Sterically demanding macrocyclic Eu(iii) complexes for selective recognition of phosphate and real-time monitoring of enzymatically generated adenosine monophosphate

The design of molecular receptors that bind and sense anions in biologically relevant aqueous solutions is a key challenge in supramolecular chemistry. The recognition of inorganic phosphate is particularly challenging because of its high hydration energy and pH dependent speciation. Adenosine monophosphate (AMP) represents a valuable but elusive target for supramolecular detection because of its structural similarity to the more negatively charged anions, ATP and ADP. We report two new macrocyclic Eu(iii) receptors capable of selectively sensing inorganic phosphate and AMP in water. The receptors contain a sterically demanding 8-(benzyloxy)quinoline pendant arm that coordinates to the metal centre, creating a binding pocket suitable for phosphate and AMP, whilst excluding potentially interfering chelating anions, in particular ATP, bicarbonate and lactate. The sensing selectivity of our Eu(iii) receptors follows the order AMP > ADP > ATP, which represents a reversal of the order of selectivity observed for most reported nucleoside phosphate receptors. We have exploited the unique host–guest induced changes in emission intensity and lifetime for the detection of inorganic phosphate in human serum samples, and for monitoring the enzymatic production of AMP in real-time.


Luminescence experiments
Luminescence spectra were recorded on a Camlin Photonics luminescence spectrometer with FluoroSENS version 3.4.7.2024 software. Emission spectra were obtained using a 40 µL or 100 µL Hellma Analytics quartz cuvette (Art no. 111-10-K-40). Excitation light was set at 332 nm and emission recorded in the range 400 -720 nm using an integration time of 0.5 seconds, increment of 1.0 nm, excitation slit of 0.2 nm and emission slit of 0.5 nm.
Quantum yields were measured using quinine sulfate in 0.05 M H2SO4 as a standard (Φem= 0.59, ex = 350 nm). [1] Emission lifetime measurements were performed on the FluoroSENS instrument. Measurements were taken of 1 mL of 0.1 absorbance samples of Eu(III) complexes in 10 mM HEPES at pH 7.0, unless stated otherwise. Measurements were obtained by indirect excitation of the Eu(III) ion via the quinoline antennae using a short pulse of light at 322 nm followed by monitoring the integrated intensity of the light emitted at 615 nm, with 500 data points collected over a 10 millisecond time period. The decay curves were plotted in Origin Labs 2019 version 9.6.0.172, and fitted to the equation: where I is the intensity at time, t, following excitation, A0 is the intensity when decay has ceased, A1 is the pre-exponential factor and k is the rate constant for the depopulation of the excited state.
The hydration state, q, of the Eu(III) complexes was determined using the modified Horrocks equation [2] : where H2O and D2O are the emission lifetime times in water and D2O, respectively, and n is the number of carbonyl-bound amide NH groups.
Plate reader data was obtained on a BMG Labtech CLARIOstar microplate reader using black Fisherbrand™ 384-well plates, using a total volume of 40 μL per well.

Anion binding titrations
Anion binding titrations were carried out in duplicate in degassed 10 mM HEPES buffer at pH 7.0. Stock solutions of anions (e.g. inorganic phosphate, AMP) containing Eu(III) complex (5 µM) were made up at 0.4, 4 and 40 mM anion. The appropriate anion stock solution was added incrementally to 100 µL of Eu(III) complex (5 µM) and the emission spectrum was recorded after each addition. The ratio of emission bands 605 -630 nm/ 585 -600 nm (ΔJ = 2 / ΔJ = 1) was plotted as a function of anion concentration. The data was analysed using a nonlinear leastsquares curve fitting procedure, based on a 1:1 binding model described by the equation:

Microplate-based enzyme simulations
Different ratios of a solution of enzymatic substrate and product(s) (e.g. cAMP and AMP, respectively) containing a known concentration Eu(III) complex (500 nM unless stated otherwise) in 10 mM HEPES at pH 7.0 were added to a 384-well plate, in triplicate, to a total well volume of 30 µL. The plate was incubated for 10 minutes prior to reading. Time-resolved emission intensities were recorded in the range 605 -630 nm (integration time of 60 -400 µs) with excitation at 292 -366 nm. The mean of the triplicate intensity values was plotted against the percentage of enzymatic product(s). Error bars indicate the standard error in the mean value.

Phosphodiesterase reaction monitoring
Phosphodiesterase

Computational details
Density functional theory (DFT) computations were performed using the wB97M-V functional [3] along with the 6-31G* basis set. [4] A large-core quasi-relativistic effective core potential (ECP), ECP52MWB, [5] was used for treating the core along with the 4f 6 shell of Eu(III) and the associated (7s6p5d)/[5s4p3d] basis set was used for the valence electrons. All computations were performed using spin-restricted orbitals using a pseudo-singlet configuration. Solvation in water was modelled using a conductor-like polarisable continuum model [6] . All computations were carried out in Q-Chem 5.4. [7] The level of theory was verified against a previously reported crystal structure [8] showing that the bond lengths for coordination around the europium atom are reproduced with a mean absolute error below 0.04 Å. reaction mixture was stirred at 80C for 24 hours. The mixture was filtered over celite and washed with EtOAc (3 × 10 mL). The organic fractions were combined, and the solvent removed under reduced pressure. A dark orange solid was collected as the crude material, which was purified by column chromatography (silica gel; 1:4 EtOAc/hexane) to give 8hydroxyquinoline-2-carbaldehyde 1 as a yellow solid (0.657 g, 60%). 1  The spectroscopic data were in agreement with those reported previously. [9]
EuCl3.6H2O (8.2 mg, 0.022 mmol) was added and the mixture stirred for 30 minutes, after which time the reaction was again basified to pH 8. 5

8-((3-Iodobenzyl)oxy)quinoline-2-carbaldehyde (7)
To a solution of aldehyde 1 (0.500 g, 2.89 mmol) in acetonitrile (25 mL) was added potassium carbonate (1.20 g, 8.65 mmol) and 3-iodobenzyl bromide (1.20 g, 4.16 mmol) and the resulting yellow solution was stirred at room temperature for 24 hours. The resulting orange solution was centrifugated at 120 rpm for 3 minutes. The solution was decanted, and the solid pellet was washed twice with dichloromethane (3 × 15 mL) and the solvent was evaporated under reduced pressure. The resulting residue was partitioned between dichloromethane (10 mL) and saturated aqueous sodium chloride (10 mL). The aqueous phase was extracted with dichloromethane (3 × 10 mL) and the combined organic phases were dried over MgSO4, filtered and concentrated under reduced pressure. The crude material was purified by column chromatography (silica gel; 20 -80% EtOAc/petroleum ether) to give the desired aldehyde 7

(8-((3-Iodobenzyl)oxy)quinolin-2-yl)methyl methanesulfonate (9)
To a solution of alcohol 8 (100 mg, 0.256 mmol) in anhydrous THF (10 mL) was added triethylamine (54 µL, 0.384 mmol) and methanesulfonyl chloride (2.0 µL, 0.28 mmol) to give a yellow solution which was stirred at 30 °C for 2 hours under a nitrogen atmosphere. The solvent was removed under reduced pressure and the residue was partitioned between dichloromethane (20 mL) and saturated sodium chloride solution (10 mL). The aqueous layer was extracted with dichloromethane (3 × 15 mL) and the organic layers combined, dried over MgSO4, filtered and evaporated under reduced pressure to give the desired mesylate ester 9 (85 mg, 71%) as a yellow oil, which was used immediately in the next step. 1                  [a] The binding energies are intended as a general measure of affinity. Evaluation of free energy differences needed to reproduce the binding constants in Table 2 of the main text is beyond the scope of this work, requiring consideration of entropic effects, solvation, conformational flexibility, and the influence of pH.