WO2019105421A1 - 核苷类似物、制备方法及应用 - Google Patents

核苷类似物、制备方法及应用 Download PDF

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WO2019105421A1
WO2019105421A1 PCT/CN2018/118259 CN2018118259W WO2019105421A1 WO 2019105421 A1 WO2019105421 A1 WO 2019105421A1 CN 2018118259 W CN2018118259 W CN 2018118259W WO 2019105421 A1 WO2019105421 A1 WO 2019105421A1
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group
analog
compound according
base
mixture
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PCT/CN2018/118259
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English (en)
French (fr)
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刘斌
赵陆洋
陈予想
杨波
简杰
陈方
颜钦
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深圳市瀚海基因生物科技有限公司
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Priority to EP18884565.5A priority Critical patent/EP3733683A4/en
Priority to CN201880077576.4A priority patent/CN111741967A/zh
Publication of WO2019105421A1 publication Critical patent/WO2019105421A1/zh
Priority to US16/882,849 priority patent/US11512106B2/en
Priority to US17/964,696 priority patent/US20230114671A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/14—Pyrrolo-pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to the field of compounds, and in particular, to the preparation and use of nucleoside analogs, nucleoside analogs.
  • DNA sequencing is a basic tool for biological research and drug research.
  • sequencing by synthesis SBS
  • sequencing by side SBL
  • the SBS nucleic acid sequencing platform on the market including the second and third generation sequencing platforms, mostly uses DNA polymerase, based on the principle of base complementary pairing, to add nucleotide analogs to the template (base extension), and to detect nucleotide similarity.
  • the signal is combined with a signal emitted after the template is introduced or a change in the physicochemical signal is brought about to achieve the determination of the nucleic acid sequence.
  • nucleotide analog having a stable structure, stable detectable signal, high reaction efficiency, and effective inhibition of nucleotide binding to a template at the next position has been a problem to be solved or to be improved.
  • L 1 , L 2 , and L 3 are each independently a covalent bond or a covalent linking group;
  • B is a base or a base derivative;
  • R 1 is an -OH or a phosphate group; and
  • R 2 is H or a cleavage group;
  • R 3 is a detectable group or a targeting group;
  • R 5 is a polymerase reaction inhibiting group;
  • R 4 is H or -OR 6 , wherein R 6 is H or a cleavable group; Cutting groups or cleavable bonds.
  • nucleoside The acid analog is attached to the 3' end of the newly synthesized strand (complementary to the template strand) by the action of an enzyme, base pairing with the template strand, and these nucleotide analogs contain an inhibitory group or a related structure, which can prevent The next base, nucleotide or nucleotide analog is ligated to prevent multiple base pairings on the same template strand in a single reaction in one cycle, and can be effectively applied to nucleic acid sequence determination.
  • the compounds are suitable for use in a variety of sequencing platforms, including, but not limited to, the HiSeq/MiSeq/NextSeq/NovaSeq platform for Illumina, BGI SEQ 50/500 for BGI, the Ion Torrent platform from ThermoFisher, and the Sequel platform from PacBio.
  • sequencing platforms including, but not limited to, the HiSeq/MiSeq/NextSeq/NovaSeq platform for Illumina, BGI SEQ 50/500 for BGI, the Ion Torrent platform from ThermoFisher, and the Sequel platform from PacBio.
  • the fluorescent molecule/extension product/template-primer complex appears as a bright spot on the image. Any factor that affects the luminescent behavior of the fluorescent molecule directly affects the application of the compound.
  • the intramolecular quenching that is, the reduction potential of the base in the same dNTP molecule affects the luminescence behavior of the fluorescent molecule in the molecule, the base and fluorescence in the compound
  • the degree of freedom of the dNTP molecule is mainly determined by the rigidity and length of the linker between the base and the fluorescent molecule in the dNTP molecule. In general, the shorter the linker, the stronger the rigidity and the more the degree of freedom. Small, the smaller the quenching between molecules.
  • the compounds of the above structure designed by the inventors have balanced considerations and verified intramolecular and intermolecular quenching, and are particularly suitable for use in edge synthesis sequencing platforms.
  • a dNTP analog is presented.
  • the dNTP analog is selected from at least one of the following: a dATP analog, which is a compound of the formula (I) shown above, B is a base A; dCTP is similar
  • the dCTP analog is a compound of the formula (I) shown above, B is a base C;
  • a dGTP analog the dGTP analog is a compound of the formula (I) shown above, and B is a base.
  • G and a dTTP analog, the dTTP analog is a compound of the formula (I) shown above, and B is a base T.
  • the dNTP analog comprises one, two, three or all four of a dATP analog, a dCTP analog, a dGTP analog, and a dTTP analog.
  • an NTP analog is presented.
  • the NTP analogue is selected from at least one of the following: an ATP analog, a CTP analog, a GTP analog, and a UTP analog, a so-called ATP analog, a CTP analog, a GTP analog
  • the UTP analogs are the compounds of the formula (I) shown above, each of which is a base A, C, G and U, respectively.
  • the NTP analog comprises one, two, three or all four of an ATP analog, a CTP analog, a GTP analog, and a UTP analog.
  • a dNTP analog mixture comprises a dATP analog, a dCTP analog, a dGTP analog, and a dTTP analog, the so-called dATP analog, dCTP analog, dGTP analog and dTTP analog are in front.
  • the compounds of formula (I) are shown, each B being a base A, C, G and T, respectively, and at least three of the so-called dATP analogs, dCTP analogs, dGTP analogs and dTTP analogs Species have different detectable groups or targeting groups, respectively.
  • a dNTP analog mixture comprises a combination of any two of the following nucleotide analogs: dATP analog, dCTP analog, dGTP analog and dTTP analog, said dATP, dCTP,
  • the dGTP and dTTP analogs are the compounds of the formula (I) shown above, each of which is a base A, C, G and T, respectively, wherein the two nucleotide analogues in the combination have different detectable a group or a targeting group.
  • the NTP analog mixture comprises an ATP analog, a CTP analog, a GTP analog, a compound of any of the preceding embodiments, and a UTP analog, said ATP, CTP, GTP and UTP analogs Is a compound of the formula (I) shown above, wherein each B is a base A, C, G and U, respectively, wherein the three nucleotide analogues in the combination have different detectable groups or targets Group.
  • a mixture of NTP analogs is presented.
  • the NTP analog mixture comprises a combination of any two of the following nucleotide analogs: an ATP analog, a CTP analog, a GTP analog is a compound of any of the preceding examples and a UTP analog
  • ATP adenosine triphosphate
  • CTP CTP
  • GTP GTP
  • UTP UTP analog
  • the so-called ATP, CTP, GTP and UTP analogs are the compounds of the formula (I) shown above, each of which is a base A, C, G and U, respectively, wherein the two nucleosides in the combination Acid analogs have different detectable groups.
  • a dNTP analog, a NTP analog, a dNTP analog mixture or a mixture of NTP analogs in any of the preceding embodiments in nucleic acid sequencing or controllable polymerase chain reaction or base extension reaction Use in.
  • kits for nucleic acid sequencing or a controllable chain polymerase reaction comprises: a dNTP analog, a NTP analog, a dNTP analog mixture or a mixture of the preceding NTP analogs in any of the preceding embodiments.
  • the kit can be used for DNA and/or RNA nucleic acid sequencing.
  • a method of determining a nucleic acid sequence comprises: (a) placing a mixture of the first nucleic acid template-primer complex, one or more nucleotide analogs, and a DNA polymerase under conditions suitable for base extension a nucleotide analog is conjugated to the first nucleic acid template-primer complex to obtain an extension product; the nucleotide analog is selected from the group consisting of dNTP analogs, NTP analogs, dNTP analog mixtures or Mixture of NTP analogs.
  • the method utilizes the nucleotide analog having the above structural features, can effectively convert the biochemical change into a stable photoelectric signal and collect, and realize the effective and accurate determination of the base sequence of the nucleic acid template.
  • a method of extending a primer comprises placing a polymerase, a nucleic acid template-primer complex, and one or more nucleotide analogs in a reaction vessel to bind the nucleotide analog to the nucleic acid template-primer complex Above, thereby obtaining an extension product; the nucleotide analogue is selected from a compound of any of the foregoing structural formulas such as formula (I).
  • This method can extend primers accurately and efficiently.
  • a reaction mixture comprises a template to be tested, a primer paired with at least a portion of the template strand to be tested, a DNA polymerase and a mixture of dNTP analogs, NTP analogs, dNTP analogs in any of the preceding embodiments Or a mixture of NTP analogs.
  • the method comprises: synthesizing SPDP using dithiodipyridine and mercaptopropionic acid; And synthesizing the first connecting structure with ethyl chloroformate, the first connecting structure is DNTP and SPDP were ligated to obtain dNTP-SPDP; the target compound was synthesized using the so-called first ligation structure and dNTP-SPDP.
  • a fourteenth aspect of the invention there is provided a process for the preparation of a compound of formula (I) of any of the preceding examples.
  • the structure of dNTP-MPSSK is as follows: use And hexapeptide synthesis second connection structure, the second connection structure is The so-called hexapeptide is H-Pro-Lys(Fmoc)-Pro-Asp-Asp-OH; the second linking structure and dNTP-MPSSK are mixed to prepare a compound.
  • FIG. 2 is a diagram showing detection results of an extended product according to an embodiment of the present invention.
  • FIG. 3 is a diagram showing the results of detection of products after extension according to an embodiment of the present invention.
  • FIG. 4 is a schematic diagram showing the read length distribution of a read fragment obtained by applying a compound according to an embodiment of the present invention
  • FIG. 5 is a schematic diagram showing the read length distribution of a read segment obtained by sequencing verification of two fluorescently labeled nucleotide analogs according to an embodiment of the present invention
  • Figure 6 is a flow diagram showing the application of a nucleotide analog comprising a capping step to base extension/sequencing according to an embodiment of the invention
  • Figure 7 is a flow diagram showing the pairing of nucleotide analogs with virtual blocking groups without capping in accordance with an embodiment of the present invention
  • Figure 8 is a flow diagram of pairing of nucleotide analogs with virtual blocking groups without capping in accordance with an embodiment of the present invention
  • FIG. 9 is a flowchart of base blocking pairing of virtual blocking according to an embodiment of the present invention.
  • FIG. 10 is a flow chart showing the application of a two-color virtual blocking nucleotide analog to SBS sequencing according to an embodiment of the present invention
  • FIG. 11 is a flow chart showing the application of a two-color virtual blocking Capless nucleotide analog to SBS sequencing according to an embodiment of the present invention
  • FIG. 12 is a flow chart of a two-color sequencing method according to an embodiment of the present invention.
  • FIG. 13 is a flow chart of a four-color sequencing method according to an embodiment of the present invention.
  • reagents, detection instruments and the like in the examples can be prepared, prepared or obtained by a person skilled in the art unless otherwise specified.
  • Base and “B” are equivalent, and refer to a base, and the base includes A, T, C, G, U and derivatives thereof.
  • controlled chain polymerase reaction refers to a polymerase chain reaction which can control the reaction to proceed continuously or discontinuously, for example, a nucleic acid sequence determination reaction based on the principle of edge synthesis sequencing.
  • substituted means that one or more hydrogen atoms in a given structure are replaced by a particular substituent.
  • an optional substituent group can be substituted at each substitutable position of the group.
  • the substituents may be substituted at the various positions, either identically or differently.
  • C 1 - 6 alkyl refers particularly to the disclosure independently methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl.
  • alkyl or "alkyl group” as used herein, denotes a saturated straight or branched monovalent hydrocarbyl group containing from 1 to 20 carbon atoms, wherein the alkyl group may optionally be Substituted by one or more substituents described herein. Unless otherwise specified, an alkyl group contains from 1 to 20 carbon atoms. In one embodiment, the alkyl group contains from 1 to 12 carbon atoms; in another embodiment, the alkyl group contains from 1 to 6 carbon atoms; in yet another embodiment, the alkyl group contains 1 - 4 carbon atoms; also in one embodiment, the alkyl group contains 1-3 carbon atoms.
  • alkyl as such or as part of a substituent means, unless otherwise specified, a straight C chain, or a branched C chain, which may be fully saturated or single or multiple. a saturated bond and includes monovalent, divalent, and polyvalent groups of a specified carbon, where alkane refers to an unsaturated carbon chain, and saturated group means, but is not limited to, methyl, ethyl, n-propyl, and iso- Propyl, n-butyl, tert-butyl, isobutyl, (cyclohexyl)methyl, n-pentyl or its isomer, n-hexyl or its isomer, n-heptyl or Isomer, n-octyl or isomer thereof, where unsaturated group means, but not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,3-pentadienyl
  • alkylene as used herein or as a substituent is derived from an alkyl group and a saturated alkyl group, which means, but is not limited to, 1 to 24 carbon atoms (eg, -CH 2 CH 2 CH 2 CH 2 -) an unsaturated alkyl derivative of a carbon chain, preferably a carbon chain of 1 to 10 carbon atoms.
  • heteroalkyl refers to a straight or branched chain and which contains at least one carbon atom and one hetero atom (eg, O, N, P, Si and S). ), N and S are preferred here.
  • the heteroalkyl group is not a cyclic group.
  • examples thereof include -CH 2 -NH-OCH 3 , -CH 3 -O-Si(CH 3 ) 3 .
  • the heteroalkane also contains a triple bond or a double bond group.
  • heteroalkylene is itself or as part of a substituent derived from an alkyl group and a saturated alkyl group. Examples thereof include, but are not limited to, unsaturated derivatives of -CH 2 -CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 - chains, for heteroalkenes
  • the hetero atom may occupy one or both of the chain ends (eg, an alkyleneoxy group, an alkylenedioxy group, a sub-substituted amino group, an alkylene diamino group).
  • cycloalkyl and “cycloheteroalkyl” are themselves or as part of a substituent which is a cyclic form of alkyl and heteroalkyl, which is not meant to be an aromatic group, and includes, but is not limited to, examples of cycloalkyl groups.
  • acyl refers to -C(O)R, wherein R is a substituent, possibly an alkyl group, a cycloalkyl group, a heteroalkyl group, and the like.
  • aryl is an unsaturated group, an aryl group or the like
  • aromatic heterocyclic group means a hetero atom (for example, O, N, P, Si and S).
  • examples include, but are not limited to, phenyl, naphthyl, imidazolyl, pyrimidinyl and the like.
  • detectable label refers to a group that is capable of specifically releasing a signal or specifically recognizing (eg, a fluorescent dye, a detectable dye).
  • a) four dyes, ROX, Alexa532, Cy5, IF700 are labeled A, T, G, C, respectively;
  • a dye Cy5 is labeled A, T, C, G, and then according to different feeding times The distinction between A, T, G, and C.
  • polymerase any natural or non-natural enzyme or catalyst that enables the polymerization of DNA to occur.
  • the polymerase is 9° NN polymerase or a variant thereof, E. coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenas enzyme, Taq DNA polymerase, Bacillus stearothermophilus DNA polymerase, Bst 2.0 DNA polymerization. Enzyme, phi29 DNA polymerase, T7 DNA polymerase.
  • nucleotide or “nucleotide analog” is meant a nucleoside-5'-polyphosphate compound, or a related analog thereof, the base of the nucleotide comprising A, T, G, C, U or Its analog contains 2, 3, 4, 5, 6, 7, 8 or more phosphoric acids. Nucleotides may be modified at base positions and/or phosphate positions.
  • a nucleotide analog is applied as follows, the nucleotide analog comprising an inhibitory group capable of virtually blocking the 3' end of the base and fluorescently labeled for use in the second or third generation (Single molecule) side synthesis (SBS), four kinds of fluorescent bases A, T, G, C with a fluorescent group ATTO647N, added in four times.
  • SBS single molecule side synthesis
  • L 1 , L 2 , and L 3 are each independently a covalent bond or a covalent linking group;
  • B is a base or a base derivative;
  • R 1 is an -OH or a phosphate group; and
  • R 2 is H or a cleavage group;
  • R 3 is a detectable group or a targeting group;
  • R 5 is a polymerase reaction inhibiting group;
  • R 4 is H or -OR 6 , wherein R 6 is H or a cleavable group; Cutting groups or cleavable bonds.
  • targeting group is meant a group/structure/compound that is capable of reacting with a particular group or compound, thereby providing a detectable signal, including by coupling, specific binding and/or click chemistry. Reaction, etc.
  • biotinyl which can be combined with a detectable group bearing streptavidin (streptomycin, SA), such as azide, can occur with a detectable group with DBCO such as DBCO-fluorescent dye
  • SA detectable group bearing streptavidin
  • DBCO such as DBCO-fluorescent dye
  • the chemical reaction is clicked to provide a detectable signal for the specific reaction at a particular location, such as a nucleic acid sequence determination reaction.
  • nucleoside The acid analog is attached to the 3' end of the newly synthesized strand (complementary to the template strand) by the action of an enzyme, base pairing with the template strand, and these nucleotide analogs contain an inhibitory group or a related structure, which can prevent The next base, nucleotide or nucleotide analog is ligated to prevent multiple base pairings on the same template strand in a single reaction in one cycle, and can be effectively applied to nucleic acid sequence determination.
  • nucleotide analogs are suitable for use in a variety of sequencing platforms, including, for example, but not limited to, HiSeq/MiSeq/NextSeq/NovaSeq platform for Illumina, BGI SEQ 50/500 for BGI, ThermoFisher's Ion Torrent platform and PacBio's Sequel platform.
  • the so-called phosphate group is a monophosphate group, a bisphosphonate group, a triphosphate group or a polyphosphoric acid group.
  • B is cytosine or a derivative thereof, thymine or a derivative thereof, adenine or a derivative thereof, guanine or a derivative thereof, hypoxanthine or a derivative thereof, deaza adenine or a derivative thereof, deazaguanine or a derivative thereof, deazahypoxanthine or a derivative thereof, 7-methylguanine or a derivative thereof, 5,6-dihydrouracil or a derivative thereof, 5-A Cytosine or a derivative thereof or 5-hydroxymethylcytosine or a derivative thereof.
  • B is divalent cytosine or a derivative thereof, bivalent ornithril and its derivatives, dimethylpyrimidine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, a divalent hypoxanthine or a derivative thereof, a divalent xanthine or a derivative thereof, a divalent deaza adenine or a derivative thereof, a divalent deazaguanine or a derivative thereof, and a divalent deaza Astragalus or its derivatives, divalent 7-methylguanine or its derivatives, divalent 5,6-dihydrouracil or its derivatives, divalent 5-methylcytosine or derivative or divalent 5 - hydroxymethylcytosine or a derivative thereof.
  • R 6 is H, optionally substituted alkyl or amine.
  • R 6 is H, C 1-5 alkyl or -NH 2 .
  • R 2 is selected from the group consisting of H, At least one of them.
  • Detectable groups include, but are not limited to, Cy2 groups, Cy3 groups, Cy5 groups, Cy7 groups, Hoechst 33258 groups, Hoechst 33342 groups, Hoechst 34580 groups, PO-PRO-1 groups, PO-PRO- 1-DNA group, POPO-1 group, DAPI-DNA group, DAPI group, SYTO 45-DNA group, Alexa Fluor@350 group, Alexa Fluor@450 group, Alexa Fluor@430 group, Alexa Fluor@488, Alexa Fluor@532, Alexa Fluor@546, Alexa Fluor@555, Alexa Fluor@568, Alexa Fluor@594, Alexa Fluor@610, Alexa Fluor@633 group, Alexa Fluor@635 group, Alexa Fluor@635 group, Alexa Fluor@647 group, Alexa Fluor@660 group, Alexa Fluor@680 group, Alexa Fluor@700 group, Alexa Fluor @750 ⁇ ,Alexa Fluor@790, APC, APC-Seta-750
  • R 3 is a detectable group, and in some examples, R 3 is a fluorescent dye.
  • R3 is a targeting group, in some examples, R3 is a group click chemistry reaction, in some instances, R 3 is azido, in some instances R3 group is biotin.
  • R 3 is a fluorescent dye, and in some examples, R3 is an Alexa Fluor@350 group, an Alexa Fluor@450 group, an Alexa Fluor@430 group, an Alexa Fluor@488 group, and an Alexa Fluor@532 Group, Alexa Fluor@546 group, Alexa Fluor@555 group, Alexa Fluor@568 group, Alexa Fluor@594 group, Alexa Fluor@610 group, Alexa Fluor@633 group, Alexa Fluor@635 base Mission, Alexa Fluor@635, Alexa Fluor@647, Alexa Fluor@660, Alexa Fluor@680, Alexa Fluor@700, Alexa Fluor@750, Alexa Fluor@790 .
  • R 3 is FAMTM group, TETTM group, JOETM group, VICTM group, HEXTM group, NEDTM group, PET group, ROXTM group, TAMRATM group, TETTM group, Texas Red A group, Rhodamine 6G (R6G) group, Cy5 group.
  • R 3 is (B in the formula is boron),
  • ROX-labeled Dibenzocyclooctyne (ROX-DBCO or DBCO-ROX) Structure of ROX-DBCO combined with dATP analogue with targeting group azide
  • ATTO Rho6G marked SHA (ATTO Rho6G-SHA or SHA-ATTO Rho6G) Structure of ATTO Rho6G-SHA combined with dTTP analogue
  • ATTO647N labeled tetrazine (ATTO647N-tetrazine or tetrazine-ATTO647N) Structure of ATTO647N-tetrazine combined with dGTP analogue:
  • R 3 is ATTO390 group, ATTO425 group, ATTO430LS group, ATTO465 group, ATTO 488 group, ATTO490LS group, ATTO495 group, ATTO514 group, ATTO520 group, ATTO532 group, ATTO550 Group, ATTO565 group, ATTO590 group, ATTO594 group, ATTO610 group, ATTO620 group, ATTO633 group, ATTO635 group, ATTO647 group, ATTO647 group, ATTO647N group, ATTO655 group, ATTO665 group , ATTO700, ATTO725, ATTO740, ATTO680, ATTOOxa12, ATTORho3B, ATTO Rho6G, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14 ATTO Rho101 group, ATTO Thio12 group.
  • R 3 is a BO-PRO-1 group, a BO-PRO-3 group, a BOBO-1 group, a BOBO-3 group, a BODIPY 630 650-X group, a BODIPY 650/665-X group , BODIPY FL group, BODIPYR6G group, BODIPY TMR-X group, BODIPY TR-X group, BODIPY TR-X pH7.0 group, BODIPYTR-X-phallacidin group, BODIPY-DiMe group, BODIPY- Phenyl group, BODIPY-TMSCC group, C3-Indocyanine group, C3-Oxacyanine group, C3-Thiacyanine-Dye (EtOH) group, C3-Thiacyanine-Dye (PrOH) group, C5-Indocyanine group, C5-Oxacyanine group, C5-Thiacyanine group, C7-Indocyanine group
  • R 3 is CF450M group, CF405S group, CF488A group, CF543 group, CF555 group, CFP, group CFSE group, CF350 group, CF485 group, Chlorophyll-A group, Chlorophyll-B group, Chromeo488 group, Chromeo 494 group, Chromeo 505 group, Chromeo 546 group, Chromeo642 group, Cryptolight CF1 group, Cryptolight CF2 group, Cryptolight CF3 group, Cryptolight CF4 group, Cryptolight CF5 group , Cryptolight CF6 group, Crystal Violet group, Cumarin 153 group, Cy2 group, Cy3 group, Cy3.5 group, Cy3B group, Cy5ET group, Cy5 group, Cy5.5 group, Cy7 group , DAPI group.
  • R 3 is a DsRed-Express group, a DsRed-Express 2 group, a DsRed-Express T1 group, a DY350XL group, a DY-480 group, a DY-480XL Megastokes group, a DY485 group, and a DY485XL Megastokes Group, DY490 group, DY490XL Megastokes group, DY-500 group, DY-500XLMegastokes group, DY-520 group, DY520 Megastokes group, DY-547 group, DY549P1 group, DY-554 group , DY-555 group, DY-557 group, DY-590 group, DY-615 group, DY-630 group, DY-631 group, DY-633 group, DY-635 group, DY -636 group, DY-647 group, DY-649P1 group, DY-650 group, DY651 group, DY-656 group
  • R 3 is Dylight488 group, Dylight 594 group, Dylight 633 group, Dylight 649 group, Dylight 680 group, Hilyte Fluor 488 group, Hilyte Fluor 555 group, Hilyte Fluor 647 group, Hilyte Fluor 680 group, Hilyte Fluor 750 Group, HiLyte Plus 555 group, HiLyte Plus 647 group, HiLyte Plus 750 group, Hoechst 33258 group, Hoechst 33342 group.
  • R 3 is a PromoFluor-350 group, a PromoFluor-405 group, a PromoFluor-415 group, a PromoFluor-488 group, a PromoFluor-488Premium group, a PromoFluor-488LS group, a PromoFluor-500LSS group, PromoFluor-505 group, PromoFluor-555 group, PromoFluor-590 group, PromoFluor-510LSS group, PromoFluor-514LSS group, PromoFluor-520LSS group, PromoFluor-532 group, PromoFluor-546 group, PromoFluor- 610 group, PromoFluor-633 group, PromoFluor-647 group, PromoFluor-670 group, PromoFluor-680 group, PromoFluor-700 group, PromoFluor-750 group, PromoFluor-770 group, PromoFluor-780 base Mission, PromoFluor-350
  • R 3 is QD525, QD565, QD585, QD605, QD655, QD705, QD800, QD903, QDpbS950, QDot525, QDot545 Group, QDot565 group, QDot585 group, QDot605 group, QDot625 group, QDot655 group, QDot705 group, QDot800 group, QpyMe2 group, QSY7 group, QSY9 group, QSY21 group, QSY35 group, Rhodamine 700perchlorate, Rhodamine, Rhodamine 6G, Rhodamine 101, Rhodamine 123, Rhodamine B, Rhodamine Green, Rhodamine pH-Probe 585-7.0, Rhodamine pH-Probe 585-7.5, Rhodamine phalloidin , Rhodamine Red-X group, Rhodamine TagpH-Probe 585-7.0 group.
  • R 3 is SYBR Green, SYPRO Ruby, SYTO 9 , SYTO 11 , SYTO 13 , SYTO 16 , SYTO 17 , SYTO 45, SYTO 59, SYTO 60, SYTO61, SYTO62, SYTO82, SYTORNASelect, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, Texas red, Texas redDHPE, Texas red-X Group.
  • L 1 is L 1 A -L 1 B -L 1 C -L 1 D -L 1 E , wherein L 1 A , L 1 B , L 1 C , L 1 D , L 1 E are a single bond, or a substituted or unsubstituted alkylene, or a substituted or unsubstituted heteroalkylene, or a substituted or unsubstituted cycloalkylene, or a substituted or unsubstituted isocycloalkylene, or substituted or not Substituted aryl alkene, in general, here L 1 A , L 1 B , L 1 C , L 1 D , L 1 E , at least one is not a bond. Or L 1 is L 1 A -L 1 B -L 1 C -L 1 D -L 1 E
  • L 1 A , L 1 B , L 1 C , L 1 D , L 1 E are an independent bond, or a substituted or unsubstituted C 1-8 alkylene, or a substituted or unsubstituted 2 to 8 membered heterocycloalkane Alkene, or a substituted or unsubstituted C 3-8 cycloalkylene, or a substituted or unsubstituted 3 to 8 membered isocycloalkylene, or a substituted or unsubstituted C 6 -8 arylene.
  • L 1 A , L 1 B , L 1 C , L 1 D , L 1 E at least one is not a bond.
  • L 2 is L 2 A -L 2 B -L 2 C -L 2 D , wherein L 2 A , L 2 B , L 2 C , L 2 D are an independent bond, or a substitution or An unsubstituted alkyl group, or a substituted or unsubstituted heteroalkylene, or a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted isocycloalkyl group, or a substituted or unsubstituted aryl group, in general, here L 2 A , L 2 B , L 2 C , L 2 D , at least one is not a bond.
  • L 3 is L 3 A -L 3 B -L 3 C -L 3 D or L 3 A L 3 B L 3 C L 3 D , wherein L 3 A , L 3 B , L 3 C , L 3 D is an independent bond, or a substituted or unsubstituted alkyl group, or a substituted or unsubstituted heteroalkylene, or a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted isocycloalkyl group, or substituted Or an unsubstituted aryl group, in which, here, L 3 A , L 3 B , L 3 C , L 3 D , at least one is not a bond.
  • L 1 can be selected as follows:
  • g1 is an integer between 0 and 10
  • R L1 is NH or O
  • g1 is preferably 1,4.
  • g2 is 0, 1, 2, 3, 4 or 5, R L1 is NH or O; and g2 is preferably 1, 4.
  • R L1a , R L1b are independently H, -CH3, -CX3, -CHX2, -CH2X, -CN, -Ph, C 1-6 alkyl, 2 to 6 alkyl or 3 to 6 cycloalkyl
  • X is Cl, Br, I.
  • L 2 can select but is not limited to is as follows: Wherein R L2a and R L2b are each independently H, C 1-5 alkyl chain, 3-6 membered cycloalkyl or phenyl group, preferably H, methyl, ethyl, 5-6 membered cycloalkyl or phenyl Wherein h1, h2, h3 are each independently an integer between 0-6, preferably 0, 1 or 2;
  • R L2c and R L2d are each independently H or a C 1-6 alkyl chain
  • h4, h5, h6 are independently 0, 1, 2, 3, 4, 5 or 6, wherein h4 is preferably 0, 1 Or 2, h5 is preferably 5 or 6, and h6 is preferably 4, 5 or 6.
  • h7 and h8 are each independently 1, 2, 3, 4, 5 or 6, preferably 1, 2 or 3.
  • removal bond refers to a bond or group capable of decomposing (separation, hydrolysis, stable bond cleavage) by a single monovalent or divalent bond.
  • the resulting resection key or excision group may be due to various external stimuli (eg, enzymes, nucleophiles, reducing agents, light irradiation, oxygen reagents, acid reagents, etc.), usually in these stimulating elements (eg TCEP, THPP, In the presence of laser irradiation, Pd(0), etc., the excision bond or the excision group is broken.
  • cleavable group or cleavable bond can be selected as follows:
  • light ablation bond or "photocleavage group” (eg, o-nitrobenzyl) can cause linkage breakage under conditions of light stimulation.
  • inhibitor refers to a group or covalent bond capable of virtually blocking the 3' end of a base, such as by blocking the 3' end of the base by steric hindrance and/or charge of the molecule, for example Under certain conditions, the inhibition acts with manganese ions so that no free manganese ions act in the polymerization pairing process.
  • Repressing groups include, but are not limited to, a charged group (eg, having a positively charged group, a negatively charged group, or a positively and negatively charged group).
  • a group comprising two or more charged groups may be selected from the group consisting of -COOH, -PO 4 , -SO 4 , -SO 3 , -SO 2 .
  • Which prioritizes the following structure wherein each of R 10 and R 11 is independently H or C 1-6 alkyl, and each a, and b are independently an integer of 0 or 1-5, and each a and b are independently 1 or 2.
  • R 5 is
  • C is a group containing at least one selected from the group consisting of an optionally substituted ethynyl group, an optionally substituted disulfide bond, an optionally substituted amide alkyl group, an optionally substituted aryl group, An optionally substituted alkoxy group, an optionally substituted azide group, an optionally substituted straight or branched alkyl group.
  • C is a group containing at least one selected from the group consisting of an ethynyl group, a disulfide bond, an amide group, an alkyleneoxy group, an aryl group, an azide group, optionally substituted by a hydroxy group, Linear or branched alkylene.
  • C is selected from at least one of the following:
  • L 1 , L 2 and L 3 are each independently selected from the group consisting of an independent bond, an optionally substituted alkyl group, an optionally substituted alkylalkenyl group, an optionally substituted heteroalkylene group, At least one of a substituted cycloalkyl group, an optionally substituted isocycloalkyl group, and an optionally substituted aryl alkene is selected.
  • L 1 , L 2 and L 3 are each independently selected from the group consisting of an independent bond, an optionally substituted C 1-8 alkyl group, an optionally substituted C 1-8 alkyl alkene, optionally substituted A C 2-8 heteroalkylene, an optionally substituted C 3-8 cycloalkylene, an optionally substituted C 3-8 isocycloalkylene , or an optionally substituted C 6-8 aryl alkene.
  • L 1 is selected from at least one of an independent bond, an optionally substituted C1-C3 alkyl group, and an optionally substituted C1-C3 alkylene.
  • L 1 comprises at least one selected from the group consisting of: Wherein, each R 7 is independently an amide bond or O, and each p 1 is independently an integer between 0 and 1 to 10.
  • each p 1 is independently 1, 4; each p 2 is independently 0, 1 to 5 An integer between, preferably, each p 2 is independently 1, 4; each R 8 , R 9 is independently selected from H, -CH 3 , -CX 3 , -CHX 2 , -CH 2 X, -CN, - At least one of Ph, C 1-6 alkyl, C 2-6 alkyl and C 3-6 cycloalkyl, wherein X is Cl, Br, I; each p 3 is independently between 0 and 1-4 The integer. According to an embodiment of the invention, each p 3 is independently 1, 2 .
  • L 2 comprises at least one selected from the group consisting of:
  • each of Rx, Ry, R A and R B is independently H, a C 1-6 alkyl chain, a C 3-10 cycloalkyl group, a C 5-10 aryl group, and each x, y, z is independently 0 or 1 An integer of ⁇ 6.
  • each Rx, Ry is independently H, C 3-5 cycloalkyl, C 5-6 cycloalkyl, phenyl.
  • L 2 is one selected from the group consisting of:
  • each of Ra and Rb is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkyl, substituted or unsubstituted isocycloalkyl, substituted or unsubstituted Aryl.
  • L 3 is selected from one of the following structures: Wherein n1, n2, n3, n4, n5, and n6 are each independently 0 or an integer of 1-7.
  • L 3 has one selected from the following structures:
  • R 3 is at least one selected from the group consisting of a dye, a click chemical reactive group, an azide group, and a biotin group.
  • R 5 comprises at least one charged group, the charged groups comprising -COOH, -PO 4 , -SO 4 , -SO 3 , -SO 2 .
  • the so-called compound has at least one of the following structures:
  • each of Ra and Rb is independently selected from the group consisting of H, an optionally substituted alkyl group, an optionally substituted heteroalkylene, an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, and an optionally substituted At least one of aryl groups.
  • each of Ra and Rb is independently H, C 1-6 alkyl, 3-6 atom heteroalkyl, C 2-6 alkenyl, 3-6 atom heteroalkenyl, respectively.
  • a C 3-6 cycloalkyl group a heterocyclic group of 3 to 6 atoms, a phenyl group, a heteroaryl group of 5 to 6 atoms, wherein each C 1-6 alkyl group, 3 to 6 atoms Heteroalkyl, C 2-6 alkenyl, 3-6 atom heteroalkenyl, C 3-6 cycloalkyl, 3-6 atom heterocyclic, phenyl and 5-6 atoms
  • the heteroaryl group is independently unsubstituted or substituted by 1, 2 or 3 halogens, C 1-6 alkyl, C 2-6 alkenyl, CN, NO 2 .
  • B in the compound is a boron atom
  • a dNTP analog comprising at least one selected from the group consisting of a dATP analog, a dCTP, a dGTP, and a dTTP analog, the four nucleotide analogs being selected from the above
  • each B is a base A, C, G and T.
  • an NTP analog is proposed.
  • the NTP analogue is selected from at least one of the following: an ATP analog, a CTP analog, a GTP analog, and a UTP analog, a so-called ATP analog, a CTP analog, a GTP analog
  • the UTP analogs are the compounds of the formula (I) shown above, each of which is a base A, C, G and U, respectively.
  • a mixture of dNTP analogs is proposed.
  • the dNTP analog mixture includes a dATP analog, a dCTP analog, a dGTP analog, and a dTTP analog
  • the so-called dATP analog, dCTP analog, dGTP analog, and dTTP analog are the structural formulas shown above ( Compounds of I), each of which is a base A, C, G and T, respectively, and at least three of the so-called dATP analog, dCTP analog, dGTP analog and dTTP analog have different detectable a group or a targeting group.
  • the dNTP analog mixture comprises a combination of any two of the following nucleotide analogs: dATP analog, dCTP analog, dGTP analog and dTTP analog, said dATP, dCTP,
  • the dGTP and dTTP analogs are the compounds of the formula (I) shown above, each of which is a base A, C, G and T, respectively, wherein the two nucleotide analogues in the combination have different detectable a group or a targeting group.
  • a mixture of NTP analogs is proposed.
  • the NTP analog mixture comprises an ATP analog, a CTP analog, a GTP analog as the former compound and a UTP analog, and the so-called ATP, CTP, GTP and UTP analogs are the structural formulas shown above as in formula (I).
  • the compounds, each B, are bases A, C, G, and U, respectively, wherein the three nucleotide analogs in the combination have different detectable groups or targeting groups.
  • a mixture of NTP analogs is proposed.
  • the NTP analog mixture comprises a combination of any two of the following nucleotide analogs: ATP analogs, CTP analogs, GTP analogs are the former compounds and UTP analogs, said ATP, CTP, GTP and UTP
  • the analogs are the compounds of the formula (I) shown above, each B being a base A, C, G and U, respectively, wherein the two nucleotide analogues in the combination have different detectable groups .
  • a mixture of dNTP analogs, NTP analogs, dNTP analogs or NTP analogs in any of the preceding embodiments is proposed in nucleic acid sequencing or controlled polymerase chain reaction or base extension reaction the use of.
  • a kit for nucleic acid sequencing or a controllable chain polymerase reaction comprises: a dNTP analog, a NTP analog, a dNTP analog mixture or a mixture of NTP analogs in any of the preceding embodiments.
  • the kit can be used for DNA and/or RNA sequencing.
  • the kit further comprises: a cleavage reagent, said cleavage reagent acting on a cleavable group or a cleavable linkage; and/or a DNA polymerase.
  • a method of preparing a nucleotide analog of any of the preceding embodiments comprising: synthesizing SPDP using dithiodipyridine and mercaptopropionic acid; using formula (10-2)
  • the compound and the ethyl chloroformate are combined to form a first linking structure, the first linking structure is a compound represented by the formula (10-7);
  • the dNTP and the SPDP are linked to obtain dNTP-SPDP; and the first linking structure and dNTP-SPDP are used for synthesis.
  • dNTP-SPDP can be obtained by a condensation reaction of a compound represented by formula (31-4) with dNTP, and further, an exchange reaction of dNTP-SPDP with a compound of formula (10-7) is carried out to obtain a target compound.
  • the dNTP is dATP, which can be purchased or synthesized by itself.
  • dATP can be obtained by the following steps: 1) coupling a compound of the formula (10-51) with trifluoroacetylpropynylamine to obtain a compound of the formula (10-52); 2) The compound represented by the formula (10-52) and pyrophosphate are subjected to a substitution reaction to obtain the dATP.
  • the compound of the formula (31-4) can be purchased or can be obtained by the following procedure: dithiodipyridine and mercaptopropionic acid are exchanged, followed by esterification with NBS.
  • the compound of the compound of the formula (10-7) is commercially available or can be obtained by transesterification of a compound represented by the formula (10-6) with NH 2 OH.
  • the compound of the formula (10-6) is commercially available or can be obtained by: a) condensing a compound represented by the formula (10-4) with NHS to obtain a formula (10-5).
  • the compound shown in the formula (b-5) is transesterified with H-Asp-Asp-OH to give a compound of the formula (10-6).
  • the compound of the formula (10-4) is commercially available, and can also be obtained by subjecting a compound represented by the formula (10-3) to trifluoroacetic acid ester hydrolysis reaction.
  • the compound of the formula (10-3) is commercially available, and can also be obtained by subjecting a compound represented by the formula (10-2) to esterification with MsCl, followed by transesterification with KSCOCH 3 .
  • the compound represented by the formula (10-2) is commercially available, and can also be obtained by subjecting a compound represented by the formula (10-1) to transesterification with ethyl chloroformate, followed by reduction reaction with NaBH 4 .
  • the dithiodipyridine was dissolved in 30 ml of ethanol, 300 ul of acetic acid was added, and 636 mg of mercaptopropionic acid (dissolved in 15 ml of ethanol) was added dropwise to the mixed solution, and the mixture was dripped in 30 min, stirred at room temperature overnight, and dried.
  • the nuclear magnetic spectrum (H spectrum) of SPDP is shown in Fig. 1.
  • the oil layer was washed with a saturated aqueous solution of ammonium chloride (500 mL*2) and washed with a saturated aqueous solution of sodium chloride (500 mL*2). Dry over anhydrous sodium sulfate and spin dry to give a dark brown solid.
  • Compound 10-12 Compound 10-52 (500 mg) was mixed with Proton sponge (431 mg) in a 100 mL round bottom flask and completely dissolved using anhydrous acetonitrile (10 mL), then acetonitrile was removed on a rotary evaporator and the procedure was repeated 3 times. The resulting mixture was placed on a high vacuum oil pump and pumped overnight. The next day, a magnetic stir bar was added to the mixture, dissolved in PO (OMe) 3 (10 mL), and the mixture was pumped on a high vacuum oil pump for 5 minutes, and the operation was repeated 3 times using a three-way argon gas.
  • the reaction solution was diluted with deionized water (120 mL) and then transferred to a flash chromatography column containing resin.
  • the solution was washed with 300 mL of deionized water, TEAB solution (concentration: 0.1 M, 0.2 M, 0.4 M, 0.6 M, 0.8 M, 1 M, each concentration of 300 mL), and the product was detected by UV. .
  • the TEAB buffer and water were removed using a rotary evaporator at 27 °C.
  • a magnetic stirrer and concentrated aqueous ammonia 100 mL
  • ammonia water was removed at 27 ° C using a rotary evaporator.
  • another method of producing a nucleotide analog of any of the preceding embodiments which comprises: synthesizing dNTP-MPSSK; synthesizing a compound represented by formula (31-4) and a hexapeptide a second linking structure, the second linking structure is a compound represented by the formula (11-10), and the hexapeptide is H-Pro-Lys(Fmoc)-Pro-Asp-Asp-OH; mixing the second linking structure and dNTP-MPSSK to prepare the target compound.
  • the oil layer was washed with a saturated aqueous solution of ammonium chloride (500 mL*2) and washed with a saturated aqueous solution of sodium chloride (500 mL*2). Dry over anhydrous sodium sulfate and spin dry to give a dark brown solid.
  • Compound 11-7 Compound 11-6 (500 mg) was mixed with Proton Sponge (431 mg) in a 100 mL round bottom flask and completely dissolved using anhydrous acetonitrile (10 mL), then acetonitrile was removed on a rotary evaporator and repeated. Three times, the resulting mixture was placed on a high vacuum oil pump and pumped overnight. The next day, a magnetic stir bar was added to the mixture, dissolved in PO (OMe) 3 (10 mL), and the mixture was pumped on a high vacuum oil pump for 5 minutes, and the operation was repeated 3 times using a three-way argon gas.
  • the reaction solution was diluted with deionized water (120 mL) and then transferred to a flash chromatography column containing resin.
  • the solution was washed with 300 mL of deionized water, TEAB solution (concentration: 0.1 M, 0.2 M, 0.4 M, 0.6 M, 0.8 M, 1 M, each concentration of 300 mL), and the product was detected by UV. .
  • the TEAB buffer and water were removed using a rotary evaporator at 27 °C.
  • a magnetic stirrer and concentrated aqueous ammonia 100 mL
  • ammonia water was removed at 27 ° C using a rotary evaporator to obtain Compound 11-7.
  • HPLC component of Compound 10 was added together with the HPLC component of Compound 7, stirred at ambient temperature for 15 minutes, concentrated, and then purified by HPLC (buffer A 0.1 M TEAB, Buffer B acetonitrile) to give Compound 11.
  • nucleotide analog that does not require capping when used
  • those skilled in the art will include, by way of example, the synthetic process of the following examples, the description of the specific reaction conditions of the above examples, and the combination of conventional knowledge.
  • the so-called no-capping method includes the use of the compound without the need for a capping step, the addition of a capping agent, and the like.
  • the compound of the formula (29-7) can be further reacted with a base and a derivative thereof, a nucleic acid or a deoxynucleic acid and a derivative thereof to obtain a compound of the formula (I), and the examples are as follows:
  • the compound represented by the formula (30-4) can be further reacted with a base and a derivative thereof, a nucleic acid or a deoxynucleic acid and a derivative thereof to obtain a compound of the formula (I), and the examples are as follows:
  • the compound represented by the formula (31-7), (31-10), (31-13) can be reacted with the compound represented by the formula (31-4) used in the alternative 2 to obtain a corresponding target.
  • the compound, at the same time, the compound represented by the formula (31-7), (31-10), (31-13) can be substituted for the compound represented by the formula (31-4) used in the example 2 to carry out the base and R.
  • the following is a synthetic route to a compound of the labeled dye with a label capable of linking/binding to a nucleotide analog with a targeting group.
  • a nucleic acid sequence determining method comprising: (a) placing a first nucleic acid template-primer complex, one or more nucleotide analogs, and a mixture of DNA polymerases The nucleotide analog is conjugated to the first nucleic acid template-primer complex under conditions suitable for base extension to obtain an extension product; the nucleotide analog is selected from the preceding dNTP analog or the preceding NTP analog or The previous dNTP analog mixture or the previous NTP analog mixture.
  • the method according to an embodiment of the present invention can quickly and accurately determine the nucleic acid sequence of a sample. This method utilizes the compound of any of the above examples for base extension, and enables nucleic acid sequence determination.
  • the first nucleic acid template-primer complex can be freed in a liquid phase environment or can be immobilized at a specific location.
  • the first nucleic acid template-primer complex is attached to a solid support. For example, it is fixed on a chip or on a microsphere.
  • the method further comprises: (b) a cleavable group or a cleavable bond acting on the extension product to obtain a second nucleic acid template-primer complex; (c) replacing the second nucleic acid template primer complex
  • the first template-primer complex is subjected to (a) and (b) at least once.
  • (a) further comprises detecting the extension product to obtain a signal corresponding to the nucleotide analog bound to the first nucleic acid template-primer complex.
  • the method further comprises determining the nucleic acid sequence based on the so-called signal.
  • the nucleotide analog used comprises a targeting group, (a) comprising the addition of a detectable compound to obtain an extension product, said so-called detectable compound having a specific group capable of interacting with the targeting group Specific binding.
  • the R 3 group of the nucleotide analog used is a targeting group, such as compound (31), (a) comprising: binding a nucleotide analog to the action of a polymerase On the template-primer complex, a Dibenzocyclooctyne (DBCO)-labeled fluorescent molecule is added such that the extension product (template-primer complex bound to the nucleotide analog) is labeled with a fluorescent molecule to enable the production of a detectable signal.
  • DBCO Dibenzocyclooctyne
  • the site of action of step (b) cleavage is the position of the C group in any of the above nucleotide analogs, for example, for a compound of formula (3), which may pass through L2-R5 in solution.
  • the space folding, charge, etc. make the base 3'-OH of the compound not exposed/difficult to bind to nucleotides or the like (virtual inhibition), and the C group thereof contains a disulfide bond, and the base 3 can be made by cutting the disulfide bond.
  • '-OH is exposed and is able to bind to the next nucleotide.
  • the method comprises placing a polymerase, a nucleic acid template-primer complex, and one or more nucleotide analogs in a reaction vessel to bind the nucleotide analog to the nucleic acid template-primer complex Above, thereby obtaining an extension product; the nucleotide analog is selected from the foregoing compounds.
  • the method according to an embodiment of the present invention can extend the primer accurately and quickly.
  • a mixture comprising a template to be tested, a primer paired with at least a portion of a template strand to be tested, a DNA polymerase and a preceding dNTP analog or a preceding NTP analog or a preceding dNTP Mixture of analogs or a mixture of previous NTP analogs.
  • the so-called primer paired with at least a part of the template chain to be tested may be in a combined state or an independent state.
  • the template, the primer polymerase and the preceding dNTP analog or the preceding NTP analog or the previous dNTP analog mixture or the previous NTP analog mixture are in buffer.
  • a nucleotide analog is applied as follows, the nucleotide analog comprising an inhibitory group capable of virtually blocking the 3' end of the base and fluorescently labeled for use in the second or third generation (single molecule)
  • SBS synthesis synthesis sequencing
  • G A fluorescent base band ATTO647N, C, T fluorescent base band Cy3B, G and T combination, A and C combination, divided into two additions.
  • a nucleotide analog is applied as follows, the nucleotide analog comprising an inhibitory group capable of virtually blocking the 3' end of the base and fluorescently labeled for use in the second or third generation (single molecule)
  • the G base does not have a fluorescent dye
  • the A base has ATTO647N
  • the C base has Cy3B
  • the T base has ATTO647N and Cy3B at the same time.
  • the four bases A, T, G, and C were mixed and added at one time.
  • a nucleotide analog is applied as follows, the nucleotide analog comprising an inhibitory group capable of virtually blocking the 3' end of the base and fluorescently labeled for use in the second or third generation (single molecule)
  • the G base has ATTO647N/Cy5
  • the A base has Texred/ROX
  • the T base is connected to Alex Fluor 488
  • the C base is connected to Cy3/ATTO532
  • A, T, G, C are four. Base mixing, one addition.
  • a nucleotide analog is applied as follows, the nucleotide analog comprising an inhibitory group capable of virtually blocking the 3' end of the base and fluorescently labeled for use in the second or third generation (single molecule)
  • SBS synthesis-sequence sequencing
  • A, T, G, and C bases are connected to biotin
  • ATTO647N is linked to streptavidin
  • A, T, G, and C are added four times, each time the base is added, and then ATTO647N- is added. Streptomycin.
  • the nucleotide analog is applied as follows, the nucleotide analog comprising an inhibitory group capable of virtually blocking the 3' end of the base, which is applied to the second or third generation (single molecule) synthesis side sequencing (SBS), A base with azide group, ROX with Dibenzocyclooctyne, T base with PBA, ATTO Rho6G with SHA, G base with TCO, ATTO647N with tetrazine, C base with biotin, Alexa Flour 488 is linked to a streptomycin group, and four bases of A, T, G, and C are mixed, and added at a time, followed by mixing of ROX-Dibenzocyclooctyne, ATTO Rho6G-SHA, ATTO647N-tetrazine, and Alexa Flour488-streptomycin.
  • SBS single molecule synthesis side sequencing
  • Targeting pairing with azido and Dibenzocyclooctyne targeted pairing of PBA with SHA, targeted pairing of TCO-based and tetrazine, and targeted pairing of biotinyl and streptomycin-based to obtain signals for base recognition , to achieve sequencing.
  • the nucleotide analog is applied as follows, the nucleotide analog comprising an inhibitory group capable of virtually blocking the 3' end of the base, which is applied to the second or third generation (single molecule) synthesis side sequencing (SBS), G, A base is linked to biotinyl, ATTO647N is linked to streptomycin, C, T is linked to PBA, ATTORho6G is connected to SHA, G is combined with T, A and C are combined, and base is added in 2 times. Next, each time the base combination was added, ATTO647N-streptomycin, ATTO Rho6G-SHA mixture was added. Targeted pairing with PBA and SHA, targeted pairing of biotinyl and streptomycin-based, and two reactions per round of reaction to obtain signals for base recognition, sequencing, and definition of one round of reactions including four bases An extension of the time.
  • SBS single molecule synthesis side sequencing
  • dNTP-5-OSSV 3 -Atto532 comprising V 3 -linker, such as the compound represented by the specific compound (11-13), wherein Dye is Atto532, in order to verify the ability of the base extension reaction, in solution
  • the base extension reaction was carried out and detected by capillary electrophoresis (first generation sequencer, ABI 3100).
  • Reaction process Primer is hybridized to the template to be tested - adding reaction buffer, base and enzyme - to carry out the extension reaction - to terminate the reaction process - the product is added to the 3100 assay.
  • the first base of the template for extension is a complementary base corresponding to the compound to be tested.
  • the Primer is labeled with fluorescence, and the reaction efficiency is quantitatively estimated by measuring the position of the primer in the capillary electrophoresis before and after the reaction, and the intensity of the fluorescent signal.
  • Reaction efficiency corresponding product peak area / (residual reactant peak area + corresponding product peak area).
  • Figure 2 shows the results of the reaction of the compound. By calculation, the reaction efficiency was >90% under solution-tested reaction conditions.
  • the first three bases in the template strand are kept the same, and the continuous addition is verified by adding the nucleotide analog to be tested.
  • the condition determines the ability of the compound to block the binding of the next base to the template strand.
  • Figure 3 shows the block effect of the compound on homopolymer. Under the experimental conditions, no extension of two or more bases in one reaction was found, indicating that the compound has better inhibition/termination ability.
  • each round of reaction includes four base extension reactions added in the order of C, T, A, G, each reaction includes: base extension, photographing, excision of side chains including dyes (acting on cleavable groups) / key) and cleaning.
  • the reaction performance of the compound was determined by data processing including image processing and base recognition calculations on the extension of the fixed strand determined on the chip.
  • Figure 4 shows the read length distribution of the reads obtained by sequencing with this compound.
  • the compound has a reversible termination function, which can effectively prevent the binding of the next base in one reaction and does not hinder the next base. Binding/extension in the next reaction can be used to achieve nucleic acid sequencing.
  • the abscissa of Figures 2 and 3 is the base size, the ordinate is the fluorescence intensity; the abscissa of Figure 4 is the read length and the ordinate is the scale.
  • the inventors also used two fluorescently labeled bases for sequencing validation.
  • the addition of two nucleotide analog mixtures is included in one round of reaction, and the combination of the mixture and the detectable label is A-Atto647N and T-AttoRho6g, C-Atto532 and G-Atto647N, for example, specific compound (11-13)
  • the indicated compounds were replaced with A, T, C, and G as needed, and sequenced by a synthetic multi-target (template) with a hybridization length of 33 nt, and 76 rounds were added in the order of AT mixed CG mixing, after each round of reaction, Photographs were taken using a 640 nm laser and then photographed using a 532 nm laser.
  • the resulting original read length distribution is shown in Figure 5, which shows that the main peak of the read length of the read obtained after 76 reactions is 32 bp.
  • the monochrome sequencing method comprises: (a) required elements: i) a set of nucleic acids (primers/probes); ii) a nucleotide polymerase; iii) a hybridizable nucleic acid or nucleic acid analog Sample template strand; iv) a set of labeled nucleotide markers comprising the following parts, bases, ribose or deoxyribose rings, resectable -SS-bonds, 3'-blocking inhibitory A group and a detectable label, where the label is linked to a base via a -SS-linker, for example, having the structure shown by the specific compound (3), wherein the base is A, T, G or C.
  • step (b) The specific steps are as follows.
  • the template chain to be tested is fixed on the chip, DNA polymerase is added, and dNTPs-SS-linker-Dye is added respectively (A, T, G, C; linker is not limited, For example, it can be V1-linker, V3-linker, etc.; the same below), so that dNTPs-SS-linker-Dye (A, T, G, C) is paired with the template chain;
  • the unpaired fluorescent molecules are washed out, Detecting the paired fluorescent molecules;
  • step adding TCEP or THPP to remove the disulfide bond, thereby removing the fluorophore attached to the template strand, bare 3'-OH, preparing for the next round of base pairing;
  • Cap reagent is added, the sulfhydryl group on the template is captured, and the excess Cap reagent is rinsed for the next round of base addition. Then, repeat the
  • the monochrome sequencing method comprises: (a) required elements: i) a set of nucleic acids; ii) a nucleotide polymerase; iii) a sample template capable of hybridizing nucleic acids or nucleic acid analogs Chain; iv) a set of labeled nucleotide analogs comprising the following parts, bases, ribose or deoxyribose rings, resectable -SS-bonds, 3'-blocking inhibitor groups and one A detectable label, where the label is linked to a base by a -SS-linker, such as a compound having the structure shown by formula (11-13), wherein Base is A, T, C or G. .
  • the specific steps are as follows.
  • the first step the template chain to be tested is fixed on the chip, DNA polymerase is added, and dNTPs-SS-linker-Dye (A, T, G, C) is added respectively, thereby dNTPs- SS-linker-Dye (A, T, G, C) is paired with the template strand;
  • the second step is to wash out the paired fluorescent molecules and detect the paired fluorescent molecules;
  • the third step is to add TCEP or THPP to remove the disulfide bonds. , thereby removing the fluorophore attached to the template strand, bare 3'-OH, preparing for the next round of base pairing; then, repeating the first, second, third, to ten times, 100 times, even 1000 times.
  • the monochrome sequencing method comprises: (a) the required elements: i) a set of nucleic acids; ii) a nucleotide polymerase; iii) a sample template strand capable of hybridizing nucleic acids or nucleic acid analogs; a group of labeled nucleotide analogs comprising the following parts, bases, ribose or deoxyribose rings, resectable -SS-bonds, 3'-blocking inhibitor groups and a detectable a label, where the label is linked to a base via a -SS-linker, for example, having the structure shown by the specific compound (19), hereinafter referred to as dNTPs-SS-linker-biotin;
  • TCEP or THPP is added to remove the disulfide bond, thereby removing the fluorophore attached to the template strand, bare 3'-OH, preparing for the next round of base pairing;
  • the fourth step adding Cap
  • the reagent captures the sulfhydryl group on the template, rinses the excess Cap reagent, and performs the next round of base addition. Then, repeats the first, second, third, and fourth steps 30 times, 100 times, or even 1000 times.
  • the cap reagent can contain IO. a compound or composition of the NH and/or IO-OH structure.
  • the two-color sequencing method comprises: (a) the required elements: i) a set of nucleic acids; ii) a nucleotide polymerase; iii) a sample template strand capable of hybridizing nucleic acids or nucleic acid analogs; A set of labeled nucleotide analogs comprising the following parts, bases, ribose or deoxyribose rings, resectable -SS-bonds, 3'-blocking inhibitor groups and a detectable label
  • the label is linked to the base by -SS-linker, for example, the structure shown by the specific compound (4), and is represented by dNTP-5-SS-linker-Dye.
  • the specific steps are as follows: First, adding DNA polymerase, dATP-5-SS-linker-Dye, dTTP-5-SS-linker-Dye, resulting in one of dNTP-5-SS-linker-Dye Or two ends of the template chain to be tested, flushing the paired bases without pairing, laser 1, 2 excitation, photography; the second step, adding TCEP or THPP to remove the disulfide bond, thereby removing the template chain The upper fluorophore, bare 3'-OH, prepare for the next round of base pairing.
  • Cap reagent is added to cap the HS group.
  • dGTP-5-SS-linker-Dye is added.
  • dCTP-5-SS-linker-Dye and DNA polymerase the sixth step, repeat the second-four steps; repeat the above steps at least once, ten times, fifty times, one hundred times, one thousand times.
  • the two-color sequencing method comprises: (a) the required elements: i) a set of nucleic acids; ii) a nucleotide polymerase; iii) a sample template strand capable of hybridizing nucleic acids or nucleic acid analogs; A set of labeled nucleotide analogs comprising the following parts, bases, ribose or deoxyribose rings, resectable -SS-bonds, 3'-blocking inhibitor groups and a detectable label
  • the label is linked to the base by -SS-linker, for example, having the structure shown by the specific compound (7), and is represented by dNTP-5-OSS-linker-Dye.
  • the specific steps are as follows: In the first step, DNA polymerase, dATP-5-OSS-linker-Dye, dTTP-5-OSS-linker-Dye is added, resulting in the two dNTP-5-OSS-linker-Dye One or two of them are connected to one end of the template chain to be tested, the paired bases that are not paired are washed, the laser is excited by 1, 2, and the camera is photographed; the second step is to remove the disulfide bond by adding TCEP or THPP, thereby removing the The fluorophore attached to the template strand, bare 3'-OH, ready for the next round of base pairing, the fourth step, adding dGTP-5-OSS-linker-Dye, dCTP-5-OSS-linker-Dye Repeat the first, second, and third steps with the DNA polymerase; repeat the above steps at least once, ten times, fifty times, one hundred times, one thousand times.
  • a two-color sequencing (FRET) method utilizes a mixture of nucleotide analogs without detectable labels and nucleotide analogs with detection labels for sequencing, including: (a) Required elements: i a set of nucleic acids; ii) a nucleotide polymerase; iii) a sample template strand capable of hybridizing a nucleic acid or nucleic acid analog; iv) a set of labeled nucleotide analogs such as a specific compound formula (89) , (90), (91), and (92), (89), (90), and (92) are represented by dNTP-5-ON 3 -V 10 -Dye, a compound of the formula (91) To have no detectable label, the following is expressed in cold dGTP.
  • the four-color sequencing method comprises: (a) required elements: i) a set of nucleic acids; ii) a nucleotide polymerase; iii) a sample template strand capable of hybridizing nucleic acids or nucleic acid analogs; a group of labeled nucleotide-like markers comprising the following parts, bases, ribose or deoxyribose rings, resectable -SS-links, 3'-blocking inhibitor groups and one detectable
  • the label here is linked to the base by -SS-linker.
  • it may have the structure shown by the compound (1), wherein the Base may be replaced with A, T, C, G, and Dye as needed.
  • Cy3b, Rox, Cy5, Alexa488, the following is represented by dNTP-5-SS-linker-Dye, and the linkers of different nucleotide analogs may be the same or different.
  • the four-color sequencing method comprises: (a) required elements: i) a set of nucleic acids; ii) a nucleotide polymerase; iii) a sample template strand capable of hybridizing nucleic acids or nucleic acid analogs; iv) a set of markers a nucleotide analog comprising the following parts, a base, a ribose or deoxyribose ring, a resectable bond/group, an inhibitor group blocked at the 3' end, and a detectable label, where the label is passed
  • the linker is linked to a base, for example, may have a structure of a compound represented by the formula (27), and is represented by a dNTP-5-OSS-linker-targeting group in which an azide group is a targeted group. It can be replaced with other targeted groups as needed, wherein the bases can be replaced with corresponding bases as needed.
  • the third step adding TCEP or THPP to remove the disulfide bond, thereby removing the fluorescent group attached to the template strand, bare 3'-OH, ready for the next round of base pairing
  • the fourth step repeat the first, second and third steps at least once, ten times, fifty times, one hundred times, one thousand times.
  • the description with reference to the terms "one embodiment”, “some embodiments”, “example”, “specific example”, or “some examples” and the like means a specific feature described in connection with the embodiment or example.
  • a structure, material or feature is included in at least one embodiment or example of the invention.
  • the schematic representation of the above terms is not necessarily directed to the same embodiment or example.
  • the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.
  • various embodiments or examples described in the specification, as well as features of various embodiments or examples may be combined and combined.

Abstract

涉及一种具有式（I）结构的核苷酸类似物，该核苷酸类似物的制备方法及其在核酸序列测定等方面的应用。

Description

核苷类似物、制备方法及应用
优先权信息
本申请请求2017年11月30日向中国国家知识产权局提交的、专利申请号为201711239701.0的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及化合物领域，具体地，本发明涉及核苷类似物、核苷类似物的制备方法和应用。
背景技术
DNA测序是一种生物研究和药物研究的基本手段。对核酸分子进行序列测定，可以采用边合成边测序(SBS)或者边连接边测序(SBL)等方式。
目前市面上的SBS核酸测序平台，包括二代和三代测序平台，多数利用DNA聚合酶、基于碱基互补配对原则将核苷酸类似物加到模板上(碱基延伸)，检测核苷酸类似物结合上模板之后发出的信号或者带来的物理化学信号方面的改变，以实现核酸序列的测定。
获得具有稳定结构、稳定可检测信号、反应效率高以及能有效抑制下一个位置核苷酸结合到模板的核苷酸类似物，一直是待解决或有待改进的难题。
发明内容
在本发明的第一方面，提出了一种化合物，其具有式(I)所示结构式：
Figure PCTCN2018118259-appb-000001
其中，L 1、L 2、L 3分别独立地为共价键或共价连接基团；B为碱基或者碱基衍生物；R 1为-OH或者磷酸基团；R 2为H或者可切割基团；R 3为可检测基团或靶向基团；R 5为聚合酶反应抑制基团；R 4为H或-OR 6，其中R 6为H或可切割基团；C为可切割基团或者可切割键。
试验测试发现，上述化合物(又可称为可逆终止基-可检测标记核苷酸或者核苷酸类似物)，应用在测序过程中，在测序的每个循环(cycle)过程中，该核苷酸类似物通过酶的作用连接在新合成的链上(与模板链互补)的3’端，与模板链的碱基配对，并且这些核苷酸类似物含有抑制基团或者相关结构，能够阻止下一个碱基、核苷酸或者核苷酸类似物连接上去，从而阻止了在一个cycle中的一次反应中同一模板链上发生多个碱基配对的情况，能有效地应用于核酸序列测定。该化合物适用于多种测序平台，例如包括但不限于用于Illumina公司的HiSeq/MiSeq/NextSeq/NovaSeq平台，华大基因的BGISEQ50/500，ThermoFisher公司的Ion Torrent平台和PacBio公司的Sequel平台。
一般地，对于带有可检测基团/标记是荧光分子的化合物，应用该化合物进行碱基延伸和/或PCR和/或测序时，对于利用成像***进行信号采集的测序平台，例如对延伸产物进行图像采集，荧光分子/延伸产物/模板-引物复合物在图像上表现为亮斑。任何影响荧光分子的发光行为的因素，直接影响该化合物的应用结果。
在设计上述化合物结构时，发明人发现，对于分子内淬灭即同一个dNTP分子里的碱基的还原电势对该分子中的荧光分子的发光行为产生的影响，化合物中的的碱基和荧光染料之间的距离越远，越不发生分子内淬灭；而对于分子间淬灭即一个dNTP分子中的荧光分子受别的dNTP分子、核酸链等的影响产生的淬灭，主要与dNTP分子自由度有关，该dNTP分子的自由度主要由该dNTP分子中的碱基和荧光分子之间的连接结构(linker)的刚柔性和长度决定，总体上，linker越短刚性越强、自由度越小，分子间的淬灭越小。
发明人设计的上述结构的化合物，平衡考虑以及验证了分子内和分子间淬灭，特别适合用于边合成边测序平台。
在本发明的第二方面，提出了一种dNTP类似物。根据本发明的实施例，所称的dNTP类似物选自下列的至少之一：dATP类似物，dATP类似物为前面所示的结构式如式(I)的化合物，B为碱基A；dCTP类似物，dCTP类似物为前面所示的结构式如式(I)的化合物，B为碱基C；dGTP类似物，dGTP类似 物为前面所示的结构式如式(I)的化合物，B为碱基G；和dTTP类似物，dTTP类似物为前面所示的结构式如式(I)的化合物，B为碱基T。
在一些示例中，该dNTP类似物包括dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物中的一种、两种、三种或者全部四种。
在本发明的第三方面，提出了一种NTP类似物。根据本发明的实施例，该NTP类似物选自下列的至少之一：ATP类似物，CTP类似物，GTP类似物，和UTP类似物，所称的ATP类似物、CTP类似物、GTP类似物和UTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和U。
在一些示例中，该NTP类似物包括ATP类似物、CTP类似物、GTP类似物和UTP类似物中的一种、两种、三种或者全部四种。
在本发明的第四方面，提出了一种dNTP类似物混合物。根据本发明的实施例，该dNTP类似物混合物包括dATP类似物，dCTP类似物，dGTP类似物，和dTTP类似物，所称的dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和T，并且所称的dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物中的至少三种分别具有不同的可检测基团或者靶向基团。在本发明的第五方面，提出了一种dNTP类似物混合物。根据本发明的实施例，该dNTP类似物混合物，包括下述任意两种核苷酸类似物的组合：dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物，所称的dATP、dCTP、dGTP和dTTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和T，其中，组合中两种核苷酸类似物具有不同的可检测基团或者靶向基团。
在本发明的第六方面，提出了一种NTP类似物混合物。根据本发明的实施例，该NTP类似物混合物包括ATP类似物、CTP类似物、GTP类似物为前面任一实施例中的化合物和UTP类似物，所称的ATP、CTP、GTP和UTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和U，其中，组合中三种核苷酸类似物具有不同的可检测基团或者靶向基团。在本发明的第七方面，提出了一种NTP类似物混合物。根据本发明的实施例，该NTP类似物混合物包括下述任意两种核苷酸类似物的组合：ATP类似物、CTP类似物、GTP类似物为前面任一实施例中的化合物和UTP类似物，所称的ATP、CTP、GTP和UTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和U，其中，组合中两种核苷酸类似物具有不同的可检测基团。
在本发明的第八方面，提出了前面任一实施例中的dNTP类似物、NTP类似物、dNTP类似物混合物或者NTP类似物混合物在核酸测序或者可控聚合酶链式反应或者碱基延伸反应中的用途。
在本发明的第九方面，提出了一种用于核酸测序或者可控链式聚合酶反应的试剂盒。根据本发明的实施例，该试剂盒包括：前面任一实施例中的dNTP类似物、NTP类似物、dNTP类似物混合物或者前面的NTP类似物混合物。该试剂盒能够用于DNA和/或RNA核酸序列测定。
在本发明的第十方面，提出了一种测定核酸序列的方法。根据本发明的实施例，该方法包括：(a)将第一核酸模板-引物复合物、一种或多种核苷酸类似物以及DNA聚合酶的混合物置于适于碱基延伸的条件下，使核苷酸类似物结合到第一核酸模板-引物复合物上，获得延伸产物；核苷酸类似物选自前面任一实施例中的dNTP类似物、NTP类似物、dNTP类似物混合物或者NTP类似物混合物。该方法利用具有上述结构特点的核苷酸类似物，能够有效地将生化变化转化为稳定的光电信号并采集，实现核酸模板碱基序列的有效和准确测定。
在本发明的第十一方面，提出了一种延伸引物的方法。根据本发明的实施例，该方法包括将聚合酶、核酸模板-引物复合物以及一种或多种核苷酸类似物置于反应容器内，使核苷酸类似物结合到核酸模板-引物复合物上，从而获得延伸产物；核苷酸类似物选自前面任一结构式如式(I)的化合物。该方法能准确、有效地延伸引物。
在本发明的第十二方面，提出了一种反应混合物。根据本发明的实施例，该反应混合物包含待测模板、与待测模板链的至少一部分配对的引物，DNA聚合酶和前面任一实施例中的dNTP类似物、NTP类似物、dNTP类似物混合物或者NTP类似物混合物。
在本发明的第十三方面，提出了一种制备上述任一示例中结构式如式(I)的化合物的方法。根据本发明的实施例，包括：利用二硫二吡啶和巯基丙酸合成SPDP；利用
Figure PCTCN2018118259-appb-000002
和氯代甲酸乙酯合成第一连接结构，第一连接结构为
Figure PCTCN2018118259-appb-000003
连接dNTP和SPDP，获得dNTP-SPDP；利用所称的第一连接结构和dNTP-SPDP，合成目标化合物。
在本发明的第十四方面，提出了一种制备前面任一示例中结构式如式(I)的化合物的方法。根据本发明的实施例，包括：合成dNTP-MPSSK，dNTP-MPSSK的结构如下所示：
Figure PCTCN2018118259-appb-000004
利用
Figure PCTCN2018118259-appb-000005
和六肽合成第二连接结构，第二连接结构为
Figure PCTCN2018118259-appb-000006
所称的六肽为H-Pro-Lys(Fmoc)-Pro-Asp-Asp-OH；混合第二连接结构和dNTP-MPSSK，以制得化合物。
附图说明
图1为本发明实施例的SPDP的核磁图谱；
图2为本发明实施例的延伸产物检测结果图；
图3为本发明实施例的延伸后产物检测结果图；
图4为本发明实施例的化合物应用在测序后得到读段的读长分布示意图；
图5为本发明实施例的两种荧光标记的核苷酸类似物进行测序验证获得的读段的读长分布示意图；
图6为本发明实施例的包含加帽步骤的核苷酸类似物应用于碱基延伸/测序的流程图；
图7为本发明实施例的无需加帽的带有虚拟阻断基团的核苷酸类似物配对的流程图；
图8为本发明实施例的无需加帽的带有虚拟阻断基团的核苷酸类似物配对的流程图；
图9为本发明实施例的虚拟阻断的碱基靶向配对的流程图；
图10为本发明实施例的双色虚拟阻断的核苷酸类似物应用于SBS测序的流程图；
图11为本发明实施例的双色虚拟阻断Capless核苷酸类似物应用于SBS测序的流程图；
图12为本发明实施例的双色测序方法流程图；
图13为本发明实施例的四色测序方法流程图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
实施例中的试剂、检测仪器等，如无特殊说明，本领域技术人员可自己配制、制备或者通过市售途径获取。
本文中，除非另有说明，“Base”和“B”等同，都指碱基，碱基包括A、T、C、G、U及其衍生物。
所称的“可控链式聚合酶反应”指可以控制使反应连续进行或不连续进行的聚合酶链式反应，例如基于边合成测序原理的核酸序列测定反应。
除非另外说明，本文所使用的所有科技术语为与所属领域技术人员的通常理解相同的含义。涉及的所有专利和公开出版物通过引用方式整体并入本申请。在所结合的科技文献、专利文献和类似材料的一 篇或多篇与本申请不同或相矛盾的情况下(包括但不限于所定义的术语、术语应用、所描述的技术等)，以本申请为准。
除非另外说明，应当应用本文所使用得下列定义。化学元素与元素周期表CAS版，和《化学和物理手册》，第75版，1994一致。此外，有机化学一般原理可参考"Organic Chemistry",Thomas Sorrell,University Science Books,Sausalito:1999,和"March's Advanced Organic Chemistry”by Michael B.Smith and Jerry March,John Wiley&Sons,New York:2007中的描述，其全部内容通过引用并入本文。
除非另有说明或者上下文中有明显的冲突，本文所使用的冠词“一”、“一个(种)”和“所述”旨在包括“至少一个”或“一个或多个”。
所称“任选取代的”与“取代或非取代的”可以交换使用。一般而言，术语“取代的”表示所给结构中的一个或多个氢原子被具体取代基所取代。除非其他方面表明，一个任选的取代基团可以在基团各个可取代的位置进行取代。当所给出的结构式中不只一个位置能被选自具体基团的一个或多个取代基所取代，那么取代基可以相同或不同地在各个位置取代。
本文中，所采用的描述方式“各…独立地为”与“…各自独立地为”和“…独立地为”可以互换，均应做广义理解，其既可以是指在不同基团中，相同符号之间所表达的具体选项之间互相不影响，也可以表示在相同的基团中，相同符号之间所表达的具体选项之间互相不影响。
本文中，说明化合物的取代基时按照基团种类和/或范围进行表述。包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C 1- 6烷基”特别指独立公开的甲基、乙基、C 3烷基、C 4烷基、C 5烷基和C 6烷基。
本文中的连接取代基。当该结构清楚地需要连接基团时，针对该基团所列举的马库什变量应理解为连接基团。例如，如果该结构需要连接基团并且针对该变量的马库什基团定义列举了“烷基”或“芳基”，则应该理解，该“烷基”或“芳基”分别代表连接的亚烷基基团或亚芳基基团。
本文使用的术语“烷基”或“烷基基团”，表示含有1至20个碳原子，饱和的直链或支链一价烃基基团，其中，所述烷基基团可以任选地被一个或多个本发明描述的取代基所取代。除非另外详细说明，烷基基团含有1-20个碳原子。在一实施方案中，烷基基团含有1-12个碳原子；在另一实施方案中，烷基基团含有1-6个碳原子；在又一实施方案中，烷基基团含有1-4个碳原子；还在一实施方案中，烷基基团含有1-3个碳原子。
术语“烷基”其本身或做为取代基的一部分是指(除特别说明)一条直的C链，或带有支链的C链，其可以是完全饱和，也可以是单或多个不饱和的键并且包括指定碳的一价，二价和多价基团，在这里，烷指不饱和碳链，饱和基团是指，但不限于，甲基，乙基，正丙基，异丙基，正丁基，叔丁基，异丁基，(环已基)甲基，正戊基或其同分异构体，正已基或其同分异构体，正庚基或其同分异构体，正辛基或其同分异构体，在这里不饱和基团是指，但不限于，乙烯基，2-丙烯基，巴豆基，2-异戊烯基，2-(丁二烯基)，2,3-戊二烯基，3-(1,4-戊二烯基)，戊基，1-丙炔基，3-丙炔基。
术语“烷基烯”其本身或作为取代基是从烷基和饱和烷基衍生基团，其指的是，但不限于，1到24个碳原子(如-CH 2CH 2CH 2CH 2-)碳链的不饱和烷基衍生物，优先1到10个碳原子的碳链。
术语“杂烷基”其本身或作为取代基一部分是指一条直链或者带有支链的链，并且其至少包含1个碳原子和一个杂原子(如：O，N，P，Si与S)，在这里优选N与S。在这里，杂烷基不是一个环状基团。实例包括，但不限于，-CH 2-CH 2-O-CH 3,-CH 2-CH 2-NH-CH 3,-CH 2-CH 2-N(CH 3)-CH 3,-CH 2-S-CH 2-CH 3,-S(O)-CH 3,-CH 2-CH 2-S(O)-CH 3,-CH＝CH-N(CH 3),-O-CH 3,-O-CH 2-CH 3与-CN。对于含有2个杂原子的结构，其实例包含，-CH 2-NH-OCH 3,-CH 3-O-Si(CH 3) 3。在这里杂烷也包含三键或双键基团。
术语“杂烷基烯”其本身或作为取代基一部分是从烷基和饱和烷基的衍生基团。其实例包含但不限-CH 2-CH 2-S-CH 2-CH 2-与-CH 2-S-CH 2-CH 2-NH-CH 2-链的不饱和衍生物，对于杂烷烯，杂原子可以占据链末端中的一个或两个(如，亚烷基氧基，亚烷基二氧基，亚完基氨基，亚烷基二氨基)。
术语“环烷基”和“环杂烷基”其本身或作为取代基一部分，其是烷基和杂烷基的环状形式，这里指的不是芳香基，对于环烷基实例包含但不限于环丙基，环丁基，环戊基，环己基，1-环己烯基，3-环已烯基，环庚基；对于环杂烷基实例包含但不限于1-(1，2，5，6-四氢吡啶基)，1-哌啶基，2-哌啶基，3-哌啶基，4-吗啉基，3-吗啉基，2-四氢呋喃基，3-四氢呋喃基等。
术语“酰基”是指-C(O)R,其中R是取代基，可能是烷基，环烷基，杂烷基等。
术语“芳香基”是不饱和的基，芳香基等，术语“芳杂环基”是指杂环中含有一个杂原子(如：O，N，P，Si与S)。对于芳香杂烷基，实例包含但不限于苯基，萘基，咪唑基，嘧啶基等。
术语“可检测标记”或“可检测基团”是指能特异性释放信号或特异性识别的基团(如荧光染料，可检测染料)。在有些实例中，a)四种染料，ROX，Alexa532，Cy5，IF700分别标记A，T，G，C；b)一种染料Cy5标记A，T，C，G，然后根据加料时间不同，实现A，T，G，C的区分。
对于术语“聚合酶”是指能使DNA聚合反应发生的任何天然的或非天然的酶或催化剂。在一些实例中，聚合酶是9°NN聚合酶或者它的变种，E.Coli DNA聚合酶I，Bacteriophage T4 DNA聚合酶，Sequenas酶，Taq DNA聚合酶，Bacillus stearothermophilus DNA聚合酶，Bst 2.0 DNA聚合酶，phi29 DNA聚合酶，T7 DNA聚合酶。
对于术语“核苷酸”或“核苷酸类似物”是指核苷-5'-聚磷酸化合物，或其相关类似物，核苷酸的碱基包含A，T，G，C，U或其类似物，其含的磷酸个数为2，3，4，5，6，7，8或更多个磷酸。核苷酸可能在碱基位置和/或磷酸位置被改性。对于测序方法，在一些实例中，核苷酸类似物的应用方法如下，该核苷酸类似物包含能够虚拟阻断碱基3'端的抑制基团且带荧光标记，其应用于二代或三代(单分子)边合成边测序(SBS)中，A，T，G，C四种荧光碱基带一种荧光基团ATTO647N,分四次加入。
化合物、化合物组合
在一些实施例中，提出了一种化合物，其具有式(I)所示结构式：
Figure PCTCN2018118259-appb-000007
其中，L 1、L 2、L 3分别独立地为共价键或共价连接基团；B为碱基或者碱基衍生物；R 1为-OH或者磷酸基团；R 2为H或者可切割基团；R 3为可检测基团或靶向基团；R 5为聚合酶反应抑制基团；R 4为H或-OR 6，其中R 6为H或可切割基团；C为可切割基团或者可切割键。
所称的“靶向基团”包括能够与特定的基团或化合物发生反应的基团/结构/化合物，进而能够提供可检测信号，发生反应包括通过偶联、特异性结合和/或点击化学反应等。例如为生物素基，可以与带有链霉亲和素(链霉素，SA)的可检测基团结合，例如为叠氮，可以与带有DBCO的可检测基团例如DBCO-荧光染料发生点击化学反应，进而可以为特定位置的反应是否实现提供可检测信号，例如核酸序列测定反应。
试验测试发现，上述化合物(又可称为可逆终止基-可检测标记核苷酸或者核苷酸类似物)，应用在测序过程中，在测序的每个循环(cycle)过程中，该核苷酸类似物通过酶的作用连接在新合成的链上(与模板链互补)的3’端，与模板链的碱基配对，并且这些核苷酸类似物含有抑制基团或者相关结构，能够阻止下一个碱基、核苷酸或者核苷酸类似物连接上去，从而阻止了在一个cycle中的一次反应中同一模板链上发生多个碱基配对的情况，能有效地应用于核酸序列测定。
上述核苷酸类似物(即式(I)所示化合物)适用于多种测序平台，例如包括但不限于用于Illumina公司的HiSeq/MiSeq/NextSeq/NovaSeq平台，华大基因的BGISEQ50/500，ThermoFisher公司的Ion Torrent平台和PacBio公司的Sequel平台。
根据本发明的实施例，所称的磷酸基团为单磷酸基团、双磷酸基团、三磷酸基团或多聚磷酸基团。
根据本发明的实施例，B为胞嘧啶或其衍生物、胸腺嘧啶或其衍生物、腺嘌呤或其衍生物、鸟嘌呤或其衍生物、次黄嘌呤或其衍生物、脱氮腺嘌呤或其衍生物、脱氮鸟嘌呤或其衍生物、脱氮次黄嘌呤或其衍生物、7-甲基鸟嘌呤或其衍生物、5，6-二氢尿嘧啶或其衍生物、5-甲基胞嘧啶或其衍生物或5-羟甲基胞嘧啶或其衍生物。
根据本发明的实施例，B为二价胞嘧啶或其衍生物、二价鸟嘧啶及其衍生物、二甲腺嘧啶或其衍生物、二价胸腺嘧啶或其衍生物、二价尿嘧啶或其衍生物、二价次黄嘌呤或其衍生物、二价黄嘌呤或其衍生物、二价脱氮腺嘌呤或其衍生物、二价脱氮鸟嘌呤或其衍生物、二价脱氮次黄嘌呤或其衍生物、二价7-甲基鸟嘌呤或其衍生物、二价5，6-二氢尿嘧啶或其衍生物、二价5-甲基胞嘧啶或者衍生物或二价5-羟甲基胞嘧啶或其衍生物。
根据本发明的实施例，R 6为H、任选取代的烷基或胺基。
根据本发明的实施例，R 6为H、C 1～5烷基或者-NH 2。
根据本发明的实施例，R 2选自H、
Figure PCTCN2018118259-appb-000008
Figure PCTCN2018118259-appb-000009
中的至少之一。
“可检测基团”包括但不限于Cy2基团，Cy3基团，Cy5基团，Cy7基团，Hoechst33258基团，Hoechst33342基团，Hoechst34580基团，PO-PRO-1基团，PO-PRO-1-DNA基团,POPO-1基团,DAPI-DNA基团，DAPI基团，SYTO 45-DNA基团，Alexa Fluor@350基团，Alexa Fluor@450基团，Alexa Fluor@430基团，Alexa Fluor@488基团，Alexa Fluor@532基团，Alexa Fluor@546基团，Alexa Fluor@555基团，Alexa Fluor@568基团，Alexa Fluor@594基团，Alexa Fluor@610基团，Alexa Fluor@633基团，Alexa Fluor@635基团，Alexa Fluor@635基团，Alexa Fluor@647基团，Alexa Fluor@660基团，Alexa Fluor@680基团，Alexa Fluor@700基团，Alexa Fluor@750基团，Alexa Fluor@790基团，APC,APC-Seta-750,AsRed2,ATTO390,ATTO425,ATTO430LS,ATTO465,ATTO488,ATTO490LS,ATTO495,ATTO514,ATTO520，ATTO532,ATTO550,ATTO565，ATTO590，ATTO594，ATTO645,ATTO680的荧光基团。
在一些实例中，R 3是可检测基团，在一些实例中，R 3是荧光染料。在一些实例中，R3是靶向基团，在一些实例中R3是点击化学反应基团，在一些实例中R 3是叠氮基，在一些实例中R3是生物素基。
在一些实例中，R 3是荧光染料，在一些实例中，R3是Alexa Fluor@350基团，Alexa Fluor@450基团，Alexa Fluor@430基团，Alexa Fluor@488基团，Alexa Fluor@532基团，Alexa Fluor@546基团，Alexa Fluor@555基团，Alexa Fluor@568基团，Alexa Fluor@594基团，Alexa Fluor@610基团，Alexa Fluor@633基团，Alexa Fluor@635基团，Alexa Fluor@635基团，Alexa Fluor@647基团，Alexa Fluor@660基团，Alexa Fluor@680基团，Alexa Fluor@700基团，Alexa Fluor@750基团，Alexa Fluor@790基团。
在一些实例中，R 3是FAMTM基团，TETTM基团，JOETM基团，VICTM基团，HEXTM基团，NEDTM基团，PET基团，ROXTM基团，TAMRATM基团，TETTM基团，Texas Red基团，Rhodamine 6G(R6G)基团，Cy5基团。
在一些实例中，R 3是
Figure PCTCN2018118259-appb-000010
Figure PCTCN2018118259-appb-000011
(该式中的B为硼元素)、
Figure PCTCN2018118259-appb-000012
以下示例可以或者容易与靶向基团结合(偶联或特异性结合)的可检测基团或化合物。ROX标记的Dibenzocyclooctyne(DBCO)(ROX-DBCO或者DBCO-ROX)
Figure PCTCN2018118259-appb-000013
ROX-DBCO与带靶向基团为叠氮的 dATP类似物结合后的结构
Figure PCTCN2018118259-appb-000014
ATTO Rho6G标记的SHA(ATTO Rho6G-SHA或者SHA-ATTO Rho6G)
Figure PCTCN2018118259-appb-000015
ATTO Rho6G-SHA与dTTP类似物结合后的结构
Figure PCTCN2018118259-appb-000016
ATTO647N标记的四嗪(ATTO647N-四嗪或者四嗪-ATTO647N)
Figure PCTCN2018118259-appb-000017
ATTO647N-四嗪与dGTP类似物结合后的结构：
Figure PCTCN2018118259-appb-000018
在一些实例中，R 3是ATTO390基团，ATTO425基团，ATTO430LS基团,ATTO465基团，ATTO 488基团,ATTO490LS基团,ATTO495基团，ATTO514基团,ATTO520基团,ATTO532基团,ATTO550基团,ATTO565基团,ATTO590基团，ATTO594基团,ATTO610基团,ATTO620基团,ATTO633基团,ATTO635基团,ATTO647基团,ATTO647基团,ATTO647N基团,ATTO655基团,ATTO665基团,ATTO700基团,ATTO725基团,ATTO740基团,ATTO680基团,ATTOOxa12基团,ATTORho3B基团,ATTO Rho6G基团,ATTO Rho11基团,ATTO Rho12基团,ATTO Rho13基团,ATTO Rho14基团,ATTO Rho101基团,ATTO Thio12基团。
在一些实例中，R 3是BO-PRO-1基团,BO-PRO-3基团,BOBO-1基团,BOBO-3基团,BODIPY630 650-X基团,BODIPY650/665-X基团,BODIPY FL基团,BODIPYR6G基团,BODIPY TMR-X基团,BODIPY TR-X基团,BODIPY TR-X pH7.0基团,BODIPYTR-X-phallacidin基团,BODIPY-DiMe基团,BODIPY-Phenyl基团,BODIPY-TMSCC基团,C3-Indocyanine基团,C3-Oxacyanine基团,C3-Thiacyanine-Dye(EtOH)基团,C3-Thiacyanine-Dye(PrOH)基团,C5-Indocyanine基团,C5-Oxacyanine基团,C5-Thiacyanine基团,C7-Indocyanine基团,C7-Oxacyanine基团,C454T,C-Phycyanin基团,Calcein基团,Calcein-red-orang基团,Calcium-Crimson基团,Calcium Green-1基团,Calcium Orange基团。
在一些实例中，R 3是CF450M基团,CF405S基团,CF488A基团,CF543基团,CF555基团,CFP,基团CFSE基团,CF350基团,CF485基团,Chlorophyll-A基团,Chlorophyll-B基团,Chromeo488基团,Chromeo494基团,Chromeo505基团,Chromeo546基团,Chromeo642基团,Cryptolight CF1基团,Cryptolight CF2基团,Cryptolight CF3基团,Cryptolight CF4基团,Cryptolight CF5基团,Cryptolight CF6基团,Crystal Violet基团,Cumarin153基团,Cy2基团,Cy3基团,Cy3.5基团,Cy3B基团,Cy5ET基团,Cy5基团,Cy5.5基团，Cy7基团,DAPI基团。
在一些实例中，R 3是DsRed-Express基团,DsRed-Express2基团,DsRed-Express T1基团,DY350XL基团,DY-480基团,DY-480XL Megastokes基团,DY485基团,DY485XL Megastokes基团,DY490基团,DY490XL Megastokes基团,DY-500基团,DY-500XLMegastokes基团,DY-520基团,DY520 Megastokes基团,DY-547基团,DY549P1基团,DY-554基团,DY-555基团,DY-557基团,DY-590基团,DY-615基团,DY-630基团,DY-631基团,DY-633基团,DY-635基团,DY-636基团,DY-647基团,DY-649P1基团,DY-650基团,DY651基团,DY-656基团,DY-673基团,DY-675基团,DY-676基团,DY-680基团,DY-681基团,DY-700基团,DY-701基团,DY-730基团,DY-731基团,DY-750基团,DY-751基团,DY-776基团,DY-782基团,Dye-28基团,Dye-33基团,Dye-45基团,Dye-304基团,Dye-1041基团。
在一些实例中，R 3是Dylight488基团，Dylight594基团，Dylight633基团，Dylight649基团，Dylight680 基团，Hilyte Fluor488基团，Hilyte Fluor555基团，Hilyte Fluor647基团，Hilyte Fluor680基团，Hilyte Fluor750基团，HiLyte Plus555基团，HiLyte Plus647基团，HiLyte Plus750基团，Hoechst33258基团，Hoechst33342基团。
在一些实例中，R 3是PromoFluor-350基团，PromoFluor-405基团，PromoFluor-415基团，PromoFluor-488基团，PromoFluor-488Premium基团，PromoFluor-488LS基团，PromoFluor-500LSS基团，PromoFluor-505基团，PromoFluor-555基团，PromoFluor-590基团，PromoFluor-510LSS基团，PromoFluor-514LSS基团，PromoFluor-520LSS基团，PromoFluor-532基团，PromoFluor-546基团，PromoFluor-610基团，PromoFluor-633基团，PromoFluor-647基团，PromoFluor-670基团，PromoFluor-680基团，PromoFluor-700基团，PromoFluor-750基团，PromoFluor-770基团，PromoFluor-780基团，PromoFluor-840基团。
在一些实例中，R 3是QD525基团，QD565基团，QD585基团，QD605基团，QD655基团，QD705基团，QD800基团，QD903基团，QDpbS950基团，QDot525基团，QDot545基团，QDot565基团，QDot585基团，QDot605基团，QDot625基团，QDot655基团，QDot705基团，QDot800基团，QpyMe2基团，QSY7基团，QSY9基团，QSY21基团，QSY35基团，Rhodamine700perchlorate基团，Rhodamine基团，Rhodamine6G基团，Rhodamine101基团，Rhodamine123基团，RhodamineB基团，RhodamineGreen基团，Rhodamine pH-Probe585-7.0基团，Rhodamine pH-Probe585-7.5基团，Rhodamine phalloidin基团，Rhodamine Red-X基团，Rhodamine TagpH-Probe 585-7.0基团。
在一些实例中，R 3是SYBR Green基团，SYPRO Ruby基团，SYTO9基团，SYTO11基团，SYTO13基团，SYTO16基团，SYTO17基团，SYTO45基团，SYTO59基团，SYTO60基团，SYTO61基团，SYTO62基团，SYTO82基团，SYTORNASelect基团，SYTOX Blue基团，SYTOX Green基团，SYTOX Orange基团，SYTOX Red基团，Texas red基团，Texas redDHPE基团，Texas red-X基团。
在一些实施例中，L 1是L 1 A-L 1 B-L 1 C-L 1 D-L 1 E，其中L 1 A，L 1 B，L 1 C，L 1 D，L 1 E是独立的一个键，或取代或非取代的烷基烯，或取代或非取代杂烷基烯，或取代或非取代环烷基烯，或取代或非取代异环烷基烯，或取代或非取代芳基烯，总之，在这里L 1 A，L 1 B，L 1 C，L 1 D，L 1 E，至少有一个不是一个键。或L 1是L 1 A-L 1 B-L 1 C-L 1 D-L 1 E
其中L 1 A，L 1 B，L 1 C，L 1 D，L 1 E是一个独立的键，或取代或非取代C 1-8烷基烯，或取代或非取代2到8元杂烷基烯，或取代或非取代C 3-8环烷基烯，或取代或非取代3到8元异环烷基烯，或取代或非取代C 6- 8芳基烯。总之，在这里L 1 A，L 1 B，L 1 C，L 1 D，L 1 E，至少有一个不是一个键。
在一些实施例中，L 2是L 2 A-L 2 B-L 2 C-L 2 D，其中L 2 A，L 2 B，L 2 C，L 2 D是一个独立的键，或取代或非取代的烷基，或取代或非取代杂烷基烯，或取代或非取代环烷基，或取代或非取代异环烷基，或取代或非取代芳基，总之，在这里L 2 A，L 2 B，L 2 C，L 2 D，至少有一个不是一个键。
在一些实施例中，L 3是L 3 A-L 3 B-L 3 C-L 3 D或L 3 AL 3 BL 3 CL 3 D，其中L 3 A，L 3 B，L 3 C，L 3 D是一个独立的键，或取代或非取代的烷基，或取代或非取代杂烷基烯，或取代或非取代环烷基，或取代或非取代异环烷基，或取代或非取代芳基，总之，在这里L 3 A，L 3 B，L 3 C，L 3 D，至少有一个不是一个键。
在一些实施例中，L 1可选择如下结构：
Figure PCTCN2018118259-appb-000019
其中，g1为0～10之间的整数，R L1为NH或O；g1优选1，4。
Figure PCTCN2018118259-appb-000020
其中g2为0,1,2,3,4或5，R L1为NH或O；g2优选1，4。
Figure PCTCN2018118259-appb-000021
其中，R L1a，R L1b分别独立为H、-CH3、-CX3、-CHX2、-CH2X、-CN、-Ph、C 1-6烷基、2到6元烷基或3到6环烷基，其中X为Cl，Br，I。
Figure PCTCN2018118259-appb-000022
其中，g3为0，1，2，3，4，优先1，2。
在一些实施例中，L 2可以选择但不限于的结构如下：
Figure PCTCN2018118259-appb-000023
其中R L2a、R L2b分别独立地为H、C 1-5烷基链、3-6元环烷基或苯基，优选H，甲基、乙基、5-6元环烷基或苯基；其中h1、h2、h3分别独立地为0-6之间的整数，优选为0、1或2；
Figure PCTCN2018118259-appb-000024
其中R L2c、R L2d分别独立地为H或C 1-6烷基链，h4、h5、h6分别独立地为0，1，2，3，4，5或6，其中h4优选为0、1或2，h5优选为5或6，h6优选为4、5或6。
Figure PCTCN2018118259-appb-000025
其中，h7、h8分别独立地为1、2、3、4、5或6，优选1、2或3。
术语“切除键”或切除基团是指这个键或基团能分解(分离，水解，稳定的键断裂)长单独的一价或二价键。造成切除键或切除基团可能是由于各种外部刺激(如，酶，亲核试剂，还原试剂，光照射，氧试剂，酸试剂等的刺激)，通常在这些刺激元素(如TCEP,THPP,激光照射，Pd(0)等)的存在下，切除键或切除基团发生断裂。
在一些实施例中，所称的可切割基团或可切割键可以选择如下：
Figure PCTCN2018118259-appb-000026
等。
术语“光切除键”或“光切除基团”(例如，邻硝基苄基)能在光照刺激的条件下，发生连接键断裂。
术语“抑制基团”(inhibition)是指能虚拟阻断碱基3'端的基团或者共价键，如通过分子的空间位阻和/或电荷的作用等阻断碱基3'端，例如在一定的条件下，inhibition与锰离子作用，使得没有游离的锰离子作用在聚合配对的过程中。
“抑制基团”包括但不限于一个带电荷的基团(如，带一个正电荷基团，一个带负电荷基团，或者一个同时带正负电荷基团)。或者包括2个或者多个带电荷的基团带电荷的基团可以从下面基团中选择，-COOH，-PO 4，-SO 4,-SO 3,-SO 2。其中优先以下结构
Figure PCTCN2018118259-appb-000027
其中，各R 10与和R 11分别独立地为H或者C 1-6烷基，各a,和b分别独立地的为0或者1-5的整数，各a和b分别独立地为1或2。
在一些应用实例中，R 5是
Figure PCTCN2018118259-appb-000028
根据本发明的实施例，C为含有选自下列至少之一的基团：任选取代的乙炔基、任选取代的二硫键、任选取代的酰胺烷基、任选取代的芳基、任选取代的烷氧基、任选取代的叠氮基、任选取代的直链或支 链烷基。
根据本发明的实施例，C为含有选自下列至少之一的基团：任选被羟基取代的乙炔基、二硫键、酰胺基团、亚烷氧基、芳基、叠氮基团、直链或者支链亚烷基。
根据本发明的实施例，C选自下列至少之一：
Figure PCTCN2018118259-appb-000029
Figure PCTCN2018118259-appb-000030
根据本发明的实施例，L 1、L 2和L 3分别独立地选自独立的键，任选取代的烷基，任选取代的烷基烯基，任选取代的杂烷基烯，任选取代的环烷基，任选取代的异环烷基以及任选取代的芳基烯的至少之一。
根据本发明的实施例，L 1、L 2和L 3分别独立地选自独立的键、任选取代的C 1-8烷基，任选取代的C 1-8烷基烯，任选取代的C 2-8杂烷基烯，任选取代的C 3-8环烷基烯，任选取代的C 3-8异环烷基烯，或任选取代的C 6-8芳基烯。
根据本发明的实施例，L 1选自独立的键、任选取代的C1～C3烷基和任选取代的C1-C3烷基烯中的至少之一。
根据本发明的实施例，L 1包括选自下列的至少之一：
Figure PCTCN2018118259-appb-000031
Figure PCTCN2018118259-appb-000032
其中，各R 7独立为酰胺键或O，各p 1独立为0、1～10之间的整数，优选地，各p 1独立为1、4；各p 2独立为0、1～5之间的整数，优选地，各p 2独立为1、4；各R 8，R 9分别独立地选自H、-CH 3、-CX 3、-CHX 2、-CH 2X、-CN、-Ph、C 1-6烷基、C 2～6烷基和C 3～6环烷基中的至少一种，其中X为Cl、Br、I；各p 3独立为0、1～4之间的整数。根据本发明的实施例，各p 3独立为1、2。
根据本发明的实施例，L 2包括选自下列的至少之一：
Figure PCTCN2018118259-appb-000033
Figure PCTCN2018118259-appb-000034
其中，各Rx、Ry、R A、R B独立为H、C 1～6烷基链、C 3-10环烷基、C 5-10芳基，各x、y、z独立为0或者1～6的整数。
根据本发明的实施例，各Rx、Ry独立为H、C 3-5环烷基、C 5-6环烷基、苯基。
根据本发明的实施例，L 2为选自如下结构的一种：
Figure PCTCN2018118259-appb-000035
其中，各Ra与Rb分别独立地为H、取代或非取代的烷基、取代或非取代杂烷基烯、取代或非取代环烷基、取代或非取代异环烷基、取代或非取代芳基。
根据本发明的实施例，L 3选自以下结构的一种：
Figure PCTCN2018118259-appb-000036
Figure PCTCN2018118259-appb-000037
其中，n1，n2，n3，n4，n5，n6分别独立地为0或者1～7的整数。
根据本发明的实施例，L 3具有选自以下结构的一种：
Figure PCTCN2018118259-appb-000038
Figure PCTCN2018118259-appb-000039
根据本发明的实施例，R 3选自染料、点击化学反应基团、叠氮基和生物素基中的的至少一种。
根据本发明的实施例，R 5包括至少一个带电荷的基团，带电荷的基团包括-COOH、-PO 4、-SO 4、-SO 3、-SO 2。
根据本发明的实施例，所称的化合物具有下列结构至少之一：
Figure PCTCN2018118259-appb-000040
Figure PCTCN2018118259-appb-000041
Figure PCTCN2018118259-appb-000042
其中，各Ra与Rb分别独立地选自H、任选取代的烷基、任选取代的杂烷基烯、任选取代的环烷基、任选取代的异环烷基和任选取代的芳基中的至少一种。根据本发明的实施例，各Ra与Rb分别独立地为H、C 1-6烷基、3-6个原子的杂烷基、C 2-6烯基、3-6个原子的杂烯基、C 3-6环烷基、3-6个原子组成的杂环基、苯基、5-6个原子组成的杂芳基，其中，各C 1-6烷基、3-6个原子的杂烷基、C 2-6烯基、3-6个原子的杂烯基、C 3-6环烷基、3-6个原子组成的杂环基、苯基和5-6个原子组成的杂芳基独立地未被取代或被1、2或3个卤素、C 1-6烷基、C 2-6烯基、CN、NO 2取代。
根据本发明的实施例，其具有下列结构至少之一：
Figure PCTCN2018118259-appb-000043
Figure PCTCN2018118259-appb-000044
Figure PCTCN2018118259-appb-000045
Figure PCTCN2018118259-appb-000046
Figure PCTCN2018118259-appb-000047
Figure PCTCN2018118259-appb-000048
Figure PCTCN2018118259-appb-000049
Figure PCTCN2018118259-appb-000050
该化合物中的B为硼原子)、
Figure PCTCN2018118259-appb-000051
Figure PCTCN2018118259-appb-000052
Figure PCTCN2018118259-appb-000053
Figure PCTCN2018118259-appb-000054
Figure PCTCN2018118259-appb-000055
Figure PCTCN2018118259-appb-000056
Figure PCTCN2018118259-appb-000057
Figure PCTCN2018118259-appb-000058
Figure PCTCN2018118259-appb-000059
Figure PCTCN2018118259-appb-000060
Figure PCTCN2018118259-appb-000061
Figure PCTCN2018118259-appb-000062
Figure PCTCN2018118259-appb-000063
Figure PCTCN2018118259-appb-000064
Figure PCTCN2018118259-appb-000065
Figure PCTCN2018118259-appb-000066
Figure PCTCN2018118259-appb-000067
Figure PCTCN2018118259-appb-000068
根据本发明的实施例，提供一种dNTP类似物，该dNTP类似物包括选自下列的至少之一：dATP类似物、dCTP、dGTP和dTTP类似物，该四种核苷酸类似物选自上述任一实施例中的化合物，各自的B为碱基A、C、G和T。
根据本发明的实施例，提出了一种NTP类似物。根据本发明的实施例，该NTP类似物选自下列的至少之一：ATP类似物，CTP类似物，GTP类似物，和UTP类似物，所称的ATP类似物、CTP类似物、GTP类似物和UTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和U。
根据本发明的实施例，提出了一种dNTP类似物混合物。根该dNTP类似物混合物包括dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物，所称的dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和T，并且所称的dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物中的至少三种分别具有不同的可检测基团或者靶向基团。
根据本发明的实施例，该dNTP类似物混合物，包括下述任意两种核苷酸类似物的组合：dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物，所称的dATP、dCTP、dGTP和dTTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和T，其中，组合中两种核苷酸类似物具有不同的可检测基团或者靶向基团。
根据本发明的实施例，提出了一种NTP类似物混合物。该NTP类似物混合物包括ATP类似物、CTP类似物、GTP类似物为前面的化合物和UTP类似物，所称的ATP、CTP、GTP和UTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和U，其中，组合中三种核苷酸类似物具有不同的可检测基团或者靶向基团。根据本发明的实施例，提出了一种NTP类似物混合物。该NTP类似物混合物，包括下述任意两种核苷酸类似物的组合：ATP类似物、CTP类似物、GTP类似物为前面的化合物和UTP类似物，所称的ATP、CTP、GTP和UTP类似物为前面所示的结构式如式(I)的化合物，各自的B分别为碱基A、C、G和U，，其中，组合中两种核苷酸类似物具有不同的可检测基团。
根据本发明的实施例，提出了前面任一实施例中的dNTP类似物、NTP类似物、dNTP类似物混合物或者NTP类似物混合物在核酸测序或者可控聚合酶链式反应或者碱基延伸反应中的用途。
根据本发明的实施例，提出了一种用于核酸测序或者可控链式聚合酶反应的试剂盒。该试剂盒包括：前面任一实施例中的dNTP类似物、NTP类似物、dNTP类似物混合物或者、NTP类似物混合物。该试剂盒能用于DNA和/或RNA序列测定。
根据本发明的实施例，该试剂盒进一步包括：切断试剂，所称的切断试剂可作用于可切割基团或者可切割键；和/或DNA聚合酶。
化合物制备
根据本发明的实施例，提供一种制备前面任一实施例中的核苷酸类似物的方法，该方法包括：利用 二硫二吡啶和巯基丙酸合成SPDP；利用式(10-2)所示化合物和氯代甲酸乙酯合成第一连接结构，第一连接结构为式(10-7)所示化合物；连接dNTP和SPDP，获得dNTP-SPDP；利用第一连接结构和dNTP-SPDP，合成化合物。
具体地，dNTP-SPDP可通过式(31-4)所示化合物与dNTP发生缩合反应获得，进一步地，将dNTP-SPDP与式(10-7)的化合物发生交换反应，以便获得目标化合物，
Figure PCTCN2018118259-appb-000069
(dNTP-SPDP)。在一些示例中，dNTP为dATP，该dATP可以购买或自己合成获得。在一个示例中，dATP可通过如下步骤获得：1)将式(10-51)所示化合物和三氟乙酰丙炔胺发生偶联反应，以便获得式(10-52)所示化合物；2)将式(10-52)所示化合物和焦磷酸盐发生成取代反应，以获得该dATP。
式(31-4)所示化合物可定制购买，也可通过如下步骤获得的：二硫二吡啶和巯基丙酸发生交换反应，之后再与NBS发生酯化反应。
式(10-7)所示化合物的化合物可商业购买，也可通过将式(10-6)所示的化合物与NH 2OH发生酯交换反应而获得。
式(10-6)所示的化合物可商业购买，也可通过如下方式获得的：a)将式(10-4)所示的化合物与NHS发生缩合反应，以便得到式(10-5)所示的化合物；b)将式(10-5)所示化合物与H-Asp-Asp-OH发生酯交换反应，以便得到式(10-6)所示的化合物。
式(10-4)所示的化合物可商业购买，也可通过将式(10-3)所示的化合物与三氟乙酸发生酯水解反应获得的。
式(10-3)所示的化合物可商业购买，也可通过将式(10-2)所示的化合物与MsCl发生酯化反应，之后再与KSCOCH 3发生酯交换反应获得的。
式(10-2)所示的化合物可商业购买，也可通过将式(10-1)所示的化合物与氯代甲酸乙酯发生酯交换反应，之后再与NaBH 4发生还原反应获得的。
进一步具体说明如下：合成式(I)所示化合物的方法，以下(1)-(3)无先后顺序要求：
(1)SPDP的合成
Figure PCTCN2018118259-appb-000070
二硫二吡啶溶于30ml的乙醇中,加入300ul的乙酸，向混合溶液中逐滴加入636mg的巯基丙酸(溶于15ml的乙醇)，30min内滴完，室温下搅拌过夜，旋干，过硅胶柱，(EA/HX＝2/3-1/1)产物旋干，NHS活化，过硅胶柱(EA/HX＝1/4-1/2)，得到产物为白色固体。SPDP的核磁图谱(H谱)见图1。
(2)碱基和R 3之间的结构(V 1-linker)的合成
Figure PCTCN2018118259-appb-000071
化合物10-2 1.02g化合物10-1(2.35mmol)溶于12mL THF中，在0℃时，0.4mL三乙胺与0.3mL的氯代甲酸乙酯加入体系中，在Ar的氛围下，搅拌10分钟，0.27g的NaBH 4(7.1mmol)分多次加入，搅拌的过程中，23mL的乙醇在10分钟内慢慢加入，在常温下，搅拌至反应到达室温，再加入10％HCl(10mL)酸化。将有机相用真空拉干。上柱纯化，得到0.97g化合物10-2。
化合物10-3 0.97g化合物10-2(2.35mmol)溶于9mLCH 2Cl 2中，加入0.7mL三乙胺(5.0mmol)将反应体系冷却至0℃。0.28mL MsCl(3.6mmol)在15min中慢慢的滴入。用TLC板检测至起始原料消失后，反应物用(2*25mL)的水清洗，用无水Na 2SO 4干燥。拉干成固体。将0.54g的KSCOCH 3(4.7mmol)加入以上固体的乙腈(6mL)中，在室温下，反应12小时。用5％-20％EtOAc/正己烷的体系，过柱纯化。得到0.57g褐色油状固体化合物10-3。
化合物10-4 0.29g化合物10-3(0.61mmol)溶于2mLCH 2Cl 2中,向这个体系中，加入2mL的三氟乙酸，在常温下，反应30min，然后用(2*20mL)的饱和食盐水洗涤，有机层用无水Na 2SO 4干燥，得到化合物10-4(0.22g,88％产率)。
化合物10-6将化合物10-4(0.22g,0.53mmol)溶于3mL乙腈中，DCC(0.12g，0.58mmol),NHS(0.07g,0.63mmol)加入到反应体系中，在常温下，搅拌1个小时，过滤完反应生成的白色固体DCU后，加入H-Asp-Asp-OH(0.13g，0.52mmol)，其2.4mL(0.25M)K 2HPO 4与1mL乙腈的混合液溶解它，再加入0.15mL DIPEA保证其pH在8左右。用HPLC纯化，拿到化合物10-6(0.25g，75％产率)。
化合物10-7 0.023mg化合物10-6溶于0.5mL 50％MeCN/H 2O混合液中，用0.5mL的1M NH 2OH处理此混合液，此反应在室温下搅拌10min后，洋浦HPLC进行纯化，拿到化合物10-7。
(3)化合物dATP-SPDP的合成
Figure PCTCN2018118259-appb-000072
化合物10-52在1L的三口烧瓶中，依次加入化合物10-51(70g)、DMF(700mL)、三氟乙酰丙炔胺(21.5g)，TEA(35.8g)、Pd(PPh 3) 4(13.7g)、CuI(4.6g)，使用氮气置换反应体系三次，在35℃，氮气保护中搅拌反应3-4h，TLC监控反应，展开剂(EA:Hex＝1:1)。将反应液倒入1.2L水中，再使用EA(500mL*2)萃取，合并油层，油层用饱和氯化铵水溶液洗涤500mL*2，再用饱和氯化钠水溶液(500mL*2)洗涤，油层使用无水硫酸钠干燥，旋干得到99g深褐色固体。粗品采用DCM 300mL，再缓慢加入Hex200mL，于0℃静置过夜重结晶，过滤得到淡黄色固体产品，用(EA/Hex＝1/5,v/v)的混合溶剂(100mL)洗涤淋洗固体，35℃真空干燥固体，合并母液，继续重结晶，最终得到淡黄色固体产品43.6g，摩尔产率为60％。
化合物10-12将化合物10-52(500mg)与Proton sponge(431mg)混合于100mL圆底烧瓶中并使用无水乙腈(10mL)完全溶解，然后在旋转蒸发仪上除去乙腈，重复操作3次，将所得混合物放置于高真空油泵上抽过夜。次日，在混合物中加入磁力搅拌子，使用PO(OMe) 3(10mL)溶解，然后将混合液在高真空油泵上抽5分钟，使用三通换氩气，重复操作3次。接着将其放置于冰水浴中搅拌5-10分钟，将POCl 3(0.14mL)在1分钟左右加入到反应体系中，然后维持在冰水浴中将反应搅拌3个小时。在100mL圆底烧瓶中将(Bu 3N) 2PPi(1383mg)溶解于无水DMF(10mL)，加入磁力搅拌子并将其在高真空油泵上抽5分钟，使用三通换氩气，重复3次。接着加入Bu3N(1.17mL)并将其放置于冰水浴中搅拌5-10分钟，将步骤2中的混合液在2-3分钟内滴加到步骤3准备好的反应液中(可使用少量PO(OMe) 3清洗瓶子，并将清洗液滴加到步骤3准备好的反应液中)。滴加完毕后将反应体系保持在冰水浴中搅拌2个小时.反应完毕后在冰水浴中将配好的TEAB(1mol，10mL)加入到反应体系中，接着搅拌1个小时。将使用去离子水浸泡的DEAE树脂装入闪式层析柱中(约10cm长)。将反应液使用去离子水稀释(120mL)，然后将其转移到装有树脂的闪式层析柱中。依次用300mL去离子水，TEAB溶液(浓度分别为0.1M，0.2M，0.4M，0.6M，0.8M，1M，每一种浓度300mL)淋洗，采用UV监测馏分，0.4M开始有产物出来。在27℃下使用旋转蒸发仪除去TEAB缓冲液和水。往旋干得到的混合物中加入磁力搅拌子以及浓氨水(100mL)，搅拌24小时后，在27℃下使用旋转蒸发仪除去氨水。
化合物10-13将SPDP(0.5mL，0.1M在无水DMF，1.25eq)加入到化合物10-12的水溶液中，将pH调到8.5左右，反应10min，用HPLC进行纯化(buffer A 0.1M TEAB,Buffer B乙腈)，化合物10-13的质谱M/Z＝739.15。
(4)化合物dATP-V 1-S-S-ATTO647N的合成
Figure PCTCN2018118259-appb-000073
化合物10-8将化合物10-7的HPLC组分与化合物13的HPLC组分加入在一起，在常温下，搅拌15分钟，浓缩，然后HPLC纯化(buffer A 0.1M TEAB,Buffer B乙腈)，得到化合物10-8，
化合物10-9将化合物10-8(约16umol)用2mL 20％piperidine/MeCN和1mL 20％piperdine/MeCN处理15分钟，除去Fmoc保护基，再将反应上HPLC纯化(buffer A 0.1M TEAB,Buffer B乙腈)，得到化合物10-9的HPLC组分。
化合物10-10将ATTO647N-NHS(0.36mL,36umol,0.1M在无水DMF中)加入到化合物10-9(17.6mL)的水和DMF的混合溶液中，用HPLC检测，待原料反应完了之后，HPLC纯化bufferA0.1M TEAB，Buffer B乙腈)，得到化合物10-10。
根据本发明的实施例，提供另一种制备前面任一实施例中的核苷酸类似物的方法，该方法包括：合成dNTP-MPSSK；利用式(31-4)所示化合物和六肽合成第二连接结构，第二连接结构为式(11-10)所示的化合物，所称的六肽为H-Pro-Lys(Fmoc)-Pro-Asp-Asp-OH；混合第二连接结构和dNTP-MPSSK，以制得目标化合物。
以下具体示例该另一制备方法，以下(1)和(2)无先后顺序要求：
(1)dNTP-MPSSK的合成
Figure PCTCN2018118259-appb-000074
化合物11-2在氮气保护下，将化合物11-1(10.2g,29.2mmol,1.0eq)溶解在吡啶(80mL)中，将TBSCl(9.8g,35.1mmol,2.2eq)溶解在吡啶(30mL)中，在冰水浴中0℃滴加(控制0℃反应副产物双保护的明显会少)。滴加完毕后，在20℃下搅拌12小时。TLC跟踪监测反应。反应结束后，加入100mL水淬灭反应。在旋转蒸发仪上45℃除去吡啶，大约旋蒸至80mL,将混合液倒入800mL水中，有白色固体析出，抽滤，用正己烷/乙酸乙酯(50mL,V正己烷:V乙酸乙酯＝8:1)淋洗3次，50℃鼓风10小时烘干得到白色固体化合物11-2(10.8g,80％)。
化合物11-3在1L的三口烧瓶中，依次加入2(10.8g)、DMF(200mL)、炔丙醇(21.5g)，TEA(35.8g)、Pd(PPh 3) 4(13.7g)、CuI(4.6g)，使用氮气置换反应体系三次，在35℃，氮气保护中搅拌反应3-4h，TLC监控反应，展开剂(EA:Hex＝1:1)。将反应液倒入1.2L水中，再使用EA(500mL*2)萃取，合并油层，油层用饱和氯化铵水溶液洗涤500mL*2，再用饱和氯化钠水溶液(500mL*2)洗涤，油层使用无水硫酸钠干燥，旋干得到99g深褐色固体。粗品采用DCM 300mL，再缓慢加入Hex200mL，于0℃静置过夜重结晶，过滤得到淡黄色固体产品，用(EA/Hex＝1/5,v/v)的混合溶剂(100mL)洗涤淋洗固体，35℃真空干燥固体，合并母液，继续重结晶，最终得到淡黄色固体产品12.6g化合物11-3，
化合物11-4将化合物11-3(10.8g,23.1mmol,1.0eq)溶于60mLDMSO中，依次加入30mL醋酸和60mL醋酸酐。混合物在25℃搅拌12小时。TLC跟踪监测反应。反应结束后，加入50mL水淬灭反应，用40mL×2 EtOAc萃取，合并有机相用50mL×2水洗两遍，用饱和NaHCO 3溶液洗涤有机相至pH为7左右。分离有机相，饱和NaCl溶液(100mL)洗涤，无水Na 2SO 4干燥，38℃旋蒸除去溶剂，得到黄色油状粗产品化合物11-4。
化合物11-5将化合物11-4(0.48mmol)溶于5mL DCM中，加入环已烷(2.88mmol)，SO 2Cl(0.96mmol)在室温下搅拌1个小时，有TLC对反应体系进行检测，拉干体系，在4mL的DMF中溶解，加入p-MePhSO 2SK(1.44ml)在常温下反应一个小时，反应液用EA萃取，用饱和食盐水洗涤，过柱，得到化合物11-5。
化合物11-6将化合物11-5(7.39mmol,1.0eq)溶解于40mL四氢呋喃中，加入三乙胺三氢氟酸盐(7.14g,44.4mmol,6eq)，25℃搅拌反应12小时。TLC跟踪监测反应。25℃旋蒸除去溶剂。加入25mL乙酸乙酯和15mL水，分离有机相，水相继续用EtOAc(10mL×3)萃取三次，直至水相中没有产品。合并有机相，38℃旋蒸除去溶剂得粗品4g。粗产品过柱，依次用DCM，DCM:MeOH(100:1/90:1/80:1/60:1/50:1/40:1/30:1/20:1)分离得到棕黄色固体化合物11-6(2.0g,63％)。
化合物11-7化合物11-6(500mg)与质子海绵(Proton sponge)(431mg)混合于100mL圆底烧瓶中并使用无水乙腈(10mL)完全溶解，然后在旋转蒸发仪上除去乙腈，重复操作3次，将所得混合 物放置于高真空油泵上抽过夜。次日，在混合物中加入磁力搅拌子，使用PO(OMe) 3(10mL)溶解，然后将混合液在高真空油泵上抽5分钟，使用三通换氩气，重复操作3次。接着将其放置于冰水浴中搅拌5-10分钟，将POCl 3(0.14mL)在1分钟左右加入到反应体系中，然后维持在冰水浴中将反应搅拌3个小时。在100mL圆底烧瓶中将(Bu 3N) 2PPi(1383mg)溶解于无水DMF(10mL)，加入磁力搅拌子并将其在高真空油泵上抽5分钟，使用三通换氩气，重复3次。接着加入Bu3N(1.17mL)并将其放置于冰水浴中搅拌5-10分钟，将步骤2中的混合液在2-3分钟内滴加到步骤3准备好的反应液中(可使用少量PO(OMe) 3清洗瓶子，并将清洗液滴加到步骤3准备好的反应液中)。滴加完毕后将反应体系保持在冰水浴中搅拌2个小时。反应完毕后在冰水浴中将配好的TEAB(1mol，10mL)加入到反应体系中，接着搅拌1个小时。将使用去离子水浸泡的DEAE树脂装入闪式层析柱中(约10cm长)。将反应液使用去离子水稀释(120mL)，然后将其转移到装有树脂的闪式层析柱中。依次用300mL去离子水，TEAB溶液(浓度分别为0.1M，0.2M，0.4M，0.6M，0.8M，1M，每一种浓度300mL)淋洗，采用UV监测馏分，0.4M开始有产物出来。在27℃下使用旋转蒸发仪除去TEAB缓冲液和水。往旋干得到的混合物中加入磁力搅拌子以及浓氨水(100mL)，搅拌24小时后，在27℃下使用旋转蒸发仪除去氨水，得到化合物11-7。
(2)碱基和R 3之间的结构(V 3-linker)的合成
Figure PCTCN2018118259-appb-000075
化合物11-9称取0.178mg(0.2mmol)六肽溶于0.1M的K 2HPO 4(5ml)中，加入SPDP 327mg(0.4mmol，4ml的DMF)，常温下反应2h，HPLC监控反应至六肽完全反应，HPLC纯化后冻干。化合物11-9的质谱，M/Z＝1087.3929。
化合物11-10 0.046mg化合物11-9溶于0.5mL 50％MeCN/H 2O混合液中，用1mL的1M NH 2OH处理此混合液，此反应在室温下搅拌10min后，洋浦HPLC进行纯化，拿到化合物10。
(3)dNTP-5-O-S-S-V 3-Dye的合成
Figure PCTCN2018118259-appb-000076
化合物11-11
将化合物10的HPLC组分与化合物7的HPLC组分加入在一起，在常温下，搅拌15分钟，浓缩，然后HPLC纯化(buffer A 0.1M TEAB,Buffer B乙腈)，得到化合物11。
化合物11-12
将化合物11-11(约16umol)用2mL 20％piperidine/MeCN和1mL 20％piperdine/MeCN处理15分钟，除去Fmoc保护基，再将反应上HPLC纯化(buffer A 0.1M TEAB,Buffer B乙腈)，得到化合物11-12的HPLC组分。
化合物11-13
将Dye-NHS(0.36mL,36umol,0.1M在无水DMF中)加入到化合物11-12(17.6mL)的水和DMF的混合溶液中，用HPLC检测，待原料反应完了之后，HPLC纯化(buffer A 0.1M TEAB,Buffer B乙腈)，得到化合物11-13。
以下示例一种使用时无需加帽的核苷酸类似物(capless核苷酸类似物)的合成路线，本领域技术人员通过以下示例的合成过程、上述示例具体反应条件的说明以及结合常规知识包括试验手段，能够制备得capless核苷酸类似物。所称的无需加帽包括使用该化合物时无需加帽步骤、加入加帽试剂等处理。
方案1
Figure PCTCN2018118259-appb-000077
Figure PCTCN2018118259-appb-000078
式(29-7)所示化合物可以进一步与碱基及其衍生物、核酸或脱氧核酸及其衍生物进行反应，以便得到式(I)所示化合物，示例如下：
Figure PCTCN2018118259-appb-000079
Figure PCTCN2018118259-appb-000080
方案2
Figure PCTCN2018118259-appb-000081
式(30-4)所示的化合物可以进一步与碱基及其衍生物、核酸或脱氧核酸及其衍生物进行反应，以便得到式(I)所示化合物，示例如下：
Figure PCTCN2018118259-appb-000082
方案3
Figure PCTCN2018118259-appb-000083
其中，式(31-7)、(31-10)、(31-13)所示的化合物可替换方案2中所用到的式(31-4)所示的化合物进行反应，以便获得相应的目标化合物，同时，式(31-7)、(31-10)、(31-13)所示的化合物可替换实施例2中所用到的式(31-4)所示的化合物进行碱基和R 3之间的结构(V 3-linker)的合成，以便得到相应的V 3-linker。
以下示例带标记的染料的化合物的合成路线，所带的标记能够与带靶向基团的核苷酸类似物连接/结合。
方案1
Figure PCTCN2018118259-appb-000084
方案2
Figure PCTCN2018118259-appb-000085
化合物的应用
根据本发明的实施例，提供一种核酸序列测定方法，该方法包括：(a)将第一核酸模板-引物复合物、一种或多种核苷酸类似物以及DNA聚合酶的混合物置于适于碱基延伸的条件下，使核苷酸类似物结合到第一核酸模板-引物复合物上，获得延伸产物；核苷酸类似物选自前面的dNTP类似物或者前面的NTP类似物或者前面的dNTP类似物混合物或者前面的NTP类似物混合物。根据本发明实施例的方法能快速、准确地测定出样本的核酸序列。该方法利用上述任一实施例中的化合物进行碱基延伸，能够实现核酸序列测定。
第一核酸模板-引物复合物可以游离于液相环境中，也可以固定在特定位置。在一些示例中，第一核酸模板-引物复合物连接在固相载体上。例如，固定在芯片上或者微球上。
在一些示例中，该方法还包括：(b)作用于延伸产物上的可切割基团或可切割键，获得第二核酸模板-引物复合物；(c)以第二核酸模板引物复合物替代第一模板-引物复合物，进行(a)和(b)至少一次。
在一些示例中，(a)还包括：检测延伸产物，以获得对应于结合到该第一核酸模板-引物复合物上的核苷酸类似物的信号。
在一些示例中，该方法还包括：基于所称的信号，确定核酸序列。
在一些示例中，使用的核苷酸类似物包含靶向基团，(a)包括加入可检测化合物来获得延伸产物，所称的可检测化合物带有特异性基团能够与该靶向基团特异性结合。在一个具体示例中，使用的核苷酸类似物的R 3基团为靶向基团，例如化合物(31)，(a)包括：在聚合酶的作用下，使核苷酸类似物结合到模板-引物复合物上，加入Dibenzocyclooctyne(DBCO)标记的荧光分子，使得延伸产物(结合了核苷酸类似物的模板-引物复合物)带有荧光分子标记，以能够产生可检测信号。
在一个示例中，步骤(b)切割的作用位点为上述任一核苷酸类似物中的C基团位置，例如对结构式(3)的化合物，该化合物在溶液中可能通过L2-R5的空间折叠、电荷等使该化合物的碱基3’-OH不暴露/难以结合核苷酸等(虚拟抑制)，其C基团包含二硫键，通过切断二硫键，能够使碱基的3’-OH暴露出来，能够结合下一个核苷酸。
根据本发明的实施例，该方法包括将聚合酶、核酸模板-引物复合物以及一种或多种核苷酸类似物置于反应容器内，使核苷酸类似物结合到核酸模板-引物复合物上，从而获得延伸产物；核苷酸类似物选自前面的化合物。根据本发明实施例的方法能准确、快速地延伸引物。根据本发明的实施例，提供一种混合物，该混合物包含待测模板、与待测模板链的至少一部分配对的引物，DNA聚合酶和前面的dNTP类似物或者前面的NTP类似物或者前面的dNTP类似物混合物或者前面的NTP类似物混合物。所称的与待测模板链的至少一部分配对的引物与待测模板可以是结合状态也可以是独立状态。
根据本发明的实施例，模板、引物聚合酶以及前面的dNTP类似物或者前面的NTP类似物或者前面的dNTP类似物混合物或者前面的NTP类似物混合物在缓冲液中。
在一些实例中，核苷酸类似物的应用方法如下，该核苷酸类似物包含能够虚拟阻断碱基3'端的抑制基团且带荧光标记，其应用于二代或三代(单分子)边合成边测序(SBS)中，G，A荧光碱基带ATTO647N，C，T荧光碱基带Cy3B，G与T组合，A与C组合，分2次加入。
在一些实例中，核苷酸类似物的应用方法如下，该核苷酸类似物包含能够虚拟阻断碱基3'端的抑制基团且带荧光标记，其应用于二代或三代(单分子)边合成边测序(SBS)中，G碱基不带荧光染料，A碱基带ATTO647N,C碱基带Cy3B，T碱基同时带ATTO647N与Cy3B。将A，T，G，C四种碱基混合，一次加入。
在一些实例中，核苷酸类似物的应用方法如下，该核苷酸类似物包含能够虚拟阻断碱基3'端的抑制基团且带荧光标记，其应用于二代或三代(单分子)边合成边测序(SBS)中，G碱基带ATTO647N/Cy5,A碱基带Texred/ROX，T碱基接Alex Fluor 488，C碱基接Cy3/ATTO532，将A，T，G，C四种碱基混合，一次加入。
在一些实例中，核苷酸类似物的应用方法如下，该核苷酸类似物包含能够虚拟阻断碱基3'端的抑制基团且带荧光标记，其应用于二代或三代(单分子)边合成边测序(SBS)中，A，T，G，C碱基接生物素，ATTO647N接链酶素，A，T，G，C分四次加入，每一次加入碱基，接着加入ATTO647N-链霉素。
在一些实例中，核苷酸类似物的应用方法如下，该核苷酸类似物包含能够虚拟阻断碱基3'端的抑制基团，其应用于二代或三代(单分子)边合成边测序(SBS)中，A碱基带叠氮基，ROX接Dibenzocyclooctyne,T碱基带PBA，ATTO Rho6G接SHA，G碱基带TCO基，ATTO647N接四嗪，C碱基接生物素基，Alexa Flour 488接链霉素基，将A，T，G，C四种碱基混合，一次加入，接下来将ROX-Dibenzocyclooctyne，ATTO Rho6G-SHA，ATTO647N-四嗪，Alexa Flour488-链霉素混合加入。以利用叠氮基和Dibenzocyclooctyne的靶向配对、PBA与SHA的靶向配对、TCO基和四嗪的靶向配对、生物素基与链霉素基的靶向配对，来获取信号实现碱基识别，实现测序。
在一些实例中，核苷酸类似物的应用方法如下，该核苷酸类似物包含能够虚拟阻断碱基3'端的抑制基团，其应用于二代或三代(单分子)边合成边测序(SBS)中，G，A碱基接生物素基，ATTO647N接链霉素，C，T碱基接PBA，ATTORho6G接SHA，将G与T组合，A与C组合，分2次加入碱基，紧接着每次碱基组合的加入，加入ATTO647N-链霉素，ATTO Rho6G-SHA混合液。以利用PBA与SHA的靶向配对、生物素基与链霉素基的靶向配对以及每轮反应包含两次反应来获取信号实现碱基识别，实现测序，定义一轮反应包括四种碱基的一次延伸。
利用上述实施例制备包含V 3-linker的dNTP-5-O-S-S-V 3-Atto532，例如具体化合物(11-13)所示的化合物，其中Dye为Atto532，为验证碱基延伸反应的能力，在溶液中进行碱基延伸反应并使用毛细管电泳的方法检测(一代测序仪，ABI3100)。
反应过程：将引物(Primer)与待测序列模板杂交——加入反应缓冲液、碱基和酶——进行延伸反应——终止反应过程——将产物加入3100检测。
根据待检测核苷酸类似物，延伸用的模板的第一个碱基为对应待测化合物的互补碱基。Primer上标有荧光，通过测定反应前后primer在毛细管电泳中所迁移的位置，以及荧光信号的强度，进行定量估算反应效率。反应效率＝对应产物峰面积/(残留反应物峰面积+对应产物峰面积)。图2为该化合物的反应结果。通过计算，在溶液验证的反应条件下其反应效率>90％。
进一步地，为了检测模板为或者包含多聚体(homopolymer)时该核苷酸类似物的表现，将模板链中前三个碱基保持相同，通过添加待测核苷酸类似物，验证连续添加状况，确定该化合物的阻断(block)下一个碱基结合到模板链的能力。图3为该化合物对homopolymer的block作用结果，在实验条件下未发现一次反应中两个或两个以上碱基的延伸，说明该化合物的抑制/终止能力较好。
为验证待测核苷酸类似物在单分子平台上的表现，发明人通过使用人工合成的多靶(20种模板核酸的混合)杂交在测序芯片上，进行性能评估。60轮反应，每轮反应包括以C、T、A、G的顺序添加的四次碱基延伸反应，每次反应包括：碱基延伸、拍照、切除侧链包括染料(作用于可切割基团/键)和清洗。通过数据处理包括图像处理和碱基识别计算芯片上确定的固定链的延伸状况，确定该化合物的反应性能。图4显示利用该化合物进行测序获得的读段的读长分布，可以看出该化合物具有可逆终止的功能，既能在一次反应中有效阻止下一个碱基的结合也能不妨碍下一个碱基在下一次反应中的结合/延伸，能用于实现核酸测序。其中，图2，3的横坐标是碱基大小(base size)，纵坐标是fluorescence intensity荧光强度；图4横坐标是读长，纵坐标是比例。
利用上述方法，发明人对其余具体化合物进行测试，包括结构式(41)、(42)、(44)-(52)、(54)-(74)的化合物，结果显示这些化合物单独地都能应用于测序。结构式(41)、(42)、(44)-(52)、(54)-(74)的化合物的反应效率分别为94.5％、96.2％、100.4％、96.4％、83.5％、13.8％、16.8％、68.9％、76.2％、64.0％、80.6％、94.1％、88.9％、80.8％、40.6％、92.1％、86.1％、93.2％、94.6％、87.8％、95.7％、50.2％、103.2％、91.5％、58.6％、107.3％、90.5％、97.1％、98.4％、98.4％、86.0％和93.0％。
发明人还使用两种荧光标记的碱基进行测序验证。一轮反应中包含两次核苷酸类似物混合物的加入，混合物以及可检测标记采用的组合为A-Atto647N和T-AttoRho6g，C-Atto532和G-Atto647N，例如具体化合物(11-13)所示的化合物，根据需要替换碱基为A、T、C、G，通过杂交长度在33nt的合成多靶(模板)进行测序，以AT混合CG混合的顺序添加76轮，每轮反应后，先使用640nm激光拍照，再使用532nm激光拍照。最终获得的原始读长分布如图5，显示76次反应后获得的读段的读长的主峰为32bp。
以下示例具体化合物应用于单色或多色测序，测序结果与上述示例结果类似(未显示)。
如图6所示，单色测序方法包含：(a)需要要素：i)一组核酸(引物/探针)；ii)一种核苷酸聚合酶；iii)一个能杂交核酸或核酸类似物的样品模板链；iv)一组带有标记的核苷酸标记物，其包含以下几部 分，碱基，核糖或脱氧核糖环，可切除的-S-S-键，3’端阻断的inhibition基团和一个可以检测的标记，在这里标记是通过-S-S-linker与碱基相连，例如具有具体化合物(3)所示的结构，其中的碱基为A，T，G或C。
(b)其具体步骤如下，第一步，将待测模板链固定在芯片上，加入DNA聚合酶，再分别加dNTPs-S-S-linker-Dye(A，T，G，C；linker不限制，例如可以为V1-linker、V3-linker等；下同)，从而dNTPs-S-S-linker-Dye(A，T，G，C)与模板链配对；第二步，冲洗完没有配对的荧光分子，检测已经配对的荧光分子；第三步，加入TCEP或THPP切除双硫键，从而除去了接在模板链上的荧光基团，裸露3’-OH,为下一轮碱基配对做准备；第四步，加入Cap试剂，捕获模板上巯基，冲洗多余的Cap试剂，进行下一轮碱基添加。然后，反复重复第一，二，三，四到30次，100次，甚至1000次。
如图7或图8所示，单色测序方法包含：(a)需要要素：i)一组核酸；ii)一种核苷酸聚合酶；iii)一个能杂交核酸或核酸类似物的样品模板链；iv)一组带有标记的核苷酸类似物，其包含以下几部分，碱基，核糖或脱氧核糖环，可切除的-S-S-键，3’端阻断的inhibition基团和一个可以检测的标记，在这里标记是通过-S-S-linker与碱基相连，例如具有式(11-13)所示结构的化合物，其中Base为A,T,C或G。。
(b)其具体步骤如下，第一步，将待测模板链固定在芯片上，加入DNA聚合酶，再分别加dNTPs-S-S-linker-Dye(A，T，G，C)，从而dNTPs-S-S-linker-Dye(A，T，G，C)与模板链配对；第二步，冲洗完没有配对的荧光分子，检测已经配对的荧光分子；第三步，加入TCEP或THPP切除双硫键，从而除去了接在模板链上的荧光基团，裸露3’-OH,为下一轮碱基配对做准备；第然后，反复重复第一，二，三，到30次，100次，甚至1000次。
如图9所示，单色测序方法包含：(a)需要要素：i)一组核酸；ii)一种核苷酸聚合酶；iii)一个能杂交核酸或核酸类似物的样品模板链；iv)一组带有标记的核苷酸类似物，其包含以下几部分，碱基，核糖或脱氧核糖环，可切除的-S-S-键，3’端阻断的inhibition基团和一个可以检测的标记，在这里标记是通过-S-S-linker与碱基相连，例如具有具体化合物(19)所示的结构，以下表示为dNTPs-S-S-linker-biotin；
(b)其具体步骤如下：第一步，加入DNA聚合酶导致dNTP-5-S-S-biotin四种中的一种接到待测模板链的一端；第二步，加入ATTO647N标记的链霉素，由于链霉素与biotin的作用，ATTO647N会标记连在待测模板链的dNTPs-S-S-linker-biotin，然后冲洗，拍照；(在这个过程中，不同的碱基都是一样，重复这一步骤。第三步，加入TCEP或THPP切除双硫键，从而除去了接在模板链上的荧光基团，裸露3’-OH,为下一轮碱基配对做准备；第四步，加入Cap试剂，捕获模板上巯基，冲洗多余的Cap试剂，进行下一轮碱基添加。然后，反复重复第一，二，三，四步骤30次，100次，甚至1000次。cap试剂可以是包含I-O＝NH和/或I-O-OH结构的化合物或组合物。
如图10所示，双色测序方法包含：(a)需要要素：i)一组核酸；ii)一种核苷酸聚合酶；iii)一个能杂交核酸或核酸类似物的样品模板链；iv)一组带有标记的核苷酸类似物，其包含以下几部分，碱基，核糖或脱氧核糖环，可切除的-S-S-键，3’端阻断的inhibition基团和一个可以检测的标记，在这里标记是通过-S-S-linker与碱基相连，例如具有具体化合物(4)所示的结构，以下表示为dNTP-5-S-S-linker-Dye。
其具体步骤如下：第一步，加入DNA聚合酶，dATP-5-S-S-linker-Dye，dTTP-5-S-S-linker-Dye，导致dNTP-5-S-S-linker-Dye两种中的一种或两种接到待测模板链的一端,冲洗完没有配对的配对的碱基，激光1，2激发，照相；第二步，加入TCEP或THPP切除双硫键，从而除去了接在模板链上的荧光基团，裸露3’-OH,为下一轮碱基配对做准备，第四步，加入Cap试剂对HS基进行加帽，第五步，加入dGTP-5-S-S-linker-Dye，dCTP-5-S-S-linker-Dye与DNA聚合酶，第六步，重复第二-四步；重复以上步骤至少一次、十次、五十次、一百次、一千次。
如图11所示，双色测序方法包含：(a)需要要素：i)一组核酸；ii)一种核苷酸聚合酶；iii)一个能杂交核酸或核酸类似物的样品模板链；iv)一组带有标记的核苷酸类似物，其包含以下几部分，碱基，核糖或脱氧核糖环，可切除的-S-S-键，3’端阻断的inhibition基团和一个可以检测的标记，在这里标记是通过-S-S-linker与碱基相连，例如具有具体化合物(7)所示的结构，以下表示为dNTP-5-O-S-S-linker-Dye。
(b)其具体步骤如下：第一步，加入DNA聚合酶，dATP-5-O-S-S-linker-Dye，dTTP-5-O-S-S-linker-Dye，导致该两种dNTP-5-O-S-S-linker-Dye中的一种或两种接到待测模板链的一端,冲洗完没有配对的配对的碱基，激光1，2激发，照相；第二步，加入TCEP或THPP切除双硫键，从而除去了接在模板链上的荧光基团，裸露3’-OH,为下一轮碱基配对做准备，第四步，加入dGTP-5-O-S-S-linker-Dye，dCTP-5-O-S-S-linker-Dye与DNA聚合酶，重复第一，二，三步；重复以上步骤至少一次、十次、五十次、一百次、一千次。
如图12所示，双色测序(FRET)方法，该方法利用混合不带可检测标记的核苷酸类似物和带检测标记的核苷酸类似物进行测序，包含：(a)需要要素：i)一组核酸；ii)一种核苷酸聚合酶；iii)一个能 杂交核酸或核酸类似物的样品模板链；iv)一组带有标记的核苷酸类似物例如为具体化合物式(89)、(90)、(91)和(92)所示的化合物，(89)、(90)和(92)以下以dNTP-5-O-N 3-V 10-Dye表示，式(91)的化合物为不带可检测标记，以下以cold dGTP表示。
(b)其具体步骤如下：第一步，加入DNA聚合酶，dATP-5-O-N 3-V 10-Cy5,dTTP-5-O-N 3-V 10-Cy3b,cold dGTP,dTTP-5-O-N 3-V 10-Cy3b(Cy5)导致该4种中的至少一种接到待测模板链的一端,冲洗完没有配对的配对的碱基，激光1，2激发，照相；第二步，加入TCEP或THPP切除双硫键，从而除去了接在模板链上的荧光基团，裸露3’-OH,为下一轮碱基配对做准备，第四步，重复第一，二，三步；；重复以上步骤至少一次、十次、五十次、一百次、一千次。
如图13所示，四色测序方法包含：(a)需要要素：i)一组核酸；ii)一种核苷酸聚合酶；iii)一个能杂交核酸或核酸类似物的样品模板链；iv)一组带有标记的核苷酸类似标记物，其包含以下几部分，碱基，核糖或脱氧核糖环，可切除的-S-S-键，3’端阻断的inhibition基团和一个可以检测的标记，在这里标记是通过-S-S-linker与碱基相连，例如，可以具有化合物(1)所示的结构，其中，Base根据需要可以替换为A，T，C，G，Dye可以根据需要替换为Cy3b、Rox、Cy5、Alexa488，以下以dNTP-5-S-S-linker-Dye表示，不同的核苷酸类似物的linker可以相同也可以不同。
(b)其具体步骤如下：第一步，加入DNA聚合酶，dATP-5-S-S-linker-Cy3b，dTTP-5-S-S-linkerRox，dGTP-5-S-S-linker-Cy5和dCTP-5-S-S-linker-Alexa488，四种dNTP-5-S-S-linker-Dye中的至少一种配对到待测模板链的一端,冲洗完没有配对的配对的碱基；拍照；第三步，加入TCEP或THPP切除双硫键，从而除去了接在模板链上的荧光基团，裸露3’-OH,为下一轮碱基配对做准备，第三步，加入Cap试剂，对HS进行Cap保护，对下一轮碱基测序做准备；第四步，重复第一，二，三步至少一次、十次、五十次、一百次、一千次。
四色测序方法包含：(a)需要要素：i)一组核酸；ii)一种核苷酸聚合酶；iii)一个能杂交核酸或核酸类似物的样品模板链；iv)一组带有标记的核苷酸类似物，其包含以下几部分，碱基，核糖或脱氧核糖环，可切除键/基团，3’端阻断的inhibition基团和一个可以检测的标记，在这里标记是通过linker与碱基相连，例如，可以具有式(27)所示化合物的结构，以下表示为dNTP-5-O-S-S-linker-靶向基团，该结构中叠氮基团为被靶向基团，可以根据需要替换成其他的被靶向基团，其中的碱基可以根据需要替换成相应的碱基。
(b)加入四种dNTP-5-O-S-S-linker-靶向基团，其中的至少一种接到待测模板链的一端，冲洗完没有配对的配对的碱基；第二步，加入ROX标记的Dibenzocyclooctyne、ATTO Rho6G标记的SHA、ATTO647N标记的四嗪和Alexa488标记链霉素混合物，使得该混合物靶向结合到待测模板上带靶向基团的核苷酸类似物，冲洗没有结合的带靶向标记的荧光染料，拍照；第三步，加入TCEP或THPP切除双硫键，从而除去了接在模板链上的荧光基团，裸露3’-OH，为下一轮碱基配对做准备，第四步，重复第一、二和三步至少一次、十次、五十次、一百次、一千次。在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (49)

  1. 一种化合物，其具有式(I)所示结构式：
    Figure PCTCN2018118259-appb-100001
    其中，L 1、L 2、L 3分别独立地为共价键或共价连接基团；
    B为碱基或者碱基衍生物；
    R 1为-OH、磷酸基团或者核苷酸；
    R 2为H或者可切割基团；
    R 3为可检测基团或者靶向基团；
    R 5为抑制基团；
    R 4为H或-OR 6，其中R 6为H或可切割基团；
    C为可切割基团或者可切割键。
  2. 根据权利要求1所述的化合物，其特征在于，所述磷酸基团为单磷酸基团、双磷酸基团、三磷酸基团或多聚磷酸基团。
  3. 根据权利要求1所述的化合物，其特征在于，B为胞嘧啶或其衍生物、胸腺嘧啶或其衍生物、腺嘌呤或其衍生物、鸟嘌呤或其衍生物、次黄嘌呤或其衍生物、脱氮腺嘌呤或其衍生物、脱氮鸟嘌呤或其衍生物、脱氮次黄嘌呤或其衍生物、7-甲基鸟嘌呤或其衍生物、5，6-二氢尿嘧啶或其衍生物、5-甲基胞嘧啶或其衍生物或5-羟甲基胞嘧啶或其衍生物。
  4. 根据权利要求1-3任一所述的化合物，其特征在于，B为二价胞嘧啶或其衍生物、二价鸟嘧啶及其衍生物、二甲腺嘧啶或其衍生物、二价胸腺嘧啶或其衍生物、二价尿嘧啶或其衍生物、二价次黄嘌呤或其衍生物、二价黄嘌呤或其衍生物、二价脱氮腺嘌呤或其衍生物、二价脱氮鸟嘌呤或其衍生物、二价脱氮次黄嘌呤或其衍生物、二价7-甲基鸟嘌呤或其衍生物、二价5，6-二氢尿嘧啶或其衍生物、二价5-甲基胞嘧啶或者衍生物或二价5-羟甲基胞嘧啶或其衍生物。
  5. 根据权利要求1-4任一所述的化合物，其特征在于，B为
    Figure PCTCN2018118259-appb-100002
  6. 根据权利要求1所述的化合物，其特征在于，R 6为H、任选取代的烷基或胺基。
  7. 根据权利要求6所述的化合物，其特征在于，R 6为H、C 1～5烷基或者-NH 2。
  8. 根据权利要求1所述的化合物，其特征在于，R 2选自H、
    Figure PCTCN2018118259-appb-100003
    Figure PCTCN2018118259-appb-100004
    中的至少之一。
  9. 根据权利要求1所述的化合物，其特征在于，C为含有选自下列至少之一的基团：
    任选取代的乙炔基、任选取代的二硫键、任选取代的酰胺烷基、任选取代的芳基、任选取代的烷氧基、任选取代的叠氮基、任选取代的直链或支链烷基。
  10. 根据权利要求9所述的化合物，其特征在于，C为含有选自下列至少之一的基团：
    任选被羟基取代的乙炔基、二硫键、酰胺基团、亚烷氧基、芳基、叠氮基团、直链或者支链亚烷基。
  11. 根据权利要求10所述的化合物，其特征在于，C选自下列至少之一：
    Figure PCTCN2018118259-appb-100005
  12. 根据权利要求1-10任一项所述的化合物，其特征在于，L 1、L 2和L 3分别独立地选自独立的键，任选取代的烷基，任选取代的烷基烯基，任选取代的杂烷基烯，任选取代的环烷基，任选取代的异环烷基以及任选取代的芳基烯的至少之一。
  13. 根据权利要求1-10任一项所述的化合物，其特征在于，L 1、L 2和L 3分别独立地选自独立的键、任选取代的C 1-8烷基，任选取代的C 1-8烷基烯，任选取代的C 2-8杂烷基烯，任选取代的C 3-8环烷基烯，任选取代的C 3-8异环烷基烯，或任选取代的C 6-8芳基烯。
  14. 根据权利要求1-10任一项所述的化合物，其特征在于，L 1选自独立的键、任选取代的C1～C3烷基和任选取代的C1-C3烷基烯中的至少之一。
  15. 根据权利要求1-10任一项所述的化合物，其特征在于，L 1为L 1 A-L 1 B-L 1 C-L 1 D-L 1 E，L 2为L 2 A-L 2 B-L 2 C-L 2 D-L 2 E和/或L 3为L 3 A-L 3 B-L 3 C-L 3 D-L 3 E，其中，
    L 1 A、L 1 B、L 1 C、L 1 D、L 1 E、L 2 A、L 2 B、L 2 C、L 2 D、L 2 E、L 3 A、L 3 B、L 3 C、L 3 D和L 3 E分别独立地选自独立的键，取代或非取代的烷基烯，取代或非取代杂烷基烯，取代或非取代环烷基烯，取代或非取代异环烷基烯，取代或非取代芳基烯，
    L 1 A、L 1 B、L 1 C、L 1 D和L 1 E中至少有一个不是独立的键，
    L 2 A、L 2 B、L 2 C、L 2 D和L 2 E中至少有一个不是独立的键，
    L 3 A、L 3 B、L 3 C、L 3 D和L 3 E中至少有一个不是独立的键。
  16. 根据权利要求15所述的化合物，其特征在于，
    各L 1 A、L 1 B、L 1 C、L 1 D、L 1 E、L 2 A、L 2 B、L 2 C、L 2 D、L 2 E、L 3 A、L 3 B、L 3 C、L 3 D和L 3 E分别独立地选自独立的键、C 2-6烷基烯、2-6个原子组成的杂烷基烯、C 3-6环烷基烯、2-6个原子组成的杂环烷基烯，C 6-10芳基烯，其中，各C 2-6烷基烯、2-6个原子组成的杂烷基烯、C 3-6环烷基烯、2-6个原子组成的杂环烷基烯，C 6-10芳基烯独立地未被取代或被1、2、3、4、5个C 1-6烷基、卤素、硝基、C 1-6卤代烷基取代。
  17. 根据权利要求1-16任一项所述的化合物，其特征在于，L 1包括选自下列的至少之一：
    Figure PCTCN2018118259-appb-100006
    其中，各R 7独立为酰胺键或O，
    各p 1独立为0、1～10之间的整数，优选地，各p 1独立为1、4；
    各p 2独立为0、1～5之间的整数，优选地，各p 2独立为1、4；
    各R 8，R 9分别独立地选自H、-CH 3、-CX 3、-CHX 2、-CH 2X、-CN、-Ph、C 1-6烷基、C 2～6烷基和C 3～6环烷基中的至少一种，其中X为Cl、Br、I；
    各p 3独立为0、1～4之间的整数，优选地，各p 3独立为1、2。
  18. 根据权利要求1-17任一项所述的化合物，L 2包括选自下列的至少之一：
    Figure PCTCN2018118259-appb-100007
    其中，各Rx、Ry、R A、R B独立为H、C 1～6烷基链、C 3-10环烷基、C 5-10芳基，各x、y、z独立为0或者1～6的整数，
    优选地，各Rx、Ry独立为H、C 3-5环烷基、C 5-6环烷基、苯基。
  19. 根据权利要求1-17任一项所述的化合物，其特征在于，L 2为选自如下结构的一种：
    Figure PCTCN2018118259-appb-100008
    Figure PCTCN2018118259-appb-100009
    其中，各Ra与Rb分别独立地为H、取代或非取代的烷基、取代或非取代杂烷基烯、取代或非取代环烷基、取代或非取代异环烷基、取代或非取代芳基。
  20. 根据权利要求1-19任一项所述的化合物，其特征在于，L 3选自以下结构的一种：：
    Figure PCTCN2018118259-appb-100010
    其中，n1，n2，n3，n4，n5，n6分别独立地为0或者1～7的整数。
  21. 根据权利要求1-20任一项所述的化合物，其特征在于，L 3具有选自以下结构的一种：
    Figure PCTCN2018118259-appb-100011
  22. 根据权利要求1-21任一项所述的化合物，其特征在于，R 3选自染料、点击化学反应基团、叠氮基和生物素基中的的至少一种。
  23. 根据权利要求1-22任一项所述的化合物，其特征在于，R 5包括至少一个带电荷的基团，所述带电荷的基团包括-COOH、-PO 4、-SO 4、-SO 3、-SO 2。
  24. 根据权利要求1-23任一项所述的化合物，其特征在于，R 5具有选自如下基团中的一种结构：
    Figure PCTCN2018118259-appb-100012
    其中，各R 10和R 11独立为H或者C 1-6烷基，各a和b独立地为0或者1-5的整数；
    优选地，各a和b分别独立地为1或2。
  25. 根据权利要求1-24任一项所述的化合物，其特征在于，R 5包括选自如下基团中的一种：
    Figure PCTCN2018118259-appb-100013
  26. 根据权利要求1-25任一项所述的化合物，其特征在于，其具有下列结构至少之一：
    Figure PCTCN2018118259-appb-100014
    Figure PCTCN2018118259-appb-100015
    其中，各Ra与Rb分别独立地选自H、任选取代的烷基、任选取代的杂烷基烯、任选取代的环烷基、任选取代的异环烷基和任选取代的芳基中的至少一种；
    优选地，各Ra与Rb分别独立地为H、C 1-6烷基、3-6个原子的杂烷基、C 2-6烯基、3-6个原子的杂烯基、C 3-6环烷基、3-6个原子组成的杂环基、苯基、5-6个原子组成的杂芳基，其中，各C 1-6烷基、3-6个原子的杂烷基、C 2-6烯基、3-6个原子的杂烯基、C 3-6环烷基、3-6个原子组成的杂环基、苯基和5-6个原子组成的杂芳基独立地未被取代或被1、2或3个卤素、C 1-6烷基、C 2-6烯基、CN、NO 2取代。
  27. 根据权利要求1-26任一项所述的化合物，其特征在于，其具有下列结构至少之一：
    Figure PCTCN2018118259-appb-100016
    Figure PCTCN2018118259-appb-100017
    Figure PCTCN2018118259-appb-100018
    Figure PCTCN2018118259-appb-100019
    Figure PCTCN2018118259-appb-100020
    Figure PCTCN2018118259-appb-100021
    Figure PCTCN2018118259-appb-100022
    Figure PCTCN2018118259-appb-100023
    Figure PCTCN2018118259-appb-100024
    Figure PCTCN2018118259-appb-100025
    Figure PCTCN2018118259-appb-100026
    Figure PCTCN2018118259-appb-100027
    Figure PCTCN2018118259-appb-100028
    Figure PCTCN2018118259-appb-100029
    Figure PCTCN2018118259-appb-100030
    Figure PCTCN2018118259-appb-100031
    Figure PCTCN2018118259-appb-100032
    Figure PCTCN2018118259-appb-100033
    Figure PCTCN2018118259-appb-100034
    Figure PCTCN2018118259-appb-100035
    Figure PCTCN2018118259-appb-100036
    Figure PCTCN2018118259-appb-100037
    Figure PCTCN2018118259-appb-100038
  28. 一种dNTP类似物，其特征在于，包括选自下列的至少之一：
    dATP类似物，所述dATP类似物为权利要求1～27中任一项所述的化合物，B为碱基A；
    dCTP类似物，所述dCTP类似物为权利要求1～27中任一项所述的化合物，B为碱基C；
    dGTP类似物，所述dGTP类似物为权利要求1～27中任一项所述的化合物，B为碱基G；和
    dTTP类似物，所述dTTP类似物为权利要求1～27中任一项所述的化合物，B为碱基T。
  29. 一种NTP类似物，其特征在于，包括选自下列的至少之一：
    ATP类似物，所述ATP类似物为权利要求1～27中任一项所述的化合物，B为碱基A；
    CTP类似物，所述CTP类似物为权利要求1～27中任一项所述的化合物，B为碱基C；
    GTP类似物，所述GTP类似物为权利要求1～27中任一项所述的化合物，B为碱基G；和
    UTP类似物，所述UTP类似物为权利要求1～27中任一项所述的化合物，B为碱基U。
  30. 一种dNTP类似物混合物，其特征在于，包括：
    dATP类似物，所述dATP类似物为权利要求1～27中任一项所述的化合物，B为碱基A；
    dCTP类似物，所述dCTP类似物为权利要求1～27中任一项所述的化合物，B为碱基C；
    dGTP类似物，所述dGTP类似物为权利要求1～27中任一项所述的化合物，B为碱基G；和
    dTTP类似物，所述dTTP类似物为权利要求1～27中任一项所述的化合物，B为碱基T，
    其中，dATP类似物、dCTP类似物、dGTP类似物和dTTP类似物中的至少三种分别具有不同的可检测基团。
  31. 一种dNTP类似物混合物，其特征在于，包括下述任意两种核苷酸类似物的组合：
    dATP类似物，所述dATP类似物为权利要求1～27中任一项所述的化合物，B为碱基A；
    dCTP类似物，所述dCTP类似物为权利要求1～27中任一项所述的化合物，B为碱基C；
    dGTP类似物，所述dGTP类似物为权利要求1～27中任一项所述的化合物，B为碱基G；和
    dTTP类似物，所述dTTP类似物为权利要求1～27中任一项所述的化合物，B为碱基T，
    其中，组合中两种核苷酸类似物具有不同的可检测基团。
  32. 一种NTP类似物混合物，其特征在于，包括：
    ATP类似物，所述ATP类似物为权利要求1～27中任一项所述的化合物，B为碱基A；
    CTP类似物，所述CTP类似物为权利要求1～27中任一项所述的化合物，B为碱基C；
    GTP类似物，所述GTP类似物为权利要求1～27中任一项所述的化合物，B为碱基G；和
    UTP类似物，所述UTP类似物为权利要求1～27中任一项所述的化合物，B为碱基U，
    其中，ATP类似物、CTP类似物、GTP类似物和UTP类似物中的至少三种分别具有不同的可检测基团。
  33. 一种NTP类似物混合物，其特征在于，包括下述任意两种核苷酸类似物的组合：
    ATP类似物，所述ATP类似物为权利要求1～27中任一项所述的化合物，B为碱基A；
    CTP类似物，所述CTP类似物为权利要求1～27中任一项所述的化合物，B为碱基C；
    GTP类似物，所述GTP类似物为权利要求1～27中任一项所述的化合物，B为碱基G；和
    UTP类似物，所述UTP类似物为权利要求1～27中任一项所述的化合物，B为碱基U，
    其中，组合中两种核苷酸类似物具有不同的可检测基团。
  34. 权利要求28所述的dNTP类似物或者权利要求29所述的NTP类似物或者权利要求30所述的dNTP类似物混合物或者权利要求31所述的dNTP类似物混合物或者权利要求32所述的NTP类似物混合物或者权利要求33所述的NTP类似物混合物在核酸测序或者可控聚合酶链式反应或者碱基延伸反应中的用途。
  35. 一种用于核酸测序或者可控链式聚合酶反应的试剂盒，包括：
    权利要求28所述的dNTP类似物或者权利要求29所述的NTP类似物或者权利要求30所述的dNTP类似物混合物或者权利要求31所述的dNTP类似物混合物或者权利要求32所述的NTP类似物混合物或者权利要求33所述的NTP类似物混合物。
  36. 根据权利要求35所述的试剂盒，其特征在于，进一步包括：
    切断试剂，所述切断试剂可作用于可切割基团或者可切割键；和/或
    DNA聚合酶。
  37. 根据权利要求36所述的试剂盒，其特征在于，所述试剂盒包括切断试剂，所述切断试剂为TCEP/THPP。
  38. 一种测定核酸序列的方法，其特征在于，包括：
    (a)将第一核酸模板-引物复合物、一种或多种核苷酸类似物以及DNA聚合酶的混合物置于适于碱基延伸的条件下，使所述核苷酸类似物结合到所述第一核酸模板-引物复合物上，获得延伸产物；
    所述核苷酸类似物选自权利要求28所述的dNTP类似物或者权利要求29所述的NTP类似物或者权利要求30所述的dNTP类似物混合物或者权利要求31所述的dNTP类似物混合物或者权利要求32所述的NTP类似物混合物或者权利要求33所述的NTP类似物混合物。
  39. 根据权利要求38所述的方法，其特征在于，所述第一核酸模板-引物复合物连接在固相载体上。
  40. 根据权利要求38所述的方法，其特征在于，所述第一核酸模板-引物复合物固定在芯片上或者 微球上。
  41. 根据权利要求38所述的方法，其特征在于，进一步包括：
    (b)作用于所述延伸产物上的可切割基团或可切割键，获得第二核酸模板-引物复合物；
    (c)以所述第二核酸模板引物复合物替代所述第一模板-引物复合物，进行(a)和(b)至少一次。
  42. 根据权利要求38-41任一所述的方法，其特征在于，(a)还包括：
    检测所述延伸产物，以获得对应于结合到该第一核酸模板-引物复合物上的核苷酸类似物的信号。
  43. 根据权利要求42所述的方法，其特征在于，进一步包括：
    基于所述信号，确定所述核酸序列。
  44. 根据权利要求38所述的方法，其特征在于，所述核苷酸类似物包含靶向基团，(a)包括加入可检测化合物来获得所述延伸产物，所述可检测化合物带有特异性基团能够与所述靶向基团特异性结合。
  45. 一种延伸引物的方法，其特征在于，包括将聚合酶、核酸模板-引物复合物以及一种或多种核苷酸类似物置于反应容器内，使所述核苷酸类似物结合到所述核酸模板-引物复合物上，从而获得延伸产物；所述核苷酸类似物选自权利要求1至27任一项所述的化合物。
  46. 一种反应混合物，其特征在于，包含待测模板、与待测模板链的至少一部分配对的引物，DNA聚合酶和权利要求28所述的dNTP类似物或者权利要求29所述的NTP类似物或者权利要求30所述的dNTP类似物混合物或者权利要求31所述的dNTP类似物混合物或者权利要求32所述的NTP类似物混合物或者权利要求33所述的NTP类似物混合物。
  47. 根据权利要求46所述的反应混合物，其特征在于，所述模板、引物聚合酶以及权利要求28所述的dNTP类似物或者权利要求29所述的NTP类似物或者权利要求30所述的dNTP类似物混合物或者权利要求31所述的dNTP类似物混合物或者权利要求32所述的NTP类似物混合物或者权利要求33所述的NTP类似物混合物在缓冲液中。
  48. 一种制备权利要求1-27任一项所述化合物的方法，其特征在于，包括：
    利用二硫二吡啶和巯基丙酸合成SPDP；
    利用
    Figure PCTCN2018118259-appb-100039
    和氯代甲酸乙酯合成第一连接结构，所述第一连接结构为
    Figure PCTCN2018118259-appb-100040
    连接dNTP和所述SPDP，获得dNTP-SPDP；
    利用所述第一连接结构和所述dNTP-SPDP，合成所述化合物。
  49. 一种制备权利要求1-27任一项所述化合物的方法，其特征在于，包括：
    合成dNTP-MPSSK，所述dNTP-MPSSK的结构如下所示：
    Figure PCTCN2018118259-appb-100041
    利用
    Figure PCTCN2018118259-appb-100042
    和六肽合成第二连接结构，所述第二连接结构为
    Figure PCTCN2018118259-appb-100043
    所述六肽为H-Pro-Lys(Fmoc)-Pro-Asp-Asp-OH；
    混合所述第二连接结构和所述dNTP-MPSSK，以制得所述化合物。
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