Synthesis and Characterization of Two-Photon Absorbing Photosensitizers for Application to Photodynamic Therapy : Targeting of Retinoblastoma

Depuis quelques années, l’Institut Curie s’est engagé, aux cotés de l’association Rétinostop,dans la lutte contre le rétinoblastome, une tumeur rétinienne chez l’enfant en bas âge. Nous pensons que l’utilisation de la Photothérapie Dynamique (PDT)permettrait d’éviter les effets secondaires néfastes auxquels les thérapies utilisées actuellement donnent lieu. Dans ce cadre, les travaux au laboratoire se sont portés sur la préparation de photo sensibilisateurs (PS) glycoconjugués permettant de cibler les cellules tumorales par interactions spécifiques avec les récepteurs lectiniques surexprimés par ces dernières.Afin d’éviter les dommages liés à l’exposition à la lumière des tissus sains à proximité de la tumeur, notre intérêt s’est récemment porté sur l’Absorption à Deux Photons (ADP), phénomène localisé et permettant l’excitation sélective duPS dans le volume de la tumeur. Cette technique permettrait d’épargner les tissus rétiniens fragiles.Ce rapport présente les travaux effectués au laboratoire sur le développement de PS absorbant à deux photons (PS-ADP) glycoconjugués pour le ciblage du rétinoblastome. Ceux-ci sont articulés autour de trois concepts moléculaires.Les oligomères de porphyrines ont été rapportés comme de bons candidats pour la PDT à Absorption Deux Photons (PDT-ADP). La préparation d’analogues glycoconjugués a été réalisée et leurs propriétés photophysiques et biologiques analysées. Ces composés présentent une haute section efficace ADP et de bons rendements quantiques de production d’oxygène singulet. Une étude complémentaire est en cours de façon à améliorer leurs propriétés d’internalisation cellulaire.Ces composés possèdent également des propriétés de fluorescence sensibles à la température et pourraient constituer un point de départ intéressant pour le développement de sondes fluorescentes ratiométriques de températures.Les hybrides conjugués de porphyrine et triphénylamines (PTP) présentent également des propriétés prometteuses en vue de leur application à la PDT-ADP.Nous proposons une étude de l’influence de plusieurs groupements sur ces propriétésparmi lesquels les substituants de la triphénylamine, les substituants des phénylesméso de la porphyrine (triéthylène glycol ou diéthylène glycol mannosyles)et un atome de zinc internalisé dans le macrocycle tétrapyrrolique. Les hybrides préparés présentent un excellent rendement quantique de production d’oxygènesingulet dans les solvants polaires et une bonne section efficace ADP. Des difficulté sont été rencontrés quant à leur internalisation cellulaire.Une seconde famille d’hybrides méthylés a été préparée s’appuyant sur les propriétés d’exaltation des triphénylamines cationiques par interaction avec l’ADN.Ces composés présentent de faibles propriétés de fluorescence et de photosensibilisation dans l’eau. Dans le glycérol l’émission de fluorescence est restaurée grâce à un transfert d’énergie entre les deux entités. Ces composés possèdent une bonne phototoxicité in vitro et permettent l’imagerie de fluorescence de cellules.The Institut Curie is strongly committed to the development of new treatments ofchildhood eye cancer (or retinoblastoma). Existing therapies often lead to severeside effects which could be avoided by the use a milder treatment techniques. Webelieve Photodynamic Therapy (PDT) provides a promissing alternative as a nonmutagenic and efficient therapy. Over the last decade, our laboratory has developpeda new strategy for the targeting of retinoblastoma through the synthesis ofglycoconjugated photosensitizers (PS) displaying a strong affinity for membranereceptors known as lectins overexpressed by the tumor cells.In order to avoid the damages to healthy tissues close to the tumour, we haveadapted our targeting strategy to Two-Photon Absorption Photodynamic Therapy(TPA-PDT). Two-Photon Absorption (TPA) is a localized excitation mechanismwhich allows strict control of the PDT effect preventing the killing of the retiniancells next to the tumor.This work presents the progress in the development of retinoblastoma targetedTPA-PDT. Three fields have been investigated.Porphyrin oligomers have been reported to possess high two-photon cross sectionsand have shown high potential for their application to TPA-PDT.We synthesizedglycoconjugated analogs of these oligomers and analyzed their photophysicaland biological properties. These compounds show high TPA cross section as wellas high singlet oxygen production quantum yields making them good candidatesfor TPA-PDT. A supplementary study is underway to enhance their cellular internalisation.They also display temperaure dependent fluorescence emission that could allowtheir use as fluorescent ratiometric temperature probes.Porphyrin-triphenylamine hybrids (PTP) are also promissing as two-photonabsorbing photosensitizers (TPA-PS). We studied the influence of different moieties,such as triphenylamine substitutents, meso-phenyl substitutent (glycosylatedor triethylene glycol) and the intern zinc atom, on their photophysical and biologicalproperties. Excellent singlet oxygen generation in polar solvent as well asgood TPA cross sections were measured. Difficulties concerning their cellular internalisationwere encountered and are currently being solved.A second family of cationic hybrids was prepared. Their behaviour relies onthe fluorescence exaltation properties of methylated triphenylamines upon bindingto DNA. These compounds display low emitting and photosensitizing propoertiesin water which can be restored by interaction with DNA. An energy transfer fromthe triphenylamine to the porphyrin was observed in glycerol. The methylatedhybrids show good in vitro phototoxicity toward cancer cells and allow fluorescenceimaging applications

Controlling the Reactivity of Enzymes in Mechanochemistry: Inert Surfaces Protect β-Glucosidase Activity During Ball Milling

Although cellulose has been identified as the foremost candidate for the replacement of fossil fuels, its recalcitrant nature prevents the full deployment of technologies based on its saccharification. We recently reported a possible strategy to resolve this conundrum: using cellulases under “RAging” - a solvent-free process that utilizes enzymes under mechanochemical conditions - to achieve fast, efficient hydrolytic depolymerization of cellulosic materials into glucose. β-Glucosidases catalyze the last and often limiting step of this process, i.e. the formation of glucose from cellobiose. Here, we reveal the high sensitivity of β-glucosidases to ball milling, as well as an unexpected stabilization effect of inert surfaces, enabling the protection of β-glucosidases under mechanochemical treatment. This approach provides an unexpected strategy to control the reactivity of enzymes under mechanochemical conditions. Finally, our results also provide the very first demonstration of enzymatic equilibrium under mechanochemical conditions

Regioselective Epoxidations by Cytochrome P450 3A4 Using a Theobromine Chemical Auxiliary to Predictably Produce N-Protected β- or γ- Amino Epoxides

N-Protected β- and γ-amino epoxides are useful chiral synthons. We report here that the enzyme cytochrome P450 3A4 can catalyze the formation of such compounds in a regio- and stereo-selective manner, even in the presence of multiple double bonds or aromatic substituents. To this end, the theobromine chemical auxiliary is used not only to control the selectivity of the enzyme, but also as a masked amine and to facilitate product recovery. Theobromine predictably directed epoxidation at the double bond of the fourth carbon from the theobromine group. Unlike with most catalysts, the selectivity did not depend on electronic or steric factors but rather on the position of the olefin relative to the theobromine group

Mechanoenzymatic Breakdown of Chitinous Material to N-Acetylglucosamine: The Benefits of a Solvent-Free Environment

We report an innovative and efficient mechanoenzymatic method to hydrolyze chitin to N-acetylglucosamine monomer using chitinase under the recently developed RAging methodology, i.e. a combination of mechanochemical ball milling activation and accelerated aging repeated over many cycles, in the absence of bulk solvent. Our result show that the activity of chitinases increases several times by switching from traditional solvent-based conditions of enzymatic catalysis to solvent-free RAging, which operates on moist solid substrates. Importantly, RAging is also highly efficient for the production of N-actylglucosamine directly from shrimp and crab shell biomass without any other processing, except for a gentle wash with aqueous acetic acid.<br /

Influence of carbohydrate biological vectors on the two-photon resonance of porphyrin oligomers.

International audienceWe propose a spectroscopic study of π-conjugated porphyrin dimers and a triphenylamine centered trimer bearing monoethyleneglycol-peracetylated α-mannose targeting moieties, which were synthesized for application to two-photon absorption photodynamic therapy (2PA-PDT). The collected spectroscopic data are explained through comparison with previous results obtained on similar nonvectorized analogues. It appears that the different shifts and variations in linear and nonlinear absorption and emission bands are mainly due to an increased twist between the tetrapyrrolic units. The 2PA properties of the triphenylamine-centered trimer have been studied, and two fluorescence emission bands are observed that seem to originate from two different 2PA excitation pathways. These results have important implication for the design of new vectorized photosensitizers for 2PA-PDT

Vinyl-diazine triphenylamines and their N-methylated derivatives: synthesis, photophysical properties and application for staining DNA

International audienceIn this paper we describe the synthesis of vinyl-diazine triphenylamines. These compounds exhibit strong green-yellow fluorescence in dichloromethane solution, important emission solvatochromism and acidochromism. Regioselective N-alkylation of these dyes provides cationic compounds that exhibit affinity for double-stranded DNA. Binding to the biopolymer results in a strong bathochromic shift and increase of the emission intensity

Efficient Enzymatic Hydrolysis of Biomass Hemicellulose in the Absence of Bulk Water

Current enzymatic methods for hemicellulosic biomass depolymerization are solution-based, typically require a harsh chemical pre-treatment of the material and large volumes of water, yet lack in efficiency. In our study, xylanase (E.C. 3.2.1.8) from Thermomyces lanuginosus is used to hydrolyze xylans from different sources. We report an innovative enzymatic process which avoids the use of bulk aqueous, organic or inorganic solvent, and enables hydrolysis of hemicellulose directly from chemically untreated biomass, to low-weight, soluble oligoxylosaccharides in &gt;70% yields

Small molecule restores itaconate sensitivity in Salmonella enterica: a potential new approach to treat bacterial infections

In the context of increasing global antibiotic resistance, the need for alternative therapeutic targets is great. Although new antibiotics and resistance inhibitors provide temporary solutions, they are bound to become obsolete. In this work, we propose a new approach, coined “bacterio‐modulation” that aims to restore macrophage potency towards bacterial strains that are able to survive in phagolysosomes. One key defense in the macrophage's arsenal is itaconate, an endogenous molecule with antimicrobial activity. Some intracellular pathogens have evolved to produce itaconate‐degrading enzymes, which are required for intracellular proliferation and to promote pathogenicity. We herein present the first molecule able to resensitize Salmonella enterica to itaconat

Carbohydrate-conjugated porphyrin dimers: synthesis and photobiological evaluation for a potential application in one-photon and two-photon photodynamic therapy.

International audienceWe report the synthesis of bioconjugated zinc porphyrin dimers 1a-e designed as photosensitizers for one-photon and two-photon excited photodynamic therapy. These macrocycles are substituted with carbohydrate units (glucose, mannose, lactose) in order to target tumor cells over-expressing lectin membrane receptors. Polarity, singlet oxygen production and in vitro photocytotoxicity are studied to determine their photodynamic therapy potentiality

Glycoconjugated porphyrin dimers as robust ratiometric temperature sensors

International audienceWe report the properties of glycoconjugated porphyrin dimers behaving as highly sensitive ratiometric temperature sensors in water. This effect results from interactions between carbohydrate and water altering molecular relaxation kinetics leading to temperature sensitive dual emission. These dimers are robust ratiometric fluorescent probes over a large temperature window (20-90 °C