Dr Maxime Klausen
email: mklausen@ed.ac.uk

Dr Mireille Blanchard-Desce
email: mireille.blanchard-desce@u-bordeaux.fr

Two-Photon Uncaging of Bioactive Compounds: Starter Guide to an Efficient IR Light Switch

Keywords: photolabile protecting groups, two-photon absorption, molecular engineering, infrared dyes, two-photon uncaging, drug delivery, photolysis

Triggering physiological responses with a light switch has become a reality with the development of smart molecular probes such as photolabile protecting groups (PPGs), able to "uncage" biological ligands on demand. To make the light switch virtually harmless and confine the excitation to the single-cell level, the caged ligands can be released using two-photon (2P) absorption and 2P microscopy using red/infrared light. This exceptional level of precision however comes at the cost of a reduced photosensitivity and a poor compatibility of early PPGs with 2P excitation. This review aims to provide a tutorial guidebook to the design of 2Psensitive PPGs suitable for optobiology by discussing challenges, strategies and progress in uncaging of bioactive compounds. To do so, we first recall the photo-physical principles governing 2P absorption, and the resulting ground rules in the design of efficient 2P absorbing organic dyes. We then detail how following these guidelines has led to tremendous progress in the development of a new generation of caged compounds, and the implications in the fields of biophotonics, from neurology to targeted therapy.

Introduction

At the frontier with biology, the design of molecular probes containing light-responsive moieties allows the use of light as an input, and collect valuable information on tissues (imaging), control physiological signals (optoneurology), or deliver bioactive ligands (therapy) as output. Among such tools, some excited chromophores undergo a photochemical cascade leading to the cleavage of a covalent bond. From the point of view of the released function, this cleavable chromophore can thus be described as a photolabile protecting group (PPG). This "protection-deprotection" strategy is suitable for masking not only chemical, but also biological functions in bioactive ligands; in which case the molecule, trapped by the PPG in the form of an inactive precursor, is often said to be metaphorically "caged" by this covalent bond. Shining light on the caged compound releases the ligand under its active form by restoring its original functionality, as a conventional deprotection reaction would do.

Although PPGs have opened avenues towards the control of physiological responses in tissues with light, the criteria for their use in biological environments are dire. In particular, the need to increase spatial resolution and avoid the photo-toxicity of UV light gradually led the research towards a promising alternative technique: two-photon excitation (2PE). This non-linear optical phenomenon makes it possible to excite a chromophore by simultaneous absorption of two halfenergy photons instead of one, which doubles the wavelength of irradiation (Figure 1). The trigger then becomes red or near-infrared (NIR) light, whose wavelengths penetrate better into tissues and reduce the risk of photodamage. Shortly after the introduction of the first 2P microscopes, [START_REF] Denk | Two-photon laser scanning fluorescence microscopy[END_REF] and even before their use in cell biology became widely accessible, Adams and Tsien predicted in 1993 the substantial progress that would rise from the combination of 2P microscopes and the photorelease of biomolecules. [START_REF] Adams | Controlling Cell Chemistry with Caged Compounds[END_REF] In almost 30 years, with the advent of Ti:Sapphire lasers and 2P microscopy techniques, the field of 2P uncaging has not only come into being, but has also largely grown. The capacity for a chromophore to absorb two photons simultaneously is however dictated by very different physical rules from the traditional singlephoton (1P) irradiation, and even to this day, the 2P photosensitivity of PPGs described in literature remains a barrier to widespread biological applications. The aim of this review is to provide guidelines and rationality to the design of new 2P-sensitive "cages" based on theoretical rules, and empirical evidence found in literature. To this end, we will first introduce the fundamental notions of photophysics at play in 2P absorption, and provide explanation for the design of 2P absorbing chromophores. We will then review how such engineering strategies were applied to the main families of PPGs, and the implications in selected biological applications.

Figure 1. Simplified Jablonski diagrams and key features of the 1PA (left) and 2PA phenomena (right). The photo-deprotection phenomenon is illustrated in cuvettes with the case of an active substrate (triangle) protected with a PPG (circle). and the resulting volume of photorelease in each case. The center picture illustrate the difference in spatial resolution between both types of excitation with the example of red-emissive dye excited with a focalised light beam (405 nm) in 1PA (top) and a pulsed laser beam (1050 nm) in 2PA (bottom)

Molecular engineering of two-photon dyes: from theory to application

From linear to non-linear optics

Light-matter interactions must be considered from two complementary perspectives. When light, as an electromagnetic wave, interacts with matter, its electric field 𝑬 ⃗ ⃗ also causes a displacement of the charges within the material, and therefore the creation of dipoles. The resulting macroscopic polarization field 𝑷 ⃗⃗ is directly related to the amplitude of 𝑬 ⃗ ⃗ in the light beam, and can generally be expressed as a Taylor polynome (Equation ( 1)).

𝑷 ⃗⃗ = 𝑷 𝟎

⃗⃗⃗⃗⃗ + 𝜀 0 𝛘 (𝟏) 𝑬 ⃗ ⃗ + 𝜀 0 𝛘 (𝟐) 𝑬 ⃗ ⃗ 2 + 𝜀 0 𝛘 (𝟑) 𝑬 ⃗ ⃗ 3 + ⋯

In which 0 is the vacuum permittivity constant, and  (n) is a tensor of rank n + 1 describing the electrical susceptibility of the n th order of the material. At the molecular level, the resulting dipole moment 𝝁 ⃗ ⃗ can be expressed in a similar way according to the local field 𝑭 ⃗ ⃗ (Equation ( 2)).

𝝁 ⃗ ⃗ = 𝝁 𝟎 ⃗⃗⃗⃗ + 𝛂𝑭 ⃗ ⃗ + 𝛃𝐅 2 + 𝛄F 3 + ⋯

In this expression, 𝝁 𝟎 ⃗⃗⃗⃗ is the permanent dipole moment of the molecule, and  is the polarisability. In linear optics, only this first-order term is taken into account. Other terms can be neglected, and the resulting polarisation expression is linear. When the intensity of 𝑬 ⃗ ⃗ increases, this approximation of linearity is no longer valid. Higher order terms such as those involving the quadratic hyperpolarisability (or second order polarizability) ,cubic hyperpolarisability (or third order polarizability)  etc., become significant in the Taylor polynome. This is the field of non-linear optics (NLO). [START_REF] Butcher | The Elements of Nonlinear Optics[END_REF][START_REF] Shen | The Principles of Nonlinear Optics[END_REF][START_REF] Sutherland | Handbook of nonlinear optics[END_REF] Quadratic hyperpolarisability  governs non-resonant second-order NLO phenomena disrupting the ground state without promoting an electron to an electronic excited state, such as second harmonic generation (SGH). [START_REF] Campagnola | Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms[END_REF] Cubic hyperpolarisability  reflects third-order phenomena, such as third harmonic generation (TGH) and the 2P absorption (2PA).

Indeed, in a quantum physics description, if the incident energy is high enough to fill the gap between the ground state and the excited state, and if the oscillation is efficient enough to "push" the electron towards a higher energy MO, resonant NLO can occur with the simultaneous absorption of two photons (or more [START_REF] Sutherland | Handbook of nonlinear optics[END_REF]). The chromophore is then promoted from its ground state S0 to an excited state, as illustrated on Figure 1. This was first theorised by Maria Göppert-Mayer in 1929, [START_REF] Göppert | Über die Wahrscheinlichkeit des Zusammenwirkens zweier Lichtquanten in einem Elementarakt[END_REF][START_REF] Göppert-Mayer | Über Elementarakte mit zwei Quantensprüngen[END_REF] but could only be observed experimentally in 1961 by Kaiser and Garrett [START_REF] Kaiser | Two-Photon Excitation in Ca2+[END_REF] after the development of the first pulsed lasers. The real part of hyperpolarisability  will influence the nonlinear refractive index of the medium, while its imaginary part will define the 2PA cross section 2. [START_REF] Terenziani | Enhanced Two-Photon Absorption of Organic Chromophores: Theoretical and Experimental Assessments[END_REF][START_REF] Andraud | Lanthanide Complexes for Nonlinear Optics: From Fundamental Aspects to Applications[END_REF] Absorption cross-sections n define the absorption capacities of 1PA and 2PA dyes as the effective "absorption surface" (or section) on which photons can be captured as light passes.

As exemplified in the case of 1PA (Figure 2), each molecule absorbs on a fraction /S of the surface beam S. On the totality of the infinitesimal section considered, the total absorbed surface , is equal to this fraction multiplied by the number of chromophores in the corresponding volume, and therefore the concentration of the sample. In 2PA, the cross-section 2 therefore quantifies the capacity of a dye to absorb two photons simultaneously, and is expressed in Göppert-Mayer (1 GM = 10 -50 cm 4 s photon -1 ) as a tribute to its original theoretician. In a common model describing the 2PA phenomenon, the absorption of a first photon causes the creation of a very short-lived (10 -15 s) non-resonant excited state, called "virtual state". To further promote the electron on a real electronic level, the absorption of the second photon must occur on the hundreds of attoseconds to tens of femtoseconds timescale, [START_REF] Shen | The Principles of Nonlinear Optics[END_REF] during the lifetime of the virtual state, which explains the use of pulsed lasers. The probability to encounter a second photon on this timeframe also depends on the spatial and temporal overlap with the incident photons, which implies a quadratic dependence on light intensity. Thus, by applying hypotheses equivalent to Beer-Lambert's law, [START_REF] Rumi | Two-photon absorption: an overview of measurements and principles[END_REF] the light intensity attenuation following 2PA can be described according to Equation ( 3), where I is the intensity expressed as photon flux, ℓ the optical path, C N the number of molecules per cm 3 , and  and 2 respectively the 1P and 2P absorption cross-sections at a given wavelength.

∂I ∂ℓ =-C N σ I -C N 𝜎 2 I² (3)
Considering that the energy of photons used for 2PA is largely insufficient to resonate alone with an excited state and cause 1PA, only the second quadratic term of Equation ( 3) remains.

This quadraticity confines the excitation exclusively to the focal point of the laser, and provides spatial resolution on the femtolitre level, i.e. the size of E. coli bacteria or individual human synapses. Although it is theoretically possible to use two photons of different energy, [START_REF] Hales | Nondegenerate two-photon absorption spectra of organic materials[END_REF][START_REF] Hales | Resonant enhancement of two-photon absorption in substituted fluorene molecules[END_REF][START_REF] Cirloganu | Extremely nondegenerate two-photon absorption in direct-gap semiconductors[END_REF] 2PE experimental setups generally use two photons of identical "degenerate" energy hexc/2, which consequently doubles the excitation wavelength used in 1PE, and shifts it to red or NIR wavelengths. Phototoxicity of such wavelengths is far lesser than UV radiations and their tissue penetration is largely improved thanks to poorer absorption of endogenic chromophores and scattering. With a penetration of up to a few cm without particular damage, [START_REF] Weissleder | A clearer vision for in vivo imaging[END_REF] this wavelength range is called the "biological transparency window". [START_REF] Bashkatov | Optical properties of human skin, subcutaneous and mucous tissues in the wavelength range from 400 to 2000 nm[END_REF] As selection rules differ in NLO, the excited state reached upon quasi-simultaneous double absorption may be different from the one achieved by single-photon absorption. Selection rules dictate whether an electronic transition is allowed or forbidden based on the overlap between the two MOs involved and the so-called transition dipole moment gx (Equation ( 4)).

 gx  ∫  g 𝜇* x 𝑑 3 𝑟 ∞ -∞ (4)
 g and * x are the wave functions respectively associated with the MO of the ground state S0

and the excited state Sx, 𝜇̂ is the dipole moment operator which corresponds to the charge of the electron multiplied by its position vector, and 𝑑 3 𝑟 indicates that the integral is calculated over the entire space coordinates. Transitions are called forbidden if the norm of their transition dipole moment is zero. In 2PA, selection rules have important consequences on the absorption wavelength and the absorption capacity of a chromophore.

From quantum models to ground rules

Based on the physical foundations of NLO, several ground rules apply to the development of non-linear materials. Among these, two key factors influence particularly the 2PA response of a material: internal charge transfer (ICT) and centrosymmetry. The latter is known to be a restrictive parameter towards even order polarisabilities (such as ). As a third order phenomenon, 2PA can theoretically be observed with any chromophore irrespective of its symmetry. The symmetry of the dye will however alter the nature of the electronic transitions observed based on their "allowed" or "forbidden" nature. Quantum models [START_REF] Butcher | The Elements of Nonlinear Optics[END_REF][START_REF] Buckingham | Permanent and Induced Molecular Moments and Long-Range Intermolecular Forces[END_REF][START_REF] Takahashi | Anharmonic oscillator modeling of nonlinear susceptibilities and its application to conjugated polymers[END_REF][START_REF] Orr | Perturbation theory of the non-linear optical polarization of an isolated system[END_REF] have been developed to rationalise such properties with regards to the symmetry of orbitals and wavefunctions. In particular, "essential states models" [START_REF] Mazumdar | A four-level "essential states" model of third order optical nonlinearity in π-conjugated polymers[END_REF][START_REF] Birge | A theoretical analysis of the two-photon properties of linear polyenes and the visual chromophores[END_REF][START_REF] Dirk | A simplified three-level model describing the molecular third-order nonlinear optical susceptibility[END_REF][START_REF] Kuzyk | Theory of molecular nonlinear optics[END_REF] allow the description of the 2PA cross-section 2 as a function of the complex tensor of the microscopic hyperpolarisability  Such models consider that, in a perturbative description [START_REF] Orr | Perturbation theory of the non-linear optical polarization of an isolated system[END_REF] of  the main contributions are provided by the lower-energy 1P-allowed excited state of the linear optical response, and by a few additional (i.e. "essential") states that are strongly dipole-coupled to it. Therefore, only the interaction between the ground state |𝑔⟩, the final two-photon-allowed excited state |𝑓⟩ and the major one-photon-allowed state |𝑖⟩ are considered. Each of those electronic states MOs belong to a certain symmetry group, and consequently, selection rules may apply restrictions to certain transitions (Figure 3). In non-centrosymmetric systems (Figure 3 (a)), the transition to the first excited state is generally both 1P-and 2P-allowed. Only two states, |𝑔⟩ and |𝑓⟩, can be considered in this description of the 2P phenomenon ("two-state model").

In the case of centrosymmetric chromophores (Figure 3 moment. Thanks to this double inversion, the transition 𝑔 → 𝑓 is 2P-allowed while being symmetry-forbidden in 1PA. This inversion of the selection rules between 1PA and 2PA can be generalised to all centrosymmetric chromophores, in which 2PA max is located at higher energies than 21PA max . Therefore, three "essential states" are considered in this description. The key to essential state models is to rely on the lifetime of the virtual state and its potential interaction with the lowest-lying resonant excited state. Indeed, the uncertainty principle states that the lifetime 𝛥𝑡 of a photon ℏ𝜔 in this intermediate state will be superior to the ratio ℏ 𝛥𝐸 ⁄ ,

where 𝛥𝐸 is the so-called "detuning resonance energy", i.e. the difference between the energy level of the virtual state and that of the 1P resonant state |𝑖⟩ (Figure 3). Consequently, if 𝛥𝐸 decreases, the lifetime of the virtual state increases, which increases the probability to absorb a second photon, and therefore the 2 of the chromophore.

As stated above, the 2PA cross-section is directly correlated to the cubic hyperpolarisability of the material, and more specifically its imaginary part Im(𝛄(-𝜔, 𝜔, -𝜔, 𝜔)). Although the demonstration goes beyond the scope of this review, [START_REF] Terenziani | Enhanced Two-Photon Absorption of Organic Chromophores: Theoretical and Experimental Assessments[END_REF][START_REF] Orr | Perturbation theory of the non-linear optical polarization of an isolated system[END_REF][START_REF] Meath | On the importance of permanent moments in multiphoton absorption using perturbation theory[END_REF][START_REF] Nakano | A proposal of new organic third-order nonlinear optical compounds. Centrosymmetric systems with large negative third-order hyperpolarizabilities[END_REF][START_REF] Przhonska | Two-Photon Absorption in Near-IR Conjugated Molecules: Design Strategy and Structure-Property Relations[END_REF] the expression of the fourth rank tensor  [START_REF] Orr | Perturbation theory of the non-linear optical polarization of an isolated system[END_REF] introduced in the context of the essential states model provides a proportionality relation for 2 [START_REF] Nakano | A proposal of new organic third-order nonlinear optical compounds. Centrosymmetric systems with large negative third-order hyperpolarizabilities[END_REF][START_REF] Zojer | Tuning the two-photon absorption response of quadrupolar organic molecules[END_REF] (Equation ( 5)).

In Equation ( 5), the imaginary unit is called j for the sake of clarity, the transition dipole moments associated with the transition 𝑥 → 𝑦 are called xy, and the difference between static dipole moments of states |𝑥⟩ and |𝑦⟩ is written xy for a better distinction. Exy is the energy difference between both states, and xy is a damping factor associated with the transition. The three components of this expression contribute differently to the value of 2. The D-term is named for its "dipolar" contribution, as it involves mainly static dipole moments Mxy. The second positive contribution is called "Two-photon-term" (T-term) as it is always present in 2PA. Finally, the negative-term (N-term) can be subtracted to this expression when the photon ℏ𝜔 is too close to the resonant energy, [START_REF] Przhonska | Two-Photon Absorption in Near-IR Conjugated Molecules: Design Strategy and Structure-Property Relations[END_REF] which is usually not the case. Thus, by neglecting the N-term, considering that the damping factor  is identical for all transitions, [START_REF] Zojer | Tuning the two-photon absorption response of quadrupolar organic molecules[END_REF] and isolating the imaginary part, Equation ( 5) can be further simplified to the following expression. [

Equation ( 5) and ( 6) therefore create a distinction between two cases, depending on the existence or not of a difference between dipole moments in the ground state and in the excited state Mgi; and therefore on the symmetry of the chromophore.

In non-centrosymmetric dipolar chromophores (Figure 3 (a)), the presence of static dipole moments in the ground state |𝑔⟩ and in the excited state |𝑓⟩ (Mgi ≠ 0) implies that the dipolar term of Equation ( 5) and ( 6) will be non-zero, and that both the D-and the T-term will contribute to the 2. However, as shown in Figure 3, the intermediate state |𝑖⟩ is merged with or higher than |𝑓⟩, (𝐸 𝑔𝑖 -ℏ𝜔) ≥ ℏ𝜔. Therefore, assuming that the static and transition dipoles are all of the same order of magnitude, the D-term contribution to the expression of 2 will generally be greater than that of the T-term.

In centrosymmetric chromophores however (Mgi = 0), the dipolar term is zero, and the 2PA cross-section is directly governed by the T-term. The lower energy level of |𝑖⟩ compared |𝑓⟩ brings out two direct consequences. First, when energy gap between the virtual state and the 1P-allowed state |𝑖⟩ decreases (Figure 3 (b) and (c)), the denominator of the T-term 𝛥𝐸² decreases quadratically. Consequently, very high 2 values can be reached in centrosymmetric "quadrupolar" compounds, which is mathematically impossible in the case of dipoles. Secondly, and as opposed to non-centrosymmetric chromophores, 𝛥𝐸 < ℏ𝜔. Therefore, for equal dipole moments, i.e. equal -conjugated structure and push-pull strength, the T-term in a centrosymmetric chromophore will necessarily be greater than the D-term in its noncentrosymmetric analogue. Therefore, provided that the detuning energy is small enough, the 2P response of a quadrupolar compound will be inherently stronger than the one of its corresponding dipole.

Based on Equation ( 5) and ( 6), general trends in the molecular engineering of 2P absorbing materials have been established. In practice, some terms of the equation such as  and  are difficult to access experimentally, [START_REF] Rumi | Structure-Property Relationships for Two-Photon Absorbing Chromophores: Bis-Donor Diphenylpolyene and Bis(styryl)benzene Derivatives[END_REF] but the following rules can be followed: [START_REF] Przhonska | Two-Photon Absorption in Near-IR Conjugated Molecules: Design Strategy and Structure-Property Relations[END_REF][START_REF] Pawlicki | Two-Photon Absorption and the Design of Two-Photon Dyes[END_REF][START_REF] He | Multiphoton Absorbing Materials: Molecular Designs, Characterizations, and Applications[END_REF][START_REF] Collini | Cooperative effects to enhance two-photon absorption efficiency: intraversus inter-molecular approach[END_REF][START_REF] Kogej | Mechanisms for enhancement of two-photon absorption in donor-acceptor conjugated chromophores[END_REF] (i) Increasing the static and transition dipole moments. According to Equation ( 6), the increase in gi leads to a quadratic increase of 2, as does the increase of transition dipole moments . In practice, this can be done in two ways. The first is to tune the electron-donating (ED) or electron-withdrawing (EW) capacity of the end-groups (and/or the core, if relevant) of the molecule. The intramolecular charge transfer (ICT) is therefore a major driving force of 2PA, and any modification in this favour should affect the 2 positively. The second is to increase the length of the -conjugated system. Indeed, according to Equation (4), the transition dipole moment is calculated by integration over the three coordinates of the radius r, i.e. the distance over which the charge moves during a transition. Intuitively,  will increase when its vector coordinates are located farther away from the origin, which is achieved with longer conjugation. There is however an upper limit to this chain length beyond which the wavefunction becomes incoherent, especially because of the loss of planarity in the molecule. [START_REF] Pawlicki | Two-Photon Absorption and the Design of Two-Photon Dyes[END_REF] (ii) Prepare centrosymmetric chromophores. For equal donor and acceptor groups, a quadrupole (D--A--D or A--D--A) will have a higher 2P response than the corresponding dipole (D--A or A--D).

(iii) Approaching resonance for the virtual state. In centrosymmetric systems, if the 1Pallowed state is located near the virtual state (i.e., the wavelength of the laser), the 2PA cross section can be significantly increased, as observed for instance on porphyrin derivatives. [START_REF] Drobizhev | Resonance enhancement of twophoton absorption in porphyrins[END_REF] This effect is called intermediate state resonance enhancement (ISRE). [START_REF] Hales | Resonant enhancement of two-photon absorption in substituted fluorene molecules[END_REF][START_REF] Drobizhev | Resonance enhancement of twophoton absorption in porphyrins[END_REF][START_REF] Kamada | Two-photon absorption properties of symmetric substituted diacetylene: drastic enhancement of the cross section near the one-photon absorption peak[END_REF]] However, at wavelengths close to 1P resonance, linear absorption may not be negligible any longer, and additional factors such as 1PA line shape and 2PA to 1PA ratio may have to be taken into account to determine the actual 2P response.

(iv)

Minimising the bandwidth. The damping factor  influences the bandwidth of the transition; and a sharper band will result in a higher 2 max than a flattened band.

Importantly, these key factors are interdependent, and they often need to be considered holistically to anticipate effects on 2. In certain cases, quantitative predictions remain hard to achieve to this day, as the current theoretical models fail to provide a complete understanding of the transitions observed experimentally. As an example, in non-centrosymmetric chromophores, intense transitions can sometimes be observed in the lower wavelengths of the 2PA spectrum without being prominent at 2PA/2 in linear absorption, which is sometimes attributed to important vibronic contributions. In centrosymmetric systems, the theoretical parity selection rule, albeit predicting the nature of the 2P-allowed transitions, sometimes falls short of quantitation and intensity prediction. Quantum chemical calculations can provide assistance in this case, but experimental measurements of the 2PA spectra remain the most reliable source of quantitative information. [START_REF] Sheik-Bahae | Sensitive measurement of optical nonlinearities using a single beam[END_REF][START_REF] Xu | Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm[END_REF] Nonetheless, since the first demonstration of the phenomenon on organic chromophores, [START_REF] Peticolas | Double Photon Excitation in Organic Crystals[END_REF] the rational design of 2P-responsive chromophores has progressed dramatically. [START_REF] Pawlicki | Two-Photon Absorption and the Design of Two-Photon Dyes[END_REF][START_REF] He | Multiphoton Absorbing Materials: Molecular Designs, Characterizations, and Applications[END_REF][START_REF] Kim | Small-Molecule Two-Photon Probes for Bioimaging Applications[END_REF] They are composed mainly of three components: an electron-donor group (D), and an electronwithdrawing group (A) linked via a polarizable -bridge (or "core", for centrosymmetric systems).

Engineering of 2P absorbing molecules

The ICT is generated by ED and EW groups located either at the ends, or on the core of the chromophore. Hammett coefficients [START_REF] Hansch | A survey of Hammett substituent constants and resonance and field parameters[END_REF] can be useful indexes to evaluate the strength of such groups, and their effect on the 2PA cross-section. The most common terminal groups are dialkyl-or diphenylamino moieties, that provide high donating strength and retain a certain stability to oxidation. [START_REF] Mongin | Synthesis, Fluorescence, and Two-Photon Absorption of a Series of Elongated Rodlike and Banana-Shaped Quadrupolar Fluorophores: A Comprehensive Study of Structure-Property Relationships[END_REF] Oxygen groups (-OH and -OR) have lower donor capacity, but phenolates are commonly used to design pH-responsive dyes. 4 (a)). The addition of dibutylamino (2) and diphenylamino (3) donor groups on stilbene 1 increased the 2 value respectively by 100 GM and 330 GM. [START_REF] Ehrlich | Twophoton absorption and broadband optical limiting with bis-donor stilbenes[END_REF] Deeper structural changes were then carried out. [START_REF] Albota | Design of Organic Molecules with Large Two-Photon Absorption Cross Sections[END_REF] Replacing the vinyl bond of stilbene 1 with a pdivinylbenzene core, which resulted in a jump of 500 GM for compound 4, thus highlighting the importance of an extended -conjugated system. Importantly, a dramatic increase in 2 was reported upon addition of EW nitrile groups on the divinylbenzene core of this centrosymmetric chromophore. 2PA cross-sections as high as 4000 GM have been reported in this work by molecular engineering of dyes taking advantage of all the driving forces mentioned above.

In centrosymmetric systems such as 4, terminal ED groups are often more efficient than terminal EW groups; and conversely an acceptor character is generally more favourable at the core of the system. [START_REF] Mongin | Synthesis, Fluorescence, and Two-Photon Absorption of a Series of Elongated Rodlike and Banana-Shaped Quadrupolar Fluorophores: A Comprehensive Study of Structure-Property Relationships[END_REF] In this sense, bis-acceptor A--D--A compound 6a is much less 2Presponsive than D--A--D compounds like 6c, or even than its oxygenated counterpart 6b.

Significant 2PA cross-sections have nonetheless been achieved with bis-acceptor structures, such as bis-pyridinium quadrupoles. [START_REF] Kamada | Cationic Two-Photon Absorption Chromophores with Double-and Triple-Bond Cores in Symmetric/Asymmetric Arrangements[END_REF] The -conjugated bridge is the key element enabling the charge transfer between the aforementioned ED and EW groups. Four factors are taken into account in the choice of the link: its donor or acceptor character (vide supra), size, conformation (rigid or flexible, plane or not), and its nature (alkyne, alkene, aryl, etc ...). The length positively influences the 2 by transferring the electrons over longer distances, which increases dipole moments. In this sense, replacing the phenyl ring in quadrupole 4

(Figure 4 (a)) with a biphenyl pattern (compound 7) allows a 60% increase in the 2PA crosssection.

To optimise the orbital overlap, and therefore the electronic coupling in a -conjugated system, planarity is also essential. This is demonstrated by the replacement of the biphenyl bridge in 7 by a constrained 2,7-fluorenyl core in 8, which prevents free rotation and causes an additional 30% increase of the 2 value. Thus, fluorene and dihydrophenanthrene cores are commonly used to block the conformation and maximize planarity. Their benzylic position(s) are commonly functionalised with bulky chains for enhanced solubility and -stacking prevention.

In a similar way, the nature of the -conjugated linkers between constitutive modules of the chromophore is important, and one of the most striking examples is the comparison of transvinylic bridges with ethynyl bridges. In general, the conjugation character is less pronounced in triple bonds than double bonds because the orbital overlap of sp hybridized carbons with neighbouring sp 2 carbons is less favourable. Compounds 8 and 6c show a perceptible, albeit limited, effect on 2 in this sense. However, in some particular cases, especially in highly sterically-hindered chromophores, a more rigid acetylenic bridge can freeze the conformation of the chromophore and enhance conjugation, as compared to a more freely-rotating double bond which can move away from the plane. The geometry of the chromophore can therefore affect immensely its 2PA properties. This feature has been notably highlighted in the case of porphyrin dimers, [START_REF] Drobizhev | Understanding Strong Two-Photon Absorption in π-Conjugated Porphyrin Dimers via Double-Resonance Enhancement in a Three-Level Model[END_REF][START_REF] Frampton | Synthesis, Crystal Structure, and Nonlinear Optical Behavior of β-Unsubstituted meso-meso E-Vinylene-Linked Porphyrin Dimers[END_REF] porphyrin arrays with controlled dihedral angle, [START_REF] Ahn | Relationship between Two-Photon Absorption and the π-Conjugation Pathway in Porphyrin Arrays through Dihedral Angle Control[END_REF] and bis-acceptor acetylenic quadrupoles with inhibited rotation. [START_REF] Kamada | Enhanced Two-Photon Absorption of Chromophores Confined in Two-Dimensional Nanospace[END_REF] The abovementioned elements can be combined within non-symmetric dipolar structures, [START_REF] Pawlicki | Two-Photon Absorption and the Design of Two-Photon Dyes[END_REF][START_REF] He | Multiphoton Absorbing Materials: Molecular Designs, Characterizations, and Applications[END_REF][START_REF] Kim | Small-Molecule Two-Photon Probes for Bioimaging Applications[END_REF] The benefits of centrosymmetric chromophores over dipoles, theorised by Equation ( 6), was confirmed experimentally, sometimes with spectacular results. [START_REF] Drobizhev | Understanding Strong Two-Photon Absorption in π-Conjugated Porphyrin Dimers via Double-Resonance Enhancement in a Three-Level Model[END_REF][START_REF] Yao | Donor-Acceptor-Donor Fluorene Derivatives for Two-Photon Fluorescence Lysosomal Imaging[END_REF][START_REF] Sadowski | π-Expanded Dipyrrolonaphthyridinediones with Large Two-Photon Absorption Cross-Section Values[END_REF] The comparison of two chromophores bearing identical D and A groups embedded in the same conjugated structure, but arranged in different symmetries, allows a simple illustration of this fact. Even considering the increase in conjugation caused by the introduction of symmetry, the 2PA cross-section of dipolar compound 9 is multiplied by a factor of 7 when the push-pull system is embedded in an equivalent quadrupolar compound 10 (Figure 4 (b)). As explained by symmetry selection rules, the 2PA max of compound 10 is also shifted away from 21PA max , while this transition remains allowed in the case of dipole 9. Even larger differences in 2 max between quadrupoles and their corresponding dipoles have been reported. [START_REF] Kamada | Cationic Two-Photon Absorption Chromophores with Double-and Triple-Bond Cores in Symmetric/Asymmetric Arrangements[END_REF] The 7-fold enhancement of 2 in compound 10 is thus superior to the number of branches (i.e.

(dipole A--D) or symmetrical chromophores (quadrupole: A--A, D--D, A--D--A , D-- A--D or octupole D(--A)3, A(--D)3.
2). Studies then focused on the potential benefits of multi-branched systems with more complex symmetry, for which more complex theoretical models had to be developed to take into account inter-branch interactions such as exciton couplings. [START_REF] Beljonne | Role of Dimensionality on the Two-Photon Absorption Response of Conjugated Molecules: The Case of Octupolar Compounds[END_REF][START_REF] Lee | Two-Photon Absorption and Nonlinear Optical Properties of Octupolar Molecules[END_REF] Adding an axis of delocalisation for the charge transfer provides compounds with multidirectional conjugation called octupoles with favourable cooperative effects between the axes. Such cooperation can be evidenced by the determination of 2/n where n is the number of branches. Comparing 4,4'

-bis(diphenylamino)stilbene (DPAS) 3 [START_REF] Ehrlich | Twophoton absorption and broadband optical limiting with bis-donor stilbenes[END_REF] with equivalent octupole 11 [START_REF] Drobizhev | Strong Cooperative Enhancement of Two-Photon Absorption in Dendrimers[END_REF] thus proves that the cross-section value is greater than three times 340 GM (Figure 5). are shown for comparison. The inset on the spectrum shows the dependence of the 2 max on the total number of stilbene branches with a linear fit (solid line), and with the thermodynamic limit of a collection of non-interacting chromophores (dashed line). Adapted with permission from ref.

[57] © The Optical Society.

In the same design strategy, further increasing the number of conjugation paths in the molecule enables the accumulation of tens of 2P absorbing dipolar branches in dendritic structures (Figure 5). Multi-branched compound 12 reached a 2 of 11 000 GM with a 2/n value of 393 GM, [START_REF] Drobizhev | Dendrimer molecules with record large two-photon absorption cross section[END_REF] which is still higher than the constitutive stilbene unit alone (compound 3). However, this strategy is limited by a saturation of the cooperative effects which generally occurs beyond the second generation, as demonstrated by comparing the different generations of dendrimer 12 (G0, G1 and G2). In these compounds, the cross-sections of the dendrimers are slightly larger than the theoretical case where stilbene sub-units contribution would simply add together (Figure 5, see inset on the spectrum), which denotes a beneficial cooperative effect between branches. However, although a marked increase in 2/n is observed for the G0 dendrimer, subsequent increase in branching is less beneficial with a 2/n saturating around 350 GM. It should also be noted that these interactions can be deleterious and degrade the response in 2PA.

Such design strategies have allowed many research groups to synthesize chromophores capable of efficiently reaching the excited state by 2P irradiation. It is worth mentioning that several other advanced strategies allow the enhancement of the 2PA response, such as the preparation of dyes with a singlet diradical nature. [START_REF] Kamada | Strong Two-Photon Absorption of Singlet Diradical Hydrocarbons[END_REF][START_REF] Kamada | Impact of Diradical Character on Two-Photon Absorption: Bis(acridine) Dimers Synthesized from an Allenic Precursor[END_REF] Importantly, relaxation processes usually remain independent from the mode of excitation (i.e.

1PA or 2PA), and from the particular excited state reached upon absorption. It is therefore commonly accepted that 2P dyes follow Kasha's rule as they would in 1PA, and that the excited state energy can then be used for different applications, including photochemistry and uncaging with same rate constants and efficiencies. Anti-Kasha effects have been reported for a number of excited state phenomena, including bond-breaking reactions, [START_REF] Demchenko | Breaking the Kasha Rule for More Efficient Photochemistry[END_REF] but remain uncommon in 2P uncaging (2PU) applications to this day. Thus, the above-mentioned guidelines have been widely applied to the design of PPGs sensitive to red/NIR light.

Photolabile protecting groups: structures, strategies and state of the art

Historically, the first application of light-induced bond cleavage was organic synthesis. In 1962, Barltrop[61] and Barton[62] both reported successively the first use of PPGs for the deprotection of carboxylic acids and amino-acids in peptide synthesis. In this early work, carbamate 13 is cleaved under UV light irradiation, triggering the release of deprotected glycine (Gly) along with the alcohol 14 as a PPG residue formed after a decarboxylation reaction (Scheme 1). In synthesis, PPGs provide "greener" and milder alternatives than standard chemical deprotection, while being orthogonal to all standard protecting groups, and have been used for instance in total synthesis. This was exemplified by the investigation of J. [START_REF] Kaplan | Rapid photolytic release of adenosine 5'triphosphate from a protected analog: utilization by the sodium:potassium pump of human red blood cell ghosts[END_REF] were the first to study the release of bioactive compounds other than amino-acids for biological purposes with use of ortho-nitrobenzyl (oNB) PPGs (Scheme 1). This work introduced for the first time the figurative term of "caged" ATP for compound 28, and of "uncaging". [START_REF] Kaplan | Rapid photolytic release of adenosine 5'triphosphate from a protected analog: utilization by the sodium:potassium pump of human red blood cell ghosts[END_REF] Photo-delivery of bioactive molecules is a very large field, and the term "cage" is sometimes deemed confusing and controversial, especially in comparison with non-covalent molecular cages and cavitands. Nonetheless, "uncaging" is now commonly accepted to describe a photoinduced bond cleavage, and even tends to spread beyond the scope of biological applications.

Controlling physiological phenomena is a challenging task with many constraints, and a PPG must meet a series of specifications to be applicable in biology (Figure 6).

(i)

The chromophore must absorb light efficiently. This efficiency is expressed by the absorption coefficient , or by 2 in the case of 2PE.

(ii)

The excitation wavelength must be compatible with physiological media in order to avoid absorption of light by biological chromophores and potential photodamage.

(iii) The photolysis reaction must be clean. The masked ligand, noted Z in the rest of this section, must be released quantitatively (i.e. with a chemical yield close to 100%) and effectively (i.e. with high photochemical quantum yield u). The quantum yield of the photocleavage reaction u ("u" standing for "uncaging") is defined as the number of molecules released per 100 photons absorbed.

(iv) Compounds must be compatible with a photolysis in aqueous oxygenated solution.

The chemically modified "caged" messenger, must be designed to be soluble in physiological media, and depending on the application, it may be necessary to go through other cell barriers, or to bind to specific receptors (cancer cells, enzyme active sites etc...).

(v)

In its caged form, the biomolecule must be inert in biological media, and stable "in the dark" (i.e. prior to irradiation) in this environment. Both the chemical intermediates and the photolysis residue must also be inert and non-toxic to the biological environment.

(vi) In biological media, the caged compound must regain its activity on a timescale compatible with the cellular process involved. This is particularly important in the case of neuronal applications in which the re-activation kinetics must be fast enough to generate a concentration peak in biomessenger, and activate the appropriate cell receptors. It must also be faster than the diffusion phenomenon (110-900 s, depending on the nature of the medium, the cage and the ligand). [START_REF] Kiskin | Two-photon excitation and photolysis by pulsed laser illumination modelled by spatially non-uniform reactions with simultaneous diffusion[END_REF] Among these criteria, the hydrosolubility of such aromatic chromophores is one of the most critical points of development, along with the photosensitivity and uncaging efficiency. As such, the photocleavage efficiency of a PPG at a specific wavelength is dictated both by its capacity to absorb light ( or 2), and by its intrinsic photochemical efficiency in a specific medium, i.e. its uncaging quantum yield u. Similarly to the definition of brightness for fluorophores (f or 2f), the overall "photosensitivity" is hence determined by the product of both abovementioned parameters. The so-called "uncaging cross-section" is noted u (expressed in M -1 cm -1 ) for 1PE, and u (expressed in GM) for 2PE, and expressed as:

ε u = ε Φ u (7) δ u = 𝜎 2 Φ u (8)
Since the first examples of photodeprotection in the 1970s, many families of cleavable compounds have been reported (Figure 6). [START_REF] Klán | Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy[END_REF][START_REF] Pelliccioli | Photoremovable protecting groups: reaction mechanisms and applications[END_REF][START_REF] Herrmann | Using photolabile protecting groups for the controlled release of bioactive volatiles[END_REF][START_REF] Weinstain | Visible-to-NIR-Light Activated Release: From Small Molecules to Nanomaterials[END_REF] Scheme 2. Photofragmentation mechanism reported for oNB derivatives in aqueous media.

The synthetic accessibility of oNB derivatives as well as their ability to protect a wide range of chemical functions (alcohols, phenols, thiols, phosphates, acids, or amines via a carbamate function) make them both user-convenient and versatile. [START_REF] Adams | Controlling Cell Chemistry with Caged Compounds[END_REF][START_REF] Klán | Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy[END_REF] However, certain drawbacks have been highlighted, such as the toxicity of the nitroso byproduct 25. [START_REF] Adams | Controlling Cell Chemistry with Caged Compounds[END_REF] This derivative can also act as a "trap" for light radiation by internal filter effect, and react with the chemical species present in the medium, in particular endogenic amines. This reactivity was suspected in early reports by Kaplan et al. who described a quantitative chemical yield for the deprotection of inorganic phosphates, but a dramatic drop to 25% for the release of ATP. [START_REF] Kaplan | Rapid photolytic release of adenosine 5'triphosphate from a protected analog: utilization by the sodium:potassium pump of human red blood cell ghosts[END_REF] Amino acids caged by oNB derivatives also have moderate stabilities to hydrolysis, [START_REF] Furuta | Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis[END_REF] and exhibit slow activation kinetics. Katz and Dalva thus report that the release of glutamate by an oNB derivative only generates the action potential in synapses after 15 to 30 ms, which limits the applications in neurology. [START_REF] Katz | Scanning laser photostimulation: a new approach for analyzing brain circuits[END_REF] Early structural modifications aimed to overcome such drawbacks, but also to improve water solubility and absorption capacities (Figure 7). As a general trend, substituents on the benzyl position tend to affect the uncaging quantum efficiency, while modifying the ring affects mostly the 1P and 2PA capacities. As such, the addition of a methyl at the benzylic position causes a strong increase of u for the release of phosphates [START_REF] Kaplan | Rapid photolytic release of adenosine 5'triphosphate from a protected analog: utilization by the sodium:potassium pump of human red blood cell ghosts[END_REF] or acids. [START_REF] Barzynski | Zur Photolyse von makromolekularen o-Nitrobenzylderivaten[END_REF] The resulting 1P photosensitivity of NPE-[Ac] is more than 3 times higher than parent compound oNB-[Ac].

Many benzyl-substituted derivatives (phenyl, [START_REF] Barltrop | Photosensitive protective groups[END_REF] nitrile [START_REF] Aujard | o-Nitrobenzyl Photolabile Protecting Groups with Red-Shifted Absorption: Syntheses and Uncaging Cross-Sections for One-and Two-Photon Excitation[END_REF]) were reported without necessarily improving the photolytic efficiency, but the -carboxy-ortho-nitrobenzyl CNB-[Glu] notably improved solubility in biological media.[86] Historically, oNB derivatives were among the first to be investigated under 2PE with lasers at the end of the 1990s for the photorelease of calcium ions, [START_REF] Brown | Photolysis of Caged Calcium in Femtoliter Volumes Using Two-Photon Excitation[END_REF][START_REF] Momotake | The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells[END_REF] followed by glutamate and acetic acid (Figure 7). A new generation of -extended oNB cages was developed over the last ten years by following the molecular engineering principles presented in section 2.3. Jullien and coworkers [START_REF] Aujard | o-Nitrobenzyl Photolabile Protecting Groups with Red-Shifted Absorption: Syntheses and Uncaging Cross-Sections for One-and Two-Photon Excitation[END_REF] were the first to report both -extensions and tuning of the auxochromic moieties on oNB cages for the release of phenol and carboxylic acid derivatives (Figure 8). Among relevant examples in their library of compounds, such modifications showed a poor influence on the 2PU properties. Substitution of the benzyl carbon with a bromine atom (30) induced a 2 to 3-fold increase in uncaging quantum yields and u, while the nitrile derivative 31 proved inefficient. Interestingly, the stronger amino donor in compound 32 made the photolysis too slow to extract a u. In this work, the authors were also the first to extend the -conjugated system of such cages, by preparing nitrobiphenyls (NBPs), but also stilbene and tolane derivatives 33 and 34.

Figure 8. Examples of modified oNB derivatives reported by Jullien and co-workers for the photorelease of a caged fluorophore, and their photochemical properties in MeCN:TRIS buffer (1:1, v:v). [START_REF] Aujard | o-Nitrobenzyl Photolabile Protecting Groups with Red-Shifted Absorption: Syntheses and Uncaging Cross-Sections for One-and Two-Photon Excitation[END_REF] Although the nitrodiphenylacetylene 34 proved the most efficient in the library, all u values remained in the order of magnitude of the parent oNV. Later on, Boinapally et al. also reported extended oNB derivatives with a similar structure to styrene 33, but locking the double bond inside a constrained dihydronaphthalene structure. [START_REF] Boinapally | Caged Glutamates with π-Extended 1,2-Dihydronaphthalene Chromophore: Design, Synthesis, Two-Photon Absorption Property, and Photochemical Reactivity[END_REF] This resulted in a quantitative chemical yield for the photorelease reaction, and a u of 1% in MeOD. Following their work on the NPPOC family and its monomethylated analogue, [START_REF] Specht | New Photoremovable Protecting Groups for Carboxylic Acids with High Photolytic Efficiencies at Near-UV Irradiation. Application to the Photocontrolled Release of L-Glutamate[END_REF] Goeldner and co-workers also explored this strategy and expanded DMNPB's -conjugated system by synthesizing NBP derivatives. [START_REF] Gug | Photolabile Glutamate Protecting Group with High One-and Two-Photon Uncaging Efficiencies[END_REF][START_REF] Specht | The donor-acceptor biphenyl platform: A versatile chromophore for the engineering of highly efficient two-photon sensitive photoremovable protecting groups[END_REF] The series of structural modifications can be summarised on a roadmap (Figure 9). The first para-alkoxy biphenyl analogue PENB-[Glu] undergoes a significant drop in u, which is more than compensated by the expected increase in 2. A photosensitivity of 3.1 GM is obtained with this strategy. The methoxy donor can be replaced by a diethylene glycol chain to reach the solubility threshold of 5 mM required for use in neuroscience. [START_REF] Specht | The donor-acceptor biphenyl platform: A versatile chromophore for the engineering of highly efficient two-photon sensitive photoremovable protecting groups[END_REF] PENB retains the advantages of parent DMNPB while improving significantly the u. In contrast, this strategy proved counterproductive in the case of the On this occasion, they also reported that the photolytic mechanism in an aqueous media is no longer a solvolysis but a rearrangement [ 11). [START_REF] Fedoryak | Synthesis of a caged glutamate for efficient one-and two-photon photorelease on living cells[END_REF] The photolysis reaction of MDNI is also less clean, leading to a mixture of nitro and nitroso-type by-products, and a chemical yield in released species of only As the photochemistry of ortho-nitroanilides is extremely sensitive to substituents, the extension of the -conjugate system has not been a popular strategy so far, and MNI-[Glu]

remains by far, and despite its low u, the most widely used reference in neuroscience for all the reasons stated above. These PPGs suffer however from a lack of versatility in terms of masked functions, and of synthetic accessibility because of poorly selective nitration reactions.

-arylcarbonyl compounds: the phenacyl family

Although the first mentions of the phenacyl photochemistry date back to 1973, [START_REF] Sheehan | Phenacyl photosensitive blocking groups[END_REF] this class of compound was introduced to uncaging by Givens et al. in the early 1990s to address some of the drawbacks of oNBs. [START_REF] Givens | Photochemistry of .alpha.-keto phosphate esters: photorelease of a caged cAMP[END_REF] Actetophenone derivatives are synthetically accessible and present a photoreactivity based on their n→* transition, which promotes either abstraction of a hydrogen atom, or of an electron from a donor molecule or neighbouring substituent. The photolysis mechanism and by-products thus depend strongly on the substituents present on the aromatic ring. The photochemical cascade leads to the release of the group located at the position of the carbonyl.

The first phenacyls used an unsubstituted acetophenone pattern and suffered from poor solubility in physiological media. [START_REF] Givens | Photochemistry of .alpha.-keto phosphate esters: photorelease of a caged cAMP[END_REF] The introduction of a hydroxyl group at the paraposition solved this problem while maintaining good photocleavage quantum yields. Givens and Park were the first to implement this structural modification on PPGs, which induces an equilibrium between the neutral p-hydroxyphenacyl (pHP) compound, absorbing at 282 nm, and its anionic form pHP -at physiological pH. The superior donating capacity of the phenolate causes the appearance of a red-shifted absorption band at 330 nm. This PPG was able to release various phosphates, including cyclic adenosine monophosphate cAMP (Scheme 5), in aqueous media with an uncaging quantum yield of 37%. [START_REF] Givens | p-Hydroxyphenacyl ATP1: A new phototrigger[END_REF] Further experiments were extended to other biological messengers, such as GABA and glutamate, [START_REF] Givens | New Photoactivated Protecting Groups. 7. p-Hydroxyphenacyl: A Phototrigger for Excitatory Amino Acids and Peptides1[END_REF] or peptides, [START_REF] Givens | New Phototriggers 9: p-Hydroxyphenacyl as a C-Terminal Photoremovable Protecting Group for Oligopeptides[END_REF] with quantum yields ranging between 12 and 40%. This pH-sensitive phenol-based strategy was later extended to other families of PPGs (vide infra).

The postulated photolysis mechanism in aqueous media (Scheme 5) involves the formation of a diradical in the triplet state 47 upon absorption of light followed by intersystem crossing (ISC).

Thus, the active species is immediately released from the excited triplet state, which places phenacyl's kinetics among the fastest (10 9 s -1 ), and makes them ideal for neurological studies.

The remaining spirodiketone byproduct 48, called "photo-Favorskii" intermediate, further

hydrolyses to p-hydroxyphenylacetic acid ( 49), [START_REF] Givens | The Photo-Favorskii Reaction of p-Hydroxyphenacyl Compounds Is Initiated by Water-Assisted, Adiabatic Extrusion of a Triplet Biradical[END_REF] 12). [START_REF] Houk | Two-Photon Activation of p-Hydroxyphenacyl Phototriggers: Toward Spatially Controlled Release of Diethyl Phosphate and ATP[END_REF] Therefore, the use of green laser excitation considerably limits further 2P applications. A mono-and di-methoxy derivative such as 50 effectively showed a 1PA band shifted to 350 nm, but reduced the u down to 3 or 4% respectively for the release of GABA via an ester bond. No 2PA measurements were performed in these studies. [START_REF] Conrad | New Phototriggers:1 Extending the p-Hydroxyphenacyl π-π* Absorption Range[END_REF] Despite a certain lack of versatility in the early structures of such derivatives, P. Singh et al.

successfully incorporated phenacyl moieties within -extended naphthalene [START_REF] Roy | Stepwise dual stimuli triggered dual drug release by a single naphthalene based two-photon chromophore to reverse MDR for alkylating agents with dual surveillance in uncaging steps[END_REF] and carbazole rings (Figure 12). [START_REF] Venkatesh | Photocaging of Single and Dual (Similar or Different) Carboxylic and Amino Acids by Acetyl Carbazole and its Application as Dual Drug Delivery in Cancer Therapy[END_REF] In the "smart" naphthalene derivative 51, glutathione activation in vitro switches on the ICT by cleaving the S-O bond and releasing SO2. The resulting extended pHP derivative can be further activated by 1P and 2PE to release the anticancer agent chlorambucil via the same cleavage mechanism. The naphthalene skeleton hereby shifts the 2PE to the red, with a maximum 2 of 77 GM at 650 nm. This reasonable 2PA cross-section is combined with an excellent u, in accordance with related pHP derivative, to give a u of 21 GM. This value is among the highest to date for any 2P-active PPG, and obtained after very limited structural modification. Interestingly, the uncaging quantum yield was reported to increase in low pH environments. In a similar strategy, mono-and bisacetylcarbazoles with higher ED groups were prepared. Acetylcarbazole PPGs such as 52 are able to release respectively one or two similar (Z = Z ') or different (Z ≠ Z') substrates upon excitation. As in the case of standard phenacyls, it was postulated that photolysis takes place from the triplet excited state, but that the bond cleavage can be either heterolytic, or homolytic followed by electron transfer. Chemically quantitative photolysis was reported on a dozen of compounds, with quantum yields in the 10-15% range in aqueous buffer with 30% of acetonitrile. Such compounds have been designed

and applied to anticancer therapy, but no 2P measurements were carried out so far. [START_REF] Venkatesh | Photocaging of Single and Dual (Similar or Different) Carboxylic and Amino Acids by Acetyl Carbazole and its Application as Dual Drug Delivery in Cancer Therapy[END_REF][START_REF] Venkatesh | Bis-Acetyl Carbazole: A Photoremovable Protecting Group for Sequential Release of Two Different Functional Groups and Its Application in Therapeutic Release[END_REF] Figure 12. Structures of PPGs containing the phenacyl photosensitive unit and corresponding 1P and 2PU properties.

Excited-state weakened rings: the coumarin family

During the course of their investigations on light-induced formation of benzyl carbocations, Givens and Matuszewski showed in the 1980s that coumarin-4-ylmethylphosphates undergo a C-O bond heterolysis leading to the release of the phosphate and the formation of a by-product from the trapping of the carbocation. [START_REF] Givens | Photochemistry of phosphate esters: an efficient method for the generation of electrophiles[END_REF] Direct heterolytic bond cleavage in the excited state is related to two intertwined particularities of certain electron-rich cycles with meta-or "pseudo-meta" substitution (Scheme 6). [START_REF] Zimmerman | Mechanistic Organic Photochemistry. II. 1,2 Solvolytic Photochemical Reactions[END_REF] First, upon promotion of an electron to the excited state, a substantial charge density is displaced from the ED group at the 7-position of the ring to the 4-position, which ends up being strongly enriched. [START_REF] Furuta | Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis[END_REF] Secondly, and as a result, while carbon C(4) possesses a very weak bonding coefficient in its ground state orbital, its antibonding contribution in the excited state is significant because of the charge transfer. Thus, when C( 4) is substituted with an sp 2 benzylic carbon, the σ-antibonding orbital mixes with the chromophore's LUMO. Finally, if a leaving group Z is present in this position, the C-Z bond is weakened in the excited state, and cleaved with rate constants of the order of 10 9 to 10 10 s - 1 . [START_REF] Schade | Deactivation Behavior and Excited-State Properties of (Coumarin-4-yl)methyl Derivatives. 1. Photocleavage of (7-Methoxycoumarin-4-yl)methyl-Caged Acids with Fluorescence Enhancement[END_REF] Such distinctive electronic attributes were first reported on meta-enriched benzyl acetates, and is commonly called "Zimmerman meta-effect". [START_REF] Zimmerman | Mechanistic Organic Photochemistry. II. 1,2 Solvolytic Photochemical Reactions[END_REF] This rationale has been used recently in the design of several new photosensitive moieties [START_REF] Šolomek | Searching for Improved Photoreleasing Abilities of Organic Molecules[END_REF] such as BODIPY [START_REF] Sitkowska | Green-Light-Sensitive BODIPY Photoprotecting Groups for Amines[END_REF][START_REF] Sitkowska | Red-light-sensitive BODIPY photoprotecting groups for amines and their biological application in controlling heart rhythm[END_REF] of fluorenyl [START_REF] Reinfelds | A New Photocage Derived from Fluorene[END_REF] derivatives, but coumarin-4-ylmethyls remain the most widely used in 2PE among them.

The resulting photocleavage mechanism [START_REF] Klán | Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy[END_REF][START_REF] Schade | Deactivation Behavior and Excited-State Properties of (Coumarin-4-yl)methyl Derivatives. 1. Photocleavage of (7-Methoxycoumarin-4-yl)methyl-Caged Acids with Fluorescence Enhancement[END_REF][START_REF] Schmidt | Kinetics Study of the Photocleavage of (Coumarin-4-yl)methyl Esters[END_REF] is then an excited state solvolysis (Scheme 6) occurring from the zwitterionic forms 54a, representing formally the Zimmerman effect in the singlet excited state, and 54b. The C-Z bond usually undergoes a heterolytic cleavage yielding an unseparated stabilised ion pair represented as 55a and 55b. Homolytic cleavage followed by electron transfer may also be observed in certain conditions, but should be disfavoured compared to an ionic mechanism. [START_REF] Zimmerman | The Meta Effect in Organic Photochemistry: Mechanistic and Exploratory Organic Photochemistry[END_REF] Scheme 6. Photo-solvolysis mechanism reported for coumarin-4-ylemethyl PPGs, and representation of the Zimmerman meta-effect occuring upon excitation of meta-enriched rings.

Competitive recombination of this ion pair may occur and inhibit the efficiency of the photolysis by reforming the starting material 53. Solvatation will otherwise separate the ions, thus exposing carbocation 56 to nucleophilic attack. In aqueous media, 56 is trapped by water, to yield the hydroxymethylcoumarin byproduct 57, along with the protonated molecule ZH.

The overall mechanism can be assimilated to an SN1 from the excited state. The photo-release efficiency is therefore highly dependent on the nature of the leaving group. The recombination step can be kinetically very competitive in the case of certain caged functional groups, such as phenols, that may also undergo photo-Claisen rearrangement. [START_REF] Wong | Control of an Unusual Photo-Claisen Rearrangement in Coumarin Caged Tamoxifen through an Extended Spacer[END_REF] Years after the first report on coumarinylmethyl photosensitivity, [START_REF] Givens | Photochemistry of phosphate esters: an efficient method for the generation of electrophiles[END_REF] Furuta et al. developed the 6-bromo-7-hydroxycoumarin (BHC) analogue, and pioneered the field of 2PU by demonstrating for the first time utility of PPGs in 2P neurological experiments. The orthobromo phenol pattern lowers the pKa of the hydroxyl group to shift the equilibrium towards the phenolate form at physiological pH. In this ambivalent strategy, the anionic form promotes the ICT and induces a bathochromic shift of the absorption band while improving strongly the solubility in water; while the more lipophilic neutral form is more likely to cross the lipid bilayers in biological media and provides increased stability for storage. [START_REF] Furuta | Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis[END_REF] Moreover, as the triplet state is not unlikely to be involved in the photolysis mechanism, the bromine substituent increases the efficiency of the cleavage by promoting ISC via heavy atom effect. BHC-[Ac]

was reported with a long-standing record 2PU photosensitivity of 2 GM (Figure 13). Yet, amino-acids caged via their carboxylic acid functionality were shown to be slightly unstable to hydrolysis. This could be further improved by caging glutamate via a carbamate function on the -nitrogen, which affected both the u and kinetics of BHC-[Glu], and relegated the u below 1 GM. BHC-[Glu] still remains a reference compound for the release of amino-acids to this day.

Many structural analogues of BHC, modified with different methoxy, [START_REF] Givens | Photochemistry of phosphate esters: an efficient method for the generation of electrophiles[END_REF][START_REF] Schade | Deactivation Behavior and Excited-State Properties of (Coumarin-4-yl)methyl Derivatives. 1. Photocleavage of (7-Methoxycoumarin-4-yl)methyl-Caged Acids with Fluorescence Enhancement[END_REF] [START_REF] Suzuki | Coumarin-4-ylmethoxycarbonyls as Phototriggers for Alcohols and Phenols[END_REF] amino [START_REF] Cürten | Synthesis, Photophysical, Photochemical and Biological Properties of Caged GABA, 4-[[(2H-1-Benzopyran-2-one-7-amino-4-methoxy) carbonyl]amino] Butanoic Acid[END_REF] and dialkylamino [START_REF] Hagen | Coumarinylmethyl Esters for Ultrafast Release of High Concentrations of Cyclic Nucleotides upon One-and Two-Photon Photolysis[END_REF][START_REF] Hess | Protecting group for carboxylic acids that can be photolyzed by visible light[END_REF][START_REF] Senda | Synthesis and Photocleavage of 7-[Bis(carboxymethyl)aminocoumarin-4-yl]methyl-Caged Neurotransmitters[END_REF][START_REF] Shembekar | Coumarin-Caged Glycine that Can Be Photolyzed within 3 μs by Visible Light[END_REF] electron-donating patterns, were reported, which highlighted an important versatility in this family of PPGs (Figure 13). Changes at the 7-position were the most extensively studied in literature, and 7-diethylaminocoumarin derivatives (DEAC) have gained popularity for 1P and 2PU of neuro-messengers. [START_REF] Hagen | Coumarinylmethyl Esters for Ultrafast Release of High Concentrations of Cyclic Nucleotides upon One-and Two-Photon Photolysis[END_REF][START_REF] Hess | Protecting group for carboxylic acids that can be photolyzed by visible light[END_REF][START_REF] Senda | Synthesis and Photocleavage of 7-[Bis(carboxymethyl)aminocoumarin-4-yl]methyl-Caged Neurotransmitters[END_REF][START_REF] Shembekar | Coumarin-Caged Glycine that Can Be Photolyzed within 3 μs by Visible Light[END_REF] With such derivatives, Shembekar et al. reported uncaging of glycine with a u of 12%, more than three times higher than BHC, and with a 1PA band shifted to 390 nm. The neurotransmitter is reported to be biologically inert, and the half-reaction time is 2.5 μs. [START_REF] Shembekar | Coumarin-Caged Glycine that Can Be Photolyzed within 3 μs by Visible Light[END_REF] The loss of solubility generated by the diethylamino group was solved with the use of two carboxymethyl chains in the work of Hagen et al. on 7-dicarboxymethylaminocoumarin (DCMAC). [START_REF] Hagen | Coumarinylmethyl Esters for Ultrafast Release of High Concentrations of Cyclic Nucleotides upon One-and Two-Photon Photolysis[END_REF] The 2P photolysis of caged cyclic guanosine monophosphate (cGMP) is reported as about 1.3 times faster than BHC-[Glu] under the same conditions. No extrapolated u value was determined considering the difference in leaving group. A sulfonated variant of DCMAC was also reported. [START_REF] Nadler | Exclusive photorelease of signalling lipids at the plasma membrane[END_REF] The extension of the -conjugated system at the 7-position with an electron-donating p-dimethylaminostyryl group was also examined. [START_REF] Bao | Styryl Conjugated Coumarin Caged Alcohol: Efficient Photorelease by Either One-Photon Long Wavelength or Two-Photon NIR Excitation[END_REF] A 2PA cross-section of 309 GM was measured at 800 nm for compound 58 (Figure 13), thus validating the expected effect on 2 as compared with DEAC. However, the uncaging quantum yield proved 60 times lower than the parent compound under the same irradiation conditions, which limits the u value to only 0.24 GM.

Strong absorption shifts were recorded after chemical transformations on the endocyclic EW carbonyl (C(2)) of the lactone ring. Costa et al. [START_REF] Fonseca | Thionated coumarins and quinolones in the light triggered release of a model amino acid: synthesis and photolysis studies[END_REF] followed by Jullien et al. [START_REF] Fournier | Coumarinylmethyl Caging Groups with Redshifted Absorption[END_REF][START_REF] Fournier | A Blue-Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation[END_REF] first reported the thionation of DEAC into 7-diethylaminothiocoumarin (DEATC, Figure 13).

Thiocarbonyls are known to provide strong bathochromic shifts due to lower energy transitions, [START_REF] Maciejewski | The photophysics, physical photochemistry, and related spectroscopy of thiocarbonyls[END_REF] and the maximum absorption of DEATC was measured at 472 nm. The compound was used in vivo for orthogonal activation of Cyclofen at 488 nm. [START_REF] Fournier | A Blue-Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation[END_REF] The u of DEATC is, however, almost an order of magnitude lower than for unmodified DEAC, and suffers from bad photostability of its photolysis by-product. [START_REF] Fournier | Coumarinylmethyl Caging Groups with Redshifted Absorption[END_REF] The library of compounds synthesised by Fournier et al. in this work contains a number of green-absorbing analogues, including dicyanovinylcoumarins used since then by Marchán et al. in a photo-activatable RGD-targeted drug delivery system. [START_REF] Gandioso | A Green Light-Triggerable RGD Peptide for Photocontrolled Targeted Drug Delivery: Synthesis and Photolysis Studies[END_REF] Although no 2P data was reported to date on C(2) modified compounds, it is worth mentioning that the 1PA curves can sometimes extend beyond 650 nm. This significant bathochromic shift was notably evidenced by Marchán et al. on C(2)cyano-(4-nitrophenyl)-methylene derivatives [START_REF] Gandioso | Development of Green/Red-Absorbing Chromophores Based on a Coumarin Scaffold That Are Useful as Caging Groups[END_REF] (Figure 13), but causes a dramatic drop in uncaging efficiency, with a u falling in the 10 -6 range in methanol.

The 3-position of the coumarin backbone is another strategic position in terms of molecular engineering since it is at the enriched end of the -conjugated system, and many commercially available dyes exploit this push-pull pattern. Ellis-Davies et al. were the first to extend the conjugated system of coumarinylmethyl PPGs in this position by adding an electronwithdrawing acrylamide moiety to DEAC. [START_REF] Olson | Optically Selective Two-Photon Uncaging of Glutamate at 900 nm[END_REF] The pronounced push-pull character shifts the 1PA band to 450 nm, which explained the acronym "DEAC450" given to the PPG. The 2PA band of this dipolar compound is also shifted to 21PA max , i.e. 900 nm. Notably, DEAC450 also shows a marked increase in uncaging quantum yield, reaching a value of 39% for carboxylic acids (Glu, [START_REF] Olson | Optically Selective Two-Photon Uncaging of Glutamate at 900 nm[END_REF] GABA [START_REF] Ellis-Davies | Wavelength-Selective One-and Two-Photon Uncaging of GABA[END_REF]), and up to 78% for phosphates (cAMP, [START_REF] Olson | Spectral Evolution of a Photochemical Protecting Group for Orthogonal Two-Color Uncaging with Visible Light[END_REF] cGMP [START_REF] Agarwal | Intracellular Uncaging of cGMP with Blue Light[END_REF]). A solubility of 0.25 mM in biological medium was reported after modulation of the solubilising units on the amide end (aspartate, [START_REF] Olson | Optically Selective Two-Photon Uncaging of Glutamate at 900 nm[END_REF][START_REF] Olson | Spectral Evolution of a Photochemical Protecting Group for Orthogonal Two-Color Uncaging with Visible Light[END_REF] or oligoethyleneglycol [START_REF] Ellis-Davies | Wavelength-Selective One-and Two-Photon Uncaging of GABA[END_REF]).

Further modifications at the 3-position were performed by Chitose et al. with the extension of electron-depleted [START_REF] Chitose | Design, Synthesis, and Reaction of π-Extended Coumarin-based New Caged Compounds with Two-photon Absorption Character in the Near-IR Region[END_REF] and electron-rich [START_REF] Chitose | Design and Synthesis of a Caged Carboxylic Acid with a Donor-π-Donor Coumarin Structure: One-photon and Two-photon Uncaging Reactions Using Visible and Near-Infrared Lights[END_REF] benzene moieties. The best results were obtained with the dimethylaminobenzene derivative, reportedly thanks to a D--D character within the PPG conjugated structure, with a 2PA cross-section of 18 GM at 680 nm in toluene, and an uncaging quantum yield of 16% for benzoic acid release. Even higher 2 values may be reached at lower wavelengths for such derivatives. [START_REF] Chitose | Design and Synthesis of a Caged Carboxylic Acid with a Donor-π-Donor Coumarin Structure: One-photon and Two-photon Uncaging Reactions Using Visible and Near-Infrared Lights[END_REF] Preserving the double bond extension present in DEAC450 and the dimethylaminobenzene ED group introduced by Chitose et al., Lin et al.

introduced styryl moieties at the 3-position. [START_REF] Lin | Coumarin Photocaging Groups Modified with an Electron-Rich Styryl Moiety at the 3-Position: Long-Wavelength Excitation, Rapid Photolysis, and Photobleaching[END_REF] Postulating an altered mechanism in which the carbocation is trapped by an enriched styryl double bond in an intramolecular cyclisation, the authors prepared a library of extended coumarin and thiocoumarin derivatives with absorptions shifted up to 515 nm and uncaging quantum yields reaching up to 70%. In spite of a lowered u compared to DEAC450, compound 59 proved the most efficient upon 2PE thanks to an enhanced ICT and an increased 2. Klausen et al. also investigated the effect of ICT on extended coumarins by introducing strongly EW thiazolyl-type moieties at the 3-position. [START_REF] Klausen | Dual-wavelength efficient two-photon photorelease of glycine by π-extended dipolar coumarins[END_REF] Interestingly, an inverse correlation between the EW strength and the u values was shown.

However, introducing an intermediate fluorenyl -bridge between the coumarin backbone and the EW extension increased dramatically both the 2 and the u thanks to a mitigated ICT strength. This possible correlation between the "space-density" of ICT and the uncaging quantum yield was supported by DFT calculations. Following this molecular engineering strategy, compound 60 was the first PPG reported with a 2P-photosensitivity above 100 GM.

Without any direct influence on the 2, other attempts have been reported to improve the u of coumarin PPG rather than its 2PA capacity. Grafting an extra methyl group on benzylic carbon is expected to provide stabilisation of the intermediate carbocation 56 during the course of the photolysis mechanism. First reported by Weis et al., [START_REF] Weis | del Campo, Ethyl substituted coumarin-4-yl derivatives as photoremovable protecting groups for amino acids with improved stability for SPPS[END_REF] this modification was further exploited by Specht et al. who replaced the methyl group with an oligoethylene glycol chain improving water solubility. [START_REF] Goegan | Synthesis and Characterization of Photoactivatable Doxycycline Analogues Bearing Two-Photon-Sensitive Photoremovable Groups Suitable for Light-Induced Gene Expression[END_REF] The u reported for the release of aminodoxycycline via a carbamate function is almost doubled compared to parent DEAC (21%), which is consistent with a stabilised carbocation, and increases the u value to 4 GM.

To this day, coumarins are among the most widely used PPGs in neurobiology, but also in materials science, [START_REF] Huang | Photocleavable coumarin crosslinkers based polystyrene microgels: phototriggered swelling and release[END_REF][START_REF] Stegmaier | Photoresponsive Surfaces with Two Independent Wavelength-Selective Functional Levels[END_REF] or in therapy. [START_REF] Skwarczynski | Development of first photoresponsive prodrug of paclitaxel[END_REF][START_REF] Karthik | Photoresponsive Coumarin-Tethered Multifunctional Magnetic Nanoparticles for Release of Anticancer Drug[END_REF][START_REF] Herzig | Light-induced antibiotic release from a coumarin-caged compound on the ultrafast timescale[END_REF] Coumarin cages po interesting release kinetics (of the order of 10 8 s -1 ), and above all an exceptional degree of structural modularity.

The quantum efficiency is therefore strongly dependent on the caged functionality; and very large disparities are reported between carbamates, acids, and phosphates, or even between two different amino acids. [START_REF] Senda | Synthesis and Photocleavage of 7-[Bis(carboxymethyl)aminocoumarin-4-yl]methyl-Caged Neurotransmitters[END_REF] Figure 13. Roadmap of the structural modifications performed on the coumarin backbone, and their consequences on the 1P and 2PU properties. Values are reported for photolysis in a phosphate buffer; b phosphate buffer with 50% MeCN; c phosphate buffer with 90% MeCN d MeOH:H2O (9:1, v:v) e MeCN:H2O (9:1, v:v).

The quinoline family

Following the first reports on 2P-sensitive coumarin PPGs, [START_REF] Furuta | Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis[END_REF] a new family of heterocyclic cages based on a quinoline skeleton was reported. Fedoryak and Dore introduced the 8-Bromo-7-hydroxyquinoline (BHQ) PPG in 2002 for uncaging of carboxylic acid, with a u of 0.59 GM at 740 nm (Figure 14). [START_REF] Fedoryak | Brominated Hydroxyquinoline as a Photolabile Protecting Group with Sensitivity to Multiphoton Excitation[END_REF] Carboxylates, phosphates and diols were successfully deprotected under the same conditions with u ranging between 0.4 and 0.9 GM. [START_REF] Zhu | 8-Bromo-7-hydroxyquinoline as a Photoremovable Protecting Group for Physiological Use: Mechanism and Scope[END_REF] These early studies showed that the BHQ-[Ac] cage is cleaved more rapidly than its BHC-[Ac] analogue, and that both solubility and dark stability to hydrolysis are compatible with studies in a physiological media, although their dark stability is much lower. [START_REF] Fedoryak | Brominated Hydroxyquinoline as a Photolabile Protecting Group with Sensitivity to Multiphoton Excitation[END_REF] Although no evidence of meta-donation was reported in quinoline, the fragmentation mechanism was also first described as an heterolytic cleavage of the C-Z bond followed by excited-state solvolysis; which was supported by the incorporation of 18 O atoms into the byproduct upon photolysis in H2 18 O. [START_REF] Zhu | 8-Bromo-7-hydroxyquinoline as a Photoremovable Protecting Group for Physiological Use: Mechanism and Scope[END_REF] At first, this SN1-type mechanism was postulated to occur from a singlet state, in which case the heavy atom effect implemented by the 8-bromo substitution would be detrimental to the photolysis efficiency. In order to increase the u, other substituents able to lower the pKa of the 7-hydroxy group, as reported in previous PPGs, but unable to promote ISC to the triplet state were considered. [START_REF] Davis | Substituent Effects on the Sensitivity of a Quinoline Photoremovable Protecting Group to One-and Two-Photon Excitation[END_REF] Structural modifications carried out in this study by Dore et al. in 2009 along with their effects on u and u are summarised on Figure 14.

A chlorinated analogue of BHQ was prepared in accordance with this strategy, but surprisingly, the u was divided by 3. This decrease was first attributed to the concomitant increase in fluorescence, which is competitive towards photolysis, but eventually highlighted an important role for the bromine atom of BHQ in the photocleavage process. Nitrile substitution caused a very strong decrease in pKa, thus shifting the equilibrium almost exclusively to the anionic form in physiological media. This modification preserved the u, and tripled the 1P photosensitivity, but did not affect 2PU significantly. Finally, an 8-nitro substitution completely quenched the photochemistry of the quinoline, presumably because of a low-energy triplet state. The ED capacity at the 7-position of the quinoline backbone was then modulated by replacing the hydroxyl group with dimethylamino or thiol groups. The effect on the 2PA response was positive, especially when a chlorine substituent was added at the 4-position, but strongly counterproductive on the uncaging quantum yield. [START_REF] Davis | Substituent Effects on the Sensitivity of a Quinoline Photoremovable Protecting Group to One-and Two-Photon Excitation[END_REF] Out of all compounds studied in this library, BHQ retained the highest u.

Thanks to this study and the comparison between bromo-and chloroquinoline, the photolysis mechanism of 8-halogeno-7-hydroxyquinolines could be elucidated (Scheme 7). in order to increase the ICT in the dipolar push-pull system, [START_REF] Tran | Two-Photon "Caging" Groups: Effect of Position Isomery on the Photorelease Properties of Aminoquinoline-Derived Photolabile Protecting Groups[END_REF] but also to incorporate the photosensitive pattern within quadrupolar [START_REF] Dunkel | Quinolinederived two-photon sensitive quadrupolar probes[END_REF][START_REF] Tran | Quinoline-Derived Two-Photon Sensitive Quadrupolar Probes[END_REF] or octupolar [START_REF] Dalko | Multiphoton Activable Quinoline Derivatives, Their Preparation and Their Uses[END_REF][START_REF] Picard | Octupolar chimeric compounds built from quinoline caged acetate moieties: a novel approach for 2-photon uncaging of biomolecules[END_REF] symmetric systems (Figure 15 (a)).

In the dipolar strategy, an EW carboxylic acid was introduced on 8-DMAQ. Surprisingly, and in a similar way to coumarin-benzothiazolyl PPG 60, although 5-carboxy-8DMAQ is 6 times less effective in 2PU than the parent compound 8-DMAQ under the same conditions, the introduction of an intermediate phenyl bridge eventually validated the strategy by tripling the value of the u in the case of compound 65. [START_REF] Tran | Two-Photon "Caging" Groups: Effect of Position Isomery on the Photorelease Properties of Aminoquinoline-Derived Photolabile Protecting Groups[END_REF] The design of quinoline-based centrosymmetric systems led to various outcomes. Quinoline dimer 66 showed slightly lower performance than 8-DMAQ alone, with only 0.40 GM at 730 nm. [START_REF] Dunkel | Quinolinederived two-photon sensitive quadrupolar probes[END_REF] The extension of the conjugation with a 2,7-fluorenyl core in compound 67 allowed a marked improvement of the u, [START_REF] Tran | Quinoline-Derived Two-Photon Sensitive Quadrupolar Probes[END_REF] however, it remains in the range of dipolar compound 65 which limits the benefit of this modification. Nonetheless, it should be noted that these values were evaluated by comparative photolysis at 730 nm. Yet, as observed on the 2PA spectrum of compound 67 (Figure 15 (c)), and in accordance with the parity selection rule, the maximum of the 2P-allowed band in these centrosymmetric compounds is located at shorter wavelengths, with a 2 max value of 25 GM at 690 nm for 67. ). Reprinted with permission from ref. [START_REF] Petit | Substitution Effect on the Oneand Two-photon Sensitivity of DMAQ "Caging" Groups[END_REF]. Copyright 2012 American Chemical Society. c) 2PA spectrum (red) of quadrupole 67 in MeCN. Linear absorption spectrum (black) is given for comparison. Reprinted from ref. [START_REF] Tran | Quinoline-Derived Two-Photon Sensitive Quadrupolar Probes[END_REF] with permission from John Wiley and Sons. d) 2PA spectrum (red) of octupole 68 in THF. Linear absorption spectrum (black) is given for comparison. Reprinted from ref. [START_REF] Picard | Octupolar chimeric compounds built from quinoline caged acetate moieties: a novel approach for 2-photon uncaging of biomolecules[END_REF] with permission from The Royal Society of Chemistry.

Octupolar compound 68 reported by Picard et al. was designed as a combination of three quinoline moieties coupled at the C(8) positions to a triphenylamine group via ethynyl bridges.

Although the 2 of 68 reached 390 GM at 730 nm (Figure 15 (d)), the uncaging quantum yield was reduced by several orders of magnitude, which considerably limited the 2P photosensitivity, and proved to be an example of counterproductive molecular engineering. [START_REF] Picard | Octupolar chimeric compounds built from quinoline caged acetate moieties: a novel approach for 2-photon uncaging of biomolecules[END_REF] As such, reports show that the ethynyl bridges used in multipolar quinoline-based compounds tend to impede the photochemical efficiency. [START_REF] Tran | Quinoline-Derived Two-Photon Sensitive Quadrupolar Probes[END_REF][START_REF] Picard | Octupolar chimeric compounds built from quinoline caged acetate moieties: a novel approach for 2-photon uncaging of biomolecules[END_REF] Similar quinoline trimers synthesised by Dalko et al.

were indeed reported with higher u values between 0.5 and 2 GM without triple-bond linkage. [START_REF] Dalko | Multiphoton Activable Quinoline Derivatives, Their Preparation and Their Uses[END_REF] 3.6. Uncaging via photo-isomerisation: the 2-hydroxycinnamyl family Ortho-hydroxycinnamic (oHC) esters were introduced by Porter and co-workers in 1988 for reversible light-induced protease inhibition. [START_REF] Turner | Photoreactivation of irreversibly inhibited serine proteinases[END_REF] In this early work, this is achieved by photocleavage of ester bonds in synthetic inhibitor compounds, which restores the enzyme activity.

Uncagers of the oHC family hold several interesting features, such as good cell permeability due to a certain hydrophilic/lipophilicity ambivalence. [START_REF] Gagey | Two-Photon Uncaging with Fluorescence Reporting: Evaluation of the o-Hydroxycinnamic Platform[END_REF] The peculiarity of their fragmentation mechanism also lies in the formation of a highly fluorescent coumarin dye 71

during the course of the photorelease (Scheme 8), starting from a non-fluorescent cinnamic acid derivative. This is explained by the stepwise formation of the 69-cis isomer upon excitation, which enables spontaneous intramolecular cyclisation followed by elimination of the alcohol from hemiester 70. This generation of luminescence opens the door to convenient photolysis monitoring, and to theranostic applications. Yet, oHC compounds suffer from slower activation kinetics than other derivatives (10 -2 s -1 range), [START_REF] Gagey | Two-Photon Uncaging with Fluorescence Reporting: Evaluation of the o-Hydroxycinnamic Platform[END_REF] which is their main limiting factor. It should be noted that the thermal cyclisation step can be accelerated when using 2PE because of localised heating at the focal point, which makes the isomerisation step rate-determining. Scheme 8. Reported mechanism for the photorelease of alcohols with oHC derivatives involving sequential E-Z photo-isomerisation and lactonisation.

The influence of substituents on the -conjugated system of the oHC platform was studied by Jullien et al. for photodeprotection of model alcohols (EtOH, BuOH). [START_REF] Gagey | Two-Photon Uncaging with Fluorescence Reporting: Evaluation of the o-Hydroxycinnamic Platform[END_REF][START_REF] Gagey | Two-Photon Uncaging with the Efficient 3,5-Dibromo-2,4-dihydroxycinnamic Caging Group[END_REF][START_REF] Gagey | Alcohol Uncaging with Fluorescence Reporting: Evaluation of o-Acetoxyphenyl Methyloxazolone Precursors[END_REF] Compound 72 (Figure 16) is another example of the abovementioned bromophenol pattern. Indeed, the 3,5dibromo-4-hydroxy electron-donating motif shifts the excitation band of the compound to 350 nm at physiological pH, while increasing its water solubility via the phenol/phenolate equilibrium. Without significantly altering the u, the increase in electron density on the aromatic ring (73 and 74) boosts the ICT, and consequently the u, which reaches 4.7 GM for compound 74. A bis-carboxylated analogue of compound 74 has been reported since then with a u of 18% and a solubility in the millimolar range in aqueous buffer, but no 2PU experiment was carried out. [START_REF] Duan | Water-soluble o-hydroxycinnamate as an efficient photoremovable protecting group of alcohols with fluorescence reporting[END_REF] Substituents on the cinnamate double bond have a significant effect on the kinetics, which is doubled in the case of the compound 75. [START_REF] Gagey | Alcohol Uncaging with Fluorescence Reporting: Evaluation of o-Acetoxyphenyl Methyloxazolone Precursors[END_REF] However, such derivatives sometimes proved unstable at room temperature because of a spontaneous lactonisation triggering the alcohol release. [START_REF] Gagey | Two-Photon Uncaging with Fluorescence Reporting: Evaluation of the o-Hydroxycinnamic Platform[END_REF] Steric hindrance can also be the cause of hypsochromic shifts of the absorption band due to a loss of planarity.

The extension of the -conjugated system of oHCs has been reported on several occasions.

Replacing the phenol ring with a naphthol (76) caused a very strong increase in photoisomerisation quantum yield, but the u did not increase accordingly. Recently, Paul et al.

also extended the -conjugated system of oHC derivatives with an EW heterocycle (R1 = Me, R3 = 2-benzothiazolyl). [START_REF] Paul | ESIPT-induced fluorescent o-hydroxycinnamate: a self-monitoring phototrigger for prompt image-guided uncaging of alcohols[END_REF] The resulting cage was used for 1PU of methylsalicylate in HeLa cells, with an estimated quantum yield of 10%, without performing 2P measurements.

Interestingly, P. Singh et al. fused the oHC pattern with a carbazole heterocycle, [START_REF] Venkatesh | One-and Two-Photon Uncaging: Carbazole Fused o-Hydroxycinnamate Platform for Dual Release of Alcohols (Same or Different) with Real-Time Monitoring[END_REF] as they previously reported for phenacyl derivatives. [START_REF] Venkatesh | Photocaging of Single and Dual (Similar or Different) Carboxylic and Amino Acids by Acetyl Carbazole and its Application as Dual Drug Delivery in Cancer Therapy[END_REF] All reported derivatives showed an uncaging quantum yield in the 4.5% range in a water/acetonitrile mixture (7:3, v:v). The sequential alcohol release upon irradiation of compound 77 can be monitored in a first instance by an increase in its green emission intensity, then by a blue shift of the emission band due to reduced ICT in the final fused-biscoumarin by-product. Subsequent z-scan 2PA measurements showed that the 2P photosensitivity of all derivatives was in the 0.4 GM range. The values are about an order of magnitude lower than for amino derivative 74, but different photolysis media and measurement techniques prevent direct comparison. Eventually, compound 72 was used in zebrafish embryos under 2PE at 750 nm, [START_REF] Gagey | Two-Photon Uncaging with the Efficient 3,5-Dibromo-2,4-dihydroxycinnamic Caging Group[END_REF] which validated the use of oHC cages in vivo. Several key advantages were highlighted in this family of PPGs for biological applications, however, they are exclusively suited for the release of alcohols, and for the study of physiological phenomena compatible with slow substrate release.

Other photosensitive structures

Although the literature surrounding 2PU is focused almost entirely on the above-mentioned structures, a multitude of other photo-cleavable moieties have been reported. [START_REF] Klán | Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy[END_REF] In the midst of organic PPGs, it is worth mentioning that the ruthenium (II) bipyridine complex RuBi, developed in the mid-2000s, [START_REF] Nikolenko | Two-photon uncaging of neurochemicals using inorganic metal complexes[END_REF] is able to release one of its amine ligands by excitation in its metal-ligand charge transfer (MLCT) band, located at 450 nm and under 2PE at 800 nm in neurons. [START_REF] Salierno | A fast ruthenium polypyridine cage complex photoreleases glutamate with visible or IR light in one and two photon regimes[END_REF] Comparative photolysis experiments showed that RuBi-[Glu] triggered the action potential twice faster than MNI-[Glu], with a u of 0.14 GM. Bond cleavage is completed in less than 50 ns, which places the complex among the fastest cages available. [START_REF] Salierno | A fast ruthenium polypyridine cage complex photoreleases glutamate with visible or IR light in one and two photon regimes[END_REF] Pyridinium moieties were also reported to undergo photochemical cleavage, [START_REF] Tatsuo | Selective removal of electron-accepting p-tolueneand naphthalenesulfonyl protecting groups for amino function via photoinduced donor acceptor ion pairs with electron-donating aromatics[END_REF] and were used in an antenna-sensitized 2PU system. [START_REF] Korzycka | Two-photon sensitive protecting groups operating via intramolecular electron transfer: uncaging of GABA and tryptophan[END_REF] It is worth highlighting that several more photocleavable structures [START_REF] Klán | Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy[END_REF] including BODIPY, [START_REF] Sitkowska | Green-Light-Sensitive BODIPY Photoprotecting Groups for Amines[END_REF][START_REF] Sitkowska | Red-light-sensitive BODIPY photoprotecting groups for amines and their biological application in controlling heart rhythm[END_REF] fluorenes, [START_REF] Reinfelds | A New Photocage Derived from Fluorene[END_REF] bimanes, [START_REF] Chaudhuri | Bimane: A Visible Light Induced Fluorescent Photoremovable Protecting Group for the Single and Dual Release of Carboxylic and Amino Acids[END_REF] hydroxyinden-3ylmethyls, [START_REF] Sasaki | Structural Transformation of the 2-(p-Aminophenyl)-1-hydroxyinden-3-ylmethyl Chromophore as a Photoremovable Protecting Group[END_REF] xanthenes [START_REF] Šebej | Fluorescein Analogues as Photoremovable Protecting Groups Absorbing at ∼520 nm[END_REF] and methylene blue derivatives [START_REF] Dao | Methylene blue as a far-red light-mediated photocleavable multifunctional ligand[END_REF] have been reported in literature, but have neither been developed for nor tested under 2PE, which opens more avenues in terms of molecular engineering of 2P cages.

Antenna-sensitized uncaging

Rather than optimising the photochemical properties and uncaging performance by direct structural modification of the PPG backbone, the light absorption capacity can be indirectly improved by introducing a second chromophore acting as an energy-harvesting antenna. The design of highly efficient 2P-absorbing antennas following the rules described in section 2.3 thus bypasses the issue encountered during the direct modification of PPGs. The energy of the antenna in its excited state must be transferred to the photochemistry module via an appropriate photophysical sensitization process such as Förster Resonance Energy Transfer (FRET), photoinduced electron transfer (PET) or triplet-triplet energy transfer (TT-ET). [START_REF] Klán | Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy[END_REF][START_REF] Klausen | Tandem Systems for Two-Photon Uncaging of Bioactive Molecules[END_REF] Although beyond the scope of this review, several multi-chromophoric systems for 1P [START_REF] Papageorgiou | An antenna triplet sensitiser for 1-acyl-7-nitroindolines improves the efficiency of carboxylic acid photorelease[END_REF][START_REF] Smirnova | Synthesis of Caged Nucleosides with Photoremovable Protecting Groups Linked to Intramolecular Antennae[END_REF][START_REF] Papageorgiou | An antenna-sensitised 1-acyl-7-nitroindoline that has good solubility properties in the presence of calcium ions and is suitable for use as a caged L-glutamate in neuroscience[END_REF] and 2PU [START_REF] Korzycka | Two-photon sensitive protecting groups operating via intramolecular electron transfer: uncaging of GABA and tryptophan[END_REF][START_REF] Pirrung | Sensitized two-photon photochemical deprotection[END_REF][START_REF] Diaz | Cooperative Veratryle and Nitroindoline Cages for Two-Photon Uncaging in the NIR[END_REF][START_REF] Hammer | Sensitized Two-Photon Activation of Coumarin Photocages[END_REF] have been reported and used successfully by following this alternative strategy.

Biological applications and underlying challenges

The intrinsic advantages of 2PU can be grasped by considering the spatio-temporal dimensions involved in physiological processes. Indeed, upon light excitation, ligand-receptor interactions must be reproduced artificially with utmost precision and specificity, and in this sense, 2PU applications all have different requirements. A wide panel of biological functions can be considered, and as a full overview of this domain goes beyond the scope of this manuscript, this section will focus on two leading applications: the controlled release of neurotransmitters and of drugs.

Uncaging of neurotransmitters: optoneurobiology

The nervous system consists of a network of polarized neuronal cells, carrying information through chemoelectric activity. Nerve impulses are converted to chemical message and back via small organic molecules acting as neurotransmitters. The challenge of 2PU in optoneurobiology is to mimic artificially the mechanics of a chemical synapse. [START_REF] Svoboda | Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience[END_REF] This implies a photolysis performed on the millisecond scale, and the release of thousands of neurotransmitters in the sub-micrometric synaptic cleft to cause the required millimolar concentration jump and trigger the action potential. [START_REF] Warther | Twophoton uncaging: New prospects in neuroscience and cellular biology[END_REF][START_REF] Ellis-Davies | Two-Photon Uncaging of Glutamate[END_REF] In this sense, the excitation volume of focalised lasers is perfectly adapted since the beam can be modelled by two Gaussian widths at half axial and lateral half-length respectively of the order of 1 μm and 0.30 μm. [START_REF] Matsuzaki | Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons[END_REF] Synapses can be excited individually by the laser, [START_REF] Branco | Dendritic Discrimination of Temporal Input Sequences in Cortical Neurons[END_REF] which greatly facilitates the study of molecular and biological dynamics.

To preserve the three-dimensional resolution at the cellular level, both diffusion and photolysis kinetics are critical points to consider since the spatial confinement can be lost when the diffusional exchange is faster than the photolysis reaction itself. To that end, the excited intermediates must remain concentrated within the irradiated volume and complete the photolytic reaction before they are replaced by a caged neuromessenger in the ground state; which implies rate constants greater than 10 5 s -1 . [START_REF] Trigo | Laser photolysis of caged compounds at 405nm: Photochemical advantages, localisation, phototoxicity and methods for calibration[END_REF] The kinetics of the photolysis is also a preponderant factor to prevent potential photo-desensitisation of the neurons by prolonged laser pulses. The size of the PPG also bears important implications, as small objects will be more compatible with the sub-micrometric synaptic cleft. Finally, mimicking the concentration jump of a chemical synapse implies that the caged neurotransmitter must be soluble at high concentrations of the order of 1 mM in physiological media.

Examples of caged neurotransmitters, hormones and neuromediators, or calcium ligands developed for neuroscience are legion, and some of them are now commercially available.

RuBi-caged glutamate, [START_REF] Fino | RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines[END_REF] or MNI-[Glu] [START_REF] Matsuzaki | Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons[END_REF][START_REF] Branco | Dendritic Discrimination of Temporal Input Sequences in Cortical Neurons[END_REF][START_REF] Carter | State-Dependent Calcium Signaling in Dendritic Spines of Striatal Medium Spiny Neurons[END_REF][START_REF] Shoham | Rapid neurotransmitter uncaging in spatially defined patterns[END_REF][START_REF] Nikolenko | Two-photon photostimulation and imaging of neural circuits[END_REF][START_REF] Nikolenko | SLM microscopy: scanless two-photon imaging and photostimulation using spatial light modulators[END_REF][START_REF] Lutz | Holographic photolysis of caged neurotransmitters[END_REF][START_REF] Zahid | Holographic Photolysis for Multiple Cell Stimulation in Mouse Hippocampal Slices[END_REF][START_REF] Abrahamsson | Thin Dendrites of Cerebellar Interneurons Confer Sublinear Synaptic Integration and a Gradient of Short-Term Plasticity[END_REF] are now commonly used in neuroscience for the mapping of nerve pathways that can be chemically identified by the presence of neuromodulators, [START_REF] Matsuzaki | Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons[END_REF][START_REF] Nikolenko | Two-photon photostimulation and imaging of neural circuits[END_REF] for structural study of neurons and their activation sequences, [START_REF] Branco | Dendritic Discrimination of Temporal Input Sequences in Cortical Neurons[END_REF][START_REF] Carter | State-Dependent Calcium Signaling in Dendritic Spines of Striatal Medium Spiny Neurons[END_REF] or for the study of synaptic plasticity. [START_REF] Abrahamsson | Thin Dendrites of Cerebellar Interneurons Confer Sublinear Synaptic Integration and a Gradient of Short-Term Plasticity[END_REF][START_REF] Araya | Input transformation by dendritic spines of pyramidal neurons[END_REF] Optoneurobiology has been a major driving application in the research of new high-performance 2P-sensitive structures. Important work has also been carried out to develop wavelength-selective "orthogonal" PPGs for multimodal photo-activation, [START_REF] Kantevari | Two-color, two-photon uncaging of glutamate and GABA[END_REF] or to reduce the interaction of caged neurotransmitters with synaptic receptors. [START_REF] Richers | Cloaked Caged Compounds: Chemical Probes for Two-Photon Optoneurobiology[END_REF]

Uncaging of drugs: targeted therapy

In drug-delivery applications, the time, space and performance scales involved in cellular responses are somewhat less demanding as the main objective is to reduce off-target effects.

However, other key factors such as dark stability, clearance and biological barriers must be taken into account. Photoactivation can be performed either at the molecular level with socalled "pro-drugs", or with the use of light-triggered nanomedicine. [START_REF] Karimi | Smart Nanostructures for Cargo Delivery: Uncaging and Activating by Light[END_REF][START_REF] Beauté | Photo-triggered polymer nanomedicines: From molecular mechanisms to therapeutic applications[END_REF] Cytotoxic anticancer agents have been caged with different 2P-sensitive PPGs, [START_REF] Venkatesh | Photocaging of Single and Dual (Similar or Different) Carboxylic and Amino Acids by Acetyl Carbazole and its Application as Dual Drug Delivery in Cancer Therapy[END_REF][START_REF] Skwarczynski | Development of first photoresponsive prodrug of paclitaxel[END_REF] and sometimes incorporated in molecular theranostic system. [START_REF] Gandioso | A Green Light-Triggerable RGD Peptide for Photocontrolled Targeted Drug Delivery: Synthesis and Photolysis Studies[END_REF][START_REF] Venkatesh | Three-Arm, Biotin-Tagged Carbazole-Dicyanovinyl-Chlorambucil Conjugate: Simultaneous Tumor Targeting, Sensing, and Photoresponsive Anticancer Drug Delivery[END_REF] No direct 2P experiments have been reported in either case, but such structures are known to be 2P-sensitive (vide supra).

Recently, DEAC has been used at the nanoscale level for the photorelease of chlorambucil and doxorubicin, respectively with silica [START_REF] Lin | Anticancer Drug Release from a Mesoporous Silica Based Nanophotocage Regulated by Either a One-or Two-Photon Process[END_REF] and gold nanoparticles upon 2PE. [START_REF] Liang | Near infrared light responsive hybrid nanoparticles for synergistic therapy[END_REF] Aminocoumarin derivatives have also been incorporated in the structure of IR-disrupted micelles [START_REF] Babin | A New Two-Photon-Sensitive Block Copolymer Nanocarrier[END_REF] and nanocomposites [START_REF] Ji | Coumarin-containing photo-responsive nanocomposites for NIR light-triggered controlled drug release via a two-photon process[END_REF] for cargo release.

Summary and Outlook

The design of organic dyes compatible with the range of wavelengths of the "biological transparency windows" is arguably one of the most prevalent challenges in optobiology nowadays. The intricacy of such work further intensifies in the case of PPGs. Shifting the 1PE band of such light-cleavable moieties to biologically compatible wavelength is possible, but highly challenging. So far, only a couple of red-absorbing BODIPY [START_REF] Sitkowska | Red-light-sensitive BODIPY photoprotecting groups for amines and their biological application in controlling heart rhythm[END_REF] coumarin [START_REF] Gandioso | Development of Green/Red-Absorbing Chromophores Based on a Coumarin Scaffold That Are Useful as Caging Groups[END_REF] and phenothiazinium [START_REF] Dao | Methylene blue as a far-red light-mediated photocleavable multifunctional ligand[END_REF] PPGs have been reported, and unfortunately, such massive bathochromic shifts often come at the cost of reduced bond-cleavage efficiencies and quantum yields. In addition, the tridimensional resolution of the excitation, and therefore the precision of the photo-activation, also remain very limited. The non-linear nature of 2PA bypasses this drawback, while providing easy access to red and IR wavelengths. Highly responsive 2P dyes are now commonly accessible via the molecular engineering strategies described above.

Reports on PPGs with 2PA cross-sections of several hundreds of GM are now common, and u records have increased by orders of magnitude over the last few years, from BHC (2 GM) [START_REF] Furuta | Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis[END_REF] to NDPA (20 GM) [START_REF] Schelkle | Light-Induced Protein Dimerization by One-and Two-Photon Activation of Gibberellic Acid Derivatives in Living Cells[END_REF] and extended coumarin derivatives (440 GM). [START_REF] Klausen | Dual-wavelength efficient two-photon photorelease of glycine by π-extended dipolar coumarins[END_REF] Following the seminal work carried out on the oNB family, coumarin PPGs have lately appeared as the most promising and popular dyes thanks to their versatility, and may open up even more avenues to both specialists and newcomers to the field. Other original designs have been reported recently with the incorporation of the less-common phenacyl and o-hydroxycinnamate families into more conjugated structures. This strategy instigated new prospects for these families of dyes, and holds great potential for future research. In addition, a dormant potential resides in the number of new photosensitive structures that were reported without focus on 2P applications, and further collaboration between research groups should be encouraged in order to investigate their response under non-linear excitation.

However, many challenges remain, as the optimisation of PPGs to 2PE is a viable but tedious strategy. Extensions of the -conjugated systemand therefore the molecular weightof the dyes often lead to poor water solubility, and excessive modification may thus be counterproductive. In this regard, although size and symmetry empirically show an increase in 2PA, the ratio of 2 max to molecular weight of dyes rarely exceeds 1 or 2. Large PPGs are also more expensive and difficult to synthesize, less atom-economical, and less suited to certain applications. Furthermore, many examples relate an unpredictable u parameter upon structural modification. Such considerations must be kept in mind, and the key is eventually to find the optimal balance between performance, size, and practicality.

Figure 2 .

 2 Figure 2. Illustration of the concept of absorption cross-section for a dye in solution. The incident beam I0 can be decomposed, along the optical pathway, into an infinity of irradiated sections S of infinitesimal length dℓ. At each position x of the optical path, the light intensity is Ix before the section, and Ix -dI at the exit. The absorption cross-section of each molecule is represented as dark green discs.

Figure 3 .

 3 Figure 3. Energy diagram following the essential states model in the case of (a) noncentrosymmetric chromophores; (b) a centrosymmetric chromophore with high detuning energy; (c) a centrosymmetric chromophore with small detuning energy.

Figure 4 .

 4 Figure 4. a) Roadmap of the structural modifications performed on centrosymmetric chromophores and stilbene derivatives, and evolution of the 2 as a function of ICT, and conjugation (length and planarity).[31,44,45] Non = n-C9H18. b) Comparison of 2 max and 2PA max values in dipole 9 quadrupole 10 of equivalent strength.[START_REF] Pokladek | Linear Optical and Third-Order Nonlinear Optical Properties of Some Fluorenyl-and Triarylamine-Containing Tetracyanobutadiene Derivatives[END_REF]

Figure 5 .

 5 Figure 5. Effect of the degree of branching on 2 and 2/n values in compound 3, corresponding octupole D(--D)3 11[44] and different generations (G0, G1 and G2) of the multibranched compound 12.[56] The 2PA spectra of dendrimers 12-G0 (squares), -G1 (downward triangles), and -G2 (upward triangles) and constitutive stilbene unit DPAS (circles), and the linear absorption of dendrimer 12-G2 (dashed curve, half abscissa values)

Figure 6 .

 6 Figure 6. Summary of the specifications required for use of PPGs in biology, and of the main families of photosensitive moieties classified by type of mechanism. The Z group represents the biological substrate protected by the PPG (note that each PPG family can uncage specific chemical functions).

Figure 7 .

 7 Figure 7. Structures and key photophysical properties of the first PPGs of the nitrobenzyl family cleaved by 1P or 2PE.

[ 81 ]

 81 In this pioneer work, Tsien and co-workers report the first u value of 0.03 GM for oNV-[Ac].[81] Its homobenzyl analogue DMNPB-[Glu] later proved approximately 6 times more efficient when irradiated at the maximum of its 2PA band, however such photosensitivities remain very limited.[89] Limited 2P photosensitivities were also recorded for two photocleavable calcium chelating ligands, dimethoxy-nitrophene DMN-[Ca][92] and nitrodibenzofurane derivative NDBF-[Ca],[91] able to release Ca 2+ ions in vitro upon ligand cleavage. An extended dibenzofuran backbone allows a 60-fold increase in 2PU cross-section in NDBF-[Ca] compared to DMN-[Ca] thanks to a large increase in u, but reported u values remain below 1 GM. This class of compounds was later extended with 2-(4-nitrophenyl)benzofuran (NPBF) derivatives, which reportedly released a model carboxylic acid (benzoic acid) with a u of 9% in DMSO, and a model calcium ligand (ethylene glycoltetraacetic acid tetraethyl ester) with a u of 30% in benzene.[START_REF] Komori | Design and synthesis of a new chromophore, 2-(4-nitrophenyl)benzofuran, for two-photon uncaging using near-IR light[END_REF] These results gave extrapolated u values between 1.6 and 16 GM in their respective solvents, which highlights the importance of the leaving group and solvent in the photolysis reaction. NPBF has also recently been used for 2P-photorelease of the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical for targeted anticancer therapy.[START_REF] Yamada | Photochemical generation of the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical from caged nitroxides by near-infrared two-photon irradiation and its cytocidal effect on lung cancer cells[END_REF]

Figure 9 . 3 . 2 .Figure 10 .

 93210 Figure 9. Roadmap of the structural modifications performed on NPPOC derivatives in order to optimise their 2PU cross-section. R = di(ethyleneglycol) ; Z = gibberellic acid.

70%.[ 108 , 121 ]Figure 11 :

 10812111 Figure 11: Structures of the most common 2P-sensitive NI-caged neurotransmitters.

Figure 14 .

 14 Figure 14. Evolution of the uncaging quantum yield (blue) and the 2PU cross-section (green)in substituted quinoline PPGs for the release of acetic acid.[START_REF] Davis | Substituent Effects on the Sensitivity of a Quinoline Photoremovable Protecting Group to One-and Two-Photon Excitation[END_REF]

[ 179 ]Scheme 7 .

 1797 Scheme 7.Reported mechanism for the photo-solvolysis of BHQ derivatives.

Figure 15 .

 15 Figure 15. a) Structures of dipolar, quadrupolar and octupolar quinoline derivatives and the resulting effect of the modifications on the 2PU properties. b) 2PA spectrum of dipole 8DMAQ-[Ac] in MeCN/Tris buffer (1/1). Reprinted with permission from ref.[START_REF] Petit | Substitution Effect on the Oneand Two-photon Sensitivity of DMAQ "Caging" Groups[END_REF]. Copyright 2012 American Chemical Society. c) 2PA spectrum (red) of quadrupole 67 in MeCN. Linear absorption spectrum (black) is given for comparison. Reprinted from ref.[START_REF] Tran | Quinoline-Derived Two-Photon Sensitive Quadrupolar Probes[END_REF] with permission from John Wiley and Sons. d) 2PA spectrum (red) of octupole 68 in THF. Linear absorption spectrum (black) is given for comparison. Reprinted from ref.[START_REF] Picard | Octupolar chimeric compounds built from quinoline caged acetate moieties: a novel approach for 2-photon uncaging of biomolecules[END_REF] with permission from The Royal Society of Chemistry.

Figure 16 .

 16 Figure 16. Structures and photochemical properties of oHC compounds caging model alcohols.

Two-photon uncaging

  remains a young but quickly expanding field, and exciting opportunities may arise if certain barriers are broken down by future research. Any work towards a rationalisation of the uncaging quantum yield in different PPG families, and towards a consensus over the measurement techniques used for uwhich still prevents direct performance comparison in some cases, would be greatly beneficial to the field. In addition, the translational potential of such probes is considerable but not entirely fulfilled, and the eye of all communities involved in their development is of utmost importance for further progress towards high clinical relevance. New modular and point-of-care laser sources are being developed for more clinically friendly use of 2PA; and future progress in this domain, combined with the development of 2P-sensitive caged bio-modulators, may open new exciting avenues in biology and light therapies.

  Early examples of photo-deprotection of glycine[START_REF] Barltrop | Photosensitive Protecting Groups[END_REF] and of phenol intermediate 15[START_REF] Li | Total Synthesis of Nominal Diazonamides-Part 1: Convergent Preparation of the Structure Proposed for (-)-Diazonamide A[END_REF] for synthetic purposes, and of ATP in biological applications.[START_REF] Kaplan | Rapid photolytic release of adenosine 5'triphosphate from a protected analog: utilization by the sodium:potassium pump of human red blood cell ghosts[END_REF] Thus grew the idea of preparing inactive photosensitive precursors of bioactive substances, and releasing them in vitro or in vivo upon irradiation. Schlaeger et al. with cyclic adenosine monophosphate (cAMP)[START_REF] Engels | Synthesis, structure, and reactivity of adenosine cyclic 3',5'phosphate-benzyltriesters[END_REF] and Kaplan et al. with adenosine triphosphate (ATP)

Li et al. on the structure of (-)-Diazonamide A, which led them to prepare compound 16 by a chemical cascade triggered by the photodeprotection of 15.[63] Scheme 1.

  Applied successfully in optobiology until the early 2000s, these early PPGs nonetheless proved to have insufficient 2P photosensitivity for practical use without tissue damage. As laser powers above the order of ten milliwatts are poorly tolerated by tissues,[START_REF] Kiskin | The efficiency of two-photon photolysis of a "caged" fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals[END_REF][START_REF] Hopt | Highly Nonlinear Photodamage in Two-Photon Fluorescence Microscopy[END_REF] high 2P photosensitivities u are required in compensation, and a minimal threshold of 3 to 30 GM has been established to perform 2PU under physiologically compatible conditions.[START_REF] Pelliccioli | Photoremovable protecting groups: reaction mechanisms and applications[END_REF] Ogden et al. thus reported that photodamage to micrometric synapses could occur with average powers above 5 mW with 5 ms exposures. At this maximum power, using a pulsed laser of 200 fs and 76 MHz, and taking into account the diffusion coefficient a caged neurotransmitter molecule in water, they estimated that the

minimum 2PU cross-section needed to achieve 50% steady-state photolysis was as high as 31 GM.

[START_REF] Kiskin | The efficiency of two-photon photolysis of a "caged" fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals[END_REF]

The recent years have been focused on the development of a second generation of PPGs based on such original structures with a 2PU cross-section falling in or beyond this range.

[START_REF] Bort | From One-Photon to Two-Photon Probes: "Caged" Compounds, Actuators, and Photoswitches[END_REF][START_REF] Abe | Design and Synthesis of Two-Photon Responsive Chromophores for Near-Infrared Light-Induced Uncaging Reactions[END_REF][START_REF] Chitose | Design and synthesis of two-photon responsive chromophores for application to uncaging reactions[END_REF][START_REF] Jakkampudi | Caged Compounds for Two-Photon Uncaging[END_REF]

  compounds 35 and 36. Indeed, replacing the alkoxy donor with a stronger ED and hydrophilic phenol group creates competitive photochemical pathways in compound 35, which released glutamate in only 10% chemical yield. Similar observations were made on the stilbene derivative 36 which released only 48% of glutamate.[START_REF] Gug | Photolabile Glutamate Protecting Group with High One-and Two-Photon Uncaging Efficiencies[END_REF][START_REF] Specht | The donor-acceptor biphenyl platform: A versatile chromophore for the engineering of highly efficient two-photon sensitive photoremovable protecting groups[END_REF] Both compounds proved unsuitable for use as PPG for this reason. Similar modifications proved however beneficial in the case of

	the dialkylamino and nitrodiphenylacetylene derivatives EANBP-[Glu] and NDPA-[Gib].
	Following the work carried out on PENB, Donato et al. further replaced the alkoxy donor with
	a stronger dialkylamino ED group on the NBP. This structural change made of EANBP-[Glu]

the first PPG to break the 10 GM mark.

[START_REF] Donato | Water-Soluble, Donor-Acceptor Biphenyl Derivatives in the 2-(o-Nitrophenyl)propyl Series: Highly Efficient Two-Photon Uncaging of the Neurotransmitter γ-Aminobutyric Acid at λ=800 nm[END_REF]

Reactivation of caged Glu occurs over a period of about 5 μs after the light pulse, and the two diethylene glycol chains on the nitrogen atom provide excellent 10 mM solubility in physiological media, which makes it compatible with neuroscience experiments. The absorption band also undergoes an 80 nm bathochromic shift, both in the 1P and 2PA spectra. This shift was also reported in other aminobiphenyl PPGs used for calcium uncaging.

[START_REF] Jakkampudi | Design and Synthesis of a 4-Nitrobromobenzene Derivative Bearing an Ethylene Glycol Tetraacetic Acid Unit for a New Generation of Caged Calcium Compounds with Two-Photon Absorption Properties in the Near-IR Region and Their Application in Vivo[END_REF]

Acknowledgements

MK thanks the Ministère de l'Enseignement Supérieur et de la Recherche and the University of Bordeaux for PhD grant, and the Engineering and Physical Sciences Research Council (EPSRC, United Kingdom) Interdisciplinary Research Collaboration (grant EP/R018669/1) for research funding. MBD gratefully acknowledges the University of Bordeaux and the Conseil Régional d'Aquitaine for funding ("Chaire d'Accueil d'excellence" grant).