Click Synthesis of Tweezer Dyads for H-Bond Assisted Cooperativity in Tandem Uncaging Systems.

"Tandem" uncaging systems, in which a photolabile protecting group (PPG) is sensitized by an energy-harvesting antenna, may increase the photosensitivity of PPGs by several orders of magnitude for two-photon (2P) photorelease. Yet, they remain poorly accessible because of arduous multi-step synthesis. In this work, we design efficient tandem uncaging systems by (i) using a convenient assembly of the building blocks relying on click chemistry, (ii) H-bonding induced proximity thus facilitating (iii) photoinduced electron transfer (PeT) as a cooperative mechanism. A strong two-photon absorber electron-donating quadrupolar antenna and various electron-accepting PPGs (mDEAC, MNI or MDNI) were clicked stepwise onto a "tweezer-shaped" pyrido-2,6-dicarboxylate platform whose H-bonding and p-stacking abilities were exploited to keep the antenna and the PPGs in close proximity. The different electron acceptor ability of the PPGs led to dyads with wildly different behaviors. Whilst the MDNI and MNI dyads showed poor dark stability or no photo-uncaging ability due to their too high electron accepting character, the mDEAC dyad benefited from optimum redox potentials to promote PeT and slow down charge recombination, resulting in enhanced uncaging quantum yield (Fu=0.38) compared to mDEAC (Fu=0.014). The unique resulted in large 2P photo-sensitivity in the near-infrared window (240 GM at 710 nm).

A dual action coumarin-camptothecin polymer for light responsive drug release and photodynamic therapy.

A light-responsive polymer allowing the controlled release of camptothecin and the generation of reactive oxygen species (ROS) is reported. The polymer was prepared by controlled copolymerisation of water-soluble N,N-dimethyl acrylamide with a bromocoumarin methacrylate monomer. The lipophilic chemotherapy agent camptothecin was caged onto the coumarin unit via a photo-cleavable carbonate ester enabling light-triggered cargo release. The polymer showed good biocompatibility in the dark, and high cancer cell killing activity mediated both by the photo-release of camptothecin and ROS generation.

Riboflavin-Vancomycin Conjugate Enables Simultaneous Antibiotic Photo-Release and Photodynamic Killing against Resistant Gram-Positive Pathogens.

Decades of antibiotic misuse have led to alarming levels of antimicrobial resistance, and the development of alternative diagnostic and therapeutic strategies to delineate and treat infections is a global priority. In particular, the nosocomial, multidrug-resistant "ESKAPE" pathogens such as Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus spp (VRE) urgently require alternative treatments. Here, we developed light-activated molecules based on the conjugation of the FDA-approved photosensitizer riboflavin to the Gram-positive specific ligand vancomycin to enable targeted antimicrobial photodynamic therapy. The riboflavin-vancomycin conjugate proved to be a potent and versatile antibacterial agent, enabling the rapid, light-mediated, killing of MRSA and VRE with no significant off-target effects. The attachment of riboflavin on vancomycin also led to an increase in antibiotic activity against S. aureus and VRE. Simultaneously, we evidenced for the first time that the flavin subunit undergoes an efficient photoinduced bond cleavage reaction to release vancomycin, thereby acting as a photoremovable protecting group with potential applications in drug delivery.

Fluorophore-Tagged Poly-Lysine RAFT Agents: Controlled Synthesis of Trackable Cell-Penetrating Peptide-Polymers.

The conjugation of a fluorophore and a variety of cell-penetrating peptides onto a RAFT agent allowed for the synthesis of polymers of defined sizes with quantifiable cell-uptake. Each peptide-RAFT agent was used to polymerize acrylamide, acrylate, and styrene monomers to form high or low molecular weight polymers (here 50 or 7.5 kDa) with the peptide having no influence on the RAFT agent's control. The incorporation of a single fluorophore per polymer chain allowed cellular analysis of the uptake of the size-specific peptide-polymers via flow cytometry and confocal microscopy. The cell-penetrating peptides had a direct effect on the efficiency of polymer uptake for both high and low molecular weight polymers, demonstrating the versatility of the strategy. These "all-in-one", synthetically accessible RAFT agents allow highly controlled preparation of synthetic peptide-polymer conjugates and subsequent quantification of their delivery into cells.

Solid-Phase Synthesis of s-Tetrazines.

An efficient synthesis of s-tetrazines by solid-phase methods is described. This synthesis route was compatible with different solid-phase resins and linkers and did not require metal catalysts or high temperatures. Monosubstituted tetrazines were routinely synthesized using thiol-promoted chemistry, using dichloromethane as a carbon source, while disubstituted unsymmetrical aryl or alkyl tetrazines were synthesized using readily available nitriles. This efficient approach enabled the synthesis of s-tetrazines in high yields (70-94%), eliminating the classical solution-phase problems of mixtures of symmetrical and unsymmetrical tetrazines, with only a single final purification step required, and paves the way to the rapid synthesis of s-tetrazines with various applications in bioorthogonal chemistry and beyond.

Two-Photon Absorption: An Open Door to the NIR-II Biological Window?

The way in which photons travel through biological tissues and subsequently become scattered or absorbed is a key limitation for traditional optical medical imaging techniques using visible light. In contrast, near-infrared wavelengths, in particular those above 1000 nm, penetrate deeper in tissues and undergo less scattering and cause less photo-damage, which describes the so-called "second biological transparency window". Unfortunately, current dyes and imaging probes have severely limited absorption profiles at such long wavelengths, and molecular engineering of novel NIR-II dyes can be a tedious and unpredictable process, which limits access to this optical window and impedes further developments. Two-photon (2P) absorption not only provides convenient access to this window by doubling the absorption wavelength of dyes, but also increases the possible resolution. This review aims to provide an update on the available 2P instrumentation and 2P luminescent materials available for optical imaging in the NIR-II window.

Design of Photosensitizing Agents for Targeted Antimicrobial Photodynamic Therapy.

Photodynamic inactivation of microorganisms has gained substantial attention due to its unique mode of action, in which pathogens are unable to generate resistance, and due to the fact that it can be applied in a minimally invasive manner. In photodynamic therapy (PDT), a non-toxic photosensitizer (PS) is activated by a specific wavelength of light and generates highly cytotoxic reactive oxygen species (ROS) such as superoxide (O2-, type-I mechanism) or singlet oxygen (1O2*, type-II mechanism). Although it offers many advantages over conventional treatment methods, ROS-mediated microbial killing is often faced with the issues of accessibility, poor selectivity and off-target damage. Thus, several strategies have been employed to develop target-specific antimicrobial PDT (aPDT). This includes conjugation of known PS building-blocks to either non-specific cationic moieties or target-specific antibiotics and antimicrobial peptides, or combining them with targeting nanomaterials. In this review, we summarise these general strategies and related challenges, and highlight recent developments in targeted aPDT.

Fluorescent phosphorus dendrimers excited by two photons: synthesis, two-photon absorption properties and biological uses.

Different types of two-photon absorbing (TPA) fluorophores have been synthesized and specifically functionalized to be incorporated in the structure of phosphorus dendrimers (highly branched macromolecules). The TPA fluorophores were included in the periphery as terminal functions, in the core, or in the branches of the dendrimer structures, respectively. Also the functionalization in two compartments (core and surface, or branches and surface) was achieved. The consequences of the location of the fluorophores on the fluorescence and TPA properties have been studied. Several of these TPA fluorescent dendrimers have water-solubilizing functions as terminal groups, and fluorophores at the core or in the branches. They have been used as fluorescent tools in biology for different purposes, such as tracers for imaging blood vessels of living animals, for determining the phenotype of cells, for deciphering the mechanism of action of anticancer compounds, and for safer photodynamic therapy.

Dual-wavelength efficient two-photon photorelease of glycine by π-extended dipolar coumarins.

Photolabile protecting groups (PPGs) releasing bioactive compounds upon two-photon excitation have emerged as increasingly popular tools to control and study physiological processes. Yet the limited two-photon photosensitivity of many cages is still a critical issue for applications. We herein report the design, synthesis and photophysical study of polarized extended coumarinyl derivatives which show large two-photon sensitivity (up to 440 GM) at two complementary wavelengths in the NIR spectral range. DFT calculations demonstrate that subtle tuning of polarization in the ground-state and confinement of the photo-induced intramolecular charge transfer upon excitation is responsible for enhancing two-photon absorption while maintaining large uncaging efficiency. These findings open a new engineering route towards efficient coumarinyl PPGs.

Tandem Systems for Two-Photon Uncaging of Bioactive Molecules.

Photodeprotection of biological ligands with the combination of photolabile protecting groups (PPGs) and two-photon (2P) excitation techniques offers the possibility to control physiological phenomena with utmost spatial and temporal precision. Yet, designing PPGs with sufficient 2P photosensitivity still remains challenging. Direct enhancement of the 2P absorption properties usually relies on modifications of the PPG structure, which sometimes results in unpredictable and counterproductive effects on the uncaging quantum yield. In that respect, combining known PPGs with a suitable two-photon light-harvesting antenna within a modular architecture offers a promising alternative. In this Minireview, we highlight this versatile strategy, with a discussion of different tandem 2P uncagers, relying on photoinduced electron transfer (PET), triplet- energy transfer (TT-ET), or Förster resonance energy transfer (FRET) sensitization to enhance the 2P sensitivity of PPGs.

Cooperative Veratryle and Nitroindoline Cages for Two-Photon Uncaging in the NIR.

Tandem uncaging systems in which a two-photon absorbing module and a cage moiety, linked via a phosphorous clip, that act together by Förster resonance energy transfer (FRET) have been developed. A library of these compounds, using different linkers and cages (7-nitroindolinyl or nitroveratryl) has been synthesized. The investigation of their uncaging and two-photon absorption properties demonstrates the scope and versatility of the engineering strategy towards efficient two-photon cages and reveals surprising cooperative and topological effects. The interactions between the 2PA module and the caging moiety are found to promote cooperative effects on the 2PA response while additional processes that enhance the uncaging efficiency are operative in well-oriented nitroindoline-derived dyads. These synergic effects combine to lead to record two-photon uncaging cross-section values (i.e., up to 20 GM) for uncaging of carboxylic acids.

Fluorescent periodic mesoporous organosilica nanoparticles dual-functionalized via click chemistry for two-photon photodynamic therapy in cells.

The synthesis of ethenylene-based periodic mesoporous organosilica nanoparticles for two-photon imaging and photodynamic therapy of breast cancer cells is described. A dedicated two-photon absorbing fluorophore possessing four triethoxysilyl groups and having large two-photon absorption in the near IR region, and azidopropyltriethoxysilane were incorporated into the structure. The mesoporous nanoparticles of 100 nm diameter were further functionalized by means of click chemistry with a propargylated fluorescent bromo-quinoline photosensitizer able to generate singlet oxygen. The photophysical properties and two-photon absorption properties of the nanoparticles were investigated evidencing complementary contribution of the two dyes. Both dyes contribute to the two-photon absorption response of the mesoporous nanoparticles while efficient FRET from the two-photon fluorophore to the quinoline sensitizer is observed. The dual-functionalized nanoparticles were incubated with MCF-7 breast cancer cells. Two-photon confocal imaging demonstrated the endocytosis of the nanoparticles within cancer cells. Moreover, brief two-photon irradiation (3 scans of 1.57 s) at 760 nm at high laser power (3 W) was shown to induce 40% of cancer cell death demonstrating the potential of the dual-functionalized mesoporous organosilica nanoparticles for two-photon photodynamic therapy.