Transforming Escherichia coli Proteomembranes into Artificial Chloroplasts Using Molecular Photocatalysis

Abstract During the light‐dependent reaction of photosynthesis, green plants couple photoinduced cascades of redox reactions with transmembrane proton translocations to generate reducing equivalents and chemical energy in the form of NADPH (nicotinamide adenine dinucleotide phosphate) and ATP (adenosine triphosphate), respectively. We mimic these basic processes by combining molecular ruthenium polypyridine‐based photocatalysts and inverted vesicles derived from Escherichia coli. Upon irradiation with visible light, the interplay of photocatalytic nicotinamide reduction and enzymatic membrane‐located respiration leads to the simultaneous formation of two biologically active cofactors, NADH (nicotinamide adenine dinucleotide) and ATP, respectively. This inorganic‐biologic hybrid system thus emulates the cofactor delivering function of an active chloroplast.


General Information
Chemicals and Materials: All reactions were performed under air if not otherwise stated. All solvents utilized for synthetic purposes were of technical grade and were redistilled prior to use. Oxygen-free solvents were obtained by bubbling argon through these liquids (2 min per 1 mL of solvent) using a common cannula piercing a septum. Sephadex TM LH-20 was obtained from GE Healthcare. Size exclusion chromatography was performed on Sephadex TM LH-20 using methanol as mobile phase. Inversion of cell membranes were performed with the French-Press Pressure Cell SLM Amic (SLM Instruments), membrane separation was performed by ultracentrifugation using an Optima XPN-100 ultracentrifuge (Beckman Coulter).
UV-vis absorption spectroscopy was performed on a JASCO V-670 UV-vis-NIR Spectrophotometer using gas-tight quartz glass cuvettes (d = 10.0 mm, Hellma). Enzyme kinetics were monitored at the Ultrospec 2100 pro (Amersham Biosciences) spectrophotometer using polystyrene cuvettes (d = 10.0 mm, Sarstedt). Microtiter plate based enzymatic read out was performed with the Tecan Infinite® M200 multimode plate reader (Tecan, Crailsheim, Germany) using transparent 96x well microtiter plates (Sarstedt). Emission spectroscopy was performed on a JASCO FP-8500 Fluorescence Spectrometer with gas-tight quartz glass cuvettes (d = 10.0 mm, Hellma). Fluorescence measurements (using AO as pH gradient sensor) were performed in an Amicon Bowman series 2 fluorescence spectrometer (spectronic industries) with single use fluorescence polystyrene makro cuvettes (ratiolab GmbH) High resolution mass spectrometry (HRMS) was performed using a Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer solariX (Bruker Daltonics) equipped with a 7.0 T superconducting magnet and interfaced to an Apollo II Dual ESI/MALDI source. For all measurements trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) was used as matrix. Spectra were analyzed with the DataAnalysisViewer 4.2 from Bruker and transferred to Origin 9.0. Spectra simulation was performed with mMass Version 5.5.0 and transferred to Origin 9.0 as well. 1 H (400 MHz) and 31 P-NMR (162 MHz) spectra were recorded on a Bruker Avance 400 MHz at room temperature and processed with MestReNova software (Version 12.0.0). The shift values are given in ppm and are referenced to the corresponding solvent residual peaks (1.94 ppm (quintet) for CD3CN and 3.31 ppm (quintet) for d4-MeOD).
Irradiation setup: For all catalysis runs and irradiation experiments analyzed in-situ via UV-vis or emission spectroscopy the irradiation setup consisted of a custom-made reactor equipped with four ventilators to exclude heating of the samples and one blue light emitting LED-stick (max = 465nm, 45-50 mW/cm 2 ) in the center of this reactor. [4] The cuvettes were placed on top of the LED-stick. For the photobiocatalytic ATP formation the GC vials containing the photoactive components were irradiated with one blue light emitting LED-stick (max = 465nm, 45-50 mW/cm 2 ) inside an argon filled glovebox (see chapter 2 for more details) at a distance of ca. 1cm.

Experimental details
The photobiocatalytic ATP generation was performed inside an argon filled glovebox using an aqueous buffer solution as solvent (50 mM BTP, 140 mM choline chloride, 5 mM MgCl2, 5 mM Na3PO4, 1 mM NAD + , 150 µM ADP and 0.2 M triethanolamine (TEOA) at pH 8.0). The amount of the vesicle solution used for the experiments was adjusted to 650 µg/mL total protein concentration. To the vesicle-buffer mixture 500 µM α-D-glucose and Hexokinase (HK, 1 U) as coupling enzyme were added. This mixture was kept on ice until degassing of the buffer with argon (2 min per mL) in an argon filled Schlenk flask. In detail, the experimental approach was as follows: First, glass vials (VWR Screw Vial, 4 mL, 45 x 14.15 mm) were charged with the respective amount of Ru complexes (5 and 25 µM solutions were evaluated) via MeOH or MeOH/H2O stock solutions using Hamilton micro syringes. After evaporation using pressured air, the glass vials were introduced into an argon filled glovebox. Here, the complexes were dissolved in 2.0 mL degassed buffer solution (see above) and then the GC-vials were sealed with a gas-tight lid. After an incubation period of 5 min, the samples were irradiated with one LED-stick (max = 465 nm, 45-50 mW/cm 2 ) from the bottom of the glass vials. Typically, every 15 min (if the photobiocatalysis experiments were performed for a duration of 1.5 h) or every 30 min (if the photobiocatalysis experiments were performed for a duration of 4 h) a 100 µL aliquot of the solution was taken and stored on ice outside of the glovebox until further use. The enzymatic readout was performed as follows: After irradiation of the reaction mixture all collected samples were boiled for 15 min at 95 °C to inactivate all enzymes avoiding reactions disturbing the following enzymatic readout (see Scheme 1). The denatured proteins were removed by centrifugation (20 min 20 • 800 g, 4 °C) and 50 µL of the supernatants were taken for the enzymatic quantification of the generated glucose-6-phosphate (G6P). The readout was performed in two steps in a 96x well microtiter plate (assay volume 100 µl). First, the enzyme diaphorase was added (0.8 U) to the reaction mixture containing 0.5 mM INT (3-(4-Iodophenyl)-2-(4-nitrophenyl)-5-phenyl-2H-tetrazol-3-ium chloride), 2.5 mM NADP + and 25 mM Tris-HCl (pH 7.5). After incubation for 30 min at room temperature, the absorbance at 492 nm (E1) was determined in a Tecan microplate reader. Afterwards, the enzyme G6P-DH (1 U) was added and incubated for 1 h at room temperature for conversion of the present G6P to 6-phospho--lactone and generation of NADPH from NADP + . The hereby formed NADPH is then used by diaphorase together with INT to yield additional formazan and NADP + (see Scheme 1). Subsequently, the absorbance at 492 nm is measured (E2). The difference of the absorbance values (Δ(E2-E1)) is used for the calculation of the G6P amount (stoichiometrically equivalent to produced ATP with the reaction equilibrium being strongly on the product site [5] ) based on a parallel performed calibration based on a serial dilution of G6P.

S5
Scheme S1: Graphical representation of the enzymatic G6P readout using the diaphorase catalyzed reductive ring opening reaction of the INT tetrazolium ring yielding the strongly absorbing formazan product ( = 492 nm). This process allows to quantify the amount of reduced nicotinamides (NADH and NADPH) by comparing the obtained values with those from an additional G6P calibration. The G6P itself, produced in the photobiocatalytic experiments, originates from the Hexokinase catalyzed phosphorylation of glucose.
The NAD + independent photoacidification of inverted E.coli vesicles using acridine orange (AO) as sensor for a proton gradient formation was performed as follows: To assess the capacity of the Ru complexes 1, 3 and 5 to induce proton gradient formation, a slightly varied photocatalysis buffer (50 mM BTP; 140 mM choline chloride; 5 mM MgCl2; 5 mM Na3PO4; 0.2 M TEOA, pH 8.0) was used without added ADP, NAD + and Hexokinase enzyme. The same amount of inverted E. coli vesicles (total protein concentration 650 µg/mL) was added to the buffer as used for the photobiocatalysis experiments. The buffer containing the vesicles was first poured into a one-necked round bottom schlenk flask, which was already set under an argon atmosphere. After bubbling argon through the suspension (2 min per mL), the schlenk flask was introduced into a glovebox. Here, glass vials equipped with the corresponding amount of Ru complex (20 µM; via MeOH stock solutions using Hamilton micro syringes and pressured air to remove all volatiles) were filled with 2.49 mL of the degassed buffer and charged with 5 µM AO by adding 10 µL of 1.25 mM degassed aqueous AO stock solution. The final yellow suspension was subsequently transferred into a sealable gas-tight quartz glass cuvette (d = 10.0 mm, Hellma) equipped with a septum on the top. Then the sample was irradiated with one LED-stick ( = 465 nm, 45-50 mW/cm 2 ) at room temperature from the bottom of the cuvette for the corresponding time in a home-made reactor as described in chapter 2. Afterwards the cuvette was placed into an Amicon fluorescence spectrometer (exc = 495 nm; det = 530 nm). After detecting a stable fluorescence signal, the proton gradient was abolished by addition of an argon degassed aqueous NH4Cl stock-solution (to reach a final concentration of 2.5 mM NH4Cl) using a needle and puncturing the septum of the cuvette.

Preparation of the inverted vesicles from Escherichia coli (E. coli) cells.
Inverted vesicles were prepared as described previously. [6] In brief, E. coli TK2309 or Pseudomonas putida KT 2440 cells were cultivated overnight in 500 mL LB medium (10 g tryptone/L; 5 g yeast extract/L; 10 g NaCl/L), harvested by centrifugation, resuspended in 50 mL of TCDG buffer (10 mM Tris-HCl, pH 7.5; 140 mM choline chloride; 0.5 mM dithiothreitol; 10 % (v/v) glycerol), and washed three times. Afterwards, the cells were suspended in a French Press buffer containing additionally to the TCDG buffer protease inhibitor cocktail tablets (Roche Diagnostics) and a trace element of DNase (Roche Diagnostics). Inversion of cell membranes was done by a single passage through a French Press at 1 • 100 psi. Cell debris was removed by centrifugation and membranes were collected by ultracentrifugation (200 • 000 g; 90 min; 4 °C). Membrane pellets were resuspended in fresh TCDG buffer and stored at -80 °C at a concentration of 22 µg/µL before use (E. coli). The total protein content was quantified by the Qubit 4 Fluorometer (Thermo Fisher Scientific) according to the manufacturer's instructions.

Luminol-luminescence based evaluation of ATP-synthesis with inverted E. coli membrane vesicles.
The qualitative evaluation of the ATP synthesis was done as described elsewhere. [6] In brief, 96x well microtiter plates (assay volume 200 µL) containing a buffered vesicle solution (50 mM BTP; 140 mM choline chloride; 5 mM MgCl2; 150 µM ADP; 5 mM inorganic phosphate (Pi)) were mixed with a luciferase/luminol solution (ATP-determination kit, Thermo Fisher Scientific) in a ratio 10:1. ATPsynthesis was initiated by the injection of NADH to a final concentration of 500 µM. Luminescence was monitored in a kinetic cycle by Tecan infiniteM200 plate reader. As a control, the experiment was also performed in the absence of inverted vesicles. To identify optimal conditions for an in vitro ATP synthesis, different concentrations of NADH (250-1000 µM, final concentration) were injected to the buffered vesicle solution (175 µg/mL) and resulting ATP levels were quantified after 2 min based on an ATP standard calibration. In an additional setup, an increasing amount of vesicles (0-550 µg/mL total protein concentration) were added to the reaction mixture and ATP levels were quantified after injection of NADH to a final concentration of 500 µM.

Acridine orange assay to examine proton gradient formation across vesicular membrane.
In every acridine orange (AO) assay a volume of 1 mL (in polystyrene makro-cuvettes) was used. To the assay buffer (50 mM BTP; 140 mM choline chloride; 5 mM MgCl2), 1 µM AO and 650 µg/mL (total protein concentration) of inverted vesicles were added and the reaction mixture was incubated at room temperature for 30 min. Afterwards, the AO fluorescence was constantly monitored in an Amicon fluorescence spectrometer (exc = 495 nm; det = 530 nm). Proton gradient formation was initiated by addition of NADH (final concentration 500 µM), ATP (1 mM final concentration) or buffer as control to the cuvette. Proton gradient was abolished by addition of NH4Cl to a final concentration of 2.5 mM.

S7
The photostability of the various Ru complexes utilized for the photobiocatalytic experiments (1)(2)(3)(4)(5) was determined in H2O:DMSO = 9:1 (v:v) under air. The samples were prepared as follows: starting from MeOH or MeOH/H2O stock solutions of the different Ru complexes, an appropriate volume was transferred into a glass vial (VWR Screw Vial, 4 mL, 45 x 14.15 mm) using Hamilton micro-syringes. After evaporation of the solvent, the Ru complex was dissolved in 3 mL of H2O:DMSO = 9:1 (v/v, 2700 µL:300 µL) and then transferred into a cuvette. Irradiation was performed in a custom-made reactor as described in chapter 1. [4] The stepwise photobiocatalysis experiments used to verify the biological activity of nicotinamide reduction products was performed as follows: First, 2.5 mL of buffered solutions (50 mM BTP, 140 mM choline chloride, 5 mM MgCl2, 5 mM Na3PO4, 0.2 M TEOA, pH 8.0, 1 mM NAD + ) containing 25 µM Ru complex 1, 2, 3 or 4 were prepared inside of an argon filled glovebox in glass vials (VWR Screw Vial, 4 mL, 45 x 14.15 mm). Prior to use, the buffer solution was hence degassed for 2 min per mL. The solution was then transferred into sealable cuvettes. Irradiation for 2 h was performed using the setup described in chapter 1 and followed by UV-vis absorption and emission spectroscopy to verify NADH formation. After these 2 h of irradiation, 1 mL of the samples was transferred into polystyrene makro-cuvettes equipped with a small stirring bar followed by the addition of AO (1 µM, final concentration). After stirring the sample for ca. 2 min to achieve a homogeneous distribution of AO, AO luminescence was detected as described above. Afterwards, 100 µL of a buffered solution (50 mM BTP, 140 mM choline chloride, 5 mM MgCl2, 5 mM Na3PO4, 0.2 M TEOA, pH 8.0) containing 440 µg/mL (total protein concentration) inverted E. coli vesicles was added to the stirred solution. This resulted in a strong luminescence increase for the blank experiment as well as for Ru complexes generating no or only less NADH, whereas only low luminescence increase was observed for the samples containing much NADH (assessed by emission spectroscopy).
The photostability of acridine orange (AO) was assessed using UV-vis absorption as well as emission spectroscopy. Therefore, a suitable amount of AO•HCl•ZnCl2 (M = 438.10 g/mol) was dissolved in deionized water and transferred into a 10 mm x 10 mm cuvette. Irradiation was performed with one LED-stick (max = 465 nm, 45-50 mW/cm 2 ) from the bottom of the cuvettes inside the custom-made reactor described in chapter 1. The emission spectra were recorded after excitation of AO with exc = 495 nm.
The photostability of the vesicles was examined by the analysis of proton gradient formation using an AO-assay. Inverted E. coli vesicles (650 µg/mL total protein concentration) were added to the AO-assay buffer (50 mM BTP; 140 mM choline chloride; 5 mM MgCl2) and irradiated with one blue light emitting LED-stick (max = 465nm, 45-50 mW/cm 2 ) for 3 days. After this time, the proton gradient formation was tested by the AO assay (1 µM acridine orange final concentration) as described above.
The emission quenching of acridine orange (AO) by Ru complex 1 was determined in air-equilibrated deionized water. Therefore, two solutions were prepared. The first solution only contained 1 µM AO, the second solution contained 1 µM AO and 100 µM 1. The addition of suitable volumes of the second S8 solution to the first one allowed to determine the emission quenching of AO by various equivalents of 1. The emission spectra were recorded after excitation of AO with exc = 495 nm.
The formate driven NAD + reduction was determined via UV-vis absorption spectroscopy. Therefore, a 5 µM solution of the Ru complexes (1, 2, 3 and 7) in H2O:DMSO =9:1 (v/v, Vtotal = 3 mL) containing 100 mM NaHCO2 and 200 µM NAD + was prepared under argon atmosphere. Solvents were degassed as discussed in chapter 1. After recording a starting spectrum, the cuvettes, serving as the reaction vessels, were placed into a pre-warmed water bath (40 °C). Catalytic turnover was measured by recording an UV-vis spectrum every 10 min and determined using the extinction coefficient of NADH at 340 nm ( = 6230 L/mol*cm). [7] In detail, the absorbance at 340 nm of the starting spectrum was subtracted from all absorbances at 340 nm of the spectra recorded upon heating the sample. The increase in absorbance at this wavelength was used to determine the turnover numbers (TONs) by dividing the concentration of NADH by the concentration of the utilized catalyst (5 µM).
The emission spectroscopic detection of the light switch effect for compounds 3 and 5 in the presence of inverted E.coli vesicles was performed as follows: To a 10 µM solution (V = 2.5 mL) of the respective complexes dissolved either in deionized water (condition i) or in the typical buffer (50 mM BTP, 140 mM choline chloride, 5 mM MgCl2, 5 mM Na3PO4, condition ii), a buffered suspension of E.coli vesicles (total protein concentration 650 µg/mL; 50 mM BTP, 140 mM choline chloride, 5 mM MgCl2, 5 mM Na3PO4) was added. For condition i, vesicle addition was performed in steps of 20 µL until Vadded,total = 180 µL was reached. For condition ii, vesicle addition was performed first in smaller steps (Vadded,total = 5 µL, 10 µL, 20 µL) then in steps of 20 µL until Vadded,total = 100 µL was reached and then in larger steps of 100 µL until Vadded,total = 400 µL was reached. In all cases the excitation was performed at the maximum of the MLCT band (exc = 447 nm for 3 and exc = 449 nm for 5, respectively) and emission was recorded from 500 -900 nm. After addition of the last vesicle aliquot and an incubation period of 1 h at room temperature, the emission spectrum was recorded again to test for a kinetically impeded complex-vesicle interaction (which was not observed, see Figure S12).
The UV-vis spectroscopic detection of supramolecular interactions between the Ru complexes 1, 2, 3, 4 and 5 with the inverted E.coli vesicles was performed as follows: First, a 10 µM aqueous buffered solution (V = 1.6 mL; buffer: 50 mM BTP, 140 mM choline chloride, 5 mM MgCl2, 0.2 M TEOA, pH 8.0) of the respective complexes was prepared and then an UV-vis absorption spectrum was recorded. After that, 55 µL of the inverted E.coli vesicles (22 µg/µL total protein concentration, i.e. 730 µg/mL final total protein concentration) were added and then incubated for 1 h at room temperature. After centrifugation (21 • 000 g, 75 min, 4 °C), the supernatant was analyzed by UV-vis spectroscopy again. The loss of the Ru complex by interaction with the inverted E.coli vesicles was determined by the absorbance loss at the MLCT maximum of the Ru complexes.

Calculation of the energy efficiency of the photobiocatalytic process:
Based on the utilized volume of the photobiocatalytic solution (V = 2.0 mL) and the concentration of RuRh complex 1 (5 µM) as well as the inner diameter of the GV vial (1.2 cm, r = 0.6 cm) and the absorbance of 5 µM 1 at 465 nm if d = 1 cm, i.e. 0.09, the following initial parameters can be calculated: The height of the solution inside the GC vial is 2 cm 3 / (0.6 cm 2 x ) = 1.8 cm.
Consequently the absorbance of light in the system is E = 0.09 x 1.8 = 0.16.
Application of E = Iog(I0/I) reveals that with E = 0.16 ca 30% of all incoming photons of  = 465 nm are absorbed by complex 1 in the utilized setup for photobiocatalysis.
Assuming that the distance of 1 cm between the GC vial and the LED stick as well as scattering of LED light by the round shaped geometry of the GC vial reduces the total photonic power input from 45 mW/cm 2 (measured directly on top of the LED stick) to 20 mW/cm 2 gives a total amount of photonic power input into the GC vial of ca. 22 mW considering the bottom area of the GC vial of (0.6 cm) 2 x  = 1.1 cm 2 . 1.5 h (5400 s) of irradiation thus yields a photonic energy input of 22 mW x 5400 s = 119 J from which only 30 % is absorbed, i.e. ca 36 J.
As over the course of 1.5 h ca. 20 µM ATP were formed in 2.0 mL solution, ca 4.0 x 10 -8 mol ATP are present when the experiment is finished. Considering the energy of the ATP → ADP + Pi conversion with -30.5 kJ/mol, [8] 4.0 x 10 -8 mol x 30.5 x 10 3 J/mol = 1.2 x 10 -3 J of photonic energy were stored.
Dividing this value by 36 J of photonic energy that was absorbed by complex 1, an absorbanceconsidered energy efficiency of ca. 3.3 x 10 -3 % is obtained. S10

Utilized Ru complexes and their acronyms
In the course of the experiments, five different Ru complexes (1)(2)(3)(4)(5) were analyzed with respect to their photobiocatalytic response in the presence of inverted E.coli vesicles. The ester protected derivatives 6 and 7 served as the molecular precursors for compounds 2 and 5, which were overall negatively charged under the utilized catalysis conditions (pH 8.0). Scheme S2: Molecular structures and corresponding acronyms of the Ru complexes utilized for photobiocatalytic experiments (1-5) as well as synthetic precursors (6 and 7) towards the free phosphonic acid derivatives 2 and 5. The acidic moieties are highlighted by blue circles. [9] Figure S4 for the corresponding blank experiment, i.e. in the absence of any Ru complex.   S19 Figure S12: Kinetic analysis of formazan absorbance (black) due to not consumed NADH remaining in the photobiocatalysis solution ( E1-value, see Scheme S1) and ATP formation using 5 µM 2 under the typically utilized conditions (N = 2).     . The addition of NH4Cl is indicated by the black arrow. A: The above described buffer solution additionally containing 20 µM of Ru complex 5 was irradiated for 10 min, then analyzed by luminescence spectroscopy. B: The above described buffer solution, additionally containing 20 µM of Ru complex 1, was irradiated for 10 min, then analyzed by luminescence spectroscopy. C: The above described buffer solution, additionally containing 20 µM of Ru complex 3, was irradiated for 10 min, then analyzed by luminescence spectroscopy. D: The diagram compares the relative luminescence intensities (normalized to the emission intensity prior to NH4Cl addition) prior to (gray bars) and after addition of NH4Cl (hatched bars) for the given Ru complexes. 7 (28 mg, 14 µmol) was dissolved in dichloromethane and degassed with an argon-flow. To the solution TMSBr (46 µL, 350 µmol, 25 eq.) was added under argon and the mixture was stirred for 12 h at room temperature. After that time methanol was added and the mixture was further stirred for 6 h at room temperature. Diethyl ether was added, and the product was filtered off to yield a dark red powder (22 mg, 95 %, calculated for the bromide salt). 1 H NMR (400 MHz, CD3CN:D2O = 9:1, v:v) δ 10.14 (s, 2H), 9.98 (s, 2H), 9.44 (5) 7.5 mg 6 (0.0044 mmol) were dissolved in 5 mL dry CH3CN under argon in a 25 mL one-necked roundbottom flask. After addition of 100 µL freshly distilled TMSBr, the mixture was stirred for 16 h at room temperature. Then the mixture was heated to 40 °C for 0.5 h. Afterwards the reaction vessel was opened under air and 1 mL of a 1:1 (v:v) mixture of acetone:H2O was added to the solution leading to the gradual formation of a precipitate. Next, the mixture was stirred for 1 h at room temperature and then all volatiles were evaporated. The quantitative formation of 5 was determined by using 1 H-NMR spectroscopy showing that all protecting ethyl ester groups were cleaved.  PF6)2 (6) [(bpy PEt )2Ru(phenO2)](PF6]2 (50 mg, 33 µmol) was dissolved in 10 mL acetonitrile, acetic acid was added (2 mL) and the solution was degassed with argon. The mixture was heated to 80 °C and one equivalent of 1,10-phenantroline-5,6-diamin (7.0 mg, 33 µmol) was added. After 6 and 18 h two more equivalents of the diamine were added to the reaction mixture, respectively. After cooling to room temperature, the tpphz formed by self-condensation of the diamine was filtered off and the solvent was removed under reduced pressure. The crude oil was taken up in a minimum amount of methanol and upon addition of an aqueous solution of ammonium hexafluorophosphate the product could be filtered off.    (Figures S19-20) and previous literature reports. [11,12] Figure S21: 1 H-NMR spectra of 1 in d4-MeOD (spectra 1-3 show a dilution series with c(1) = 3.4 (bottom), 1.7 (center) and 1.0 mM (top), respectively). No signals were omitted by cutting the spectrum between 8.9 and 9.4 ppm (see Figure S17).  Figure S17). Figure S23: H,H-COSY spectrum of 1 (c = 3.4 mM) in d4-MeOD covering the aromatic region in which the bpy based signals occur (the signal numbering is based on the molecular structure of 1 depicted in Figure S17).          For the UV-vis spectra 10 µM of the complexes were used, for the emission spectra the concentration was set to 5 µM. Although 5 was much better soluble in water compared to DMSO, no higher water content could be used in order to not fully quench the emission of 5 due to the well-known light-switch effect. [13] Figure S37: UV-vis (panel A) and emission spectra (panel B) of 6 (red curves) and 7 (black curves) in DMSO (c = 5 µM).