Fluorescent dyes

Abstract

Provided are fluorescent dye phosphoramidite compounds compatible with automated oligonucleotide synthesis, solid supports, and labeled polynucleotides incorporating the compounds. The compounds allow for the universal incorporation of fluorescent labels into any position of a polynucleotide.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application No. 63 / 052,862, filed on July 16, 2020, the entire contents of which are expressly incorporated herein by reference. Background of the Invention

[0004] Fluorescent dye is one of the most commonly used labels for modifying oligonucleotides, because they provide sensitive detection in a variety of applications from PCR to sequencing. The preparation of polynucleotides labeled with fluorescent dyes is generally completed by post-synthesis conjugation, for example, by reacting activated dye intermediates with amino derivatives of polynucleotides. This method suffers from certain shortcomings, including low conjugation yields and the need to carry out additional purification of the conjugated product. Fluorescent dyes are incorporated into synthetic polynucleotides via automated phosphoramidite synthesis to provide a more convenient method. However, there are few phosphoramidite derivatives of fluorescent dyes that allow fluorescent dyes to be added to polynucleotides as a part of automated solid phase synthesis that are commercially available. In addition, there are few such derivatives that can allow the incorporation of fluorescent dye moieties at any position of synthetic polynucleotides.

[0005] Therefore, a need remains for fluorescent dyes that are compatible with automated phosphoramidite oligonucleotide synthesis conditions, can be incorporated into any position of a polynucleotide, and provide high endpoint fluorescent signals in PCR applications.

[0006] Overview

[0007] This summary is provided to introduce some concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] In one aspect, the present disclosure provides a compound represented by Formula I or a stereoisomer, salt or tautomer thereof:

[0009]

[0010] in:

[0011] X is H, halogen or C1-C5 alkyl; R 1 , R 2 , R 3 and R 4 R is independently H or an optionally substituted C1-C6 alkyl group; 5 , R 6 , R 7 , R 8 and R 9 are independently H, halogen or optionally substituted C1-C6 alkyl; L1 is an optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 50 Heteroalkylene; L 2 and L 3 is independently an optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 30 Heteroalkylene; Q 1 is a hydroxyl protecting group; Z is CH, N, NHC(O)N or OC(O)N; Y is OH, OP(OCH2CH2CN)NR 10 R 11 or a solid support; and R 10 and R 11 and C1-C6 alkyl are independently optionally substituted.

[0012] In some embodiments, X is Cl, Br, or F. In some embodiments, X is Cl. In some embodiments, X is F. In some embodiments, X is optionally substituted methyl or optionally substituted ethyl. In some embodiments, X is o-MeC6H4CH2.

[0013] In some embodiments, the solid support is controlled pore glass or polystyrene.

[0014] In some embodiments, the compound is a compound of Formula (IA) or a stereoisomer, salt, or tautomer thereof:

[0015]

[0016] In some embodiments, R 1 is H. In some embodiments, R 2 In some embodiments, R 3 In some embodiments, R 4 In some embodiments, the compound is a compound of formula (IB) or a stereoisomer, salt or tautomer thereof:

[0017]

[0018] In some embodiments, L 1 is C2-C6 alkylene or -(CH2CH2O) m CH2CH2-, wherein m is an integer from 1 to 10. In some embodiments, L 1 is C2 alkylene. In some embodiments, Z is OC(O)N.

[0019] In some embodiments, the compound is a compound of Formula (IC) or a stereoisomer, salt, or tautomer thereof:

[0020]

[0021] wherein m is an integer from 1 to 10.

[0022] In some embodiments, the compound is a compound of Formula (ID) or a stereoisomer, salt, or tautomer thereof:

[0023]

[0024] Where W is NH or O, and L 4 is an optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 50 Heteroalkylene.

[0025] In some embodiments, Q 1 is trimethylsilyl, TBDMS, acetyl, dimethoxytrityl or trityl. 10 and R 11 In some embodiments, R 5 In some embodiments, R 7 In some embodiments, R 8 In some embodiments, R 6 and R 9 It's H.

[0026] In some embodiments, the compound is a compound of Formula (IE) or a stereoisomer, salt, or tautomer thereof:

[0027]

[0028] On the other hand, the disclosure provides polynucleotides containing the residues of the compounds of the disclosure. In some embodiments, the polynucleotides of the labeling are prepared using automated phosphoramidite synthesis. In some embodiments, the polynucleotides of the labeling also include a fluorescence quencher. In some embodiments, the polynucleotides of the labeling also include a BHQ type fluorescence quencher. In some embodiments, the polynucleotides of the labeling are connected to a solid support. In some embodiments, the solid support is a controlled pore glass bead, a polystyrene bead, a magnetic bead or a microplate.

[0029] In another aspect, the present disclosure provides a method for preparing a labeled conjugate of a ligand, comprising contacting the ligand with a compound of the present invention in a suitable solvent under conditions sufficient to covalently link the compound to the ligand, thereby forming a labeled conjugate, wherein Y is OP(OCH2CH2CN)NR 10 R 11 And R10 and R 11 and C1-C6 alkyl are independently optionally substituted.

[0030] In some embodiments, the ligand is a polynucleotide or a solid support. In some embodiments, the ligand is a polynucleotide. In some embodiments, the conditions sufficient to covalently link the compound to the ligand are automated phosphoramidite oligonucleotide synthesis conditions.

[0031] In another aspect, the present disclosure provides kits, such as PCR diagnostic kits, comprising the labeled polynucleotides disclosed herein.

[0032] Details

[0033] Provided herein are fluorescent dye derivatives, such as phosphoramidites, that can be incorporated into oligonucleotides via standard automated oligonucleotide synthesis. Also provided are solid supports modified with fluorescent dyes and polynucleotides comprising fluorescent dyes.

[0034] In this specification and the appended claims, the words "comprise" and "include" and variations thereof, such as "comprises," "comprising," "includes," and "including," are intended to convey that additional elements or integers not specifically recited may be included where the context permits. As used herein, the term "consisting of" is intended to mean including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements are present. The term "consisting essentially of means that a composition, method, or structure may include additional ingredients, steps, and / or parts, but only if the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0035] The terms “a” and “an” and “the” and similar terms are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0036] Unless otherwise specified herein or context clearly contradicts, all methods described herein can be performed in any suitable order. The use of any and all embodiments or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention to which protection is otherwise claimed.

[0037] The grouping of the alternative elements or embodiments of the present invention disclosed herein should not be construed as limiting. Each group member can be quoted and claimed individually or in any combination with other members of the group or other elements found herein. It is expected that one or more members of the group may be included in the group or deleted from the group for convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to include the modified group in this article, thereby satisfying the written description of all Markush groups used in the appended claims.

[0038] Unless otherwise indicated, nucleic acids or oligonucleotides are written left to right in 5' to 3' orientation.

[0039] As used herein, the term "amplification" refers to any means of generating at least a partial sequence of at least one target nucleic acid or its sequence complement, generally in a template-dependent manner, including but not limited to a wide range of techniques for linear or exponential amplification of nucleic acid sequences. Non-limiting exemplary amplification methods include polymerase chain reaction (PCR), reverse transcriptase PCR, real-time PCR, nested PCR, multiplex PCR, quantitative PCR (Q-PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), rolling circle amplification (RCA), strand displacement amplification (SDA), ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLPA), ligation followed by Q-replicase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplex amplification, digital amplification, etc. Descriptions of this technique can be found in other materials by Ausubel et al.; PCR Primers: A Laboratory Manual, Diffenbach, ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, NJ (2002); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990).

[0040] As used herein, the term "base" refers to a nitrogen-containing heterocyclic moiety capable of forming a hydrogen bond, such as a Watson-Crick type hydrogen bond, with a complementary nucleotide base or nucleotide base analog, such as a purine, a 7-deazapurine or a pyrimidine. Typical bases are the naturally occurring bases adenine, cytosine, guanine, thymine and uracil. Bases also include analogs of naturally occurring bases, such as deazaadenine, 7-deaza-8-azaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, inosine, muscimol, nitropyrrole, nitroindole, 2-aminopurine, 2,6-diaminopurine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-chloro-6-aminopurine, xanthine, hypoxanthine, and the like.

[0041] As used herein, the term "complementarity" refers to the ability of polynucleotide sequences to hybridize to base pairs and from base pairs to each other. Base pairs are generally formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide chains. Complementary polynucleotide chains can be base-paired in a Watson-Crick manner (e.g., A to T, A to U, C to G) or in any other manner that allows the formation of a duplex. The "complementarity" percentage of a probe sequence and a target sequence is the "identity" percentage of a probe sequence and a target sequence or the complement of a target sequence. When determining the "complementarity" degree between a probe and a target sequence, the "complementarity" degree is expressed as the identity percentage between the probe sequence and the complement of the sequence of the target sequence that best matches it or the sequence of the target sequence. Exemplary probes are polynucleotides as described herein.

[0042] As used herein, the term "duplex" refers to a double-stranded hybrid complex formed by annealing (hybridization) of complementary (or partially complementary) single-stranded polynucleotides, such as DNA, RNA, LNA or peptide nucleic acid (PNA).

[0043] As used herein, "fluorescence quenching" refers to any process that reduces the fluorescence intensity of a fluorescent sample, i.e., a fluorescent polynucleotide probe. A variety of molecular interactions can lead to quenching. Non-limiting examples include excited state reactions, molecular rearrangements, energy transfer, ground state complex formation, and collisional quenching.

[0044] As used herein, "halogen" refers to F, Cl, Br or I.

[0045] The terms "hybridize" and "hybridization" are used herein to refer to "specific hybridization", which in some embodiments is the preferential binding, duplexing or annealing of a nucleic acid molecule to a specific nucleotide sequence under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target sequence and to other sequences to a lesser extent or not at all. "Stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization are sequence-dependent and are different under different environmental parameters. A detailed guide to nucleic acid hybridization can be found in, for example, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I, Ch.2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, NY. The degree of hybridization of a polynucleotide to a target sequence, also known as hybridization intensity, is determined by methods well known in the art. A preferred method is to determine the T of a given hybridization duplex. m This can be achieved by subjecting the duplex formed in solution to gradually increasing temperatures and monitoring the denaturation of the duplex, for example, by the absorption of ultraviolet light, which increases as the unstacking of the base pairs accompanies the denaturation. m It is usually defined as the temperature at which half of the DNA strands are in the single-stranded (ssDNA) state. m It depends on various parameters, such as the length of the hybridizing complementary strand sequences, their specific nucleotide sequence, base composition, base modifications and the concentration of the complementary strands.

[0046] As used herein, the terms "label" and "detectable label" are used interchangeably and refer to a moiety that, when attached to a biomolecule, nucleoside, nucleotide, or polynucleotide, renders such biomolecule, nucleoside, nucleotide, or polynucleotide detectable by a suitable detection means. Exemplary labels include fluorophores, chromophores, radioisotopes, spin labels, enzyme labels, chemiluminescent labels, electrochemiluminescent compounds, magnetic labels, microspheres, colloidal metals, immunolabels, ligands, enzymes, and the like.

[0047] As used herein, the term "modified nucleotide base" or "modified base" refers to a base that does not have the structure of a naturally occurring base and is therefore a non-naturally occurring base. As used herein, the term "modified sugar" refers to a sugar or sugar analog that does not have the structure of a naturally occurring sugar such as ribose or deoxyribose sugar and is therefore a non-naturally occurring sugar.

[0048] As used herein, the term "naturally occurring" in the context of nucleic acid molecules refers to an RNA or DNA molecule (single- or double-stranded) having a naturally occurring nucleotide sequence and containing only naturally occurring components such as bases, sugars, nucleosides, and nucleotides.

[0049] As used herein, the term "nucleoside" refers to a molecule consisting of a nitrogenous base of the type mentioned herein, which is bound to a sugar of the type mentioned herein, for example, to a ribose or deoxyribose sugar, via a β-glycosidic linkage. Examples of nucleosides include adenosine, cytidine, guanosine, thymidine, uridine, and inosine.

[0050] As used herein, the term "nucleotide" refers to the phosphate ester of a nucleoside, either as an independent monomer or as a subunit within a polynucleotide. Nucleotide monomers include, for example, nucleotide 5'-monophosphate, nucleotide 5'-diphosphate, nucleotide 5'-triphosphate, and nucleotide 3'-monophosphate. Nucleotide triphosphates are sometimes expressed as "NTPs," "dNTPs" (2'-deoxypentose) or "ddNTPs" (2', 3'-dideoxypentose), to specifically indicate the structural features of ribose sugar. "Nucleotide 5'-triphosphate" refers to a nucleotide having a triphosphate group at the 5' position. The triphosphate group may include sulfur substitutions of one or more phosphate oxygen atoms, such as α-thionucleotide 5'-triphosphates. Nucleotide monophosphates, nucleotide diphosphates, or nucleotide triphosphates may be used as substrates for nucleic acid processing enzymes that catalyze the modification of nucleic acids or nucleic acid intermediates.

[0051] As used herein, the term "oligonucleotide" broadly refers to a single chain consisting of a deoxyribose or ribose sugar ring mainly or completely composed of about 2 to about 300 naturally occurring or modified nucleotide monomer units, for example, substituted by A, C, G, T or U bases, and which is connected by conventional phosphate backbone moieties. More specifically, the term refers to a single-stranded deoxyribonucleotide within the above-mentioned size range. In some embodiments, an oligonucleotide may include one or more modified bases and / or sugars. In addition to the nucleotide monomer units, an oligonucleotide may incorporate one or more detectable labels and / or one or more reactive groups.

[0052] As used herein, the term "plurality" means more than one.

[0053] As used herein, the term "polynucleotide" generally refers to an oligonucleotide comprising about 10 to about 300 nucleotide monomer units. In addition to the nucleotide monomer units, the polynucleotide may incorporate one or more detectable labels and / or one or more reactive groups.

[0054] As used herein, the term "primer" refers to a polynucleotide or modified polynucleotide that is effective as a starting point for synthesizing a polynucleotide chain complementary to a target nucleic acid chain. For example, primers for PCR include forward and reverse primers, wherein the forward primer contains a sequence complementary to a region of the target nucleic acid chain and guides the synthesis of the complementary chain. The reverse primer contains a sequence complementary to the opposite chain and guides the synthesis of the opposite chain along the target nucleic acid chain.

[0055] As used herein, the term "probe" refers to a labeled oligonucleotide or a labeled modified oligonucleotide containing a sequence complementary to a target nucleic acid sequence region, allowing the probe to form a duplex with the target sequence and generate a detectable signal indicating the presence of the target sequence region. The detectable signal is generated directly or indirectly during or after hybridization. In certain applications, such as during primer extension in 5'-nuclease PCR, the probe lacks an extendable 3' hydroxyl group to prevent polymerase-mediated extension of the probe. In certain embodiments, the probe includes Probe, TaqMan Probe, Probes, molecular beacons (for example, those disclosed in Tyagi, Sanjay & Kramer, Fred. (2012) Molecular Beacons in Diagnostics. F1000 medicine reports. 4.10.10.3410 / M4-10), and the like.

[0056] As used herein, the term "protecting group", "protective group" or "protected form" refers to a labile chemical modification of a functional group (e.g., a hydroxyl group) intended to retain its functionality and / or to obtain chemical selectivity in subsequent reactions. Protecting groups are removed from the final product by deprotection treatment (e.g., treatment with an acid).

[0057] As used herein, the term "solid support" refers to any insoluble material, including particles (e.g., beads), fibers, monoliths, membranes, filters, plastic strips, arrays, microplates, etc. In some embodiments, the solid support is a solid support suitable for automated phosphoramidite oligonucleotide synthesis, such as polystyrene and controlled pore glass (CPG).

[0058] In one aspect, provided herein is a fluorescent dye compound represented by Formula I or a stereoisomer, salt or tautomer thereof:

[0059]

[0060] in:

[0061] X is H, halogen or optionally substituted C1-C5 alkyl;

[0062] R 1 , R 2 , R 3 and R 4 is independently H or optionally substituted C1-C6 alkyl;

[0063] R 5 , R 6 , R 7 , R 8 and R 9 are independently H, halogen or optionally substituted C1-C6 alkyl;

[0064] L 1 is selected from optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 50 a heteroalkylene linker group;

[0065] L 2 and L 3 is independently selected from optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 30 a heteroalkylene linker group;

[0066] Q 1 is a hydroxyl protecting group, such as a protecting group compatible with automated oligonucleotide synthesis;

[0067] Z is CH, N, NHC(O)N or OC(O)N;

[0068] Y is OH, OP(OCH2CH2CN)NR 10 R 11 or a solid support; and

[0069] R 10 and R 11 and C1-C6 alkyl are independently optionally substituted.

[0070] In some embodiments, Formula I comprises a phosphoramidite group (ie, Y is OP(OCH2CH2CN)NR 10 R 11 ) and a hydroxyl group protected with an acid labile protecting group such as a trityl or dimethoxytrityl group. In some embodiments, Q 1 is a dimethoxytrityl group. In some embodiments, R 10and R 11 It is isopropyl.

[0071] In some embodiments of Formula I, X is Cl, Br, or F. In some embodiments, X is Cl.

[0072] In some embodiments, the compound is a compound of Formula (IA) or a stereoisomer, salt, or tautomer thereof:

[0073]

[0074] In some embodiments, R 1 is H. In some embodiments, R 2 In some embodiments, R 3 In some embodiments, R 4 In some embodiments, R 1 is H, and R 2 , R 3 and R 4 Each of them is a methyl group.

[0075] In some embodiments of Formula (I), the compound is a compound of Formula (IB) or a stereoisomer, salt, or tautomer thereof:

[0076]

[0077] In the formulae disclosed herein, the linker group L 1 It may contain one or more heteroatoms selected from N, O, S, P and combinations thereof. 1 PEG 2-10 In some embodiments, L 1 is C2-C6 alkylene or -(CH2CH2O) m CH2CH2-, wherein m is an integer from 1 to 10. In other embodiments, L 1 is an optionally substituted alkylene group, such as -CH2CH2-. In some embodiments, L 1 is an alkylene group optionally substituted by a methyl group.

[0078] In some embodiments, Z is OC(O)N.

[0079] In some embodiments of Formula (I), (IA), and (IB), the compound is a compound of Formula (IC) or a stereoisomer, salt, or tautomer thereof:

[0080]

[0081] wherein m is an integer from 1 to 10.

[0082] In some embodiments of Formula (I), the compound is a compound of Formula (ID) or a stereoisomer, salt, or tautomer thereof:

[0083]

[0084] Where W is NH or O, and L 4 is an optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 50 Heteroalkylene.

[0085] In the formula shown in this article, Q 1 represents a hydroxyl protecting group. Examples of such protecting groups are known in the art (see, e.g., Peter GMWuts, Greene's protective groups in organic synthesis (2006)). Suitable hydroxyl protecting groups include base labile groups and acid labile groups. In some embodiments, Q is a hydroxyl protecting group compatible with automated phosphoramidite oligonucleotide synthesis conditions, such as a trityl or dimethoxytrityl group. In some embodiments, Q 1 It is trimethylsilyl, TBDMS, acetyl, dimethoxytrityl or trityl.

[0086] In some embodiments, R 5 is methyl or a halogen such as Cl or F. In some embodiments, R 7 is methyl or a halogen such as Cl or F. In some embodiments, R 8 is methyl or a halogen such as Cl or F. In some embodiments, R 7 is H. In some embodiments, R 6 and R 9 It's H.

[0087] In some embodiments, the compound is a compound of Formula (IE) or a stereoisomer, salt, or tautomer thereof:

[0088]

[0089] In some embodiments of Formula (I), Y is a solid support such as controlled pore glass or polystyrene. In some embodiments of Formula (I), Y optionally comprises a linking group that connects the controlled pore glass or polystyrene to the rest of the structure.

[0090] In another aspect, the present disclosure provides a compound of Formula II or a stereoisomer, salt or tautomer thereof:

[0091]

[0092] wherein X is H, halogen or optionally substituted C1-C5 alkyl;

[0093] R 1 , R 2 , R 3 and R 4 are independently H or optionally substituted C1-C6 alkyl; and

[0094] R 5 , R 6 , R 7 , R 8 and R 9 is independently H, halogen or optionally substituted C1-C6 alkyl.

[0095] In some embodiments of Formula II, X is Cl, Br, or F. In some embodiments, X is Cl. In some embodiments, X is F.

[0096] In some embodiments of Formula II, R 1 is H. In some embodiments, R 2 In some embodiments, R 3 In some embodiments, R 4 In some embodiments, R 1 is H, and R 2 , R 3 and R 4 Each of them is a methyl group.

[0097] As used herein, the terms "alkyl", "alkenyl" and "alkynyl" include straight chain, branched and cyclic monovalent hydrocarbon groups, and combinations of these, which, when unsubstituted, contain only C and H. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. The total number of carbon atoms in each such group is sometimes described herein, for example, when the group can contain up to ten carbon atoms, it can be represented as 1-10C, C1-C 10 , C-C10 or C1-10.

[0098] As used herein, the terms "heteroalkyl", "heteroalkenyl" and "heteroalkynyl" refer to the corresponding hydrocarbons in which one or more chain carbon atoms have been replaced by heteroatoms. Exemplary heteroatoms include N, O, S and P. When heteroatoms are allowed to replace carbon atoms, for example, in a heteroalkyl group, although still written as, for example, C3-C10, the number describing the group represents the sum of the number of carbon atoms in the ring or chain and the number of such heteroatoms included as a replacement for the carbon atoms in the described ring or chain.

[0099] Typically, alkyl, alkenyl, and alkynyl substituents contain 1-10 carbon atoms (alkyl) or 2-10 carbon atoms (alkenyl or alkynyl). Preferably, they contain 1-8 carbon atoms (alkyl) or 2-8 carbon atoms (alkenyl or alkynyl). Sometimes they contain 1-6 carbon atoms (alkyl) or 2-6 carbon atoms (alkenyl or alkynyl). Individual groups may include more than one type of multiple bond, or more than one multiple bond; when such groups contain at least one carbon-carbon double bond, they are included in the definition of the term "alkenyl", and when such groups contain at least one carbon-carbon triple bond, they are included in the term "alkynyl".

[0100] As used herein, the terms "alkylene," "alkenylene," and "alkynylene" include straight chain, branched chain, and cyclic divalent hydrocarbon groups and combinations thereof.

[0101] Alkyl, alkenyl and alkynyl groups may be optionally substituted to the extent that such substitution makes chemical sense. Typical substituents include, but are not limited to, halogen (F, Cl, Br, I), =O, =N-CN, =N-OR, =NR, OR, NR2, SR, S02R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R and NO2, wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C8 10 Aryl or C5-C 10 wherein each R is optionally substituted by halogen (F, Cl, Br, I), =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SO2R', SO2NR'2, NR'SO2R', NR'CONR'2, NR'C(O)OR', NR'C(O)R', CN, C(O)OR', C(O)NR'2, OC(O)R', C(O)R', and NO2, wherein each R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl, or C5-C 10 Heteroaryl. Alkyl, alkenyl and alkynyl groups may also be replaced by C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl or C5-C 10 The heteroaryl groups are substituted, each of which may be substituted with substituents appropriate to the particular group.

[0102] Although "alkyl" as used herein includes cycloalkyl and cycloalkylalkyl groups, the term "cycloalkyl" is used herein to describe a carbocyclic non-aromatic group connected via a ring carbon atom, and "cycloalkylalkyl" is used to describe a carbocyclic non-aromatic group connected to a molecule via an alkyl linker. Similarly, "heterocyclyl" is used to identify a non-aromatic ring group containing at least one heteroatom as a ring member and connected to a molecule via a ring atom, which can be C or N; and "heterocyclylalkyl" can be used to describe such a group connected to another molecule via an alkylene linker. As used herein, these terms also include rings containing one double bond or two double bonds, as long as the ring is not aromatic.

[0103] "Aromatic" or "aryl" substituents or moieties refer to monocyclic or fused bicyclic moieties having the well-known characteristics of aromaticity; examples include phenyl and naphthyl. Similarly, the terms "heteroaromatic" and "heteroaryl" refer to such monocyclic or fused bicyclic ring systems containing one or more heteroatoms as ring members. Suitable heteroatoms include N, O and S, the inclusion of which allows aromaticity in 5- and 6-membered rings. General heteroaromatic systems include monocyclic C5-C6 aromatic groups, such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl and imidazolyl, and fused bicyclic moieties formed by fusing one of these monocyclic groups with a phenyl ring or any heteroaromatic monocyclic group to form a C8-C10 bicyclic group, such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl etc. Any monocyclic or fused bicyclic system with aromatic characteristics in terms of the electronic distribution of the whole ring system is included in this definition. It also includes bicyclic groups, wherein the ring at least directly connected to the rest of the molecule has aromatic characteristics. Generally, the ring system contains 5-12 ring member atoms. Preferably, monocyclic heteroaryl groups contain 5-6 ring members, and bicyclic heteroaryl groups contain 8-10 ring members.

[0104] The aryl and heteroaryl moieties may be substituted with various substituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C 12aryl, C1-C8 acyl, and hetero forms of these, each of which can itself be further substituted; other substituents for aryl and heteroaryl moieties include halogen (F, Cl, Br, I), OR, NR2, SR, S02R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R, and NO2, wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C7-C 12 Arylalkyl or C6-C 12 The term "arylalkyl" refers to a substituent group that is generally substituted with ...

[0105] As used herein, "optionally substituted" means that the particular group described may have one or more hydrogen substituents substituted by non-hydrogen substituents. In some optionally substituted groups or moieties, all hydrogen substituents are substituted by non-hydrogen substituents, and the non-hydrogen substituents are, for example, C1-C6 alkyl, C2-C6 heteroalkyl, alkynyl, halogen (F, Cl, Br, I), N3, OR, NR2, SR, S02R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R, oxo and NO2, wherein each R is independently H, C1-C6 alkyl or C2-C6 heteroalkyl. When an optional substituent is connected via a double bond such as carbonyl oxygen or oxo (=O), the group occupies two available valences, so the total number of substituents that can be included is reduced according to the number of available valences.

[0106] Salts, stereoisomers and tautomers of the compounds disclosed herein are also within the scope of the present disclosure. As used herein, "stereoisomer" or "stereoisomers" refers to compounds with different chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. As used herein, "tautomers" refers to alternative forms of compounds with different proton positions, such as enol-ketone and imine-enamine tautomers. As used herein, the "salt" of a compound refers to the ion of a compound associated with a counterion ion. Salts of compounds can be formed by neutralization reactions of acids and bases. Salts can be derived from a variety of organic and inorganic counterions well known in the art, and include sodium, potassium, calcium, magnesium, ammonium and tetraalkylammonium as examples only; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochlorides, hydrobromides, tartrates, mesylates, acetates, maleates and oxalates. Although the structures of the compounds disclosed herein may be shown in only one resonance form, it is understood that all resonance forms are included.

[0107] The dyes of the present disclosure can be prepared by any suitable method. For example, the dyes can be prepared according to the methods shown in the following reaction schemes I and II.

[0108] On the other hand, provided herein are polynucleotides labeled with fluorescent dyes, which are prepared by automated oligonucleotide synthesis using fluorescent dye phosphoramidites disclosed herein. The polynucleotides comprise portions derived from compounds of the present disclosure. As used herein, "fluorescent dye labeled polynucleotides" or "labeled polynucleotides" refer to polynucleotides prepared by automated oligonucleotide synthesis from compounds of the formula disclosed herein.

[0109] The polynucleotides of the labels disclosed herein may include one or more additional parts. In some embodiments of the present disclosure, the polynucleotides of the labels include a minor groove binder. In some embodiments, the polynucleotides of the labels include an intercalator. In some embodiments, the polynucleotides of the labels include a second fluorophore and / or a fluorescence quencher.

[0110] Generally, the polynucleotides labeled with dyes disclosed herein are polynucleotides wherein the backbone comprises 2'-deoxyribose or ribose. However, the polynucleotides of the labeling may comprise one or more modifications. In some embodiments, the polynucleotides comprise sugar modifications, such as modified sugars. Various sugar modifications are useful. Some non-limiting sugar modifications include arabinose, d-arabinohexitol, 2-fluoroarabinose, xylulose, hexose or bicyclic sugars.

[0111] The polynucleotide of the mark of the present disclosure may include one or more backbone modifications. In some embodiments, the polynucleotide includes backbone modifications. In some embodiments, the backbone modification is selected from the sugar phosphate backbone, locked nucleic acid backbone, peptide backbone, phosphotriester backbone, phosphoramidate backbone, siloxane backbone, carboxymethyl ester backbone, acetamidate backbone, carbamate backbone, thioether backbone, bridged methylene phosphonate backbone, thiophosphate backbone, methylphosphonate backbone, alkylphosphonate backbone, phosphate backbone, alkylthiophosphonate backbone, dithiophosphate backbone, carbonate backbone, phosphotriester backbone, carboxymethyl ester backbone, methylthiophosphate backbone, dithiophosphate backbone, backbone with ethoxy bond, and any two or more combinations of the foregoing. In a specific embodiment of the present disclosure, the backbone modification is the sugar phosphate backbone modified.

[0112] The polynucleotides of the labels disclosed herein may include one or more modified or non-natural bases. Modified bases include modified thymine and cytosine bases (e.g., those disclosed in U.S. Pat. Nos. 9,598,455 and 9,598,456), 2,6-diaminopurine bases, universal bases, etc. The polynucleotides of the labels disclosed herein may include non-nucleoside segments or non-nucleoside monomers (e.g., linkers such as poly(ethylene glycol) linkers).

[0113] In some embodiments, the polynucleotides disclosed herein are probes, such as 5'-nuclease PCR probes. In certain embodiments, the polynucleotides further comprise one or more additional labels, such as fluorescent quenchers. As will be appreciated by those of ordinary skill in the art, the position of the label within the oligonucleotide may vary and is not limited to the disclosure herein.

[0114] In some embodiments, provided herein is a modified polynucleotide comprising a dye portion as a fluorophore at one end of its sequence and a fluorescence quencher at the other end of its sequence, such that the fluorescence quencher inhibits the fluorescence signal of the fluorophore in the complete probe (i.e., the oligonucleotide used as the probe) via an energy transfer mechanism such as fluorescence resonance energy transfer ("FRET"). When the polymerase extends the primer along the template to which the probe is also hybridized, the 5'-nuclease activity of the polymerase cuts the probe, thereby diffusing the fluorophore away from the fluorescence quencher, so that a fluorescence signal is now detected. The signal increases proportionally with each PCR cycle with the amount of the probe cut, and therefore increases proportionally with the amount of the amplified product (e.g., amplicon, target sequence). This allows direct detection and quantification of the target DNA sequence.

[0115] In some embodiments, the dye moiety is at least one nucleotide position away from the polynucleotide sequence end of the label and the fluorescence quencher is connected to the base at least one nucleotide position away from the other end of the polynucleotide modified. In some embodiments, the dye moiety and the fluorescence quencher are located inside the probe. As will be appreciated by those of ordinary skill in the art, the position of the fluorophore and / or fluorescence quencher in the probe can vary and is not limited.

[0116] In some embodiments, the dye moiety and the fluorescence quencher are not at the ends of the FRET probe. In some embodiments, the emission spectrum of the dye overlaps significantly with the absorption spectrum of the fluorescence quencher. However, when the quenching involves a collision mechanism, or the overlap increases due to reaction conditions or probe structure, for example, such spectral overlap is less important or not required.

[0117] There is a wealth of practical guidance in the art for selecting an appropriate fluorophore-quencher for a particular probe. See, for example, FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971). Quenchers that can be used for inclusion in the probes disclosed herein include disazo quenchers (e.g., those disclosed in U.S. Pat. No. 6,790,945), quenchers available from Biosearch Technologies, Inc. (Black Hole TM Quenchers: BHQ-1, BHQ-2 and BHQ-3), TAMRA, carboxytetramethylrhodamine, 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl), Quencher, Quencher, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)-I and 2-[6-(1,3-dihydro-2H-isoindol-2-yl)-9-{2-[(4-[(2,5-dioxopyrrolidin-1-yl)oxy]carbonylpiperidin-1-yl)sulfonyl]phenyl}-3H-xanthene-3-ylidene]-2,3-dihydro-1H-isoindolium chloride (2-[6-(1,3-dihydro-2H-isoindol-2-yl)-9-{2-[(4-[(2,5-dioxopyrrolidin-1-yl)oxy]carbonylpiperidin-1-yl)sulfonyl]phenyl}-3H-xanthene-3-ylidene]-2,3-dihydro-1H-isoindolium chloride

[0118] piperidin-1-yl)sulfo-nyl]phenyl}-3H-xanthen-3-ylidene]-2,3-dihydro-1H-isoin dolium chloride) (QSY21) and other quenchers known in the art.

[0119] On the other hand, disclosed herein is a method for preparing a labeled conjugate of a ligand, which includes contacting the ligand with a compound of the formula provided herein in a suitable solvent, under conditions sufficient to covalently link the compound to the ligand, thereby forming a dye-labeled conjugate. Suitable ligands include biomolecules (e.g., polynucleotides, oligonucleotides, proteins, antibodies, peptides or polysaccharides), synthetic polymers (e.g., polymers with olefinic backbones, such as polyacrylic acid) and solid supports (e.g., controlled pore glass or polystyrene).

[0120] In some embodiments, the part is a polynucleotide. In some embodiments, the condition that is enough to covalently link the compound of the present disclosure to the part, i.e., oligonucleotide or polynucleotide, is the phosphoramidite oligonucleotide synthesis condition of automation. The phosphoramidite oligonucleotide synthesis condition of automation for synthesizing and deprotecting synthetic oligonucleotides is well known in the art, and for example, in the current scheme (Current Protocols in Nucleic Acid Chemistry) in nucleic acid chemistry, volume 1, Beaucage et al. edit, John Wiley & Sons, described in 2002, and its disclosure is incorporated herein by reference.

[0121] The phosphoramidite method of oligonucleotide synthesis such as DNA is considered to be the standard synthesis method used in most automated synthesizers. The structural unit for synthesis is generally referred to as a nucleotide structural unit, a monomer or a nucleoside phosphoramidite, which is an activated nucleoside derivative (phosphoramidite). Acid-cleavable protecting groups, generally dimethoxytrityl (DMT) groups, are used to protect the 5'-end of nucleosides, and β-cyanoethyl groups are used to protect 3'-phosphite moieties. Monomers can also include other groups for protecting other parts such as reactive primary amines in core bases. Protective groups are selected to prevent branching or other unwanted side reactions from occurring during the synthesis process. Skilled technicians will be able to easily select protecting groups with properties suitable for use under specific synthesis and deprotection and / or cleavage conditions. For example, in Greene & Wuts, "Protective Groups In Organic Chemistry," 3rd edition, John Wiley & Sons, 1999 (hereinafter referred to as "Green & Wuts") teaches a variety of amine protecting groups.

[0122] Typically, oligonucleotides are synthesized on a solid support such as controlled pore glass (CPG) or a polystyrene packed column, membrane or similar material. Oligonucleotides are typically synthesized from the 3' end to the 5' end. The first nucleotide structural unit or monomer is typically fixed to the support via a linker such as a long chain alkylamine controlled pore glass (LCAA-CPG).

[0123] In some embodiments, the synthesis method using a phosphoramidite reagent involves multiple rounds of: (i) DMT deprotection to reveal the free hydroxyl group, which can be achieved, for example, by treatment with 2.5% or 3% dichloroacetic acid or trichloroacetic acid in dichloromethane; (ii) coupling of a nucleoside or other phosphoramidite reagent to the free hydroxyl group, which can be performed, for example, in acetonitrile containing tetrazole (e.g., 0.45M or 0.5M tetrazole); (iii) oxidation, which can be performed, for example, by treatment with I2 / 2,6-lutidine / H2O; and capping, which can be performed, for example, by treatment with 6.5% acetic anhydride in tetrahydrofuran (THF) and activation with 10% 1-methylimidazole (NMI) in THF.

[0124] Other conditions for carrying out the various steps in the synthesis are also known in the art and can be used herein. For example, phosphoramidite coupling can be carried out in acetonitrile containing 0.25M 5-ethylthio-1H-tetrazolyl, 0.25M 4,5-dicyanoimidazole (DCI) or 0.25M 5-benzylthio-1H-tetrazolyl (BTT). Oxidation can be carried out using 0.1M, 0.05M or 0.02M I2 in THF / H2O / pyridine (7:2:1). End-capping can be carried out by treating with THF / lutidine / acetic anhydride and then by treating with 16% NMI in THF.

[0125] Removal of any protecting groups and cleavage from synthetic reagents is generally accomplished by treatment with concentrated ammonium hydroxide at 60°C for 1-12 hours, although nucleoside phosphoramidites protected with groups that can be removed under milder conditions such as by treatment with concentrated ammonium hydroxide at room temperature for 4-17 hours or with 0.05 M potassium carbonate in methanol, or with 25% tert-butylamine in water / ethanol are also known and can be used.

[0126] The term "cleavage" with respect to solid phase oligonucleotide synthesis refers to the destruction of the bond connecting the oligonucleotide to the solid support. In some embodiments, cleavage involves the hydrolysis of the succinate bond between the 3' hydroxyl group of the attached oligonucleotide and the solid support.

[0127] The term "deprotection" as used herein refers to the removal of a protecting group from an exocyclic amine of a heterocyclic base of an oligonucleotide. Typically, deprotection involves the hydrolysis of an amide moiety consisting of an exocyclic amine and an amino protecting group such as a benzoyl or isobutyryl group. Various deprotection techniques and methods are known in the art.

[0128] On the other hand, provided herein are kits comprising labeled polynucleotides or compounds of the present disclosure. In some embodiments, the kit is a PCR diagnostic kit comprising one or more polynucleotides of the present disclosure. In some embodiments, the kit is an automated molecular diagnostic kit.

[0129] Although each element of the present invention is described herein as comprising multiple embodiments, it should be understood that each embodiment of a given element of the present invention can be used with each embodiment of the other elements of the present invention, unless otherwise specified, and that each such use is intended to form a different embodiment of the present invention.

[0130] The referenced patents, patent applications, and scientific literature cited herein are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification should be resolved in favor of the latter. Similarly, any conflict between the definition of a word or phrase as understood in the art and the definition of a word or phrase specifically taught in this specification should be resolved in favor of the latter.

[0131] The present invention is illustrated by the following examples. These examples are included for illustrative purposes only and are not intended to limit the present invention. Example

[0132] Proton ( 1 H, 400MHz) and phosphorus ( 31 P, 160MHz) nuclear magnetic resonance (NMR) spectra were obtained on a Bruker Biospin 400 instrument. NMR samples were prepared in DMSO-d6 and CD3CN, and the residual protonated solvent was used as an internal chemical shift standard. LCMS data were obtained by electrospray ionization (ESI) on Agilent 1200 series (LC / MSD Trap XCT Plus) and Agilent 1260infinity (6130 quadrupole LC / MS) instruments. Biotage Isolera LS and Teledyne ISCO Automated chromatography was performed on a Combi Flash instrument on silica gel 60. Analytical thin layer chromatography was performed on aluminum-backed silica gel 60 F254 and the plates were visualized under UV light (254 and 365 nm). All reagents were from commercial sources unless otherwise stated.

[0133] Polynucleotides comprising compounds of the present disclosure were synthesized on a MerMade-12 oligonucleotide synthesizer using a standard 200 nmol DNA protocol with DMT removal-coupling-capping-oxidation-capping cycles. The coupling time of the dye phosphoramidites of the present disclosure can be adjusted accordingly.

[0134] For polynucleotides containing a 5' dye, the synthesis is complete after the last dye phosphoramidite coupling cycle. In the case of internal incorporation of the dye phosphoramidite, more nucleoside monomers are added after dye coupling. The final DMT group remains on the polynucleotide.

[0135] The fully assembled polynucleotides were cleaved from the solid support and deprotected by 30% ammonium hydroxide at 55°C for 10-12 hours. After removal of ammonia, the oligonucleotides were analyzed and purified by reverse phase HPLC (RP-HPLC) on a C18 Gemini column with a linear gradient elution of acetonitrile / 0.1M triethylammonium bicarbonate at pH 7. After removal of the DMT group, the polynucleotides were purified a second time. The excitation and emission spectra of the labeled oligonucleotides were recorded on an Agilent Cary fluorometer (200 nM oligonucleotides, 0.1 M Tris buffer, pH 8).

[0136] An exemplary synthesis of an exemplary fluorescent dye phosphoramidite (eg, Compound D) of the present disclosure is shown in Reaction Scheme I.

[0137] First, starting alcohol compound A (for example, the compound of Table I) in the presence of a base, original position is acylated with bis (4-nitrophenyl) carbonate, then replaces 4-nitrophenyl ester with secondary amine nucleophilicity, forms carbamate bond to produce intermediate product C.Product can be separated by flash chromatography.Under the anhydrous condition excluding acid, standard phosphitylation is used that compound C is converted into phosphoramidite D.Protection reaction mixture and product are protected from the influence of light and air (oxygen).Use silica gel flash chromatography to purify product.

[0138] Reaction Scheme I.

[0139]

[0140] Alcohol compounds A, such as the compounds of Table 1, can be prepared according to the method shown in Reaction Scheme II.

[0141] Reaction Scheme II.

[0142]

[0143] Table 1.

[0144]

[0145] Preparation of Precursor Alcohol A (Compound 19A).

[0146]

[0147] Under a dry, inert atmosphere, 2,3-dihydro-2,3,3-trimethyl-1-(2-hydroxyethyl)-indol-5-ol (prepared according to the steps of WO 2019 / 217470; 28.0 mmol) and 4-fluororesorcinol (56.0 mmol) were suspended in 50 mL of methanesulfonic acid. The solution was heated at 55 °C until it became homogeneous. 2,4,5-Trimethylbenzaldehyde (61.6 mmol) in methanesulfonic acid (15 mL) was added to the solution, and the mixture was heated at 55 °C for 1.5 hours. The reaction flask was placed in an ice bath, and 150 g of crushed ice and 125 ml of water were added. The mixture was extracted three times with THF / CHCl3 (1:2), and the combined organic layers were extracted with saturated NaHCO3 and dried over anhydrous Na2SO4. Silica gel chromatography using acetonitrile / water provided 3.67 mmol (13%) of dye compound 19A with excitation / emission maxima of 528 nm / 546 nm, respectively.

[0148] Preparation of an exemplary phosphoramidite (Compound 19D).

[0149]

[0150] Under an argon atmosphere, triethylamine was added to a solution of compound 19A in anhydrous N,N-dimethylformamide, and then BNPC was added at one time. The reaction was incubated for 40 minutes. The connector compound L1 was added to the reaction mixture, and the reaction mixture was incubated at ambient temperature for 2.5 hours. The reaction mixture was transferred to a separatory funnel and the reaction flask was rinsed with ethyl acetate. The solution was extracted with water. The aqueous layer was discarded, and the organic layer was further washed with deionized water and a saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate for 18 hours under stirring. The crude product was purified by flash chromatography to produce compound 19C.

[0151] Compound 19C was dissolved in anhydrous dichloromethane under argon, and anhydrous N,N-diisopropylethylamine was added via a syringe under stirring. The reaction flask was placed in an ice bath for 10 minutes to cool the reaction mixture to <10°C. Pam-Cl was added dropwise via a syringe within 1 minute, and the reaction mixture was removed from the ice. The reaction mixture was protected from light by wrapping the flask with aluminum foil, and it was gradually warmed to room temperature over 2.5 hours. The reaction mixture was diluted with ethyl acetate and transferred to a separatory funnel under argon. After transfer, the reaction mixture was extracted with 10% saturated sodium bicarbonate solution. The water layer was discarded, and the organic layer was extracted with saturated sodium chloride to remove water. The organic layer was dried over anhydrous sodium sulfate under argon and protected from light. After drying, the solution was filtered and the solvent was evaporated to produce a red foamy crude product. The crude product was purified by flash chromatography. The product compound 19D was analyzed by HPLC, LC-MS and NMR.

[0152] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

1. A compound of formula I or a stereoisomer, salt or tautomer thereof: in: X is H, halogen or C1-C5 alkyl; R 1 , R 2 , R 3 and R 4 is independently H or optionally substituted C1-C6 alkyl; R 5 , R 6 , R 7 , R 8 and R 9 are independently H, halogen or optionally substituted C1-C6 alkyl; L 1 is an optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 50 heteroalkylene; L 2 and L 3 is independently an optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 30 heteroalkylene; Q 1 is trimethylsilyl, TBDMS, acetyl, dimethoxytrityl or trityl; Z is CH, N, NHC(O)N or OC(O)N; Y is OH, OP(OCH2CH2CN)NR 10 R 11 or a solid support; and R 10 and R 11 is independently an optionally substituted C1-C6 alkyl group, wherein optionally substituted alkyl refers to an alkyl optionally substituted by F, Cl, Br, I, =O, =N-CN, =N-OR, =NR, OR, NR2, SR, S02R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R or NO2, wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C8 10 Aryl or C5-C 10 wherein each R is optionally substituted by F, Cl, Br, I, =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SO2R', SO2NR'2, NR'SO2R', NR'CONR'2, NR'C(O)OR', NR'C(O)R', CN, C(O)OR', C(O)NR'2, OC(O)R', C(O)R', and NO2, wherein each R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl, or C5-C 10 Heteroaryl, wherein optionally substituted heteroalkylene refers to heteroalkylene optionally substituted with C1-C6 alkyl, C2-C6 heteroalkyl, alkynyl, F, Cl, Br, I, N3, OR, NR2, SR, S02R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R, oxo, and NO2, wherein each R is independently H, C1-C6 alkyl, or C2-C6 heteroalkyl, Wherein, heteroalkylene, heteroalkyl, heteroalkenyl, heteroalkynyl and heteroacyl refer to the corresponding hydrocarbons in which one or more chain carbon atoms have been replaced by heteroatoms selected from N, O, S and P, and Herein, heteroaryl refers to a monocyclic or fused bicyclic aromatic ring system containing one or more heteroatoms selected from N, O and S as ring members.

2. The compound of claim 1, or a stereoisomer, salt or tautomer thereof, wherein X is Cl, Br or F.

3. The compound or stereoisomer, salt or tautomer thereof of any one of claims 1-2, wherein the solid support is controlled pore glass or polystyrene.

4. The compound or stereoisomer, salt or tautomer thereof according to any one of claims 1 to 2, wherein the compound or stereoisomer, salt or tautomer thereof is a compound or stereoisomer, salt or tautomer thereof of formula (IA):

5. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 1 It's H.

6. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 2 It's methyl.

7. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 3 It's methyl.

8. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 4 It's methyl.

9. The compound or stereoisomer, salt or tautomer thereof of claim 1, wherein the compound or stereoisomer, salt or tautomer thereof is a compound or stereoisomer, salt or tautomer thereof of formula (IB):

10. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein L 1 is C2-C6 alkylene or -(CH2CH2O) m CH2CH2-, wherein m is an integer from 1 to 10.

11. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein L 1 It is a C2 alkylene group.

12. The compound of any one of claims 1-2, or a stereoisomer, salt or tautomer thereof, wherein Z is OC(O)N.

13. The compound or stereoisomer, salt or tautomer thereof according to any one of claims 1 to 2, wherein the compound or stereoisomer, salt or tautomer thereof is a compound or stereoisomer, salt or tautomer thereof of formula (IC): wherein m is an integer from 1 to 10.

14. The compound or stereoisomer, salt or tautomer thereof of claim 1, wherein the compound or stereoisomer, salt or tautomer thereof is a compound or stereoisomer, salt or tautomer thereof of (ID): Where W is NH or O, and L 4 is an optionally substituted C2-C 10 Alkylene or optionally substituted C2-C 50 Heteroalkylene.

15. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 10 and R 11 It is isopropyl.

16. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 5 It's methyl.

17. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 7 It's methyl.

18. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 8 It's methyl.

19. The compound according to any one of claims 1 to 2, or a stereoisomer, salt or tautomer thereof, wherein R 6 and R 9 It's H.

20. The compound of claim 1 or a stereoisomer, salt or tautomer thereof, wherein the compound or a stereoisomer, salt or tautomer thereof is a compound of formula (IE) or a stereoisomer, salt or tautomer thereof:

21. A labeled polynucleotide prepared using automated phosphoramidite synthesis, which is formed from the compound of any one of claims 1 to 20, or a stereoisomer, salt or tautomer thereof.

22. The labeled polynucleotide of claim 21, wherein the labeled polynucleotide further comprises a fluorescence quencher.

23. The labeled polynucleotide of claim 21, wherein the labeled polynucleotide is attached to a solid support.

24. The labeled polynucleotide of claim 23, wherein the solid support is controlled pore glass beads, polystyrene beads, magnetic beads, or a microplate.

25. A method for preparing a labeled conjugate of a ligand, comprising contacting the ligand with a compound according to any one of claims 1 to 20 in a suitable solvent under conditions sufficient to covalently link the compound to the ligand, thereby forming a labeled conjugate; wherein Y is OP(OCH2CH2CN)NR 10 R 11 And R 10 and R 11 and C1-C6 alkyl are independently optionally substituted.

26. The method of claim 25, wherein the ligand is a polynucleotide or a solid support.

27. The method of claim 25, wherein the ligand is a polynucleotide.

28. The method of claim 25, wherein the conditions sufficient to covalently attach the compound to the ligand are automated phosphoramidite oligonucleotide synthesis conditions.

29. An automated molecular diagnostic kit comprising the labeled polynucleotide of claim 21 or claim 22.

30. The kit of claim 29, wherein the kit is a PCR diagnostic kit.

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