A biocompatible redox MRI probe based on a Mn( ii )/Mn( iii ) porphyrin
TL;DR: Water soluble fluorinated Mn-porphyrin derivative Mn-3 acts as an ascorbate specific turn-on MRI probe, which in turn can be re-oxidized by oxygen, and the relaxivity increase from the oxidized to the reduced form is considerably improved at medium frequencies.
Abstract: For the development of redox responsive MRI probes based on the MnIII/MnII couple, stable complexation of both reduced and oxidized forms of the metal ion and appropriate tuning of the redox potential in the biologically relevant range are key elements. The water soluble fluorinated Mn-porphyrin derivative Mn-3 satisfies both requirements. In aqueous solutions, it can reversibly switch between MnIII/MnII oxidation states. In the presence of ascorbic acid or β-mercaptoethanol, the MnIII form undergoes reduction, which is slowly but fully reversed in the presence of air oxygen. A UV-Vis kinetic study of MnIII/MnII reduction under oxygen-free conditions yielded second-order rate constants, k2, of 46.1 M−1 s−1 and 13.8 M−1 s−1 for the reaction with ascorbic acid and β-mercaptoethanol, respectively. This could correspond, in the absence of oxygen, to a half-life of a few minutes in blood plasma and a few seconds in circulating immune cells where ascorbic acid reaches 20–40 μM and a few mM concentrations, respectively. In contrast to expectations based on the redox potential, reduction with glutathione or cysteine does not occur. It is prevented by the coordination of the glutathione carboxylate group(s) to MnIII in the axial position, as was evidenced by NMR data. Therefore, MnIII-3 acts as an ascorbate specific turn-on MRI probe, which in turn can be re-oxidized by oxygen. The relaxivity increase from the oxidized to the reduced form is considerably improved at medium frequencies (up to 80 MHz) with respect to the previously studied Mn-TPPS4 analogues; at 20 MHz, it amounts to 150%. No in vitro cytotoxicity is detectable for Mn-3 in the typical MRI concentration range. Finally, 19F NMR resonances of MnIII-3 are relatively sharp which could open further opportunities to exploit such complexes as paramagnetic 19F NMR probes.
Summary (2 min read)
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- After several extractions of the crude with dichloromethane/water, the organic layer was dried with anhydrous sodium sulfate and concentrated under vacuum giving 3 in 82% yield (Scheme 1).
- The absence of the sharp sextet characteristic of the free aqueous Mn2+ cation even at a very high amplification, led to the conclusion that the MnIII-3 sample was free of aqueous Mn2+ .
- If some MnII-3 were present in the sample, a broad signal at giso ~2.0 would be observable at room temperature,62 which was not the case.
Results and Discussion
- Synthesis of the biocompatible porphyrin-based probes Reagents and conditions: i) DMF, NaH, room temperature, 2h; ii) acetic acid/sodium acetate, Mn(II)acetate tetrahydrate, 80ºC, 2h.
- In order to obtain free base porphyrin 2, nucleophilic aromatic substitution of 1 using mono methyl protected polyethylene glycol with Mw = ~500 g/mol (PEG500) was performed, in DMF as solvent and NaH as base, at room temperature for two hours.
- The product was obtained in 65% yield, after purification, and fully characterized by 1H, 19F NMR spectroscopy and ESI-FIA-TOF mass spectrometry (see Figs. 1, S1, S2 and S3, ESI).
Proton and 19F NMR
- The proton NMR spectrum of MnIII-3 in CDCl3 (Fig. S6a) shows a broad signal at -22.76 ppm corresponding to the -pyrrole protons, characteristic of MnIII high-spin meso-porphyrins.
- 63, 64 The PEG chain CH2 protons at 3.66 ppm are unshifted and hardly broadened relative to the signal at 3.65 ppm for free PEG due to their long distance from the paramagnetic center.
- This set of signals corresponds to the fluorine atoms at the meta positions of the meso-phenyl groups in the five possible combinations of the chain lengths of the four polydisperse PEG chains with n = 10- 11.
- The broad signal around -126.2 ppm results from the overlap of the broader signals of the fluorine atoms at the ortho position, which are closer to the metal center.
- 65, 66 Upon reduction of MnIII-3 to MnII-3, all the 19F resonances are completely broadened out, in accordance with the efficient paramagnetic relaxation of MnII (Fig. S6c).
- The relaxation properties have been investigated for both the oxidized MnIII and the reduced MnII form of the porphyrin complex 3. MnII-3 was obtained by adding 30 equivalents of ascorbic acid to the MnIII analogue.
- Full conversion of MnIII-3 to MnII-3 was evidenced by recording UV-Vis spectra before the relaxivity measurements.
- The NMRD profiles recorded at 25 °C for the reduced and for the oxidized form are presented in Fig.
- A new water-soluble fluorinated porphyrin derivative conjugated with PEG chains was synthesized.
- Interestingly, 19F NMR resonances of MnIII-3 are relatively sharp which could open further possibilities to exploit such complexes as paramagnetic 19F NMR probes.93.
- The present MnII/MnIII-porphyrin couple satisfies many of the criteria required for a redox imaging probe: good water solubility, biocompatibility, non-toxicity, thermodynamic and kinetic stability in both metal oxidation states, a redox half-cell potential accessible to biologically relevant reducing agents with fast kinetics (adapted to the imaging time scale), and a strong “turn on” relaxivity response upon reduction.
- 80, 81 Therefore, the role of ascorbic acid should not be ignored either in maintaining the extracellular redox state.
- Furthermore, MRI quantification of ascorbate could be also achieved by separating the effect of the in vivo concentration of the probe from the redox effect on the observed contrast.
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...An ideal contrast agent is one that is able to affect radiofrequency pulses and enhance image contrast on relaxation time weighted MR image (properties that are present in co-ordinated metal ions) [11,34]....
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Q1. What have the authors contributed in "A biocompatible redox mri probe based on a mn( ii )/mn( iii ) porphyrin" ?
Pinto et al. this paper proposed a new water-soluble fluorinated porphyrin derivative conjugated with PEG chains, which is capable of stabilizing both MnIII and MnII oxidation states in a biologically relevant range of redox potentials.
Q2. What have the authors stated for future works in "A biocompatible redox mri probe based on a mn( ii )/mn( iii ) porphyrin" ?
Interestingly, 19F NMR resonances of MnIII-3 are relatively sharp which could open further possibilities to exploit such complexes as paramagnetic 19F NMR probes. In this respect, Mn-porphyrin redox responsive probes can be advantageous. The present MnII/MnIII-porphyrin couple satisfies many of the criteria required for a redox imaging probe: good water solubility, biocompatibility, non-toxicity, thermodynamic and kinetic stability in both metal oxidation states, a redox half-cell potential accessible to biologically relevant reducing agents with fast kinetics ( adapted to the imaging time scale ), and a strong “ turn on ” relaxivity response upon reduction. Their redox sensitive MRI probe might have potential to report, with fast kinetics ( few minutes half-life ), on extracellular ascorbate without the interference of the cysteine-cystine buffer.