Process for making an asymmetric fluorophore
Inventors
ARNATT, CHRISTOPHER • Elliott, John
Assignees
United States Department of Commerce
Publication Number
US-9951271-B2
Publication Date
2018-04-24
Expiration Date
2036-10-14
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Abstract
The present invention relates to a process for making an asymmetric fluorophore. The asymmetric fluorophore is useful as a stain for staining live cells or fixed cell and provides whole-cell staining of such cells.
Core Innovation
The present invention relates to a process for making an asymmetric fluorophore that is useful as a stain for staining live cells or fixed cells and provides whole-cell staining of such cells. The asymmetric fluorophore includes a BODIPY-based, whole-cell stain that can be used directly in live or fixed cells. It is characterized by aqueous solubility, ability to cross cellular membranes, and capability to efficiently stain live and fixed cells under similar conditions.
The asymmetric fluorophore is an activated BODIPY-NHS ester that reacts with primary amines on protein molecules within the cytoplasm, cellular membrane, or cell surface, enabling it to remain inside the cell for long periods, thereby labeling cells under both living and fixed conditions. Whole-cell staining with this fluorophore can be used for cell tracking, counting, morphology, phenotyping, and high content screening.
The invention also provides a process for synthesizing the asymmetric fluorophore by sequentially decarboxylating a starting compound, condensing it with 2-pyrrole aldehyde, difluorinating, hydrolyzing, and succinimating to produce the asymmetric fluorophore. This compound features high quantum yield, small Stokes shift, high photostability, and insensitivity to cellular environment. Its asymmetric structure offers favorable physiochemical properties such as cell membrane permeability and aqueous solubility.
Claims Coverage
The patent presents two main independent claims outlining the process for making the asymmetric fluorophore and the process for whole-cell staining using the asymmetric fluorophore. The inventive features cover the key synthetic steps and the cell staining mechanism.
Process for making an asymmetric fluorophore
The inventive process comprises: 1. decarboxylating a compound of Formula 1 to form Formula 2, 2. condensing the compound of Formula 2 with 2-pyrrole aldehyde to form Formula 3, 3. difluorinating the compound of Formula 3 in presence of boron trifluoride ethyl etherate to form Formula 4, 4. hydrolyzing the compound of Formula 4 to form Formula 5, and 5. succinimating the compound of Formula 5 by coupling with N-hydroxysuccinimide to form the asymmetric fluorophore of Formula 6.
Process for whole-cell staining using the asymmetric fluorophore
The inventive staining process includes: decarboxylating the starting compound to form Formula 2, converting it to the asymmetric fluorophore (Formula 6) by the steps above, combining the asymmetric fluorophore with a cell, and forming a radical by reacting with primary amines in cellular compounds. This radical irreversibly couples to cellular components via an amide linkage, effectively staining the live or fixed cell where the stained cellular compound remains a part of the cell after staining.
The claims cover both the detailed multi-step synthetic process to produce the asymmetric fluorophore and the application of this fluorophore in whole-cell staining through covalent binding to cellular compounds, enabling effective fluorescent labeling of live and fixed cells.
Stated Advantages
The asymmetric fluorophore provides efficient whole-cell staining for both live and fixed cells under similar staining conditions.
It exhibits high quantum yield, sharp fluorescence peak, high photostability, and insensitivity to the cellular staining environment.
The fluorophore is non-toxic at effective concentrations and does not cause cytotoxicity during prolonged exposure.
Its asymmetrical molecular structure offers improved aqueous solubility and cellular membrane permeability.
It can be used to stain cells without requiring permeabilization and is compatible with monitoring live cell dynamics over extended periods.
The staining is persistent, allowing cell tracking and phenotyping with low background fluorescence and selective staining of intracellular amines.
Compared to standards like CFSE and FNHS, it provides brighter staining and more selective intracellular labeling.
Documented Applications
Whole-cell fluorescent staining to determine cell count, morphology, or phenotype in live or fixed cells.
Cell tracking and monitoring dynamic changes in live cells over extended periods.
Use in high content screening (HCS) and quantitative fluorescence microscopy for cellular analysis.
Fluorescent study of cells for labeling and imaging in biological research.
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