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-10994029-B2
Publication Date
2021-05-04
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
Embodiments of conformationally restricted cyanine fluorophores, as well as methods of making and using these compounds, are disclosed. The fluorophores have a chemical structure according to Formula I, including variations such as Formula IA, IB, IC, ID, II, and III, with substituents R1-R11, Y1, and Y2 that can include hydrogen, deuterium, alkyl, heteroalkyl, sulfonate, amino, conjugatable moieties, targeting agents, or drugs.
The conformational restriction is achieved by modification of the cyanine compound such that the molecule loses flexibility in the central conjugated polymethine bridge region, resulting in improved spectroscopic properties such as increased fluorescence quantum yield, extended fluorescence lifetimes, and red-shifted absorption and emission maxima relative to unrestricted cyanines. Methods for making these conformationally restricted pentamethine and heptamethine cyanine fluorophores involve key synthetic steps including chemoselective olefin metathesis, intramolecular Michael addition, and cyclization cascades.
The problem being addressed relates to the limitation of existing far-red and near-infrared cyanine fluorophores, which typically exhibit modest fluorescence quantum yields due to excited-state trans- to cis-polyene rotation deactivation. Although synthetic strategies exist for conformationally restricting trimethine cyanines in the green spectral region by installing fused rings, these strategies are not applicable to far-red cyanines due to the synthetic complexity involved. Consequently, prior to the present disclosure, no suitable synthetic methods or compounds providing conformational restriction in far-red and near-IR cyanines were known.
Claims Coverage
The patent contains multiple independent claims focused on compounds, pharmaceutical compositions, synthetic methods, and methods of use comprising conformationally restricted cyanine fluorophores.
compound having a chemical structure according to Formula I
A compound having a chemical structure according to Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, comprising a cyanine fluorophore with defined substituents R1-R11, Y1, and Y2, where A is a specific core structure ensuring conformational restriction.
conformational restriction in cyanine fluorophores
The compounds have sulfonate, carboxylate, or groups comprising a conjugatable moiety, targeting agent, or drug at positions R3 and/or R6, and Y1 and Y2 are often C(Rc)2 groups where Rc can be alkyl or conjugatable moieties. This structural design provides conformational restriction and tailored functionality.
pharmaceutical composition including conformationally restricted cyanine fluorophore
Pharmaceutical compositions comprising a compound according to Formula I and a pharmaceutically acceptable carrier suitable for administration.
method for making conformationally restricted pentamethine cyanine fluorophores
A method comprising: combining a compound according to Formula IV with 3-buten-1-yl trifluoromethanesulfonate to produce compound V; combining compounds V and VI with N-((1E,3Z)-3-(phenylamino)propo-1-en-1-yl)aniline to form compound VII; reacting compound VII with 3,3-dimethoxy-1-propene in the presence of a ruthenium catalyst to form compound VIII; and cyclizing compound VIII with acid or BBr3 to produce compound IX.
method for making conformationally restricted heptamethine cyanine fluorophores
A method comprising: combining compound IV with 3-buten-1-yl trifluoromethanesulfonate to form compound V; combining compound X with 3-buten-1-yl trifluoromethanesulfonate to form compound XI; combining compounds V and XI with (1E,4E)-1,5-bis(dimethylamino)-penta-1,4-dien-3-one to form compound XII; reacting compound XII with 3,3-dimethoxy-1-propene and a ruthenium catalyst to form compound XIII; and treating compound XIII with acid to give compound XIV.
functional group modifications of conformationally restricted cyanine fluorophores
Further synthetic steps include triflation of compound XIV, followed by palladium-catalyzed coupling with boronic acids or amination with amines to provide diverse substituents; subsequent N- to O-rearrangements under basic conditions enable conjugation to biomolecules, targeting agents, or drugs.
method of using conformationally restricted cyanine fluorophores with targeting agents
Methods of combining the compound with a sample comprising a target binding a targeting agent included in the compound, then irradiating with visible or near-infrared light to produce and detect fluorescence for imaging.
method for detecting reactive oxygen species (ROS)
Combining the compound with a reducing agent to form a reduced compound, contacting a sample where ROS oxidizes it back to fluorescent form, irradiating with light, and detecting fluorescence indicating ROS presence.
The independent claims cover chemical compounds with conformationally restricted cyanine fluorophores, pharmaceutical compositions thereof, synthetic routes for preparing these compounds, and methods of use including imaging targets and detecting reactive oxygen species. The inventive features focus on the molecular structure conferring conformational restriction, improved fluorescence properties, synthetic methods involving cyclization cascades and olefin metathesis, and functional uses with targeting agents or as sensors.
Stated Advantages
Improved quantum yields and extended fluorescence lifetimes relative to corresponding non-restricted cyanines, with increases up to at least 5 times.
Red-shifted absorption and emission maxima compared to non-restricted cyanines by 10-50 nm.
Superior recovery from hydride reduction enabling efficient photoactivation without requiring high thiol or deoxygenated buffers.
Enhanced fluorescence stability across different solvents and insensitivity to temperature variations, enabling better fluorescence lifetime imaging microscopy (FLIM).
Resistance to polyene-heteroatom adduct formation, providing stability suitable for chemical and single-molecule imaging contexts.
Facilitation of high-quality single-molecule localization microscopy with excellent photon counts and localization precision.
Documented Applications
Imaging applications that include in vitro, ex vivo, and in vivo localization and tracking of cellular components using fluorescence microscopy.
Targeted imaging of biological samples comprising targets such as tumor biomarkers using compounds conjugated with targeting agents like antibodies or phalloidin.
Single-molecule localization microscopy techniques such as PALM, dSTORM, and TRABI-Biplane imaging for super-resolution visualization.
Fluorescence-guided tumor visualization and surgical excision via systemic or local administration of the compounds followed by targeted irradiation with far-red or near-infrared light.
Detection and measurement of reactive oxygen species (ROS) in samples by reduction and subsequent fluorescence reactivation upon ROS oxidation.
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