Ratiometric Detection of ATP by Fluorescent Cyclophanes with Bellows‐Type Sensing Mechanism

Abstract Pyrene‐based cyclophanes have been synthesized with the aim to realize a bellows‐type sensing mechanism for the ratiometric detection of nucleotide concentrations in a buffered aqueous solution. The sensing mechanism involves the encapsulation of a nucleobase between two pyrene rings, which affects the monomer‐excimer equilibrium of the receptor in the excited state. The nature of the spacer and its connection pattern to pyrene rings have been varied to achieve high selectivity for ATP. The 1,8‐substituted pyrene‐based cyclophane with the 2,2’‐diaminodiethylamine spacer demonstrates the best selectivity for ATP showing a 50‐fold increase in the monomer‐excimer emission ratio upon saturation with the nucleotide. The receptor can detect ATP within the biological concentrations range over a wide pH range. NMR and spectroscopic studies have revealed the importance of hydrogen bonding and stacking interactions for achieving a required receptor selectivity. The probe has been successfully applied for the real‐time monitoring of creatine kinase activity.


Synthesis of compounds
1,6-Dibromopyrene 1,8-dibromopyrene synthesized according to literature methods without modifications. [1] Already published procedures were used to synthesize pyrene-1,8-dicarbaldehyde or pyrene-1,6-dicarbaldehyde. [2] The general conditions for the dialdehyde synthesis are the following: A solution of n-butyllithium in hexane (2.5M, 6 equiv) was added dropwise to a solution of a mixture of 1,6-and 1,8-dibromopyrene (1:1 molar ration obtained from the previous reaction) in anhydrous THF under an atmosphere of argon at -50°C. Reaction was stirred for 20 min at this temperature and then warmed to 25°C and stirred for additional 1 h. The reaction mixture was cooled to -40°C and DMF (6 equiv) was added dropwise to the solution. The mixture was stirred at room temperature for 12 h before water was added. THF was removed under reduced pressure. The resulting residue was suspended in 100 ml of H2O and 200 ml DCM and the aqueous phase was extracted three times with 200 ml of CH2Cl2. The organic phases were combined and dried over NaSO4. Solvents were removed under reduced pressure. The residue was purified by a column chromatography on silica gel using eluent CH2Cl2-hexane 1:1 with decreasing hexane portion to pure DCM. The first fraction contained pyrene-1,6-dicarbaldehyde (25% yield), the second fraction contained pyrene-1,8-dicarbaldehyde (21% yield).

S2
The general method for the synthesis of pyrene-macrocycles The synthesis of macrocyclic receptors was adapted from the method described by Fabbrizzi. [3] Pyrene-1,8-dicarbaldehyde or pyrene-1,6-dicarbaldehyde (3 mM, 774 mg) was added to a round bottom flask, then 400 ml of acetonitrile and 40 ml of methanol were added. The flask was placed in an oil bath and heated to 50 o C with stirring, until the dialdehyde was completely dissolved. The appropriate amine (3 mmol) was dissolved in 100 ml of acetonitrile. The resulting solution was slowly added from the dropping funnel to the pyrene-1,8-dicarbaldehyde or pyrene-1,6-dicarbaldehyde solution with stirring and heating at 50 o C. The reaction was achieved by heating at 50 o C for 72 h. The solvent was removed under reduced pressure without heating. To the residue was added 300 ml of methanol and 30 mmoles of sodium borohydride, the flask was placed in an oil bath and heated to 50 o C for 3 hours, then the reaction was kept overnight at room temperature. Methanol was removed under reduced pressure. The resulting solid was suspended in 100 ml of H2O and 100 ml mixture chloroform-ethanol 10:100 and the aqueous phase was extracted with three portions 100 ml of mixture chloroform-ethanol 10:100. The organic phases were collected and dried with NaSO4. Solvents were removed under reduced pressure. The residue was purified by chromatography on silica gel using eluent ethanol-chloroform 1:1 to ethanolchloroform-ammonia 100:100:5. 6,16-dioxa-3,9,13,19-tetraaza-1,11(1,6)-dipyrenacycloicosaphane The product is a pale yellow powder. Yield: 43%. M.p. 176-179 0 C. 1   Potentiometric studies of receptor 1.
All solutions were prepared in 0.05M NaCl solution with ca. 0.5 mM concentration of compounds in deionised water. For titrations standard 0.1M solution of NaOH was used. The potentiometric titrations were carried out on a Mettler Toledo G20 Titrator equipped with a DGi 102-Mini pH-electrode. The electrode was calibrated with standard calibrating solutions from Mettler Toledo. The reaction vessel was kept at constant temperature 23°C. The value of K'w was determined from data obtained in the alkaline range of the titration, and found to be equal to 13-14.00 in our experimental conditions. The titration experiment was carried out as follows: in the reaction vessel was placed a solution of a compound (and calculated amount of HCl); after stirring the solution for 5 minutes the titrations was started. The experiment was repeated 3-5 times. For the experiment in the presence of an anion, the corresponding amount of its solution was added prior to the titrations. The obtained data was imported to the HYPEQUAD 2008 program and fitted to obtained protonation constants. [4]  Fluorescence studies

Relationship between pH and fluorescence intensity
The solution with a desired pH value were prepared by adjusting 50 mM solution of acetic acid with an appropriate amount of sodium hydroxide. Receptor concentration 10 -5 M was achieve by addition of a receptor in DMSO to an aqueous solution with the fixed pH. Content of DMSO in the final solution was 6% vol. Excitation at 350 nm, slit 2:1.

Competitive binding of ATP
The competition experiment was conducted as follows: 10 -5 M solution of the receptor in a buffered solution (50 mM MES, pH 6.2, 6% DMSO) was treated first with excess ATP (50 equiv) dissolved in the same buffer and fluorescence was measured before and after addition. The ratio of intensities (I/I0) represents the relative fluorescence increase after addition of ATP (marked and "None" below in Figure). The following experiments were done with a competing nucleotide and a mixture of a competing nucleotide with ATP. For example, the fluorescence the receptor was measured a) in the presence of 100 equiv of GTP and b) in the presence of 100 equiv of GTP together with 50 equiv of ATP (see Figure below). "None" corresponds to fluorescence changes after addition of 50 equiv of ATP. "GTP" corresponds to fluorescence changes after addition of 100 equiv. of GTP. "GTP+ATP" corresponds to fluorescence changes after addition 100 equiv. of GTP and 50 equiv. of ATP.

Fluorescence titration of receptors with nucleotides
The receptors (0.01 mM) were dissolved in a buffered solution (50mM MES, pH 6.2, 6%DMSO) and then titrated with nucleotides (0.02M) followed by fluorescence measurements of each titration point. S12

UV-Vis studies
UV-Vis titrations were carried out were carried out in the same manner as the fluorescence titrations. To a receptor solution with 10 -5 M concentration, a solution of the receptor (10 -5 M)and a nucleotide (0.02 M) was added in portions. At each step a spectrum was measured. All the spectra were combined by using HypSpec program and fitted to yield the binding constants.

X-ray studies
Single clear light colourless plate crystals of 19Kat_OSH01_2 recrystallised from a mixture of methanol and TCM by solvent layering. A suitable crystal with dimensions 0.39 × 0.25 × 0.11 mm 3 was selected and mounted on a mylar loop in perfluoroether oil on a SuperNova, Dual, Cu at home/near, Atlas diffractometer. The crystal was kept at a steady T = 152.95(10) K during data collection. The structure was solved with the ShelXT (Sheldrick, 2015) solution program using dual methods and by using Olex2 [5] as the graphical interface.

S21
A clear light colourless plate-shaped crystal with dimensions 0.39 × 0.25 × 0.11 mm 3 was mounted on a mylar loop in perfluoroether oil. Data were collected using a SuperNova, Dual, Cu at home/near, Atlas diffractometer equipped with a Cryojet Oxford Instruments low-temperature device operating at T = 152.95(10) K. Data were measured using  scans using Cu K radiation. The diffraction pattern was indexed and the total number of runs and images was based on the strategy calculation from the program CrysAlisPro (Rigaku, V1.171.40.53, 2019). The maximum resolution that was achieved was  = 63.693° (0.86 Å).
The diffraction pattern was indexed and the total number of runs and images was based on the strategy calculation from the program CrysAlisPro The structure was solved and the space group P21/c (# 14) determined by the ShelXT (Sheldrick, 2015) structure solution program using using dual methods and refined by full matrix least squares minimisation on F 2 using version 2018/3 of ShelXL 2018/3 (Sheldrick, 2015). All non-hydrogen atoms were refined anisotropically. Hydrogen atom positions were calculated geometrically and refined using the riding model. Most hydrogen atom positions were calculated geometrically and refined using the riding model, but some hydrogen atoms were refined freely.