Class of stable heptamethine cyanine fluorophores and biomedical applications thereof
Inventors
Schnermann, Martin John • Nani, Roger Rauhauser • Shaum, James Blaine
Assignees
US Department of Health and Human Services
Publication Number
US-10876003-B2
Publication Date
2020-12-29
Expiration Date
2034-11-05
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Abstract
Embodiments of C4′-alkyl-ether heptamethine cyanine fluorophores according to general formula I, and pharmaceutically acceptable salts thereof, are disclosed. Methods of making and using the C4′-alkyl-ether heptamethine cyanine fluorophores also are disclosed.
Core Innovation
This disclosure concerns a synthetic method for making cyanine fluorophores, particularly stable heptamethine cyanine (Cy7) fluorophores, embodiments of Cy7 fluorophores made by the disclosed method and pharmaceutical salts thereof, and methods of using the Cy7 fluorophores. Embodiments of the disclosed Cy7 fluorophores have a structure according to formula I, which includes C4′-alkyl-ether heptamethine cyanine fluorophores with substituents that provide useful optical and chemical properties for near-infrared imaging.
The problem being solved is that many near-infrared heptamethine cyanines suffer from chemical stability issues at the C4′ position and challenging synthesis. Standard synthetic approaches such as C4′-chloride exchange fail with alkoxide nucleophiles due to poor kinetics and side reactions, leading to scarcity of C4′-O-alkyl ether cyanines that are desirable for stability and bioconjugation. There is a need for stable, near-IR heptamethine cyanine fluorophores and effective synthetic methods for making such fluorophores.
Claims Coverage
The claims comprise one independent claim directed to a compound of formula I and additional independent claims directed to methods of using the compound. The main inventive features involve the chemical structure of stable C4′-alkyl-ether heptamethine cyanine fluorophores and their uses in near-infrared fluorescence detection.
Compound comprising C4′-alkyl-ether heptamethine cyanine structure
A compound or pharmaceutically acceptable salt according to formula I, in which the heptamethine cyanine includes C4′-alkyl-ether substituents providing enhanced stability and bioconjugatable functionality. Specific substituents include variations of R2 (e.g., methyl), alkyl groups at R14-R17, and sulfonate or alkyl groups at R4, R7, R9, and R12.
Biomolecule-containing and drug substituents enabling bioconjugation
The fluorophore can include a biomolecule-containing group at the R3 position such as antibodies, peptides, proteins, nucleic acids, carbohydrates, haptens, or receptor ligands. Alternatively, R3 may be a drug or a drug moiety enabling pharmaceutical conjugation and targeting.
Stable C4′-alkyl-ether heptamethine cyanine variants
Specific compounds wherein R4 and R9 are n-propyl or sulfonate groups, R7 and R12 are hydrogen or sulfonate salts, and R3 includes maleimidyl or succinimidyl containing groups enabling further conjugation, including fluorescent properties with absorbance and emission maxima suitable for near-IR imaging.
Method of fluorescence detection using the compound
A method comprising contacting a biological sample with the compound of formula I, irradiating with light in the near-infrared range, and detecting fluorescence, wherein fluorescence indicates presence of the compound in the biological sample. This includes in vivo administration and imaging techniques such as binding to targets like antigens or tumors, and fluorescence-guided surgery or diagnostics.
The claims cover compositions of stable C4′-alkyl-ether heptamethine cyanine fluorophores with varied bioconjugatable groups and their uses in near-IR fluorescence detection and imaging, including in vivo applications targeting biomolecules or drugs, providing improved stability and imaging properties.
Stated Advantages
C4′-alkyl-ether Cy7 fluorophores exhibit superior chemical stability, particularly resistance to thiol nucleophiles, compared to traditional phenol- or thiol-substituted heptamethine cyanines.
They demonstrate optical properties ideal for near-infrared imaging, including absorbance maxima around 750-800 nm, emission maxima around 775-850 nm, small Stokes shifts, and high extinction coefficients.
The synthetic method enables preparation of previously inaccessible C4′-O-alkyl ether cyanines under mild conditions with improved substrate scope and bioconjugation suitability.
The fluorophores facilitate in vivo fluorescence imaging due to improved tissue penetration and reduced autofluorescence in the near-IR range.
Documented Applications
In vitro, in vivo, or ex vivo fluorescence imaging of biological samples for detection of biomolecules or drugs using near-infrared fluorescence.
Fluorescence-guided surgery, particularly cancer surgery, enabling visualization and excision of tumors through near-IR imaging.
Flow cytometry and fluorescence-activated cell sorting (FACS) of cells bound by the fluorophore conjugated to biomolecules.
Clinical diagnostics and monitoring, including detection of tumor antigens, receptors, or nucleic acid sequences using fluorophore conjugates.
Imaging and location tracking of drugs conjugated to the fluorophores within bodily fluids or tissues.
Construction of antibody-fluorophore conjugates for targeted imaging, such as conjugates of panitumumab for EGFR-expressing tumors and folate conjugates for folate receptor-expressing cells.
Use in nucleic acid or protein fluorescent tagging during sequencing, immunoassays, or other molecular biology techniques.
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