Nucleoside analogue, preparation method and application
阅读说明：本技术 核苷类似物、制备方法及应用 (Nucleoside analogue, preparation method and application ) 是由 刘斌 赵陆洋 陈予想 杨波 简杰 陈方 颜钦 于 2018-11-29 设计创作，主要内容包括：涉及一种具有式(I)结构的核苷酸类似物,该核苷酸类似物的制备方法及其在核酸序列测定等方面的应用。<Image he="267" wi="700" file="DDA0002648263640000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(Relates to a nucleotide analogue with a structure shown in a formula (I), a preparation method of the nucleotide analogue and application of the nucleotide analogue in aspects of nucleic acid sequence determination and the like.)
A compound having the formula shown in formula (I):
wherein L is1、L2、L3Each independently is a covalent bond or a covalent linking group;
b is a base or a base derivative;
R1is-OH, a phosphate group or a nucleotide;
R2is H or a cleavable group;
R3a detectable group or a targeting group;
R5is an inhibiting group;
R4is H OR-OR6Wherein R is6Is H or a cleavable group;
c is a cleavable group or a cleavable bond.
The compound of claim 1, wherein the phosphate group is a monophosphate group, diphosphate group, triphosphate group, or polyphosphate group.
The compound of claim 1, wherein B is cytosine or a derivative thereof, thymine or a derivative thereof, adenine or a derivative thereof, guanine or a derivative thereof, hypoxanthine or a derivative thereof, deazaadenine or a derivative thereof, deazaguanine or a derivative thereof, deazahypoxanthine or a derivative thereof, 7-methylguanine or a derivative thereof, 5, 6-dihydrouracil or a derivative thereof, 5-methylcytosine or a derivative thereof, or 5-hydroxymethylcytosine or a derivative thereof.
A compound according to any one of claims 1 to3, wherein B is divalent cytosine or a derivative thereof, divalent guanine and a derivative thereof, dimethyladenine or a derivative thereof, divalent thymine or a derivative thereof, divalent uracil or a derivative thereof, divalent hypoxanthine or a derivative thereof, divalent xanthine or a derivative thereof, divalent deazaadenine or a derivative thereof, divalent deazaguanine or a derivative thereof, divalent deazahypoxanthine or a derivative thereof, divalent 7-methylguanine or a derivative thereof, divalent 5, 6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine or a derivative thereof, or divalent 5-hydroxymethylcytosine or a derivative thereof.
A compound according to any one of claims 1 to4, wherein B is
A compound of claim 1, wherein R is6Is H, optionally substituted alkyl or amine.
A compound of claim 6, wherein R is6Is H, C1～5Alkyl or-NH2。
A compound of claim 1, wherein R is2Selected from H,
The compound of claim 1, wherein C is a group comprising at least one selected from the group consisting of:
optionally substituted ethynyl, optionally substituted disulfide, optionally substituted amidoalkyl, optionally substituted aryl, optionally substituted alkoxy, optionally substituted azido, optionally substituted linear or branched alkyl.
The compound of claim 9, wherein C is a group comprising at least one selected from the group consisting of:
ethynyl optionally substituted with hydroxyl, disulfide, amide, alkyleneoxy, aryl, azide, straight or branched alkylene.
The compound of claim 10, wherein C is selected from at least one of:
a compound according to any one of claims 1 to 10 wherein L is1、L2And L3Each independently selected from at least one of an independent bond, optionally substituted alkyl, optionally substituted alkylalkenyl, optionally substituted heteroalkylene, optionally substituted cycloalkyl, optionally substituted isocycloalkyl, and optionally substituted arylalkenyl.
A compound according to any one of claims 1 to 10 wherein L is1、L2And L3Each independently selected from the group consisting of an independent bond, optionally substituted C1-8Alkyl, optionally substituted C1-8Alkyl alkenes, optionally substituted C2-8Heteroalkylene, optionally substituted C3-8Cycloalkylene, optionally substituted C3-8Isocycloalkylalkenes, or optionally substituted C6-8An arylalkene.
A compound according to any one of claims 1 to 10 wherein L is1At least one selected from the group consisting of an independent bond, an optionally substituted C1-C3 alkyl, and an optionally substituted C1-C3 alkyl alkene.
A compound according to any one of claims 1 to 10 wherein L is1Is L1 A-L1 B-L1 C-L1 D-L1 E，L2Is L2 A-L2 B-L2 C-L2 D-L2 EAnd/or L3Is L3 A-L3 B-L3 C-L3 D-L3 EWherein, in the step (A),
L1 A、L1 B、L1 C、L1 D、L1 E、L2 A、L2 B、L2 C、L2 D、L2 E、L3 A、L3 B、L3 C、L3 Dand L3 EEach independently selected from the group consisting of an independent bond, a substituted or unsubstituted alkyl alkene, a substituted or unsubstituted heteroalkyl alkene, a substituted or unsubstituted cycloalkyl alkene, a substituted or unsubstituted isocycloalkyl alkene, a substituted or unsubstituted aryl alkene,
L1 A、L1 B、L1 C、L1 Dand L1 EAt least one of which is not a separate key,
L2 A、L2 B、L2 C、L2 Dand L2 EAt least one of which is not a separate key,
L3 A、L3 B、L3 C、L3 Dand L3 EAt least one of which is not a separate bond.
The compound of claim 15,
each L1 A、L1 B、L1 C、L1 D、L1 E、L2 A、L2 B、L2 C、L2 D、L2 E、L3 A、L3 B、L3 C、L3 DAnd L3 EEach independently selected from the group consisting of an independent bond, C2-6Alkyl alkene, hetero alkyl alkene composed of 2-6 atoms, C3-6Cycloalkylene, heterocycloalkylalkene of 2 to6 atoms, C6-10Arylalkenes of which each C2-6Alkyl alkene, hetero alkyl alkene composed of 2-6 atoms, C3-6Cycloalkylene, heterocycloalkylalkene of 2 to6 atoms, C6-10The arylalkenes are independently unsubstituted or substituted by 1,2,3,4, 5C1-6Alkyl, halogen, nitro, C1-6Haloalkyl substitution.
A compound according to any one of claims 1 to16 wherein L is1Comprising at least one selected from the group consisting of:
wherein each R is7Independently an amide bond or O, or a pharmaceutically acceptable salt thereof,
each p1Independently an integer of 0,1 to 10, preferably each p1Independently 1, 4;
each p2Independently an integer of 0,1 to5, preferably each p2Independently 1, 4;
each R8，R9Are respectively and independently selected from H and H-CH3、-CX3、-CHX2、-CH2X、-CN、-Ph、C1-6Alkyl radical, C2～6Alkyl and C3～6At least one of cycloalkyl, wherein X is Cl, Br, I;
each p3Independently an integer of 0,1 to4, preferably each p3Independently 1 and 2.
The compound according to any one of claims 1-17, L2Comprising at least one selected from the group consisting of:
wherein each of Rx, Ry and RA、RBIndependently is H, C1～6Alkyl chain, C3-10Cycloalkyl radical, C5-10Aryl, each of x, y, and z is independently 0 or an integer of 1 to6,
preferably, each Rx, Ry is independently H, C3-5Cycloalkyl radical, C5-6Cycloalkyl, phenyl.
The compound of any one of claims 1-17, wherein L is2Is one selected from the following structures:
The compound of any one of claims 1-19, wherein L is3Is selected from the group consisting ofOne of the following structures: :
wherein n1, n2, n3, n4, n5 and n6 are respectively and independently 0 or an integer of 1-7.
The compound of any one of claims 1-20, wherein L is3Having one of the following structures:
the compound of any one of claims 1-21, wherein R is3At least one selected from the group consisting of a dye, a click chemistry reactive group, an azido group, and a biotin group.
The compound of any one of claims 1-22, wherein R is5Comprising at least one charged group comprising-COOH, -PO4、-SO4、-SO3、-SO2。
The compound of any one of claims 1-23, wherein R is5Has a structure selected from the group consisting of:
wherein each R is10And R11Independently is H or C1-6Alkyl, each a and b is independently 0 or an integer from 1 to 5;
preferably, each of a and b is independently 1 or 2.
The compound of any one of claims 1-24, wherein R is5Comprises one selected from the following groups:
a compound according to any one of claims 1 to 25, characterized in that it has at least one of the following structures:
wherein each Ra and Rb is independently selected from at least one of H, optionally substituted alkyl, optionally substituted heteroalkylene, optionally substituted cycloalkyl, optionally substituted isocycloalkyl, and optionally substituted aryl;
preferably, each Ra and Rb is independently H, C1-6Alkyl, 3-6 atom heteroalkyl, C2-6Alkenyl, heteroalkenyl of 3 to6 atoms, C3-6Cycloalkyl, heterocyclic group consisting of 3 to6 atoms, phenyl, heteroaryl group consisting of 5 to6 atoms, wherein each C1-6Alkyl, 3-6 atom heteroalkyl, C2-6Alkenyl, heteroalkenyl of 3 to6 atoms, C3-6Cycloalkyl, heterocyclyl consisting of 3 to6 atoms, phenyl and heteroaryl consisting of 5 to6 atoms are independently unsubstituted or substituted by 1,2 or 3 halogen, C1-6Alkyl radical, C2-6Alkenyl, CN, NO2And (4) substitution.
A compound according to any one of claims 1 to 26, characterized in that it has at least one of the following structures:
a dNTP analog comprising at least one selected from the group consisting of:
a dATP analogue which is a compound according to any one of claims 1 to 27, B is base a;
a dCTP analogue which is a compound according to any one of claims 1 to 27, B is base C;
a dGTP analogue which is a compound according to any one of claims 1 to 27, B is base G; and
a dTTP analog which is a compound of any one of claims 1 to 27, wherein B is a base T.
An NTP analogue comprising at least one member selected from the group consisting of:
an ATP analogue, the ATP analogue being a compound according to any one of claims 1 to 27, B being base A;
a CTP analog which is a compound of any one of claims 1 to 27, B is base C;
a GTP analogue, the GTP analogue being a compound according to any one of claims 1 to 27, B being a base G; and
a UTP analogue which is a compound as claimed in any one of claims 1 to 27, B being the base U.
A dNTP analog mixture comprising:
a dATP analogue which is a compound according to any one of claims 1 to 27, B is base a;
a dCTP analogue which is a compound according to any one of claims 1 to 27, B is base C;
a dGTP analogue which is a compound according to any one of claims 1 to 27, B is base G; and
a dTTP analog which is a compound of any one of claims 1 to 27, B is a base T,
wherein at least three of the dATP analog, dCTP analog, dGTP analog and dTTP analog have different detectable groups, respectively.
A dNTP analog mixture comprising a combination of any two of the following nucleotide analogs:
a dATP analogue which is a compound according to any one of claims 1 to 27, B is base a;
a dCTP analogue which is a compound according to any one of claims 1 to 27, B is base C;
a dGTP analogue which is a compound according to any one of claims 1 to 27, B is base G; and
a dTTP analog which is a compound of any one of claims 1 to 27, B is a base T,
wherein the two nucleotide analogs in the combination have different detectable groups.
A NTP analogue mixture comprising:
an ATP analogue, the ATP analogue being a compound according to any one of claims 1 to 27, B being base A;
a CTP analog which is a compound of any one of claims 1 to 27, B is base C;
a GTP analogue, the GTP analogue being a compound according to any one of claims 1 to 27, B being a base G; and
a UTP analogue which is a compound according to any one of claims 1 to 27, B is a base U,
wherein at least three of the ATP analogue, the CTP analogue, the GTP analogue and the UTP analogue have different detectable groups respectively.
A mixture of NTP analogues comprising a combination of any two of the following nucleotide analogues:
an ATP analogue, the ATP analogue being a compound according to any one of claims 1 to 27, B being base A;
a CTP analog which is a compound of any one of claims 1 to 27, B is base C;
a GTP analogue, the GTP analogue being a compound according to any one of claims 1 to 27, B being a base G; and
a UTP analogue which is a compound according to any one of claims 1 to 27, B is a base U,
wherein the two nucleotide analogs in the combination have different detectable groups.
Use of a dNTP analogue of claim 28 or the NTP analogue of claim 29 or the dNTP analogue mixture of claim 30 or the dNTP analogue mixture of claim 31 or the NTP analogue mixture of claim 32 or the NTP analogue mixture of claim 33 in nucleic acid sequencing or controlled polymerase chain reaction or base extension reaction.
A kit for nucleic acid sequencing or controlled chain polymerase reaction comprising:
a dNTP analogue of claim 28 or the NTP analogue of claim 29 or the dNTP analogue mixture of claim 30 or the dNTP analogue mixture of claim 31 or the NTP analogue mixture of claim 32 or the NTP analogue mixture of claim 33.
The kit of claim 35, further comprising:
a cleaving reagent that can act on the cleavable group or cleavable bond; and/or
A DNA polymerase.
The kit of claim 36, wherein the kit comprises a cleavage reagent that is TCEP/THPP.
A method for determining a nucleic acid sequence, comprising:
(a) subjecting a mixture of a first nucleic acid template-primer complex, one or more nucleotide analogs, and a DNA polymerase to conditions suitable for base extension such that the nucleotide analogs bind to the first nucleic acid template-primer complex to obtain an extension product;
the nucleotide analogue is selected from a dNTP analogue of claim 28 or the NTP analogue of claim 29 or the dNTP analogue mixture of claim 30 or the dNTP analogue mixture of claim 31 or the NTP analogue mixture of claim 32 or the NTP analogue mixture of claim 33.
The method of claim 38, wherein the first nucleic acid template-primer complex is attached to a solid support.
The method of claim 38, wherein the first nucleic acid template-primer complex is immobilized on a chip or on a microsphere.
The method of claim 38, further comprising:
(b) acting on the cleavable group or cleavable bond on the extension product to obtain a second nucleic acid template-primer complex;
(c) performing (a) and (b) at least once with the second nucleic acid template primer complex replacing the first template-primer complex.
The method of any one of claims 38-41, wherein (a) further comprises:
detecting the extension product to obtain a signal corresponding to the nucleotide analog bound to the first nucleic acid template-primer complex.
The method of claim 42, further comprising:
based on the signal, the nucleic acid sequence is determined.
The method of claim 38, wherein the nucleotide analog comprises a targeting moiety, and (a) comprises adding a detectable compound having a specificity moiety capable of specifically binding to the targeting moiety to obtain the extension product.
A method of extending a primer, comprising placing a polymerase, a nucleic acid template-primer complex, and one or more nucleotide analogs in a reaction vessel, such that the nucleotide analogs bind to the nucleic acid template-primer complex, thereby obtaining an extension product; the nucleotide analogue is selected from the compounds of any one of claims 1 to 27.
A reaction mixture comprising a test template, a primer for pairing with at least a portion of a strand of the test template, a DNA polymerase and a dNTP analog of claim 28 or an NTP analog of claim 29 or a dNTP analog mixture of claim 30 or a dNTP analog mixture of claim 31 or an NTP analog mixture of claim 32 or an NTP analog mixture of claim 33.
The reaction mixture of claim 46, wherein said template, primer polymerase and dNTP analogs of claim 28 or NTP analogs of claim 29 or dNTP analogs mixture of claim 30 or dNTP analogs mixture of claim 31 or NTP analogs mixture of claim 32 or NTP analogs mixture of claim 33 are in a buffer.
A process for preparing a compound according to any one of claims 1 to 27, comprising:
synthesizing SPDP by using dithiodipyridine and mercaptopropionic acid;
by using
Connecting dNTP and the SPDP to obtain dNTP-SPDP;
synthesizing the compound using the first linking structure and the dNTP-SPDP.
A process for preparing a compound according to any one of claims 1 to 27, comprising:
synthesizing dNTP-MPSSK, wherein the structure of the dNTP-MPSSK is shown as follows:
by usingAnd hexapeptide synthesis of a second linking structure
mixing the second linking structure and the dNTP-MPSSK to produce the compound.
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