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EP-4735536-A1 - POLY-SARCOSINE SOLUBILIZED DYES AND METHODS FOR USING THE SAME

EP4735536A1EP 4735536 A1EP4735536 A1EP 4735536A1EP-4735536-A1

Abstract

Dyes, including tandem dyes, are provided. Aspects of embodiments of the dyes include a fluorophore and a water-solubilizing poly-sarcosine group. Also provided are methods of using the dyes, as well as kits that include such dyes.

Inventors

  • BARTHOLOMEW, GLENN P.

Assignees

  • Becton, Dickinson and Company

Dates

Publication Date
20260506
Application Date
20240612

Claims (15)

  1. 1 . A tandem dye comprising: a donor fluorophore; an acceptor fluorophore; a water-solubilizing poly-sarcosine group.
  2. 2. The tandem dye according to Claim 1 , wherein the water-solubilizing polysarcosine group comprises a polymer having from 2 to 40 residues.
  3. 3. The tandem dye according to Claim 2, wherein the polymer is selected from the group consisting of a poly-sarcosine polymer, an N-modified polysarcosine polymer and a poly-sarcosine-type co-polymer.
  4. 4. The tandem dye according to Claim 3, wherein the polymer is a polysarcosine polymer.
  5. 5. The tandem dye according to any of the preceding claims, wherein at least one of the donor and acceptor fluorophore comprises an organic dye.
  6. 6. The tandem dye according to any of the preceding claims, wherein both the donor fluorophore and acceptor fluorophore comprise an organic dye.
  7. 7. The tandem dye according to any of the preceding claims, wherein at least one of the donor and acceptor fluorophore comprises a pendant watersolubilizing poly-sarcosine group.
  8. 8. The tandem dye according to any of the preceding claims, further comprising a non-conjugated polymeric backbone comprising non-conjugated repeat units, wherein the donor and acceptor fluorophore are linked to the nonconjugated polymeric backbone.
  9. 9. The tandem dye according to Claim 8, wherein the non-conjugated repeat units comprise a plurality of amino acid residues.
  10. 10. The tandem dye according to Claims 8 or 9, wherein the non-conjugated polymeric backbone comprises a water-solubilizing poly-sarcosine group bound thereto.
  11. 1 1 . The tandem dye according to any of Claims 8 to 10, wherein at least one of the donor and acceptor fluorophores is linked to the non-conjugated polymeric backbone by a water-solubilizing poly-sarcosine group.
  12. 12. The tandem dye according to any of Claims 8 to 11 , wherein the nonconjugated polymeric backbone comprises a water-solubilizing poly-sarcosine group.
  13. 13. A labeled specific binding member comprising: a tandem dye according to any of the preceding claims; and a specific binding member.
  14. 14. A method comprising contacting a sample with a labeled specific binding member according to Claim 13.
  15. 15. A kit comprising: a tandem dye according to an of Claims 1 to 12 or a labeled specific binding member according to Claim 13.

Description

POLY-SARCOSINE SOLUBILIZED DYES AND METHODS FOR USING THE SAME CROSS-REFERENCE TO RELATED APPLICATION Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63/524,017 filed June 29, 2023; the disclosure of which application is incorporated herein by reference in its entirety. INTRODUCTIO Fluorescent dyes are compounds which, when irradiated with light of a wavelength which they absorb, emit light of a (usually) different wavelength. Fluorescent dyes find use in a variety of applications in biochemistry, biology and medicine, e.g., in diagnostic kits, in microscopy or in drug screening. Fluorescent dyes are characterized by a number of parameters allowing a user to select a suitable dye depending on the desired purpose. Parameters of interest include the excitation wavelength maximum, the emission wavelength maximum, the Stokes shift, the extinction coefficient, the fluorescence quantum yield and the fluorescence lifetime. Dyes may be selected according to the application of interest in order to, e.g., allow penetration of exciting radiation into biological samples, to minimize background fluorescence and/or to achieve a high signal-to-noise ratio. Molecular recognition involves the specific binding of two molecules. Molecules which have binding specificity for a target biomolecule find use in a variety of research and diagnostic applications, such as the labelling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separations and chromatography. Target biomolecules may be detected by labelling with a fluorescent dye. Polyethylene glycol (PEG) groups have been attached to fluorescent dyes (as well as small molecules, nucleotides, peptides, proteins, liposomes, and nanoparticles) to improve solubility, stability, and pharmacokinetic properties. However, some naive individuals can have pre-existing antibodies that can bind to PEG and induce an immune response (Chen et al., above). Many common methods of synthesizing PEG and other polymers generate polydisperse compositions with broad distributions of molecular weights. Such polydispersity can disadvantageously inhibit control over the properties of the polymers. SUMMARY Dyes, including tandem dyes, are provided. Aspects of embodiments of the dyes include a fluorophore and a water-solubilizing poly-sarcosine group. Also provided are methods of using the dyes, as well as kits that include such dyes. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows chemical structures of polysarcosine, N-modified polysarcosine, and a polysarcosine copolymer. FIG. 2 shows the chemical structures of polysarcosine and methyl-polyethylene glycol. FIG. 3 shows the chemical structures of exemplary cyanine, rhodamine, coumarin, and BODIPY dyes. FIG. 4 shows examples of a dye and a tandem dye that include a polymeric donor chromophore, poly-sarcosine groups, and an antibody. The bottom structure includes an acceptor dye, thereby making the structure a tandem dye. FIG. 5 shows examples of structures having a dye connected to a biomolecule through a linker that includes a poly-sarcosine group. FIG. 6 shows a solid-phase synthesis of poly-sarcosine groups. FIG. 7 shows synthetic options wherein a hydroxyl group attached to a polysarcosine group is converted to different substituted amine groups. FIG. 8 shows different options for the placement of poly-sarcosine groups within a compound. FIG. 9 shows poly-sarcosine groups attached to a polyfluorene or BODIPY chromophore. FIG. 10 shows SSC data for donor-acceptor complexes that used PEGylated dyes and Sarcosine-functionalized dyes covalently attached to hCD4 and used as a cell stain. The sarcosine-substituted dyes provide roughly 10% greater signal. FIG. 11 shows pendant poly-sarcosine groups attached to a BODIPY dye or a cyanine dye. DEFINITIONS "Alkyl" refers to a monoradical, branched or linear, non-cyclic, saturated hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, t-butyl, octyl, decyl, cyclopentyl, and cyclohexyl. In some cases, the alkyl group has 1 to 24 carbon atoms, e.g., 1 to 12, 1 to 6, or 1 to 3. "Alkenyl" refers to a monoradical, branched or linear, non-cyclic hydrocarbonyl group that comprises a carbon-carbon double bond. Exemplary alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl. “Alkynyl" refers to a monoradical, branched or linear, non-cyclic hydrocarbonyl group that comprises a carbon-carbon triple bond. Exemplary alkynyl groups include ethynyl and n-propynyl. "Cycloalkyl” refers to a monoradical, cyclic, saturated hydrocarbon group. Similarly, “cycloalkenyl” refers to a monoradical and cyclic group having carbon-carbon double bond whereas "cycloalkynyl” refers to a monoradical an