Porphyrins

There are several methods available to measure respiration of living samples, which can be globally divided into two groups: O2-dependent quenching of porphyrin-based phosphors (Seahorse Bioscience XF respirometer and Luxcel MitoXpress) and amperometric O₂ sensors (Clark electrodes, including the widely adopted Oroboros system)1 ,42 (link). Historically, the amperometric approach has been the main method used to assess mitochondrial respiration in C. elegans. For the amperometric approach, nematodes are delivered into a single respiratory chamber, which is separated from two half-cells by O₂-permeable material. In this way, only O₂ can diffuse from the assay medium through the membrane. When a small voltage is applied to the half-cells, O₂ is reduced by electrons at the cathode yielding hydrogen peroxide. Subsequently, H₂O₂ oxidizes the Ag (silver) of the Ag/AgCl anode, which results in an electrical current that is proportional to the O₂ pressure – and thus concentration – in the experimental respiratory chamber.
Apart from the detection modality, differences of the XF respirometric method appear at the level of number of worms per assay, replicates, multiple, or real-time measurements and the ability to inject compounds during an experiment (Table 1). The Clark electrode approach requires thousands (~2000-5000) of worms in a single chamber to obtain an estimation of the oxygen consumption rate43 (link). Performing multiple measurements, biological replicates and comparing conditions provide the biggest challenges within the Clark electrode method as the traditional set-up only allows the measurements of one sample at a time. In contrast, a XF96 respirometer requires ~10-20 worms per well to acquire a reproducible oxygen consumption rate, measurements can be easily and quickly (in the order of minutes) repeated in an automated way and since XF respirometers can analyse whole plates at the same time, about 96 conditions/replicates can be tested at once. An additional difference is the presence of drug-injection ports that can be programmed to inject compounds in all 96 wells at time points that are specified a priori during an XF respirometer experiment. Clark electrode systems also allow injection of compounds, and even offer flexibility with respect to the timing, dosing and number of additions as compounds are injected manually during the course of the assay. However, precise timing of manual additions between replicate experiments may be challenging.
More similar to the Seahorse XF respirometer method is the Luxcel MitoXpress O₂ consumption assay, which relies on O₂-dependent quenching of porphyrin-based phosphor. The MitoXpress kits provide a way of performing real-time analysis of cellular respiration, via an oxygen-quenching fluorophore system. Worms are placed into the wells of a 96- or 384-well plate, the kit reagents are added, and measurements are made in a fluorometric plate reader. Multiple conditions and replicates can be tested side-by-side in the wells of a single plate, but repeated measurements over time are more challenging as there is typically no automatized mixing system integrated in the plates or plate-readers to restore basal O₂ levels. In addition, single estimation of the OCR takes >90 minutes, while careful estimations of the OCR in the XF respirometer approach takes only 2-5 minutes of measuring time. Finally, the use of compounds to assess multiple aspects of mitochondrial function related to oxygen consumption is limited since compounds need to be injected manually immediately prior to the start of the experiment.

All solvents and reagents were obtained from commercial sources and used as received. Palladium porphyrins Pd-1-OBu and Pd-1-OH were synthesized as described previously (43 ). Column chromatography was performed on Selecto silica gel (Fisher) or aluminum oxide (neutral, Brockmann I, ~150 mesh, 58 Å). Preparative gel permeation chromatography was performed on S-X1 (Biorad) beads, using THF as a mobile phase, unless otherwise stated. ¹H and ¹³C NMR spectra were recorded on a Bruker DPX-400 spectrometer. Mass spectra were obtained on a MALDI-TOF Voyager-DE RP BioSpectrometry workstation, using α-cyano-4-hydroxycinnamic acid as the matrix.
Detailed description of synthetic procedures, ¹H and ¹³C NMR, MALDI-TOF, absorption and emission spectra and Stern-Volmer oxygen quenching plots can be found in Supporting Information.
Quartz fluorometric cells (Starna, Inc., 1 cm optical path length) were used in optical experiments. Optical spectra were recorded on a Perkin-Elmer Lambda 35 UV–vis spectrophotometer. Steady-state fluorescence and phosphorescence measurements were performed on a SPEX Fluorolog-2 spectrofluorometer (Jobin-Yvon Horiba), equipped with an infrared-enhanced R2658P PMT (Hamamatsu). Emission spectra were obtained using solutions with absorption at the excitation maximum of approximately 0.05 OD. Quantum yields of emission of all of the synthesized compounds were measured relative to the fluorescence of tetraphenylporphyrin φ_fl = 0.11 in deoxygenated C₆H₆ (37 ).
The system for oxygen titrations was described previously (43 , 61 (link)). Time-resolved phosphorescence measurements were performed using an in-house-constructed phosphorometer (57 ), modified for time-domain operation. For phosphorescence measurements, solutions were deoxygenated by argon bubbling (Airgas, grade 5.5), while monitoring changes in the phosphorescence lifetimes. Aqueous solutions were deoxygenated by a glucose/glucose oxidase/catalase enzymatic system (5 (link)) or by prolonged purging with argon.