Introduction

The detection of the superoxide radical anion (O2 •-) in biological systems is still an ambitious goal. So far, the method of choice to evaluate the O2 •-level in those systems is the use of hydroethidine (HE) as a fluorogenic probe. Since HE was not designed for the superoxide detection, we aim to develop new HE-based probes to understand the mechanism between HE and O2 •-, to modulate its site-specific detection and to improve its ability to detect the superoxide radical in biological systems.

The superoxide radical anion 1.The reactive oxygen species and their implication in the oxidative stress

In biological systems, the reactive oxygen species (ROS) are a set of chemical species that are byproducts of the molecular oxygen, but whose are more reactive than O2 itself. ROS are also well known under the designation "free radicals", since a lot of those species possess an unpaired electron in their valence orbital. However, this latter term is restrictive to designate those species since a lot of ROS are diamagnetic molecules as well. [1] Reactive oxygen species (ROS) are normally generated in every aerobe. ROS are involved in the normal cellular metabolism by playing a key role in numerous physiological processes and notably in signaling process. An example in which the signaling process mediated by ROS is necessary is the immune response, which allows to organisms to fight against exogenous bacteria. The importance of the ROS species as antibacterial agent is notably underlined by the fact that mammal species, which cannot produce NO • died rapidly of infection, while they can survive in pathogen-free condition. [2] However, ROS possess equally deleterious effects against cells. Indeed, ROS cause also damage to biomolecules such as protein, DNA or lipids, including the lipid peroxidation. [3] Those damages are normal and can be easily counterbalanced by repairing process. [4] However, it is important to note that the deleterious effects are not only govern by the oxidant properties of the ROS, but are also essentially mediated by signaling processes, such as the triggering of apoptosis.

It is important to note that ROS are produced and regulated by pro-oxidants and antioxidants respectively, which can be enzymes but not only. When an imbalance in favor of the pro-oxidant occurred that lead to disruption of the signaling process and eventually to uncontrolled damage to biomolecule in an organism, the organism is said under oxidative stress. [1] Almost all diseases are mediated by ROS. The ROS mediated processes of some disease are well described in the literature with, for example, the artherosclerosis and ischemia/reperfusion injury. However, it is important to note that the oxidative stress, that may result from the disease, is in most of the case a consequence of the disease and not a cause. [1] This may eventually explained why antioxidant-based treatments on cardiovascular diseases has been disappointed so far. [5] However, some authors reported that antioxidant-based treatment helped to reduced pathologies associated to the oxidative stress, which result as a side effect from cancers. [6,7]

Formation and reaction of the superoxide radical by biological relevant species

In biological systems, the superoxide radical anion (O2 •-) is proximal species at the origin of the formation of most ROS. The O2 •-is the product form by the monoelectronic reduction of the molecular oxygen. [8] Figure 1 formation of the O2 •-by monoelectronic reduction of the molecular oxygen. O2 •-is notably and essentially formed within mitochondria as a byproduct of the cellular respiration. The production of which is believed to occur essentially at the level of complex I of electron transport chain (ETC). [9] Indeed, during the aerobic metabolism process. The molecular oxygen, used as combustive, end up normally to be reduced by 4 electrons to give water. However, the incomplete reduction of the O2 lead to the formation of O2 •-. It is estimated that this phenomenon occurred to 1 to 3% of the dioxygen. [8] But this data are likely to be overestimated by 10-100-fold. [8]

Figure 2 Expected Formation of O2 •-from leak of ETC within mitochondria

The superoxide radical is also extensively formed in neutrophilic cells upon the activation of transmembrane enzymatic complex Nox2 located in macrophage plasma membranes. This phenomenon is described as the oxidative burst. It is important to note that activated macrophages produce O2 •-to a far greater extent than mitochondria. Which can be simply explained by the fact that the sole role of the Nox2 enzyme, contains in the NADPH oxidase of phagocytes, is to produce O2 •- upon its activation, while the mitochondrial superoxide production appears just as a byproduct of the cellular respiration through leakage of electrons from the ETC. The superoxide is also naturally produced in cells that contains xanthine oxidase, such as hepatocytes. [10] In which the oxidation-reduction reaction between O2 and HX catalyzed by XO gives O2 •-. HX is in turn oxidized successively in xanthine and then uric acid. The superoxide radical is dismutated by the superoxide dismutase (SOD) at diffusion controlled rate to yield hydrogen peroxide and dioxygen. [8] SOD enzyme, which catalyzes this reaction in biological systems, exists under different forms: MnSOD inside the mitochondrial matrix and Cu-ZnSOD in the cytosol and the inner-membrane space (FeSOD can also be found). The product of this reaction, the H2O2 is extensively used as signaling molecules. Furthermore, it is important to note that without the presence of the SOD the superoxide cannot dismutate by itself. This underline the weak reactivity of O2 •-. This weak reactivity can also be evaluated in cells. Where indeed, the O2 •-by itself is quite inert against biomolecules.

However, in biological model at physiological pH, the O2 •-formed is rapidly pronated, due to its pKa (4.9) and the pH of the mitochondria, which give therefore the hydroperoxyl radical (HOO • ). [11] It is important to note that HOO • can react with O2 •-to give hydrogen peroxide but with a rate constant lower than the SOD, which makes this pathway disfavor in biological systems.

Regarding the reaction of the superoxide radical with other ROS. It is important to note the O2 •-can also be scavenged by NO, at diffusion control rates, to give peroxynitrite. A compound that can react with the CO2 to give the highly toxic nitrosoperoxycarbonate (ONOOCO2 -) that can be harmful through its hemolytic fission.

The general pathway of the generation of ROS starting from the superoxide radical is shown in the Figure 5. O2 •-can notably react with the Fe 3+ according to the reaction of Haber-Weiss Chemistry to give the hydroxyl radical, which radical can notably scavenged by the gluthatiation.

Figure 5 Formation and consumption of the superoxide radical anion by biological relevant species according literature. [12] The superoxide by itself is harmless toward biomolecules. [13] The oxidative damages are mostly mediated by the hydrogen peroxide and hydroxyl radical. [8] H2O2 can be generated by other pathways, which didn't involve superoxide. Therefore, measuring the production of the latter is not a reliable method to assess the superoxide production. H2O2 is also regulated by some enzymes such as catalase or gluthathion peroxidase.

state-of-the-art in detection techniques of O2 •-

Evaluation of the superoxide by spin trapping technique associated to EPR spectroscopy

Although, the role of the superoxide is broadly understood, its detection remains a challenge. This detection is of major importance to attribute certainly its role in the biological event. [14] The direct detection of O2 •-is not possible since O2 •-possess a very short lifetime, a low steady state concentration (nM) and unfavorable spectroscopic properties. Therefore, indirect methods of detections are required to evaluate its level.

Even though the spin trapping technique associated to EPR spectroscopy is the most reliable technique to detect free radicals, the applicability of which is imitated in biological systems for the superoxide detection. Indeed, the reaction of the superoxide with nitrones, used as spin trap, lead to adducts that are rapidly reduced by biological relevant reductants to give an EPR-silent hydroxylamine. Among those nitrone, the DIPPMPO that is notably very efficient to detect the superoxide in cell free systems (Figure 6). [15] Figure 6 Reaction of a nitrone and the superoxide radical anion and bioreduction of the spin-adduct.

The spin trapping technique can also be employed to assay the ROS level in cells by monitoring the redox status of the endogenous iron sulfur clusters. [16] This method is all the more efficient since it doesn't require the internalization of an exogenous compound within cells. However, this method didn't give information of the particular superoxide level. Therefore, additional method required to assays specifically the superoxide level in cells.

Fluorescent based probes for the ROS detection

Another technique, widely used to assess the ROS level in biological systems, is the use of small molecules as fluorescent dyes. A wild rang of such molecules have been already developed, designed and synthesized. [START_REF] Sabnis | Handbook of Fluorescent Dyes and Probes: Sabnis/Handbook of Fluorescent Dyes and Probes[END_REF] Small molecules can be used as fluorescent probes to detect ROS, if the extent of the oxidant generated is proportional to the amount of the detected marker in the system investigated. [START_REF] Zielonka | [END_REF] It is also important for the ROS probe to be as inert as possible. Therefore, those probes must be used at a concentration that didn't affect the mitochondrial respiration or the redox status of the cells. On the other hand, these probes also have to be used at the highest concentration possible to compete with the endogenous scavengers of the ROS studied. [START_REF] Zielonka | [END_REF] The superoxide produced within mitochondria used to be assayed through the hydrogen peroxide level in the extracellular milieu measured with the amplex red probe (since H2O2 can readily cross membranes by passive diffusion). [9] Indeed, amplex red associated with horseradish peroxidase reacts with H2O2 to form the fluorescent resorufin (Figure 7). [19] However, it is important to note that this method of detection is limited, since there is several pathways that yield H2O2 which are not mediated by the disproportionation of O2 •-by SOD. The dichlorodihydrofluorescein (DCFH) is also a probe frequently used to evaluate "the cellular oxidative stress" by fluorescence. [20] The non-fluorescent DCFH permeates membranes to be oxidized inside cells. Even though, this chemiluminescent probe is essentially used to evaluate the H2O2 level, as amplex Red, the DCFH does not react directly with H2O2. [21,22] [16] Indeed, DCFH reacts with peroxidase and other hemeprotein at high oxidation state. This reaction leads to dichlorofluorescein DCF, a green fluorescent compound that can be conveniently quantified notably by fluorescence life imaging. However, it is important to note that the radical intermediate of this compound can reduce the molecular oxygen to give O2 •-, which can in turn induce an increase of the level of ROS in the studied systems (Figure 8). Another fluorescent probe that was notably used to detect O2 •-is the lucigenin, which probe is a dication that can react with the superoxide to give a stable compound. However, this probe has been discarded for the superoxide detection, since the radical cation of this probe can reduce the molecular oxygen to produce O2 •-.

Hydroethidine (HE) as a fluorescent probe 1.3.1 HE reacts with O2 •-to form a specific marker

Hydroethidine (HE) used as a fluorescent probe is the gold standard in the superoxide detection. Indeed, HE reacts directly with the superoxide radical to give the 2-Hydroxyethidium (2-OH-E + ). [23] The 2-OH-E + is a specific-product of the reaction between HE and O2 •-. Both HE and 2-OH-E + are fluorescent. However, the fluorescence properties (excitation and emission wavelength) of the superoxide marker (2-OH-E + ) and the probe (HE) differ markedly. Interestingly, the hydroxylated superoxide marker also possesses the advantage of being a stable compound (Figure 10). In cell free system, the stoichiometry between HE and O2 •-is expected to be 1:2. Indeed, a first reaction involves the oxidation of HE by 1 electron to give the radical cation HE •+ . The second reaction of HE •+ with O2 •-is even more interesting, since in this step HE •+ traps the O2 •-to yield the hydroxylated marker. Indeed, preliminary data from Medical college of Wisconsin has been proved that the oxygen at the C-2 position is originate from the direct reaction with O2 •-. Furthermore, the rate constant of the reaction between HE and O2 •-that leads to the radical cation has been calculated by Zielonka et al. to be 2 × 10 6 . [24] The rate constant for the second step, which involve the radical cation and the O2 •-is 7.6 × 10 8 .(future work).

One of the advantages of HE over the other fluorogenic probes is that the radical cation of the HE probes didn't reduce the dioxygen to produce the superoxide. This is of major importance to measure accurately the formation of superoxide. Furthermore, it is important to note that to measure the superoxide radical, the HE probe has to be in excess compared of its oxidation product since the concentration of the 2-OH-E + is not only controlled by the concentration of superoxide but also by the concentration of the probe.

Interestingly, it has been reported that the radical cation can also trap the chloride atom contained into hypochlorous form from the myeloperoxidase to yield the 2-Cl-E + . [25] The formation of the 2-Cl-E + is all the more interesting since this compound is none fluorescent and therefore didn't interfere with the measurement of O2 •-. The additional reaction is all the more interesting since, it allows to the HE probe to measure the presence of other ROS by HPLC.

Limitation in the application of HE in the superoxide detection in cellular system

The 2-OH-E + is not the only red-fluorescent product that results from the oxidation of the HE probe. Indeed, HE can also be oxidized according to a non-specific pathway to give E + . E + and the 2-OH-E + shares similar fluorescence properties. [26] Therefore, in the systems in which E + is preponderant, the superoxide level cannot be assayed directly by fluorescence. In those systems, the superoxide marker (2-OH-E + ) and the ethidium (E + ) need to be separated by HPLC to assay the superoxide level. Indeed, HE can be oxidized by one electron with serval biological relevant species such as heme protein (including notably the cytochrome C (Fe 3+ )), hydroxyl radical or the peroxynitrite, etc. The resulting radical cation intermediate can therefore react with the O2 •-with rate that can compete with the SOD to give the hydroxylated marker. However, HE •+ can be self-scavenged to form dimers. Those dimers are non-fluorescent, so they don't represent a problem. Moreover, a detection of high levels of dimers by HPLC can be even interesting since it gives insight of the redox status of the transitions metals into cells, and which highlights thereby the presence of a strong one-electron oxidant in the biological system studied. [27] However, HE •+ can be also disproportionated or oxidized by another electron to give E + . Therefore, in mitochondria, an increase of the presence of O2 •-is likely to be accompanied by an increase of E + . Which meant that the increase of the red fluorescence that can be observed in under fluorescence life imaging may not reflect an increase directly of the superoxide (Figure 11).

Moreover, in regular cells 1 , the E + level is likely to be 10-to-100 fold higher compared to the 2-OH-E + level making the distinction between the superoxide marker and the non-specific oxidation of the probe non-reliable if taken directly by direct fluorescence techniques, such as fluorescence life imaging. Unfortunately, despite the fact that, those methods of assay the superoxide level has been discarded, most authors continue to assess the superoxide directly by fluorescence.

In cellular models, even by using the HPLC method, the 2-OH-E + level cannot be converted to a O2 •- level for several reasons (i) the stoichiometry between the 2-OH-E + and O2 •-in cell-free system (that is theoretically 1:2) is still not accurate and poorly reproducible. [28] (ii) in cellular systems, the radical cation intermediate HE •+ are likely to be generated by other oxidants than O2 •-. (iii) the HE oxidation products might uncouple the mitochondria and inhibit the complex IV due to their positive charge. [29] Therefore an increase of the 2-OH-E + level and other oxidation products level can trigger a domino effect leading to an additional increase of the 2-OH-E + level. HE is also a photosensitive molecule. Indeed, under light HE yield E + . HE is also prone to the oxidation in the dark in presence of oxygen at room temperature. [30] Finally, even if the 2-OH-E + is stable compounds, this superoxide marker are likely to be accompanied by the unreacted probe (HE) in servals fold-higher concentration, which may undergo further oxidation including into 2-OH-E + . For this reason the superoxide assessment needs to be as fast as possible.

Finally, it is important to note that the oxidation of HE by heme protein is a limitation for the superoxide detection in vivo due to the large amount of heme protein in blood. Therefore, there is an unmet need to develop new HE-based probes that can be more reliable for the superoxide detection in vivo.

State of the art in the synthesis of HE and its derivatives

Figure 12 Approaches to the synthesis of Ethidium bromide (the precursor of HE) describe in the literature

The synthetic route leading to Ethidium bromide (E + ) have been widely described in the literature (the precursor of HE under its oxidized form). This synthesis is notably well described by Lion et al. [31] This synthesis started from the biphenyl 1. The formation of the phenanthdrine core was achieved according a Bischler Napieralski reaction in the presence of POCl3. This synthetic route shows the compatibility for the Ethidium moiety for the ethyloxycarbonyl protecting group.

Synthesis of HE derivatives functionalized on their phenyl group

Figure 13 A recent approaches to the synthesis of an ethidium derivatives. [32] Another synthetic route have been recently described by Chu et al. to synthesize a HE-derivatives. [32] This synthetic route started biphenyl 11. This synthetic route was a model for the development of new HE -derivatives. This approach shows notably the treatment that are compatible with the probe. Therefore, one of the advantages of this synthetic route is notably to show the compatibility of the ethidium derivatives with the click chemistry reaction.

The HE-derivatives that were developed by Chu et al. have been used in vivo to detect the ROS production. Their aims were to incorporate a F 18 as radiotracer, therefore They craft it on the phenyl in position 5. It is important to note, that the functionalization of this position didn't affect the reaction between the HE-based probe and the superoxide radical anion. However, recently, another improved PET radiotracer have been developed by Wilson et al. [33] These authors synthesized this improved probe starting from the commercially 3,8-diamino-6-phenylphenanthridine (DAPP) in 4 steps by methylation of the aza aminogroup and the reduction of the phenanthridine core. We also favored this more direct approaches to synthesize the most of our HE-derivatives to avoid waste of resources, time and effort. The commercially available 3,8-diamino-6-phenylphenanthridine (DAPP) can be used as starting material to synthesize readily a wild range of HE-derivatives (Figure 14). The DAPP scaffold can be notably functionalized at three amino groups. The exocyclic amino-function groups are relatively good reactant in basic condition, while imino group have way poorer nucleophilic character. Therefore, to functionalize specifically the amino group in position 5, it is necessary to protect upstream the exocyclic amino-function groups. [34] DAPP-derivatives protected with different carbamates were already described in the literature. The tert-butoxycarbonyl protecting group (Boc) was notably used to achieve the development of a chemical microarrays bearing a phenanthdridinium moiety as fluorescent stain. [35] However, this DAPP-derivatives is reported to be unstable. [34] The protection of the exocyclic amino group of the DAPP can also be performed with the benzyl carbamate protecting groups, the protection of the amino group with such groups was already reported for the ethidium by Luedke et al. Other carbamate-based protecting group like ethoxycarbonyl (EOC) or allyloxycarbonyl (Alloc) were also reported. It is important to note Cbz and Alloc-DAPP derivatives should be preferred over the EOC-derivatives of the DAPP due to a better solubility in the nitrobenzene, which is the optimal solvent to perform the quaternisation of the aza amino group. Typically, the quaternization is typically performed through the substitution on the triflate group of the desired moiety. The substitution on idode group was also described in the literature but this approach conducts to the desired product in poor yield with a lot of side products.

Approaches to the synthesis of HE-derivatives

Aims of the thesis.

The use of the currently available HE-based probes to investigate free radical processes in functional biological systems is challenging and limited by four major problems: (i) Limited knowledge of the mechanism of formation of 2-OH-E + and of non-specific oxidation products (E + and several dimers), (ii) Non-selective biodistribution of HE, (iii) Degradation of HE by heme proteins, (iv) Interference of commercial mitochondria-targeted HE analog (MitoSOX) with mitochondrial function, (v) Requirement of HPLC analyses.

The first aim of the thesis was to improve the biodistribution of HE by introducing (i) an 3acetoxymethoxycarbonyl group for in cell esterase-assisted accumulation, (ii) linking HE to mesoporous silica for extracellular detection, (iii) a triphenylphosphonium group with different linker lengths for targeting mitochondria. The Chapter 3 is devoted to the characterization and uses of the MitoHE derivatives in cell experiments, with a focus on the effect of the linker. The Chapter 4 is focused on a detailed study on the reactivity of HE and derivatives with the superoxide radical anion. Finally, the Chapter 5 is devoted to the synthesis of new probes that allow a shift of fluorescence.

Chapter 2: Development of novel ethidium-based probes for sitespecific detection of superoxide.

Introduction

Hydroethidine is a neutral lipophilic molecule at physiological pH and is taken up by cells via passive diffusion through the cell membrane. It is generally assumed that HE is distributed evenly over the whole cells and extracellular milieu. [32] While this may be an advantage as superoxide from various intracellular/extracellular localizations can be detected using a single probe, the determination of the source of O2 •-can be achieved only by using biochemical treatments. The use of fluorescence microscopy would be inappropriate, due to the fluorescence spectral overlap of different oxidation products. Therefore, the use of HE as a superoxide probe is not enough to determine precisely the subcellular compartments of the superoxide formation. To fill this unmet need, the mitochondria targeted analog MitoSOX (Mito-HE) [36] and hydropropidine (HPr + ) [37] have notably been developed to detect the mitochondrial and extracellular superoxide, respectively (Figure 15). The MitoSOX probe consists of a HE moiety linked to a triphenylphosphonium cation (TPP) through a 6-carbon alkyl linker in place of the ethyl group. The TPP moiety is used as a cargo to accumulate the probe inside the mitochondrial matrix in response to the negative mitochondrial membrane potential. Hydropropidine consists of a hydroethidine moiety linked to a diethylmethylammonium cation through a 3-carbon alkyl chain linker in place of the ethyl group. This highly localized positive charge of the tetraalkylammonium cation prevents the internalization of the probe within the cell. One of the goals of this work was to extend the palette of site-specific HE derivatives and develop new, improved probes for site-specific measurements of extracellular, cytosolic and mitochondrial superoxide (Figure 15).

HE based probe for site-specific detection of O2 •-in the extracellular milieu

HE-based probes can be readily and reliably used to detect superoxide produced by activated neutrophils, with fluorescence measurements upon validation of the model by HPLC. Hydropropidine (HPr + ) has been recently developed by Michalski et al. as cell-impermeant HE-based probe to detect selectively the extracellular superoxide. [37] This probe was subsequently applied in high throughput screening (HTS) experiments aimed at the discovery of novel inhibitors of NADPH oxidase-2. [38,[START_REF] Zielonka | NADPH Oxidases[END_REF] In that sole purpose, HPr + was found to be a better probe than HE for serval reasons: (i) Since the activation of Nox2 generates O2 •-in the cytosol as well as in the extracellular milieu, the extracellular O2 •-production can be assayed without having to internalize exogenous molecules into cells by using the HPr + probe. Due to its impermeable membrane character, HPr + can be used in greater extent than HE or MitoSOX without affecting the mitochondrial respiration. This is of major importance since the rate of the reaction between the HE-based probe and the superoxide is concentration-dependent. (ii) For the same reason, the use of HPr + instead of HE or MitoSOX limits the nonspecific oxidation of the probe induced by the intracellular heme proteins such as cytochrome c (located within the mitochondria). (iii) Finally, the fluorescence yield of the superoxide marker product can be increased upon binding to DNA, included in the reaction medium. [37] However, one of the limitations of the HPr + probe is its non-specific oxidation by the extracellular heme proteins present, for example, in blood. We propose to overcome the limitations of the ethidiumbased probes for O2 •-by embedding the probes in mesoporous silica particles. Heme proteins are responsible for the rapid consumption of HE (and its derivatives), leading to the formation of E + and dimers. We want to explore the potential to graft HE in mesoporous particles to block the interaction of HE with the protein heme centers to protect the probe from heme protein-mediated oxidation and extend its applicability to blood and in vivo systems (Figure 16).

Figure 16

The ideal extracellular probe. Schematic representation of the proposed grafting of HE on mesoporous silica particles and its protection against heme proteins. (scheme drawn by Dr. M. Hardy).

Synthesis of HE-based probe anchored in SBA-15 silica

The goal of this chapter deals with the design, the synthesis and the characterization of the HE probe anchored inside the pores of the silica. For that purpose, the CMO team (ICR) prepared an appropriate functionalized silica allowing the grafting of the HE probes. Indeed, in previous work, they demonstrated the efficiency of the nanostructured hybrid silica (i.e SBA-15) to improve the stability of radicals. [START_REF] Vibert | [END_REF][41][42] Thus, the preparation of the ethidium precursor allowing the grafting on SBA-15 type silica was performed. We would like to thank the master students Ikram Yagoub, Anita Liang and Djouher Jamali for their contributions to this part of the project.

One of our issues was to find an appropriate protective group for the exocyclic amine groups of the ethidium moiety, which would be capable of withstanding the various steps of grafting process and enable an easy deprotection once ethidium is grafted onto the silica. Following several unsuccessful attempts, the azide-alkyne click chemistry approach has been chosen as this reaction is straightforward and compatible with HE. [32] The most classic click reaction is the copper-catalyzed reaction via 1,2,3triazole. [43,44] Moreover, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) have been successfully used for the synthesis of functionalized silica. [45] For grafting the HE probe, we decided to use [3 + 2] cycloaddition reaction (Huisgen cycloaddition) between an azide and an alkyne.

Post-grafting deprotection Synthesis of SBA-E + (ethyl carbamate protection) The SBA-E + was prepared in a four-step synthesis sequence (Figure 17). The protection of the 3,8diaminophenanthridine (DAPP) was performed by adapting the procedures from the literature. [46,47] Figure 17 Synthesis of SBA-E + via carbamate protection. Reagents and conditions. i, Ethyl chloroformate, pyridine, 0 °C to rt., overnight, 91%. ii, pent-4-yn-1-yl triflate, nitrobenzene, 50 °C, 48h, 26%. iii, SBA-N3, CuSO4, sodium ascorbate, dry THF, 70 °C, 24h. iv, HBr solution (48 %), reflux, 24h. (synthesis performed by Ikram Yagoub).

The synthetic sequence began with the protection of the two exocyclic amino functions using the ethyl chloroformate protective groups, which led to the compound 19 in 91% yield. Then, the freshly prepared, pent-4-yn-1-yl triflate reacted with the compound 19 in nitrobenzene leading to the alkyne 20 in 26% yield. The cycloaddition reaction was performed using 0.5 equiv. of CuSO4•5H2O and 5 equiv. of sodium ascorbate to afford compound 21. The last step was performed using HBr solution (48 %) under reflux for 24 h leading to the desired product. The major advantage of this method is the suitable yield of the alkylation in step two. Indeed, various reports mentioned the alkylation of the phenanthridine as a difficult step. [48,49] The major disadvantage of this approach is the degradation of the synthesized silica-E + during post-grafting deprotection using relatively strong conditions (HBr, reflux). Indeed, it was difficult to confirm that the system is fully intact after such treatment. In addition, the monitoring of the reaction in solid state synthesis was difficult. Therefore, we decided to explore the deprotection of the ethidium moiety before grafting.

Deprotection before grafting Synthesis of SBA-E + (amide protection) Thomas at al. used chloroacetyl chloride substituents as protective groups for different ligands binding to RNA hairpin loops. [50] This procedure was adapted to prepare the SBA-HE in a five-step synthetic sequence (scheme 7). Scheme 7. Synthesis of SBA-E + . Reagents and conditions. i, chloroacetyl chloride, TEA, benzene, 0 °C to rt., overnight, 80%. ii, pent-4-yn-1-yl triflate, nitrobenzene, 50 °C, 48h, 3%. iii, thiourea, EtOH, reflux, 24h, 86%. (iv) SBA-N3, CuSO4, sodium ascorbate, dry DMF, 52 °C, 24h. (synthesis performed by Ikram Yagoub).

After the protection step leading to compound 22 in 80 % yield, the alkylation reaction was completed in 3% yield. The deprotection was performed using thiourea in EtOH solution under reflux and led to the corresponding alkyne 24 in 86 % yield. Then, the click reaction was performed to afford the SBA-E + . The advantage of this pathway of synthesis is the deprotection step (in good yield) occurring before the grafting. The major issue is the very low yield of alkylation in step two (only 3 % yield). This yield is not suitable for big scale synthesis.

Synthesis of SBA-E + (Allyl carbamate protection)

The preparation of SBA-E + was performed in a four-steps synthesis (Scheme 8). Scheme 8. Synthesis of SBA-E + via Alloc protection. Reagent and condition: i, allyl chloroformate, dry pyridine, 0 °C to rt. 48h, 97%. ii, pent-4-yn-1-yl triflate, nitrobenzene, 50 °C, 72h, 87%. iii, TBAF, dry THF, reflux, 70°C, 72h, 88%. iv, SBA-N3, CuSO4, sodium ascorbate, dry DMF, 52 °C, 4 days. v, NaBH4, MeOH, rt, quant. (synthesis performed by Anita Liang).

The compound 25 was obtained by adapting the procedure developed by Huber et al.. [50] Then, the alkylated product 26 was obtained in 87% yield. The alloc groups were deprotected using tetra-nbutylammonium fluoride (TBAF) according to the literature method to give the desired product 24 with 88% yield. [51] The cycloaddition reaction allowed to graft compound 24 onto silica to obtain SBA-E + . According to the thermogravimetric analysis (TGA) (experimental part), the loading of the ethidium moiety corresponds to 0.35 mmol/g that corresponds to 25 % grafting. The SBA-HE was obtained by reduction of the ethidium moiety using sodium borohydride dissolved in MeOH at room temperature. The color change was easily observable (dark red to brown). The SBA-HE was obtained in quantitative yield. This approach was selected as the approach of choice for the synthesis of silica in which the ethidium moiety, either in the oxidized or reduced form, is anchored inside the pores of the silica.

Fluorescence properties of SBA-HE and its non-specific oxidation products.

Upon preparation of the silica-HE and silica-E + particles, their fluorescence properties have been characterized. The results showed similar fluorescence properties of HE and E + , to the ones observed for the non-covalently bound ('free') HE and E + , respectively (Figure 18 B andA). These data provide a proof of concept that hydroethidine can be anchored inside the pores of mesoporous silica keeping the same fluorescent properties. The reactivity towards superoxide radical anion of this material, the preparation of nanoparticle-based HE will be investigated in the future.

AM-HE a cytosolic superoxide probe

We aim to develop an HE-based probe functionalized with an ester group that can, upon its internalization in cell, undergo hydrolysis reaction catalyzed by intracellular esterases. The esterification should lead to the formation of a HE-derivatives bearing a carboxyl group, which is expected to be sequestrated inside the cytosol. Indeed, the carboxylate moiety of the probe, due to its negative charge, should prevent its efflux from cells and its internalization within mitochondria. Preliminary results have shown that the regular esters (ethyl ethanoate, methyl ethanoate) didn't hydrolyze in the presence of esterases. Therefore, we decided to develop HE-derivatives bearing an activated ester. We have successfully synthesized and characterized a novel derivative of HE, AM-HE, devoted to the measurement of cytosolic O2 •-. This probe contains an acetoxymethyl (AM) ester group, which is known to be stable in the absence of esterases, but undergoes a rapid cleavage upon accumulation in cells, due to the abundance of cellular esterases (Figure 19). [52] Figure 19 Subcellular distribution expected for AM-HE, its hydrolysis product and their superoxide markers

Synthesis and investigations of AM-HE derivatives

AM-HE was synthesized by a convergent route, which involves the coupling of the activated ester 30 with the Cbz-protected DAPP (Scheme 20). Therefore, the two exocyclic amino functions of the DAPP were protected by reacting DAPP with Cbz-Cl in basic conditions and led to the compound 28 in 90% yield on the one hand. On the other hand, the activated ester was synthesized by reacting glycolic acid with bromomethyl acetate to give the compound 29 in 72% yield. The hydroxyl group of the compound 29 was activated with triflate to react with the compound 28 leading to the corresponding alkylated phenanthridinium 31 in 58% yield. Finally, the deprotection of the carbamate groups were performed in the presence of H2/Pd(OH)2 leading to Am-HE in a 45% yield. Scheme 20. Synthesis of Am-HE. Reagents and conditions. a, lutidine, CH2Cl2; b, pyridine, rt, 5h; i, benzyl chloroformate, K2CO3, MeCN, 12h, rt, 90%; ii, PhNO2, 12h, 40°C, 58%; iii, H2/Pd(OH)2, EtOH, DMF, 45%. (Am-HE was prepared for the first time by Dr. M. Hardy). HE-COOH, the result of the AM-HE hydrolysis, which is expected to be membrane-impermeant in its basic form, was synthesized in 3 steps from the protected DAPP 29. The synthesis began by the quaternization of the nitrogen contained in the phenanthridine core of 23 by reacting 23 with ethyl bromoethanoate leading to the formation of the compound 32 in a 45% yield. Both Cbz and ester groups were hydrolyzed in strong acidic conditions (HBr 48%) to give the phenanthridinium 33 in a 63% yield. The final step was the reduction of the phenanthridinium core using NaBH4 to afford HE-COOH in quantitative yield.

Evaluation of the performance of the AM-HE probe in cell free system

The ability of the AM-HE probe to detect O2 •-has been evaluated in a cell-free system. These experiments were performed by Dr. J. Zielonka (MCW) and monitored by LC-MS systems. The incubation of AM-HE in the presence of esterases showed that AM-HE is efficiently converted in HE-COOH. Indeed, after 20 min, more than 95% of the AM-HE was converted into HE-COOH (Figure 22 A,B). Moreover, in the presence of the HX/XO system to generate the superoxide radical anion, AM-HE led to the corresponding marker of O2 •-(2-OH-E + -AM) and its corresponding hydrolyzed product (2-OH-E + -COOH) (Figure 22 C,D). In addition, LC-MS/MS analyses showed that AM-HE can be firstly hydrolyzed into HE-COOH and then reacts with O2 •-to yield 2-OH-E + -COOH (Figure 4). We evidenced that HE-AM probe can be hydrolyzed by esterases and allowed to detect superoxide either in its ester or hydrolyzed form. The subcellular redistribution of the hydrolyzed formed of the AM-HE probe and its reactivity toward O2 •-in biological systems are still under investigation.

Fine tuning of Mito-HE alkyl chain linker for mitochondrial superoxide detection 2.4.1 Introduction

Since O2 •-in most types of cells is believed to be formed primarily inside the mitochondria as a side product of the ETC, we decided to explore the potential to improve the existing mitochondrial-targeted superoxide probe MitoSOX. [36] The MitoSOX probe consists of a hydroethidine moiety which is linked to a triphenylphosphonium cation (TPP) through a 6-carbon alkyl linker. The TPP moiety is used to accumulate the probe inside the mitochondrial matrix in response to the negative mitochondrial membrane potential. [53] Our focus went on the modulation of the alkyl chain linker, which affects the physicochemical properties (lipophilicity and size) of the probe, to improve the probe uptake and modulate the site of mitochondrial sequestration. There is a need to improve the MitoSOX probe since it has been reported that MitoSOX inhibits mitochondrial complex IV. [29] Modulating the length of the alkyl chain linker between the molecule of interest and its cargo is a well-established strategy to modulate the extent of site-specific accumulation of the targeted agent, without affecting the desired activity of the molecule of interest. [54,55] Indeed, Murphy and coworkers developed this strategy and reported the fine-tuning of the mitochondria-targeted antioxidant MitoQ by modulation of its size and lipophilicity. [55] We adapted this approach to fine-tune the chemical structure of Mito-HE probe with the goal to improve the superoxide detection efficiency without affecting mitochondrial respiration. In this work, MitoSOX probe will be referred to as Mito6-HE. In addition to Mito6-HE, three homologs (Miton-HE) having a different carbon number (n) in their alkyl linker (n = 2, 4, 10) were synthesized and evaluated.

Synthesis of Miton-HE variants

The Miton-HE variants were prepared in a four-step synthetic sequence (Scheme 23). The synthetic sequence started with the protection of the two exocyclic amino functions of DAPP by the benzyl carbamate protective groups leading to the compound 28 in 90% yield. The corresponding freshly prepared ω-bromoalkyl trifluoromethanesulfonate 35 reacted with the compound 28 in nitrobenzene leading to the corresponding phenanthridinium salt 36. The nucleophilic substitution of 36 by the triphenylphosphine in acetonitrile led to the phenanthridinium dications 37 which were ions exchanged from bromide to TFA -on reverse column chromatography. Finally, the deprotection of the carbamate groups were performed in the presence of H2/Pd(OH)2 leading to the Miton-HE derivatives. The reaction iii didn't proceed as expected in the case of 3a (3 for n = 2), numerous unknown side products were obtained including the elimination product (DAPP). Indeed, the proximity of the two positive charges in addition to the steric hindrance of the TPP and the phenanthridinium moieties may have disfavored the formation of 4a (4 for n = 2). Thus, another synthesis route was adopted to prepare Mito2-HE (Scheme 2). The reduction of the bromoethidium 3a in the presence of sodium borohydride followed by the nucleophilic substitution of the bromide by the triphenylphosphine in acetonitrile led to the mixture of 4a and 6 in 36% yield. Finally, the deprotection of the carbamate groups were performed as described above to give Mito2-HE in 81% yield.

The superoxide markers 2-OH-Miton-E + were prepared by reacting Miton-HE with Fremy's salt. The procedure was simplified as compared to the one described by Zielonka et al. [56] Moreover, the dimers and the ethidium products were prepared by oxidizing Miton-HE with ferricyanide and chloranil, respectively adapting the procedure described by Zielonka et al. (Scheme 24). [27,56] Scheme 24. Synthesis of oxidation products of Miton-HE. Reagents and conditions. i, potassium nitrosodisulfonate, DMSO, water, 0°C, 10 min, 90°C; ii, K3Fe(CN)6, DMSO, water, 0°C, 10 min. iii, Chloranil, DMSO, MeOH, 0°C, 10 min.

Spectroscopic properties of Miton-HE and their oxidation products

Absorption properties

Due to the instability of Mito2-HE oxidation products in protic solvents, their absorption spectra were recorded in acetonitrile (figure 1 andTable 2).

I
Clearly, the absorption properties of Miton-HE hydroxylation and oxidation products are not affected by the length of the aliphatic chains. The position of the absorption maxima and the extinction coefficient values for the Mito2-HE oxidation products determined in acetonitrile appear to be in the same range compared to the other derivatives. In the case of n = 4, 6 and 10, 2-OH-Miton-HE, Miton-E + and Miton-E + -Miton-E + exhibit a strong absorption band in the UV range with an additional absorption band in the visible region between 400 and 600 nm in ethanol as well as in aqueous solution at pH 7.4.

The absorption band in the UV range for 2-OH-Miton-E + appears less intense and wider compared to the non-hydroxylated oxidation products of Miton-HE. In aqueous solution at pH 7.4, the UV band exhibit 3 maxima at (275, 302 and 327 nm for Mito4-E + in aqueous solution at pH=7.4, the values are similar for n = 6 and n = 10). The hydroxylated markers exhibit also an additional small band at 393 nm (for Mito4-E + in aqueous solution at pH=7.4, the values are in the same range for n = 6 and n = 10).

Furthermore, concerning the influence of solvents on the spectral properties of these products. In aqueous solution, the maxima wavelengths absorptions are blue shifted compared to those collected in EtOH. In ethanol, the extinction coefficient values for Miton-E + and Miton-E + -Miton-E + appear to be about 1.2 fold greater than those calculated in aqueous solutions. The extinction coefficients of 2-OH-Miton-E + are approximately equal in these two solvents.

It is important to note that the absorption properties were taken essentially to characterize our oxidation products.

Moreover, even if the absorbance is taken on the more selective absorption band in the visible, the intracellular concentration of the ethidium moiety is usually much higher (up to 10 fold) than the corresponding hydroxylated products. Therefore, the fluorescence intensity is mostly due to the ethidium and cannot be attributed to the superoxide.

Fluorescence properties of Miton-HE

The 5. (experimental section). As already reported [16,36] , the excitation and the emission maxima for Mito6-E + are 520 nm and 588 nm respectively. These maxima for 2-OH-Mito6-E + are blue-shifted approximately 20 nm relative to which of Mito6-E + . 2-OH-Mito6-E + has also a more selective excitation wavelength in the range of 385-405 nm. However, the emission spectra of 2-OH-Mito6-E + exhibit a significant overlap with those of Miton-E + . These overlaps make the differentiation between the hydroxylation products and the two-electron oxidation products challenging. Furthermore, 2-OH-Miton-E + and Miton-E + (n = 4, 10) exhibit fluorescence properties similar to those for n = 6.

These results suggest that the length of the alkyl linker do not affect the fluorescence properties of the Miton-HE two-electron oxidation and hydroxylation products. Therefore, similar conclusions can be drawn for Mito6-HE and its homologs (Miton-HE). Since the Miton-E + levels are likely to be at least 10fold greater than the 2-OH-Miton-E + level in cells. [56] O2 •-cannot be monitored directly by fluorescence or confocal microscopy. This also applies to the shorter excitation wavelength (388 nm) proposed previously, because the nonspecific oxidation products still fluoresce, when present at higher concentrations. [16] Thus, to be measured without artifactual signals, the superoxide marker needs to be extracted from cells and separated from Miton-E + by HPLC.

Study of the stability of the Mito2-HE oxidation products in water

The instability of the Mito2-HE oxidation products was discovered during HPLC analyses of their aqueous solutions, formed upon dilution of the stock solutions prepared in DMSO. We observed that Mito2-HE oxidation products (2-OH-Mito2-E + , Mito2-E + and Mito2-E + -Mito2-E + ) decompose spontaneously to give respectively 2-OH-DAPP, DAPP, DAPP-DAPP and vinyl-triphenylphosphonium (vinyl-TPP) in H2O, MeOH or in EtOH. HPLC analyses showed that within less than 1 h in these protic solvents, 10 µM of Mito2-HE oxidation products yield quantitatively their elimination products (Figure 25). The mechanism of elimination is proposed in (Figure 26) Nevertheless, Mito2-HE oxidation products can be relatively stabilized in acidic condition in 0.1% formic acid in water or in non-protic solvents like DMSO.

Evaluation of the performance of the Miton-HE derivatives in cell free system

The efficiency of the Miton-HE derivatives probes to react with O2 •-has been evaluated by UV-HPLC (290 nm) in the cell-free system using hypoxanthine/xanthine oxidase system to generate a flux of O2 •- at 0.5 µM/min (Figure 27). The flux of O2 •-was determined as described in the experimental part. The presence of the superoxide markers of the Miton-HE derivatives was confirmed by incubating Miton-HE with Fremy's salt. The presence of the non-specific oxidation products were confirmed by incubating Miton-HE with chloranil and potassium ferricyanide. Our results showed that in the presence of the HX/XO system to generate O2 •-, the formation of the superoxide markers 2-OH-Mito4-E + , 2-OH-Mito6-E + and 2-OH-Mito10-E + is proportional to the time of incubation. However, in the case of the Mito2-HE probe, superoxide-dependent oxidation is followed by the conversion of the 2-OH-Mito2-E + into 2-OH-DAPP. Therefore, the Miton-HE derivatives were all able to detect the O2 •-in cell free system. Indeed, the modulation of side chain does not affect the ability of the HE-derivatives to react with O2 •-. When Mito10-HE was used at 10 M concentration, after 1 hour, 3 µM of 2-OH-Mito10-E + were formed. When Mito4-HE was used, for that same period, 4.8 µM of 2-OH-Mito4-E + were generated. For Mito6-HE the amount of 2-OH-Mito6-E + was 2.4 µM. Consequently, the reactivity with superoxide radical anion using the Miton-HE probes appears to be similar. Indeed, as expected, there is no clear correlation between the alkyl chain linker and the superoxide reactivity. All these probes are able to detect the superoxide radical. Thus, their ability to detect superoxide radical anion was next investigated in cellular models. Chapter 3: Assessment of the applicability of Miton-HE probes for site-specific detection of O2 •-in cellular models.

The cellular models for stimulation of mitochondrial superoxide generation.

The pancreatic cancer cells were used for testing the performance of Miton-HE probes in detecting mitochondrial O2 •-. It has been previously shown that these cells upon treatment with mitochondrial toxins increase the production of mitochondrial O2 •-, which can be followed using commercially available Mito6-HE probe, in combination with HPLC-based analyses of the superoxide-specific product. [16] For evaluation of the Miton-HE probes, we used approaches to stimulate mitochondrial superoxide production. In the first approach, we used a mitochondria-targeted nitropyridinium cation, Mito-NP, as a mitochondrial redox cycling agent. Mito-NP has been developed by Dr. Zielonka in the Medical College of Wisconsin as a potential anticancer agent. Mito-NP was designed to stimulate mitochondrial O2 •-formation and this has been demonstrated in pancreatic and colon cancer cells. The redox cycling by Mito-NP involves one-electron reduction of the nitropyridinium cation to the corresponding pyridinyl radical, with subsequent transfer of the electron to molecular oxygen to form O2 •-(Figure 28). The second approach involved treatment of cells with atovaquone (ATO), an anti-malaria drug, containing a hydroxynaphthoquionone moiety. ATO is known to inhibit mitochondrial complex III activity. [57] and based on its chemical structure, we reasoned that it would also exhibit redox cycling activity, with the formation of semiquinone-type radical as the intermediate involved in electron shuttling to molecular oxygen (Figure 29). Therefore, we used two chemically distinct compounds, both expected to significantly stimulate mitochondrial superoxide production.

Mitochondrion

Figure 29 Redox cycling mediated by atovaquone, which leads to the production of O2 •-within mitochondria.

The HPLC method is more reliable than the fluorescence life imaging method

The cells were cultured, treated, collected and processed as described in the experimental part, according to the method extensively described by Zielonka et al.. [START_REF] Zielonka | NADPH Oxidases[END_REF] For all our experiments, the HLPC method was used to assess the superoxide production in cellular models with the newly synthesized probes and commercially available HE and Mito6-HE. Indeed, as already mentioned in the introduction, this method is the only one that enables the distinction between the superoxide marker and the ethidium-type products. It has been proposed that the superoxide-specific product is not only generated by intracellular superoxide, but also produced in small amount due to the autooxidation. [30,[START_REF] Zielonka | [END_REF] However the majority of the literature data and our own results clearly indicate that stimulation of cellular superoxide production increases the detected level of the hydroxylated product. Therefore, the 2-OH-E + level, measured by HPLC or LC-MS, remains a valid and best indication of the cellular/mitochondrial superoxide. [16,28]

Calculation method used to assay the indication of the superoxide in cells by using the HE-derivative probes.

The detected concentration of 2-OH-E + (or its mitochondria-targeted analogs) in cell lysates divided to the total lysate protein concentration was used as a measure of superoxide level/production. The nonspecific oxidation of the probe was followed by monitoring the levels of E + -type products and one-electron oxidation of the probes was followed by quantitation of the E + -E + -type dimers. The dimeric products analogous to HE-HE and HE-E + dimers were also detected. However due to the lack of authentic standards, as well as their lower stability, a quantitative analysis of those products was not performed.

With the assumption that the probe intercepts only a small fraction (<10%) of the total flux of the cellular superoxide produced, the data can be also 'normalized' to the intracellular level of the probe, by calculation of the 2-OH-E + /HE ratio. In the same way, the levels of E + and E + -E + -type products were 'normalized' to the probe level and expressed as the E + /HE and E + -E + /HE ratios, respectively. This calculation method is important when the cellular level of the probe is significantly affected by the treatment of interest, as the concentration of the probe is one of the factors controlling the amount of the products formed.

Assessment of the mitochondrial O2

•-production in MiaPaCa-2

Comparison of the ability to detect the mitochondrial O2 •- production of Miton-HE variants or HE in MitoNP treated MiaPaCa-2

The MiaPaCa-2 pancreatic cancer cells were used as the cellular model to assess the ability of the Miton-HE probes to detect mitochondrial O2 •-. [60] The stimulation of MiaPaCa-2 cells with the novel redox cycling compound developed in the Medical College of Wisconsin, Mito-NP, leads to induction of mitochondrial superoxide generation via a redox cycling mechanism. [61] The concentration used and time of treatment was optimized in the Medical College of Wisconsin, in order to selectively and potently induce oxidation of mitochondrial peroxiredoxin-3, but not cytosolic peroxiredoxin-1. The ability of Miton-HE probes to trap the mitochondrial O2 •-was assayed in Mito-NP-treated MiaPaCa-2 cells under the conditions of induction of mitochondrial, but not cytosolic superoxide generation. All the probes tested were compared using the same batch and passage of cells, to minimize the number of variables. The HPLC profiles of the probes' oxidation products are shown in Figure 30 (for HE) and Figure 33 (for Miton-HE probes).

HE confirms that Mito-NP stimulates superoxide production in MiaPaCa-2 cells

Before testing the performance of the Mito-HE probes, HE probe was used to ensure the experimental conditions selected, based on H2O2-dependent oxidation of mitochondrial peroxiredoxin-3, result in significant stimulation of superoxide production. The data shown in Figure 30 confirm, that treatment of MiaPaCa-2 cells with 10 µM Mito-NP for 24 h results in increased production of O2 •-, reflected in statistically significant increase in the detected level of 2-OH-E + . In addition to increased 2-OH-E + , the other oxidation products, including E + and E + -E + were elevated by the treatment with Mito-NP. This suggests that in addition to O2 •-, other stronger oxidant(s) are induced by the treatment.

Effect of the length of the alkyl linker on the uptake of the Miton-HE probes.

As indicated earlier, probe concentration/availability is one of the factors controlling the amount of the products formed in cells. Therefore, for comparison of the different Miton-HE probes, intracellular probe level was first evaluated. The data presented in Figure 33 show that the Miton-HE probes levels in MiaPaCa-2 cells can be correlated with the length of the alkyl linker. Clearly, the longer the carbon chain linker the higher is the cellular probe level. Similar observations on the effect of the length of the alkyl linker and compound lipophilicity on the extent of cellular and mitochondrial uptake were reported previously for mitoquinone and mito-metformin. [54] Although the data obtained represent the total cellular level of the probes and their products formed, due to the presence of the TPP moiety, the Miton-HE probes are expected to be concentrated in mitochondria, driven by the negative plasma and mitochondrial membranes potentials. [53] Due to a lower value of the plasma membrane potential, as compared to the mitochondrial membrane potential, the transport of the probes through the plasma membrane is expected to be the ratelimiting step for their internalization into the mitochondrial matrix.

Of note, the level of the probe was also affected by the treatment with Mito-NP. This can be due to increased probe consumption via oxidation by the oxidants induced by Mito-NP and/or by decreased rate of probe uptake, due to ROS-induced depolarization of the mitochondrial membrane. Decreased probes' availability due to the treatment results in their lowering ability to compete for O2 •-with other cellular targets (e.g. MnSOD). Therefore, the levels of the oxidation products, in addition to the absolute values (normalized to total cellular protein level) were also graphed normalized to the probes' levels (Figure 33).

Result on the superoxide detection for the Miton-HE variants in MiaPaCa-2 cells

While all the synthesized probes were detectable in cell extracts, when analyzed by HPLC, only in case of longer lengths of the linker (n = 6 and 10), the oxidation products were present in sufficient amount for quantitative analyses. In case of the shortest linker (n = 2), the intracellular level of the 2-OH-Mito2-E + and its elimination product (2-OH-DAPP) were below the limit of detection, while for n = 4, the intracellular level of the 2-OH-Mito4-E + was detectable but below the limit of quantification. For n = 6 and 10, the Figure 31 and Figure 32 show that the 2-OH-Mito6-E + and 2-OH-Mito10-E + signals respectively were quantifiable by HPLC-FLD. However, the intracellular levels of these products, when normalized to the total amount of cellular protein were not increased in Mito-NP-treated cells, when compared to control cells. As Mito-NP decreased the probe availability, the data were also normalized to the cellular level of the probe.

As shown in Figure 33, the 2-OH-Mito6-E + /Mito6-HE and 2-OH-Mito10-E + /Mito10-HE values were markedly increased in extracts from the Mito-NP-treated cells relative to the untreated control cells. Therefore, Mito6-HE and Mito10-HE detects the mitochondrial superoxide in MiaPaCa-2 cells. Furthermore, it is important to note that the increase of both levels are in the same range (29% and 34% for n = 6 and n = 10 respectively), which suggests that the efficiency of Mito6-HE and Mito10-HE to detect mitochondrial O2 •-in MiaPaCa-2 cells is similar and validates the proposed normalization approach.

Discussion on the result of the non-specific oxidation of the Miton-HE probes

markedly increased in Mito-NP-treated cell, when compared to the untreated control cells. The level of Miton-E + products was high enough to be detectable for the alkyl linker length of 4, 6 and 10. Only in case of the shortest alkyl linker used (n = 2) the corresponding oxidation product was not observed.

On the other hand, in case of the dimeric products, the Miton-E + -Miton-E + dimer was detectable and quantifiable only for the longest linker tested (n = 10). In this case, the Mito10-E + -Mito10-E + level and the Mito10-E + -Mito10E + /Mito10-HE-Mito10-HE ratio were increased in treated cells relative to the control indicating that Mito10-HE is oxidized by one electron oxidant(s) stimulated in mitochondria of MiaPaCa-2 cells by Mito-NP. A andB) and with a diode-array detector at 290 nm (C,D). Initially, the fluorescence detector parameters were excitation at 358 nm and emission at 440 nm. At 5.4 min after the injection, they were changed to be excitation at 490 nm and emission at 567 nm. Note (figure 13 A), the top hplc traces is the injection of a standard of Mito6-HE (10 µM) (it is not an injection of a cell lysate).

Stimulation of the superoxide production in MiaPaCa-2 cells with atovaquone

To further validate the utility of the probes to detect mitochondrial superoxide, their performance was tested in MiaPaCa-2 cells treated with mitochondrial complex III inhibitor and redox cycler, atovaquone (ATO). Prior the cell experiments, the possibility of direct oxidation of the HE probe by ATO was tested in aqueous solution at pH = 7.4. (Figure 34). The results indicate that the direct reaction does not occur under the experimental conditions used for cell-based assays. To optimize the conditions for efficient mitochondrial superoxide stimulation by ATO, its concentration-dependence (0 to 40 µM with 1 hour pretreatment) was investigated in MiaPaCa-2 cells. Higher concentrations of ATO were not tested due to the solubility limitation. O2 •-production was monitored with the HE probe (10 µM during 1 h). Figure 36 shows that the intracellular level of 2-OH-E + was markedly increased relative to the untreated control cells when cells were treated with ATO concentrations of 20 µM and 40 µM. These results confirm that ATO induces O2 •-formation in MiaPaCa-2 cells. Furthermore, the same trend was followed for the E + and the E + -E + levels, suggesting the formation of one-electron oxidizing species upon treatment.

Since 40 µM ATO induced the highest amount of O2 •-while not inducing the cell death, this concentration was selected for further experiments using the Miton-HE probes.

Effect of ATO on probes' availability in MiaPaCa-2

Analyses of the intracellular levels of Miton-HE probes, confirms previous observation that the longer the alkyl chain length, the higher intracellular level of the probe. Also, similar to the Mito-NP treatment, addition of ATO resulted in decreased detected Miton-HE probe levels.

Mito6-HE and Mito10-HE report mitochondrial superoxide induced by atovaquone in MiaPaCa-2 cells

HPLC/FLD analyses of the cell lysates reveal that the 2-OH-Mito6-E + level is markedly increased in atovaquone treated cells relative to the untreated control cells. The same conclusion applies also for the ratio 2-OH-Mito10-E + /Mito10-HE ratio level that is markedly increased relative to the untreated control cells (Figure 37). Similar to the results observed for Mito-NP used as the superoxide stimulant, 2-OH-Mito2-E + was not detected and the 2-OH-Mito4-E + was present at the concentration below the limit of quantification. Clearly, the conclusion that the probes with the long alkyl linkers (n = 6 or 10) perform best in detection of mitochondrial superoxide hold true, regardless of the stimulant used.

The similar conclusions as in case of Mito-NP, can be also drawn for the other oxidation products detected: Miton-E + can be quantified for n = 4, 6, 10 and the dimeric product (Miton-E + -Miton-E + ) is only detectable for n = 10.

Conclusion

Assessment of the applicability of Miton-HE probes for site-specific detection of O2 •-in MiaPaCa-2 has been performed. The mitochondrial superoxide production was stimulated with two mechanistically and structurally different agents (mitochondria-targeted nitro-pyridinium cation and atovaquone). Similar conclusions were drawn from both experimental models. The longer is the alkyl linker, the greater is the intracellular probe level. The increased mitochondrial superoxide was detectable in case of the probes with the long alkyl linkers (n = 6 or 10), consistent with their highest cellular accumulation, stability of the oxidation products, and most favorable fluorescence detection parameters under the HPLC conditions used.

Chapitre 4 Design, synthesis and application of chemical analogs of HE for mechanistic studies of the reaction between HE and O2 •-

4.1 Proposed mechanism between HE and the O2 •-.

One of the objectives of this project was to develop and synthesize new molecules for the purpose of investigation of the oxidation mechanism of HE in the presence of O2 •-. As already mentioned in the introduction, the first step of this reaction is the formation of radical cation HE •+ . The formation of this radical cation during one-electron oxidation of HE is well established and it has been already spectroscopically characterized. [24] Furthermore, DFT calculations of HE However, the detailed mechanism of the reaction between HE •+ and O2 •-leading to the 2-OH-E + remains to be established. We propose that the reaction of HE •+ with O2 •-leads to the intermediate 40, followed by aromatisation of the phenyl group. The subsequent step may involve the fragmentation of the O-O bond mediated by the anchimeric assistance of the nitrogen atom (N5), which leads to an intermediate that can be the immino-quinone derivative of HE reported through a tautomeric equilibrium. [62] Finally, we believe that this intermediate can be subsequently deprotonated at the C-6 position to yield 2-OH-E + (Figure 39).

Figure 39 Proposed mechanism for the reaction between HE and O2 •-.

In order to study the mechanism described in Figure 1, we decided to investigate the effects of the replacement of the hydrogen atoms localized at the position C-6 and on the exocyclic amino groups by methyl groups, on the formation of 2-OH-E + and on the kinetics of reaction with superoxide. Thus, we designed and prepared three HE derivatives as model system, MeHE, TMeHE and TMeHPr + (Figure 40). TMeHPr + was designed for the mechanistic studies as a water-soluble analog of TMeHE, due to low aqueous solubility of TMeHE.

Synthesis of MeE

MeE was obtained in 40% yield by reaction of E + with methyl magnesium bromide in CH2Cl2 for 3 days at 40°C (Figure 41). MeE bears a methyl group in position 6 at the place of the labile hydrogen atom in HE. In contrast to HE, the formation of an ethidium cation is not possible following the one-electron oxidation. Unfortunately, MeE was not sufficiently stable in aqueous solutions to evaluate its reactivity towards superoxide radical anion. However, in organic solvent the probe was stable, enabling generation and characterization of its radical cation.

Synthesis and development of TMeHE derivatives

The synthesis of Tetramethyl-ethidium (TMeE + ) was reported by Le Bret and Chalvet in 1977. [63] Inspired by this work, we aimed to develop a reduced analog of TMeE + named TMeHE. Therefore, TMeHE was synthesized in two-steps using the commercially available ethidium cation (E + ) (Figure 42). The tetramethylation of the exocyclic amines of ethidium bromide was performed at 180°C in the presence of trimethylphosphate. This step was adapted from the previously described procedure. In this reaction the N,N,N',N'-tetramethylated derivative was obtained in 65% yield as purple crystals (Figure 43), which was subsequently reduced by sodium borohydride in ethanol to afford TMeHE in quantitative yield. The superoxide marker 2-OH-TMeE + and the corresponding ethidium product (TMeE + ) were prepared by reacting TMeHE with Fremy's salt and ferricyanide respectively (experimental part). It is interesting to note that for the first time, the superoxide marker has been crystallized (Figure 43) and the crystal structure determined. This provides a convincing evidence of the position of the hydroxyl group (the site of hydroxylation of the radical cation by superoxide radical anion).

2.1 Superoxide detection of TMeHE in a cellular model RAW 264.7

Our partners from Lodz University of Technology confirmed that TMeHE reacts with superoxide in cellfree system using the hypoxanthine/xanthine oxidase system. Therefore, we evaluated the ability of TMeHE probe to detect O2 •-in activated RAW 264.7 macrophages, a widely used murine cell line. The superoxide production was stimulated using the phorbol myristate acetate (PMA). The cells were incubated with the probes (TMeHE or HE) at the final concentration of 10 µM for 1 h. Cells were

TMe-E + 2-OH-TMe-E +

collected and lysed as described in the experimental part according to the literature method already mentioned. [START_REF] Zielonka | NADPH Oxidases[END_REF] The HPLC traces recorded for extracts from control (non-stimulated) and PMAstimulated cells for both probes are shown in (Figure 44). In case of both probes, clear increase in the yield of the hydroxylated oxidation product can be observed. The Figure 45 shows the results of quantitative analyses of the cell lysates, including the levels of the probes (HE, TMeHE), the superoxide markers (2-OH-E + , 2-OH-TMeE + ) and the non-specific oxidation products of the probes (E + and TMeE + ).

When HE was used as the probe, the dimers were present essentially under the intermediate form (HE-E + ), which were detected but couldn't have been quantified due to the lack of isolated standards.

Those data show that the intracellular level of the TMeHE probe was higher than the HE probe. This is probably due to the higher lipophilicity of the TMeHE probe compared to HE.

Therefore, this result suggests that the extent of the probe that is adsorbed in the plasma membrane is expected to be higher in the case of the TMeHE probe compared to HE probe. The 2-OH-TMeE + level and the 2-OH-TMeE + /TMeHE ratio level were markedly increased in PMA stimulated cells relative to the untreated control cells. Those results show that the TMeHE probe is able to detect the superoxide radical anion in RAW 264.7 cells. Furthermore, the 2-OH-TMeE + and the 2-OH-E + levels in PMA treated cells were in the same range. However, the background level of 2-OH-TMeE + was higher than the one of 2-OH-E + . The origin of this observation is under investigation.

Since these results show that the TMeHE can react with O2 •-to yield the 2-OH-TMeE + , we can draw the conclusion that the hydrogen atoms located in the exocylic amino groups of HE are not indispensable for the HE-based probe to yield the 2-hydroxylated derivatives.

Development of TMeHPr + .

TMeHPr + was designed and synthesized to perform the calculation of the rate constant of the reaction between the tetramethylated-hydroethidine derivatives and the O2 •-in aqueous solution. TMeHPr + is the tetra-methylated analog of hydropropidine (HPr + ), a water-soluble analog of HE (chapter 2). This strategy had to be considered since the TMeHE was not soluble in aqueous solutions.

TMeHPr + has been synthesized following two approaches (Figure 46). The first approach leads to TMeHPr + in 4 steps with 13% overall yield starting from the DAPP. The second approach yielded this final compound in 6 steps with 8% overall yield. The first synthetic route began with the reductive amination of the two exocyclic amino groups of DAPP affording the compound 43 in 92% yield. 3iodopropyl trifluoromethanesulfonate (freshly prepared) reacted with 43 in nitrobenzene to afford the phenanthridinium salt 44 in 16% yield. The substitution of the iodide group of 44 by N,Ndiethylmethylamine in methanol led to TMePr ++ in 85% yield. Finally, the reduction of TMePr ++ by sodium borohydride in MeOH led to TMeHPr + in a quantitative yield. The second synthetic sequence started with the Cbz-protection of the two exocyclic amino functions to afford compound 28 in 90% yield. 3-Iodopropyl trifluoromethanesulfonate was reacted with compound 28 in nitrobenzene leading The synthesis described above (Pathway 1) is a straightforward route to prepare functionalized tetramethyl-ethidium derivatives. It is worth to mention that the TMeHPr + can be obtained in two steps from the commercially available, but relatively expensive, propidium (Pr ++ ).

Pulse radiolysis analyses of TMeHPr +

To test the influence of the methylation of the amino groups on the rates of decay and the rates of reaction with superoxide of the HE radical cation, pulse radiolysis experiments of HE, HPr + and TMeHPr + were performed by Dr. R. Michalski (Lodz University of Technology). Pulse radiolysis of TMeHPr + in saturated solution of N2O containing NaN3 allows one electron oxidation of the probe, which leads to the formation of the radical cation TMeHPr •++ . This radical cation has been spectroscopically characterized (Figure 47 A). TMeHPr •++ possess a strong absorption band that is red-shifted relative to HE •+ and HPr •++ , with maxima located at 480 nm. Therefore, in the pulse radiolysis experiment, the decay of TMeHPr •++ was followed by monitoring the changes in absorbance at 480 nm. The first pulse radiolysis experiment has been performed to measure the rate constant of the decay of TMeHPr •++ in the absence of O2 •-, which corresponds thereby to the disproportionation of TMeHPr •++ . The secondorder rate constant for TMeHPr •++ was calculated to be (3.3 ± 0.2) × 10 5 M -1 s -1 .

The kinetics of the decay of TMeHPr •++ was also compared with those of the already existent probes HPr + and HE. Therefore, the decay rate of HPr •2+ and HE •+ were calculated by our partners to be (3.3 ± 0.1) × 10 8 M -1 s -1 and (2.0 ± 0.2) × 10 8 M -1 s -1 , respectively. These results suggest that the presence of the bulky tetra methyl groups decreased the rate of decay of the radical cation by three order of magnitude. This can be explained since the steric hindrance induced by the methyl groups prevent the formation of dimers and should also limit the disproportionation of the radical cation since this reaction requires close proximity of both phenanthridine moieties.

TMeHPr + was also used to evaluate the reactivity of HE •+ with O2 •-. Considering that the rate of TMeHPr •++ decay is 10 3 -fold smaller compared to those of HPr •++ and HE •+ , the steady state concentration of TMeHPr •++ are likely to be much more markedly affected depending of the presence of superoxide compared to HPr •++ and HE •+ . The second-order rate constant TMeHPr •2+ with O2 •-was calculated to be 5.3 ± 0.1 × 10 8 M -1 s -1 . The example of fitting of the single-exponential decay functions to the kinetic trace obtained at 480 nm for TMeHPr •++ is shown in (Figure 47 B). Analogous rate constant has also been determined under the same conditions for the HPr + probe. Indeed, even if the steady state concentration of HPr •2+ is likely to be smaller than TMeHPr •++ . The determined value of the second-order rate constant of the reaction of HPr •2+ with superoxide should be more relevant to HE probe, since the four methyl groups in TMeHPr •++ affect the electron density of the phenanthridine core. Therefore, the second-order rate constant for reaction of HPr •2+ with superoxide was determined under the same experimental conditions and is equal to (7.4 ± 0.1) × 10 8 M -1 s -1 . Therefore, those results show that the second order rate constant of the HE-based radical cation and O2 •-is not significantly affected by the methylation of exocyclic aromatic amine groups. To confirm the improvement of the stability of the radical cation of TMeHPr + compared to the nontetramethylated probe, the oxidation of TMeHPr •++ was next investigated in the presence of ferricyanide. HPLC traces of the reaction mixtures between TMeHPr + and a successive addition of ferricyanide have shown that there was no formation of dimeric products (Figure 48 A). These results validate that the bulky tetramethyl group shield the radical cation at the C-2 position from its selfscavenging. Therefore, in the presence of this strong one-electron oxidant, the sole product that is formed is the non-specific oxidation product TMePr ++ . Furthermore, the incubation of TMeHPr + (50 µM) in presence of an increasing concentration of Fe(CN)6 3-(0 -200 µM) in aqueous solution monitored by absorbance have also shown that stoichiometry of the reaction that involves TMeHPr + and Fe(CN)6 3- is 1:2 (Figure 48 B). This stoichiometry further confirms the absence of the formation of dimer.

Spectrophotometric properties of TMeHE oxidation products

The absorption spectra of the solutions of 2-OH-TMeE + and TMeE + in ethanol and in aqueous solutions at pH 7.4 are shown in Figure 49 A and B, respectively. The absorption maxima and the extinction coefficient values for these products in aqueous solution at pH 7.4 Table 1 and in ethanol are shown in the experimental part The spectroscopic properties of TMeHPr + oxidation products have also been studied by our research partners (data not shown), and were similar to those obtained for the TMeHE oxidation product and are displayed in the Table 3 (experimental part). The shape of the absorption spectra for the 2-OH-TMeE + and TMeE + are similar to those already discussed for Miton-HE oxidation products. However, it is important to note that the absorption properties of the TMeE + and 2-OH-TMeE + are red shifted compared to those of HE and its analogs functionalized on the side chain. The influence of the solvents is also similar as those reported previously. The maxima wavelengths absorption for the TMeHE oxidation products collected in aqueous solution are blue shifted compared to those collected in EtOH and with a lower absorbance.

Fluorescence properties of TMeHE oxidation products

The fluorescence properties of the non-specific oxidation of the probe TMeE + was reported recently by Luedtke et al.. [64] These authors reported that the functionalization of the amino groups with EWG tends to decrease the excitation and emission maxima for the E + derivatives. In their series of molecules, the TMeE + was the only ethidium derivative in which the exocyclic amines were functionalized with donor groups. Unlike the other molecules of the series, the fluorescence spectrum of TMeE + was red-shifted compared to ethidium. Those authors propose that this red-shift is attributed to the increase of the HOMO as well as the decrease of the LUMO. Along similar lines, a red shift compared to E + were also reported recently with the MitoNeo, in which the exocyclic amine functionalized neopentyl groups induce an inductive donor effect as well. [65] Therefore, the fluorescence spectra have been collected without DNA by Dr. R. Therefore, those data show that there is a better shift of fluorescence between the 2-hydroxylated superoxide marker and the non-specific oxidation product of the probe for the tetramethylated-HE based probe compared to the original one.

This better shift could be explained since TME + and 2-OH-TME + possess respectively 0 and 1 hydrogen donors, while E + and 2-OH-E + possess respectively 4 and 5 respectively. Therefore, a bigger variation on the physicochemical properties as the fluorescence properties or the lipophilicity was expected for the non-specific oxidation and hydroxylated-superoxide marker products of the TMeHE-base probes compared to the non-methylated one. However, it is important to note that the TME + and 2-OH-TME + emission spectra are broad. Therefore, since the non-specific oxidation of the probe is likely to be present 10-100-fold higher than the superoxide marker, the various oxidation products of HE-analogs functionalized with alkylated inductive donor groups also need to be separate by HPLC to give the indication of the superoxide in cellular models. Fortunately, since there is a bigger variation for the lipophilicity for these two compounds, the separation of the 2-OH-TMeE + from TMeE + is easier compared to that of 2-OH-E + from E + . This easier separation allows the development of HPLC methods with a shorter time of run. This is of major importance, since the HE-based probes are prone to the autooxidation among cell treatments. [30] 500 600 700 800

TMeE + A B E TMeE + was initially described as an ethidium analog, which can bind externally to the DNA but cannot intercalate between the pair-bases of the double helix. [66,67] The absence of the intercalating character was tentatively explained by the absence of hydrogen donor, due to the methylation of the amino groups. [63] Based on those assumptions Ryosuke et al. collected the fluorescence intensity for a TMeE +like in presence and in absence of a clay surface to understand by which process the fluorescence intensity of the ethidium derivatives are enhanced in the presence of DNA. [68] Their results show that there was an enhancement of the fluorescence intensity for a TMeE + -like in presence of a clay surface. [68] They claim that this clay surface has similar properties to DNA. Based on their results, those authors conclude that the main cause of the intercalating-dependent fluorescence of ethidium is related to the non-radiative deactivation induces by water molecules on the amino groups through hydrogen bonding. Therefore, the increase of fluorescence of the TMeE + -like in the clay surface refute the common belief idea of this phenomenon tentatively explained in 1977, which are, however, still based on the shielding of the amino groups from water molecules. [69] Indeed, it was believed that the proton exchange of the amino groups with water molecules, whose occurs in the excited state S1, was the factor that contributes the most to the quenching of the fluorescence of the phenanthridiniumderivatives. Another factor that was previously reported as preponderant and that were reconsidered to have a minor contribution, by Ryosuke et al., is the conformation restriction of the phenyl ring in C-6 position. However, we didn't find so far any clue in the literature on how the suppression of degree of freedom of the ethidium derivatives by binding affects the fluorescence enhancement, which eventually may explain why the fluorescence of the ethidium based is enhanced by external binding to the DNA. Nevertheless, we record the emission spectra of the 2-OH-TMeE+ in the presence and in the absence of DNA, our results show that the fluorescence intensity was more than 10-fold increase in the presence of DNA (Figure 49 E). Therefore, those results show that the fluorescence intensity of the marker is enhanced in the presence of DNA. However, we don't know if this enhancement result from an external binding to the DNA or this enhancement is allowed by an eventual partial intercalating character of the 2-OH-TMeE + that may notably be allowed by the hydrogen bond resulting from the hydroxyl group. [67] Chapter 5 Synthesis of new probes that allow a shift of fluorescence

One of the major limitations of the HE-based assay for O2 •-is the requirement of HPLC-based separation due to fluorescence spectral overlapping between E + and 2-OH-E + . This significantly affects the throughput of the assay, requires sample destruction (extraction of the probe and oxidation products), and can be carried out only in a limited number of biological laboratories. In addition, HPLC-based analyses required significantly higher amount of biological materials and lack to opportunity for realtime and monitoring of superoxide generation. There is an urgent, but yet unfulfilled need for novel approached for specific, fluorescence-based detection of O2 •-. Since the TMeHE-derivatives did not yield an enough shift of fluorescence emission bands between the non-specific oxidation product and the superoxide-marker, additional strategies have been explored to selectively detect O2 •-by fluorescence using novel derivatives of HE (called Shift-HE). The ultimate goal of this approach is to avoid the need of HPLC-based separation and to detect specifically and reliably O2 •-using fluorescence spectroscopy in biological systems. Consequently, we propose to take advantage of the introduction of the hydroxyl group adjacent to the amino group in the superoxide-specific product, to trigger a further intramolecular rearrangement (cyclisation) that will result in the release of a product with fluorescence parameters easily distinguishable from that of non-specific (ethidium-type) oxidation products (Figure 50). This "Turn-ON" strategy requires three principal's conditions: i) Reactivity of the Shift-HE towards superoxide must be similar to that of HE and must lead to the formation of 2-OH-E + derivatives; ii) intramolecular cyclisation must occur due to the introduction of the hydroxyl group; iii) intramolecular cyclisation must lead to the release of a fluorophore possessing specific fluorescent properties, easily distinguishable from that of ethidium-type product, with the use of standard fluorescent techniques available in most biological laboratories.

HE-derivatives functionalized with EWG

Preliminary data have shown that the HE-derivatives in which the exocyclic amino groups were functionalized with electron-withdrawing groups (EWG) were unreactive towards O2 •-generated by the enzymatic system HX/XO. As an example, we synthesized and tested the Pyrrole-HE derivative (Figure 51), and were unable to observe any oxidation of the probe or consumption of O2 •-, generated by the HX/XO system. We also attempted to take advantage of the functionalization of the carbamate groups of the HE-derivatives to allow a favored 5-member ring intramolecular cyclization. We prepared Carbamate-HE derivatives (Cbz-HE and Ph-HE). The reaction with the Fremy's salt yield to the 2hydroxylated superoxide marker in poor yield and no formation of the 2-OH markers were observed when HX/XO system was used to generate the superoxide (Figure 52).

Thus, due to the lack of reactivity towards superoxide of these derivatives, these strategies had to be reconsidered and revised.

Figure 51 Pyrrole-HE. (Pyrrole-HE have been prepared by the SREP-team).

Figure 52 EWG-HE derivatives.

8-Alloc-HE by adapting the procedure of Luedtke et al. (Figure 53). [70] In conclusion, the functionalization by any EWG groups on the exocyclic amines need to be proscribed for the superoxide detection. As these results suggest that even derivatization of a single amine group of HE using EWG significantly lowers its reactivity towards superoxide, we next aimed to functionalize the exocyclic amine with help of an alkyl linker, to prevent the electronic deactivation of the phenanthridine core. We attempted to take advantage of the reported spontaneous intramolecular cyclization of the phenol group on the amide or carboxylate, although favored by a Thorpe-Ingold effect induced by a trimethyl-lock. [71]

Investigation of HE derivatives containing self-immolative moieties releasing phenolic products

We firstly investigate the expected 6-exo-trig cyclization with newly synthesized HE-derivatives, which bear alcohols or phenols as the expected leaving group. The ability of those HE-derivatives to react with O2 •-and to cyclize upon the formation of the 2-hydroxylated marker was then investigated to generate a proof of concept for further syntheses of HE derivatives carrying fluorophores as the leaving groups. The HE derivatives were synthesized by reacting the correspondent aryl or alkyl 2bromoacetate in the presence of K2CO3 in neat conditions (Figure 55). Therefore, a first HE-derivative in which exocyclic amino group were functionalized with an ester group separated by a methylene group were developed. After reduction, under mild conditions (to avoid possible reduction of the ester moiety), those compounds were assayed using the superoxide generating system HX/XO. HE-CH2COOEt did react with O2 •-to give the hydroxylated-superoxide marker that was assigned by LC/MS. However, no trace of the cyclization product has been detected by LC/MS (Figure 56).This could be explained by the fact that phenolic group is a better leaving group than alkyl alcohol. Therefore, additional probes were developed in which the R1 group has been replaced by a phenyl group to obtain a better leaving group. HE-CH2COOPh has been synthesized by reducing the corresponding ethidium as described in the Figure 55. LC/MS analysis of the incubation mixture of HE-CH2COOPh in the presence of Fremy's salt reveal the formation of a product with an exact mass and an isotopic pattern consistent with the expected corresponding cyclized hydroxylated superoxide product. Therefore, the efficiency of HE-CH2COOPh to react with the superoxide radical has been investigated using the enzymatic system (HX/XO). While the same cyclized hydroxylated product has been detected, its yield was low.

To enhance the reaction with the superoxide radical we functionalized the exocyclic amines with donor methyl groups. Me2E + has been synthesized starting from the ethidium bromide. The first step consisted of the protection of the exocylic amino group with acetyl groups in pyridine to give the compound 48 that was used for further derivatization without any purification. The intermediate 48 was then methylated with MeI in basic condition in the presence of DMF to give 49. Finally, the intermediate 49 was deprotected under acidic condition to yield Me2E + with an overall yield of 25%. The exocyclic amine of Me2E + was functionalized in the presence of phenyl bromoacetate and K2CO3 in neat conditions as described in the Figure 55. This compound was also assayed in the presence of the superoxide generating system (HX/XO) by LC/MS. However, the results showed that the superoxide marker was still present in trace concentration and there were no traces of the cyclization product (data not shown).

Therefore, we tried to improve the efficiency of this reaction by improving the solubility of the probe in aqueous solution. Thus, we developed a hydropropidine-based probe in which the exocyclic aminegroups were functionalized. This compound has been synthesized starting from the commercially available propidium. Hence, the ability of the resulting HPr + -Ch2COOPh to react with O2 •-was evaluated. LC/MS analysis suggests that HPr + -Ch2COOPh reacts with O2 •-to give the desired cyclized compound (Figure 57).

Based on this encouraging result we developed new HE-based probe in which the R1 group is functionalized with a fluorescent leaving group.

Development of self-immolative HE-coumarin probe

We aim to prepare a Turn-ON HE-based probe that can release a fluorescent 7-hydroxycoumarin (umbelliferone) upon the formation of the 2-hydroxylated superoxide marker. One of the advantage will be to suppress the fluorescence overlapping of 7-hydroxycoumarin with the HE-derivatives and its oxidation products. Indeed, the fluorescence spectrum of the 7-hydroxy-coumarin collected in aqueous solution at physiological pH possesses maxima of excitation and emission at 325 and 465 nm respectively. [72] The assessment of the superoxide level directly by fluorescence should be therefore envisioned.

The compound 50 was prepared by adapting the method described by Zhou et al.. [73] Since reacting 50 with ethidium bromide yield the desired compound in low yield, an halogen exchange (Br to I) has been performed in the presence of NaI to afford 51 in 92% yield. Then, the compound 51 was reacted in the presence of ethidium bromide to afford 52. Finally, the selective reduction of the imine has been performed by reducing 52 with NaBH4 in MeCN to afford 53.

Figure 59 Synthesis of coumarin-HE. Reagents and conditions. i, Bromoacetyl Bromide, pyridine, DCM, 2 h, -15°C to rt, quant.; ii, NaI, acetone, 3 h, rt, 92%; Ethidium bromide/propidium idodide, K2CO3, PhNO2, reflux, overnight, 90%; NaBH4, MeCN, 10 min.

Unfortunately, the compound 53a failed to detect the superoxide in the cell-free system and in PMAstimulated neutrophils. Indeed, the major compound detected was the starting material 53a without a trace of the superoxide marker.

To verify that the solubility in water was not an issue, the hydropropidine analog was also prepared (53b). Unfortunately, the probe hydrolyzed at physiological pH avoiding the possibility to have a superoxide-specific fluorescence. Indeed, the carboxylic acid derivative was observed.

We decided to explore another approach consisting in developing HE-derivatives that bear a single fluorophore on the exocyclic amino group located at the C-3 position.

Half-HE

The exocyclic amino group at the C-8 position of the amino group is more reactive that that at the 3-position. Thus, to avoid the protection step, one of our first strategies was to develop HEderivatives that incorporate a phenanthridine core bearing a single exocyclic amino group at the C-3 position. Half-HE was then designed for that purpose. This compound has been synthesized by the SREP laboratory (Dr. M. Mehmet) from ethidum bromide. The selective elimination of the amino group at C-8 position was performed by reacting ethidium in the presence of sodium nitrite in acidic condition, the driving force of this reaction was the release of the N2 molecule. The resulting Half-E + was then reduced with sodium borohydride to afford Half-HE. The efficiency of the Half-HE probe to react with the superoxide has also been investigated. Thus, the Half-HE probe in the presence of the superoxide generating system yield both the hydroxylated-marker and the non-specific-oxidation of the probe (Figure 60). It is important to note that the shift of fluorescence between the 2-hydroxylated marker and the nonspecific oxidation product of the Half-HE probe was not markedly improved compared to the already existing HE (data not shown).

In order to minimize the yield of the non-specific oxidation of the Half-HE into Half-E + , we prepared the deuterated analog (Half-DE). It has been reported that the isotopic effect induced by the carbondeuterium bond at the C-6 position slows down the non-specific oxidation of the HE probe. [74] Unfortunately, in the case of Half-HE, the isotopic effect was not observed (Figure 61). The opposite effect was obtained. The reaction between Half-HE and the superoxide radical anion is less specific than in the case of HE. The lower electronic density of the phenanthridine core due to the absence of amino group at the C-8 position can be the explanation. Therefore, we investigated the possibility to develop a HE-derivative functionalized on a single amino-group by electron donor groups. Thus, the 3-pyrrolidine-E + was developed. This compound was synthesized by reacting ethidium bromide with 1,4-diodobutane in basic condition in a mixture of DMF and MeCN and afford the compound 8-pyrrolidine-E + in 51% yield.

Figure 62 Selective synthesis of 8-pyrrolidine-E +

The structure of this compound has been confirmed by nOe experiments, through a correlation mark of the H7 of the phenanthridine core and the proton of the pyrrolidine (experimental part). Indeed, the highly shielded doublet in aromatic region was attributed to the H7 based on the literature of ethidium derivatives. [64] The 8-pyrrolidine-E + was next functionalized with 7-aminocoumarin as a fluorophore group. The amide group bearing the nitrogen will hamper the non-specific oxidation of the probe. Since the ester group of the phenanthridine linked to a carboxyl group were likely to undergo cleavage in protic solvent we further grafted the aminocoumarin via an amide linker. Furthermore, the coumarin that we used was also functionalized with a methyl group on the Michael position to improve the stability of this moiety. This compound was next investigated using the superoxide generated system HX/XO. The LC/MS analyses over the time show that Pyr-coum-HE reacts with superoxide to give the hydroxylated marker. However, no trace of the cyclized product has been detected. The HPLC-FLD show the presence of the amino-coumarin, however, in trace concentration.

Conclusion

New ethidium based probes and their oxidation products have been synthesized and characterized by fluorescence spectroscopy. Therefore, in order to prevent the non-specific oxidation of the probe by heme proteins, HE have been successfully grafted onto a SBA-15 type silica via the azide-alkyne click chemistry approach. These mesoporous Silica-HE derivatives can be interesting to assess the extracellular level of O2 •-in ex-vivo models for example.

Furthermore, the mitochondria-targeted derivatives of HE has been fine-tuned by modulating the length of the alkyl chain linker. Our results show that those probes were able to detect O2 •-and the longer is the linker the greater is the intracellular level of the probe in cells (MiaPaCa-2).

The amino group of the phenanthridine core of HE has also been functionalized with several functions (using Ethidium Bromide as starting material). This functionalization allows the modulation of the spectroscopic properties of the HE-derivatives.

The oxidation of the new HE-derivatives, in which the amino groups of the phenanthridine core were functionalized, yield also the hydroxylated marker. Notably, for the tetramethylated derivative, the position of the hydroxyl group has been confirmed to be at the position 2 by NMR and X-ray diffraction.

The applicability of TMe-HE probe for the detection of O2 •-has been assessed in PMA-stimulated RAW 264.7. The kinetic properties of the radical cation of TMe-HE has been determined by Pulse radiolysis by our partners at Lodz University of technology. The lifetime of the radical cation, which react with O2 •-to give the hydroxylated marker, has been increased markedly compared to the nonetetramethylated original HE.

The cyclization of the HE probes were possible however the shift of the emission spectra between the 2-hydroxylated marker and the non-specific oxidation of the probe was not enough to make the distinction between both compounds directly by fluorescence. However, it is important to note that this cyclisation didn't affect the cyclic conjugated system.

Experimental Part

EXPERIMENTAL PROCEDURES

Materials

All reactions under dry argon atmosphere were performed using oven-dried or flame-dried glassware and solvents were added via syringe. CH2Cl2 was distilled under dry argon atmosphere in the presence of P2O5. All reagents were used as received without further purification. The reactions were monitored by TLC on silica gel Merck 60F254 and by HPLC/MS using C18 reverse phase column. Hydrogenation of compounds was performed under continuous-flow conditions using a high-pressure hydrogenator (H-Cube Mini, Thales Nanotechnology Inc.). Crude materials were purified by flash chromatography on Merck Silica gel 60 (0.040-0.063 mm) unless otherwise indicated. 31 P NMR, 1 H NMR and 13 C NMR spectra were recorded with a Bruker DPX 300 or 400 spectrometers at 121.49 or 161.98, 300.13 or 400.13 and 75.54 or 100.61 MHz respectively. 31 P NMR was taken in CDCl3 using 85% H3PO4 as an external standard with broad-band 1 H decoupling. Signal splitting patterns were described as: singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint.), multiplet (m). Chemical shifts (δ) are reported in ppm and coupling constant J values in Hertz. HRMS analyses were performed at the spectropole faculty of the Aix-Marseille Université on a QStar Elite (Applied Biosystems SCIEX) with API as a ionisation source. HPLC/MS experiments were performed using an Agilent 1260 infinity system coupled with a 6120 simple quadripole. This system was equipped with a C18 column (Zorbax 1.8 µM, 3 × 50 mm) that was equilibrated with 10% MeCN (containing 0.1% (v/v) formic acid) in 0.1% formic acid aqueous solution at the flow rate of 0.40 mL/min. After sample injection, the MeCN mobile phase concentration was increased to 40% over 3 min. Then, increased to 100% over 8 min and maintained at this concentration over a period of 6 min, unless otherwise indicated.

Synthesis of 3,8-Bis(carbethoxyamino)-6-phenylphenanthridine 19

19 was synthesized by adapting the literature method. [49] Ethyl chloroformate (0.4 g, 3.8 mmol) was added dropwise to a solution of DAPP (0.5 g, 1.8 mmol) in dry pyridine (5 mL) under inert atmosphere at 0°C. The reaction mixture was stirred at room temperature overnight. The dark red solution was poured into cold water and the yellow precipitate was filtered.

Synthesis of 3,8-bis(2-chloroacetamido)-5-(pent-4-yn-1-yl)-6-phenylphenanthridin-5-ium 22

Chloroacetyl chloride (0.396 g, 3.5 mmol) was slowly added over 20 min to a solution of DAPP (0.4 g, 1.4 mmol) in benzene (4 mL) and TEA (0.21 g, 2.1 mmol) at 0°C. The mixture was then warmed to room temperature and stirred for overnight under inert atmosphere. After total conversion, 1 mL of DMF was added to the crude material. The crude material was then washed with water, then dissolved in THF, dried over Na2SO4 and concentrated in vacuo to yield 22 as a dark oil (0.185 g, 32 %

General procedure A :

Triflates were prepared by adapting the procedure described by R. Armstrong-Chong et al. [75] To a solution of corresponding alcohol (2.4 mmol) in DCM (4 mL/mmol) were added lutidine (1.4 eq) and trifluoromethanesulfonic anhydride (1.2 eq) at -15°C under inert atmosphere. The reaction mixture was then allowed to stir at 0°C. After 1 h, 80 mL of DCM was added to the reaction mixture. The organic layer was washed twice with NH4Cl sat. (25 mL), twice with brine (50 mL), dried over Na2SO4 and distilled under reduced pressure to afford the corresponding triflates as an orange oil. The compound was used without further purifications.

2-Bromoethyl-trifluoromethanesulfonate 35a

35a was obtained from 2-bromoethanol (0.3 g, 2.4 mmol) following the general procedure A (0.62 g, 100% yield). 1 H NMR (CDCl3, 400 MHz): δ 4.74 (2H, t, J = 6.5), 3.60 (2H, t, J = 6.5).

4-bromobutyl-trifluoromethanesulfonate 35b

35b was obtained from 4-Bromobutanol (0.3 g, 2.0 mmol) following the general procedure A (0.62 g, 100% yield).

10-bromodecyl-trifluoromethanesulfonate 35d

35d was obtained from 10-bromodecanol (0.4 g, 1.7 mmol) following the general procedure A (0.62 g, 100% yield). 1 H NMR (CDCl3, 400 MHz): δ 4.55 (2H, t, J = 6.5 Hz), 3.42 (2H, t, J = 6.9 Hz), 1.90-1.80 (4H, m), 1.49-1.26 (12H, m).

3-Iodopropyl-trifluoromethanesulfonate

To a solution of 3-iodopropanol (0.4 g, 2.1 mmol) in DCM (8 mL), lutidine (0.35 mL, 3.0 mmol) and trifluoromethanesulfonic anhydride (0.43 mL, 2.6 mmol) were added at -15°C under inert atmosphere. The reaction mixture was then allowed to stir at 0°C. After 1 h, 2 mL of NH4Cl and 100 mL of DCM was added to the reaction mixture.

General procedure B :

A stirring suspension of corresponding phenanthridine in nitrobenzene (2.8 mL/mmol) was warm to 40°C and maintained at this temperature until complete dissolution. Then the corresponding triflate (2.5 eq) in nitrobenzene (2.0 mL/mmol) was added to the mixture. After 4h, the reaction was cooled to room temperature before complete conversion. Then, the starting material was recovered by column chromatography (DCM to DCM/EtOH: 99.5/0.5 to 98/2) and the desired product was eluted from the column using 3% Ethanol in DCM as eluent. The solvent was removed under vacuum to afford a phenanthridinium salt as a deep yellow solid.

Synthesis

General procedure D

Hydrogenation of protected phenanthridine under continuous-flow conditions was performed using a high-pressure hydrogenator. The corresponding phenanthridine in EtOH (50 mL/ mmol) was introduced into the reactor by a pump at 75° C, 40 bar of hydrogen pressure with a flow rate of 1 mL/min over a Pd(OH)2 CatCart®. The reaction was monitored by LC-MS. After total conversion, the crude material was concentrated under vacuum to afford the desired HE-derivative. The crude materials could be further purified on reverse phase flash chromatography using water and acetonitrile supplemented with 0.1% of TFA as eluent.

3,8-Diamino-5-(2-triphenylphosphonium-ethyl)-6-phenylphenanthridine (Mito2-HE)

Mito2-HE was obtained from a mixture of 39a and 39a' (131 mg, 0.13 mmol), following the general procedure D. After total conversion, the crude material was concentrated under vacuum to afford Mito2-HE as an orange powder (68.9 mg, 81%).

HRMS

3,8-Diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridine (HPr + )

HPr + were obtained from the phenanthridinium salt 5 (126 mg, 0.14 mmol), following the general procedure D. Purification of the crude material by flash chromatography on a reversed phase C18 column (0.1% TFA in H2O/MeCN 90/10 vol.) afforded HPr + as a white powder (68.2 mg, 93%).

General procedure E : synthesis of superoxide marker

To a solution containing HE-derivative in DMSO (40 µL/µmol) at 0°C was rapidly added (40 µL/µmolHEderivative) of fremy's salt solution in water (0.1 M). The resulting mixture was vortexed and shielded from light. After 10 min, the crude material was centrifuged and the supernatant was purified by semipreparative HPLC using a mixture of 0.1% [v/v] TFA in water and 0.1% [v/v] TFA in MeCN as mobile phase. 2-OH-Mito6-E + was obtained from the Mito6-HE (3 mg, 4 µmol), following the general procedure E. The supernatant was purified by semi-preparative HPLC, using a Beckman C18 column (Beckmann Ultrasphere, 250 µm, 10 mm × 5 mm) equilibrated with 20% MeCN in aqueous solution. The compounds were eluted by increasing the content of MeCN from 20% to 60% at a flow rate of 4 mL/min over 14 min. The pure fractions collected at 12.5 min were combined to afford 2-OH-Mito6-E + as a red solid (1.5 mg, 43%).

3,8-Diamino-5-(10-triphenylphosphoniumdecyl)-2-hydroxy-6-phenylphenanthridinium

(2-OH-Mito10-E + ) 2-OH-Mito10-E + was obtained from the Mito10-HE (7.8 mg, 9,36 µmol), following the general procedure E. The supernatant was purified by HPLC, using a Kinetex C18 column (Phenomenex, 5 µm, 250 mm × 21.2 mm) equilibrated with 10% MeCN in aqueous solution. The compounds were eluted by increasing the content of MeCN from 10% to 100% over 25 min at a flow rate of 13 mL/min to afford 2-OH-Mito10-E + as a red solid (6.0 mg, 71%

3,8-Tetramethylamino-5-[3-(diethylmethylammonio)propyl]-2-hydroxy-6-phenylphenanthridine (2-OH-TMePr ++ )

2-OH-TMePr ++ was obtained from the TMeHPr + (5.0 mg, 8.5 µmol), following the general procedure E. The supernatant was purified by HPLC, using a Kinetex C18 column (Phenomenex, 5 µm, 250 mm × 21.2 mm) equilibrated with 10% MeCN in aqueous solution. The compounds were eluted by increasing the content of MeCN from 10% to 100% over 25 min at a flow rate of 13 mL/min to afford 2-OH-TMePr ++ as a red solid (2.5 mg, 41%

General procedure F : synthesis the standard of non-specific oxidation of the probe

To a solution containing Miton-HE in DMSO (0.62 mL/µmol) at 0°C was rapidly added the same volume of a chloranil solution in MeOH (2 mM). After 4h, the methanol was removed under vacuum and the crude material was purified by semi-preparative HPLC using a mixture of 0.1% [v/v] TFA in water and 0.1% [v/v] TFA in MeCN as mobile phase.

3,8-Diamino-5-(2-triphenylphosphonium-ethyl)-6-phenylphenanthridinium (Mito2-E + )

Mito2-E + was obtained from the Mito2-HE (6.79 mg, 9.6 µmol), following the general procedure F. The crude material was purified by HPLC, using a Kinetex C18 column (Phenomenex, 5 µm, 250 mm × 21.2 mm) equilibrated with 10% MeCN in aqueous solution. The compounds were eluted by increasing the content of MeCN from 10% to 100% over 25 min at a flow rate of 13 mL/min to afford Mito2-E + as a violet powder (5.7 mg, 73%). HRMS calcd. for C39H34N3P 2+ [M] 2+ 287.6240, found 287.6240.

3,8-Diamino-5-(6-triphenylphosphonium-hexyl)-6-phenylphenanthridinium (Mito6-E + )

Mito6-E + was obtained from the Mito6-HE (2.2 mg, 3.0 µmol), following the general procedure F. Purification of the crude material by HPLC afford Mito6-E + as a violet powder (1.23 mg, 52%).

General procedure G : synthesis of Miton-E + -E +

To a solution containing Miton-HE in DMSO (20 µL/µmol) on ice was rapidly added the same volume of a solution of Potassium Ferricyanide in water (1 M). The resulting mixture was vortexed and shielded from the light on ice. After 10 min EtOH (400 µL) was added to the mixture. Then, the crude material was centrifuged and purified by semi-preparative HPLC using a mixture of 0.1% [v/v] TFA in water and 0.1% [v/v] TFA in MeCN as mobile phase.

2-Bis-[3,8-Diamino-5-(2-triphenylphosphonium-ethyl)-6-phenylphenanthridinium] (Mito2-E + -E + )

Mito2-E + -E + was obtained from the Mito2-HE (7.5 mg, 0.011 mmol), following the general procedure G. The supernatant was purified by semi-preparative HPLC, using a Beckman C18 column (Beckmann Ultrasphere, 250 µm, 10 mm × 5 mm) equilibrated with 20% MeCN in aqueous solution. The compounds were eluted, at a flow rate of 4 mL/min, by increasing the content of MeCN from 20% to 32% over 6 min and then from 32% to 49% over 10.5 min. The pure fractions collected at 16 min were combined to afford Mito2-E + -E + as a violet solid (5.4

2-Bis-[Diamino-5-(4-triphenylphosphonium-butyl)-6-phenylphenanthridium] (Mito4-E + E + )

Mito4-E + -E + was obtained from the Mito4-HE (2.2 mg, 3 µmol), following the general procedure G. The supernatant was purified by HPLC, using a Kinetex C18 column (Phenomenex, 5 µm, 250 mm × 21.2 mm) equilibrated with 37% MeCN in aqueous solution. The compounds were eluted, at a flow rate of 10 mL/min, by increasing the content of MeCN from 37% to 44% over 6 min and then from 44% to 50% over 11.5 min. The pure fractions collected at 15.5 min were combined to afford Mito4-E + -E + as a violet solid (0.9 mg, 31%). HRMS calcd. for C82H74N6P 4+ [M] 4+ 301.1357, found 301.1366.

2-Bis-[3,8-diamino-5-(10-triphenylphosphoniumdecyl)-6-phenylphenanthridinium] (Mito10-E + E + )

Mito10-E + -E + was obtained from the Mito10-HE (5.4 mg, 6.7 µmol), following the general procedure G. The supernatant was purified by HPLC, using a Kinetex C18 column (Phenomenex, 5 µm, 250 mm × 21.2 mm) equilibrated with 10% MeCN in aqueous solution. The compounds were eluted by increasing the content of MeCN from 10% to 100% over 25 min at a flow rate of 13 mL/min to afford Mito10-E + E + as a violet solid (0.9 mg, 15%). HRMS calcd for C94H98N6P2 4+

3,8-Bis-benzyloxycarbonylamino-5-[3-(diethylmethylammonio)propyl]-6phenylphenanthridinium (46)

To a solution of phenanthridinium salt 4 (0.15 g, 0.18 mmol) in MeOH (10 mL) N,N-diethylmethylamine (0.1 mL, 0.9 mmol) was added. The reaction mixture was then refluxed for 1 day under inert atmosphere and monitored by HPLC. After total conversion, the reaction was cooled to room temperature and concentrated. The crude material was purified by flash chromatography on a reversed phase C18 column (0.1% TFA in H2O/MeCN 90/10 vol.) to afford the phenanthridinium salt 46 as a yellow solid (126 mg, 75%). ESI-MS for C43H46N4O4 2+ , [M] 2+ , 341.4.

General procedure H : Reduction of the Ethidium moieties

The reduction of the Ethidium moieties were prepared by adapting the procedure described by J. Zielonka et al. [56] Synthesis of 3,8-diamino-5-(pent-4-yn-1-yl)-6-phenylphenanthridin-5-ium via Allyl carbamate protection (E + -pent-4-yne, 24) 26 (0.885 g, 1.17 mmol) was dissolved in dry THF (14 mL) and tetra-n-butylammonium fluoride (TBAF, 3.8 g, 14.5 mmol) was added, the mixture was heated under reflux for 3 days. The reaction was quenched by addition of 80 mL NH4Cl sat. (80 mL), then, extracted three times with DCM (25 mL) and concentrated by rotary evaporator. Next, 100 mL diethyl ether was added, the dark purple precipitate was filtered and the solid was dissolved with DCM/EtOH, concentrated under vacuum to give dark purple oil. The product was purified on flash chromatography silica column and eluted with DCM/EtOH (86/14, v:v). The column eluent was concentrated in vacuum to give 24 as a dark purple oil, yield (0.25 g, 49 %). In a twin-neck round-bottom flasks, E + -pent-4-yne (0.068 g, 0.176 mmol) was dissolved in dry DMF (8 mL) with SBA-N3 (0.085 g) and was stirred at rt. for 15-20 min under argon atmosphere. Then, sodium ascorbate (1.56 g, 45 equiv., 7.9 mmol) and CuSO4•5H2O (0.459 g, 10.5 equiv., 1.8 mmol) were added. The reaction was stirred at 50 °C for 4 days. After the reaction, the solution was poured into DMF (25 mL) and the mixture was filtered, washed twice with DMF (20 mL), EtOH (2×25 mL), HCl (10%, 2×20 mL), H2O (2×20 mL) and Et2O (3×20 mL). The product SBA-E + as a deep brown solid is obtained, yield 0.176g. 29 Si CP-MAS NMR (79.5 MHz): δ (ppm) -58.6 (T 2 ), -64.3 (T 3 ),-91.3 (Q 2 ), -99.6 (Q 3 ), -108.4 (Q 4 ).

ESI-MS

General procedure K

The corresponding phenanthridine was added to the corresponding alkyl or aryl bromoacetate and K2CO3. Then the solid mixture was melted at 80°C. The reaction was monitored by HPLC, after complete conversion. The reaction was cooled to rt while stirring and then Et2O was rapidly added to the reaction mixture. The reaction was then washed with Et2O, filtered. The residue was dissolved with DCM (supplemented with 2% EtOH), collected and concentrated in vacuo. The compound was after eventually purified by flash chromatography to afford the corresponding phenanthridine as pink-red solid.

5-ethyl-3,8-bis(methyl(2-oxo-2-phenoxyethyl)amino)-6-phenylphenanthridin-5-ium (46c)

46c was obtained from 45c (6.5 mg, 0.015 mmol), following the general procedure K. Purification of the crude material by flash chromatography on a reverse phase C18 (H2O/MeCN from 9/1 to 4/6) afforded 46c (3.2 mg, 34%). 8-Alloc-E + was prepared by adapting the procedure described by Luedtke and al. [70] To a solution of DAPP (280 mg, 0.71 mmol) in acetone and Phosphate Buffer (0.05 M, pH = 7.4) was added allyl chloroformate (0.08 mL, 0.71 mmol), while stirring. The reaction mixture was warmed to 32°C. After 5 h, the reaction was cool down. The crude material was extracted with DCM and purified by flash chromatography (DCM/EtOH: 8/2) to afford 8-Alloc-E + as an orange solid (175 mg, 0.51%).

ESI-MS

ESI-MS

General procedure L

The phenanthridine derivative was added to the corresponding alkyl or aryl bromoacetate and K2CO3 (3 eq). Then the solid mixture was melted at 50°C. The reaction was monitored by HPLC, after complete conversion. The reaction was cooled to rt while stirring and then Et2O was rapidly added to the reaction mixture. The reaction was then washed with Et2O, filtered. The residue was dissolved with DCM (supplemented with 2% EtOH), collected and concentrated in vacuo. The compound was after eventually purified by flash chromatography to afford the corresponding phenanthridine as pink-red solid.

General procedure M : Reduction of the Ethidium moieties

To a solution of the corresponding ethidium derivative in MeCN (5 mL) at 0°C under inert atmosphere and shield from the light was added a suspension of NaBH4 (2eq) in MeCN (5 mL) drop by drop until the red color just disappears. The crude material was then concentrated under vacuum. Finally, the crude material was dissolved with the lowest volume of water/acetonitrile: 3/1 supplemented with 1% formic acid. Eventually, DMSO was added to form the column deposit. The Hydroethidine derivative was therefore purified on reverse phase flash chromatography using water and acetonitrile supplemented with 0.1% of formic acid as eluent. *taken in acetonitrile due to the low stability of 2-OH-Mito2-E + , Mito2-E + and Mito2-E + -Mito2-E + in protic solvents. Mito10-HE and their oxidation products were eluted by increasing the content of MeCN (v/v) from 20% to 65% over 5 min (at a flow rate of 1.5 ml/min) and from 65% to 100% over 2 min. The absorption detector was used to measure Mito10-HE (at 370 nm; retention time: 4.72 min), Mito10-E + (at 290 nm; retention time: 5.80 min), Mito10-E + -Mito10-E + (at 290 nm; retention time: 6.19 min) and o-MitoPh-CH3 (at 240 nm; retention time: 5.34 min). A fluorescence detector (initial excitation at 358 nm and emission at 440 nm) was used to monitor Mito10-HE. At 5.4 min after the injection, the fluorescence parameters were changed (excitation at 490 nm and emission at 567 nm) for sensitive detection of 2-OH-Mito10-E + (retention time: 5.69 min) and Mito10-E + (retention time: 5.80 min).

Fluorescence excitation and emission maxima

TMeHE and their oxidation products were eluted by increasing the content of MeCN (v/v) from 20% to 40% over 1 min (at a flow rate of 1.5 mlmL/min), from 40% to 49% over 2 min and then from 49% to 100% over 2 min. The absorption detector was used to measure DAPP (at 290 nm; retention time: 2.0 min), 2-OH-TMeE + (at 370 nm; retention time: 2.9 min), and TMeHE (at 370 nm, retention time: 2.4 min). A fluorescence detector (initial excitation at 358 nm and emission at 400 nm) was used to monitor TMeHE. At 2.7 min after the injection, the fluorescence parameters were changed (excitation at 490 nm and emission at 608 nm) for sensitive detection of 2-OH-TMeE + (retention time: 2.9 min), they were changed again (excitation at 555 nm and emission at 625 nm) for sensitive detection of TMeE+ (retention time: 4.8 min).

Procedure for experiment in cell free system

To an 1.5 mL Eppendorf tube containing 120 µL of PBS (0.5 M, pH = 7.4) on ice, was successively added 20 µL of DTPA solution (1 mM in PBS), 20 µL of Hypoxanthine solution (5 mM in PB), 20 µL of HEderivative probe (0.5 mM in PBS/DMSO: 9/1 [v/v]) and 20 µL of xanthine oxidase solution (0.1 U/mL). The resulting mixture was vortexed, shield from the light and incubated at room temperature for 25 min. After 25 min, the mixture was vortexed, centrifuged for 1 min. Then, a volume of 35 µL of the resulting supernatant was transferred to HPLC vials for analysis. The samples (25 μl) were injected into a reverse phase column (Luna Omega Polar C18, 100 mm × 2.1 mm, 1.6 μm) equilibrated with 20% (v/v) MeCN, 80% (v/v) water containing 0.1% (v/v) formic acid. TM-HE-PPh2-alcyne and their oxidation products were eluted by increasing the content of MeCN (v/v) from 20% to 70% over 8.5 min at a flow rate of 0.18 ml/min, from 70% to 100% over 0.5 min at a flow rate of 0.18 ml/min, kept at this level for 4.5 min. The flow rate was then raised to 0.35 ml/min over 1 min, then raised to 0.450 ml/min over 2.5 min and kept at this level for 1 min. Eppendorf tube was then vortexed and incubated at room temperature for another 30 min. The solution was analyzed every 30 min.

HPLC traces of Miton-HE probes in MIA PaCa-2 stimulated by atovaquone (1 h, 40 µM)

Figure 65 HPLC analysis of cell lysates. HPLC traces recorded with a fluorescence detector using parameters: excitation at 358 nm and emission at 440 nm (A), excitation at 490 nm and emission at 567 nm (B) and with a diode-array detector at 290 nm (C) and at 242 nm (D). Figure 66 HPLC analysis of cell lysates. HPLC traces recorded with a fluorescence detector using parameters: excitation at 358 nm and emission at 440 nm (A), excitation at 490 nm and emission at 567 nm (B) and with a diode-array detector at 290 nm (C).

Figure 3

 3 Figure 3 formation of superoxide in stimulated macrophages.

Figure 4

 4 Figure 4 Formation of O2 •-from molecular oxygen and hypoxanthine in the presence of the xanthine oxidase enzyme

Figure 7

 7 Figure 7 Reaction of amplex red with hydrogen peroxide in presence of HRP to give resorufin

Figure 8

 8 Figure 8 Reaction of DCFH with hydrogen peroxide in presence of peroxidase lead to green fluorescent compound

Figure 9

 9 Figure 9 Reaction of the lucigenin and the superoxide to give a specific marker.

Figure 10

 10 Figure 10 Reaction of HE probe with O2 •-that yied the specific and stable fluorescent marker 2-OH-E +

Figure 11

 11 Figure 11 Pathway of the reaction that involve HE and O2 •-and other biological relevant species in cellular models.

Figure 14 Des

 14 Figure 14 Des Approach to synthesis HE-derivatives from DAPP

Figure 15

 15 Figure 15 Predicted biodistribution of new HE-derivatives and chemical structures of the existing probes for site-specific detection of O2 •-.

Figure 18 ( 3

 183 Figure 18 (A) Qualitative absorption spectra of HE (brown), 2-OH-E + (black) and E + (black). Qualitative fluorescence emission spectra of HE (blue), 2-OH-E + (yellow), E + (red). (B) Qualitative fluorescence excitation and emission spectra of SBA-HE (ex: 380 nm, em: 435 nm), 2-OH-SBA-E + (ex: 490 nm, 600 nm), SBA-E + (ex: 500 nm, em: 600 nm). in suspension in Phosphate Buffer. C) Thermogravimetric analysis of SBA-E + (red) and SBA-N3 (blue).

Figure 21

 21 Figure 21 Synthesis of HE-COOH. Reagents and conditions. i, BrCH2COOEt, PhNO2, 12h, 40°C 45%; ii, HBr, overnight, reflux 63%; NaBH4, MeOH, 10 min, 0°C, quant. (HE-COOH was prepared for the first time by Dr. M. M Ayhan).

Figure 22 (

 22 Figure22 (A, B) Mass spectral analyses of products formed from AM-HE probe in the presence and in the absence of esterases. (C, D) LC-MS/MS analyses of products formed from AM-HE probe using the HX/XO system alone or in the presence of horsh radish peroxidase (HRP) or in the presence of esterases.

  AB). The absorption spectra for Miton-HE (n = 2, 4, 6 and 10), 2-OH-Miton-E + , Miton-E + and Miton-E + -Miton-E + (n = 4, 6, 10) in ethanol and in aqueous solution at pH 7.4 are shown in figure II, Figure III and figure IV AB, respectively. The absorption maxima and the extinction coefficient values for these products are shown in the experimental section (Table

  fluorescence spectra for 2-OH-Miton-E + and Miton-E + (n = 4, 6, 10) in presence of DNA are shown in figure I C, figure II C, figure III C, and figure IV D respectively. The positions of the excitation and emission maxima for 2-OH-Miton-E + and Miton-E + (n = 4, 6, 10) are shown in the Table

Figure I Figure

 I Figure I Spectroscopic properties of Mito2-HE and its oxidation and products. UV-Vis absorption spectra of 2-OH-Mito2-E + (10 µM), Mito2-E + (10 µM), Mito2-E + -Mito2-E + (10 µM) in acetonitrile (A) and Mito2-HE (10 µM) in aqueous solution of Tris buffer (10 mM, pH 7.4) and EDTA (1 mM)(B). Fluorescence excitation/emission matrices for 2-OH-Mito2-E + (10 µM) and Mito2-E + (10 µM) in acetonitrile at room temperature (C) in three-dimensional (upper) and two-dimensional (lower) spaces. Excitation and emission spectra of 2-OH-Mito2-E + (10 μM) and Mito2-E + (10 μM) (D).

Figure

  Figure III Spectroscopic properties of Mito6-HE and its oxidation products. UV-Vis absorption spectra of Mito6-HE (10 µM), 2-OH-Mito6-E + (10 µM), Mito6-E + (10 µM), Mito6-E + -Mito6-E + (10 µM) in ethanol (A) and in aqueous solution of Tris buffer (10 mM, pH 7.4) and EDTA (1 mM)(B). Fluorescence excitation/emission matrices for 2-OH-Mito6-E + (0.5 µM) and Mito6-E + (2 µM) in the presence of DNA (0.1 mg/ml) in aqueous solution of Tris buffer (10 mM, pH 7.4) and EDTA (1 mM) at room temperature (C) in three-dimensional (upper) and two-dimensional (lower) spaces. Excitation and emission spectra of 2-OH-Mito6-E + (0.5 μM) and Mito6-E + (2 μM) (D).

Figure

  Figure IV Spectroscopic properties of Mito10-HE and its oxidation products. The UV-Vis absorption spectra of Mito10-HE (10 µM), 2-OH-Mito10-E + (10 µM), Mito10-E + (10 µM), Mito10-E + -Mito10-E + (10 µM) in ethanol (A) and in aqueous solution of Tris buffer (10 mM, pH 7.4) and EDTA (1 mM)(B). Fluorescence excitation/emission matrices for 2-OH-Mito10-E + (1 µM) and Mito10-E + (2 µM) in the presence of DNA (0.1 mg/ml) in aqueous solution of Tris buffer (10 mM, pH 7.4) and EDTA (1 mM) at room temperature (C) in three-dimensional (upper) and two-dimensional (lower) spaces. Excitation and emission spectra of 2-OH-Mito10-E + (1 μM) and Mito10-E + (2 μM) (D).

Figure 25

 25 Figure25 HPLC analyses of the products formed from Mito2-E + after 45 minutes of incubation in water or ethanol.

Figure 26

 26 Figure 26Proposed elimination mechanism of Mito2E + and 2-OH-Mito2E + in water.

Figure 27

 27 Figure 27 HPLC analyses of products formed from Fremy's salt, chloranil or O2 •-mediated oxidation of Miton-HE. For Mito2-HE (A), Mito4-HE (B), Mito6-HE (C) or Mito10-HE.

Figure 28

 28 Figure 28 Redox cycling mediated by MitoNP, which leads to the production of O2 •-within mitochondria.

Figure 30 Figure 31

 3031 Figure 30 Measurement of O2 •-, in the cell lysates of MiaPaCa-2 stimulated by MitoNP (24h, 10 µM) using the HE probe. The data were expressed as the concentration (normalized to total cellular protein, left), the ratio of each product to the detected level of the probe (right). T-test vs. control: *p<0.05, **p<0.01, ***p<0.001.

Figure 32

 32 Figure 32 HPLC analysis of cell lysates. HPLC analysis of cell lysates of the cell experiment of Mito10-HE in MiaPaCa-2. HPLC traces have been recorded with a fluorescence detector (A and B) and with a diode-array detector at 290 nm (C,D). Initially, the fluorescence detector parameters were excitation at 358 nm and emission at 440 nm. At 5.4 min after the injection, they were changed to be excitation at 490 nm and emission at 567 nm. Note (figure13 A), the top hplc traces is the injection of a standard of Mito6-HE (10 µM) (it is not an injection of a cell lysate).

Figure 33

 33 Figure 33 Measurement of O2 •-, in MiaPaCa-2 stimulated by MitoNP (24h, 10 µM) using the Miton-HE probe in the cell lysates. The data were expressed as the concentration (normalized to total cellular protein, left), the ratio of each product to the detected level of the probe (right). T-test vs. control: *p<0.05, **p<0.01, ***p<0.001. This experiment was repeated with the same conclusion.

Figure 34

 34 Figure 34 Reactivity of HE (10 µM) with Atovaquone (40 µM) in phosphate Buffer 0.05 M.

Figure 35 HPLCFigure 36

 3536 Figure 35 HPLC analysis of cell lysates. HPLC traces recorded with a fluorescence detector using parameters: excitation at 358 nm and emission at 440 nm (A), excitation at 490 nm and emission at 567 nm (B) and with a diode-array detector at 290 nm (C).

Figure 37

 37 Figure 37 Measurement of O2 •-, in MIA PaCa-2 stimulated by atovaquone (1h, 40 µM) using the Miton-HE probe in the cell lysates. The data were expressed as the concentration (normalized to total cellular protein, left), the ratio of each product to the detected level of the probe (right). T-test vs. control: *p<0.05, **p<0.01, ***p<0.001.

  •+ and HPr •+ performed by Dr. A. Sikora et col. (Lodz University of Technology, Poland) have shown that the spin density is highly localized in the carbon at the C-2 and the C-12 position of the phenanthridine core. Similar spin distributions were calculated for the radical cations of MeE and TMeHE (Figure38). This supports the mechanism, in which superoxide radical anion reacts with the radical cation of dihydroethidium based probe, through a direct attack at the C-2 carbon atom, to yield 2-hydroxylated cationic product.

Figure 38

 38 Figure 38 Chemical structures of (A) HPr •2+ , (B) HE •+ , (C) MeE •+ , and (D) TMeHE •+ , and appropriate spin density maps obtained from DFT quantum mechanical calculations using 6-311+G(d,p) basis set.

Figure 40

 40 Figure 40 Model molecules to explore the mechanism of the reaction of HE with superoxide and other oxidants.

Figure 41

 41 Figure 41 Synthesis of MeE. (molecule obtained from the Srep team)

Figure 42

 42 Figure 42 Synthesis of TMeHE. (synthesis performed by the Srep team).

Figure 43 X

 43 Figure 43 X-Ray structures of TMeE + and 2-OH-TMeE + .

Figure 44 HPLCFigure 45

 4445 Figure 44 HPLC analysis of cell lysates of HE vs TMHE in PMA stimulated RAW 264.7. HPLC traces of HE-treated cells are shown using the fluorescence parameters: excitation at 358 nm and emission at 440 nm, the excitation and emission parameters were switched at 2.4 min to 490 and 567 nm respectively for sensitive detection of 2-OH-E+ (intensity ×1.5 for this part) (A). HPLC traces of HE-treated cells recorded with the diode area detector at 290 nm (B). HPLC traces of TMeHEtreated cells are shown at 370 nm (C) and (D). The HPLC traces displayed corresponds to the mean value based on the 2-OHderivatives level.

Figure 46

 46 Figure 46 Synthetic pathways leading to TMeHPr + .

Figure 47

 47 Figure 47 Transition absorption spectrum recorded after pulse radiolysis of TMeHPr + (50 µM) in N2O saturated solution containing PBS (50 mM, pH 7.4) with NaN3 (0.1 M). The spectrum was recorded 9 µs after the electron pulse. (B) Monitoring of the absorbance of TMeHPr •2+ at 480 nm in the presence of O2 •-after pulse radiolysis of O2-saturated aqueous solution containing TMeHPr + (180 µM), NaN3 (3 mM), formate (50 mM), and PBS. The first order kinetics calculated is shown in black.

Figure 48 (

 48 Figure 48 (A) HPLC traces of the incubation of TMeHPr + (50 µM) in presence of an increasing concentration of Fe(CN)6 3-(0 -110 µM) (B) UV-Vis absorption spectra of the incubation of TMeHPr + (50 µM) in presence of an increasing concentration of Fe(CN)6 3-(0 -200 µM), in aqueous solution. (This experiment and the graph have been performed by Radoslow Michalski)

  Michalski. The fluorescence spectra for 2-OH-TMeE + and TMeE + without DNA, in aqueous solution supplemented with 20% of MeCN are shown Figure 49 C and D respectively. The positions of the excitation and emission maxima were 500 and 640 nm for 2-OH-TMeE + and 560 and 665 nm for TMeE + . Analogous data were also collected in the presence of DNA and in the absence of MeCN and shown in Figure 49 E. The positions of the excitation and emission maxima collected was therefore 480 and 598 nm for 2-OH-TMeE + and 565 and 623 nm for TMeE + .

Figure 49

 49 Figure 49 Spectroscopic properties of the oxidation products of TMeHE. The UV-Vis absorption spectra 2-OH-TMeE + (10 µM), TMeE + (10 µM) in ethanol (A) and in aqueous solution of Tris buffer (10 mM, pH 7.4) and EDTA (1 mM)(B). Fluorescence excitation/emission matrices for 2-OH-TMeE + (10 µM) and TMeE + (10 µM) in the absence of DNA in phosphate buffer (50 mM, pH 7.4) containing 0.1 mM DTPA and 20% MeCN, (C) and (D) respectively (experiment and 2D figure performed by Dr. R. Michaleski, PhD). Fluorescence Emission spectra of 2-OH-TMeE + (10 μM) and TMeE + (50 μM) (E).

Figure 50

 50 Figure 50 Strategy to develop a new Shift-HE derivative.

Figure 53 .

 53 Figure 53. Synthesis of 8-Alloc-HE.

Figure 54 HPLC analyses of products formed from 8 -

 548 Figure 54 HPLC analyses of products formed from 8-Alloc-HE probe in the presence of the superoxide generating system HX/XO.

Figure 55

 55 Figure 55 Synthesis of the HE-derivatives bearing alcohols as leaving groups.

Figure 56

 56 Figure 56 Synthesis of E + -CH2COOEt (A) and HPLC traces of the reaction HE-CH2COOEt in the presence of HX/XO at 290 nm (B) and taken on fluorescence (excitation : 490 nm, emission : 564 nm).

Figure 57

 57 Figure 57 Synthesis of Me2-E + .

Figure 58 HPLC

 58 Figure 58 HPLC traces of the oxidation of HPr + -CH2COOPh with superoxide over the time. The data were collected at 290 nm (A) and by fluorescence (excitation: 490 nm, emission: 564 nm).

Figure 60

 60 Figure 60 Synthesis of half-HE. (i) Hypophosphorous acid, 12 h, 0°C, 15%.; (ii) NaBH4, MeOH, 10 min, (iii) NaBD4, D2O, DCM.

Figure 61 HPLC

 61 Figure 61 HPLC analyses of products formed from Half-HE probe in the presence of the superoxide generating system HX/XO.

Figure 63

 63 Figure 63Synthesis of a monocoumarine. Reagents and conditions: Bromoacetyl Bromide, pyridine, DCM, 2 h, -15°C to rt, 60%; ii, NaI, acetone, 3 h, rt, 92%; iii, K2CO3, PhNO2, reflux, overnight, 90%; iv, NaBH4, MeCN, 10 min.

Figure 64 HPLC

 64 Figure 64 HPLC analyses of products formed from 8-Pyr-Coum-HE probe in the presence of the superoxide generating system HX/XO.

of 3 , 8 -

 38 bis((ethoxycarbonyl)amino)-5-(pent-4-yn-1-yl)-6-phenylphenanthridin-5-ium 20

31 P

 31 -Mito4-E+ was obtained from the Mito4-HE (7.5 mg, 10,4 µmol), following the general procedure E. The supernatant was purified by HPLC, using a Kinetex C18 column (Phenomenex, 5 µm, 250 mm × 21.2 mm) equilibrated with 10% MeCN in aqueous solution. The compounds were eluted by increasing the content of MeCN from 10% to 100% over 25 min at a flow rate of 13 mL/min to afford 2-OH-Mito4-E + as a red solid (3.4 mg, 39%). HRMS calcd. for C41H38N3OP 2+[M] 2+ 309.6371, found 309.6371. NMR (CD3OD, 121.5 MHz): δ 23.58. 1 HNMR (CD3OD, 300 MHz): δ 8.39 (d, J = 9.2 Hz, 1H), 7.93-7.88 (m, 4H), 7.78-7.66 (m, 17H), 7.55 (dd, J = 9.2 Hz, J = 2.4 Hz, 1H), 7.51-7.48 (m, 2H), 6.43 (d, J = 2.4 Hz, 1H), 4.58 (t, J = 7.6 Hz, 2H), 3.27-3.20 (m, 2H), 2.35-2.11 (m, 2H), 1.62-48 (m, 2H).

5-( 2 -

 2 (acetoxymethoxy)-2-oxoethyl)-3,8-diamino-2-hydroxy-6-phenylphenanthridin-5-ium 2-OH-AM-E + was obtained from the AM-HE (5 mg, 11 µmol), following the general procedure E. The supernatant was purified by HPLC, using a Kinetex C18 column (Phenomenex, 5 µm, 250 mm × 21.2 mm). Pure fractions were collected and combined to afford 2-OH-AM-E + as a red solid (2.3 mg, 36%).ESI-MS for C24H22N3O5+ , [M] + , 432.2.

31 P

 31 NMR (CD3OD, 121.5 MHz): δ 20.58. 1 H NMR (CD3OD, 300 MHz): δ 8.71-859 (2H, m), 7.99-7.94 (3H, m), 7.86-7.59 (21H, m), 7.41 (1H, dd, J = 9 Hz, J = 1.8 Hz), 7.25-7.15 (1H, m), 6.59 (1H, d, J = 1.8 Hz), 6.38 (1H, d, J = 2.4 Hz), 5.04-4.97 (2H, m), 4.10-3.89 (2H, m).

31 P

 31 NMR (CD3OD, 121 MHz): δ 23.53. 1 H NMR (MeOD d4, 400 MHz) : δ 8.77 (2H, s), 8.64 (2H, d, J = 9.0 Hz), 7.98-7.92 (7H, m), 7.85-7.72 (38H, m), 7.68-7.64 (2H, m), 7.63-7.57 (4H, m), 6.55 (2H, d, J = 2.3 Hz), 4.73-4.63 (4H, m), 3.38-3.28 (4H, m), 4.40-2.26 (4H, m), 1.74-1.60 (4H, m).

1 H

 1 NMR (CD3OD, 400 MHz): δ 10.53 (1H, s), 10.03 (1H, s), 9.10-9.08 (1H, m), 9.03 (1H, d, J = 9.2 Hz), 8.96 (1H, d, J = 9.3 Hz), 8.30 (1H, dd, J = 9.3 Hz, J = 2.3 Hz), 7.96 (1H, d, J = 2.3 Hz), 7.92-7.78 (4H, m), 7.75-7.70 (2H, m), 7.47-7.29 (10H, m), 5.32 (2H, s), 5.14 (2H, s), 4.85-4.77 (2H, m), 4.85-4.79 (6H, m), 2.97 (3H, m), 2.55-2.40 (2H, m), 1.29 (6H, t, J = 7.2 Hz). 1 H NMR (CDCl3, 400 MHz): δ 9.45, 8.83 (1H, d, J = 9.0 Hz), 8.73 (1H, d, J = 9.0 Hz), 8.68 (1H, d, J = 9.1 Hz), 8.54-8.37 (2H, m), 7.84-7.77 (3H, m), 7.76-7.69 (2H, m), 7.50-7.45 (4H, m), 7.44-7.33 (8H, m), 5.28 (2H, s), 5.17 (2H, s), 5.0-4.8 (2H, m), 3.55-3.40 (2H, m), 3.40-3.27 (4H, m), 2.97 (3H, s), 2.64-2.49 (2H, m), 1.32-1.20 (6H, m). 13 C NMR (CDCl3, 100 MHz): δ 163.

1 H

 1 NMR (CDCl3, 400 MHz) δ 7.71 (1H, d, J = 9.5 Hz), 7.53 (1H, d, J = 9.5 Hz), 7.17-7.15 (1H, m), 7.10 (1H, d(broad)), 6. (1H, dd, J = 9.7 Hz, J = 2.2 Hz), 6.43 (1H, d, J = 9.5 Hz), 3.95 (s). 2-iodo-N-(4-methyl-2-oxo-2H-chromen-7-yl)acetamide 54 was obtained from 53 (200 mg, 0.68 mmol), following the general procedure J to afford 54 (220 mg, 95%). ESI-MS (m/z) for [C12H10INO3][M] + , 344.0.

  (m/z) for [C39H36N3O4 + ], [M] + , 610.0. 1 H NMR (CDCl3, 400 MHz) δ 8.66 (1H, d, J = 9.9 Hz), 8.64 (1H, d, J = 9.9 Hz), 7.75-7.63 (4H, m), 7.60 (1H, s(broad)), 7.52 (2H, d, J = 7.2 Hz), 7.43-7.32 (4H, m), 7.24-7.07 (5H, m), 7.00 (2H, d, J = 7.2 Hz), 6.90-6.84 (1H, m), 6.35 (1H, d, J = 2.3 Hz), 2.23-5.14 (2H, m), 4.85 (2H, s), 4.27 (2H, s), 3.89-3.81 (4H, m), 3.67-3.59 (4H, m), 3.45 (3H, s), 3.07 (3H, s), 1.53 (3H, t, J = 7.0 Hz).

  (m/z) for [C25H28N3O2 + ], [M] + , 401.2. 1 H NMR (MeOD, 300 MHz) δ 8.76 (1H, d, J = 2.5 Hz), 8.73 (1H, d, J = 2.0 Hz), 8.14 (1H, dd, J = 9.3 Hz, J = 2.5 Hz), 7.84-7.75 (3H, m), 7.71 (1H, d, J = 2.2 Hz), 7.67-7.61 (m, 2H), 6.00-5.87 (1H, m), 5.32 (1H, dd, J = 17.2 Hz, J = 1.5 Hz), 5.20 (1H, dd, J = 10.5 Hz, J = 1.5 Hz), 4.70 (2H, q, J = 7.1 Hz), 4.58 (2H, dt, J = 5.5 Hz, J = 1.5 Hz), 1.54 (3H, t, J = 7.1 Hz).

3-( 3 , 8 -

 38 bis((2-oxo-2-((2-oxo-2H-chromen-7-yl)oxy)ethyl)amino)-6-phenylphenanthridin-5(6H)-yl)-N,N-diethyl-N-methylpropan-1-aminium 52b 52b was obtained from propidium iodide (10 mg, 0.015 mmol) and 51 and (50 mg, 0.014 mmol), following the general procedure L. Purification of the crude material by flash chromatography on a reverse phase C18 (H2O/MeCN from 9/1 to 5/95) afforded 52b (6.4 mg, 41%). ESI-MS (m/z) for [C37H35N4O3 + ], [M] + , 583.2. 1 H NMR (DMSO-d6, 300 MHz) δ 10.44 (s, 1H), 7.80 (1H, d, J = 1.7 Hz), 7.73 (1H, d, J = 8.8 Hz), 7.55 (1H, dd, J = 8.8 Hz, J = 1.7 Hz), 7.46 (1H, d, J = 8.2 Hz), 7.40 (1H, d, J = 8.2 Hz), 7.24-7.07 (6H, m), 6.45 (1H, d, J = 8.2 Hz), 6.35-6.24 (2H, m), 6.07-5.97 (2H, m), 5.43 (1H, s(broad)), 3.97-3.91 (2H, m), 3.25-3.10 (5H, m), 2.40 (3H, s), 1.95-1.8 (4H, m), 1.05 (3H, t, J = 7.0 Hz).

3-( 3 , 8 -

 38 bis((2-oxo-2-phenoxyethyl)amino)-6-phenylphenanthridin-5(6H)-yl)-N,N-diethyl-Nmethylpropan-1-aminium 47d 47d was obtained from 46d (15 mg, 0.017 mmol), following the general procedure M. Purification of the crude material by flash chromatography on a reverse phase C18 (H2O/MeCN from 9/1 to 6/4) afforded 47d (10 mg, 73%).ESI-MS(m/z) for [C43H47N4O4 + ][M] + , 683.5. 1 H NMR (MeCN-d3, 300 MHz) δ 7.53 (1H, d, J = 8.3 Hz), 7.50 (1H, d, J = 8.3 Hz), 7.45-7.35 (4H, m), 7.31-7.24 (2H, m), 7.23-7.16 (5H, m), 7.09 (2H, d, J = 8.0 Hz), 7.00 (2H, d, J = 8.0 Hz), 6.66 (1H, dd, J = 8.8 Hz, J = 2.0 Hz), 6.49 (1H, s(broad)), 6.19 (1H, dd, J = 8.0 Hz, J = 1.5 Hz), 6.09 (1H, s(broad)), 5.38 (1H, s), 5.04-4.90 (2H, m), 4.23 (2H, d, J = 5.5 Hz), 4.18 (2H, d, J = 6.0 Hz), 3.58-3.51 (1H, m), 3.26-3.03 (7H, m), 2.69 (s, 3H), 1.12-1.05 (6H, t, J = 6.4 Hz).

  from 46c (3.2 mg, 0.046 mmol), following the general procedure M. Purification of the crude material by flash chromatography on a reverse phase C18 (H2O/MeCN from 9/1 to 4/6) afforded 47c (1.1 mg, 38%).ESI-MS(m/z) for [C39H37N3O4], [MH] + , 612.4. 1 H NMR (CDCl3, 400 MHz) δ 7.62 (1H, d, J = 8.8 Hz), 7.57 (1H, d, J = 8.3 Hz), 7.35-7.30 (4H, m), 7.25-7.13 (7H, m), 7.01 (2H, d, J = 8.3 Hz), 6.88 (2H, d, J = 8.0 Hz), 6.73 (1H, dd, J = 8.8 Hz, J = 1.5 Hz), 6.47 (1H, d, J = 2.2 Hz), 8.28 (1H, dd, J = 8.4 Hz, J = 2.3 Hz), 6.11 (1H, d, J = 2.2 Hz), 5.41 (1H, s), 3.50-3.40 (m, 1H), 3.30-3.20 (1H,m), 3.19 (3H, s), 3.13 (3H, s), 1.20 (3H, t, J = 7.1 Hz).

  was obtained from E + -CH2COOEt (15 mg, 0.026 mmol), following the general procedure M. Purification of the crude material by flash chromatography on a reverse phase C18 (H2O/MeCN from 9/1 to 1/1) afforded HE-CH2COOEt (3.2 mg, 24%).

1 H

 1 NMR (CDCl3, 400 MHz) δ 8.52 (1H, s), 7.46 (1H, d, J = 10.5 Hz), 7.3 (1H, J = 10.5 Hz), 7.21-7.11 (m, 5H), 8.55 (1H, dd, J = 8.5 Hz, J = 2.5 Hz), 6.34 (1H, d, J = 2.5 Hz), 6.07 (1H, d(broad), J = 7.9 Hz), 6.03 (1H, d, J = 2.1 Hz), 5.34 (1H, s), 4.19 (2H, q, J = 7.1 Hz), 4.13 (2H, q, J = 7.1 Hz), 3.91 (1H, s), 3.85 (1H, s), 3.43-3.36 (1H, m), 3.26-3.17 (1H, m), 1.25 (3H, t, J = 7.1 Hz), 1.19 (3H, t, J = 7.1 Hz), 1.15 (3H, t, J = 7.1 Hz). bis(2-oxo-2H-chromen-7-yl) 2,2'-((5-ethyl-6-phenyl-5,6-dihydrophenanthridine-3,8diyl)bis(azanediyl))diacetate 53a

Figure 67

 67 Figure67HPLC analysis of cell lysates. HPLC traces recorded with a fluorescence detector using parameters: excitation at 358 nm and emission at 440 nm (A), excitation at 490 nm and emission at 567 nm (B) and with a diode-array detector at 290 nm (C and D).

Figure 68

 68 Figure68HPLC analysis of cell lysates. HPLC traces recorded with a fluorescence detector using parameters: excitation at 358 nm and emission at 440 nm (A), excitation at 490 nm and emission at 567 nm (B) and with a diode-array detector at 290 nm (C and D).

* E + /HE TMeE + /TMeHE to

  the corresponding phenanthridinium cation 45 in 39% yield. The substitution of the iodide group of 45 by N,N-diethylmethylamine in methanol led to the dication 46 in 75% yield. The deprotection of the carbamate groups of 46 by hydrogenation (Pd(OH)2) afforded HPr + in a 93% yield. Finally, the methylation of the two exocyclic amino groups of HPr + in the presence of trimethylphosphate led to TMePr ++ in 32% yield.

**

  The precipitate was dissolved with THF and concentrated in vacuum to give 19 as a yellow solid (0.69 g, 97 %). ESI-MS (m/z) for [C25H23N3O4], [MH] + , 430.4. 1 H NMR (300.13 MHz, MeOD-d4) δ (ppm) 8.67 (1H, d, J = 9.1 Hz), 8.59 (1H, d, J = 9.1 Hz), 8.23 (2H, dd, J = 2.2 Hz, 9.1 Hz), 7.98 (1H, dd, J = 2.2 Hz, 9.1 Hz), 7.81 (1H, dd, J = 2.2 Hz, 9.1 Hz), 7.72-7.69 (2H, m), 7.64-7.60 (3H, m), 4.28-4.14 (4H, m), 1.32 (6H, dt, J = 19.3 Hz, 7.1 Hz).

  Allyl chloroformate (1.49 g, 12.33 mmol) was added dropwise to the solution. The mixture was then warmed to room temperature and stirred for 2 days. At the end of the reaction, the dark red solution was poured into cold water and the yellow precipitate was filtered. The precipitate was washed with pentane and filtered, then the solid was dissolved with DCM/EtOH and concentrated in vacuo to afford 25 as a yellow solid (1.23 g, 96.8 %).

	DAPP (0.8 g, 2.80 mmol) was dissolved in dry pyridine (6.5 mL) and the flask was cooled to 0 °C under
	inert atmosphere. ESI-MS (m/z) for [C27H23N3O4], [MH] + , 454.4. 1 H NMR (300 MHz, DMSO-d6): δ (ppm) 10.09 (s, 1H), 10.06
	(s, 1H), 8.75 (d, J = 9.08 Hz, 1H), 8.65 (d, J = 9.08 Hz, 1H), 8.29 (s, 1H), 8.21 (s, 1H), 7.99 (d, J = 8.95 Hz,
	1H), 7.81 (d, J = 8.85 Hz, 1H), 7.71-7.68 (m, 2H), 7.61-7.57 (m, 3H), 6.10-5.90 (m, 2H), 5.44-5.31 (m,
	2H), 5.29-5.20 (m, 2H), 4.68 (dt, J = 5.5, 1.28 (×2) Hz, 2H), 4.61 (dt, J = 5.41, 1.38 (×2) Hz, 2H).
	3,8-Bis-benzyloxycarbonylamino-6-phenylphenanthridine 28
	To a suspension of DAPP (0.8 g, 2.8 mmol) and potassium carbonate (0.8 g, 5.8 mmol) in MeCN (15 mL)
	was added dropwise benzyl chloroformate (1,1 mL, 7.7 mmol) at 0°C under inert atmosphere. The
	reaction mixture was stirred overnight at room temperature. The crude material was filtered, washed
	with MeCN and dissolved in THF. After concentration under reduced pressure, the crude product was
	dissolved in DCM and filtered. The filtrate was dried over Na2SO4 and the solvent distilled under
	reduced pressure. Distillation of the volatiles under reduced pressure (10 -3 Torr) afforded 28 as a pale ). ESI-MS (m/z) for [C23H17N3O2], [MH] + , 438.2. 1 H NMR (400 MHz, DMSO-d6): δ (ppm) 10.67 (s, 2H), 8.83 (d, J = 9.05 yellow solid (1.4 g, 90% yield). ESI-MS for [C35H27N3O4], [MH] + , 554.4.
	Hz, 1H), 8.73 (d, J = 9.05 Hz, 1H), 8.43 (s, 1H), 8.34 (s, 1H), 8.20 (dd, J = 9.05, 2.20 Hz, 1H), 7.88 (dd, J = 1 H NMR (DMSO d6, 300 MHz) δ 10.18 (2H, 2s), 8.76 (1H, d, J = 9.3 Hz), 8.67 (1H, d, J = 9.1 Hz), 8.31 (1H, 8.93, 2.08 Hz, 1H), 7.73-7.71 (m, 2H), 7.63-7.59 (m, 3H), 4.36 (s, 2H), 4.28 (s, 2H). d, J = 2.1 Hz), 8.25 (1H, d, J = 2.3 Hz), 8.01 (1H, dd, J = 2.1 Hz, J = 9.1 Hz), 7.83 (1H, dd, J = 2.3 Hz, J = 9.3
	Hz), 7.75-7.69 (2H, m), 7.82-7.58 (3H, m), 7.52-7.32 (10H, m), 5.23 (2H, s), 5.16 (2H, s). 13 C NMR (DMSO
	d6, 75 MHz) δ 161.3, 153.3, 143.3, 139.4, 138.0, 136.4, 135.9, 135.8, 129.6, 129.5, 128.8, 128.7, 128.5,
	128.4, 128.3, 126.9, 125.3, 123.1, 122.8, 122.7, 119.6, 119.1, 117.9, 116.8, 67.9, 67.2.
	3,8-bis(((allyloxy)carbonyl)amino)-5-(pent-4-yn-1-yl)-6-phenylphenanthridin-5-ium 25

  J = 85.6 Hz, 117.9, 106.1, 66.8, 66.5, 54.8, 30.2, 30.1, 28.8, 28.7, 28.6, 28.5, 28.2, 26.1, 22.2 (d, J = 4.4 Hz), 21.3 (d, J = 51.3 Hz).

	37b was obtained from the phenanthridinium salt 37b (0.12 g, 0.14 mmol), following the general 37d was obtained from the phenanthridinium salt 36d (65 mg, 0.07 mmol), following the general
	procedure C as a deep yellow solid (146 mg, 91%). ESI-MS for C57H50N3O4P 2+ , [M] 2+ , 436.0. 1 HNMR procedure C. Purification of the crude material by flash chromatography on a reverse phase C18 (0.1%
	(MeOD, 300 MHz): δ 8.86 (1H, s), 8.85 (1H, d, J = 9.6 Hz), 8.81 (1H, d, J = 9.8 Hz), 8.19 (1H, dd, J = 8.2 TFA in H2O/MeCN from 90/10 to 30/70) afforded the phenanthridinium salt 37d as a deep orange solid
	Hz), 7.89-7.50 (12H, m), 7.38-7.2 (12H, m), 5.04 (2H, s), 5.02 (2H, s), 4.85-4.73 (2H, m), 3.54 (2H, t, J = (68 mg, 82%).
	14.2 Hz), 2.46-2.26 (2H, m), 1.84-1.64 (2H, m). 13 CNMR (MeOD, 75 MHz): δ 164.6, 155.5, 155.2, 143.8,
	141.6, 137.7, 137.5, 136.52 (d, J = 3.3 Hz), 135.6, 135.0 (d, J = 9.9 Hz),133.2, 133.1, 132.6, 132.2, 131.7 ESI-MS for C63H62N3O4P 2+ , [M] 2+ , 478.0. 31 P NMR (CD3OD, 162 MHz): δ 23.76. 1 H NMR (CD3OD, 400
	(d, J = 12.6 Hz), 131.0, 130.6, 130.2, 130.0, 129.8, 129.7, 129.4, 129.3, 129.0, 128.8, 127.1, 126.7, 124.8, MHz): δ 9.00 (1H, d, J = 1.2 Hz), 8.97 (1H, d, J = 9.3 Hz), 8.92 (1H, d, J = 9.3 Hz), 8.26 (1H, dd, J = 9.3 Hz,
	123.0, 119.7 (d, J = 86.9 Hz), 119.3, 107.5, 68.1, 68.0, 55.3, 30.8, 22.4 (d, J = 50.6 Hz), 21.1 (d, J = 3.3 J = 2.2 Hz), 7.94-7.85 (5H, m), 7.84-7.71 (17H, m), 7.71-7.65 (2H, m), 7.47-7.43 (2H, m), 7.40-7.28 (8H,
	Hz). m), 5.27 (2H, s), 5.13 (2H, s), 4.68 (2H, t, J = 8.1 Hz,), 3.42-3.35 (2H, m), 2.10-2.00 (2H, m), 1.72-1.60
	(2H, m), 1.58-1.50 (2H, m), 1.42-1.17 (10H, m). 13 C NMR (CD3OD, 100 MHz): δ 162.9, 154.1, 153.8,
	142.6, 140.1, 136.2, 134.9 (d, J = 2.9 Hz), 134.4, 133.3 (d, J = 9.5 Hz), 131.4, 131.3, 130.8, 130.1 (d, J =
	12.5 Hz), 129.4, 129.0, 128.2, 128.1, 128.0, 127.8, 127.8, 127.6, 125.6, 125.0, 123.2, 121.6, 121.5, 3,8-Bis-benzyloxycarbonylamino-5-(6-triphenylphosphonium-hexyl)-6-phenylphenanthridinium 37c 118.56 (d,
	37c was obtained from the phenanthridinium salt 36c (300 mg, 0.34 mmol), following the general
	procedure C. Purification of the crude material by flash chromatography on a reverse phase C18 (0.1%
	TFA in H2O/MeCN from 90/10 to 30/70) afforded the phenanthridinium salt 37c as a deep yellow solid
	(375 mg, 95%). ESI-MS for C59H54N3O4P 2+ , [M] 2+ , 449.8. 31 P NMR (CD3OD, 162 MHz): δ 23.71. 1 H NMR
	(CD3OD, 400 MHz): δ 9.01 (1H, s), 8.89 (1H, d, J = 9.2 Hz), 8.93 (1H, d, J = 9.2 Hz), 8.27 (1H, dd, J = 9.2
	Hz, J = 2.2 Hz), 7.90 (1H, d, J = 2.2 Hz), 7.89-7.63 (22H, m), 7.40-7.31 (10H, m), 5.14 (4H, s), 4.68 (2H, t,
	J = 7.6 Hz), 3.43-3.34 (2H, m), 2.09-1.98 (2H, m), 1.75-1.61 (2H, m), 1.60-1.50 (2H, m), 1.48-1.38 (2H,
	m). 13 C NMR (CD3OD, 75 MHz): δ 164.6, 155.7, 155.4, 144.0, 141.7, 137.8, 137.6, 136.50 (d, J = 2.8 Hz),
	135.9, 134.9 (d, J = 9.9 Hz), 132.9, 132.4, 131.7 (d, J = 12.6 Hz), 130.9, 129.8, 129.7, 129.6, 129.4, 129.2,
	129.1, 127.3, 126.6, 124.8, 123.2, 120.0 (d, J = 86.4 Hz), 107.6, 68.3, 68.1, 56.1, 30.7, 30.0, 26.9, 23.2
	(d, J = 4.3 Hz), 23.1 (d, J = 51.0 Hz).
	20 was obtained from the phenanthridine 19 (0.92 g, 0.92 mmol) following the general procedure B to
	afford 20 as a deep yellow solid (100 mg, 26 % yield). ESI-MS (m/z) for [C30H30N3O4] + , [M] + , 496.4. 1 H 3,8-Bis-benzyloxycarbonylamino-5-(10-triphenylphosphonium-decyl)-6-phenylphenanthridinium NMR (400 MHz, MeOD-d4): δ (ppm) 8.89 (d, J = 9.17 Hz, 1H), 8.83 (d, J = 9.70 Hz, 2H), 8.18 (dd, J = 8.93, 1.59 Hz, 1H), 7.91 (d, J = 8.92 Hz, 1H), 7.87-7.82 (m, 3H), 7.72-7.70 (m, 2H), 4.88 (t, J = 8.93 Hz, 2H), 37d
	2.37-2.34 (m, 2H), 2.27-2.23 (m, 3H), 2.12 (t, J = 2.45 Hz,1H). 13 C NMR (100 MHz, MeOD-d4): δ (ppm)
	163.25, 154.23, 153.98, 142.61, 140.12, 134.35, 131.38, 131.21, 130.75, 129.51, 129.03, 128.13,

  calcd. for C39H35N3P [M] + 576.2563, found 576.2561. 31 P NMR (CD3OD, 162 MHz): δ 23.46.

	1H), 5.68 (s, 2H), 5.63 (s, 1H), 4.14 (s, 2H), 2.05 (s, 3H). 13 CNMR (MeOD, 300 MHz): δ 171.1, 170.1,
	145.5, 143.2, 137.8, 129.8, 129.3, 128.4, 125.4, 124.9, 120.4, 119.1.
	1 H
	NMR (CD3OD, 400 MHz): δ 7.93-7.54 (18H, m), 7.26-7.02 (4H, m), 8.90 (2H, d, J = 7.1 Hz), 6.76 (1H, dd,
	J = 8.4 Hz, J = 0.73 Hz), 6.61 (2H, d, J = 12.9 Hz), 4.61 (1H, s), 4.15-3.90 (2H, m), 3.80-3.69 (1H, m), 3.65-

3,8-Diamino-5-(2-triphenylphosphonium-ethyl)-2-hydroxy-6-phenylphenanthridinium (2-OH-Mito2- E + )

  MHz): δ 20.58.1 H NMR (CD3OD, 300 MHz): δ 8.43 (1H, d, J = 9.2 Hz), 7.99-7.94 (4H, m), 7.82-7.56 (20H, m), 6.38 (1H, d, J = 2.3 Hz), 5.05-4.98 (2H, m), 4.02-3.92 (2H, m).

	P NMR (CD3OD, 121.5
	2-OH-Mito2-E + was obtained from the Mito2-HE (5.0 mg, 0.0072 mmol), following the general
	procedure E. The supernatant was purified by semi-preparative HPLC, using a Beckman C18 column
	(Beckmann Ultrasphere, 250 µm, 10 mm × 5 mm) equilibrated with 20% MeCN in aqueous solution.
	The compounds were eluted by increasing the content of MeCN from 20% to 50% over 16 min at a
	flow rate of 4 mL/min. The pure fractions collected at 15 min were combined to afford 2-OH-Mito2-E +
	as a red solid (5.5 mg, 94%). HRMS calcd. for C39H34N3OP 2+ [M] 2+ 295.6214, found 295.6213.

  ). HRMS calcd. for C47H50N3OP 2+ [M] 2+ 351.6840, found 351.3839. 31 P NMR (CD3OD, 121 MHz): δ 23.77. 1 H NMR (CD3OD, 300 MHz): δ 8.39 (1H, d, J = 9.3 Hz), 7.92-7.86 (5H, m), 7.82-7.12 (18H, m), 7.58-7.54 (3H, m), 7.39 (1H, s), 6.45 (1H, d, J = 2.3 Hz), 4.51 (2H, t, J = 7.7 Hz), 3.42-3.30 (2H, m), 1.97-1.87 (2H, m), 1.70-1.62 (2H, m), 1.57-1.50 (2H, m), 1.31-1.14 (10H, m).

  ). HRMS-ESI m/z calc. for C31H42N4O 2+ [M] 2+ 243.1674, found 243.1679. 1 H NMR (CD3OD, 400 MHz): δ 8.60 (H10, d, J = 9.4 Hz), 8.1 (H1, s), 7.88-7.83 (3H, H9, m), 7.76-7.72 (2H, m), 7.42 (H4, s), 6.33 (H7, d, J = 2.6 Hz), 4.75-4.69 (2H, m), 3.37-3.30 (4H, m), 3.27 (2H, t, J = 8.6 Hz), 3.16 (6H, s), 2.97 (3H, s), 2.94 (6H, s), 2.47-2.37 (2H, m), 1.28 (6H, t, J = 7.3 Hz). 13 C NMR (CD3OD, 100 MHz):

	δ 157.9 (C6), 154.6 (C2), 151.3 (C8), 147.4 (C3), 133.6, 132.8, 131.3, 130.0, 129.5 (C11), 127.8 (C14), 127.2
	(C13), 126.3 (C9), 124.43 (C10), 124.35 (C12), 109.0 (C7), 107.3 (C1), 106.1 (C4), 58.1, 57.3, 51.5, 48.0, 43.2,
	40.1.

  mg, 63%). HRMS calcd. for C78H66N6P2 4+ [M] 4+ 287.1201, found 287.1200.

	31 PNMR (CD3OD, 162 MHz): δ 20.73, 1 HNMR (CD3OD, 400 MHz): δ 8.77 (1H, s), 8.71-8.59 (2H, m), 7.99-
	7.94 (5H, m), 7.82-7.59 (32H, m), 6.46 (1H, d, J = 2.4 Hz), 5.18-5.07 (2H,m), 5.06-4.96 (1H,m), 4.23-4.02
	(3H, m).

  [M] 4+ 343.1827 found 343.1828. J = 291.6 Hz), 107.4 (C4), 68.1 (C6),60.6, 59.6, 59.5, 49.8, 49.4, 48.8, 48.5, 22.6, 9.7.

	139.9 (C14), 132.3 (C13), 132.1, 131.5, 129.3, 129.1 (C1), 128.5 (C10), 125.1 (C12), 122.6 (C9), 120.8 (C7),
	119.3 (q,
	31 P NMR (MeOD d4, 121.5 MHz): δ 23.78. 1 H NMR (MeOD d4, 300 MHz): δ 8.74 (2H, s), 8.61 (2H, d, J =
	9.3 Hz), 7.90-7.57 (50H, m), 7.57 (2H, dd, J = 9.3 Hz, J = 2.4 Hz), 6.53 (2H, d, J = 2.4 Hz), 4.71-4.64 (m,
	4H), 3.09 (m, 4H), 1.72-163 (m, 4H), 1.56-143 (m, 4H), 1.38-1.13 (m, 24H).

  1, 153.7, 143.2, 139.4, 135.5, 135.2, 133.8, 132.1, 131.2, 130.3, 130.0, 128.63, 128.59, 128.5, 128.4, 128.13, 128.10, 128.0, 125.3, 123.3, 122.3, 121.3, 106.4, 67.54, 67.48, 57.1, 56.9, 51.1, 47.4, 22.1, 7.8.

  (m/z) for [C24H22N3] + , [MH] + , 352.4. 1 H NMR (300.13 MHz, MeOD-d4): δ (ppm) 8.57 (d, J = 9.25, 1H), 8.50 (d, J = 9.06, 1H), 7.78-7.75 (m, 3H), 7.60-7.52 (m, 3H), 7.41-7.34 (m, 2H), 6.45 (d, J = 2.45, 1H), 4.68 (t, J = 8.31, 2H), 2.29-2.24 (m, 2H), 2.15-2.08 (m, 3H). 13 C NMR (75.47 MHz, MeOD-d4): δ (ppm) 160.49, 152.82, 149.50, 136.36, 133.46, 132.16, 130.77, 130.02, 127.73, 129.52, 126.68, 125.91, 123.71, 121.45, 119.59, 110.27, 99.73, 82.24, 71.29, 54.80, 54.02, 28.26, 16.30, 13.91. Synthesis of SBA-E + via Allyl carbamate deprotection (15)

13 C

 13 CP-MAS NMR (101.6 MHz): δ (ppm) 145.8, 129.1, 127.0, 71.3, 53.6, 37.2, 31.0, 22.1, 16.4, 9.7.

Table 3 Absorption maxima (λmax) and extinction coefficient values (ε) for TMeHE oxidation products in ethanol and aqueous solution (10 mM Tris, 1 mM EDTA).

 3

	Ethanol	Aqueous, pH 7
	λmax

Table 4 Absorption maxima (λmax) and extinction coefficient values (ε) for TMeHPr + oxidation products in ethanol and aqueous solution (PBS (50 mM), pH 7.4 with 0.1 mM). Data collected and calculated by Radoslow Michalski, PhD. Aqueous, pH 7 λmax

 4

			ε (λmax)
		(nm)	(L mol -1 cm -1 )
	TMeHPr +	278	1.6 × 10 4
		360	1.4 × 10 4
	2-OH-TMePr ++	282	2.8 × 10 4
		479	5.0 × 10 3
	TMePr ++	334	2.1 x 10 4
		547	5.0 × 10 3

Table 5 Fluorescence excitation and emission maxima for 2-OH-Miton-E + and Miton-E + in the presence of DNA in aqueous solution of Tris buffer and EDTA.

 5

	n	2-OH-Miton-E +		Miton-E +
		λexc	λem	λexc	λem
		(nm)	(nm)	(nm)	(nm)
	4	390	569	520	593
		500
	6	390	565	520	588
		500
	10	380	565	520	588
		500

In this work the regular cells will refers as cell that didn't produce superoxide through activated Nox2, without xanthine and xanthine oxidase or others particular enzyme that produce O2 •-.

The data obtained with the non-targeted probe, HE (Figure30) suggest the formation of additional oxidants in Mito-NP-treated cells. This is also observed in case of tested Mito-HE probes (Figure33) suggesting the mitochondrial localization of the oxidant(s) production. The MitonE + /MitonHE ratio is

Acknowledgments

This work has been realized in team "Structure et Réactivité des Espèces Paramagnétiques" under the direction of Olivier Ouari and Micael Hardy.

3,8-Diamino-5-(10-triphenylphosphoniumdecyl)-6-phenylphenanthridine (Mito10-HE) and 3,8-Diamino-5-(10-triphenylphosphoniumdecyl)-6-phenylphenanthridinium (Mito10-E + )

Mito10-HE and Mito10-E + were obtained from the phenanthridinium salt 37d (79 mg, 0.067 mmol), following the general procedure D. Purification of the crude material by flash chromatography on a reverse phase C18 (H2O/MeCN from 90/10 to 69/31) afforded Mito10-HE as an orange powder (38 mg, 71%) and Mito10-E + as a violet powder (15 mg, 24%).

General procedure I

The functionalized coumarin was synthesized by adapting the procedure described in literature. [73] To a solution of 2-Hydroxycourmarine and pyridine (1.5 eq) in DCM (0.2 M) was added dropwise Bromoacetyl bromide (1.5 eq) at -15°C. After 30 min the reaction was then allowed to stir at room temperature. Then, the mixture was partitioned between DCM and water, the product was extracted with DCM. The combined organics layers were washed with water and dried over Na2SO4. The solvent was removed under vacuum to afford the desired coumarin. 2-oxo-2H-chromen-7-yl 2-bromoacetate 50 was obtained from 7-hydroxycoumarin (1.6 g, 9.9 mmol), following the general procedure I to afford 50 ( 2.7 g, 95%).

General procedure J

A solution of the coumarin derivatives and NaI (3eq) in acetone (0.3 M) was stirred at room temperature for 3 h. The reaction was monitored by LC/MS. After the end of the reaction, the mixture was partitioned between DCM and water, the product was extracted with DCM. The combined organics layers were washed with water and dried over Na2SO4. The solvent was removed under vacuum to afford the desired coumarin. 2-oxo-2H-chromen-7-yl 2-iodoacetate 51 51 was obtained from 50 (625 mg, 2.2 mmol), following the general procedure J to afford 51 (500 mg, 69%). 53a was obtained from 52a (10 mg, 0.012 mmol), following the general procedure M. Purification of the crude material by flash chromatog/raphy on a reverse phase C18 (H2O/MeCN from 9/1 to 55/45) afforded 53a (4.2 mg, 48%).

General procedure N

The deprotection of protected exocyclic amino group have also been performed by adapting the literature method described by Chu et al. [32] A solution of corresponding phenanthridine in HBr (25 mL/mmol) was heated under reflux for 3 days, then neutralized with Na2CO3 to pH 8, extracted twice with DCM, the combined organic layer was washed with water, NaCl (sat.) and dried over Na2SO4, and concentrated under reduced pressure. The crude material was eventually further purified by C-18 reverse phase flash chromatography using aqueous solution of TFA 0.1% and acetonitrile supplemented with TFA 0.1%. Note : the coefficient extinction for the Miton-HE derivatives appear to be 3-fold lower, but they was all in the same rang.

Extinction coefficients

Table 1 Absorption maxima (λmax) and extinction coefficient values (ε) for Miton-HE and their oxidation products in

Table 2 Absorption maxima (λmax) and extinction coefficient values (ε) for Miton-HE and their oxidation products in organic solvents (taken in ethanol unless otherwise indicated). n Miton-HE 2-OH-Miton

Cell culture and treatment conditions

Cell culture and treatment conditions

The MIA PaCa-2 cell line was obtained from the American Type Culture Collection (Manassas, VA), where cells are regularly authenticated. MIA PaCa-2 cells were obtained over the last five years, stored in liquid nitrogen, and used within 20 passages after thawing. Cells were grown at 37°C in 5% CO2. Cells were maintained in DMEM (CAT#11965, Invitrogen, San Diego, CA) containing 10% (v/v) fetal bovine serum.

MIA PaCa-2 or RAW 264.7 cells were cultured in 100 mm dishes and were treated with HE, TMeHE or Miton-HE. Cells were treated with phorbol 12-myristate 13-acetate (PMA), MitoNP (10 µM) 24 h or ATO (10, 20 or 40 µM) 1 h before the addition of HE or Miton-HE to a final concentration of 10 µM. Cells were incubating for 1 hour at 37°C in 5% CO2. After incubation, an aliquot of 100 µL of the medium was collected and immediately frozen in liquid nitrogen. Then, the medium was discarded and the cells were washed twice with ice-cold Dulbecco's phosphate buffered saline (DPBS), and scrape-harvested into 1 mL of DPBS. The samples were centrifuged on minicentrifuge for 1 min. Then, the supernatant was discarded. The cell pellet was then snap frozen in liquid nitrogen.

For HE and TMe-HE experiment,

Frozen cell pellets were placed on ice and were syringe lysed using 10 strokes through a 28 ga needle in 150 µL of 0.1% Triton X-100 in ice-cold phosphate buffered saline containing 1 μM of DAPP as internal standards. Probes and its oxidation products were then extracted by adding 100 µL of the resulting mixture in 100 µL of 0.1% formic acid in MeCN. The samples were incubated on ice for 1 h, and then centrifuged for 30 min at 20 000 g at 4°C. A volume of 100 µL of the supernatant was then transferred to a fresh tube containing 100 µL of 0.1% formic acid in water. This solution was centrifuged for an additional 15 min at 20 000 g at 4°C. A volume of 150 µL of the resulting supernatant was transferred to HPLC vials for analysis.

For Miton-HE experiment,

Frozen cell pellets were placed on ice and were syringe lysed using 10 strokes through a 28 ga needle in 150 µL of 0.1% Triton X-100 in ice-cold phosphate buffered saline containing 1 μM of o-MitoPhCH3 as internal standards (alternatively, 2-OH-Mito6-HE was used as internal standard for Mito2-HE and Mito4-HE experiments). Probes and its oxidation products were then extracted by adding 50 µL of the resulting mixture in 150 µL of 0.1% formic acid in MeCN. The samples were incubated on ice for 1 h, and then centrifuged for 30 min at 20 000 g at 4°C. A volume of 100 µL of the supernatant was then transferred to a fresh tube containing 100 µL of 0.1% formic acid in water. This solution was centrifuged for an additional 15 min at 20 000 g at 4°C. A volume of 150 µL of the resulting supernatant was transferred to HPLC vials for analysis.

HPLC-based measurements of HE, Miton-HE and their oxidation products.

HPLC analyses were performed using an Agilent 1100 system (North Billerica, MA) equipped with absorption and fluorescence detectors and refrigerated autosampler (4°C). The samples (50 μl) were injected into a reverse phase column (Phenomenex, Kinetex C18, 100 mm × 4.6 mm, 2.6 μm) equilibrated with 20% (v/v) MeCN, 80% (v/v) water containing 0.1% (v/v) trifluoroacetic acid. HE and their oxidation products were eluted by increasing the content of MeCN (v/v) from 20% to 56% over 4.5 min at a flow rate of 1.5 ml/min. The absorption detector was used to measure DAPP (at 290 nm; retention time: 2.5 min), HE-E + (at 290 nm; retention time: 4.1 min), E + -E + (at 290 nm; retention time: 4.3 min), and HE (at 370 nm, retention time: 1.6 min). A fluorescence detector (initial excitation at 358 nm and emission at 440 nm) was used to monitor HE. At 2.3 min after the injection, the fluorescence parameters were changed (excitation at 490 nm and emission at 567 nm) for sensitive detection of 2-OH-E + (retention time: 3.2 min) and E + (retention time: 3.3 min).

Mito2-HE and their oxidation products were eluted by increasing the content of MeCN (v/v) from 20% to 60% over 6.7 min (at a flow rate of 1.5 ml/min) and from 60% to 100% over 0.3 min. The absorption detector was used to measure 2-OH-DAPP (at 290 nm; retention time: 2.73 min), DAPP (at 290 nm; retention time: 3.04 min), Mito2-HE (at 242 nm; retention time: 4.45 min), Mito2-E + (at 290 nm; retention time: 6.07 min) and Mito2-E + -Mito2-E + (at 290 nm; retention time: 7.43 min). A fluorescence detector (initial excitation at 490 nm and emission at 590 nm) was used to monitor 2-OH-DAPP (retention time: 2.75 min). At 3.0 min after the injection, the fluorescence parameters were changed (excitation at 358 nm and emission at 440 nm) for sensitive detection of Mito2-HE (retention time: 4.45 min). At 5.0 min after the injection, the fluorescence parameters were changed (excitation at 490 nm and emission at 590 nm) for sensitive detection of 2-OH-Mito2-E + (retention time: 6.19 min) and 2-OH-Mito6-E + (retention time: 7.98 min).

Mito4-HE and their oxidation products were eluted by increasing the content of MeCN (v/v) from 20% to 55% over 5 min (at a flow rate of 1.5 ml/min) and from 55% to 100% over 2 min. The absorption detector was used to measure Mito4-HE (at 370 nm; retention time: 3.60 min), Mito4-E + (at 290 nm; retention time: 4.57 min) and Mito4-E + -Mito4-E + (at 290 nm; retention time: 5.11 min). A fluorescence detector (initial excitation at 358 nm and emission at 440 nm) was used to monitor Mito4-HE. At 3.63 min after the injection, the fluorescence parameters were changed (excitation at 490 nm and emission at 567 nm) for sensitive detection of 2-OH-Mito4-E + (retention time: 4.50 min), Mito4-E + (retention time: 4.59 min) and 2-OH-Mito6-E + (retention time: 5.03 min).

Mito6-HE and their oxidation products were eluted by increasing the content of MeCN (v/v) from 20% to 60% over 5 min (at a flow rate of 1.5 ml/min) and from 60% to 100% over 2 min. The absorption detector was used to measure Mito6-HE (at 370 nm; retention time: 3.88 min), Mito6-E + (at 290 nm; retention time: 4.77 min), Mito6-E + -Mito6-E + (at 290 nm; retention time: 5.15 min) and o-MitoPh-CH3 (at 240 nm; retention time: 5.43 min). A fluorescence detector (initial excitation at 358 nm and emission at 440 nm) was used to monitor Mito6-HE. At 4.4 min after the injection, the fluorescence parameters were changed (excitation at 490 nm and emission at 567 nm) for sensitive detection of 2-OH-Mito6-E + (retention time: 4.68 min) and Mito6-E + (retention time: 4.77 min).

Résumé

The superoxide radical anion (O2 •-) is the proximal species at the origin of the formation of most Free Radicals, which are well known to be at the origin of the oxidative stress. Free radicals are involved in the aging process and almost all diseases. The detection of O2 •-is of special interest to understand the chemical processes involved in the diseases and to monitor the O2 •-production in assay for drug discovery. In this project, we aim to improve the tools that allow the detection of O2 •-. Indeed, currently Hydroethidine (HE), the gold standard in the superoxide detection, suffers from serval limitations. Indeed, the use of HE to detect O2 •-requires harsh post-treatment conditions. Here we aim to design and develop new HE-derivatives, which allow a more direct detection.

Abstract

L'anion radicalaire superoxyde (O2 •-) est à l'origine de la formation de la plupart des radicaux libres, qui sont bien connus pour être à l'origine du stress oxydatif. Les radicaux libres sont impliqués dans le processus de vieillissement et dans presque toutes les maladies. La détection de O2 •-présente donc un intérêt particulier afin de mieux comprendre les processus chimiques impliqués dans les maladies, elle permet également de développer de nouveaux médicaments en surveillant la production de O2 •-grâce à des tests cellulaires. Dans ce projet, nous visons à améliorer les outils permettant la détection de O2 •- . En effet, l'étalon-or Hydroethidine (HE) souffre de certaines limitations, qui font que la détection de O2 •-nécessite un post-traitement drastique. Ici, nous visons donc à développer de nouveaux dérivés de HE pour permettre une détection plus directe.

Abstract

Superoxide radical anion (O2 •-) is the proximal species at the origin of the formation of most Reactive Oxygen Species (ROS). ROS are involved in both physiological redox signaling processes and pathological processes leading to oxidative damage to biomolecules. Therefore, the detection and the quantification of this radical anion are of major interest. So far, Hydroethidine (HE), use as a fluorogenic probe, is the gold standard to evaluate the O2 •-level in biological systems. O2 •-reacts with HE to give 2-hydroxyethidium (2-OH-E + ) as a specific marker, this reaction is mediated by the formation of a radical cation intermediate HE •+ . However, in biological models, HE can also react with heme proteins or other biological relevant species leading to Ethidium (E + ). Unfortunately, the fluorescence spectra of 2-OH-E + and E + overlap, which limits the measurement of the O2 •-level directly by fluorescence. The conventional assay of the O2 •-level requires thereby HPLC separation. Since HE was not designed for the superoxide detection, we aim in this project to develop new HE-based probes to understand the mechanism between HE and the superoxide, to limit the non-specific oxidation of the probe, to modulate its site-specific detection and to improve its ability to detect O2 •-in biological systems. These new probes and their oxidation products have been synthesized and characterized by fluorescence spectroscopy. The efficiency of some newly synthesized HE-based probes to react with O2 •-has been evaluated by HPLC in the cell-free system using hypoxanthine/xanthine oxidase and in intact MiaPaCa-2 or RAW 264.7 cells. Pulse radiolysis of some new synthesized HE-derivatives have also been performed by the partners of the project. Their results show that the lifetime of the radical cation, which traps O2 •-, of the newly synthesized TMeHPr + has been increased compared to the gold standard.