New photocleavable pyranine derivatives, their method of preparation and their uses

Abstract

The present invention relates to new caged pyranine fluorophores of the following formula (I), (I) or a salt thereof, and to composition comprising thereof. or a salt thereof. Another object of the invention relates to a method for measuring a light intensity of a light source in a medium using caged pyranine fluorophores or composition comprising thereof. The present invention also concerns a new method for preparing caged pyranine fluorophores.

Description

[0001] New photocleavable pyranine derivatives, their method of preparation and their uses Filed of the invention The present invention relates to new photocleavable pyranine derivatives useful as photochemical tool for measuring light parameters of a light source such as light intensity. The present invention also relates to a method for preparing the photocleavable pyranine. Background of the invention Light is both a trigger and a reactant, which is endowed with attractive features. Indeed, it is essentially non-invasive, can be delivered with exclusive temporal and spatial resolution, and be used as well for introducing a perturbation and reading out its impact. Moreover, many light sources are nowadays available to deliver various photon fluxes in different wavelength ranges. Light has accordingly found ubiquitous exploitation in many academic and applied fields of chemistry and biology. For instance, chemists have recently implemented light to drive photocatalysis where reactants are converted into products by dual action of a light-activated photocatalyst which activates at its turn a metallic catalyst (see Cambié et al. Chem. Rev. 2016, 116, 10276-10341 and Ravelli, D. et al. Chem. Soc. Rev. 2009, 38, 1999-2011). Biologists have introduced optogenetics, which provides powerful tools to optically manipulate or record physiological processes at the cellular level (G. Miesenbock. Science, 326:395–399, 2009). Despite its attractive features, light as a trigger or a reactant often suffers from the lack of characterization of the light sources, which makes difficult to compare the results obtained in various groups as well as to secure reproducibility. Light meters in which a detector integrates the overall photon flux over an illuminated surface are not often available in the laboratories of chemistry and biology (see Mergerle, U. et al. Photochem. Photobiol. Sci. 2010, 9, 1400-1406). Moreover, their proper use requests technical expertise and they do not give access to the spatial profile of the light excitation nor to the light flux per unit of surface. In contrast, actinometers can yield the photon flux density at a sample from imaging the time dependence of the extent of their photochemical reaction (see Kuhn, H. J. et al. Pure Appl. Chem. 2004, 76, 2105-2146 and Reinfields et al. ChemPhotoChem 2019, 3, 441-449). However, most of them rely on absorbance for reporting, which is not enough sensitive to measure light intensity in several geometries (e.g. for calibrating illumination of microscopes). Fluorescent actinometers are attractive to overcome this limitation. Most photoactivatable fluorophores switch from a dark to a bright state under illumination. Obtained by grafting a caging group which acts as a fluorescence quencher or as dark precursors in which fluorescence is restored upon light irradiation, they have been exploited in numerous fields (investigation of cell lineage, spatiotemporal interrogation of fluid flows, monitoring of dynamic processes, reconstruction of images with subdiffraction resolution (P. Klán et al., Chem. Rev., 2013, 113, 119-191). However, they have only been seldom characterized in order to yield quantitative information on the absolute value of the light intensity (Kiskin, N. I. et al., Eur. Biophys. J., 2002, 30, 588-604. Haugland, R. P. et al. have described in 1996 photoactivable caged fluorophores comprising a 8- hydroxypyren-1,3,6-trisulfonic acid scaffold (HPTS) substituted with a photolabile protecting group (US 5,514,710). The photolysis of the protecting group results in liberation of a free hydroxypyrenesulfonic acid dye. However, the synthesis of these caged fluorophores required tedious purifications. Further, the three ionizable sulfonic acid groups present on the fluorophores of Haugland et al. render them suitable for application in aqueous solutions but non usable in organic solvents nor for biological applications requiring internalization into cells. There is thus a need for new photoactivable derivatives, easy to synthesize, that can be used efficiently in various environments such as organic solvents or biological medium, to calibrate light intensity from different sources, in particular to measure light intensity. Summary of the invention Surprisingly, the inventors have developed new caged fluorophores as photoactivable tool, which can be implemented to calibrate light intensity with one- and two-photon excitation under different geometries. The photoactivable derivatives of the invention may be used for example in a biological medium for allowing cell labeling or in organic solvents, in particular to characterize light sources in photocatalysis and in fields of daily life. According to a first aspect, the present invention relates to new caged pyranine fluorophores of the following formula (I): or a salt thereof, wherein: X is an aryl or a heterocyclyl, the aryl and heterocyclyl being optionally substituted with one or more, preferably one to five substituents selected in the group consisting of C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, C1-C6 haloalkoxy, oxo, OH, NRR’, halogen, CN, NO2, C(O)OH, C(O)-O-C1-C6 alkyl, -C(O)H, C(O)-C1- C6 alkyl, C(O)-aryl and aryl, with R and R’ being each independently H or C1-C6 alkyl, L is a linker corresponding to a divalent radical derived from a C1-C10 aliphatic chain, wherein one or more, preferably 1, 2, 3, or 4, methylene unit(s) of the C1-C10 aliphatic chain are optionally replaced by –O– or – C(=O)–, wherein said C1-C10aliphatic chain is optionally substituted with one or more, preferably one, substituent selected from OH, C1-C6haloalkyl, C1-C6alkoxy, NR’’R’’’, halogen, C(O)OH, C(O)-O-C1-C6alkyl, C(O)H, C(O)-C1-C6alkyl, C(O)-O-aryl, C(O)-aryl and aryl, with R’’ and R’’’ being each independently H or C1- C6 alkyl, and R1, R2and R3each independently represent NR4R5or OR6, R4and R5each independently represent a C4-C18 alkyl, wherein up to 4 methylene units of the C4-C18 alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least two methylene units of the C4-C18alkyl are not replaced, or R4and R5together with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing R4and R5, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, such as a methyl, R6represents H, a C1-C18 alkyl or an aryl, said C1-C18 alkyl and aryl being optionally substituted with one or more, preferably one or two, substituents selected in the group consisting of OH, halogen, C1-C6 haloalkyl, C1-C6alkoxy and C1-C6haloalkoxy, provided that X is not when all of R1, R2and R3are OH and L is -CH2-. According to a second aspect, the present invention relates to a composition comprising a compound of formula (I) as defined above and a medium, the medium being preferably selected from an organic solvent and an aqueous medium. According to a third aspect, the present invention relates to the use of a compound of the following formula (II) or a composition comprising thereof to measure light intensity: X or a salt thereof, wherein X is an aryl or a heterocyclyl, the aryl and heterocyclyl being optionally substituted with one or more, preferably one to five substituents selected in the group consisting of C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, C1-C6haloalkoxy, oxo, OH, NRR’, halogen, CN, NO2, C(O)OH, C(O)-O-C1-C6alkyl, -C(O)H, C(O)-C1- C6alkyl, C(O)-aryl and aryl, with R and R’ being each independently H or C1-C6alkyl, L is a linker corresponding to a divalent radical derived from a C1-C10 aliphatic chain, wherein one or more, preferably 1,2,3, or 4, methylene unit(s) of the C1-C10 aliphatic chain are optionally replaced by –O– or – C(=O)–, wherein said C1-C10 aliphatic chain is optionally substituted with one or more, preferably one, substituent selected from OH, C1-C6 haloalkyl, C1-C6 alkoxy, NR’’R’’’, halogen, C(O)OH, C(O)-O-C1-C6 alkyl, C(O)H, C(O)-C1-C6alkyl, C(O)-O-aryl, C(O)-aryl and aryl, with R’’ and R’’’ being each independently H or C1- C6alkyl, Ra, Rband Rceach independently represent NRiRiior OR6, and Riand Riieach independently represent H or C1-C18 alkyl, wherein up to 4 methylene units of the C4-C18 alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least one methylene units of the C4-C18alkyl are not replaced, or Riand Riitogether with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing Riand Rii, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, such as a methyl, R6represents a H, C1-C18 alkyl or an aryl, said C1-C18 alkyl and aryl being optionally substituted with one or more, preferably one or two, substituents selected in the group consisting of OH, halogen, C1-C6 haloalkyl, C1-C6 alkoxy and C1-C6 haloalkoxy. According to a fourth aspect, the present invention relates to a method for measuring a light intensity of a light source in a medium comprising the steps of: (a) Adding a compound of formula (II) as defined above to a medium, (b) Illuminating the medium comprising the compound of formula (II) obtained at step (a) with a light source for a time t1, (c) Measuring the increase of fluorescence during the time t1 and determining the light intensity. According to a fifth aspect, the present invention relates to a method for preparing a compound of formula (II) as defined above, comprising the steps of: (i) Reacting 8-hydroxypyren-1,3,6-trisulfonic acid trisodium salt (HPTS.3Na) with a compound of formula (III): X-L-LG (III) in which X and L are as defined above for compound (II) and LG is a leaving group, in order to form the following trisodium salt (IIa): wherein the HPTS.3Na is not converted into the corresponding free sulfonic acid before reacting with the compound of formula (III), (ii) optionally converting the salt (IIa) obtained at step (a) into a compound of formula (II) or a salt thereof in which Ra, Rband Rceach independently represent NRiRiior OR6or (ii’) optionally converting the salt (IIa) obtained at step (a) into a compound of formula (II) in which Ra, Rband Rcrepresents OH or a salt thereof other than the trisodium salt (IIa). Definitions According to the present invention, the article “a” or “an” means one or more than one, as for example in the expression “composition comprising a compound of formula (I)” means one or more than one compound of formula (I). In the illustrations of the present disclosure, wavy lines represent the point of attachment to the rest of the molecule. The term “halogen”, as used in the present invention, refers to a fluorine, bromine, chlorine or iodine atom. The term “Cx-Cyaliphatic chain" designates a linear or branched hydrocarbon chain, completely saturated or containing one or more unsaturations, but not aromatic, comprising from x to y carbon atoms, preferably from 1 to 6 carbon atoms. According to the present invention, the term "aliphatic chain" includes linear or branched, alkyl, alkenyl or alkynyl groups. The term “Cx-Cyalkyl”, as used in the present invention, refers to a linear or branched monovalent saturated hydrocarbon chain containing from x to y carbon atoms, for example from 1 to 18 or from 1 to 6, including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n- pentyl, n-hexyl, and the like. The term “Cx-Cy alkenyl”, as used in the present invention, refers to a linear or branched monovalent unsaturated hydrocarbon chain containing from x to y carbon atoms, for example from 2 to 6, and comprising at least one double bond including, but not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like. The term “Cx-Cyalkynyl”, as used in the present invention, refers to a linear or branched monovalent unsaturated hydrocarbon chain containing from x to y carbon atoms, for example from 2 to 6, and comprising at least one triple bond including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like. The term “Cx-Cy alkoxy”, as used in the present invention, refers to a group of formula O-Cx-Cyalkyl, in which the Cx-Cy alkyl moiety is as defined above. It can be a methoxy group also refers as O-methyl. The term "aryl", as used in the present invention, refers to an aromatic hydrocarbon group preferably comprising from 6 to 12 carbon atoms and comprising one or more fused rings, such as, for example but not limited to, a phenyl or naphthyl group. Advantageously, it is a phenyl group. The term “heterocyclyl” as used in the present invention, refers to an aromatic or non-aromatic, saturated or unsaturated, monocyclic or polycyclic group (including fused, bridged or spiro rings) comprising preferably from 5 to 10 atoms in the ring, in which one or several, notably one or two, advantageously one, atom(s) of the ring are each independently a heteroatom selected in the group consisting of a sulfur atom, an oxygen atom and a nitrogen atom, the other atom(s) of the ring being carbon atom(s). It is understood that the expression “4 to 6-membered heterocyclyl” refers to a heterocyclyl group as defined herein comprising from 4 to 6 atoms in the ring. In particular, the heterocyclyl is a 2H-1-benzopyran group (also called 2H-chromene). The term "Cx-Cy haloalkyl" refers to a Cx-Cy alkyl chain as defined above wherein one or more hydrogen atoms are replaced by a halogen atom selected from fluorine, chlorine, bromine or iodine, preferably a fluorine atom. For example, it is a CF3 group. The term "C1-C6haloalkoxy" refers to a C1-C6alkoxy as defined above wherein one or more hydrogen atoms are replaced by a halogen atom selected from fluorine, chlorine, bromine or iodine, preferably a fluorine atom. For example, it is a O-CF3group. In the context of the present invention, “unsaturated” means that the hydrocarbon chain may contain one or more unsaturation(s), i.e. a double bond C=C or a triple bond C≡C, advantageously one. The terms “coumarin derivative” and “2-benzopyronyl group” both refers to the following group, linked to the rest of the molecule via one of its carbon atoms: The term “leaving group” refers to an atom or group of atoms that are easily able to break away from a molecule with a lone pair, breaking the bond between it and the molecule. Such a leaving group can be in particular a halogen atom, a sulfonate, a N-succinimidyloxy, a 4-nitro-phenyloxy, pentafluorophenoxy or a N-benzotriazoloxy. The sulfonate is in particular a group –OSO2-RLG with RLG representing a (C1- C6)alkyl, aryl, aryl-(C1-C6)alkyl or (C1-C6)alkyl-aryl group, the said group being optionally substituted with one or several halogen atoms such as fluorine atoms. The sulfonate can be notably a mesylate (CH3- S(O2)O-), a triflate (CF3-S(O)2O-) or a tosylate (p-Me-C6H4-S(O)2O-). The term “light intensity” refers to the number of incident photon(s) from a light source on a sample that is illuminated. It is often measured in Watt (W). Alternatively, it can be expressed in mole of photon per unit of time (E.s-1). The term “light power” may be used also in place of “light intensity”, in particular in the case of a light source with a two-photon excitation. The term “photon flux density” refers to the number of photons provided by a light source and passing through a particular area per unit of time. It is expressed in mole of photon per unit of time and unit of surface (E.m-2.s-1). The term “molar absorption coefficient (ε)” refers to the measure of the ability of a chemical species to absorb light at a given wavelength. The SI unit of molar absorption coefficient is the square meter per mole (m2 / mol). It can also be expressed in M−1⋅cm−1or L⋅mol−1⋅cm−1. The term “uncaging” in the context of the present invention refers to the cleavage under illumination of a photolabile group from the molecule to which said photolabile group is attached. In particular, the uncaging as used herein refers to the transformation of a caged pyranine derivative of formula (I) into the corresponding free pyranine derivative. In other terms, the uncaging refers to the cleavage of the group X-L leading to the liberation of a free pyranine derivative. The position bearing the group X-L in the caged pyranine derivative of formula (I) bears an OH group after the uncaging. The term “quantum yield of uncaging” refers to the probability of liberating a molecule protected by a photolabile protecting group after light absorption. The term “uncaging cross section (σ)” refers to the product of the molar absorption coefficient by the quantum yield of uncaging. The term “characteristic time of photolysis / photoconversion (τ)” refers to the time extracted from applying a monoexponential fitting function on the time evolution of the initial time-normalized concentration of a photosensitive molecule or a molecule protected by a photolabile protecting group upon illumination. In the context of the present invention, the characteristic time is obtained from the following function: in which t is the time of acquisition, A is the amplitude of the increase of the fluorescence signal occurring upon illumination, and τ is the characteristic time. Detailed description Compound of formula (I) According to the present invention, X is an aryl or a heterocyclyl, the aryl and heterocylyl being optionally substituted with one or more, preferably 1, 2, 3, 4 or 5, substituents selected in the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, OH, oxo, NRR’, halogen, CN, NO2, C(O)OH, C(O)-O-C1-C6 alkyl, - C(O)H, C(O)-C1-C6 alkyl, C(O)-aryl and aryl, with R and R’ being each independently H or C1-C6 alkyl. According to some embodiments, X is an aryl (e.g. phenyl) or a heterocyclyl (e.g.2H-1-benzopyranyl, in particular 2H-1-benzopyran-4-yl), unsubstituted or substituted with one or more, preferably one, two or three, substituents selected in the group consisting of C1-C6alkyl, such as methyl, C1-C6alkoxy, such as methoxy, C1-C6haloalkyl, such as trifluoromethyl, oxo, OH, NH2, NH-C1-C6alkyl, N(C1-C6alkyl)2, halogen, CN, NO2and aryl, such as phenyl. According to some embodiments, X is selected in the group consisting of: which Rxand Ryare each independently H or C1-C6 alkyl, notably a methyl, Rxis a phenyl or a C1-C6alkyl, notably a methyl, Rxis H or a C1-C6 alkyl such as methyl, Ryis H, a C1-C6 alkyl such as methyl, a O- C1-C6 alkyl such as O-methyl, F, CF3, OCF3 or CN, Rwis H, a C1-C6 alkyl such as methyl, a O-C1-C6 alkyl such as O-methyl or F and Rzis H or F, Rxis H, a C1-C6alkyl such as methyl, a O-C1-C6alkyl such as O-methyl or CN, Ryis H, a C1-C6 alkyl such as methyl or a O-C1-C6 alkyl such as O-methyl, Rzis H, a C1-C6 alkyl such as a methyl, a O-C1-C6alkyl such as a O-methyl, Cl, F, OH, NH2, N(C1-C6alkyl)2or phenyl, in particular , Rxis H, phenyl or a O-C1-C6alkyl such as a O-methyl, Ryis H, a O-C1-C6alkyl such as a O-methyl, a halogen such as Br, phenyl or NO2, Rzis H, a O-C1-C6alkyl such as a O-methyl, N(Et)2or OH and Rwis H or Br, Rxand Ryare each independently H or O-methyl, and which Rxis H or an aryl such as a phenyl, Ryis H, a O-C1-C6alkyl such as a O-methyl, a halogen such as Br or an aryl such as a phenyl, Rzis H, OH, a O-C1-C6 alkyl such as a O-methyl, N(Alkyl)2 or an aryl such as a phenyl, and Rwis H, N(Alkyl)2 or an aryl such as a phenyl, in particular In a particular embodiment, X is a phenyl group substituted with one or more, preferably one, two or three, substituents selected in the group consisting of C1-C6 alkyl, C1-C6 alkoxy and NO2, notably C1-C6 alkoxy and NO2.Preferably, X is a phenyl group substituted with at least one NO2 group. In this embodiment, at least one of R1, R2and R3is in particular not OH; more particularly, all of R1, R2and R3is not OH. More preferably, X corresponds to the group of formula (A): in which the wavy line represents the point of attachment to the rest of the molecule. In this embodiment, at least one of R1, R2and R3is in particular not OH; more particularly, all of R1, R2and R3is not OH. In another particular embodiment, X is a 2-benzopyronyl group, also called coumarin derivative, substituted with one or more, preferably one, two or three, notably two, substituents selected in the group consisting of C1-C6alkyl, OH and O- C1-C6alkyl, such as O-methyl. More preferably X corresponds to the group of formula (B): (B) in which the wavy line represents the point of attachment to the rest of the molecule. According to the present invention, X is linked to the rest of the molecule, i.e. to the pyranine moiety, through a linker L corresponding to a divalent radical derived from a C1-C10aliphatic (e.g. C1-C6aliphatic) chain, wherein one or more, preferably 1, 2, 3 or 4, methylene unit(s) of the C1-C10 aliphatic chain are optionally replaced by ––O– or –C(=O)–, wherein said C1-C10 aliphatic chain is optionally substituted with one or more, preferably one, substituent selected from OH, C1-C6 haloalkyl, C1-C6 alkoxy, NR’’R’’’, halogen, C(O)OH, C(O)-O-C1-C6 alkyl, C(O)H, C(O)-C1-C6 alkyl, C(O)-O-aryl, C(O)-aryl and aryl, with R’’ and R’’’ being each independently H or C1-C6alkyl, in particular selected from OH, C1-C6haloalkyl, such as trifluoromethyl, C1-C6alkoxy, such as methoxy, NH2, halogen, C(O)OH and C(O)-aryl, such as C(O)-phenyl. For chemical reason, it is obvious that, in the C1-C10 aliphatic chain an oxygen cannot be linked to another oxygen atom. In the same way an ester (C(O)O) function cannot be linked to another ester function. In particular embodiments, L is a divalent radical –(CH2)n- with n being an integer from 1 to 4, preferably 1 or 2, unsubstituted or substituted with one or more, notably one, substituent selected from OH, C1-C6 haloalkyl, such as trifluoromethyl, C1-C6alkoxy, such as methoxy, NH2, halogen, C(O)OH and C(O)-aryl, such as C(O)-phenyl. Preferably, L is -(CH2)nwith n being 1 or 2, preferably 1. According to some embodiments, the moiety X-L is selected from the group consisting of: Rxand Ryare each independently H or methyl, Rxand Ryare each independently H or O-methyl, Rxis H, a C1-C6 alkyl such as a methyl, a O-C1-C6 alkyl such as a O-methyl or CN, Ryis H, a C1-C6 alkyl such as a methyl or a O-C1-C6 alkyl such as a O-methyl, Rzis H, a C1-C6 alkyl such as a methyl, a O-C1-C6 alkyl such as a O-methyl, Cl, F, OH, NH2, N(C1-C6alkyl)2 or phenyl, in particular Rwis H, methyl, CF3, CCl3, CBr3, CN, COOH or phenyl, Preferably X In the compound of formula (I), X-L is a photolabile protecting group that is removable under light exposition. In particular, according to the present invention, X is a photolabile protecting group that is removable under light exposition at wavelengths (λ) ranging from 300 nm to 800 nm. According to the present invention, the pyranine moiety of the compound of formula (I) is substituted by sulfonamide groups, i.e. R1, R2and R3each independently represent NR4R5, or by sulfonic acid groups or sulfonic ester groups, i.e R1, R2and R3each independently represent OR6, wherein R4and R5each independently represent a C4-C18 alkyl, wherein up to 4 methylene units of the C4- C18 alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least two methylene units of the C4-C18 alkyl are not replaced, or R4and R5together with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing R4and R5, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, such as a methyl, R6represents H, a C1-C18 alkyl or an aryl, said C1-C18 alkyl and aryl being optionally substituted with one or more, preferably one or two, substituents selected in the group consisting of OH, halogen, C1-C6haloalkyl, C1-C6alkoxy and C1-C6haloalkoxy. Preferably, the pyranine moiety of the compound of formula (I) is substituted by sulfonamide or sulfonic ester groups. According to some embodiments, R1, R2and R3each independently represent NR4R5, with R4and R5as defined above. In particular, R4and R5are each independently a C4-C18 alkyl, wherein up to 4 methylene units of the C4- C18 alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least two methylene units of the C4-C18alkyl are not replaced. For chemical reason, it is obvious that an oxygen or nitrogen atom cannot be linked to another oxygen or nitrogen atom. In the same way an ester (C(O)O) or amide (C(O)N) function cannot be linked to another ester or amide function. In this embodiment, R4and R5are preferably each independently a C4-C18 alkyl, notably a C6-C18 alkyl, notably a C8-C16 alkyl, or a –(CH2)p-O-CH3 group in which p is an integer from 2 to 16, preferably from 4 to 16, in particular from 6 to 14. Advantageously, R4and R5together with the nitrogen atom to which they are bonded form a 4 to 6- membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing R4and R5, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18alkyl, preferably C1-C12alkyl, more preferably C1-C6 alkyl, such as a methyl. Preferably, R4and R5together with the nitrogen atom to which they are bonded form a 5 or 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing R4and R5, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18alkyl, preferably C1-C12alkyl, more preferably C1-C6alkyl, such as a methyl. In a more preferred embodiment, R4and R5together with the nitrogen atom to which they are bonded form a 5 or 6-membered heterocyclyl group, not comprising another heteroatom in addition to the nitrogen atom bearing R4and R5, unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, preferably C1-C12alkyl, more preferably C1-C6 alkyl, such as methyl, more preferably unsubstituted. Preferably, R1, R2and R3each independently represent a group of formula in which m is 1 or 2, and preferably m is 2. In another more preferred embodiment, R4and R5together with the nitrogen atom to which they are bonded form a 5 or 6-membered heterocyclyl group, comprising one heteroatom selected from O and N, preferably N, in addition to the nitrogen atom bearing R4and R5, unsubstituted or substituted by one or more, preferably one, C1-C18alkyl, preferably C1-C12alkyl, more preferably C1-C6alkyl, such as methyl. According to this embodiment, advantageously, R1, R2and R3each independently represent a group of formula , in which Y is O, S or NR7, preferably NR7, R7being H or C1-C6 alkyl, and m is 1 or 2, and preferably m is 2. According to some other embodiments, R1, R2and R3each independently represent OR6, wherein R6is as defined above. In some embodiments, R6is H, provided that X is not when L is -CH2-. Preferably, R6represents a C1-C18 alkyl, preferably C1-C12, more preferably C1-C6, in particular a methyl, optionally substituted with one or more, preferably one or two, substituents selected in the group consisting of OH, halogen, C1-C6haloalkyl, C1-C6alkoxy and C1-C6haloalkoxy, preferably OH, halogen and C1-C6haloalkyl. More preferably, R6represents a C1-C6alkyl, such as a methyl, substituted with one or two substituents selected in the group consisting of halogen and C1-C6haloalkyl, such as trifluoromethyl. Even more preferably, R6represents a C1-C6alkyl, such as a methyl, substituted with one or two, preferably one, C1-C6 haloalkyl, such as trifluoromethyl. In each of the above-mentioned embodiments, R1, R2and R3preferably are identical. The compound of formula (I) may notably be selected in the group consisting of: thereof such as a sodium salt, preferably

[0002] , , and a salt thereof such as a sodium salt. According to preferred embodiments, the compound of formula (I) corresponds to a compound of formula (I’) : or a salt thereof, wherein: X is an aryl or a heterocyclyl, the aryl and heterocyclyl being optionally substituted with one or more, preferably one to five substituents selected in the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6haloalkoxy, oxo, OH, NRR’, halogen, CN, NO2, C(O)OH, C(O)-O-C1-C6alkyl, -C(O)H, C(O)-C1- C6alkyl, C(O)-aryl and aryl, with R and R’ being each independently H or C1-C6alkyl, L is a linker corresponding to a divalent radical derived from a C1-C10aliphatic chain, wherein one or more, preferably 1, 2, 3, or 4, methylene unit(s) of the C1-C10aliphatic chain are optionally replaced by –O– or – C(=O)–, wherein said C1-C10 aliphatic chain is optionally substituted with one or more, preferably one, substituent selected from OH, C1-C6 haloalkyl, C1-C6 alkoxy, NR’’R’’’, halogen, C(O)OH, C(O)-O-C1-C6 alkyl, C(O)H, C(O)-C1-C6 alkyl, C(O)-O-aryl, C(O)-aryl and aryl, with R’’ and R’’’ being each independently H or C1- C6alkyl, and R1, R2and R3each independently represent NR4R5or OR6a, R4and R5each independently represent a C4-C18alkyl, wherein up to 4 methylene units of the C4-C18alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least two methylene units of the C4-C18 alkyl are not replaced, or R4and R5together with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing R4and R5, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18alkyl, such as a methyl, R6arepresents a C1-C18alkyl or an aryl, said C1-C18alkyl and aryl being optionally substituted with one or more, preferably one or two, substituents selected in the group consisting of OH, halogen, C1-C6haloalkyl, C1-C6 alkoxy and C1-C6 haloalkoxy. The preferred embodiments recited above for X, L, R1, R2and R3in compound of formula (I) can be applied to the compound of formula (I’), except that R6(corresponding to R6ain formula (I’)) cannot be H. The compound of formula (I) may notably be selected in the group consisting of: Composition comprising a compound of formula (I) The present invention also relates to a composition comprising a compound of formula (I) as defined in the present disclosure and a medium. According to some embodiments, the medium comprises an organic solvent, preferably a polar solvent including without being limited to, alcohol such as methanol or ethanol, acetonitrile or acetone. According to these embodiments, the medium may consist in an organic solvent as defined above. In a variant, the medium comprises, or consist in, a mixture of an organic solvent as defined above and an aqueous solution. The aqueous solution in mixture with the organic solvent is preferably an aqueous buffer, more preferably a basic aqueous buffer. In the context of the present invention, a basic aqueous buffer refers to an aqueous buffer having a pH ranging from 7.0 to 9.5, in particular from 7.2 to 8.5. Example of suitable basic aqueous buffer include PBS (phosphate buffered saline) buffer having a pH of 7.4 and Trizma® buffer having a pH of 8.4. Typically, the organic solvent is in mixture with an aqueous solution, notable an aqueous buffer according to a ratio organic solvent / aqueous solution ranging from 25:1 to 1:1 (v / v), preferably from 20:1 to 10:1 (v / v). According to these embodiments, in which the medium comprises an organic solvent, optionally in a mixture with an aqueous solution such as a basic aqueous buffer, the compound of formula (I) is preferably hydrophobic. Hence, the compound of formula (I) according to these embodiments preferably has R1, R2and R3each independently representing: - NR4R5with R4and R5each being a C4-C18 alkyl, or - a group of formula m is 1 or 2. Preferably, in these embodiments, the compound of formula (I) has R1, R2and R3each independently representing a group of formula in which m is preferably 2. Alternatively, the compound of formula (I) according to these embodiments may have R1, R2and R3each independently representing OR6with R6representing a hydrophobic chain typically an unsubstituted C2- C18alkyl, preferably C4-C18alkyl, A composition in which the medium comprises an organic solvent is particularly suitable for characterizing a light source used in photocatalysis, which are most often performed in organic solvents. According to some other embodiments, the medium is an aqueous medium which does not comprise an organic solvent. According to these embodiments, the aqueous medium is typically a biological medium such as a medium for culturing cells, or an aqueous buffer such as a basic aqueous buffer. Example of suitable basic aqueous buffer include PBS (phosphate buffered saline) buffer having a pH of 7.4 and Trizma® buffer having a pH of 8.4. According to these embodiments, in which the medium is an aqueous medium deprived from an organic solvent the compound of formula (I) is preferably hydrophilic, with the understanding that the compound of formula (I) is hydrophilic enough to be able to cross biological barriers such as cell membranes. Hence, the compound of formula (I) according to these embodiments preferably has R1, R2and R3each independently representing: - a group of formula , in which Y is O, S or NR7, preferably NR7, R7being H or C1-C6alkyl, and m is 1 or 2, and preferably m is 2, or - OR6with R6representing H (at the condition that X is not of formula (A) when L is -CH2-), a methyl or a longer chain such as a C2-C12alkyl, preferably C2-C6alkyl, provided that said chain is substituted by at least one polar group such as OH, halogen or C1-C6 haloalkyl, in particular a C1-C6 haloalkyl, such as a trifluoromethyl. A composition in which the medium is an aqueous medium deprived from an organic solvent and comprising a compound of formula (I) able to cross biological barrier is particularly suitable for characterizing / calibrating a light source used in a biological sample. For example, it can be used for optogenetic applications where accurate measurement of light intensity at depth in a light-scattering medium is demanded, or to perform cell lineage by labelling a mother cell engaged in active division. Applications The present invention relates to the use of a compound of the following formula (II), preferably a compound of formula (I) as defined above, to calibrate and thus characterize a light source: or a salt thereof, wherein X is an aryl or a heterocyclyl, the aryl and hetereocyclyl being optionally substituted with one or more, preferably one to five substituents selected in the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, OH, oxo, NRR’, halogen, CN, NO2, C(O)OH, C(O)-O-C1-C6 alkyl, -C(O)H, C(O)-C1- C6 alkyl, C(O)-aryl and aryl, with R and R’ being each independently H or C1-C6 alkyl, L is a linker corresponding to a divalent radical derived from a C1-C10aliphatic chain, wherein one or more, preferably 1,2,3,4, methylene unit(s) of the C1-C10aliphatic chain are optionally replaced by –O– or – C(=O)–, wherein said C1-C10aliphatic chain is optionally substituted with one or more, preferably one, substituent selected from OH, C1-C6 haloalkyl, C1-C6 alkoxy, NR’’R’’’, halogen, C(O)OH, C(O)-O-C1-C6 alkyl, C(O)H, C(O)-C1-C6 alkyl, C(O)-O-aryl, C(O)-aryl and aryl, with R’’ and R’’’ being each independently H or C1- C6 alkyl, Ra, Rband Rceach independently represent NRiRiior OR6, and Riand Riieach independently represent H or C1-C18alkyl, wherein up to 4 methylene units of the C4-C18alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least one methylene units of the C4-C18alkyl are not replaced, or Riand Riitogether with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing Riand Rii, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, such as a methyl, R6represents a H, C1-C18alkyl or an aryl, said C1-C18alkyl and aryl being optionally substituted with one or more, preferably one or two, substituents selected in the group consisting of OH, halogen, C1-C6haloalkyl, C1-C6 alkoxy and C1-C6 haloalkoxy. X and L will be preferably as defined above for the compound of formula (I). According to some embodiments, Ra, Rband Rceach independently represent NRiRii, with Riand Riias defined above. In particular, Riand Riiare each independently H or C1-C18alkyl, wherein up to 4 methylene units of the C4-C18alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least two methylene units of the C4-C18 alkyl are not replaced. For chemical reason, it is obvious that an oxygen or nitrogen atom cannot be linked to another oxygen or nitrogen atom. In the same way an ester (C(O)O) or amide (C(O)N) function cannot be linked to another ester or amide function. In this embodiment, Riand Riiare preferably each independently H or C1-C18 alkyl, H or C4-C18 alkyl, notably H or C6-C18 alkyl, notably H or C8- C16 alkyl, or H or –(CH2)p-O-CH3 group in which p is an integer from 2 to 16, preferably from 4 to 16, in particular from 6 to 14. Advantageously, Riand Riitogether with the nitrogen atom to which they are bonded form a 4 to 6- membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing R4and R5, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, preferably C1-C12alkyl, more preferably C1-C6 alkyl, such as a methyl. Preferably, Riand Riitogether with the nitrogen atom to which they are bonded form a 5 or 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing R4and R5, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18alkyl, preferably C1-C12alkyl, more preferably C1-C6 alkyl, such as a methyl. In a more preferred embodiment, Riand Riitogether with the nitrogen atom to which they are bonded form a 5 or 6-membered heterocyclyl group, not comprising another heteroatom in addition to the nitrogen atom bearing Riand Rii, unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, preferably C1-C12alkyl, more preferably C1-C6alkyl, such as methyl, more preferably unsubstituted. Preferably, Ra, Rband Rceach independently represent a group of formula in which m is 1 or 2, and preferably m is 2. In another more preferred embodiment, Riand Riitogether with the nitrogen atom to which they are bonded form a 5 or 6-membered heterocyclyl group, comprising one heteroatom selected from O and N, preferably N, in addition to the nitrogen atom bearing Riand Rii, unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, preferably C1-C12alkyl, more preferably C1-C6 alkyl, such as methyl. According to this embodiment, advantageously, Ra, Rband Rceach independently represent a group of formula , in which Y is O, S or NR7, preferably NR7, R7being H or C1-C6alkyl, and m is 1 or 2, and preferably m is 2. According to some other embodiments, Ra, Rband Rceach independently represent OH or OR6, wherein R6is as defined above. In each of the above-mentioned embodiments, Ra, Rband Rcpreferably are identical. In the context of the present invention, characterizing a light source means determining light parameters of the light source at a given wavelength, said parameters including light intensity and photon flux density. In particular, the present invention relates to the use of a compound of formula (II) or a composition comprising thereof to measure light intensity of a light source. The composition comprising a compound of formula (II) for this use is as defined above for the composition comprising a compound of formula (I). In some embodiments, the compound of formula (II) is a compound of formula (I), for use in measuring light intensity of a light source. Thus, the present invention provides a method for determining light parameters of the light source, in particular for measuring light intensity of a light source, in a medium, the method comprising the steps of: (a) Adding a compound of formula (II) as defined above to a medium, (b) Illuminating the medium comprising the compound of formula (II) obtained at step (a) with the light source for a time t1, (c) Measuring the increase of fluorescence during the time t1 and determining the light parameters, in particular the light intensity. It is understood that steps (b) and (c) are achieved simultaneously. The method is notably achieved for a light source at a given wavelength. The light source used in the method has notably a wavelength at one photon comprised in the range from 300 nm to 800 nm, notably from 400 nm to 700 nm, i.e. in the visible range, or a wavelength at two photons in the range from 700 nm to 900 nm. According to some embodiments, the method is achieved for a light source having a light intensity ranging from 10-4E.m-2.s-1to 104E.m-2.s-1. The medium may be as described above. In some preferred embodiments, the medium is an aqueous medium deprived of an organic solvent, for example a basic aqueous buffer, useful to characterize a light source used in a biological medium, such as in cells. In step (b), illumination of the medium leads to the uncaging of the compound of formula (II). The uncaging yield typically ranges from 20% to 100%, in particular from 20% to 90%. In step (c), light parameters, such as light intensity, are typically determined by using calibration data of said light parameters such as light intensity in function of photoconversion parameters of the compound of formula (II) used in the method. Photoconversion parameters of the compound of formula (I) includes molar absorption coefficient ε and uncaging cross section σ (with one-photon illumination) or δ (with two- photon illumination). Indeed, a compound of formula (II) at a given wavelength has specific photoconversion parameters, i.e. a specific molar absorption coefficient and a specific uncaging cross section. These photoconversion parameters may be calculated by the skilled person using methods well- known in the art. In particular, the molar absorption coefficient of a compound of formula (II) may be extracted from the slope of the dependence of the absorbance of the medium comprising the compound of formula (II) on its concentration. The uncaging cross section of a compound of formula (II) may be retrieved from fitting the time dependence of the concentration of the compound upon illumination and analyzing the extracted characteristic times. In step (c), the time evolution of the normalized fluorescence signal measured upon illumination at a given wavelength of a medium comprising a compound of formula (I) is typically monoexponential and may be represented by the equation (1): ^(^)= 1 + ^(1 − ^^^⁄^) (1) in which t is the duration of acquisition, A is the amplitude of the increase of the fluorescence signal occurring upon illumination, and τ is the characteristic time associated to uncaging. According to some embodiments, step (c) of the method comprises the following sub-steps: (c1) Measuring the increase of fluorescence during the time t1, (c2) Calculating the characteristic time τ from the time variation of the fluorescence signal, (c3) Calculating the light parameters, in particular light intensity, from the characteristic time τ calculated in step (c2). Preferably, in step (c2), the characteristic time τ is calculated by using the equation (1) as described above, provided that time evolution of the fluorescence signal measured at step (c1) is monoexponential. Preferably, in step (c3), the light intensity of the light source with one-photon used to illuminate the medium comprising a compound of formula (I) is calculated by using the equation (2) : in which τ is the characteristic time σ is the uncaging cross section with one-photon of the compound of formula (I) I is the light intensity. Alternatively, the light power of the light source with two-photon (involving laser pulses over time) used to illuminate the medium comprising a compound of formula (I) is calculated by using the equation (2a) : in which τP is the duration of the laser pulses, h is the Planck constant equal to 6,626x10-34Joule, c is the light velocity in a vacuum, δ is the uncaging cross section with two-photons of the compound of formula (I), T is the period of the laser pulses, λ is the excitation wavelength, V is the volume of the container comprising the medium, Vexc is the focal volume resulting from laser focusing, and P is the power of the light source. In the above described method or use, the compound of formula (II) is preferably selected in the group consisting of: In some embodiments of the above described method or use, the compound of formula (II) is a compound of formula (I’) as described above, in particular selected in the group consisting of: In some other embodiments of the above described method or use, the compound of formula (II) is : thereof such as a sodium salt. Method of preparation of a compound of formula (II) The present invention also relates to a method for preparing a compound of formula (II): or a salt thereof, wherein X is an aryl or heterocyclyl, the aryl or heterocyclyl being optionally substituted with one or more, preferably one to five substituents selected in the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, oxo, OH, NRR’, halogen, CN, NO2, C(O)OH, C(O)-O-C1-C6 alkyl, -C(O)H, C(O)-C1- C6 alkyl, C(O)-aryl and aryl, with R and R’ being each independently H or C1-C6 alkyl, L is a linker corresponding to a divalent radical derived from a C1-C10 aliphatic chain, wherein one or more, preferably 1,2,3,4, methylene unit(s) of the C1-C10aliphatic chain are optionally replaced by –O– or – C(=O)–, wherein said C1-C10aliphatic chain is optionally substituted with one or more, preferably one, substituent selected from OH, C1-C6haloalkyl, C1-C6alkoxy, NR’’R’’’, halogen, C(O)OH, C(O)-O-C1-C6alkyl, C(O)H, C(O)-C1-C6 alkyl, C(O)-O-aryl, C(O)-aryl and aryl, with R’’ and R’’’ being each independently H or C1- C6 alkyl, Ra, Rband Rceach independently represent NRiRiior OR6, and Riand Riieach independently represent H or C1-C18 alkyl, wherein up to 4 methylene units of the C4-C18 alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least one methylene units of the C4-C18alkyl are not replaced, or Riand Riitogether with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing Riand Rii, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, such as a methyl, R6represents a H, C1-C18alkyl or an aryl, said C1-C18alkyl and aryl being optionally substituted with one or more, preferably one or two, substituents selected in the group consisting of OH, halogen, C1-C6haloalkyl, C1-C6 alkoxy and C1-C6 haloalkoxy, comprising the steps of: (i) Reacting 8-hydroxypyren-1,3,6-trisulfonic acid trisodium salt (HPTS.3Na) with a compound of formula (III): X-L-LG (III) in which X and L is as defined above and LG is a leaving group, in order to form the following trisodium salt (IIa): wherein the HPTS.3Na is not converted into the corresponding free sulfonic acid before reacting with the compound of formula (III), (ii) optionally converting the salt (IIa) obtained at step (a) into a compound of formula (II) or a salt thereof in which Ra, Rband Rceach independently represent NRiRiior OR6or (ii’) optionally converting the salt (IIa) obtained at step (a) into a compound of formula (II) in which Ra, Rband Rcrepresents OH or a salt thereof other than the trisodium salt (IIa). In the compound of formula (II), the substituents X, L and R6are typically as described above for compound of formula (I), with the same preferred embodiments, and Riand Riiare typically as described above for R4and R5respectively in compound of formula (I), with same preferred embodiments. In particular, in the compound of formula (II), Ra, Rband Rcare preferably identical. More preferably, Ra, Rband Rcare NRiRii, preferably Riand Riitogether with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18alkyl, such as a methyl. According to preferred embodiments, LG is selected in the group consisting of halogen, preferably Br, and sulfonate ester group such as mesylate or tosylate group. The compound of formula (III) is preferably the following compounds: According to preferred embodiments, step (i) is achieved in a polar solvent, such as acetonitrile, especially at the temperature of ebullition of the solvent until a complete conversion of HPTS.3Na is observed. In the context of the present invention, in step (i) the HPTS.3Na is not converted into the corresponding free sulfonic acid before reacting with the compound of formula (III). Step (i) may be achieved with or without a catalyst. In the methods of the present invention, the purification of the final compound is fast and easy and enables to obtain a greater yield than in methods of the prior art. According to preferred embodiments, step (ii) comprises the following sub-steps: (ii-1) activating the SO3Na groups of the trisodium salt (IIa) obtained at step (a) with a halide compound to afford the corresponding sulfonyl halide groups, (ii-2) when necessary, reacting each sulfonyl halide groups with a reagent of formula RaH, RbH or RcH, in which Ra, Rband Rceach independently represent NRiRiior OR6, (ii-3) optionally salifying the compound of formula (II) in which Ra, Rband Rceach independently represent NRiRiior OR6obtained in step (ii-2) in order to form a salt thereof. The halide compound used in step (ii-1) is typically a chlorinating agent such as oxalyl chloride or thionyl chloride. In the method of preparation of a compound of formula (II), an intermediate compound obtained at the end of a reacting step or the final compound obtained at the end of the reaction can be separated from the reaction medium by methods well known to the person skilled in the art, such as by extraction, evaporation of the solvent or by precipitation or crystallisation (followed by filtration). Said compound can be also purified if necessary by methods well known in the art, such as by recrystallisation, by distillation, by chromatography on a column chromatography (for instance on silica gel) or by high performance liquid chromatography (HPLC). Description of the figures Figure 1: UV-Vis absorption spectra of the free and caged pyranine derivatives. Absorption spectra of the free (dotted lines) and caged (solid lines) pyranine derivatives 3a-4a (A), 3b-4b (B), 3c-4c (C) 3d-4d (D) HPTS-4e and 4g (E, solid line: 4e; dashed line: 4g) and 3f-4f (F). The spectra were recorded at 2 µM concentration in 10 mM pH 7.4 PBS (A) (for 3a-4a), in 10 mM pH 7.4 PBS (B) (for 3b-4b), in 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v) (C) and (D) (for 3c-4c and 3d-4d), in 10 mM pH = 8.4 Trizma buffer (E) (for HPTS-4e-4g) and in 10 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 1 (v / v) (F) (for 3f-4f). T = 293 K. Figure 2: Determination of the proton exchange constants of the free pyranine derivatives 3a and 3b. A,B: pH-dependence of the absorption (solid lines) and emission (dotted lines; λexc = 480 nm) spectra of 3a (A) and 3b (B) recorded at 2 μM in 10 mM universal buffer. pH = 9.02, 8.46, 7.47, 7.11, 6.90, 6.71, 6.25, 5.85, 5.32, 5.00, 4.55, 4.05, 2.90 for 3a and pH = 9.01, 8.44, 7.47, 6.83, 6.50, 5.97, 5.47, 5.11, 4.86, 4.40, 3.98, 3.47, 3.04, 2.69, 2.41 for 3b. T = 293 K. Figure 3: (A): Preliminary evidence for uncaging in 4a. Temporal evolution of the fluorescence emission at ^em= 540 nm upon homogeneous illumination of 2 ^M 4a in 10 mM pH 7.4 PBS contained in a 54 ^L cuvette (3 mm optical pathlength) with the LED at ^exc=(365±25) nm (2.5×10-8E.s-1; 1.4×10-3E.m-2.s-1). Markers: experimental data, solid line: monoexponential fit to Eq. (^ (^) = The characteristic time τ = 460 s has been retrieved from A at T = 293 K;(B): Preliminary evaluation of the uncaging yield in 4a. Comparison of the fluorescence emission spectra recorded at a same 2 ^M concentration in 10 mM pH 7.4 PBS from the free pyranine derivative 3a (solid line), the caged pyranine derivative 4a (dotted line), and the caged pyranine derivative 4a after illuminating with the LED at ^exc=(365±25) nm (2.5×10-8E.s-1; 1.4×10-3E.m-2.s-1) up to the stabilization of the fluorescence signal (dashed line recorded after time t = 3500 s). T = 293 K. Figure 4 : (A),(C),(E): Preliminary evidence for uncaging in 4b. Temporal evolution of the fluorescence emission at ^em= 540 nm upon homogeneous illumination of 2 ^M 4b in 10 mM pH 7.4 PBS contained in a 54 ^L cuvette (3 mm optical pathlength) with the LED at ^exc=(365±25) nm (A: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; C: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; E: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1). Markers: experimental data, solid line: biexponential fit to Eq. (^ (^) = ^^+ characteristic times τ1= 107 s and τ2 = 1855 s, τ1 = 216 s and τ2 = 804 s, and τ1 = 432 s and τ2 = 1950 s have been retrieved from (A), (C) and (E) respectively at T = 293 K; (B),(D),(F): Preliminary evaluation of the uncaging yield in 4b. Comparison of the fluorescence emission spectra recorded at a same 2 ^M concentration in 10 mM pH 7.4 PBS from the free pyranine derivative 3b (solid line), the caged pyranine derivative 4b (dotted line), and the caged pyranine derivative 4b after illuminating with the LED at ^exc=(365±25) nm (B: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; D: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; F: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1) up to the stabilization of the fluorescence signal (dashed line recorded after time t; B: t = 3500 s; D: t = 5000 s; F: t = 8000 s). T = 293 K. Figure 5 : (A),(C),(E): Preliminary evidence for uncaging in 4c. Temporal evolution of the fluorescence emission at λem= 540 nm upon homogeneous illumination of 2 μM 4c in 1 mM pH = 7.4 PBS / acetonitrile 1 / 19 (v / v) contained in a 54 μL cuvette (3 mm optical pathlength) with the LED at λexc=(365±25) nm (A: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; C: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; E: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1). Markers: experimental data, solid line: monoexponential fit to Eq. (^(^)= The characteristic time τ = 42 s, τ = 84 s and τ = 193 s have been retrieved from (A), (C), and (E) respectively at T = 293 K; (B),(D),(F): Preliminary evaluation of the uncaging yield in 4c. Comparison of the fluorescence emission spectra recorded at a same 2 μM concentration in 1 mM pH = 7.4 PBS / acetonitrile 1 / 19 (v / v) from the free pyranine derivative 3c (solid line), the caged pyranine derivative 4c (dotted line), and the caged pyranine derivative 4c after illuminating with the LED at λexc=(365±25) nm (B: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; D: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; F: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1) up to the stabilization of the fluorescence signal (dashed line recorded after time t; B: t = 1000 s; D: t = 2000 s; F: t = 4000 s). T = 293 K. Figure 6 : (A),(C),(E): Preliminary evidence for uncaging in 4d. Temporal evolution of the fluorescence emission at λem= 540 nm upon homogeneous illumination of 2 μM 4d in (A: 10 mM pH = 7.4 PBS / acetonitrile 1 / 1 (v / v); C: 2 mM pH = 7.4 PBS / acetonitrile 1 / 4 (v / v); E: 1 mM pH = 7.4 PBS / acetonitrile 1 / 19 (v / v)) contained in a 54 μL cuvette (3 mm optical pathlength) with the LED at λexc=(365±25) nm at 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1. Markers: experimental data, solid line: biexponential fit to Eq. (^(^)= ^^+ ^^^^^ / ^^) (a) and monoexponential fit to Eq. (^(^)= ^^+ ^^^^ / ^) (c,e); The characteristic times τ1= 44 s and τ2= 1170 s, τ = 44 s, and τ = 46 s have been retrieved from (A), (C), and (E) respectively at T = 293 K; (B),(D),(F): Preliminary evaluation of the uncaging yield in 4d. Comparison of the fluorescence emission spectra recorded at a same 2 μM concentration in (B: 10 mM pH = 7.4 PBS / acetonitrile 1 / 1 (v / v); D: 2 mM pH = 7.4 PBS / acetonitrile 1 / 4 (v / v); F: 1 mM pH = 7.4 PBS / acetonitrile 1 / 19 (v / v)) from the free pyranine derivative 3d (solid line), the caged pyranine derivative 4d (dotted line), and the caged pyranine derivative 4d after illuminating with the LED at λexc=(365±25) nm at 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1up to the stabilization of the fluorescence signal (dashed line recorded after time t; B: t = 6000 s; D: t = 2000 s; F: t = 1000 s). T = 293 K. Figure 7: (A),(C),(E): Preliminary evidence for uncaging in 4d. Temporal evolution of the fluorescence emission at λem= 540 nm upon homogeneous illumination of 0.2 μM 4d in 10 mM pH = 7.4 PBS / acetonitrile 1 / 1 (v / v) contained in a 54 μL cuvette (3 mm optical pathlength) with the LED at λexc=(365±25) nm (A: Markers: experimental data, solid line: biexponential fit to Eq. (^(^)= ^^+ ^ and monoexponential fit to Eq. (^(^)= ^^+ ^^^^ / ^) (E); The characteristic times τ1 = 46 s and τ2 = 1061 s, τ1 = 92 s and τ2 = 814 s, and τ = 262 s have been retrieved from (A), (C), and (E) respectively at T = 293 K; (B),(D),(F): Preliminary evaluation of the uncaging yield in 4d. Comparison of the fluorescence emission spectra recorded at a same 2 μM concentration in 10 mM pH = 7.4 PBS / acetonitrile 1 / 1 (v / v) from the free pyranine derivative 3d (solid line), the caged pyranine derivative 4d (dotted line), and the caged pyranine derivative 4d after illuminating with the LED at λexc=(365±25) nm (B: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; D: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; F: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1) up to the stabilization of the fluorescence signal (dashed line recorded after time t = 4000 s). T = 293 K. Figure 8. A,C,E: Preliminary evidence for uncaging in 4e. Temporal evolution of the fluorescence emission at ^em= 500 nm upon homogeneous illumination of 2 ^M 4e in 10 mM pH = 8.4 Trizma buffer contained in a 54 ^L cuvette (3 mm optical pathlength) with the LED at ^exc=(365±25) nm (A: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; C: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; E: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1). Markers: experimental data, solid line: monoexponential fit to Eq. (^(^)= characteristic times τ = 82 s, τ = 184 s and τ = 468 s have been retrieved from A, C, and E respectively at T = 293 K; B,D,F: Preliminary evaluation of the uncaging yield in 4e. Comparison of the fluorescence emission spectra recorded at a same 2 ^M concentration in 10 mM pH = 8.4 Trizma buffer from the free pyranine derivative HPTS (solid line), the caged pyranine derivative 4e (dotted line), and the caged pyranine derivative 4e after illuminating with the LED at ^exc=(365±25) nm (B: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; D: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; F: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1) up to the stabilization of the fluorescence signal (dashed line recorded after time t; B: t = 1000 s; D: t = 2000 s; F: t = 4000 s). T = 293 K. Figure 9. A,C,E: Preliminary evidence for uncaging in 4f. Temporal evolution of the fluorescence emission at ^em= 540 nm upon homogeneous illumination of 2 ^M 4f in 10 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 1 (v / v) contained in a 54 ^L cuvette (3 mm optical pathlength) with the LED at ^exc=(365±25) nm (A: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; C: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; E: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1). Markers: experimental data, solid line: exponential fit to Eq. (^(^)= (A,C) and biexponential fit to Eq. (^(^)= ^^+ ^^^^^ / ^^) (E); The characteristic times τ = 91 s, τ = 199 s, τ1 = 360 s and τ2 = 3630 s, have been retrieved from A, C, and E respectively at T = 293 K; B,D,F: Preliminary evaluation of the uncaging yield in 4f. Comparison of the fluorescence emission spectra recorded at a same 2 ^M concentration in 10 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 1 (v / v) from the free pyranine derivative 3f (solid line), the caged pyranine derivative 4f (dotted line), and the caged pyranine derivative 4f after illuminating with the LED at λexc=(365±25) nm (B: 2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1; D: 1.1×10-8E.s-1, 6.2×10-4E.m-2.s-1; F: 5.1×10-9E.s-1, 2.8×10-4E.m-2.s-1) up to the stabilization of the fluorescence signal (dashed line recorded after time t = 4000 s). T = 293 K. Figure 10: A,C,E Preliminary evidence for uncaging in 4g. Temporal evolution of the fluorescence emission at ^em= 510 nm upon homogeneous illumination of 2 μM 4g in 10 mM pH = 8.4 Trizma buffer contained in a 54 μL cuvette (3 mm optical pathlength) with the LED at λexc=(365±25) nm (A: 1.9 ×10-8E.s-1, 7.6 ×10-5E.m-2.s-1; C: 4.2 ×10-8E.s-1, 1,7 ×10-4E.m-2.s-1; E: 6.3 ×10-8E.s-1, 2.5 ×10-4E.m-2.s-1). Markers: experimental data, solid line: exponential fit to Eq. (^ (^) = characteristic times τ = 33 s, τ = 10 s and τ = 7 s, have been retrieved from A, C, and E respectively at T = 293 K; B,D,F: Preliminary evaluation of the uncaging yield in 4g. Comparison of the fluorescence emission spectra recorded at a same 2 μM concentration in 10 mM pH = 8.4 Trizma buffer from the free pyranine derivative HPTS (solid line), the caged pyranine derivative 4g (dotted line), and the caged pyranine derivative 4g after illuminating with the LED at λexc=(365±25) nm (B:1.9 ×10-8E.s-1, 7.6 ×10-5E.m-2.s-1; D: 4.2 ×10-8E.s-1, 1.7 ×10-4E.m-2.s-1; F: 6.3 ×10-8E.s-1, 2.5 ×10-4E.m-2.s-1) up to the stabilization of the fluorescence signal (dashed line recorded after time t = 250 s). T = 293 K. Figure 11: Quantitative analysis of 4b, 4d, 4e and 4g, uncaging at 365 nm from absorption and fluorescence reporting. A: Temporal evolution of the normalized absorbance at 480 nm (circles) and normalized emission at 540 nm (squares; λexc = 480 nm) of 2 µM 4b in 10 mM pH = 8.4 Trizma buffer contained in a 1×1 cm2quartz cuvette exposed to irradiation at constant light intensity equal to 7.210-8E.s-1at 365 nm. ; B: Temporal evolution of the normalized absorbance at 524 nm (circles) and normalized emission at 556 nm (squares; λexc= 480 nm) of 2 µM 4d in 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v) contained in a 1×1 cm2quartz cuvette exposed to irradiation at constant light intensity equal to 7.2 10-8E.s-1at 365 nm; C: Temporal evolution of the normalized absorbance at 456 nm (circles) and normalized emission at 510 nm (squares; λexc = 450 nm) of of 2 µM 4e in 10 mM pH = 8.4 Trizma buffer contained in a 1×1 cm2quartz cuvette exposed to irradiation at constant light intensity equal to 7.2×10-8E.s-1at 365 nm D : Temporal evolution of the normalized absorbance at 450 nm (circles) and normalized emission at 510 nm (squares; λexc= 450 nm) of 5 µM 4g in 10 mM pH = 8.4 Trizma buffer contained in a 1×1 cm2quartz cuvette exposed to irradiation at constant light intensity equal to 5.4×10-9E.s-1at 365 nm. Figure 12: Measurement of the uncaging cross section of 4b, 4d, 4e and 4g at 365 nm. Light intensity- dependence of the inverse of the characteristic time τ retrieved from the monoexponential fit of the fluorescence rise under 365 nm illumination for 4b (A), 4d (B), 4e (C) and 4g (D) in Figure 13. Markers: Experimental data; Solid line: Linear fit. The extracted slopes are 12.9, 38.9, 16.3 and 462 m2.mol-1for 4b, 4d, 4e and 4g, respectively. T = 293 K. Figure 13: Measurement of the uncaging cross section of 4b, 4d, 4e and 4g at 365 nm. A: Time evolution of the normalized fluorescence emission at 540 nm from 2 µM 4b in 10 mM pH = 7.4 PBS contained in a 1×1 cm2quartz cuvette upon irradiation at constant light intensity at 365 nm at various light intensities (in 10-8E.s-1): 3.7 (circles), 7.3 (squares), 14.6 (triangles), 21.9 (diamonds) and 29.2 (pentagons). Markers: Experimental data; Solid lines: Monoexponential fit with Eq. (3); the characteristic times 691 s, 267 s, 136 s, 87 s and 59 s have been retrieved at the corresponding light intensities; B: Time evolution of the normalized fluorescence emission at 540 nm from 2 µM 4d in 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v) contained in a 54 μL cuvette (3 mm optical pathlength) upon irradiation at constant light intensity at 365 nm at various light intensities (in 10-9E. s-1): 6.0 (circles), 12.0 (squares), 23.9 (triangles), 35.9 (diamonds) and 47.9 (pentagons). Markers: Experimental data; Solid lines: Monoexponential fit with Eq. (3); the characteristic times 119 s, 49 s, 18 s, 11 s and 8 s have been retrieved at the corresponding light intensities; C: Time evolution of the normalized fluorescence emission at 510 nm from 2 µM 4e in 10 mM pH = 8.4 Trizma buffer contained in a 54 μL cuvette (3 mm optical pathlength) upon irradiation at constant light intensity at 365 nm at various light intensities (in 10-9E.s-1): 6.0 (circles), 12.0 (squares), 23.9 (triangles), 35.9 (diamonds) and 47.9 (pentagons). Markers: Experimental data; Solid lines: Monoexponential fit with Eq. (3); the characteristic times 257 s, 113 s, 46 s, 30 s and 20 s have been retrieved at the corresponding light intensities; D: Time evolution of the normalized fluorescence emission at 510 nm from 2 µM 4g in 10 mM pH = 8.4 Trizma buffer contained in a 54 μL cuvette (3 mm optical pathlength) upon irradiation at constant light intensity at 365 nm at various light intensities (in 10-9E.s-1): 1.2 (circles), 2.6 (squares), 4.9 (triangles), 11.2 (diamonds) and 15.1 (pentagons) . Markers: Experimental data; Solid lines: Monoexponential fit with Eq. (3); the characteristic times 27 s, 12 s, 8 s, 4 s and 3 s have been retrieved at various light intensities. T = 293 K. Figure 14: Measurement of the uncaging cross section of 4b, 4d, 4e and 4g at 300, 365, 405 and 440 nm. A: Time evolution of the normalized fluorescence emission at 540 nm from 2 µM 4b in 10 mM pH = 7.4 PBS contained in a 1×1 cm2quartz cuvette upon irradiation at constant light intensity at 300 (circles), 365 (squares), 405 (triangles), and 440 (diamonds) nm at the following light intensities (in 10-5E.m-2.s-1): 1.8, 4.3, 5.8, and 6.3 respectively. Markers: Experimental data; Solid lines: Monoexponential fit with Eq.(^ (^) = 1 + characteristic times 16360 s, 2820 s, 9370 s, and 4420 s have been retrieved at various excitation wavelength respectively from A. B: Time evolution of the normalized fluorescence emission at 540 nm from 2 µM 4d in 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v) contained in a 1×1 cm2quartz cuvette upon irradiation at constant light intensity at 300 (circles), 365 (squares), 405 (triangles), and 440 (diamonds) nm at the following light intensities (in 10-5E.m-2.s-1): 2.7, 6.5, 8.7, and 9.5 respectively. Markers: Experimental data; Solid lines: Monoexponential fit with Eq. (^(^)= 1 + characteristic times 1760 s, 1800 s, 1210 s, and 5190 s have been retrieved at various excitation wavelength respectively from B. C: Time evolution of the normalized fluorescence emission at 510 nm from 2 µM 4e in 10 mM pH = 8.4 Trizma buffer contained in a 1×1 cm2quartz cuvette upon irradiation at constant light intensity at 300 (circles), 365 (squares), and 405 (triangles) nm at the following light intensities (in 10-5E.m-2.s-1): 6.2, 15, and 20 respectively. Markers: Experimental data; Solid lines: Monoexponential fit with Eq. ( ^(^)= 1 + the characteristic times 2820 s, 2070 s and 5350 s have been retrieved at various excitation wavelength respectively from C. D: Time evolution of the normalized fluorescence emission at 510 nm from 2 µM 4g in 10 mM pH = 8.4 Trizma buffer contained in a 1×1 cm2quartz cuvette upon irradiation at constant light intensity at 300 (circles), 365 (squares), 405 (triangles) and 440 (diamonds) nm at the following light intensities (in 10-6E.m-2.s-1): 1.0, 2.4, 3.2 and 1.7 respectively Markers: Experimental data; Solid liness: Monoexponential fit with Eq.(3) the characteristic times 763 s, 385 s, 294 s and 12578 s have been retrieved at various excitation wavelength respectively from C. T = 293 K. Figure 15. Evaluation of the kinetics and yield associated to uncaging of 4b, 4d, 4e and 4g upon illumination at 365 nm by HPLC. Time evolution of the composition of solutions of 4b (A; 2 μM in 10 mM pH = 7.4 PBS), 4d (B; 2 μM in 1 mM pH = 7.4 PBS buffer / acetonitrile 1 / 19 v / v), 4e (C; 2 μM in10 mM pH = 8.4 Trizma buffer) and 4g (D; 5 μM in 10 mM pH = 8.4 Trizma buffer, upon illumination at 365 nm at 1.0 10-7E.s-1for 4b, 4d and 4e) and 5.4×10-9E.s-1(for 4g). Markers: concentrations of the caged compounds 4b, 4d, 4e and 4g (squares) and photoreleased compounds 3b, 3d, and HPTS (circles) as extracted from the peak areas in the HPLC chromatograms; Solid lines: Monoexponential fit with Eq. (3); The characteristic times 83 s, 101 s 484 s and 92 s for 4b, 4d, 4e and 4g and 136, 486 sand 71 s for 3d and HPTS respectively have been retrieved from A, B, C and D. T = 293 K. Figure 16: Photostability of 3b, 3d and HPTS. Time evolution of the normalized fluorescence emission from 2 μM solution of 3b (λem= 540 nm; circles) in 10 mM pH 7.4 PBS, 3d (λem= 540 nm; squares) in 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v) and HPTS (λem= 500 nm; triangles) in 10 mM pH = 8.4 Trizma buffer contained in a 54 μL cuvette (3 mm optical pathlength) upon homogeneous illumination with the LED at λexc=(365±25) nm (2.5×10-8E.s-1, 1.4×10-3E.m-2.s-1) at the following times (s): 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, 2400, 3000 and 3600. Markers: Experimental data, lines: linear fit. The extracted slopes are -8.3×10-4, -3.7×10-3and -1.8×10-3s-1and the characteristic times 1205 s, 270 s and 555 s have been retrieved from the slopes of the linear fit for 3b, 3d, HPTS respectively. T = 293 K. Figure 17: Measurement of the photon flux density in a liquid film of 4b, 4e and 4g. A,B: Images of a liquid film of 2 ^M 4b in 10 mM pH 7.4 PBS before illumination (A) and after illumination (B) at 405 nm (laser power 10% (for 405 nm) and 100% (for 488 nm); frame: 256×256 pixel2; zoom factor: 5; dwell time: 1.7 μs); C: Time evolution of the fluorescence signal extracted from integrating the fluorescence level over the ROI (white frame in A).Markers: Fluorescence signal as extracted from integrating the fluorescence level over the ROI in A; Solid line: Monoexponential fit with Eq. (3) The characteristic time 13 μs has been retrieved from processing the fluorescence signal over the white zone in A. D,E: Images of a liquid film of 1 ^M 4e in 10 mM pH = 8.4 Trizma buffer before illumination (D) and after illumination (E) at 405 nm (laser power: 20% (for 405 nm) and 100% (for 488 nm); frame: 1024×1024 pixel2; zoom factor: 1; dwell time: 1.03 μs); F: Time evolution of the fluorescence signal extracted from integrating the fluorescence level over the ROI (white frame in D). Markers: Fluorescence signal as extracted from integrating the fluorescence level over the ROI in D; Solid line: Monoexponential fit with Eq. (3); the characteristic time 13 μs has been retrieved from processing the fluorescence signal over the white zone in D. G,H: Images of a liquid film of 2 ^M 4g in 10 mM pH = 8.4 Trizma buffer before illumination (G) and after illumination (H) at 405 nm (laser power: 1% (for 405 nm) and 100% (for 488 nm); frame: 1024×1024 pixel2; zoom factor: 1; dwell time: 1.03 μs); I: Time evolution of the fluorescence signal extracted from integrating the fluorescence level over the ROI (white frame in G). Markers: Fluorescence signal as extracted from integrating the fluorescence level over the ROI in G; Solid line: Monoexponential fit with Eq. (3); the characteristic time 2 μs has been retrieved from processing the fluorescence signal over the white zone in G. T = 293 K. Figure 18: Evaluation of the kinetics of uncaging for 4e and 4g with two-photon activation. Temporal evolution of the normalized fluorescence emission from 3 µM solutions of 4e (A) and 4g (C) in 10 mM pH = 8.4 Trizma buffer upon irradiation at λ = 750 nm at laser powers P (mW), 8.8 (circles), 12.3 (squares), 17.6 (triangles) and 22.0 (pentagons) for 4e and 7 (circles), 11.3 (squares), 14.7 (triangles) and 23.1 (pentagons) for 4g; Dependence of the inverse of the characteristic time ^ retrieved from the time evolutions displayed in A, C on the applied laser power P for 4e (B) and 4g (D). From the linear fits applied in B and D (solid lines), we retrieved the power exponent and the uncaging cross sections with two-photon excitation from the slope and the intercept at the origin respectively. Figure 19: Measurement of the photon flux density in living zebra fish embryos. A,D: Bright field image of living zebra fish embryos conditioned with 2 μM DMSO (A) and 1 μM 4b (D) in water at 405 nm and 488 nm (laser power: 2%; frame: 256×256 pixel2; zoom factor: 5; dwell time: 0.52 μs); B,E: Fluorescence image of living zebra fish embryos conditioned with 2 μM DMSO (B) and 1 μM 4b (E) in water before illumination at 405 nm (laser power: 10%; frame: 256×256 pixel2; zoom factor: 5; dwell time: 1.7 μs); C,F: Time evolution of the fluorescence signal extracted from integrating the fluorescence level over the ROI (white frame in B and E). Markers: Fluorescence signal as extracted from integrating the fluorescence level over the ROI in B and E respectively; Solid lines: Monoexponential fit with Eq. (3); the characteristic time 5.3 μs has been retrieved from processing the fluorescence signal over the white zone in E. G: Distribution of light intensity I in living zebra fish embryos conditioned with 1 μM 4b in water. Bars: Frequency of light intensity extracted from different ROIs of 10 embryos (sample size: 88 ROIs, minimum bin: 3.2×104E.m-2.s-1, maximum bin: 13×104E.m-2.s-1and bin size: 1.25×104E.m-2.s-1). Solid lines: Fit with Eq. ^(^) = ^^+ ^^^^{−[(^ − ^^)⁄^ ]^}. T = 293 K. Figure 20: Measurement of the photon flux density in living Hela cells. A,C: Images of living Hela cells conditioned with 4a (A) or 4b (C) under illumination at 365 nm; B,D: Time evolution of the fluorescence signal at 365 nm of living Hela cells conditioned with 4a (B) or 4b (D) and of the surrounding pH 7.4 PBS buffer (0.01 M phosphate, 0.154 M NaCl) under illumination at 365 nm (the circle and square markers in B and D are associated with the frames delimited by the solid and dotted lines in A and C respectively). Markers: Fluorescence signal as extracted from integrating the fluorescence level over the ROIs; Solid lines: Monoexponential fit with Eq. (3); the characteristic time 1.7 s has been retrieved from processing the fluorescence signal over the frames delimited by the solid line in C. T = 298 K. Figure 21: Measurement of the photon flux density in living Hela cells and in 4b solution in pH 7.4 PBS buffer (0.01 M phosphate, 0.154 M NaCl). A,C: Images of living Hela cells conditioned with 4b under illumination at 365 nm; B,D: Time evolution of the fluorescence signal at 365 nm of living Hela cells conditioned with 4b and of the surrounding pH 7.4 PBS solution under illumination at 365 nm (regions of interest - ROIs: white solid and dotted frames in A and C respectively). Markers: Fluorescence signal as extracted from integrating the fluorescence level over the ROIs delimited by the solid (disks) and dotted (circles) line; Solid lines: Monoexponential fit with Eq. (^(^)= ^^+ ^^^^ / ^); the characteristic times 0.9 and 2.5 s have been retrieved in B and D from processing the fluorescence signal over the zones delimited by a solid white line in A and C; E: Images of 4b solution in pH 7.4 PBS buffer under illumination at 365 nm; F: Time evolution of the fluorescence signal at 365 nm of 4b solution in pH 7.4 PBS buffer under illumination at 365 nm (regions of interest - ROIs: white solid and dotted frames). Markers: Fluorescence signal as extracted from integrating the fluorescence level over the ROIs delimited by the solid (disks) and dotted (circles) line; Solid lines: Monoexponential fit with Eq. (^ (^) = ^^+ ^^^^ / ^); the characteristic times 3.2 and 3.4 s have been retrieved in F from processing the fluorescence signal over the green and red zones respectively in E. T = 293 K. Examples 1. Material and methods General information: 4,5-dimethoxy-2-nitrobenzyl bromide and trisodium 8-hydroxypyrene-1,3,6- trisulfonate (HPTS) were purchased from Acros Organics. Commercially available reagents were used as obtained.1H and13C NMR spectra were recorded at 300 K on a Bruker AM 300 spectrometer; chemical shifts are reported in ppm with protonated solvent as internal reference (1H, CHCl3 in CDCl37.26 ppm, CHD2SOCD3 in CD3SOCD32.50 ppm, CHD2COCD3 in CD3COCD32.05 ppm;13C,13CDCl3 in CDCl377.0 ppm,13CD3SOCD3 in CD3SOCD339.5 ppm,13CD3COCD3 in CD3COCD329.9 ppm),19F, C7H5F3 in CHD2SOCD3 -63.7 ppm. Mass spectra (chemical ionization and electronic impact with NH3or CH4) were performed by the Service de Spectrométrie de Masse de Chimie ParisTech and mass spectra high resolution were performed by the Service de Spectrométrie de Masse de l’Institut de Chimie Organique et Analytique (Orléans). Column chromatography was performed on silica gel 60 (0.040–0.063 nm; Merck). Analytical thin-layer chromatography (TLC) was conducted on Merck silica gel 60 F254 pre-coated plates. Preparation of the universal buffer at 10 mM concentration: To 100 mL of Milli-Q water were dissolved 82 mg of sodium acetate (CH3COONa), 120 mg of sodium dihydrogenophosphate (NaH2PO4) and 518.2 mg of 2-(cyclohexylamino)ethane-1-sulfonic acid (CHES). The pH of the initial solution (5.90) solution was then adjusted to pH = 9 with 1 M NaOH. UV / Vis absorption and fluorescence spectrometers UV / Vis absorption spectra were recorded on a UV / Vis spectrophotometer (Cary 300 UV-Vis, Agilent Technologies, Santa Clara, CA) at 293 K equipped with a Peltier 1×1 thermostatic cell holder (Agilent Technologies). Samples were contained either in 1 cm × 1 cm (3 mL; the cuvette content was stirred) or in 0.3 cm × 0.3 cm (54 µL; the cuvette content was not stirred) quartz cuvettes (Hellma Optics, Jena, Germany). Fluorescence measurements were acquired on a LPS 220 spectrofluorometer (PTI, Monmouth Junction, NJ), equipped with a TLC50 cuvette holder (Quantum Northwest, Liberty Lake, WA) thermoregulated at 293 K. Photochemical experiments Light sources: In order to photoconvert the reactants and track the evolution of fluorescent products via absorbance and fluorescence under time constant illumination, the following light sources were used: (i) For the photoconversion under various light intensities, 365 nm and 405 nm Light Emitting Diodes (LEDs) monochromatically filtered to generate a spatially homogeneous source of illumination; (ii) The Xenon lamp of the LPS 220 spectrofluorometer (PTI, Monmouth Junction, NJ) for photoconversion experiments at various wavelengths. The light intensity delivered from the light sources has been calibrated by using α-(4- diethylamino)phenyl)-N-phenylnitrone as an actinometer (see P. F. Wang et al., New J. Chem., 1996, 20, 895-907) and rhodamine B as a fluorescent reporter. Data processing: The time evolution of the recorded signal S(t) (absorbance and fluorescence emission) has been processed either with - a monoexponential function ^ ^(^)= ^(∞)+ ^^^^(3) - or a biexponential function ^ ^ ^ ^(^) = ^(∞) + ^^^^^ ^ + ^^^^^(4) HPLC: 3 mL of a solution of the caged pyranine (4a and 4b: 2 ^M in 10 mM pH = 7.4 PBS; 4d: 2 ^M in 1 mM pH = 7.4 PBS / acetonitrile 1 / 19 (v / v); 4e: 2 ^M in 10 mM pH = 8.4 Trizma buffer; 4f: 2 ^M in 10 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 1 (v / v) and 4g: 5 ^M in 10 mM pH = 8.4 Trizma buffer were placed in a quartz cuvette (10x10 mm) and submitted to irradiation at 365 nm under stirring. Aliquots of 30 μL were drawn at regular intervals and were subsequently analyzed by high-pressure liquid chromatography using an Agilent 1260 Infinity LC System equipped with autosampler and diode array detector. Chromatographic separations were carried out by using an Atlantis T3 (for 4a-d and 4f) (3x150 mm, particle size 3 ^m; Waters Corporation, Milford, MA) and C18 column (for 4e and 4g) (4.6x50 mm, particle size 2.7 ^m; Poroshell 120) operating at 0.7 mL / min (for 4a-d and 4f) and 1.5 mL / min (for 4e and 4g) and thermostated at 25°C. Detection of the caged and uncaged pyranines was performed at 405 and 480 nm respectively.15 μL (for 4a-d, 4e, 4g and 4f), 25 μL (for 4b) were injected in the chromatographic system. The compounds 4a-d and 4f were eluted with a mobile phase composed of two solvents A (methanol) and B (ammonium acetate buffer, pH 5.5) whereas 4e was eluted with two solvents C (5 mM tetrabutylammonium chloride in methanol), D (5 mM tetrabutylammonium chloride and 5 mM ammonium acetate in H2O, pH 5.5) and 4g was eluted with two solvents A (methanol) and E (10 mM triethylamine in H2O, pH 5.5 was adjusted with glacial acetic acid). A gradient was used to optimize the separation of the analytes. For 4a, the gradient used was as follow: Initially, the column was equilibrated with a mobile phase consisting of 45% A and 55% B. Six minutes after the injection, the proportion of A was linearly increased to 95 % and for one additional minute to 98%. After this step, composition of the mobile phase was set to initial condition within 0.5 min and the column was equilibrated for 9.5 min prior to next injection. Retention times for the caged (4a) and free pyranine (3a) were tR= 6 min and tR= 2.3 min respectively in these experimental conditions. For 4b, the gradient used was as follow: Initially, the column was equilibrated with a mobile phase consisting of 70% A and 30% B. Six minutes after the injection, the proportion of A was linearly increased to 95% and for one additional minute to 98%. After this step, composition of the mobile phase was set to initial condition within 0.5 min and the column was equilibrated for 9.5 min prior to next injection. Retention times for the caged (4b) and free pyranine (3b) were tR= 5.9 min and tR= 1.9 min respectively in these experimental conditions. For 4d, the gradient used was as follow: Initially, the column was equilibrated with a mobile phase consisting of 70% A and 30% B. Six minutes after the injection, the proportion of A was linearly increased to 95% and for 1.5 additional minute to 98%. After this step, composition of the mobile phase was set to initial condition within 0.5 min and the column was equilibrated for 9.5 min prior to next injection. Retention times for the caged (4d) and free pyranine (3d) were tR = 8.7 min and tR = 5.9 min respectively in these experimental conditions. For 4e, the gradient used was as follow: Initially, the column was equilibrated with a mobile phase consisting of 50% C and 50% D. Four minutes after the injection, the proportion of C was linearly increased to 60%. After this step, composition of the mobile phase was set to initial condition within 0.5 min and the column was equilibrated for 5.5 min prior to next injection. Retention times for the caged (4e) and free pyranine (HPTS) were tR= 3.9 min and tR= 1.2 min respectively in these experimental conditions. For 4f, the gradient used was as follow: Initially, the column was equilibrated with a mobile phase consisting of 70% A and 30% B. Six minutes after the injection, the proportion of A was linearly increased to 95% and for 1.5 additional minute to 98%. After this step, composition of the mobile phase was set to initial condition within 0.5 min and the column was equilibrated for 9.5 min prior to next injection. Retention times for the caged (4f) and free pyranine (3f) were tR= 7.6 min and tR= 4.9 min respectively in these experimental conditions. For 4g, the gradient used was as follow: Initially, the column was equilibrated with a mobile phase consisting of 10% A and 90% E. Two minutes after the injection, the proportion of A was linearly increased to 20% and for four additional minutes to 70%. After this step, composition of the mobile phase was set to initial condition within 0.5 min and the column was equilibrated for 5.5 min prior to next injection. Retention times for the caged (4g) and free pyranine (HPTS) were tR= 5.1 min and tR= 1.6 min respectively in these experimental conditions. Cells experiments Experiments on cultured cells were performed on HeLa cells (ATCC) grown on DMEM (Cytiva Hyclone) supplemented with 9% (v / v) fetal bovine serum (FBS - GIBCO) at 37 °C within a 5% CO2 atmosphere. The caged pyranine derivatives 4a-b were solubilized at 10 mM in DMSO then diluted in pH 7.4 PBS solution. For microscopic imaging, the cells were seeded in ^Dish (Ibidi) according to the manufacturer protocol. The cells were washed with pH 7.4 PBS, and treated with the pH 7.4 PBS solution containing 4a or 4b. After incubation for 1 h in the dark, the cells were washed with plain pH 7.4 PBS prior to illumination to avoid any possible interference, which could originate from photoreleasing the free fluorophore in the incubating solution. Living zebra fish embryos experiments Experiments were performed on living zebra fish embryos grown on water medium at 37 °C within a 5% CO2 atmosphere. The caged pyranine derivatives 4b were solubilized at 10 mM in DMSO then diluted in water. For confocal microscopic imaging, the embryos were washed with water, and treated with water solution containing 4b at 5 hpf. After incubation for overnight in the dark, the embryos were washed with plain water prior to illumination to avoid any possible interference, which could originate from photoreleasing the free fluorophore from the surface of the embryos. 2. Synthesis of pyranines 4a- g The caged trisulfonamide pyranine derivatives 4a-d were synthesized in four steps according to the method described in Finkler, B. et al. Photochem. Photobiol. Sci., 2014, 13, p.548-562 (see Scheme 1). In the first step, the ester 1 was produced in 92% yield from acylating the HPTS phenol with acetic anhydride. In the next step, its sulfonic acid groups were converted to chlorosulfonyl groups by reacting with oxalyl chloride to form the compound 2, which was used for the next step without any further purification. The free trisulfonamide pyranine derivatives 3a-d were then obtained by condensation of the compound 2 with the amines in fair to good yields (50-70%). The free pyranine derivative of trisulfonate ester 3f was obtained by condensing compound 2 with trifluoroethanol in good yields (74%). Finally, the caged trisulfonamide pyranine derivatives 4a-d were synthesized by reacting the free pyranine derivatives 3a-d with 4,5-dimethoxy-2-nitrobenzylbromide in the presence of potassium carbonate in 54-80% yield. Scheme 1: Pathway 1 for the synthesis of compounds 4a-d The caged trisulfonamide pyranine derivative 4d was also synthesized by the method of the invention (Schemes 2 and 3). Here, the HPTS phenol was first etherified by 4,5-dimethoxy-2-nitrobenzylbromide in the presence of a phase transfer catalyst to yield caged-HPTS 4e with 90% yield. Then, 4d was obtained in 50% yield from converting the sulfonic acid groups to chlorosulfonyl groups with oxalyl chloride to afford compound A, followed by reaction with piperidine. The caged trisulfoester pyranine 4f was synthesized in 83% yield using the method of the invention, by reacting compound A with trifluoroethanol. Scheme 2: Pathway 2 for the synthesis of compounds 4e, 4d and 4f The caged pyranine derivative 4g was also synthesized by the method of the invention (Scheme 3). Here, the HPTS phenol was first etherified by 6,7-dimethoxycoumarinyl-4-methyl in the presence of a phase transfer catalyst to yield caged-HPTS 4g with 74% yield.

[0003] 3: Pathway 2 for the synthesis of compound 4g 8-Acetoxypyrene-1,3,6-trisulfonyl chloride 2 The compound 1 (1.0 g, 1.8 mmol) was suspended in anhydrous dichloromethane (80 mL). Oxalyl chloride (1.35 mL, 16 mmol, 9 eq) was added followed by 10 drops of anhydrous dimethylformamide. The resulting mixture was stirred at room temperature under inert atmosphere for 18 h. The solvent was evaporated. The crude was dissolved in anhydrous dichloromethane (3x20 mL) and then the solution was concentrated under reduced pressure. Compound 2 was obtained as a yellow / orange powder and used without further purification for the next step. Or The compound 1 (2.5 g, 4.5 mmol) was suspended in thionyl chloride (20 mL) and 10 drops of anhydrous dimethylformamide was added. The resulting mixture was stirred at 76°C for 5 h. The reaction mixture was then cooled down to room temperature and poured into ice with vigorous stirring. A yellow / orange color precipitate was obtained with effervescence of SO2gas. The crude precipitate was filtered and dried under reduced pressure. Compound 2 was obtained as a yellow / orange powder and used without further purification for the next step. 8-Hydroxypyrene-N’,N’’,N’’’-trishydroxyethyl-1,3,6-trisulfonamide 3a (intermediate) Compound 2 (0.140 g, 0.25 mmol) was dissolved in anhydrous dichloromethane (10 mL). The solution was then cooled to 0 °C and a solution of ethanolamine (0.137 g, 2.25 mmol, 9 eq.) in anhydrous dichloromethane (5 mL) was added dropwise. The reaction mixture was stirred under inert atmosphere for 48 h at room temperature, yielding a red precipitate. The crude residue was purified by flash chromatography using acetone / cyclohexane (75 / 25 v / v) as eluent. The pure product was further recrystallized using acetone / pentane (70 / 30 v / v), yielding 3a as orange powder (70 mg, 48 % yield).1H-NMR (300 MHz, acetone-d6): δ 9.36 (1H, s), 9.29 (1H, d, J = 10 Hz), 9.20 (1H, d, J = 10 Hz), 9.06 (1H, d, J = 10 Hz), 8.86 (1H, d, J = 10 Hz), 8.50 (1H, s), 7.12 (3H, m) 3.50 (6H, m), 3.12 (6H, m).13C-NMR (75 MHz, DMSO-d6): δ 157.1, 137.9, 131.6, 130.4 (2C), 130.2, 128.1, 127.9, 126.0, 125.9, 125.8, 122.5, 120.5, 120.3, 117.2, 116.2, 59.9 (2C), 59.8, 45.0, 44.9 (2C). HRMS (ESI): m / z calc. for C22H26N3O10S3: 588.077483, found: 588.078556 [M+H]+. 8-Hydroxypyrene-N’,N’’,N’’’-tris-N-methylpiperazine-1,3,6-trisulfonamide 3b (intermediate) Compound 2 (1.0 g, 1.8 mmol) was dissolved in anhydrous dichloromethane (25 mL). The solution was then cooled to 0°C before the addition of N-methylpiperazine (3.60 mL, 32 mmol, 18 eq.). The reaction mixture was stirred under inert atmosphere for 48 h at room temperature. Then dichloromethane was evaporated under vacuum. The crude mixture was then washed with cyclohexane (3x20 mL) to remove the excess N-methylpiperazine. The crude residue was purified by recrystallization in absolute ethanol / n- pentane (70 / 30 v / v), yielding 3b as a red / orange powder (760 mg, 60 % yield).1H-NMR (300 MHz, DMSO-d6): δ 9.18 (1H, d, J = 10 Hz), 8.98 (1H, s), 8.97 (1H, d, J = 10 Hz), 8.87 (1H, d, J = 10 Hz), 8.81 (1H, d, J = 10 Hz), 8.23 (1H, s), 3.19 (8H, m), 3.07 (4H, m), 2.36 (8H, m), 2.26 (4H, m), 2.12 (6H, s), 2.04 (3H, s).13C NMR (75 MHz, DMSO-d6): δ 158.3, 134.2, 133.1, 131.8, 130.3, 128.6, 127.0, 126.6, 126.4, 126.3, 125.9, 122.6, 121.2, 120.2, 117.7, 117.6, 53.6 (4C), 53.50 (2C), 45.1 (4C), 45.0 (2C), 44.9 (3C). HRMS (ESI): m / z calc. for C31H41N6O7S3: 705.219337, found: 705.219684 [M+H]+. 8-Hydroxypyrene-N,N,N’,N’,N’’,N’’-hexamethyl-1,3,6-trisulfonamide 3c (intermediate) Compound 2 (1.0 g, 1.8 mmol) was dissolved in anhydrous dichloromethane (25 mL). The solution was then cooled to 0°C before the addition of dimethylamine (2 M in THF, 9 mL, 32 mmol, 18 eq.). The reaction mixture was stirred under inert atmosphere for 48 h at room temperature. Then dichloromethane was evaporated under vacuum and 1 M hydrochloric acid (10 mL) was added. The aqueous phase was extracted with dichloromethane (3×25 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was purified by recrystallization in absolute ethanol / n-pentane (70 / 30 v / v), yielding 3c as a bright yellow powder (630 mg, 65 % yield).1H NMR (300 MHz, DMSO-d6): δ 9.27 (1H, d, J = 10 Hz), 9.15 (1H, d, J = 10 Hz), 9.02 (1H, d, J = 10 Hz), 8.98 (1H, s), 8.87 (1H, d, J = 10 Hz), 8.30 (1H, s), 2.83 (6H, s), 2.81 (12H, s).13C NMR (300 MHz, DMSO-d6): δ 154.6, 133.7, 132.6, 131.5, 129.8, 128.3, 128.0, 127.8, 126.1, 125.9, 125.5, 123.8, 121.6, 120.6, 119.5, 116.0, 37.3 (2C), 37.2 (4C). MS (CI): m / z calc. for C22H25N3O7S3: 539.09, found: 540.33 [M+H]+. 8-Hydroxypyrene-N’,N’’,N’’’-tris-piperidine-1,3,6-trisulfonamide 3d (intermediate) Compound 2 (1.0 g, 1.8 mmol) was dissolved in anhydrous dichloromethane (25 mL). The solution was then cooled to 0 °C before the addition of piperidine (3.20 mL, 32 mmol, 18 eq.). The reaction mixture was stirred under inert atmosphere for 48 h at room temperature. Then dichloromethane was evaporated under vacuum and 1 M hydrochloric acid (20 mL) was added. The aqueous phase was extracted with dichloromethane (3×25 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was then washed with cyclohexane / absolute ethanol (7 / 3 v / v; 3×15 mL), yielding 3d as a bright yellow powder (840 mg, 71 % yield).1H NMR (300 MHz, DMSO-d6): δ 9.27 (1H, d, J = 10 Hz), 9.12 (1H, d, J = 10 Hz), 9.03 (1H, s), 8.99 (1H, d, J = 10 Hz), 8.86 (1H, d, J = 10 Hz), 8.33 (1H, s), 3.16 (12H, m), 1.50 (12H, m), 1.36 (6H, m).13C NMR (75 MHz, DMSO-d6): δ 154.8, 134.4, 132.4, 131.2, 129.8, 128.7, 128.4, 128.3, 126.1, 125.9, 125.5, 123.7, 121.5, 120.7, 119.3, 116.1, 46.2 (2C), 46.1 (4C), 24.9 (6C), 22.7 (3C). HRMS (ESI): m / z calc. for C31H38N3O7S3: 660.186640, found: 660.187152 [M+H]+. Tris(2,2,2-trifluoroethyl) 8-hydroxypyrene-1,3,6-trisulfonate 3f (intermediate) To a solution of trifluoroethanol (1.3 mL, 18 mmol) in anhydrous DCM (35 mL), anhydrous triethylamine (4.6 mL, 33 mmol) was added and cooled down to 0°C in an ice bath. The solution of compound 2 (1.3 g, 2.3 mmol) in anhydrous DCM (120 mL) was then added dropwise at 0°C. The resulting mixture was then slowly warmed to room temperature and stirred under inert atmosphere for 40 h. The organic phase was then separated and extracted with 1 M HCl (x3) and saturated NaCl (x2) solution. The crude residue was obtained by evaporating the organic phase dried over Na2SO4 under reduced pressure. It was then purified by flash chromatography using n-pentane / ethyl acetate (70 / 30 v / v) as eluent, yielding 3f as a dark red powder (1.2 g, 74 % yield).1H-NMR (300 MHz, acetone-d6): δ 9.34 (1H, s), 9.23 (1H, d, J = 10 Hz), 9.16 (1H, d, J = 10 Hz), 8.98 (1H, d, J = 10 Hz), 8.92 (1H, d, J = 10 Hz), 8.56 (1H, s), 4.89 (6H, m).13C NMR (75 MHz, DMSO-D6): δ 134.3, 132.8, 132.4, 131.9, 129.3, 128.9, 125.7, 125.4, 127.9, 124.3, 120.6, 116.9 (2C, q, JCF = 276 Hz), 127.8, 124.3, 120.5, 116.8 (1C, q, JCF = 276 Hz), 123.4, 123.1, 122.5, 121.8, 120.4, 118.9, 117.3 (2C), 66.1, 65.6, 65.1, 64.6 (2C, q, JCF= 36 Hz), 66.0, 65.5, 65.0, 64.6 (q, JCF= 36 Hz).19F NMR (282 MHz, DMSO-d6): δ -75.1 to -75.2 (m, 3CF3). MS (ESI): m / z calc. for C22H13F9O10S3: 702.94, found: 702.84 [M-H]-. 8-(4,5-Dimethoxy-2-nitrobenzyloxy)pyrene-N’,N’’,N’’’-trishydroxyethyl-1,3,6-trisulfonamide 4a (comparative) Potassium carbonate (0.023 g, 0.17 mmol, 1.2 eq.) and 4,5-dimethoxy-2-nitrobenzyl bromide (0.05 g, 0.17 mmol, 1.2 eq.) were added to a solution of compound 3a (0.08 g, 0.14 mmol) in acetonitrile (10 mL). The reaction mixture was then heated to reflux and stirred for 24 h in the dark. The obtained precipitate was washed with dichloromethane (3×10 mL) followed by acetone (3×10 mL). The precipitate was filtered and dried under a vacuum pump, yielding 4a as a muted red powder (0.079 g, 73 % yield).1H-NMR (300 MHz, DMSO-d6): δ 9.26 (1H, d, J = 10 Hz), 9.16 (1H, d, J = 10 Hz), 9.14 (1H, s), 9.03 (1H, d, J = 10 Hz), 8.75 (1H, d, J = 10 Hz), 8.50 (1H, s), 7.79 (1H, s), 7.62 (1H, s), 5.97 (2H, s), 3.96 (3H, s), 3.92 (3H, s), 3.29 (6H, m), 2.91 (6H, m).13C NMR (75 MHz, CDCl3,): δ 153.1, 153.0, 148.3, 140.1, 137.9, 132.8, 132.7, 130.5, 129.9, 127.9, 127.6, 125.8, 125.3, 125.0, 124.8, 124.4, 122.7, 121.6, 120.3, 112.1, 112.0, 108.5, 68.4, 59.9 (3C), 56.3, 56.2, 45.2 (3C). HRMS (ESI): m / z calc. for C31H35N4O14S3: 783.130614, found: 783.129715 [M+H]+. 8-(4,5-Dimethoxy-2-nitrobenzyloxy)pyrene-N’,N’’,N’’’-tris-N-methylpiperizine-1,3,6-trisulfonamide 4b (compound of the invention) Potassium carbonate (0.017 g, 0.12 mmol, 1.2 eq.) and 4,5-dimethoxy-2-nitrobenzyl bromide (0.033 g, 0.12 mmol, 1.2 eq.) were added to a solution of compound 3b (0.07 g, 0.10 mmol) in acetonitrile (10 mL). The reaction mixture was heated to reflux and stirred for 24 h in the dark. After cooling, the reaction mixture was poured into water and extracted with dichloromethane (3×25 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was then purified by recrystallization in dichloromethane / n-pentane (50 / 50 v / v), yielding pure 4b as a dark red powder (49 mg, 54 % yield).1H-NMR (300 MHz, CDCl3): δ 9.32 (1H, d, J= 10 Hz), 9.29 (1H, d, J = 10 Hz), 9.25 (1H, s), 9.15 (1H, d, J = 10 Hz), 8.86 (1H, d, J = 10 Hz), 8.39 (1H, s), 7.83 (1H, s), 7.35 (1H, s), 5.96 (2H, s), 4.01 (3H, s), 3.98 (3H, s), 3.28 (8H, m), 3.19 (4H, m), 2.44 (12H, m), 2.22 (6H, s), 2.21 (3H, s).13C NMR (75 MHz, CDCl3,): δ 153.9, 153.3, 148.8, 140.0, 133.7, 132.5, 131.9, 130.6, 129.8, 129.7, 128.3, 126.4, 126.2, 125.8, 125.5, 125.1, 123.4, 123.2, 122.3, 113.2, 110.5, 108.6, 69.1, 56.6, 56.5, 54.2 (4C), 54.1 (2C), 45.8 (2C), 45.7 (4C), 45.7 (2C), 45.6. HRMS (ESI): m / z calc. for C40H50N7O11S3: 900.272495, found: 900.271354 [M+H]+. 8-((4,5-Dimethoxy-2-nitrobenzyl)oxy)pyrene-N,N,N’,N’,N’’,N’’-hexamethyl-1,3,6-trisulfonamide 4c (comparative) Potassium carbonate (0.15 g, 1.1 mmol, 1.2 eq.) and 4,5-dimethoxy-2-nitrobenzyl bromide (0.30 g, 1.1 mmol, 1.2 eq.) were added to a solution of compound 3c (0.50 g, 0.92 mmol) in acetonitrile (10 mL). The reaction mixture was heated to reflux and stirred for 24 h in the dark. After cooling, the precipitate was filtered and washed with acetone, yielding pure 4c as yellow powder (0.54 g, 79 % yield).1H-NMR (300 MHz, CDCl3): δ 9.32 (1H, d, J = 10 Hz), 9.30 (1H, d, J = 10 Hz), 9.17 (1H, S), 9.14 (1H, d, J = 10 Hz), 8.86 (1H, d, J = 10 Hz), 8.41 (1H, s), 7.83 (1H, s), 7.37 (1H, s), 5.95 (2H, s), 4.02 (3H, s), 4.00 (3H, s), 2.94 (12H, s), 2.87 (6H, s).13C NMR (75 MHz, CDCl3): δ 153.9, 153.3, 148.8, 140.1, 134.1, 132.6, 131.9, 130.2, 129.8, 128.4, 126.4, 126.4, 125.9, 125.5, 125.1, 123.5, 123.2, 122.4, 113.1, 112.8, 110.7, 108.6, 69.1, 56.6, 56.5, 37.7 (2C), 37.6 (2C), 37.5 (2C). HRMS (ESI): m / z calc. for C31H35N4O11S3: 735.145897, found: 735.145803 [M+H]+. 8-(4,5-Dimethoxy-2-nitrobenzyloxy)pyrene-N’,N’’,N’’’-tris-piperidine-1,3,6-trisulfonamide 4d (compound of the invention) Potassium carbonate (0.025 g, 0.18 mmol, 1.2 eq.) and 4,5-dimethoxy-2-nitrobenzyl bromide (0.05 g, 0.18 mmol, 1.2 eq.) were added to a solution of compound 3d (0.10 g, 0.15 mmol) in acetonitrile (10 mL). The reaction mixture was heated to reflux and stirred for 24 h in the dark. After cooling, the reaction mixture was poured into water and extracted with dichloromethane (3×25 mL). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude residue was then purified by flash chromatography using cyclohexane / ethyl acetate (70 / 30 v / v) as eluent, yielding 4d as a light yellow powder (80 mg, 62 % yield).1H-NMR (300 MHz, CDCl3): δ 9.30 (1H, d, J= 10 Hz), 9.29 (1H, d, J = 10 Hz), 9.22 (1H, s), 9.13 (1H, d, J = 10 Hz), 8.85 (1H, d, J = 10 Hz), 8.39 (1H, s), 7.83 (1H, s), 7.36 (1H, s), 5.95 (2H, s), 4.01 (3H, s), 3.98 (3H, s), 3.27 (8H, m), 3.17 (4H, m), 1.62 (12H, m) 1.47 (6H, m).13C NMR (75 MHz, CDCl3): δ 153.8, 153.2, 148.8, 140.1, 134.6, 132.3, 131.7, 130.7, 130.6, 130.1, 128.2, 126.5, 126.3, 125.9, 125.5, 124.9, 123.5, 123.1, 122.2, 113.0, 110.6, 108.6, 69.1, 56.6, 56.5, 46.7 (4C), 46.6 (2C), 25.4 (4C), 25.3 (2C), 23.5 (2C), 23.4. HRMS (ESI): m / z calc. for C40H47N4O11S3: 855.239798, found: 855.239384 [M+H]+. Trisodium 8-(4,5-Dimethoxy-2-nitrobenzyloxy)pyrene -1,3,6-trisulfonic acid 4e (synthesized according to the method of the invention) Potassium carbonate (0.17 g, 1.2 mmol, 1.2 eq.), tetrabutyl ammonium chloride (0.89 g, 3.2 mmol, 3.2 eq.) and 4,5-dimethoxy-2-nitrobenzyl bromide (0.33 g, 1.2 mmol, 1.2 eq.) were added to trisodium 8- hydroxypyrene-1,3,6-trisulfonic acid (HPTS, 0.52 g, 1 mmol) suspended in acetonitrile (20 mL). The resulting reaction mixture was then refluxed for 24 h. After the suspension was cooled down to room temperature, acetonitrile was removed under reduced pressure. The residue was washed with dichloromethane (5×15 mL) and dried under vacuum, yielding 4e as a light yellow powder (0.65 g, 90 %).1H-NMR (300 MHz, DMSO-d6): δ 9.15 (1H, d, J = 10 Hz), 9.06, (1H, d, J = 10 Hz), 9.04 (1H, s), 8.97 (1H, d, J = 10 Hz), 8.40 (1H, d, J = 10 Hz), 8.33 (1H, d, J = 10 Hz), 7.80 (1H, s), 7.68 (1H, s), 5.82 (2H, s), 3.99 (3H, s), 3.92 (3H, s).13C NMR (75 MHz, DMSO-d6): δ 153.2, 150.6, 147.9, 143.2, 140.1, 139.8, 139.7, 127.8, 127.5, 127.2, 126.3, 126.2, 125.5, 125.0, 124.4, 123.9, 120.9, 119.8, 119.7, 111.3, 109.2, 108.4, 67.5, 56.2, 56.1. HRMS (ESI): m / z calc. for C25H16NNa2O14S3: 695.953379, found: 695.952248 [M-Na]-. 8-(4,5-Dimethoxy-2-nitrobenzyloxy)pyrene -1,3,6-trisulfonyl chloride A (intermediate in the method of the invention) The compound 4e (0.10 g, 0.14 mmol) was suspended in anhydrous dichloromethane (10 mL). Oxalyl chloride (0.11 mL, 1.3 mmol, 9 eq) was added followed by 7 drops of anhydrous dimethylformamide. The resulting mixture was stirred at room temperature under inert atmosphere for 18 h. The solvent was evaporated. The crude residue was dissolved in anhydrous dichloromethane (3x10 mL) and then the solution was concentrated under reduced pressure. Compound A was obtained as a yellow / orange powder and used without further purification for the next step. Or Compound 4e (2.5 g, 3.5 mmol) was suspended in thionyl chloride (20 mL) and 10 drops of anhydrous dimethylformamide was added. The resulting mixture was stirred at 76°C for 5 h. The reaction mixture was then cooled down to room temperature and poured into ice with vigorous stirring. A yellow / orange color precipitate was obtained with effervescence of SO2gas. The crude precipitate was filtered and dried under reduced pressure. Compound A was obtained as a yellow / orange powder and used without further purification for the next step. 8-(4,5-Dimethoxy-2-nitrobenzyloxy)pyrene-N’,N’’,N’’’-tris-piperidine-1,3,6-trisulfonamide 4d (compound of the invention, synthesized according to the method of the invention) Compound A (0.10 g, 0.14 mmol) was dissolved in anhydrous dichloromethane (10 mL). The solution was then cooled to 0 °C before the addition of piperidine (0.12 mL, 1.3 mmol, 9 eq.). The reaction mixture was stirred under inert atmosphere for 48 h at room temperature. Then the reaction mixture was filtered and the filtrate was evaporated under reduced pressure. The crude residue was then washed with cyclohexane / absolute ethanol (7 / 3 v / v; 3×10 mL), yielding 4d as a bright yellow powder (60 mg, 50 % yield). Tris(2,2,2-trifluoroethyl) 8-((4,5-dimethoxy-2-nitrobenzyl)oxy)pyrene-1,3,6-trisulfonate 4f (compound of the invention, synthesized according to the method of the invention) To a solution of trifluoroethanol (0.8 mL, 11 mmol) in anhydrous DCM (25 mL), anhydrous triethylamine (2.8 mL, 20 mmol) was added and cooled down to 0°C in an ice bath. The solution of compound A (1.0 g, 1.4 mmol) in anhydrous DCM (120 mL) was then added dropwise at 0°C. The resulting mixture was then slowly warmed to room temperature and stirred under inert atmosphere for 40 h. The crude precipitate was obtained with the addition of n-pentane to the reaction mixture. The crude residue was then purified by washing with n-pentane / DCM (60 / 40 v / v; 3x30mL), yielding 4f as a light yellow powder (1.05 g, 83 % yield).1H-NMR (300 MHz, DMSO-d6): δ 9.27 (1H, s), 9.21 (1H, d, J = 10 Hz), 9.11 (1H, d, J = 10 Hz), 9.01 (1H, d, J = 10 Hz), 8.94 (1H, d, J = 10 Hz), 8.68 (1H, s), 7.82 (1H, s), 7.63 (1H, s), 6.10 (2H, s), 5.07 (6H, m), 4.01 (3H, s), 3.93 (3H, s).13C NMR (75 MHz, DMSO-d6): δ 154.6, 153.3, 148.31, 140.0, 133.3, 132.4, 131.8, 131.7, 129.2, 127.9, 124.2, 120.5, 116.8 (2C, q, JCF = 276 Hz), 127.8, 124.2, 120.5, 116.8 (1C, q, JCF = 276 Hz), 127.7, 125.9, 125.7, 125.5, 125.0, 124.9, 124.3, 123.0, 122.9, 120.5, 114.6, 111.8, 108.5, 68.8, 66.2, 65.7, 65.2, 64.7 (3C, q, JCF = 36 Hz), 56.4, 56.2.19F NMR (282 MHz, DMSO-d6): δ -75.2 to -75.4 (m, 3CF3). MS (CI, NH3): m / z calc. for [M+NH4]+: 917.04, found: 917.18 [M+NH4]+. Trisodium 8-(4-(oxymethyl)-6,7-dimethoxy-2H-chromen-2-one)pyrene-1,3,6-trisulfonic acid 4g (compound of the invention, synthesized according to the method of the invention) Potassium carbonate (39 mg, 0.29 mmol, 1.5 eq.) and 6,7-dimethoxy-4-bromomethyl coumarin (86 mg, 0.29 mmol, 1.5 eq.) were added to trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, 100 mg, 0.19 mmol) in acetonitrile / H2O (v / v, 9 / 3, 12 mL). The resulting reaction mixture was then heated at 80°C for 24 h in the dark. After cooling at to room temperature, the precipitate was filtered, washed with acetonitrile / H2O (v / v, 9 / 1) and finally with dichloromethane and dried in vacuo over P2O5. The pure coumpound 4g was obtained as a light yellow powder (105 mg, 74 %).1H-NMR (300 MHz, DMSO-d6): δ 9.16 (1H, d, J = 9 Hz), 9.05, (1H, d, J = 9 Hz), 9.02 (1H, s), 8.98 (1H, d, J = 9 Hz), 8.43 (1H, d, J = 9 Hz), 8.35 (1H, d, J = 9 Hz), 7.39 (1H, s), 7.17 (1H, s), 6060 (s,1H), 5.85 (2H, s), 3.90 (3H, s), 3.80 (3H, s).13C NMR (75 MHz, DMSO-d6): δ 160.4, 152.8, 151.5, 150.4, 149.2, 145.9, 143.3, 140.2, 139.9, 127.8, 127.5, 126.4, 126.2, 125.5, 125.1, 124.5, 124.1, 121.2, 119.9, 119.9, 109.7 (2C), 109.4, 106.0, 100.4, 66.9, 56.2, 56.0 HRMS (ESI): m / z calc. for C28H19O14S3: 674.9942, found: 674.9936 [M-H]-. 3. Absorption and emission properties of the free (3a-d) and caged pyranines (4a-e) All the free or caged pyranine derivatives are readily soluble in dimethyl sulfoxide to produce stock solutions. Hence, they were first dissolved in dimethyl sulfoxide and then diluted in their respective solvents for all experimental studies. The hydrophilic free and caged pyranine derivatives were diluted in 10 mM pH = 7.4 PBS buffer for 4a-b or 10 mM pH = 8.4 Trizma buffer for 4e and 4g. At the corresponding pH, the phenol group of the free pyranine derivative is fully ionized (vide infra), which maximizes fluorescence emission upon exciting at the 488 nm excitation wavelength, which is widely available on commercially available optical setups. The hydrophobic free and caged pyranine derivatives were diluted in 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v) for 4c-d and 10 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 1 (v / v) for 4f. Here the organic solvent enables for good solubility whereas a basic aqueous buffer is requested to both promote the liberation of the free pyranine derivative and cause phenol ionization of the free pyranine. At neutral pH, the free pyranine derivatives 3a-d, f and HPTS exhibit a strong absorption band in the 440 – 550 nm wavelength range peaking at 500 nm, which is red-shifted by 50 nm in comparison to HPTS (see Figure 1). Upon decreasing pH (see Figure 2), this absorption band drops and a new absorption band appears at 420 nm as anticipated from the gradual protonation of the phenolate yielding the phenol. The dependence of the absorbance at 488 nm and 420 nm on pH was utilized to extract the ground state pKa of the phenol function in the hydrophilic pyranine derivative 3a-b. A pKa of 6.0±0.1 was found for 3a. For 3b, the dependence of the absorbance on pH was better accounted by taking into account two proton exchange constants.4.7±0.1 and 6.6±0.1, were found which are interpreted as arising from ionization of the phenol and a protonated N-methyl nitrogen respectively. The pKaof 3a is lower than the one of the parent HPTS (7.3) as anticipated from the absence of any destabilizing electrostatic interaction in the phenolate form. It is even lower in 3b as expected from the stabilizing interaction between the negative charge of the phenolate and the positive charge of the ammonium. All the spectroscopic parameters are reported in Table 1 below. Pyranine ^ ^^^ (nm) ^(^^^^(mM-1cm-1) ^^^^(nm) atives^^^ ^)^^deriv^^3a 489 29 545 4a 421 9 538 3b 500 23 552 4b 432 21 543 3c 522 10 556 4c 417 39 -- 3d 524 10 557 4d 419 36 -- HPTS 456 24 507 4e 401 17 507 4g 401 25 510 3f 535 26 560 4f 426 22 -- Table 1. Absorption and emission properties of the free (3a-d,f, HPTS) and caged (4a-g) pyranine derivatives. ^ and ^^^^^^designate the maximum of light absorption, the molar absorption coefficient at and the maximum of fluorescence emission respectively. Solvents: 10 mM pH = 7.4 PBS buffer (for 3a-b and 4a-b), 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v) (for 3c-d and 4c-d), 10 mM pH = 8.4 Trizma buffer (for HPTS,4e and 4g), and 10 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 1 (v / v) (for 3f and 4f). 4. Preliminary evidence and evaluation for uncaging 4a-g A first series of illumination experiments were carried out to establish the relevance of the design of the caged pyranine derivatives 4a-4g and to fix optimal conditions for their reliable use as fluorescent actinometers for measuring light intensity. These experiments were carried out by following the time evolution of the fluorescence signal at 540 nm from applying time constant and spatially homogeneous light at 365 nm on solutions of the caged pyranine derivatives 4a-g contained in 54 ^L cuvettes (3 mm optical pathlength). Figures 3-10 display the results. Depending on the nature of the caged pyranine derivative and on the experimental conditions, we observed an increase of the fluorescence signal by a factor 3.4 to 15, which mainly occurred at the 50-500 s time scale at (0.1-2.5) 10-8E.s-1light intensity and (0.6-14) 10-4E.m-2.s-1photon flux density. The range of the uncaging yield was further evaluated from comparing the fluorescence intensities of solutions of the free pyranine derivatives 3a-d,f and HPTS with the ones of solutions of the caged pyranine derivatives 4a-g at the same concentration after uncaging for giving the following results: 71% yield for 4a, 34% yield for 4b, 55% yield for 4c, 73% yield for 4d, 71% yield for 4e, 95% yield for 4f and 17% yield for 4g. Moreover, this first series of uncaging experiments enable to select optimal experimental conditions in which the time evolution of the fluorescence signal of the illuminated solution could be reliably fitted with a monoexponential fitting function to extract the characteristic time ^, and to retrieve first values of the uncaging cross sections (see Table 2), to fix optimal conditions for using the caged pyranine derivatives 4a-g as fluorescent actinometers for measuring light intensity. - Regarding the hydrophobic caged pyranine derivative 4d: A value of 15.6±1.6 m2.mol-1has been retrieved for the associated cross section for 4d in pH = 7.4 PBS buffer: acetonitrile 1:1 (v:v). - Comparison with the comparative caged pyranine derivative 4c: The uncaging of 4c was performed at light intensities ranging from 0.5 to 2.510-8E.s-1((2.8-14).10-4E.m-2.s-1) in pH = 7.4 PBS buffer: acetonitrile 1:19 (v:v) (Figure 5). In the examined range of timescales (0-4.103s), a monoexponential fitting function was applied and a characteristic time essentially inversely proportional to the light intensity was retrieved, thus allowing to determine the associated cross section for 4c which is 18.3±2.0 m2.mol-1. Yet, a departure from the monoexponential behavior at the longest times was observed for 4c. Since the dimethylamino substituents in 4c are less bulky than the piperidine ones in 4d, it is likely that deaggregation is slower in 4c than in 4d. - Regarding the hydrophilic comparative caged pyranine 4a: 4a was dissolved in 10 mM pH 7.4 PBS (Figure 3). Under illumination at 365 nm at 2.5×10-8E.s-1light intensity and 1.4×10-3E.m-2.s-1photon flux density, an increase of its fluorescence by a factor 3 was observed and an uncaging yield of 70 % was computed from comparing the final signal with the one of the free pyranine 3a at the same concentration in the same solvent. Moreover, from fitting the time fluorescence emission with a monoexponential function, a value of 1.6±0.2 m2.mol-1was retrieved for the associated cross section. - Regarding the hydrophilic cage pyranine 4b: 4b was dissolved in 10 mM pH 7.4 PBS and the resulting solution was submitted to light intensities ranging from 0.5 to 2.510-8E.s-1((2.8-14) 10-4E.m-2.s-1) (Figure 4). The drop of light intensity governs a change from a biexponential to a monoexponential time evolution of the fluorescence signal without any significant modification of both the contrast between the final and initial fluorescence signal (around 6) and the uncaging yield was found to be around 60 %. - Regarding the hydrophilic cage pyranine 4e: Upon illumination of 4e in 10 mM pH = 8.4 Trizma buffer at 365 nm, an increase of fluorescence by a factor up to 6 was observed, which is associated to an uncaging yield of 70 % from comparing the final signal with a reference HPTS solution at the same concentration in the same solvent (Figure 8). Moreover, the time fluorescence emission from the aqueous solution of 4e in 10 mM pH = 8.4 Trizma buffer is satisfactorily accounted by a monoexponential fit at light intensities ranging from 0.5 to 2.510-8E.s-1((2.8- 14) 10-4E.m-2.s-1). The retrieved characteristic time is essentially inversely proportional to the light intensity, which yields 8.6±1.0 m2.mol-1for the associated cross section. As an outcome of this first series of experiments, complementary experiments were performed on the most promising caged fluorophores for calibration of light intensity in various environments: (i) 4d for applications in organic solvents and hydrophobic media. Indeed, it exhibits an uncaging cross section similar to 4c but it does not suffer from any departure from a monoexponential time evolution of the fluorescence signal observed with the latter at long times, which makes data processing less robust; (ii) 4e and 4g for applications in aqueous solutions; (iii) 4b for applications requiring permeation through cell bilayers. Indeed, as it will be illustrated later on, in contrast to 4b, 4a revealed unable to cross the cell membranes (vide infra). 5. Determination of light parameters of 4b, 4d, 4e and 4g The time evolution of the absorption and emission spectra of the caged pyranine derivatives 4b, 4d, 4e and 4g upon illumination in a 1×1 cm2quartz cuvette with a LED emitting at 365 nm in 10 mM pH = 7.4 PBS, 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v), and 10 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 1 (v / v) respectively has been implemented (see Figure 11). The acquisition times (in seconds) are: 0, 60, 300, 600, 1200, 2400, 3600. As anticipated from uncaging at the considered pH, one observes the gradual decay of the absorption band associated with the caged pyranine derivatives 4d, 4e and 4g, and the rise of the absorption band associated with the anionic form of the free pyranine 3d and HPTS. The presence of sharp isosbestic points further suggests the absence of any observable intermediate in the conversion of the caged into the free pyranine at the considered time scale. As expected from caging the phenolic OH group, all the caged pyranine derivatives are weakly fluorescent or non-fluorescent upon excitation at 450-480 nm. Under illumination at 365 nm, one observes the gradual rise of the fluorescence emission identical to the one of the free pyranine. The kinetics of the time evolutions of the absorbance and fluorescence signals were then analyzed. They were satisfactorily fitted with monoexponential functions and the extracted characteristic times were in line for both observables. Hence, 535 s and 530 s, 173 s and 134 s, 535 s and 298 s and 82 s and 72 s were found with the absorbance and the fluorescence for 4b, 4d, 4e and 4g respectively. In a second series of experiments for quantitative characterization of the uncaging of 4b, 4d, 4e and 4g their uncaging cross section at 365 nm was measured by using fluorescence for reporting. The same experiments of time evolution of the absorption and emission spectra of the caged pyranine derivatives 4b, 4d, 4e and 4g displayed in Figure 11 were reproduced upon irradiation at various light intensities at 365 nm. Then, by applying the monoexponential fitting function given in Eq. (1) to the temporal rises of the normalized fluorescence the characteristic times of the photoconversion ^ were retrieved: in which t is the time of acquisition, A is the amplitude of the increase of the fluorescence signal occurring upon illumination, and τ is the characteristic time. As displayed in Figure 12A-D, the inverse of the characteristic times of the photoconversion linearly depends on the light intensity. This behavior demonstrates that the rate limiting step of uncaging is photochemical at least up to 0.01 E.m-2.s-1light intensity. Eq. (linear fit) was used to retrieve the values of the uncaging cross sections from the slope: 12.9, 38.9, 16.3 and 462 m2.mol-1were found for 4b, 4d, 4e and 4g, respectively (see Table 2 below). Equipped with the values of the uncaging cross sections at 365 nm, the range of excitation wavelengths for uncaging by using a Xe lamp as a light source was expanded. Hence, the time evolution of the fluorescence signal from solutions of 4b, 4d, 4e and 4g, upon applying illumination from 300 to 440 nm was recorded (see Figure 14). After monoexponential fitting, the corresponding relaxation times were retrieved, which were converted into values of the uncaging cross section from knowledge of the photon flux density measured with a powermeter by further using the value of the uncaging cross sections at 365 nm as a reference. The results are displayed in Table 3. Combined with the absorption spectra of 4b, 4d, 4e and 4g displayed in Figure 1, they have been used to retrieve the uncaging quantum yields of 4b, 4d, 4e and 4g at several wavelengths (see Table 3 below). In the last series of experiments for quantitative characterization of the uncaging of 4b, 4d, 4e and 4g, their uncaging cross section at 365 nm was measured by using HPLC to quantitate both the disappearance of the caged pyranines and the formation of the photoreleased pyranine upon illumination at 365 nm. Figure 15A-D displays the results. As expected, the peak associated to the caged pyranine derivative gradually disappears and it tends towards zero. At the same rate, the peak from the free pyranine grows and saturates at concentrations indicating an uncaging yield in the (19-67) % range. After monoexponential fitting of the time concentration evolutions, the characteristic times associated with the decay of the caged pyranine derivatives were retrieved and the rise of the photoreleased pyranine derivatives and subsequently the uncaging cross sections: 35.2±3.5, 28.9±2.9 and 21.5±2.1, 6.0±0.6 and 6.0±0.6, 606±61 and 785±78 m2.mol-1were respectively found for 4b, 4d, 4e and 4g respectively in fair agreement with the values retrieved from the preceding fluorescence experiments (see Table 2). The kinetics of photobleaching were evaluated upon submitting 2 ^M solutions of the free pyranine derivatives 3b, 3d and HPTS to LED illumination at 365 nm (see Figure 16). We could extract an estimate of their photobleaching cross section: 0.3, 1.2, 0.6 and 0.8 m2.mol-1were retrieved for 3b, 3d, and HPTS respectively, which suggests that no significant photobleaching should occur upon using the caged pyranine derivatives for measuring light intensity. Pyranine ^ (m2.mol-1) ^ (m2.mol-1) ^ (m2.mo-1 2 -1derivatives I II III l ) ^ (m .mol ) 4a 1.6±0.2 - - 1.6±0.2 4b 7.5±0.7 12.9±1.3 35.2±3.5 18.5±1.9 4c 18.3±2.0 - - 18.3±1.9 4d 15.6±1.6 38.9±3.7 25.2±2.5 26.6±2.7 4e 8.4±0.7 16.3±0.6 6.0±0.6 10.2±1.1 4g 560±56 462±11 695±70 572±57 Table 2. Photoconversion parameters of 4a-e,g. ^I, ^II, and ^III, ^ respectively designate the uncaging cross sections of the caged pyranine derivatives extracted from analyzing (i) the time dependence of the rise of the fluorescence emission at 540 nm for solutions of 4a-e, g, at 500 nm for the solution of 4e, and at 510 nm for the solution of 4g, under monochromatic illumination at 365 nm; (ii) the light intensity-dependence of the inverse of the associated relaxation time τ for 4b, 4d, 4e and 4g at 365 nm; (iii) the time evolution of the concentrations in caged pyranine derivative and its photoreleased free pyranine derivative measured by HPLC under illumination at 365 nm. ^ is the mean value of ^I, ^II, and ^III. T = 293 K. Caged pyranine ^exc ε( ^) ^( ^) ^ 103× ^( ^) derivatives (nm) (mM-1cm-1) (m2.mol-1) 300 24 7.7±0.7 1.4±0.1 365 14 18.5±1.9 5.7±0.6 4b 405 18 4.1±0.4 1.0±0.1 440 18 8.0±0.8 1.9±0.2 300 32 66±7 9.0±0.9 365 17 26.6±2.7 6.8±0.7 4d 405 27 29.4±2.9 4.7±0.5 440 4 6.3±0.6 6.8±0.6 300 6.5 18.2±1.8 12.2±1.2 4e 365 16 10.2±1.1 2.8±0.3 405 16 2.9±0.3 0.8±0.1 300 10 674±67 293±30 365 25 572±57 100±10 4g 405 22 575±58 114±12 440 0.6 17±2 123±13 Table 3. Dependence of the photoconversion parameters of 4b, 4d, 4e and 4g on the wavelength of light excitation. The molarabsorption coefficients ε( ^) were retrieved from the spectra recorded at 2 µM concentration in 10 mM pH = 7.4 PBS buffer (for4b), 1 mM pH = 8.4 Trizma buffer / acetonitrile 1 / 19 (v / v) (for 4d), and 10 mM pH = 8.4 Trizma buffer (for 4e and 4g) contained in a 1×1 cm2quartz cuvette. The uncaging cross sections ^( ^) of the caged pyranine derivatives have been extracted from analyzing the rise of the fluorescence emission at 540 nm of solutions of 4b, 4d, 4e, 4f (see legend of the Figure 14) under monochromatic illumination at 300, 365, 405, and 440 nm as representatives of 300 nm – 440 nm range by further using the value of the uncagingcross sections at 365 nm as a reference. The quantum yield of uncaging ^( ^) of the caged pyranine derivatives have beenextracted from the relation ^( ^) = ^( ^) / [2.3ε( ^)]. T = 293 K. 6. Applications Characterization of light intensity at 405 nm on a confocal microscope (using a liquid film sandwiched between two glass plates; Figure 17) The possibility of measuring the light intensity of a confocal microscope which exploits photon flux densities up to 104E.m-2.s-1has been investigated utilizing the caged pyranine derivatives 4b, 4e and 4g in liquid thin films. The liquid thin films were prepared with 2 μM 4b in 10 mM pH 7.4 PBS buffer, 1 μM 4e in 10 mM pH = 8.4 Trizma buffer and 2 μM 4g in 10 mM pH = 8.4 Trizma buffer, each solution sandwiched between two glass slides. Figure 17A-H displays the results. Figure 17A,D,G and 17B,E,H provide the fluorescence images of the thin films before and after scanning the field of view with the focused 405 nm laser respectively. The illumination increases the fluorescence intensity, which is in line with photoreleasing the free pyranine derivatives. The characteristic times were retrieved from the time evolution of the fluorescence signals integrated over the ROI (white frame). The uncaging time was processed to extract the uncaging cross section of 0, 4.2, and 565 m2.mol-1respectively for 4b, 4e and 4g from further exploiting the applied light power (independently measured with a power meter) and the beam waist of the laser in the focal plane (see Table 4). The latter values of the uncaging cross sections are in the range measured from the previous cuvette experiments (see Table 3). Hence, the reasonable agreement between the values of the uncaging cross sections over a wide range of photon flux densities suggests that the three caged pyranines 4b, 4e and 4g can be implemented to extract the photon flux density in a variety of optical setups. SampleCaged pyranines^exc (nm) ^ ^ (m2.mol-1) 4b 405 6.0±1.2 Thin film 4e 405 4.2±0.8 4g 405 565±44Table 4. Photoconversion parameters of 4b, 4e and 4g in thin films.Quantitative analysis of uncaging of 4e and 4g with two-photon excitation A series of experiments on 4e and 4g were performed to measure their cross section for uncaging with two-photon excitation. For designing the measurement protocol, advantage was taken of the increase of the fluorescence signal upon uncaging. Figures 18A and C respectively display the temporal evolution of the fluorescence emission from 3 µM solution of 4e and 4g in 10 mM pH = 8.4 Trizma buffer upon irradiation at 750 nm at (7-23) mW laser powers. One observes a continuous increase, which is in line with the realization of the uncaging process observed above. The characteristic time ^ was retrieved from applying a monoexponential fitting function and we verified that the dependence of the inverse of ^ with the applied laser power was quadratic as expected from Eq.(2) (Figures 18B and D). More precisely, we found 2.1 and 2.0 for the power exponent for 4e and 4g respectively. Hence, it was concluded that the observed uncaging behavior did result from two-photon absorption. The associated uncaging cross sections with two-photon excitation was extracted. The values 4.210-3and 4.210-2GM were retrieved for 4e and 4g respectively. Light intensity at a targeted location in a biological sample (cultured cells, Zebrafish embryo) with one- and two-photon excitation (relevance for optogenetics) The caged pyranine derivative 4b was used for measuring light intensity in living zebra fish embryos. The living zebra fish embryos at 5 hpf were conditioned for over-night with 1 ^M solutions of 4b and 2 μM of DMSO in water. The embryos were then washed with water to avoid any possible interference, which could originate from photoreleasing the free fluorophore in the surface of the embryos and placed in an agarose mold prior to light scanning with a confocal microscope by using dual illumination at 405 nm (for 4b uncaging) and 488 nm (for imaging the liberated free pyranine 3b). In the control experiment shown in Figure 19A-C, illumination of embryos solely conditioned with 2 μM DMSO does not promote any rise of the fluorescence emission. In contrast, we observed a rise of the fluorescence signal upon illumination for the embryos conditioned with 1 μM 4b (Figure 19D-F) suggesting 4b uncaging to occur with release of the free pyranine derivative 3b. The characteristic time ^ was retrieved from applying a monoexponential function on the time evolutions of the fluorescence signal from illuminated 4b conditioned embryos. The uncaging cross section of 4b measured in confocal microscopy (Table 4) and Eq. (1) was then exploited to extract the distribution of light intensity I in living zebra fishes (see in Figure 19G). As a last application, the caged pyranine derivatives 4a and 4b were used for measuring light intensity in cultured cells. Living Hela living cells were conditioned for 1 h with 1 ^M solutions of 4a and 4b in pH 7.4 PBS buffer (0.01 M phosphate, 0.154 M NaCl). Their external medium was subsequently washed prior to illumination to avoid any possible interference, which could originate from photoreleasing the free fluorophore in the incubating solution. Then the Hela cells were imaged with an epifluorescence by using 365 nm LED illumination. Figure 20A-D displays the results. As shown in Figure 20A, 365 nm illumination of the conditioned cells does not promote any rise of the fluorescence emission. In fact, the time evolution of the fluorescence signal is essentially similar to the behavior observed upon illuminating the background. This behavior suggests that the caged pyranine derivative 4a does not cross the cell membranes so as to be discarded for its use in biological media. In contrast, 365 nm illumination of the cells conditioned with the caged pyranine 4b causes an increase of the fluorescence signal, which significantly departs from the corresponding behavior observed in the background (see Figure 21A-D). Moreover, we observed that the characteristic times retrieved from processing the fluorescence increase with a monoexponential function from illuminated cells and 4b solution in pH 7.4 PBS buffer under identical illuminations conditions were essentially similar (Figure 21E-F). Hence, we could adopt the uncaging cross section of 4b in pH 7.4 PBS buffer to extract the value of the photon flux density applied on cells and found 0.03 E.m-2.s-1.

Claims

CLAIMS 1. A compound of the following formula (I)or a salt thereof, wherein: X is an aryl or a heterocyclyl, the aryl and heterocycly, being optionally substituted with one or more, preferably one to five substituents selected in the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1- C6 haloalkyl, C1-C6 haloalkoxy, OH, oxo, NRR’, halogen, CN, NO2, C(O)OH, C(O)-O-C1-C6 alkyl, - C(O)H, C(O)-C1-C6 alkyl, C(O)-aryl and aryl, with R and R’ being each independently H or C1-C6 alkyl, L is a linker corresponding to a divalent radical derived from a C1-C10 aliphatic chain, wherein one or more, preferably 1, 2, 3, or 4, methylene unit(s) of the C1-C10aliphatic chain are optionally replaced by –O– or –C(=O)–, wherein said C1-C10aliphatic chain is optionally substituted with one or more, preferably one, substituent selected from OH, C1-C6haloalkyl, C1-C6alkoxy, NR’’R’’’, halogen, C(O)OH, C(O)-O-C1-C6 alkyl, C(O)H, C(O)-C1-C6 alkyl, C(O)-O-aryl, C(O)-aryl and aryl, with R’’ and R’’’ being each independently H or C1-C6 alkyl, and R1, R2and R3each independently represent NR4R5or OR6, R4and R5each independently represent a C4-C18 alkyl, wherein up to 4 methylene units of the C4-C18 alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least two methylene units of the C4-C18alkyl are not replaced, or R4and R5together with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing R4and R5, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, such as a methyl,R6represents a H, C1-C18alkyl or an aryl, said C1-C18alkyl and aryl being optionally substituted with one or more, preferably one or two, substituents selected in the group consisting of OH, halogen, C1-C6 haloalkyl, C1-C6 alkoxy and C1-C6 haloalkoxy, provided that X is notwhen all of R1, R2and R3are OH and L is -CH2-.

2. The compound of claim 1, wherein X is selected from the group consisting of:Rxand Ryare each independently H or C1-C6alkyl, notably a methyl,Rxis a phenyl or a C1-C6 alkyl, notably a methyl,Rxis H or a C1-C6alkyl such as methyl, Ryis H, a C1-C6alkyl such as methyl, a O-C1-C6alkyl such as O-methyl, F, CF3, OCF3or CN, Rwis H, a C1-C6alkyl such as methyl, a O-C1-C6alkyl such as O-methyl or F and Rzis H or F,Rxis H, a C1-C6 alkyl such as methyl, a O-C1-C6 alkyl such as O-methyl or CN, Ryis H, a C1-C6alkyl such as methyl or a O-C1-C6alkyl such as O-methyl, Rzis H, a C1-C6alkyl such as a methyl, a O-C1-C6alkyl such as a O-methyl, Cl, F, OH, NH2, N(C1-C6alkyl)2or phenyl,Rxis H, phenyl or a O-C1-C6 alkyl such as a O-methyl, Ryis H, a O-C1-C6 alkyl such as a O-methyl, a halogen such as Br, phenyl or NO2, Rzis H, a O-C1-C6alkyl such as a O-methyl, N(Et)2or OH and Rwis H or Br,which Rxand Ryare each independently H or O-methyl, andwhich Rxis H or an aryl such as a phenyl, Ryis H, a O-C1-C6 alkyl such as a O- methyl, a halogen such as Br or an aryl such as a phenyl, Rzis H, OH a O-C1-C6 alkyl such as a O- methyl, N(Alkyl)2 or an aryl such as a phenyl, and Rwis H (Alkyl)2 or an aryl such as a phenyl, the wavy line representing the point of attachment to the rest of the molecule.

3. The compound of claim 1 or 2, wherein X corresponds to the group of formula (A) or (B)in which the wavy line represents the point of attachment to the rest of the molecule.

4. The compound of any one of claims 1 to 3, wherein L is a divalent radical –(CH2)n- with n being an integer from 1 to 4, preferably 1 or 2.

5. The compound of any one of claims 1 to 4, wherein R1, R2and R3each independently represent a group of formula in which m is 1 or 2, and preferably m is 2.

6. The compound of any one of claims 1 to 4, wherein R1, R2and R3each independently represent a group of formula, in which Y is O, S or NR7, preferably NR7, R7being H or C1-C6 alkyl, and m is 1 or 2, and preferably m is 2.

7. The compound of any one of claims 1 to 4, wherein R1, R2and R3each independently represents OR6, R6representing a H, a C1-C6alkyl, such as a methyl, substituted with one or two substituents selected in the group consisting of halogen and C1-C6haloalkyl, such as trifluoromethyl.

8. The compound of any one of claims 1 to 7, wherein R1, R2and R3are identical.

9. The compound of any one of claims 1 to 8, selected in the group consisting of:

10. A composition comprising a compound of formula (I) as defined in any one of claims 1 to 9 and a medium.

11. The composition according to claim 10, wherein the medium is an organic solvent, in particular a polar solvent, and the compound of formula (I) is as defined in claim 5.

12. The composition according to claim 10, wherein the medium is an aqueous medium, such as a biological medium, preferably a medium for culturing cells, and the compound of formula (I) is as defined in claim 6.

13. Use of a compound of formula (II) or a composition comprising thereof to measure light intensity:or a salt thereof, wherein X and L are as defined in any one of claims 1 to 4, Ra, Rband Rceach independently represent NRiRiior OR6, Riand Riieach independently represent H or a C1-C18alkyl, wherein up to 4 methylene units of the C4- C18alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least one methylene unit of the C4-C18 alkyl are not replaced, or Riand Riitogether with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing Riand Rii, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18alkyl, such as a methyl, and R6is as defined in any one of claims 1, 7 and 8.

14. A method for measuring a light intensity of a light source in a medium comprising the steps of: (a) Adding a compound of formula (II) as defined in claim 13 to a medium, (b) Illuminating the medium comprising the compound of formula (II) obtained at step (a) with a light source for a time t1,(c) Measuring the increase of fluorescence during the time t1 and determining the light intensity.

15. A method for preparing a compound of formula (II):or a salt thereof, wherein X and L are as defined in any one of claims 1 to 4, Ra, Rband Rceach independently represent NRiRiior OR6, and Riand Riieach independently represent H or a C1-C18 alkyl, wherein up to 4 methylene units of the C4- C18 alkyl are optionally replaced by O, C(O), NH or N-C1-C6alkyl, provided that at least one methylene unit of the C4-C18 alkyl are not replaced, or Riand Riitogether with the nitrogen atom to which they are bonded form a 4 to 6-membered heterocyclyl group optionally comprising, in addition to the nitrogen atom bearing Riand Rii, one or more, preferably one or two, heteroatom(s) selected from N, S and O, preferably N, said heterocyclyl group being unsubstituted or substituted by one or more, preferably one, C1-C18 alkyl, such as a methyl, R6is as defined in any one of claims 1, 7 and 8, comprising the steps of: (i) Reacting 8-hydroxypyren-1,3,6-trisulfonic acid trisodium salt (HPTS.3Na) with a compound of formula (III): X-L-LG (III) in which X is as defined in any one of claims 1 to 4, L is as defined above and LG is a leaving group, in order to form the following trisodium salt (IIa):N wherein the HPTS.3Na is not converted into the corresponding free sulfonic acid before reacting with the compound of formula (III), (ii) optionally converting the salt (IIa) obtained at step (a) into a compound of formula (II) or a salt thereof in which Ra, Rband Rceach independently represent NRiRiior OR6or (ii’) optionally converting the salt (IIa) obtained at step (a) into a compound of formula (II) in which Ra, Rband Rcrepresents OH or a salt thereof other than the trisodium salt (IIa).

16. The method of claim 15, wherein step (ii) comprises the following sub-steps: (ii-1) activating the SO3Na groups of the trisodium salt (IIa) obtained at step (a) with a halide compound to afford the corresponding sulfonyl halide groups, (ii-2) reacting each sulfonyl halide groups with a reagent of formula RaH, RbH or RcH, in which Ra, Rband Rceach independently represent NRiRiior OR6, (ii-3) optionally salifying the compound of formula (II) in which Ra, Rband Rceach independently represent NRiRiior OR6obtained in step (ii-2) in order to form a salt thereof.

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