles in order to tune

the optical properties and optimize the PET efﬁciencies of the

indicators (Figure 2). Whereas 2,4-dimethylpyrrol is commer-

cially available, other pyrrols can be conveniently prepared from

the respective ketones and 3-phenyl-2H-azirene.^[28]A different

route is necessary for preparation of furan-fused pyrrols.^[29]

2.2. Spectral and Electrochemical Properties of the New

Fluoroionophores

In this contribution we report a palette of K⁺ﬂuoroiono-

phores based on BODIPY dyes having tuneable spectral proper-

ties, good brightness, insensitivity to pH in the relevant con-

ditions and tuneable sensitivities. They incorporate a selective,

yet conveniently accessible aza-crown-ether receptor. We will

report on preparation and properties of novel materials (solid

state optical sensors and potassium-sensitive nanoparticles)

which enable numerous applications in science and technology

exemplary demonstrated for measurement in fetal bovine

serum samples.

FI 1 prepared from 2,4-dimethylpyrrol is a commonly used

BODIPY dye with absorption in the blue and emission in the

green region of the electromagnetic spectrum (Figure 3 and

Table 1). A bathochromic shift of about 70 nm is obtained

via extension of the aromatic system to tetraphenyl-BODIPY

FI 2. Rigidization results in an even further bathochromic

shift of the absorption and emission (about 70 nm), higher

molar absorption coefﬁcients and ﬂuorescence quantum yields

(Table 1). As expected FI 4 absorbs at longer wavelength com-

pared to FI 3 due to the electron-donating character of the

methoxy-group. FI 3 and FI 4 can be efﬁciently excited using

red light and show emission in the red/NIR part of the spec-

trum. FI 5 includes a fused furan ring and belongs to the so-

called Keio Fluors variation of BODIPYs.^[30]This dye class has

extraordinarily high molar absorption coefﬁcients and high

quantum yields. Indeed, the indicator FI 5 demonstrates ε of

2. Results and Discussion

2.1. Synthesis of BODIPY Fluoroionophores

The new ﬂuoroionophores (FIs) represent a highly modular

system consisting of two building blocks—the receptor and the

chromophore (Figure 1). The spectral properties are controlled

by the pyrroles and the sensitivity is controlled by the receptor

which enables high ﬂexibility of the design. The synthesis of

the receptor (o-(2-methoxyethoxy)phenylaza-[18]crown-6 lariat

ether) was performed as described by Ast et al.^[16](Figure S1,

−1

195 600 ^mcm⁻¹and QY of 60% in the “on” state (Table 1).

BODIPY indicators are well known for their sharp and narrow

absorption and emission spectra^[31]which is also the case for

all new ﬂuoroionophores except FI 2. It shows a very broad

absorption and consequently a lower molar absorption coef-

ﬁcient. This can be attributed to the rotation of the phenyl

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

Figure 2. a) Synthesis of different pyrroles and BODIPY indicators. b) Synthetic pathway to FI 5.

rings, since aggregation of the dye was not observed. FI 2

already shows an appreciable quantum yield of 35% in solution

when protonated with triﬂuoracetic acid whereas the quantum

yields for BODIPYs FI 1, 3, 4, and 5 in solution are signiﬁ-

cantly higher. Luminescence lifetimes in the “on”-state (fully

protonated) vary from 3.1 to 5.1 ns. To conclude, all the new

ﬂuoroionophores feature excellent ﬂuorescence brightness

(BS) which is particularly high for FI 5.

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

for the excited state (E_1/2red*) from the reduction potential in

the ground state (E_1/2red) and the energy of the excited state

(E₀₀) clearly show that FI 1–3 in the excited state are more pow-

erful oxidants than FI 4 and FI 5. In the same conditions, the

oxidation potential of the free aza-crown receptor (E_1/2ox(rec))

was estimated to be 0.817 V. Thus, ΔG of the electron transfer

reaction (ΔG_PET= E_1/2ox(rec)–E_1/2red*(FI)^[32]) is negative for

FI 1–FI 3 but close to zero for FI 4 and FI 5. An increase in

_1/2ox(rec) by at least 0.1 V is detectable in presence of K⁺

(Figure S2g, Supporting Information) indicating the increase

in ΔG of quenching reaction. It should be mentioned that the

absolute values are just a very rough estimation due to the fact

that the ion pairing energy describing charge generation and

separation within the electron-transfer complex is neglected

and E₀₀was estimated from ﬂuorescence maxima and not on

the edge of the spectra.

2.3. Polymer-Based Sensing Materials

In order to prepare solid state sensing materials the

ﬂuoroionophores have to be immobilized in a hydrogel matrix

(Figure 4). This was achieved by simply dissolving the dyes

and the polymer in organic solvent and coating the resulting

“cocktail” onto a transparent inert polyethylene terephthalate

support. Hydromed D4 was chosen as a suitable matrix due

to its capability to take up about 100% water, good ion perme-

ability, biocompatibility, and commercial availability. As can be

seen from Figure 4 immobilized FI 1 leaches continuously out

of the sensor matrix into the analyzed solution. Leaching of the

dye can be attributed to very hydrophilic nature of the receptor

which is particularly pronounced in presence of K⁺due to the

charged nature of the resulting complex. In contrast to FI 1,

indicators FI 2–5 are signiﬁcantly more hydrophobic which

completely eliminates leaching (Figure 4 and Figure S3, Sup-

porting Information). Due to instability of FI 1-based sensors

only materials based on FI 2–5 were characterized. The calibra-

tion curves for the sensors were obtained in 20 × 10⁻³m TRIS

buffer at pH of 7.4 (representing the pH of blood) with dif-

ferent KCl concentrations (Figure 5). As can be seen, the ﬂuo-

rescence intensity greatly increases in presence of the analyte

due to decreased efﬁciency of PET from the amino group of the

receptor to the chromophore. For FI 2 and FI 3 the relative ﬂuo-

rescence enhancement in presence of K⁺is higher than for FI 4

and 5. This behavior is attributed to the electron-donating effect

Figure 3. a) Normalized absorption and b) emission spectra of the

ﬂuoroionophores in dichloromethane.

Cyclovoltammetric measurements (Figure S2, Supporting

Information, and Table 1) show that FI 2 and FI 3 are more

difﬁcult to oxidize than FI 4 and FI 5 which indicates more

electron-rich character of the latter. FI 1 bearing 4 electron-

donating methyl groups instead of electron-withdrawing aryls

shows signiﬁcantly lower oxidation potential compared to

other FIs. The trend in the ground state reduction potentials

is less evident. However, the reduction potentials estimated

Table 1. Photophysical and electrochemical properties of the ﬂuoroionophores in solutions and immobilized in a hydrogel matrix (D4).

Dye

QY in

THF^a)

QY in D4 with

1 ^mKCl

QY in D4 with

0.1 m HCl

Lifetime in

THF^a)[ns]

_1/2ox

[V]

E_1/2red

[V]

E_1/2ox*^b)

[V]

E_1/2red*^c)

[V]

λmax abs (ε)

λmax em

[nm]

BS, ε·QY

[nm] ([^m⁻¹cm⁻¹])

FI 1

FI 2

FI 3

FI 4

FI 5

504 (53 600)

571 (29 900)

640 (109 300)

655 (87 200)

670 (195 600)

513

615

660

682

688

0.66

0.35

0.68

0.53

0.60

35 376

10 465

74 324

46 216

117 360

n.d.

0.20

0.47

0.42

n.d.

0.64

0.76

0.46

0.41

5.1

3.5

5.0

4.1

3.1

0.44

0.93

1.08

0.80

0.88

1.15

1.07

1.06

0.79

0.92

−1.27

−0.95

−0.82

−1.03

−0.88

−1.98

−1.09

−0.80

−1.02

−0.92

^a)Contains 3% v/v triﬂuoroacetic acid; ^b)Calculated as E_1/2ox* = E_1/2ox – E₀₀; E₀₀is estimated from the λmax em; ^c)Calculated as E_1/2red* = E_1/2red + E₀₀; E₀₀is estimated

from the λmax em.

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

of the methoxy groups in both indicators (FI 4 and 5) which

decrease the PET efﬁciency in the absence of K⁺. Therefore,

these ﬂuoroionophors show emission in the absence of K⁺

whereas the emission of FI 2 and 3 is “switched off” almost

completely in the same conditions (Figure 5c,f and Figure S4b,d,

Supporting Information). This ﬁndings show good correlation

with the results of the electrochemical investigation.

The emission spectra (Figure 5) show only a minor

bathochromic shift in presence of K⁺and upon protonation of

the receptor in acidic media, which indicates that the PET effect

is predominant over intramolecular charge transfer (ICT).

Comparison of the very similar rigid chromophores FI 3

and FI 4 reveals that systematic tuning of the PET efﬁciency

is easily possible. Signiﬁcant increase in the dynamics for FI 4

and FI 5 is expected if further electron-withdrawing sub-

stituents (halogens, sulphonamides) are introduced into the

chromophore. The normalized F/F₀values at 5 × 10⁻³m K⁺

(typical extracellular concentration), 150 × 10⁻³m K⁺(typical

intracellular concentration), and at saturation of the sensor

(1 ^mK⁺) as well as Kd values for each sensing material are listed

in Table 2. The Kd value of the indicators inside the polymer

membrane were determined using the Benesi–Hildebrand

equation as described in literature^[27](Equation (S1), Sup-

porting Information). Almost ideal linear ﬁt (Figure 5e,f) indi-

cates a 1:1 complexation behavior. K_dcan be calculated using

the slope of the ﬁt. The relative ﬂuorescence enhancement in

case of FI 2 and 3 is very good at low K⁺concentrations and the

Figure 4. a) Scheme of a polymer-based sensing material. The ﬂuoroiono-

phores are physically immobilized in a polymer hydrogel matrix. b) Nor-

malized absorption maxima of the ﬂuoroionophores immobilized in

hydrogel D4. 100 × 10⁻³m KCl solution was pumped through a ﬂow-

through cell for 24 h.

Figure 5. a,d) F/F₀calibration curves for FI 2–5 immobilized in hydrogel D4. The values of F and F₀were taken at λ = 605, 645, 668, and 685 nm for

FI 2, FI 3, FI 4, and FI 5, respectively. b,e) Exemplar Benesi–Hildebrand plot for FI 3 and FI 4 for the determination of Kd via the slope. Benesi–Hilde-

brand plots for FI 2 and FI 5 are shown in the Supporting Information. R²of the linear ﬁt is >0.989 for all materials. c,f) Exemplar normalized emission

spectra of FI 3 and FI 4 with different K⁺concentrations and protonation with 0.1 m HCl. The inset in (a) shows the photographic image (λ_exc365 nm)

of hydrogel D4 foil based on FI 3 with the emission switched on with 150 × 10⁻³m K⁺.

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Table 2. Fluorescence enhancement factors and K_dvalues for all sensing materials.

(FLIM) measurements which is of high

interest for biological applications.

F/F₀at 5 × 10⁻³m K⁺

F/F₀at 150 × 10⁻³m K⁺

F/F₀at 1 m K⁺

K_d[×10⁻³m]

24.1

Sensor material

FI 2 in D4

2.8

2.5

1.2

1.0

1.3

1.7

2.5

14.1

16.9

2.9

18.4

2.4. Selectivity of the Sensor

FI 3 in D4

25.5

67.7

FI 4 in D4

3.7

37.1

Inertness to changes of pH in the physiologi-

cally relevant range is essential for practical

applications of the sensors. As can be seen

(Figure 7a), the calibration curves are iden-

tical at pH 7.1, 7.4, and 7.7. Investigation of

the acid–base equilibrium of the aromatic

amine of the receptor (FI 3 in D4) reveals an

apparent pK_avalue of 3.2 (Figure 7b). Thus,

FI 5 in D4

2.3

3.4

102.7

177.4

15.6

23.0 (at 800 × 10⁻³m)

FI 3 in RL100

FI 3 in PS/PVP

FI 3 in D4^a)

6.7

5.0

11.9

26.0

17.5

66.8

^a)With 150 × 10⁻³m Na⁺background.

dynamic range extends beyond 150 × 10⁻³m making these two

ﬂuoroionophores promising for both intra- and extracellular

measurements.

the receptor can be used in the pH range of 5.5–9 without

any pH cross-talk, i.e., it is suitable for intra- or extracellular

measurements.^[33]

Since the polymer environment can affect the photophysical

properties of the indicators, the QYs of the ﬂuoroionophores in

hydrogel D4 were also investigated (Table 1). FI 2 and 3 show

an increased QY in foil when fully protonated (PET off) which

can be attributed to a more rigid environment where nonemis-

sive deactivation processes are minimized. However, the QY in

1 ^mKCl is lower than for the protonated form indicating that

K⁺does not fully inhibit the PET effect with these indicators. FI

4 and 5 have a lower QY in foil than in solution, but the QY of

the protonated indicator dyes and the dyes in 1 ^mK⁺are similar

indicating an efﬁcient inhibition of the PET effect by K⁺. In

general, all indicators show a high QY upon complexation with

K⁺in the sensor matrix. Combined with a high molar absorp-

tion coefﬁcient this results in very bright sensors. Therefore,

indicator dyes can be used for preparation of thin sensor layers

which subsequently improves the response time of the sensor.

Since the ﬂuorescence of FI 4 and FI 5 is not fully quenched

in the absence of K⁺(both free and complexed forms are emis-

sive), self-referenced measurements of the luminescence life-

time become possible (Figure 6 and Figure S5, Supporting

Information). This enables ﬂuorescence lifetime imaging

FI 3 shows no response to Ca²⁺or Mg²⁺(Figure 7c). On the

other hand, the indicator dye shows high cross sensitivity to

NH₄since the size of NH₄is comparable to that of K⁺. How-

ever, NH₄does not occur in biological samples in an inter-

fering concentration (except urine) whereas sodium is the main

ion present in the extracellular space (150 × 10⁻³m). Therefore,

the high K⁺/Na⁺selectivity of the sensors is of great impor-

tance. In fact, FI 3 shows a ﬂuorescence enhancement of 1.3 at

150 × 10⁻³m Na⁺compared to 16.89 at 150 × 10⁻³m K⁺and to

2.46 at 5 × 10⁻³m K⁺. When simulating extracellular conditions

(150 × 10⁻³m Na⁺background) the F/F₀and Kd value are only

slightly increased compared to 150 × 10⁻³m Na⁺background

(Table 2). It should be considered that in most applications the

concentration of Na⁺does not vary drastically (Figure S6, Sup-

porting Information).

To demonstrate sufﬁcient selectivity over Na⁺ions, we meas-

ured the response of a sensor at different K⁺concentrations

in presence of 110 × 10⁻³and 130 × 10⁻³m Na⁺. Buffer solu-

tions with different K⁺and Na⁺concentrations were pumped

through a ﬂow through cell to imitate extracellular K⁺measure-

ments. Figure 8 shows that the response of the sensor is fast

and fully reversible (a–c). Variation of the Na⁺background from

110 × 10⁻³to 130 × 10⁻³m at constant K⁺concentration (e–f)

does not result is a noticeable cross-talk. Therefore, it is pos-

sible to reliably measure K⁺at varying background of Na⁺in

the concentration range typical for extracellular measurements.

The sensor shows a fast response and recovery (t₉₀= 10 s) and

no hysteresis at any concentration. Leaching or aggregation

of the dye was not observed either. Interestingly, hydrophobic

perylene-based pH indicators previously showed slow response

and hysteresis when physically embedded in hydrogel D4.^[34]

We do not observe such behavior for the potassium ﬂuoroiono-

phores. Therefore, it is likely that the hydrophobic part of the

ﬂuoroionophore remains ﬁxed in the hydrophobic domains of

the hydrogel whereas much more hydrophilic receptor is local-

ized in the hydrophilic domains.

2.5. Water-Dispersible Nanoparticles

Analyte-sensitive nanoparticles represent versatile analytical

tools that are attractive for sensing and imaging in small

Figure 6. Calibration of FI 4 and FI 5 immobilized in hydrogel D4 using

ﬂuorescence decay time read-out.

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

Figure 7. a) Calibration curve for FI 3 in hydrogel D4 at different pH values. b) Determination of the pKa of the receptor for FI 3 in hydrogel D4.

c) Exemplar determination of the selectivity for FI 3 immobilized in hydrogel D4.

volumes such as cells. Therefore, additionally to planar sensor

foils we prepared two different kinds of nanosensors based on

commercially available polymers. RL100 is a copolymer of dif-

ferent acrylates with quaternary ammonium groups which are

responsible for the excellent cell-penetrating properties of the

nanobeads.^[35]It is frequently used for drug delivery due to

the positive charge and its nonbiodegradability.^[36–38]Particles

can be prepared in a very simple procedure by dissolving the

polymer and the indicator dye in an organic solvent (e.g., ace-

tone) followed by precipitation in water, forming particles with

a positive charge outside and the lipophilic indicator entrapped

inside.^[35,39]The particles show an average size of ≈30 nm and

due to the positive charge on the surface the nanoparticle show

water uptake and ion permeability.

Poly(styrene-block-vinylpyrrolidone) (PS/PVP) is a highly

versatile particle platform for optical sensors.^[40]These com-

mercially-available uncharged particles have an average size

of 245 nm and core–shell architecture, which can be used

Figure 9a shows a calibration curve for FI 3 entrapped in the

particles. The sensor material has a higher K_d(177.4 × 10⁻³m)

compared to hydrogel D4 (67.7 × 10⁻³m) which can be attrib-

uted to the positive charge of the particles lowering the equi-

librium of positively charged K⁺ions inside the particle. Fur-

thermore, the polarity of the polymer is different to Hydrogel

D4 affecting the PET efﬁciency. It should be emphasized that

the dynamic range, the spectral properties and the cell penetra-

tion of this material match the requirements for intracellular

measurements.

Figure 9. a) Sensing properties of FI 3 immobilized in positively charged

RL100 nanoparticles dispersed in an aqueous solution. The inset shows

a photographic image of the aqueous dispersions of the nanoparticles at

varying K⁺concentration under 365 nm excitation. b) Sensing properties

of PS-PVP nanoparticles stained with FI 3. Benesi–Hildebrand plots for

both materials are shown in Figure S3 of the Supporting Information.

Figure 8. Normalized ﬂuorescence of FI 3 in hydrogel D4 ﬁxed in a ﬂow-

cell while pumping solutions with different K⁺and Na⁺concentrations

through it.

Adv. Funct. Mater. 2016,

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for incorporation of (indicator) dyes into these two domains.

Physical entrapment of the dyes is achieved via swelling the

particle with an organic solvent, adding the indicator and then

removing the solvent.

We entrapped FI 3 in the shell in order to allow interac-

tion of the analyte with the indicator. The calibration of the

sensor material shows a large dynamic range and a K_dof

15.6 × 10⁻³m K⁺(Figure 9b). The most attractive feature of

this material is the possibility of incorporating a second dye

in the core (e.g., an oxygen indicator for dual sensing or an

inert dye for referencing). Also the neutral nature of the par-

ticles enhances the sensing options in complex media (e.g.,

cell cultures) since they do not absorb interfering species, are

not incorporated by cells and do not aggregate in samples with

high ion concentration.^[40]

2.6. Fiber-Optic Sensors

To demonstrate the possible application of the newly developed

sensing materials, we fabricated a ﬁber-optic sensor suitable

for read-out with a commercially available phase ﬂuorometer

(Firesting, Figure 10). In order to ensure a reliable performance

the ﬂuorescence intensity of the FI 3 was referenced against

luminescence of Egyptian blue^[41]using the so-called dual life-

time referencing (DLR) scheme.^[42]Brieﬂy, the overall phase

shift is determined only by the ratio of the intensities of the

ﬂuoroionophore and the analyte-insensitive long-lived refer-

ence luminophore. The sensor material was mounted on the

top of an optical ﬁber using a metal ferrule (Figure 10). Fetal

bovine serum (FBS) was used to demonstrate an application

of this compact sensor in a complex sample solution. For cali-

bration different FBS samples were prepared by spiking them

with KCl solution and determining the real K⁺concentration

via ICP-OES since the obtained FBS already contains a certain

amount of K⁺. The optical sensor shows reproducible perfor-

mance in serum (Figure 10a). The calibration curve in the low

mM range can be ﬁtted linearly (R²= 0.998, Figure 10b). Three

serum samples with unknown K⁺concentration were pre-

pared and measured with the calibrated optode and reference

measurements were performed again on ICP-OES. The values

obtained with the optodes (21.42 × 10⁻³, 23.52 × 10⁻³, and

32.59 × 10⁻³m K⁺for samples 1, 2, and 3, respectively) showed

excellent match with the reference data (21.53 × 10⁻³, 23.71 ×

10⁻³, and 32.27 × 10⁻³m, respectively) demonstrating ability of

the sensor to accurately measure K⁺concentration in a complex

sample media within the mM range. It should be also consid-

ered that optical components of Firesting currently show only

limited compatibility with the emission spectrum of the FI 3

so that simple modiﬁcation of the emission ﬁlter is expected to

dramatically (about 10-fold) improve already very good signal-

to-noise ratio.

Figure 10. a) Recorded phase shift values of the referenced sensor in FBS

samples with different K⁺concentrations. b) Calibration curve of the ref-

erenced sensor. The K⁺concentrations were determined using ICP-OES.

The inset shows the phase ﬂuorometer (PyroScience Firesting) and an

optical plastic ﬁber with a sensor spot attached to the distal end of the

ﬁber with help of a metal cap.

a selective aza-crown receptor and bright and photostable

tetraaryl-BODIPY chromophores. Several representatives show

absorption and emission in the red/NIR part of the spectrum,

high molar absorption coefﬁcients and ﬂuorescence quantum

yields. For the ﬁrst time, the K⁺sensors were obtained by

simple immobilization of the ﬂuoroionophores into a stable and

biocompatible hydrogel. Despite the simple design, the sensors

show no leaching of the indicators, and feature fast and repro-

ducible response. The “off”–“on” enhancement factor can be

tuned over a broad range via introduction of electron-donating

and electron-withdrawing substituents into the chromophore.

The materials show excellent response to K⁺in aqueous solu-

tion with a very good selectivity over possible competing ions

(e.g., Na⁺) and no pH dependency in physiologically relevant

conditions. Importantly, the new materials are applicable in

a variety of formats, including planar sensor foils and spots,

ﬁber-optic (micro)sensors, and water-dispersible nanosensors

and reliably operate even in complex samples such as serum.

3. Conclusions

In this contribution we presented a straightforward and

efﬁcient strategy leading to optical K⁺sensors with tune-

able properties. A palette of new ﬂuoroionophores combines

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

(www.pyroscience.com) with at a modulation frequency of 4 kHz.

procedure^[15]and subsequent preparation of compound S6 was

performed according to Ast et at.^[16]

The nanoparticles are prepared via a simple procedure and

enable extra- and intracellular quantiﬁcation of potassium. We

believe that the above properties of the new high-performance

materials will make them valuable analytical tools in biomed-

ical research and diagnostics.

3-Phenyl-2H-Azirene: Synthesis was performed according to literature

using styrene as starting material.^[43]

2-(4-Propylphenyl)-4-Phenylpyrrol (2): 4′-Propylacetophenone (5.00 g,

30.8 mmol, 1 eq) was dissolved in 90 mL dry THF and cooled down to

−78 °C. LDA (2 ^min THF) (17.05 mL, 1.1 eq) and 3-phenyl-2H-azirene

(3.61 g, 30.8 mmol, 1 eq) were added dropwise. The mixture was stirred

at −78 °C for 3 h, warmed up to RT, quenched with water and neutralized

with diluted HCl. THF was removed under vacuum and the mixture was

extracted with DCM, dried over Na₂SO₄and the solvent removed under

vacuum. Puriﬁcation was performed by column chromatography (silica

gel, eluent: CH + DCM = 1 + 1) to obtain the product as white crystals

(5.687 g, 18.6%). ¹H NMR (300 MHz, CD₂Cl₂) δ 8.49 (bs, 1H), 7.64 – 7.54

(m, 2H), 7.49 – 7.39 (m, 2H), 7.44 – 7.32 (m, 2H), 7.23 (d, J = 7.9 Hz,

3H), 7.16 – 7.08 (m, 1H), 6.87 – 6.79 (m, 1H), 2.63 (t, J = 7.7 Hz, 2H),

1.67 (p, J = 7.4 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H). DI-EI: m/z: [M⁺] calcd

for C₁₉H₁₉N, 261.1518; found, 261.1508.

5-Chloro-3-Phenyl-1,4-Dihydroindeno[1,2-b] Pyrrole (3): The synthesis

of 3 was performed analogously to that of 2 but 1.0 g (6.0 mmol) of

5-chloro-1-indanone and 0.70 g (6.0 mmol) of 3-phenyl-2H-azirene were

used instead. The product was isolated as white crystals (486 mg, 30%).

¹H NMR (300 MHz, CD₂Cl₂) δ 8.56 (bs, 1H), 7.66 – 7.59 (m, 2H), 7.48

(s, 1H), 7.43 – 7.36 (m, 2H), 3.76 (s, 2H). DI-EI: m/z: [MH⁺] calcd for

C₁₇H₁₂ClN, 265.0658; found, 265.0656.

4,5-Dihydro-7-Methoxy-3-Phenylbenzo[g]indole (4): The synthesis of

4 was performed analogously to that of 2 but 5.06 g (28.7 mmol) of

6-methoxy-1-tetralone and 3.36 g (28.7 mmol) of 3-phenyl-2H-azirene

were used instead. The product was isolated as greenish crystals

(2.04 g, 25.8%). ¹H NMR (300 MHz, CD₂Cl₂) δ 8.40 (bs, 1H), 7.51 – 7.44

(m, 2H), 7.43 – 7.35 (m, 2H), 7.29 – 7.20 (m, 1H), 7.13 (d, J = 8.4 Hz, 1H),

6.93 (d, J = 2.7 Hz, 1H), 6.84 (s, 1H), 6.76 (dd, J = 8.4, 2.6 Hz, 1H),

3.81 (s, 3H), 2.97 – 2.89 (m, 4H). DI-EI: m/z: [M⁺] calc for C₁₉H₁₇NO,

275.1310; found, 275.1305.

4. Experimental Section

Materials and Methods: ¹H NMR spectra were recorded on a 300 MHz

instrument from Bruker. MALDI-TOF mass spectra were recorded on a

Micromass TofSpec 2E in refectron mode at an accelerating voltage of

+20 kV. Absorption measurements were performed on a Cary 50 UV–

vis spectrophotometer from Varian. Luminescence spectra, calibrations,

quantum yields and lifetimes (TCSPC) were measured on a Fluorolog-3

luminescence spectrometer (Horiba). Calibrations were performed

using a peristaltic pump and a home-made ﬂow through cell. Quantum

yields were measured using the absolute method in an integrating

sphere. QY and lifetimes of the indicators in solution were acquired in

THF in presence triﬂuoroacetic acid (0.3 × 10⁻³m) to fully protonate the

indicator. QYs for the foils were measured at 1000 × 10⁻³m aqueous

KCl and 0.1 ^maqueous HCl. Leaching was investigated by recording the

absorption spectra of a foil during continuous pumping a 100 × 10⁻³

KCl solution (20 × 10⁻³m TRIS, pH 7.4) through the ﬂow-through cell

(10 mL min⁻¹). pKa determination was performed using a 20 × 10⁻³

universal buffer (citrate, acetate, BIS-TRIS, and TRIS); pH was adjusted

with HCl with help of a digital pH meter (Seven Easy, Mettler Toledo,

www.mt.com) calibrated at 25 °C with standard buffers of pH 7.0 and

4.0 (WTW, www.wtw.com). K⁺concentrations of the fetal bovine serum

were quantiﬁed with an axially viewed ICP-OES (Ciros Vision EOP, Spectro,

Germany) using a cross-ﬂow nebulizer, a Scott type spray chamber and

a standard ICP torch with a 2.5 mm inner diameter injector. 1200 W RF

power, 12 l min⁻¹outer gas ﬂow, 0.7 l min⁻¹intermediate gas ﬂow and

0.83 l min⁻¹nebulizer gas ﬂow were used. K 404.721 nm emission line

was used for quantiﬁcation. Scandium (Sc 361.384 nm) was used as an

internal standard at a concentration of 1 mg l⁻¹. Serum measurements

with the optode were performed with a Firesting phase ﬂuorometer

Fluoroionophores: Fluoroionophore FI 1: N-(4-Formyl-2-methoxy-

ethoxyphenyl) aza-[18]crown-6 ether (357 mg, 0.809 mmol, 0.5 eq)

and 2,4-dimethylpyrrole (169 mg, 1.78 mmol, 2.2 eq) were dissolved in

5 mL of anhydrous dichloromethane and 1 drop of triﬂuoroacetic acid

was added. The mixture was shielded from light and stirred at RT for

48 h, DDQ (367 mg, 1.61 mmol, 2 eq) was added. After stirring for

another 60 min, N,N-diisopropylethylamine (2.15 mL, 12.3 mmol,

15 eq) and BF₃OEt₂(1.50 mL, 12.3 mmol, 15 eq) were added and

stirred for 60 min. The mixture was extracted with water, dried over

Na₂SO₄and the solvent removed in vacuo. The ﬁnal product was

puriﬁed by column chromatography and was obtained as purple crystals

Electrochemical measurements were performed using

a VMP3

electrochemical workstation (Biologic). The measurements were carried

out at room temperature. A 1 mm diameter gold disc was employed as

the working electrodes. A platinum wire served as the counter electrode.

Measurements were performed using a Ag/AgCl reference electrode

(BAS Inc). Ferrocene was used as reference.

−1

Eudragit RL-100 copolymer (poly-(ethylacrylate-co-methylmethacrylate-

co-trimethyl-aminoethyl methacrylate), M.W. ≈ 150 000 Da, 8.8%–12%

of quaternary ammonium groups) was from Degussa, Germany (www.

evonik.com). Hydrochloric acid 37% (HCl), sodium sulfate anhydrous

(Na₂SO₄), and all other solvents including the deuterated solvents were

from VWR (www.vwr.com). Polyurethane hydrogel (Hydromed D4) was

purchased from AdvanSource biomaterials (www.advbiomaterials.

com). Poly(ethylene terephthalate) (PET) support Melinex 505

was obtained from Pütz (www.puetz-folien.com). Poly(styrene-

blockvinylpyrrolidone) emulsion in water (38% w/w emulsion in water),

lithium diisopropylamide (LDA) (2 ^min THF), 2,3-dichloro-5,6-dicyano-p-

benzoquinone (DDQ), triﬂuoracetic acid, Pd(dppf)Cl₂, boron triﬂuoride

etherate (BF₃OEt₂), water-free dichloromethane, and fetal bovine serum

were purchased from Aldrich (www.sigmaaldrich.com). 5-Bromo-2-

furaldehyde was purchased from ABCR (www.abcr.de). Buffer substances,

KCl, NaCl, CaCl₂, NH₄Cl, MgCl₂were from Roth (www.carlroth.com).

Silica gel (0.04–0.063 mm) was acquired from Acros Organics (www.

ﬁshersci.com). Silanized Egyptian blue microparticles (trimethylsilyl

form) were prepared according to a literature procedure.^[41]All other

chemicals were purchased from TCI Europe (www.tcichemicals.com).

PMMA ﬁbers (∅ 1 mm) were from Ratioplast (www.ratioplast.com).

(117 mg, 22%). UV–vis (DCM): λ_max(ε), nm (m cm⁻¹) = 504 (53600).

¹H NMR (300 MHz, CDCl₃) δ 7.25 (m, 1H), 7.06 – 6.82 (m, 2H), 6.00

(s, 2H), 4.35 – 4.14 (m, 2H), 3.80 – 3.23 (m, 29H), 2.55 (s, 6H), 1.47 (s,

6H). MALDI-TOF: m/z: [MH⁺] calcd for C₃₄H₄₉N₃O₇, 660.364; found,

660.389.

Fluoroionophore FI 2: The synthesis of FI

2 was performed

analogously to that of FI 1 but 100 mg (0.226 mmol) of N-(4-formyl-2-

methoxyethoxyphenyl) aza-[18]crown-6 ether and 120 mg (0.459 mmol)

of 2-(4-propylphenyl)-4-phenylpyrrole were used instead. The product

was isolated as purple crystals (58 mg, 26%). UV–vis (DCM): λ_max

−1

(ε), nm (m cm⁻¹) = 571 (29900). H NMR (300 MHz, CD₂Cl₂) δ 7.68

(d, J = 7.8 Hz, 4H), 7.21 (d, J = 7.9 Hz, 4H), 6.91 – 6.81 (m, 10H),

6.49 (s, 2H), 3.65 – 3.27 (m, 31H), 2.62 – 2.54 (m, 4H), 1.66 – 1.58

(m, 4H), 0.91 (t, J = 7.3 Hz, 6H). MALDI-TOF: m/z: [MK⁺] calcd for

C₆₀H₆₈BF₂N₃O₇K, 1030.4766; found, 1030.4915.

Fluoroionophore FI 3: The synthesis of FI

3 was performed

analogously to that of FI 1 but 256 mg (0.582 mmol) of N-(4-formyl-2-

methoxyethoxyphenyl) aza-[18]crown-6 ether and 299 mg (1.164 mmol)

of 5-chloro-3-phenyl-1,4-dihydroindeno[1,2-b]pyrrole were used instead.

The product was isolated as green crystals (125 mg, 21%). UV–vis

−1

(DCM): λ_max(ε), nm (m cm⁻¹) = 640 (109300), 589 (20000). H NMR

(300 MHz, CD₂Cl₂) δ 8.32 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 7.1 Hz, 4H),

7.06 – 6.85 (m, 10H), 6.75 – 6.57 (m, 2H), 6.47 – 6.29 (m, 1H),

Synthesis:N-(4-Formyl-2-Methoxyethoxyphenyl)aza-[18]crown-6

Ether:

Synthesis of compound S3 was performed according to literature

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3.78 – 3.34 (m, 31H), 1.27 (s, 4H). MALDI-TOF: m/z: [MH⁺] calcd for

C₅₆H₅₅BF₂Cl₂N₃O₇, 1000.3488; found, 1000.3410.

tube and dried via vacuum and 5 mL water-free DCM was added under

an Ar atmosphere. N,N-Diisopropylethylamine (305 μL, 1.75 mmol,

4 eq) and BF₃OEt₂(220 μL, 1.75 mmol, 4 eq) were added to the solution

and after 60 min stirring, the solution was extracted with H₂O, dried over

Na₂SO₄and concentrated under vacuum. The crude product was puriﬁed

using column chromatography (silica gel, eluent: DCM + MeOH =

100 + 1 to 100 + 20) to obtain the dye as green blue crystals (35 mg,

Fluoroionophore FI 4: The synthesis of FI

4 was performed

analogously to that of FI 1 but 100 mg (0.226 mmol) of N-(4-formyl-2-

methoxyethoxyphenyl) aza-[18]crown-6 ether and 124 mg (0.452 mmol)

of 4,5-dihydro-7-methoxy-3-phenylbenzo[g]indole were used instead.

The product was isolated as green crystals (41 mg, 18%). UV–vis

−1

8.3%). UV–vis (DCM): λ_max(ε), nm (m cm⁻¹) = 670 (195600), 616

(DCM): λ_max(ε), nm (M⁻¹cm⁻¹) = 655 (87200), 601 (22000). H NMR

(48500). ¹H NMR (300 MHz, CDCl₃) δ 7.80 (d, J = 2.2 Hz, 2H), 7.66 (dd,

J = 8.6, 2.2 Hz, 2H), 7.16 (d, J = 4.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.87

(s, 2H), 6.34 (s, 2H), 4.22 (s, 2H), 3.94 (s, 6H), 3.82 – 3.49 (m, 26H),

3.43 (s, 3H). MALDI-TOF: m/z: [MNa⁺] calcd for C₄₈H₅₀BCl₂F₂N₃O₁₁Na,

986.2789; found, 986.4067.

Planar Sensor Films: An appropriate amount of the indicator was

dissolved in a hydrogel D4 stock solution (10 wt% in THF). Sensor ﬁlms

were prepared by knife coating of these “sensor cocktails” onto dust-free

PET foils (25 μm wet ﬁlm thickness). Dye concentrations for calibrations

and QY determination were 0.2 wt% and for leaching experiment 1 wt%

in respect to the polymer.

Fiber-Optic Sensor: 0.5 mg FI 3, 20 mg of silanized Egyptian blue and

100 mg hydrogel D4 were dissolved in 1 g THF. The “cocktail” was knife

coated onto a dust-free PET foil (75 μm wet ﬁlm thickness). A sensor

spot (≈2 mm diameter) was stamped out and ﬁxed with a metal cap on

a 1 m PMMA ﬁber.

RL100 Particles: 100 mg Eudragit RL100 were dissolved in 50 mL

acetone, and indicator dye FI 3 (1 mg) was added. 250 mL water was

added quickly under vigorous stirring (3 s); acetone was removed using

rotary evaporator and the particle dispersion was further concentrated to

a volume of 50 mL. For calibrations 0.5 mL of particle dispersion were

added to 2 mL of KCl aqueous solutions.

(300 MHz, CD₂Cl₂) δ 8.74 (d, J = 8.9 Hz, 2H), 7.11 – 6.70 (m, 17H), 3.89 (s,

6H), 3.73 – 3.34 (m, 31H), 2.91 – 2.68 (m, 4H), 2.44 – 2.32 (m, 4H).

MALDI-TOF: m/z: [MNa⁺] calcd for C₆₀H₆₅BF₂N₃O₉Na, 1020.4792;

found, 1020.4977.

5-(3-Chloro-4-Methoxyphenyl)-Furan-2-Carbaldehyde (5): 5-Bromo-

2-furaldehyde (3.00 g, 0.017 mol, 1 eq), 3-chloro-4-methoxyphenyl)

boronic acid (3.20 g, 0.017 mol, 1 eq) and Na₂CO₃(60 mL of 2

solution) and) were dissolved in 300 mL toluene and 60 mL ethanol.

The mixture was degassed for 20 min by vigorously stirring under heavy

Ar ﬂow. After addition of the catalyst [1,1′-bis(diphenylphosphino)

ferrocene] dichloropalladium(II) (Pd(dppf)Cl₂) (20.0 mg, 0.15 mol%) the

reaction mixture was heated up to 80 °C and stirred for 18 h under inert

atmosphere. After cooling, the organic phase was washed with water

and brine, dried over Na₂SO₄and evaporated. The resulting residue

was puriﬁed by column chromatography (silica gel, eluent: CH + DCM =

1 + 1 to 1 + 5) to obtain 5-(4-methoxyphenyl)-furan-2-carbaldehyde as a

yellow solid (2.89 g, 71.1%). ¹H NMR (300 MHz, CDCl₃) δ 9.63 (s, 1H),

7.84 (d, J = 2.2 Hz, 1H), 7.71 (dd, J = 8.6, 2.2 Hz, 1H), 7.31 (d, J = 3.7 Hz,

1H), 6.99 (d, J = 8.6 Hz, 1H), 6.74 (d, J = 3.7 Hz, 1H), 3.96 (s, 3H).

Ethyl-2-Azidoacetate (6): Ethyl 2-bromoacetate (11.50 mL, 0.10 mol,

1 eq) and NaN₃(13.20 g, 0.20 mol, 2 eq) were stirred in 300 mL acetone

and 100 mL H₂O. After 1 h the reaction solution was extracted with DCM

and brine (3×), the organic phase dried with Na₂SO₄and concentrated

under vacuum and yields in a colorless liquid (12.53 g, 93.9%). ¹H

NMR (300 MHz, CDCl₃) δ 4.27 (q, J = 7.1 Hz, 2H), 3.87 (s, 2H), 1.32

(t, J = 7.1 Hz, 3H).

PS/PVP Particles: 213 mg of the PS/PVP emulsion (38% emulsion in

water) was diluted with 25 mL H₂O and 20 mL EtOH. FI 3 (1 mg, 1 wt%)

was dissolved in 20 mL EtOH and was added dropwise under vigorous

stirring into the emulsion of the polymer. The emulsion was concentrated

under reduced pressure to remove all ethanol and partly water. It was

then diluted with water up to 10 mL overall volume. For calibration 20 μL

of the solution were added to 1980 μL buffered KCl solution.

2-(3-Chloro-4-Methoxyphenyl)-4H-furo[3,2-b] Pyrrole-5-Carboxylic Acid

Ethyl Ester (7): 5-(3-Chloro-4-methoxyphenyl)-furan-2-carbaldehyde

(2.02 g, 8.54 mmol, 1 eq) and ethyl 2-azidoacetate (4.0 mL, 34.7 mmol,

4 eq) were dissolved in 120 mL anhydrous ethanol and cooled down to

0 °C. Sodium ethoxide (20 w% in ethanol, 25 mL, 34.7 mmol, 4 eq) was

added dropwise over 30 min and stirred for 3 h. The reaction mixture

was poured on 250 mL sat. NH₄Cl solution, the precipitate collected

and washed with H₂O. The intermediate product was dissolved in

85 mL toluene, reﬂuxed for 4 h. A precipitation forms after cooling

down to RT and the solvent was removed using rotary evaporator.

Column chromatography (silica gel, eluent: CH + EE = 5 + 1 to 1 + 1)

was performed and the product was obtained as an orange solid (1.44 g,

Supporting Information

Supporting Information is available from the Wiley Online Library or

from the author.

52.7%). H NMR (300 MHz, CDCl₃) δ 8.86 (bs, 1H), 7.73 (s, 1H), 7.58

Acknowledgements

(d, J = 8.6 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.79 (s, 1H), 6.59 (s, 1H),

4.36 (q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H). DI-EI:

m/z: [M⁺] calcd for C₁₆H₁₄NO₄Cl, 319.0611; found, 319.0599.

The authors would like to thank Georg Michelitsch, Bianca Hörler, Josef

Lechner, Nicole Steinmann, and Lukas Heupl for support in synthesis of

the indicators and receptors. The authors gratefully acknowledge Stefan

Freudenberger and Nika Mahne from the Institute for Chemistry and

Technology of Materials (TU Graz) for the help with the cyclovoltametry

measurements. Helmar Wiltsche and Monika Winkler of the Institute of

Analytical Chemistry and Food Chemistry (TU Graz) are thanked for the

help with the ICP-OES measurements. Financial support from European

Commission (“Schema” Project No. 614002) is gratefully acknowledged.

2-(3-Chloro-4-Methoxyphenyl)-4H-furo[3,2-b] Pyrrole-5-Carboxylic Acid

(8): 2-(3-Chloro-4-methoxyphenyl)-4H-furo[3,2-b] pyrrole-5-carboxylic

acid ethyl ester (780 mg, 2.44 mmol, 1 eq) was dissolved in 25 mL

ethanol and a NaOH (7 mL of 2.5 ^msolution) was added and the mixture

was reﬂuxed for 1 h. After cooling, HCl conc. was added resulting in a

green precipitate. The resulting precipitate was ﬁltered and washed with

H₂O and dried in the oven at 60 °C overnight (548 .7 mg, 70%). 1H

NMR (300 MHz, DMSO-d6) δ 11.60 (bs, 1H), 7.87 (d, J = 2.2 Hz, 1H),

7.74 (dd, J = 8.7, 2.2 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.11 (s, 1H), 6.71

(s, 1H), 3.89 (s, 3H). DI-EI: m/z: [M+] calcd for C₁₄H₁₀NO₄Cl, 291.0298;

found, 291.0299.

Received: July 27, 2016

Published online:

Fluoroionophore FI 5: 2-(3-Chloro-4-methoxyphenyl)-4H-furo[3,2-b]

pyrrole-5-carboxylic acid (127.9 mg, 0,438 mmol, 1 eq) and N-(4-formyl-

2-methoxyethoxyphenyl) aza-[18]crown-6 ether (96.2 mg, 0.218 mmol,

0.5 eq) were dissolved in 3 mL conc. triﬂuoroacetic acid under Ar

atmosphere. The mixture was stirred at 50 °C for 1 h, POCl₃(0.50 mL)

was added, the stirred for 10 min at 50 °C and precipitated slowly into

cold water. The precipitate was collected and transferred into a Schlenk

[1] J. M. Burnell, B. H. Scribner, B. T. Uyeno, M. F. Villamil, J. Clin.

Invest. 1956, 35, 935.

[2] J. R. Elkinton, A. W. Winkler, J. Clin. Invest. 1944, 23, 93.

[3] B. Hille, Prog. Biophys. Mol. Biol. 1970, 21, 1.

10 wileyonlinelibrary.com

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Funct. Mater. 2016,

DOI: 10.1002/adfm.201603822

[4] H. Eagle, Science 1955, 122, 501.

[5] P. K. Whelton, J. He, J. A. Cutler, F. L. Brancati, L. J. Appel,

D. Follmann, M. J. Klag, JAMA 1997, 277, 1624.

[6] D. C. Gadsby, R. Niedergerke, S. Page, Nature 1971, 232, 651.

[7] A. L. Hodgkin, Biol. Rev. 1951, 26, 339.

[8] M. Näbauer, S. Kääb, Cardiovasc. Res. 1998, 37, 324.

[9] J. K. Tusa, H. He, J. Mater. Chem. 2005, 15, 2640.

[10] T. L. Doane, C. Burda, Chem. Soc. Rev. 2012, 41, 2885.

[11] M. Schäferling, Angew. Chem. Int. Ed. 2012, 51, 3532.

[12] M. J. Ruedas-Rama, J. D. Walters, A. Orte, E. A. H. Hall, Anal. Chim.

Acta 2012, 751, 1.

[26] W. Namkung, P. Padmawar, A. D. Mills, A. S. Verkman, J. Am.

Chem. Soc. 2008, 130, 7794.

[27] X. Zhou, F. Su, Y. Tian, C. Youngbull, R. H. Johnson, D. R. Meldrum,

J. Am. Chem. Soc. 2011, 133, 18530.

[28] W. Zhao, E. M. Carreira, Chem. Eur. J. 2006, 12, 7254.

[29] S. K. Yang, X. Shi, S. Park, T. Ha, S. C. Zimmerman, Nat. Chem.

2013, 5, 692.

[30] K. Umezawa, Y. Nakamura, H. Makino, D. Citterio, K. Suzuki, J. Am.

Chem. Soc. 2008, 130, 1550.

[31] N. Boens, V. Leen, W. Dehaen, Chem. Soc. Rev. 2012, 41,

1130.

[13] A. P. de Silva, T. S. Moody, G. D. Wright, Analyst 2009, 134, 2385.

[14] K. Meuwis, N. Boens, F. C. De Schryver, J. Gallay, M. Vincent,

Biophys. J. 1995, 68, 2469.

[32] A. Weller, Pure Appl. Chem. 1968, 16, 125.

[33] C. Deutsch, J. S. Taylor, D. F. Wilson, Proc. Natl. Acad. Sci. USA

1982, 79, 7944.

[15] H. He, M. A. Mortellaro, M. J. P. Leiner, R. J. Fraatz, J. K. Tusa,

J. Am. Chem. Soc. 2003, 125, 1468.

[34] D. Aigner, S. M. Borisov, P. Petritsch, I. Klimant, Chem. Commun.

2013, 49, 2139.

[16] S. Ast, T. Schwarze, H. Müller, A. Sukhanov, S. Michaelis,

J. Wegener, O. S. Wolfbeis, T. Körzdörfer, A. Dürkop, H.-J. Holdt,

Chem. Eur. J. 2013, 19, 14911.

[17] R. D. Carpenter, A. S. Verkman, Eur. J. Org. Chem. 2011, 2011, 1242.

[18] M. Magzoub, P. Padmawar, J. A. Dix, A. S. Verkman, J. Phys. Chem.

B 2006, 110, 21216.

[35] A. Fercher, S. M. Borisov, A. V. Zhdanov, I. Klimant, D. B. Papkovsky,

ACS Nano 2011, 5, 5499.

[36] S. Das, P. K. Suresh, R. Desmukh, Nanomed. Nanotechnol. Biol.

Med. 2010, 6, 318.

[37] U. K. Sharma, A. Verma, S. K. Prajapati, H. Pandey, A. C. Pandey,

Appl. Nanosci. 2014, 5, 143.

[19] P. Padmawar, X. Yao, O. Bloch, G. T. Manley, A. S. Verkman, Nat.

Methods 2005, 2, 825.

[38] K. Dillen, J. Vandervoort, G. Van den Mooter, A. Ludwig, Int. J.

Pharm. 2006, 314, 72.

[20] X. Zhou, F. Su, W. Gao, Y. Tian, C. Youngbull, R. H. Johnson,

D. R. Meldrum, Biomaterials 2011, 32, 8574.

[21] X. Kong, F. Su, L. Zhang, J. Yaron, F. Lee, Z. Shi, Y. Tian,

D. R. Meldrum, Angew. Chem. Int. Ed. 2015, 54, 12053.

[22] T. Hirata, T. Terai, T. Komatsu, K. Hanaoka, T. Nagano, Bioorg. Med.

Chem. Lett. 2011, 21, 6090.

[23] B. Sui, X. Yue, M. G. Tichy, T. Liu, K. D. Belﬁeld, Eur. J. Org. Chem.

2015, 2015, 1189.

[24] J.-P. Malval, I. Leray, B. Valeur, New J. Chem. 2005, 29, 1089.

[25] M. Baruah, W. Qin, R. A. L. Vallée, D. Beljonne, T. Rohand,

W. Dehaen, N. Boens, Org. Lett. 2005, 7, 4377.

[39] S. M. Borisov, T. Mayr, G. Mistlberger, K. Waich, K. Koren,

P. Chojnacki, I. Klimant, Talanta 2009, 79, 1322.

[40] S. M. Borisov, T. Mayr, I. Klimant, Anal. Chem. 2008, 80, 573.

[41] S. M. Borisov, C. Würth, U. Resch-Genger, I. Klimant, Anal. Chem.

2013, 85, 9371.

[42] I. Klimant, C. Huber, G. Liebsch, G. Neurauter, A. Stangelmayer,

O. S. Wolfbeis, in New Trends in Fluorescence Spectroscopy, Springer

Series on Fluorescence (Eds: P. B. Valeur, D. J.-C. Brochon),

Springer, Berlin 2001, pp. 257–274.

[43] A. G. Hortmann, D. A. Robertson, B. K. Gillard, J. Org. Chem. 1972,

37, 322.