Modified nucleoside or nucleotide

Abstract

The present invention provides a modified nucleoside or nucleotide in which the 3'-OH group of the modified nucleoside or nucleotide is reversibly blocked. [Solution] A compound of formula (A) or a salt thereof is provided. TIFF2026086551000395.tif33149 (In formula (A), R is a reversible blocking group, and R is TIFF2026086551000396.tif25149 (R' is selected from H, monophosphate, diphosphate group, triphosphate group, or tetraphosphate group.)

Description

[Technical Field]

[0001] This invention relates to the field of nucleic acid sequencing. In particular, this invention relates to modified nucleosides or nucleotides in which the 3'-OH group of the modified nucleoside or nucleotide is reversibly blocked. [Background technology]

[0002] The advent of NGS sequencing overcomes the drawbacks of Sanger's high cost and time requirements, significantly accelerating the application of gene sequencing technology. Currently, NGS sequencing is widely applied in fields such as prenatal screening, tumor diagnosis, tumor treatment, and animal and plant breeding, driving advancements in science and medicine.

[0003] Deoxyribonucleoside triphosphate (dNTP) analogs supporting reversible blocking groups are crucial raw materials in NGS sequencing. The incorporation of reversible blocking groups allows for the retention of the 3'-OH group in dNTPs, overcoming the weaknesses of Sanger sequencing and ensuring the accuracy of base recognition. Deoxyribonucleoside triphosphate (dNTP) analogs with reversible blocking groups can be considered the most decisive technology in NGS sequencing.

[0004] Currently, many dNTP compounds carrying reversible blocking groups have been reported. Reversible blocking of dNTPs is mainly achieved through two approaches. The first approach is based on the direct introduction of a reversible blocking group to the 3'-OH of the dNTP. The advantage of such modified dNTPs is that blocking the 3'-OH ensures blocking efficiency in sequencing. The second approach is based on blocking the polymerase by base modification rather than blocking the 3'-OH. The advantage of this approach is that the blocking group can be selected from a wider range, rather than being limited to polymerases.

[0005] Generally, the two approaches to reversible blocking have their own advantages and disadvantages, but the method of directly introducing the base blocking into the 3'-OH group exhibits greater certainty and significantly higher blocking efficiency. Therefore, such a scheme is primarily used in NGS sequencing currently on the market. [Overview of the Initiative]

[0006] Azidomethyl is a highly effective 3'-OH reversible blocking group with excellent advantages such as good stability, mild removal conditions, and a fast removal rate. The escaping reaction of azidomethyl on the 3'-OH is essentially a cascade reaction in which the azido group undergoes a Staudinger reaction under the action of a phosphorus reagent to produce an intermediate with a methyleneamino group on the 3'-OH, which then undergoes a rapid hydrolysis reaction to liberate the 3'-OH. In such a cascade reaction, both the Staudinger reaction and the hydrolysis reaction proceed very rapidly, and therefore azidomethyl can be removed at a very rapid rate (as shown in Figure 1).

[0007] The azide group is a special type of chemical group in which three nitrogen atoms in its chemical structure form a conjugated structure and lie on the same plane. The three nitrogen atoms are not located in a straight line, but rather at a specific angle. The conjugated structure formed by the three nitrogen atoms in the azide group can be represented by several resonance structures, which contributes to its stability.

[0008] Azide groups are generally stable. However, due to their repeating structure of three nitrogen atoms, azide groups can react under suitable reaction conditions and rapidly release nitrogen, thus possessing very high reactivity and a tendency towards explosion. The widespread use of azide groups in various click reactions is precisely based on their characteristics.

[0009] Based on the rapid reactivity of azide groups, the inventors designed a class of azide-supported 3'-OH blocking groups that can undergo cascading reactions. The 3'-OH blocking group undergoes a Staudinger reaction to release an amine group, which attacks the ester / carbonate protecting group on the 3'-OH and liberates the 3'-OH (as shown in Figure 2). Similarly, all groups that can release a nucleophilic atom under suitable conditions, such as -S-SR, -OCOR, and -OCONHR, are applicable to such designs.

[0010] The present invention aims to develop a class of dNTP analogs that have ester and carbonate groups supported on a 3'-OH group, which are useful in NGS sequencing. Such dNTP analogs have a general structural formula shown in Figure 3, which has a 3'-OH group protected by a reversible blocking group, and bulk structures including 2'-deoxyuridine triphosphate, 2'-deoxythymosine triphosphate, 2'-deoxycytidine triphosphate, 2'-deoxyadenosine triphosphate, 7-deaza-2'-deoxyadenosine triphosphate, 2'-deoxyguanosine triphosphate, and 7-deaza-2'-deoxyguanosine triphosphate.

[0011] Therefore, in a first aspect of the present invention, the present invention provides a compound of formula (A) or a salt thereof, [ka] During the ceremony, R is a reversible blocking group, and R is [ka] Selected from, The heteroaryls are as follows: [ka] Selected from, Each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''. Each X is independently selected from O, NH, S, each Y is independently selected from a direct bond, O, NH, S, CH, CH2, C(CH3)2, R is -N3, -SS-C1-C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH2, -OCOR m , -OCONHR m and is selected from, each R m is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl) or aromatic alkyl (e.g., phenyl C1-C6 alkyl), R 1 is -N3, -SS-C1-C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH2, -OCOR m , -OCONHR m and is selected from, each R m is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl) or aromatic alkyl (e.g., phenyl C1-C6 alkyl), R 2 R 3 and R 4 are each independently selected from O, NH, S, CH, CH2, C(CH3)2,

Chemical formula

Chemical formula

Chemical formula

[0012] In some embodiments, R is [ka] Selected from.

[0013] In some embodiments, R is [ka] Selected from.

[0014] In some embodiments, R is [ka] Selected from.

[0015] In some embodiments, R is [ka] Selected from.

[0016] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0017] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0018] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0019] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0020] In some embodiments, X is O.

[0021] In some embodiments, each Y is selected either directly or independently of CH2.

[0022] In some embodiments, R 0 The alkyl group is -N3 or -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl).

[0023] In some embodiments, R 0 It is -N3.

[0024] In some embodiments, R 1 The alkyl group is -N3 or -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl).

[0025] In some embodiments, R 1 It is -N3.

[0026] In some embodiments, R 2 , R 3 , and R 4 Each of these is independently selected from O, NH, S, CH, CH2, and C(CH3)2, and satisfies the following condition: R 2 If O or S is selected, R 3 and R 4 is CH2; R 3 If O or S is selected, R 2 and R 4 is CH2; R 4 If O or S is selected, R 2 and R 3 is CH2; R 2 If is C(CH3)2, then R 3 and R 4 is CH2; R 3 If is C(CH3)2, then R 2 and R 4 is CH2; R 4 If is C(CH3)2, then R 2 and R 3 If is CH2, then R 2 CH is, R 3 and R 4 It is CH2.

[0027] In some embodiments, R 2 , R 3 , and R 4 CH or CH2 are selected independently.

[0028] In some embodiments, R 2 and R 3 CH is, R 4 It is CH2.

[0029] In some embodiments, each heteroaryl may be independently substituted by one R'', where each R'' is [ka] Selected independently from, preferably R'' is [ka] That is the case.

[0030] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] One is selected from the above, and the remaining R''(one or more) are independently selected from -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, and alkoxy (e.g., C1-C6 alkoxy).

[0031] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] The remaining R''(one or more) are independently selected from nitro, aliphatic alkyl (e.g., C1-C6 alkyl), F, I, Br, and Cl.

[0032] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] The remaining R''(one or more) are independently selected from nitro and C1-C6 alkyl groups, preferably the remaining R''(one or more) are C1-C6 alkyl groups.

[0033] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 of any one of is selected from [Chemical formula] [Chemical formula]

[0034] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 of any one of is selected from [Chemical formula] [Chemical formula]

[0035] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 Among R 5 , R 7 or R 9 is [Chemical formula] [Chemical formula]

[0036] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R9 One of the following is [ka] One is selected from the above, and the remaining four are independently selected from H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (e.g., C1-C6 alkoxy), and C1-C6 alkyl-C(=O)-NH2-.

[0037] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 One of the following is [ka] The remaining four are independently selected from H, nitro, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkyl-C(=O)-NH2-.

[0038] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 R 5 or R 9 teeth, [ka] The remaining four are independently selected from H, nitro, methoxy, and acetamide.

[0039] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 R 5 or R 9 teeth, [Chemical formula] where R 7 is selected from H, nitro, methoxy, acetamide, and the remaining three are H.

[0040] In some embodiments, any one of R x and R y is selected from [Chemical formula] and the other is selected from H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (e.g., C1-C6 alkoxy).

[0041] In some embodiments, any one of R x and R y is selected from [Chemical formula] and the other is H.

[0042] In some embodiments, any one of R x and R y is [Chemical formula] and the other is H.

[0043] In some embodiments, R z is selected from [Chemical formula] In some embodiments, R

[0044] is selected from z is [Chemical formula] That is the case.

[0045] In some embodiments, R 10a , R 10b , and R 10c One of these is -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), -ONH2, -OCOR m ,-OCONHR m One is selected from, and the other two are independently selected from H, aliphatic alkyl (e.g., C1-C6 alkyl such as methyl, ethyl, isopropyl, tert-butyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), and cycloalkyl (e.g., C3-C6 cycloalkyl), and each R m The element is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl).

[0046] In some embodiments, R 10a , R 10b , and R 10c One of these is a -N3 or -SS-C1~C6 alkyl group (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), and the other two are independently selected from H and aliphatic alkyl groups (e.g., C1~C6 alkyl groups such as methyl, ethyl, isopropyl, and tertbutyl).

[0047] In some embodiments, R 10a , R 10b , and R 10c One of these is either -N3 or -SS-methyl, and the other two are independently selected from H and methyl, respectively.

[0048] In some embodiments, R 10a , R 10b , and R 10cAny one of them is -N3, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl or -SS-isobutyl, and the other two are each independently selected from H and methyl.

[0049] In some embodiments, R 11a and R 11b are each independently selected from H and aliphatic alkyl (for example, C1-C6 alkyl such as methyl, ethyl, isopropyl, tert-butyl, etc.).

[0050] In some embodiments, R 11a and R 11b are H.

[0051] In some embodiments, R 12 is selected from -N3, -SS-C1-C6 alkyl (for example, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH2, -OCOR m , -OCONHR m , and each R m is independently selected from aliphatic alkyl (for example, C1-C6 alkyl), cycloalkyl (for example, C3-C6 cycloalkyl) or aromatic alkyl (for example, phenyl C1-C6 alkyl).

[0052] In some embodiments, R 12 is -N3 or -SS-C1-C6 alkyl (for example, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl).

[0053] In some embodiments, R 12 is -N3.

[0054] In some embodiments, n is selected from 1, 2, 3.

[0055] In some embodiments, n is 2.

[0056] In some embodiments, m1 is 1.

[0057] In some embodiments, each m2 is independently selected from 0 or 1.

[0058] In some embodiments, [ka] Overall, [ka] Selected from.

[0059] In some embodiments, R' is a triphosphate group ( [ka] )

[0060] In some embodiments, Z is O.

[0061] In some embodiments, Base is [ka] Selected from.

[0062] In a second aspect of the present invention, the present invention provides a compound of formula (I) or a salt thereof, [ka] During the ceremony, X is selected from O, NH, and S. Y is selected from direct bonds, O, NH, S, CH, CH2, and C(CH3)2. R 0 -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH2, -OCOR m ,-OCONHR m Selected from, each R mThis is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl), n is selected from 1, 2, 3, 4, and 5. R' is H, monophosphate group ( [ka] ), diphosphate group ( [ka] ), triphosphate group ( [ka] ) or tetraphosphate group ( [ka] ) are selected from, Each Z is selected independently from O, S, and BH. The base is selected from a base, a deaza base, or a tautomer thereof. For example, the base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine, or a tautomer thereof.

[0063] In some embodiments, X is O.

[0064] In some embodiments, Y is CH2.

[0065] In some embodiments, R 0 The alkyl group is -N3 or -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl).

[0066] In some embodiments, R 0 It is -N3.

[0067] In some embodiments, n is selected from 1, 2, and 3.

[0068] In some embodiments, n is 2.

[0069] In some embodiments, R' is a triphosphate group ( [ka] )

[0070] In some embodiments, Z is O.

[0071] In some embodiments, Base is [ka] Selected from.

[0072] In a third aspect of the present invention, the present invention provides a compound of formula (II) or a salt thereof, [ka] During the ceremony, X is selected from O, NH, and S. Y is selected from direct bonds, O, NH, S, CH, CH2, and C(CH3)2. R 1 -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH2, -OCOR m ,-OCONHR m Selected from, each R m This is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl), R 2 , R 3 , and R 4 Each of these is independently selected from O, NH, S, CH, CH2, and C(CH3)2. [ka] is a single bond [ka] or double bond [ka] This represents, R' is H, monophosphate group ( [ka] ), diphosphate group ( [ka] ), triphosphate group ( [ka] ) or tetraphosphate group ( [ka] ) are selected from, Each Z is selected independently from O, S, and BH. The base is selected from a base, a deaza base, or a tautomer thereof. For example, the base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine, or a tautomer thereof.

[0073] In some embodiments, X is O.

[0074] In some embodiments, Y is a direct bond.

[0075] In some embodiments, R 1 The alkyl group is -N3 or -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl).

[0076] In some embodiments, R 1 It is -N3.

[0077] In some embodiments, R 2 , R 3 , and R4 Each of these is independently selected from O, NH, S, CH, CH2, and C(CH3)2, and satisfies the following condition: R 2 If O or S is selected, R 3 and R 4 is CH2; R 3 If O or S is selected, R 2 and R 4 is CH2; R 4 If O or S is selected, R 2 and R 3 is CH2; R 2 If is C(CH3)2, then R 3 and R 4 is CH2; R 3 If is C(CH3)2, then R 2 and R 4 is CH2; R 4 If is C(CH3)2, then R 2 and R 3 If is CH2, then R 2 CH is, R 3 and R 4 It is CH2.

[0078] In some embodiments, R 2 , R 3 , and R 4 CH or CH2 are selected independently.

[0079] In some embodiments, R 2 and R 3 CH is, R 4 It is CH2.

[0080] In some embodiments, R' is a triphosphate group ( [ka] )

[0081] In some embodiments, Z is O.

[0082] In some embodiments, Base is [ka] Selected from.

[0083] In a fourth aspect of the present invention, the present invention provides a compound of formula (III) or a salt thereof, [ka] During the ceremony, A' is, [ka] Selected from, The heteroaryls are as follows: [ka] Selected from, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', Each R'' represents H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (e.g., C1-C6 alkoxy), [ka] Selected independently from, X is selected from O, NH, and S. R 5 , R 6 , R 7 , R 8 , and R 9 These include H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (e.g., C1-C6 alkoxy), [ka] Each is independently selected from R 5 , R 6 , R 7 , R 8 , and R 9 Isn't it also H? or R 5 , R 6 , R 7 , R 8 , and R 9 These include H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (e.g., C1-C6 alkoxy), C1-C6 alkyl-C(=O)-NH2-, [ka] Each is independently selected from R 5 , R 6 , R 7 , R 8 , and R 9 It is not H at the same time, R 10a , R 10b , R 10c , R 11a , R 11b , and R 12 These are H, -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), -ONH2, -OCOR m ,-OCONHR m Each R is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl such as methyl, ethyl, isopropyl, tert-butyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), and cycloalkyl (e.g., C3-C6 cycloalkyl), and each R m R is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl), 10a , R 10b , and R10c It is not H at the same time, m1 is selected from 1, 2, 3, 4, 5, and 6. m2 is selected from 0, 1, 2, 3, 4, 5, and 6. R' is H, monophosphate group ( [ka] ), diphosphate group ( [ka] ), triphosphate group ( [ka] ) or tetraphosphate group ( [ka] ) are selected from, Each Z is selected independently from O, S, and BH. The base is selected from a base, a deaza base, or a tautomer thereof. For example, the base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine, or a tautomer thereof.

[0084] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0085] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0086] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0087] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0088] In some embodiments, the heteroaryl is as follows: [ka] Selected from.

[0089] In some embodiments, the heteroaryl is as follows: [ka] Selected from.

[0090] In some embodiments, each heteroaryl may be independently substituted by one R'', where each R'' is [ka] Selected independently from, preferably R'' is [ka] That is the case.

[0091] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] One is selected from the above, and the remaining R''(one or more) are independently selected from -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, and alkoxy (e.g., C1-C6 alkoxy).

[0092] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] The remaining R''(one or more) are independently selected from nitro, aliphatic alkyl (e.g., C1-C6 alkyl), F, I, Br, and Cl.

[0093] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] The remaining R''(one or more) are independently selected from nitro and C1-C6 alkyl groups, preferably the remaining R''(one or more) are C1-C6 alkyl groups, and more preferably the remaining R''(one or more) are methyl groups.

[0094] In some embodiments, R 13 , R 14 , R 15 , and R 16 R 16 teeth, [ka] And R 13 , R14 , and R 15 Each of these is independently selected from H, nitro, and C1-C6 alkyl (e.g., methyl).

[0095] In some embodiments, R 13 , R 14 , R 15 , and R 16 R 16 teeth, [ka] And R 13 , R 14 , and R 15 H is H.

[0096] In some embodiments, R 17 , R 18 , and R 19 R 19 teeth, [ka] And R 17 and R 18 Each of these is independently selected from H, nitro, and C1-C6 alkyl (e.g., methyl).

[0097] In some embodiments, R 17 , R 18 , and R 19 R 19 teeth, [ka] And R 17 and R 18 H is H.

[0098] In some embodiments, R 20 , R 21 , and R 22 R 22 teeth, [ka] And R 20 and R21 Each of these is independently selected from H, nitro, and C1-C6 alkyl (e.g., methyl).

[0099] In some embodiments, R 20 , R 21 , and R 22 R 22 teeth, [ka] And R 20 and R 21 H is H.

[0100] In some embodiments, R 23 , R 24 , R 25 , and R 26 R 26 teeth, [ka] And R 23 , R 24 , and R 25 Each of these is independently selected from H, nitro, and C1-C6 alkyl (e.g., methyl).

[0101] In some embodiments, R 23 , R 24 , R 25 , and R 26 R 26 teeth, [ka] And R 23 , R 24 , and R 25 H is H.

[0102] In some embodiments, R 27 , R 28 , and R 29 R 29 teeth, [ka] And R27 and R 28 Each of these is independently selected from H, nitro, and C1-C6 alkyl (e.g., methyl).

[0103] In some embodiments, R 27 , R 28 , and R 29 R 29 teeth, [ka] And R 27 and R 28 H is H.

[0104] In some embodiments, R 30 , R 31 , and R 32 R 32 teeth, [ka] And R 30 and R 31 Each of these is independently selected from H, nitro, and C1-C6 alkyl (e.g., methyl).

[0105] In some embodiments, R 30 , R 31 , and R 32 R 32 teeth, [ka] And R 30 and R 31 H is H.

[0106] In some embodiments, R 33 , R 34 , and R 35 R 34 or R 35 teeth, [ka] And R 33The element is selected from H, nitro, and C1-C6 alkyl (e.g., methyl).

[0107] In some embodiments, R 33 , R 34 , and R 35 R 34 or R 35 teeth, [ka] And R 33 It is either H or methyl.

[0108] In some embodiments, X is O.

[0109] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 One of the following is [ka] One is selected from the above, and the remaining four are independently selected from H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, and alkoxy (e.g., C1-C6 alkoxy).

[0110] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 One of the following is [ka] The remaining four are independently selected from H, nitro, and C1-C6 alkyl groups.

[0111] In some embodiments, R 5 , R 6, R 7 , R 8 , and R 9 R 5 or R 9 teeth, [ka] The remaining four are selected independently from H and nitro.

[0112] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 One of the following is [ka] One is selected from the above, and the remaining four are independently selected from H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (e.g., C1-C6 alkoxy), and C1-C6 alkyl-C(=O)-NH2-.

[0113] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 One of the following is [ka] The remaining four are independently selected from H, nitro, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkyl-C(=O)-NH2-.

[0114] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 R 5 or R 9teeth, [ka] The remaining four are independently selected from H, nitro, methoxy, and acetamide.

[0115] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 R 5 or R 9 teeth, [ka] And R 7 The first three are selected from H, nitro, methoxy, and acetamide, and the remaining three are H.

[0116] In some embodiments, R 10a , R 10b , and R 10c One of these is -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), -ONH2, -OCOR m ,-OCONHR m One is selected from, and the other two are independently selected from H, aliphatic alkyl (e.g., C1-C6 alkyl such as methyl, ethyl, isopropyl, tert-butyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), and cycloalkyl (e.g., C3-C6 cycloalkyl), and each R m The element is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl).

[0117] In some embodiments, R 10a , R 10b , and R 10cOne of these is -N3 or -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), and the other two are independently selected from H and C1~C6 alkyl (e.g., methyl, ethyl, isopropyl, tertbutyl).

[0118] In some embodiments, R 10a , R 10b , and R 10c One of these is either -N3 or -SS-methyl, and the other two are independently selected from H and methyl, respectively.

[0119] In some embodiments, R 10a , R 10b , and R 10c One of these is -N3, -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl, and the other two are independently selected from H and methyl, respectively.

[0120] In some embodiments, R 11a and R 11b Each of the elements is independently selected from H and aliphatic alkyl groups (e.g., C1-C6 alkyl groups such as methyl, ethyl, isopropyl, and tert-butyl).

[0121] In some embodiments, R 11a and R 11b H is H.

[0122] In some embodiments, R 12 -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH2, -OCOR m ,-OCONHR m Selected from, each R m The element is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl).

[0123] In some embodiments, R 12 The alkyl group is -N3 or -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl).

[0124] In some embodiments, R 12 It is -N3.

[0125] In some embodiments, m1 is 1.

[0126] In some embodiments, m2 is selected from 0 or 1.

[0127] In some embodiments, [ka] Overall, [ka] Selected from.

[0128] In some embodiments, R' is a triphosphate group ( [ka] )

[0129] In some embodiments, Z is O.

[0130] In some embodiments, Base is [ka] Selected from.

[0131] In a fifth aspect of the present invention, the present invention provides a compound of formula (IV) or a salt thereof, [ka] During the ceremony, A is [ka] Selected from, The heteroaryls are as follows: [ka] Selected from, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', Each R'' represents H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (e.g., C1-C6 alkoxy), [ka] Selected independently from, R 5 , R 6 , R 7 , R 8 , R 9 , R x , R y , and R z These include H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, alkoxy (e.g., C1-C6 alkoxy), [ka] Each is independently selected from R 5 , R 6 , R 7 , R 8 , and R 9 It is not H at the same time, R 10a , R 10b , R 10c , R 11a , R 11b , and R 12These are H, -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH2, -OCOR m ,-OCONHR m Each R is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl such as methyl, ethyl, isopropyl, tert-butyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), and cycloalkyl (e.g., C3-C6 cycloalkyl), and each R m R is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl), 10a , R 10b , and R 10c It is not H at the same time, X is selected from O, NH, and S. m1 is selected from 1, 2, 3, 4, 5, and 6. m2 is selected from 0, 1, 2, 3, 4, 5, and 6. R' is H, monophosphate group ( [ka] ), diphosphate group ( [ka] ), triphosphate group ( [ka] ) or tetraphosphate group ( [ka] ) are selected from, Each Z is selected independently from O, S, and BH. The base is selected from a base, a deaza base, or a tautomer thereof. For example, the base is selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine, or a tautomer thereof.

[0132] In some embodiments, A is [ka] Selected from.

[0133] In some embodiments, A is [ka] Selected from.

[0134] In some embodiments, A is [ka] That is the case.

[0135] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0136] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0137] In some embodiments, the heteroaryl is as follows: [ka] Selected from the above, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R''s.

[0138] In some embodiments, each heteroaryl may be independently substituted by one R'', where each R'' is [ka] Selected independently from, preferably R'' is [ka] That is the case.

[0139] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] One is selected from the above, and the remaining R''(one or more) are independently selected from -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, and alkoxy (e.g., C1-C6 alkoxy).

[0140] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] The remaining R''(one or more) are independently selected from nitro, aliphatic alkyl (e.g., C1-C6 alkyl), F, I, Br, and Cl.

[0141] In some embodiments, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is [ka] The remaining R''(one or more) are independently selected from nitro and C1-C6 alkyl groups, and preferably the remaining R''(one or more) are C1-C6 alkyl groups.

[0142] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 One of the following is [ka] One is selected from the above, and the remaining four are independently selected from H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, and alkoxy (e.g., C1-C6 alkoxy).

[0143] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 One of the following is [ka] The remaining four are independently selected from H, nitro, and C1-C6 alkyl groups.

[0144] In some embodiments, R 5 , R 6 , R 7 , R 8 , and R 9 R 7 teeth, [ka] And the remaining four are H.

[0145] In some embodiments, R x and R yOne of the following is [ka] One is selected from, and the other is selected from H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), F, I, Br, Cl, and alkoxy (e.g., C1-C6 alkoxy).

[0146] In some embodiments, R x and R y One of the following is [ka] The other is H.

[0147] In some embodiments, R z teeth, [ka] Selected from.

[0148] In some embodiments, R z teeth, [ka] That is the case.

[0149] In some embodiments, R 10a , R 10b , and R 10c One of these is -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH2, -OCOR m ,-OCONHR mOne is selected from, and the other two are independently selected from H, aliphatic alkyl (e.g., C1-C6 alkyl such as methyl, ethyl, isopropyl, tert-butyl), aromatic alkyl (e.g., phenyl C1-C6 alkyl), and cycloalkyl (e.g., C3-C6 cycloalkyl), and each R m The element is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl).

[0150] In some embodiments, R 10a , R 10b , and R 10c One of these is -N3 or -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), and the other two are independently selected from H and aliphatic alkyl (e.g., C1~C6 alkyl such as methyl, ethyl, isopropyl, tertbutyl in particular).

[0151] In some embodiments, R 11a and R 11b Each of the elements is independently selected from H and aliphatic alkyl groups (e.g., C1-C6 alkyl groups such as methyl, ethyl, isopropyl, and tert-butyl).

[0152] In some embodiments, R 11a and R 11b H is H.

[0153] In some embodiments, R 12 -N3, -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH2, -OCOR m ,-OCONHR m Selected from, each R m The element is independently selected from aliphatic alkyl (e.g., C1-C6 alkyl), cycloalkyl (e.g., C3-C6 cycloalkyl), or aromatic alkyl (e.g., phenyl C1-C6 alkyl).

[0154] In some embodiments, R 12 The alkyl group is -N3 or -SS-C1~C6 alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl).

[0155] In some embodiments, R 12 It is -N3.

[0156] In some embodiments, X is O.

[0157] In some embodiments, m1 is 1.

[0158] In some embodiments, m2 is selected from 0 or 1.

[0159] In some embodiments, R' is a triphosphate group ( [ka] )

[0160] In some embodiments, Z is O.

[0161] In some embodiments, Base is [ka] Selected from.

[0162] In a sixth aspect of the present invention, the present invention provides the following compounds or salts thereof. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0163] In some embodiments, the above-described compound or salt thereof may carry an additional detectable label (e.g., a fluorescent label).

[0164] In some embodiments, additional detectable labels supported by a compound or salt thereof are introduced by affinity reagents (e.g., antibodies, aptamers, affimers, nottins), the affinity reagents support the detectable labels, and the affinity reagents can specifically recognize and bind to epitopes of the compound or salt thereof.

[0165] In some embodiments, additional detectable labels (e.g., fluorescent labels) are optionally linked to the compound or a salt thereof via a linker.

[0166] In some embodiments, additional detectable labels (e.g., fluorescent labels) are optionally linked to the base of the compound or its salt via a linker.

[0167] In some embodiments, if an additional detectable label (e.g., a fluorescent label) is optionally linked to the base of the compound or its salt via a linker, the structure of the base is as follows: [ka] Selected from, preferably, [ka] Selected from.

[0168] In some embodiments, the linker is either a severable linker or a non-severable linker.

[0169] In some embodiments, the cleavable linker is selected from a linker that can be cleaved by electrophilic reactions, a linker that can be cleaved by nucleophilic reactions, a linker that can be cleaved by photodegradation, a linker that can be cleaved under reducing conditions, a linker that can be cleaved under oxidizing conditions, a safety-catch linker, a linker that can be cleaved by a desorption mechanism, or any combination thereof.

[0170] In some embodiments, the linker has the structure of formula (B), [ka] During the ceremony, R 36 , R 37 , R 38 , R 39Each of the following elements is independently selected from H, -N3, N3-C1~C6 alkyl, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1~C6 alkyl), cycloalkyl (e.g., C3~C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1~C6 alkyl), F, I, Br, Cl, and alkoxy (e.g., C1~C6 alkoxy), and the C1~C6 alkyl is optionally substituted with other C1~C6 alkyl elements. p is selected from 1, 2, 3, 4, 5, or 6. q is selected from any integer between 1 and 12.

[0171] In some specific embodiments, R 36 , R 37 , R 38 , and R 39 One of the elements is an N3-C1~C6 alkyl group, and the C1~C6 alkyl group is optionally substituted with another C1~C6 alkyl group. The others are independently selected from H, -N3, nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C1~C6 alkyl), cycloalkyl (e.g., C3~C6 cycloalkyl), aromatic alkyl (e.g., phenyl C1~C6 alkyl), F, I, Br, Cl, and alkoxy (e.g., C1~C6 alkoxy).

[0172] In some specific embodiments, R 36 The N3-C1~C6 alkyl group is optionally substituted with another C1~C6 alkyl group, and R 37 , R 38 , and R 39 H and C1-C6 alkyl groups are selected independently.

[0173] In some specific embodiments, R 36 R is an N3-methyl that is optionally substituted with methyl, 37 , R 38 , and R 39 H is H.

[0174] In some specific embodiments, p is selected from 1, 2, or 3.

[0175] In some specific embodiments, p is 1.

[0176] In some specific embodiments, q is selected from 2, 3, 4, 5, or 6.

[0177] In some specific embodiments, q is 4.

[0178] In some embodiments, the methyl terminus of the linker of formula (B) is connected to the base, and its amino terminus is connected to an additional detectable label (e.g., a fluorescent label). For example, [ka] That is the case.

[0179] In some embodiments, the linker of formula (B) has the following structure [ka] It has.

[0180] In some embodiments, the linker has the structure of formula (C): [ka] It has.

[0181] In some embodiments, the alkynyl terminus of the linker of formula (C) is connected to the Base, and its amino terminus is connected to an additional detectable label (e.g., a fluorescent label). For example, [ka] That is the case.

[0182] In some embodiments, the linker has the structure of formula (D): [ka] It has.

[0183] In some embodiments, the alkynyl terminus of the linker of formula (D) is connected to the Base, and its amino terminus is connected to an additional detectable label (e.g., a fluorescent label). For example, [ka] That is the case.

[0184] In some embodiments, the detectable labels are as follows: [ka] [ka] Selected from.

[0185] In some embodiments, different bases result in different detectable labels (e.g., fluorescent labels).

[0186] In some embodiments, a compound or salt thereof supporting an additional detectable label has the following structure: [ka] It has.

[0187] In some embodiments, a compound or salt thereof supporting an additional detectable label has the following structure: [ka] It has.

[0188] In some embodiments, a compound or salt thereof supporting an additional detectable label has the following structure: [ka] It has.

[0189] In some embodiments, a compound or salt thereof supporting an additional detectable label has the following structure: [ka] It has.

[0190] In a seventh aspect of the present invention, the present invention provides a method for halting nucleic acid synthesis, comprising the step of incorporating such compound or salt thereof into a nucleic acid molecule.

[0191] In some embodiments, the incorporation of the compound or a salt thereof is achieved by terminal transferase, terminal polymerase, or reverse transcriptase.

[0192] In some embodiments, the method includes the step of incorporating a compound or a salt thereof into a nucleic acid molecule that terminates by using a polymerase.

[0193] In some embodiments, the method includes the step of performing a nucleotide polymerization reaction using polymerase under conditions in which polymerase can carry out the nucleotide polymerization reaction, thereby incorporating the compound or a salt thereof into the 3' end of a nucleic acid molecule.

[0194] In an eighth aspect of the present invention, the present invention provides a method for preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising the step of incorporating such a compound or a salt thereof into a growing complementary polynucleotide, wherein the incorporation of the compound or a salt thereof prevents any subsequent nucleotides from being introduced into the growing complementary polynucleotide.

[0195] In some embodiments, the incorporation of the compound or a salt thereof is achieved by terminal transferase, terminal polymerase, or reverse transcriptase.

[0196] In some embodiments, the method includes the step of incorporating a compound or a salt thereof into a growing complementary polynucleotide by using a polymerase.

[0197] In some embodiments, the method includes the step of carrying out a nucleotide polymerization reaction using a polymerase under conditions in which the polymerase can carry out the nucleotide polymerization reaction, thereby incorporating a compound or a salt thereof into the 3' end of a growing complementary polynucleotide.

[0198] In a ninth aspect of the present invention, the present invention is a method for determining the sequence of a target single-stranded polynucleotide, 1) A step of monitoring the sequential incorporation of nucleotides complementary to a target single-stranded polynucleotide into a growing nucleic acid chain, wherein at least one of the incorporated complementary nucleotides is a compound or a salt thereof as described above, and the compound or salt thereof carries an additional detectable label (e.g., a fluorescent label), 2) A step of detecting a detectable sign and This provides a method that includes this.

[0199] In some embodiments, additional detectable labels (e.g., fluorescent labels) are optionally linked to the compound or a salt thereof via a linker.

[0200] In some embodiments, the linker is as described above.

[0201] In some embodiments, additional detectable labels are as described above.

[0202] In some embodiments, if the base is different, the detectable label (e.g., fluorescent label) supported by the compound or its salt is different.

[0203] In some embodiments, the reversible blocking group (R) and detectable label in the compound or its salt are removed before introducing the following complementary nucleotide.

[0204] In some embodiments, the reversible blocking group (R) and the detectable label are removed simultaneously.

[0205] In some embodiments, the reversible blocking group (R) and the detectable label are removed sequentially. For example, the reversible blocking group is removed after the detectable label is removed, or the detectable label is removed after the reversible blocking group is removed.

[0206] In some embodiments, the method involves the following steps: (a) A step of preparing a plurality of different nucleotides, wherein the plurality of different nucleotides are the above-mentioned compounds or salts thereof, and each nucleotide carries an additional detectable label that can be distinguished from an additional detectable label supported by another nucleotide being detected. (b) The step of incorporating multiple different nucleotides into a sequence complementary to the target single-stranded polynucleotide, (c) A step of detecting additional detectable labels supported by the nucleotides in step (b) to determine the type of incorporated nucleotide, (d) A step of removing the reversible blocking group and detectable label supported by the nucleotide in step (b), (e) a step which optionally repeats steps (b) to (d) once or more times, This determines the sequence of the target single-stranded polynucleotide.

[0207] In some embodiments, the method involves the following steps: (1) A step of preparing a first nucleotide, a second nucleotide, a third nucleotide, and a fourth nucleotide, wherein at least one of the four nucleotides is a compound or a salt thereof as described above, the bases contained in the four nucleotides are different from each other, the four nucleotides carry an additional detectable label (e.g., a fluorescent label), preferably the additional detectable label carried by the four nucleotides is introduced by an affinity reagent (e.g., an antibody, aptamer, affimer, nottin), the affinity reagent carries the detectable label, the affinity reagent can specifically recognize and bind to the epitope of each nucleotide, or preferably the four nucleotides are optionally linked to the additional detectable label via a linker, more preferably the bases of the four nucleotides are optionally linked to the additional detectable label via a linker, and most preferably the additional detectable labels carried by the four nucleotides are different from each other. (2) The steps include contacting four types of nucleotides with a target single-stranded polynucleotide, removing nucleotides not incorporated into the growing nucleic acid chain, detecting a detectable label supported by nucleotides incorporated into the growing nucleic acid chain, and removing reversible blocking groups and detectable labels supported by nucleotides incorporated into the growing nucleic acid chain, Optionally, method (3): includes a step of repeating step (2) once or multiple times.

[0208] In some embodiments, the method involves the following steps: (a) A step of preparing a mixture comprising a double-stranded nucleic acid chain, at least one compound or salt thereof as described above, a polymerase and an excision reagent, wherein the double-stranded nucleic acid chain comprises a growing nucleic acid chain and a nucleic acid chain to be sequenced, the compound or salt thereof supports an additional detectable label (e.g., a fluorescent label), preferably the additional detectable label supported by the compound or salt thereof is introduced by an affinity reagent (e.g., an antibody, aptamer, affimer, nottin), the affinity reagent supports the detectable label, the affinity reagent can specifically recognize and bind to an epitope of the compound or salt thereof, or preferably the compound or salt thereof is optionally linked to the additional detectable label via a linker, or more preferably the base of the compound or salt thereof is optionally linked to the additional detectable label via a linker. (b) Steps (i), (ii) and (iii) below: Step (i): Using polymerase, incorporate the compound or a salt thereof into a growing nucleic acid chain to form a nucleic acid intermediate containing a reversible blocking group and a detectable label. Step (ii): A step of detecting a detectable label contained in the nucleic acid intermediate. Step (iii): Remove reversible blocking groups and / or detectable labels contained in the nucleic acid intermediate by using an excision reagent. A step that performs a reaction including [a specific element], and optionally repeats the step once or multiple times. Includes.

[0209] In some embodiments, the removal of reversible blocking groups and detectable labels is performed simultaneously, or sequentially (for example, the reversible blocking groups are removed first, or the detectable labels are removed first).

[0210] In some embodiments, the excision reagent used to remove the reversible blocking group is the same as the one used to remove the detectable label.

[0211] In some embodiments, the excision reagent used to remove the reversible blocking group is different from the one used to remove the detectable label.

[0212] In some embodiments, the double strand is connected to a support.

[0213] In some embodiments, the growing nucleic acid strand is a primer.

[0214] In some embodiments, the primers are annealed to the nucleic acid strand being sequenced to form a double helix.

[0215] In some embodiments, the double chain, the compound or a salt thereof, and the polymerase together form a reaction system containing a solution phase and a solid phase.

[0216] In some embodiments, the bases contained in the compound or its salt are different from each other.

[0217] In some embodiments, the additional detectable labels supported by the compound or a salt thereof are different from each other.

[0218] In some embodiments, the compound or a salt thereof is incorporated into a growing nucleic acid chain using polymerase under conditions in which the polymerase can carry out a nucleotide polymerization reaction, thereby forming a nucleic acid intermediate containing a reversible blocking group and a detectable label.

[0219] In some embodiments, the polymerase is selected from KOD polymerase or its variants (e.g., KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391).

[0220] In some embodiments, prior to any step of detecting a detectable label contained in the nucleic acid intermediate, the solution phase of the reaction system in the previous step is removed and the double strand linked to the support is retained.

[0221] In some embodiments, the excision reagent comes into contact with the double-stranded or growing nucleic acid strand in a reaction system containing a solution phase and a solid phase.

[0222] In some embodiments, the excision reagent can remove reversible blocking groups and additional detectable labels supported by compounds incorporated into the growing nucleic acid chain without affecting phosphate diester bonds on the double-stranded skeleton.

[0223] In some embodiments, the solution phase of the reaction system in this step is removed after an optional step of removing reversible blocking groups and / or additional detectable labels contained in the nucleic acid intermediate.

[0224] In some embodiments, a cleaning operation is performed after any step including a removal operation.

[0225] In some embodiments, after step (ii), the method further includes the steps of determining the type of compound incorporated into the nucleic acid chain growing in step (i) according to the signal detected in step (ii), and determining the type of nucleotide at the corresponding position in the nucleic acid chain being sequenced, based on the principle of complementary base pairing.

[0226] In a tenth aspect of the present invention, the present invention provides a kit comprising at least one compound or a salt thereof as described above.

[0227] In some embodiments, the kit comprises a first compound, a second compound, a third compound, and a fourth compound, where each of the first, second, third, and fourth compounds is independently one of the above-described compounds or a salt thereof.

[0228] In some embodiments, the first compound is adenine, 7-deazaadenine, or tautomers thereof (for example, [ka] ) is selected from the following, and in the second compound, the base is thymine, uracil or their tautomers (e.g., [ka] ) are selected from the following, and in the third compound, the base is cytosine or its tautomer (e.g., [ka] ) are selected from the following, and in the fourth compound, the base is guanine, 7-deazaguanine or their tautomers (e.g., [ka] ) will be selected from.

[0229] In some embodiments, the first, second, third, and fourth compounds support additional detectable labels.

[0230] In some embodiments, additional detectable labels supported by the first, second, third, and fourth compounds are introduced by affinity reagents (e.g., antibodies, aptamers, affimers, nottins) that carry the detectable labels and can specifically recognize and bind to epitopes of the first, second, third, or fourth compounds.

[0231] In some embodiments, the first, second, third, and fourth compounds are optionally linked to additional detectable labels via a linker.

[0232] In some embodiments, the base of the first, second, third, or fourth compound is optionally linked to an additional detectable label via a linker.

[0233] In some embodiments, the bases contained in the first, second, third, and fourth compounds are different from each other.

[0234] In some embodiments, the additional detectable labels supported by the first, second, third, and fourth compounds are different from each other.

[0235] In some embodiments, the linker is as described above.

[0236] In some embodiments, the detectable labels are as described above.

[0237] In some embodiments, the kit further includes reagents for pre-treating nucleic acid molecules, supports for linking nucleic acid molecules to be sequenced, reagents for linking nucleic acid molecules to be sequenced to the supports (e.g., by covalent or non-covalent bonding), primers for initiating the nucleotide polymerization reaction, polymerase for carrying out the nucleotide polymerization reaction, one or more buffer solutions, one or more washing solutions, or any combination thereof.

[0238] In an eleventh aspect of the present invention, the present invention provides the use of such compounds or salts thereof or such kits for determining the sequence of a target single-stranded polynucleotide. [Brief explanation of the drawing]

[0239] [Figure 1] As an example of the present invention, a reaction for removing azidomethyl is illustrated. [Figure 2] As an example of the present invention, a nucleophilic cyclization cascade reaction for reversible cleavage is illustrated. [Figure 3] As an example of the present invention, a reversible blocking nucleotide analog having a 3'-OH label is shown in the figure. [Modes for carrying out the invention]

[0240] Embodiments of the present invention are described in detail below by specific embodiments, but should not be construed as limitations on the present invention.

[0241] Unless otherwise specified, the above groups and substituents have common meanings in the field of medicinal chemistry.

[0242] Throughout this specification, substituents of the compounds disclosed herein are disclosed according to the type or range of the group. It should be noted that the present invention includes each independent partial combination of every possible member of these types and ranges of groups. For example, the term “C1-C6 alkyl” specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl groups.

[0243] In addition, unless otherwise specifically noted, the expressions "each of ... is selected independently from..." and "each of ... is selected independently from..." are interchangeable when used throughout this specification and should both be interpreted in a broad sense, that is, they may mean that specific choices represented by the same or different symbols are independent of each other in different groups, or that specific choices represented by the same or different symbols are independent of each other in the same group.

[0244] The term "aliphatic alkyl" refers to any linear or branched saturated group containing 1 to 20 carbon atoms, for example, C1-C 12 This refers to alkyl, preferably C1-C6 alkyl groups.

[0245] The term "C1-C6 alkyl" refers to any linear or branched saturated group containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-amyl, tert-amyl, n-hexyl, etc.

[0246] The term "alkoxy" refers to any of the above alkyl groups (e.g., C1-C6 alkyl groups) that are connected to the rest of the molecule by an oxygen atom (-O-).

[0247] The term "cycloalkyl" refers to saturated cyclic hydrocarbyl compounds with 3 to 10 membered monocyclic rings, such as C3-C8 cycloalkyls, preferably C3-C6 cycloalkyls.

[0248] The term "C3-C6 cycloalkyl" refers to saturated cyclic hydrocarbyl compounds with 3 to 6 members in a monocyclic ring system. C3-C6 cycloalkyls may also include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and others.

[0249] The term "aromatic alkyl" refers to arylalkyl or heteroarylalkyl, where alkyl is defined as described above.

[0250] The term "heteroaryl" refers to an aromatic heterocycle, generally a 5, 6, 7, or 8-membered heterocycle containing 1 to 3 heteroatoms selected from N, O, or S, and the heteroaryl ring may optionally be further fused with aromatic or non-aromatic carbocyclic or heterocycles. Non-exclusive examples of heteroaryls include, for example, pyridyl, pyrazinyl, pyrimidinyl, pyridadinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, thioxazolyl, pyrrolyl, phenyl-pyrrolyl, furanil, phenyl-furanil, oxazolyl, isoxazolyl, pyrazolyl, thiophenyl, benzofuranil, benzothiophenyl, benzo-1,3-dioxolane (benzodioxane), isodihydroindolyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, 1,2,3-triazolyl, 1-phenyl-1,2,3-triazolyl, 2,3-dihydroindolyl, 2,3-dihydrobenzofuranil, 2,3-dihydrobenzothiophenyl, benzopyranil, 2,3-dihydrobenzoxazinyl, and 2,3-dihydroquinoxalinyl.

[0251] In this invention, [ka] is a group connected to a heteroaryl group [ka] This refers to [the above]. Other similar structures can be understood by referring to the above content and relating them accordingly.

[0252] In this invention, R is [ka] The heteroaryls may be selected from the above, and each heteroaryl may be the same or different. Furthermore, if each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', this means that the substituents R'' on different heteroaryls may be the same or different, and the substituents R'' on the same heteroaryl may be the same or different.

[0253] In this invention, "each R'' is independently selected from H, -N3, etc., and is not H" means that if the heteroaryl is substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'' substituents, at least one R'' substituent is not H.

[0254] The term "aryl" refers to a carbocyclic aromatic group having 6 to 14 carbon atoms, for example, C6-C 10 This means aryl, preferably phenyl.

[0255] It will be apparent to those skilled in the art from all the above descriptions that any group having a compound name such as "phenyl C1-C6 alkyl" should be understood to be constructed from a part from which it is derived, for example, from a phenyl-substituted C1-C6 alkyl group, as defined above.

[0256] As used herein, the term “salt of a compound of formula (A), formula (I), formula (II), formula (III), or formula (IV)” may be exemplified by organic addition salts of anion-forming organic acids, including but not limited to formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, α-ketoglutarate, α-glycerophosphate, alkylsulfonate, or arylsulfonate, preferably, alkylsulfonate is methanesulfonate or ethanesulfonate, and arylsulfonate is benzenesulfonate or p-toluenesulfonate. Alternatively, the term also means inorganic salts thereof, including but not limited to hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate, and carbonate, sulfate, or phosphate.

[0257] In this invention, R is [ka] You may choose from the following, in the formula, [ka] is a single bond [ka] or double bond [ka] Represents R 2 , R 3 , and R 4 Each of these is independently selected from O, NH, S, CH, CH2, and C(CH3)2. 2 and R 3 R is only available when both are CH. 2 and R 3 The bond between them is a double bond, R 2 and R 3 If each atom or group other than CH is independently selected from O, NH, S, CH2, C(CH3)2, then R 2 and R 3It should be understood that the bond between them is a single bond. For example, the expression "R 2 , R 3 , and R 4 " is independently selected from CH or CH2" is R 2 and R 3 Both are CH, and R 2 and R 3 The bond between them is a double bond, R 4 CH2 is, R 3 and R 4 The bond between them is a single bond, or R 3 and R 4 Both are CH, and R 3 and R 4 The bond between them is a double bond, R 2 CH2 is, R 2 and R 3 This means that the bond between them is a single bond. Similarly, Y and R 2 Between or R 3 and R 4 The single and / or double bonds between them can be understood by referring to the above information and relating them accordingly.

[0258] The term "direct bond" means that the groups on both sides are directly connected. For example, [ka] In this case, if Y is a direct bond, [ka] teeth, [ka] It will become.

[0259] Formula (B) of the present invention [ka] Regarding the compound, the expression "R 36 " is N3-C1~C6 alkyl, and C1~C6 alkyl is optionally substituted with C1~C6 alkyl" is R36 This means that is an N3-C1~C6 alkyl group, and that hydrogen atoms on the C1~C6 alkyl group may be further replaced by other C1~C6 alkyl groups. For example, R 36 If is N3-methyl and the methyl group is optionally substituted with another methyl group, the compound of formula (B) has the following structure [ka] It may have that. Other similar expressions can be understood in correspondence with the above content.

[0260] In the method of the present invention, any substance consisting of two strands, namely a growing nucleic acid strand and a sequencing nucleic acid strand, may be called a "double-stranded" substance regardless of the length of the growing nucleic acid strand or the sequencing nucleic acid strand, and the sequencing nucleic acid strand may be longer than the length of the growing nucleic acid strand.

[0261] In the method of the present invention, the nucleic acid molecule to be sequenced may be any target nucleic acid molecule. In some preferred embodiments, the nucleic acid molecule to be sequenced may include deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, or any combination thereof. In the method of the present invention, the nucleic acid molecule to be sequenced is not limited to any particular type. In some preferred embodiments, the nucleic acid molecule to be sequenced may be DNA or RNA. In some preferred embodiments, the nucleic acid molecule to be sequenced may be genomic DNA, mitochondrial DNA, chloroplast DNA, mRNA, cDNA, miRNA, or siRNA. In some preferred embodiments, the nucleic acid molecule to be sequenced may be linear or cyclic. In some preferred embodiments, the nucleic acid molecule to be sequenced may be double-stranded or single-stranded. For example, the nucleic acid molecule to be sequenced may be single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or a hybrid of DNA and RNA. In some preferred embodiments, the nucleic acid molecule to be sequenced is single-stranded DNA. In some preferred embodiments, the nucleic acid molecule to be sequenced is double-stranded DNA.

[0262] In the method of the present invention, the nucleic acid molecules to be sequenced are not limited by their source. In some preferred embodiments, the nucleic acid molecules to be sequenced can be obtained from any source, such as any cell, tissue, or organism (e.g., viruses, bacteria, fungi, plants, and animals). In some preferred embodiments, the nucleic acid molecules to be sequenced originate from mammals (e.g., humans, non-human primates, rodents, or canids), plants, birds, reptiles, fish, fungi, bacteria, or viruses.

[0263] Methods for extracting or obtaining 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 and chloroform extraction. For detailed descriptions of such methods, see, for example, J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Short Protocols in Molecular Biology, 3rd edition, John Wiley & Sons, Inc., 1995. In addition, various commercial kits can be used to extract nucleic acid molecules from various sources (e.g., cells, tissues, or organisms).

[0264] In the method of the present invention, the nucleic acid molecule to be sequenced is not limited by its length. In some preferred embodiments, the length of the nucleic acid molecule to be sequenced may 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, or at least 2000 bp. In some preferred embodiments, the length of the nucleic acid molecule to be sequenced may be 10-20 bp, 20-30 bp, 30-40 bp, 40-50 bp, 50-100 bp, 100-200 bp, 200-300 bp, 300-400 bp, 400-500 bp, 500-1000 bp, 1000-2000 bp, or greater than 2000 bp. In some preferred embodiments, the nucleic acid molecules to be sequenced may have a length of 10 to 1000 bp to facilitate high-throughput sequencing.

[0265] In the method for polynucleotide preparation or sequencing of the present invention, a suitable polymerase may be used to carry out the nucleotide polymerization reaction. In some exemplary embodiments, the polymerase can synthesize a new DNA strand using DNA as a template (e.g., DNA polymerase). In some exemplary embodiments, the polymerase can synthesize a new DNA strand using RNA as a template (e.g., reverse transcriptase). In some exemplary embodiments, the polymerase can synthesize a new RNA strand using DNA or RNA as a template (e.g., RNA polymerase). Therefore, in some preferred embodiments, the polymerase is selected from DNA polymerase, RNA polymerase, and reverse transcriptase. A suitable polymerase can be selected as required by the actual need to carry out the nucleotide polymerization reaction. In some preferred embodiments, the polymerization reaction is a polymerase chain reaction (PCR). In some preferred embodiments, the polymerization reaction is a reverse transcription reaction.

[0266] In the method of the present invention, KOD polymerase or a variant thereof may be used in the nucleotide polymerization reaction. KOD polymerase or a variant thereof (e.g., KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391) may result in an acceptable integration efficiency for the modified nucleosides or nucleotides of the present invention. KOD POL391 and KOD POL171 result in an acceptable integration efficiency for the modified nucleotides of the present invention. In some embodiments, the integration efficiency of KOD POL391 or KOD POL171 for the modified nucleotides of the present invention is greater than 70%, for example, 70% to 80%, 80% to 90%, or 90% to 100%.

[0267] In the method for preparing or sequencing polynucleotides of the present invention, the polymerization reaction of nucleotides is carried out under appropriate conditions. Appropriate polymerization conditions include the composition of the solution phase, the concentration of each component, the pH of the solution phase, and the polymerization temperature. Polymerization is carried out under appropriate conditions to obtain an acceptable and even higher integration efficiency.

[0268] In this invention, the hydroxyl (-OH) at the 3' position of deoxyribose in the compound of formula (A) is protected (by R), thereby halting polymerization induced by polymerase (e.g., DNA polymerase). For example, if the compound of formula (A) is introduced at the 3' end of a growing nucleic acid chain, the polymerase cannot proceed to the next round of polymerization because there is no free hydroxyl (-OH) at the 3' position of the deoxyribose in the compound, and polymerization is halted. In such a case, only one base is incorporated into the growing nucleic acid chain in each round of polymerization.

[0269] In addition, the protecting group (R) of the hydroxyl (-OH) at the 3' position of deoxyribose in the compound of formula (A) can be removed to return it to a free hydroxyl (-OH). The growing nucleic acid chain may then undergo a next round of polymerization using polymerase and the compound of formula (A) to reincorporate the bases.

[0270] In other words, the hydroxyl (-OH) at the 3' position of deoxyribose in compound (A) is reversibly blocked. When compound (A) is incorporated into the 3' end of a growing nucleic acid chain, it stops polymerization induced by polymerase, halting any further elongation of the growing nucleic acid chain. After the blocking group contained in compound (A) is removed, polymerase can continue polymerization of the growing nucleic acid chain, extending the chain.

[0271] Some embodiments described herein relate to the use of conventional detectable labels. Detection may be achieved by any suitable method, including fluorescence spectroscopy or other optical means. Preferred labels are fluorescent labels, i.e., fluorophores that emit light at a specific wavelength after absorbing energy. Many suitable fluorescent labels are known in the art. For example, Welch et al. (Chem. Eur. J. 5(3):951~960, 1999) disclose dansyl-functionalized fluorescent moieties that 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. Suitable labels for use are also disclosed by Prober et al. (Science 238:336-341, 1987), Connell et al. (BioTechniques 5(4):342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15(11):4593-4602, 1987), and Smith et al. (Nature 321:674, 1986). Other commercially available fluorescent labels include, but are not limited to, fluorescein, rhodamine (including TMR, Texas Red, and Rox), alexa, BODIPY, acridine, coumarin, pyrene, benzanthracene, and cyanine.

[0272] Multiple labels, such as bifluorophore FRET cassettes (Tet. Let. 46:8867~8871, 2000), can also be used in this application. Multi-fluorodendrimer systems (J. Am. Chem. Soc. 123:8101~8108, 2001) can also be used. Fluorescent labeling is preferred, but other forms of detectable labeling will be useful, as will be apparent to those skilled in the art. For example, microparticles including quantum dots (Empodocles et al., Nature 399:126~130, 1999), gold nanoparticles (Reichert et al., Analyse Chem. 72:6025~6029, 2000), and microbeads (Lacoste et al., Proc. Natl. Acad. Sci USA 97(17):9461~9466, 2000) can all be used.

[0273] Multicomponent labeling can also be used in this application. Multicomponent labeling relies on interaction with further compounds for detection. The most common multicomponent labeling used in biology is the biotin-streptavidin system. Biotin is used as a label bound to a nucleotide or modified nucleotide. Streptavidin is then added separately to enable detection. Other multicomponent systems can be used. For example, dinitrophenol has commercially available fluorescent antibodies that can be used for detection.

[0274] In some embodiments described herein, the loading of the above-mentioned detectable labels onto modified nucleotide or nucleoside molecules can be achieved by incorporating affinity reagents (e.g., antibodies, aptamers, affimers, and Nottins) that can specifically recognize and bind to epitopes on the modified nucleotide or nucleoside molecules. The specific principle is described in International Publication No. 2018129214A1, which is incorporated herein by reference in its entirety.

[0275] In other embodiments described herein, modified nucleotide or nucleoside molecules may be linked to the detectable label described above. In some such embodiments, the linker used may be cleavable. The use of a cleavable linker ensures that the label can be removed as needed after detection, thus avoiding any interference signals from any subsequently added labeled nucleotide or nucleoside.

[0276] In other embodiments, the linker used is not cleavable. In each situation in which the labeled nucleotide of the present invention is incorporated, it is not necessary to subsequently incorporate any nucleotide, and therefore, it is not necessary to remove the label from the nucleotide.

[0277] Those skilled in the art will be aware of the usefulness of dideoxynucleoside triphosphates in Sanger sequencing and related protocols (Sanger type), which rely on randomized chain termination at specific types of nucleotides. The same effect achieved by using ddNTPs can be achieved by using the 3'-OH protecting groups described herein, both of which prevent subsequent nucleotide incorporation; therefore, it will be recognized that the nucleotides of this application may be useful in Sanger sequencing and related protocols. Similarly, the modified nucleosides and nucleotides of this application may be useful in high-throughput sequencing, particularly in high-throughput sequencing platforms based on synthetic sequencing. By using the reversible blocking nucleotides and analogues of this application, the type of incorporated nucleotide can be determined one by one during the sequencing process.

[0278] Cleavable linkers are known in the art, and conventional chemistry can be applied to attach them to nucleotides or modified nucleotides and labels. Linkers can be cleaved by any suitable method, including exposure to acids, bases, nucleophiles, electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes, etc. Linkers such as those discussed herein can also be cleaved by the same catalysts used to cleave 3'-O-protecting groups. Suitable linkers can be adapted from standard chemical protecting groups, such as those disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Further suitable cleavable linkers for use in solid-phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100:2092~2157, 2000).

[0279] The use of the term “cleavable linker” is not intended to imply that the entire linker needs to be removed, for example, from the nucleotide or modified nucleotide. If a detectable label is bound to the nucleotide or modified nucleotide, the nucleoside cleavage site may be located on the linker in a position that ensures that a portion of the linker remains bound to the nucleotide or modified nucleotide after cleavage.

[0280] The linker may be bound to any position on the nucleotide or modified nucleotide, provided that Watson-Crick base pairing can still be performed if a detectable label is bound to the nucleotide or modified nucleotide.

[0281] A. Linker that can be cleaved by electrophilic reaction Linkers that can be cleaved by electrophilic reactions are typically proton-cleaved and include acid-sensitive cleavage. Suitable linkers include modified benzyl systems such as trityl, p-alkoxybenzyl esters, and p-alkoxybenzylamides. Other suitable linkers include tert-butyloxycarbonyl (Boc) groups and acetal systems.

[0282] The use of thiophilic metals such as nickel, silver, or mercury in the cleavage of thioacetals or other sulfur-containing protecting groups may also be considered for the preparation of suitable linker molecules.

[0283] B. Linkers that can be cleaved by nucleophilic reactions Nucleophilic cleavage is also a well-recognized method in the preparation of linker molecules. Groups such as esters that are unstable in water (i.e., can be cleaved simply at a basic pH) and groups that are unstable to non-aqueous nucleophiles can be used. Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or t-butyldimethylsilane (TBDMS).

[0284] C. Linker that can be cut by photodegradation Linkers that can be cleaved by photolysis have been widely used in carbohydrate chemistry. It is preferable that the light required to activate the cleavage does not affect other components of the modified nucleotide. For example, when a fluorophore is used as a label, it is preferable that the fluorophore absorbs light of a different wavelength than the wavelength required to cleave the linker molecule. Suitable linkers include those based on O-nitrobenzyl compounds and nitroveratril compounds. Linkers based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64:3454~3460, 1999).

[0285] D. Linker that can be cut under reducing conditions Many linkers are known to be sensitive to reductive cleavage. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction is also known in the art.

[0286] E. Linkers that can be cut under oxidizing conditions Oxidation-based methods are well known in the art. Oxidation-based methods include the oxidation of p-alkoxybenzyl groups and the oxidation of sulfur and selenium linkers. The use of aqueous iodine to cleave disulfides and other sulfur or selenium-based linkers is also within the scope of the present invention.

[0287] F. Safety Catch Linker A safety-catch linker is a linker that cleaves in two steps. In a preferred system, the first step is the generation of a reactive nucleophile, followed by a second step involving intramolecular cyclization resulting in cleavage. For example, a levulinic acid ester bond can be treated with hydrazine or photochemically to release an active amine, which can then be cyclized to cleave an ester at another location in the molecule (Burgess et al., J. Org. Chem. 62:5165~5168, 1997).

[0288] G. Linker that can be cut by a detachment mechanism Elimination reactions can also be used. For example, base-catalyzed elimination of groups such as Fmoc and cyanoethyl, as well as palladium-catalyzed reductive elimination of allyl groups, can be used.

[0289] In some embodiments, the linker may include spacer units. The length of the linker is not critical, provided that the label is held at a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and the enzyme.

[0290] In some embodiments, the linker may consist of functionalities similar to a 3'-OH protecting group. This makes the deprotection and deprotection processes more efficient, as only a single treatment is required to remove both the labeling and protecting groups. Particularly preferred linkers are phosphine-cleavable azide-containing linkers.

[0291] The present invention is further described below in conjunction with specific examples. Unless otherwise specified, the reagents used in the following examples may be commercially available. In addition, if one or more of the nucleotide analogs, dTTP, dATP, dCTP, and dGTP are prepared for each 3'-OH modification, those skilled in the art can certainly prepare and obtain the remaining one or more nucleotide analogs. [Examples]

[0292] I. Preparation example Example 1 The structure of the target compound is, [ka] That is the case.

[0293] (1) Step 1 [ka]

[0294] T-nucleoside (500 mg, 1.4 mmol, 1 eq) (available from Beijing OKeanos Tech.Co.,Ltd., catalog no. OK-N-18102) and azide protecting group (268 mg, 1.4 mmol, 1 eq) (available from Beijing OKeanos Tech.Co.,Ltd., catalog no. OK-H-20001) were added separately to a dry 100 mL round-bottom flask equipped with a rubber stopper with a magnetic stirrer and an argon balloon, followed by the addition of 20 mL of anhydrous methylene chloride as a solvent by syringe. The reaction mixture was stirred at room temperature to promote dissolution, followed by the separate addition of DCC (435 mg, 2.1 mmol, 1.5 eq) and DMAP (20 mg, 0.14 mmol, 0.1 eq), and the mixture was reacted for a further 4 hours at room temperature under a nitrogen balloon to provide a protective atmosphere. The reaction was monitored at half-hour intervals by TLC until the starting materials were substantially depleted. The reaction mixture was concentrated using a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, silica gel column diameter 3.5 cm, packing height 10 cm). The yield from this step was 95%.

[0295] 1 H NMR (400 MHz, CDCl3) δ 8.41 (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 = 6.8 Hz); 0.97 (9H, s); 0.10 (6H, s).

[0296] (2) Step 2 [ka]

[0297] The product from Step 1 (750 mg, 1.4 mmol, 1 eq) was added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer and a rubber stopper with an argon balloon, followed by the addition of 20 mL of anhydrous THF as solvent via syringe. The reaction mixture was stirred to promote dissolution, and then a 1 mol / L solution of TBAF (1.8 mL, 1.8 mmol, 1.3 eq) was slowly added dropwise via syringe in an ice bath. The flask was allowed to cool to room temperature and stirred continuously for approximately 2 hours. The reaction was monitored by TLC at half-hour intervals until the dots of the starting material on the TLC plate disappeared. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, methylene chloride / anhydrous methanol = 100 / 1 to 10 / 1, silica gel column diameter 3.5 cm, packed height 10 cm). The yield of this step was 70%.

[0298] 1 H NMR (400 MHz, DMSO-d6) δ 11.34 (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 = 6.8 Hz).

[0299] (3) Step 3 [ka]

[0300] The product from Step 2 (350 mg, 0.85 mmol, 1 eq) and a proton sponge (363.5 mg, 1.7 mmol, 2 eqs) were added to a dry 100 mL two-neck flask No. 1 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Tributylammonium pyrophosphate (932.5 mg, 1.7 mmol, 2 eqs) was added to a dry 100 mL two-neck flask No. 2 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Argon was packed into the two-neck flask No. 1, and then 15 mL of trimethyl phosphate was added while stirring to promote dissolution. The reaction was brought to 0°C, and then phosphorus oxychloride (120 μL, 1.3 mmol, 1.5 eq) was slowly added by syringe, and the mixture was continuously stirred at 0°C for 1.5 hours. Argon was packed into a two-necked flask (No. 2), and then 2 mL of anhydrous DMF and DIPEA (740 μL, 4.25 mmol, 5 eq) were added by syringe while stirring at 0°C to promote dissolution. The liquid from flask No. 1 was transferred to flask No. 2 by syringe. The mixture was maintained at 0°C and stirred continuously for 3 hours, and then the reaction was quenched by adding 20 mL of TEAB solution (0.1 mol / L). The reaction mixture was diluted with 50 mL of deionized water and then separated using a pre-prepared DEAE resin column, and eluted by gradient elution (H2O:TEAB (1 mol / L) = 10 / 1 to 0 / 1, DEAE column diameter 4.5 cm, packing height 8 cm). The fractions were identified by HPLC and MS, and those fractions containing triphosphate were collected. They were concentrated to 10 mL under vacuum and subsequently separated by preparative liquid chromatography using a gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98~98 / 2). The high-purity product solution recovered from the separation was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powder. MW = 655. The yield for this step was 50%.

[0301] 1H NMR (400 MHz, DMSO-d6) δ 11.32 (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, 1.0 Hz, 3H).

[0302] Example 2 The structure of the target compound is, [ka] That is the case.

[0303] (1) Step 1 [ka]

[0304] G-nucleoside (600 mg, 1.58 mmol, 1 eq) (available from Beijing Okeanos Tech.Co.,Ltd., catalog no. OK-N-18103) and azide protecting group (302 mg, 1.58 mmol, 1 eq) (available from Beijing Okeanos Tech.Co.,Ltd., catalog no. OK-H-20001) were added separately to a dry 100 mL round-bottom flask equipped with a rubber stopper with a magnetic stirrer and an argon balloon, followed by the addition of 20 mL of anhydrous methylene chloride as solvent by syringe. The reaction mixture was stirred at room temperature to promote dissolution, and then DCC (488 mg, 2.37 mmol, 1.5 eq) and DMAP (20 mg, 0.16 mmol, 0.1 eq) were added separately under a nitrogen balloon to provide a protective atmosphere, and the mixture was reacted at room temperature for a further 4 hours. The reaction was monitored by TLC at half-hour intervals until the starting materials were substantially depleted. The reaction mixture was concentrated using a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, silica gel column diameter 3.5 cm, packing height 10 cm). The yield from this step was 95%.

[0305] 1 H NMR (500 MHz, DMSO-d6) δ 10.65 (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, 3.6 Hz, 1H), 5.65 - 5.49 (m, 2H), 4.27 (qd, J = 4.8, 1.9 Hz, 1H), 3.86 (dd, J = 4.6, 2.2 Hz, 2H), 2.99 - 2.80 (m, 1H), 2.78 - 2.61 (m, 1H), 1.50 (d, J = 6.7 Hz, 3H), 0.88 (s, 9H), 0.07 (dd, J = 4.5, 1.3 Hz, 6H).

[0306] (2) Step 2 [ka]

[0307] The product from Step 1 (850 mg, 1.54 mmol, 1 eq) was added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer and a rubber stopper with an argon balloon. Subsequently, 20 mL of anhydrous THF was added as a solvent by syringe, and the mixture was stirred at room temperature to promote dissolution. The reaction mixture was cooled in an ice bath, and a 1 mol / L solution of TBAF (2.0 mL, 2.0 mmol, 1.3 eq) was slowly added dropwise by syringe. The flask was brought to room temperature and stirred continuously for approximately 2 hours. The reaction was monitored by TLC at half-hour intervals until the dots of the starting material on the TLC plate disappeared. The reaction mixture was concentrated in a rotary evaporator, and the product was separated by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, methylene chloride / anhydrous methanol = 100 / 1 to 10 / 1, silica gel column diameter 3.5 cm, packed height 10 cm). The yield of this step was 70%.

[0308] 1 H NMR (500 MHz, DMSO-d6) δ 10.68(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 = 6.8 Hz).

[0309] (3) Step 3 [ka]

[0310] The product from Step 2 (375 mg, 0.85 mmol, 1 eq) and a proton sponge (363.5 mg, 1.7 mmol, 2 eqs) were added to a dry 100 mL two-neck flask No. 1 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Tributylammonium pyrophosphate (932.5 mg, 1.7 mmol, 2 eqs) was added to a dry 100 mL two-neck flask No. 2 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Argon was packed into the two-neck flask No. 1, and 15 mL of trimethyl phosphate was added while stirring to promote dissolution. The reaction was brought to 0°C, and then phosphorus oxychloride (120 μL, 1.3 mmol, 1.5 eq) was slowly added by syringe, and the mixture was continuously stirred at 0°C for 1.5 hours. Argon was packed into a two-necked flask (No. 2), and then 5 mL of anhydrous DMF and DIPEA (740 μL, 4.25 mmol, 5 eq) were added by syringe while stirring at 0°C to promote dissolution. The liquid from flask No. 1 was transferred to flask No. 2 by syringe. The mixture was maintained at 0°C and stirred continuously for 3 hours, and then the reaction was quenched by adding 20 mL of TEAB solution (0.1 mol / L). The reaction mixture was diluted with 50 mL of deionized water and then separated using a pre-prepared DEAE resin column, and eluted by gradient elution (H2O / TEAB (1 mol / L) = 10 / 1 to 0 / 1, DEAE column diameter 4.5 cm, packing height 8 cm). The fraction containing triphosphates was collected, concentrated under vacuum using a rotary evaporator, and then separated by preparative liquid chromatography by gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). The high-purity product solution recovered from the separation was concentrated, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powdered solid. MW = 680. The yield of this step was 45%.

[0311] 1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 8.05 (d, J = 3.1 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.73 - 7.62 (m, 2H), 7.50 (t, J = 7.6 Hz, 1H), 6.87 (s, 2H), 6.25 - 6.19 (m, 1H), 5.72 (d, J = 4.9 Hz, 1H), 5.61 (q, J = 6.6 Hz, 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, 2.0 Hz, 3H).

[0312] Example 3 The structure of the target compound is, [ka] That is the case.

[0313] (1) Step 1 [ka]

[0314] In a dry 100 mL round-bottom flask equipped with a rubber stopper, magnetic stirrer, and argon balloon, C-nucleoside (600 mg, 1.56 mmol, 1 eq) (available from Beijing Okeanos Tech. Co., Ltd., catalog number OK-N-18104) and azide protecting group (297 mg, 1.56 mmol, 1 eq) (available from Beijing Okeanos Tech. Co., Ltd., catalog number OK-H-20001) were added separately, followed by the addition of 20 mL of anhydrous methylene chloride as solvent by syringe. The reaction mixture was stirred at room temperature to promote dissolution, and then DCC (480 mg, 2.34 mmol, 1.5 eq) and DMAP (20 mg, 0.16 mmol, 0.1 eq) were added separately under a nitrogen balloon to provide a protective atmosphere. The reaction was then allowed to proceed for a further 4 hours at room temperature. The reaction was monitored by TLC at half-hour intervals until the starting materials were substantially depleted. The reaction mixture was concentrated using a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, silica gel column diameter 3.5 cm, packing height 10 cm). The yield from this step was 95%.

[0315] 1 H NMR (400 MHz, DMSO-d6) δ 10.91 (1H, s); 8.23 ​​(1H, d, J =7.52 Hz); 7.89 (1H, d, J = 6.80 Hz); 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 =6.04 Hz), 4.42 (1H, t); 3.92 (2H, m); 2.74 (1H, m); 2.32(1H, m); 1.47 (3H, d, J = 6.7 Hz); 0.86 (9H, s); 0.09 (6 hours, s).

[0316] (2) Step 2 [ka]

[0317] The product from Step 1 (860 mg, 1.54 mmol, 1 eq) was added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer and a rubber stopper with an argon balloon, followed by the addition of 20 mL of anhydrous THF as solvent by syringe. The reaction mixture was stirred to promote dissolution, cooled in an ice bath, and then 1 mol / L solution of TBAF (2.0 mL, 2.0 mmol, 1.3 eq) was slowly added dropwise by syringe. The flask was brought to room temperature and stirred continuously for approximately 2 hours. The reaction was monitored by TLC at half-hour intervals until the dots of the starting material on the TLC plate disappeared. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, methylene chloride / anhydrous methanol = 100 / 1 to 10 / 1, silica gel column diameter 3.5 cm, packed height 10 cm). The yield of this step was 70%.

[0318] 1 7.23(1H, d, J =7.48 Hz); 6.22(1H, t); 5.57(1H, m); 5.47(1H, d, J = 6.00 Hz); 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 = 6.7 Hz).

[0319] (3) Step 3 [ka]

[0320] The product from Step 2 (377 mg, 0.85 mmol, 1 eq) and a proton sponge (363.5 mg, 1.7 mmol, 2 eqs) were added to a dry 100 mL two-neck flask No. 1 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Tributylammonium pyrophosphate (932.5 mg, 1.7 mmol, 2 eqs) was added to a dry 100 mL two-neck flask No. 2 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Argon was packed into the two-neck flask No. 1, and then 15 mL of trimethyl phosphate was added while stirring to promote dissolution. The reaction was brought to 0°C, and then phosphorus oxychloride (120 μL, 1.3 mmol, 1.5 eq) was slowly added by syringe, and the mixture was continuously stirred at 0°C for 1.5 hours. Argon was packed into a two-necked flask (No. 2), and then 2 mL of anhydrous DMF and DIPEA (740 μL, 4.25 mmol, 5 eq) were added by syringe while stirring at 0°C to promote dissolution. The mixture was maintained at 0°C and stirred continuously for 3 hours, and then the reaction was quenched by adding 20 mL of TEAB solution (0.1 mol / L). The reaction mixture was diluted with 50 mL of deionized water and then separated using a pre-prepared DEAE resin column, and eluted by gradient elution (H2O:TEAB (1 mol / L) = 10 / 1 to 0 / 1, DEAE column diameter 4.5 cm, packing height 8 cm). The fraction containing triphosphates was collected, concentrated using a rotary evaporator, and then separated by preparative liquid chromatography using gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). The high-purity product solution recovered from the separation was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powdered solid. MW = 684. The yield of this step was 40%.

[0321] (4) Step 4 [ka]

[0322] 500 mg (0.73 mmol, 1 eq) of triphosphate from step 3 and 5 ml of deionized water were added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred at room temperature to complete dissolution, and 25% aqueous ammonia (2.5 g, 36.5 mmol, 50 eq) was slowly added, followed by stirring at room temperature for approximately 6 hours. Ammonia in the solution was removed under vacuum using a rotary evaporator, and the residue was diluted to approximately 10 mL with deionized water. After separation by preparative liquid chromatography using gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98~98 / 2), the resulting high-purity product solution was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and freeze-dried in a freeze-dryer to obtain a powder solid. MW = 642. The yield of this step was 90%.

[0323] 1 H NMR (400 MHz, DMSO-d6) δ 7.99 (dd, J = 7.5, 2.2 Hz, 1H), 7.90 (d, J = 7.8 Hz, 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 = 7.5 Hz, 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, 1.4 Hz, 3H).

[0324] Example 4 The structure of the target compound is, [ka] That is the case.

[0325] (1) Step 1 [ka]

[0326] In a dry 100 mL round-bottom flask equipped with a rubber stopper, magnetic stirrer, and argon balloon, A-nucleoside (600 mg, 1.65 mmol, 1 eq) (available from Beijing Okeanos Tech. Co., Ltd., catalog no. OK-N-18101) and azide protecting group (314 mg, 1.65 mmol, 1 eq) (available from Beijing Okeanos Tech. Co., Ltd., catalog no. OK-H-20001) were added separately, followed by the addition of 20 mL of anhydrous methylene chloride as solvent by syringe. The reaction mixture was stirred at room temperature to promote dissolution, and then DCC (508 mg, 2.5 mmol, 1.5 eq) and DMAP (24 mg, 0.17 mmol, 0.1 eq) were added separately under a nitrogen balloon to provide a protective atmosphere. The reaction was then allowed to proceed for a further 4 hours at room temperature. The reaction was monitored by TLC at half-hour intervals until the starting materials were substantially depleted. The reaction mixture was concentrated using a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, silica gel column diameter 3.5 cm, packing height 10 cm). The yield from this step was 95%.

[0327] 1 H NMR (500 MHz, CDCl3) δ 8.36 (1H, s); 8.28 (1H, d, J = 4.4 Hz); 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 = 6.7 Hz); 0.94 (9H, s); 0.15 (6H, s).

[0328] (2) Step 2 [ka]

[0329] The product from Step 1 (828 mg, 1.54 mmol, 1 eq) was added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer and a rubber stopper with an argon balloon, followed by the addition of 20 mL of anhydrous THF as solvent by syringe. The reaction mixture was stirred to promote dissolution, cooled in an ice bath, and then a 1 mol / L solution of TBAF (2.0 mL, 2.0 mmol, 1.3 eq) was slowly added dropwise by syringe. The flask was brought to room temperature and stirred continuously for approximately 2 hours. The reaction was monitored by TLC at half-hour intervals until the dots of the starting material on the TLC plate disappeared. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, methylene chloride / anhydrous methanol = 100 / 1 to 10 / 1, silica gel column diameter 3.5 cm, packed height 10 cm). The yield of this step was 70%.

[0330] 1 H NMR (500 MHz, DMSO-d6) δ 10.66 (s, 1H), 8.00 (d, J = 1.5 Hz, 1H), 7.92 (ddd, J = 8.0, 3.0, 1.4 Hz, 1H), 7.82 - 7.59 (m, 2H), 7.51 (t, J = 7.5 Hz, 1H), 6.46 (s, 2H), 6.20 (dt, J = 9.5, 5.4 Hz, 1H), 5.58 (ddd, J = 15.6, 6.2, 2.5 Hz, 2H), 5.21 (td, J = 5.6, 1.8 Hz, 1H), 4.24 (dd, J = 4.3, 1.7 Hz, 1H), 3.77 - 3.55 (m, 2H), 2.91 (qd, J = 9.1, 5.8 Hz, 1H), 2.62 (dt, J = 13.7, 6.6 Hz, 1H), 1.51 (dd, J = 6.7, 1.7 Hz, 3H).

[0331] (3) Step 3 [ka]

[0332] The product from Step 2 (360 mg, 0.85 mmol, 1 eq) and a proton sponge (363.5 mg, 1.7 mmol, 2 eqs) were added to a dry 100 mL two-neck flask No. 1 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Tributylammonium pyrophosphate (932.5 mg, 1.7 mmol, 2 eqs) was added to a dry 100 mL two-neck flask No. 2 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Argon was packed into the two-neck flask No. 1, and then 15 mL of trimethyl phosphate was added while stirring to promote dissolution. The reaction was brought to 0°C, and then phosphorus oxychloride (120 μL, 1.3 mmol, 1.5 eq) was slowly added by syringe, and the mixture was continuously stirred at 0°C for 1.5 hours. Argon was packed into a two-necked flask (No. 2), and then 5 mL of anhydrous DMF and DIPEA (740 μL, 4.25 mmol, 5 eq) were added by syringe while stirring at 0°C to promote dissolution. The mixture was maintained at 0°C and stirred continuously for 3 hours, and then the reaction was quenched by adding 20 mL of TEAB solution (0.1 mol / L). The reaction mixture was diluted with 50 mL of deionized water and then separated using a pre-prepared DEAE resin column, and eluted by gradient elution (H2O:TEAB (1 mol / L) = 10 / 1 to 0 / 1, DEAE column diameter 4.5 cm, packing height 8 cm). The fraction containing triphosphates was collected and concentrated under vacuum using a rotary evaporator, and then separated by preparative liquid chromatography by gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). The high-purity product solution recovered from the separation was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powdered solid. MW = 664. The yield of this step was 40%.

[0333] 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 3.6 Hz, 1H), 8.15 (d, J = 1.5 Hz, 1H), 7.97 (d, J = 7.8 Hz, 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 = 5.3 Hz, 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, 1.5 Hz, 3H).

[0334] Example 5 The structure of the target compound,

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[0335] (1)ステップ1

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[0336] T-nucleoside (500 mg, 1.40 mmol, 1 eq) (available from Beijing Okeanos Tech.Co.,Ltd., catalog number OK-N-18102) and disulfaneol protecting group (340 mg, 1.40 mmol, 1 eq) (available from Beijing Okeanos Tech.Co.,Ltd., catalog number OK-H-21003) were added separately to a dry 100 mL round-bottom flask equipped with a rubber stopper having a magnetic stirrer and an argon balloon, followed by the addition of 20 mL of anhydrous methylene chloride as a solvent using a syringe. The reaction mixture was stirred at room temperature to promote dissolution, and then DCC (433 mg, 2.10 mmol, 1.5 eq) and DMAP (17 mg, 0.14 mmol, 0.1 eq) were added separately under a nitrogen balloon to provide a protective atmosphere, and the mixture was reacted at room temperature for a further 6 hours. The reaction was monitored by TLC at half-hour intervals until the starting materials were substantially depleted. The reaction mixture was concentrated using a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, silica gel column diameter 3.5 cm, packing height 10 cm), yielding 85%. 1 H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 4.7 Hz, 1H), 7.84 (t, J = 8.3 Hz, 1H), 7.63 - 7.57 (m, 2H), 7.52 (t, J = 7.6 Hz, 1H), 7.33 (dd, J = 11.0, 4.1 Hz, 1H), 6.47 - 6.40 (m, 1H), 5.52 - 5.45 (m, 1H), 5.31 - 5.20 (m, 1H), 4.30 (d, J = 21.8 Hz, 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 = 7.0 Hz, 3H), 1.12 - 1.07 (m, 3H), 0.96 (s, 9H), 0.17 (s, 6H).

[0337] (2) Step 2 [ka]

[0338] The product from Step 1 (690 mg, 1.19 mmol, 1 eq) was added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer and a rubber stopper with an argon balloon, followed by the addition of 20 mL of anhydrous THF as solvent by syringe. The reaction mixture was stirred at room temperature to promote dissolution, cooled in an ice bath, and then a 1 mol / L solution of TBAF (1.6 mL, 1.6 mmol, 1.3 eq) was slowly added dropwise by syringe. The flask was brought to room temperature and stirred continuously for approximately 2 hours. The reaction was monitored by TLC at half-hour intervals until the dots of the starting material on the TLC plate disappeared. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 5 / 1 to 1 / 1 to methylene chloride / anhydrous methanol = 100 / 1 to 10 / 1, silica gel column diameter 3.5 cm, packed height 10 cm). The yield of this step was 80%.

[0339] 1 H NMR (500 MHz, CDCl3) δ 8.57 (s, 1H), 7.87 - 7.80 (m, 1H), 7.60 (dd, J = 7.8, 1.9 Hz, 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 = 7.1 Hz, 1H), 4.29 (dd, J = 14.3, 2.4 Hz, 1H), 4.02 (dd, J = 4.9, 2.5 Hz, 2H), 2.59 - 2.55 (m, 2H), 2.29 - 2.19 (m, 2H), 1.94 (d, J = 0.6 Hz, 3H), 1.70 (d, J = 7.0 Hz, 3H), 1.11 (td, J = 7.4, 2.6 Hz, 3H).

[0340] (3) Step 3 [ka]

[0341] The product from Step 2 (396 mg, 0.85 mmol, 1 eq) and a proton sponge (363.5 mg, 1.7 mmol, 2 eqs) were added to a dry 100 mL two-neck flask No. 1 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Tributylammonium pyrophosphate (932.5 mg, 1.7 mmol, 2 eqs) was added to a dry 100 mL two-neck flask No. 2 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Argon was packed into the two-neck flask No. 1, and then 15 mL of trimethyl phosphate was added while stirring to promote dissolution. The reaction was brought to 0°C, and then phosphorus oxychloride (120 μL, 1.3 mmol, 1.5 eq) was slowly added by syringe, and the mixture was continuously stirred at 0°C for 1.5 hours. Argon was packed into a two-necked flask (No. 2), and then 5 mL of anhydrous DMF and DIPEA (740 μL, 4.25 mmol, 5 eq) were added by syringe at 0°C while stirring to promote dissolution. The mixture was maintained at 0°C and stirred continuously for 3 hours, and then the reaction was quenched by adding 20 mL of TEAB solution (0.1 mol / L). The reaction mixture was diluted with 50 mL of deionized water and then separated using a pre-prepared DEAE resin column, and eluted by gradient elution (H2O:TEAB (1 mol / L) = 10 / 1 to 0 / 1, DEAE column diameter 4.5 cm, packing height 8 cm). The fraction containing triphosphates was collected and concentrated under vacuum using a rotary evaporator, and then separated by preparative liquid chromatography by gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). The high-purity product solution recovered from the separation was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powdered solid. MW = 706. The yield of this step was 40%.

[0342] 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.96 (dd, J = 6.0, 1.0 Hz, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.68 - 7.56 (m, 2H), 7.46 - 7.41 (m, 1H), 6.38 - 6.27 (m, 1H), 5.60 (d, J = 5.3 Hz, 1H), 5.16 - 5.11 (m, 1H), 4.31 (d, J = 20.8 Hz, 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 = 7.0 Hz, 3H), 1.09 - 1.03 (m, 3H).

[0343] Example 6 The structure of the target compound,

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[0344] (1)ステップ1

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[0345] G-nucleoside (600 mg, 1.58 mmol, 1 eq) (available from Beijing Okeanos Tech.Co.,Ltd., catalog no. OK-N-18103) and disulfaneol protecting group (605 mg, 1.58 mmol, 1 eq) (available from Beijing Okeanos Tech.Co.,Ltd., catalog no. OK-H-21003) were added separately to a dry 100 mL round-bottom flask equipped with a rubber stopper with a magnetic stirrer and an argon balloon, followed by the addition of 20 mL of anhydrous methylene chloride as a solvent using a syringe. The reaction mixture was stirred at room temperature to promote dissolution, and then DCC (488 mg, 2.37 mmol, 1.5 eq) and DMAP (20 mg, 0.16 mmol, 0.1 eq) were added separately under a nitrogen balloon to provide a protective atmosphere, and the mixture was reacted at room temperature for a further 4 hours. The reaction was monitored by TLC at half-hour intervals until the starting materials were substantially depleted. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1-1 / 2, silica gel column diameter 3.5 cm, packing height 10 cm). The yield for this step was 80%.

[0346] 1H NMR (500 MHz, CDCl3) δ 12.00 (s, 1H), 7.93 (t, J = 9.2 Hz, 1H), 7.88 - 7.83 (m, 1H), 7.59 (dd, J = 13.6, 8.0 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 6.37 - 6.33 (m, 2H), 5.62 (s, 1H), 5.30 - 5.22 (m, 1H), 4.36 (dd, J = 10.9, 1.5 Hz, 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 = 6.9 Hz, 3H), 1.13 (d, J = 2.5 Hz, 3H), 0.92 (s, 9H), 0.12 (s, 6H).

[0347] (2) Step 2 [ka]

[0348] The product from Step 1 (932 mg, 1.54 mmol, 1 eq) was added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer and a rubber stopper with an argon balloon, followed by the addition of 20 mL of anhydrous THF as solvent via syringe. The reaction mixture was stirred to promote dissolution, cooled in an ice bath, and then 1 mol / L solution of TBAF (2.0 mL, 2.0 mmol, 1.3 eq) was slowly added dropwise via syringe. The flask was brought to room temperature and stirred continuously for approximately 2 hours. The reaction was monitored by TLC at half-hour intervals until the dots of the starting material on the TLC plate disappeared. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, methylene chloride / anhydrous methanol = 100 / 1 to 10 / 1, silica gel column diameter 3.5 cm, packed height 10 cm). The yield of this step was 70%.

[0349] 1 H NMR (400 MHz, CDCl3) δ 11.71 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.2 Hz, 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, 7.2 Hz, 2H), 2.26 - 2.20 (m, 1H), 2.06 - 1.97 (m, 1H), 1.69 (d, J = 6.9 Hz, 3H), 1.14 (t, J = 7.3 Hz, 3H).

[0350] (3)ステップ3

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[0351] The product from Step 2 (417 mg, 0.85 mmol, 1 eq) and a proton sponge (363.5 mg, 1.7 mmol, 2 eqs) were added to a dry 100 mL two-neck flask No. 1 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Tributylammonium pyrophosphate (932.5 mg, 1.7 mmol, 2 eqs) was added to a dry 100 mL two-neck flask No. 2 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Argon was packed into the two-neck flask No. 1, and then 15 mL of trimethyl phosphate was added while stirring to promote dissolution. The reaction was brought to 0°C, and then phosphorus oxychloride (120 μL, 1.3 mmol, 1.5 eq) was slowly added by syringe, and the mixture was continuously stirred at 0°C for 1.5 hours. Argon was packed into a two-necked flask (No. 2), and then 5 mL of anhydrous DMF and DIPEA (740 μL, 4.25 mmol, 5 eq) were added by syringe while stirring at 0°C to promote dissolution. The mixture was maintained at 0°C and stirred continuously for 3 hours, and then the reaction was quenched by adding 20 mL of TEAB solution (0.1 mol / L). The reaction mixture was stirred continuously for 0.5 hours, diluted with 50 mL of deionized water, and then separated using a pre-prepared DEAE resin column. Elution was performed by gradient elution (H2O:TEAB (1 mol / L) = 10 / 1 to 0 / 1, DEAE column diameter 4.5 cm, packing height 8 cm). The fraction containing triphosphate was collected, concentrated to 10 mL under vacuum, and then separated by preparative liquid chromatography using gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). The high-purity product solution recovered from the separation was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powdered solid. MW = 706. The yield of this step was 40%.

[0352] 1H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 8.03 (d, J = 5.5 Hz, 1H), 7.88 (d, J = 7.8 Hz, 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 = 5.0 Hz, 1H), 5.22 - 5.10 (m, 1H), 4.40 - 4.27 (m, 1H), 4.21 (dd, J = 11.0, 5.0 Hz, 1H), 4.01 (dd, J = 7.0, 3.7 Hz, 1H), 2.59 - 2.52 (m, 1H), 2.48 - 2.30 (m, 3H), 1.65 (d, J = 7.0 Hz, 3H), 1.07 (t, J = 5.1 Hz, 3H).

[0353] Example 7 The structure of the target compound,

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[0354] (1)ステップ1

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[0355] In a dry 100 mL round-bottom flask equipped with a rubber stopper, magnetic stirrer, and argon balloon, C-nucleoside (608 mg, 1.58 mmol, 1 eq) (available from Beijing Okeanos Tech. Co., Ltd., catalog number OK-N-18104) and disulfaneol protecting group (605 mg, 1.58 mmol, 1 eq) (available from Beijing Okeanos Tech. Co., Ltd., catalog number OK-H-21003) were added separately, followed by the addition of 20 mL of anhydrous methylene chloride as a solvent using a syringe. The reaction mixture was stirred at room temperature to promote dissolution, and then DCC (488 mg, 2.37 mmol, 1.5 eq) and DMAP (20 mg, 0.16 mmol, 0.1 eq) were added separately under a nitrogen balloon to provide a protective atmosphere. The mixture was then reacted at room temperature for a further 4 hours. The reaction was monitored by TLC at half-hour intervals until the starting materials were substantially depleted. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1-1 / 2, silica gel column diameter 3.5 cm, packing height 10 cm). The yield for this step was 90%.

[0356] 1H NMR (500 MHz, CDCl3) δ 9.53 (s, 1H), 8.35 (d, J = 6.0 Hz, 1H), 7.87 (ddd, J = 7.8, 4.4, 1.3 Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H), 7.54 - 7.50 (m, 1H), 7.41 (d, J = 6.0 Hz, 1H), 7.32 (td, J = 7.7, 1.2 Hz, 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, 0.6 Hz, 3H), 1.11 (td, J = 7.4, 3.3 Hz, 3H), 0.93 (s, 9H), 0.13 (s, 6H).

[0357] (2)ステップ2

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[0358] The product from Step 1 (938 mg, 1.54 mmol, 1 eq) was added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer and a rubber stopper with an argon balloon, followed by the addition of 20 mL of anhydrous THF as solvent by syringe. The reaction mixture was stirred to promote dissolution, cooled in an ice bath, and then 1 mol / L solution of TBAF (2.0 mL, 2.0 mmol, 1.3 eq) was slowly added dropwise by syringe. The flask was brought to room temperature and stirred continuously for approximately 2 hours. The reaction was monitored by TLC at half-hour intervals until the dots of the starting material on the TLC plate disappeared. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, methylene chloride / anhydrous methanol = 100 / 1 to 10 / 1, silica gel column diameter 3.5 cm, packed height 10 cm). The yield of this step was 80%.

[0359] 1 H NMR (400 MHz, CDCl3) δ 9.50 (s, 1H), 8.39 (s, 1H), 7.85 (t, J = 7.2 Hz, 1H), 7.61 - 7.51 (m, 3H), 7.34 (t, J = 7.3 Hz, 1H), 6.30 (s, 1H), 5.63 (d, J = 2.8 Hz, 1H), 5.27 - 5.19 (m, 1H), 4.40 (d, J = 15.5 Hz, 1H), 4.15 - 3.94 (m, 2H), 3.40 (s, 1H), 2.83 (td, J = 14.3, 4.8 Hz, 1H), 2.60 - 2.45 (m, 1H), 2.27 - 2.14 (m, 2H), 1.69 (d, J = 6.8 Hz, 3H), 1.10 (q, J = 6.8 Hz, 3H).

[0360] (3) Step 3 [ka]

[0361] The product from Step 2 (421 mg, 0.85 mmol, 1 eq) and a proton sponge (363.5 mg, 1.7 mmol, 2 eqs) were added to a dry 100 mL two-neck flask No. 1 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Tributylammonium pyrophosphate (932.5 mg, 1.7 mmol, 2 eqs) was added to a dry 100 mL flask No. 2 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Argon was packed into the two-neck flask No. 1, and then 15 mL of trimethyl phosphate was added while stirring to promote dissolution. The reaction was brought to 0°C, and then phosphorus oxychloride (120 μL, 1.3 mmol, 1.5 eq) was slowly added by syringe, and the mixture was continuously stirred at 0°C for 1.5 hours. Argon was packed into a two-necked flask (No. 2), and then 5 mL of anhydrous DMF and DIPEA (740 μL, 4.25 mmol, 5 eq) were added by syringe while stirring at 0°C to promote dissolution. The mixture was maintained at 0°C and stirred continuously for 3 hours, and then the reaction was quenched by adding 20 mL of TEAB solution (0.1 mol / L). The reaction mixture was stirred continuously for 0.5 hours. The reaction mixture was diluted with 50 mL of deionized water and then separated using a pre-prepared DEAE resin column, and eluted by gradient elution (H2O:TEAB (1 mol / L) = 10 / 1 to 0 / 1, DEAE column diameter 4.5 cm, packing height 8 cm). The fraction containing triphosphates was collected, concentrated under vacuum using a rotary evaporator, and then separated by preparative liquid chromatography using gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). The high-purity product solution recovered from the separation was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powdered solid. MW = 733. The yield of this step was 45%.

[0362] (4) Step 4 [ka]

[0363] 537 mg (0.73 mmol, 1 eq) of triphosphate from step 3 and 5 ml of deionized water were added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer. The mixture was stirred at room temperature to complete dissolution, followed by the addition of 25% aqueous ammonia (2.5 g, 36.5 mmol, 50 eq), and the mixture was stirred at room temperature for approximately 2 hours. The ammonia in the solution was removed under vacuum using a rotary evaporator, and the residue was diluted to approximately 10 mL with deionized water. After separation by preparative liquid chromatography using gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98~98 / 2), the high-purity product solution recovered from the separation was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powder solid. MW = 691. The yield of this step was 90%.

[0364] 1 H NMR (400 MHz, DMSO-d6) δ 7.97 (dd, J = 7.4, 6.2 Hz, 1H), 7.85 (d, J = 8.7 Hz, 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 = 7.4 Hz, 1H), 5.53 (d, J = 5.0 Hz, 1H), 5.17 - 5.10 (m, 1H), 4.31 (d, J = 19.8 Hz, 1H), 4.13 - 4.01 (m, 2H), 2.46 - 2.23 (m, 4H), 1.65 (d, J = 7.0 Hz, 3H), 1.09 - 1.03 (m, 3H).

[0365] Example 8 The structure of the target compound is, [ka] That is the case.

[0366] (1) Step 1 [ka]

[0367] In a dry 100 mL round-bottom flask equipped with a rubber stopper, magnetic stirrer, and argon balloon, A-nucleoside (577 mg, 1.58 mmol, 1 eq) (available from Beijing Okeanos Tech. Co., Ltd., catalog no. OK-N-18101) and disulfaneol protecting group (605 mg, 1.58 mmol, 1 eq) (available from Beijing Okeanos Tech. Co., Ltd., catalog no. OK-H-21003) were added separately, followed by the addition of 20 mL of anhydrous methylene chloride as solvent by syringe. The reaction mixture was stirred at room temperature to promote dissolution, and then DCC (488 mg, 2.37 mmol, 1.5 eq) and DMAP (20 mg, 0.16 mmol, 0.1 eq) were added separately under a nitrogen balloon to provide a protective atmosphere. The reaction was then allowed to proceed for a further 4 hours at room temperature. The reaction was monitored by TLC at half-hour intervals until the starting materials were substantially depleted. The reaction mixture was concentrated using a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1-1 / 2, silica gel column diameter 3.5 cm, packing height 10 cm). The yield from this step was 85%.

[0368] 11H NMR (500 MHz, CDCl3) δ 8.34 (s, 1H), 8.24 (d, J = 11.2 Hz, 1H), 7.87 (ddd, J = 7.8, 4.4, 1.3 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.32 (td, J = 7.7, 1.2 Hz, 1H), 6.58 (t, J = 7.1 Hz, 1H), 6.21 (s, 2H), 5.68 - 5.61 (m, 1H), 5.27 (p, J = 7.0 Hz, 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, 0.6 Hz, 3H), 1.11 (td, J = 7.4, 3.3 Hz, 3H), 0.93 (s, 9H), 0.13 (t, J = 1.7 Hz, 6H).

[0369] (2) Step 2 [Chemical formula]

[0370] The product from Step 1 (907 mg, 1.54 mmol, 1 eq) was added to a dry 100 mL round-bottom flask equipped with a magnetic stirrer and a rubber stopper with an argon balloon, followed by the addition of 20 mL of anhydrous THF as solvent via syringe. The reaction mixture was stirred to promote dissolution, cooled in an ice bath, and then 1 mol / L solution of TBAF (2.0 mL, 2.0 mmol, 1.3 eq) was slowly added dropwise via syringe. The flask was brought to room temperature and stirred continuously for approximately 2 hours. The reaction was monitored by TLC at half-hour intervals until the dots of the starting material on the TLC plate disappeared. The reaction mixture was concentrated in a rotary evaporator, followed by product separation by silica gel column chromatography (hexane / EA = 10 / 1 to 1 / 1, methylene chloride / anhydrous methanol = 100 / 1 to 10 / 1, silica gel column diameter 3.5 cm, packed height 10 cm). The yield of this step was 80%.

[0371] 1 H NMR (500 MHz, CDCl3) δ 8.37 (s, 1H), 7.93 (s, 1H), 7.89 - 7.85 (m, 1H), 7.65 - 7.61 (m, 1H), 7.56 (td, J = 7.7, 1.3 Hz, 1H), 7.39 - 7.33 (m, 1H), 6.37 (dd, J = 9.3, 5.3 Hz, 1H), 6.02 (s, 2H), 5.83 (t, J = 5.5 Hz, 1H), 5.31 - 5.23 (m, 1H), 4.48 (d, J = 14.2 Hz, 1H), 4.09 - 3.97 (m, 2H), 3.33 - 3.28 (m, 1H), 2.63 (dd, J = 14.0, 4.9 Hz, 1H), 2.41 - 2.31 (m, 2H), 1.73 (d, J = 7.0 Hz, 3H), 1.17 (td, J = 7.4, 2.3 Hz, 3H).

[0372] (3) Step 3 [ka]

[0373] The product from Step 2 (404 mg, 0.85 mmol, 1 eq) and a proton sponge (363.5 mg, 1.7 mmol, 2 eqs) were added to a dry 100 mL two-neck flask No. 1 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Tributylammonium pyrophosphate (932.5 mg, 1.7 mmol, 2 eqs) was added to a dry 100 mL two-neck flask No. 2 equipped with a magnetic stirrer and a three-way valve with an argon balloon. Argon was packed into the two-neck flask No. 1, and then 15 mL of trimethyl phosphate was added while stirring to promote dissolution. The reaction was brought to 0°C, and then phosphorus oxychloride (120 μL, 1.3 mmol, 1.5 eq) was slowly added by syringe, and the mixture was continuously stirred at 0°C for 1.5 hours. Argon was packed into a two-necked flask (No. 2), and then 5 mL of anhydrous DMF and DIPEA (740 μL, 4.25 mmol, 5 eq) were added by syringe while stirring at 0°C to promote dissolution. The mixture was maintained at 0°C and stirred continuously for 3 hours, and then the reaction was quenched by adding 20 mL of TEAB solution (0.1 mol / L). The reaction mixture was stirred continuously for 0.5 hours, diluted with 50 mL of deionized water, and then separated using a pre-prepared DEAE resin column, and eluted by gradient elution (H2O:TEAB (1 mol / L) = 10 / 1 to 0 / 1, DEAE column diameter 4.5 cm, packing height 8 cm). The fraction containing triphosphates was collected and concentrated under vacuum using a rotary evaporator, and then separated by preparative liquid chromatography by gradient elution (CH3CN:TEAB (0.1 mol / L) = 2 / 98 to 98 / 2). The high-purity product solution recovered from the separation was concentrated under vacuum using a rotary evaporator, transferred to a plastic centrifuge tube, and then freeze-dried in a freeze-dryer to obtain a powdered solid. MW = 715. The yield of this step was 40%.

[0374] 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.15 (s, 1H), 8.05 (d, J = 6.5 Hz, 1H), 7.69 (t, J = 8.2 Hz, 1H), 7.60 - 7.54 (m, 2H), 7.27 (s, 2H), 6.25 - 6.18 (m, 1H), 5.70 (t, J = 5.0 Hz, 1H), 5.22 - 5.10 (m, 1H), 4.40 - 4.27 (m, 1H), 4.21 (dd, J = 11.0, 5.0 Hz, 1H), 4.01 (dd, J = 7.0, 3.7 Hz, 1H), 2.59 - 2.52 (m, 1H), 2.48 - 2.30 (m, 3H), 1.65 (d, J = 7.0 Hz, 3H), 1.07 (t, J = 5.1 Hz, 3H).

[0375] Example 9 (1) Step 1 [ka]

[0376] The product was synthesized in the same manner as in Step 1 of Example 4, by substituting 2-(1-azidoethyl)benzoic acid with 2-(1-azidoethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20002), yielding 85% yield and MW=539.

[0377] 1H NMR (400 MHz, DMSO-d6) δ 8.86 (dd, J = 4.7, 1.7 Hz, 1H), 8.38 (dt, J = 7.9, 1.7 Hz, 1H), 8.33 (s, 1H), 8.16 (d, J = 0.9 Hz, 1H), 7.58 (dd, J = 7.9, 4.7 Hz, 1H), 7.32 (s, 2H), 6.47 (ddd, J = 8.5, 6.1, 2.9 Hz, 1H), 5.66 (dq, J = 6.2, 2.0 Hz, 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, 6.3 Hz, 1H), 2.87 - 2.68 (m, 1H), 1.61 (d, J = 6.7 Hz, 3H), 0.86 (s, 9H), 0.04 (d, J = 3.9 Hz, 6H).

[0378] (2) Step 2 [ka]

[0379] The product was synthesized in the same manner as in Step 2 of Example 4, by substituting 2-(1-azidoethyl)benzoic acid with 2-(1-azidoethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20002), yielding 75% yield and MW=425.

[0380] 1H NMR (500 MHz, DMSO-d6) δ 8.86 (dd, J = 4.8, 1.8 Hz, 1H), 8.45 - 8.31 (m, 2H), 8.16 (d, J = 1.1 Hz, 1H), 7.59 (dd, J = 7.9, 4.7 Hz, 1H), 7.38 (s, 2H), 6.47 (ddd, J = 9.4, 5.8, 4.2 Hz, 1H), 5.71 - 5.62 (m, 1H), 5.57 (ddd, J = 7.5, 4.8, 2.8 Hz, 1H), 5.33 (p, J = 6.7 Hz, 1H), 4.34 (ddt, J = 7.6, 4.1, 2.2 Hz, 1H), 3.83 - 3.60 (m, 2H), 3.09 (ddd, J = 14.5, 9.0, 5.9 Hz, 1H), 2.85 - 2.65 (m, 1H), 1.61 (dd, J = 6.7, 2.7 Hz, 3H).

[0381] (3) Step 3 [ka]

[0382] The product was synthesized in the same manner as in Step 3 of Example 4, by substituting 2-(1-azidoethyl)benzoic acid with 2-(1-azidoethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20002), yielding 40% yield and MW=665.

[0383] 1H NMR (400 MHz, DMSO-d6) δ 8.85 (d, J = 3.6 Hz, 1H), 8.66 (d, J = 3.4 Hz, 1H), 8.40 (d, J = 7.3 Hz, 1H), 8.15 (s, 1H), 7.58 (dd, J = 7.9, 4.8 Hz, 1H), 7.31 (s, 2H), 6.54 - 6.45 (m, 1H), 5.77 (d, J = 5.1 Hz, 1H), 5.37 - 5.28 (m, 1H), 4.44 (d, J = 4.5 Hz, 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, 2.1 Hz, 3H).

[0384] Example 10 [ka]

[0385] The product was synthesized in the same manner as in steps 1-3 of Example 2, with 2-(1-azidoethyl)benzoic acid replaced by 2-(1-azidoethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20002). The yield of the final step was 40%, and the MW = 681.

[0386] 1H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 8.84 (d, J = 4.6 Hz, 1H), 8.39 - 8.29 (m, 1H), 8.02 (d, J = 5.5 Hz, 1H), 7.57 (dd, J = 7.8, 4.8 Hz, 1H), 6.71 (s, 2H), 6.28 - 6.16 (m, 1H), 5.73 (d, J = 4.9 Hz, 1H), 5.35 - 5.28 (m, 1H), 4.36 (dd, J = 10.4, 6.0 Hz, 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, 2.6 Hz, 3H).

[0387] Example 11 [ka]

[0388] The product was synthesized in the same manner as in steps 1-4 of Example 3, with 2-(1-azidoethyl)benzoic acid replaced by 2-(1-azidoethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20002). The yield for the last two steps was 30%, and the MW = 641.

[0389] 1H NMR (400 MHz, DMSO-d6) δ 8.83 (d, J = 3.5 Hz, 1H), 8.33 (d, J = 7.8 Hz, 1H), 7.95 (dd, J = 7.4, 2.9 Hz, 1H), 7.55 (dd, J = 7.9, 4.7 Hz, 1H), 7.30 (s, 1H), 7.08 (s, 1H), 6.37 - 6.34 (m, 1H), 5.82 (d, J = 7.4 Hz, 1H), 5.54 (d, J = 4.9 Hz, 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, 1.7 Hz, 3H).

[0390] Example 12 [ka]

[0391] The product was synthesized in the same manner as in steps 1-3 of Example 1, with 2-(1-azidoethyl)benzoic acid replaced by 2-(1-azidoethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20002). The yield of the final step was 45%, and the MW = 656.

[0392] 1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 8.84 (dd, J = 4.6, 1.1 Hz, 1H), 8.34 (d, J = 7.9 Hz, 1H), 7.98 (s, 1H), 7.56 (dd, J = 7.9, 4.8 Hz, 1H), 6.39 - 6.32 (m, 1H), 5.63 (d, J = 5.2 Hz, 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, 1.3 Hz, 3H).

[0393] Example 13 (1) Step 1 [ka]

[0394] The product was synthesized in the same manner as in Example 1, Step 1, by substituting 2-(1-azidoethyl)benzoic acid with 2-(azidomethyl)-4-nitrobenzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21004). The yield of this step was 85%, and the MW = 560.

[0395] 1H NMR (500 MHz, CDCl3) δ 8.43 (d, J = 2.3 Hz, 1H), 8.34 - 8.23 ​​(m, 2H), 8.20 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 1.2 Hz, 1H), 6.45 (dd, J = 9.3, 5.2 Hz, 1H), 5.54 (d, J = 5.9 Hz, 1H), 5.04 - 4.84 (m, 2H), 4.28 (q, J = 1.8 Hz, 1H), 4.12 - 3.92 (m, 2H), 3.48 (s, 1H), 2.60 (dd, J = 14.0, 5.3 Hz, 1H), 2.28 (ddd, J = 14.0, 9.3, 6.1 Hz, 2H), 1.95 (d, J = 1.2 Hz, 3H), 0.97 (s, 9H), 0.18 (d, J = 1.1 Hz, 6H).

[0396] (2) Steps 2-3 [ka]

[0397] The product was synthesized in the same manner as in steps 2-2-3 of Example 1, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)-4-nitrobenzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21004). The yield of the final step was 40%, and the MW = 686.

[0398] 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.41 (d, J = 2.0 Hz, 1H), 8.33 (dd, J = 8.6, 2.2 Hz, 1H), 8.23 ​​(d, J = 8.6 Hz, 1H), 7.95 (s, 1H), 6.36 (dd, J = 9.0, 6.0 Hz, 1H), 5.65 (d, J = 5.2 Hz, 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, 5.9 Hz, 1H), 1.84 (s, 3H).

[0399] Example 14 [ka]

[0400] The product was synthesized in the same manner as in steps 1-3 of Example 2, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)-4-nitrobenzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21004). The yield of the final step was 40%, and the MW = 711.

[0401] 1 H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 8.42 (d, J = 2.3 Hz, 1H), 8.34 (dd, J = 8.6, 2.3 Hz, 1H), 8.25 (d, J = 8.6 Hz, 1H), 8.03 (s, 1H), 6.82 (s, 2H), 6.23 (dd, J = 9.2, 5.9 Hz, 1H), 5.76 (d, J = 4.2 Hz, 1H), 4.99 (s, 2H), 4.39 (t, J = 4.8 Hz, 1H), 4.25 - 4.00 (m, 2H), 3.31 - 3.14 (m, 1H), 2.59 (dd, J = 14.0, 6.4 Hz, 1H).

[0402] Example 15 [ka]

[0403] The product was synthesized in the same manner as in steps 1-4 of Example 3, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)-4-nitrobenzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21004). The yield for the last two steps was 30%, and the MW = 671.

[0404] 1 H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 2.1 Hz, 1H), 8.33 (dd, J = 8.5, 2.3 Hz, 1H), 8.23 ​​(d, J = 8.6 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.28 (s, 1H), 7.07 (s, 1H), 6.37 (dd, J = 9.1, 5.6 Hz, 1H), 5.81 (d, J = 7.5 Hz, 1H), 5.56 (d, J = 5.4 Hz, 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).

[0405] Example 16 [ka]

[0406] The product was synthesized in the same manner as in steps 1-3 of Example 4, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)-4-nitrobenzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21004). The yield of the final step was 40%, and the MW = 695.

[0407] 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.42 (d, J = 1.9 Hz, 1H), 8.34 (dd, J = 8.5, 2.2 Hz, 1H), 8.29 (d, J = 8.6 Hz, 1H), 8.15 (s, 1H), 7.29 (s, 2H), 6.51 (dd, J = 9.0, 6.1 Hz, 1H), 5.77 (d, J = 5.3 Hz, 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).

[0408] Example 17 [ka]

[0409] The product was synthesized in the same manner as in steps 1-3 of Example 1, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20003). The yield of the final step was 45%, and the MW = 641.

[0410] 1 H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.99 (dd, J = 9.6, 8.7 Hz, 2H), 7.68 (td, J = 7.6, 1.1 Hz, 1H), 7.61 - 7.50 (m, 2H), 6.35 (dd, J = 9.3, 5.7 Hz, 1H), 5.60 (d, J = 5.3 Hz, 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, 5.7 Hz, 1H), 1.84 (s, 3H).

[0411] Example 18 [ka]

[0412] The product was synthesized in the same manner as in steps 1-3 of Example 2, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20003). The yield of the final step was 40%, and the MW = 666.

[0413] 1 H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.03 (d, J = 9.8 Hz, 2H), 7.69 (td, J = 7.6, 1.2 Hz, 1H), 7.60 - 7.51 (m, 2H), 6.67 (s, 2H), 6.23 (dd, J = 9.4, 5.8 Hz, 1H), 5.68 (d, J = 4.9 Hz, 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).

[0414] Example 19 [ka]

[0415] The product was synthesized in the same manner as in steps 1-4 of Example 3, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20003). The yield for the last two steps was 30%, and MW = 626.

[0416] 1 H NMR (400 MHz, DMSO-d6) δ 8.00 (dd, J = 7.8, 1.1 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.68 (td, J = 7.6, 1.4 Hz, 1H), 7.61 - 7.50 (m, 2H), 7.29 (s, 1H), 7.07 (s, 1H), 6.37 (dd, J = 9.2, 5.5 Hz, 1H), 5.82 (d, J = 7.5 Hz, 1H), 5.51 (d, J = 5.4 Hz, 1H), 4.81 (s, 2H), 4.32 (s, 1H), 4.13 - 3.97 (m, 2H), 2.41 (dd, J = 13.8, 5.6 Hz, 1H), 2.30 (ddd, J = 14.4, 7.5, 4.6 Hz, 1H).

[0417] Example 20 [ka]

[0418] The product was synthesized in the same manner as in steps 1-3 of Example 4, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20003). The yield of the final step was 40%, and the MW = 650.

[0419] 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.15 (s, 1H), 8.05 (d, J = 6.5 Hz, 1H), 7.69 (t, J = 8.2 Hz, 1H), 7.60 - 7.54 (m, 2H), 7.27 (s, 2H), 6.49 (dd, J = 8.8, 5.8 Hz, 1H), 5.71 (d, J = 5.6 Hz, 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).

[0420] Example 21 [ka]

[0421] The product was synthesized in the same manner as in steps 1-3 of Example 1, with 2-(1-azidoethyl)benzoic acid replaced by 2-(1-azidoethyl)-4-methoxybenzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21005). The yield of the final step was 45%, and the MW = 685.

[0422] 1 H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.00 - 7.90 (m, 2H), 7.11 (d, J = 2.6 Hz, 1H), 7.04 (dd, J = 8.8, 2.6 Hz, 1H), 6.39 - 6.29 (m, 1H), 5.74 - 5.68 (m, 1H), 5.55 (d, J = 5.1 Hz, 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, 1.4 Hz, 3H).

[0423] Example 22 [ka]

[0424] The product was synthesized in the same manner as in steps 1-3 of Example 3, with 2-(1-azidoethyl)benzoic acid replaced by 2-(1-azidoethyl)-4-methoxybenzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21005). The yield for the last two steps was 30%, and the MW = 670.

[0425] 1 H NMR (400 MHz, DMSO-d6) δ 8.00 - 7.89 (m, 2H), 7.32 (s, 1H), 7.11 (d, J = 2.5 Hz, 2H), 7.05 (dd, J = 8.8, 2.6 Hz, 1H), 6.41 - 6.31 (m, 1H), 5.82 (d, J = 7.5 Hz, 1H), 5.74 - 5.68 (m, 1H), 5.49 (d, J = 4.5 Hz, 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, 1.5 Hz, 3H).

[0426] Example 23 [ka]

[0427] The product was synthesized in the same manner as in steps 1-3 of Example 4, with 2-(1-azidoethyl)benzoic acid replaced by 2-(1-azidoethyl)-4-methoxybenzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21005). The yield of the final step was 40%, and the MW = 694.

[0428] 1 H NMR (400 MHz, DMSO-d6) δ 8.61 (d, J = 3.4 Hz, 1H), 8.16 (d, J = 1.4 Hz, 1H), 8.02 (dd, J = 8.7, 1.7 Hz, 1H), 7.30 (s, 2H), 7.12 (d, J = 2.5 Hz, 1H), 7.07 (dd, J = 8.8, 2.6 Hz, 1H), 6.51 - 6.46 (m, 1H), 5.78 - 5.72 (m, 1H), 5.68 (d, J = 5.2 Hz, 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, 2.0 Hz, 3H).

[0429] Example 24 (1) Steps 1-2 [ka]

[0430] The product was synthesized in the same manner as in Steps 1-2 of Example 1, with 2-(1-azidoethyl)benzoic acid replaced by 2-(2-azidopropan-2-yl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20004). The yield of the final step was 90%, and MW = 430.

[0431] 1H NMR (400 MHz, CDCl3) δ 9.12 (s, 1H), 8.31 (dd, J = 4.8, 1.7 Hz, 1H), 7.39 (dd, J = 7.7, 1.6 Hz, 1H), 6.96 (dd, J = 7.3, 5.2 Hz, 1H), 5.97 (t, J = 7.2 Hz, 1H), 5.33 (dd, J = 5.1, 2.7 Hz, 1H), 4.02 (d, J = 2.3 Hz, 1H), 3.70 (d, J = 2.4 Hz, 2H), 2.72 (s, 1H), 2.25 (dd, J = 10.7, 5.1 Hz, 2H), 1.60 (s, 3H), 1.45 (s, 6H).

[0432] (2) Step 3 [ka]

[0433] The product was synthesized in the same manner as in Step 3 of Example 1, by substituting 2-(1-azidoethyl)benzoic acid with 2-(2-azidopropan-2-yl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20004), with a yield of 35% and MW=670.

[0434] 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.67 (dd, J = 4.7, 1.4 Hz, 1H), 8.02 (dd, J = 7.7, 1.4 Hz, 1H), 7.96 (s, 1H), 7.47 (dd, J = 7.7, 4.8 Hz, 1H), 6.29 (dd, J = 9.2, 5.7 Hz, 1H), 5.61 (d, J = 5.3 Hz, 1H), 4.31 (s, 1H), 4.19 - 4.03 (m, 2H), 2.56 - 2.52 (m, 1H), 2.36 (dd, J = 13.9, 5.6 Hz, 1H), 1.84 (s, 3H), 1.70 (d, J = 3.8 Hz, 6H).

[0435] Example 25 (1) Steps 1-2 [ka]

[0436] The product was synthesized in the same manner as in Steps 1-2 of Example 1, with 2-(1-azidoethyl)benzoic acid replaced by 2-(2-(azidomethyl)phenyl)acetic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20005). The yield of the final step was 90%, and the MW = 415.

[0437] 1H NMR (500 MHz, CDCl3) δ 8.08 (s, 1H), 7.46 (d, J = 1.1 Hz, 1H), 7.39 - 7.32 (m, 3H), 7.30 (d, J = 6.8 Hz, 1H), 6.20 (dd, J = 8.5, 5.9 Hz, 1H), 5.37 - 5.35 (m, 1H), 4.40 (s, 2H), 4.08 (q, J = 2.4 Hz, 1H), 3.90 (qd, J = 11.8, 2.6 Hz, 2H), 3.76 (s, 2H), 2.48 - 2.33 (m, 2H), 1.92 (d, J = 1.0 Hz, 3H).

[0438] (2) Step 3 [ka]

[0439] The product was synthesized in the same manner as in Step 3 of Example 1, by substituting 2-(1-azidoethyl)benzoic acid with 2-(2-(azidomethyl)phenyl)acetic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20005). The yield of this step was 40%, and the MW = 655.

[0440] 1 H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.91 (s, 1H), 7.43 - 7.28 (m, 4H), 6.26 (dd, J = 9.4, 5.6 Hz, 1H), 5.34 (d, J = 5.4 Hz, 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, 5.5 Hz, 1H), 1.82 (s, 3H).

[0441] Example 26 [ka]

[0442] The product was synthesized in the same manner as in steps 1-3 of Example 1, with 2-(1-azidoethyl)benzoic acid replaced by 2-(2-(1-azidoethyl)phenyl)acetic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20006). The yield of the final step was 40%, and the MW = 669.

[0443] 1 H NMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 7.87 (s, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.35 (dt, J = 7.9, 4.2 Hz, 1H), 7.30 (d, J = 4.0 Hz, 2H), 6.26 (dd, J = 9.5, 5.6 Hz, 1H), 5.33 (d, J = 5.4 Hz, 1H), 5.05 (q, J = 6.7 Hz, 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, 1.3 Hz, 3H).

[0444] Example 27 (1) Steps 1-2 [ka]

[0445] The product was synthesized in the same manner as in Steps 1-2 of Example 1, with 2-(1-azidoethyl)benzoic acid replaced by 2-(2-azidoethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21006). The yield of the final step was 90%, and the MW = 415.

[0446] 1 H NMR (500 MHz, CDCl3) δ 8.40 (s, 1H), 7.98 (d, J = 7.8 Hz, 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 = 2.4 Hz, 1H), 4.02 (d, J = 2.5 Hz, 2H), 3.54 (t, J = 7.0 Hz, 2H), 3.27 (t, J = 7.1 Hz, 2H), 2.60 - 2.50 (m, 2H), 1.95 (s, 3H).

[0447] (2) Step 3 [ka]

[0448] The product was synthesized in the same manner as in Step 3 of Example 1, by substituting 2-(1-azidoethyl)benzoic acid with 2-(2-azidoethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21006). The yield of this step was 40%, and the MW = 655.

[0449] 1 H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 7.98 - 7.85 (m, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 6.34 (dd, J = 9.3, 5.7 Hz, 1H), 5.57 (d, J = 5.3 Hz, 1H), 4.30 (s, 1H), 4.08 - 4.02 (m, 2H), 3.57 (t, J = 6.9 Hz, 2H), 3.21 (t, J = 6.9 Hz, 2H), 2.55 - 2.52 (m, 1H), 2.43 - 2.27 (m, 1H), 1.84 (s, 3H).

[0450] Example 28 (1) Steps 1-2 [ka]

[0451] The product was synthesized in the same manner as in steps 1-2 of Example 2, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21007). The yield of the final step was 90%, and the MW = 427.

[0452] 1 H NMR (400 MHz, CDCl3) δ 8.52 (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).

[0453] (2) Step 3 [ka]

[0454] The product was synthesized in the same manner as in Step 3 of Example 2, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21007). The yield of this step was 30%, and the MW = 667.

[0455] 1H NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.83 (dd, J = 4.8, 1.5 Hz, 1H), 8.40 (dd, J = 7.9, 1.7 Hz, 1H), 8.00 (d, J = 6.6 Hz, 1H), 7.59 (dd, J = 7.9, 4.8 Hz, 1H), 6.73 (s, 2H), 6.23 (dd, J = 9.3, 5.8 Hz, 1H), 5.72 (d, J = 4.8 Hz, 1H), 4.85 (s, 2H), 4.36 (d, J = 4.9 Hz, 1H), 4.27 - 4.16 (m, 1H), 4.01 - 3.93 (m, 1H), 3.26 - 3.18 (d, J = 14.3 Hz, 1H), 2.56 (dd, J = 13.7, 5.3 Hz, 1H).

[0456] Example 29 [ka]

[0457] The product was synthesized in the same manner as in steps 1-3 of Example 1, with 2-(azidoethyl)benzoic acid replaced by 2-(azidomethyl)nicotinic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21007). The yield of the final step was 40%, and the MW = 642.

[0458] 1H NMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 8.81 (dd, J = 4.8, 1.7 Hz, 1H), 8.39 (dd, J = 7.9, 1.7 Hz, 1H), 7.89 (d, J = 4.2 Hz, 1H), 7.58 (dd, J = 7.9, 4.8 Hz, 1H), 6.35 (dd, J = 9.0, 6.0 Hz, 1H), 5.59 (d, J = 4.8 Hz, 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 = 2.7 Hz, 3H).

[0459] Example 30 [ka]

[0460] The product was synthesized in the same manner as in steps 1-3 of Example 5, with 2-(1-(ethyldisulfaneol)ethyl)benzoic acid replaced with 2-(1-(methyldisulfaneol)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20007). The yield of the final step was 30%, and the MW = 692.

[0461] 1 H NMR (400 MHz, D2O) δ 7.90 - 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, 3.0 Hz, 1H), 4.41 - 4.24 (m, 2H), 2.65 - 2.52 (m, 2H), 1.98 (d, J = 15.2 Hz, 3H), 1.94 (s, 3H), 1.68 (d, J = 7.2 Hz, 3H).

[0462] Example 31 [ka]

[0463] The product was synthesized in the same manner as in steps 1-3 of Example 6, with 2-(1-(ethyldisulfaneol)ethyl)benzoic acid replaced with 2-(1-(methyldisulfaneol)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20007). The yield of the final step was 30%, and the MW = 717.

[0464] 1 H NMR (400 MHz, D2O) δ 8.14 (t, J = 3.1 Hz, 1H), 7.82 (dd, J = 8.1, 3.1 Hz, 1H), 7.55 (q, J = 3.2 Hz, 2H), 7.38 - 7.34 (m, 1H), 6.31 - 6.22 (m, 1H), 5.76 (d, J = 4.1 Hz, 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, 3.2 Hz, 3H), 1.62 (dd, J = 7.2, 3.3 Hz, 3H).

[0465] Example 32 [ka]

[0466] The product was synthesized in the same manner as in steps 1-4 of Example 7, with 2-(1-(ethyldisulfaneol)ethyl)benzoic acid replaced with 2-(1-(methyldisulfaneol)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20007). The yield for the last two steps was 28%, and the MW = 677.

[0467] 1 H NMR (400 MHz, D2O) δ 8.08 - 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, 2.3 Hz, 1H), 5.73 - 5.64 (m, 1H), 5.13 - 5.02 (m, 1H), 4.60 (d, J = 12.6 Hz, 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, 2.2 Hz, 3H), 1.69 (dd, J = 7.2, 2.1 Hz, 3H).

[0468] Example 33 [ka]

[0469] The product was synthesized in the same manner as in steps 1-3 of Example 8, with 2-(1-(ethyldisulfaneol)ethyl)benzoic acid replaced with 2-(1-(methyldisulfaneol)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20007). The yield of the final step was 35%, and the MW = 701.

[0470] 1H NMR (400 MHz, D2O) δ 8.53 (dd, J = 5.2, 1.8 Hz, 1H), 8.18 - 8.10 (m, 1H), 7.83 (d, J = 7.8 Hz, 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, 5.6 Hz, 1H), 1.93 (dd, J = 25.1, 1.9 Hz, 3H), 1.60 (dd, J = 6.9, 4.0 Hz, 3H).

[0471] Example 34 (1) Step 1~ [ka]

[0472] The product was synthesized in the same manner as in Example 5 Step 1, by substituting 2-(1-(ethyldisulfaneyl)ethyl)benzoic acid with 2-(1-(isobutyldisulfaneyl)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21008), with a yield of 79% and MW=608.

[0473] 1H NMR (400 MHz, CDCl3) δ 8.66 - 8.39 (m, 1H), 7.89 - 7.80 (m, 1H), 7.63 - 7.47 (m, 3H), 7.33 (td, J = 8.8, 4.5 Hz, 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 = 6.8 Hz, 2H), 0.98 - 0.93 (m, 9H), 0.87 - 0.81 (m, 4H), 0.19 - 0.13 (m, 6H).

[0474] (2) Steps 2-3 [ka]

[0475] The product was synthesized in the same manner as in steps 2-3 of Example 5, with 2-(1-(ethyldisulfaneol)ethyl)benzoic acid replaced with 2-(1-(isobutyldisulfaneol)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21008). The yield of the final step was 32%, and the MW = 734.

[0476] 1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 7.96 (dd, J = 8.2, 0.9 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 3.9 Hz, 2H), 7.46 - 7.41 (m, 1H), 6.33 (td, J = 9.7, 5.6 Hz, 1H), 5.63 - 5.55 (m, 1H), 5.18 - 5.08 (m, 1H), 4.30 (d, J = 23.4 Hz, 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).

[0477] Example 35 [ka]

[0478] The product was synthesized in the same manner as in steps 1-3 of Example 6, with 2-(1-(ethyldisulfaneol)ethyl)benzoic acid replaced with 2-(1-(isobutyldisulfaneol)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21008). The yield of the final step was 40%, and the MW = 759.

[0479] 1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 8.03 (d, J = 6.9 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 2.8 Hz, 2H), 7.49 - 7.38 (m, 1H), 6.66 (s, 2H), 6.27 - 6.15 (m, 1H), 5.69 (dd, J = 8.2, 4.0 Hz, 1H), 5.16 (q, J = 6.9 Hz, 1H), 4.39 - 4.26 (m, 1H), 4.19 (dd, J = 10.6, 5.1 Hz, 1H), 4.01 (dd, J = 11.4, 5.9 Hz, 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, 6.6 Hz, 1H), 1.65 (d, J = 6.8 Hz, 3H), 0.85 - 0.74 (m, 6H).

[0480] Example 36 [ka]

[0481] The product was synthesized in the same manner as in steps 1-4 of Example 7, with the yield of the last two steps being 30% and MW = 719.

[0482] 1H NMR (400 MHz, DMSO-d6) δ 7.98 (dd, J = 9.4, 7.6 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 4.1 Hz, 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, 1.0 Hz, 1H), 5.53 (d, J = 4.4 Hz, 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 = 2.5 Hz, 3H), 0.78 (d, J = 2.6 Hz, 3H).

[0483] Example 37 [ka]

[0484] The product was synthesized in the same manner as in steps 1-3 of Example 8, with 2-(1-(ethyldisulfanale)ethyl)benzoic acid replaced with 2-(1-(isobutyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21008), and the final product yielded 40% and MW = 743.

[0485] 1H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J = 8.3 Hz, 1H), 8.14 (d, J = 5.9 Hz, 1H), 7.94 - 7.87 (m, 1H), 7.64 (d, J = 3.9 Hz, 2H), 7.46 (dt, J = 8.1, 4.2 Hz, 1H), 7.32 (s, 2H), 6.51 - 6.44 (m, 1H), 5.75 (s, 1H), 5.18 (q, J = 7.0 Hz, 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 = 6.7 Hz, 3H), 0.78 (d, J = 6.7 Hz, 3H).

[0486] Example 38 (1) Step 1 [ka]

[0487] The product was synthesized in the same manner as in Step 1 of Example 5, with 2-(1-(ethyldisulfanayl)ethyl)benzoic acid replaced with 2-(1-(isopropyldisulfanayl)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21009). The yield of this step was 80%, and the MW = 594.

[0488] 1H NMR (500 MHz, CDCl3) δ 8.51 (s, 1H), 7.85 (ddd, J = 12.8, 7.8, 1.2 Hz, 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, 5.3 Hz, 1H), 5.50 (t, J = 5.3 Hz, 1H), 5.28 - 5.15 (m, 1H), 4.32 (dd, J = 36.3, 1.3 Hz, 1H), 4.07 - 3.96 (m, 2H), 2.59 (td, J = 13.5, 5.3 Hz, 1H), 2.47 - 2.16 (m, 2H), 1.95 (s, 3H), 1.70 (dd, J = 7.0, 1.0 Hz, 3H), 1.17 - 1.06 (m, 6H), 0.97 (s, 9H), 0.18 (s, 6H).

[0489] (2) Step 2 [ka]

[0490] The product was synthesized in the same manner as in Step 2 of Example 5, by substituting 2-(1-(ethyldisulfanale)ethyl)benzoic acid with 2-(1-(isopropyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21009), with a yield of 90% and MW=594.

[0491] 1H NMR (500 MHz, CDCl3) δ 8.84 (s, 1H), 7.84 (ddd, J = 7.4, 5.8, 1.3 Hz, 1H), 7.65 - 7.49 (m, 3H), 7.33 (td, J = 7.7, 1.2 Hz, 1H), 6.37 - 6.27 (m, 1H), 5.61 (tt, J = 4.7, 2.5 Hz, 1H), 5.22 (dq, J = 13.9, 7.0 Hz, 1H), 4.30 (dq, J = 19.8, 2.4 Hz, 1H), 4.03 (dd, J = 5.7, 2.7 Hz, 2H), 2.60 - 2.31 (m, 3H), 1.95 (d, J = 1.1 Hz, 3H), 1.70 (d, J = 7.0 Hz, 3H), 1.15 - 1.07 (m, 6H).

[0492] (3) Step 3 [ka]

[0493] The product was synthesized in the same manner as in Step 3 of Example 5, by substituting 2-(1-(ethyldisulfanale)ethyl)benzoic acid with 2-(1-(isopropyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21009). The yield of this step was 40%, and the MW = 720.

[0494] 1H NMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 7.98 (dd, J = 6.6, 0.9 Hz, 1H), 7.85 (dd, J = 7.7, 2.4 Hz, 1H), 7.61 (t, J = 6.4 Hz, 2H), 7.45 - 7.40 (m, 1H), 6.36 - 6.31 (m, 1H), 5.60 (d, J = 5.3 Hz, 1H), 5.11 (q, J = 6.9 Hz, 1H), 4.30 (d, J = 20.0 Hz, 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 = 7.0 Hz, 3H), 1.06 (d, J = 5.7 Hz, 6H).

[0495] Example 39 [ka]

[0496] The product was synthesized in the same manner as in steps 1-3 of Example 6, with 2-(1-(ethyldisulfanale)ethyl)benzoic acid replaced with 2-(1-(isopropyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21009). The yield of the final step was 40%, and the MW = 745.

[0497] 1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 7.98 (d, J = 7.9 Hz, 1H), 7.88 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 3.3 Hz, 2H), 7.47 - 7.40 (m, 1H), 6.63 (d, J = 25.9 Hz, 2H), 6.27 - 6.16 (m, 1H), 5.68 (t, J = 5.3 Hz, 1H), 5.32 (t, J = 4.7 Hz, 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, 1.7 Hz, 1H), 1.65 (d, J = 6.9 Hz, 3H), 1.13 (d, J = 7.9 Hz, 6H).

[0498] Example 40 [ka]

[0499] The product was synthesized in the same manner as in steps 1-4 of Example 7, with 2-(1-(ethyldisulfanale)ethyl)benzoic acid replaced with 2-(1-(isopropyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21009). The yield for the last two steps was 30%, and the MW = 705.

[0500] 1H NMR (400 MHz, DMSO-d6) δ 8.00 - 7.93 (m, 1H), 7.84 (dd, J = 7.6, 3.3 Hz, 1H), 7.62 (d, J = 3.7 Hz, 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 = 7.3 Hz, 1H), 5.52 (d, J = 5.2 Hz, 1H), 5.11 (q, J = 7.0 Hz, 1H), 4.30 (d, J = 19.7 Hz, 1H), 4.14 - 3.97 (m, 2H), 2.47 - 2.38 (m, 2H), 2.34 - 2.31 (m, 1H), 1.64 (d, J = 7.0 Hz, 3H), 1.10 (d, J = 2.1 Hz, 6H).

[0501] Example 41 (1) Step 1 [ka]

[0502] The product was synthesized in the same manner as in Example 5 Step 1, by substituting 2-(1-(ethyldisulfanale)ethyl)benzoic acid with 2-(1-(tert-butyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21010), with a yield of 80% and MW=608.

[0503] 1H NMR (400 MHz, CDCl3) δ 8.73 (d, J = 5.8 Hz, 1H), 7.84 (ddd, J = 7.8, 3.5, 1.5 Hz, 1H), 7.70 - 7.45 (m, 3H), 7.32 (td, J = 7.6, 1.4 Hz, 1H), 6.46 (td, J = 9.5, 5.3 Hz, 1H), 5.57 - 5.35 (m, 1H), 5.16 (p, J = 7.1 Hz, 1H), 4.28 (dd, J = 17.9, 1.7 Hz, 1H), 4.13 - 3.90 (m, 2H), 2.58 (ddd, J = 14.7, 9.8, 5.3 Hz, 1H), 2.23 (dtd, J = 16.2, 6.2, 2.7 Hz, 1H), 1.98 - 1.92 (m, 3H), 1.69 (d, J = 6.7 Hz, 3H), 1.22 (d, J = 11.2 Hz, 8H), 0.96 (s, 9H), 0.17 (s, 6H).

[0504] (2) Step 2 [ka]

[0505] The product was synthesized in the same manner as in Step 2 of Example 5, by substituting 2-(1-(ethyldisulfanayl)ethyl)benzoic acid with 2-(1-(tert-butyldisulfanayl)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21010). The yield of this step was 90%, and the MW = 494.

[0506] 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H), 7.83 (dt, J = 7.9, 1.6 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.55 (dtd, J = 7.8, 4.0, 1.4 Hz, 2H), 7.32 (td, J = 7.5, 1.4 Hz, 1H), 6.31 (ddd, J = 8.4, 6.0, 2.8 Hz, 1H), 5.59 (dt, J = 5.4, 2.5 Hz, 1H), 5.14 (dq, J = 18.4, 7.0 Hz, 1H), 4.27 (dt, J = 8.4, 2.5 Hz, 1H), 4.01 (d, J = 2.6 Hz, 2H), 2.65 - 2.48 (m, 2H), 2.35 (s, 2H), 1.94 (d, J = 1.2 Hz, 3H), 1.68 (dd, J = 6.9, 1.4 Hz, 3H), 1.22 (d, J = 2.9 Hz, 9H).

[0507] (3) Step 3 [ka]

[0508] The product was synthesized in the same manner as in Step 3 of Example 5, by substituting 2-(1-(ethyldisulfanayl)ethyl)benzoic acid with 2-(1-(tert-butyldisulfanayl)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21010). The yield of this step was 40%, and the MW = 734.

[0509] 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 4.6 Hz, 2H), 7.44 - 7.39 (m, 1H), 6.37 - 6.29 (m, 1H), 5.56 (t, J = 5.0 Hz, 1H), 5.07 - 4.98 (m, 1H), 4.29 (d, J = 28.0 Hz, 1H), 4.14 - 3.99 (m, 2H), 2.39 (dd, J = 13.9, 5.9 Hz, 1H), 2.31 (dd, J = 14.2, 5.7 Hz, 1H), 1.83 (s, 3H), 1.63 (dd, J = 7.0, 1.1 Hz, 3H), 1.19 (d, J = 2.5 Hz, 9H).

[0510] Example 42 (1) Step 1 [ka]

[0511] The product was synthesized in the same manner as in Step 1 of Example 5, with 2-(1-(ethyldisulfanale)ethyl)benzoic acid replaced with 4-acetamido-2-(1-(methyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21002). The yield of this step was 80%, and the MW = 623.

[0512] 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.68 (s, 1H), 7.64 - 7.58 (m, 2H), 7.46 (s, 1H), 6.42 (dd, J = 9.3, 5.1 Hz, 1H), 5.46 (d, J = 5.9 Hz, 1H), 5.42 - 5.36 (m, 1H), 4.29 (d, J = 1.8 Hz, 1H), 4.04 - 3.95 (m, 2H), 2.57 (dd, J = 13.8, 5.3 Hz, 1H), 2.29 - 2.23 (m, 1H), 2.22 (s, 3H), 2.06 (s, 3H), 1.94 (d, J = 1.2 Hz, 3H), 1.68 (d, J = 7.0 Hz, 3H), 0.95 (s, 9H), 0.17 (s, 6H).

[0513] (2) Step 2 [ka]

[0514] The product was synthesized in the same manner as in Step 2 of Example 5, by substituting 2-(1-(ethyldisulfanale)ethyl)benzoic acid with 4-acetamido-2-(1-(methyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21002), with a yield of 75% and MW=509.

[0515] 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.89 (dd, J = 8.5, 5.6 Hz, 1H), 7.66 (d, J = 9.5 Hz, 2H), 7.54 (d, J = 7.2 Hz, 1H), 7.45 (s, 1H), 6.29 (t, J = 7.1 Hz, 1H), 5.59 - 5.56 (m, 1H), 5.41 - 5.35 (m, 1H), 4.29 - 4.25 (m, 1H), 4.01 (d, J = 2.2 Hz, 2H), 3.01 - 2.93 (m, 1H), 2.58 - 2.55 (m, 1H), 2.23 (s, 3H), 2.09 (d, J = 5.6 Hz, 3H), 1.95 (d, J = 1.2 Hz, 3H), 1.68 (s, 3H).

[0516] (3) Step 3 [ka]

[0517] The product was synthesized in the same manner as in Step 3 of Example 5, by substituting 2-(1-(ethyldisulfanale)ethyl)benzoic acid with 4-acetamido-2-(1-(methyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21002), with a yield of 35% and MW=749.

[0518] 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 10.52 (d, J = 3.1 Hz, 1H), 7.97 (d, J = 4.1 Hz, 1H), 7.92 - 7.82 (m, 2H), 7.69 (d, J = 8.8 Hz, 1H), 6.37 - 6.26 (m, 1H), 5.55 (d, J = 5.3 Hz, 1H), 5.34 - 5.25 (m, 1H), 4.27 (d, J = 16.7 Hz, 1H), 4.12 - 4.05 (m, 2H), 2.49 - 2.46 (m, 1H), 2.35 (td, J = 13.9, 5.7 Hz, 1H), 2.11 (d, J = 12.7 Hz, 3H), 2.08 (s, 3H), 1.84 (s, 3H), 1.61 (d, J = 6.9 Hz, 3H).

[0519] Example 43 (1) Step 1 [ka]

[0520] The product was synthesized in the same manner as in Step 1 of Example 7, by substituting 2-(1-(ethyldisulfanale)ethyl)benzoic acid with 4-methoxy-2-(1-(methyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21001). The yield of this step was 80%, and the MW = 623.

[0521] 1H NMR (500 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.24 (d, J = 7.5 Hz, 1H), 7.92 (dd, J = 8.7, 4.0 Hz, 1H), 7.24 (d, J = 7.5 Hz, 1H), 7.10 (d, J = 2.6 Hz, 1H), 7.00 (ddd, J = 8.8, 2.6, 1.4 Hz, 1H), 6.19 (q, J = 6.3 Hz, 1H), 5.57 (d, J = 8.0 Hz, 3H), 5.46 - 5.37 (m, 1H), 5.37 - 5.27 (m, 1H), 4.37 (dd, J = 21.7, 2.9 Hz, 1H), 3.95 (dd, J = 11.3, 3.2 Hz, 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).

[0522] (2) Step 2 [ka]

[0523] The product was synthesized in the same manner as in Step 2 of Example 7, by substituting 2-(1-(ethyldisulfanale)ethyl)benzoic acid with 4-methoxy-2-(1-(methyldisulfanale)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21001), with a yield of 85% and MW=509.

[0524] 1H NMR (500 MHz, DMSO-d6) δ 10.91 (s, 1H), 8.35 (d, J = 7.5 Hz, 1H), 7.92 (dd, J = 8.8, 4.1 Hz, 1H), 7.24 (d, J = 7.5 Hz, 1H), 7.10 (d, J = 2.7 Hz, 1H), 7.00 (ddd, J = 8.8, 2.7, 1.3 Hz, 1H), 6.22 (dt, J = 7.9, 6.0 Hz, 1H), 5.43 (dt, J = 6.6, 2.1 Hz, 1H), 5.39 - 5.30 (m, 1H), 5.26 (t, J = 5.3 Hz, 1H), 4.29 (dd, J = 18.4, 2.7 Hz, 1H), 3.85 (s, 3H), 3.72 (dd, J = 5.5, 3.2 Hz, 2H), 2.68 - 2.55 (m, 1H), 2.34 (dt, J = 14.1, 7.0 Hz, 1H), 2.16 - 2.08 (m, 6H), 1.65 (d, J = 7.0 Hz, 3H).

[0525] (3) Steps 3-4 [ka]

[0526] The product was synthesized in the same manner as in steps 3-4 of Example 7, with 2-(1-(ethyldisulfaneol)ethyl)benzoic acid replaced with 4-methoxy-2-(1-(methyldisulfaneol)ethyl)benzoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21001). The total yield of these two steps was 30%, and the MW = 707.

[0527] 1H NMR (400 MHz, DMSO) δ 7.96 - 7.90 (m, 2H), 7.29 (s, 1H), 7.09 (d, J = 2.3 Hz, 2H), 7.00 (dd, J = 8.8, 2.5 Hz, 1H), 6.34 (dd, J = 15.3, 6.6 Hz, 1H), 5.81 (d, J = 7.5 Hz, 1H), 5.47 (d, J = 4.3 Hz, 1H), 5.33 (q, J = 7.0 Hz, 1H), 4.28 (d, J = 17.7 Hz, 1H), 4.13 - 3.97 (m, 2H), 3.85 (s, 3H), 2.43 - 2.22 (m, 2H), 2.13 (d, J = 10.6 Hz, 3H), 1.64 (d, J = 7.0 Hz, 3H).

[0528] Example 44 [ka]

[0529] The product was synthesized in the same manner as in steps 1-3 of Example 5, by substituting 2-(1-(ethyldisulfaneyl)ethyl)benzoic acid with 3-(azidomethyl)furan-2-carboxylic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-21011), with a yield of 45% and MW=631 in the final step.

[0530] 1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 8.01 (d, J = 1.6 Hz, 1H), 7.91 (d, J = 22.7 Hz, 1H), 6.83 (d, J = 1.6 Hz, 1H), 6.33 (dd, J = 9.4, 5.7 Hz, 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).

[0531] Example 45 [ka]

[0532] The product was synthesized in the same manner as in steps 1-3 of Example 5, with 2-(1-(ethyldisulfaneol)ethyl)benzoic acid replaced with 4-azidobutanoic acid (available from Beijing Okeanos Tech. Co., Ltd., catalog number: OK-H-20008). The yield of the final step was 20%, and the MW = 593.

[0533] 1 H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 7.90 (d, J = 0.9 Hz, 1H), 6.24 (dd, J = 9.5, 5.6 Hz, 1H), 5.33 (d, J = 5.5 Hz, 1H), 4.09 (s, 1H), 4.04 - 3.95 (m, 2H), 3.38 (t, J = 6.8 Hz, 2H), 2.44 (t, J = 7.3 Hz, 2H), 2.41 - 2.33 (m, 1H), 2.18 (dd, J = 13.8, 5.6 Hz, 1H), 1.82 (s, 3H), 1.81 - 1.75 (m, 2H).

[0534] II. Examples of Tests The inventors have found that the nucleotide analogs of this application provide both excellent blocking and excellent polymerization effects. Sequencing performed using these nucleotide analogs yielded excellent results with both high mapping rates and low error rates.

[0535] Specifically, the inventors evaluated and tested the nucleotide analogs prepared in the above preparation example using a high-throughput sequencer.

[0536] 1. Evaluation of blocking effect Nucleotide substrates: Fluorescently labeled standard hot dNTPs (4 types) and standard cold dNTPs (4 types) having the structures shown below, all available from the MGISEQ-2000RS high-throughput sequencing kit (FCL SE50, MGI Tech Co., Ltd., catalog number 1000012551). For each test, only one of the nucleotide analog cold dNTPs of the present invention (dTTP, dATP, dCTP, and dGTP) was used. For ease of presentation, only the test results for dTTP are provided in Table 1 below for each type of 3'-OH modification.

[0537] Sequencing was performed using the above-mentioned nucleotide substrates and the MGISEQ-2000RS high-throughput sequencing kit (FCL SE50) in accordance with the MGISEQ2000 sequencer operating protocol.

[0538] [ka]

[0539] (1) DNA nanospheres are prepared using an Escherichia coli (Ecoli) sequencing library.

[0540] (2) Load the DNA nanospheres onto the MGISEQ2000 sequencing chip.

[0541] (3) Insert the loaded sequencing chip into the MGISEQ2000 sequencer and set the sequencing process.

[0542] (4) Perform test round 1: Insert a standard hot dNTP, take a photograph to record the signal value, then cut the blocking group with thpp reagent, 65°C for 1 minute.

[0543] (5) Perform test round 2: Incorporate a standard cold dNTP, then a standard hot dNTP, take a photograph to record the signal value, then cut the blocking group with thpp reagent, 65°C for 1 minute.

[0544] (6) Perform test round 3: Insert standard hot dNTPs, take a photograph to record the signal value, then cut the blocking group with thpp reagent, 65°C for 1 minute.

[0545] (7) Conduct test round 4: Incorporate the cold dNTP nucleotide analog of the present invention (only one type of cold dNTP is incorporated into each test), then incorporate the standard hot dNTP, take a photograph to record the signal value, then cleave the blocking group with thpp reagent at 65°C for 1 minute.

[0546] (8) Conduct test round 5: Install a standard hot dNTP and take a picture to record the signal value.

[0547] (9) Evaluate the integration efficiency and cutting efficiency. The results are shown in Table 1.

[0548] Formula for calculating embedded efficiency:

number

[0549] During the ceremony, EI (Integration Efficiency) is the ratio of the integration efficiency values ​​for the test nucleotide and the comparison nucleotide. C1 is the signal value for test round 1. C2 is the signal value for test round 2. C3 is the signal value for test round 3. C4 is the signal value for test round 4.

[0550] Formula for calculating cutting efficiency:

number

[0551] During the ceremony, Ec (cleavage efficiency) is the ratio of cleavage efficiency values ​​for the test nucleotide and the comparison nucleotide. EI is the ratio of the integration efficiency values ​​for the test nucleotide and the comparison nucleotide. C3 is the signal value for test round 3. C5 is the signal value for test round 5. CGT is the signal for base C, base G, and base T in test round 3.

[0552] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0553] 2. Evaluation of sequencing effects Sequence example 1 Nucleotide substrates: Fluorescently labeled standard hot dNTPs (four types, all available from the MGISEQ-2000RS high-throughput sequencing kit (FCL SE50), MGI Tech Co., Ltd., catalog number 1000012551), and nucleotide analog cold dNTPs of the present invention having the structures shown below (four types, named AEB).

[0554] [ka]

[0555] Sequencing was performed using the above-mentioned nucleotide substrates and the MGISEQ-2000RS high-throughput sequencing kit (FCL SE50) in accordance with the MGISEQ2000 sequencer operating protocol.

[0556] (1) DNA nanospheres are prepared using an E. coli sequencing library.

[0557] (2) Load DNA nanospheres onto the MGISEQ2000 sequencing chip.

[0558] (3) The loaded sequencing chip is mounted in the MGISEQ2000 sequencer, the sequencing process is set, hot dNTP integration: 60°C for 2 minutes, cold dNTP integration: 60°C for 2 minutes, signal acquisition, blocking substrate disconnection: 65°C for 2 minutes.

[0559] (4) Perform a base call analysis on the offline data and output the results of sequencing metrics including mapping rate, error rate, and Q30. The results are shown in Table 2.

[0560] [Table 7]

[0561] Sequence example 2 Nucleotide substrates: Fluorescently labeled standard hot dNTPs (four types, all available from the MGISEQ-2000RS high-throughput sequencing kit (FCL SE50), MGI Tech Co., Ltd., catalog number 1000012551), and cold dNTPs of the present invention having the structures shown below (four types, named SSEB).

[0562] [ka]

[0563] The sequencing procedure was the same as that described in Sequencing Example 1. The results of the base call analysis are shown in Table 3.

[0564] [Table 8]

[0565] Sequence example 3 Nucleotide substrates: Fluorescently labeled standard hot dNTPs (four types, all available from the MGISEQ-2000RS high-throughput sequencing kit (FCL SE50), MGI Tech Co., Ltd., catalog number 1000012551), and cold dNTPs of the present invention having the structures shown below (four types, named AZBN).

[0566] [ka]

[0567] The sequencing procedure was the same as that described in Sequencing Example 1. The results of the base call analysis are shown in Table 4.

[0568] [Table 9]

[0569] Sequencing Example 4: Sequencing using a hot T-SSEB 1. Synthesis of nucleotide analog dTTP (named Hot T-SSEB) carrying a fluorescent group.

[0570] (1) Step 1 [ka]

[0571] Iodized nucleoside substrate T (Okeanos Tech, catalog number OK-N-16001, 1 g) was dissolved in DMF, followed by the addition of Pd(PPh3)4 (10 mol%), CuI (15 mol%), triethylamine (3 eq), and substrate propargylamine (Okeanos Tech, catalog number OK20A410, 1.5 eq). The mixture was reacted at 60°C for 12 hours. The reaction was quenched with water, extracted by DCM, concentrated, and purified by column chromatography to obtain 1.1 g of the product as a white solid.

[0572] MS[ES(-)], m / z 491.1. 1 H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 10.00 (t, J = 5.5 Hz, 1H), 7.94 (s, 1H), 6.12 (dd, J = 7.6, 5.9 Hz, 1H), 5.28 (d, J = 4.1 Hz, 1H), 4.26 - 4.12 (m, 3H), 3.88 (q, J = 2.8 Hz, 1H), 3.81 (dd, J = 11.5, 2.6 Hz, 1H), 3.73 (dd, J = 11.5, 3.1 Hz, 1H), 2.17 (ddd, J = 13.2, 6.0, 2.8Hz, 1H), 2.05 (ddd, J = 13.3, 7.7, 5.8 Hz, 1H), 0.87 (s, 9H), 0.08 (d, J = 1.8 Hz, 6H).

[0573] (2) Step 2 [ka]

[0574] The nucleoside (300 mg) from Step 1 was dissolved in 10 mL of DMF, followed by the addition of DCC (1.2 eq) and DMAP (10% mol). The mixture was stirred for 30 minutes, and then the disulfaneyl carboxylic acid substrate (OKeanos Tech, catalog no. OK20A420) (1.5 eq) was added. The mixture was stirred for 12 hours and subjected to direct column chromatography to obtain 359 mg of the product as a white solid.

[0575] MS[ES(-)], m / z 700.3. 1 H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 10.03 (t, J = 5.5 Hz, 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 = 0.6 Hz, 3H), 1.65 (dd, J = 7.0, 1.0 Hz, 3H), 0.90 (s, 9H), 0.13 (d, J = 1.2 Hz, 6H).

[0576] (3) Step 3 [ka]

[0577] The nucleoside (300 mg) from Step 2 was dissolved in 10 mL of THF, followed by the addition of TBAF (2 eq, 1 M in THF) at 0°C. The mixture was stirred at 0°C for 30 minutes and then warmed with stirring for 4 hours to return to room temperature. The mixture was subjected directly to column chromatography to obtain 200 mg of the product as a white solid.

[0578] MS[ES(-)], m / z 587.2. 1 H NMR (400 MHz, DMSO-d6) δ 11.68 (d, J = 3.2 Hz, 1H), 10.06 (t, J = 5.6 Hz, 1H), 8.23 ​​(d, J = 3.2 Hz, 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 = 5.2 Hz, 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 = 9.2 Hz, 3H), 1.68 - 1.59 (m, 3H).

[0579] (4) Step 4 [ka]

[0580] The nucleoside (200 mg) from Step 3 was dissolved in 5 mL of trimethyl phosphate. Phosphorus oxychloride (1.5 eq) was added to the mixture at 0°C while stirring for 120 minutes. The reaction mixture was added to a DMF solution (5 mL) of tributylammonium pyrophosphate (2 eq) while continuously stirring at 0°C for 3 hours. The reaction was quenched with 0.1 M TEAB buffer and separated by preparative HPLC reversed-phase column chromatography (C18, mobile phase: 0.1 M TEAB-acetonitrile). After concentration, the residue was added to 3 mL of concentrated aqueous ammonia and reacted for 2 hours, followed by preparative HPLC reversed-phase column chromatography (C18, mobile phase: 0.1 M TEAB-acetonitrile) to obtain 120 mg of the product as a white solid.

[0581] MS[ES(-)], m / z 745.5. 1 H NMR (400 MHz, D2O) δ 8.51 (s, 1H), 7.90 (dd, J = 7.9, 1.4 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.67 (td, J = 7.6, 1.4 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 6.45 (td, J = 9.2, 5.7 Hz, 1H), 5.75 (t, J = 4.7 Hz, 1H), 5.15 - 5.07 (m, 1H), 4.72 - 4.64 (m, 1H), 4.39 (d, J = 3.3 Hz, 2H), 4.07 (s, 2H), 2.82 - 31P NMR (162 MHz, D2O) δ -9.79 (dd, J = 20.3, 9.6 Hz, 1P), -11.68 (d, J = 19.2 Hz, 1P), -22.82 (td, J = 19.2, 6.1 Hz, 1P).

[0582] (5) Step 5 [ka]

[0583] In a dry 20 mL round-bottom flask equipped with a magnetic stirrer, dye-linker solid (Okeanos Tech, OK-F-20211) (9.3 mg, 1 eq.) was added and dissolved with an appropriate amount of DMF (3 ml). Subsequently, TNTU solid (7 mg, 0.02 mmol, 2 eq.) and DIPEA (2.6 mg, 0.02 mmol, 2 eq.) were added sequentially. The DIPEA was diluted in 0.5 ml of DMF in a 1.5 ml PE tube and added dropwise to the mixture while stirring for approximately 1 hour. The mixture was sampled, dissolved in acetonitrile, and monitored for AF532-V4 feedstock depletion by HPLC and MS. Solid hot T-mSSEB substrate (15 mg, 0.02 mmol, 1 eq.) was added to the mixture at room temperature while stirring for 1 hour. The mixture was sampled, dissolved in acetonitrile, and the reaction progress was monitored by HPLC and MS. The reaction was allowed to proceed overnight until the complete consumption of the NHS ester was achieved, followed by quenching with 0.1 M TEAB buffer and separation by preparative HPLC reverse-phase column chromatography (C18, mobile phase: 0.1 M TEAB-acetonitrile). The desired fraction was concentrated and added to 3 mL of concentrated aqueous ammonia, reacted for 2 hours, and subsequently purified by preparative HPLC reverse-phase column chromatography (C18, mobile phase: 0.1 M TEAB-acetonitrile) to obtain 10 mg of solid.

[0584] MS[ES(-)], m / z 1815.0. 1H NMR (400 MHz, DMSO-d6) δ 9.06 (d, J = 6.7 Hz, 1H), 8.80 (d, J = 47.0 Hz, 1H), 8.28 (t, J = 7.2 Hz, 1H), 8.18 - 8.00 (m, 1H), 7.86 (d, J = 7.7 Hz, 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 = 3.5 Hz, 1H), 5.21 - 5.14 (m, 2H), 4.34 (d, J = 19.3 Hz, 1H), 4.26 (dd, J = 10.5, 3.9 Hz, 1H), 4.18 (dd, J = 10.6, 4.5 Hz, 2H), 4.09 (d, J = 3.5 Hz, 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 = 7.6 Hz, 4H), 2.57 (t, J = 7.1 Hz, 5H), 2.46 - 2.37 (m, 1H), 2.05 (d, J = 16.8 Hz, 3H), 2.01 - 1.93 (m, 4H), 1.86 - 1.81 (m, 3H), 1.77 - 1.72 (m, 1H), 1.65 (d, J = 6.9 Hz, 3H).

[0585] 2. Sequencing performed using nucleotide analogs containing the hot T-SSEB synthesized above. Nucleotide substrates: fluorescently labeled standard hot dNTPs (MGISEQ-2000RS high-throughput sequencing kit (FCL SE50), available from MGI Tech Co., Ltd., catalog number 1000012551), hot dTTP replaced with hot T-SSEB synthesized above having the structure shown below, and four types of nucleotide analog cold dNTPs of the present invention having the structure shown below (named SSEB).

[0586] [ka]

[0587] The sequencing procedure was the same as that described in Sequencing Example 1. The results of the base call analysis are shown in Table 5.

[0588] [Table 10]

[0589] Sequence example 5: Sequence using SS-Hot G 1. Synthesis of dGTP (named SS-Hot G), a nucleotide analog carrying a fluorescent group. [ka]

[0590] The product was synthesized in the same manner as in sequencing example 4, but the iodized G-nucleoside substrate was used instead of the iodized T-nucleoside substrate, and the azidocarboxylic acid substrate was used instead of the disulfaneolcarboxylic acid substrate, both of which were available from OKeanos Tech Co., Ltd. A new dye-linker was required and was available from MyChem LLC Co., Ltd. (catalog number 110920Cy5). 10 mg of the final product was obtained.

[0591] MS[ES(-)], m / z 1575.1 H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 8.49 (s, 1H), 8.34 (t, J = 13.1 Hz, 2H), 8.07 (t, J = 5.5 Hz, 1H), 7.92 (dd, J = 7.2, 4.6 Hz, 2H), 7.80 (d, J = 1.7 Hz, 2H), 7.70 (d, J = 7.4 Hz, 1H), 7.67 - 7.62 (m, 4H), 7.49 (t, J = 7.4 Hz, 2H), 7.30 (d, J = 8.3 Hz, 3H), 6.58 (t, J = 12.3 Hz, 1H), 6.41 (s, 2H), 6.36 - 6.27 (m, 3H), 6.26 - 6.12 (m, 2H), 5.60 (dd, J = 13.1, 6.4 Hz, 3H), 4.86 (s, 2H), 4.48 (dd, J = 29.1, 16.7 Hz, 2H), 4.35 - 4.27 (m, 2H), 4.07 (d, J = 4.8 Hz, 4H), 3.59 (s, 2H), 3.50 (d, J = 5.7 Hz, 2H), 3.34 - 3.29 (m, 2H), 2.78 (t, J = 6.8 Hz, 2H), 2.13 (t, J = 6.7 Hz, 2H), 2.07 (t, J = 7.2 Hz, 2H), 1.68 (s, 12H), 1.53 - 1.48 (m, 8H), 1.35 (dt, J = 15.8, 8.1 Hz, 4H).

[0592] 2. Sequencing performed using nucleotide analogs containing SS-Hot G synthesized as described above. Nucleotide substrates: fluorescently labeled standard hot dNTPs (MGISEQ-2000RS high-throughput sequencing kit (FCL SE50), available from MGI Tech Co., Ltd., catalog number 1000012551), hot dGTP replaced with SS-hot G synthesized above; four types of cold dNTPs (named SS-cold) of the present invention having the structures shown below.

[0593] [ka]

[0594] The sequencing procedure was the same as that described in Sequencing Example 1. The results of the base call analysis are shown in Table 6.

[0595] [Table 11]

Claims

【Request Item 1】 【Chemistry 1】 (In the formula, R is a reversible blocking group, and R is 【Chemistry 2】 Selected from, Preferably, R is 【Transformation 3】 Selected from, More precisely, R is 【Chemistry 4】 Selected from, The aforementioned heteroaryl is as follows: 【Transformation 5】 Selected from, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', Preferably, the heteroaryl is: 【Transformation 6】 Selected from, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', More preferably, the heteroaryl is: 【Transformation 7】 Selected from, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', Each X is independently selected from O, NH, and S. Preferably, X is O, Each Y is a direct bond, O, NH, S, CH, CH ２ , C (CH ３ ) ２ Selected independently from, Preferably, each Y is directly bonded or CH ２ Selected independently from, R ０ is -N ３ , -SS-C １ ~C ６ alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tert-butyl), -ONH ２ , -OCOR ｍ , -OCONHR ｍ selected from, each R ｍ is independently selected from aliphatic alkyl (e.g., C １ ~C ６ alkyl), cycloalkyl (e.g., C ３ ~C ６ cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ alkyl), Preferably, R ０ Ha-N ３ or -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), More specifically, R ０ Ha-N ３ And, R １ Ha-N ３ , -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH ２ , -OCOR ｍ ,-OCONHR ｍ Selected from, each R ｍ is an aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ Selected independently from alkyl, Preferably, R １ Ha-N ３ or -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), More specifically, R １ Ha-N ３ And, R ２ , R ３ , and R ４ is O, NH, S, CH, CH ２ , C (CH ３ ) ２ Each is independently selected from, Preferably, R ２ , R ３ , and R ４ are each independently selected from O, NH, S, CH, CH ２ , C(CH ３ ), ２ and satisfy the following conditions: When R ２ is selected from O or S, R ３ and R ４ are CH ２ ; When R ３ is selected from O or S, R ２ and R ４ are CH ２ ; When R ４ is selected from O or S, R ２ and R ３ are CH ２ ; When 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 are CH ２ ; When Y is CH, R ２ is CH, R ３ and R ４ are CH ２ , More specifically, R ２ , R ３ , and R ４ CH, CH ２ Each is independently selected from, Most preferably, R ２ and R ３ are CH, and R ４ is CH ２ and 【Transformation 8】 is a single bond 【Chemistry 9】 or double bond 【Chemistry 10】 This represents, Each R'' is H, -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ Alkoxy), 【Chemistry 11】 Selected independently of H, Preferably, if each heteroaryl is independently substituted by one R'', then each R'' is 【Chemistry 12】 Selected independently from, preferably R'' is 【Chemistry 13】 Is it, Alternatively, preferably, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is 【Chemistry 14】 Selected from, the remaining R'' is -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ They are selected independently from each of the alkoxys, Alternatively, more preferably, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is 【Chemistry 15】 And the remaining R'' is nitro, aliphatic alkyl (e.g., C １ ~C ６ Either alkyl, F, I, Br, or Cl is selected independently from each of these, Or, most preferably, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is 【Chemistry 16】 And the remaining R' is nitro, C １ ~C ６ Each alkyl group is independently selected, preferably the remaining R'' is C １ ~C ６ It is alkyl, R ５ , R ６ , R ７ , R ８ , R ９ , R ｘ , R ｙ , and R ｚ H, -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ Alkoxy), C １ ~C ６ Alkyl-C(=O)-NH ２ - 【Chemistry 17】 Each is independently selected from, R ５ , R ６ , R ７ , R ８ , and R ９ It is not H at the same time, Preferably, R ５ , R ６ , R ７ , R ８ , and R ９ One of the following is [Chemistry 18] One is selected from these, and the remaining four are H, -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ Alkoxy), C １ ~C ６ Alkyl-C(=O)-NH ２ - Each is independently selected from, More specifically, R ５ , R ６ , R ７ , R ８ , and R ９ One of the following is 【Chemistry 19】 The remaining four are H, Nitro, and C. １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Alkyl-C(=O)-NH ２ - Each is independently selected from, More preferably, R ５ , R ６ , R ７ , R ８ , and R ９ R ５ or R ９ teeth, 【Chemistry 20】 The remaining four are independently selected from H, nitro, methoxy, and acetamide. Most preferably, R ５ , R ６ , R ７ , R ８ , and R ９ R ５ or R ９ teeth, 【Chemistry 21】 And R ７ The first three are selected from H, nitro, methoxy, and acetamide, and the remaining three are H. Preferably, R ｘ and R ｙ One of the following is 【Chemistry 22】 One is selected from the other, and the other is H, -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ Selected from alkoxy, More specifically, R ｘ and R ｙ One of the following is 【Chemistry 23】 One is selected from the other, and the other is H. Most preferably, R ｘ and R ｙ One of the following is 【Chemistry 24】 The other is H, Preferably, R ｚ teeth, 【Chemistry 25】 Selected from, More specifically, R ｚ teeth, 【Chemistry 26】 And, R １０ａ , R １０ｂ , R １０ｃ , R １１ａ , R １１ｂ , and R １２ H, -N ３ , -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), -ONH ２ , -OCOR ｍ ,-OCONHR ｍ , aliphatic alkyl (e.g., especially methyl, ethyl, isopropyl, tert-butyl, etc.) １ ~C ６ Alkyl), aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Each of the cycloalkyl groups is independently selected, and each R ｍ is an aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ Independently selected from alkyl, R １０ａ , R １０ｂ , and R １０ｃ It is not H at the same time, Preferably, R １０ａ , R １０ｂ , and R １０ｃ Any one of these is -N ３ , -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), -ONH ２ , -OCOR ｍ ,-OCONHR ｍ One of these is selected, and the other two are H, aliphatic alkyl (e.g., C such as methyl, ethyl, isopropyl, tert-butyl). １ ~C ６ Alkyl), aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Each of the cycloalkyl groups is independently selected, and each R ｍ is an aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ Selected independently from alkyl, More specifically, R １０ａ , R １０ｂ , and R １０ｃ Any one of these is -N ３ or -SS-C １ ~C ６ The other two are alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), and the other two are H, and aliphatic alkyl (e.g., C, particularly methyl, ethyl, isopropyl, tertbutyl, etc.). １ ~C ６ Each alkyl group is independently selected, Most preferably, R １０ａ , R １０ｂ , and R １０ｃ Any one of these is -N ３ -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl, where the other two are independently selected from H or methyl. Preferably, R １１ａ and R １１ｂ is H or aliphatic alkyl (e.g., especially C such as methyl, ethyl, isopropyl, tert-butyl). １ ~C ６ Each alkyl group is independently selected, More specifically, R １１ａ and R １１ｂ H is, Preferably, R １２ Ha-N ３ , -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH ２ , -OCOR ｍ ,-OCONHR ｍ Selected from, each R ｍ is an aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ Selected independently from alkyl, More specifically, R １２ Ha-N ３ or -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), Most preferably, R １２ Ha-N ３ And, n is selected from 1, 2, 3, 4, or 5, preferably n is selected from 1, 2, or 3, and more preferably n is 2. each m １ is independently selected from 1, 2, 3, 4, 5, and 6, preferably m １ is 1, each m ２ is independently selected from 0, 1, 2, 3, 4, 5, and 6, preferably each m ２ It is selected independently from 0 or 1. Most preferably, 【Chemistry 27】 Overall, 【Chemistry 28】 Selected from, R' is H, monophosphate group ( 【Chemistry 29】 ), diphosphate group ( 【Transformation 30】 ), triphosphate group ( 【Chemistry 31】 ) or tetraphosphate group ( 【Chemistry 32】 ) are selected from, Preferably, R' is a triphosphate group ( 【Transformation 33】 ) and Each Z is independently selected from O, S, and BH, and preferably Z is O. Base is selected from a base, a deaza base, or a tautomer thereof. For example, Base may be selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine, or a tautomer thereof. Preferably, Base is 【Transformation 34】 (Selected from) A compound of formula (A) or a salt thereof.

2. Equation (I) 【Chemistry 35】 (In the formula, X is selected from O, NH, and S. Preferably, X is O, Y is a direct bond, O, NH, S, CH, CH ２ , C (CH ３ ) ２ Selected from, Preferably, Y is CH ２ And, R ０ Ha-N ３ , -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH ２ , -OCOR ｍ ,-OCONHR ｍ Selected from, each R ｍ is an aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ Selected independently from alkyl, Preferably, R ０ Ha-N ３ or -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), More specifically, R ０ Ha-N ３ And, n is selected from 1, 2, 3, 4, and 5. Preferably, n is selected from 1, 2, and 3. More preferably, n is 2, R' is H, monophosphate group ( 【Transformation 36】 ), diphosphate group ( 【Chemistry 37】 ), triphosphate group ( 【Transformation 38】 ) or tetraphosphate group ( 【Chemistry 39】 ) are selected from, Preferably, R' is a triphosphate group ( 【Chemistry 40】 ) and Each Z is selected independently from O, S, and BH. Preferably, Z is O, Base is selected from a base, a deaza base, or a tautomer thereof. For example, Base may be selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine, or a tautomer thereof. Preferably, Base is 【Chemistry 41】 A compound or salt thereof according to claim 1, having a structure represented by (selected from).

3. Formula (III) 【Chemistry 42】 (In the formula, A' is, 【Chemistry 43】 Selected from, The aforementioned heteroaryl is as follows: 【Chemistry 44】 Selected from, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', Preferably, the heteroaryl is: 【Chemistry 45】 Selected from, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', More preferably, the heteroaryl is: 【Chemistry 46】 Selected from, each heteroaryl may be independently substituted with one or more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', Or more preferably, the heteroaryl is: 【Chemistry 47】 Selected from, Most preferably, the heteroaryl is: 【Chemistry 48】 Selected from, Each R'' is H, -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ Alkoxy), 【Chemistry 49】 Selected independently from, Preferably, if each heteroaryl is independently substituted by one R'', then each R'' is [Transformation 50] Selected independently from, preferably R'' is 【Chemistry 51】 Is it, Alternatively, preferably, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is 【Chemistry 52】 Selected from, the remaining R'' is -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ They are selected independently from each of the alkoxys, Alternatively, more preferably, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is 【Chemistry 53】 And the remaining R'' is nitro, aliphatic alkyl (e.g., C １ ~C ６ Either alkyl, F, I, Br, or Cl is selected independently from each of these, Or, most preferably, each heteroaryl may be independently substituted with more (e.g., 2, 3, 4, 5, or 6, preferably 2) R'', one of the R'' is 【Chemistry 54】 And the remaining R' is nitro, C １ ~C ６ Each alkyl group is independently selected, preferably the remaining R'' is C １ ~C ６ It is alkyl, and more preferably the remaining R'' is methyl. R １３ , R １４ , R １５ , and R １６ R １６ teeth, 【Transformation 55】 And R １３ , R １４ , and R １５ H, Nitro, C １ ~C ６ Each alkyl (e.g., methyl) is independently selected, Preferably, R １３ , R １４ , R １５ , and R １６ R １６ teeth, 【Transformation 56】 And R １３ , R １４ , and R １５ H is, R １７ , R １８ , and R １９ R １９ teeth, 【Chemistry 57】 And R １７ and R １８ H, Nitro, C １ ~C ６ Each alkyl (e.g., methyl) is independently selected, Preferably, R １７ , R １８ , and R １９ R １９ teeth, 【Chemistry 58】 And R １７ and R １８ H is, R ２０ , R ２１ , and R ２２ R ２２ teeth, 【Chemistry 59】 And R ２０ and R ２１ H, Nitro, C １ ~C ６ Each alkyl (e.g., methyl) is independently selected, Preferably, R ２０ , R ２１ , and R ２２ R ２２ teeth, 【Transformation 60】 And R ２０ and R ２１ H is, R ２３ , R ２４ , R ２５ , and R ２６ R ２６ teeth, 【Chemistry 61】 And R ２３ , R ２４ , and R ２５ H, Nitro, C １ ~C ６ Each alkyl (e.g., methyl) is independently selected, Preferably, R ２３ , R ２４ , R ２５ , and R ２６ R ２６ teeth, 【Transformation 62】 And R ２３ , R ２４ , and R ２５ H is, R ２７ , R ２８ , and R ２９ R ２９ teeth, 【Transformation 63】 And R ２７ and R ２８ H, Nitro, C １ ~C ６ Each alkyl (e.g., methyl) is independently selected, Preferably, R ２７ , R ２８ , and R ２９ R ２９ teeth, 【Chemistry 64】 And R ２７ and R ２８ H is, R ３０ , R ３１ , and R ３２ R ３２ teeth, 【Transformation 65】 And R ３０ and R ３１ H, Nitro, C １ ~C ６ Each alkyl (e.g., methyl) is independently selected, Preferably, R ３０ , R ３１ , and R ３２ R ３２ teeth, 【Chemical Formula 66】 And R ３０ and R ３１ H is, R ３３ , R ３４ , and R ３５ R ３４ or R ３５ teeth, 【Transformation 67】 And R ３３ H, Nitro, C １ ~C ６ Selected from alkyl groups (e.g., methyl), Preferably, R ３３ , R ３４ , and R ３５ R ３４ or R ３５ teeth, 【Transformation 68】 And R ３３ is H or methyl, X is selected from O, NH, and S. Preferably, X is O, R ５ , R ６ , R ７ , R ８ , and R ９ H, -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ Alkoxy), C １ ~C ６ Alkyl-C(=O)-NH ２ - 【Transformation 69】 Each is independently selected from, R ５ , R ６ , R ７ , R ８ , and R ９ It is not H at the same time, Preferably, R ５ , R ６ , R ７ , R ８ , and R ９ One of the following is 【Transformation 70】 One is selected from these, and the remaining four are H, -N ３ , nitro, amino, sulfo, carboxyl, aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl, aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), F, I, Br, Cl, alkoxy (e.g., C １ ~C ６ Alkoxy), C １ ~C ６ Alkyl-C(=O)-NH ２ - Each is independently selected from, More specifically, R ５ , R ６ , R ７ , R ８ , and R ９ One of the following is 【Chemistry 71】 The remaining four are H, Nitro, and C. １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Alkyl-C(=O)-NH ２ - Each is independently selected from, More preferably, R ５ , R ６ , R ７ , R ８ , and R ９ R ５ or R ９ teeth, 【Chemistry 72】 The remaining four are independently selected from H, nitro, methoxy, and acetamide. Most preferably, R ５ , R ６ , R ７ , R ８ , and R ９ R ５ or R ９ teeth, 【Transformation 73】 And R ７ The first three are selected from H, nitro, methoxy, and acetamide, and the remaining three are H. R １０ａ , R １０ｂ , R １０ｃ , R １１ａ , R １１ｂ , and R １２ H, -N ３ , -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), -ONH ２ , -OCOR ｍ ,-OCONHR ｍ , aliphatic alkyl (e.g., especially methyl, ethyl, isopropyl, tert-butyl, etc.) １ ~C ６ Alkyl), aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Each of the cycloalkyl groups is independently selected, and each R ｍ is an aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ Independently selected from alkyl, R １０ａ , R １０ｂ , and R １０ｃ It is not H at the same time, Preferably, R １０ａ , R １０ｂ , and R １０ｃ Any one of these is -N ３ , -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, or -SS-isobutyl), -ONH ２ , -OCOR ｍ ,-OCONHR ｍ One of these is selected, and the other two are H, aliphatic alkyl (e.g., C such as methyl, ethyl, isopropyl, tert-butyl). １ ~C ６ Alkyl), aromatic alkyl (e.g., phenyl C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Each of the cycloalkyl groups is independently selected, and each R ｍ is an aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ Selected independently from alkyl, More specifically, R １０ａ , R １０ｂ , and R １０ｃ Any one of these is -N ３ or -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, -SS-isobutyl), and the other two are H and C. １ ~C ６ Alkyl (e.g., methyl, ethyl, isopropyl, tert-butyl) is independently selected from each of these, Most preferably, R １０ａ , R １０ｂ , and R １０ｃ Any one of these is -N ３ These are -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl, and -SS-isobutyl, and the other two are independently selected from H and methyl, respectively. Preferably, R １１ａ and R １１ｂ C is H, aliphatic alkyl (e.g., especially methyl, ethyl, isopropyl, tert-butyl, etc.) １ ~C ６ Each alkyl group is independently selected, More specifically, R １１ａ and R １１ｂ H is, Preferably, R １２ Ha-N ３ , -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), -ONH ２ , -OCOR ｍ ,-OCONHR ｍ Selected from, each R ｍ is an aliphatic alkyl (e.g., C １ ~C ６ Alkyl), cycloalkyl (e.g., C ３ ~C ６ Cycloalkyl) or aromatic alkyl (e.g., phenyl C １ ~C ６ Selected independently from alkyl, More specifically, R １２ Ha-N ３ or -SS-C １ ~C ６ Alkyl (e.g., -SS-methyl, -SS-ethyl, -SS-isopropyl, -SS-tertbutyl), Most preferably, R １２ Ha-N ３ And, I understand １ is selected from 1, 2, 3, 4, 5, and 6, preferably m １ is 1, I understand ２ is selected from 0, 1, 2, 3, 4, 5, and 6, preferably m ２ It is selected from 0 or 1, Most preferably, 【Chemistry 74】 Overall, 【Chemistry 75】 Selected from, R' is H, monophosphate group ( 【Transformation 76】 ), diphosphate group ( 【Chemical 77】 ), triphosphate group ( 【Transformation 78】 ) or tetraphosphate group ( 【Chemistry 79】 ) are selected from, Preferably, R' is a triphosphate group ( 【Chemistry 80】 ) and Each Z is selected independently from O, S, and BH. Preferably, Z is O, Base is selected from a base, a deaza base, or a tautomer thereof. For example, Base may be selected from adenine, 7-deazaadenine, thymine, uracil, cytosine, guanine, 7-deazaguanine, or a tautomer thereof. Preferably, Base is 【Chemistry 81】 A compound or salt thereof according to claim 1, having a structure represented by (selected from).

4. The aforementioned compound is as follows: 【Chemistry 82】 【Chemistry 83】 【Chemical 84】 【Chemical 85】 【Chemical 86】 【Transformation 87】 【Chemical 88】 【Chemical 89】 [Chemical 90] 【Chemistry 91】 【Chemistry 92】 【Chemistry 93】 【Chemical 94】 【Chemical 95】 【Chemistry 96】 A compound or salt thereof according to claim 1, selected from the above.

5. The compound or a salt thereof is supported with an additional detectable label (e.g., a fluorescent label), Preferably, the additional detectable label supported by the compound or a salt thereof is introduced by an affinity reagent (e.g., an antibody, aptamer, affimer, nottin), the affinity reagent supports the detectable label, and the affinity reagent can specifically recognize and bind to the epitope of the compound or a salt thereof. Preferably, the additional detectable label (e.g., a fluorescent label) is optionally linked to the compound or a salt thereof via a linker. Preferably, the additional detectable label (e.g., a fluorescent label) is optionally linked to the Base of the compound or salt thereof via a linker. Preferably, the linker is a severable linker or a non-severable linker. Preferably, the cleavable linker is selected from the group consisting of a linker that can be cleaved by an electrophilic reaction, a linker that can be cleaved by a nucleophilic reaction, a linker that can be cleaved by photodegradation, a linker that can be cleaved under reducing conditions, a linker that can be cleaved under oxidizing conditions, a safety-catch linker, a linker that can be cleaved by a desorption mechanism, or any combination thereof. Preferably, the detectable marker is: 【Chemistry 97】 Selected from, Preferably, the compound or salt thereof according to any one of claims 1 to 4, wherein the detectable label (e.g., fluorescent label) differs when the Base is different.

6. A method for halting nucleic acid synthesis, comprising the step of incorporating a compound or a salt thereof described in any one of claims 1 to 5 into a nucleic acid molecule that halts synthesis, Preferably, the incorporation of the compound or a salt thereof is achieved by a terminal transferase, terminal polymerase, or reverse transcriptase. Preferably, the step includes incorporating the compound or a salt thereof into the nucleic acid molecule to terminate it using polymerase. Preferably, the polymerase is used to carry out the nucleotide polymerization reaction under conditions in which the polymerase can carry out the nucleotide polymerization reaction, thereby including the step of incorporating the compound or a salt thereof into the 3' end of the nucleic acid molecule, thereby terminating the reaction. method.

7. A method for preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, comprising the step of incorporating a compound or salt thereof according to any one of claims 1 to 5 into a growing complementary polynucleotide, wherein the incorporation of the compound or salt thereof prevents any subsequent nucleotides from being introduced into the growing complementary polynucleotide. Preferably, the incorporation of the compound or a salt thereof is achieved by a terminal transferase, terminal polymerase, or reverse transcriptase. Preferably, the process includes the step of incorporating the compound or a salt thereof into the growing complementary polynucleotide by using a polymerase. Preferably, the process includes carrying out the nucleotide polymerization reaction using a polymerase under conditions in which the polymerase can perform the nucleotide polymerization reaction, thereby incorporating the compound or a salt thereof into the 3' end of the growing complementary polynucleotide. method.

8. A method for determining the sequence of a target single-stranded polynucleotide, 1) A step of monitoring the incorporation of nucleotides complementary to the target single-stranded polynucleotide in a growing nucleic acid chain, wherein at least one incorporated complementary nucleotide is a compound or salt thereof according to any one of claims 1 to 5, and the compound or salt thereof carries an additional detectable label (e.g., a fluorescent label), 2) A step of detecting the detectable label and determining the incorporated nucleotide, Preferably, the additional detectable label (e.g., a fluorescent label) is optionally linked to the compound or a salt thereof via a linker. Preferably, the linker is as defined in claim 5, Preferably, the additional detectable marker is as defined in claim 5, Preferably, if the Base is different, the detectable label (e.g., fluorescent label) supported by the compound or its salt will be different. Preferably, the reversible blocking group (R) and the detectable label in the compound or its salt are removed before the introduction of the next complementary nucleotide. Preferably, the reversible blocking group and the detectable label are removed simultaneously, or Preferably, the reversible blocking group and the detectable label are removed sequentially, for example, the reversible blocking group is removed after the detectable label is removed, or the detectable label is removed after the reversible blocking group is removed, in the step of A method that includes this.

9. The following steps: (a) A step of preparing a plurality of different nucleotides, wherein the plurality of different nucleotides are a compound or salt thereof according to any one of claims 1 to 5, and each nucleotide carries an additional detectable label that can be distinguished from an additional detectable label carried by another nucleotide being detected. (b) The step of incorporating the plurality of different nucleotides into a sequence complementary to the target single-stranded polynucleotide, (c) A step of detecting the additional detectable label supported by the nucleotide in step (b) to determine the type of incorporated nucleotide, (d) A step of removing the reversible blocking group and the detectable label supported by the nucleotide in step (b), (e) a step in which steps (b) to (d) are optionally repeated once or multiple times, The method according to claim 8, wherein the sequence of the target single-stranded polynucleotide is determined as a result.

10. The following steps: (1) A step of preparing a first nucleotide, a second nucleotide, a third nucleotide, and a fourth nucleotide, wherein at least one of the four nucleotides is a compound or salt thereof according to any one of claims 1 to 5, the Base contained in the four nucleotides are different from each other, the four nucleotides are supported with an additional detectable label (e.g., a fluorescent label), preferably the additional detectable label supported by the four nucleotides is introduced by an affinity reagent (e.g., an antibody, aptamer, affimer, nottin), the affinity reagent is supported with the detectable label, the affinity reagent is able to specifically recognize and bind to the epitope of each nucleotide, or preferably the four nucleotides are optionally linked to the additional detectable label via a linker, or more preferably the Base of the four nucleotides is optionally linked to the additional detectable label via a linker, most preferably the additional detectable label supported by the four nucleotides are different from each other, (2) The process includes contacting the four types of nucleotides with a target single-stranded polynucleotide, removing nucleotides not incorporated into the growing nucleic acid chain, detecting the detectable label supported by the nucleotides incorporated into the growing nucleic acid chain, and removing the reversible blocking group and the detectable label supported by the nucleotides incorporated into the growing nucleic acid chain. The method according to claim 8, optionally comprising the step of (3) repeating step (2) once or more times.

11. The following steps: (a) A step of preparing a mixture comprising a double-stranded, at least one compound or salt thereof according to any one of claims 1 to 5, a polymerase and an excision reagent, wherein the double-stranded comprises a nucleic acid strand to be grown and a nucleic acid strand to be sequenced, the compound or salt thereof supports an additional detectable label (e.g., a fluorescent label), preferably the additional detectable label supported by the compound or salt thereof is introduced by an affinity reagent (e.g., an antibody, aptamer, affimer, nottin), the affinity reagent supports the detectable label, the affinity reagent can specifically recognize and bind to an epitope of the compound or salt thereof, or preferably the compound or salt thereof is optionally linked to the additional detectable label via a linker, or more preferably the Base of the compound or salt thereof is optionally linked to the additional detectable label via a linker. (b) Steps (i), (ii), and (iii) below: Step (i): Using polymerase to incorporate the compound or a salt thereof into the growing nucleic acid chain to form a nucleic acid intermediate containing the reversible blocking group and the detectable label. Step (ii): A step of detecting the detectable label contained in the nucleic acid intermediate, Step (iii): A step of removing the reversible blocking group and / or the detectable label contained in the nucleic acid intermediate by using the excision reagent, Preferably, the removal of the reversible blocking group and the removal of the detectable label are performed simultaneously, or the removal of the reversible blocking group and the removal of the detectable label are performed sequentially (for example, the reversible blocking group is removed first, or the detectable label is removed first), Preferably, the excision reagent used to remove the reversible blocking group is the same as the one used to remove the detectable label. Preferably, the excision reagent used to remove the reversible blocking group is different from the one used to remove the detectable label. A step in which a reaction including is performed, and optionally the step is repeated once or multiple times. The method according to claim 8, including the method described in claim 8.

12. The aforementioned double strand is connected to a support, Preferably, the growing nucleic acid chain is a primer. Preferably, the primer is annealed to the nucleic acid strand to be sequenced to form the double helix, Preferably, the double chain, the compound or a salt thereof, and the polymerase together form a reaction system containing a solution phase and a solid phase. Preferably, the Base contained in the compound or its salt is different from one another. Preferably, the additional detectable labels supported by the compound or a salt thereof are different from each other. Preferably, the compound or a salt thereof is incorporated into the growing nucleic acid chain using the polymerase under conditions in which the polymerase can carry out a nucleotide polymerization reaction, thereby forming a nucleic acid intermediate containing a reversible blocking group and the detectable label. Preferably, the polymerase is selected from KOD polymerase or its variants (e.g., KOD POL151, KOD POL157, KOD POL171, KOD POL174, KOD POL376, KOD POL391), Preferably, before any step of detecting the detectable label contained in the nucleic acid intermediate, the solution phase of the reaction system in the previous step is removed, and the double strand linked to the support is retained. Preferably, the excision reagent comes into contact with the double-stranded or growing nucleic acid strand in the reaction system containing the solution phase and the solid phase. Preferably, the excision reagent can remove the reversible blocking group and the additional detectable label supported by the compound incorporated into the growing nucleic acid chain without affecting the phosphate diester bond on the double-stranded skeleton. Preferably, after any step of removing the reversible blocking group and / or additional detectable label contained in the nucleic acid intermediate, the solution phase of the reaction system in this step is removed. Preferably, a washing operation is performed after any step including a removal operation. Preferably, the method according to claim 11, further comprising, after step (ii), a step of determining the type of compound incorporated into the growing nucleic acid chain in step (i) according to the signal detected in step (ii), and a step of determining the type of nucleotide at the corresponding position in the nucleic acid chain being sequenced based on the principle of complementary base pairing.

13. A kit comprising at least one compound or salt thereof as described in any one of claims 1 to 5, Preferably, the kit comprises a first compound, a second compound, a third compound, and a fourth compound, wherein each of the first, second, third, and fourth compounds is independently a compound or a salt thereof as described in any one of claims 1 to 5. Preferably, in the first compound, Base is adenine, 7-deazaadenine, or tautomers thereof (for example, 【Chem.98】 ) is selected from the above, and in the second compound, the Base is thymine, uracil or a tautomer thereof (for example, 【Chem.99】 ) are selected from the above, and in the third compound, the Base is cytosine or its tautomer (for example, 【Chemistry 100】 ) selected from the above, and in the fourth compound, the Base is guanine, 7-deazaguanine or tautomers thereof (for example, 【Chemistry 101】 ) are selected from, Preferably, the first, second, third, and fourth compounds carry additional detectable labels. Preferably, the additional detectable label supported by the first, second, third, and fourth compounds is introduced by an affinity reagent (e.g., an antibody, aptamer, affimer, or Nottin), the affinity reagent supports the detectable label, and the affinity reagent can specifically recognize and bind to the epitope of the first, second, third, or fourth compound. Preferably, the first, second, third, and fourth compounds are optionally linked to the additional detectable label via a linker. Preferably, the Base of the first, second, third, or fourth compound is optionally linked to the additional detectable label via a linker. Preferably, the Base contained in the first, second, third, and fourth compounds is different from one another. Preferably, the additional detectable labels supported by the first, second, third, and fourth compounds are different from each other. Preferably, the linker is as defined in claim 5, Preferably, the kit wherein the detectable label is as defined in claim 5.

14. The kit according to claim 13, further comprising: a reagent for pre-treating nucleic acid molecules; a support for linking nucleic acid molecules to be sequenced; a reagent for linking the nucleic acid molecules to be sequenced to the support (for example, by covalent or non-covalent bonding); a primer for initiating a nucleotide polymerization reaction; a polymerase for carrying out the nucleotide polymerization reaction; one or more buffer solutions; one or more washing solutions; or any combination thereof.

15. Use of a compound or salt thereof according to any one of claims 1 to 5, or a kit according to claim 13 or 14, for determining the sequence of a target single-stranded polynucleotide.

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