Poloxamer stabilizing reagent

Abstract

This specification provides biotechnology reagents containing poloxamer excipients. In particular, haloalkane-conjugated fluorophores are provided in solutions or lyophilized formulations containing poloxamer, enabling storage, accurate distribution / measurement, reconstitution, and addition to complex biological samples without the need for organic solvents.

Description

[Technical Field]

[0001] Cross-reference of related applications This invention claims priority under U.S. Provisional Patent Application No. 63 / 495,690, filed on 12 April 2023, which is incorporated herein by reference in its entirety.

[0002] This specification provides biotechnology reagents containing poloxamer excipients. In particular, haloalkane-conjugated fluorophores are provided in solutions or lyophilized formulations containing poloxamer, enabling storage, accurate distribution / measurement, reconstitution, and addition to complex biological samples without the need for organic solvents. [Background technology]

[0003] HALOTAG is a modified haloalkane dehalogenase designed to covalently bind to haloalkane (e.g., chloroalkane) ligands (see Los et al. ACS Chem. Biol. 2008, 3, 6, 373-382; the whole is incorporated by reference). HALOTAG ligands need to be easily and non-toxicly stored, reconstituted, and added to various systems (e.g., cells, animals). Currently, HALOTAG ligands are supplied as solids or DMSO solutions. Although widely used, these formulations have problems with stability, storage, accurate distribution (especially when trace amounts are required), and the need for organic solvents. This can be problematic for biological systems, particularly in animal models where amplification occurs. What is needed now is a lyophilized formulation of HALOTAG ligand that is highly stable, easily reconstituted in aqueous buffers across a wide range of concentrations, and can be added to everything from simple biological samples to animal model systems without the need for organic solvents. [Overview of the project]

[0004] This specification provides biotechnology reagents containing poloxamer excipients. In particular, haloalkane-conjugated fluorophores are provided in solutions or lyophilized formulations containing poloxamer, enabling storage, accurate distribution / measurement, reconstitution, and addition to complex biological samples without the need for organic solvents.

[0005] In some embodiments, provided herein are (a) formula R-linker-AX (wherein R is a low molecular weight fluorophore, the linker is a polyatomic linear or branched chain, and A is (CH2) 4-20 (b) A composition comprising a compound containing (X is a halogen) and a poloxamer polymer.

[0006] In some embodiments, the fluorophores are low molecular weight (e.g., molecular weight less than 3,000 daltons, <2,500 daltons, <2,000 daltons, <1,500 daltons, <1,000 daltons, <900 daltons, <800 daltons, <700 daltons, <600 daltons). In certain embodiments, the fluorophores are hydrophobic low molecular weight with low solubility in aqueous solutions. In some embodiments, the fluorophores (e.g., bound to a haloalkane) dissolve in aqueous solutions at concentrations of 1 mM or less (e.g., 1 mM, 800 μM, 600 μM, 400 μM, 200 μM, 100 μM, 75 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, or less) in the absence of poloxamers. In some embodiments, fluorophores (e.g., bound to haloalkanes) dissolve in aqueous solutions at concentrations of 200 μM or less (e.g., 200 μM, 100 μM, 75 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, or less) when poloxamers are absent. In some embodiments, fluorophores (e.g., bound to haloalkanes) dissolve in aqueous solutions at concentrations of 2 μM or less (e.g., 2 μM, 1 μM, 0.75 μM, 0.50 μM, 0.1 μM, or less) when poloxamers are absent. In some embodiments, in the presence of poloxamer, fluorophores (e.g., bound to haloalkanes) dissolve in aqueous solutions at concentrations of 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 500 μM, 750 μM, 1 mM, 1.5 mM, 2 mM, or higher, or any range in between.In some embodiments, particularly with respect to fluorophores with low solubility (e.g., bound to haloalkanes), the compound dissolves in aqueous solution at a concentration of at least 1 nM (e.g., 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 500 μM, 750 μM, 1 mM, 1.5 mM, 2 mM, or higher, or any range of concentrations in between) in the presence of poloxamer.

[0007] In some embodiments, the fluorophore is rhodol or rhodamine dye (Beija et al. Chem. Soc. Rev., 2009, 38, 2410-2433; the whole is incorporated by reference), or a variant or derivative thereof. In some embodiments, rhodol or rhodamine dye is [ka] [ka] Selected from. In some embodiments, the fluorophore is rhodamine.

[0008] In some embodiments, the linker is a polyatomic linear or branched chain containing C, N, S, or O, and optionally includes one or more ring structures. In some embodiments, the linker includes a cleavable moiety. In some embodiments, the cleavable moiety is selected from allyl heteroatom groups and propargyl heteroatom groups.

[0009] In some embodiments, the poloxamer is selected from poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, and poloxamer 407. In some embodiments, the poloxamer is poloxamer 407.

[0010] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a protein, or any combination thereof. In some embodiments, the buffer is selected from phosphate buffer, tricine, and 2-(N-morpholino)ethanesulfonic acid. In some embodiments, the surfactant is selected from polysorbate 20, polysorbate 40, and polysorbate 80. In some embodiments, the reducing agent is selected from thiourea and 6-aza-2-thiothymine. In some embodiments, the salt is selected from sodium chloride and sodium phosphate. In some embodiments, the radical scavenger is selected from ascorbic acid and sodium ascorbate. In some embodiments, the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexane-tetraacetic acid. In some embodiments, the protein is selected from bovine serum albumin, gelatin, and the polypeptide fraction of highly purified porcine skin collagen.

[0011] In some embodiments, the composition is in the form of a lyophilized powder, a cake, or a plastic film.

[0012] In some embodiments, the composition is a solution. In some embodiments, poloxamer is present in the solution at a concentration of 0.1–20% (e.g., 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any value or range in between (e.g., 1–10%, 2.5–9%)).

[0013] In some embodiments, the haloalkane-bonded fluorophore is stabilized against thermal decomposition, chemical decomposition, photo-induced decomposition, or any combination thereof (for example, compared to the same haloalkane-bonded fluorophore without the poloxamer).

[0014] In some embodiments, provided herein is a linker of the formula R-linker-AX (wherein R is a low molecular weight fluorophore, linker is a polyatomic linear or branched chain, and A is (CH2) 4-20A method for preserving a compound (where X is a halide), the method comprising contacting the compound with poloxamer. In some embodiments, contacting the compound with poloxamer comprises dissolving the compound in an organic solvent to form a first solution, and mixing the first solution with poloxamer to form a mixture. In some embodiments, the mixing step comprises dissolving the poloxamer in a second solution, and mixing the second solution with the first solution. In some embodiments, the method further comprises contacting a solid substrate with the mixture. In some embodiments, the solid substrate is a plate or surface made of glass, metal, or plastic. In some embodiments, the solid substrate is paper or a fiber matrix. In some embodiments, the method comprises drying the mixture. In some embodiments, the drying step comprises freeze-drying, air-drying, drying by a rotary evaporator, or vacuum drying. In some embodiments, drying is carried out in an inert atmosphere at ambient temperature. In some embodiments, drying is carried out at a temperature of approximately -80°C to approximately 70°C (e.g., -80°C, -75°C, -70°C, -65°C, -60°C, -55°C, -50°C, -45°C, -40°C, -35°C, -30°C, -25°C, -20°C, -15°C, -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or a range in between). [Brief explanation of the drawing]

[0015] [Figure 1] A graph showing the change in purity over time at varying temperatures for a reconstituted lyophilized composition containing a chloroalkyl-fluorophore compound and a poloxamer 407 polymer, for stability testing. [Figure 2] Graphs and figures showing the results of functional tests of reconstituted lyophilized compositions containing chloroalkyl-fluorophore compounds and poloxamer 407 polymers compared to standard solid-state chloroalkyl-fluorophore compounds. [Figure 3]Figure A shows a comparison of the results of 100 μL DPBS reconstitution between 9-mg and 3-mg p-407 formulations in 100 nmol JF-552 (left vial: 3-mg p-407 formulation reconstituted with 100 μL DPBS for 4 hours, showing a turbid suspension. Right vial: 9-mg p-407 formulation reconstituted with 100 μL DPBS for 4 hours, maintaining a clear solution). Figure B shows a comparison of the results of 100 μL DPBS reconstitution between 9-mg and 3-mg p-407 formulations in 100 nmol JF-669 (left vial: 3-mg p-407 formulation reconstituted with 100 μL DPBS, yielding only a turbid suspension. Right vial: 9-mg p-407 formulation reconstituted with 100 μL DPBS, yielding a clear solution). [Figure 4] This graph shows the solubility of dye-chloroalkyl ligand complexes lyophilized in the presence of various excipients and reconstituted in DPBS at pH 7.0. The graph also shows the change in clarity over time in wells containing JF552-chloroalkane or JF669-chloroalkane. Poloxamer, cyclodextrin, or pullulan were used as excipients. Pullulan and cyclodextrin were insoluble and therefore could not achieve the desired clarity. [Figure 5] This shows the solubility of dye-chloroalkyl ligand complexes lyophilized in the presence of poloxamer and reconstituted in DPBS at pH 7.0, physiological saline, Tris at pH 8.0, bicarbonate at pH 9.5, or citrate at pH 6.0. The graph shows the change in clarity over time in each well containing the poloxamer-containing JF669-chloroalkyl compound, reconstituted in DPBS at pH 7.0, physiological saline, Tris at pH 8.0, bicarbonate at pH 9.5, or citrate at pH 6.0. [Figure 6] This study analyzes the stability of dye-chloroalkyl ligand complexes that were freeze-dried in the presence of various excipients and reconstituted in DPBS at pH 7.0. [Figure 7]These are fluorescence images of fluorophore-HaloTag® ligand prepared in 2.5% poloxamer 407 (1 nmol of ligand per vial), lyophilized, and reconstituted. Each sample is excited and emits light at different wavelengths in the UV / VIS spectrum. The lyophilized ligand was resuspended in 1 mL of cell medium to prepare a 1 μM 5x working concentration solution, which was then added to cells at a final concentration of 200 nM. The upper panel shows the "signal" of cells that stably express HaloTag® and are localized in the nucleus, while the lower panel shows the "background" of parental cells that do not express HaloTag® but are stained with HaloTag® ligand. [Modes for carrying out the invention]

[0016] definition Methods and materials similar to or equivalent to those described herein may be used, but several preferred methods, compositions, apparatus, and materials are described herein. However, prior to describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific molecules, compositions, methods, or protocols described herein, as these may vary according to conventional experimentation and optimization. It should also be understood that the terminology used in the specification is for the purpose of describing specific embodiments or embodiments and is not intended to limit the scope of the embodiments described herein.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field to which this invention pertains. In case of any conflict, however, this specification, including its definitions, shall prevail. Accordingly, in relation to the embodiments described herein, the following definitions apply:

[0018] In this specification, the term “polymer” refers to an organic compound in which two or more repeating units are covalently bonded within a chain that may be linear or branched. Generally, polymers consist of one or more repeating units that are covalently linked to form a linear main chain. The repeating units may be identical or different. Thus, a -AAAA- type structure in which A is a repeating unit is a polymer and is also known as a homogeneous polymer. -ABAB- or -AAABAAAB- type structures (in which A and B are repeating units) are also polymers and are sometimes referred to as copolymers. In this specification, the term “polymer” explicitly includes chains consisting of only two repeating units, such as disaccharides, and also includes chains consisting of more repeating units, such as oligosaccharides and polysaccharides. The term “polymer” also includes non-carbohydrate polymers such as synthetic polymers (and oligomers consisting of only two monomer units). In some embodiments, the polymer (e.g., polysaccharide) and oligomer (e.g., oligosaccharide) are limited to a predetermined length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 750, 1000, or more, or in a range between these (e.g., 2 to 10, 5 to 25, 10 to 50, over 100, etc.)).

[0019] In this specification, the term "poloxamer" refers to a nonionic triblock copolymer in which a central hydrophobic chain, polyoxypropylene (poly(propylene oxide)), is surrounded on both sides by hydrophilic chains, polyoxyethylene (poly(ethylene oxide)). Poloxamers are, [ka] It has the general structure shown above, where a=2-130 and b=15-67. Specific poloxamers are designated with a three-digit number ("poloxamer 407" or "P407"). Multiplying the first two digits by 100 gives the approximate molecular weight of the polyoxypropylene core, and multiplying the last digit by 10 gives the polyoxyethylene content (for example, P407 = a poloxamer with a polyoxypropylene molecular weight of 4000 g / mol and a polyoxyethylene content of 70%). Poloxamers are also known by the trade names PLURONIC®, KOLLIPHOR, and SYNPERONIC.

[0020] In this specification and in the accompanying claims, the singular forms "a," "an," and "the" refer to multiple objects unless the context explicitly indicates otherwise. For example, a reference to "polymer" refers to one or more polymers and their equivalents known to those skilled in the art.

[0021] Where used herein, the term “contains” and its linguistic variations indicate the presence of described features, elements, method steps, etc., without excluding the presence of additional features, elements, method steps, etc. Conversely, the term “consistes of” and its linguistic variations indicate the presence of described features, elements, method steps, etc., excluding undescribed features, elements, method steps, etc., except for impurities that are usually associated with them. The phrase “essentially consists of” means, in addition to the listed features(plural), elements(plural), method steps(plural), etc., any additional features(plural), elements(plural), method steps(plural), etc., that do not substantially affect the fundamental properties of the composition, system, or method. Many embodiments herein are described using the non-limiting expression “contains.” Such embodiments encompass multiple embodiments of the closed form “consistes of” and / or “essentially consists of,” which may alternatively be claimed or described using such wording.

[0022] As used herein, the term "low molecular weight" refers to compounds having a low molecular weight (e.g., <3000 daltons, <2500 daltons, <2000 daltons, <1500 daltons, <1000 daltons, <900 daltons, <800 daltons, <700 daltons, <600 daltons) and a size (e.g., length, width, diameter, etc.) of approximately 1 nm. Larger structures, such as peptides, proteins, and nucleic acids, do not qualify as low molecular weight compounds.

[0023] In this specification, the term "fluorophore" refers to a chemically fluorescent moiety that can re-emit light upon photoexcitation (for example, excited by light of a first wavelength (or wavelength range) and emitting light of a second wavelength (or wavelength range)). Fluorophores generally include structures in which multiple aromatic rings are bonded together, or planar or cyclic molecules having numerous π bonds.

[0024] In this specification, the term "rhodamine" refers to a related group of dyes (xanthene derivatives) that constitute a subset of triallylmethane dyes, and their basic chemical structure is as follows: [ka] This includes those with various modifications as illustrated herein.

[0025] This specification provides biotechnology reagents containing poloxamer excipients. In particular, haloalkane-conjugated fluorophores are provided in solutions or lyophilized formulations containing poloxamer, enabling storage, accurate distribution / measurement, reconstitution, and addition to complex biological samples without the need for organic solvents.

[0026] In some embodiments, compositions containing poloxamer can stabilize haloalkane-linked fluorophores and suppress their degradation compared to compositions without poloxamer. In some embodiments, the compositions improve the efficiency of reconstitution of haloalkane-linked fluorophores. In some embodiments, the compositions improve dynamic solubility (for example, compared to compositions without poloxamer and / or paper or fiber matrix or other surfaces).

[0027] In some embodiments, the compounds provided herein are those of the following formula (I). R-Linker-AX, Here, R is a low molecular weight fluorophore and A is (CH2) 4-20 Here, X is a halide, AX is a substrate of the dehalogenase, and the linker is a polyatomic linear or branched chain containing C, N, S, or O, and optionally has one or more cyclic structures.

[0028] In some embodiments, R is a fluorescent low molecular weight moiety. In some embodiments, the fluorescent moiety is a fluorophore. Suitable fluorophores used as the fluorescent moiety herein are stilbazolium derivatives (Marquesa et al. Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling. ChemRxiv. Cambridge: Cambridge Open Engage;2021;the whole is incorporated by reference), xanthene derivatives (e.g., fluorescein, rhodamine, rhodol, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., Cascade Blue), oxazine derivatives (e.g., Nile Red, Nile Blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavin, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA FLUOR (Invitrogen), DYLIGHT FLUOR (Thermo This includes, but is not limited to, those listed below, such as Scientific (Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dye (CYANDYE, LLC), SETAU and SQUARE dye (SETA BioMedicals), QUASAR and CAL FLUOR dye (Biosearch Technologies), and SURELIGHT DYES (APC, RPE, PerCP, hycobilisomes) (Columbia Biosciences).

[0029] In some embodiments, the small molecule fluorophore (R) is a rhodamine dye. In some embodiments, the rhodamine dye is [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula]

[0030] Other rhodamine and / or rhodol dyes are also included within the scope of the embodiments herein.

[0031] In some embodiments, the structure of the fluorophore (R) is [Chemical formula] where Y is C, O or Si, and when Y is C or Si, it is substituted with two CH3 groups. Here, R 1 , R 2 , R 3 , R 4 and R 5 are each independently H or F (e.g., R 1 ~R 5 are all H. R 1 ~R 5 are all F. R 1 and R 2 are F, and R 3 is H. R 1 ~R 3 are F, and R 4 ~R5 H is R 1 and R 2 F is R 3 ~R 5 (For example, is H). In some embodiments, azetidine is further substituted at the 3-position with one or two non-halogen substituents (e.g., CO2H, CH3, F, etc.). In some embodiments, the exemplary compounds described herein are [ka] It has the structure (where R 1ー5 (and Y are defined as described above, and Y and azetidine are optionally substituted as described above, with alternative linkers and AX groups also included in the range).

[0032] In some embodiments, the structure of the fluorophore (R) is [ka] And, Here, Y is C, O, or Si, and if Y is C or Si, it is substituted with two CH3 groups. Here, R 1 , R 2 , R 3 , R 4 and R 5 Each is independently either H or F (for example, R 1 ~R 5 All of them are H. 1 ~R 5 All of them are F. 1 and R 2 F is R 3 H is R 1 ~R 3 F is R 4 ~R 5 H is R 1 and R 2 F is R 3 ~R 5 (For example, H is...) Here each [ka] This includes azetidine or fully deuterated pyrrolidine. In some embodiments, if azetidine is present, it is further substituted at the 3-position with one or two non-halogen substituents (e.g., CO2H, CH3, F, etc.). In some embodiments, the exemplary compounds described herein are [ka] It has the structure of R. 1ー5 , Y and [ka] It is defined as described above, and Y and azetidine are optionally substituted as described above, with alternative linkers and AX groups also included in the range.

[0033] In some embodiments, the fluorophore(R) is a fluorescent moiety. A fluorescent moiety is a component that, when a compound containing it binds to a target (e.g., a haloalkane binding to a modified dehalogenase), produces an enhanced fluorescence signal (e.g., 10x, 31x, 50x, 100x, 310x, 500x, 1000x, or more). Exemplary fluorescent moieties for use in the embodiments of this specification include the following fluorophores from the JANELIA FLUOR family: [ka] [ka] Furthermore, see also other JANELIA FLUOR dyes described herein (see, for example, U.S. Patents 9,933,417, 10,018,624, 10,161,932, and 10,495,632, each of which is incorporated herein by reference in its entirety). For the use and design of fluorescent functional groups, dyes, probes, and substrates, see, for example, Grimm et al. Nat Methods. 3117 Oct;14(10):987-994.; Wang et al. Nat Chem. 3120 Feb;12(2):165-172; which are incorporated herein by reference in their entirety.

[0034] In some embodiments, the linker is a polyatomic linear or branched chain containing C, N, S, or O, or a group containing one or more rings, e.g., saturated or unsaturated rings (e.g., one or more aryl rings, heteroaryl rings, or any combination thereof). In some embodiments, the linker includes combinations of -O(CH2)2-, -(CH2)O-, -CH2-, -NHC(O)O-, -OC(O)NH-, NHC(O)-, and -C(O)NH-. In some embodiments, the linker is 5 to 50 atoms long (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or in between). In some embodiments, the length of the linker that bonds the alkyl halide to the fluorophore allows for optimization of the spatial proximity and geometric arrangement of the two (e.g., the bonding of the modified dehalogenase to the alkyl halide, or the function of the functional moiety (e.g., fluorescein)). The scope of embodiments herein is not limited by the types of linkers available. The fluorophore and AX may be directly bonded (e.g., the linker consists of a single covalent bond) or bonded via a suitable linker. The embodiments are not limited to any particular linker group. Various linker groups are intended, and suitable linkers include alkyl groups, methylene carbon chains, ethers, polyethers, alkylamide linkers, peptide linkers, modified peptide linkers, poly(ethylene glycol) (PEG) linkers, streptavidin-biotin or avidin-biotin linkers, polyamino acids (e.g., polylysine), functionalized PEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93 / 06868, and Tomalia et al.). This may include, but is not limited to, those described in al., Angew. Chem. Int. Ed. Engl. 29:138-175 (1990) (these are incorporated herein by reference in their entirety), PEG-chelate polymers (W94 / 08629, WO94 / 09056, and WO96 / 26754, these are incorporated herein by reference in their entirety), oligonucleotide linkers, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfides, or combinations thereof.In some embodiments, the linker is cleavable, for example, enzymatically (e.g., at the TEV protease site), chemically, or by photoinduction. In some embodiments, the cleavable linker comprises an allyl heteroatom group or a propargyl heteroatom group and includes, for example, a linker described in U.S. Patent Application No. 16 / 813,295 (which is incorporated herein by reference in its entirety).

[0035] In some embodiments, AX is a haloalkane (also known as an alkyl halide). In some embodiments, AX is a chloroalkane. In some embodiments, AX is a substrate of a modified dehalogenase that can be stably bound (e.g., covalently) to the haloalkane substrate (see, for example, U.S. Patents 7,425,436, 7,429,472, 7,867,726, 7,888,086, 7,935,803, U.S. Patent Reissue RE42,931, 8,168,405, 8,202,700, and 8,257,939, all of which are incorporated herein by reference in their entirety). One such modified dehalogenase is the commercially available HaloTag protein.

[0036] The haloalkyl compounds in the compositions and methods of this specification include a haloalkyl group, a linker, and a functional group. Exemplary compounds include rhodamine dyes bonded to a haloalkane. Limiting examples of these compounds include: [ka] These include (where alternative linkers and AX groups are also included in the scope).

[0037] In some embodiments, the fluorophore of the compounds herein is a rhodamine-based dye, a fluorescein-based dye, or a coumarin-based dye. The fluorophore may also include other groups to which it is added (e.g., biotin, bapta, etc.). In some embodiments, the fluorophore(R) of the compounds herein is a pro-fluorophore or is fluorescent (e.g., the ligand becomes fluorescent upon binding with HALOTAG). Non-limiting examples of fluorophore(R) within the scope of this specification are: [ka] [ka] This includes, for example, non-limiting examples of compounds containing the above fluorophore(R) include: [ka] [ka] This includes, among others. (Here, alternative linkers and AX groups are also included in the scope).

[0038] These exemplary compounds can be modified to have different linkers, alkyl halides, rhodamine dyes, and the like, as described herein.

[0039] In some embodiments, the compositions herein further include poloxamer. In certain embodiments, the presence of poloxamer achieves stabilization against degradation of the compound, improved solubility of the compound in water or aqueous solution, and the like. In some embodiments, by stabilizing the haloalkyl compound, increasing the water solubility of the haloalkyl compound, and / or improving the reconstitution efficiency of the haloalkyl compound in non-organic buffers, the compositions herein enable the use of haloalkyl compounds in point-of-care, pre-packaged, and / or solid-phase systems, methods, and assays where untreated and / or organic-phase compounds are more unsuitable.

[0040] In some embodiments, the compositions described herein comprise one or more poloxamers. A poloxamer is a nonionic triblock copolymer in which a central poly(propylene oxide) block is sandwiched between poly(ethylene oxide) blocks on both sides. Poloxamers are also known by certain trademark names, including Pluronic® and Kolliphor®. Exemplary poloxamers include poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, and poloxamer 407. In some embodiments, the liquid compositions herein contain poloxamer in concentrations of 0.1% to 20% (e.g., 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or values ​​or ranges between them (e.g., 1-10%, 2.5-9%)).

[0041] In addition to haloalkane-linked fluorophores and poloxamers, the compositions of this specification may further comprise other polymers or excipient components.

[0042] Other polymers may be naturally derived biopolymers or synthetic polymers. In some embodiments, the polymer is a naturally derived biopolymer. Suitable naturally derived biopolymers include carbohydrates, such as disaccharides (e.g., trehalose, maltose, and sucrose), polysaccharides (e.g., pullulan, dextran, and cellulose), and unsulfated glycosaminoglycans (e.g., hyaluronic acid). Mixtures of naturally derived biopolymers may also be used. The polymer may be a derivative of a naturally derived polymer, such as a functionalized cellulose (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.).

[0043] In some embodiments, the other polymer is pullulan, a polysaccharide containing maltotriose repeating units. Maltotriose is a trisaccharide containing three glucose units linked via α-1,4 glycosidic bonds. In pullulan polymers, the maltotriose units are linked to each other via α-1,6 glycosidic bonds. Pullulan is naturally produced from starch by the fungus Aureobasidium pullulans and is commonly found in quantities of approximately 4.5 x 10⁻⁶. 4 ~about 6 x10 5 It has a mass range of Da and is commercially available from various suppliers (CAS number 9057-02-7).

[0044] In some embodiments, the other polymer is dextran, a complex branched polysaccharide containing glucose repeating units. Linear linkages are generally formed by α-1,6 glycosidic bonds, and branching generally occurs from α-1,3 bonds. Naturally occurring dextran can have molecular weights ranging from about 9 kDa to about 2000 kDa. Dextran can be synthesized from sucrose by certain bacteria, including leuconostoc mesembroids and Streptococcus mutans. Commercially available dextran produced by leuconostoc mesembroids (CAS number 9004-54-0) is available from numerous suppliers, including Sigma Aldrich, and can have a diverse molecular weight range ranging from about 1 kDa to about 670 kDa.

[0045] In some embodiments, the other polymer is a cyclic saccharide polymer such as a cyclodextrin. Typical cyclodextrins are α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, which have 6, 7, and 8 glucopyranose units. The glucopyranose units may be functionalized. An example of a cyclodextrin is hydroxypropyl-β-cyclodextrin.

[0046] In some embodiments, the other polymer is a non-sulfated glycosaminoglycan. A glycosaminoglycan is a linear polysaccharide having repeating disaccharide units, each repeating unit comprising one amino sugar (N-acetylglucosamine or N-acetylgalactosamine) and one of either a uronic acid (glucuronic acid or iduronic acid) or galactose. An exemplary non-sulfated glycosaminoglycan is hyaluronic acid, where the repeating disaccharide comprises N-acetylglucosamine, glucurone, and glucuronic acid linked alternately via β-(1→4) and β-(1→3) glycosidic bonds. Polymers of hyaluronic acid have a size range of 5 kDa to 20,000 kDa.

[0047] In some embodiments, the other polymer is cellulose, a polysaccharide consisting of linear repeating β-1,4 linked D-glucose units. Natural fibers can contain up to 10,000 glucose units and have a molecular weight exceeding 1000 Da.

[0048] In some embodiments, the other polymer is a synthetic polymer. The synthetic polymer may be a homopolymer, copolymer, or block copolymer (e.g., diblock copolymer, triblock copolymer, etc.). Non-limiting examples of suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyacrylates. Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), and hydroxypropyl Methacrylate (HPMA), poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acid), polyanhydride, polyorthoester, poly(esteramide), polyamide, poly(ester ether), polycarbonate, polyalkylene (e.g., polyethylene and polypropylene), polyalkylene glycol (e.g., poly(ethylene glycol) (PEG) and polypropylene glycol (PPG)), and their copolymers (e.g., poloxamer), polyalkylene terephthalate (e.g., poly(ethylene terephthalate), etc.), polyvinyl alcohol (PVA), polyvinyl ether, polyvinyl ester (e.g., poly(vinyl acetate), etc.), polyvinyl halide (e.g., poly(vinyl chloride) (PVC), etc.), polyvinylpyrrolidone,Polysiloxane, polystyrene (PS), polyurethane, derivatized cellulose (e.g., alkylcellulose, hydroxyalkylcellulose, cellulose ether, cellulose ester, nitrocellulose, hydroxypropylcellulose, carboxymethylcellulose, etc.), acrylic acid polymers ("polyacrylic acid") (e.g., poly(methyl(meth)acrylate)(PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly( Hexyl (meth)acrylate, poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polydioxanone and its copolymers (e.g., polyhydroxyalkanoate, polypropylene fumarate), polyoxymethylene, poly(ortho)ester, poly(butyric acid), poly(valeric acid), poly(lactide-co -Caprolactone), trimethylene carbonate, polyvinylpyrrolidone (PVP), poly(1-vinylpyrrolidone-co-vinylacetate) (PVP-VA), poly(4-vinylpyridine), poly(4-vinylpyridine-co-butyl methacrylate), poly(4-vinylpyridine-co-styrene), poly[4-vinylpyridinium poly(hydrogen fluoride)], methacrylate (p(MAA-co-MMA)) copolymer, poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate), Examples include poly(1-vinylpyrrolidone-co-styrene), poly(4-vinylpyridinium-p-toluenesulfonate), hydroxypropyl acetate succinate (HPMC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), poly(ethylene-alto-propylene) (PEP), 2-methylacrylamide glucopyranose (MAG), dimethyl adipimidate (DMA), polyvinylcaprolactam-polyvinyl acetate, and any mixtures and copolymers thereof.

[0049] In some embodiments, the other synthetic polymer is a polyalkylene glycol. In some embodiments, the synthetic polymer is a polyalkylene glycol copolymer. In some embodiments, the synthetic polymer is a block copolymer (e.g., another poloxamer) comprising at least one poly(propylene oxide) block and at least one poly(ethylene oxide) block.

[0050] In some embodiments, the compound (e.g., a haloalkyl compound (e.g., R-linker-AX)) and poloxamer may be present in the composition in a weight ratio of about 0.000001:1 to about 1:1 (e.g., 0.000001:1, 0.000002:1, 0.000005:1, 0.00001:1, 0.00002:1, 0.00005:1, 0.0001:1, 0.0002:1, 0.0005:1, 0.001:1, 0.002:1, 0.005:1, 0.01:1, 0.02:1, 0.05:1, 0.1:1, 0.2:1, 0.5:1, 1:1, or a range in between).

[0051] In some embodiments, the composition includes a buffer such as a phosphate buffer, borate buffer, acetate buffer, or citrate buffer, or other common buffers, e.g., bicine, tricine, tris(hydroxymethyl)aminomethane (Tris), N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino)ethanesulfonic acid (MES), Dulbecco's phosphate-buffered saline (DPBS), carbonate buffer (e.g., sodium carbonate and sodium bicarbonate solution), citrate buffer, etc. In some embodiments, the composition comprises a phosphate buffer. In some embodiments, the composition comprises tricine. In some embodiments, the composition comprises 2-(N-morpholino)ethanesulfonic acid. The composition may also comprise any combination of buffers. In some embodiments, the dry compositions herein are reconstituted in a solution comprising a buffer (for example, a desired pH (e.g., 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or higher, or lower)).

[0052] In some embodiments, the composition further comprises a detergent or surfactant (in addition to, for example, haloalkane-linked fluorophores and poloxamers). In some embodiments, the detergent or surfactant is present in an amount of about 0.01 mol% to 5 mol% (e.g., 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, or any range in between (e.g., 0.1 to 0.5%)). Exemplary surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants. Examples of nonionic detergents include Brij 35, Triton® surfactants (e.g., Triton® X series (octylphenol ethoxylates such as Triton® X-100, Triton® X-100R, Triton® X-114)), octyl glucoside, polyoxyethylene (9) dodecyl ether, digitonin, and octylphenyl polyethylene glycol (IGEPAL). CA630), n-octyl-β-D-glucopyranoside (betaOG), n-dodecyl-β-D-maltoside, Tween® 20 (polysorbate 20 or polyethylene glycol (20) sorbitan monolaurate), Tween® 40 (polysorbate 40 or polyethylene glycol (20) sorbitan monopalmitate), Tween® 80 (polysorbate 80 or polyethylene glycol (20) sorbitan monooleate), polidokanol, n-dodecyl-β-D-maltoside (DDM), nonidet P40 substitute, N Examples include P-40 nonylphenyl polyethylene glycol, C12E8 (octaethylene glycol-n-dodecyl monoether), hexaethylene glycol mono-n-tetradecyl ether (C14E06), octyl-β-thioglucopyranoside (octylthioglucoside, OTG), Pluronic® F-68 (poloxamer 188), Pluronic® F-127 (poloxamer 407), saponins, emulgen, polyethylene glycol trimethylnonyl ether, and polyoxyethylene 10-lauryl ether (C12E10).Examples of ionic detergents (anionic or cationic) include deoxycholic acid, sodium cholate, sodium dodecyl sulfate (SDS), N-lauroyl sarcosine, and cetyltrimethylammonium bromide (CTAB). Examples of amphoteric reagents include chaps, twitter ion 3-14, and 3-[(3-coramidopropyl)dimethylammonio]-1-propanesulfonic acid. In some embodiments, the surfactant is polysorbate 20. The composition may also include any combination of surfactants.

[0053] In some embodiments, the composition may further include reducing agents such as dithiothreitol (DTT), 2-mercaptoethanol (BME), cysteamine, (2S)-2-amino-1,4-dimercaptobutane (DTBA), thiourea, and 6-aza-2-thiothymine (ATT), in addition to (e.g., haloalkane-linked fluorophores and poloxamers). In some embodiments, the reducing agent is thiourea. In some embodiments, the reducing agent is ATT. The composition may also include any combination of reducing agents.

[0054] In some embodiments, the composition may further include salts such as sodium chloride, potassium chloride, magnesium chloride, and sodium phosphate (in addition to, for example, haloalkane-linked fluorophores and poloxamers). In some embodiments, the salt is sodium chloride. In some embodiments, the salt is sodium phosphate. The composition may also include any combination of salts.

[0055] In some embodiments, the composition may further contain radical scavengers such as ascorbic acid and sodium ascorbate (in addition to, for example, haloalkane-linked fluorophores and poloxamers). In some embodiments, the composition may further contain metal chelating agents such as citric acid, ethylenediaminetetraacetic acid, and trans-1,2-diaminocyclohexanetetraacetic acid. In some embodiments, the composition contains ascorbic acid. In some embodiments, the composition contains sodium ascorbate. In some embodiments, the composition contains citric acid. In some embodiments, the composition contains trans-1,2-diaminocyclohexanetetraacetic acid. The composition may contain any combination of radical scavengers and / or chelating agents.

[0056] In some embodiments, the composition may further contain proteins (in addition to, for example, haloalkane-linked fluorophores and poloxamers). For example, the composition may contain a carrier protein. In some embodiments, the protein may be bovine serum albumin (BSA). In some embodiments, the protein may be a polypeptide fraction of highly purified porcine collagen (e.g., Prionex). In some embodiments, the protein may be gelatin. The composition may also contain any combination of proteins.

[0057] In some embodiments, the composition may further contain a solvent (in addition to, for example, haloalkane-linked fluorophores and poloxamers). Some compositions are completely dry with any solvent completely removed, while others may contain a solvent or some residual solvent. In some embodiments, the composition may contain an organic solvent such as methanol, ethanol, isopropanol, ethylene glycol, propylene glycol, or any combination thereof. For example, the composition may contain a combination of ethanol and propylene glycol.

[0058] As described above, the composition may include any combination of the above-mentioned components (in addition to the haloalkane-linked fluorophores and poloxamers). For example, in some embodiments, the composition may include a protein, a buffer, and a reducing agent. In some embodiments, the composition may include a protein, a buffer, and a metal chelating agent.

[0059] The compositions herein may be in the form of lyophilized powders or cakes. Such compositions can be prepared by lyophilizing a mixture of the components of the composition, as further described below. Powdered products may be supplied in containers such as bottles, vials, snap tubes, microtiter plates, paper or fiber matrix or other solid material supports, or lab-on-a-chips. Powdered products may be dispensed into multiple snap tubes. Each tube contains a predetermined amount of the composition that can be dissolved in an appropriate amount of solution and used directly for the assay of interest.

[0060] The composition may be in the form of a rigid yet plastic material, such as a “drop” cast or film. Such a composition can be prepared by coating a surface with a solution containing the composition components (e.g., haloalkane-linked fluorophores and poloxamers) and then drying the composition. Drying can be carried out, for example, by air drying, room temperature drying, high temperature drying (e.g., about 30°C to about 70°C, or about 30°C to about 40°C, e.g., about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C), drying in an inert atmosphere, or drying under reduced pressure. The drop cast or film may be supplied in containers such as bottles, vials, snap tubes, microtiter plates, paper or fiber matrix or other solid material supports, or lab-on-a-chips.

[0061] In some embodiments, the composition is in the form of a solution (e.g., an aqueous solution). When the composition is a solution, the pH of the composition may be about 5.5 to 8.0, for example, about 6.5 to about 7.5. In some embodiments, the pH of the composition is about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0062] The composition may be provided in other forms, such as tablets or capsules (including soluble tablets or capsules that can be added to samples such as buffers or biological samples). The composition may also be provided as a pre-formed film formed on a surface, such as the wells of a 96-well plate. This allows the composition to be directly dissolved in an appropriate amount of solution and used directly in the assay of interest.

[0063] The compositions of this disclosure can be used in any conventional method in which haloalkyl compounds (e.g., HALOTAG ligands) have been used. For example, they can be used in HALOTAG-based methods to detect or isolate one or more molecules in a sample, such as enzymes, cofactors of enzymatic reactions, enzyme substrates, enzyme inhibitors, enzyme activators, etc. Samples may include animals (e.g., vertebrates), plants, fungi, physiological fluids (e.g., blood, plasma, urine, mucus secretions), cells, cell lysates, cell supernatants, or purified fractions of cells (e.g., intracellular organelle fractions).

[0064] This specification provides a method for stabilizing haloalkyl-linked fluorophores, the method comprising contacting the compound with an effective amount of poloxamer and / or paper or fiber matrix to form a composition. Haloalkyl-linked fluorophores can be stabilized against thermal decomposition, chemical decomposition, photo-induced decomposition, or any combination thereof.

[0065] In some embodiments, the compositions herein contain haloalkyl-bonded fluorophores at temperatures ranging from about -80°C to about 80°C, about -75°C to about 80°C, about -70°C to about 80°C, about -65°C to about 80°C, about -60°C to about 80°C, about -55°C to about 80°C, about -50°C to about 80°C, about -45°C to about 80°C, about -40°C to about 80°C, about -35°C to about 80°C, about -30°C to about 80°C, about -25°C to about 80°C, about -20°C to about 80°C, about -15°C to about 80°C, about -10°C to about 80°C, about -5°C to about 80°C, about 0°C to about 80°C, and about -80°C. It is stabilized against decomposition in the temperature range of ℃ to approximately 75℃, approximately -80℃ to approximately 70℃, approximately -80℃ to approximately 65℃, approximately -80℃ to approximately 60℃, approximately -80℃ to approximately 55℃, approximately -80℃ to approximately 50℃, approximately -80℃ to approximately 45℃, approximately -80℃ to approximately 40℃, approximately -80℃ to approximately 35℃, approximately -80℃ to approximately 30℃, approximately -80℃ to approximately 25℃, approximately -20℃ to approximately 60℃, approximately -20℃ to approximately 55℃, approximately -20℃ to approximately 50℃, approximately -20℃ to approximately 45℃, approximately -20℃ to approximately 40℃, approximately -20℃ to approximately 35℃, approximately -20℃ to approximately 30℃, or approximately -20℃ to approximately 25℃.

[0066] In some embodiments, the compositions herein contain haloalkyl-bonded fluorophores compared to compositions that do not contain polymers or paper or fiber matrices, for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 5 It stabilizes against decomposition over 0, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360 days, 1 year, 2 years, 3 years, 4 years, or 5 years.

[0067] In some embodiments, the composition increases the half-life of the haloalkyl-bonded fluorophores to degradation by at least about 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 times compared to compositions that do not contain polymers or paper or fiber matrices.

[0068] This specification also provides a method for improving the solubility of a haloalkyl-linked fluorophore, comprising contacting the haloalkyl-linked fluorophore with an effective amount of poloxamer and / or a paper or fiber matrix. Here, the solubility of the haloalkyl-linked fluorophore is improved compared to the haloalkyl-linked fluorophore that has not been in contact with poloxamer. The solubility of the haloalkyl-linked fluorophore in aqueous solution may be improved compared to the corresponding haloalkyl-linked fluorophore that has not been in contact with poloxamer and / or a paper or fiber matrix. In the presence of poloxamer, the solubility of the haloalkyl-linked fluorophore may be improved after reconstitution of the lyophilized powder, dropcast film or "droplet," or after rehydration of the paper or fiber matrix or other solid support material in which the haloalkyl-linked fluorophore is located.

[0069] In some embodiments, the composition enhances the solubility of haloalkyl-linked fluorophores in aqueous solutions, for example, those further containing components such as pure water or buffers, salts, proteins, reducing agents, radical scavengers, surfactants, or any combination of such components. In some embodiments, the composition enhances the solubility of haloalkyl-linked fluorophores in aqueous buffers, such as phosphate-buffered saline (PBS) with a pH of about 6.5 to about 7.5 (e.g., pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or any range in between), or other suitable buffers. In some embodiments, the composition enhances the solubility of haloalkyl-linked fluorophores in biological or environmental fluids, such as biological samples from subjects or culture media (e.g., cell culture media).

[0070] This specification also provides a method for increasing the reconstitution rate of haloalkyl-linked fluorophores, comprising contacting the haloalkyl-linked fluorophores with an effective amount of poloxamer and / or paper or fiber matrix. Here, the reconstitution rate of the haloalkyl-linked fluorophores is improved compared to haloalkyl-linked fluorophores that have not been contacted with poloxamer and / or paper or fiber matrix.

[0071] In some embodiments, the composition enhances the reconstitution rate of haloalkyl-linked fluorophores in aqueous solutions, such as pure water or buffer solutions, salts, proteins, reducing agents, surfactants, or any combination of such components. In some embodiments, the composition enhances the reconstitution rate of haloalkyl-linked fluorophores in aqueous buffers, such as phosphate-buffered saline (PBS) with a pH of about 6.5 to about 7.5 (e.g., pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5, or any range in between), or other suitable buffers. In some embodiments, the composition enhances the solubility of compounds in biological or environmental fluids, such as biological samples from subjects or culture media (e.g., tissue culture media).

[0072] The composition may have any combination of the properties disclosed herein. For example, the composition may have the increased solubility, improved reconstitution rate, increased stability, and / or increased half-life described herein. The composition may have any one of the disclosed properties, or any combination thereof, and may also have other improved properties.

[0073] In embodiments of the methods described herein, the contact step may include the steps of: dissolving a haloalkyl-bonded fluorophore in a first solvent to form a first solution; mixing the first solution with a poloxamer and / or paper or fiber matrix to form a mixture; and drying the mixture. In some embodiments, the contact step includes the steps of: dissolving a haloalkyl compound in a first solvent to form a first solution; dissolving a poloxamer in a second solvent to form a second solution; mixing the first solution and the second solution to form a mixture; and drying the mixture. In some embodiments, the contact step includes the steps of: dissolving a haloalkyl compound in a solvent to form a first solution; applying the first solution to paper or fiber matrix; and drying the paper or fiber matrix. In some embodiments, the contact step includes the steps of: dissolving the compound in a first solvent to form a first solution; dissolving the poloxamer in a second solvent to form a second solution; mixing the first solution and the second solution to form a third solution; applying the third solution to paper or a fiber matrix; and drying the paper or fiber matrix.

[0074] In some embodiments, the drying step includes freeze-drying. In some embodiments, the drying step includes air-drying. In some embodiments, the drying step includes drying at ambient temperature in an inert atmosphere (e.g., nitrogen or argon). In some embodiments, the drying step includes drying at a high temperature (e.g., 30°C). In some embodiments, the drying step includes vacuum drying. In some embodiments, one or all of the solutions used in the method may be deoxygenated. Deoxygenation can be achieved by methods such as degassing the solution under reduced pressure or bubbling an inert gas (e.g., nitrogen or argon) into the solution.

[0075] The composition can be tested by using it as a substrate for HALOTAG.

[0076] In certain embodiments, the compositions disclosed herein are provided as part of a kit. The compositions may be housed in a single container. In some embodiments, the kit may further include, in addition to the modified dehalogenase (HALOTAG), appropriate reagents and instructions for use that enable the user to perform assays using it. The kit may also include one or more buffers, such as those disclosed herein. The kit may include instructions for storage of the compositions and / or the single container in which the compositions are housed. Instructions for use included in the kits of this disclosure may be affixed to the packaging or included as accompanying documentation. Instructions are typically in writing or printed form, but are not limited thereto. This disclosure considers any medium that can store and communicate such instructions to an end user. Such mediums include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term “instructions for use” may include the address of an internet site providing the instructions for use. [Examples]

[0077] During the development of the embodiments described herein, experiments were conducted to confirm that polymers and / or excipients enhance the clarity of solutions containing haloalkyl-linked fluorophores, and / or increase the concentration of haloalkyl-linked fluorophores in the solution, and / or remain present after the dissolution of dried haloalkyl-linked fluorophores. These experiments confirmed that poloxamers result in improved solution clarity and increased concentrations of multiple haloalkyl-linked fluorophores in the solution.

[0078] Example 1 During the development of the embodiments herein, experiments were conducted to demonstrate the lyophilization and reconstitution of the compositions herein, which include haloalkyl-fluorophore compounds and polymer excipients.

[0079] Chloroalkyl-JANELIA FLUOR 554 (CA-JFX554) and chloroalkyl-JANELIA FLUOR 650 (CA-JFX650) were each dissolved in 100% methanol to a final concentration of 500 μM. Next, 2020 μl each of the CA-JFX554 solution and CA-JFX650 solution were taken and diluted in 101 ml of 2.5% poloxamer 407 solution to a final concentration of 10 μM of the haloalkyl-fluorophore compound. For lyophilization, 100 μl of the polymer / compound solution was dispensed into 2 ml amber tubes.

[0080] Purity tests were conducted on the haloalkyl-fluorophore compounds (Figure 1). Even after 18 days at room temperature, the lyophilized ligands maintained a purity of over 95%. Furthermore, even when the lyophilized ligands were stored in an FBS-containing aqueous medium at 4°C, a purity of over 95% was maintained after 18 days. Accelerated stability tests predict that a minimum purity of 90% will be maintained even after storage at -20°C for over 4 years.

[0081] Functionality tests were performed by staining cells with lyophilized ligand and solid standard form ligand, and quantifying the staining intensity at saturated and near-saturated concentrations (Figure 2). The lyophilized ligand was dissolved by adding 1 mL of phenol red-free cell culture medium to a vial to obtain a 1 μM stock solution. On the other hand, since the solid ligand is insoluble in water, it was first dissolved in DMSO to a concentration of 200 μM, and then diluted 1:200 with phenol red-free cell culture medium to obtain a 1 μM stock solution. The dye was added to cells at the indicated final concentration. Experiments conducted during the development of embodiments of the present invention demonstrated that the lyophilized ligand exhibited similar staining intensity to the solid form at both saturated and near-saturated concentrations under the same staining conditions (37°C for 30 minutes, with medium change).

[0082] Example 2 Preparation of JF dye HaloTag ligand 90 mg of P-407 was added to a 100 mL round-bottom flask and dissolved by stirring at 70-75°C. 1 μmol of JF dye HaloTag ligand (JF-669 or JF-552) was dissolved in approximately 3.5 mL of ethanol, and the resulting solution was transferred to the dissolved P-407. The resulting mixture was stirred at 65°C to obtain a homogeneous solution. The obtained solution was concentrated under reduced pressure and then redissolved in 10 mL of ultrapure water. Next, the resulting aqueous solution was dispensed into 10 1 mL vials of 1-dram material, frozen, and then lyophilized to obtain a formulation cake containing 100 nmol of ligand per vial (Figure 3A-B).

[0083] Example 3 Solubility analysis of JF dye HaloTag ligand Lyophilized chloroalkyl-JANELIA FLUOR 552 (CA-JF552) and chloroalkyl-JANELIA FLUOR 669 (CA-JF669) vial samples containing various excipients, prepared before lyophilization, were each dissolved in 100 μL of aqueous buffer to obtain a final concentration of 1 mM. Subsequently, 70 μL each of the CA-JF 552 and CA-JF669 solutions were transferred to the wells of a transparent 96-well plate, and transparency analysis was performed using 850 nm [bandwidth 3.5 nm] optical density absorbance measurement (OD850) with a Tecan Spark plate reader (Figures 4 and 5). The target absorbance limit for transparency measurement was determined to evaluate ligand solubility using a known turbidity sample similarly prepared with poloxamer.

[0084] A transparency target value of <0.1 was used. Experiments conducted during the development of the embodiments described herein demonstrated that the visual transparency of the sample corresponds to an absorbance value of 0.1 or less.

[0085] Example 4 Stability analysis of JF dye HaloTag ligand Lyophilized chloroalkyl-JANELIA FLUOR 552 (CA-JF552) and chloroalkyl-JANELIA FLUOR 669 (CA-JF669) vial samples, prepared with various excipients, were dissolved in 100 μL aqueous buffer to obtain a final concentration of 1 mM. HPLC analysis of the haloalkyl-fluorophore compounds was performed, and the HaloTag ligand content in the solution was quantified by comparing the area of ​​the ligand peak at 254 nm. HPLC tests were performed using a ThermoFisher Vanquish HPLC system, a ThermoFisher Accucore RP-MS (50 × 2.1 mm, 2.6 μm) reversed-phase HPLC column, a 0.1% trifluoroacetic acid aqueous solution / acetonitrile mobile phase, and a column oven temperature of 30°C. Measurements were performed after complete dissolution of the samples in Corning DPBS pH 7.0, and the stability of the lyophilized HaloTag ligand was confirmed by comparing it with the initial ethanol stock solution used to prepare the formulation ligand (Figure 6).

[0086] Example 5 The functionality of various formulated lyophilized HaloTag® ligands that excite and emit light across the entire UV / VIS spectrum was tested by parallel staining experiments using cells that stably express HaloTag® localized in the nucleus and parental cells that do not express HaloTag®. The lyophilized ligand was dissolved by adding 1 mL of phenol red-free cell culture medium to a vial to obtain a 1 μM stock solution. The dye was added to the cells at a final concentration of 200 nM. Experiments conducted during the development of the embodiments described herein demonstrated that the preparation of lyophilized HaloTag® ligands in poloxamer is effective for various fluorescent HaloTag® ligands across the entire UV-VIS spectrum (Figure 7).

Claims

1. A composition, (a) A haloalkyl bonded fluorophore comprising the formula R-linker-A-X, wherein R is a fluorophore, the linker is a polyatomic linear or branched chain, and A is (CH 2 ) 4ー20 X is a halide, and the haloalkyl bonded fluorophore, (b) The composition comprising poloxamer.

2. The composition according to claim 1, wherein the linker functional portion is selected from the group consisting of nucleic acid molecules, amino acids, peptides, receptor proteins, glycoproteins, antibodies, lipids, haptens, receptor ligands, fluorophores, and photocatalysts.

3. The composition according to claim 2, wherein the fluorophore is a low molecular weight fluorophore.

4. The composition according to claim 3, wherein the low molecular weight fluorophore is a rhodamine dye.

5. The rhodamine dye is, 【Chemistry 1-1】 [Chemistry 1-2] A composition according to claim 4, selected from the following.

6. The composition according to claim 1, wherein the linker is a polyatomic linear or branched chain containing C, N, S, or O, and optionally includes one or more ring structures.

7. The composition according to claim 6, wherein the linker includes a severable portion.

8. The composition according to claim 7, wherein the cleavable portion is selected from an allyl heteroatom group and a propargyl heteroatom group.

9. The composition according to claim 1, wherein the poloxamer is selected from poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, and poloxamer 407.

10. The composition according to claim 9, wherein the poloxamer is poloxamer 407.

11. The composition according to claim 1, further comprising a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a protein, or a combination thereof.

12. The composition according to claim 11, wherein the buffer is selected from phosphate buffer, tricine, and 2-(N-morpholino)ethanesulfonic acid.

13. The composition according to claim 11, wherein the surfactant is selected from polysorbate 20, polysorbate 40, and polysorbate 80.

14. The composition according to claim 11, wherein the reducing agent is selected from thiourea and 6-aza-2-thiothymine.

15. The composition according to claim 11, wherein the salt is selected from sodium chloride and sodium phosphate.

16. The composition according to claim 11, wherein the radical scavenger is selected from ascorbic acid and sodium ascorbate.

17. The composition according to claim 11, wherein the chelating agent is selected from citric acid and trans-1,2-diaminocyclohexanetetraacetic acid.

18. The composition according to claim 11, wherein the protein is selected from bovine serum albumin, gelatin, and a polypeptide fraction of highly purified porcine collagen.

19. The composition according to any one of claims 1 to 18, wherein the composition is in the form of a freeze-dried powder, a cake, or a plastic film.

20. The composition according to any one of claims 1 to 18, wherein the composition is a solution.

21. The composition according to claim 19 or 20, wherein the haloalkyl-bonded fluorophore is stabilized against thermal decomposition, chemical decomposition, photo-induced decomposition, or a combination thereof.

22. A method for preserving a haloalkyl bonded fluorophore comprising the formula R-linker-A-X, wherein R is a fluorophore, the linker is a polyatomic linear or branched chain, and A is (CH 2 ) 4ー20 The storage method wherein X is a halogen, and the method includes contacting the compound with a poloxamer.

23. The method according to claim 22, wherein contacting the haloalkyl-bonded fluorophore with the poloxamer comprises dissolving the haloalkyl-bonded fluorophore in an organic solvent to form a first solution, and mixing the first solution with the poloxamer to form a mixture.

24. The method according to claim 23, wherein the mixing step includes dissolving the poloxamer in a second solution and mixing the second solution with the first solution.

25. The method according to claim 24, further comprising contacting a solid substrate with the mixture.

26. The method according to claim 25, wherein the solid substrate is a plate or surface made of glass, metal, or plastic.

27. The method according to any one of claims 22 to 26, further comprising drying the mixture.

28. The method according to claim 27, wherein the drying step includes freeze-drying.

29. The method according to claim 27, wherein the drying step includes air drying.

30. The method according to claim 27, wherein the drying is carried out in an inert atmosphere at ambient temperature.

31. The method according to claim 27, wherein the drying step includes vacuum drying.

32. The method according to claim 27, wherein the drying is carried out at a temperature of about -80°C to about 70°C.

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