Fluorescence-based Ca 2+ -sensors

A wide range of fluorescence sensors have been developed to measure the most relevant biological alkali metal ions (Li⁺, Na⁺, K⁺) and alkaline earth metal ions (Mg²⁺, Ca²⁺).^[123]

The basic structure of fluorescence-based ion-sensors consists of a ligand and a fluorophore. The ligand binds selectively to the respective ion, which affects the fluorescence properties of the fluorophore.^[265] Derivatives of ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) were designed as selective Ca²⁺

ligands. Based on this, TSIEN developed 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) which shows a high affinity to Ca²⁺.^[266] Structurally, the ethylene bridge between the oxygen and the nitrogen of EGTA is replaced in BAPTA by benzene rings (see Figure 4.25).^[266] The development of BAPTA as a high-affinity ligand

66

and optical indicator for Ca²⁺ analysis has been a major step in the detection of cellular Ca²⁺.^[266]

EGTA BAPTA

Figure 4.25 Structural formula of ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA).

The structural modification of the ligand system leads to alterations of properties, like a shift in the basicity of the nitrogen atoms.^[264] The EGTA nitrogen atom has a pKa value of 8.5 – 9.5. The aromatic rings of the BAPTA molecule change the pKa value to 6.0 – 6.3.^[264] This basicity shift leads to a decisive change in the complexation of Ca²⁺ at a physiological pH.^[264] Under these conditions, the nitrogen atoms of the BAPTA molecule are deprotonated, in contrast to the nitrogen atoms of the EGTA molecule which are protonated. Due to this, no displacement of a H⁺ in the coordination sphere of BAPTA is required for the coordination of Ca²⁺, which accelerates the Ca²⁺ absorption of BAPTA in two or three orders of magnitude.^[264] Crystal structures of 5,5-difluorinated BAPTA with complexed Ca²⁺ show BAPTA as an octadentate ligand, where the four carboxyl groups, the oxygen atoms of the ethylene glycol bridge and the nitrogen atoms, which act as a LEWIS acid are involved.^[267] The functionalisation of the aromatic rings with substituents makes it possible to adjust the ligand binding properties by controlling the basicity of the coordinating nitrogen.^[265] Electron donating groups, like alkyl chains (-C_nH_2n+1) and heteroatoms (N, O), increase the basicity of the Ca²⁺-binding nitrogen, which leads to a higher affinity (smaller K_d). Electron withdrawing groups, such as halogen atoms (F, Cl, -Br), nitro (-NO₂) and nitrile (-CN) groups lead to a decrease in the basicity and to a lower binding affinity (larger K_d).^[265] This allows the synthesis of a variety of BAPTA-based ligands with a broad distribution of Kd values. For additional ligand motifs with modified K_d values, one of the aromatic rings can be replaced by other functionalisation (see Figure 4.26). However, the thereof developed aminophenol triacetic acid (APTRA) does not show the desired affinity for Ca²⁺ and is also not selective towards other divalent ions such as Mg²⁺.^[265] In contrast, 2-(2′-morpholino-2′-oxoethoxy)-N,N-bis(hydroxycarbonylmethyl)-aniline (MOBHA) is a possible chelating unit for low affinity Ca²⁺-sensors.[123,268,269]

67

BAPTA APTRA MOBHA

Figure 4.26 Structural formula of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), aminophenol triacetic acid (APTRA), 2-(2′-morpholino-2′-oxoethoxy)-N,N-bis(hydroxycarbonylmethyl)aniline (MOBHA).

BAPTA shows a fluorescence maximum in the range of ~250 nm which is shifting by Ca²⁺-binding to two maxima in a range of 200 – 300 nm (see Figure 4.27).^[270] These fluorescence maxima result in an excitation wavelength < 200 nm, which is highly toxic for biological systems. Due to the need of new biological compatible Ca²⁺-sensors, it is necessary to shift the fluorescence wavelength to higher wavelengths.

Figure 4.27 Fluorescence spectra of different Ca²⁺-sensors with and without Ca²⁺.^[270] Reprinted from E. Carafoli, C. B. Klee, Calcium as a Cellular Regulator, Copyright (1999) Oxford University Press. Reproduced with permission of the Licensor through PLSclear

Such a fluorescence shift can be obtained by the attachment of a fluorophore to the chelating unit.^[264] This attachment leads to a change in the electronic system of BAPTA and the Ca²⁺-binding effects transfer to the electronic system of the fluorophore.^[264,270] In Figure 4.28, variations of Ca²⁺-sensors based on BAPTA are shown.^[265] The used fluorophores of the presented Ca²⁺-sensors are attached via different motifs.

68

Fura-red Rhod-2

Calcium orange STDBT

Figure 4.28 Selection of Ca²⁺-sensors.^[265]

Two different working principles of the fluorescent Ca²⁺-sensors were found, the photoinduced electron transfer (PET) and the photoinduced charge transfer (PCT).[113,265,271]

PET based sensors have an electron donating group (D) in their structure which is part of the ligand system. In the BAPTA ligand, D is represented by the nitrogen atoms. The D is connected by a bridge to the conjugated aromatic system of a fluorophore and due to an electron transfer, the fluorescence of the fluorophore is quenched by the D (see Figure 4.29).[113,265,271]

An example for this type of Ca²⁺-sensors is Rhod-2, shown in Figure 4.28. In the Ca²⁺ free case, an electron from the highest occupied molecular orbital (HOMO) of the fluorophore is excited to the lowest unoccupied molecular orbital (LUMO) of the fluorophore. Subsequently, the D transfers an electron from its HOMO to the partially filled HOMO of the fluorophore in a radiation free manner (see Figure 4.29).[113,265,271]

69

Figure 4.29 Schematic functional principle of a PET based Ca²⁺-sensors. After fluorophore excitation in the Ca²⁺

unbound state, the fluorescence is suppressed by electron transfer of the D to the fluorophore. The binding of Ca²⁺

leads to an increase of the fluorescence, due to the missing electron transfer of the D.[113,265,271]

Therefore, a natural fluctuation of the K_d is present in complex biological systems.^[265,271]

The PET type sensors are also named non-ratiometric sensors because the evaluation considers only one excitation wavelength and one emission wavelength.^[265,271]

The PCT based Ca²⁺-sensors show, in contrast to the PET based Ca²⁺-sensors, a different fluorophore attachment to the ligand unit. The fluorophore is directly linked to the Ca²⁺

coordinating unit without a bridge.[113,265,271]

Here, the fluorophore can be an electron acceptor (A) or an electron donating group (D) and is connected to the electronic counterpart, the Ca²⁺-binding unit, via an aromatic system (see Figure 4.30).[113,265,271]

Depending on the distribution of D and A in the sensor, different fluorescence behaviours are observed by binding Ca²⁺. As a result of the structure, an outright charge transfer process from D to A takes place at an excitation of the aromatic system in an unbound

70

state.[113,265,271]

The binding of Ca²⁺ alters the energy levels and affects the properties of the fluorophore. It is distinguished between the role of the chelator and the fluorophore as D or A. If the chelator acts as an A, the binding of Ca²⁺ leads to a decrease of the energy levels S₀ and S₁ (see Figure 4.30 a). Here, a greater decrease of the S₁ state occurs, which results in a red shift in the absorption and the fluorescence spectra. The opposite case shows a blue shift of the spectra (see Figure 4.30 b). In addition, the PST principle may result in a change in molar extinction (), fluorescence quantum yields (F) and excited-state lifetime ().[113,265,271] change of the ground state (S0) and the excited state (S1). Depending on the A/D distribution a) a decrease of the energy difference between S₀ and S₁ results from the Ca²⁺ binding and leads to a red shift of the fluorescence or b) an increase of the energy difference between S0 and S1 results from the binding of Ca²⁺ and leads to a blue shift of the fluorescence.[113,265,271]

Graphic is in accordance toYAN and OHEIM et al.^[265,271]

To determine the Ca²⁺ concentration, two different absorption or emission wavelengths are observed and a ratiometric value (R = F(1)/F(2)) is calculated. From the respective ratios of Ca²⁺-free (Rmin) and Ca²⁺-saturated (Rmax), the following Formula 4.5 shows the relationship of the Ca²⁺ concentration.^[265,271]

4.5

These types of Ca²⁺-sensors are called ratiometric sensors because a ratio of two different absorption or emission wavelengths is calculated.^[265,271] Compared to the non-ratiometric sensors, the ratiometric sensors show less sensitivity to factors that can influence the fluorescence measurements. Such factors include the dye concentration, the optical path length and the sensitivity of the instrument.^[265] A dependency of the sensors of the solvent polarity, ionic strength, viscosity and temperature is still existing. However, in the literature, the non-ratiometric sensors like Fura-2 are used for most experiments.^[265] Based

71 on optimised fluorescence measurements, the ratiometric sensors could be more common in the future due to their advantages.