We report the use of bioorthogonal reactions as an original strategy in photodynamic therapyt oa chieve conditional phototoxicity and specific subcellular localization simultaneously.O ur novel halogenated BODIPY-tetrazine probes only become efficient photosensitizers (F D % 0.50) through an intracellular inverse-electron-demand Diels-Alder reaction with as uitable dienophile.A binitio computations reveal an activation-dependent change in decaychannels that controls 1 O 2 generation. Our bioorthogonal approach also enables spatial control. As aproof-of-concept, we demonstrate the feasibility of the selective activation of our dormant photosensitizer in cellular nuclei, causing cancer cell death upon irradiation. Thus,o ur dual biorthogonal, activatable photosensitizers open new venues to combat current limitations of photodynamic therapy.Photodynamic therapy (PDT) is aw ell-established medical treatment for several maladies [1] that is based on the direct or indirect generation of cytotoxic reactive oxygen species (ROS) under exposure of ap hotosensitizer (PS) to light. [2] Light-controlled treatments,s uch as PDT,s hould overcome the medicinal challenge of side effects.However,the reality is that classical PSs have dark toxicity and al ack of selectivity, which causes undesirable adverse effects.Optogenetics [3] and photopharmacology [4] hold the promise to solve off-target issues,a lthough, unlike PDT,t hey are not yet at the stage of clinical development. Therefore,t here is ac lear need for improved PS designs that increase selectivity and minimize collateral injury to healthy tissue.A long these lines,s everal groups have demonstrated the potential of second-generation PSs,w hose therapeutic properties had been improved by either controllable activation [5] or specific delivery of the PSs. [6] Herein, we envision at hird-generation of PSs that combine both modulation of singlet oxygen ( 1 O 2 )production and controlled localization through ab ioorthogonal inverse-electron-demand Diels-Alder (iEDDA) tetrazine cycloaddition reaction.Since the discovery of the suitability of tetrazines for the bioorthoonal ligation pool in 2008, [7] this approach has become ap owerful bioconjugation method for imaging, [8] detection, [9] diagnostics, [10] and bioorthogonal release reactions. [11] However, its potential has not yet been exploited in PDT.Indeed, to the best of our knowledge,n one of the bioorthogonal reactions has ad irect application in the modulation of ROSproduction. Therefore,taking advantage of such ar eaction and inspired by the pioneering work of Weissleder and co-workers on superbright bioorthogonal boron dipyrromethene (BODIPY)/tetrazine turn-on probes, [12] we explore novel bioorthogonal turn-on probes for the controllable generation of 1 O 2 from the straightforward functionalization of the 4,4-difluoro-4-bora-3a,4a-diazas-indacene core. [13] BODIPY-based PSs have excellent photophysical properties, [14] and the quantum yield of their triplet excited state could be enhanced by the incorporation of ...
Photodynamic therapy (PDT) leads to cancer remission via the production of cytotoxic species under photosensitizer (PS) irradiation. However,c oncomitantd amage and dark toxicity can both hinder itsu se. With this in mind, we have implemented av ersatile peptide-based platform of bioorthogonally activatable BODIPY-tetrazine PSs. Confocal microscopy and phototoxicity studies demonstrated that the incorporation of the PS, as ab ifunctionalm odule, into a peptidee nabled spatiala nd conditional controlo fs inglet oxygen (1 O 2)g eneration.C omparing subcellular distribution, PS confined in the cytoplasmic membrane achieved the highest toxicities (IC 50 = 0.096 AE 0.003 mm)a fter activation and without apparent dark toxicity.O ur tunable approach will inspiren ovel probes towards smart PDT.
Photoswitchable oligonucleotides can determine specific biological outcomes by light-induced conformational changes. In particular, artificial probes activated by visible-light irradiation are highly desired in biological applications. Here, we report two novel types of visible-light photoswitchable peptide nucleic acids (PNAs) based on the molecular transducers: hemithioindigo and tetra-ortho-fluoroazobenzene. Our study reveals that the tetra-ortho-fluoroazobenzene–PNA conjugates have promising properties (fast reversible isomerization, exceptional thermal stability, high isomer conversions and sensitivity to visible-light irradiation) as reversible modulators to control oligonucleotide hybridization in biological contexts. Furthermore, we verified that this switchable modification delivers a slightly different hybridization behavior in the PNA. Thus, both melting experiments and strand-displacement assays showed that in all the cases the trans-isomer is the one with superior binding affinities. Alternative versions, inspired by our first compounds here reported, may find applications in different fields such as chemical biology, nanotechnology and materials science.
Ribosomes of Trypanosoma bruceL a parasitic, flagellated protozoan (order Kinetoplastida), were identified on sucrose density gradients by their radioactively labeled nascent peptides. Ultraviolet absorption revealed only cytoplasmic ribosomes which served as internal sedimentation markers.Synthesis on cytoplasmic ribosomes was completely inhibited by cycloheximide. In the presence of this antibiotic, nascent peptides were associated with ribosomes of lower sedimentation coefficient than the cytoplasmic ribosomes. Chloramphenicol blocked synthesis on these ribosomes which are probably the mitochondrial ribosomes.These ribosomes differed from the cytoplasmic ribosomes in several ways. Their sedimentation coefficient was about 72S rather than 84S. The stability of the 72S ribosomes was less sensitive to pancreatic ribonuclease and low Mg ÷+ concentrations, dissociating below 0. l mM Mg ÷+. The 72S ribosomes were more sensitive to elevated KCI concentrations, dissociating above 0.25 M.Protein synthetic activity associated with the 725 class of ribosomes was found in trypanosomes grown in rats. Under these conditions no cytochromes or fully active Krebs cycle is present in these cells and respiration is insensitive to cyanide.Mitochondrial ribosomes have been observed in a wide spectrum of organisms ranging from fungi to mammals. These ribosomes differ from their cytoplasmic counterparts in antibiotic sensitivity, monomer stability at various ion concentrations, sedimentation coefficient, and the molecular weights of their RNA species (see reference no. 3 for review). The properties of mitochondrial ribosomes from different organisms differ greatly but two distinct classes seem to emerge. The first class has a sedimentation coefficient in the 70-80S range and contains RNA species of approximately 16S and 23S. This class of mitochondrial ribosomes is found in fungi and protozoa (2,3,6,7,17). The second class, frequently referred to as "miniribosomes," has a sedimentation coefficient in the 50-60S range and RNA species which are approximately 12S and 16S. "'Miniribosomes" have been found in higher animals "from shark to man (11 13, 19). It is noteworthy that the protein synthetic activity of mitochondrial ribosomes, regardless of source, is insensitive to cycloheximide (an inhibitor of cytoplasmic ribosomes) but is sensitive to inhibitors of bacterial protein synthesis
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