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

Abstract

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.

Fig. 1. (a) Structures of cationic Eu(iii) complexes designed to bind phosphate and AMP in water, (b) DFT optimised geometry of [Eu·Bn]⁺ showing the anion binding site occupied by a single water molecule.

Fig. 2. Absorption (blue) and emission (pink) spectrum of [Eu·mBOH₂]⁺ (5 μM) in 10 mM HEPES at pH 7.0 and 295 K, λ_exc = 322 nm.

Fig. 3. Selective emission response of [Eu·Bn]⁺ for phosphate. (a and b) Emission enhancement of [Eu·Bn]⁺ (5 μM) with phosphate (1 mM) compared with citrate, lactate, acetate, sulfate and bicarbonate (1 mM each); (c) variation in emission spectra of [Eu·Bn]⁺ upon incremental addition of phosphate; (d) plot of fraction bound (determined from ΔJ = 2/ΔJ = 1 intensity ratio) versus phosphate concentration, showing the fit to a 1 : 1 binding isotherm. Measured in 10 mM HEPES at pH 7.0 and 295 K, λ_exc = 322 nm.

Fig. 4. Selective emission enhancement of [Eu·mBOH₂]⁺ for AMP over ATP, ADP and cAMP. (a) Change in emission spectra of [Eu·mBOH₂]⁺ (5 μM) in the presence of 1 mM AMP over ATP, ADP and cAMP. (b) Change in intensity of the ΔJ = 2 emission band of [Eu·mBOH₂]⁺ (5 μM) with AMP, ATP, ADP and cAMP (1 mM each); (c) plot of fraction bound (determined from ΔJ = 2/ΔJ = 1 intensity ratio) versus AMP concentration, showing the fit to a 1 : 1 binding isotherm. Measured in 10 mM HEPES at pH 7.0 and 295 K, λ_exc = 322 nm.

Fig. 5. Molecular geometries of [Eu·mBOH₂]⁺ bound to (a) inorganic phosphate and AMP in (b) a monodentate and (c) a bidentate manner involving formation of a cyclic boronic ester. Bond distances of Eu(iii) to donor atoms are shown: N(m) – macrocycle, N(q) – quinoline, O(ac) – acetate, O(et) – ether, O(P) – phosphate.

Fig. 6. Measurement of phosphate in human serum. (a) Variation in time-resolved emission spectrum of [Eu·Bn]⁺ (20 μM) and (b) linear increase in time-resolved emission of the ΔJ = 2 band upon addition of phosphate in human serum. Measured at pH 7.0 and 295 K, λ_exc = 322 nm, integration time = 60–400 μs.

Fig. 7. Real-time monitoring of phosphodiesterase (PDE) activity. (a) Simulation of the PDE reaction, recording the time-resolved emission of [Eu·mBOH₂]⁺ with increasing % AMP. Conditions: 100 μM cAMP + AMP, 5 mM MgCl₂, 5 μM [Eu·mBOH₂]⁺, 10 mM HEPES at pH 7.0. (b) Real-time monitoring of the PDE-catalysed conversion of cAMP to AMP using the time-resolved emission of [Eu·mBOH₂]⁺, comparing the change in rate of reaction upon activation with calmodulin (2 μM) and CaCl₂ (60 μM). Conditions: 200 μM cAMP, 4 μL PDE (0.1 mg mL⁻¹), 5 mM MgCl₂, 10 μM [Eu·mBOH₂]⁺, 10 mM HEPES at pH 7.0, λ_exc = 292–366 nm, λ_em = 615–625 nm, integration time = 60–400 μs.