Polymeric tandem dyes with linker groups

Abstract

The present invention relates to polymer tandem dyes having a linker group. Disclosed herein are compounds useful as fluorescent dyes or color dyes. In certain embodiments, the compounds have the following structure (I): (I) or a stereoisomer, tautomer, or salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , L 4 , M 1 , M 2 , m and n are as defined herein. Also provided are methods relating to the preparation and use of the compounds.

Description

[0001] This application is a divisional application of patent application 202080005874.X. Technical Field

[0002] This disclosure generally relates to dimerized and polymerized chromophore compounds having spacer groups (e.g., polymer compounds containing fluorescent dye moieties), methods for their preparation, and their use in various analytical methods. Background Technology

[0003] Fluorescent and / or staining dyes are known to be particularly suitable for applications requiring highly sensitive detection reagents. Dyes that can preferentially label specific components or parts in a sample allow researchers to determine the presence, amount, and / or location of those specific components or parts. Furthermore, they enable the monitoring of the spatial and temporal distribution of a specific system in different environments.

[0004] Fluorescence and colorimetric methods are extremely prevalent in chemistry and biology. These methods provide useful information about the presence, structure, distance, orientation, complexation, and / or location of biomolecules. Furthermore, time-resolved methods are increasingly used in dynamic and kinetic measurements. As a result, numerous strategies have been developed for fluorescently or color-labeling biomolecules such as nucleic acids and proteins. Since the analysis of biomolecules generally takes place in aqueous environments, the focus has been on developing and using dyes compatible with aqueous systems that can elucidate desired spatial information and biomolecular interactions.

[0005] Accordingly, techniques involving resonance energy transfer have been developed to reveal this structural information. In particular, Förster resonance energy transfer ("FRET"—sometimes used interchangeably with fluorescence resonance energy transfer) technology produces reliable measurements of changes in biomolecular distances and interactions. Resonance energy transfer techniques are relatively inexpensive and allow for rapid measurement; however, FRET is hampered by several limitations, including chromophore orientation and localization, and energy transfer masking due to free fluorophores and undesirable pH sensitivity. Additionally, known FRET reagents (e.g., modified phycoerythrin or Brilliant Violet™) suffer from a lack of photostability, undesirable physical characteristics (e.g., size, cell permeability), and high batch-to-batch variability (requiring equipment recalibration).

[0006] There is a need in the art for water-soluble dyes, particularly resonance energy transfer dyes, which exhibit increased molar brightness and / or increased FRET emission signals. Ideally, the dyes and biomarkers should be strongly stained or fluorescent and should be available in a variety of colors and fluorescence wavelengths. This disclosure fulfills this need and provides other related advantages. Summary of the Invention

[0007] In summary, embodiments of this disclosure generally relate to compounds used as water-soluble fluorescent and / or coloring dyes and / or probes, enabling the visual detection of analyte molecules such as biomolecules, and reagents used for their preparation. In particular, in some embodiments, the compounds of this disclosure are useful because they enable FRET fluorescence emission associated with them. Methods for the visual detection of analyte molecules using dyes are also described.

[0008] Currently disclosed implementations of dyes include those using a linker (e.g., "L"). 4 The dye covalently links two or more fluorescent and / or stained moieties (i.e., chromophores). Compared to previously reported protein-based dimerization and / or polymerization dyes, the dyes in this paper are significantly brighter, enabling FRET absorption and emission resulting from intramolecular interactions, and allowing robust reproducibility using readily available methods known in the art (i.e., automated DNA synthesis methods).

[0009] The water-soluble fluorescent or coloring dyes of this disclosure are strongly coloring and / or fluorescent, enabling FRET processes (e.g., absorption, emission, Stokes shift) and allowing for easy observation by visual inspection or other means. In some embodiments, compounds can be observed without prior light or chemical or enzymatic activation. By appropriately selecting dyes as described herein, valuable spatial information about visually detectable analyte molecules and target molecules in various colors can be obtained.

[0010] In one embodiment, a compound having the following structure (I) is provided:

[0011]

[0012] (I)

[0013] Or its stereoisomers, tautomers or salts, wherein R 1 R 2 R 3 R 4 R 5 L 1 L 2 L 3 L 4 M 1 M 2 m and n are as defined in this paper.

[0014] Another embodiment provides a compound having the following structure (II):

[0015]

[0016] (II)

[0017] Or its stereoisomers, salts or tautomers, wherein R 1 R 2 R 3 R 4 R 5 L 1 L 2 L 3 L 4 L 5 L 6 L 7a L 7b M 1 M 3 q, w, m and n are as defined in this paper.

[0018] Another embodiment provides a compound having the following structure (III):

[0019]

[0020] (III)

[0021] Or its stereoisomers, salts or tautomers, wherein R 1 R 2 R 3 R 4 R 5 L 1 L 2 L 3 L 4 L 8 M 1 M 2 m and n are as defined in this paper.

[0022] One embodiment provides a compound having the following structure (IV):

[0023] (IV)

[0024] Or its stereoisomers, salts or tautomers, wherein R 1 R 2 R 3 R 4 R 5 L 1 L 2 L 3 L 4 L 5 L 6 L 7a L 7b L 8 M 1 M 2m and n are as defined in this paper.

[0025] Another embodiment provides a polymer compound comprising an acceptor chromophore and a donor chromophore, the acceptor chromophore having an acceptor transition dipole moment and covalently linked to the polymer backbone, and the donor chromophore having a donor transition dipole moment and covalently linked to the polymer backbone, wherein the acceptor chromophore and the donor chromophore have a molecular weight greater than about 1 × 10⁻⁶. 10 The J-value and the polymer compound adopts a conformation in solution under physiological conditions in which the effective distance between the acceptor chromophore and the donor chromophore is less than about 50.0 nm and the acceptor transition dipole and the donor transition dipole are substantially parallel or substantially antiparallel.

[0026] The foregoing embodiments describe compounds for a variety of applications, including dyes used as FRET, fluorescence, and / or coloring agents in various analytical methods.

[0027] Accordingly, another embodiment provides a method for staining a sample, the method comprising adding one of the aforementioned compounds to the sample in an amount sufficient to produce an optical response when the sample is illuminated at an appropriate wavelength.

[0028] In other embodiments, this disclosure provides a method for visually detecting and analyzing molecules, including:

[0029] (a) Providing one of the aforementioned compounds (e.g., a compound with structure (I), (II), (III), or (IV); and

[0030] (b) Detecting compounds through their visual properties.

[0031] Other disclosed methods include methods for visually detecting biomolecules, said methods comprising:

[0032] (a) Mixing one of the aforementioned compounds (e.g., compounds of structure (I), (II), (III), or (IV)) with one or more biomolecules; and

[0033] (b) Detecting compounds through their visual properties.

[0034] Other embodiments involve compositions comprising at least one of the aforementioned compounds (e.g., compounds of structure (I), (II), (III), or (IV)) and one or more biomolecules. Use of the compositions in analytical methods for detecting one or more analytes (e.g., biomolecules) is also provided.

[0035] These and other aspects of this disclosure become more apparent upon reference to the details described below. Attached Figure Description

[0036] In the accompanying drawings, the same reference numerals identify similar elements. The dimensions and relative positions of elements in the drawing are not necessarily drawn to scale, and some of these elements are enlarged and positioned to improve the readability of the drawing. Furthermore, the specific shapes of the elements drawn are not intended to convey any information about the true shape of that particular element, but are chosen solely for ease of identification in the drawing.

[0037] Figure 1 The diagram shows exemplary donor emission spectra and acceptor excitation (or absorption) spectra and their overlap, which are used to determine the J-value.

[0038] Figure 2 The fluorescence emission spectrum of compound I-1 is shown, using Cy3 as the donor chromophore and AF680 as the acceptor. Excitation was performed at 488 nm.

[0039] Figure 3 The fluorescence emission spectrum of compound I-2 is described, using AF555 as the donor chromophore and AF680 as the acceptor. Excitation was performed at 488 nm.

[0040] Figure 4 The fluorescence emission spectrum of compound I-3 is shown, using Cy3 as the donor chromophore and AF647 as the acceptor. Excitation was performed at 488 nm.

[0041] Figure 5 The fluorescence emission spectrum of compound I-4 is shown, using AF555 as the donor chromophore and AF647 as the acceptor. Excitation was performed at 488 nm.

[0042] Figure 6 The fluorescence emission spectrum of compound I-5 is described, using Cy3 as the donor chromophore and AF594 as the acceptor. Excitation was performed at 488 nm.

[0043] Figure 7 The fluorescence emission spectrum of compound I-6 is described, using AF555 as the donor chromophore and AF594 as the acceptor. Excitation was performed at 488 nm.

[0044] Figure 8 The fluorescence emission spectrum of compound I-7 is shown, using Cy3 as the donor chromophore and AF700 as the acceptor. Excitation was performed at 488 nm.

[0045] Figure 9 The fluorescence emission spectrum of compound I-8 is described, using AF555 as the donor chromophore and AF700 as the acceptor. Excitation was performed at 488 nm.

[0046] Figure 10 This diagram shows the positional overlap of the fluorescence emission spectra of compounds I-1, I-3, I-5, and I-7.

[0047] Figure 11 This is an overlay of the emission spectra of compounds containing the dual-luciferin moiety, excited at 350 nm. Compound I-10 (relative to compound A) shows increased fluorescence, which is a result of FRET excitation from the donor AF350 moiety. Invention Details

[0049] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments of this disclosure. However, those skilled in the art will understand that this disclosure may be practiced without these details.

[0050] Unless the context otherwise requires, throughout the specification and claims, the word "comprising" and its variations such as "containing" and "including" shall be interpreted in an open-ended sense, that is, "including, but not limited to".

[0051] Throughout this specification, the phrase "in one embodiment" or "an embodiment" means that a particular property, structure, or feature described in that embodiment is included in at least one embodiment of this disclosure. Therefore, the phrase "in one embodiment" or "in an embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, a particular property, structure, or feature may be combined in any suitable manner in one or more embodiments.

[0052] The term "about" when used in conjunction with the stated value or range has the meaning reasonably understood by one of ordinary skill in the art, that is, to be slightly larger or smaller than the stated value or range, falling within ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0053] "Amino" refers to the -NH2 group.

[0054] "Carboxyl group" refers to the -CO2H group.

[0055] "Cyano" refers to the -CN group.

[0056] "Formyl group" refers to the -C(=O)H group.

[0057] "Hydroxyl" or "hydroxyl group" refers to the -OH group.

[0058] "Imine" refers to the =NH group.

[0059] "Nitro" refers to the -NO2 group.

[0060] "Oxo" refers to the =O group.

[0061] "Hydrosulfur", "thiol" or "sulfur" refers to the -SH group.

[0062] "Thio" refers to the =S group.

[0063] "Alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, without unsaturation, and having one to twelve carbon atoms (C1-C2). 12 Alkyl groups (one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl) and connected to the remainder of the molecule by single bonds, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specified in the specification, alkyl groups are optionally substituted.

[0064] "alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that links the rest of a molecule to residues. It consists only of carbon and hydrogen, is unsaturated, and has one to twelve carbon atoms, such as methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butynylene, propynylene, n-butynylene, etc. The alkylene chain is linked to the rest of the molecule and to residues via single bonds. The connection points between the alkylene chain and the rest of the molecule, and between the alkylene chain and residues, can be via one carbon or any two carbons in the chain. Unless otherwise specified in the specification, alkylene is optionally substituted.

[0065] "Alkenyl" or "alkenyl chain" refers to a straight or branched divalent hydrocarbon chain that links the rest of a molecule to residues. It consists only of carbon and hydrogen, contains at least one carbon-carbon double bond, and has two to twelve carbon atoms, such as vinylidene, propenide, n-butenyl, etc. The alkenyl chain is connected to the rest of the molecule by a single bond and to residues by either a double or single bond. The connection points between the alkenyl chain and the rest of the molecule, and between the alkenyl chain and residues, can be through one or any two carbons in the chain. Unless otherwise specified in the specification, the alkenyl group is optionally substituted.

[0066] "Inyynyl" or "Inyynyl chain" refers to a straight or branched divalent hydrocarbon chain that links the rest of the molecule to residues. It consists only of carbon and hydrogen, contains at least one carbon-carbon triple bond, and has two to twelve carbon atoms, such as vinylene, allylene, n-buteneyl, etc. The ynylyl chain is connected to the rest of the molecule by single bonds and to residues by double or single bonds. The connection points between the ynyl chain and the rest of the molecule, and between the ynyl chain and residues, can be through one carbon or any two carbons in the chain. Unless otherwise specified in the specification, the ynylyl group is optionally substituted.

[0067] "Alkyl ether" refers to any alkyl group as defined above, wherein at least one carbon-carbon bond is replaced by a carbon-oxygen bond. The carbon-oxygen bond may be terminal (e.g., alkoxy group) or internal (i.e., COC). Alkyl ethers include at least one carbon-oxygen bond, but may include more than one. For example, polyethylene glycol (PEG) includes the meaning of alkyl ether. Unless specifically stated otherwise in the specification, the alkyl ether group is optionally substituted. For example, in some embodiments, the alkyl ether is replaced with an alcohol or -OP (=R a (R) b )R c Replace, where R a R b and R c Each is defined as a compound with structure (I).

[0068] "Alkoxy" refers to the formula -OR a Group, wherein R a It is an alkyl group containing one to twelve carbon atoms as defined above. Unless otherwise specified in the specification, the alkoxy group is optionally substituted.

[0069] "Heteroalkylene" refers to an alkylene group as defined above, containing at least one heteroatom (e.g., Si, N, O, P, or S) within or at the end of the alkylene chain. In some embodiments, the heteroatom is within the alkylene chain (i.e., the heteroalkylene group contains at least one carbon-[heteroatom]-carbon bond, where x is 1, 2, or 3). In other embodiments, the heteroatom is at the end of the alkylene group and thus connects the alkylene group to the rest of the molecule (e.g., M). a -HAM b M a and M b Each is a separate part of the molecule (H is a heteroatom and A is an alkylene group). Unless otherwise specified in the specification, the heteroalkylene group is optionally substituted. Exemplary heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide), and the linking groups for "C linker," "HEG linker," and "PEG 1K linker" are as follows:

[0070]

[0071] The multimers of the aforementioned C-linker, HEG linker, and / or PEG 1K linker are included in various embodiments of the heteroalkylene linker. In some embodiments of the PEG 1K linker, n ranges from 19 to 25, for example, n is 19, 20, 21, 22, 23, 24, or 25. The multimer may comprise, for example, the following structures:

[0072]

[0073] Where x is 0 or a greater than 0 integer, for example, x is in the range of 0-100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0074] "Heteroalkenyl" is a heteroalkylene group as defined above, containing at least one carbon-carbon double bond. Unless otherwise specified in the specification, heteroalkenyl groups are optionally substituted.

[0075] "Heteroyneyl" is a heteroalkylene group containing at least one carbon-carbon triple bond. Unless otherwise specified in the specification, heteroalkylene groups are optionally substituted.

[0076] When referring to "heteroatom linkers," "heteroatom" refers to a linker group consisting of one or more heteroatoms. Exemplary heteroatom linkers include single atoms selected from O, N, P, and S, and multiple heteroatoms having, for example, the formula -P(O - (=O)O- or -OP(O) - )(=O)O- linkers and their polymers and combinations.

[0077] "Phosphate / ester" refers to -OP(=O)(R a )R b Group, wherein R a It is OH, O - OR c ;and R b It is OH, O - OR c Thiophosphate / ester group or other phosphate / ester group, wherein R c It is a balanced ion (e.g., Na+). + wait).

[0078] "Phosphoalkyl" refers to -OP (=O)(R a )R b Group, wherein R a It is OH, O - OR c ;and R b It is an -O alkyl group, wherein R c It is a balanced ion (e.g., Na+). +(etc.). Unless otherwise specifically stated in the specification, the phosphate alkyl group is optionally substituted. For example, in some embodiments, the -O alkyl portion of the phosphate alkyl group is optionally substituted with one or more hydroxyl, amino, hydrogen sulfide, phosphate / ester, thiophosphate / ester, phosphate alkyl, thiophosphate alkyl, phosphate alkyl ether or thiophosphate alkyl ether.

[0079] "Phosphoalkyl ether" refers to -OP(=O)(R a )R b Group, wherein R a It is OH, O - OR c ;and R b It is an -O alkyl ether, wherein R c It is a balanced ion (e.g., Na+). + (etc.). Unless otherwise specifically stated in the specification, the phosphate alkyl ether group is optionally substituted. For example, in some embodiments, the -O alkyl ether portion of the phosphate alkyl ether group is optionally substituted with one or more hydroxyl, amino, hydrogen sulfide, phosphate / ester, thiophosphate / ester, phosphate alkyl, thiophosphate alkyl, phosphate alkyl ether or thiophosphate alkyl ether.

[0080] "Thiophosphate / ester" refers to -OP (=R a (R) b )R c Group, wherein R a Is it O or S, R? b It is OH, O - S - OR d or SR d ;and R c It is OH, SH, O - S - OR d SR d phosphate / ester group or other thiophosphate / ester group, wherein R d It is a balanced ion (e.g., Na+). + (etc.) and the condition is: i) R a It is S; ii) R b It is S - or SR d ;iii)R c It's SH, S - or SR d ; or a combination of iv), i), ii) and / or iii).

[0081] "Thiophosphate alkyl" refers to -OP (=R) a (R) b )Rc Group, wherein R a Is it O or S, R? b It is OH, O - S - OR d or SR d ;and R c It is an -O alkyl group, wherein R d It is a balanced ion (e.g., Na+). + (etc.) and the condition is: i) R a It is S; ii) R b It is S - or SR d ; or iii)R a It is S and R b It is S - or SR d Unless otherwise specified in the specification, the thiophosphate alkyl group is optionally substituted. For example, in some embodiments, the -O alkyl portion of the thiophosphate alkyl group is optionally substituted with one or more hydroxyl, amino, hydrogen sulfide, phosphate / ester, thiophosphate / ester, phosphate alkyl, thiophosphate alkyl, phosphate alkyl ether or thiophosphate alkyl ether.

[0082] "Thiophosphate alkyl ether" refers to -OP (=R a (R) b )R c Group, wherein R a Is it O or S, R? b It is OH, O - S - OR d or SR d ;and R c It is an -O alkyl ether, wherein R d It is a balanced ion (e.g., Na+). + (etc.) and the condition is: i) R a It is S; ii) R b It is S - or SR d ; or iii)R a It is S and R b It is S - or SR d Unless otherwise specified in the specification, the thiophosphate alkyl ether group is optionally substituted. For example, in some embodiments, the -O alkyl ether portion of the thiophosphate alkyl group is optionally substituted with one or more hydroxyl, amino, hydrogen sulfide, phosphate / ester, thiophosphate / ester, phosphate alkyl, thiophosphate alkyl, phosphate alkyl ether or thiophosphate alkyl ether.

[0083] "Carbocyclic ring" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing 3 to 18 carbon atoms. Unless otherwise specified in the specification, the carbocyclic ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems, and may be partially or fully saturated. Non-aromatic carbocyclic residues include cycloalkyl groups, while aromatic carbocyclic residues include aryl groups. Unless otherwise specified in the specification, the carbocyclic group is optionally substituted.

[0084] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbon ring, which may include fused or bridged ring systems having three to fifteen carbon atoms, preferably three to ten carbon atoms, and is saturated or unsaturated and connected to the remainder of the molecule by a single bond. Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl groups include, for example, adamantyl, norcamphenyl, decahydronaphthyl, 7,7-dimethyl-bicyclo-[2.2.1]heptyl, etc. Unless otherwise specifically stated in the specification, cycloalkyl groups are optionally substituted.

[0085] "Aryl" refers to a ring system comprising at least one carbocyclic aromatic ring. In some embodiments, the aryl group comprises 6 to 18 carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems. Aryl groups include, but are not limited to, those derived from the following: anthracene, acenaphthene, phenanthrene, anthracene, azulene, benzene, benzoxene, fluoranthene, fluorene, asymmetric indole, symmetric indole, indene, indene, naphthalene, phenanthracene, heptamethrin, pyrene, and triphenylene. Unless otherwise specifically stated in the specification, the aryl group is optionally substituted.

[0086] "Heterocycle" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing one to twelve carbon atoms and one to six heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, a heterocycle can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heterocycle may optionally be oxidized; the nitrogen atom may optionally be quaternized; and the heterocycle may be partially or fully saturated. Examples of aromatic heterocycles are listed below in the definition of heteroaryl (i.e., heteroaryl is a subset of heterocycles). Examples of non-aromatic heterocycles include, but are not limited to, dioxacyclopentyl, thienyl[1,3]dithiaalkyl, decahydroisoquinolinyl, imidazolinyl, imidazoalkyl, isothiazolyl, isoxazolyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperidinyl, 2-oxopiperidinyl, 2-oxopiperylyl, oxazolyl, piperidinyl, piperazine, 4-piperidinoneyl, pyrrolylyl, pyrazolylyl, pyrazolopyrimidinyl, quininecycloyl, thiazoalkyl, tetrahydrofuranyl, trioxaneyl, trithiohexaneyl, triazineyl, tetrahydropyranyl, thiomorpholinyl, thiozamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless otherwise specified in the specification, the heterocyclic group is optionally substituted.

[0087] "Heteroaryl" refers to a 5- to 14-membered ring system comprising one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen, and sulfur, and at least one aromatic ring. For certain embodiments of this disclosure, the heteroaryl residue may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl residue may optionally be oxidized; the nitrogen atom may optionally be quaternized. Examples include, but are not limited to, azazyl, acridine, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxacyclopentadienyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxazolyl, 1,4-benzodioxane, benzonaphthofuranyl, benzooxazolyl, benzodioxacyclopentadienyl, benzodioxacyclohexadienyl, and benzopyranyl. Benzopyranone, benzofuran, benzofuranone, benzothiophene (benzothiophene), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, benzooxazolinone, benzoimidazolium thionyl, carbazole, cenyl, dibenzofuran, dibenzothiophene, furanyl, furanone, isothiazolyl, imidazoyl, indazole, indole, indazole, isoindole, indolinyl, iso... Indololinyl, isoquinolinyl, indazinyl, isoxazolyl, naphridinyl, oxadiazolyl, 2-oxoazapyridine, oxazolyl, vinyl oxide, 1-pyridinyl oxide, 1-pyrimidinyl oxide, 1-pyrazinyl oxide, 1-pyridazinyl oxide, 1-phenyl-1H-pyrroleyl, phenazinyl, phenothiazinyl, phenotoxazinyl, phthalazinyl, pteridinyl, pteridinoneyl, purine, pyrroleyl, pyrazolyl, pyridinyl, pyridinyl, pyridinoneyl, pyridinyl Zinyl, pyrimidinyl, pyrimidinoneyl, pyridazinyl, pyrroloyl, pyrido[2,3-d]pyrimidinoneyl, quinazolinyl, quinazolinoneyl, quinoxalinyl, quinoxalinoneyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-d]pyrimidin-4-oneyl, thieno[2,3-d]pyrimidin-4-oneyl, triazolyl, tetrazolyl, triazinyl, and thienyl (i.e., thienyl). Unless otherwise specified in the specification, heteroaryl groups are optionally substituted.

[0088] The term "-alkyl / ...alkyl" refers to a specific structural feature (e.g., alkyl, aryl, heteroalkyl) that is linked to the rest of the molecule by a single bond and to a residue by a single bond. In other words, the term "-alkyl / ...alkyl" refers to a specific structural feature with the meaning described herein, which is a linker between the molecule and a residue. The attachment point of the "-alkyl / ...alkyl" chain to the rest of the molecule and to the residue can be through one atom of the chain or any two atoms within the chain. For example, alkylene and heteroalkylene refer to a linker comprising both an alkylene moiety and a heteroalkylene moiety.

[0089] "Fused" refers to a ring system containing at least two rings, wherein the two rings share at least one common ring atom, such as two shared ring atoms. In the case of a heterocyclic or heteroaryl ring, the shared ring atom can be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tetracyclic, and so on.

[0090] "Conjugation" refers to the overlap of one p-orbital with another p-orbital across a σ bond. Conjugation can occur in cyclic or acyclic compounds. "Degree of conjugation" refers to the overlap of at least one p-orbital with another p-orbital across a σ bond. For example, 1,3-butadiene has one degree of conjugation, while benzene and other aromatic compounds generally have multiple degrees of conjugation. Fluorescent and coloring compounds generally contain at least one degree of conjugation.

[0091] "Fluorescence" refers to the ability of molecules to absorb light of a specific frequency and emit light of different frequencies. Fluorescence is well known to those skilled in the art.

[0092] "Coloring" refers to the absorption of light within the coloring spectrum by molecules (i.e., red, yellow, blue, etc.).

[0093] "FRET" refers to Förster resonance energy transfer, which is a physical interaction in which energy is transferred from the excitation of one part (e.g., the first chromophore or "donor") to an adjacent part (e.g., the second chromophore or "acceptor"). "FRET" is sometimes used interchangeably with fluorescence resonance energy transfer (i.e., when each chromophore is a fluorescent part). Generally, FRET requires (1) the excitation or absorption spectrum of the acceptor chromophore to overlap with the emission spectrum of the donor chromophore; (2) the transition dipole moments of the acceptor and donor chromophores to be substantially parallel (i.e., at about 0° or 180°); and (3) the acceptor and donor chromophores to be spatially proximate (i.e., close to each other). The energy transfer from the donor to the acceptor occurs through non-radiative dipole-dipole coupling, and the distance between the donor and acceptor chromophores is generally much smaller than the wavelength of light.

[0094] A "donor" or "donor chromophore" refers to a chromophore (e.g., a fluorophore) that is induced into an excited electronic state and can transfer its excitation or absorption energy to a nearby acceptor chromophore in a nonradiative manner through long-range dipole-dipole interactions. Without being theoretically constrained, it is believed that the energy transfer occurs because the oscillating dipoles of the respective chromophores have similar resonance frequencies. Donors and acceptors with these similar resonance frequencies are called "donor-acceptor pairs," which are used interchangeably with "FRET moieties," "FRET pairs," and "FRET dyes."

[0095] "Acceptor" or "acceptor chromophore" refers to a chromophore (e.g., a fluorophore) to which excitation or absorption energy from a donor chromophore is transferred via nonradiative transfer through long-range dipole-dipole interactions.

[0096] "Stokes shift" refers to the difference in the position (e.g., wavelength) of the band maximum between the excitation or absorption spectrum and the emission spectrum of an electronic transition (e.g., from an excited state to an unexcited state or vice versa). In some embodiments, the compound has a Stokes shift greater than 25 nm, greater than 30 nm, greater than 35 nm, greater than 40 nm, greater than 45 nm, greater than 50 nm, greater than 55 nm, greater than 60 nm, greater than 65 nm, greater than 70 nm, greater than 75 nm, greater than 80 nm, greater than 85 nm, greater than 90 nm, greater than 95 nm, greater than 100 nm, greater than 110 nm, greater than 120 nm, greater than 130 nm, greater than 140 nm, greater than 150 nm, greater than 160 nm, greater than 170 nm, greater than 180 nm, greater than 190 nm, or greater than 200 nm.

[0097] The "J-value" is calculated as the integral of the spectral overlap between the emission spectrum of the donor chromophore and the excitation or absorption spectrum of the acceptor chromophore. The emission spectrum of the donor chromophore is a spectrum produced when the donor chromophore is excited with a preferred excitation or absorption wavelength. The preferred excitation or absorption wavelengths of the donor chromophores are located at or near their respective maximum excitation or absorption wavelengths, which are well known to those skilled in the art (e.g., Pacific Blue has a maximum excitation or absorption at about 401 nm, and FITC has a maximum excitation or absorption at about 495 nm). Exemplary examples of the "J-value" are shown herein. Figure 1 .

[0098] A "linker" is a continuous chain of at least one atom, such as carbon, oxygen, nitrogen, sulfur, phosphorus, or combinations thereof, that connects a portion of a molecule to another portion of the same molecule or to different molecules, parts, or solid carriers (e.g., particles). Linkers can connect molecules via covalent bonds or other means, such as ionic bonds or hydrogen bonds.

[0099] "Physiological conditions" refers to a solution or medium having a temperature ranging from about 20 to 40°C, an atmospheric pressure of about 1 atm (101 kPa or 14.7 psi), a pH of about 6 to 8, a glucose concentration of about 1 to 20 mM, an atmospheric oxygen concentration, and / or gravity. "Physiological conditions" includes solutions or media having a subset of these properties (e.g., a temperature ranging from 20 to 40°C and a pH of about 6 to 8). The conditions may also include buffer components or systems, including but not limited to phosphates, bicarbonates, hemoglobin, and / or proteins.

[0100] The term "biomolecule" refers to any variety of biological substances, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers, and mixtures thereof. More specifically, the term is intended to include, but is not limited to, RNA, DNA, oligonucleotides, modified or derived nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), antibodies, antigens and toxins, as well as bacteria, viruses, blood cells, and tissue cells. The visually detectable biomolecules of this disclosure (e.g., compounds having the structure (I), (II), (III), or (IV) of the biomolecule to which they are attached) are prepared as follows: the biomolecule is contacted with a compound having reactive groups such that the biomolecule can be attached to the compound via any available atom or functional group on the biomolecule, such as an amino, hydroxyl, carboxyl, or hydrogen sulfide group, as further described herein.

[0101] A "reactive group" is a part capable of reacting with a second reactive group (e.g., a "complementary reactive group") to form one or more covalent bonds, for example, through substitution, oxidation, reduction, addition, or cycloaddition reactions. Exemplary reactive groups are provided in Table 1 and include, for example, nucleophiles, electrophiles, dienes, dienophiles, aldehydes, oximes, hydrazones, alkynes, amines, azides, acyl azides, acyl halides, nitriles, nitrile ketones, hydrogen sulfides, disulfides, sulfonyl halides, isothiocyanates, imine esters, activated esters, ketones, α,β-unsaturated carbonyl groups, alkenes, maleimides, α-haloimides, epoxides, aziridines, tetrazides, tetrazolium, phosphine, biotin, thiocyclopropane, etc.

[0102] "Bioconjugation" or "bioconjugation reaction," and related variations, refers to a chemical reaction that forms a stable covalent bond between two molecules. The term "bioconjugated compound" is generally used when one of the molecules is a biological molecule (e.g., an antibody), but it can also be used to describe the formation of a covalent bond with a non-biological molecule (e.g., a polymer resin). The product or compound obtained by the above reaction strategy is a "conjugated compound," "bioconjugated compound," or its grammatical equivalent.

[0103] The terms "visible" and "visually detectable" are used herein to refer to a substance that can be observed by visual inspection without prior light exposure or chemical or enzymatic activation. The visually detectable substance absorbs and emits light in the spectral range of about 300 to about 900 nm. Preferably, the substance is strongly colored, preferably having at least about 40,000, more preferably at least about 50,000, even more preferably at least about 60,000, still more preferably at least about 70,000, and most preferably at least about 80,000 nm. -1 cm -1The molar extinction coefficient. The compounds disclosed herein can be detected by visual observation or by means of optical detection devices, including but not limited to absorption spectrophotometers, transmission optical microscopes, digital cameras, and scanners. Visually detectable substances are not limited to those that emit and / or absorb visible light. Substances that emit and / or absorb light in the ultraviolet (UV) region (about 10 nm to about 400 nm), infrared (IR) region (about 700 nm to about 1 mm), and other regions of the electromagnetic spectrum are also included in the scope of "visually detectable" substances.

[0104] For the purposes of this disclosure, the term "photostable visual dye" refers to a chemical component that is visually detectable as defined above and does not significantly change or decompose upon exposure to light. Preferably, the photostable visual dye does not exhibit significant bleaching or decomposition after exposure to light for at least 1 hour. More preferably, the visual dye is stable after exposure to light for at least 12 hours, even more preferably at least 24 hours, still more preferably at least 1 week, and most preferably at least 1 month. Non-limiting examples of photostable visual dyes suitable for the compounds and methods of this disclosure include azo dyes, indigo dyes, quinacridone pigments, dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole, quinophthalone, and truarycarbonium.

[0105] As used herein, the term "perylene derivative" is intended to include any visually detectable substituted perylene. However, the term is not intended to include perylene itself. The terms "anthracene derivative," "naphthalene derivative," and "pyrene derivative" are used similarly. In some preferred embodiments, the derivative (e.g., perylene, pyrene, anthracene, or naphthalene derivative) is an imide, diimide, or hydrazide imide derivative of perylene, anthracene, naphthalene, or pyrene.

[0106] The polymer compounds of various embodiments of this disclosure are intended for a wide range of analytical applications, such as biochemical and biomedical applications, where it is necessary to determine the presence, location, spatial interactions, or amount of a particular analyte (e.g., a biomolecule). Therefore, in another aspect, this disclosure provides a method for visually detecting biomolecules, comprising: (a) providing a biological system having visually detectable biomolecules, said molecules comprising compounds of the embodiments disclosed herein (e.g., compounds of structures (I), (II), (III), or (IV)) linked to the biomolecules; and (b) detecting the biomolecules by their visual properties. For the purposes of this disclosure, the phrase "detecting said biomolecules by their visual properties" means observing the biomolecules with the naked eye or with the aid of optical detection devices, including but not limited to absorption spectrophotometers, transmission optical microscopes, digital cameras, and scanners, without illumination or chemical or enzymatic activation. Densimeters can be used to quantify the amount of visually detectable biomolecules present. For example, the relative amounts of biomolecules in two samples can be measured by measuring relative optical density. If the stoichiometry of the dye / biomolecule is known, and the extinction coefficient of the dye molecule is known, then the absolute concentration of the biomolecule can also be determined from a measurement of optical density. As used herein, the term "biological system" refers to any solution or mixture containing one or more biomolecules in addition to visually detectable biomolecules. Non-limiting examples of said biological systems include cells, cell extracts, tissue samples, electrophoresis gels, test mixtures, and hybridization reaction mixtures.

[0107] "Solid carrier" or "solid carrier residue" means any solid substrate known in the art for molecular solid-phase carriers. For example, "microparticles" means any various small particles used to attach to the compounds disclosed herein, including but not limited to glass beads, magnetic beads, polymer beads, non-polymer beads, etc. In some embodiments, the microparticles comprise polystyrene beads.

[0108] A "base-pairing moiety" refers to a heterocyclic moiety capable of hybridizing with a complementary heterocyclic moiety via hydrogen bonds (e.g., Watson-Crick base pairing). Base-pairing moieties include both natural and non-natural bases. Non-limiting examples of base-pairing moieties are RNA and DNA bases such as adenosine, guanosine, thymidine, cytosine, and uridine and their analogues.

[0109] Embodiments of this disclosure are also intended to cover all isotopically labeled structural (I) or (II) compounds, wherein one or more atoms are replaced by atoms with different atomic masses or mass numbers. Examples of isotopes capable of being incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, respectively, for example... 2 H, 3 H, 11 C, 13 C, 14C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I.

[0110] Isotope-labeled structural (I) or (II) compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes similar to those described below and in the examples below, replacing the previously used unlabeled reagent with an appropriately isotope-labeled reagent.

[0111] "Stable compound" and "stable structure" refer to compounds that are robust enough to undergo separation from the reaction mixture to a useful degree of purity and to be formulated as an effective therapeutic agent.

[0112] "Optional" or "optionally" means that the event or environment subsequently described may or may not occur, and the description includes both the occurrence and non-occurrence of said event or environment. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted and unsubstituted alkyl groups.

[0113] "Salt" includes acid and base addition salts.

[0114] "Acid addition salts" refer to salts formed with inorganic acids, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, and salts formed with organic acids, such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, camphoric acid, camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, viscous acid, and gentian. Acids, including glucoheponic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, 2-oxoglutamate, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, mesylate, mucoacid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-para-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, undecenoic acid, etc.

[0115] "Base addition salts" refer to salts prepared by adding inorganic or organic bases to free acids. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary, and tertiary amines; substituted amines, including naturally substituted amines; cyclic amines; and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dianophenoxylate, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, ammonium salts, phenethylbenzylamine, N,N'-dibenzylethylenediamine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. The particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0116] Crystallization can produce solvates of the compounds described herein. Embodiments of this disclosure include all solvates of the described compounds. As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the disclosed compound and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of this disclosure can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., and corresponding solvated forms. The compounds of this disclosure may be true solvates, while in other cases the compounds of this disclosure may retain only foreign water or other solvents, or a mixture of water and some foreign solvents.

[0117] Embodiments of the disclosed compounds (e.g., compounds of structures I, II, III, or IV) or their salts, tautomers, or solvates may contain one or more stereocenters and may thus produce enantiomers, diastereomers, and other stereoisomers, which may be defined in absolute stereochemistry as (R)- or (S)- or (D)- or (L)- for amino acids. Embodiments of this disclosure are intended to include all of the said possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and stepwise crystallization. Conventional techniques used to prepare / separate individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). In the case of compounds described herein containing an olefinic double bond or other geometrically asymmetric centers, and unless otherwise specified, it is desirable that the compounds include E and Z geometric isomers. Similarly, it is desirable that all tautomer forms be included.

[0118] "Stereoisomers" are compounds composed of identical atoms bonded by the same bonds, but having different three-dimensional structures that are not interchangeable. This disclosure contemplates various stereoisomers and mixtures thereof, including "enantiomers," which refer to two stereoisomers whose molecules are mirror images of each other that are not superimposed.

[0119] "Tautomerism" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This disclosure includes tautomerisms of any of the said compounds. Various tautomer forms of the compounds can be readily derived by those skilled in the art.

[0120] The chemical nomenclature and structural diagrams used in this paper are modified forms of the IUPAC nomenclature system, and the nomenclature was performed using ACD / Name version 9.07 software and / or ChemDraw Ultra version 11.0 software (CambridgeSoft). Common names familiar to those skilled in the art are also used.

[0121] As described above, in one embodiment of this disclosure, compounds are provided for use as FRETs, fluorescent dyes, and / or coloring dyes in various analytical methods. In other embodiments, compounds are provided as synthetic intermediates for preparing compounds used as FRETs, fluorescent dyes, and / or coloring dyes. In general terms, embodiments of this disclosure relate to dimers, trimers, and higher polymers of FRET, fluorescent, and / or coloring moieties. The FRET, fluorescent, and / or coloring moieties are linked by linking portions. Without being theoretically limited, it is believed that linkers help maintain sufficient spatial distance / proximity between donor-acceptor pairs to reduce or eliminate intramolecular quenching, and maintain sufficient proximity to facilitate nonradiative energy transfer.

[0122] Accordingly, in some embodiments the compound has the following structure (A):

[0123]

[0124] (A)

[0125] Where L is a linker sufficient to maintain steric separation between one or more (e.g., each) M groups, thereby reducing or eliminating intramolecular quenching, and R... 1 R 2 R 3 L 1 L 2 L 3 and n are defined as in structures (I), (II), (III), or (IV).

[0126] In other embodiments, compounds having the following structure (I) are provided:

[0127]

[0128] (I)

[0129] Or its stereoisomers, salts or tautomers, wherein:

[0130] M 1 and M 2 Each specific value of is an independent chromophore, provided that M . 1 and M 2 At least one of them is a FRET donor, and M 1 and M 2 The other one is the corresponding FRET receptor;

[0131] L 1 The specific values ​​of are arbitrary connectives, provided that L 1 At least one specific value is that oxygen is present and is contained;

[0132] L 2 and L3 Each specific value is independently an optional alkylene, alkenylene, ynylene, heteroalkylene, heteroalkenylene, heteroynylene, or heteroatom linker;

[0133] L 4 Each specific value is independently an alkylene or heteroalkylene linker;

[0134] R 1 Each specific value is independently H, alkyl, or alkoxy;

[0135] R 2 and R 3 Each of these can be independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP (=R) a (R) b )R c Q, or its protected form, or L';

[0136] R 4 The specific values ​​are independently OH, SH, and O. - S - OR d or SR d ;

[0137] R 5 Each specific value is independently oxidized, thiolated, or absent;

[0138] R a Is it O or S?

[0139] R b It is OH, SH, O - S - OR d or SR d ;

[0140] R c It is OH, SH, O - S - OR d ,OL',SR d Alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate / ester, thiophosphate / ester, phosphate-alkyl, thiophosphate-alkyl, phosphate-alkyl ether or thiophosphate-alkyl ether;

[0141] R d They are balanced ions;

[0142] Each specific value of Q is independently a portion containing a reactive group or its protected form, the reactive group being capable of forming a covalent bond with the analyte molecule, the target portion, the solid support, or the complementary reactive group Q';

[0143] Each specific value of L' is independently a linker containing a covalent bond with Q, a linker containing a covalent bond with the target moiety, a linker containing a covalent bond with the analyte molecule, a linker containing a covalent bond with the solid support, a linker containing a covalent bond with the solid support residue, a linker containing a covalent bond with the nucleoside, or a linker containing a covalent bond with another structure (I) compound.

[0144] Each specific value of m is an independent integer of 0 or greater; and

[0145] n is an integer of 1 or greater.

[0146] In a more specific implementation, in M 1 and M 2 L, when selected from fluorescein, pyrene and perylene 4 At least one specific value is a heteroalkylene group.

[0147] Some other embodiments provide compounds having the following structure (II):

[0148]

[0149] (II)

[0150] Or its stereoisomers, salts or tautomers, wherein:

[0151] M 1 and M 3 Each specific value of is an independent chromophore, provided that M . 1 and M 3 At least one of them is a FRET donor, and M 1 and M 3 The other one is the corresponding FRET receptor;

[0152] L 1 L 7a and L 7b Each specific value is an independently optional connector;

[0153] L 2 L 3 L 5 and L 6 Each specific value is independently an optional alkylene, alkenylene, ynylene, heteroalkylene, heteroalkenylene, heteroynylene, or heteroatom linker;

[0154] L 4 Each specific value is independently an alkylene or heteroalkylene linker;

[0155] R 1 Each specific value is independently H, alkyl, or alkoxy;

[0156] R 2 and R 3 Each of these can be independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP (=R) a (R) b )R c Q, or its protected form, or L';

[0157] R 4 The specific values ​​are independently OH, SH, and O. - S - OR d or SR d ;

[0158] R 5 Each specific value is independently oxidized, thiolated, or absent;

[0159] R a Is it O or S?

[0160] R b It is OH, SH, O - S - OR d or SR d ;

[0161] R c It is OH, SH, O - S - OR d ,OL',SR d Alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate / ester, thiophosphate / ester, phosphate-alkyl, thiophosphate-alkyl, phosphate-alkyl ether or thiophosphate-alkyl ether;

[0162] R d They are balanced ions;

[0163] Each specific value of Q is independently a portion containing a reactive group or its protected form, the reactive group being capable of forming a covalent bond with the analyte molecule, the target portion, the solid support, or the complementary reactive group Q';

[0164] Each specific value of L' is independently a linker containing a covalent bond with Q, a linker containing a covalent bond with the target moiety, a linker containing a covalent bond with the analyte molecule, a linker containing a covalent bond with the solid support, a linker containing a covalent bond with the solid support residue, a linker containing a covalent bond with the nucleoside, or a linker containing a covalent bond with another compound of structure (II).

[0165] Each specific value of m is an independent integer of 0 or greater; and

[0166] n is an integer of 1 or greater;

[0167] q is an integer of 0 or greater, provided that at least one specific value of q is an integer of 1 or greater; and

[0168] w is an integer of 0 or greater, provided that at least one specific value of q is an integer of 1 or greater.

[0169] Some other embodiments provide compounds having the following structure (III):

[0170]

[0171] (III)

[0172] Or its stereoisomers, salts or tautomers, wherein:

[0173] M 1 and M 2 Each specific value of is an independent chromophore, provided that M . 1 At least two specific values ​​of M are independently FRET donors, and M 2 At least one specific value is related to M 1 At least two specific values ​​each form a FRET receptor for a FRET pair;

[0174] L 1 The specific values ​​of are arbitrary connectives, provided that L 1 At least one specific value is that oxygen is present and is contained;

[0175] L 2 and L 3 Each specific value is independently an optional alkylene, alkenylene, ynylene, heteroalkylene, heteroalkenylene, heteroynylene, or heteroatom linker;

[0176] L 4 Each specific value is independently an alkylene or heteroalkylene linker;

[0177] L 8 Each specific value is an independent rigid connection.

[0178] R 1 Each specific value is independently H, alkyl, or alkoxy;

[0179] R 2 and R 3 Each of these can be independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP (=R) a (R) b )R c Q, or its protected form, or L';

[0180] R 4 The specific values ​​are independently OH, SH, and O. - S - OR d or SR d ;

[0181] R 5 Each specific value is independently oxidized, thiolated, or absent;

[0182] R a Is it O or S?

[0183] R b It is OH, SH, O - S - OR d or SR d ;

[0184] R c It is OH, SH, O - S - OR d ,OL',SR d Alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate / ester, thiophosphate / ester, phosphate-alkyl, thiophosphate-alkyl, phosphate-alkyl ether or thiophosphate-alkyl ether;

[0185] R d They are balanced ions;

[0186] Each specific value of Q is independently a portion containing a reactive group or its protected form, the reactive group being capable of forming a covalent bond with the analyte molecule, the target portion, the solid support, or the complementary reactive group Q';

[0187] Each specific value of L' is independently a linker containing a covalent bond with Q, a linker containing a covalent bond with the target moiety, a linker containing a covalent bond with the analyte molecule, a linker containing a covalent bond with the solid support, a linker containing a covalent bond with the solid support residue, a linker containing a covalent bond with the nucleoside, or a linker containing a covalent bond with another compound of structure (III).

[0188] Each specific value of m is an independent integer of 0 or greater; and

[0189] n is an integer of 1 or greater.

[0190] In a more specific implementation, in M 1 and M 2 L, when selected from fluorescein, pyrene and perylene 4 At least one specific value is a heteroalkylene group.

[0191] One specific embodiment provides a compound having the following structure (IV):

[0192]

[0193] (IV)

[0194] Or its stereoisomers, salts or tautomers, wherein:

[0195] M 1 and M 3 Each specific value of is an independent chromophore, provided that M . 1 At least two specific values ​​of M are independently FRET donors, and M 3 At least one specific value is related to M 1 At least two specific values ​​each form a FRET receptor for a FRET pair;

[0196] L 1 L 7a and L 7b Each specific value is an independently optional connector;

[0197] L 2 L 3 L 5 and L 6 Each specific value is independently an optional alkylene, alkenylene, ynylene, heteroalkylene, heteroalkenylene, heteroynylene, or heteroatom linker;

[0198] L 4 Each specific value is independently an alkylene or heteroalkylene linker;

[0199] L 8 Each specific value is an independent rigid connection.

[0200] R 1 Each specific value is independently H, alkyl, or alkoxy;

[0201] R 2 and R 3 Each of these can be independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP (=R) a (R) b )R c Q, or its protected form, or L';

[0202] R 4 The specific values ​​are independently OH, SH, and O. - S - OR d or SR d ;

[0203] R 5 Each specific value is independently oxidized, thiolated, or absent;

[0204] R a Is it O or S?

[0205] R b It is OH, SH, O - S - OR d or SR d ;

[0206] R c It is OH, SH, O - S - OR d ,OL',SR d Alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate / ester, thiophosphate / ester, phosphate-alkyl, thiophosphate-alkyl, phosphate-alkyl ether or thiophosphate-alkyl ether;

[0207] R d They are balanced ions;

[0208] Each specific value of Q is independently a portion containing a reactive group or its protected form, the reactive group being capable of forming a covalent bond with the analyte molecule, the target portion, the solid support, or the complementary reactive group Q';

[0209] Each specific value of L' is independently a linker containing a covalent bond with Q, a linker containing a covalent bond with the target moiety, a linker containing a covalent bond with the analyte molecule, a linker containing a covalent bond with the solid support, a linker containing a covalent bond with the solid support residue, a linker containing a covalent bond with the nucleoside, or a linker containing a covalent bond with another structure (IV) compound.

[0210] Each specific value of m is an independent integer of 0 or greater; and

[0211] n is an integer of 1 or greater.

[0212] In some of the foregoing embodiments, L 4 At least one specific value is a heteroalkylene group. In some embodiments, L 4 The specific values ​​are heteroalkylene groups. In more specific embodiments, the heteroalkylene group comprises olefin oxides. In some related embodiments, the heteroalkylene group comprises ethylene oxides.

[0213] In the implementation, L 4At least one specific value is an alkylene group. In some more specific embodiments, at least one alkylene group is an ethylene group. In some embodiments, each specific value of the alkylene group is an ethylene group.

[0214] In some more specific embodiments, the compound has the following structure (IA):

[0215]

[0216] (IA)

[0217] in:

[0218] Each specific value of z is an integer independently between 1 and 100; and

[0219] Each specific value of m is an integer from 0 to 6.

[0220] In other embodiments, the compound has the following structure (IIA):

[0221]

[0222] (IIA)

[0223] in:

[0224] Each specific value of z is an integer independently between 1 and 100; and

[0225] Each specific value of m is an integer from 0 to 6.

[0226] In some embodiments, the compound has the following structure (IIIA):

[0227]

[0228] (IIIA)

[0229] in:

[0230] L 4 It is -(OCH2CH2) z -;

[0231] Each specific value of z is an integer independently between 1 and 100; and

[0232] Each specific value of m is an integer from 0 to 6.

[0233] In some other embodiments, the compound has the following structure (IVA):

[0234]

[0235] (IVA)

[0236] in:

[0237] L 4 It is -(OCH2CH2) z -;

[0238] Each specific value of z is an integer independently between 1 and 100; and

[0239] Each specific value of m is an integer from 0 to 6.

[0240] In some embodiments, the specific values ​​of z are independently integers from 1 to 30. In some embodiments, the specific values ​​of z are independently 3 or 6. In other embodiments, the specific values ​​of m are independently integers from 2 to 4. In some embodiments, the specific values ​​of m are 2.

[0241] In some more specific embodiments, the compound has the following structure (IB):

[0242]

[0243] (IB)

[0244] in:

[0245] x 1 x 2 x 3 and x 4 Each specific value is an integer from 0 to 6.

[0246] In some embodiments, the compound has the following structure (IIB):

[0247]

[0248] (IIB)

[0249] in:

[0250] x 1 x 2 x 3 and x 4 Each specific value is an integer from 0 to 6.

[0251] In some more specific embodiments, the compound has the following structure (IIIB):

[0252]

[0253] (IIIB)

[0254] in:

[0255] x 1 x 2 x 3x 4 x 5 and x 6 Each specific value is an integer from 0 to 6.

[0256] In some embodiments, the compound has the following structure (IVB):

[0257]

[0258] (IVB)

[0259] in:

[0260] x 1 x 2 x 3 ,x 4 x 5 and x 6 Each specific value is an integer from 0 to 6.

[0261] In some implementations, one or more specific values ​​of z are integers from 3 to 6. In certain specific implementations, x 1 and x 3 All specific values ​​of x are 0, and x 2 and x 4 Each specific value of x is 1. In some implementations, x 1 x 2 x 3 and x 4 Each of its specific values ​​is 1.

[0262] In some implementations, x 1 x 3 and x 5 All specific values ​​of x are 1, and x 2 x 4 and x 6 All specific values ​​of x are 0. In some implementations, x 1 x 2 x 3 x 4 x 5 and x 6 Each of its specific values ​​is 1.

[0263] In one particular implementation, L 4 It has the following structure:

[0264]

[0265] in:

[0266] Each specific value of p is an independent integer from 0 to 6; and

[0267] Each specific value of y is an integer from 1 to 100.

[0268] In some implementations, R 3 It includes the following structure:

[0269]

[0270] in:

[0271] Each specific value of p is an independent integer from 0 to 6; and

[0272] Each specific value of y is an integer from 1 to 100.

[0273] Various linkers and substituents (e.g., M) in compounds with structures (I), (II), (III), or (IV) 1 M 2 M 3 Q, R 1 R 2 R 3 R c L 1 L 2 L 3 L 4 L 5 L 5 L 6 L 7a L 7b R 8 The compounds may be optionally substituted with one or more substituents. For example, in some embodiments, optional substituents are selected to optimize the water solubility or other properties of the compounds of structures (I), (II), (III), or (IV). In some embodiments, each alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphate alkyl, thiophosphate alkyl, phosphate alkyl ether, and thiophosphate alkyl ether in the compounds of structures (I), (II), (III), or (IV) may be optionally substituted with one or more substituents selected from hydroxyl, alkoxy, alkyl ether, alkoxyalkyl ether, hydrogen sulfide, amino, alkylamino, carboxyl, phosphate / ester, thiophosphate / ester, phosphate alkyl, thiophosphate alkyl, phosphate alkyl ether, and thiophosphate alkyl ether.

[0274] In some implementations, L 1 or L 7b Each specific value is independently an optional alkylene, alkenylene, ynylene, heteroalkylene, heteroalkenylene, heteroynylene, or heteroatom linker. In some embodiments, L 1 or L 7bIt is a linker containing functional groups capable of being formed by the reaction of two complementary reactive groups (e.g., azide and alkyne). In some embodiments, L 1 or L 7b Each specific value is independently optional: alkylene, alkenylene, ynylene, heteroalkylene, heteroalkenylene, heteroynylene, alkyleneheteroarylalkylene, alkyleneheterocyclicalkylene, alkylenecarbocyclicalkylene, heteroalkyleneheteroarylalkylene, heteroalkyleneheterocyclicalkylene, heteroalkylenecarbocyclicalkylene, heteroalkyleneheteroarylheteroalkylene, heteroalkyleneheterocyclicheteroalkylene, heteroalkyleneheterocyclicheteroalkylene, heteroalkylenecarbocyclicheteroalkylene. Alkylene, alkyleneheteroarylene, alkyleneheterocyclic, alkylenecarbamocyclic, heteroarylene, heterocyclic, carbamocyclic, alkyleneheteroarylene, alkyleneheterocyclic, heteroarylene, alkylenecarbamocyclic, carbamocyclic, heteroarylene, heteroarylene, heteroarylene, heteroarylene, heteroarylene, carbamocyclic, carbamocyclic, or heteroatom linkers. 1 or L 7b It is optional and can be replaced.

[0275] Optional connector L 1 and L 7b Can be used as M 1 M 2 and M 3 The attachment point between part and the rest of the compound. For example, in some embodiments, synthetic precursors of compounds with structures (I), (II), (III), or (IV) are prepared, and M is attached using any kind of readily available method known in the art, such as a method called "click chemistry". 1 M 2 and / or M 3 Partially linked to the synthetic precursor. Any rapid and substantially irreversible reaction intended to this end can be used to connect M... 1 M 2 Or M 3 Or both may attach to the synthetic precursor to form compounds of structure (I), (II), (III), or (IV). Exemplary reactions include copper-catalyzed reactions of azides and alkynes to form triazoles (Huisgen 1,3-dipolar cycloaddition), reactions of dienes and dienophiles (Diels-Alder), strain-promoted acetylene-nitroketone cycloaddition, reactions of strained alkenes with azides, tetrazides, or tetraazoles, [3+2] cycloaddition of alkenes and azides, reverse-demand Diels-Alder reactions of alkenes and tetrazides, photoreactions of alkenes and tetraazoles, and various substitution reactions, such as the replacement of leaving groups by nucleophilic attack on electrophilic atoms. In some embodiments, L is formed 1 or L 7bThe reaction can take place in an aqueous environment.

[0276] Accordingly, in some implementations L 1 or L 7b The specific values ​​are linkers containing functional groups that can be formed through the reaction of two complementary reactive groups, such as functional groups of products of one of the aforementioned "click" reactions. In various embodiments, for L 1 or L 7b At least one specific value of the functional group, which can be formed by the reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, hydrogen sulfide, disulfide, sulfonyl halide, isothiocyanate, imine ester, activated ester, ketone, α,β-unsaturated carbonyl, olefin, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazolium, phosphine, biotin, or thiacyclopropane functional group with a complementary reactive group.

[0277] In other embodiments, for L 1 or L 7b At least one specific value, the functional group can be formed by the reaction of alkyne and azide.

[0278] In other implementations, for L 1 or L 7b At least one specific value, the functional group comprises an olefin, ester, amide, thioester, disulfide, carbocyclic, heterocyclic, or heteroaryl group. In some more specific embodiments, for L 1 or L 7b At least one specific value, L 1 or L 7b It is a linker containing a triazole functional group.

[0279] In some implementations, L 7b -M 3 At least one specific value of contains the following structure:

[0280]

[0281] Where L a and L b Each is an optional connector, independent of the others.

[0282] In some implementations, L 1 -M 1 or L 1 -M 2 At least one specific value of contains one of the following structures:

[0283] or

[0284] Where L a and L b Each is an optional connector, independent of the others.

[0285] In some implementations, L 7b -M 3 At least one specific value of contains the following structure:

[0286]

[0287] Where L a and L b Each is an optional connector, independent of the others.

[0288] In some implementations, L a or L b It may be either yes or neither. In some implementations, L a or L b Yes, or both exist. In some more specific implementations, L a and L b When present, each is independently an alkylene or heteroalkylene. In other embodiments, L a and L b Independently possessing one of the following structures:

[0289] ; or .

[0290] In other different implementations, L 1 or L 7b Each specific value is independently an optional alkylene or heteroalkylene linker.

[0291] In some implementations, L 1 At least one specific value of has one of the following structures:

[0292] ; ; or ,

[0293] in

[0294] a, b, and c are each an integer in the range 1-6.

[0295] In some implementations, L 1 Each specific value has one of the following structures:

[0296] ; ; or ,

[0297] in

[0298] a, b, and c are each an integer in the range 1-6.

[0299] In some implementations, L 1 At least one specific value of has one of the following structures:

[0300] ; ; or .

[0301] In some implementations, L 1 Each specific value has one of the following structures:

[0302] ; ; or .

[0303] In some implementations, L 7a It includes optionally substituted 5-7 member heteroaryl linkers. In some embodiments, L 7a Each specific value is independently an optionally substituted 5-7 member heteroaryl linker. In some more specific embodiments, L 7a It is a 6-membered heteroaryl group. In some more specific embodiments, L 7a It contains 2 N atoms and 2 O atoms. In some embodiments, L 7a The specific values ​​of L are replaced. In some implementations, L 7a It is substituted with at least one oxygen. In some more specific embodiments, L 7a It has one of the following structures:

[0304]

[0305] In other embodiments, R 4 The specific values ​​of OH and O are independent. - OR d The meaning of "OR" should be understood. d "and"SR d "Expected to refer to O that binds to cations" - and S - For example, a disodium salt of a phosphate group can be represented as:

[0306] ,

[0307] Where R a It is sodium (Na) + ).

[0308] In other embodiments, R 5 The specific values ​​are for oxygenation.

[0309] In some different embodiments of any of the aforementioned compounds, R 1 It's H.

[0310] In various other implementations, R 2 and R 3 Each is independently OH or -OP (=R) a (R) b )R c In some different implementations, R 2 Or R 3 Is it OH or -OP (=R) a (R) b )R c And R 2 or R 3 The other is Q or a linker containing a covalent bond with Q. In some implementations, R 2 and R 3 Each is independently -OP(=R) a (R) b )R c In certain specific implementations, R c It is OL'. In some of those embodiments, L' is a heteroalkylene linker connected to: Q, the targeting moiety, the analyte molecule, the solid support, the solid support residue, the nucleoside, or other compound with structures (I), (II), (III), or (IV). In some embodiments, L' comprises an oxidized olefin or a phosphodiester moiety, or a combination thereof. In some embodiments, L' has the following structure:

[0311] ,

[0312] in:

[0313] m'' and n'' are independent integers from 1 to 10;

[0314] R e It is H, an electron pair or a balanced ion;

[0315] L'' is R e Or with the following direct links or linkers: Q, target moiety, analyte molecule, solid support, solid support residue, nucleoside or another compound of structure (I), (II), (III) or (IV).

[0316] In other embodiments, specific values ​​of Q are independently portions containing reactive groups capable of forming covalent bonds with analyte molecules or solid supports (e.g., controlled porous glass or polystyrene beads). In other embodiments, specific values ​​of Q are independently portions containing reactive groups capable of forming covalent bonds with complementary reactive groups Q'. For example, in some embodiments, Q' is present on another compound with structure (I), (II), (III), or (IV) (e.g., R). 2 or R 3 The reaction of compound (I), (II), (III), or (IV) with another compound (I), (II), (III), or (IV) results in a dimer of compound (I), (II), (III), or (IV) covalently bonded. Polymeric compounds (I), (II), (III), or (IV) can also be prepared in a similar manner and included within the scope of embodiments of this disclosure.

[0317] The type of Q group and the connectivity of the Q group with the rest of the compound in structure (I), (II), (III), or (IV) are not particularly restricted, provided that Q contains a portion with suitable reactivity to form the desired bond.

[0318] In some embodiments, Q is the portion that is not easily hydrolyzed under aqueous conditions, but is sufficiently reactive to bond with corresponding groups (e.g., amines, azides, or alkynes) on analyte molecules (e.g., biomolecules) or solid supports.

[0319] Some embodiments of compounds with structures (I), (II), (III), and / or (IV) include a Q group generally used in the field of bioconjugates. For example, in some embodiments, Q comprises a nucleophilic, electrophilic, or cycloaddition reactive group. In some more specific embodiments, Q comprises a thiosulfate, disulfide, activated ester, isothiocyanate, azide, alkyne, olefin, diene, dienophile, acyl halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group. In some embodiments, the activated ester is an N-succinimide ester, an imine ester, or a polyfluorophenyl ester. In other embodiments, the alkyne is an alkyl azide or an acyl azide. In some embodiments, Q comprises a maleimide functional group.

[0320] The exemplary Q section is provided in Table I below.

[0321] Table 1. Exemplary Q Section

[0322]

[0323]

[0324] It should be noted that in some embodiments in which Q is SH, the SH portion will tend to form a disulfide bond with another hydrogen sulfide group on another compound of structure (I), (II), (III), or (IV). Accordingly, some embodiments include compounds of structure (I), (II), (III), or (IV) in the form of a disulfide dimer, the disulfide bond being derived from the SH Q group.

[0325] In some other implementations, R 2 or R 3 One of them is OH or -OP (=R) a (R) b )R c , and R 2 or R 3 Another component is a linker containing a covalent bond with the analyte molecule or a linker containing a covalent bond with a solid carrier. For example, in some embodiments, the analyte molecule is a nucleic acid or a polymer thereof, or an amino acid or a polymer thereof. In other embodiments, the analyte molecule is an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer, or prion. In various embodiments, the solid carrier is a polymer bead or a non-polymer bead. In some embodiments, the targeting portion is an antibody or a cell surface receptor antagonist.

[0326] In certain specific implementations, R 2 or R 3 It has one of the following structures:

[0327]

[0328]

[0329] In some implementations, R 2 or R 3 One of them is OH or -OP (=R) a (R) b )R c And R 2 or R 3 Another one contains the following structure:

[0330] .

[0331] The value of m is another variable that can be selected based on the desired distance between chromophores, FRET interaction, fluorescence, and / or color intensity. In some embodiments, specific values ​​of m are independent integers from 1 to 10. In other embodiments, specific values ​​of m are independent integers from 1 to 5, such as 1, 2, 3, 4, or 5.

[0332] The fluorescence intensity can also be adjusted by selecting different n values. In some embodiments, n is an integer from 1 to 100. In other embodiments, n is an integer from 1 to 10.

[0333] M 1 M 2 and M 3 The selection is based on desired optical properties, such as desired Stokes shift, absorption / emission overlap, specific color, and / or fluorescence emission wavelengths. In some embodiments, M 1 M 2 Or M 3 The specific values ​​of M are the same; however, it is important to note that M 1 M 2 Or M 3 The specific values ​​of M do not need to be the same. 1 M 2 Or M 3 Some embodiments include a compound, wherein M 1 M 2 Or M 3 The specific values ​​are not the same. Some implementations include compounds, wherein each of their respective M... 1 M 2 Or M 3 The specific values ​​are the same.

[0334] In some implementations, M 1 M 2 and M 3 Selected to have absorption and / or emission characteristics for use in FRET methods. For example, in the described embodiment, different M 1 M 2 and M 3 Partially selected so that M 1 It exhibits radiation absorption at one wavelength, which induces M through the FRET mechanism. 2 Or M 3 Radiation emission at different wavelengths. Exemplary M 1 M 2 and M 3 Some of these can be appropriately selected by those skilled in the art based on the desired terminal application.

[0335] Each of their own M 1 M 2 and M 3 From M 1 M 2 Or M 3 Any position (i.e., an atom) on M is attached to the rest of the molecule. Those skilled in the art will recognize that M... 1 M2 Or M 3 Means of attaching to the rest of the molecule. Exemplary methods include the "click" reaction described herein.

[0336] In some implementations, M 1 M 2 Or M 3 It is the FRET, fluorescent, or colored portion. Any fluorescent and / or colored portion can be used to form FRET donor-acceptor pairs, for example, those known in the art and generally used in colorimetric, UV, and / or fluorescence assays. In some embodiments, M 1 M 2 M 3 Or all of the specific values ​​are independently fluorescent or colored. M is useful in various embodiments of this disclosure. 1 M 2 Or M 3 Examples of such derivatives include, but are not limited to: xanthones (e.g., fluorescein, rhodamine, Oregon Green, eosin, or Texas Red); cyanine blue derivatives (e.g., cyanine blue, indigo, oxacarbonyl cyanine, thiacarbonyl cyanine, or benzocyanine); squaraine derivatives and cyclically substituted squaraines, including Seta, SeTau, and square dyes; naphthalene derivatives (e.g., dansyl sulfonyl and prodan derivatives); coumarin derivatives; and oxadiazole derivatives (e.g., ...). Examples include pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole; anthracene derivatives (e.g., anthraquinones, including DRAQ5, DRAQ7, and CyTRAK orange); pyrene derivatives such as cascade blue; oxazine derivatives (e.g., Nile red, Nile blue, cresol purple, oxazine 170); acridine derivatives (e.g., proflavin, acridine orange, acridine yellow); aryl methine derivatives: auramine, crystal violet, malachite green; and tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, or bilirubin). Other exemplary M 1 M 2 Or M 3 Some of these include: cyanine dyes, xanthate / ester dyes (e.g., Hex, Vic, Nedd, Joe, or Tet); Yakima Yellow; Redmond Red; Tamra; Texas Red; and Alexa Fluor® dyes.

[0337] In any of the other embodiments described above, M 1 M 2 Or M 3 Or each specific value independently contains two or more aryl or heteroaryl rings or combinations thereof, such as three or more or four or more aryl or heteroaryl rings or combinations thereof, or even five or more aryl or heteroaryl rings or combinations thereof. In some embodiments, M 1 M 2 M3 Each specific value may independently contain a hexaaryl or heteroaryl ring or a combination thereof. In other embodiments, the rings are fused. For example, in some embodiments, M 1 M 2 Or M 3 Or each of the specific values ​​independently contains two or more fused rings, three or more fused rings, four or more fused rings, five or more fused rings, or even six or more fused rings.

[0338] In some implementations, M 1 M 2 M 3 Or all of them may be ring-shaped. For example, in some embodiments, M 1 M 2 M 3 Or it may be entirely composed of carbon rings. In other embodiments, M 1 M 2 M 3 Or all of them may be heterocyclic. In the other embodiments described above, M 1 M 2 M 3 Each specific value may independently contain an aryl moiety. In some of these embodiments, the aryl moiety is polycyclic. In other more specific instances, the aryl moiety is a fused polycyclic aryl moiety, which may contain at least two, at least three, at least four, or even more than four aryl rings.

[0339] In any other embodiment of the compound with structures (I), (II), (III), or (IV) mentioned above, M 1 M 2 M 3 Each specific value may independently contain at least one heteroatom. For example, in some embodiments, the heteroatom is nitrogen, oxygen, or sulfur.

[0340] In any of the foregoing, or still more, embodiments, M 1 M 2 M 3 All or all of the specific values ​​independently contain at least one substituent. For example, in some embodiments the substituent is fluorine, chlorine, bromine, iodine, amino, alkylamino, arylamino, hydroxyl, hydrogen sulfide, alkoxy, aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, carboxyl, sulfonate / ester group, amide, or formyl.

[0341] In some implementations, M 1 and M 2 At least one combination of or M 1 and M 3 At least one combination of these is a J-value greater than approximately 1 × 10⁻⁶.10 FRET pairs.

[0342] The disclosed compounds can be used as high quantum-efficiency fluorescent and / or coloring dyes. This is due to the donor moiety (e.g., M...). 1 Emission spectrum and receptor portion (e.g., M) 2 Or M 3 The absorption or excitation spectra overlap. Accordingly, some embodiments provide a J-value greater than about 1 × 10⁻⁶. 11 FRET pairs. In a more specific embodiment, the FRET pairs have a value greater than about 1 × 10⁻⁶. 12 The J-value. In some implementations, the FRET pair has a value greater than about 1.2 × 10⁻⁶. 12 The J-value. In some implementations, the FRET pair has a value greater than about 1.5 × 10⁻⁶. 12 The J-value.

[0343] In some more specific implementations, M 1 M 2 Or M 3 Each specific value independently comprises a fused polycyclic aryl moiety, wherein the moiety comprises at least two fused rings.

[0344] In certain specific implementations, M 1 M 2 Or M 3 Each specific value is independently selected from dimethylamino zirconia, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terrylene, biphenyl, porphyrin, benzo[a]pyrene, (fluorophenyl-dimethyl-difluoroborazon-diaza-indarsen)phenyl, (di-fluorophenyl-difluoroborazon-diaza-indarsen)phenyl, tetraphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthalene, bi-anthracene, squaric acid, squaric acid onyx, 9,10-ethynylanthracene, and the ter-naphthalene moiety.

[0345] In some implementations, M 1 M 2 Or M 3 The specific values ​​are independently selected from p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthate, cyclohexane, rubrogene, benzoxene, cyanine blue, peryleneimide, perylene amide, and their derivatives. In some embodiments, M 1 M 2 Or M 3 The specific values ​​are independently selected from coumarin dyes, halogen dyes, dipyrrole-methylboron difluoride dyes, ruthenium bipyridine dyes, thiazole orange dyes, polyacetylenes, and N-aryl-1,8-naphthalenediamide dyes. In some embodiments, M 1 M 2Or M 3 The specific values ​​are independently selected from coumarin dyes, boron-dipyrrole methylene, rhodamine, cyan blue, pyrene, perylene, perylene-imide, 6-FAM, 5-FAM, 6-FITC, 5-FITC, and their derivatives.

[0346] In some implementations, M 1 M 2 Or M 3 Each specific value independently has one of the following structures:

[0347] ; ; ; ; ; ; ; ; ; or .

[0348] In some implementations, M 1 M 2 Or M 3 Each specific value independently has one of the following structures:

[0349] ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;

[0350] ; ; ; ; ; or .

[0351] In some implementations, L 8 Each specific value independently comprises one or more fused aryl or heteroaryl ring systems. In some embodiments, L 8 Each specific value independently comprises one or more fused bicyclic or tricyclic aryl or heteroaryl ring systems. In some embodiments, the one or more fused bicyclic or tricyclic aryl or heteroaryl ring systems have one of the following structures:

[0352] ; ; ; or ,

[0353] in:

[0354] a 1 a 2 and a 3 Each specific value is independently a 5, 6, or 7-membered carbon ring or heterocycle; and

[0355] L 6 It is a direct-connect key or connector.

[0356] In certain specific implementations, L 8 Each specific value independently has one of the following structures:

[0357] ; ; or .

[0358] In some implementations, L 8 Each specific value independently comprises a phosphate diester. In some embodiments, L 8 At least one specific value of L includes ethylene oxide. In a more specific embodiment, L 8 At least one specific value of contains one of the following structures:

[0359] or

[0360] in:

[0361] g is an integer in the range 1-10; and

[0362] z' is an integer in the range 1-30.

[0363] In some of the foregoing embodiments, z' is 3, 6, or 11-28. In some embodiments, g ranges from 2 to 5. In other, more specific embodiments, L 8 At least one specific value of contains the following structure:

[0364] .

[0365] In some implementations, L 8 Each specific value contains the following structure:

[0366] .

[0367] In some specific embodiments, the compounds are selected from those in Table 2. In other specific embodiments, the compounds are selected from those in Table 3. The compounds in Tables 2 and 3 were prepared according to the procedures described in the examples and their identities were confirmed by mass spectrometry.

[0368] Table 2. Exemplary compounds with structures I, II, III, and IV

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379] Table 3. Exemplary precursor compounds

[0380]

[0381]

[0382] As used in Tables 2 and 3 above and throughout this disclosure, F and F' refer to fluorescein moieties having the following structures, respectively:

[0383]

[0384] As used in Table 2 above and throughout this disclosure, Bs refers to a portion having the following structure:

[0385]

[0386] Bs

[0387] As used in Table 2 above and throughout this disclosure, FITC refers to a portion having the following structure:

[0388]

[0389] FITC

[0390] As used in Table 2 above and throughout this disclosure, AF555 refers to a portion having the following structure:

[0391]

[0392] As used in Table 2 above and throughout this disclosure, Cy3 refers to a portion having one of the following structures:

[0393]

[0394] Cy3

[0395] As used in Table 2 above and throughout this disclosure, AF700 refers to the fluorescent dye Alexa Fluor 700, which has CAS Registry Number 1246956-22-8.

[0396] As used in Table 2 above and throughout this disclosure, AF680 refers to a portion having the following structure:

[0397]

[0398] AF680

[0399] As used in Table 2 above and throughout this disclosure, AF647 refers to a portion having the following structure:

[0400]

[0401] AF647

[0402] As used in Table 2 above and throughout this disclosure, AF350 refers to a portion having the following structure:

[0403]

[0404] AF350

[0405] As used in Table 2 above and throughout this disclosure, PB refers to a portion having the following structure:

[0406]

[0407] PB

[0408] As used in Table 2 above and throughout this disclosure, AF594 refers to a portion having the following structure:

[0409]

[0410] AF594

[0411] As shown in Table 2 above and throughout this disclosure, dT refers to the following structure:

[0412]

[0413] dT

[0414] in:

[0415] R is H or a direct key.

[0416] Alternatively, for any embodiment of the dye portion disclosed herein, the oxidative portion It can be replaced by a direct bond to the rest of the molecule. In some of the foregoing embodiments, L 1 It is an alkylene group, such as methylene or ethylene.

[0417] The currently disclosed dye combinations are "tunable," meaning that by appropriately selecting any of the variables in the aforementioned compounds, those skilled in the art can obtain compounds with desired and / or predetermined FRET properties (e.g., fluorescence emission signal, Stokes shift). The "tunability" of the compounds allows users to easily obtain compounds with desired Stokes shift, fluorescence signal, and / or color for use in specific tests or for identifying specific analytes. Although all variables can affect the FRET properties of a compound, it is believed that M... 1 M 2 M 3 L 4 The appropriate selection of m and n plays a crucial role in the molar fluorescence of a compound. Accordingly, one embodiment provides a method for obtaining a compound with desired FRET fluorescence properties, the method comprising selecting M with known interaction properties. 1 M 2 and M 3 Partial preparation of the M 1 M 2 and M 3 Compounds with partial structures (I), (II), (III), or (IV), and selected L 4 Appropriate variables for m and n are used to obtain the desired FRET characteristics (e.g., Stokes shift, donor emission reduction).

[0418] In some embodiments, the FRET fluorescence emission signal can be expressed as a fold increase or decrease relative to the FRET fluorescence emission signal of the parent chromophore (e.g., monomer). In some embodiments, the FRET fluorescence emission signal of the compounds of the present invention is 1.1×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, or greater than 10× of the parent chromophore / fluorophore. Various embodiments include by appropriately selecting L... 4 Compounds with a desired fluorescence fold increase relative to the parent fluorophore were prepared using m and n.

[0419] For ease of illustration, various compounds containing a phosphorus moiety (e.g., phosphates / esters, etc.) are described as being in anionic form (e.g., -OPO(OH)O). - -OPO3 2- Those skilled in the art will readily understand that forms of charge that are pH-dependent and uncharged (e.g., protonated, or salts such as sodium or other cations) are also included within the scope of embodiments of this disclosure.

[0420] Compositions comprising any of the foregoing compounds and one or more analyte molecules (e.g., biomolecules) are provided in various other embodiments. In some embodiments, the use of said composition in analytical methods for detecting one or more analyte molecules is also provided.

[0421] In other embodiments, the compound can be used in various analytical methods. For example, in some embodiments, this disclosure provides a method for staining a sample, the method comprising adding to the sample any of the compounds of the foregoing embodiments (e.g., compounds of structures (I), (II), (III), or (IV)) in an amount sufficient to produce an optical response when the sample is illuminated at an appropriate wavelength.

[0422] In some embodiments of the aforementioned method, R 2 It is a linker, comprising a covalent link to an analyte molecule such as a biomolecule. For example, a nucleic acid or a polymer thereof, or an amino acid or a polymer thereof (e.g., a polynucleotide or polypeptide). In further embodiments, the biomolecule is an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer, or prion.

[0423] In other embodiments of the aforementioned method, R 2 It is a connector that comprises a covalent bond with a solid carrier, such as microparticles (e.g., controlled-pore glass or polystyrene beads). For example, in some embodiments, the microparticles are polymer beads or non-polymer beads.

[0424] In even more embodiments, the optical response is a fluorescence response.

[0425] In other embodiments, the sample comprises cells, and some embodiments further include observing the cells by flow cytometry.

[0426] In further embodiments, the method also includes distinguishing the fluorescence response from the fluorescence response of a second fluorophore having different optical properties for detection.

[0427] In other embodiments, this disclosure provides a method for visually detecting and analyzing molecules, such as biomolecules, comprising:

[0428] (a) Providing a polymer compound (e.g., structure (I), (II), (III) or (IV)) according to the foregoing embodiments, wherein the polymer compound comprises a covalent bond (e.g., R) with the analyte molecule. 2 or R 3 One is a linker containing a covalent bond with the analyte molecule, and R 2 or R 3 The other one is H, OH, alkyl, alkoxy, alkyl ether, or -OP (=R). a (R) b )R c );and

[0429] (b) Detecting compounds through their visual properties.

[0430] In some embodiments, the analyte molecule is a nucleic acid or a polymer thereof, or an amino acid or a polymer thereof (e.g., a polynucleotide or polypeptide). In many other embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion.

[0431] In other embodiments, a method for visually detecting and analyzing molecules, such as biomolecules, is provided, the method comprising:

[0432] (a) Mixing any of the foregoing polymer compounds with analyte molecules, wherein the compound contains a covalent bond with Q selected from Table 1; and

[0433] (b) Formation of bioconjugates of polymeric compounds and analyte molecules; and

[0434] (c) Detect the bioconjugate by its visual properties.

[0435] Some embodiments provide the use of the composition in analytical methods for detecting one or more analyte molecules.

[0436] In addition to the methods described above, embodiments of compounds (I), (II), (III), and (IV) are also used in various disciplines and methods, including but not limited to: endoscopic imaging for the identification of cancerous and other tissues; single-cell and / or single-molecule analytical methods, such as the detection of polynucleotides with little or no amplification; cancer imaging, such as conjugating compounds (I), (II), (III), and (IV) with antibodies or sugars or other moieties that preferentially bind to cancer cells; imaging during surgical procedures; binding histones to identify various diseases; and drug delivery, such as replacing the M moieties in compounds (I), (II), (III), and (IV) with an active pharmaceutical ingredient. 1 M 2 and / or M 3Partial; and / or contrast agents used in dental and other surgeries, for example, through preferential binding of compounds of structures (I), (II), (III) and (IV) to various bacterial flora and / or organisms.

[0437] It should be understood that any embodiment of the compounds with structures (I), (II), (III), and (IV) described above, and the reference to R in the compounds with structures (I), (II), (III), or (IV) described above, are also understood. 1 R 2 R 3 R 4 R 5 L 1 L 2 L 3 L 4 L 5 L 6 L 7a L 7b L 8 M 1 M 2 M 3 Any specific choice of the variables m and / or n can be independently combined with other embodiments and / or variables of the compounds in structures (I), (II), (III), and (IV) to form embodiments of this disclosure not specifically disclosed above. Furthermore, if in a particular embodiment and / or claim any specific R... 1 R 2 R 3 R 4 R 5 L 1 L 2 L 3 L 4 L 5 L 6 L 7a L 7b L 8 M 1 M 2 M 3 If the variables m and / or n list the options, it should be understood that each individual option may be removed from the specific implementation and / or claims and the remaining list of options shall be considered to be within the scope of this disclosure.

[0438] It should be understood that combinations of substituents and / or variables in the formulas described herein are permissible, provided that such contribution yields a stable compound.

[0439] Those skilled in the art will also recognize that the functional groups of the intermediate compounds described herein may require protection by suitable protecting groups. These functional groups include hydroxyl, amino, mercapto, and carboxylic acids. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidine, and guanidinyl groups include tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for mercapto groups include -C(O)-R'' (where R'' is alkyl, aryl, or arylalkyl), p-methoxybenzyl, triphenylmethyl, etc. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups may be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. Those skilled in the art will understand that the protecting group can also be a polymer resin such as Wang resin, Rink resin, or 2-chlorotriphenylmethyl-chloride resin.

[0440] Furthermore, all the compounds of this disclosure existing in the form of free bases or acids can be converted into their salts by treatment with suitable inorganic or organic bases or acids using methods known to those skilled in the art. The salts of the compounds of this disclosure can be converted into their free base or acid forms using standard techniques.

[0441] The reaction schemes described below illustrate exemplary methods for preparing the compounds of this disclosure. It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known to them. It should also be understood that those skilled in the art can prepare other compounds (I), (II), (III), or (IV) without specific description below by using appropriate starting components and modifying the synthetic parameters as needed in a manner similar to that described below. Typically, the starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized from sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this disclosure.

[0442] In particular, methods for preparing the compounds disclosed herein (e.g., structures (I), (II), (III) and (IV)) can be found in, for example, PCT Pub. Nos. WO 2015 / 027176, WO 2016 / 138461, WO 2016 / 183185, WO2017 / 173348, WO 2017 / 173355, WO 2017 / 177065, WO 2017 / 196954, WO 2017 / 214165 and WO2018 / 022925, all of which are incorporated herein by reference in their entirety.

[0443] Reaction Scheme I

[0444]

[0445] Reaction scheme I illustrates an exemplary method for preparing intermediates for the preparation of compounds with structures (I), (II), (III), and (IV), wherein R 1 L 2 and L 3 As defined above, R 2 and R 3 As defined above or its protected variant, and L is an optional linker. Referring to reaction scheme 1, compound a can be purchased or prepared by methods well known to those skilled in the art. The reaction of a with MX (where X is a halogen such as bromine) under Suzuki coupling conditions known in the art yields compound b. Compound b can be used to prepare compounds of structures (I), (II), (III), or (IV), as described below.

[0446] Reaction Scheme II

[0447]

[0448] Reaction scheme II describes an alternative method for preparing intermediates used in the preparation of compounds with structures (I), (II), (III), and (IV). Referring to reaction scheme II, where R... 1 L, L 2 L 3 G is as defined above, while R... 2 and R 3 As defined above or its protective variant, a commercially available or well-prepared compound of structure c reacts with M-G' to produce a compound of structure d. Here, G and G' represent functional groups with complementary reactivity (i.e., functional groups that form covalent bonds in the reaction). G' can be attached to M or be part of the structural framework of M. G can be any number of the functional groups described herein, such as alkynes or amines.

[0449] Compounds with structures (I), (II), (III), or (IV) can be prepared from one of structures b or d by reacting with a phosphoridamide compound having structure (e) below under well-known automated DNA synthesis conditions:

[0450] ,

[0451] (e)

[0452] Each X is an independent desired monomer unit (e.g., a dye-containing portion, a linker, etc.).

[0453] DNA synthesis methods are well known in the art. In short, two alcohol groups, such as the R groups of intermediate b or d described above, are involved. 2 and R 3 The phosphoramidyl group is functionalized with a dimethoxytriphenylmethyl (DMT) group and a 2-cyanoethyl-N,N-diisopropylaminophosphamide group, respectively. The phosphoramidyl group is typically coupled to an alcohol group in the presence of an activator such as tetrazolium, followed by oxidative oxidation of the phosphorus atom with iodine. The dimethoxytriphenylmethyl group can be removed with an acid (e.g., chloroacetic acid) to expose the free alcohol, which can react with the phosphoramidyl group. The 2-cyanoethyl group can be removed by treatment with ammonia after oligomerization.

[0454] The preparation of phosphorous amides for use in oligomerization methods is also well known in the art. For example, primary alcohols (e.g., R...) 3 It can be protected to a DMT group by reacting with DMT-Cl. Secondary alcohols (e.g., R...) 2 The phosphorus amide is then functionalized to phosphorus amide by reacting with a suitable reagent, such as 2-cyanoethyl N,N-diisopropylchlorophosphamide. Methods for preparing phosphorus amides and their oligomerization are well known in the art and are described in more detail in the examples.

[0455] Compounds with structures (I), (II), (III), and (IV) were prepared as follows: intermediates b or d and e were oligomerized according to the well-known phosphorus amide chemistry described above. The desired number of repeating units m and n were incorporated into the molecule by coupling the phosphorus amide to the desired number of times.

[0456] In various other embodiments, compounds for preparing compounds of structures (I), (II), (III), and (IV) are provided. These compounds can be prepared as monomers, dimers, and / or oligomers as described above, and then M can be synthesized via any number of synthetic methods (e.g., the "click" reaction described above). 1 M 2 and / or M 3 Partial covalent linkage to compounds to form compounds with structures (I), (II), (III), and (IV).

[0457] The efficiency of the FRET process depends in part on the characteristics of the chromophores. In particular, high-efficiency FRET requires a large overlap (i.e., J-value) between the absorption spectra of the donor chromophore and the emission spectra of the acceptor chromophore. Additionally, the distance and orientation of the chromophores play a crucial role. FRET efficiency is inversely proportional to the sixth power of the distance between the chromophores, and the angles of the transition dipole moments should be substantially parallel or antiparallel (i.e., close to 0° or 180°). Accordingly, in some embodiments, the covalent attachment of the first and second chromophores to the polymer backbone is selected such that the distance between the first and second chromophores is minimized and the transition dipole moments are substantially aligned. The efficiency of FRET can be expressed by the following formula:

[0458]

[0459] Where E FRET R is the FRET efficiency, R is the distance between chromophores, and Ro is expressed by the following formula:

[0460]

[0461] Where J is the spectral overlap between the acceptor absorption spectrum and the donor emission spectrum, Q o It is the donor quantum efficiency, n -4 It is the mediator index (constant) between the donor and recipient, while K 2 It is a dipole orientation match.

[0462] Accordingly, one embodiment provides a polymer compound comprising an acceptor chromophore and a donor chromophore, wherein the acceptor chromophore has an acceptor transition dipole moment and is covalently linked to the polymer backbone, and the donor chromophore has a donor transition dipole moment and is covalently linked to the polymer backbone, wherein the acceptor chromophore and the donor chromophore have a molecular weight greater than about 1 × 10⁻⁶. 10 The J-value, and the polymer compound adopts a conformation in solution under physiological conditions in which the effective distance between the acceptor chromophore and the donor chromophore is less than about 50.0 nm and the acceptor transition dipole and the donor transition dipole are substantially parallel or substantially antiparallel.

[0463] In some embodiments, the recipient chromophore and the donor chromophore have a density greater than about 1 × 10⁻⁶. 11 The J-value. In some embodiments, the acceptor chromophore and the donor chromophore have a J-value greater than about 1 × 10⁻⁶. 12 The J-value. In some more specific embodiments, the acceptor chromophore and the donor chromophore have a J-value greater than about 1.2 × 10⁻⁶. 12 The J-value. In some more specific embodiments, the acceptor chromophore and the donor chromophore have a J-value greater than about 1.5 × 10⁻⁶. 12 The J-value.

[0464] In some embodiments, the effective distance between the acceptor chromophore and the donor chromophore is less than about 25.0 nm. In some embodiments, the effective distance between the acceptor chromophore and the donor chromophore is less than about 10.0 nm.

[0465] In some embodiments, the acceptor chromophore is a fluorescent dye moiety. In some embodiments, the acceptor chromophore has a maximum emission in the range of about 480 nm to about 740 nm. In more specific embodiments, the acceptor chromophore has a maximum emission at about 488 nm, about 500 nm, about 506 nm, about 519 nm, about 522 nm, about 528 nm, about 564 nm, about 573 nm, about 591 nm, about 603 nm, about 616 nm, about 622 nm, about 640 nm, about 650 nm, about 666 nm, about 679 nm, 714 nm, or about 731 nm.

[0466] In some embodiments, the receptor chromophore has one of the following structures:

[0467] ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; or

[0468] .

[0469] In some embodiments, the donor chromophore is a fluorescent dye moiety. In some embodiments, the donor chromophore has a maximum absorbance at about 350 nm, about 405 nm, or about 488 nm. In some more specific embodiments, the donor chromophore has one of the following structures:

[0470] ; ; or .

[0471] In some implementations, both the acceptor chromophore and the donor chromophore are fluorescent dye moieties.

[0472] In some embodiments, the angle between the acceptor and donor transition dipole moments ranges from 120° to 180°. In some more specific embodiments, the angle between the acceptor and donor transition dipole moments ranges from 0° to 60°. In some more specific embodiments, the polymer compound further comprises a first acceptor chromophore covalently attached to the proximal end of the polymer backbone, a second acceptor chromophore covalently attached to the distal end of the polymer backbone, and a donor chromophore covalently attached between the proximal and distal ends of the polymer backbone.

[0473] In a more specific embodiment, the first acceptor chromophore and the second acceptor chromophore are identical. In some embodiments, the polymer backbone comprises a phosphate / ester linker. In more embodiments, the polymer backbone comprises an olefin oxide linker. In some embodiments, the olefin oxide is ethylene oxide.

[0474] In some embodiments, the polymer compound has a molecular weight of less than 20,000 g / mol. In some embodiments, the polymer compound has concentrations of less than 19,000 g / mol, 18,500 g / mol, 18,000 g / mol, 17,500 g / mol, 17,000 g / mol, 16,500 g / mol, 16,000 g / mol, 15,500 g / mol, 15,000 g / mol, 14,500 g / mol, 14,000 g / mol, 13,500 g / mol, 13,000 g / mol, 12,500 g / mol, 11,500 g / mol, 11,000 g / mol, 10,500 g / mol, 10,000 g / mol, 9,500 g / mol, 9,000 g / mol, 8,500 g / mol, 8,000 g / mol, 7,500 g / mol, 7,000 g / mol. Molecular weights of g / mol, 6,500 g / mol, 6,000 g / mol, 5,500 g / mol, 5,000 g / mol, 4,500 g / mol, 4,000 g / mol, 3,500 g / mol, 3,000 g / mol, 2,500 g / mol, 2,000 g / mol, 1,500 g / mol, or 1,000 g / mol.

[0475] In some embodiments, the polymer compound is not a peptide or protein. In other embodiments, the polymer backbone does not have amide bonds.

[0476] The following embodiments are provided for illustrative purposes and are not intended to be limiting. Detailed Implementation

[0477] Example

[0478] General methods

[0479] Mass spectrometry analysis was performed on a Waters / Micromass Quattro micro MS / MS system (MS single mode) using MassLynx 4.1 acquisition software. The mobile phase used for dye LC / MS was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8. Phosphoramide and precursor molecules were also analyzed using a Waters Acquity UHPLC system with a 2.1 mm x 50 mm Acquity BEH-C18 column maintained at 45 °C using an acetonitrile / water mobile phase gradient. The molecular weights of monomer intermediates were obtained using ionization-enhanced ionization with α-ionium cation infusion on a Waters / Micromass Quattro micro MS / MS system (MS single mode). Excitation and emission characterization experiments were recorded on a Cary Eclipse spectrophotometer.

[0480] All reactions were carried out in furnace-dried glassware under a nitrogen atmosphere unless otherwise specified. Commercially available DNA synthesis reagents were purchased from Glen Research (Sterling, VA). Anhydrous pyridine, toluene, dichloromethane, diisopropylethylamine, triethylamine, acetic acid, pyridine, and THF were purchased from Aldrich. All other chemicals were purchased from Aldrich or TCI and used as is without additional purification.

[0481] Example 1

[0482] Synthetic polymer backbone and derivatization

[0483] Synthesize representative polymer compounds

[0484] Polymers were synthesized at 0.5–1 µmol scales using an Applied Biosystems 394 DNA / RNA synthesizer. Polymer compounds were synthesized directly on CPG beads or polystyrene solid supports. Synthesis was performed in the 3'–5' direction using standard solid-phase DNA synthesis methods (i.e., β-cyanoethylphosphamide coupling chemistry). Monomer reagents (e.g., Fluoroside phosphoramide, C2-alkyl phosphoramide, hexadecimalyl glycol phosphoramide, aminophosphamide, and acetylenic phosphoramide) were dissolved in acetonitrile and dichloromethane to prepare 0.1 M stock solutions. Monomer reagents were added sequentially using the following synthetic cycle:

[0485] 1) Remove the 5'-dimethoxytriphenylmethyl protecting group with dichloromethane or dichloroacetic acid from toluene;

[0486] 2) Couple the next phosphoramidite monomer reagent with the activator reagent (0.2 M ETT) in acetonitrile;

[0487] 3) Oxidation of phosphate / ester (III) with iodine / pyridine / water to form stable phosphate / ester (V); and

[0488] 4) End-cap the unreacted 5'-hydroxyl group with acetic anhydride / 1-methylimidazole / acetonitrile.

[0489] Repeat the synthesis cycle until the desired polymer compound (e.g., the compound in Table 2 or 3) is assembled. Upon completion, remove the terminal monomethoxytriphenylmethyl (MMT) or dimethoxytriphenylmethyl (DMT) groups with dichloromethane or dichloroacetic acid in toluene. After synthesis, treat the support with 20% diethylamine / acetonitrile for 15 minutes.

[0490] The polymer was then cleaved from the solid support at 55°C with a concentrated ammonium hydroxide aqueous solution for 2 hours. The construct was characterized by ESI-MS to confirm its quality and purity. Concentration was determined by UV chromatography.

[0491] Chromophore coupling to amine / acetylene polymer compounds

[0492] Representative polymer constructs are synthesized to possess orthogonal reactive groups (e.g., amines and alkynes), allowing the incorporation of different dye moieties. For example, click chemistry is used to attach a first fluorophore to the alkyne of the polymer, and a second fluorophore is attached to the amine of the polymer in a subsequent synthetic step. Alternatively, the fluorophore can be incorporated during the DNA synthesis step: phosphorous amide containing the fluorophore is incorporated during step 2 described above.

[0493] For post-synthetic modification, a 100 mM sodium ascorbate solution and a 500 mM phosphate buffer solution at pH 7.6 were prepared. The first desired dye moiety containing the azide functional group was dissolved in dimethyl sulfoxide ("dye solution") at a concentration of 100 mM.

[0494] A 100 mM sodium ascorbate solution (i.e., a 2.5:1:1 mixture of 100 mM sodium ascorbate / 100 mM THTPA / 50 mM copper sulfate) is prepared as follows: add equal volumes of THTPA and copper sulfate and let the mixture stand for 5-10 minutes, then add sodium ascorbate.

[0495] Phosphate buffer, dye solution, polymer compound, and additional DMSO were mixed together, followed by sodium ascorbate solution. The prepared reaction had a final polymer concentration of 0.5 mM, a phosphate buffer concentration of 100 mM, 50-60% DMSO, and a 5-10 times excess of dye. The reaction was placed overnight in the dark on a shaker or vortex mixer. The sample was diluted with water and desalted with G-25 Sephadex resin. The sample was collected and characterized by ESI-MS to confirm quality and purity. The polymer compound was lyophilized after sample analysis.

[0496] The addition of a second desired dye moiety (e.g., fluorophore or other label) to a representative polymer compound is achieved using NHS-ester / amine coupling chemistry. Initially, a sodium tetraborate buffer solution at pH 9 ("borate buffer") is prepared. The second dye moiety, containing the NHS-ester, is dissolved in DMSO at a concentration of 100–200 mM. The prepared reaction has a polymer concentration of 1–2 mM, a borate buffer concentration of 100 mM, 30–75% DMSO, and a 5–10-fold excess of dye. The reaction is incubated overnight in the dark on a shaker or vortex mixer. The sample is diluted with water and desalted using G-25 Sephadex resin. The sample is collected and characterized by ESI-MS to confirm quality and purity. Additionally, FRET characteristics are determined using UV and fluorescence spectroscopy.

[0497] Example 2

[0498] Compound emission characterization

[0499] Representative structural (III) polymer compounds (compounds I-1, I-3, I-5, and I-7) and structural (IV) polymer compounds (compounds I-2, I-4, I-6, and I-8) were prepared according to the method described in Example 1 of this document. The structural (III) compounds were synthesized, wherein the donor chromophore M... 2 Part of it is Cy3. Synthetic structure (IV) compound, in which the donor chromophore M 3 Part of it is AF555. It is well established in the art that Cy3 and AF555 are spectrally equivalent as distinct species, and this study allows for a preliminary evaluation of spectral differences in more complex environments. Two M values ​​are present in each compound. 1 Some of the respective chromophores are selected from AF680, AF647, AF594 and AF700 and are the same in each compound.

[0500] The test compounds were prepared in solution at concentrations ranging from 1 to 10 µM and pH 7.0. The samples were measured using an excitation wavelength of 488 nm, and the obtained spectra are shown below. Figures 2 to 9As data show, the emission signals of the acceptor chromophores are substantially similar; however, some implementations exhibit better energy transfer from the donor to the acceptor chromophore, evidenced by reduced relative fluorescence from the donor fluorophore, see, for example... Figure 4 relative to Figure 5 A smaller emission peak (better energy transfer).

[0501] Figure 10 A positional overlay of the emission spectra of compounds of structure (III) is provided, and the Stokes shift ranges observed for different embodiments of the invention are shown. Based on the maximum emission relative to the excitation wavelength, the Stokes shifts of compounds I-7, I-1, I-3, and I-5 are 243 nm, 226 nm, 191 nm, and 134 nm, respectively.

[0502] Example 3

[0503] Confirmed valid FRET transmission

[0504] To demonstrate the high FRET efficiency of the embodiments of the present invention, the emission spectrum of compound I-10 containing donor and acceptor chromophores is compared with that of a corresponding compound that does not contain a donor chromophore but contains the same type and number of acceptor chromophores.

[0505] Compound I-10 was prepared according to the method described in Example 1 of this document, and was synthesized to possess the donor chromophore AF350. The acceptor chromophores at both ends of the molecule are the fluorescein derivative FITC.

[0506] Comparative compounds of difluorescein that do not contain donor chromophores, compound A is shown below:

[0507]

[0508] Compound A

[0509] Where F is the fluorescein moiety:

[0510] .

[0511] The test compound was prepared in solution at a concentration of 350 nM and pH 7.0. The sample was evaluated using an excitation wavelength of 350 nm, and the resulting overlapping spectra are shown in [Figure number missing]. Figure 11 For the maximum emission at 520 nm, the measured Stokes shift of compound I-10 is 170 nM.

[0512] Compound I-10 showed a ~5.5-fold increase in fluorescence compared to compound A, demonstrating an efficient transfer of excitation energy from the donor chromophore (AF350) to the acceptor FITC moiety, thus providing the observed increase.

[0513] All US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent publications mentioned in this specification and / or listed on the application data page, including US patent application No. 62 / 906,591 filed on September 26, 2019, are incorporated herein by reference in their entirety and without contradiction.

[0514] It should be understood from the foregoing that although specific embodiments of this disclosure have been described herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, this disclosure is not limited except by the appended claims.

Claims

1. Compounds having the following structure (I): (I) or its salts or tautomers, wherein: The compound comprises: a first acceptor chromophore covalently attached to the proximal end of the backbone; The second acceptor chromophore is covalently attached to the distal end of the scaffold; and the donor chromophore is covalently attached between the proximal and distal ends of the scaffold. M 1 and M 2 Each specific value of is an independent chromophore, provided that M . 1 and M 2 At least one of them is a FRET donor, M 1 and M 2 The other one is the corresponding FRET receptor, in which M 1 and M 2 Each of them independently possesses one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; or ; L 1 The specific values ​​are linkers, which comprise a continuous chain of atoms selected from carbon, oxygen, and nitrogen, provided that L 1 At least one specific value contains oxygen; L 2 and L 3 Each specific value is independently optional C1-C 12 Alkylene or C1-C 12 Heteroalkylene linkers; L 4 Each specific value is independently C1-C 12 Alkylene or C1-C 12 Heteroalkylene linkers; R 1 Each specific value is independently H, alkyl, or alkoxy; R 2 and R 3 Each is independently OH, SH, -OP (=R) a (R) b )R c Q, or its protected form, or L'; R 4 The specific values ​​are independently OH, SH, and O. - S - OR d or SR d ; R 5 Each specific value is independently oxidized, thiolated, or absent; R a Is it O or S? R b It is OH, SH, O - S - OR d or SR d ; R c It is OH, SH, O - S - OR d ,OL',SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate / ester, thiophosphate / ester, phosphate-alkyl, thiophosphate-alkyl, phosphate-alkyl ether or thiophosphate-alkyl ether; R d They are balanced ions; Each specific value of Q independently has one of the following structures: ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; or -NH2; Each X is an independent halogen; Each specific value of L' is independently a linker containing a covalent bond with Q, a linker containing a covalent bond with the target moiety, a linker containing a covalent bond with the analyte molecule, a linker containing a covalent bond with the solid support, a linker containing a covalent bond with the solid support residue, a linker containing a covalent bond with the nucleoside, or a linker containing a covalent bond with another compound of structure (III). Each specific value of m is an integer from 0 to 6 independently; and n is an integer from 1 to 10.

2. The compound of claim 1, wherein the compound has the following structure (IA): (IA) in: L 4 It is -(OCH2CH2) z -; Each specific value of z is an integer independently between 1 and 100; and Each specific value of m is an integer from 0 to 6.

3. The compound of claim 2, wherein each specific value of z is independently an integer from 1 to 30.

4. The compound of any one of claims 2-3, wherein each specific value of z is independently 3 or 6.

5. The compound of claim 1, wherein each specific value of m is independently an integer from 2 to 4.

6. The compound of claim 1, wherein each specific value of m is 2.

7. The compound of claim 1, wherein L 1 It includes one of the following structures: ; ; or , in a, b, and c are each an integer in the range 1-6.

8. The compound of claim 1, wherein R 4 The specific values ​​of OH and O are independent. - OR d .

9. The compound of claim 1, wherein R 5 The specific values ​​are for oxygenation.

10. The compound of claim 1, wherein R 1 The specific values ​​of are H.

11. The compound of claim 1, wherein R 2 or R 3 One of them is OH or -OP (=R) a (R) b )R c , and R 2 or R 3 The other is Q or a linker containing a covalent bond with Q.

12. The compound of claim 1, wherein R 2 and R 3 Each independently contains -OP(=R) a (R) b )R c .

13. The compound of claim 12, wherein R c She is an office lady (OL).

14. The compound of claim 13, wherein L' is a linker to a heteroalkylene group, Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside, or other compound with structure (I).

15. The compound of claim 13 or 14, wherein L' comprises an oxidized olefin or a phosphate diester moiety or a combination thereof.

16. The compound of claim 13, wherein L' has the following structure: ， in: m'' and n'' are independent integers from 1 to 10; R e It is H, an electron pair or a balanced ion; L'' is R e Or with the following direct links or linkers: Q, target moiety, analyte molecule, solid support, solid support residue, nucleoside or another (III) compound.

17. The compound of claim 1, wherein R 2 or R 3 One of them is OH or -OP (=R) a (R) b )R c And R 2 or R 3 The other is a linker containing covalent bonds with the analyte molecule or a linker containing covalent bonds with the solid support.

18. The compound of claim 17, wherein the analyte molecule is a nucleic acid or a polymer thereof, or an amino acid or a polymer thereof.

19. The compound of claim 17, wherein the analyte molecule is an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer, or prion.

20. The compound of claim 17, wherein the solid support is a polymer bead or a non-polymer bead.

21. The compound of claim 1, wherein R 2 or R 3 It has one of the following structures: ； ； ； ； ； ； ； ； or .

22. The compound of claim 1, wherein R 2 or R 3 One of them is OH or -OP (=R) a (R) b )R c And R 2 or R 3 Another one contains the following structure: 。 23. The compound of claim 1, wherein the FRET pair has a concentration greater than 1 × 10⁻⁶. 11 The J-value.

24. The compound of claim 1, wherein the compound is selected from: in: Bs has the following structure: ； FITC has the following structure: ； Cy3 has the following structure: ； The AF680 has the following structure: ； AF647 has the following structure: ； The AF350 has the following structure: ； PB has the following structure: ； AF594 has the following structure: ； and dT has the following structure: ， in: R is H or a direct key.

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