Frédéric Bolze

We synthesized new para-dialkylaminonitrobiphenyl (ANBP) derivatives, s-ANBP and t-ANBP, functionalized with dimethyltrityl and phosphoramidite groups for incorporation into DNA backbones as photocleavable linkers via solid-phase synthesis. Both derivatives exhibited excellent chemical stability under diverse conditions, including acidic, alkaline, and high-salt environments and elevated temperatures. Their incorporation into DNA influenced duplex stability and antisense oligonucleotide (ASO) dissociation efficiency, depending on the number of ANBP units and adjacent nucleotide deletions. The s-/t-ANBPconjugated DNA showed efficient one-photon photolysis at 415 nm and enhanced two-photon absorption for extended π-system in t-ANBP, with δ_uΦ_u values of 1.6 GM (740 nm) and 2.7 GM (800 nm). ANBP-conjugated DNA was used to construct a 3D DNA nanocage capable of light-triggered ASO 4625 release, validated by an in vitro RNA digestion assay, confirming antisense functionality. This platform demonstrates precise, light-mediated therapeutic delivery and offers potential for broader applications in drug delivery and clinical use.

The emerging field of photopharmacology is a promising chemobiological methodology for optical control of drug activities that could ultimately solve the off-target toxicity outside the disease location of many drugs for the treatment of a given pathology. The use of photolytic reactions looks very attractive for a light-activated drug release but requires to develop photolytic reactions sensitive to red or near-infrared light excitation for better tissue penetration. This review will present the concepts of triplet-triplet annihilation upconversion-based photolysis and their recent in vivo applications for light-induced drug delivery using photoactivatable nanoparticles.

Liposome-based nanoparticles able to release, via a photolytic reaction, a payload anchored at the surface of the phospholipid bilayer were prepared. The liposome formulation strategy uses an original drug-conjugated blue light-sensitive photoactivatable coumarinyl linker. This is based on an efficient blue light-sensitive photolabile protecting group modified by a lipid anchor, which enables its incorporation into liposomes, leading to blue to green light-sensitive nanoparticles. In addition, the formulated liposomes were doped with triplet–triplet annihilation upconverting organic chromophores (red to blue light) in order to prepare red light sensitive liposomes able to release a payload, by upconversion-assisted photolysis. Those light-activatable liposomes were used to demonstrate that direct blue or green light photolysis or red light TTA-UC-assisted drug photolysis can effectively photorelease a drug payload (Melphalan) and kill tumor cells in vitro after photoactivation.

Fluorogenic bioorthogonal reactions are promising tools for tracking small molecules or biomolecules in living organisms. Two-photon excitation, by shifting absorption towards the red, significantly increases the signal-to-noise ratio and decreases photodamages, while allowing to image about 10 times deeper than with a confocal. However, efficient two-photon excitable fluorogenic probes are currently lacking. We report here the design and synthesis of fluorogenic probes based on a two-photon excitable fluorophore and a tetrazine quenching moiety. These probes react with bicyclo[6.1.0]no-4-yn-9yl)methanol (BCN) with good to impressive kinetic rate constant (up to 1.1x103 M-1.s-1) and emit in the red window with moderate to high turn-on. TDDFT allowed to rationalize both the kinetic and fluorogenic performance of the different probes. The best candidate displays a 13.8-fold turn-on measured by quantifying fluorescence intensities in live cells under one-photon excitation, whereas a value of 3 is sufficient for high contrast live-cell imaging. In addition, live-cell imaging under two-photon excitation confirmed that there was no need for washing to monitor the reaction between BCN and this probe since a 8.0-fold turn-on was measured under two-photon excitation. Finally, the high two-photon brightness of the clicked adduct (>300 GM) allows the use of a weak laser power compatible with in vivo imaging.

In this study, we synthesized two phosphoramidites based on (2,7-bis-{4-nitro-8-[3-(2-propyl)-styryl]}-9,9-bis-[1-(3,6-dioxaheptyl)]-fluorene (BNSF) and (4,4’-bis-{8-[4-nitro-3-(2-propyl)-styryl]}-3,3’-di-methoxybiphenyl (BNSMB) structures as visible light-cleavable linkers for oligonucleotide conjugation. In addition to commercial ultraviolet (UV) photocleavable (PC) linker, the BNSMB linker was further applied as a building component to construct photoregulated DNA devices as duplex structures which are functionalized with fluorophores and quenchers. Selective cleavage of PC and BNSMB is achieved in response to ultraviolet (UV) and visible light irradiations as two inputs respectively. This leads to controllable dissociation of pieces of DNA fragments which is followed by changes of fluorescence emission as signal outputs in of system. By tuning the number and position of the photocleavable molecules, fluorophores and quenchers, various DNA devices were developed in which they mimic functions of Boolean logic gates and achieve logic operations in AND, OR, NOR and NAND gates in response to two different wavelengths of light inputs. By sequence design, the photolysis products can be precisely programmed in DNA devices and triggered release in a selective or sequential manner. Thus, this photoregulated DNA device shows potentials as a wavelength dependent drug delivery system for selective control over the release of multiple individual therapeutic oligonucleotide-based drugs. We believe that our work not only enriches the library of photocleavable phosphoramidite available for bio-conjugation, but also pave the way of developing spatiotemporal-controlled, orthogonal regulated DNA-based logic circuits for a range of applications in materials science, polymer, chemistry and biology.

Photolytic reactions allow the optical control of the liberation of biological effectors by photolabile protecting groups. The development of versatile technologies enabling the use of deep-red or NIR light excitation still represents a challenging issue, in particular for light-induced drug release (eg; light induced prodrug activation). Here, we present light-sensitive biocompatible lipid nanocapsules able to liberate an antitumoral drug through photolysis. We demonstrate that original photon upconverting nanoparticles (LNC-UCs) chemically conjugated to a coumarin-based photocleavable linker can quantitatively and efficiently release a drug by upconversion luminescence-assisted photolysis using a deep-red excitation wavelength. In addition, we were also able to demonstrate that such nanoparticles are stable in the dark, without any drug leakage in the absence of light. These findings open new avenues to specifically liberate diverse drugs using deep- red or NIR excitations for future therapeutic applications in nanomedicine.

Red to NIR absorption and emission wavelengths are key requirements for intravital bioimaging. One of the way to reach such excitation wavelengths is to use two-photon excitation. Unfortunately, there is still a lack of two-photon excitable fluorophores that are both efficient and biocompatible. Thus, we design a series of biocompatible quadrupolar dyes in order to study their ability to be used for live-cell imaging, and in particular for two-photon microscopy. Hence, we report the synthesis of 5 probes based on different donor cores (phenoxazine, acridane, phenazasiline and phenothiazine) and the study of their linear and non-linear photophysical properties. TD-DFT calculations were performed and were able to highlight the structure-property relationship of this series. All these studies highlight the great potential of three of these biocompatible dyes for two-Photon microscopy, as they both exhibit high two-photon cross-sections (up to 3 650 GM) and emit orange to red light. This potential was confirmed through live-cell two-photon microscopy experiments, leading to images with very high brightness and contrast.

The use of caged compounds, defined as photolabile precursors of biological effectors, have proven to be attractive in various fields of biology. Photolytical reactions have been widely used to allow a rapid and efficient concentration jump of various biological effectors within organized biological systems. During the last two decades, the challenge was to overcome the difficulty that only high energy UV light was used to induce photochemical reactions on photoremovable protecting groups (PPGs). Infrared-sensitive PPGs should be able to improve in vivo applications of caged compounds. The present review is focused on recent strategies enabling the use of excitation wavelength inside the photo-therapeutical window (600–1000 nm) to efficiently and specifically cleave a chemical bond.

In this study, we report the synthesis, linear and non-linear photophysical studies and live cell imaging of two two-photon activatable probes based on a silafluorene core: SiFluo-V and SiFluo-L. Thanks to their quadrupolar (A-π-D-π-A) design, these probes exhibit respectively good to impressive two-photon cross sections (from 210 GM to 2150 GM). TD-DFT calculations support the experimental evidence that SiFluo-L displays far better two-photon absorption properties than SiFluo-V. Moreover, SiFluo-L possesses all requirements for bioimaging as it is water soluble, cell-permeant and and presents a low cytotoxicity (IC80≥10 µM). It labels mitochondria in live-cell imaging at low laser power with high brightness, contrast and photostability. This study demonstrates that silafluorene is a promising core to develop new two-photon fluorophores for live cell imaging.

With the aim of developing new molecular theranostic agents, a π-extended Zn(II) porphyrin as photosensitizer for photodynamic therapy (PDT) linked to two GdDOTA-type complexes for magnetic resonance imaging (MRI) detection was synthesized. The relaxivity studies revealed a much higher relaxivity value per Gd ion for this medium sized molecule (19.32 mM–1 s–1 at 20 MHz and 298 K) compared to clinical contrast agents—a value which strongly increases in the presence of bovine serum albumin, reaching 25.22 mM–1 s–1. Moreover, the photophysical studies showed the strong ability of the molecule to absorb light in the deep red (670 nm, ε ≈ 60000 M–1 cm–1) and in the near-infrared following two-photon excitation (920 nm, σ2 ≈ 650 GM). The conjugate is also able to generate singlet oxygen, with a quantum yield of 0.58 in DMSO. Promising results were obtained in cellular studies, demonstrating that the conjugate is internalized in HeLa cells at micromolar concentration and leads to 70% of cell death following 30 min irradiation at 660 nm. These results confirm the potential of the designed molecule as an imaging and therapeutic agent.

The use of photolabile protecting groups (PPGs) has been growing in emphasis for decades, and they nowadays enable cutting-edge results in numerous fields ranging from organic synthesis to neurosciences. PPGs are chemical entities that can be conjugated to a biomolecule to hide its biological activity, forming a stable so called “caged compound”. This conjugate can be simply cleaved by light and therefore, the functionality of the biomolecule is restored with the formation of a PPG by-product. However, there is a sizeable need of PPGs able to quantify the “uncaging” process. In this review, we will discuss several strategies leading to an acute quantification of the uncaging events by fluorescence. In particular, we will focus on how molecular engineering of PPG could open new opportunities by an easy access to photoactivation protocols

Despite the advantages of photodynamic therapy (PDT) over chemotherapy or radiotherapy such as low side effects, lack of treatment resistance and spatial selectivity inherent to light activation of the drug, several limitations especially related to the photosensitiser (PS) prevent PDT from becoming widespread in oncology. Herein, new folic acid- and biotin-conjugated PSs for tumour-targeting PDT are reported, with promising properties related to PDT such as intense absorption following one-photon excitation in the red or two-photon excitation in the near-infrared, and also high singlet oxygen quantum yield (close to 70% in DMSO). Cellular studies demonstrated that both targeted PSs induced phototoxicity, the folate-targeted PS being the most effective one with 80% of cell death following 30 min of irradiation and a phototoxicity four times higher than that of the non-targeted PS. This result is in accordance with the uptake of the folate-targeted PS in HeLa cells, mediated by the folate receptors. Moreover, this folate-targeted PS was also phototoxic following two-photon excitation at 920 nm, opening new perspectives for highly selective PDT treatment of small and deep tumours.

A molecular theranostic agent designed for photodynamic therapy (PDT) treatment in the near-infrared and for imaging tissue tumors with magnetic resonance imaging (MRI) is reported. It consists of a linear pi-conjugated Zn(II) porphyrin dimer linked at each extremity to a GdDOTA-type complex. This agent has shown very promising potential for PDT applications with good singlet oxygen generation in DMSO and high linear absorption in the near- infrared (lambda(max) = 746 nm, epsilon approximate to 10(5) M-1 cm(-1)). Moreover, this molecule has a propensity for two-photon excited PDT with high two-photon cross sections (similar to 8000 GM in 880-930 nm range), which should allow for deeper tumor treatments and higher spatial precision as compared to conventional one-photon PDT. Regarding the MRI contrast agent properties, the molecule has shown superior relaxivity (14.4 mM(-1) s(-1) at 40 MHz, 298 K) in comparison to clinical contrast agents and the ability to be internalized in cells, thanks to its amphiphilic character. Irradiation of HeLa cells using either one-photon (740 nm) or two-photon excitation (910 nm) has led in both cases to important cell death.

We report the synthesis and photolytic properties of caged 9aminodoxycycline derivatives modified with 2-{4'-bis-[2-(2methoxyethoxy)ethyl]-4-nitrobiphenyl-3-yl}prop-1-oxy (EANBP) and PEG7-ylated (7-diethylamino-2-oxo-2H-chromen-4-yl)methyl (PEG7-DEACM) groups. 9-Aminodoxycycline is a tetracycline analogue capable of activating transcription through the inducible TetOn transgene expression system and can be regioselectively coupled to two-photon-sensitive photo-removable protecting groups by carbamoylation. The EANBP-based caged 9aminodoxycycline showed complex photochemical reactions but did release 10% of 9-aminodoxycycline. However, 9-(PEG7DEACMamino)doxycycline exhibited excellent photolysis efficiency at 405 nm with quantitative release of 9aminodoxycycline and a 0.21 uncaging quantum yield. Thanks to the good two-photon sensitivity of the DEACM chromophore, 9aminodoxycycline release by two-photon photolysis is possible, with calculated action cross-sections of up to 4.0 GM at 740 nm. Therefore, 9-(PEG7-DEACMamino)doxycycline represents a very attractive tool for the development of a light-induced gene expression method in living cells.

Three zwitterionic meso-substituted A(3)B- and AB(2)C-porphyrins containing one sulfonato alkylpyridinium substituent and three or two alkoxy-substituted phenyl groups were synthesized in good yield and fully characterized as to their physicochemical properties by a variety of techniques. This new series of inner salt donor-acceptor meso-substituted porphyrin derivatives were prepared for possible application as amphiphilic probes for membrane insertion in the area of combined second-harmonic and two-photon fluorescence cellular microscopy. To this end, the linear and nonlinear optical properties of the compounds were characterized, together with their electrochemical and spectroelectrochemical properties in non-aqueous media. The neutral design of such molecules enabled us to determine their second order non-linear properties, both by Electric Field Induced Second Harmonic Generation and Hyper-Rayleigh Scattering. Two-photon absorption cross sections of these dyes were also measured by the two-photon induced fluorescence method. The zwitterionic nature of the inner salt results in very specific solvent-dependent redox-properties, which could be rationalized in terms of solvent-dependent ion-pairing. The overall data electrochemical and photophysical data indicates that these new porphyrinic systems should be good probes for membrane potential sensing.

A molecular theranostic agent for magnetic resonance imaging (MRI) and photodynamic therapy (PDT) consisting of four [GdDTTA]− complexes (DTTA4− = diethylenetriamine-N,N,N″,N″-tetraacetate) linked to a meso-tetraphenylporphyrin core, as well as its yttrium(III) analogue, was synthesized. A variety of physicochemical methods were used to characterize the gadolinium(III) conjugate 1 both as an MRI contrast agent and as a photosensitizer. The proton relaxivity measured in H2O at 20 MHz and 25 °C, r1 = 43.7 mmol–1 s–1 per gadolinium center, is the highest reported for a bishydrated gadolinium(III)-based contrast agent of medium size and can be related to the rigidity of the molecule. The complex displays also a remarkable singlet oxygen quantum yield of ϕΔ = 0.45 in H2O, similar to that of a meso-tetrasulfonated porphyrin. We also evidenced the ability of the gadolinium(III) conjugate to penetrate in cancer cells with low cytotoxicity. Its phototoxicity on Hela cells was evaluated following incubation at low micromolar concentration and moderate light irradiation (21 J cm–2) induced 50% of cell death. Altogether, these results demonstrate the high potential of this conjugate as a theranostic agent for MRI and PDT.

The convergent synthesis and characterization of a potential theranostic agent, [DPP-ZnP-GdDOTA]-, which combines a diketopyrrolopyrrole-porphyrin component DPPZnP as a two-photon photosensitizer for photodynamic therapy (PDT) with a gadolinium(III) DOTA complex as a magnetic resonance imaging probe, is presented. [DPP-ZnP-GdDOTA]- has a remarkably high longitudinal water proton relaxivity(19.94 mm-1 s-1 at 20 MHz and 258C) for a monohydrated molecular system of this size. The Nuclear Magnetic Relaxation Dispersion (NMRD) profile is characteristic of slow rotation, related to the extended and rigid aromatic units integrated in the molecule and to self-aggregation occurring in aqueous solution. The two-photon properties were examinedand large two-photon absorption cross-sections around 1000 GM were determined between 910 and 940 nm in DCM with 1% pyridine and in DMSO. Furthermore, the newconjugate was able to generate singlet oxygen, with quantumyield of 0.42 and 0.68 in DCM with 1% pyridine and DMSO, respectively. Cellular studies were also performed. The [DPP-ZnP-GdDOTA]- conjugate demonstrated low dark toxicity and was able to induce high one-photon and moderate two-photon phototoxicity on cancer cells.

Abstract Herein the synthesis, spectroscopic characterization, two‐photon absorption and electrochemical properties of 3,6‐disubstituted carbazole tweezers is reported. A dimer resulting from a Glaser homocoupling was isolated during a Sonogashira coupling reaction between a diethynyl‐carbazole spacer and a 5‐bromo‐triarylporphyrin and the properties of this original compound were compared with the 3,6‐disubstituted carbazole bisporphyrin tweezers. The dyads reported herein present a two‐photon absorption maximum at 920 nm with two‐photon absorption cross‐section in the 1200 GM range. Despite a strong linear absorption in the Soret region and moderate fluorescence quantum yield, they both lead to a high brightness reaching 30 000 M −1 cm −1 .

Five new non-ionic water-soluble two-photon dyes for fluorescence microscopy built around a diketopyrrolopyrrole central core were designed, prepared and characterized by 1H and 13C NMR, UV-Visible spectroscopy, HRMS and IR. Excitation and emission wavelengths were tuned by modifying the lengths of the conjugated systems and the nature of the substituents surrounding the diketopyrrolopyrrole central core. These fluorescent dyes are highly photostable and present high one- and two-photon brightness. Their application in confocal and two-photon excited fluorescence microscopy was performed on HeLa cell cultures with low power excitation.

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Two-photon (TP) excitation of organic chromophores is of great interest for decades, as applications of such phenomena from 3-dimentional (3D) microfabrication to optical limiting and optical data storage, are of increasing importance. More recently, two-photon excitation found important applications in biology, notably in two-photon excited microscopy (TPEM) or two-photon photodynamic therapy (2P-PDT). Nevertheless, these techniques were using dyes or sensitizers designed for one-photon processes with low two-photon response. The lack of efficient molecules specifically designed for two-photon applications has led us to design new chromophores for biological applications with increased sensitivity to two-photon excitation and specifically added properties useful in biological media, such as water solubility. Here we describe the molecular engineering of such dyes mainly for cell and small animal observation by TPEM and their conjugation to magnetic nanoparticles and bio-nanoparticles such as viruses.We will then focus on the possibility to use photochemical reaction for cell triggering by two-photon photorelease of biologically active substances the so-called two-photon uncaging.

Two-photon (TP) excitation of organic chromophores is of great interest for decades, as applications of such phenomena from 3-dimentional (3D) microfabrication to optical limiting and optical data storage, are of increasing importance. More recently, two-photon excitation found important applications in biology, notably in two-photon excited microscopy (TPEM) or two-photon photodynamic therapy (2P-PDT). Nevertheless, these techniques were using dyes or sensitizers designed for one-photon processes with low two-photon response. The lack of efficient molecules specifically designed for two-photon applications has led us to design new chromophores for biological applications with increased sensitivity to twophoton excitation and specifically added properties useful in biological media, such as water solubility. Here we describe the molecular engineering of such dyes mainly for cell and small animal observation by TPEM and their conjugation to magnetic nanoparticles and bio-nanoparticles such as viruses. We will then focus on the possibility to use photochemical reaction for cell triggering by two-photon photorelease of biologically active substances the so-called two-photon uncaging.

Light‐responsive biologically active compounds offer the possibility to study the dynamics of biological processes. Phototriggers and photoswitches have been designed, providing the capability to rapidly cause the initiation of wide range of dynamic biological phenomena. We will discuss, in this article, recent developments in the field of light‐triggered chemical tools, specially how two‐photon excitation, “caged” fluorophores, and the photoregulation of protein activities in combination with time‐resolved x‐ray techniques should break new grounds in the understanding of dynamic biological processes.

A π‐extended [2‐(2‐nitrophenyl)propoxy]carbonyl (NPPOC) derivative has been prepared as an efficient UV and near‐IR photolabile protecting group for glutamate. This glutamate cage compound exhibits efficient photorelease upon one‐photon excitation (εΦ=990 M⁻¹ cm⁻¹ at 315 nm). In addition, it also shows efficient photorelease in activation of glutamate receptors in electrophysiological recordings. Combined with a high two‐photon uncaging cross‐section (δΦ=0.45 GM at 800 nm), its overall properties make this new cage—3‐(2‐propyl)‐4′‐methoxy‐4‐nitrobiphenyl (PMNB)—for glutamate a very promising tool for two‐photon neuronal studies.

Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood-brain barriers. They are characterized by two-photon absorption cross-sections of 100-150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity. Microsc. Res. Tech., 2007. (c) 2007 Wiley-Liss, Inc.

Two series of water-soluble blue fluorescent two-photon excited chromophores for bio-imaging based on bis-stilbenic structure were synthesized. Wadsworth-Emmons reaction was used to build one dimensional D-π-aromatic core-π-D structures. The water solubility is induced by three oligo-ethylene glycol moieties in positions 3, 4, and 5 of the peripheral phenyl ring. Their two-photon absorption (TPA) cross-section in the range of 150 GM at 700 nm have been measured by the way of their two-photon excited fluorescence (TPEF) properties. It is 10 to 100 times higher than commercial dyes commonly used in bio-imaging. This makes these new TPA chromophores good candidates for in-vivo two-photon excited (TPE) microscopy.

“Organic Chemistry, where now?” This article reports the outcome of the first edition of ESYOP, a symposium devoted to the future of organic chemistry. The collective answer proposed to the above question has been elaborated by thirty-year-old French-speaking researchers. The challenges reported may be structured in three interdependent themes: quest for simplicity, nature as a guide, design of molecular structures capable of autonomy and adaptability. In the future, organic chemistry may be a science devoted to the synthesis of ‘intelligent' molecular systems inspired by Nature by using the simplest means.