A Coumarin–Porphyrin FRET Break-Apart Probe for Heme Oxygenase-1

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–L1. We designed Fe–L1 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–L2, 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.


Photophysical Characterisation
Sample preparation: Stock solutions of L 1 , Fe-L 1 , L 2 and Fe-L 2 analogues were made in DMSO with a concentration range of 1 -10 mM and stored at -20 °C in the dark. Samples were thawed to room temperature directly before use. All samples were diluted to 20 µM in PBS buffer pH = 7.4 (ThermoFisher, tissue culture grade, 10010023) and 10 µM in CHCl3 buffer for the photophysical measurements. Total concentration of DMSO in samples run for photophysical measurements was < 1 %.
Absorption spectroscopy: UV-Visible absorption spectra were measured using an Agilent Technologies Cary 60 Spectrophotometer operating with WinUV software. The sample was held in a quartz cuvette with a path length of 1 cm. Absorption spectra were recorded against a baseline of pure solvent in a optically matched cuvette with a scan rate of 600 nm / min and a data interval of 1.0 nm. Extinction coefficients were calculated from the Beer Lambert Law (Equation 1).

A = εcl Eq 1.
Where A = the absorbance at a particular wavelength, is the extinction coefficient, c is the concentration and l is the path length (width of the quartz cuvette, 1 cm).
Absorbance spectra to quantify the HO-1 activity were measured in transparent 96-well plates with a Beckman Coulter Paradigm plate reader. Fluorescent spectra were obtained in a quartz cuvette using a Cary Eclipse Fluorescence Spectrophotometer (Agilent).

Fluorescence spectroscopy: Emission and excitation spectra were acquired on an Agilent
Technologies Carry Eclipse Fluorescence Spectrophotometer, in quartz cuvettes with a path length of 1 cm. Emission and excitation spectra were collected with a scan rate of 120.0 nm / min, a delay interval of 1.0 nm and band-passes of 5 nm unless stated otherwise. FRET efficiency (E) was calculated according to Equation 2.
Where E is the FRET efficiency, AD and ED is the absorbance and the emission of the donor fluorophore respectively, and ADA and EDA are the absorbance and emission of the donor fluorophore in the presence of the acceptor. Porphyrin emission and excitation spectra after excitation at the coumarin donor fluorophore were recorded using a 395 nm filter to remove 2 ,-light from the fluorescence emission and I JK L from the excitation spectrum.

Quantum yields:
The fluorescence quantum yields of L 1 -DME and L 2 were determined relative to tetraphenyl porphyrin (TPP) ( NOP = 0.11) in toluene. 1 Solutions of the reference and the sample were prepared so that the absorbance intensity at 514 nm was ≤ 0.1. All measurements were recorded under aerated conditions at room temperature in PBS buffer (pH = 7.4) and chloroform. Absorbance and emission spectrum were run consecutively with identical instrumentation parameters. The quantum yield for the free-base and transitionmetal-based porphyrins were calculated from Equation 3.
Where ∅ is the quantum yield, I is the integrated intensity of the emission spectrum, A is the absorbance at the excitation wavelength and n is the refractive index of the solvent. 'a' refers to the sample, and 'b' refers to the standard.

Preparation of human blood-derived macrophages:
Human blood-derived macrophages were prepared from normal human volunteers exactly as previously described (PMID: 32667970).
Briefly, favourable institutional Ethical opinion and consent were obtained, 50 mL of blood aseptically withdrawn. Peripheral blood mononuclear cells were separated by Ficoll-Hypaque and cultured at 2 x 10 5 cells per well in a 24-well plate (1.8 cm 2 culture surface per well).
Macrophages were cultured overnight in 10% autologous serum and IMDM and then stimulated with hemin 20 μM (Sigma Aldrich 51280, concentration chosen for pathophysiological representativeness).