Heme oxygenase-1 (HO-1) is a vital enzyme in humans that primarily regulates free heme concentrations. The overexpression of HO-1 is commonly associated with cardiovascular and neurodegenerative diseases including atherosclerosis and ischemic stroke. Currently, there are no known chemical probes to detect HO-1 activity, limiting its potential as an early diagnostic/prognostic marker in these serious diseases. Reported here are the design, synthesis, and photophysical and biological characterization of a coumarin–porphyrin FRET break-apart probe to detect HO-1 activity, Fe–L 1 . We designed Fe–L 1 to “break-apart” upon HO-1-catalyzed porphyrin degradation, perturbing the efficient FRET mechanism from a coumarin donor to a porphyrin acceptor fluorophore. Analysis of HO-1 activity using Escherichia coli lysates overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC–MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of Fe–L 2 , we have shown that close structural analogues of heme are required to maintain HO-1 activity. It is anticipated that this work will act as a foundation to design and develop new probes for HO-1 activity in the future, moving toward applications of live fluorescent imaging.

o-Aminophenol-N,N,O-triacetate, known as APTRA, is one of the most well-established ligands for targeting magnesium ions but, like other aminocarboxylate ligands, it binds Ca much more strongly than Mg. The synthesis of an O-phosphinate analogue of APTRA is reported here, namely o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate, referred to as APDAP. Metal binding studies monitored using UV-visible spectroscopy show that the affinity of APDAP for Ca is reduced by over two orders of magnitude compared to APTRA, and for Zn by over three orders of magnitude, whereas the affinity for Mg is attenuated to a much lesser extent, by a factor of only about 7. The selectivity towards Mg is thus substantially improved. DFT calculations support the notion that longer P-O and P-C bonds in APDAP (compared to corresponding C-O and C-C bonds in APTRA) favour a larger angle at the metal, an effect that is less unfavourable for smaller ions like Mg than for larger ions such as Ca. Derivatives of APDAP can be anticipated that will offer improved sensing of Mg in the presence of Ca, in the physiologically important millimolar concentration range.

In a macrocyclic terbium complex incorporating a biaryl sensitiser, the observed variation of emission lifetime is shown to be determined by the solubility of oxygen in the solvent system and the relative energy of the chromophore excited state, rather than any dependence on solvent viscosity.

Four alkynyl-1-naphthyl fluorophores have been synthesised with tri- or pentadentate ligating groups suited to the binding of magnesium. Their photophysical and binding properties for magnesium, calcium and zinc ions have been assessed using absorption, emission and excitation spectroscopy. Each compound has a pK value between 6.2 and 5.1 and exhibits no significant pH response around pH 7.2. Enhanced selectivity for Mg over Ca was observed with a pentadentate phosphinate substituted system, compared to its carboxylate analogue, due to a 10-fold lowering of affinity for the Ca ion. In each case, the overall dissociation constants for Mg fall in the low mm range.

A series of three europium(III) complexes has been created in which an APTRA moiety has been integrated into the sensitising chromophore (APTRA=o-aminophenol-N,N,N-triacetate). The constitutionally isomeric complexes EuL and EuL feature the APTRA unit linked to a metal-bound pyridine ring through an alkynyl unit, differing according to the disposition of the APTRA substituents relative to the C≡C unit (para-N and para-O). In EuL , the APTRA ring is directly bonded to the Eu-coordinated pyridine (para-O). The metal binding affinities for magnesium, calcium and zinc ions have been measured by using emission and excitation spectroscopy. The pyridylalkynylaryl systems, EuL and EuL , offer superior affinity and selectivity for Mg . The Mg affinities are surprisingly very different from prior studies on structurally related systems that incorporate organic fluorophores as reporters, as opposed to the macrocyclic Eu complex moiety. A much-reduced affinity for calcium and zinc-possibly arising from the lower donor ability of the aryl N or O atoms arising from extended conjugation-means that magnesium ion concentrations can be measured directly in serum for the first time, by using such an approach. An apparent dissociation constant for magnesium binding of K =2.4 mm was calculated in the serum background.