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Assignees
Member
Rice UniversityRice University is a leading research university in Houston, Texas, recognized for its emphasis on scientific discovery, innovation, and interdisciplinary collaboration. The institution is committed to academic excellence, impactful research, and community engagement, offering robust undergraduate and graduate programs in engineering, natural sciences, social sciences, humanities, business, and the arts. Rice is distinguished by its history of collaboration with organizations such as NASA, fostering advances in space science, biotechnology, energy research, and artificial intelligence.
Rice University is a leading research university in Houston, Texas, recognized for its emphasis on scientific discovery, innovation, and interdisciplinary collaboration. The institution is committed to academic excellence, impactful research, and community engagement, offering robust undergraduate and graduate programs in engineering, natural sciences, social sciences, humanities, business, and the arts. Rice is distinguished by its history of collaboration with organizations such as NASA, fostering advances in space science, biotechnology, energy research, and artificial intelligence.
Publication Number
US-11913884-B2
Publication Date
2024-02-27
Expiration Date
Abstract
Disclosed herein are photoactivable fluorophores comprising one or more thiocarbonyl groups as well as conjugates and compositions thereof. The present disclosure also provides methods of preparing photoactivatable fluorophores as well as methods of imaging using the photoactivatable fluorophores, conjugates, and compositions of the present disclosure.
Core Innovation
The present disclosure provides photoactivatable fluorophores comprising a fluorophore core structure or an analog thereof and one or more thiocarbonyl groups, as well as conjugates and compositions thereof. The disclosure also provides methods of preparing photoactivatable fluorophores and methods of imaging using the photoactivatable fluorophores, conjugates, and compositions of the present disclosure. In some embodiments, the fluorophore core structure is selected from examples including xanthene, acridine, phenoxazine, phenazine, dioxine, 2,3-dihydro-1H-indene, 2,3-dihydro-1H-inden-1-one, cyanine, pyrene, and other exemplified cores, and at least one thiocarbonyl group may be in conjugation with the core structure.
The background describes limitations of prior photoactivatable fluorophores, namely that many photocaged fluorophores are relatively large and can only be efficiently cleaved by irradiation with ultraviolet light, which dramatically limits their application for biological studies. Alternative approaches have required a light-capturing sensitizer or an expensive multi-photon light source. The disclosure identifies a need for minimally-modified visible light-photoactivatable probes across a broad spectral range.
The disclosure provides a general strategy of single-atom sulfur-for-oxygen substitution (introduction of thiocarbonyl groups) in fluorophores to induce a photoinduced electron transfer (PET)-quenching state that is reversible by photochemical oxidation of the thiocarbonyl group to its oxo derivative, thereby restoring strong fluorescence. The disclosure further provides methods of preparing such photoactivatable fluorophores by converting one or more carbonyl groups into thiocarbonyl groups [procedural detail omitted for safety], and methods of obtaining images of biological material contacted with the photoactivatable fluorophores by irradiating the material to convert thiocarbonyl groups into carbonyl groups and performing fluorescence imaging.
Claims Coverage
Independent claim 1 is directed to a photoactivatable fluorophore; the claim recites multiple core structural embodiments and detailed substituent definitions, and the specification includes claims directed to conjugates and compositions that reference the photoactivatable fluorophore.
Photoactivatable fluorophore comprising one or more thiocarbonyl groups
A photoactivatable fluorophore comprising a fluorophore core structure or an analog thereof and one or more thiocarbonyl groups, provided that the fluorophore core structure or analog thereof is not 2H-chromene or 4a,9,9a,10-tetrahydroacridine.
Fluorophore core structure selection
The fluorophore core structure is exemplified as xanthene, acridine, phenoxazine, phenazine, dioxine, 2,3-dihydro-1H-indene, 2,3-dihydro-1H-inden-1-one, cyanine, pyrene, or an analog of any of these core structures, and additional cores such as 2,3-dihydrophenalene, 2,3-dihydro-1H-phenalen-1-one, 1H-phenalene-1,3(2H)-dione, or 1,8-naphthalimide are exemplified.
Defined structural formulae with X and R substituents
The claim recites multiple alternative structural formulae in which atoms or groups (e.g., X1, X2, X3, X4, X5, X6, X7, X8) and substituents (e.g., R1-R15, Ra, Rb, Rc, Rd, Re, Rf, etc.) are defined with specified allowable classes (hydrogen; halo; alkyl(C≤8); aryl(C≤12); alkoxy; alkylamino; dialkylamino; amido; —C(O)Rh; —C(S)NHRg; formation of arenes or heteroarenes by taking substituents together; and provision for pharmaceutically acceptable salts or tautomers).
Conjugates and compositions incorporating the photoactivatable fluorophore
Conjugates of the formula T-L-E, wherein E is a photoactivatable fluorophore according to the claim, L is a covalent bond or a linker, and T is a targeting moiety; and compositions comprising a photoactivatable fluorophore or conjugate and an excipient are recited.
Claim 1 covers photoactivatable fluorophores defined by the presence of one or more thiocarbonyl groups on a wide range of fluorophore core structures with detailed substituent and structural definitions; related inventive aspects include defined structural alternatives, recited conjugates (T-L-E) using the fluorophores, and compositions containing such conjugates or fluorophores.
Stated Advantages
Minimal molecular modification: the strategy requires a single-atom sulfur-for-oxygen substitution, representing a minimal change to fluorophore structures.
High efficiency of unmasking using visible light: thio-caged fluorophores can be converted to their strongly fluorescent oxo analogues upon irradiation with visible light in air.
Ease of design and synthesis: the disclosure states ease of design and synthesis as a significant advantage over certain prior photoactivatable dyes.
Superior optical properties and broad spectral applicability: thio-caged fluorophores exhibit advantageous optical properties and can be prepared across a broad spectral range, enabling applications such as super-resolution imaging.
Documented Applications
Imaging biological material contacted with a photoactivatable fluorophore by irradiating the material to convert thiocarbonyl groups into carbonyl groups and performing fluorescence imaging.
Super-resolution imaging techniques, explicitly including Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM).
Imaging of biological targets including proteins, lipids, RNA, DNA, and small molecules, and labeling components of cells such as organelles (for example, lipid droplets in adipocytes).
Use in vivo, ex vivo, or in vitro imaging, including imaging performed on patients and tissue samples as described in the specification.
Conjugation to targeting moieties to increase imaging selectivity, where targeting moieties include nucleic acid sequences, peptides, proteins, antibodies, antibody fragments, small molecules, or functional groups that target subcellular organelles.
Formulations and compositions for administration and delivery of the photoactivatable fluorophores or conjugates, including compositions comprising an excipient and routes for formulation as described in the specification.
Diagnostic use: methods comprising fluorescence imaging using the disclosed photoactivatable fluorophores to diagnose a disease or disorder are described.
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