Conformational restriction of cyanine fluorophores in far-red and near-IR range
Inventors
Schnermann, Martin J. • Michie, Megan S.
Assignees
US Department of Health and Human Services
Publication Number
US-11707537-B2
Publication Date
2023-07-25
Expiration Date
2038-08-24
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Abstract
Conformationally restricted cyanine fluorophores, as well as methods of making and using the compounds, are described. The conformationally restricted cyanine fluorophores have a chemical structure according to Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof: wherein A is and wherein each “*” designates an attachment point of A.
Core Innovation
Conformationally restricted cyanine fluorophores and methods of making and using these compounds are described. The compounds have a chemical structure according to Formula I or stereoisomers or pharmaceutically acceptable salts thereof, wherein A is a specified conformationally restricting moiety attached to the polymethine bridge of the cyanine structure. The substituents R1-R11 and Y1, Y2 include hydrogen, deuterium, alkyl, heteroalkyl, various functional groups, or groups comprising conjugatable moieties, targeting agents, or drugs.
The background identifies that single molecule localization microscopy (SMLM) techniques such as PALM and dSTORM require fluorophores with high photon yields in the on state and low background fluorescence in the off state. While trimethine cyanines emitting in the green region have had their photoisomerization pathway obstructed via fused ring systems to improve quantum yields, extending this synthetic strategy to far-red cyanines (pentamethine and heptamethine emitting) is challenging due to the complexity of the fused ring system required. Existing far-red fluorophores like Cy5 and Alexa 647 have modest quantum yields below 0.2 in aqueous solutions, with excited state deactivation via trans- to cis-polyene rotation limiting brightness.
The invention solves the problem by providing conformationally restricted cyanine fluorophores in the far-red and near-infrared range with improved quantum yields, extended fluorescence lifetimes, and red-shifted absorption/emission spectra compared to non-restricted cyanines. Methods of making these restricted cyanines involve synthetic strategies including cyclization cascades of dialdehyde precursors accessed through chemoselective olefin metathesis, intramolecular Michael addition, and dihydropyran ring-forming cascades. The compounds are applicable to imaging, including methods combining the compounds with targets for visualization using light in the visible or near-infrared range.
Claims Coverage
The patent includes multiple independent claims directed to compounds of a defined chemical structure (Formula I) and methods of synthesis and use. The main inventive features cover conformational restriction in cyanine fluorophores, incorporation of targeting agents or drugs, and methods for synthesis and imaging applications.
Conformationally restricted cyanine fluorophores with defined chemical structure
The compounds have a chemical structure according to Formula I with a conformationally restricting moiety A joined to the polymethine bridge, with specific substituents R1-R11 and Y1, Y2 which may include H, deuterium, alkyl, heteroalkyl, sulfonate, amino groups, conjugatable moieties, targeting agents, or drugs. The restriction leads to improved photophysical properties in the far-red and near-infrared spectrum.
Conjugation to targeting agents or drugs at specified substituents
At least one substituent of R3, R6, R9-R11, Y1, or Y2 comprises a group containing a targeting agent (e.g., antibodies, ligands, peptides, nucleic acid strands) or a drug moiety, which can be conjugated via groups such as carboxylates, amides, or alkyl linkers, enabling specific binding and biomedical applications.
Methods of synthesis involving cyclization cascades and olefin metathesis
Methods for making the compounds include combining intermediates via cross-metathesis with 3-buten-1-yl trifluoromethanesulfonate, formation of cyanine fluorophores through condensation with bis vinylogous amides, cyclization reactions in acid or boron tribromide, and functional group modifications such as triflation, palladium-catalyzed coupling, or amination.
Methods of using the compounds for imaging a target in samples and subjects
The compounds bearing targeting agents are combined with samples comprising a target, and the target is imaged by visualizing fluorescence after irradiation with light in the visible, far-red, or near-infrared range. The methods may include prior reduction of the compound and are applicable in vitro, ex vivo, or in vivo, including imaging and excision of tumors in subjects.
Methods of detecting reactive oxygen species (ROS) using reduced compounds
The compounds are combined with a reducing agent to form a reduced species, which upon contact with ROS is oxidized back to the fluorescent cyanine, enabling detection by fluorescence emission following irradiation, thus providing a method to detect the presence of reactive oxygen species in samples.
The claims cover conformationally restricted cyanine fluorophores with defined chemical structures incorporating targeting agents or drugs, efficient synthetic routes for pentamethine and heptamethine cyanines, and methods for use in fluorescence imaging and ROS detection, demonstrating a comprehensive inventive scope over composition, synthesis, and application.
Stated Advantages
Improved quantum yields and extended fluorescence lifetimes relative to corresponding non-restricted cyanine fluorophores.
Red-shifted absorption and emission maxima by at least 10-30 nm relative to non-restricted cyanines.
Enhanced fluorescence recovery after hydride reduction, enabling PALM-like SMLM with high photon counts without requiring high thiol, deoxygenated buffer.
Improved labeling properties including decreased aggregation and better antibody labeling due to inclusion of sulfonate groups.
Cell permeability in some embodiments and suitability for live-cell localization and tracking.
Documented Applications
Use in single molecule localization microscopy (SMLM) including PALM and dSTORM techniques for imaging cellular components with molecular resolution.
Fluorescence-based imaging of targets in vitro, ex vivo, and in vivo, including visualization of tissue samples, biological fluids, and tumors in subjects.
Fluorescence-guided tumor visualization and excision using targeted irradiation in the far-red or near-infrared range.
Fluorescent labeling of cellular components using targeting agents such as phalloidin conjugates to bind F-actin for cellular structure visualization.
Detection of reactive oxygen species in biological samples via fluorescence activation of oxidized cyanine fluorophores from reduced precursors.
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