WO2022083686A1 - 修饰的核苷或核苷酸 - Google Patents

修饰的核苷或核苷酸 Download PDF

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WO2022083686A1
WO2022083686A1 PCT/CN2021/125262 CN2021125262W WO2022083686A1 WO 2022083686 A1 WO2022083686 A1 WO 2022083686A1 CN 2021125262 W CN2021125262 W CN 2021125262W WO 2022083686 A1 WO2022083686 A1 WO 2022083686A1
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alkyl
group
cycloalkyl
independently selected
compound
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PCT/CN2021/125262
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English (en)
French (fr)
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徐讯
滕波
章文蔚
陈奥
李汉东
鄢胜壹
卓世添
沈亮
张泳辉
高南风
赵杰
廖莎
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深圳华大生命科学研究院
深圳华大基因科技有限公司
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Priority to EP21882101.5A priority Critical patent/EP4234566A1/en
Priority to KR1020237017194A priority patent/KR20230107259A/ko
Priority to JP2023548993A priority patent/JP2023546741A/ja
Priority to US18/033,037 priority patent/US20240067673A1/en
Priority to CN202180071993.XA priority patent/CN116507736A/zh
Priority to CA3199278A priority patent/CA3199278A1/en
Priority to AU2021363336A priority patent/AU2021363336A1/en
Publication of WO2022083686A1 publication Critical patent/WO2022083686A1/zh

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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
    • 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
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of nucleic acid sequencing.
  • the present invention relates to modified nucleosides or nucleotides whose 3'-OH is reversibly blocked.
  • NGS sequencing has overcome the disadvantages of high cost and long sequencing time of Sanger sequencing, and has greatly promoted the application of gene sequencing technology.
  • NGS sequencing has been deeply applied in prenatal screening, tumor diagnosis, tumor treatment, animal and plant breeding and other fields, driving the progress of science and technology and medicine.
  • Nucleoside triphosphate (dNTP) analogs with reversible blocking groups are key raw materials in NGS sequencing. Due to the introduction of the reversible blocking group, the 3'-OH group in dNTP can be retained, which overcomes the shortcomings of Sanger sequencing and ensures the accuracy of base recognition. It can be said that nucleoside triphosphate analogs (dNTPs) with reversible blocking groups are the most critical technology in NGS sequencing.
  • the realization of reversible blocking of dNTP is mainly achieved through two categories of ideas.
  • the first type of idea is to directly introduce a reversible blocking group at the 3'-OH of dNTPs.
  • the advantage of this type of modified dNTPs is that the blocking of 3'-OH ensures the blocking efficiency in sequencing.
  • Another idea is that 3'-OH is not blocked, but the polymerase is blocked by base modification.
  • the advantage of this strategy is that the modification selectivity of the blocking group is wider and is not limited by the polymerase. .
  • Azidomethyl is a kind of efficient 3'-OH reversible blocking group, which has great advantages such as good stability, mild excision conditions, and fast excision speed.
  • a careful analysis of the 3'-OH azide methyl excision reaction is essentially a cascade reaction.
  • the azide group undergoes the Staudinger reaction under the action of the phosphorus reagent to generate an intermediate with a 3'-OH methyleneamine group, which then undergoes a rapid hydrolysis reaction to release the 3'-OH group.
  • both the Staudinger reaction and the hydrolysis reaction are very fast, so the excision of the azidomethyl group is extremely fast (as shown in Figure 1).
  • Azide group is a special kind of chemical group, three nitrogen atoms in its chemical structure form a conjugated structure and are in a plane. But at the same time, the three nitrogen atoms did not form a straight line, but formed a polyline structure with a certain angle.
  • the conjugated structure formed by three nitrogen atoms in the azide group has multiple resonance formulas, which is beneficial to the stability of its structure.
  • the azide group has some stability. However, due to the superimposed structure of three nitrogen atoms, under suitable reaction conditions, the azide group can easily react and release nitrogen gas quickly, so the azide group has very active reactivity and explosion risk. It is based on this characteristic of the azide group that it is widely used in various click reactions.
  • 3'-OH blocking groups Based on the fast reaction characteristics of azide groups, the inventors designed a class of 3'-OH blocking groups with azide groups that can react in series. After this type of 3'-OH blocking group uses the Staudinger reaction to release the amino group, the amino group attacks the ester group/carbonate protecting group of the 3'-OH group to release 3'-OH (as shown in Figure 2).
  • groups that can release nucleophilic atoms under suitable conditions are suitable for this design, such as -S-SR group, -OCOR, -OCONHR and so on.
  • the present invention aims to develop a class of dNTP analogs with an ester group at 3'-OH and carbonate ester for NGS sequencing.
  • the general structural formula of this type of dNTP analog is shown in Figure 3, and the 3'-OH is protected by a reversible blocking group, including 2'deoxyuridine triphosphate, 2'deoxythymidine triphosphate, and 2'deoxycytidine triphosphate , The main structure of 2'deoxyadenosine triphosphate, 7-deaza-2'deoxyadenosine triphosphate, 2'deoxyguanosine triphosphate and 7-deaza-2'deoxyguanosine triphosphate.
  • the present invention provides a compound represented by formula (A) or a salt thereof,
  • R is a reversible blocking group, R is selected from
  • the heteroaryl group is selected from the following:
  • each heteroaryl group is independently optionally substituted by 1 or more (such as 2, 3, 4, 5, 6, preferably 2) R";
  • each X is independently selected from O, NH, S;
  • each Y is independently selected from direct bond, O, NH, S, CH, CH2 , C( CH3 ) 2 ;
  • R 0 is selected from -N 3 , -SS-C1-C6 alkyl (such as -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH 2 , - OCOR m , -OCONHR m , wherein each R m is independently selected from aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), or aromatic alkyl (such as phenyl) C1-C6 alkyl);
  • R 1 is selected from -N 3 , -SS-C1-C6 alkyl (such as -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH 2 , - OCOR m , -OCONHR m , wherein each R m is independently selected from aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), or aromatic alkyl (such as phenyl) C1-C6 alkyl);
  • R 2 , R 3 , R 4 are each independently selected from O, NH, S, CH, CH 2 , C(CH 3 ) 2 ;
  • Each R" is independently selected from H, -N3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic Family alkyl (such as phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (such as C1-C6 alkoxy), and not H at the same time;
  • R 5 , R 6 , R 7 , R 8 , R 9 , R x , R y , R z are each independently selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic alkyl (such as phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (such as C1- C6 alkoxy), and R 5 , R 6 , R 7 , R 8 and R 9 are not H at the same time;
  • R 10a , R 10b , R 10c , R 11a , R 11b , R 12 are each independently selected from H, -N 3 , -SS-C1-C6 alkyl (such as -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl or -SS-isobutyl), -ONH 2 , -OCOR m , -OCONHR m , aliphatic alkyl (such as C1-C6 alkyl, specifically methyl Me, ethyl Et, isopropyl iPr, tert-butyl tBu), aromatic alkyl (such as phenyl C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), wherein each R m independently selected from aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such
  • n is selected from 1, 2, 3, 4, 5;
  • each m 1 is independently selected from 1, 2, 3, 4, 5, 6;
  • each m is independently selected from 0, 1, 2 , 3, 4, 5, 6;
  • R' is selected from H, a monophosphate group diphosphate group triphosphate group or tetraphosphate group
  • Each Z is independently selected from O, S, BH;
  • Base is selected from base, deaza base or its tautomer, for example Base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine or its tautomers.
  • R is selected from
  • R is selected from
  • R is selected from
  • R is selected from
  • heteroaryl group is selected from the group consisting of:
  • each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • the heteroaryl group is selected from the group consisting of: Each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • the heteroaryl group is selected from the group consisting of: Each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • the heteroaryl group is selected from the group consisting of: Each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • X is O.
  • each Y is independently selected from a direct bond, CH2 .
  • R 0 is -N 3 or -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl).
  • R 0 is -N 3 .
  • R 1 is -N 3 or -SS-C1-C6 alkyl (eg, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl).
  • R 1 is -N 3 .
  • R 2 , R 3 , R 4 are each independently selected from O, NH, S, CH, CH 2 , C(CH 3 ) 2 , and the following conditions are met: when R 2 is selected from O, S , R 3 and R 4 are CH 2 ; when R 3 is selected from O, S, R 2 and R 4 are CH 2 ; when R 4 is selected from O, S, R 2 and R 3 are CH 2 ; R 2 is C (CH 3 ) 2 , R 3 and R 4 are CH 2 ; when R 3 is C(CH 3 ) 2 , R 2 and R 4 are CH 2 ; when R 4 is C(CH 3 ) 2 , R 2 and R 3 is CH 2 ; when Y is CH, R 2 is CH , and R 3 and R 4 are CH 2 .
  • R 2 , R 3 , R 4 are each independently selected from CH, CH 2 .
  • R 2 and R 3 are CH and R 4 is CH 2 .
  • each R" is independently selected from Preferably, R" is
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R"
  • one R" of the multiple R" selected from The remaining R" are each independently selected from -N3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic Alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R", one R" of the multiple R" for The remaining R" are each independently selected from nitro, aliphatic alkyl (eg, C1-C6 alkyl), F, I, Br, Cl.
  • multiple R" of the multiple R" for The remaining R" are each independently selected from nitro, aliphatic alkyl (eg, C1-C6 alkyl), F, I, Br, Cl.
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R"
  • one R" of the multiple R" for The remaining R" are each independently selected from nitro, C1-C6 alkyl, preferably, the remaining R" is C1-C6 alkyl.
  • any one of R 5 , R 6 , R 7 , R 8 , R 9 is selected from The remaining four are each independently selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), Aromatic alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • any one of R 5 , R 6 , R 7 , R 8 , R 9 is selected from The remaining four are each independently selected from H, nitro, C1-C6 alkyl.
  • R 5 , R 6 , R 7 , R 8 , R 9 , R 5 , R 7 or R 9 is selected from The remaining four are each independently selected from H, nitro.
  • R 5 , R 6 , R 7 , R 8 , R 9 , R 5 or R 9 is The remaining four are each independently selected from H, nitro, methoxy, acetamido.
  • R 7 is selected from H, nitro, methoxy, acetamido, and the remaining three are H.
  • any one of Rx, Ry is selected from The other is selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • any one of Rx, Ry is selected from The other is H.
  • any one of Rx, Ry is The other is H.
  • R z is selected from
  • Rz is
  • any one of R 10a , R 10b , R 10c is selected from -N 3 , -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-iso propyl, -SS-tert-butyl or -SS-isobutyl), -ONH 2 , -OCOR m , -OCONHR m , and the other two are independently selected from H, aliphatic alkyl (such as C1-C6 alkane) group, such as methyl, ethyl, isopropyl, tert-butyl), aromatic alkyl (such as phenyl C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), wherein each R m is independently selected from aliphatic alkyl (eg C1-C6 alkyl), cycloalkyl (eg C3-C6
  • any one of R 10a , R 10b , R 10c is -N 3 or -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-isopropyl base, -SS-tert-butyl or -SS-isobutyl), the other two are independently selected from H, aliphatic alkyl (such as C1-C6 alkyl, specifically such as methyl, ethyl, isopropyl) , tert-butyl).
  • aliphatic alkyl such as C1-C6 alkyl, specifically such as methyl, ethyl, isopropyl
  • any one of R 10a , R 10b , R 10c is -N 3 or -SS-methyl, and the other two are each independently selected from H, methyl.
  • any one of R 10a , R 10b , R 10c is -N 3 , -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl, or - SS-isobutyl, the other two are each independently selected from H, methyl.
  • R 11a , R 11b are each independently selected from H, aliphatic alkyl (eg, C1-C6 alkyl, specifically, methyl, ethyl, isopropyl, tert-butyl).
  • aliphatic alkyl eg, C1-C6 alkyl, specifically, methyl, ethyl, isopropyl, tert-butyl.
  • R 11a , R 11b are H.
  • R 12 is selected from -N 3 , -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl) , -ONH 2 , -OCOR m , -OCONHR m , wherein each R m is independently selected from aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl) or aromatic Alkyl (eg phenyl C1-C6 alkyl).
  • aliphatic alkyl such as C1-C6 alkyl
  • cycloalkyl such as C3-C6 cycloalkyl
  • aromatic Alkyl eg phenyl C1-C6 alkyl
  • R 12 is -N 3 or -SS-C1-C6 alkyl (eg, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl).
  • R 12 is -N 3 .
  • n is selected from 1, 2, 3.
  • n is 2.
  • m 1 is 1.
  • each m2 is independently selected from 0, 1.
  • R' is a triphosphate group
  • Z is O.
  • Base is selected from
  • the present invention provides a compound represented by formula (I) or a salt thereof,
  • X is selected from O, NH, S;
  • Y is selected from direct bond, O, NH, S, CH, CH2 , C( CH3 ) 2 ;
  • R 0 is selected from -N 3 , -SS-C1-C6 alkyl (such as -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH 2 , - OCOR m , -OCONHR m , wherein each R m is independently selected from aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), or aromatic alkyl (such as phenyl) C1-C6 alkyl);
  • n is selected from 1, 2, 3, 4, 5;
  • R' is selected from H, a monophosphate group diphosphate group triphosphate group or tetraphosphate group
  • Each Z is independently selected from O, S, BH;
  • Base is selected from base, deaza base or its tautomer, for example Base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine or its tautomers.
  • X is O.
  • Y is CH2 .
  • R 0 is -N 3 or -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl).
  • R 0 is -N 3 .
  • n is selected from 1, 2, 3.
  • n is 2.
  • R' is a triphosphate group
  • Z is O.
  • Base is selected from
  • the present invention provides a compound represented by formula (II) or a salt thereof,
  • X is selected from O, NH, S;
  • Y is selected from direct bond, O, NH, S, CH, CH2 , C( CH3 ) 2 ;
  • R 1 is selected from -N 3 , -SS-C1-C6 alkyl (such as -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH 2 , - OCOR m , -OCONHR m , wherein each R m is independently selected from aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), or aromatic alkyl (such as phenyl) C1-C6 alkyl);
  • R 2 , R 3 , R 4 are each independently selected from O, NH, S, CH, CH 2 , C(CH 3 ) 2 ;
  • R' is selected from H, a monophosphate group diphosphate group triphosphate group or tetraphosphate group
  • Each Z is independently selected from O, S, BH;
  • Base is selected from base, deaza base or its tautomer, for example Base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine or its tautomers.
  • X is O.
  • Y is a direct bond
  • R 1 is -N 3 or -SS-C1-C6 alkyl (eg, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl).
  • R 1 is -N 3 .
  • R 2 , R 3 , R 4 are each independently selected from O, NH, S, CH, CH 2 , C(CH 3 ) 2 , and the following conditions are met: when R 2 is selected from O, S , R 3 and R 4 are CH 2 ; when R 3 is selected from O, S, R 2 and R 4 are CH 2 ; when R 4 is selected from O, S, R 2 and R 3 are CH 2 ; R 2 is C (CH 3 ) 2 , R 3 and R 4 are CH 2 ; when R 3 is C(CH 3 ) 2 , R 2 and R 4 are CH 2 ; when R 4 is C(CH 3 ) 2 , R 2 and R 3 is CH 2 ; when Y is CH, R 2 is CH , and R 3 and R 4 are CH 2 .
  • R 2 , R 3 , R 4 are each independently selected from CH, CH 2 .
  • R 2 and R 3 are CH and R 4 is CH 2 .
  • R' is a triphosphate group
  • Z is O.
  • Base is selected from
  • the present invention provides a compound represented by formula (III) or a salt thereof,
  • A' is selected from
  • the heteroaryl group is selected from the following:
  • each heteroaryl group is independently optionally substituted by 1 or more (such as 2, 3, 4, 5, 6, preferably 2) R";
  • Each R" is independently selected from H, -N3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic Family alkyl (such as phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (such as C1-C6 alkoxy),
  • X is selected from O, NH, S;
  • R 5 , R 6 , R 7 , R 8 , R 9 are each independently selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkane base (such as C3-C6 cycloalkyl), aromatic alkyl (such as phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (such as C1-C6 alkoxy), and R 5 , R 6 , R 7 , R 8 and R 9 are not H at the same time;
  • R 10a , R 10b , R 10c , R 11a , R 11b , R 12 are each independently selected from H, -N 3 , -SS-C1-C6 alkyl (such as -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl or -SS-isobutyl), -ONH 2 , -OCOR m , -OCONHR m , aliphatic alkyl (such as C1-C6 alkyl, specifically methyl , ethyl, isopropyl, tert-butyl), aromatic alkyl (such as phenyl C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), wherein each R m is independently selected from Aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycl
  • n 1 is selected from 1, 2, 3, 4, 5, 6;
  • n 2 is selected from 0, 1, 2, 3, 4, 5, 6;
  • R' is selected from H, a monophosphate group diphosphate group triphosphate group or tetraphosphate group
  • Each Z is independently selected from O, S, BH;
  • Base is selected from base, deaza base or its tautomer, for example Base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine or its tautomers.
  • heteroaryl group is selected from the group consisting of:
  • each heteroaryl group is independently optionally substituted with 1 or more (e.g. 2, 3, 4, 5, 6, preferably 2) R".
  • the heteroaryl group is selected from the following: Each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • the heteroaryl group is selected from the group consisting of: Each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • the heteroaryl group is selected from the group consisting of: Each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • heteroaryl group is selected from the group consisting of:
  • heteroaryl group is selected from the group consisting of:
  • each R" is independently selected from Preferably, R" is
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R"
  • one R" of the multiple R" selected from The remaining R" are each independently selected from -N3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic Alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R", one R" of the multiple R" for The remaining R" are each independently selected from nitro, aliphatic alkyl (eg, C1-C6 alkyl), F, I, Br, Cl.
  • multiple R" of the multiple R" for The remaining R" are each independently selected from nitro, aliphatic alkyl (eg, C1-C6 alkyl), F, I, Br, Cl.
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R"
  • one R" of the multiple R" for The remaining R" is each independently selected from nitro, C1-C6 alkyl, preferably, the remaining R" is C1-C6 alkyl, more preferably, the remaining R" is methyl.
  • R 13 , R 14 , R 15 , R 16 , R 16 is R 13 , R 14 , R 15 are each independently selected from H, nitro, C1-C6 alkyl (eg, methyl).
  • R 13 , R 14 , R 15 , R 16 , R 16 is R 13 , R 14 and R 15 are H.
  • R 17 and R 18 are each independently selected from H, nitro, C1-C6 alkyl (eg, methyl).
  • R 17 and R 18 are H.
  • R 20 , R 21 , R 22 , R 22 is R 20 and R 21 are each independently selected from H, nitro, C1-C6 alkyl (eg, methyl).
  • R 20 , R 21 , R 22 , R 22 is R 20 and R 21 are H.
  • R 23 , R 24 , R 25 , R 26 , R 26 is R 23 , R 24 , R 25 are each independently selected from H, nitro, C1-C6 alkyl (eg, methyl).
  • R 23 , R 24 , R 25 , R 26 , R 26 is R 23 , R 24 and R 25 are H.
  • R 27 and R 28 are each independently selected from H, nitro, C1-C6 alkyl (eg, methyl).
  • R 27 and R 28 are H.
  • R 30 , R 31 , R 32 , R 32 is R 30 , R 31 are each independently selected from H, nitro, C1-C6 alkyl (eg, methyl).
  • R 30 among R 30 , R 31 , R 32 , R 32 is R 30 and R 31 are H.
  • R 33 is selected from H, nitro, C1-C6 alkyl (eg methyl).
  • R 33 is H or methyl.
  • X is O.
  • any one of R 5 , R 6 , R 7 , R 8 , R 9 is selected from The remaining four are each independently selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), Aromatic alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • any one of R 5 , R 6 , R 7 , R 8 , R 9 is The remaining four are each independently selected from H, nitro, C1-C6 alkyl.
  • R 5 , R 6 , R 7 , R 8 , R 9 , R 5 or R 9 is The remaining four are each independently selected from H, nitro.
  • R 5 , R 6 , R 7 , R 8 , R 9 , R 5 or R 9 is The remaining four are each independently selected from H, nitro, methoxy, acetamido.
  • R 7 is selected from H, nitro, methoxy, acetamido, and the remaining three are H.
  • any one of R 10a , R 10b , R 10c is selected from -N 3 , -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-iso propyl, -SS-tert-butyl or -SS-isobutyl), -ONH 2 , -OCOR m , -OCONHR m , and the other two are independently selected from H, aliphatic alkyl (such as C1-C6 alkane) group, such as methyl, ethyl, isopropyl, tert-butyl), aromatic alkyl (such as phenyl C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), wherein each R m is independently selected from aliphatic alkyl (eg C1-C6 alkyl), cycloalkyl (eg C3-C6
  • any one of R 10a , R 10b , R 10c is -N 3 or -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-isopropyl group, -SS-tert-butyl or -SS-isobutyl), and the other two are independently selected from H, C1-C6 alkyl (such as methyl, ethyl, isopropyl, tert-butyl).
  • any one of R 10a , R 10b , R 10c is -N 3 or -SS-methyl, and the other two are each independently selected from H, methyl.
  • any one of R 10a , R 10b , R 10c is -N 3 , -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl, - SS-isobutyl, the other two are each independently selected from H, methyl.
  • R 11a , R 11b are each independently selected from H, aliphatic alkyl (eg, C1-C6 alkyl, specifically, methyl, ethyl, isopropyl, tert-butyl).
  • aliphatic alkyl eg, C1-C6 alkyl, specifically, methyl, ethyl, isopropyl, tert-butyl.
  • R 11a , R 11b are H.
  • R 12 is selected from -N 3 , -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl) , -ONH 2 , -OCOR m , -OCONHR m , wherein each R m is independently selected from aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl) or aromatic Alkyl (eg phenyl C1-C6 alkyl).
  • aliphatic alkyl such as C1-C6 alkyl
  • cycloalkyl such as C3-C6 cycloalkyl
  • aromatic Alkyl eg phenyl C1-C6 alkyl
  • R 12 is -N 3 or -SS-C1-C6 alkyl (eg, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl).
  • R 12 is -N 3 .
  • m 1 is 1.
  • m 2 is selected from 0, 1.
  • R' is a triphosphate group
  • Z is O.
  • Base is selected from
  • the present invention provides a compound represented by formula (IV) or a salt thereof,
  • A is selected from
  • the heteroaryl group is selected from the following:
  • each heteroaryl group is independently optionally substituted by 1 or more (such as 2, 3, 4, 5, 6, preferably 2) R";
  • Each R" is independently selected from H, -N3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic Family alkyl (such as phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (such as C1-C6 alkoxy),
  • R 5 , R 6 , R 7 , R 8 , R 9 , R x , R y , R z are each independently selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic alkyl (such as phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (such as C1- C6 alkoxy), and R 5 , R 6 , R 7 , R 8 and R 9 are not H at the same time;
  • R 10a , R 10b , R 10c , R 11a , R 11b , R 12 are each independently selected from H, -N 3 , -SS-C1-C6 alkyl (such as -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH 2 , -OCOR m , -OCONHR m , aliphatic alkyl (such as C1-C6 alkyl, specifically methyl, ethyl, isopropyl , tert-butyl), aromatic alkyl (such as phenyl C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), wherein each R m is independently selected from aliphatic alkyl (such as C1 -C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl) or aromatic al
  • X is selected from O, NH, S;
  • n 1 is selected from 1, 2, 3, 4, 5, 6;
  • n 2 is selected from 0, 1, 2, 3, 4, 5, 6;
  • R' is selected from H, a monophosphate group diphosphate group triphosphate group or tetraphosphate group
  • Each Z is independently selected from O, S, BH;
  • Base is selected from base, deaza base or its tautomer, for example Base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine or its tautomers.
  • A is selected from
  • A is selected from
  • A is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-phenyl
  • heteroaryl group is selected from the group consisting of:
  • each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • the heteroaryl group is selected from the group consisting of: Each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • the heteroaryl group is selected from the group consisting of: Each heteroaryl group is independently optionally substituted with 1 or more (eg 2, 3, 4, 5, 6, preferably 2) R".
  • each R" is independently selected from Preferably, R" is
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R"
  • one R" of the multiple R" selected from The remaining R" are each independently selected from -N3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic Alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R", one R" of the multiple R" for The remaining R" are each independently selected from nitro, aliphatic alkyl (eg, C1-C6 alkyl), F, I, Br, Cl.
  • multiple R" of the multiple R" for The remaining R" are each independently selected from nitro, aliphatic alkyl (eg, C1-C6 alkyl), F, I, Br, Cl.
  • each heteroaryl group is independently optionally substituted with multiple (eg, 2, 3, 4, 5, 6, preferably 2) R"
  • one R" of the multiple R" for The remaining R" are each independently selected from nitro, C1-C6 alkyl, preferably, the remaining R" is C1-C6 alkyl.
  • any one of R 5 , R 6 , R 7 , R 8 , R 9 is selected from The remaining four are each independently selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), Aromatic alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • any one of R 5 , R 6 , R 7 , R 8 , R 9 is The remaining four are each independently selected from H, nitro, C1-C6 alkyl.
  • R 5 , R 6 , R 7 , R 8 , R 9 , R 7 is The remaining four are H.
  • any one of Rx, Ry is selected from The other is selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • any one of Rx, Ry is The other is H.
  • R z is selected from
  • Rz is
  • any one of R 10a , R 10b , R 10c is selected from -N 3 , -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-iso propyl, -SS-tert-butyl), -ONH 2 , -OCOR m , -OCONHR m , and the other two are independently selected from H, aliphatic alkyl (such as C1-C6 alkyl, specifically methyl, ethyl, isopropyl, tert-butyl), aromatic alkyl (such as phenyl C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), wherein each R m is independently selected from aliphatic family of alkyl groups (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl) or aromatic al
  • any one of R 10a , R 10b , R 10c is -N 3 or -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-isopropyl group, -SS-tert-butyl), and the other two are independently selected from H, aliphatic alkyl (eg C1-C6 alkyl, specifically methyl, ethyl, isopropyl, tert-butyl).
  • R 11a , R 11b are each independently selected from H, aliphatic alkyl (eg, C1-C6 alkyl, specifically, methyl, ethyl, isopropyl, tert-butyl).
  • aliphatic alkyl eg, C1-C6 alkyl, specifically, methyl, ethyl, isopropyl, tert-butyl.
  • R 11a , R 11b are H.
  • R 12 is selected from -N 3 , -SS-C1-C6 alkyl (eg -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl) , -ONH 2 , -OCOR m , -OCONHR m , wherein each R m is independently selected from aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl) or aromatic Alkyl (eg phenyl C1-C6 alkyl).
  • aliphatic alkyl such as C1-C6 alkyl
  • cycloalkyl such as C3-C6 cycloalkyl
  • aromatic Alkyl eg phenyl C1-C6 alkyl
  • R 12 is -N 3 or -SS-C1-C6 alkyl (eg, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl).
  • R 12 is -N 3 .
  • X is O.
  • m 1 is 1.
  • m 2 is selected from 0, 1.
  • R' is a triphosphate group
  • Z is O.
  • Base is selected from
  • the present invention provides the following compounds or salts thereof,
  • the aforementioned compounds or salts thereof carry additional detectable labels (eg, fluorescent labels).
  • the additional detectable label carried by the compound or salt thereof is introduced by an affinity reagent (eg, antibody, aptamer, Affimer, Knottin) that carries the detectable label, And the affinity reagent can specifically recognize and bind to the epitope of the compound or its salt.
  • an affinity reagent eg, antibody, aptamer, Affimer, Knottin
  • the additional detectable label (eg, a fluorescent label) is optionally attached to the compound or salt thereof through a linking group.
  • the additional detectable label (eg, a fluorescent label) is optionally attached to the Base of the compound or salt thereof through a linking group.
  • the structure of Base is selected from the following preferably selected from
  • the linking group is a cleavable linking group or a non-cleavable linking group.
  • the cleavable linking group is selected from the group consisting of electrophilically cleavable linking groups, nucleophilic cleavable linking groups, photocleavable linking groups, cleavable linking groups under reducing conditions, oxidative A linking group that is cleaved under conditions, a safety handle-type linking group, a linking group that is cleaved by an elimination mechanism, or any combination thereof.
  • the linking group has the structure of formula (B):
  • R 36 , R 37 , R 38 , R 39 are each independently selected from H, -N 3 , N 3 -C1-C6 alkyl, nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), aromatic alkyl (such as phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (such as C1-C6 alkoxy base), the C1-C6 alkyl is optionally substituted by a C1-C6 alkyl;
  • p is selected from 1, 2, 3, 4, 5, 6;
  • q is selected from any integer between 1-12.
  • any one of R 36 , R 37 , R 38 , R 39 is N 3 -C1-C6 alkyl, and said C1-C6 alkyl is optionally replaced by C1-C6 alkyl substituted, and the rest are independently selected from H, -N 3 , nitro, amino, sulfo, carboxyl, aliphatic alkyl (such as C1-C6 alkyl), cycloalkyl (such as C3-C6 cycloalkyl), Aromatic alkyl (eg phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (eg C1-C6 alkoxy).
  • R 36 is N 3 -C1-C6 alkyl, and said C1-C6 alkyl is optionally substituted with C1-C6 alkyl, R 37 , R 38 , R 39 are each independently Selected from H, C1-C6 alkyl.
  • R 36 is N 3 -methyl, and the methyl group is optionally substituted with methyl, and R 37 , R 38 , R 39 are H.
  • p is selected from 1, 2, 3.
  • p is 1.
  • q is selected from 2, 3, 4, 5, 6.
  • q is 4.
  • the methyl-terminus of the linking group represented by formula (B) is attached to the Base, and the amino-terminus is attached to the additional detectable label (eg, a fluorescent label).
  • the additional detectable label e.g, a fluorescent label
  • the linking group has the structure of formula (C):
  • the alkynyl terminus of the linking group of formula (C) is attached to the Base and the amino terminus is attached to the additional detectable label (eg, a fluorescent label).
  • the additional detectable label e.g, a fluorescent label.
  • the linking group has the structure of formula (D):
  • the alkynyl terminus of the linking group represented by formula (D) is attached to the Base, and the amino terminus is attached to the additional detectable label (eg, a fluorescent label).
  • the additional detectable label e.g, a fluorescent label
  • the detectable label is selected from the group consisting of:
  • the Base is different and the detectable label (eg, fluorescent label) is different.
  • the detectable label eg, fluorescent label
  • the structure of the compound or salt thereof bearing an additional detectable label is shown below:
  • the structure of the compound or salt thereof bearing an additional detectable label is shown below:
  • the structure of the compound or salt thereof bearing an additional detectable label is shown below:
  • the structure of the compound or salt thereof bearing an additional detectable label is shown below:
  • the present invention provides a method for terminating nucleic acid synthesis, comprising: incorporating the aforementioned compound or a salt thereof into a nucleic acid molecule to be terminated.
  • incorporation of the compound or salt thereof is accomplished by a terminal transferase, terminal polymerase, or reverse transcriptase.
  • the method comprises incorporating the compound or salt thereof into the nucleic acid molecule to be terminated using a polymerase.
  • the method comprises: using a polymerase to polymerize nucleotides under conditions that allow the polymerase to polymerize the nucleotides, thereby incorporating the compound or salt thereof into the nucleic acid molecule to be terminated 3' end.
  • the present invention provides a method for preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising incorporating the aforementioned compound or a salt thereof into said growing complementary A polynucleotide wherein incorporation of the compound or salt thereof prevents the introduction of any subsequent nucleotides into the growing complementary polynucleotide.
  • incorporation of the compound or salt thereof is accomplished by a terminal transferase, terminal polymerase, or reverse transcriptase.
  • the method comprises incorporating the compound or salt thereof into the grown complementary polynucleotide using a polymerase.
  • the method comprises: using a polymerase to polymerize nucleotides under conditions that allow the polymerase to polymerize the nucleotides, thereby incorporating the compound or salt thereof into the growing complement 3' end of the polynucleotide.
  • the present invention provides a method for determining the sequence of a target single-stranded polynucleotide, comprising:
  • the additional detectable label (eg, a fluorescent label) is optionally attached to the compound or salt thereof through a linking group.
  • the linking group is as previously described.
  • the additional detectable label is as previously described.
  • the Base is different and the detectable label (eg, fluorescent label) carried by the compound or salt thereof is different.
  • the detectable label eg, fluorescent label
  • the reversible blocking group (R) and the detectable label are removed from the compound or salt thereof prior to introduction of the next complementary nucleotide.
  • the reversible blocking group and the detectable label are removed simultaneously.
  • the reversible blocking group and the detectable label are removed sequentially; eg, the reversible blocking group is removed after the detectable label is removed, or, After the reversible blocking group is removed, the detectable label is removed.
  • the method includes the steps of:
  • sequence of the target single-stranded polynucleotide is determined.
  • the method includes the steps of:
  • nucleotides (1) providing a first nucleotide, a second nucleotide, a third nucleotide and a fourth nucleotide, at least one of the four nucleotides is the aforementioned compound or a salt thereof, the four
  • the Bases contained in the nucleotides are different from each other, and the four nucleotides carry an additional detectable label (such as a fluorescent label), preferably, the additional detectable label carried by the four nucleotides is carried out by a parent.
  • the affinity reagents carry the detectable label, and the affinity reagents can specifically recognize and bind to the epitope of each nucleotide, Or preferably, the four nucleotides are optionally linked to the additional detectable label through a linking group, more preferably, the Base in the four nucleotides is optionally linked to the additional detectable label through a linking group.
  • the additional detectable labels are attached, most preferably, the additional detectable labels carried by the four nucleotides are different from each other;
  • the method includes the steps of:
  • the additional detectable label such as a fluorescent label
  • the additional detectable label carried by the compound or its salt is introduced by an affinity reagent (such as an antibody, aptamer, Affimer, Knottin), the affinity and the reagent carries the detectable label, and the affinity reagent can specifically recognize and bind to the epitope of the compound or its salt, or preferably, the compound or its salt is bound to the
  • the additional detectable label is attached, or more preferably, the Base in the compound or a salt thereof is optionally attached to the additional detectable label through a linking group;
  • the cleavage of the reversible blocking group and the cleavage of the detectable label are performed simultaneously, or alternatively, the cleavage of the reversible blocking group and the cleavage of the detectable label are separated (eg, the reversible blocking group is cleaved first, or the detectable label is cleaved first).
  • the cleavage reagent used for cleavage of the reversible blocking group and cleavage of the detectable label is the same reagent.
  • the cleavage reagents used for cleavage of the reversible blocking group and cleavage of the detectable label are different reagents.
  • the duplex is attached to a support.
  • the growing nucleic acid strand is a primer.
  • the primers form the duplex by annealing to the nucleic acid strand to be sequenced.
  • the duplex, the compound or salt thereof, and the polymerase together form a reaction system comprising a solution phase and a solid phase.
  • the compounds or salts thereof contain different Bases.
  • the additional detectable labels carried by the compounds or salts thereof are different from each other.
  • the compound or salt thereof is incorporated into a growing nucleic acid strand using a polymerase under conditions that allow the polymerase to polymerize nucleotides to form a reversible blocking group comprising a detectable label Nucleic acid intermediates.
  • the polymerase is selected from KOD polymerases or mutants thereof (eg, KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391).
  • the solution phase of the reaction system of the previous step is removed, and the duplex attached to the support is retained.
  • the excision reagent is contacted with the duplex or the growing nucleic acid strand in a reaction system comprising a solution phase and a solid phase.
  • the cleavage reagent is capable of cleaving a reversible blocking group in a compound incorporated into a growing nucleic acid strand and an additional detectable label carried by the compound without affecting the phosphates on the duplex backbone diester bond.
  • the solution phase of the reaction system for this step is removed.
  • a washing operation is performed.
  • step (ii) after step (ii), it further comprises: according to the signal detected in step (ii), determining the type of compound incorporated into the growing nucleic acid strand in step (i), and based on the principle of complementary base pairing, Determine the type of nucleotide at the corresponding position in the nucleic acid strand to be sequenced.
  • the present invention provides a kit comprising at least one of the aforementioned compounds or salts thereof.
  • the kit comprises first, second, third and fourth compounds, each of which is independently the aforementioned compound or a salt thereof.
  • Base in the first compound, Base is selected from adenine, 7-deazaadenine or a tautomer thereof (eg ); in the second compound, Base is selected from thymine, uracil or its tautomer (for example ); in the third compound, Base is selected from cytosine or its tautomer (for example ); in the fourth compound, Base is selected from guanine, 7-deazaguanine or its tautomer (for example ).
  • the first, second, third and fourth compounds carry additional detectable labels.
  • the additional detectable labels carried by the first, second, third and fourth compounds are introduced by an affinity reagent (eg, an antibody, aptamer, Affimer, Knottin) that The reagent carries the detectable label, and the affinity reagent can specifically recognize and bind to the epitope of the first, second, third or fourth compound.
  • an affinity reagent eg, an antibody, aptamer, Affimer, Knottin
  • the first, second, third and fourth compounds are optionally linked to the additional detectable label through a linking group.
  • the Base in the first, second, third and fourth compounds is optionally linked to the additional detectable label through a linking group.
  • the first, second, third and fourth compounds comprise Bases that are different from each other.
  • the additional detectable labels carried by the first, second, third and fourth compounds are different from each other.
  • the linking group is as previously described.
  • the detectable label is as previously described.
  • the kit further comprises: reagents for pretreating nucleic acid molecules; a support for ligating nucleic acid molecules to be sequenced; valence or non-covalent linkage) reagents; primers for initiating nucleotide polymerization; polymerases for carrying out nucleotide polymerization; one or more buffer solutions; one or more washing solutions; or any combination thereof.
  • the present invention provides the use of the aforementioned compound or a salt thereof or the aforementioned kit for determining the sequence of a target single-stranded polynucleotide.
  • Fig. 1 represents the excision reaction of the azidomethyl group of the embodiment of the present invention
  • Fig. 2 shows that the nucleophilic cyclization tandem reaction according to the embodiment of the present invention is used for reversible blocking excision
  • Figure 3 shows the 3'-OH-labeled reversible blocking nucleotide analogs of the examples of the present invention.
  • C1-C6 alkyl specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl groups.
  • aliphatic alkyl refers to any straight or branched chain saturated group containing 1-20 carbon atoms, eg, C1-C12 alkyl, preferably C1-C6 alkyl.
  • C1-C6 alkyl refers to any straight or branched chain saturated group containing 1-6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isopropyl Butyl, tert-butyl, sec-butyl, n-pentyl, tert-amyl, n-hexyl, etc.
  • alkoxy refers to any of the foregoing alkyl groups (eg, C1-C6 alkyl groups, etc.), which are attached to the remainder of the molecule through an oxygen atom (-O-).
  • cycloalkyl refers to a hydrocarbon having a 3-10 membered monocyclic ring system with saturated rings, eg, C3-C8 cycloalkyl, preferably C3-C6 cycloalkyl.
  • C3-C6 cycloalkyl refers to a hydrocarbon having a 3-6 membered monocyclic ring system with a saturated ring, and the C3-C6 cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • aromatic alkyl refers to arylalkyl or heteroarylalkyl. wherein alkyl is as defined above.
  • heteroaryl refers to an aromatic heterocycle, typically a 5-, 6-, 7-, 8-membered heterocycle having 1 to 3 heteroatoms selected from N, O or S; heteroaryl
  • the base ring can optionally be further fused or attached to aromatic and non-aromatic carbocyclic and heterocyclic rings.
  • Non-limiting examples of such heteroaryl groups are eg pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thioxazolyl, pyrrolyl, benzene yl-pyrrolyl, furyl, phenyl-furyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzofuranyl, benzothienyl, benzol,3-dioxolane (benzodioxin), isoindoline, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, 1,2,3-triazolyl, 1-phenyl-1, 2,3-triazolyl, 2,3-indoline, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenz
  • each heteroaryl group may be the same or different.
  • each heteroaryl group is independently optionally substituted with 1 or more (such as 2, 3, 4, 5, 6, preferably 2) R", it means that the substitution on different heteroaryl groups
  • the groups R" may be the same or different, and the multiple substituents R" on the same heteroaryl group may be the same or different.
  • each R is independently selected from H, -N3 , etc., and is not H at the same time, which means that if the heteroaryl group is composed of 1 or more (such as 2, 3, 4, 5, 6, preferably 2) R" is substituted, then at least one substituent R" is not H.
  • aryl refers to a group of a carbocyclic aromatic system having 6-14 carbon atoms, such as C6-C10 aryl, preferably phenyl.
  • any group whose name is a compound name, such as "phenyl C1-C6 alkyl”, should refer to the moiety from which it is conventionally derived, such as from benzene C1-C6 alkyl group substituted with C1-C6 alkyl group, wherein C1-C6 alkyl group is as defined above.
  • an example of the term "salt of a compound of formula (A), formula (I), formula (II), formula (III) or formula (IV)” is an organic acid formed from an anion-forming organic acid Acid addition salts, including but not limited to formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, ⁇ -ketoglutarate, ⁇ -glycerophosphate, alkylsulfonate or arylsulfonate; preferably, the alkylsulfonate is methylsulfonate or ethylsulfonate; the The aryl sulfonate is benzene sulfonate or p-toluene sulfonate.
  • Suitable inorganic salts may also be formed including, but not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate and carbonate, s
  • R can be selected from in, Indicates a single bond - or double bond
  • R 2 , R 3 , and R 4 are each independently selected from O, NH, S, CH, CH 2 , C(CH 3 ) 2 . It can be understood that between R 2 and R 3 , only when R 2 and R 3 are both CH, and between R 2 and R 3 is a double bond, when R 2 and R 3 are independently selected from other than CH When the remaining atoms or atomic groups (ie, O, NH, S, CH 2 , C(CH 3 ) 2 ) are present, there is a single bond between R 2 and R 3 .
  • R 2 , R 3 and R 4 are each independently selected from CH and CH 2 ", which means: R 2 and R 3 are both CH, and between R 2 and R 3 is a double bond, and R 4 is CH 2 , and between R 3 and R 4 is a single bond; alternatively, both R 3 and R 4 are CH, and between R 3 and R 4 is a double bond, R 2 is CH 2 , and one of R 2 and R 3 is a single key.
  • single or double bonds between Y and R2, or between R3 and R4 can be understood with reference to the foregoing.
  • R36 is N3 - C1-C6 alkyl optionally substituted by C1-C6 alkyl
  • R36 is N3 - C1-C6 alkane group
  • the H on the C1-C6 alkyl group may also be substituted by another C1-C6 alkyl group.
  • R 36 is N 3 -methyl, and the methyl group is optionally substituted by methyl
  • the structure of the compound represented by formula (B) can be can also be Other similar descriptions can be understood with reference to the foregoing.
  • the substance is composed of the two chains of the growing nucleic acid chain and the nucleic acid chain to be sequenced, it is called a "duplex", and it is the same as the growing nucleic acid chain or the nucleic acid chain to be sequenced. Regardless of chain length, the nucleic acid chain to be sequenced can be longer than the chain length of the growing nucleic acid chain.
  • the nucleic acid molecule to be sequenced can be any nucleic acid molecule of interest.
  • the nucleic acid molecule to be sequenced comprises deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or any combination thereof.
  • the nucleic acid molecule to be sequenced is not limited by its type.
  • the nucleic acid molecule to be sequenced is DNA or RNA.
  • the nucleic acid molecule to be sequenced may be genomic DNA, mitochondrial DNA, chloroplast DNA, mRNA, cDNA, miRNA, or siRNA.
  • the nucleic acid molecule to be sequenced is linear or circular. In certain preferred embodiments, the nucleic acid molecule to be sequenced is double-stranded or single-stranded.
  • the nucleic acid molecule to be sequenced can be single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or a hybrid of DNA and RNA.
  • the nucleic acid molecule to be sequenced is single-stranded DNA. In certain preferred embodiments, the nucleic acid molecule to be sequenced is double-stranded DNA.
  • the nucleic acid molecule to be sequenced is not limited by its origin.
  • the nucleic acid molecule to be sequenced can be obtained from any source, eg, any cell, tissue, or organism (eg, viruses, bacteria, fungi, plants, and animals).
  • the nucleic acid molecules to be sequenced are derived from mammals (eg, humans, non-human primates, rodents, or canines), plants, birds, reptiles, fish, Fungi, bacteria or viruses.
  • nucleic acid molecules from cells, tissues or organisms are well known to those skilled in the art. Suitable methods include, but are not limited to, ethanol precipitation, chloroform extraction, and the like. Detailed descriptions of such methods can be found, for example, in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., An In-depth Molecular Biology Laboratory Guide , 3rd Edition, John Wiley & Sons, Inc., 1995. In addition, various commercial kits can be used to extract nucleic acid molecules from various sources (eg, cells, tissues, or organisms).
  • the nucleic acid molecule to be sequenced is not limited by its length.
  • the length of the nucleic acid molecule to be sequenced can be at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1000 bp in length , or at least 2000bp.
  • the length of the nucleic acid molecule to be sequenced can be 10-20bp, 20-30bp, 30-40bp, 40-50bp, 50-100bp, 100-200bp, 200-300bp, 300-400bp, 400-500bp, 500-1000bp, 1000-2000bp, or more than 2000bp.
  • nucleic acid molecules to be sequenced may have a length of 10-1000 bp to facilitate high-throughput sequencing.
  • a suitable polymerase can be used to carry out the nucleotide polymerization reaction.
  • the polymerase is capable of synthesizing new DNA strands using DNA as a template (eg, a DNA polymerase).
  • the polymerase is capable of synthesizing new DNA strands using RNA as a template (eg, reverse transcriptase).
  • the polymerase is capable of synthesizing new RNA strands using DNA or RNA as a template (eg, RNA polymerase).
  • the polymerase is selected from the group consisting of DNA polymerases, RNA polymerases, and reverse transcriptases. According to actual needs, a suitable polymerase can be selected to carry out the nucleotide polymerization reaction.
  • the polymerization reaction is a polymerase chain reaction (PCR). In certain preferred embodiments, the polymerization reaction is a reverse transcription reaction.
  • KOD polymerase or a mutant thereof can be used to carry out nucleotide polymerization.
  • KOD polymerase or mutants thereof eg, KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391
  • KOD POL391 and KOD POL171 have acceptable polymerization efficiencies for the modified nucleotides of the present invention.
  • the polymerization efficiency of KOD POL391 or KOD POL171 for the modified nucleotides of the invention is above 70%, eg, 70%-80%, 80%-90%, or 90%-100%.
  • the polymerization reaction of nucleotides is carried out under suitable conditions.
  • suitable polymerization conditions include the composition of the solution phase and the concentration of each component, the pH of the solution phase, the polymerization temperature, and the like. Carrying out the polymerization under suitable conditions is beneficial to obtain acceptable, even high, polymerization efficiencies.
  • the hydroxyl group (-OH) at the 3' position of the deoxyribose sugar of the compound represented by the formula (A) is protected (protected by R), so that they can terminate polymerases (such as DNA polymerases) ) polymerization.
  • polymerases such as DNA polymerases
  • the protective group (R) of the hydroxyl group (-OH) at the 3' position of the deoxyribose sugar of the compound represented by the formula (A) can be removed and converted into a free hydroxyl group (-OH).
  • the growing nucleic acid chain can be subjected to the next round of polymerization using a polymerase and a compound of formula (A), and a base is introduced again.
  • the hydroxyl group (-OH) at the 3' position of the deoxyribose sugar of the compound of formula (A) is reversibly blocked: when the compound of formula (A) is incorporated into the 3' end of a growing nucleic acid chain, they The polymerase will continue to carry out polymerization, and the further extension of the growing nucleic acid chain will be terminated; and, after the blocking group contained in the compound represented by formula (A) is removed, the polymerase will be able to continue the growing nucleic acid chain. Polymerization continues to extend the nucleic acid chain.
  • Detection can be performed by any suitable method, including fluorescence spectroscopy or other optical means.
  • Preferred labels are fluorescent labels or fluorophores that emit radiation of defined wavelengths after absorbing energy.
  • Many suitable fluorescent labels are known.
  • Welch et al. (Chem. Eur. J. 5(3):951-960, 1999) disclose dansyl-functionalized fluorescent moieties, which can be used in the present invention.
  • Zhu et al. (Cytometry 28:206-211, 1997) describe the use of fluorescent labels Cy3 and Cy5, which can also be used in the present invention.
  • Prober et al. Science 238:336-341, 1987
  • fluorescent labels include, but are not limited to, fluorescein, rhodamine (including TMR, Texas Red, and Rox), alexa, fluoroboron, acridine, coumarin, pyrene, benzanthracene, and Anthocyanins.
  • multiple labels can also be used in this application, such as a dual-fluorophore FRET cassette (Tet. Let. 46:8867-8871, 2000), a multi-fluorophore dendrimer system (J.Am.Chem.Soc.123:8101) -8108, 2001).
  • fluorescent labels are preferred, other forms of detectable labels will be apparent to those of ordinary skill in the art.
  • microparticles including quantum dots (Empodocles et al., Nature 399:126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72:6025-6029, 2000) and microbeads (Lacoste et al., Proc.Natl.Acad.Sci USA 97(17):9461-9466, 2000) can also be used.
  • Multicomponent labels can also be used in this application.
  • Multicomponent labels are labels that rely on interaction with additional compounds for detection.
  • the most commonly used multicomponent labeling in biology is the biotin-streptavidin system. Biotin is used as a label attached to nucleotides or modified nucleotides. Streptavidin alone was then added to allow detection to occur.
  • Other multi-component systems can be used. For example, dinitrophenol has commercially available fluorescent antibodies for detection.
  • the modified nucleotide or nucleoside molecule can be made to carry the detectable label described above by the introduction of an affinity reagent (eg, antibody, aptamer, Affimer, Knottin), which and reagents can specifically recognize and bind to the epitope of the modified nucleotide or nucleoside molecule.
  • an affinity reagent eg, antibody, aptamer, Affimer, Knottin
  • modified nucleotide or nucleoside molecules can be linked to the detectable labels described above.
  • the linking group used is cleavable. The use of a cleavable linking group ensures that the label can be removed after detection if desired, which avoids any interfering signals with any subsequently incorporated labeled nucleotides or nucleosides.
  • the linking group used is not cleavable. Since, in each instance where a labeled nucleotide of the invention is incorporated, subsequent incorporation of the nucleotide is not required, there is no need to remove the label from the nucleotide.
  • modified nucleosides and nucleotides of the present application also have utility in high-throughput sequencing, especially in high-throughput sequencing platforms based on sequencing by synthesis, using the reversible blocking nucleosides of the present application Acids and their analogs can be detected individually during the sequencing process for the types of nucleotides incorporated.
  • Cleavable linking groups are well known in the art, and conventional chemistry can be used to attach linking groups to nucleotides or modified nucleotides and labels.
  • the linking group can be cleaved by any suitable method, including exposure to acids, bases, nucleophiles, electrophiles, free radicals, metals, reducing or oxidizing agents, light, temperature, enzymes, and the like.
  • the linking groups discussed herein can also be cleaved using the same catalysts used to cleave the 3'-O-protecting group bond.
  • Suitable linking groups can be modified from standard chemical protecting groups as disclosed in Greene & Wuts, Protective Groups in Organic Synthesis (Protective Groups in Organic Synthesis), John Wiley & Sons.
  • Suitable cleavable linking groups for solid phase synthesis are also disclosed in Guillier et al. (Chem. Rev. 100:2092-2157, 2000).
  • nucleoside cleavage site can be located at a position on the linking group that ensures that a portion of the linking group remains attached to the nucleoside after cleavage Acids or modified nucleotides remain attached.
  • the linking group can be attached anywhere on the nucleotide or modified nucleotide as long as Watson-Crick base pairing is still possible.
  • Electrophilically cleavable linking groups are typically cleaved by protons and include acid-sensitive cleavage.
  • Suitable linking groups include modified benzyl systems such as trityl, p-alkoxybenzyl esters, and p-alkoxybenzyl amides.
  • Other suitable linking groups include tert-butoxycarbonyl (Boc) groups and acetal systems.
  • thiophilic metals such as nickel, silver or mercury in the cleavage of thioacetals or other sulfur-containing protecting groups can also be considered for the preparation of suitable linking molecules.
  • Nucleophilic cleavage is also an accepted method in the preparation of linker molecules.
  • Groups that are labile in water ie, capable of simple cleavage at basic pH values
  • esters can be used, as well as groups that are labile to non-aqueous nucleophiles.
  • Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS).
  • Photolyzable linking groups are widely used in sugar chemistry.
  • the light required to activate cleavage does not affect other components in the modified nucleotide.
  • a fluorophore is used as the label, preferably the fluorophore absorbs light of a different wavelength than the light required to cleave the linker molecule.
  • Suitable linking groups include those based on O-nitrobenzyl compounds and nitroveratrol compounds. Linking groups based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64:3454-3460, 1999).
  • linking groups that are sensitive to reductive cleavage are known.
  • Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups.
  • Disulfide bond reduction is also known in the art.
  • Oxidation-based methods are well known in the art. These methods include oxidation of alkoxybenzyl groups and oxidation of sulfur and selenium linking groups. It is also within the scope of this invention to use aqueous iodine to cleave disulfides and other sulfur- or selenium-based linking groups.
  • Safety-catch linkers are those that are cleaved in two steps.
  • the first step is the creation of a reactive nucleophilic center, followed by a second step involving intramolecular cyclization, which results in cleavage.
  • the levulinate linkage can be treated with hydrazine or photochemically to release the active amine, which is then cyclized to cleave the ester elsewhere in the molecule (Burgess et al., J. Org. Chem. 62:5165 -5168, 1997).
  • Elimination reactions can also be used.
  • Base-catalyzed elimination of groups such as fluorenemethoxycarbonyl and cyanoethyl as well as palladium-catalyzed reductive elimination of the allyl system can be used.
  • the linking group may comprise a spacer unit.
  • the length of the linking group is not critical as long as the label is kept a sufficient distance from the nucleotides so as not to interfere with the interaction between the nucleotides and the enzyme.
  • the linking group may consist of a similar function to the 3'-OH protecting group. This makes the deprotection and deprotection process more efficient as only a single treatment is required to remove the label and protecting groups.
  • Particularly preferred linking groups are azide-containing linking groups that are cleavable via a phosphine.
  • the target product structure is
  • step 1 a dry 100 mL round-bottomed flask equipped with a magnet and a rubber stopper with an argon balloon, and weigh the product obtained in step 1 (750 mg, 1.4 mmol, 1 eq) into the round-bottomed flask.
  • 20 mL of anhydrous THF was drawn from a syringe as a solvent, and after stirring to dissolve it, a 1 mol/L TBAF solution (1.8 mL, 1.8 mmol, 1.3 eq) was drawn from a syringe, and slowly added dropwise to the reaction solution under ice bath conditions. The reaction flask was moved to room temperature and stirred for about 2 h.
  • the target product structure is
  • step 1 850 mg, 1.54 mmol, 1 eq
  • 20 mL of anhydrous THF was drawn from a syringe as a solvent, and after stirring at room temperature to dissolve it, a 1 mol/L TBAF solution (2.0 mL, 2.0 mmol, 1.3 eq) was drawn from a syringe, and slowly added dropwise to the reaction solution under ice bath conditions.
  • the reaction flask was moved to room temperature and stirred for about 2 h.
  • reaction was monitored every half hour by TLC until the starting point was substantially disappeared.
  • the reaction yield of this step is 70%.
  • the reaction yield of this step is 45%.
  • the target product structure is
  • step 1 Prepare a dry 100 mL round-bottomed flask equipped with a magnet and a rubber stopper with an argon balloon, and weigh the product obtained in step 1 (860 mg, 1.54 mmol, 1 eq) into the round-bottomed flask.
  • 20 mL of anhydrous THF was drawn from a syringe as a solvent, and after stirring to dissolve, a 1 mol/L TBAF solution (2.0 mL, 2.0 mmol, 1.3 eq) was drawn from a syringe, and slowly added dropwise to the reaction solution under ice bath conditions.
  • the reaction flask was moved to room temperature and stirred for about 2 h. The reaction was monitored every half hour by TLC until the starting point had essentially disappeared.
  • the reaction yield of this step is 40%.
  • the reaction yield of this step is 90%.
  • the target product structure is
  • step 1 Prepare a dry 100 mL round-bottomed flask equipped with a magnet and a rubber stopper with an argon balloon, and weigh the product obtained in step 1 (828 mg, 1.54 mmol, 1 eq) into the round-bottomed flask.
  • 20 mL of anhydrous THF was drawn from a syringe as a solvent, and after stirring to dissolve, a 1 mol/L TBAF solution (2.0 mL, 2.0 mmol, 1.3 eq) was drawn from a syringe, and slowly added dropwise to the reaction solution under ice bath conditions.
  • the reaction flask was moved to room temperature and stirred for about 2 h. The reaction was monitored every half hour by TLC until the starting point was substantially disappeared.
  • the reaction yield of this step is 40%.
  • the target product structure is
  • step 1 Prepare a dry 100 mL round-bottomed flask equipped with a magnet and a rubber stopper with an argon balloon, and weigh the product obtained in step 1 (690 mg, 1.19 mmol, 1 eq) into the round-bottomed flask.
  • 20 mL of anhydrous THF was drawn from a syringe as solvent, and after stirring at room temperature to dissolve it, a 1 mol/L TBAF solution (1.6 mL, 1.6 mmol, 1.3 eq) was drawn from a syringe, and slowly added dropwise to the reaction solution under ice bath conditions. The reaction flask was moved to room temperature and stirred for about 2 h.
  • the reaction yield of this step is 40%.
  • the target product structure is
  • step 1 Prepare a dry 100 mL round-bottomed flask equipped with a magnet and a rubber stopper with an argon balloon, and weigh the product obtained in step 1 (932 mg, 1.54 mmol, 1 eq) into the round-bottomed flask.
  • 20 mL of anhydrous THF was drawn from a syringe as a solvent, and after stirring to dissolve it, a 1 mol/L TBAF solution (2.0 mL, 2.0 mmol, 1.3 eq) was drawn from a syringe, and slowly added dropwise to the reaction solution under ice bath conditions.
  • the reaction flask was moved to room temperature and stirred for about 2 h. The reaction was monitored every half hour by TLC until the starting point was substantially disappeared.
  • the reaction yield of this step is 40%.
  • the target product structure is
  • step 1 Prepare a dry 100 mL round-bottomed flask equipped with a magnet and a rubber stopper with an argon balloon, and weigh the product obtained in step 1 (938 mg, 1.54 mmol, 1 eq) into the round-bottomed flask.
  • 20 mL of anhydrous THF was drawn from a syringe as a solvent, and after stirring to dissolve, a 1 mol/L TBAF solution (2.0 mL, 2.0 mmol, 1.3 eq) was drawn from a syringe, and slowly added dropwise to the reaction solution under ice bath conditions.
  • the reaction flask was moved to room temperature and stirred for about 2 h. The reaction was monitored every half hour by TLC until the starting point had essentially disappeared.
  • the target product structure is
  • step 1 Prepare a dry 100 mL round-bottomed flask equipped with a magnet and a rubber stopper with an argon balloon, and weigh the product obtained in step 1 (907 mg, 1.54 mmol, 1 eq) into the round-bottomed flask.
  • 20 mL of anhydrous THF was drawn from a syringe as a solvent, and after stirring to dissolve, a 1 mol/L TBAF solution (2.0 mL, 2.0 mmol, 1.3 eq) was drawn from a syringe, and slowly added dropwise to the reaction solution under ice bath conditions.
  • the reaction flask was moved to room temperature and stirred for about 2 h. The reaction was monitored every half hour by TLC until the starting point had essentially disappeared.
  • the reaction yield of this step is 40%.
  • the inventors performed evaluation tests on the nucleotide analogs prepared in the aforementioned preparation examples on a high-throughput sequencer.
  • Nucleotide substrates fluorescently labeled standard hot dNTPs (four types) and standard cold dNTPs (four types), with the following structures, both from MGISEQ-2000RS high-throughput sequencing reagent kit (FCL SE50), Shenzhen MGI Technology Co., Ltd., Item No. 1000012551; nucleotide analogs of the present invention cold dNTPs (including dTTP, dATP, dCTP, dGTP, only one type is used for each test, for convenience, for each modification of 3'-OH , the following table 1 only shows the test results of dTTP).
  • the first round of on-machine test polymerize standard hot dNTP, take pictures to record the signal value, and then use thpp reagent to excise the blocking group, 65°C for 1min.
  • the second round of on-board test polymerize standard cold dNTP, then polymerize standard hot dNTP, take pictures to record the signal value, and then use thpp reagent to excise the blocking group, 65°C for 1 min.
  • the fourth round of on-machine testing polymerize the nucleotide analogs of the present invention cold dNTP (only one cold dNTP is polymerized for each test), then polymerize standard hot dNTP, and take pictures to record the signal value. Then the blocking group was excised with thpp reagent, 65°C for 1 min.
  • the fifth round of on-board testing aggregate standard hot dNTP, and take pictures to record the signal value.
  • EI incorporation efficiency
  • C1 is the signal value of the first round of on-board testing
  • C2 is the signal value of the second round of on-board testing
  • C3 is the signal value of the third round of on-board test
  • C4 is the signal value of the fourth round of on-board test
  • Ec is the ratio of the excision efficiency between the test nucleotide and the comparison nucleotide
  • EI is the ratio of the polymerization efficiency of the test nucleotide to the comparison nucleotide
  • C3 is the signal value of the third round of on-board test
  • C5 is the signal value of the fifth round on-board test
  • CGT is the signal of C base, G base and T base in the third round.
  • Nucleotide substrate fluorescently labeled standard hot dNTPs (four kinds, from MGISEQ-2000RS high-throughput sequencing reagent kit (FCL SE50), Shenzhen Huada Zhizao Technology Co., Ltd., Item No. 1000012551); the nucleotides of the present invention
  • the analog cold dNTPs (four, designated AEB), have the following structures.
  • Nucleotide substrate fluorescently labeled standard hot dNTPs (four kinds, from MGISEQ-2000RS high-throughput sequencing reagent kit (FCL SE50), Shenzhen Huada Zhizao Technology Co., Ltd., Item No. 1000012551); the nucleotides of the present invention
  • the analog cold dNTPs (four, designated SSEB), have the following structures.
  • Nucleotide substrate fluorescently labeled standard hot dNTPs (four kinds, from MGISEQ-2000RS high-throughput sequencing reagent kit (FCL SE50), Shenzhen Huada Zhizao Technology Co., Ltd., Item No. 1000012551); the nucleotides of the present invention
  • the analog cold dNTPs (four, designated AZBN), have the following structures.
  • Iodonucleoside substrate T (OKeanos Tech, product number OK-N-16001) 1g was dissolved in DMF, added Pd(PPh3)4 (10mol%), CuI (15mol%), triethylamine (3eq) and the substrate Propargylamine (OKeanos Tech, product number OK20A410) (1.5eq) was reacted at 60 degrees for 12h. The reaction was quenched by addition of water and extracted with DCM. After concentration, 1.1 g of white solid product was obtained by column separation.
  • step 1 300 mg of the nucleoside obtained in step 1 was dissolved in 10 mL of DMF, DCC (1.2eq) and DMAP (10% mol) were added, and after the reaction was stirred for 30 minutes, the dithiocarboxylic acid substrate (OKeanos Tech, product number OK20A420) (1.5 eq). After the reaction was stirred for 12 hours, 359 mg of white solid was obtained by direct column separation.
  • step 2 300 mg of the nucleosides obtained in step 2 were dissolved in 10 mL of THF, TBAF (2eq, 1M in THF) was added at 0 degrees, the reaction was stirred at 0 degrees for 30 minutes, and then raised to room temperature and stirred for 4 hours. The reaction was directly column-separated to obtain 200 mg of white solid.
  • the reaction was separated on a preparative HPLC reversed-phase column (C18, mobile phase: 0.1M TEAB-acetonitrile), concentrated and directly added with 3 mL of concentrated ammonia water, and reacted for 2 h using a preparative HPLC reversed-phase column (C18, mobile phase: 0.1M TEAB-acetonitrile) 120 mg of white solid was isolated.
  • the hot T-mSSEB substrate solid (15 mg, 0.02 mmol, 1 eq.) was added to the above reaction system, and the reaction was stirred at room temperature. After 1 hour a sample was taken in acetonitrile and the progress of the reaction was monitored by MS and HPLC. The reaction was carried out overnight to complete consumption of the nhs ester. Quench by adding 0.1M TEAB buffer.
  • Nucleotide substrate fluorescently labeled standard hot dNTP (from MGISEQ-2000RS high-throughput sequencing reagent kit (FCL SE50), Shenzhen Huada Zhizao Technology Co., Ltd., item number 1000012551), wherein hot dTTP is obtained by using the aforementioned preparation Replaced by hot T-SSEB, the structure is as follows; the nucleotide analogs of the present invention cold dNTP (four kinds, named SSEB), the structure is as follows.
  • the sequencing process is the same as the above-mentioned sequencing example 1, and the Basecall analysis results are shown in Table 5.
  • the synthesis method was similar to that of Sequencing Example 4. Among them, the iodo G nucleoside substrate was used to replace the iodo T nucleoside substrate, and the azide carboxylic acid substrate was used to replace the dithiocarboxylic acid substrate, all provided by OKeanos Tech. The dye-linker solid needed to be replaced and was supplied by MyChem LLC (Cat. No. 110920Cy5). 10 mg of final product was obtained.
  • Nucleotide substrate fluorescently labeled standard hot dNTP (from MGISEQ-2000RS high-throughput sequencing reagent kit (FCL SE50), Shenzhen MGI Technology Co., Ltd., Item No. 1000012551), wherein hot dGTP was prepared using the SS-hot G is substituted; the nucleotide analogs of the present invention are cold dNTPs (four kinds, named as SS-cold), and the structure is as follows.

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Abstract

一种修饰的核苷或核苷酸，所述修饰的核苷或核苷酸的3'-OH被可逆阻断；同时还涉及包含所述核苷或核苷酸的试剂盒，以及基于所述核苷或核苷酸的测序方法。

Description

修饰的核苷或核苷酸 技术领域
本发明涉及核酸测序领域。具体地，本发明涉及修饰的核苷或核苷酸，所述修饰的核苷或核苷酸的3'-OH被可逆阻断。
背景技术
NGS测序的出现克服了Sanger测序成本高、测序时间长等缺点，极大地推动了基因测序技术的应用。目前，NGS测序已经在产前筛查、肿瘤诊断、肿瘤治疗、动植物育种等领域深度应用，带动了科技与医学的进步。
带有可逆阻断基团的核苷三磷酸(dNTP)类似物是NGS测序中关键原材料。由于可逆阻断基团的引入，使得dNTP中3’-OH基团可以保留，克服了Sanger测序中的缺点，同时保证了碱基识别的准确性。可以说带有可逆阻断基团的核苷三磷酸类似物(dNTP)是NGS测序中最为关键的技术。
目前已经有众多的带有可逆阻断基团的dNTP化合物被报道。实现dNTP可逆阻断主要通过两大类思路来实现。第一类思路是在dNTP的3’-OH直接引入可逆阻断基团，这类修饰的dNTP具有的优势是3’-OH的阻断保证了测序中的阻断效率。另一类思路是3’-OH不阻断，而是靠碱基的修饰进行聚合酶的阻断，这类策略的优势在阻断基团的修饰选择性更广，不受限于聚合酶。
总的来说，两类可逆阻断思路各有优缺点，但是直接在3’-OH引入基团阻断的方法更为可靠，其阻断效率明显更高。因此在目前已经商品化的NGS测序中，主要使用的这种方案。
发明内容
叠氮甲基是一类高效的3’-OH可逆阻断基团，它具有稳定性好、切除条件温和、切除速度快等巨大优势。仔细分析3’-OH叠氮甲基切除反应，其实质是一个串联反应(cascade reaction)。叠氮基团在磷试剂的作用下发生Staudinger反应后生成了中间体带有3’-OH亚甲胺基团，随后其发生快速的水解反应释放出3’-OH基团。在这个串联反应中，Staudinger反应与水解反应都十分迅速，所以叠氮甲基的切除速度极快(如图1所示)。
叠氮基团是一类特殊的化学基团，其化学结构中三个氮原子形成共轭结构，处在一个平面。但是同时三个氮原子并未形成一条直线，而是形成了具有一定角度的折线结构。叠氮基团中三个氮原子形成的共轭结构有多个共振式，有利于其结构的稳定。
总的来说，叠氮基团具有一定的稳定性。但是由于三个氮原子的叠加结构，在合适的反应条件下，叠氮基团很容易发生反应并快速释放出氮气，因此叠氮基团具有很活泼的反应性与爆炸性风险。正是基于叠氮基团这个特点，它被广泛的应用于各种Click reaction中。
基于叠氮基团快速反应的特点，发明人设计了一类带有叠氮基团，可以发生串联反应的3’-OH阻断基团。这类3’-OH阻断基团利用Staudinger反应释放出氨基后，氨基对3’-OH基团的酯基/碳酸酯保护基进行进攻释放出3’-OH(如图2所示)。以此类推，在合适条件下可以释放出亲核性原子的基团均适用于本设计，例如-S-SR基团，-OCOR,-OCONHR等。
本发明旨在开发一类在3’-OH带有酯基、碳酸酯dNTP类似物用于NGS测序。这类dNTP类似物的结构通式如图3所示，可逆阻断基团保护3’-OH，包 括2’脱氧尿苷三磷酸、2’脱氧胸腺苷三磷酸、2’脱氧胞苷三磷酸、2’脱氧腺苷三磷酸、7-脱氮-2’脱氧腺苷三磷酸、2’脱氧鸟苷三磷酸与7-脱氮-2’脱氧鸟苷三磷酸的主体结构。
为此，在本发明的第一方面，本发明提供了式(A)所示的化合物或其盐，
Figure PCTCN2021125262-appb-000001
其中：
R为可逆阻断基团，R选自
Figure PCTCN2021125262-appb-000002
Figure PCTCN2021125262-appb-000003
所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000004
Figure PCTCN2021125262-appb-000005
，各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
各X独立地选自O、NH、S；
各Y独立地选自直接键、O、NH、S、CH、CH 2、C(CH 3) 2；
R 0选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
R 1选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
R 2、R 3、R 4各自独立地选自O、NH、S、CH、CH 2、C(CH 3) 2；
Figure PCTCN2021125262-appb-000006
表示单键——或双键
Figure PCTCN2021125262-appb-000007
各R”独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如 C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、
Figure PCTCN2021125262-appb-000008
Figure PCTCN2021125262-appb-000009
且不同时为H；
R 5、R 6、R 7、R 8、R 9、R x、R y、R z各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、
Figure PCTCN2021125262-appb-000010
且R 5、R 6、R 7、R 8、R 9不同时为H；
或者，R 5、R 6、R 7、R 8、R 9、R x、R y、R z各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、C1-C6烷基-C(＝O)-NH 2-、
Figure PCTCN2021125262-appb-000011
且R 5、R 6、R 7、R 8、R 9不同时为H；
R 10a、R 10b、R 10c、R 11a、R 11b、R 12各自独立地选自H、-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)、-ONH 2、-OCOR m、-OCONHR m、脂肪族烷基(如C1-C6烷基，具体如甲基Me、乙基Et、异丙基iPr、叔丁基tBu)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)，且R 10a、R 10b、R 10c不同时为H；
n选自1、2、3、4、5；
各m 1独立地选自1、2、3、4、5、6；
各m 2独立地选自0、1、2、3、4、5、6；
R’选自H，单磷酸基团
Figure PCTCN2021125262-appb-000012
二磷酸基团
Figure PCTCN2021125262-appb-000013
三磷酸基团
Figure PCTCN2021125262-appb-000014
或四磷酸基团
Figure PCTCN2021125262-appb-000015
各Z独立地选自O，S，BH；
Base选自碱基、脱氮碱基或其互变异构体，例如Base选自腺嘌呤、7-脱氮腺嘌呤、胸腺嘧啶、尿嘧啶、胞嘧啶、鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体。
在一些实施方案中，R选自
Figure PCTCN2021125262-appb-000016
Figure PCTCN2021125262-appb-000017
在一些实施方案中，R选自
Figure PCTCN2021125262-appb-000018
Figure PCTCN2021125262-appb-000019
在一些实施方案中，R选自
Figure PCTCN2021125262-appb-000020
Figure PCTCN2021125262-appb-000021
在一些实施方案中，R选自
Figure PCTCN2021125262-appb-000022
Figure PCTCN2021125262-appb-000023
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000024
，各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000025
Figure PCTCN2021125262-appb-000026
Figure PCTCN2021125262-appb-000027
各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000028
Figure PCTCN2021125262-appb-000029
各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000030
各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，X为O。
在一些实施方案中，各Y独立地选自直接键、CH 2。
在一些实施方案中，R 0为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)。
在一些实施方案中，R 0为-N 3。
在一些实施方案中，R 1为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)。
在一些实施方案中，R 1为-N 3。
在一些实施方案中，R 2、R 3、R 4各自独立地选自O、NH、S、CH、CH 2、C(CH 3) 2，且满足以下条件：R 2选自O、S时，R 3和R 4为CH 2；R 3选自O、S时，R 2和R 4为CH 2；R 4选自O，S时，R 2和R 3为CH 2；R 2为C(CH 3) 2时，R 3和R 4为CH 2；R 3为C(CH 3) 2时，R 2和R 4为CH 2；R 4为C(CH 3) 2时，R 2和R 3为CH 2；Y为CH时，R 2为CH,R 3和R 4为CH 2。
在一些实施方案中，R 2、R 3、R 4各自独立地选自CH、CH 2。
在一些实施方案中，R 2和R 3为CH，R 4为CH 2。
在一些实施方案中，各杂芳基独立任选地被1个R”所取代时，各R”独立地选自
Figure PCTCN2021125262-appb-000031
优选地，R”为
Figure PCTCN2021125262-appb-000032
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”选自
Figure PCTCN2021125262-appb-000033
剩余R”各自独立地选自-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷 基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
Figure PCTCN2021125262-appb-000034
剩余R”各自独立地选自硝基、脂肪族烷基(如C1-C6烷基)、F、I、Br、Cl。
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
Figure PCTCN2021125262-appb-000035
剩余R”各自独立地选自硝基、C1-C6烷基，优选地，剩余R”为C1-C6烷基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个选自
Figure PCTCN2021125262-appb-000036
其余四个各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个选自
Figure PCTCN2021125262-appb-000037
其余四个各自独立地选自H、硝基、C1-C6烷基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中，R 5、R 7或R 9选自
Figure PCTCN2021125262-appb-000038
其余四个各自独立地选自H、硝基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个选自
Figure PCTCN2021125262-appb-000039
其余四个各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、C1-C6烷基-C(＝O)-NH 2-。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个为
Figure PCTCN2021125262-appb-000040
其余四个各自独立地选自H、硝基、C1-C6烷基、C1-C6烷氧基、C1-C6烷基-C(＝O)-NH 2-。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
Figure PCTCN2021125262-appb-000041
其余四个各自独立地选自H、硝基、甲氧基、乙酰胺基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
Figure PCTCN2021125262-appb-000042
R 7选自H、硝基、甲氧基、乙酰胺基，其余三个为H。
在一些实施方案中，R x、R y中，任意一个选自
Figure PCTCN2021125262-appb-000043
另一个选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些实施方案中，R x、R y中，任意一个选自
Figure PCTCN2021125262-appb-000044
另一个为H。
在一些实施方案中，R x、R y中，任意一个为
Figure PCTCN2021125262-appb-000045
另一个为H。
在一些实施方案中，R z选自
Figure PCTCN2021125262-appb-000046
在一些实施方案中，R z为
Figure PCTCN2021125262-appb-000047
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)、-ONH 2、-OCOR m、-OCONHR m，另两个各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如 C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)。
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)，另两个各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)。
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个为-N 3或-SS-甲基，另两个各自独立地选自H、甲基。
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个为-N 3、-SS-甲基、-SS-乙基、-SS-异丙基、-SS-叔丁基或-SS-异丁基，另两个各自独立地选自H、甲基。
在一些实施方案中，R 11a、R 11b各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)。
在一些实施方案中，R 11a、R 11b为H。
在一些实施方案中，R 12选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)。
在一些实施方案中，R 12为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)。
在一些实施方案中，R 12为-N 3。
在一些实施方案中，n选自1、2、3。
在一些实施方案中，n为2。
在一些实施方案中，m 1为1。
在一些实施方案中，各m 2独立地选自0、1。
在一些实施方案中，
Figure PCTCN2021125262-appb-000048
作为整体，选自
Figure PCTCN2021125262-appb-000049
Figure PCTCN2021125262-appb-000050
在一些实施方案中，R’为三磷酸基团
Figure PCTCN2021125262-appb-000051
在一些实施方案中，Z为O。
在一些实施方案中，Base选自
Figure PCTCN2021125262-appb-000052
Figure PCTCN2021125262-appb-000053
在本发明的第二方面，本发明提供了式(I)所示化合物或其盐，
Figure PCTCN2021125262-appb-000054
其中：
X选自O、NH、S；
Y选自直接键、O、NH、S、CH、CH 2、C(CH 3) 2；
R 0选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
n选自1、2、3、4、5；
R’选自H，单磷酸基团
Figure PCTCN2021125262-appb-000055
二磷酸基团
Figure PCTCN2021125262-appb-000056
三磷酸基团
Figure PCTCN2021125262-appb-000057
或四磷酸基团
Figure PCTCN2021125262-appb-000058
各Z独立地选自O，S，BH；
Base选自碱基、脱氮碱基或其互变异构体，例如Base选自腺嘌呤、7-脱氮腺嘌呤、胸腺嘧啶、尿嘧啶、胞嘧啶、鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体。
在一些实施方案中，X为O。
在一些实施方案中，Y为CH 2。
在一些实施方案中，R 0为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)。
在一些实施方案中，R 0为-N 3。
在一些实施方案中，n选自1、2、3。
在一些实施方案中，n为2。
在一些实施方案中，R’为三磷酸基团
Figure PCTCN2021125262-appb-000059
在一些实施方案中，Z为O。
在一些实施方案中，Base选自
Figure PCTCN2021125262-appb-000060
Figure PCTCN2021125262-appb-000061
在本发明的第三方面，本发明提供了式(II)所示化合物或其盐，
Figure PCTCN2021125262-appb-000062
其中：
X选自O、NH、S；
Y选自直接键、O、NH、S、CH、CH 2、C(CH 3) 2；
R 1选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
R 2、R 3、R 4各自独立地选自O、NH、S、CH、CH 2、C(CH 3) 2；
Figure PCTCN2021125262-appb-000063
表示单键——或双键
Figure PCTCN2021125262-appb-000064
R’选自H，单磷酸基团
Figure PCTCN2021125262-appb-000065
二磷酸基团
Figure PCTCN2021125262-appb-000066
三磷酸基团
Figure PCTCN2021125262-appb-000067
或四磷酸基团
Figure PCTCN2021125262-appb-000068
各Z独立地选自O，S，BH；
Base选自碱基、脱氮碱基或其互变异构体，例如Base选自腺嘌呤、7-脱氮腺嘌呤、胸腺嘧啶、尿嘧啶、胞嘧啶、鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体。
在一些实施方案中，X为O。
在一些实施方案中，Y为直接键。
在一些实施方案中，R 1为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)。
在一些实施方案中，R 1为-N 3。
在一些实施方案中，R 2、R 3、R 4各自独立地选自O、NH、S、CH、CH 2、C(CH 3) 2，且满足以下条件：R 2选自O、S时，R 3和R 4为CH 2；R 3选自O、S时，R 2和R 4为CH 2；R 4选自O，S时，R 2和R 3为CH 2；R 2为C(CH 3) 2时，R 3和R 4为CH 2；R 3为C(CH 3) 2时，R 2和R 4为CH 2；R 4为C(CH 3) 2时，R 2和R 3为CH 2；Y为CH时，R 2为CH,R 3和R 4为CH 2。
在一些实施方案中，R 2、R 3、R 4各自独立地选自CH、CH 2。
在一些实施方案中，R 2和R 3为CH，R 4为CH 2。
在一些实施方案中，R’为三磷酸基团
Figure PCTCN2021125262-appb-000069
在一些实施方案中，Z为O。
在一些实施方案中，Base选自
Figure PCTCN2021125262-appb-000070
Figure PCTCN2021125262-appb-000071
在本发明的第四方面，本发明提供了式(III)所示化合物或其盐，
Figure PCTCN2021125262-appb-000072
其中：
A’选自
Figure PCTCN2021125262-appb-000073
所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000074
Figure PCTCN2021125262-appb-000075
，各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
各R”独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、
Figure PCTCN2021125262-appb-000076
Figure PCTCN2021125262-appb-000077
X选自O、NH、S；
R 5、R 6、R 7、R 8、R 9各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、
Figure PCTCN2021125262-appb-000078
且R 5、R 6、R 7、R 8、R 9不同时为H；
或者，R 5、R 6、R 7、R 8、R 9各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、C1-C6烷基-C(＝O)-NH 2-、
Figure PCTCN2021125262-appb-000079
且R 5、R 6、R 7、R 8、R 9不同时为H；
R 10a、R 10b、R 10c、R 11a、R 11b、R 12各自独立地选自H、-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)、-ONH 2、-OCOR m、-OCONHR m、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)，且R 10a、R 10b、R 10c不同时为H；
m 1选自1、2、3、4、5、6；
m 2选自0、1、2、3、4、5、6；
R’选自H，单磷酸基团
Figure PCTCN2021125262-appb-000080
二磷酸基团
Figure PCTCN2021125262-appb-000081
三磷酸基团
Figure PCTCN2021125262-appb-000082
或四磷酸基团
Figure PCTCN2021125262-appb-000083
各Z独立地选自O，S，BH；
Base选自碱基、脱氮碱基或其互变异构体，例如Base选自腺嘌呤、7-脱氮腺嘌呤、胸腺嘧啶、尿嘧啶、胞嘧啶、鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000084
，各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所 取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000085
Figure PCTCN2021125262-appb-000086
Figure PCTCN2021125262-appb-000087
各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000088
Figure PCTCN2021125262-appb-000089
各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000090
各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000091
Figure PCTCN2021125262-appb-000092
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000093
在一些实施方案中，各杂芳基独立任选地被1个R”所取代时，各R”独立地选自
Figure PCTCN2021125262-appb-000094
优选地，R”为
Figure PCTCN2021125262-appb-000095
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”选自
Figure PCTCN2021125262-appb-000096
剩余R”各自独立地选自-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
Figure PCTCN2021125262-appb-000097
剩余R”各自独立地选自硝基、脂肪族烷基(如C1-C6烷基)、F、I、Br、Cl。
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
Figure PCTCN2021125262-appb-000098
剩余R”各自独立地选自硝基、C1-C6烷基，优选地，剩余R”为C1-C6烷基，更优选地，剩余R”为甲基。
在一些实施方案中，R 13、R 14、R 15、R 16中，R 16为
Figure PCTCN2021125262-appb-000099
R 13、R 14、R 15各自独立地选自H、硝基、C1-C6烷基(如甲基)。
在一些实施方案中，R 13、R 14、R 15、R 16中，R 16为
Figure PCTCN2021125262-appb-000100
R 13、R 14、R 15为H。
在一些实施方案中，R 17、R 18、R 19中，R 19为
Figure PCTCN2021125262-appb-000101
R 17、R 18各自独立地选自H、硝基、C1-C6烷基(如甲基)。
在一些实施方案中，R 17、R 18、R 19中，R 19为
Figure PCTCN2021125262-appb-000102
R 17、R 18为H。
在一些实施方案中，R 20、R 21、R 22中，R 22为
Figure PCTCN2021125262-appb-000103
R 20、R 21各自独立地选自H、硝基、C1-C6烷基(如甲基)。
在一些实施方案中，R 20、R 21、R 22中，R 22为
Figure PCTCN2021125262-appb-000104
R 20、R 21为H。
在一些实施方案中，R 23、R 24、R 25、R 26中，R 26为
Figure PCTCN2021125262-appb-000105
R 23、R 24、R 25各自独立地选自H、硝基、C1-C6烷基(如甲基)。
在一些实施方案中，R 23、R 24、R 25、R 26中，R 26为
Figure PCTCN2021125262-appb-000106
R 23、R 24、R 25为H。
在一些实施方案中，R 27、R 28、R 29中，R 29为
Figure PCTCN2021125262-appb-000107
R 27、R 28各自独立地选自H、硝基、C1-C6烷基(如甲基)。
在一些实施方案中，R 27、R 28、R 29中，R 29为
Figure PCTCN2021125262-appb-000108
R 27、R 28为H。
在一些实施方案中，R 30、R 31、R 32中，R 32为
Figure PCTCN2021125262-appb-000109
R 30、R 31各自独立地选自H、硝基、C1-C6烷基(如甲基)。
在一些实施方案中，R 30、R 31、R 32中，R 32为
Figure PCTCN2021125262-appb-000110
R 30、R 31为H。
在一些实施方案中，R 33、R 34、R 35中，R 34或R 35为
Figure PCTCN2021125262-appb-000111
R 33选自H、硝基、C1-C6烷基(如甲基)。
在一些实施方案中，R 33、R 34、R 35中，R 34或R 35为
Figure PCTCN2021125262-appb-000112
R 33为H或甲基。
在一些实施方案中，X为O。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个选自
Figure PCTCN2021125262-appb-000113
其余四个各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个为
Figure PCTCN2021125262-appb-000114
其余四个各自独立地选自H、硝基、C1-C6烷基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
Figure PCTCN2021125262-appb-000115
其余四个各自独立地选自H、硝基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个选自
Figure PCTCN2021125262-appb-000116
其余四个各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、C1-C6烷基-C(＝O)-NH 2-。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个为
Figure PCTCN2021125262-appb-000117
其余四个各自独立地选自H、硝基、C1-C6烷基、C1-C6烷氧基、C1-C6烷基-C(＝O)-NH 2-。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
Figure PCTCN2021125262-appb-000118
其余四个各自独立地选自H、硝基、甲氧基、乙酰胺基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
Figure PCTCN2021125262-appb-000119
R 7选自H、硝基、甲氧基、乙酰胺基，其余三个为H。
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)、-ONH 2、-OCOR m、-OCONHR m，另两个各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)。
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)，另两个各自独立地选自H、C1-C6烷基(如甲基、乙基、异丙基、叔丁基)。
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个为-N 3或-SS-甲基，另两个各自独立地选自H、甲基。
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个为-N 3、-SS-甲基、-SS-乙基、-SS-异丙基、-SS-叔丁基、-SS-异丁基，另两个各自独立地选自H、甲基。
在一些实施方案中，R 11a、R 11b各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)。
在一些实施方案中，R 11a、R 11b为H。
在一些实施方案中，R 12选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)。
在一些实施方案中，R 12为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)。
在一些实施方案中，R 12为-N 3。
在一些实施方案中，m 1为1。
在一些实施方案中，m 2选自0、1。
在一些实施方案中，
Figure PCTCN2021125262-appb-000120
作为整体，选自
Figure PCTCN2021125262-appb-000121
Figure PCTCN2021125262-appb-000122
在一些实施方案中，R’为三磷酸基团
Figure PCTCN2021125262-appb-000123
在一些实施方案中，Z为O。
在一些实施方案中，Base选自
Figure PCTCN2021125262-appb-000124
Figure PCTCN2021125262-appb-000125
在本发明的第五方面，本发明提供了式(IV)所示化合物或其盐，
Figure PCTCN2021125262-appb-000126
其中：
A选自
Figure PCTCN2021125262-appb-000127
所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000128
Figure PCTCN2021125262-appb-000129
，各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
各R”独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、
Figure PCTCN2021125262-appb-000130
Figure PCTCN2021125262-appb-000131
R 5、R 6、R 7、R 8、R 9、R x、R y、R z各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧 基)、
Figure PCTCN2021125262-appb-000132
且R 5、R 6、R 7、R 8、R 9不同时为H；
R 10a、R 10b、R 10c、R 11a、R 11b、R 12各自独立地选自H、-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)，且R 10a、R 10b、R 10c不同时为H；
X选自O、NH、S；
m 1选自1、2、3、4、5、6；
m 2选自0、1、2、3、4、5、6；
R’选自H，单磷酸基团
Figure PCTCN2021125262-appb-000133
二磷酸基团
Figure PCTCN2021125262-appb-000134
三磷酸基团
Figure PCTCN2021125262-appb-000135
或四磷酸基团
Figure PCTCN2021125262-appb-000136
各Z独立地选自O，S，BH；
Base选自碱基、脱氮碱基或其互变异构体，例如Base选自腺嘌呤、7-脱氮腺嘌呤、胸腺嘧啶、尿嘧啶、胞嘧啶、鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体。
在一些实施方案中，A选自
Figure PCTCN2021125262-appb-000137
在一些实施方案中，A选自
Figure PCTCN2021125262-appb-000138
在一些实施方案中，A为
Figure PCTCN2021125262-appb-000139
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000140
，各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000141
Figure PCTCN2021125262-appb-000142
Figure PCTCN2021125262-appb-000143
各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，所述杂芳基选自以下：
Figure PCTCN2021125262-appb-000144
Figure PCTCN2021125262-appb-000145
各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代。
在一些实施方案中，各杂芳基独立任选地被1个R”所取代时，各R”独立地选自
Figure PCTCN2021125262-appb-000146
优选地，R”为
Figure PCTCN2021125262-appb-000147
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”选自
Figure PCTCN2021125262-appb-000148
剩余R”各自独立地选自-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
Figure PCTCN2021125262-appb-000149
剩余R”各自独立地选自硝基、脂肪族烷基(如C1-C6烷基)、F、I、Br、Cl。
在一些实施方案中，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
Figure PCTCN2021125262-appb-000150
剩余R”各自独立地选自硝基、C1-C6烷基，优选地，剩余R”为C1-C6烷基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个选自
Figure PCTCN2021125262-appb-000151
其余四个各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中的任意一个为
Figure PCTCN2021125262-appb-000152
其余四个各自独立地选自H、硝基、C1-C6烷基。
在一些实施方案中，R 5、R 6、R 7、R 8、R 9中，R 7为
Figure PCTCN2021125262-appb-000153
其余四个为H。
在一些实施方案中，R x、R y中，任意一个选自
Figure PCTCN2021125262-appb-000154
另一个选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些实施方案中，R x、R y中，任意一个为
Figure PCTCN2021125262-appb-000155
另一个为H。
在一些实施方案中，R z选自
Figure PCTCN2021125262-appb-000156
在一些实施方案中，R z为
Figure PCTCN2021125262-appb-000157
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，另两个各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)。
在一些实施方案中，R 10a、R 10b、R 10c中的任意一个为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)，另两个各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)。
在一些实施方案中，R 11a、R 11b各自独立地选自H、脂肪族烷基(如C1-C6 烷基，具体如甲基、乙基、异丙基、叔丁基)。
在一些实施方案中，R 11a、R 11b为H。
在一些实施方案中，R 12选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)。
在一些实施方案中，R 12为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)。
在一些实施方案中，R 12为-N 3。
在一些实施方案中，X为O。
在一些实施方案中，m 1为1。
在一些实施方案中，m 2选自0、1。
在一些实施方案中，R’为三磷酸基团
Figure PCTCN2021125262-appb-000158
在一些实施方案中，Z为O。
在一些实施方案中，Base选自
Figure PCTCN2021125262-appb-000159
Figure PCTCN2021125262-appb-000160
在本发明的第六方面，本发明提供了以下化合物或其盐，
Figure PCTCN2021125262-appb-000161
Figure PCTCN2021125262-appb-000162
Figure PCTCN2021125262-appb-000163
Figure PCTCN2021125262-appb-000164
Figure PCTCN2021125262-appb-000165
Figure PCTCN2021125262-appb-000166
Figure PCTCN2021125262-appb-000167
Figure PCTCN2021125262-appb-000168
Figure PCTCN2021125262-appb-000169
Figure PCTCN2021125262-appb-000170
Figure PCTCN2021125262-appb-000171
Figure PCTCN2021125262-appb-000172
Figure PCTCN2021125262-appb-000173
Figure PCTCN2021125262-appb-000174
Figure PCTCN2021125262-appb-000175
Figure PCTCN2021125262-appb-000176
在一些实施方案中，前述的化合物或其盐携带额外的可检测标记(如荧光标记)。
在一些实施方案中，所述化合物或其盐携带的额外的可检测标记是通过亲和试剂(如抗体、适体、Affimer、Knottin)引入的，所述亲和试剂携带所述可检测标记，且所述亲和试剂可以特异性识别并结合所述化合物或其盐的表位。
在一些实施方案中，所述额外的可检测标记(如荧光标记)任选地通过连接基团与所述化合物或其盐连接。
在一些实施方案中，所述额外的可检测标记(如荧光标记)任选地通过连接基团与所述化合物或其盐的Base连接。
在一些实施方案中，所述额外的可检测标记(如荧光标记)任选地通过连接基团与所述化合物或其盐的Base连接时，Base的结构选自以下
Figure PCTCN2021125262-appb-000177
Figure PCTCN2021125262-appb-000178
Figure PCTCN2021125262-appb-000179
优选选自
Figure PCTCN2021125262-appb-000180
在一些实施方案中，所述连接基团为可裂解的连接基团或不可裂解的连接基团。
在一些实施方案中，所述可裂解的连接基团选自亲电裂解的连接基团、亲核裂解的连接基团、可光解的连接基团、还原条件下裂解的连接基团、氧化条件下裂解的连接基团、安全拉手型连接基团、经消除机理裂解的连接基团，或其任何组合。
在一些实施方案中，所述连接基团具有式(B)所示结构：
Figure PCTCN2021125262-appb-000181
其中：
R 36、R 37、R 38、R 39各自独立地选自H、-N 3、N 3-C1-C6烷基、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)，所述C1-C6烷基任选地被C1-C6烷基所取代；
p选自1、2、3、4、5、6；
q选自1-12之间任意整数。
在一些具体实施方案中，R 36、R 37、R 38、R 39中，任意一个为N 3-C1-C6烷基，且所述C1-C6烷基任选地被C1-C6烷基所取代，其余各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)。
在一些具体实施方案中，R 36为N 3-C1-C6烷基，且所述C1-C6烷基任选地被C1-C6烷基所取代，R 37、R 38、R 39各自独立地选自H、C1-C6烷基。
在一些具体实施方案中，R 36为N 3-甲基，且所述甲基任选地被甲基所取代，R 37、R 38、R 39为H。
在一些具体实施方案中，p选自1、2、3。
在一些具体实施方案中，p为1。
在一些具体实施方案中，q选自2、3、4、5、6。
在一些具体实施方案中，q为4。
在一些实施方案中，式(B)所示的连接基团的甲基端与所述Base连接，氨基端与所述额外的可检测标记(如荧光标记)连接。例如，
Figure PCTCN2021125262-appb-000182
Figure PCTCN2021125262-appb-000183
在一些实施方案中，式(B)所示的连接基团的结构式为
Figure PCTCN2021125262-appb-000184
在一些实施方案中，所述连接基团具有式(C)所示结构：
Figure PCTCN2021125262-appb-000185
在一些实施方案中，式(C)所示的连接基团的炔基端与所述Base连接，氨基端与所述额外的可检测标记(如荧光标记)连接。例如
Figure PCTCN2021125262-appb-000186
Figure PCTCN2021125262-appb-000187
在一些实施方案中，所述连接基团具有式(D)所示结构：
Figure PCTCN2021125262-appb-000188
在一些实施方案中，式(D)所示的连接基团的炔基端与所述Base连接，氨基端与所述额外的可检测标记(如荧光标记)连接。例如
Figure PCTCN2021125262-appb-000189
在一些实施方案中，所述可检测标记选自以下：
Figure PCTCN2021125262-appb-000190
Figure PCTCN2021125262-appb-000191
在一些实施方案中，Base不同，所述可检测标记(如荧光标记)不同。
在一些实施方案中，携带额外的可检测标记的所述化合物或其盐的结构如下所示：
Figure PCTCN2021125262-appb-000192
Figure PCTCN2021125262-appb-000193
在一些实施方案中，携带额外的可检测标记的所述化合物或其盐的结构如下所示：
Figure PCTCN2021125262-appb-000194
Figure PCTCN2021125262-appb-000195
在一些实施方案中，携带额外的可检测标记的所述化合物或其盐的结构如下所示：
Figure PCTCN2021125262-appb-000196
Figure PCTCN2021125262-appb-000197
在一些实施方案中，携带额外的可检测标记的所述化合物或其盐的结构如下所示：
Figure PCTCN2021125262-appb-000198
Figure PCTCN2021125262-appb-000199
在本发明的第七方面，本发明提供了终止核酸合成的方法，其包括：将前述的化合物或其盐掺入待终止的核酸分子中。
在一些实施方案中，所述化合物或其盐的掺入通过末端转移酶、末端聚合酶或逆转录酶来实现。
在一些实施方案中，所述方法包括：使用聚合酶，将所述化合物或其盐掺入待终止的核酸分子中。
在一些实施方案中，所述方法包括：在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述化合物或其盐掺入待终止的核酸分子的3'端。
在本发明的第八方面，本发明提供了制备在测序反应中与目标单链多核苷酸互补的生长的多核苷酸的方法，其包括将前述的化合物或其盐掺入所述生长的互补多核苷酸，其中，所述化合物或其盐的掺入防止了任何后续的核苷酸引入所述生长的互补多核苷酸中。
在一些实施方案中，所述化合物或其盐的掺入通过末端转移酶、末端聚合酶或逆转录酶来实现。
在一些实施方案中，所述方法包括：使用聚合酶，将所述化合物或其盐掺入所述生长的互补多核苷酸。
在一些实施方案中，所述方法包括：在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述化合物或其盐掺入所述生长的互补多核苷酸的3'端。
在本发明的第九方面，本发明提供了测定目标单链多核苷酸的序列的方法，其包括：
1)监测生长的核酸链中与目标单链多核苷酸互补的核苷酸的顺序掺入，其中，掺入的至少一个互补核苷酸是前述的化合物或其盐，且所述化合物或其盐 携带额外的可检测标记(如荧光标记)，以及，
2)检测所述可检测标记。
在一些实施方案中，所述额外的可检测标记(如荧光标记)任选地通过连接基团与所述化合物或其盐连接。
在一些实施方案中，所述连接基团如前所述。
在一些实施方案中，所述额外的可检测标记如前所述。
在一些实施方案中，Base不同，所述化合物或其盐携带的可检测标记(如荧光标记)不同。
在一些实施方案中，在引入下一个互补核苷酸之前，将所述化合物或其盐中的可逆阻断基团(R)和所述可检测标记除去。
在一些实施方案中，所述可逆阻断基团和所述可检测标记被同时除去。
在一些实施方案中，所述可逆阻断基团和所述可检测标记被先后除去；例如，在所述可检测标记被除去之后，所述可逆阻断基团被除去，或者，在所述可逆阻断基团被除去之后，所述可检测标记被除去。
在一些实施方案中，所述方法包括以下步骤：
(a)提供多种不同的核苷酸，其中所述多种不同的核苷酸是前述的化合物或其盐，其中，每种类型的核苷酸携带的额外的可检测标记在检测时可以与其他类型的核苷酸携带的额外的可检测标记区分开；
(b)将所述多种不同的核苷酸掺入目标单链多核苷酸的互补序列中；
(c)检测(b)的核苷酸携带的额外的可检测标记，从而确定掺入的核苷酸的类型；
(d)除去(b)的核苷酸中的可逆阻断基团和其携带的可检测标记；和
(e)任选地重复步骤(b)-(d)一次或多次；
从而确定所述目标单链多核苷酸的序列。
在一些实施方案中，所述方法包括以下步骤：
(1)提供第一核苷酸、第二核苷酸、第三核苷酸和第四核苷酸，四种核苷酸中的至少一种是前述的化合物或其盐，所述四种核苷酸包含的Base互不相同，且所述四种核苷酸携带额外的可检测标记(如荧光标记)，优选地，所述四种核苷酸携带的额外的可检测标记是通过亲和试剂(如抗体、适体、Affimer、Knottin)引入的，所述亲和试剂携带所述可检测标记，且所述亲和试剂可以特异性识别并结合每一种核苷酸的表位，或者优选地，所述四种核苷酸任选地通过连接基团与所述额外的可检测标记连接，更优选地，所述四种核苷酸中的Base任选地通过连接基团与所述额外的可检测标记连接，最优选地，所述四种核苷酸所携带的额外的可检测标记互不相同；
(2)将所述四种核苷酸与目标单链多核苷酸进行接触；除去未掺入生长的核酸链中的所述核苷酸；检测掺入生长的核酸链中的所述核苷酸所携带的所述可检测标记；除去掺入生长的核酸链中的所述核苷酸所包含的可逆阻断基团和所携带的所述可检测标记；
任选地，还包括(3)：重复(2)一次或多次。
在一些实施方案中，所述方法包括以下步骤：
(a)提供包含双链体、至少一种前述的化合物或其盐、聚合酶和切除试剂的混合物；所述双链体包含生长的核酸链以及待测序的核酸链；所述化合物或其盐携带额外的可检测标记(如荧光标记)，优选地，所述化合物或其盐携带的额外的可检测标记是通过亲和试剂(如抗体、适体、Affimer、Knottin)引入 的，所述亲和试剂携带所述可检测标记，且所述亲和试剂可以特异性识别并结合所述化合物或其盐的表位，或者优选地，所述化合物或其盐任选地通过连接基团与所述额外的可检测标记连接，或者更优选地，所述化合物或其盐中的Base任选地通过连接基团与所述额外的可检测标记连接；
(b)进行包含以下步骤(i)、(ii)和(iii)的反应，任选地，重复一次或多次：
步骤(i)：使用聚合酶，使所述化合物或其盐掺入生长的核酸链，形成包含可逆阻断基团和可检测标记的核酸中间体：
步骤(ii)：对所述核酸中间体所包含的可检测标记进行检测；
步骤(iii)：使用切除试剂将所述核酸中间体所包含的可逆阻断基团和/或所述可检测标记切除。
在一些实施方案中，对所述可逆阻断基团的切除和对所述可检测标记的切除同时进行，或者，对所述可逆阻断基团的切除和对所述可检测标记的切除分步进行(例如，先切除所述可逆阻断基团，或者先切除所述可检测标记)。
在一些实施方案中，对所述可逆阻断基团的切除和对所述可检测标记的切除使用的切除试剂是同样的试剂。
在一些实施方案中，对所述可逆阻断基团的切除和对所述可检测标记的切除使用的切除试剂是不同的试剂。
在一些实施方案中，所述双链体连接于支持物上。
在一些实施方案中，所述生长的核酸链为引物。
在一些实施方案中，所述引物通过退火至待测序的核酸链上，形成所述双链体。
在一些实施方案中，所述双链体、所述化合物或其盐、以及所述聚合酶一起形成含有溶液相和固相的反应体系。
在一些实施方案中，所述化合物或其盐包含的Base互不相同。
在一些实施方案中，所述化合物或其盐携带的额外的可检测标记互不相同。
在一些实施方案中，在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶，使所述化合物或其盐掺入生长的核酸链，形成包含可逆阻断基团和可检测标记的核酸中间体。
在一些实施方案中，所述聚合酶选自KOD聚合酶或其突变体(例如KOD POL151、KOD POL157、KOD POL171、KOD POL174、KOD POL376、KOD POL391)。
在一些实施方案中，在任意一个检测所述核酸中间体所包含的可检测标记步骤前，移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体。
在一些实施方案中，所述切除试剂与所述双链体或所述生长的核酸链在含有溶液相和固相的反应体系中接触。
在一些实施方案中，所述切除试剂能够切除掺入生长的核酸链的化合物中的可逆阻断基团和所述化合物携带的额外的可检测标记，并且不会影响双链体骨架上的磷酸二酯键。
在一些实施方案中，在任意一个切除所述核酸中间体所包含的可逆阻断基团和/或额外的可检测标记的步骤后，移除这一步骤反应体系的溶液相。
在一些实施方案中，在任意一个包含移除操作的步骤之后，进行洗涤操作。
在一些实施方案中，步骤(ii)之后，进一步包括：根据步骤(ii)检测得到的信号，确定步骤(i)中掺入生长的核酸链的化合物的类型，并基于碱基互补配对原则，确定待测序的核酸链中相应位置处的核苷酸类型。
在本发明的第十方面，本发明提供了试剂盒，其包含至少一个前述的化合物或其盐。
在一些实施方案中，所述试剂盒包含第一、第二、第三和第四化合物，所述第一、第二、第三和第四化合物各自独立地为前述的化合物或其盐。
在一些实施方案中，所述第一化合物中，Base选自腺嘌呤、7-脱氮腺嘌呤或其互变异构体(例如
Figure PCTCN2021125262-appb-000200
)；所述第二化合物中，Base选自胸腺嘧啶、尿嘧啶或其互变异构体(例如
Figure PCTCN2021125262-appb-000201
)；所述第三化合物中，Base选自胞嘧啶或其互变异构体(例如
Figure PCTCN2021125262-appb-000202
)；所述第四化合物中，Base选自鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体(例如
Figure PCTCN2021125262-appb-000203
)。
在一些实施方案中，所述第一、第二、第三和第四化合物携带额外的可检测标记。
在一些实施方案中，所述第一、第二、第三和第四化合物携带的额外的可检测标记是通过亲和试剂(如抗体、适体、Affimer、Knottin)引入的，所述亲和试剂携带所述可检测标记，且所述亲和试剂可以特异性识别并结合所述第一、第二、第三或第四化合物的表位。
在一些实施方案中，所述第一、第二、第三和第四化合物任选地通过连接基团与所述额外的可检测标记连接。
在一些实施方案中，所述第一、第二、第三和第四化合物中的Base任选地通过连接基团与所述额外的可检测标记连接。
在一些实施方案中，所述第一、第二、第三和第四化合物包含的Base互不相同。
在一些实施方案中，所述第一、第二、第三和第四化合物所携带的额外的可检测标记互不相同。
在一些实施方案中，所述连接基团如前所述。
在一些实施方案中，所述可检测标记如前所述。
在一些实施方案中，所述试剂盒还包含：用于预处理核酸分子的试剂；用于连接待测序的核酸分子的支持物；用于将待测序的核酸分子与支持物连接(例如，共价或非共价连接)的试剂；用于起始核苷酸聚合反应的引物；用于进行核苷酸聚合反应的聚合酶；一种或多种缓冲溶液；一种或多种洗涤溶液；或其任何组合。
在本发明的第十一方面，本发明提供了前述的化合物或其盐或者前述的试 剂盒用于测定目标单链多核苷酸的序列的用途。
附图说明
图1表示本发明实施例的叠氮甲基的切除反应；
图2表示本发明实施例的亲核环化串联反应用于可逆阻断切除；
图3表示本发明实施例的3’-OH标记可逆阻断核苷酸类似物。
具体实施方式
下面通过具体实施例详细描述本发明的实施方式，但是无论如何它们不能解释为对本发明的限制。
除非特殊说明，上述基团和取代基具有药物化学领域的普通含义。
在本说明书的各部分，本发明公开化合物的取代基按照基团种类或范围公开。特别指出，本发明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“C1-C6烷基”特别指独立公开的甲基、乙基、C3烷基、C4烷基、C5烷基和C6烷基。
另外，需要说明的是，除非以其他方式明确指出，在本文中通篇采用的描述方式“各…独立地为/选自”和“…各自独立地为/选自”可以互换，均应做广义理解，其既可以是指在不同基团中，相同或不同的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，相同或不同的符号之间所表达的具体选项之间互相不影响。
术语“脂肪族烷基”指的是任意的含有1-20个碳原子的直链或支链饱和基团，例如，C1-C12烷基，优选C1-C6烷基。
术语“C1-C6烷基”指的是任意的含有1-6个碳原子的直链或支链饱和基团，例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、叔戊基、正己基等。
术语“烷氧基”指的是任意上述烷基(例如C1-C6烷基等)，其通过氧原子(-O-)连接到分子的其余部分。
术语“环烷基”是指具有饱和环的3-10元单环系统的烃，例如，C3-C8环烷基，优选C3-C6环烷基。
术语“C3-C6环烷基”是指具有饱和环的3-6元单环系统的烃，C3-C6环烷基可以为环丙基、环丁基、环戊基、环己基等。
术语“芳香族烷基”指的是芳基烷基或杂芳基烷基。其中烷基如上文所定义。
术语“杂芳基”是指芳族的杂环，通常为具有1至3个选自N、O或S的杂原子的5-、6-、7-、8-元的杂环；杂芳基环可以任选地进一步稠合或连接于芳族和非芳族的碳环和杂环。所述杂芳基的非限制性的实例为例如吡啶基、吡嗪基、嘧啶基、哒嗪基、吲哚基、咪唑基、噻唑基、异噻唑基、噻噁唑基、吡咯基、苯基-吡咯基、呋喃基、苯基-呋喃基、噁唑基、异噁唑基、吡唑基、噻吩基、苯并呋喃基、苯并噻吩基、苯并1,3-二氧戊环(苯并二噁茂)、异二氢吲哚基、苯并咪唑基、吲唑基、喹啉基、异喹啉基、1,2,3-三唑基、1-苯基-1,2,3-三唑基、2,3-二氢吲哚基、2,3-二氢苯并呋喃基、2,3-二氢苯并噻吩基、苯并吡喃基、2,3-二氢苯并噁嗪基、2,3-二氢喹喔啉基等。
本发明中，
Figure PCTCN2021125262-appb-000204
表示基团
Figure PCTCN2021125262-appb-000205
与杂芳基相连。其余相似结构可参照前述内容进行理解。
本发明中，R可以选自
Figure PCTCN2021125262-appb-000206
其中，各杂芳基可以相同，也可以不同。另外，各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代时，其表示，不同的杂芳基上的取代基R”可以相同，也可以不同，而且，同一个杂芳基上的多个取代基R”可以相同，也可以不同。
本发明中，“各R”独立地选自H、-N 3等等，且不同时为H”，其表示，若所述杂芳基被1个或多个(如2、3、4、5、6个，优选2个)R”所取代，则至少有1个取代基R”不为H。
术语“芳基”指的是具有6-14个碳原子的碳环芳族体系的基团，例如C6-C10芳基，优选苯基。
从所有上述描述中，对本领域技术人员显而易见的是，其名称是复合名称的任意基团，例如“苯基C1-C6烷基”，应该指的是常规地从其衍生的部分例如从被苯基取代的C1-C6烷基来构建，其中C1-C6烷基如上文所定义。
如本文所使用，术语“式(A)、式(I)、式(II)、式(III)或式(IV)所示的化合物的盐”的例子是由形成阴离子的有机酸形成的有机酸加合盐，包括但不限于甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐、抗坏血酸盐、α-酮戊二酸盐、α-甘油磷酸盐、烷基磺酸盐或芳基磺酸盐；优选地，所述烷基磺酸盐为甲基磺酸盐或乙基磺酸盐；所述芳基磺酸盐为苯磺酸盐或对甲苯磺酸盐。也可形成合适的无机盐，包括但不限于盐酸盐、氢溴酸盐、氢碘酸盐、硝酸盐、碳酸氢盐和碳酸盐、硫酸盐或磷酸盐等。
本发明中，R可以选自
Figure PCTCN2021125262-appb-000207
其中，
Figure PCTCN2021125262-appb-000208
表示单键——或双键
Figure PCTCN2021125262-appb-000209
且R 2、R 3、R 4各自独立地选自O、NH、S、CH、CH 2、C(CH 3) 2。可以理解的是，R 2和R 3之间，只有当R 2和R 3均为CH时，R 2和R 3之间为双键，当R 2和R 3各自独立地选自除CH外的其余原子或原子团(即O、NH、S、CH 2、C(CH 3) 2)时，R 2和R 3之间为单键。例如，“R 2、R 3、R 4各自独立地选自CH、CH 2”，其表示：R 2和R 3均为CH，且R 2和R 3之间为双键，R 4为CH 2，且R 3和R 4之间为单键；或者，R 3和R 4均为CH，且R 3和R 4之间为双键，R 2为CH 2，且R 2和R 3之间为单键。类似地，Y和R 2之间，或者R 3和R 4之间的单键或双键可参照前述内容进行理解。
术语“直接键”表示其两侧的基团直接相连，例如，
Figure PCTCN2021125262-appb-000210
中，Y为直接键，则
Figure PCTCN2021125262-appb-000211
将变为
Figure PCTCN2021125262-appb-000212
对于本发明的式(B)所示化合物
Figure PCTCN2021125262-appb-000213
术语“R 36为N 3-C1-C6烷基，且所述C1-C6烷基任选地被C1-C6烷基所取代”，其指的是，R 36为N 3-C1-C6烷基，且所述C1-C6烷基上的H还可以被另外的C1-C6烷基所取代。例如，R 36为N 3-甲基，且所述甲基任选地被甲基所取代时，式(B)所示化合物的结构可以为
Figure PCTCN2021125262-appb-000214
也可以为
Figure PCTCN2021125262-appb-000215
其余类似描述可以参照前述内容进行理解。
在本发明的方法中，只要是由生长的核酸链和待测序的核酸链这两条链组成的物质，均称其为“双链体”，与生长的核酸链或待测序的核酸链的链长无关，待测序的核酸链可以比生长的核酸链的链长更长。
在本发明的方法中，待测序的核酸分子可以是任何目的核酸分子。在某些优选的实施方案中，所述待测序的核酸分子包含脱氧核糖核苷酸、核糖核苷酸、经修饰的脱氧核糖核苷酸、经修饰的核糖核苷酸、或其任何组合。在本发明的方法中，待测序的核酸分子不受其类型的限制。在某些优选的实施方案中，所述待测序的核酸分子为DNA或RNA。在某些优选的实施方案中，所述待测序的核酸分子可以为基因组DNA，线粒体DNA，叶绿体DNA，mRNA，cDNA，miRNA，或siRNA。在某些优选的实施方案中，所述待测序的核酸分子为线性的或者环状的。在某些优选的实施方案中，所述待测序的核酸分子为双链的或者单链的。例如，所述待测序的核酸分子可以为单链DNA(ssDNA)，双链DNA(dsDNA)，单链RNA(ssRNA)，双链RNA(dsRNA)，或者DNA和RNA的杂合体。在某些优选的实施方案中，所述待测序的核酸分子为单链DNA。在某些优选的实施方案中，所述待测序的核酸分子为双链DNA。
在本发明的方法中，待测序的核酸分子不受其来源的限制。在某些优选的实施方案中，待测序的核酸分子可以获自任何来源，例如，任何细胞、组织或生物体(例如，病毒，细菌，真菌，植物和动物)。在某些优选的实施方案中，待测序的核酸分子源自哺乳动物(例如，人、非人灵长类动物、啮齿类动物或犬科动物)、植物、鸟类、爬行类、鱼类、真菌、细菌或病毒。
从细胞、组织或生物体中提取或获得核酸分子的方法是本领域技术人员公知的。合适的方法包括但不限于乙醇沉淀法，氯仿抽提法等。关于此类方法的详细描述可参见例如，J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995。另外，还可使用各种商业化的试剂盒来从各种来源(例如细胞、组织或生物体)提取核酸分子。
在本发明的方法中，待测序的核酸分子不受其长度的限制。在某些优选的实施方案中，待测序的核酸分子的长度可以为至少10bp，至少20bp，至少30bp，至少40bp，至少50bp，至少100bp，至少200bp，至少300bp，至少 400bp，至少500bp，至少1000bp，或者至少2000bp。在某些优选的实施方案中，待测序的核酸分子的长度可以为10-20bp，20-30bp，30-40bp，40-50bp，50-100bp，100-200bp，200-300bp，300-400bp，400-500bp，500-1000bp，1000-2000bp，或者超过2000bp。在某些优选的实施方案中，待测序的核酸分子可具有10-1000bp的长度，以利于进行高通量测序。
在本发明的制备多核苷酸的方法或测序方法中，可使用合适的聚合酶来进行核苷酸聚合反应。在一些示例性实施方案中，所述聚合酶能够以DNA为模板合成新的DNA链(例如DNA聚合酶)。在一些示例性实施方案中，所述聚合酶能够以RNA为模板合成新的DNA链(例如反转录酶)。在一些示例性实施方案中，所述聚合酶能够以DNA或RNA为模板合成新的RNA链(例如RNA聚合酶)。因此，在某些优选的实施方案中，所述聚合酶选自DNA聚合酶，RNA聚合酶，和反转录酶。可根据实际需要，选择合适的聚合酶来进行核苷酸聚合反应。在某些优选的实施方案中，所述聚合反应为聚合酶链式反应(PCR)。在某些优选的实施方案中，所述聚合反应为反转录反应。
在本发明的方法中，可以使用KOD聚合酶或其突变体进行核苷酸聚合反应。KOD聚合酶或其突变体(例如KOD POL151、KOD POL157、KOD POL171、KOD POL174、KOD POL376、KOD POL391)对本发明的修饰的核苷或核苷酸具有可接受的聚合效率。KOD POL391和KOD POL171对本发明的修饰的核苷酸的具有可接受的聚合效率。在某些实施方案中，KOD POL391或KOD POL171对本发明的修饰的核苷酸的聚合效率在70％以上，例如70％-80％、80％-90％或90％-100％。
在本发明的制备多核苷酸的方法或测序方法中，核苷酸的聚合反应在适宜的条件下进行。适宜的聚合条件包括溶液相的组成以及各成分的浓度、溶液相的pH、聚合温度等。在适宜的条件下进行聚合，有利于获得可接受的、甚至高的聚合效率。
在本发明中，所述式(A)所示化合物的脱氧核糖3'位置处的羟基(-OH)是受保护的(被R所保护)，因此，它们能够终止聚合酶(例如DNA聚合酶)的聚合作用。例如，当式(A)所示化合物被引入生长的核酸链的3'端时，由于该化合物的脱氧核糖的3'位置处不存在游离的羟基(-OH)，聚合酶将无法继续进行下一轮的聚合反应，从而聚合反应将被终止。在这种情况下，在每一轮的聚合反应中，将有且只有一个碱基被掺入生长的核酸链。
此外，所述式(A)所示化合物的脱氧核糖3'位置处羟基(-OH)的保护基团(R)能够被去除，并转变为游离的羟基(-OH)。随后，可使用聚合酶和式(A)所示化合物对生长的核酸链进行下一轮的聚合反应，并再次引入一个碱基。
因此，所述式(A)所示化合物的脱氧核糖3'位置处羟基(-OH)被可逆阻断：当式(A)所示化合物被掺入生长的核酸链的3'端时，它们将终止聚合酶继续进行聚合作用，终止生长的核酸链的进一步延伸；并且，在式(A)所示化合物所包含的阻断基团被去除后，聚合酶将能够继续对生长的核酸链进行聚合作用，继续延伸核酸链。
本文描述的某些实施方案涉及常规可检测标记的使用。可通过任何适合的方法进行检测，包括荧光光谱学或其他光学手段。优选的标记为荧光标记即荧光团，该荧光团在吸收能量后发出限定波长的辐射。已知许多种适合的荧光标记。例如，Welch等人(Chem.Eur.J.5(3):951-960,1999)公开了丹酰基-功能化的 荧光部分，其可在本发明中使用。Zhu等人(Cytometry28:206-211,1997)描述了荧光标记Cy3和Cy5的使用，其也可以在本发明中使用。Prober等人(Science238:336-341,1987)、Connell等人(BioTechniques5(4):342-384,1987)、Ansorge等人(Nucl.AcidsRes.15(11):4593-4602,1987)和Smith等人(Nature321:674,1986)也公开了适合使用的标记。其他可商业购得的荧光标记包括但不限于荧光素、若丹明(包括TMR、德克萨斯红和Rox)、alexa、氟硼荧、吖啶、香豆素、芘、苯并蒽和花青苷。
本申请中也可以使用多重标记，例如双荧光团FRET盒(Tet.Let.46:8867-8871,2000)、也可以使用多荧光体树枝状系统(J.Am.Chem.Soc.123:8101-8108,2001)。虽然优选荧光标记，对于本领域的普通技术人员来说其他形式的可检测标记也明显适用。例如微颗粒，包括量子点(Empodocles等人,Nature 399:126-130,1999)、金纳米颗粒(Reichert等人,Anal.Chem.72:6025-6029,2000)和微珠(Lacoste等人,Proc.Natl.Acad.Sci USA 97(17):9461-9466,2000)也都可以使用。
本申请也可以使用多组分标记。多组分标记是依赖于与用于检测的另外化合物的相互作用的标记。在生物学中最常用的多组分标记是生物素-链霉亲和素系统。生物素用作与核苷酸或修饰的核苷酸相连接的标记。然后单独加入链霉亲和素使检测发生。可以使用其他多组分系统。例如，二硝基苯酚具有可商业购得的可用于检测的荧光抗体。
在本文描述的某些实施方案中，可以通过亲和试剂(如抗体、适体、Affimer、Knottin)的引入使得修饰的核苷酸或核苷分子携带上文描述的可检测标记，所述亲和试剂可以特异性识别并结合所述修饰的核苷酸或核苷分子的表位，具体原理详见WO2018129214A1。WO2018129214A1中的全部相关内容引入本申请中。
在本文描述的另外一些实施方案中，可以将修饰的核苷酸或核苷分子与上文描述的可检测标记相连接。在某些这类实施方案中，所用的连接基团可裂解。使用可裂解的连接基团确保了在需要时所述标记能够在检测后被除去，这避免了与随后并入的任何标记的核苷酸或核苷的任何干扰信号。
在另一些实施方案中，所使用的连接基团是不可裂解的。因为在并入了本发明的标记核苷酸的每个情况中，不需要随后并入核苷酸，因此不需要将标记从核苷酸中除去。
本领域技术人员了解双脱氧核苷三磷酸酯在Sanger测序法及相关方案(Sanger型)中的效用，其依赖于在特定类型核苷酸处的随机链终止。本申请中的核苷酸在Sanger法及相关方案中具有效用，因为通过使用ddNTPs实现的相同效果可以通过使用本文描述的3’-OH阻断基团来实现：均防止了后续核苷酸的掺入。同样的，本申请中的修饰的核苷和核苷酸在高通量测序中也具有效用，特别是在基于边合成边测序的高通量测序平台中，使用本申请的可逆阻断核苷酸及其类似物，可在测序过程中逐个检测掺入的核苷酸类型。
可裂解的连接基团是本领域中熟知的，并且可应用常规化学将连接基团与核苷酸或修饰的核苷酸和标记相连。可通过任何适合的方法裂解所述连接基团，包括暴露于酸、碱、亲核试剂、亲电试剂、自由基、金属、还原剂或氧化剂、光、温度、酶等。还可以使用用于断裂3’-O-保护基键的相同催化剂裂解本文中讨论的连接基团。如Greene&Wuts,Protective Groups in Organic Synthesis(有机合成中的保护基),John Wiley&Sons中所公开的，合适的连接基团可修改自标 准的化学保护基。Guillier等人(Chem.Rev.100:2092-2157,2000)中还公开了用于固相合成的合适的可裂解的连接基团。
使用术语“可裂解的连接基团”并非意味着需要除去整个连接基团，例如，从核苷酸或修饰的核苷酸中除去。当可检测标记与核苷酸或修饰的核苷酸相连接时，核苷裂解位点可位于连接基团上的位置，该位置能够确保在裂解后一部分的连接基团仍与所述核苷酸或修饰的核苷酸保持连接。
当可检测标记与核苷酸或修饰的核苷酸相连接时，连接基团可以连接在核苷酸或修饰的核苷酸上的任何位置上，只要Watson-Crick碱基配对仍然能够进行。
A.亲电裂解的连接基团
亲电裂解的连接基团典型地被质子所裂解，并包括对酸敏感的裂解。合适的连接基团包括修饰的苄基系统，诸如三苯甲基、对烃氧基苄基酯和对烃氧基苄基酰胺。其他适合的连接基团包括叔丁氧羰基(Boc)基团和缩醛系统。
为制备合适的连接分子，还可以考虑在硫缩醛或其他含硫保护基的裂解中使用诸如镍、银或汞的亲硫金属。
B.亲核裂解的连接基团
在连接分子的制备中，亲核裂解也是被公认的方法。可以使用在水中不稳定的基团(即，能够在碱性pH值下简单地裂解)，例如酯类，以及对非水性亲核试剂不稳定的基团。氟离子可用于裂解诸如三异丙基硅烷(TIPS)或叔丁基二甲基硅烷(TBDMS)的基团中的硅氧键。
C.可光解的连接基团
可光解的连接基团在糖化学中被广泛使用。优选地，激活裂解所需的光不影响修饰的核苷酸中的其他组分。例如，如果使用荧光团作为标记，优选地，该荧光团吸收与裂解所述连接分子所需的光不同波长的光。适合的连接基团包括那些基于O-硝基苄基化合物和硝基藜芦基化合物的连接基团。也可以使用基于安息香化学的连接基团(Lee等人,J.Org.Chem.64:3454-3460,1999)。
D.还原条件下的裂解
已知多种对还原裂解敏感的连接基团。使用基于钯催化剂的催化氢化已用于裂解苄基和苄氧羰基基团。二硫键还原也为本领域所知。
E.氧化条件下的裂解
基于氧化的方法为本领域所公知。这些方法包括对烃氧基苄基的氧化以及硫和硒连接基团的氧化。使用碘溶液(aqueous iodine)来使二硫化物和其他基于硫或硒的连接基团裂解也在本发明的范围内。
F.安全拉手型连接基团
安全拉手型连接基团(safety-catch linker)为那些在两步中裂解的连接基团。在优选的系统中，第一步是反应性亲核中心的产生，随后的第二步涉及分子内环化，这导致裂解。例如，可以用肼或光化学方法处理乙酰丙酸酯连接来释放活性的胺，然后所述胺被环化以使分子中其他位置的酯裂解(Burgess等人,J.Org.Chem.62:5165-5168,1997)。
G.经消除机理裂解
也可以使用消除反应。可以使用诸如芴甲氧羰基和氰基乙基的基团的碱催化的消除以及烯丙基系统的钯催化的还原消除。
在某些实施方案中，连接基团可包含间隔单元。连接基团的长度并不重要，只要所述标记与核苷酸保持足够的距离，以免干扰核苷酸与酶之间的相互 作用。
在某些实施方案中，连接基团可由与3’-OH保护基类似的功能组成。这会使脱保护和脱保护方法更加有效，因为仅需要单一处理就除去标记和保护基。特别优选的连接基团是可通过膦裂解的含叠氮化物的连接基团。
下面结合具体实施例对本发明进行进一步的解释说明。如无特别说明，以下实施例所使用的试剂均可以常规市购获得。另外，对于3'-OH的每一种修饰，如果制备了核苷酸类似物dTTP、dATP、dCTP、dGTP中的一种或几种，则本领域技术人员可以毫无疑义地制备获得剩余的一种或几种核苷酸类似物。
一、制备实施例
实施例一
目标产物结构为
Figure PCTCN2021125262-appb-000216
(1)步骤1
Figure PCTCN2021125262-appb-000217
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取T核苷(500mg,1.4mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-N-18102)和叠氮保护基(268mg,1.4mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-H-20001)分别加入圆底烧瓶中，注射器抽取20mL无水二氯甲烷作溶剂，室温条件下搅拌使其溶解。称取DCC(435mg,2.1mmol,1.5eq)和DMAP(20mg,0.14mmol,0.1eq)，分别加入反应液中，氮气球做气体保护，室温条件下继续反应约4h。每隔半小时采用TLC监测反应，直至原料基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane/EA＝10/1～1/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为95％。
1H NMR(400MHz,CDCl 3)δ8.41(1H,s)；7.97(1H,m)；7.61(3H,m)；7.40(1H,m)； 7.27(1H,s)；6.45(1H,m)；5.67(1H,m)；5.50(1H,m)；4.28(1H,m)；4.02(1H,m)；2.59(1H,m)；2.24(1H,m)；1.96(3H,s)；1.55(3H,d,J＝6.8Hz)；0.97(9H,s)；0.10(6H,s).
(2)步骤2
Figure PCTCN2021125262-appb-000218
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取步骤1得到的产物(750mg,1.4mmol,1eq)加入圆底烧瓶中。注射器抽取20mL无水THF作溶剂，搅拌使其溶解后，注射器抽取1mol/L的TBAF溶液(1.8mL,1.8mmol,1.3eq),冰浴条件下缓慢滴加入反应液中。反应瓶移至室温条件下继续搅拌约2h。每隔半小时采用TLC监测反应直至原料点基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane/EA＝10/1～1/1转二氯甲烷：无水甲醇＝100/1～10/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为70％。 1H NMR(400MHz,DMSO-d 6)δ11.34(1H,s)；7.89(1H,m)；7.78(1H,s)；7.70–7.62(2H,m)；7.50-7.46(1H,m)；6.27–6.24(1H,m)；5.58-5.55(1H,m),5.26(1H,t)；4.19(1H,br.)；3.71(2H,br.)；1.79(3H,d)；1.49(3H,d,J＝6.8Hz).
(3)步骤3
Figure PCTCN2021125262-appb-000219
准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶1号，称取步骤2中制备得到的产物(350mg,0.85mmol,1eq),质子海绵(363.5mg,1.7mmol,2eq)，加入两口瓶1号。准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶2号，称取三丁基焦磷酸铵(932.5mg,1.7mmol,2eq)加入两口瓶2号。将两口瓶1号中注入氩气，加入15mL磷酸三甲酯，搅拌使其充分溶解。反应转移至0℃环境下，注射器抽取三氯氧磷(120uL,1.3mmol,1.5eq)，缓慢加入反应体系。保持0℃搅拌1.5h。将反应瓶2注入氩气，用注射器抽取2mL无水DMF和DIPEA(740uL,4.25mmol,5eq)，0℃条件下搅拌使其溶解。用注射器将反应 瓶1中的液体转移至反应瓶2中。保持0℃，继续搅拌3h后，加入20mL TEAB溶液(0.1mol/L)淬灭反应。反应液用50mL去离子水稀释，然后用准备好的DEAE树脂柱梯度冲洗分离(H 2O:TEAB(1mol/L)＝10/1～0/1，DEAE柱直径4.5cm，装柱高度8cm)。分离得到的溶液分别用HPLC和MS鉴定，收集含有三磷酸化产物的溶液并用旋蒸仪真空浓缩到10mL后，用制备液相色谱分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中，然后用冻干机冻干至产物为粉末状固体。MW＝655。本步骤产率为50％。
1H NMR(400MHz,DMSO-d 6)δ11.32(s,1H),7.97(s,1H),7.93–7.88(m,1H),7.72–7.60(m,2H),7.52–7.46(m,1H),6.40–6.29(m,1H),5.64–5.54(m,2H),4.33(s,1H),4.17–4.01(m,2H),2.54-2.50(m,1H),2.45–2.36(m,1H),1.85(s,3H),1.49(dd,J＝6.7,1.0Hz,3H).
实施例二
目标产物结构为
Figure PCTCN2021125262-appb-000220
(1)步骤1
Figure PCTCN2021125262-appb-000221
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取G核苷(600mg,1.58mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-N-18103)和叠氮保护基(302mg,1.58mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-H-20001)分别加入圆底烧瓶中，注射器抽取20mL无水二氯甲烷作溶剂，室温条件下搅拌使其溶解。称取DCC(488mg,2.37mmol,1.5eq)和DMAP(20mg,0.16mmol,0.1eq)，分别加入反应液中，氮气球做气体保护，室温条件下继续反应约4h。采用TLC每隔半小时监测反应，直至原料基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为95％。
1H NMR(500MHz,DMSO-d 6)δ10.65(s,1H),7.99–7.84(m,2H),7.79–7.61(m, 2H),7.57–7.45(m,1H),6.47(s,2H),6.20(ddd,J＝9.1,5.8,3.6Hz,1H),5.65–5.49(m,2H),4.27(qd,J＝4.8,1.9Hz,1H),3.86(dd,J＝4.6,2.2Hz,2H),2.99–2.80(m,1H),2.78–2.61(m,1H),1.50(d,J＝6.7Hz,3H),0.88(s,9H),0.07(dd,J＝4.5,1.3Hz,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000222
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取步骤1得到的产物(850mg,1.54mmol,1eq)加入圆底烧瓶中。注射器抽取20mL无水THF作溶剂，室温搅拌使其溶解后，注射器抽取1mol/L的TBAF溶液(2.0mL,2.0mmol,1.3eq)，冰浴条件下缓慢滴加入反应液中。反应瓶移至室温条件下继续搅拌约2h。采用TLC每隔半小时监测反应，直至原料点基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane/EA＝10/1～1/1转二氯甲烷/无水甲醇＝100/1～10/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为70％。 1H NMR(500MHz,DMSO-d 6)δ10.68(H,s),7.99(1H,s),7.91(1H,m),7.67(2H,m),7.46(1H,m),6.52(2H,s),6.17(1H,m),5.58(2H,m),5.25(1H,m),4.23(1H,t),3.66(2H,t),2.89(1H,m),2.61(1H,m),1.49(3H,d,J＝6.8Hz).
(3)步骤3
Figure PCTCN2021125262-appb-000223
准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶1号，称取步骤2中制备得到的产物(375mg,0.85mmol,1eq)，质子海绵(363.5mg,1.7mmol,2eq)，加入两口瓶1号。准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶2号，称取三丁基焦磷酸铵(932.5mg,1.7mmol,2eq)加入两口瓶2号。将两口瓶1号中注入氩气，加入15mL磷酸三甲酯，搅拌使其充分溶解。反应转移至0℃环境下，注射器抽取三氯氧磷(120uL,1.3mmol,1.5eq)，缓慢加入反应体系。保持0℃搅拌1.5h。将反应瓶2注入氩气，用注射器抽取5mL无水DMF和DIPEA(740uL,4.25mmol,5eq)，0℃条件下搅拌使其溶解。用注射器将反应 瓶1中的液体转移至反应瓶2中。保持0℃，继续搅拌3h后，加入20mL TEAB溶液(0.1mol/L)淬灭反应。反应液用50mL去离子水稀释，然后用准备好的DEAE树脂柱梯度冲洗分离(H 2O/TEAB(1mol/L)＝10/1～0/1,DEAE柱直径4.5cm，装柱高度8cm)。收集含有三磷酸化产物的溶液并用旋蒸仪真空浓缩后，用制备液相色谱分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中，然后用冻干机冻干至产物为粉末状固体。MW＝680。本步骤反应产率为45％。
1H NMR(400MHz,DMSO-d 6)δ11.12(s,1H),8.05(d,J＝3.1Hz,1H),7.93(d,J＝7.6Hz,1H),7.73–7.62(m,2H),7.50(t,J＝7.6Hz,1H),6.87(s,2H),6.25-6.19(m,1H),5.72(d,J＝4.9Hz,1H),5.61(q,J＝6.6Hz,1H),4.36-4.33(m,1H),4.25-4.18(m,1H),4.06-4.01(m,1H),3.27–3.12(m,1H),2.60–2.52(m,1H),1.50(dd,J＝6.7,2.0Hz,3H).
实施例三
目标产物结构为
Figure PCTCN2021125262-appb-000224
(1)步骤1
Figure PCTCN2021125262-appb-000225
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取C核苷(600mg,1.56mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-N-18104)和叠氮保护基(297mg,1.56mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20001)分别加入圆底烧瓶中，注射器抽取20mL无水二氯甲烷作溶剂，室温条件下搅拌使其溶解。称取DCC(480mg,2.34mmol,1.5eq)和DMAP(20mg,0.16mmol,0.1eq)，分别加入反应液中，氮气球做气体保护，室温条件下继续反应约4h。采用TLC每隔半小时监测反应，直至原料基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1，硅胶柱直径3.5cm，装 柱高度10cm)。本步骤反应产率为95％。
1H NMR(400MHz,DMSO-d 6)δ10.91(1H,s)；8.23(1H,d,J＝7.52Hz)；7.89(1H,d,J＝6.80Hz)；7.66(2H,m)；7.48(1H,m)；7.23(1H,d,J＝7.48)；6.19(1H,t),5.55(1H,m)；5.44(1H,d,J＝6.04Hz),4.42(1H,t)；3.92(2H,m)；2.74(1H,m)；2.32(1H,m)；1.47(3H,d,J＝6.7Hz)；0.86(9H,s)；0.09(6H,s).
(2)步骤2
Figure PCTCN2021125262-appb-000226
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取步骤1得到的产物(860mg,1.54mmol,1eq)加入圆底烧瓶中。注射器抽取20mL无水THF作溶剂，搅拌使其溶解后，注射器抽取1mol/L的TBAF溶液(2.0mL,2.0mmol,1.3eq)，冰浴条件下缓慢滴加入反应液中。反应瓶移至室温条件下继续搅拌约2h。采用TLC每隔半小时监测反应直至原料点基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1转二氯甲烷：无水甲醇＝100/1～10/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为70％。 1H NMR(400MHz,DMSO-d 6)δ10.90(1H,s)；8.34(1H,d,J＝7.48Hz)；7.89(1H,d,J＝7.84Hz)；7.66(2H,m)；7.48(1H,m)；7.23(1H,d,J＝7.48Hz)；6.22(1H,t)；5.57(1H,m)；5.47(1H,d,J＝6.00Hz)；5.25(1H,s)；4.32(1H,m)；3.72(1H,s)；2.67(1H,m)；2.31(1H,m)；1.45(3H,d,J＝6.7Hz).
(3)步骤3
Figure PCTCN2021125262-appb-000227
准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶1号，称取步骤2中制备得到的产物(377mg,0.85mmol,1eq)，质子海绵(363.5mg,1.7mmol, 2eq)，加入两口瓶1号。准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶2号，称取三丁基焦磷酸铵(932.5mg,1.7mmol,2eq)加入两口瓶2号。将两口瓶1号中注入氩气，加入15mL磷酸三甲酯，搅拌使其充分溶解。反应转移至0℃环境下，注射器抽取三氯氧磷(120uL,1.3mmol,1.5eq)，缓慢加入反应体系。保持0℃搅拌1.5h。将反应瓶2注入氩气，用注射器抽取2mL无水DMF和DIPEA(740uL,4.25mmol,5eq)，0℃条件下搅拌使其溶解。保持0℃，继续搅拌3h后，加入20mLTEAB溶液(0.1mol/L)淬灭反应。反应液用50mL去离子水稀释，然后用准备好的DEAE树脂柱梯度冲洗分离(H 2O:TEAB(1mol/L)＝10/1～0/1，DEAE柱直径4.5cm，装柱高度8cm)。收集含有三磷酸化产物的溶液并用旋蒸仪真空浓缩，用制备液相色谱分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中，然后用冻干机冻干至产物为粉末状固体。MW＝684。本步骤反应产率为40％。
(4)步骤4
Figure PCTCN2021125262-appb-000228
准备一个干燥的装有磁子的100mL圆底烧瓶，称取步骤3中制备得到的三磷酸化产物(500mg,0.73mmol,1eq)加入5ml去离子水，室温条件下搅拌使其充分溶解，缓慢加入25％氨水溶液(2.5g,36.5mmol,50eq)。室温条件下搅拌约6h。用旋蒸仪真空除去溶液中的氨水，然后用去离子水稀释至约10mL。制备液相分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中，用冻干机冻干至产物为粉末状固体。MW＝642。本步骤反应产率为90％。
1H NMR(400MHz,DMSO-d 6)δ7.99(dd,J＝7.5,2.2Hz,1H),7.90(d,J＝7.8Hz,1H),7.71-7.63(m,2H),7.54–7.42(m,1H),7.30(s,1H),7.10(s,1H),6.41–6.28(m,1H),5.81(d,J＝7.5Hz,1H),5.65–5.50(m,2H),4.33(s,1H),4.18–3.96(m,2H),2.46–2.26(m,2H),1.49(dd,J＝6.7,1.4Hz,3H).
实施例四
目标产物结构为
Figure PCTCN2021125262-appb-000229
(1)步骤1
Figure PCTCN2021125262-appb-000230
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取A核苷(600mg,1.65mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-N-18101)和叠氮保护基(314mg,1.65mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-H-20001)分别加入圆底烧瓶中，注射器抽取20mL无水二氯甲烷作溶剂，室温条件下搅拌使其溶解。称取DCC(508mg,2.5mmol,1.5eq)和DMAP(24mg,0.17mmol,0.1eq)，分别加入反应液中，氮气球做气体保护，室温条件下继续反应约4h。采用TLC每隔半小时监测反应，直至原料基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为95％。
1H NMR(500MHz,CDCl 3)δ8.36(1H,s)；8.28(1H,d,J＝4.4Hz)；7.98(1H,m)；7.65(2H,m)；7.41(1H,m)；6.60(1H,t)；6.0(2H,br.),5.68(2H,m)；4.40(1H,br.)；4.01(2H,br.)；1.93(1H,m)；1.68(1H,m)；1.56(3H,d,J＝6.7Hz)；0.94(9H,s)；0.15(6H,s).
(2)步骤2
Figure PCTCN2021125262-appb-000231
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取步骤1得到的产物(828mg,1.54mmol,1eq)加入圆底烧瓶中。注射器抽取20mL无水THF作溶剂，搅拌使其溶解后，注射器抽取1mol/L的TBAF溶液(2.0mL,2.0mmol,1.3eq)，冰浴条件下缓慢滴加入反应液中。反应瓶移至室温条件下继续搅拌约2h。采用TLC每隔半小时监测反应，直至原料点基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1转二氯甲烷：无水甲醇＝100/1～10/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为70％。 1H NMR(500MHz,DMSO-d 6)δ10.66(s,1H),8.00(d,J＝1.5Hz,1H),7.92(ddd,J＝8.0,3.0,1.4Hz,1H),7.82–7.59(m,2H),7.51(t,J＝7.5Hz,1H),6.46(s,2H),6.20(dt,J＝9.5,5.4Hz,1H),5.58(ddd,J＝15.6,6.2,2.5Hz,2H),5.21(td,J＝5.6,1.8Hz,1H),4.24(dd,J＝4.3,1.7Hz,1H),3.77–3.55(m,2H),2.91(qd,J＝9.1,5.8Hz,1H),2.62(dt,J＝13.7,6.6Hz,1H),1.51(dd,J＝6.7,1.7Hz,3H).
(3)步骤3
Figure PCTCN2021125262-appb-000232
准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶1号，称取步骤2中制备得到的产物(360mg,0.85mmol,1eq),质子海绵(363.5mg,1.7mmol,2eq)，加入两口瓶1号。准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶2号，称取三丁基焦磷酸铵(932.5mg,1.7mmol,2eq)加入两口瓶2号。将两口瓶1号中注入氩气，加入15mL磷酸三甲酯，搅拌使其充分溶解。反应转移至0℃环境下，注射器抽取三氯氧磷(120uL,1.3mmol,1.5eq)，缓慢加入反应体系。保持0℃搅拌1.5h。将反应瓶2注入氩气，用注射器抽取5mL无水DMF和DIPEA(740uL,4.25mmol,5eq)，0℃条件下搅拌使其溶解。保持0℃，继续搅拌3h后，加入20mL TEAB溶液(0.1mol/L)淬灭反应。反应液用50mL去离子水稀释，然后用准备好的DEAE树脂柱梯度冲洗分离(H 2O:TEAB(1mol/L)＝10/1～0/1，DEAE柱直径4.5cm，装柱高度8cm)。收集含有三磷酸化产物的溶液并用旋蒸仪真空浓缩后，用制备液相色谱分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪 真空浓缩后，转移到塑料离心管中，然后用冻干机冻干至产物为粉末状固体。MW＝664.。本步骤反应产率为40％。
1H NMR(400MHz,DMSO-d 6)δ8.62(d,J＝3.6Hz,1H),8.15(d,J＝1.5Hz,1H),7.97(d,J＝7.8Hz,1H),7.72-7.65(m,2H),7.55–7.47(m,1H),7.29(s,2H),6.51-6.46(m,1H),5.74(d,J＝5.3Hz,1H),5.68–5.58(m,1H),4.44-4.41(m,1H),4.14–4.01(m,2H),3.19-3.11(m,1H),2.75–2.65(m,1H),1.51(dd,J＝6.7,1.5Hz,3H).
实施例五
目标产物结构为
Figure PCTCN2021125262-appb-000233
(1)步骤1
Figure PCTCN2021125262-appb-000234
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取T核苷(500mg,1.40mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-N-18102)和二硫保护基(340mg,1.40mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21003)分别加入圆底烧瓶中，注射器抽取20mL无水二氯甲烷作溶剂，室温条件下搅拌使其溶解。称取DCC(433mg,2.10mmol,1.5eq)和DMAP(17mg,0.14mmol,0.1eq)，分别加入反应液中，氮气球做气体保护，室温条件下继续反应约6h。采用TLC每隔半小时监测反应，直至原料基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1，硅胶柱直径3.5cm，装柱高度10cm)，本步骤反应产率为85％。 1H NMR(400MHz,CDCl 3)δ8.61(d,J＝4.7Hz,1H),7.84(t,J＝8.3Hz,1H),7.63–7.57(m,2H),7.52(t,J＝7.6Hz,1H),7.33(dd,J＝11.0,4.1Hz,1H),6.47-6.40(m,1H),5.52–5.45(m,1H),5.31–5.20(m,1H),4.30(d,J＝21.8Hz,1H),4.07–3.94(m,2H),3.51-3.44(m,1H),2.63–2.53(m,1H),2.29-2.20(m,2H),1.94(s,3H),1.69(d,J＝7.0Hz,3H),1.12–1.07(m,3H),0.96(s,9H),0.17(s,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000235
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取步骤1得到的产物(690mg,1.19mmol,1eq)加入圆底烧瓶中。注射器抽取20mL无水THF作溶剂，室温搅拌使其溶解后，注射器抽取1mol/L的TBAF溶液(1.6mL,1.6mmol,1.3eq)，冰浴条件下缓慢滴加入反应液中。反应瓶移至室温条件下继续搅拌约2h。采用TLC每隔半小时监测反应，直至原料点基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane/EA＝5/1～1/1转二氯甲烷/无水甲醇＝100/1～10/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为80％。
1H NMR(500MHz,CDCl 3)δ8.57(s,1H),7.87–7.80(m,1H),7.60(dd,J＝7.8,1.9Hz,1H),7.57–7.50(m,2H),7.39–7.29(m,1H),6.35–6.25(m,1H),5.64–5.56(m,1H),5.23(p,J＝7.1Hz,1H),4.29(dd,J＝14.3,2.4Hz,1H),4.02(dd,J＝4.9,2.5Hz,2H),2.59-2.55(m,2H),2.29–2.19(m,2H),1.94(d,J＝0.6Hz,3H),1.70(d,J＝7.0Hz,3H),1.11(td,J＝7.4,2.6Hz,3H).
(3)步骤3
Figure PCTCN2021125262-appb-000236
准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶1号，称取步骤2中制备得到的产物(396mg,0.85mmol,1eq)，质子海绵(363.5mg,1.7mmol,2eq)，加入两口瓶1号。准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶2号，称取三丁基焦磷酸铵(932.5mg,1.7mmol,2eq)加入两口瓶2号。将两口瓶1号中注入氩气，加入15mL磷酸三甲酯，搅拌使其充分溶解。反应转移至0℃环境下，注射器抽取三氯氧磷(120uL,1.3mmol,1.5eq)，缓慢加入反应体系。保持0℃搅拌1.5h。将反应瓶2注入氩气，用注射器抽取5mL无水DMF和DIPEA(740uL,4.25mmol,5eq)，0℃条件下搅拌使其溶解。保持0℃，继续搅拌3h后，加入20mL TEAB溶液(0.1mol/L)淬灭反应。反应液用50mL去离子水稀释，然后用准备好的DEAE树脂柱梯度冲洗分离(H 2O:TEAB(1mol/L)＝10/1～0/1，DEAE柱直径4.5cm，装柱高度8cm)。收集含有三磷酸化产物的溶液并用旋蒸仪真空浓缩后，用制备液相色谱分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中，然后用冻干机冻干至产物为粉末状固体。MW ＝706。本步骤反应产率为40％。
1H NMR(400MHz,DMSO-d 6)δ11.30(s,1H),7.96(dd,J＝6.0,1.0Hz,1H),7.86(d,J＝7.7Hz,1H),7.68–7.56(m,2H),7.46-7.41(m,1H),6.38–6.27(m,1H),5.60(d,J＝5.3Hz,1H),5.16-5.11(m,1H),4.31(d,J＝20.8Hz,1H),4.18–4.00(m,2H),2.58–2.51(m,1H),2.44–2.22(m,3H),1.85(s,3H),1.65(d,J＝7.0Hz,3H),1.09–1.03(m,3H).
实施例六
目标产物结构为
Figure PCTCN2021125262-appb-000237
(1)步骤1
Figure PCTCN2021125262-appb-000238
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取G核苷(600mg,1.58mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-N-18103)和二硫保护基(605mg,1.58mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-H-21003)分别加入圆底烧瓶中，注射器抽取20mL无水二氯甲烷作溶剂，室温条件下搅拌使其溶解。称取DCC(488mg,2.37mmol,1.5eq)和DMAP(20mg,0.16mmol,0.1eq)，分别加入反应液中，氮气球做气体保护，室温条件下继续反应约4h。采用TLC每隔半小时监测反应，直至原料基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/2，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为80％。
1H NMR(500MHz,CDCl 3)δ12.00(s,1H),7.93(t,J＝9.2Hz,1H),7.88–7.83(m,1H),7.59(dd,J＝13.6,8.0Hz,1H),7.53(t,J＝7.6Hz,1H),7.33(t,J＝7.5Hz,1H),6.37-6.33(m,2H),5.62(s,1H),5.30–5.22(m,1H),4.36(dd,J＝10.9,1.5Hz,1H),4.01–3.89(m,2H),3.51–3.40(m,1H),2.78–2.66(m,2H),2.33–2.22(m,2H),1.70(d,J＝6.9Hz,3H),1.13(d,J＝2.5Hz,3H),0.92(s,9H),0.12(s,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000239
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取步骤1得到的产物(932mg,1.54mmol,1eq)加入圆底烧瓶中。注射器抽取20mL无水THF作溶剂，搅拌使其溶解后，注射器抽取1mol/L的TBAF溶液(2.0mL,2.0mmol,1.3eq),冰浴条件下缓慢滴加入反应液中。反应瓶移至室温条件下继续搅拌约2h。采用TLC每隔半小时监测反应，直至原料点基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1转二氯甲烷：无水甲醇＝100/1～10/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为70％。
1H NMR(400MHz,CDCl 3)δ11.71(s,1H),7.97(d,J＝7.8Hz,1H),7.62(d,J＝7.9Hz,1H),7.52(t,J＝7.5Hz,2H),7.32(t,J＝7.2Hz,1H),6.36–6.02(m,1H),5.41–5.22(m,2H),3.03–2.97(m,3H),2.34(dd,J＝14.6,7.2Hz,2H),2.26–2.20(m,1H),2.06–1.97(m,1H),1.69(d,J＝6.9Hz,3H),1.14(t,J＝7.3Hz,3H).
(3)步骤3
Figure PCTCN2021125262-appb-000240
准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶1号，称取步骤2中制备得到的产物(417mg,0.85mmol,1eq),质子海绵(363.5mg,1.7mmol,2eq)，加入两口瓶1号。准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶2号，称取三丁基焦磷酸铵(932.5mg,1.7mmol,2eq)加入两口瓶2号。将两口瓶1号中注入氩气，加入15mL磷酸三甲酯，搅拌使其充分溶解。反应转移至0℃环境下，注射器抽取三氯氧磷(120uL,1.3mmol,1.5eq)，缓慢加入反应体系。保持0℃搅拌1.5h。将反应瓶2注入氩气，用注射器抽取5mL无水DMF和DIPEA(740uL,4.25mmol,5eq)，0℃条件下搅拌使其溶解。保持0℃，继续搅拌3h后，加入20mL TEAB溶液(0.1mol/L)淬灭反应。反应液继续搅拌0.5h。反应液用50mL去离子水稀释，然后用准备好的DEAE树脂柱梯度冲洗分离(H 2O:TEAB(1mol/L)＝10/1～0/1，DEAE柱直径4.5cm，装柱高度8cm)。收集含有三磷酸化产物的溶液并用旋蒸仪真空浓缩到10mL后，用制备液相色谱分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中，然后用冻干机冻干至产物为粉末状固体。MW＝706。本步骤反应产率为40％。
1H NMR(400MHz,DMSO-d 6)δ10.82(s,1H),8.03(d,J＝5.5Hz,1H),7.88(d,J＝ 7.8Hz,1H),7.69–7.58(m,2H),7.46-7.42(m,1H),6.72(s,2H),6.25-6.18(m,1H),5.70(t,J＝5.0Hz,1H),5.22–5.10(m,1H),4.40–4.27(m,1H),4.21(dd,J＝11.0,5.0Hz,1H),4.01(dd,J＝7.0,3.7Hz,1H),2.59–2.52(m,1H),2.48–2.30(m,3H),1.65(d,J＝7.0Hz,3H),1.07(t,J＝5.1Hz,3H).
实施例七
目标产物结构为
Figure PCTCN2021125262-appb-000241
(1)步骤1
Figure PCTCN2021125262-appb-000242
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取C核苷(608mg,1.58mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-N-18104)和二硫保护基(605mg,1.58mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21003)分别加入圆底烧瓶中，注射器抽取20mL无水二氯甲烷作溶剂，室温条件下搅拌使其溶解。称取DCC(488mg,2.37mmol,1.5eq)和DMAP(20mg,0.16mmol,0.1eq)，分别加入反应液中，氮气球做气体保护，室温条件下继续反应约4h。采用TLC每隔半小时监测反应，直至原料基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/2，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为90％。
1H NMR(500MHz,CDCl 3)δ9.53(s,1H),8.35(d,J＝6.0Hz,1H),7.87(ddd,J＝7.8,4.4,1.3Hz,1H),7.59(t,J＝7.9Hz,1H),7.54-7.50(m,1H),7.41(d,J＝6.0Hz,1H),7.32(td,J＝7.7,1.2Hz,1H),6.44-6.38(m,1H),5.50-5.48(m,1H),5.29–5.21(m,1H),4.45–4.36(m,1H),4.01(s,2H),3.50–3.44(m,1H),2.93–2.86(m,1H),2.24–2.12(m,2H),1.69(dd,J＝7.0,0.6Hz,3H),1.11(td,J＝7.4,3.3Hz,3H),0.93(s,9H),0.13(s,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000243
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取步骤1得到的产物(938mg,1.54mmol,1eq)加入圆底烧瓶中。注射器抽取20mL无水THF作溶剂，搅拌使其溶解后，注射器抽取1mol/L的TBAF溶液(2.0mL,2.0mmol,1.3eq)，冰浴条件下缓慢滴加入反应液中。反应瓶移至室温条件下继续搅拌约2h。采用TLC每隔半小时监测反应直至原料点基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1转二氯甲烷：无水甲醇＝100/1～10/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为80％。
1H NMR(400MHz,CDCl 3)δ9.50(s,1H),8.39(s,1H),7.85(t,J＝7.2Hz,1H),7.61-7.51(m,3H),7.34(t,J＝7.3Hz,1H),6.30(s,1H),5.63(d,J＝2.8Hz,1H),5.27-5.19(m,1H),4.40(d,J＝15.5Hz,1H),4.15–3.94(m,2H),3.40(s,1H),2.83(td,J＝14.3,4.8Hz,1H),2.60–2.45(m,1H),2.27–2.14(m,2H),1.69(d,J＝6.8Hz,3H),1.10(q,J＝6.8Hz,3H).
(3)步骤3
Figure PCTCN2021125262-appb-000244
准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶1号，称取步骤2中制备得到的产物(421mg,0.85mmol,1eq)，质子海绵(363.5mg,1.7mmol,2eq)，加入两口瓶1号。准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶2号，称取三丁基焦磷酸铵(932.5mg,1.7mmol,2eq)加入两口瓶2号。将两口瓶1号中注入氩气，加入15mL磷酸三甲酯，搅拌使其充分溶解。反应转移至0℃环境下，注射器抽取三氯氧磷(120uL,1.3mmol,1.5eq)，缓慢加入反应体系。保持0℃搅拌1.5h。将反应瓶2注入氩气，用注射器抽取5mL无水DMF和DIPEA(740uL,4.25mmol,5eq)，0℃条件下搅拌使其溶解。保持0℃，继续搅拌3h后，加入20mL TEAB溶液(0.1mol/L)淬灭反应。反应液继续搅拌0.5h。反应液用50mL去离子水稀释，然后用准备好的DEAE树脂柱梯度冲洗分离(H 2O:TEAB(1mol/L)＝10/1～0/1，DEAE柱直径4.5cm，装柱高度8cm)。收集含有三磷酸化产物的溶液并用旋蒸仪真空浓缩后，用制备液相色谱 分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中，然后用冻干机冻干至产物为粉末状固体。MW＝733。本步骤产率为45％。
(4)步骤4
Figure PCTCN2021125262-appb-000245
准备一个干燥的装有磁子的100mL圆底烧瓶，称取步骤3中制备得到的三磷酸化产物(537mg,0.73mmol,1eq)加入5ml去离子水，室温条件下搅拌使其充分溶解，加入25％氨水溶液(2.5g,36.5mmol,50eq)。室温条件下搅拌约2h，用旋蒸仪真空除去溶液中的氨水，然后用去离子水稀释至约10mL。制备液相分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中，然后用冻干机冻干至产物为粉末状固体。MW＝691。本步骤产率为90％。
1H NMR(400MHz,DMSO-d 6)δ7.97(dd,J＝7.4,6.2Hz,1H),7.85(d,J＝8.7Hz,1H),7.66–7.58(m,2H),7.45-7.41(m,1H),7.31(s,1H),7.10(s,1H),6.39–6.29(m,1H),5.81(d,J＝7.4Hz,1H),5.53(d,J＝5.0Hz,1H),5.17-5.10(m,1H),4.31(d,J＝19.8Hz,1H),4.13–4.01(m,2H),2.46–2.23(m,4H),1.65(d,J＝7.0Hz,3H),1.09–1.03(m,3H).
实施例八
目标产物结构为
Figure PCTCN2021125262-appb-000246
(1)步骤1
Figure PCTCN2021125262-appb-000247
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取A核苷(577mg,1.58mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-N-18101)和二硫保护基(605mg,1.58mmol,1eq)(北京欧凯纳斯生化科技有限公司，货号OK-H-21003)分别加入圆底烧瓶中，注射器抽取20mL无水二氯甲烷作溶剂，室温条件下搅拌使其溶解。称取DCC(488mg,2.37mmol,1.5eq)和DMAP(20mg,0.16mmol,0.1eq)，分别加入反应液中，氮气球做气体保护，室温条件下继续反应约4h。采用TLC每隔半小时监测反应直至原料基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/2，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为85％。
1H NMR(500MHz,CDCl 3)δ8.34(s,1H),8.24(d,J＝11.2Hz,1H),7.87(ddd,J＝7.8,4.4,1.3Hz,1H),7.60(d,J＝7.9Hz,1H),7.52(t,J＝7.6Hz,1H),7.32(td,J＝7.7,1.2Hz,1H),6.58(t,J＝7.1Hz,1H),6.21(s,2H),5.68–5.61(m,1H),5.27(p,J＝7.0Hz,1H),4.45–4.36(m,1H),4.04–3.95(m,2H),2.83–2.73(m,2H),2.31–2.18(m,2H),1.69(dd,J＝7.0,0.6Hz,3H),1.11(td,J＝7.4,3.3Hz,3H),0.93(s,9H),0.13(t,J＝1.7Hz,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000248
准备一个干燥的装有磁子和带氩气球橡皮塞的100mL圆底烧瓶，称取步骤1得到的产物(907mg,1.54mmol,1eq)加入圆底烧瓶中。注射器抽取20mL无水THF作溶剂，搅拌使其溶解后，注射器抽取1mol/L的TBAF溶液(2.0mL,2.0mmol,1.3eq)，冰浴条件下缓慢滴加入反应液中。反应瓶移至室温条件下继续搅拌约2h。采用TLC每隔半小时监测反应直至原料点基本消失。反应液用旋蒸仪浓缩后，用硅胶柱色谱法分离产物(Hexane：EA＝10/1～1/1转二氯甲烷：无水甲醇＝100/1～10/1，硅胶柱直径3.5cm，装柱高度10cm)。本步骤反应产率为80％。
1H NMR(500MHz,CDCl 3)δ8.37(s,1H),7.93(s,1H),7.89–7.85(m,1H),7.65–7.61(m,1H),7.56(td,J＝7.7,1.3Hz,1H),7.39–7.33(m,1H),6.37(dd,J＝9.3,5.3Hz,1H),6.02(s,2H),5.83(t,J＝5.5Hz,1H),5.31-5.23(m,1H),4.48(d,J＝14.2Hz,1H),4.09–3.97(m,2H),3.33-3.28(m,1H),2.63(dd,J＝14.0,4.9Hz,1H),2.41–2.31(m,2H),1.73(d,J＝7.0Hz,3H),1.17(td,J＝7.4,2.3Hz,3H).
(3)步骤3
Figure PCTCN2021125262-appb-000249
准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶1号，称取步骤2中制备得到的产物(404mg,0.85mmol,1eq)，质子海绵(363.5mg,1.7mmol,2eq)，加入两口瓶1号。准备一个干燥的装有磁子和带氩气气球三通阀的100mL两口瓶2号，称取三丁基焦磷酸铵(932.5mg,1.7mmol,2eq)加入两口瓶2号将两口瓶1号中注入氩气，加入15mL磷酸三甲酯，搅拌使其充分溶解。反应转移至0℃环境下，注射器抽取三氯氧磷(120uL,1.3mmol,1.5eq)，缓慢加入反应体系。保持0℃搅拌1.5h。将反应瓶2注入氩气，用注射器抽取5mL无水DMF和DIPEA(740uL,4.25mmol,5eq)，0℃条件下搅拌使其溶解。保持0℃，继续搅拌3h后，加入20mL TEAB溶液(0.1mol/L)淬灭反应。反应液继续搅拌0.5h。反应液用50mL去离子水稀释，然后用准备好的DEAE树脂柱梯度冲洗分离(H 2O:TEAB(1mol/L)＝10/1～0/1，DEAE柱直径4.5cm，装柱高度8cm)。收集含有三磷酸化产物的溶液并用旋蒸仪真空浓缩后，用制备液相色谱分离产物，梯度为(CH 3CN:TEAB(0.1mol/L)＝2/98～98/2)。分离得到的高纯度产物溶液用旋蒸仪真空浓缩后，转移到塑料离心管中然后用冻干机冻干至产物为粉末状固体。MW＝715。本步骤反应产率为40％。
1H NMR(400MHz,DMSO-d 6)δ8.60(s,1H),8.15(s,1H),8.05(d,J＝6.5Hz,1H),7.69(t,J＝8.2Hz,1H),7.60-7.54(m,2H),7.27(s,2H),6.25-6.18(m,1H),5.70(t,J＝5.0Hz,1H),5.22–5.10(m,1H),4.40–4.27(m,1H),4.21(dd,J＝11.0,5.0Hz,1H),4.01(dd,J＝7.0,3.7Hz,1H),2.59–2.52(m,1H),2.48–2.30(m,3H),1.65(d,J＝7.0Hz,3H),1.07(t,J＝5.1Hz,3H).
实施例九
(1)步骤1
Figure PCTCN2021125262-appb-000250
合成方法参考实施例四步骤1，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)烟酸(2-(1-azidoethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20002)，本步骤反应产率 为85％，MW＝539。
1H NMR(400MHz,DMSO-d 6)δ8.86(dd,J＝4.7,1.7Hz,1H),8.38(dt,J＝7.9,1.7Hz,1H),8.33(s,1H),8.16(d,J＝0.9Hz,1H),7.58(dd,J＝7.9,4.7Hz,1H),7.32(s,2H),6.47(ddd,J＝8.5,6.1,2.9Hz,1H),5.66(dq,J＝6.2,2.0Hz,1H),5.41–5.24(m,1H),4.44–4.23(m,1H),4.05–3.77(m,2H),3.13(ddd,J＝14.3,8.3,6.3Hz,1H),2.87–2.68(m,1H),1.61(d,J＝6.7Hz,3H),0.86(s,9H),0.04(d,J＝3.9Hz,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000251
合成方法参考实施例四步骤2，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)烟酸(2-(1-azidoethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20002)，本步骤反应产率为75％，MW＝425。
1H NMR(500MHz,DMSO-d 6)δ8.86(dd,J＝4.8,1.8Hz,1H),8.45–8.31(m,2H),8.16(d,J＝1.1Hz,1H),7.59(dd,J＝7.9,4.7Hz,1H),7.38(s,2H),6.47(ddd,J＝9.4,5.8,4.2Hz,1H),5.71–5.62(m,1H),5.57(ddd,J＝7.5,4.8,2.8Hz,1H),5.33(p,J＝6.7Hz,1H),4.34(ddt,J＝7.6,4.1,2.2Hz,1H),3.83–3.60(m,2H),3.09(ddd,J＝14.5,9.0,5.9Hz,1H),2.85–2.65(m,1H),1.61(dd,J＝6.7,2.7Hz,3H).
(3)步骤3
Figure PCTCN2021125262-appb-000252
合成方法参考实施例四步骤3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)烟酸(2-(1-azidoethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20002)，本步骤反应产率为40％，MW＝665。
1H NMR(400MHz,DMSO-d 6)δ8.85(d,J＝3.6Hz,1H),8.66(d,J＝3.4Hz,1H),8.40(d,J＝7.3Hz,1H),8.15(s,1H),7.58(dd,J＝7.9,4.8Hz,1H),7.31(s,2H),6.54 –6.45(m,1H),5.77(d,J＝5.1Hz,1H),5.37–5.28(m,1H),4.44(d,J＝4.5Hz,1H),4.14–4.02(m,2H),3.23–3.12(m,1H),2.76–2.66(m,1H),1.61(dd,J＝6.7,2.1Hz,3H).
实施例十
Figure PCTCN2021125262-appb-000253
合成方法参考实施例二步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)烟酸(2-(1-azidoethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20002))，最后一步反应产率为40％，MW＝681。
1H NMR(400MHz,DMSO-d 6)δ10.77(s,1H),8.84(d,J＝4.6Hz,1H),8.39–8.29(m,1H),8.02(d,J＝5.5Hz,1H),7.57(dd,J＝7.8,4.8Hz,1H),6.71(s,2H),6.28–6.16(m,1H),5.73(d,J＝4.9Hz,1H),5.35-5.28(m,1H),4.36(dd,J＝10.4,6.0Hz,1H),4.24–4.15(m,1H),4.01–3.97(m,1H),3.25–3.13(m,1H),2.61-2.55(m,1H),1.60(dd,J＝6.6,2.6Hz,3H).
实施例十一
Figure PCTCN2021125262-appb-000254
合成方法参考实施例三步骤1-4，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)烟酸(2-(1-azidoethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20002)，最后两步反应产率为30％，MW＝641。
1H NMR(400MHz,DMSO-d 6)δ8.83(d,J＝3.5Hz,1H),8.33(d,J＝7.8Hz,1H),7.95(dd,J＝7.4,2.9Hz,1H),7.55(dd,J＝7.9,4.7Hz,1H),7.30(s,1H),7.08(s,1H),6.37-6.34(m,1H),5.82(d,J＝7.4Hz,1H),5.54(d,J＝4.9Hz,1H),5.39–5.20(m,1H),4.39–4.32(m,1H),4.14–3.98(m,2H),2.47-2.41(m,1H),2.36–2.24(m,1H),1.59(dd,J＝6.7,1.7Hz,3H).
实施例十二
Figure PCTCN2021125262-appb-000255
合成方法参考实施例一步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)烟酸(2-(1-azidoethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20002)，最后一步反应产率为45％，MW＝656。
1H NMR(400MHz,DMSO-d 6)δ11.31(s,1H),8.84(dd,J＝4.6,1.1Hz,1H),8.34(d,J＝7.9Hz,1H),7.98(s,1H),7.56(dd,J＝7.9,4.8Hz,1H),6.39–6.32(m,1H),5.63(d,J＝5.2Hz,1H),5.33-5.26(m,1H),4.35(s,1H),4.15-4.04(m,2H),2.55-2.52(m,1H),2.46–2.38(m,1H),1.84(s,3H),1.58(dd,J＝6.6,1.3Hz,3H).
实施例十三
(1)步骤1
Figure PCTCN2021125262-appb-000256
合成方法参考实施例一步骤1，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)-4-硝基苯甲酸(2-(azidomethyl)-4-nitrobenzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21004)，该步骤反应产率为85％，MW＝560。
1H NMR(500MHz,CDCl 3)δ8.43(d,J＝2.3Hz,1H),8.34–8.23(m,2H),8.20(d,J＝8.6Hz,1H),7.57(d,J＝1.2Hz,1H),6.45(dd,J＝9.3,5.2Hz,1H),5.54(d,J＝5.9Hz,1H),5.04–4.84(m,2H),4.28(q,J＝1.8Hz,1H),4.12–3.92(m,2H),3.48(s,1H),2.60(dd,J＝14.0,5.3Hz,1H),2.28(ddd,J＝14.0,9.3,6.1Hz,2H),1.95(d,J＝1.2Hz,3H),0.97(s,9H),0.18(d,J＝1.1Hz,6H).
(2)步骤2-3
Figure PCTCN2021125262-appb-000257
合成方法参考实施例一步骤2-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)-4-硝基苯甲酸(2-(azidomethyl)-4-nitrobenzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21004)，最后一步反应产率为40％，MW＝686。
1H NMR(400MHz,DMSO-d 6)δ11.30(s,1H),8.41(d,J＝2.0Hz,1H),8.33(dd,J＝8.6,2.2Hz,1H),8.23(d,J＝8.6Hz,1H),7.95(s,1H),6.36(dd,J＝9.0,6.0Hz,1H),5.65(d,J＝5.2Hz,1H),4.97(s,2H),4.37(s,1H),4.15-4.04(m,2H),2.56-2.52(m,1H),2.44(dd,J＝14.0,5.9Hz,1H),1.84(s,3H).
实施例十四
Figure PCTCN2021125262-appb-000258
合成方法参考实施例二步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)-4-硝基苯甲酸(2-(azidomethyl)-4-nitrobenzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21004)，最后一步反应产率为40％，MW＝711。
1H NMR(400MHz,DMSO-d 6)δ11.00(s,1H),8.42(d,J＝2.3Hz,1H),8.34(dd,J＝8.6,2.3Hz,1H),8.25(d,J＝8.6Hz,1H),8.03(s,1H),6.82(s,2H),6.23(dd,J＝9.2,5.9Hz,1H),5.76(d,J＝4.2Hz,1H),4.99(s,2H),4.39(t,J＝4.8Hz,1H),4.25-4.00(m,2H),3.31–3.14(m,1H),2.59(dd,J＝14.0,6.4Hz,1H).
实施例十五
Figure PCTCN2021125262-appb-000259
合成方法参考实施例三步骤1-4，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)-4-硝基苯甲酸(2-(azidomethyl)-4-nitrobenzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21004)，最后两步反应产率为30％，MW＝671。
1H NMR(400MHz,DMSO-d 6)δ8.41(d,J＝2.1Hz,1H),8.33(dd,J＝8.5,2.3Hz,1H),8.23(d,J＝8.6Hz,1H),7.95(d,J＝7.5Hz,1H),7.28(s,1H),7.07(s,1H),6.37(dd,J＝9.1,5.6Hz,1H),5.81(d,J＝7.5Hz,1H),5.56(d,J＝5.4Hz,1H),4.98(s,2H),4.37(s,1H),4.12-3.99(m,2H),2.47–2.40(m,1H),2.36–2.26(m,1H).
实施例十六
Figure PCTCN2021125262-appb-000260
合成方法参考实施例四步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)-4-硝基苯甲酸(2-(azidomethyl)-4-nitrobenzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21004)，最后一步反应产率为40％，MW＝695。
1H NMR(400MHz,DMSO-d 6)δ8.58(s,1H),8.42(d,J＝1.9Hz,1H),8.34(dd,J＝8.5,2.2Hz,1H),8.29(d,J＝8.6Hz,1H),8.15(s,1H),7.29(s,2H),6.51(dd,J＝9.0,6.1Hz,1H),5.77(d,J＝5.3Hz,1H),5.02(s,2H),4.47(s,1H),4.14–4.00(m,2H),3.17–3.08(m,1H),2.75-2.70(m,1H).
实施例十七
Figure PCTCN2021125262-appb-000261
合成方法参考实施例一步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸((2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)苯甲酸(2-(azidomethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20003)，最后一步反应产率为45％，MW＝641。
1H NMR(400MHz,DMSO-d 6)δ11.30(s,1H),7.99(dd,J＝9.6,8.7Hz,2H),7.68(td,J＝7.6,1.1Hz,1H),7.61–7.50(m,2H),6.35(dd,J＝9.3,5.7Hz,1H),5.60(d,J＝5.3Hz,1H),4.80(s,2H),4.31(s,1H),4.18–4.00(m,2H),2.57–2.50(m,1H),2.39(dd,J＝13.9,5.7Hz,1H),1.84(s,3H).
实施例十八
Figure PCTCN2021125262-appb-000262
合成方法参考实施例二步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸((2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)苯甲酸(2-(azidomethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20003)，最后一步反应产率为40％，MW＝666。
1H NMR(400MHz,DMSO-d 6)δ10.70(s,1H),8.03(d,J＝9.8Hz,2H),7.69(td,J＝7.6,1.2Hz,1H),7.60–7.51(m,2H),6.67(s,2H),6.23(dd,J＝9.4,5.8Hz,1H),5.68(d,J＝4.9Hz,1H),4.82(s,2H),4.40–4.30(m,1H),4.21-4.16(m,1H),4.05–3.96(m,1H),3.22–3.10(m,1H),2.60–2.51(m,1H).
实施例十九
Figure PCTCN2021125262-appb-000263
合成方法参考实施例三步骤1-4，其中，将2-(1-叠氮乙基)苯甲酸((2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)苯甲酸(2-(azidomethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20003)，最后两步反应产率为30％，MW＝626。
1H NMR(400MHz,DMSO-d 6)δ8.00(dd,J＝7.8,1.1Hz,1H),7.95(d,J＝7.5Hz,1H),7.68(td,J＝7.6,1.4Hz,1H),7.61–7.50(m,2H),7.29(s,1H),7.07(s,1H),6.37(dd,J＝9.2,5.5Hz,1H),5.82(d,J＝7.5Hz,1H),5.51(d,J＝5.4Hz,1H),4.81(s,2H),4.32(s,1H),4.13–3.97(m,2H),2.41(dd,J＝13.8,5.6Hz,1H),2.30(ddd,J＝14.4,7.5,4.6Hz,1H).
实施例二十
Figure PCTCN2021125262-appb-000264
合成方法参考实施例四步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸((2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)苯甲酸(2-(azidomethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20003)，最后一步反应产率为40％，MW＝650。
1H NMR(400MHz,DMSO-d 6)δ8.60(s,1H),8.15(s,1H),8.05(d,J＝6.5Hz,1H),7.69(t,J＝8.2Hz,1H),7.60-7.54(m,2H),7.27(s,2H),6.49(dd,J＝8.8,5.8Hz,1H),5.71(d,J＝5.6Hz,1H),4.83(s,2H),4.42–4.34(m,1H),4.02–3.80(m,2H),2.72–2.63(m,1H),2.35–2.30(m,1H).
实施例二十一
Figure PCTCN2021125262-appb-000265
合成方法参考实施例一步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)-4-甲氧基苯甲酸(2-(1-azidoethyl)-4-methoxybenzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21005)，最后一步反应产率为45％，MW＝685。
1H NMR(400MHz,DMSO-d 6)δ11.30(s,1H),8.00–7.90(m,2H),7.11(d,J＝2.6Hz,1H),7.04(dd,J＝8.8,2.6Hz,1H),6.39–6.29(m,1H),5.74-5.68(m,1H),5.55(d,J＝5.1Hz,1H),4.30(s,1H),4.16–3.98(m,2H),3.86(s,3H),2.48–2.30(m,2H),1.84(s,3H),1.48(dd,J＝6.7,1.4Hz,3H).
实施例二十二
Figure PCTCN2021125262-appb-000266
合成方法参考实施例三步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)-4-甲氧基苯甲酸(2-(1-azidoethyl)-4-methoxybenzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21005)，最后两步反应产率为30％，MW＝670。
1H NMR(400MHz,DMSO-d 6)δ8.00–7.89(m,2H),7.32(s,1H),7.11(d,J＝2.5Hz,2H),7.05(dd,J＝8.8,2.6Hz,1H),6.41–6.31(m,1H),5.82(d,J＝7.5Hz,1H),5.74-5.68(m,1H),5.49(d,J＝4.5Hz,1H),4.30(s,1H),4.10-4.00(m,2H),3.86(s,3H),2.42-2.36(m,1H),2.33-2.23(m,1H),1.48(dd,J＝6.6,1.5Hz,3H).
实施例二十三
Figure PCTCN2021125262-appb-000267
合成方法参考实施例四步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(1-叠氮乙基)-4-甲氧基苯甲酸(2-(1-azidoethyl)-4-methoxybenzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21005)，最后一步反应产率40％，MW＝694。
1H NMR(400MHz,DMSO-d 6)δ8.61(d,J＝3.4Hz,1H),8.16(d,J＝1.4Hz,1H),8.02(dd,J＝8.7,1.7Hz,1H),7.30(s,2H),7.12(d,J＝2.5Hz,1H),7.07(dd,J＝8.8,2.6Hz,1H),6.51-6.46(m,1H),5.78-5.72(m,1H),5.68(d,J＝5.2Hz,1H),4.43–4.36(m,1H),4.14–4.00(m,2H),3.87(s,3H),3.18–3.08(m,1H),2.71–2.62(m,1H),1.50(dd,J＝6.6,2.0Hz,3H).
实施例二十四
(1)步骤1-2
Figure PCTCN2021125262-appb-000268
合成方法参考实施例一步骤1-2，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(2-叠氮丙-2-基)烟酸(2-(2-azidopropan-2-yl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20004)，最后一步反应产率为90％，MW＝430。
1H NMR(400MHz,CDCl 3)δ9.12(s,1H),8.31(dd,J＝4.8,1.7Hz,1H),7.39(dd,J＝7.7,1.6Hz,1H),6.96(dd,J＝7.3,5.2Hz,1H),5.97(t,J＝7.2Hz,1H),5.33(dd,J＝5.1,2.7Hz,1H),4.02(d,J＝2.3Hz,1H),3.70(d,J＝2.4Hz,2H),2.72(s,1H),2.25(dd,J＝10.7,5.1Hz,2H),1.60(s,3H),1.45(s,6H).
(2)步骤3
Figure PCTCN2021125262-appb-000269
合成方法参考实施例一步骤3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(2-叠氮丙-2-基)烟酸(2-(2-azidopropan-2-yl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20004)，该步骤反应产率为35％，MW＝670。
1H NMR(400MHz,DMSO-d 6)δ11.30(s,1H),8.67(dd,J＝4.7,1.4Hz,1H),8.02(dd,J＝7.7,1.4Hz,1H),7.96(s,1H),7.47(dd,J＝7.7,4.8Hz,1H),6.29(dd,J＝9.2,5.7Hz,1H),5.61(d,J＝5.3Hz,1H),4.31(s,1H),4.19–4.03(m,2H),2.56-2.52(m,1H),2.36(dd,J＝13.9,5.6Hz,1H),1.84(s,3H),1.70(d,J＝3.8Hz,6H).
实施例二十五
(1)步骤1-2
Figure PCTCN2021125262-appb-000270
合成方法参考实施例一步骤1-2，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(2-(叠氮甲基)苯基)乙酸(2-(2-(azidomethyl)phenyl)acetic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20005)，最后一步反应产率为90％，MW＝415。
1H NMR(500MHz,CDCl 3)δ8.08(s,1H),7.46(d,J＝1.1Hz,1H),7.39–7.32(m,3H),7.30(d,J＝6.8Hz,1H),6.20(dd,J＝8.5,5.9Hz,1H),5.37-5.35(m,1H),4.40(s,2H),4.08(q,J＝2.4Hz,1H),3.90(qd,J＝11.8,2.6Hz,2H),3.76(s,2H),2.48–2.33(m,2H),1.92(d,J＝1.0Hz,3H).
(2)步骤3
Figure PCTCN2021125262-appb-000271
合成方法参考实施例一步骤3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(2-(叠氮甲基)苯基)乙酸(2-(2-(azidomethyl)phenyl)acetic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20005)，该步骤反应产率为40％，MW＝655。
1H NMR(400MHz,DMSO-d 6)δ11.33(s,1H),7.91(s,1H),7.43–7.28(m,4H),6.26(dd,J＝9.4,5.6Hz,1H),5.34(d,J＝5.4Hz,1H),4.50(s,2H),4.09(s,1H),4.05–3.95(m,2H),3.84(s,2H),2.44–2.31(m,1H),2.18(dd,J＝13.9,5.5Hz,1H),1.82(s,3H).
实施例二十六
Figure PCTCN2021125262-appb-000272
合成方法参考实施例一步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(2-(1-叠氮乙基)苯基)乙酸(2-(2-(1-azidoethyl)phenyl)acetic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20006)，最后一步反应产率为40％，MW＝669。
1H NMR(400MHz,DMSO-d 6)δ11.29(s,1H),7.87(s,1H),7.44(d,J＝7.6Hz,1H),7.35(dt,J＝7.9,4.2Hz,1H),7.30(d,J＝4.0Hz,2H),6.26(dd,J＝9.5,5.6Hz,1H),5.33(d,J＝5.4Hz,1H),5.05(q,J＝6.7Hz,1H),4.10(s,1H),4.02–3.94(m,2H),3.90(d,J＝2Hz,2H),2.42-2.33(m,1H),2.21–2.12(m,1H),1.81(s,3H),1.45(dd,J＝6.7,1.3Hz,3H).
实施例二十七
(1)步骤1-2
Figure PCTCN2021125262-appb-000273
合成方法参考实施例一步骤1-2，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(2-叠氮乙基)苯甲酸(2-(2-azidoethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21006)，最后一步反应产率为90％，MW＝415。
1H NMR(500MHz,CDCl 3)δ8.40(s,1H),7.98(d,J＝7.8Hz,1H),7.57–7.49(m,2H),7.39–7.30(m,2H),6.36–6.29(m,1H),5.64–5.56(m,1H),4.26(d,J＝2.4Hz,1H),4.02(d,J＝2.5Hz,2H),3.54(t,J＝7.0Hz,2H),3.27(t,J＝7.1Hz,2H),2.60–2.50(m,2H),1.95(s,3H).
(2)步骤3
Figure PCTCN2021125262-appb-000274
合成方法参考实施例一步骤3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(2-叠氮乙基)苯甲酸(2-(2-azidoethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21006)，该步骤反应产率为40％，MW＝655。
1H NMR(400MHz,DMSO-d 6)δ11.28(s,1H),7.98–7.85(m,2H),7.57(t,J＝7.4Hz,1H),7.42(t,J＝7.7Hz,2H),6.34(dd,J＝9.3,5.7Hz,1H),5.57(d,J＝5.3Hz,1H),4.30(s,1H),4.08–4.02(m,2H),3.57(t,J＝6.9Hz,2H),3.21(t,J＝6.9Hz,2H),2.55–2.52(m,1H),2.43–2.27(m,1H),1.84(s,3H).
实施例二十八
(1)步骤1-2
Figure PCTCN2021125262-appb-000275
合成方法参考实施例二步骤1-2，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)烟酸(2-(azidomethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21007)，最后一步反应产率为90％，MW＝427。
1H NMR(400MHz,CDCl 3)δ8.52(s,2H),δ＝8.20(d,1H),δ＝7.90(d,1H),δ＝7.58(s,1H),δ＝7.35(dd,1H),δ＝6.26(t,1H),δ＝5.59(dq,1H),δ＝5.11(s,2H),δ＝4.61(dt,1H),δ＝3.90(m,2H),δ＝2.38(m,2H).
(2)步骤3
Figure PCTCN2021125262-appb-000276
合成方法参考实施例二步骤3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)烟酸(2-(azidomethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21007)，该步骤反应产率为30％，MW＝667。
1H NMR(400MHz,DMSO-d 6)δ10.76(s,1H),8.83(dd,J＝4.8,1.5Hz,1H),8.40(dd,J＝7.9,1.7Hz,1H),8.00(d,J＝6.6Hz,1H),7.59(dd,J＝7.9,4.8Hz,1H),6.73(s,2H),6.23(dd,J＝9.3,5.8Hz,1H),5.72(d,J＝4.8Hz,1H),4.85(s,2H),4.36(d,J＝4.9Hz,1H),4.27–4.16(m,1H),4.01–3.93(m,1H),3.26-3.18(d,J＝14.3Hz,1H),2.56(dd,J＝13.7,5.3Hz,1H).
实施例二十九
Figure PCTCN2021125262-appb-000277
合成方法参考实施例一步骤1-3，其中，将2-(1-叠氮乙基)苯甲酸(2-(1-azidoethyl)benzoic acid)替换为2-(叠氮甲基)烟酸(2-(azidomethyl)nicotinic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21007)，最后一步反应产率为40％，MW＝642。
1H NMR(400MHz,DMSO-d 6)δ11.29(s,1H),8.81(dd,J＝4.8,1.7Hz,1H),8.39(dd,J＝7.9,1.7Hz,1H),7.89(d,J＝4.2Hz,1H),7.58(dd,J＝7.9,4.8Hz,1H),6.35(dd,J＝9.0,6.0Hz,1H),5.59(d,J＝4.8Hz,1H),4.83(s,2H),4.35(s,1H),4.14–3.97(m,2H),2.46–2.36(m,2H),1.83(d,J＝2.7Hz,3H).
实施例三十
Figure PCTCN2021125262-appb-000278
合成方法参考实施例五步骤1-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(甲基二硫基)乙基)苯甲酸(2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20007)，最后一步反应产率为30％，MW＝692。
1H NMR(400MHz,D 2O)δ7.90–7.82(m,2H),7.69–7.58(m,2H),7.46–7.39(m,1H),6.47–6.35(m,1H),5.73(s,1H),5.10-5.01(m,1H),4.57(dd,J＝13.6,3.0Hz,1H),4.41–4.24(m,2H),2.65–2.52(m,2H),1.98(d,J＝15.2Hz,3H),1.94(s,3H),1.68(d,J＝7.2Hz,3H).
实施例三十一
Figure PCTCN2021125262-appb-000279
合成方法参考实施例六步骤1-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(甲基二硫基)乙基)苯甲酸(2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20007)，最后一步反应产率为30％，MW＝717。
1H NMR(400MHz,D 2O)δ8.14(t,J＝3.1Hz,1H),7.82(dd,J＝8.1,3.1Hz,1H),7.55(q,J＝3.2Hz,2H),7.38-7.34(m,1H),6.31-6.22(m,1H),5.76(d,J＝4.1Hz,1H),5.10–5.00(m,1H),4.65–4.55(m,1H),4.36-4.21(m,2H),3.02–2.94(m,1H),2.71–2.61(m,1H),1.94(dd,J＝22.2,3.2Hz,3H),1.62(dd,J＝7.2,3.3Hz,3H).
实施例三十二
Figure PCTCN2021125262-appb-000280
合成方法参考实施例七步骤1-4，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(甲基二硫基)乙基)苯甲酸(2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20007)，最后两步反应产率为28％，MW＝677。
1H NMR(400MHz,D 2O)δ8.08–8.00(m,1H),7.91–7.84(m,1H),7.72–7.59(m,2H),7.49–7.40(m,1H),6.48–6.40(m,1H),6.19(dd,J＝7.7,2.3Hz,1H),5.73–5.64(m,1H),5.13–5.02(m,1H),4.60(d,J＝12.6Hz,1H),4.36-4.27(m,2H),2.71-2.64(m,1H),2.55–2.44(m,1H),2.00(dd,J＝9.5,2.2Hz,3H),1.69(dd,J＝7.2,2.1Hz,3H).
实施例三十三
Figure PCTCN2021125262-appb-000281
合成方法参考实施例八步骤1-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(甲基二硫基)乙基)苯甲酸(2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20007)，最后一步反应产率为35％，MW＝701。
1H NMR(400MHz,D 2O)δ8.53(dd,J＝5.2,1.8Hz,1H),8.18–8.10(m,1H),7.83(d,J＝7.8Hz,1H),7.55-7.51(m,2H),7.38-7.34(m,1H),6.49-6.40(m,1H),5.81–5.73(m,1H),5.07-5.00(m,1H),4.67–4.57(m,1H),4.39–4.18(m,2H),3.06–2.95(m,1H),2.75(ddd,J＝21.2,14.2,5.6Hz,1H),1.93(dd,J＝25.1,1.9Hz,3H),1.60(dd,J＝6.9,4.0Hz,3H).
实施例三十四
(1)步骤1-
Figure PCTCN2021125262-appb-000282
合成方法参考实施例五步骤1，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丁基二硫基)乙基)苯甲酸(2-(1-(isobutyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21008)，该步骤反应产率为79％，MW＝608。
1H NMR(400MHz,CDCl 3)δ8.66–8.39(m,1H),7.89–7.80(m,1H),7.63–7.47(m,3H),7.33(td,J＝8.8,4.5Hz,1H),6.47-6.40(m,1H),5.52–5.40(m,1H),5.30–4.92(m,1H),4.36–4.21(m,1H),4.07–3.92(m,2H),2.60–2.48(m,1H),2.31–2.16(m,1H),1.94(s,3H),1.72–1.66(m,2H),1.63–1.51(m,1H),1.00(d,J＝6.8Hz,2H),0.98–0.93(m,9H),0.87–0.81(m,4H),0.19–0.13(m,6H).
(2)步骤2-3
Figure PCTCN2021125262-appb-000283
合成方法参考实施例五步骤2-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丁基二硫基)乙基)苯甲酸(2-(1-(isobutyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21008)，最后一步反应产率为32％，MW＝734。
1H NMR(400MHz,DMSO-d 6)δ11.31(s,1H),7.96(dd,J＝8.2,0.9Hz,1H),7.85(d,J＝8.3Hz,1H),7.62(d,J＝3.9Hz,2H),7.46-7.41(m,1H),6.33(td,J＝9.7,5.6Hz,1H),5.63–5.55(m,1H),5.18–5.08(m,1H),4.30(d,J＝23.4Hz,1H),4.18–4.02(m,2H),2.57–2.51(m,1H),2.43-2.29(m,1H),2.12–1.93(m,2H),1.84(s,3H),1.70–1.56(m,4H),0.85–0.71(m,6H).
实施例三十五
Figure PCTCN2021125262-appb-000284
合成方法参考实施例六步骤1-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丁基二硫基)乙基)苯甲酸(2-(1-(isobutyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21008)，最后一步反应产率40％，MW＝759。
1H NMR(400MHz,DMSO-d 6)δ10.73(s,1H),8.03(d,J＝6.9Hz,1H),7.89(d,J＝7.6Hz,1H),7.63(d,J＝2.8Hz,2H),7.49–7.38(m,1H),6.66(s,2H),6.27–6.15(m,1H),5.69(dd,J＝8.2,4.0Hz,1H),5.16(q,J＝6.9Hz,1H),4.39–4.26(m,1H),4.19(dd,J＝10.6,5.1Hz,1H),4.01(dd,J＝11.4,5.9Hz,1H),2.57-2.52(m,1H),2.48–2.41(m,1H),2.24–2.12(m,2H),1.70(dd,J＝13.2,6.6Hz,1H),1.65(d,J＝6.8Hz,3H),0.85–0.74(m,6H).
实施例三十六
Figure PCTCN2021125262-appb-000285
合成方法参考实施例七步骤1-4，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丁基二硫基)乙基)苯甲酸(2-(1-(isobutyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21008)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21008)，最后两步反应产率30％，MW＝719。
1H NMR(400MHz,DMSO-d 6)δ7.98(dd,J＝9.4,7.6Hz,1H),7.85(d,J＝7.8Hz,1H),7.62(d,J＝4.1Hz,2H),7.46-7.42(m,1H),7.33(s,1H),7.12(s,1H),6.40–6.30(m,1H),5.82(dd,J＝7.5,1.0Hz,1H),5.53(d,J＝4.4Hz,1H),5.17-5.11(m,1H),4.30(d,J＝21.7Hz,1H),4.16–4.01(m,1H),2.46–2.27(m,2H),2.15–1.98(m,2H),1.71–1.58(m,4H),0.80(d,J＝2.5Hz,3H),0.78(d,J＝2.6Hz,3H).
实施例三十七
Figure PCTCN2021125262-appb-000286
合成方法参考实施例八步骤1-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丁基二硫基)乙基)苯甲酸(2-(1-(isobutyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21008)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21008)，最后一步反应产率40％，MW＝743。
1H NMR(400MHz,DMSO-d 6)δ8.67(d,J＝8.3Hz,1H),8.14(d,J＝5.9Hz,1H),7.94–7.87(m,1H),7.64(d,J＝3.9Hz,2H),7.46(dt,J＝8.1,4.2Hz,1H),7.32(s,2H),6.51-6.44(m,1H),5.75(s,1H),5.18(q,J＝7.0Hz,1H),4.45–4.33(m,1H),4.16–4.04(m,2H),3.25–3.12(m,1H),2.71-2.57(m,1H),2.19–2.01(m,2H),1.73–1.59(m,4H),0.81(d,J＝6.7Hz,3H),0.78(d,J＝6.7Hz,3H).
实施例三十八
(1)步骤1
Figure PCTCN2021125262-appb-000287
合成方法参考实施例五步骤1，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丙基二硫基)乙基)苯甲酸(2-(1-(isopropyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21009)，该步骤反应产率为80％，MW＝594。
1H NMR(500MHz,CDCl 3)δ8.51(s,1H),7.85(ddd,J＝12.8,7.8,1.2Hz,1H),7.64–7.57(m,2H),7.57–7.50(m,1H),7.37–7.29(m,1H),6.45(ddd,J＝20.4,9.2,5.3Hz,1H),5.50(t,J＝5.3Hz,1H),5.28–5.15(m,1H),4.32(dd,J＝36.3,1.3Hz,1H),4.07–3.96(m,2H),2.59(td,J＝13.5,5.3Hz,1H),2.47–2.16(m,2H),1.95(s,3H),1.70(dd,J＝7.0,1.0Hz,3H),1.17–1.06(m,6H),0.97(s,9H),0.18(s,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000288
合成方法参考实施例五步骤2，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丙基二硫基)乙基)苯甲酸(2-(1-(isopropyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21009)，该步骤反应产率为90％，MW＝594。
1H NMR(500MHz,CDCl 3)δ8.84(s,1H),7.84(ddd,J＝7.4,5.8,1.3Hz,1H),7.65–7.49(m,3H),7.33(td,J＝7.7,1.2Hz,1H),6.37–6.27(m,1H),5.61(tt,J＝4.7,2.5Hz,1H),5.22(dq,J＝13.9,7.0Hz,1H),4.30(dq,J＝19.8,2.4Hz,1H),4.03(dd,J＝5.7,2.7Hz,2H),2.60–2.31(m,3H),1.95(d,J＝1.1Hz,3H),1.70(d,J＝7.0Hz,3H),1.15–1.07(m,6H).
(3)步骤3
Figure PCTCN2021125262-appb-000289
合成方法参考实施例五步骤3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丙基二硫基)乙基)苯甲酸(2-(1-(isopropyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21009)，该步骤反应产率为40％，MW＝720。
1H NMR(400MHz,DMSO-d 6)δ11.29(s,1H),7.98(dd,J＝6.6,0.9Hz,1H),7.85(dd,J＝7.7,2.4Hz,1H),7.61(t,J＝6.4Hz,2H),7.45-7.40(m,1H),6.36-6.31(m,1H),5.60(d,J＝5.3Hz,1H),5.11(q,J＝6.9Hz,1H),4.30(d,J＝20.0Hz,1H),4.12-4.04(m,2H),2.58–2.51(m,1H),2.46-2.29(m,2H),1.84(s,3H),1.64(d,J＝7.0Hz,3H),1.06(d,J＝5.7Hz,6H).
实施例三十九
Figure PCTCN2021125262-appb-000290
合成方法参考实施例六步骤1-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丙基二硫基)乙基)苯甲酸(2-(1-(isopropyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21009)，最后一步反应产率为40％，MW＝745。
1H NMR(400MHz,DMSO-d 6)δ10.57(s,1H),7.98(d,J＝7.9Hz,1H),7.88(d,J＝7.5Hz,1H),7.63(d,J＝3.3Hz,2H),7.47-7.40(m,1H),6.63(d,J＝25.9Hz,2H),6.27–6.16(m,1H),5.68(t,J＝5.3Hz,1H),5.32(t,J＝4.7Hz,1H),5.18–5.10(m,1H),4.37–4.28(m,1H),4.02-3.96(m,1H),2.68-2.66(m,1H),2.61-2.56(m,1H),2.33(dt,J＝3.5,1.7Hz,1H),1.65(d,J＝6.9Hz,3H),1.13(d,J＝7.9Hz,6H).
实施例四十
Figure PCTCN2021125262-appb-000291
合成方法参考实施例七步骤1-4，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(异丙基二硫基)乙基)苯甲酸(2-(1-(isopropyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21009)，最后两步反应产率为30％，MW＝705。
1H NMR(400MHz,DMSO-d 6)δ8.00–7.93(m,1H),7.84(dd,J＝7.6,3.3Hz,1H),7.62(d,J＝3.7Hz,2H),7.47–7.38(m,1H),7.28(s,1H),7.07(s,1H),6.38-6.33(m,1H),5.81(d,J＝7.3Hz,1H),5.52(d,J＝5.2Hz,1H),5.11(q,J＝7.0Hz,1H),4.30(d,J＝19.7Hz,1H),4.14–3.97(m,2H),2.47–2.38(m,2H),2.34–2.31(m,1H),1.64(d,J＝7.0Hz,3H),1.10(d,J＝2.1Hz,6H).
实施例四十一
(1)步骤1
Figure PCTCN2021125262-appb-000292
合成方法参考实施例五步骤1，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(叔丁基二硫基)乙基)苯甲酸(2-(1-(tert-butyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21010)，该步骤反应产率为80％，MW＝608。
1H NMR(400MHz,CDCl 3)δ8.73(d,J＝5.8Hz,1H),7.84(ddd,J＝7.8,3.5,1.5Hz,1H),7.70–7.45(m,3H),7.32(td,J＝7.6,1.4Hz,1H),6.46(td,J＝9.5,5.3Hz,1H),5.57–5.35(m,1H),5.16(p,J＝7.1Hz,1H),4.28(dd,J＝17.9,1.7Hz,1H),4.13–3.90(m,2H),2.58(ddd,J＝14.7,9.8,5.3Hz,1H),2.23(dtd,J＝16.2,6.2,2.7Hz,1H),1.98–1.92(m,3H),1.69(d,J＝6.7Hz,3H),1.22(d,J＝11.2Hz,8H),0.96(s,9H),0.17(s,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000293
合成方法参考实施例五步骤2，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(叔丁基二硫基)乙基)苯甲酸(2-(1-(tert-butyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21010)，该步骤反应产率为90％，MW＝494。
1H NMR(400MHz,CDCl 3)δ8.90(s,1H),7.83(dt,J＝7.9,1.6Hz,1H),7.62(d,J＝7.9Hz,1H),7.55(dtd,J＝7.8,4.0,1.4Hz,2H),7.32(td,J＝7.5,1.4Hz,1H),6.31(ddd,J＝8.4,6.0,2.8Hz,1H),5.59(dt,J＝5.4,2.5Hz,1H),5.14(dq,J＝18.4,7.0Hz,1H),4.27(dt,J＝8.4,2.5Hz,1H),4.01(d,J＝2.6Hz,2H),2.65–2.48(m,2H),2.35(s,2H),1.94(d,J＝1.2Hz,3H),1.68(dd,J＝6.9,1.4Hz,3H),1.22(d,J＝2.9Hz,9H).
(3)步骤3
Figure PCTCN2021125262-appb-000294
合成方法参考实施例五步骤3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为2-(1-(叔丁基二硫基)乙基)苯甲酸(2-(1-(tert-butyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21010)，该步骤反应产率为40％，MW＝734。
1H NMR(400MHz,DMSO-d 6)δ11.30(s,1H),7.91(d,J＝7.2Hz,1H),7.84(d,J＝8.0Hz,1H),7.62(d,J＝4.6Hz,2H),7.44-7.39(m,1H),6.37–6.29(m,1H),5.56(t,J＝5.0Hz,1H),5.07-4.98(m,1H),4.29(d,J＝28.0Hz,1H),4.14–3.99(m,2H),2.39(dd,J＝13.9,5.9Hz,1H),2.31(dd,J＝14.2,5.7Hz,1H),1.83(s,3H),1.63(dd,J＝7.0,1.1Hz,3H),1.19(d,J＝2.5Hz,9H).
实施例四十二
(1)步骤1
Figure PCTCN2021125262-appb-000295
合成方法参考实施例五步骤1，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为4-乙酰氨基-2-(1-(甲基二硫基)乙基)苯甲酸(4-acetamido-2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21002)，该步骤反应产率为80％，MW＝623。
1H NMR(400MHz,CDCl 3)δ8.29(s,1H),7.88(d,J＝8.6Hz,1H),7.68(s,1H),7.64–7.58(m,2H),7.46(s,1H),6.42(dd,J＝9.3,5.1Hz,1H),5.46(d,J＝5.9Hz,1H),5.42-5.36(m,1H),4.29(d,J＝1.8Hz,1H),4.04-3.95(m,2H),2.57(dd,J＝13.8,5.3Hz,1H),2.29–2.23(m,1H),2.22(s,3H),2.06(s,3H),1.94(d,J＝1.2Hz,3H),1.68(d,J＝7.0Hz,3H),0.95(s,9H),0.17(s,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000296
合成方法参考实施例五步骤2，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为4-乙酰氨基-2-(1-(甲基二硫基)乙基)苯甲酸(4-acetamido-2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21002)，该步骤反应产率为75％，MW＝509。
1H NMR(400MHz,CDCl 3)δ8.08(s,1H),7.89(dd,J＝8.5,5.6Hz,1H),7.66(d,J＝9.5Hz,2H),7.54(d,J＝7.2Hz,1H),7.45(s,1H),6.29(t,J＝7.1Hz,1H),5.59-5.56(m,1H),5.41-5.35(m,1H),4.29-4.25(m,1H),4.01(d,J＝2.2Hz,2H),3.01–2.93(m,1H),2.58–2.55(m,1H),2.23(s,3H),2.09(d,J＝5.6Hz,3H),1.95(d,J＝1.2Hz,3H),1.68(s,3H).
(3)步骤3
Figure PCTCN2021125262-appb-000297
合成方法参考实施例五步骤3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为4-乙酰氨基-2-(1-(甲基二硫基)乙基)苯甲酸(4-acetamido-2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21002)，该步骤反应产率为35％，MW＝749。
1H NMR(400MHz,DMSO-d 6)δ11.30(s,1H),10.52(d,J＝3.1Hz,1H),7.97(d,J＝4.1Hz,1H),7.92–7.82(m,2H),7.69(d,J＝8.8Hz,1H),6.37–6.26(m,1H),5.55(d,J＝5.3Hz,1H),5.34–5.25(m,1H),4.27(d,J＝16.7Hz,1H),4.12-4.05(m,2H),2.49-2.46(m,1H),2.35(td,J＝13.9,5.7Hz,1H),2.11(d,J＝12.7Hz,3H),2.08(s,3H),1.84(s,3H),1.61(d,J＝6.9Hz,3H).
实施例四十三
(1)步骤1
Figure PCTCN2021125262-appb-000298
合成方法参考实施例七步骤1，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为4-甲氧基-2-(1-(甲基二硫基)乙基)苯甲酸(4-methoxy-2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21001)，该步骤反应产率为80％，MW＝623。
1H NMR(500MHz,DMSO-d 6)δ10.92(s,1H),8.24(d,J＝7.5Hz,1H),7.92(dd,J＝8.7,4.0Hz,1H),7.24(d,J＝7.5Hz,1H),7.10(d,J＝2.6Hz,1H),7.00(ddd,J＝8.8,2.6,1.4Hz,1H),6.19(q,J＝6.3Hz,1H),5.57(d,J＝8.0Hz,3H),5.46–5.37(m,1H), 5.37–5.27(m,1H),4.37(dd,J＝21.7,2.9Hz,1H),3.95(dd,J＝11.3,3.2Hz,1H),3.85(s,3H),2.78–2.62(m,1H),2.38–2.26(m,1H),2.14–2.09(m,6H),0.86(s,9H),0.09(s,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000299
合成方法参考实施例七步骤2，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为4-甲氧基-2-(1-(甲基二硫基)乙基)苯甲酸(4-methoxy-2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21001)，该步骤反应产率为85％，MW＝509。
1H NMR(500MHz,DMSO-d 6)δ10.91(s,1H),8.35(d,J＝7.5Hz,1H),7.92(dd,J＝8.8,4.1Hz,1H),7.24(d,J＝7.5Hz,1H),7.10(d,J＝2.7Hz,1H),7.00(ddd,J＝8.8,2.7,1.3Hz,1H),6.22(dt,J＝7.9,6.0Hz,1H),5.43(dt,J＝6.6,2.1Hz,1H),5.39–5.30(m,1H),5.26(t,J＝5.3Hz,1H),4.29(dd,J＝18.4,2.7Hz,1H),3.85(s,3H),3.72(dd,J＝5.5,3.2Hz,2H),2.68–2.55(m,1H),2.34(dt,J＝14.1,7.0Hz,1H),2.16–2.08(m,6H),1.65(d,J＝7.0Hz,3H).
(3)步骤3-4
Figure PCTCN2021125262-appb-000300
合成方法参考实施例七步骤3-4，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为4-甲氧基-2-(1-(甲基二硫基)乙基)苯甲酸(4-methoxy-2-(1-(methyldisulfaneyl)ethyl)benzoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21001)，这两步的反应产率为30％，MW＝707。
1H NMR(400MHz,DMSO)δ7.96-7.90(m,2H),7.29(s,1H),7.09(d,J＝2.3Hz,2H),7.00(dd,J＝8.8,2.5Hz,1H),6.34(dd,J＝15.3,6.6Hz,1H),5.81(d,J＝7.5Hz,1H),5.47(d,J＝4.3Hz,1H),5.33(q,J＝7.0Hz,1H),4.28(d,J＝17.7Hz,1H),4.13 –3.97(m,2H),3.85(s,3H),2.43–2.22(m,2H),2.13(d,J＝10.6Hz,3H),1.64(d,J＝7.0Hz,3H).
实施例四十四
Figure PCTCN2021125262-appb-000301
合成方法参考实施例五步骤1-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为3-(叠氮甲基)呋喃-2-羧酸(3-(azidomethyl)furan-2-carboxylic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-21011)，最后一步反应产率为45％，MW＝631。
1H NMR(400MHz,DMSO-d 6)δ11.31(s,1H),8.01(d,J＝1.6Hz,1H),7.91(d,J＝22.7Hz,1H),6.83(d,J＝1.6Hz,1H),6.33(dd,J＝9.4,5.7Hz,1H),5.63–5.56(m,1H),4.65(s,2H),4.25(s,1H),4.10–3.99(m,2H),2.48–2.46(m,1H),2.37–2.32(m,1H),1.83(s,3H).
实施例四十五
Figure PCTCN2021125262-appb-000302
合成方法参考实施例五步骤1-3，其中，将2-(1-(乙基二硫基)乙基)苯甲酸(2-(1-(ethyldisulfaneyl)ethyl)benzoic acid)替换为4-叠氮丁酸(4-azidobutanoic acid)(北京欧凯纳斯生化科技有限公司，货号：OK-H-20008)，最后一步反应产率为20％，MW＝593。
1H NMR(400MHz,DMSO-d 6)δ11.28(s,1H),7.90(d,J＝0.9Hz,1H),6.24(dd,J＝9.5,5.6Hz,1H),5.33(d,J＝5.5Hz,1H),4.09(s,1H),4.04–3.95(m,2H),3.38(t,J＝6.8Hz,2H),2.44(t,J＝7.3Hz,2H),2.41–2.33(m,1H),2.18(dd,J＝13.8,5.6Hz,1H),1.82(s,3H),1.81–1.75(m,2H).
二、测试例
发明人发现，本申请的核苷酸类似物具有优异的阻断效果，同时具有优异的聚合效果；且利用本申请的核苷酸类似物进行测序时，测序效果优异，测序比对率高，错误率低。
具体地，发明人对前述制备实施例制备得到的核苷酸类似物在高通量测序 仪上进行了评估测试。
1、阻断效果评估
核苷酸底物：荧光标记的standard hot dNTP(四种)和standard cold dNTP(四种)，结构如下，均来自于MGISEQ-2000RS高通量测序试剂套装(FCL SE50)，深圳华大智造科技股份有限公司，货号1000012551；本发明核苷酸类似物cold dNTP(包括dTTP、dATP、dCTP、dGTP四种，每次测试只使用一种，为了表示方便，对于3'-OH的每一种修饰，下表1仅展示了dTTP的测试结果)。
按照MGISEQ2000测序仪操作规程，使用上述核苷酸底物以及MGISEQ-2000RS高通量测序试剂套装(FCL SE50)进行测序。
Figure PCTCN2021125262-appb-000303
(1)利用Ecoli测序文库制备DNA纳米球；
(2)将DNA纳米球装载到MGISEQ2000测序芯片上；
(3)将装载完成的测序芯片加载至MGISEQ2000测序仪上，设置测序流 程；
(4)第一轮上机测试：聚合standard hot dNTP，拍照记录信号值，然后用thpp试剂切除阻断基团，65℃1min。
(5)第二轮上机测试：聚合standard cold dNTP，然后聚合standard hot dNTP，拍照记录信号值，然后用thpp试剂切除阻断基团，65℃1min。
(6)第三轮上机测试：聚合standard hot dNTP，拍照记录信号值。后用thpp试剂切除阻断基团，65℃1min。
(7)第四轮上机测试：聚合本发明核苷酸类似物cold dNTP(每次测试只聚合一种cold dNTP)，然后聚合standard hot dNTP，拍照记录信号值。然后用thpp试剂切除阻断基团，65℃1min。
(8)第五轮上机测试：聚合standard hot dNTP，拍照记录信号值。
(9)评估聚合效率和切除效率，结果见表1。
聚合效率计算：
Figure PCTCN2021125262-appb-000304
其中：
EI(Incorporation efficiency)，为测试核苷酸与对比核苷酸聚合效率比值；
C1为第一轮上机测试信号值；
C2为第二轮上机测试信号值；
C3为第三轮上机测试信号值；
C4为第四轮上机测试信号值；
切除效率计算：
Figure PCTCN2021125262-appb-000305
其中：
Ec(Cleavage efficiency)，为测试核苷酸与对比核苷酸切除效率比值；
EI为测试核苷酸与对比核苷酸聚合效率比值；
C3为第三轮上机测试信号值；
C5为第五轮上机测试信号值；
CGT为第三轮中C碱基、G碱基与T碱基的信号。
表1 核苷酸类似物的聚合效率和切除效率(仅展示dTTP)
Figure PCTCN2021125262-appb-000306
Figure PCTCN2021125262-appb-000307
Figure PCTCN2021125262-appb-000308
Figure PCTCN2021125262-appb-000309
Figure PCTCN2021125262-appb-000310
2、测序效果评估
测序例1
核苷酸底物：荧光标记的standard hot dNTP(四种，来自于MGISEQ-2000RS高通量测序试剂套装(FCL SE50)，深圳华大智造科技股份有限公司，货号1000012551)；本发明核苷酸类似物cold dNTP(四种，命名为AEB)，结构如下。
Figure PCTCN2021125262-appb-000311
按照MGISEQ2000测序仪操作规程，使用上述核苷酸底物以及MGISEQ-2000RS高通量测序试剂套装(FCL SE50)进行测序。
(1)利用Ecoli测序文库制备DNA纳米球；
(2)将DNA纳米球装载到MGISEQ2000测序芯片上；
(3)将装载完成的测序芯片加载至MGISEQ2000测序仪上，设置测序流程，hot dNTP聚合：60℃2min；cold dNTP聚合：60℃2min；信号采集；切除阻断基团：65℃2min。
(4)下机数据进行basecall分析，输出测序指标，比对率，错误率，Q30等，结果见表2。
表2 Basecall分析结果
Figure PCTCN2021125262-appb-000312
Figure PCTCN2021125262-appb-000313
测序例2
核苷酸底物：荧光标记的standard hot dNTP(四种，来自于MGISEQ-2000RS高通量测序试剂套装(FCL SE50)，深圳华大智造科技股份有限公司，货号1000012551)；本发明核苷酸类似物cold dNTP(四种，命名为SSEB)，结构如下。
Figure PCTCN2021125262-appb-000314
测序过程同上述测序例1，Basecall分析结果见表3。
表3 Basecall分析结果
核苷酸类似物cold dNTP Mix SSEB
参考基因(Reference) Ecoli.fa
循环数(Cycle Number) 50
Q30(％) 91.89
Lag(％) 0.11
Runon(％) 0.23
ESR(％) 90.33
比对率(Mapping Rate,％) 98.59
平均错误率(AvgError Rate,％) 0.13
测序例3
核苷酸底物：荧光标记的standard hot dNTP(四种，来自于MGISEQ-2000RS高通量测序试剂套装(FCL SE50)，深圳华大智造科技股份有限公司，货号1000012551)；本发明核苷酸类似物cold dNTP(四种，命名为AZBN)，结构如下。
Figure PCTCN2021125262-appb-000315
测序过程同上述测序例1，Basecall分析结果见表4。
表4 Basecall分析结果
核苷酸类似物cold dNTP Mix AZBN
参考基因(Reference) Ecoli.fa
循环数(Cycle Number) 50
Q30(％) 61.39
Lag(％) 0.03
Runon(％) 0.12
ESR(％) 62.17
比对率(Mapping Rate,％) 5.75
平均错误率(AvgError Rate,％) 4.94
去除N后的平均错误率(AvgError Rate！N,％) 4.89
测序例4 使用hot T-SSEB进行测序
1、合成带有荧光基团的核苷酸类似物dTTP(命名为hot T-SSEB)
(1)步骤1
Figure PCTCN2021125262-appb-000316
碘代核苷底物T(OKeanos Tech，货号OK-N-16001)1g溶解于DMF中，加入Pd(PPh3)4(10mol％)，CuI(15mol％)，三乙胺(3eq)与底物炔丙胺(OKeanos Tech，货号OK20A410)(1.5eq)在60度条件下反应12h。加入水淬灭反应，DCM萃取。浓缩后柱分离得到白色固体产品1.1g。
MS[ES(-)],m/z 491.1. 1H NMR(400MHz,DMSO-d6)δ11.67(s,1H),10.00(t,J＝5.5Hz,1H),7.94(s,1H),6.12(dd,J＝7.6,5.9Hz,1H),5.28(d,J＝4.1Hz,1H),4.26-4.12(m,3H),3.88(q,J＝2.8Hz,1H),3.81(dd,J＝11.5,2.6Hz,1H),3.73(dd,J＝11.5,3.1Hz,1H),2.17(ddd,J＝13.2,6.0,2.8Hz,1H),2.05(ddd,J＝13.3,7.7,5.8Hz,1H),0.87(s,9H),0.08(d,J＝1.8Hz,6H).
(2)步骤2
Figure PCTCN2021125262-appb-000317
步骤1获得的核苷300mg溶解在10mL的DMF中，加入DCC(1.2eq)与DMAP(10％mol)，反应搅拌30分钟后，加入二硫羧酸底物(OKeanos Tech，货号OK20A420)(1.5eq)。反应搅拌12小时后，直接柱分离得到白色固体359mg。
MS[ES(-)],m/z 700.3. 1H NMR(400MHz,DMSO-d 6)δ11.74(s,1H),10.03(t,J＝5.5Hz,1H),7.98(s,1H),7.90–7.82(m,1H),7.68–7.58(m,2H),7.47-7.40(m,1H),6.23-6.18(m,1H),5.45-5.42(m,1H),5.20-5.12(m,1H),4.38–4.16(m,3H),3.98–3.87(m,2H),2.60-2.53(m,1H),2.40-2.31(m,1H),2.06(d,J＝0.6Hz,3H),1.65(dd,J＝7.0,1.0Hz,3H),0.90(s,9H),0.13(d,J＝1.2Hz,6H).
(3)步骤3
Figure PCTCN2021125262-appb-000318
步骤2获得的核苷300mg溶解在10mL的THF中，在0度下加入TBAF(2eq,1M in THF)，反应在0度搅拌30分钟后，升至室温搅拌4小时。反应直接柱分离得到白色固体200mg。
MS[ES(-)],m/z 587.2. 1H NMR(400MHz,DMSO-d 6)δ11.68(d,J＝3.2Hz,1H),10.06(t,J＝5.6Hz,1H),8.23(d,J＝3.2Hz,1H),7.88–7.78(m,1H),7.66–7.56(m,2H),7.43–7.39(m,1H),6.25–6.13(m,1H),5.48–5.45(m,1H),5.31(t,J＝5.2Hz,1H),5.18–5.12(m,1H),4.28–4.16(m,3H),3.76–3.67(m,2H),2.50–2.40(m,2H),2.04(d,J＝9.2Hz,3H),1.68–1.59(m,3H).
(4)步骤4
Figure PCTCN2021125262-appb-000319
步骤3获得的核苷200mg溶解在5mL的磷酸三甲酯中，在0度下加入三氯氧磷(1.5eq)，反应在0度搅拌120分钟后，将反应液加入到三丁基焦磷酸铵(2eq)的DMF(5mL)溶液中，反应在0度继续搅拌3h后，加入0.1M TEAB缓冲液淬灭。反应利用制备HPLC反相柱(C18，流动相：0.1M TEAB-乙腈)分离，浓缩后直接加入浓氨水3mL，反应2h后利用制备HPLC反相柱(C18，流动相：0.1M TEAB-乙腈)分离得到白色固体120mg。
MS[ES(-)],m/z 745.5. 1H NMR(400MHz,D 2O)δ8.51(s,1H),7.90(dd,J＝7.9,1.4Hz,1H),7.73(d,J＝7.8Hz,1H),7.67(td,J＝7.6,1.4Hz,1H),7.48(t,J＝7.5Hz,1H),6.45(td,J＝9.2,5.7Hz,1H),5.75(t,J＝4.7Hz,1H),5.15–5.07(m,1H),4.72–4.64(m,1H),4.39(d,J＝3.3Hz,2H),4.07(s,2H),2.82-2.76(m,1H),2.64–2.54(m,1H),2.02(d,J＝6.4Hz,3H),1.73(dd,J＝7.1,2.2Hz,3H)；31P NMR(162MHz,D2O)δ-9.79(dd,J＝20.3,9.6Hz,1P),-11.68(d,J＝19.2Hz,1P),-22.82(td,J＝19.2,6.1Hz,1P).
(5)步骤5
Figure PCTCN2021125262-appb-000320
称取dye-linker固体(OKeanos Tech，OK-F-20211)(9.3mg，1eq.)于带磁力搅拌子的20毫升干燥圆底烧瓶中，用适量DMF(3ml)溶解，依次加入TNTU固体(7mg，0.02mmol，2eq.)和DIPEA(2.6mg，0.02mmol，2eq.)，DIPEA称取于1.5ml PE管中并用0.5ml DMF稀释后滴加入反应体系，作为搅拌反应。约1小时后，取样溶于乙腈，通过HPLC和MS监测AF532-V4原料消耗完毕。往上述反应体系加入hot T-mSSEB底物固体(15mg，0.02mmol，1eq.)，室温搅拌反应。1小时后取样溶于乙腈，通过MS和HPLC监测反应进程。反应过夜至nhs酯消耗完毕。加入0.1M TEAB缓冲液淬灭。反应利用制备HPLC反相柱(C18，流动相：0.1M TEAB-乙腈)分离，浓缩后直接加入浓氨水3mL，反应2h后利用制备HPLC反相柱(C18，流动相：0.1M TEAB-乙腈)分离得到固体10mg。
MS[ES(-)],m/z 1815.0. 1H NMR(400MHz,DMSO-d 6)δ9.06(d,J＝6.7Hz,1H),8.80(d,J＝47.0Hz,1H),8.28(t,J＝7.2Hz,1H),8.18-8.00(m,1H),7.86(d,J＝7.7Hz,1H),7.75–7.66(m,2H),7.66–7.58(m,3H),7.58–7.40(m,4H),7.39-7.33(m,1H),7.17–7.07(m,1H),6.98(s,2H),6.79(s,2H),6.31–6.17(m,1H),5.58(d,J＝3.5Hz,1H),5.21-5.14(m,2H),4.34(d,J＝19.3Hz,1H),4.26(dd,J＝10.5,3.9Hz,1H),4.18(dd,J＝10.6,4.5Hz,2H),4.09(d,J＝3.5Hz,2H),3.99(s,2H),3.95–3.82(m,2H),3.68(s,6H),3.52(s,4H),3.33–3.24(m,2H),3.22–3.15(m,2H),3.14–3.06(m,2H),2.65(t,J＝7.6Hz,4H),2.57(t,J＝7.1Hz,5H),2.46–2.37(m,1H),2.05(d,J＝16.8Hz,3H),2.01-1.93(m,4H),1.86-1.81(m,3H),1.77-1.72(m,1H),1.65(d,J＝6.9Hz,3H).
2、使用包含前述制备的hot T-SSEB的核苷酸类似物进行测序
核苷酸底物：荧光标记的standard hot dNTP(来自于MGISEQ-2000RS高通量测序试剂套装(FCL SE50)，深圳华大智造科技股份有限公司，货号1000012551)，其中hot dTTP使用前述制备获得的hot T-SSEB替代，结构如下；本发明核苷酸类似物cold dNTP(四种，命名为SSEB)，结构如下。
Figure PCTCN2021125262-appb-000321
测序过程同上述测序例1，Basecall分析结果见表5。
表5 Basecall分析结果
Figure PCTCN2021125262-appb-000322
Figure PCTCN2021125262-appb-000323
测序例5 使用SS-hot G进行测序
1、合成带有荧光基团的核苷酸类似物dGTP(命名为SS-hot G)
Figure PCTCN2021125262-appb-000324
合成方法与测序例4类似。其中，使用碘代G核苷底物替代碘代T核苷底物，叠氮羧酸底物替代二硫羧酸底物，均由OKeanos Tech公司提供。dye-linker固体需要替换，由MyChem LLC公司提供(货号110920Cy5)。获得终产品10mg。
MS[ES(-)],m/z 1575。 1H NMR(400MHz,DMSO-d 6)δ10.54(s,1H),8.49(s,1H),8.34(t,J＝13.1Hz,2H),8.07(t,J＝5.5Hz,1H),7.92(dd,J＝7.2,4.6Hz,2H),7.80(d,J＝1.7Hz,2H),7.70(d,J＝7.4Hz,1H),7.67-7.62(m,4H),7.49(t,J＝7.4Hz,2H),7.30(d,J＝8.3Hz,3H),6.58(t,J＝12.3Hz,1H),6.41(s,2H),6.36–6.27(m,3H),6.26–6.12(m,2H),5.60(dd,J＝13.1,6.4Hz,3H),4.86(s,2H),4.48(dd,J＝29.1,16.7Hz,2H),4.35–4.27(m,2H),4.07(d,J＝4.8Hz,4H),3.59(s,2H),3.50(d,J＝5.7Hz,2H),3.34-3.29(m,2H),2.78(t,J＝6.8Hz,2H),2.13(t,J＝6.7Hz,2H),2.07(t,J＝7.2Hz,2H),1.68(s,12H),1.53–1.48(m,8H),1.35(dt,J＝15.8,8.1Hz,4H).
2、使用包含前述制备的SS-hot G的核苷酸类似物进行测序
核苷酸底物：荧光标记的standard hot dNTP(来自于MGISEQ-2000RS高通量测序试剂套装(FCL SE50)，深圳华大智造科技股份有限公司，货号1000012551)，其中hot dGTP使用前述制备获得的SS-hot G替代；本发明核苷酸类似物cold dNTP(四种，命名为SS-cold)，结构如下。
Figure PCTCN2021125262-appb-000325
Figure PCTCN2021125262-appb-000326
测序过程同上述测序例1，Basecall分析结果见表6。
表6 Basecall分析结果
Figure PCTCN2021125262-appb-000327

Claims (15)

  1. 式(A)所示的化合物或其盐，
    Figure PCTCN2021125262-appb-100001
    其中：
    R为可逆阻断基团，R选自
    Figure PCTCN2021125262-appb-100002
    Figure PCTCN2021125262-appb-100003
    优选地，R选自
    Figure PCTCN2021125262-appb-100004
    Figure PCTCN2021125262-appb-100005
    更优选地，R选自
    Figure PCTCN2021125262-appb-100006
    所述杂芳基选自以下：
    Figure PCTCN2021125262-appb-100007
    Figure PCTCN2021125262-appb-100008
    Figure PCTCN2021125262-appb-100009
    各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
    优选地，所述杂芳基选自以下：
    Figure PCTCN2021125262-appb-100010
    Figure PCTCN2021125262-appb-100011
    各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
    更优选地，所述杂芳基选自以下：
    Figure PCTCN2021125262-appb-100012
    各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
    各X独立地选自O、NH、S；
    优选地，X为O；
    各Y独立地选自直接键、O、NH、S、CH、CH 2、C(CH 3) 2；
    优选地，各Y独立地选自直接键、CH 2；
    R 0选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
    优选地，R 0为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)；
    更优选地，R 0为-N 3；
    R 1选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
    优选地，R 1为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)；
    更优选地，R 1为-N 3；
    R 2、R 3、R 4各自独立地选自O、NH、S、CH、CH 2、C(CH 3) 2；
    优选地，R 2、R 3、R 4各自独立地选自O、NH、S、CH、CH 2、C(CH 3) 2，且满足以下条件：R 2选自O、S时，R 3和R 4为CH 2；R 3选自O、S时，R 2和R 4为CH 2；R 4选自O，S时，R 2和R 3为CH 2；R 2为C(CH 3) 2时，R 3和R 4为CH 2；R 3为C(CH 3) 2时，R 2和R 4为CH 2；R 4为C(CH 3) 2时，R 2和R 3为CH 2；Y为CH时，R 2为CH,R 3和R 4为CH 2；
    更优选地，R 2、R 3、R 4各自独立地选自CH、CH 2；
    最优选地，R 2和R 3为CH，R 4为CH 2；
    Figure PCTCN2021125262-appb-100013
    表示单键
    Figure PCTCN2021125262-appb-100014
    或双键
    Figure PCTCN2021125262-appb-100015
    各R”独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、
    Figure PCTCN2021125262-appb-100016
    Figure PCTCN2021125262-appb-100017
    且不同时为H；
    优选地，各杂芳基独立任选地被1个R”所取代时，各R”独立地选自
    Figure PCTCN2021125262-appb-100018
    优选地，R”为
    Figure PCTCN2021125262-appb-100019
    或者优选地，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”选自
    Figure PCTCN2021125262-appb-100020
    剩余R”各自独立地选自-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)；
    或者更优选地，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
    Figure PCTCN2021125262-appb-100021
    剩余R”各自独立地选自硝基、脂肪族烷基(如C1-C6烷基)、F、I、Br、Cl；
    或者最优选地，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
    Figure PCTCN2021125262-appb-100022
    剩余R”各自独立地选自硝基、C1-C6烷基，优选地，剩余R”为C1-C6烷基；
    R 5、R 6、R 7、R 8、R 9、R x、R y、R z各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、C1-C6烷基-C(＝O)-NH 2-、
    Figure PCTCN2021125262-appb-100023
    且R 5、R 6、R 7、R 8、R 9不同时为H；
    优选地，R 5、R 6、R 7、R 8、R 9中的任意一个选自
    Figure PCTCN2021125262-appb-100024
    Figure PCTCN2021125262-appb-100025
    其余四个各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、C1-C6烷基- C(＝O)-NH 2-；
    更优选地，R 5、R 6、R 7、R 8、R 9中的任意一个为
    Figure PCTCN2021125262-appb-100026
    其余四个各自独立地选自H、硝基、C1-C6烷基、C1-C6烷氧基、C1-C6烷基-C(＝O)-NH 2-；
    进一步优选地，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
    Figure PCTCN2021125262-appb-100027
    其余四个各自独立地选自H、硝基、甲氧基、乙酰胺基；
    最优选地，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
    Figure PCTCN2021125262-appb-100028
    R 7选自H、硝基、甲氧基、乙酰胺基，其余三个为H；
    优选地，R x、R y中，任意一个选自
    Figure PCTCN2021125262-appb-100029
    另一个选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)；
    更优选地，R x、R y中，任意一个选自
    Figure PCTCN2021125262-appb-100030
    另一个为H；
    最优选地，R x、R y中，任意一个为
    Figure PCTCN2021125262-appb-100031
    另一个为H；
    优选地，R z选自
    Figure PCTCN2021125262-appb-100032
    更优选地，R z为
    Figure PCTCN2021125262-appb-100033
    R 10a、R 10b、R 10c、R 11a、R 11b、R 12各自独立地选自H、-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)、-ONH 2、-OCOR m、-OCONHR m、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷 基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)，且R 10a、R 10b、R 10c不同时为H；
    优选地，R 10a、R 10b、R 10c中的任意一个选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)、-ONH 2、-OCOR m、-OCONHR m，另两个各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
    更优选地，R 10a、R 10b、R 10c中的任意一个为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)，另两个各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)；
    最优选地，R 10a、R 10b、R 10c中的任意一个为-N 3、-SS-甲基、-SS-乙基、-SS-异丙基、-SS-叔丁基或-SS-异丁基，另两个各自独立地选自H、甲基；
    优选地，R 11a、R 11b各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)；
    更优选地，R 11a、R 11b为H；
    优选地，R 12选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
    更优选地，R 12为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)；
    最优选地，R 12为-N 3；
    n选自1、2、3、4、5；优选地，n选自1、2、3；更优选地，n为2；
    各m 1独立地选自1、2、3、4、5、6；优选地，m 1为1；
    各m 2独立地选自0、1、2、3、4、5、6；优选地，各m 2独立地选自0、1；
    最优选地，
    Figure PCTCN2021125262-appb-100034
    作为整体，选自
    Figure PCTCN2021125262-appb-100035
    Figure PCTCN2021125262-appb-100036
    R’选自H，单磷酸基团
    Figure PCTCN2021125262-appb-100037
    二磷酸基团
    Figure PCTCN2021125262-appb-100038
    三磷酸基团
    Figure PCTCN2021125262-appb-100039
    或四磷酸基团
    Figure PCTCN2021125262-appb-100040
    优选地，R’为三磷酸基团
    Figure PCTCN2021125262-appb-100041
    各Z独立地选自O，S，BH；优选地，Z为O；
    Base选自碱基、脱氮碱基或其互变异构体，例如Base选自腺嘌呤、7-脱氮 腺嘌呤、胸腺嘧啶、尿嘧啶、胞嘧啶、鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体；
    优选地，Base选自
    Figure PCTCN2021125262-appb-100042
  2. 权利要求1的化合物或其盐，其中，所述化合物具有式(I)所示结构，
    Figure PCTCN2021125262-appb-100043
    其中：
    X选自O、NH、S；
    优选地，X为O；
    Y选自直接键、O、NH、S、CH、CH 2、C(CH 3) 2；
    优选地，Y为CH 2；
    R 0选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
    优选地，R 0为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)；
    更优选地，R 0为-N 3；
    n选自1、2、3、4、5；
    优选地，n选自1、2、3；
    更优选地，n为2；
    R’选自H，单磷酸基团
    Figure PCTCN2021125262-appb-100044
    二磷酸基团
    Figure PCTCN2021125262-appb-100045
    三磷酸基团
    Figure PCTCN2021125262-appb-100046
    或四磷酸基团
    Figure PCTCN2021125262-appb-100047
    优选地，R’为三磷酸基团
    Figure PCTCN2021125262-appb-100048
    各Z独立地选自O，S，BH；
    优选地，Z为O；
    Base选自碱基、脱氮碱基或其互变异构体，例如Base选自腺嘌呤、7-脱氮腺嘌呤、胸腺嘧啶、尿嘧啶、胞嘧啶、鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体；
    优选地，Base选自
    Figure PCTCN2021125262-appb-100049
  3. 权利要求1的化合物或其盐，其中，所述化合物具有式(III)所示结构，
    Figure PCTCN2021125262-appb-100050
    其中：
    A’选自
    Figure PCTCN2021125262-appb-100051
    所述杂芳基选自以下：
    Figure PCTCN2021125262-appb-100052
    Figure PCTCN2021125262-appb-100053
    Figure PCTCN2021125262-appb-100054
    各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
    优选地，所述杂芳基选自以下：
    Figure PCTCN2021125262-appb-100055
    Figure PCTCN2021125262-appb-100056
    各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
    更优选地，所述杂芳基选自以下：
    Figure PCTCN2021125262-appb-100057
    各杂芳基独立任选地被1个或多个(如2、3、4、5、6个，优选2个)R”所取代；
    或者更优选地，所述杂芳基选自以下：
    Figure PCTCN2021125262-appb-100058
    Figure PCTCN2021125262-appb-100059
    最优选地，所述杂芳基选自以下：
    Figure PCTCN2021125262-appb-100060
    各R”独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、
    Figure PCTCN2021125262-appb-100061
    Figure PCTCN2021125262-appb-100062
    优选地，各杂芳基独立任选地被1个R”所取代时，各R”独立地选自
    Figure PCTCN2021125262-appb-100063
    优选地，R”为
    Figure PCTCN2021125262-appb-100064
    或者优选地，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”选自
    Figure PCTCN2021125262-appb-100065
    剩余R”各自独立地选自-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)；
    或者更优选地，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
    Figure PCTCN2021125262-appb-100066
    剩余R”各自独立地选自硝基、脂肪族烷基(如C1-C6烷基)、F、I、Br、Cl；
    或者最优选地，各杂芳基独立任选地被多个(如2、3、4、5、6个，优选2个)R”所取代时，多个R”中，1个R”为
    Figure PCTCN2021125262-appb-100067
    剩余R”各自独立地选自硝基、C1-C6烷基，优选地，剩余R”为C1-C6烷基，更优选地，剩余R”为甲基；
    R 13、R 14、R 15、R 16中，R 16为
    Figure PCTCN2021125262-appb-100068
    R 13、R 14、R 15各自独立地选自H、硝基、C1-C6烷基(如甲基)；
    优选地，R 13、R 14、R 15、R 16中，R 16为
    Figure PCTCN2021125262-appb-100069
    R 13、R 14、R 15为H；
    R 17、R 18、R 19中，R 19为
    Figure PCTCN2021125262-appb-100070
    R 17、R 18各自独立地选自H、硝基、C1-C6烷基(如甲基)；
    优选地，R 17、R 18、R 19中，R 19为
    Figure PCTCN2021125262-appb-100071
    R 17、R 18为H；
    R 20、R 21、R 22中，R 22为
    Figure PCTCN2021125262-appb-100072
    R 20、R 21各自独立地选自H、硝基、C1-C6烷基(如甲基)；
    优选地，R 20、R 21、R 22中，R 22为
    Figure PCTCN2021125262-appb-100073
    R 20、R 21为H；
    R 23、R 24、R 25、R 26中，R 26为
    Figure PCTCN2021125262-appb-100074
    R 23、R 24、R 25各自独立地选自H、硝基、C1-C6烷基(如甲基)；
    优选地，R 23、R 24、R 25、R 26中，R 26为
    Figure PCTCN2021125262-appb-100075
    R 23、R 24、R 25为H；
    R 27、R 28、R 29中，R 29为
    Figure PCTCN2021125262-appb-100076
    R 27、R 28各自独立地选自H、硝基、C1-C6烷基(如甲基)；
    优选地，R 27、R 28、R 29中，R 29为
    Figure PCTCN2021125262-appb-100077
    R 27、R 28为H；
    R 30、R 31、R 32中，R 32为
    Figure PCTCN2021125262-appb-100078
    R 30、R 31各自独立地选自H、硝基、C1-C6烷基(如甲基)；
    优选地，R 30、R 31、R 32中，R 32为
    Figure PCTCN2021125262-appb-100079
    R 30、R 31为H；
    R 33、R 34、R 35中，R 34或R 35为
    Figure PCTCN2021125262-appb-100080
    R 33选自H、硝基、C1-C6烷基(如甲基)；
    优选地，R 33、R 34、R 35中，R 34或R 35为
    Figure PCTCN2021125262-appb-100081
    R 33为H或甲基；
    X选自O、NH、S；
    优选地，X为O；
    R 5、R 6、R 7、R 8、R 9各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、C1-C6烷基-C(＝O)-NH 2-、
    Figure PCTCN2021125262-appb-100082
    且R 5、R 6、R 7、R 8、R 9不同时为H；
    优选地，R 5、R 6、R 7、R 8、R 9中的任意一个选自
    Figure PCTCN2021125262-appb-100083
    Figure PCTCN2021125262-appb-100084
    其余四个各自独立地选自H、-N 3、硝基、氨基、磺基、羧基、脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)、芳香族烷基(如苯基C1-C6烷基)、F、I、Br、Cl、烷氧基(如C1-C6烷氧基)、C1-C6烷基- C(＝O)-NH 2-；
    更优选地，R 5、R 6、R 7、R 8、R 9中的任意一个为
    Figure PCTCN2021125262-appb-100085
    其余四个各自独立地选自H、硝基、C1-C6烷基、C1-C6烷氧基、C1-C6烷基-C(＝O)-NH 2-；
    进一步优选地，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
    Figure PCTCN2021125262-appb-100086
    其余四个各自独立地选自H、硝基、甲氧基、乙酰胺基；
    最优选地，R 5、R 6、R 7、R 8、R 9中，R 5或R 9为
    Figure PCTCN2021125262-appb-100087
    R 7选自H、硝基、甲氧基、乙酰胺基，其余三个为H；
    R 10a、R 10b、R 10c、R 11a、R 11b、R 12各自独立地选自H、-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)、-ONH 2、-OCOR m、-OCONHR m、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)，且R 10a、R 10b、R 10c不同时为H；
    优选地，R 10a、R 10b、R 10c中的任意一个选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基或-SS-异丁基)、-ONH 2、-OCOR m、-OCONHR m，另两个各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)、芳香族烷基(如苯基C1-C6烷基)、环烷基(如C3-C6环烷基)，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
    更优选地，R 10a、R 10b、R 10c中的任意一个为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基，-SS-异丁基)，另两个各自独立地选自H、C1-C6烷基(如甲基、乙基、异丙基、叔丁基)；
    最优选地，R 10a、R 10b、R 10c中的任意一个为-N 3、-SS-甲基、-SS-乙基、-SS-异丙基、-SS-叔丁基、-SS-异丁基，另两个各自独立地选自H、甲基；
    优选地，R 11a、R 11b各自独立地选自H、脂肪族烷基(如C1-C6烷基，具体如甲基、乙基、异丙基、叔丁基)；
    更优选地，R 11a、R 11b为H；
    优选地，R 12选自-N 3、-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)、-ONH 2、-OCOR m、-OCONHR m，其中，各R m独立地选自脂肪族烷基(如C1-C6烷基)、环烷基(如C3-C6环烷基)或芳香族烷基(如苯基C1-C6烷基)；
    更优选地，R 12为-N 3或-SS-C1-C6烷基(如-SS-甲基,-SS-乙基,-SS-异丙基,-SS-叔丁基)；
    最优选地，R 12为-N 3；
    m 1选自1、2、3、4、5、6；优选地，m 1为1；
    m 2选自0、1、2、3、4、5、6；优选地，m 2选自0、1；
    最优选地，
    Figure PCTCN2021125262-appb-100088
    作为整体，选自
    Figure PCTCN2021125262-appb-100089
    Figure PCTCN2021125262-appb-100090
    R’选自H，单磷酸基团
    Figure PCTCN2021125262-appb-100091
    二磷酸基团
    Figure PCTCN2021125262-appb-100092
    三磷酸基团
    Figure PCTCN2021125262-appb-100093
    或四磷酸基团
    Figure PCTCN2021125262-appb-100094
    优选地，R’为三磷酸基团
    Figure PCTCN2021125262-appb-100095
    各Z独立地选自O，S，BH；
    优选地，Z为O；
    Base选自碱基、脱氮碱基或其互变异构体，例如Base选自腺嘌呤、7-脱氮腺嘌呤、胸腺嘧啶、尿嘧啶、胞嘧啶、鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体；
    优选地，Base选自
    Figure PCTCN2021125262-appb-100096
  4. 权利要求1的化合物或其盐，其中，所述化合物选自以下，
    Figure PCTCN2021125262-appb-100097
    Figure PCTCN2021125262-appb-100098
    Figure PCTCN2021125262-appb-100099
    Figure PCTCN2021125262-appb-100100
    Figure PCTCN2021125262-appb-100101
    Figure PCTCN2021125262-appb-100102
    Figure PCTCN2021125262-appb-100103
    Figure PCTCN2021125262-appb-100104
    Figure PCTCN2021125262-appb-100105
    Figure PCTCN2021125262-appb-100106
    Figure PCTCN2021125262-appb-100107
    Figure PCTCN2021125262-appb-100108
    Figure PCTCN2021125262-appb-100109
    Figure PCTCN2021125262-appb-100110
    Figure PCTCN2021125262-appb-100111
    Figure PCTCN2021125262-appb-100112
  5. 权利要求1-4任一项所述的化合物或其盐，其携带额外的可检测标记(如荧光标记)；
    优选地，所述化合物或其盐携带的额外的可检测标记是通过亲和试剂(如抗体、适体、Affimer、Knottin)引入的，所述亲和试剂携带所述可检测标记，且所述亲和试剂可以特异性识别并结合所述化合物或其盐的表位；
    优选地，所述额外的可检测标记(如荧光标记)任选地通过连接基团与所述化合物或其盐连接；
    优选地，所述额外的可检测标记(如荧光标记)任选地通过连接基团与所述化合物或其盐的Base连接；
    优选地，所述连接基团为可裂解的连接基团或不可裂解的连接基团；
    优选地，所述可裂解的连接基团选自亲电裂解的连接基团、亲核裂解的连接基团、可光解的连接基团、还原条件下裂解的连接基团、氧化条件下裂解的连接基团、安全拉手型连接基团、经消除机理裂解的连接基团，或其任何组合；
    优选地，所述可检测标记选自以下：
    Figure PCTCN2021125262-appb-100113
    Figure PCTCN2021125262-appb-100114
    优选地，Base不同，所述可检测标记(如荧光标记)不同。
  6. 终止核酸合成的方法，其包括：将权利要求1-5任一项所述的化合物或其盐掺入待终止的核酸分子中；
    优选地，所述化合物或其盐的掺入通过末端转移酶、末端聚合酶或逆转录酶来实现；
    优选地，所述方法包括：使用聚合酶，将所述化合物或其盐掺入待终止的核酸分子中；
    优选地，所述方法包括：在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述化合物或其盐掺入待终止的核酸分子的3'端。
  7. 制备在测序反应中与目标单链多核苷酸互补的生长的多核苷酸的方法，其包括将权利要求1-5中任一项所述的化合物或其盐掺入所述生长的互补多核苷酸，其中，所述化合物或其盐的掺入防止了任何后续的核苷酸引入所述生长的互补多核苷酸中；
    优选地，所述化合物或其盐的掺入通过末端转移酶、末端聚合酶或逆转录酶来实现；
    优选地，所述方法包括：使用聚合酶，将所述化合物或其盐掺入所述生长的互补多核苷酸；
    优选地，所述方法包括：在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述化合物或其盐掺入所述生长的互补多核苷酸的3'端。
  8. 测定目标单链多核苷酸的序列的方法，其包括：
    1)监测生长的核酸链中与目标单链多核苷酸互补的核苷酸的掺入，其中，掺入的至少一个互补核苷酸是权利要求1-5中任一项所述的化合物或其盐，且所述化合物或其盐携带额外的可检测标记(如荧光标记)，以及，
    2)检测所述可检测标记，确定掺入的核苷酸；
    优选地，所述额外的可检测标记(如荧光标记)任选地通过连接基团与所述化合物或其盐连接；
    优选地，所述连接基团如权利要求5所限定；
    优选地，所述额外的可检测标记如权利要求5所限定；
    优选地，Base不同，所述化合物或其盐携带的可检测标记(如荧光标记)不同；
    优选地，在引入下一个互补核苷酸之前，将所述化合物或其盐中的可逆阻断基团(R)和所述可检测标记除去；
    优选地，所述可逆阻断基团和所述可检测标记被同时除去；或者
    优选地，所述可逆阻断基团和所述可检测标记被先后除去；例如，在所述可检测标记被除去之后，所述可逆阻断基团被除去，或者，在所述可逆阻断基团被除去之后，所述可检测标记被除去。
  9. 权利要求8的方法，其包括以下步骤：
    (a)提供多种不同的核苷酸，其中所述多种不同的核苷酸是权利要求1-5任一项所述的化合物或其盐，其中，每种类型的核苷酸携带的额外的可检测标记在检测时可以与其他类型的核苷酸携带的额外的可检测标记区分开；
    (b)将所述多种不同的核苷酸掺入目标单链多核苷酸的互补序列中；
    (c)检测(b)的核苷酸携带的额外的可检测标记，从而确定掺入的核苷酸的类型；
    (d)除去(b)的核苷酸中的可逆阻断基团和其携带的可检测标记；和
    (e)任选地重复步骤(b)-(d)一次或多次；
    从而确定所述目标单链多核苷酸的序列。
  10. 权利要求8的方法，其包括以下步骤：
    (1)提供第一核苷酸、第二核苷酸、第三核苷酸和第四核苷酸，四种核苷酸中的至少一种是权利要求1-5中任一项所述的化合物或其盐，所述四种核苷酸包含的Base互不相同，且所述四种核苷酸携带额外的可检测标记(如荧光标记)，优选地，所述四种核苷酸携带的额外的可检测标记是通过亲和试剂(如抗体、适体、Affimer、Knottin)引入的，所述亲和试剂携带所述可检测标记，且所述亲和试剂可以特异性识别并结合每一种核苷酸的表位，或者优选地，所述四种核苷酸任选地通过连接基团与所述额外的可检测标记连接，或者更优选地，所述四种核苷酸中的Base任选地通过连接基团与所述额外的可检测标记连接，最优选地，所述四种核苷酸所携带的额外的可检测标记互不相同；
    (2)将所述四种核苷酸与目标单链多核苷酸进行接触；除去未掺入生长的核酸链中的所述核苷酸；检测掺入生长的核酸链中的所述核苷酸所携带的所述可检测标记；除去掺入生长的核酸链中的所述核苷酸所包含的可逆阻断基团和所携带的所述可检测标记；
    任选地，还包括(3)：重复(2)一次或多次。
  11. 权利要求8的方法，其包括以下步骤：
    (a)提供包含双链体、至少一种权利要求1-5中任一项所述的化合物或其盐、聚合酶和切除试剂的混合物；所述双链体包含生长的核酸链以及待测序的核酸链；所述化合物或其盐携带额外的可检测标记(如荧光标记)，优选地，所述化合物或其盐携带的额外的可检测标记是通过亲和试剂(如抗体、适体、 Affimer、Knottin)引入的，所述亲和试剂携带所述可检测标记，且所述亲和试剂可以特异性识别并结合所述化合物或其盐的表位，或者优选地，所述化合物或其盐任选地通过连接基团与所述额外的可检测标记连接，或者更优选地，所述化合物或其盐中的Base任选地通过连接基团与所述额外的可检测标记连接；
    (b)进行包含以下步骤(i)、(ii)和(iii)的反应，任选地，重复一次或多次：
    步骤(i)：使用聚合酶，使所述化合物或其盐掺入生长的核酸链，形成包含可逆阻断基团和可检测标记的核酸中间体：
    步骤(ii)：对所述核酸中间体所包含的可检测标记进行检测；
    步骤(iii)：使用切除试剂将所述核酸中间体所包含的可逆阻断基团和/或所述可检测标记切除；
    优选地，对所述可逆阻断基团的切除和对所述可检测标记的切除同时进行，或者，对所述可逆阻断基团的切除和对所述可检测标记的切除分步进行(例如，先切除所述可逆阻断基团，或者先切除所述可检测标记)；
    优选地，对所述可逆阻断基团的切除和对所述可检测标记的切除使用的切除试剂是同样的试剂；
    优选地，对所述可逆阻断基团的切除和对所述可检测标记的切除使用的切除试剂是不同的试剂。
  12. 权利要求11的方法，其中，所述双链体连接于支持物上；
    优选地，所述生长的核酸链为引物；
    优选地，所述引物通过退火至待测序的核酸链上，形成所述双链体；
    优选地，所述双链体、所述化合物或其盐、以及所述聚合酶一起形成含有溶液相和固相的反应体系；
    优选地，所述化合物或其盐包含的Base互不相同；
    优选地，所述化合物或其盐携带的额外的可检测标记互不相同；
    优选地，在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶，使所述化合物或其盐掺入生长的核酸链，形成包含可逆阻断基团和可检测标记的核酸中间体；
    优选地，所述聚合酶选自KOD聚合酶或其突变体(例如KOD POL151、KOD POL157、KOD POL171、KOD POL174、KOD POL376、KOD POL391)；
    优选地，在任意一个检测所述核酸中间体所包含的可检测标记步骤前，移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体；
    优选地，所述切除试剂与所述双链体或所述生长的核酸链在含有溶液相和固相的反应体系中接触；
    优选地，所述切除试剂能够切除掺入生长的核酸链的化合物中的可逆阻断基团和所述化合物携带的额外的可检测标记，并且不会影响双链体骨架上的磷酸二酯键；
    优选地，在任意一个切除所述核酸中间体所包含的可逆阻断基团和/或额外的可检测标记的步骤后，移除这一步骤反应体系的溶液相；
    优选地，在任意一个包含移除操作的步骤之后，进行洗涤操作；
    优选地，步骤(ii)之后，进一步包括：根据步骤(ii)检测得到的信号，确定步骤(i)中掺入生长的核酸链的化合物的类型，并基于碱基互补配对原 则，确定待测序的核酸链中相应位置处的核苷酸类型。
  13. 试剂盒，其包含至少一个权利要求1-5任一项所述的化合物或其盐；
    优选地，所述试剂盒包含第一、第二、第三和第四化合物，所述第一、第二、第三和第四化合物各自独立地为权利要求1-5任一项所述的化合物或其盐；
    优选地，所述第一化合物中，Base选自腺嘌呤、7-脱氮腺嘌呤或其互变异构体(例如
    Figure PCTCN2021125262-appb-100115
    )；所述第二化合物中，Base选自胸腺嘧啶、尿嘧啶或其互变异构体(例如
    Figure PCTCN2021125262-appb-100116
    )；所述第三化合物中，Base选自胞嘧啶或其互变异构体(例如
    Figure PCTCN2021125262-appb-100117
    )；所述第四化合物中，Base选自鸟嘌呤、7-脱氮鸟嘌呤或其互变异构体(例如
    Figure PCTCN2021125262-appb-100118
    )；
    优选地，所述第一、第二、第三和第四化合物携带额外的可检测标记；
    优选地，所述第一、第二、第三和第四化合物携带的额外的可检测标记是通过亲和试剂(如抗体、适体、Affimer、Knottin)引入的，所述亲和试剂携带所述可检测标记，且所述亲和试剂可以特异性识别并结合所述第一、第二、第三或第四化合物的表位；
    优选地，所述第一、第二、第三和第四化合物任选地通过连接基团与所述额外的可检测标记连接；
    优选地，所述第一、第二、第三和第四化合物中的Base任选地通过连接基团与所述额外的可检测标记连接；
    优选地，所述第一、第二、第三和第四化合物包含的Base互不相同；
    优选地，所述第一、第二、第三和第四化合物所携带的额外的可检测标记互不相同；
    优选地，所述连接基团如权利要求5所限定；
    优选地，所述可检测标记如权利要求5所限定。
  14. 权利要求13的试剂盒，其中，所述试剂盒还包含：用于预处理核酸分子的试剂；用于连接待测序的核酸分子的支持物；用于将待测序的核酸分子与支持物连接(例如，共价或非共价连接)的试剂；用于起始核苷酸聚合反应的引物；用于进行核苷酸聚合反应的聚合酶；一种或多种缓冲溶液；一种或多种洗涤溶液；或其任何组合。
  15. 权利要求1-5任一项所述的化合物或其盐或者权利要求13-14任一项所述的试剂盒用于测定目标单链多核苷酸的序列的用途。
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