Nucleosides and nucleotides with 3'-hydroxy blocking groups, and use thereof in polynucleotide sequencing methods

Abstract

To provide a reversible protecting group for use in polynucleotide sequencing methods, which exhibits long-term stability, is efficiently incorporated by a polymerase enzyme, causes blocking of secondary or further nucleotide incorporation, and has the ability to be removed under mild conditions that do not damage the polynucleotide structure.SOLUTION: Provided are methods for preparing nucleotide and nucleoside molecules having an acetal or thiocarbamate 3'-OH blocking group, and the use of fully functionalized nucleotides containing a 3'-OH blocking group for sequencing applications.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (background) (Field) The present disclosure generally relates to nucleotides, nucleosides, or nucleotides containing a 3'-hydroxy protecting group. The present invention relates to oligonucleotides and their use in polynucleotide sequencing methods. 3'-hydroxy-protected nucleotides, nucleosides, or oligonucleosides Methods for preparing the tides are also disclosed. [Background technology]

[0002] Description of Related Art Advances in molecular research are driven, in part, by the availability of information used to characterize molecules or their biological responses. In particular, the study of nucleic acids, DNA and RNA, has been driven by improvements in the techniques used to benefit from developments in techniques used for sequence analysis and the study of hybridization events. I am receiving it.

[0003] An example of a technology that has improved the study of nucleic acids is the development of fabricated arrays of immobilized nucleic acids. These arrays typically consist of high density matrices of polynucleotides immobilized on a solid support material. For example, Fodor et al., Trends Biote See ch. 12: 19-26, 1994. This is because masks protect However, to allow for the attachment of appropriately modified nucleotide phosphoramidites, The method involves the use of chemically sensitized glass surfaces that are exposed in defined areas for nucleic acid synthesis. The fabricated arrays also contain known polynucleotides in a predetermined can be prepared by "spotting" techniques onto a solid support at positions (e.g., Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).

[0004] One method for determining the nucleotide sequence of nucleic acids bound to an array is "sequencing by synthesis." This technique for determining the sequence of DNA is called "sequencing" or "SBS." Ideally, controlled sequencing of the correct complementary nucleotide on the other side of the nucleic acid being sequenced is required. This requires the incorporation of each nucleotide residue. Because bases are sequenced one at a time, multiple cycles of adding nucleotides are possible. This allows for accurate sequencing and prevents uncontrolled sequential acquisitions. The incorporated nucleotides are then subjected to removal of the label moiety and subsequent sequencing. Before the test, the sample is read using an appropriate label attached to it. Summary of the Invention [Problem to be solved by the invention]

[0005] Structural modifications ("protecting groups" or " A "blocking group" ("blocking group") is included in each labeled nucleotide added to the growing chain, blocking one nucleotide. Ensures that only the nucleotide with the protecting group is incorporated. The protecting groups are then removed under reaction conditions that do not interfere with the integrity of the DNA being sequenced. The sequencing cycle then continues with the incorporation of the next protected, labeled nucleotide. can continue.

[0006] Nucleotides, usually nucleotide triphosphates, to aid in DNA sequencing is used to incorporate additional nucleotide bases into a polynucleotide chain. To prevent the polymerase used from continuing to replicate, a 3'-hydroxy protecting group is generally added. There are many restrictions on the types of groups that can be added to nucleotides and still be appropriate. The protecting group can be easily removed from the sugar moiety without damaging the polynucleotide chain. It prevents additional nucleotide molecules from being added to the polynucleotide chain. Furthermore, modified nucleotides should be prepared using a suitable nucleotide sequence for their incorporation into a polynucleotide chain. It must be compatible with the polymerase or other suitable enzyme used. An ideal protecting group would exhibit long-term stability, be efficiently incorporated by polymerase enzymes, and Causes blockage of secondary or further nucleotide incorporation, resulting in polynucleotide structure It must be capable of being removed under mild, non-damaging conditions, preferably under aqueous conditions. It must be.

[0007] Reversible protecting groups have been described previously, e.g., Metzker et al., (Nucleic Acids Research, 22 (20): 4259-4 267, 1994) consists of eight 3'-modified 2-deoxyribonucleoside 5'-triphosphates. Synthesis and use of 3'-modified dNTPs and two DNA templates for incorporation activity WO2002 / 029003 describes a method for assaying growing DNA fragments in a polymerase reaction. Sequencing may involve the use of an allyl protecting group to cap the 3'-OH group on the A strand. It describes how to do this.

[0008] Furthermore, several reversible protecting groups have been developed and their deprotection under DNA-compatible conditions has been investigated. The method is described in International Application Publication Nos. WO2004 / 014897 and WO2014 / 1395 96, each of which is incorporated herein by reference in its entirety. To be incorporated. [Means for solving the problem]

[0009] (overview) Some embodiments of the present disclosure provide a structure covalently attached to the 3'-carbon atom. [ka] Ribose or deoxyribonucleotides with a removable 3'-OH protecting or blocking group to form A nucleotide or nucleoside containing a base, wherein: Each R 1a and R 1b are independently H, C1-C6 alkyl, C1-C6 haloalkyl C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, substituted It is optionally substituted phenyl, or optionally substituted aralkyl. Each R 2a and R 2b are independently H, C1-C6 alkyl, C1-C6 haloalkyl, cyano, or halogen; Alternatively, R 1a and R 2a along with the atoms to which they are attached, if necessary Forming a substituted 5- to 8-membered heterocyclyl group; R 3 is H, optionally substituted C2-C6 alkenyl, optionally substituted C3 C-C7 cycloalkenyl, optionally substituted C-C6 alkynyl, or substituted (C1-C6 alkylene)Si(R 4 )3; and Each R 4are independently H, C1-C6 alkyl, or optionally substituted C6-C 10 a In some embodiments, each R 1a and R 1b is H or C1-C6 alkyl, and R 2a and R 2b Both If H, then R 3 is a substituted C2-C6 alkenyl, an optionally substituted C3-C7 silyl chloroalkenyl, optionally substituted C2-C6 alkynyl, or optionally substituted Good (C1-C6 alkylene)Si(R 4 )3. In some embodiments, each R 1 a , R 1b , R 2a and R 2b If is H, then R 3 is not H.

[0010] Some embodiments of the present disclosure provide a structure covalently attached to the 3'-carbon atom. [ka] Ribose or deoxyribose with a removable 3'-OH blocking group to form A nucleoside or nucleotide containing a nucleotide, wherein: R 5 and R 6 each independently represents H, C1-C6 alkyl, C2-C6 alkenyl C2-C6 alkynyl, C1-C6 haloalkyl, C2-C8 alkoxyalkyl, Optionally substituted -(CH2) m -phenyl, optionally substituted -(CH2) n -(5- or 6-membered heteroaryl), optionally substituted -(CH) k -C3~C7 carbocyclyl or optionally substituted -(CH2) p -(3- to 7-membered heterocyclyl ru); -(CH2) m -, -(CH2) n -, -(CH2) k - and -(CH2) p -of each of which is optionally substituted; and each of m, n, k, and p is independently 0, 1, 2, 3, or 4; Pertaining to nucleosides or nucleotides.

[0011] Some embodiments of the present disclosure include the 3'-OH blocked nucleotides described herein. The present invention relates to an oligonucleotide or polynucleotide comprising the molecule.

[0012] Some embodiments of the present disclosure include a method for sequencing a target single-stranded polynucleoside in a sequencing reaction. The present invention relates to a method for preparing a growing polynucleotide complementary to a nucleotide, and Complementary polynucleotides that grow nucleotide molecules described herein that prevent the introduction of This involves the incorporation of subsequent nucleotides into a growing complementary polynucleotide. In some embodiments, the incorporation of a nucleotide is initiated by a polymerase, a terminal deoxynucleotide, or a This is achieved by cleotidyl transferase (TdT), or reverse transcriptase. In embodiments, incorporation is achieved by a polymerase (e.g., a DNA polymerase). will be done.

[0013] Some further embodiments of the present disclosure provide a method for detecting a target single-stranded polynucleotide, comprising: Regarding the method for determining the sequence: (a) a nucleoside containing a 3'-OH blocking group and a detectable label as described herein; The nucleotide is then transferred to a copy polynucleotide strand that is complementary to at least a portion of the target polynucleotide strand. Incorporating into; (b) detecting the identity of the nucleotide incorporated into the copy polynucleotide strand; and (c) Labeling and 3'-OH removal from nucleotides incorporated into the copy polynucleotide strand. Chemically removing the blocking group.

[0014] In some embodiments, the sequencing method further comprises (d) chemically removing the label. and washing the 3' blocking group from the copy polynucleotide strand. In some embodiments, such a washing step also removes unincorporated nucleosides. In some such embodiments, the incorporated nucleotide is removed. The 3' blocking group and detectable label are removed before the next complementary nucleotide is introduced. In some further embodiments, the 3' blocking group and the detectable The label is removed in a single step of a chemical reaction. The sequential integrations described include at least 50, at least 100, at least 150 , at least 200 times, or at least 250 times.

[0015] Some further embodiments of the present disclosure include a plurality of nucleotides or The present invention relates to kits containing nucleoside molecules, and packaging materials therefor. Use nucleotides, nucleosides, oligonucleotides, or kits to synthesize biological Detect, measure, or identify a system (e.g., including its processes or components) Examples of nucleotides, oligonucleotides, or kits that can be used include: Techniques include sequencing, expression analysis, hybridization analysis, genetic analysis, RN Assays, cell assays (e.g., cell binding or cell function assays), or protein assays (e.g., protein binding assays or protein activity assays). It can be used in automated equipment to perform specific techniques, such as sequencing equipment. The scanning instrument contains two detectors operating at different wavelengths to distinguish between different detectable labels. May contain more than one laser. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a line graph showing the stability of various 3' blocked nucleotides as a function of time in buffer at 65°C. [Figure 2] Figure 2A is a line graph illustrating the percentage (%) of remaining nucleotide (starting material) as a function of time for deblocking nucleotides with a 3'-AOM blocking group compared to nucleotides with a 3'-O-azidomethyl (-CHN) blocking group in solution. Figure 2B is a line graph illustrating the percentage (%) of 3'-unblocked nucleotide as a function of time for unblocking 3'-blocked nucleotides compared to various acetal blocking groups in solution. [Figure 3] Figures 3A and 3B illustrate sequencing results on an Illumina MiniSeq® instrument using fully functionalized nucleotides (ffNs) containing 3'-AOM blocking groups in the incorporation mix, and Figure 3C illustrates the sequencing error rate using fully functionalized nucleotides (ffNs) containing 3'-AOM blocking groups in the incorporation mix compared to standard ffNs containing 3'-O-azidomethyl blocking groups. [Figure 4]Figure 4A and 4B illustrate a comparison of key sequencing metrics including phasing, prephasing, and error rate using fully functionalized nucleotides containing 3'-AOM and 3'-O-azidomethyl blocking groups using two different DNA polymerases (Pol812 and Pol1901), respectively. [Figure 5] FIG. 5 is a line graph illustrating the sequence stability of fully functionalized nucleotides bearing 3′-AOM or 3′-O-azidomethyl blocking groups as a function of time in buffer at 45° C. [Figure 6] FIG. 6 is a line graph illustrating the stability of nucleosides with various 3' blocking groups as a function of time in buffer at 65°C. [Figure 7] FIG. 7 is a line graph illustrating the percentage (%) of remaining 3′-blocked nucleotides as a function of time, comparing the cleavage (unblocking) rate of the thiocarbamate 3′-blocking group dimethylthiocarbamate (DMTC) with that of the 3′-O-azidomethyl (3′-O-CH2N3) blocking group under two different conditions (Oxone® or NaIO4). DETAILED DESCRIPTION OF THE INVENTION

[0017] (Detailed explanation) Embodiments of the present disclosure are directed to sequencing applications, such as sequencing-by-synthesis (SB Nucleic acid with a 3'-OH acetal or thiocarbamate blocking group for (S) These blocking groups are known in the art. It offers better stability in solution compared to conventional 3'-OH blockers. The silyl group provides increased stability during the synthesis of fully functionalized nucleotides (ffNs) and It also has improved stability in solution during formulation, storage, and handling in sequencing equipment. The 3'-OH blocking groups described herein also provide low pre-cleavage to improve data quality. Fading, low signal attenuation can be achieved, which makes sequencing applications This allows for longer reads from

[0018] (definition) Unless otherwise defined, all technical and scientific terms used herein are defined by the The term "inc" has the same meaning as commonly understood by those skilled in the art. "including" and "include" The use of other forms such as "included" is not limiting. "having" as well as "have" and "has" The use of other forms such as "had" and "had" is not limiting. As stated in the transitional phrase of the claim, The terms "comprise(s)" and "comprises" are used in the text, whether in the body or elsewhere. and "comprising" have an open-ended meaning. That is, the above term should be interpreted as including the phrase "having at least "including at least" or "including at least For example, when used in the context of a process, In this case, the term "comprising" means that the process includes at least the It means that the compound, composition, or When used in the context of a device, the term "comprising" The compound, composition, or device contains at least the recited features or components, but may contain additional It means that additional features or ingredients may also be included.

[0019] Common organic abbreviations used herein are defined as follows: ℃ Celsius temperature dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate dTTP deoxythymidine triphosphate ddNTP Dideoxynucleotide Triphosphate ffN fully functionalized nucleotides RT room temperature Sequencing by SBS synthesis SM starting material

[0020] As used herein, the term "array" refers to an array in which different probe molecules are arranged according to their relative positions. The term refers to a population of different probe molecules attached to one or more substrates such that they can be distinguished from one another by their molecular structure. The array comprises different probes, each located at a different addressable location on the substrate. Alternatively or additionally, the array may comprise a plurality of probes, each of which may comprise a different molecule. The probe molecules may be attached to a surface of the substrate. In a liquid that can be identified according to the position of the substrate on or according to the position of the substrate. Exemplary arrays located on a surface include those described, for example, in U.S. Pat. No. 6,355,431 B1; As described in U.S. Patent No. 2002 / 0102578 and PCT Publication No. WO00 / 63437 These include, but are not limited to, arrays containing beads in wells containing For example, microfluidic devices such as fluorescence-activated cell sorters (FACS) can be used to sort liquid samples. An exemplary format that can be used in the present invention to distinguish beads within a ray is, for example: Further examples of arrays that can be used in the present invention are described in U.S. Patent No. 6,524,793. Examples include U.S. Patents 5,429,807; 5,436,327; 5,561,071; 5,583,211;5,658,734;5,837,858;5,874,219; 5,919,523;6,136,269;6,287,768;6,287,776; 6,288,220;6,297,006;6,291,193;6,346,413; Nos. 6,416,949; 6,482,591; 6,514,751 and 6,610,482 ; and WO93 / 17126; WO95 / 11995; WO95 / 35505; EP7 42287; and those described in EP799897. It will not be done.

[0021] As used herein, the term "covalently attached" "covalently bonded" or "covalently bonded" means that the bond between atoms refers to the formation of chemical bonds characterized by the sharing of electron pairs at the The coating provides a more effective surface binding than other means, such as adhesion or electrostatic interactions. In comparison, it refers to a polymer coating that forms a chemical bond with the functionalized surface of the substrate. Polymers covalently attached to surfaces can also be attached via means in addition to covalent bonds. It will be understood that.

[0022] As used herein, an "R" group represents a substituent that may be attached to the indicated atom. The R group may be substituted or unsubstituted. When two "R" groups are "bonded together," When described as forming a ring or ring system "together with the atoms to which it is bonded," the atoms, intervening bonds, In this case, the collective unit of the two R groups is the recited ring. For example, Substructures exist: [ka] And R 1 and R 2 is defined as being selected from the group consisting of hydrogen and alkyl or R 1 and R 2 together with the atom to which they are attached, When defined as forming a cyclyl, R 1 and R 2 is hydrogen or alkyl or the substructure has the structure: [ka] wherein A is an aryl ring or carbocyclyl containing the depicted double bond.

[0023] Certain radical naming conventions refer to either monoradicals or diradicals, depending on the context. For example, it is understood that a substituent may include two bonds to the remainder of the molecule. Where a point of attachment is required, it is understood that the substituent is a diradical. For example, two Substituents identified as alkyl requiring a point of attachment include -CH2-, -CH2CH2 This includes diradicals such as - and -CH2CH(CH3)CH2-. Other Radical Naming Rules clearly indicates that the radical is a diradical such as "alkylene" or "alkenylene". vinegar.

[0024] As used herein, the term "halogen" or "halo" refers to any of the radiative elements in column 7 of the periodic table of the elements. means any one of the linearly stable atoms, for example, fluorine, chlorine, bromine, or iodine; Fluorine and chlorine are preferred.

[0025] As used herein, "C" is a set of integers where "a" and "b" are integers. a ~C b " is Al The number of carbon atoms in the alkyl, alkenyl or alkynyl group, or the number of carbon atoms in the cycloalkyl or aryl refers to the number of ring atoms in the alkyl group, i.e., alkyl, alkenyl, alkynyl, cycloalkane, The chiral ring and the aryl ring can contain "a" to "b" carbon atoms. For example, a "C1-C4 alkyl" group refers to any alkyl group having from 1 to 4 carbon atoms. , i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)- and (CH3)3C-; C3-C4 cycloalkyl groups include all cyclopropanol groups having 3 to 4 carbon atoms. Similarly, "4- to 6-membered heterocyclic groups" refers to cyclopropyl and cyclobutyl groups. "Heterocyclyl" groups include all heterocyclyl groups having a total of 4 to 6 ring atoms, e.g. , azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, molar Refers to alkyl, alkenyl, alkynyl, cycloalkyl, or aryl If "a" and "b" are not specified for a group, they are as defined in these definitions. The broadest range is envisaged. As used herein, the term "C1-C6" means C1, C2, C3, C4, C5 and C6, and defined by one of two numbers For example, C1-C6 alkyl includes C1, C2, C3, C4, C5 and and C6 alkyl, C2-C6 alkyl, C1-C3 alkyl, etc. Similarly, C2-C 6Alkenyl includes C2, C3, C4, C5 and C6 alkenyl, C2-C5 alkenyl C2-C6 alkynyl includes C2, C3, C4, C5 and C6 alkenyl. and C6 alkynyl, C2-C5 alkynyl, C3-C4 alkynyl, etc. 8 Cycloalkyl is 3, 4, 5, 6, 7, and 8 carbon atoms, or C3-C7 Defined by one of two values, such as cycloalkyl or C5-C6 cycloalkyl The hydrocarbon rings include those in the range.

[0026] As used herein, "alkyl" refers to a group that is fully saturated (i.e., has no double bonds or An alkyl group is a straight or branched hydrocarbon chain containing 1 to 20 carbon atoms. (wherever it appears in this specification, it may have a number such as "1 to 20" Ranges refer to each integer within the specified range; for example, "1 to 20 carbon atoms" refers to an alkyl group. The alkyl group may be 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms. However, this definition also applies to occurrences of the term "alkyl" where no numerical range is specified. The alkyl group may also be a medium-sized alkyl group having 1 to 9 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group may also be designated as "C1-C4 alkyl" or similar designations. For example, "C1-C6 alkyl" means that there are 1 to 6 carbon atoms in the alkyl chain. That is, the alkyl chain is methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, It indicates that the butyl group is selected from the group consisting of isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, and isobutyl. Examples of aryl include, but are not limited to, butyl, tertiary butyl, pentyl, hexyl, and the like. stomach.

[0027] As used herein, "alkoxy" refers to a group in which R is alkyl as defined above. It refers to the formula -OR, for example, "C1-C9 alkoxy", and includes methoxy, ethoxy, n-propoxy, propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, Examples include, but are not limited to, sec-butoxy, and tert-butoxy.

[0028] As used herein, "alkenyl" refers to any straight or branched chain alkyl group containing one or more double bonds. An alkenyl group can have from 2 to 20 carbon atoms, but this definition includes Occurrences of the term "alkenyl" where no numerical range is specified are also covered. It may also be a medium-sized alkenyl having 2 to 9 carbon atoms. or a lower alkenyl having 2 to 6 carbon atoms. It may also be designated as "C2-C6 alkenyl" or similar designations. By way of example only, "C2-C6 alkenyl" means that there are 2 to 6 carbon atoms in the alkenyl chain, i.e. That is, the alkenyl chain is ethenyl, propen-1-yl, propen-2-yl, propen- 3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl , 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl- 1-phenyl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1 1,2-dien-4-yl, and buta-1,2-dien-4-yl. Typical alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, and the like. Examples of alkyl groups include, but are not limited to, hexyl, hexenyl, and the like.

[0029] As used herein, "alkynyl" refers to a straight or branched chain alkyl group containing one or more triple bonds. An alkynyl group can have from 2 to 20 carbon atoms, but this definition includes: Occurrences of the term "alkynyl" where no numerical range is specified are also covered. The alkynyl group may also be a medium-sized alkynyl having 2 to 9 carbon atoms. It can be a lower alkynyl having 2 to 6 carbon atoms. The alkynyl group is defined as "C2 alkynyl" or similar designations. -C6 alkynyl" means that there are 2 to 6 carbon atoms in the alkynyl chain, i.e., The alkynyl chain is ethynyl, propyn-1-yl, propyn-2-yl, or butyn-1-yl , butyn-3-yl, butyn-4-yl, and 2-butynyl Typical alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, and alkynyl groups. Examples of alkynyl include, but are not limited to, cyclohexyl, cyclohexyl, and cyclohexyl.

[0030] As used herein, "heteroalkyl" refers to a group containing one or more heteroatoms, i.e., , straight chains containing elements other than carbon in the chain backbone, including, but not limited to, nitrogen, oxygen, and sulfur Heteroalkyl groups refer to chain or branched hydrocarbon chains. Heteroalkyl groups can have 1 to 20 carbon atoms. However, this definition also covers occurrences of the term "heteroalkyl" where no numerical range is specified. Heteroalkyl groups also include medium-sized heteroalkyl groups having 1 to 9 carbon atoms. Heteroalkyl groups can also be lower heteroalkyl groups having 1 to 6 carbon atoms. A heteroalkyl group can be a "C1-C6 heteroalkyl" or similar. A heteroalkyl group may contain one or more heteroatoms. By way of example only, "C4-C6 heteroalkyl" means a heteroalkyl group having 4 to 6 carbon atoms in the heteroalkyl chain. atom and one or more heteroatoms in the backbone of the chain.

[0031] The term "aromatic" refers to a ring or ring system having a conjugated pi-electron system, including carbocyclic aromatics. This includes both aromatic (e.g., phenyl) and heteroaromatic (e.g., pyridine) groups. The term refers to monocyclic or fused-ring polycyclic (i.e. , including rings sharing adjacent pairs of atoms).

[0032] As used herein, "aryl" refers to an aromatic ring or rings containing only carbon in the ring backbone. Refers to a ring system (i.e., two or more fused rings sharing two adjacent carbon atoms). If the group is a ring system, all rings in the system are aromatic. Aryl groups are groups containing 6 to 18 carbon atoms. Although the term "aryl" may have a numerical range, this definition applies to occurrences of the term "aryl" where no numerical range is specified. In some embodiments, the aryl group has 6 to 10 carbon atoms. The aryl group is a group having C6 to C 10 aryl," "C6 or C 10 aryl" or the same Examples of aryl groups include, but are not limited to, phenyl ... Examples include aryl, naphthyl, azulenyl, and anthracenyl.

[0033] "Aralkyl" or "arylalkyl" refers to the "C 7-14 "Aralkyl" The aryl group is bonded to an alkylene group as a substituent, and is preferably benzyl, 2-phenyl, or 2-phenyl. phenylpropyl, 3-phenylpropyl, and naphthylalkyl. In some cases, an alkylene group may be joined to a lower alkylene group (i.e., C1 to C6 alkylene group).

[0034] As used herein, "heteroaryl" refers to a group containing one or more heteroatoms, i.e., Elements other than carbon, including but not limited to nitrogen, oxygen, and sulfur in the ring structure an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing When heteroaryl is a ring system, each ring in the system is aromatic. The alkyl group has 5 to 18 ring members (i.e., a ring skeleton including carbon atoms and heteroatoms). Although the term "heteroatom" may have any number (number of atoms constituting the molecule), this definition applies to the term "heteroatom" where no numerical range is specified. In some embodiments, the heteroaryl group is a 5-membered heteroaryl group. The heteroaryl group has from 1 to 10 ring members or from 5 to 7 ring members. "5- to 10-membered heteroaryl," or similar designations. Examples of heteroaryl rings include furyl, thienyl, phthalazinyl, pyrrolyl, and aryl. Xazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl , triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isopropyl Examples include, but are not limited to, androyl, and benzothienyl.

[0035] A "heteroaralkyl" or "heteroarylalkyl" is a heteroaryl group that has an alkyl group as a substituent. Examples include 2-thienylmethyl, 3 ... -Thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl alkyl, isoxazolyl, and imidazolyl. In some cases, an alkylene group may be a lower alkylene group (i.e., , C1 to C6 alkylene group).

[0036] As used herein, "carbocyclyl" refers to a non-carbon atom containing only carbon atoms in the ring system backbone. means an aromatic cyclic ring or ring system. When a carbocyclyl is a ring system, two or more rings may be At least one of the ring systems may be joined together in a fused, bridged, or spiro-connected fashion. A carbocyclyl may have any degree of saturation, as long as one or more rings is not aromatic. Thus, carbocyclyl includes cycloalkyl, cycloalkenyl, and cycloalkyl. The carbocyclyl group may have 3 to 20 carbon atoms. However, this definition also covers occurrences of the term "carbocyclyl" where no numerical range is specified. Carbocyclyl groups also include medium-sized carbocyclyl groups having 3 to 10 carbon atoms. The carbocyclyl group may also be a carbocyclyl group having 3 to 6 carbon atoms. A carbocyclyl group may be designated "C3-C6 carbocyclyl" or similar designations. Examples of carbocyclyl rings include cyclopropyl, cyclobutyl, and the like. , cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, Bicyclyl[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl These include, but are not limited to:

[0037] As used herein, "cycloalkyl" refers to a fully saturated carbocyclyl ring. or ring systems. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0038] As used herein, a "heterocyclyl" refers to a group having at least one heterocyclic group in the ring skeleton. Heterocyclyl means a non-aromatic cyclic ring or ring system containing fused, bridged, or may be joined together in a spiro bond manner. A heterocyclyl is one in which at least one of the ring systems The ring may have any degree of saturation, provided that it is not aromatic. The heterocyclyl group may be present in either the aromatic or aromatic ring. The number of ring members (i.e., the number of atoms that make up the ring structure, including carbon atoms and heteroatoms) However, this definition also covers occurrences of the term "heterocyclyl" where no numerical range is specified. The heterocyclyl group is also a medium-sized heterocyclyl having 3 to 10 ring members. The heterocyclyl group may also be a heterocyclyl having 3 to 6 ring members. Heterocyclyl groups may also be designated as "3- to 6-membered heterocyclyl" or similar designations. In preferred 6-membered monocyclic heterocyclyls, the heteroatom is O, N, or S. In preferred 5-membered monocyclic heterocyclyls, the heteroatom is selected from one to three of One or two heteroatoms are selected from O, N, or S. Examples of heterocyclyl rings Examples include azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, and iodopropyl. Midazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepa piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, 4-piperidinyl Donyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl , 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-o Xathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl , hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxo Ranyl, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolyl thiazolinyl, ... Zolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydro tetrahydrofuran, tetrahydropyranyl, tetrahydroiophenyl, tetrahydrothiopyranyl tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline. I can't.

[0039] An "O-carboxy" group refers to a group in which R is hydrogen, C1-C6 alkyl, as defined herein. , C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C 10 aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl; It refers to the "-OC(=O)R" group.

[0040] A "C-carboxy" group refers to a group in which R is hydrogen, C1-C6 alkyl, as defined herein. , C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carbocyclyl, C6-C 10 The group consisting of aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl Non-limiting examples include carboxyl (i.e., i.e., -C(=O)OH).

[0041] A "sulfonyl" group is a group in which R is hydrogen, C1-C6 alkyl, C 2~C6 alkenyl, C2~C6 alkynyl, C3~C7 carbocyclyl, C6~C 10 selected from aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl Refers to the "-SO2R" group.

[0042] A "sulfino" group refers to a "-S(=O)OH" group.

[0043] An "S-sulfonamide" group is a group consisting of R A and R B are each independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- C7 carbocyclyl, C6-C 10 Aryl, 5-10 membered heteroaryl, and 3-1 0-membered heterocyclyl, "-SO2NR A R B " refers to the group.

[0044] An "N-sulfonamide" group is R A and R b are each independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- C7 carbocyclyl, C6-C10 Aryl, 5-10 membered heteroaryl, and 3-1 0-membered heterocyclyl, "-N(R A )SO2R B " refers to the group.

[0045] A "C-amide" group is a group consisting of R A and R B are each independently hydrogen, as defined herein , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 chlorine Bocyclyl, C6-C 10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heteroaryl -C(=O) selected from cyclocyclyl N R A R B " refers to the group.

[0046] An "N-amido" group is an R A and R B are each independently hydrogen, as defined herein , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 chlorine Bocyclyl, C6-C 10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heteroaryl -N(R A )C(=O)R B " refers to the group.

[0047] An "amino" group is R A and R B are each independently hydrogen, as defined herein, C 1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 carboxy Krill, C6~C 10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heteroaryl Selected from Krill, "-NR A R B " group. Non-limiting examples include free amino ( i.e., -NH2).

[0048] An "aminoalkyl" group refers to an amino group connected via an alkylene group.

[0049] The "alkoxyalkyl" group is an alkyl group such as "C2-C8 alkoxyalkyl". It refers to an alkoxy group bonded via an alkylene group.

[0050] As used herein, a substituent refers to a group in which one or more hydrogen atoms have been replaced by another atom or group. Unless otherwise specified, when a group is considered to be "substituted," In this case, the group is selected from C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C 1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally halo, C1-C6 alkyl , C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy substituted), C3-C7-carbocyclyl-C1-C6-alkyl (optionally halo, C1-C6 Alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloal substituted with 3-10 membered heterocyclyl-C1-C6-alkyl (optionally halo, C1 -C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 halo aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy), substituted with oxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl ( C1-C6) alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy , C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5-10 membered heteroaryl alkyl (optionally halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy) aryl, and C1-C6 haloalkoxy), 5-10 membered heteroaryl (C1-C6 ) alkyl (optionally halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 halo alkyl, and C1-C6 haloalkoxy), halo, -CN, hydroxy, C1 -C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether ), aryloxy, sulfhydryl (mercapto), halo (C1-C6) alkyl (e.g. , -CF3), halo(C1-C6)alkoxy (e.g., -OCF3), C1-C6 alkyl Thio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamyl , N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido , S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, acyl , cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl -H, -SO3H, -Sulfino, -OSO2C1-4 alkyl, and oxo (=O) When a group is described as "optionally substituted," it means that the group is substituted with one or more substituents selected from among the substituents. Wherever "substituted" is described, the group may be substituted with the substituents described above. do.

[0051] As used herein, the term "hydroxy" refers to an --OH group.

[0052] As used herein, the term "cyano" group refers to a "CN" group.

[0053] As used herein, the term "azido" refers to the group --N3.

[0054] As used herein, a "nucleotide" includes a nitrogenous heterocyclic base, a sugar, and one or more amino acids. They are the monomeric units of nucleic acid sequences. In RNA, sugars are ribonucleotides. In DNA, it is deoxyribose, which means it does not have the hydroxyl group found in ribose. The nitrogen-containing heterocyclic base may be a purine or pyrimidine base. contains adenine (A) and guanine (G), and their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U). and their modified derivatives or analogs. The C-1 atom of deoxyribose is , which binds to N-1 of pyrimidines or N-9 of purines.

[0055] As used herein, a "nucleoside" is structurally similar to a nucleotide, but The phosphate moiety is missing. Examples of nucleoside analogs are those in which the label is attached to the base and the phosphate group is As used herein, the term "nucleoside" refers to a nucleoside that is not attached to a sugar molecule. It is used in its ordinary sense as understood by those skilled in the art. Examples include ribonucleosides containing a ribose moiety. Deoxyribonucleosides include those containing deoxyribose and deoxyribose moieties, Modified pentose moieties include, but are not limited to, those in which an oxygen atom is replaced with a carbon and / or or a pentose moiety in which a carbon atom is replaced by a sulfur or oxygen atom. A "side" is a monomer that can have a substituted base and / or sugar moiety. Additionally, nucleosides can be synthesized into larger DNA and / or RNA polymers and oligomers. It can be incorporated into

[0056] As used herein, the term "purine base" is used in its ordinary sense as understood by those skilled in the art. Similarly, the term "pyrimidine base" is understood by those skilled in the art to include tautomers thereof. As used herein, the term "substituted or unsubstituted" means a substituted or unsubstituted hydroxyl group, and includes tautomers thereof. A non-limiting list of phosphorus bases includes purine, adenine, guanine, hypoxanthine, oxalyl guanine, alloxanthin, 7-alkylguanine (e.g., 7-methylguanine), theo Examples of pyrimidine bases include bromine, caffeine, uric acid, and isoguanine. Cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine ( For example, 5-methylcytosine) but are not limited to these.

[0057] As used herein, an oligonucleotide or polynucleotide is defined herein as When it is said that a nucleoside or nucleotide is "comprised," it is meant to be a nucleotide as described herein. The nucleoside or nucleotide of Similarly, a nucleoside or a nucleotide can form an oligonucleotide. "incorporated" into a nucleotide or polynucleotide, or When described as part of a polynucleotide, the nucleosides or nucleoside groups described herein The nucleotide is capable of forming a covalent bond with an oligonucleotide or polynucleotide. In some such embodiments, the covalent bond is between the oligonucleotide or 3' hydroxyl group of the oligonucleotide or polynucleotide and It is formed as a phosphodiester bond between the 3' carbon atom of the nucleotide and the 5' carbon atom of the nucleotide. It is formed between the 5' phosphate group of the nucleotide described herein.

[0058] As used herein, a "derivative" or "analog" refers to a compound having a modified base moiety and / or or modified sugar moieties. Such derivatives and analogs are described, for example, in Scheit, Nucleotide A nalogs (John Wiley & Son, 1980) and Uhlm an et al., Chemical Reviews 90:543-584, 1990. Phosphorodithioates, alkylphosphonates, phosphoryl Anilidate and phosphoramidate bonds. "Nucleotide" and "modified" are used interchangeably and are used interchangeably with "nucleotide" as defined herein. and "nucleoside."

[0059] As used herein, the term "phosphate" is used in its ordinary sense as understood by those skilled in the art. and its protonated forms (e.g., [ka] As used herein, the terms "monophosphate," "diphosphate," and "triphosphate" is used in its ordinary sense as understood by those skilled in the art and includes protonated forms.

[0060] As used herein, the terms "protecting group" and "protecting group" refer to a group that is present in a molecule and is protected from Any atom or group of atoms added to a molecule to prevent it from undergoing an undesired chemical reaction The terms "protecting group" and "protecting group" are sometimes used interchangeably.

[0061] As used herein, the prefix "photo" or "photo-" refers to light or electromagnetic This term refers to radio waves, microwaves, infrared, visible, ultraviolet, X-rays, or one or more categories commonly known as the gamma-ray portion of the spectrum This may include all or part of the electromagnetic spectrum, including but not limited to Portions of the spectrum are blocked by metallic regions on the surface, such as the metals described herein. Alternatively or additionally, part of the spectrum may be glass, plastic, through interstitial regions of a surface, such as regions made of silicon dioxide, silica, or other materials described herein; In certain embodiments, radiation that can pass through metals can be used. Alternatively or additionally, the substrate may be made of glass, plastic, silica, or any other material described herein. Material-masked radiation can be used.

[0062] As used herein, the term "phasing" refers to the 3' terminator and Incomplete removal of the fluorophore and polymerase chain reaction in a given sequencing cycle This is caused by the failure of the enzyme to complete the incorporation of some of the DNA strands within the cluster. This refers to the phenomenon of SBS. Prephasing is the process by which nucleosides lack effective 3' terminators. An incorporation event is triggered by a failed termination. Fading and pre-fading reduce the signal measured in a particular cycle. The signal strength is composed of the signal from the current cycle and the noise from the previous and next cycles. As the number of cycles increases, the number of cycles per cluster affected by fading and pre-fading increases. Prephasing increases the proportion of sequences that are not identical, preventing the identification of the correct base. During sequencing (SBS), unprotected or unblocked 3'- This may be caused by the presence of trace amounts of OH nucleotides. Unreacted 3'-OH nucleotides are used during the manufacturing process or, in some cases, during storage and testing. It may be generated during the drug treatment process. The discovery of a nucleotide analogue that reduces For example, the nucleotides provided The analogues have faster SBS cycle times, lower fading and pre-phasing values. , and may result in longer sequence read lengths.

[0063] (3'-hydroxyacetal blocking group) Some embodiments of the present disclosure provide a structure covalently attached to the 3'-carbon atom. [ka] Ribose or deoxyribonucleotides with a removable 3'-OH protecting or blocking group to form A nucleotide or nucleoside molecule containing a base, wherein: Each R 1a and R 1b are independently H, C1-C6 alkyl, C1-C6 haloalkyl C1-C6 alkoxy, C1-C6 haloalkoxy, cyano, halogen, optionally substituted phenyl, or optionally substituted aralkyl; Each R 2a and R 2b are independently H, C1-C6 alkyl, C1-C6 haloalkyl , cyano, or halogen; Alternatively, R 1a and R 2aalong with the atoms to which they are attached, if necessary Forming a substituted 5- to 8-membered heterocyclyl group; R 3 is H, optionally substituted C2-C6 alkenyl, optionally substituted C3-C7 silyl chloroalkenyl, optionally substituted C2-C6 alkynyl, or optionally substituted (C 1-C6 alkylene)Si(R 4 )3; and Each R 4 are independently H, C1-C6 alkyl, or optionally substituted C6-C 10 a reel; however, each R 1a , R 1b , R 2a , and R 2b If H, then R 3 is H isn't it, Pertaining to a nucleotide or nucleoside molecule.

[0064] Some further embodiments of the present disclosure include a compound having the structure of formula (I): [ka] wherein R' is H, a monophosphate, a diphosphate, a triphosphate, a thiophosphate, phosphate ester analogs, -O- attached to reactive phosphorus-containing groups, or protected by protecting groups protected -O-; R'' is H or OH; B is a nucleobase; R 1a , R 1b , R 2a , R 2b , and R 3 Each of the above relates to a compound, as defined above. In some further embodiments, B is [ka] In some further embodiments, the nucleobase is and covalently attached to a detectable label (e.g., a fluorescent dye), e.g., B is [ka] In some such embodiments, R' is triphosphate. In such embodiments, R'' is H.

[0065] In some embodiments of the acetal blocking groups described herein, R 1a and R 1b At least one of is H. In some such embodiments, each R 1a Oh BiR 1b is H. In some other embodiments, R 1a and R 1b At least one of One is C1-C6 alkyl, for example, methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R 2a and R 2b Each of these is independently H, halogen In some such embodiments, R 2a and R 2b At least one of is H or C1-C6 alkyl. In such embodiments, each R 2a and R 2b is H. Some such In the embodiment, each R 2a and R 2b is C1-C6 alkyl, for example, methyl, ethyl, isopropyl or t-butyl. In one embodiment, each R 2a and R 2b is methyl In some such embodiments, each R 2a and R 2b is, independently, In some such embodiments, R 2a is H and R 2b is halogen or C1-C6 alkyl.

[0066] In some embodiments of the acetal blocking groups described herein, R 3 teeth, and optionally substituted C2-C6 alkenyl. Hey, R 3 is halogen, C1-C6 alkyl, C1-C6 haloalkyl, and the like. and combinations thereof. C2-C6 alkenyl (e.g., vinyl, propenyl). In terms of form, R 3 teeth [ka] In some other embodiments, R 3 is C2 to C6 substituted as needed In some such embodiments, R is alkynyl. 3 are halogens, C1~ Independently from the group consisting of C6 alkyl, C1-C6 haloalkyl, and combinations thereof C2-C6 alkynyl (e.g., ethoxylated) optionally substituted with one or more substituents selected from the following: In one embodiment, R 3 is an optionally substituted ethyni Lu( [ka] In some other embodiments, R 3 is optionally substituted (C1-C6 alkyl alkylene)Si(R 4)3. In some such embodiments, R 4 Few At least one C 1-4 In some further embodiments, R 4 Noso Each is C1-C4 alkyl, for example, methyl, ethyl, isopropyl or t-butyl. In one embodiment, R 3 is -(CH2)-SiMe3. Some alternatives In an embodiment of the present invention, R 3 is a C1-C6 alkyl.

[0067] In some alternative embodiments, R 1a and R 2a are the atoms to which they are bound. Together with the alkyl group, they form a 5- to 7-membered heterocyclyl. R 1a and R 2a together with the atoms to which they are attached form a 6-membered heterocyclyl In some such embodiments, the 6-membered heterocyclyl group forms the structure [ka] In some further embodiments, each R 1b , R 2b and R 3 At least One is H. In some other embodiments, each R 1b , R 2b and R 3 At least and the other is C1-C6 alkyl. In one embodiment, each R 1b , R 2b and R 3 is H is.

[0068] In some further embodiments, the compound of formula (I) may also be represented by formula (Ia): Represented: [ka] In the formula, each R 2c and R 2d are independently H, halogen (e.g., fluoro, chloro) , C1-C6 alkyl (e.g., methyl, ethyl, or isopropyl), or C1- C haloalkyl (e.g., -CHF2, -CH2F, or -CF3). In some such embodiments, R 1a and R 1b One of them is H. In such an embodiment, each R 1a and R 1b is H. In some other embodiments, R 1a and R 1b At least one of the groups is C1-C6 alkyl, for example, methyl, ethyl, iso propyl or t-butyl. In some embodiments, R 2a and R 2b That Each is independently H, halogen, or C1-C6 alkyl. In such an embodiment, each R 2a and R 2b is H. Some such embodiments So, R 2c and R 2d each independently represents H, a halogen, or a C1-C6 alkyl group. In some such embodiments, each R 2c and R 2d is C1~ C alkyl, for example, methyl, ethyl, isopropyl, or t-butyl. In terms of form, each R 2c and R 2d is methyl. In some such embodiments, Each R 2c and R 2dis independently halogen. In some such embodiments, R 2c is H and R 2d is H, halogen (fluoro, chloro) or C1-C6 alkyl (e.g., methyl, ethyl, isopropyl, or t-butyl). In this state, each R 1a and R 1b is H;R 2a is H;R 2b H, Haloge R is methyl or methyl; 2c is H;R 2d is H, halogen, methyl, ethyl, isopropyl, or t-butyl.

[0069] Non-limiting embodiments of the blocking groups described herein are selected from the group consisting of: These include those with structures that: [ka] , covalently attached to the 3' carbon of ribose or deoxyribose.

[0070] (3'-hydroxythiocarbamate blocking group) Some additional embodiments of the present disclosure provide a structure covalently attached to the 3'-carbon atom. [ka] Contains a ribose or deoxyribose with a removable 3'-OH blocking group to form A nucleoside or nucleotide, wherein: R 5 and R 6 each independently represents H, C1-C6 alkyl, C2-C6 alkenyl C2-C6 alkynyl, C1-C6 haloalkyl, C2-C8 alkoxyalkyl, optionally substituted -(CH2) m-phenyl, optionally substituted -(CH2) n -(5 is a 6-membered heteroaryl), optionally substituted -(CH2) k -C3-C7 carboxylic acids -aryl, or optionally substituted -(CH2) p -(3- to 7-membered heterocyclyl) ; Alternatively, R 5 and R 6 are substituted, along with the atoms to which they are attached. Forms a good 5- to 7-membered heterocyclyl; -(CH2) m -, -(CH2) n -, -(CH2) k - and -(CH2) p -of each of which is optionally substituted; and each of m, n, k, and p is independently 0, 1, 2, 3, or 4; Pertaining to nucleosides or nucleotides.

[0071] Some additional embodiments relate to compounds of formula (II): [ka] where R' is H, monophosphate, diphosphate, triphosphate, thiophosphate, phosphate ester analogs, -O- attached to a reactive phosphorus-containing group or -O- protected by a protecting group; R '' is H or OH; B is a nucleobase; R 5 and R 6 Each of the is defined above In some further embodiments, B is [ka] In some further embodiments, the nucleobase is and covalently attached to a detectable label (e.g., a fluorescent dye), e.g., B is [ka] In some such embodiments, R' is triphosphate. In such embodiments, R'' is H.

[0072] In some embodiments of the thiocarbamate blocking groups described herein, R 5 oh Yobi R 6 At least one of is H. In some such embodiments, each R 5 Oh BiR 6 is H. In some such embodiments, R 5 is H and R 6 is C1~ C6 alkyl, for example, methyl, ethyl, isopropyl, or t-butyl. In some such embodiments, R 5 is H and R 6 is C2-C6 alkenyl ( vinyl or allyl) or C2-C6 alkynyl (e.g., ethynyl or propyl) In some such embodiments, R 5 is H and R 2 teeth, optionally substituted -(CH2) m -phenyl, optionally substituted -(CH2) n -(5 or 6-membered heteroaryl), optionally substituted -(CH2) k -C3~C7 Carbocycle or optionally substituted -(CH2) p -(3- to 7-membered heterocyclyl). Some In a further embodiment, the C3-C7 carbocyclyl group is a C3-C7 cycloalkyl group. The 3- to 7-membered heterocyclyl group can be a C3-C7 cycloalkenyl. The structure may contain zero or one double bond. 5 is H R 6 is an optionally substituted -(CH2) m -phenyl, optionally substituted -(CH ) n -6-membered heteroaryl, optionally substituted -(CH2) k -C5 or C6 carboxylate Cryl, or optionally substituted -(CH2) p -(5- or 6-membered heterocyclyl). In some embodiments, m, n, k, or p is 0. In other embodiments, m, n , k or p is 1 or 2. In some other embodiments, R 5 and R 6 Few At least one is C1-C6 alkyl, for example, methyl, ethyl, isopropyl or is t-butyl. In some further embodiments, R 5 and R 6 Both are C1~ C alkyl. In one embodiment, R 5 and R 6 Both of the are methyl.

[0073] In some alternative embodiments, R 5 and R 6 are the atoms to which they are bonded. Together they form an optionally substituted 5- to 7-membered heterocyclyl. In embodiments, R 5 and R 6 along with the atoms to which they are attached, if necessary Forming a substituted piperidinyl.

[0074] Non-limiting embodiments of the 3'-O-thiocarbamate blocking groups described herein include , including those having a structure selected from the group consisting of: [ka] It is covalently attached to the 3' carbon of ribose or deoxyribose.

[0075] Additional embodiments of the present disclosure include nucleosides or nucleotides described herein. Concerning oligonucleotides or polynucleotides.

[0076] In any of the embodiments of the blocking group described herein, the group may be "optionally substituted When described as "substituted," it can be either unsubstituted or substituted.

[0077] A nucleotide or nucleoside having a 3' hydroxy blocking group as described herein In any embodiment of the invention, the nucleoside or nucleotide may optionally be linked to a linker. The linker may be covalently attached to a detectable label (e.g., a fluorophore) via a In some such embodiments, the detection Possible labels (e.g., fluorophores) are attached to the nucleoside or or is covalently attached to the nucleobase of a nucleotide. A cleavable label (e.g., a fluorophore) is attached to the nucleoside or is covalently attached to the 3' oxygen of the nucleotide. Such cleavable linkers may include an azide moiety, a disulfide moiety, an acetal moiety, In some embodiments, the 3' hydroxyl group may comprise a 3' hydroxyl group, ... The blocking group and the cleavable linker (and attached label) may be the same or substantially the same. can be removed under the same chemical reaction conditions, e.g., blocking groups and detectable labels can be , can be removed in a single chemical reaction. Possible labels are removed in two separate steps.

[0078] In some embodiments, the nucleotides or nucleosides described herein In some further aspects, the 2' deoxyribose The sugars contain one, two, or three phosphate groups at the 5' position of the sugar ring. In some embodiments, the nucleotides described herein are nucleotide triphosphates.

[0079] In some embodiments, the 3' blocked nucleotides or nucleotides described herein The nucleotides were the same as those protected with standard 3'-OH blocking groups disclosed in the prior art. Compared to nucleotides or nucleosides, the solution or sequencing application It provides excellent stability in handling reagents during the process. Methyl protecting groups, such as acetal or thiocarbamate protecting groups disclosed herein. The 3'-OH of the AK group was protected with azidomethyl under the same conditions for the same period. Compared to at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% ,80%,90%,100%,200%,300%,400%,500%,600%,7 00%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, and can provide 3000% improved stability, which reduces pre-phase values and improves sequencing. In some embodiments, stability is measured at ambient temperature or ambient temperature. In another embodiment, stability is measured at a temperature lower than the normal temperature (e.g., 4-10°C). It is measured at high temperatures such as 40°C, 45°C, 50°C, 55°C, 60°C or 65°C. In such embodiments, stability is achieved in a basic pH environment, e.g., pH 9.0, 9.2, It is measured in solutions of 9.4, 9.6, 9.8 or 10.0. In embodiments, the stability is enhanced by the ability to react with a polymerase (e.g., a DNA polymerase), terminal deoxygenation, Regardless of the presence or absence of enzymes such as synucleotidyltransferase or reverse transcriptase It is measured without

[0080] In some embodiments, the 3' blocked nucleotides or nucleotides described herein The nucleotides were then reacted with the same nucleic acid protected with a standard 3'-OH blocking group as disclosed in the prior art. Chemical cleavage for sequencing applications compared to nucleotides or nucleosides Provides excellent deblocking speed in solution during the step. For example, 3'-O-azide Methyl protecting groups, such as acetal or thiocarbamate protecting groups disclosed herein. The blocking group can be deblocked using standard deblocking reagents (e.g., tris(hydroxypropyl)phosphine). At least 5%, 10%, 20%, 30% or more of the azidomethyl-protected 3'-OH %, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% , 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000 %, 1500%, or 2000% improved deblocking rate, thereby improving the In some embodiments, the deblocking rate is increased by 100%. is measured at ambient or subambient temperatures (e.g., 4-10°C). In embodiments, the deblocking rate is 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, the deblocking rate is measured at elevated temperatures, such as at 100°C. Basic pH environment, e.g., pH 9.0, 9.2, 9.4, 9.6, 9.8, or 10.0 In some such embodiments, the deblocking reagent and the substrate (i.e., That is, the molar ratio of 3'-block nucleoside or nucleotide) is about 10:1, about 5: 1, about 2:1, or about 1:1.

[0081] In some embodiments, a palladium deblocking reagent (e.g., Pd(0)) is used. , removes the 3' acetal blocking group (e.g., AOM blocking group). The two oxygen atoms of the AOM group can also form chelate complexes. This allows removal of the deblocking reagent in close proximity to the functional group, accelerating the deblocking rate. It can be made faster.

[0082] (Deprotection of 3'-OH blocking group) The 3'-acetal blocking groups described herein can be removed or deprotected under a variety of chemical conditions. Acetal blocking groups containing vinyl or alkenyl moieties can be cleaved. [ka] In the case of ) in the presence of Pd(OAc)2 or allylic Pd(II) chloride dimer. II) complexes. ) phosphines (THP or THPP). Those containing alkynyl groups (e.g., ethynyl). For these blocking groups, they also contain phosphine ligands (e.g., THP or or THMP) in the presence of a Pd(II) complex (e.g., Pd(OAc)2 or allyl It can be removed by Pd(II) chloride dimer.

[0083] (Palladium cleavage reagent) In some embodiments, the acetal blocking groups described herein are palladium In some such embodiments, the Pd catalyst is water soluble. In some such embodiments, a Pd(0) complex (e.g., tris(3,3 ',3''-Phosphine dinetris(benzenesulfonato)palladium(0) non-sodium In some cases, Pd(0) complexes are also formed with alkenes, alcohols, and alkyl groups. In situ synthesis from the reduction of Pd(II) complexes with reagents such as amines, phosphines and metal hydrides. Suitable palladium sources include Na2PdCl4, Pd(CH3CN)2Cl2, (PdCl(C3H5))2, [Pd(C3H5)(THP)]Cl, [Pd(C3H5) )(THP)2]Cl, Pd(OAc)2, Pd(Ph3)4, Pd(dba)2, Pd (Acac)2, PdCl2(COD), in one such embodiment, the Pd(0) complex is In another embodiment, the palladium source is alicyclic palladium. Palladium(II) chloride dimer [(PdCl(C3H5))2]. In some embodiments, the Pd(0) complex can be prepared by mixing a Pd(II) complex with a phosphine. Suitable phosphines include tris(hydroxypropyl)phosphine, Sphingolipids (THP), tris(hydroxymethyl)phosphine (THMP), 1,3, 5-Triaza-7-phosphaadamantane (PTA), bis(p-sulfonatophenyl) Phenylphosphine dihydrate potassium salt, Tris(carboxyethyl)phosphine (TC EP), and triphenylphosphine-3,3',3'-trisulfonic acid trisodium salt Water-soluble phosphines such as salts are included.

[0084] In some embodiments, Pd(0) is a Pd(II) complex [(PdCl(C3H5) )2] is prepared in situ by mixing with THP. The molar ratio of is about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or In some further embodiments, the ratio of ascorbic acid to HCl may be 1:10 or 1:10. Adding one or more reducing agents, such as an acid or its salt (e.g., sodium ascorbate) In some embodiments, the cleavage mixture may comprise a primary amine, a secondary amine, or both. amines, tertiary amines, carbonates, phosphates, or borates, or combinations thereof. In some further embodiments, the buffering reagent may comprise ethanol. ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, Sodium carbonate, sodium phosphate, sodium borate, 2-dimethylaminomethanol (DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-tetramethylbenzyl methyl ether Tetramethylethylenediamine (TEMED), or N,N,N',N'-tetraethylethylenediamine In one embodiment, the buffer solution contains: In another embodiment, the buffering agent is a carbonate, a phosphate, or a borate. In one embodiment, the inorganic salts include one or more inorganic salts, such as a salt, a salt, or a combination thereof. The salt is a sodium salt.

[0085] Alternatively, the alkynyl moiety containing the blocking group can be reacted with the alkynyl group in the presence of (NH4)2MoS4. Other non-limiting conditions for cleavage of the alkynyl moiety include the use of a THPTA ligand ( Cu(II) complex with bis(3-hydroxypropyltriazolylmethyl)amine, and and ascorbate. Brominated rings containing six-membered heterocycles (e.g., tetrahydropyran) Non-limiting conditions for cleaving the locking group include cyclodextrin or Ln(OTf)3( lanthanide triflates). Blocking groups containing alkylsilane groups (e.g. Non-limiting cleavage conditions for -CH2SiMe3 include LiBF4 (lithium tetrafluoroborate), Other acetal blocking groups such as -O(CH2)O-C1-C6 alkyl are also included. The carboxylic acid group can be removed by LiBF4 or Bi(OTf)3 (bismuth triflate). Non-limiting exemplary conditions for cleaving the various blocking groups described are as follows: This is shown in Scheme 1. Scheme 1. Example of 3'-deblocking conditions [ka]

[0086] The 3'-O-thiocarbamate blocking groups described herein are capable of blocking under a variety of chemical conditions. The thiocarbamate blocking groups described herein can be removed or cleaved by Non-limiting exemplary conditions for cleaving include NaIO4 and Oxone®. ) (potassium peroxymonosulfate).

[0087] Additionally, the azide group of -CH2N3 can be converted to an amino group with a phosphine. The azido group of -CH2N3 converts such molecules to thiols, especially dithiothreitol ( can be converted to an amino group by contact with a water-soluble thiol such as DTT In one embodiment, the phosphine is THP.

[0088] (Compatible with linearization) To maximize the throughput of nucleic acid sequencing reactions, multiple template molecules are used. Parallel processing of multiple templates is advantageous because it allows for sequencing of multiple templates in parallel. This can be achieved using nucleic acid array technology. These arrays are typically immobilized on a solid support material. It consists of a high-density matrix of purified polynucleotides.

[0089] Both WO98 / 44151 and WO00 / 18957 disclose a method for immobilizing multiple identical immobilized polymers. Clusters formed from polynucleotide strands and multiple identical immobilized polynucleotide strands The amplification products are immobilized on a solid support to form an array of clones or "colonies." This paper describes a method of nucleic acid amplification that allows multiple identical immobilized complementary strands to be detected. This type of array is referred to herein as a "clustered array." The nucleic acid molecules present in the DNA colonies on the clustered array prepared according to the method are, for example, For example, as described in WO98 / 44152, Plates can be provided. The products of a solid-phase amplification reaction as described are immobilized polynucleotide strands and immobilized complements. The so-called "bridge" structure formed by the annealing of pairs of strands, with both strands 5' Strong edge support provides a better template for nucleic acid sequencing To generate a template that is at least partially single-stranded, It is preferred to remove substantially all or at least part of one of the immobilized strands in the structure. Therefore, the portion of the template that is single-stranded is preferably ligated to the sequencing primer. The immobilization of one strand within a "bridged" double-stranded nucleic acid structure is available for hybridization. The process of removing all or part of a strand is called "linearization." Linearization can involve enzymatic There are various methods, including but not limited to cleavage, photochemical cleavage, or chemical cleavage. Non-limiting examples of linearization methods are described in PCT Publication No. WO2007 / 010251, U.S. Pat. Publication No. 2009 / 0088327, U.S. Patent Publication No. 2009 / 0118128, and and U.S. Patent Application No. 62 / 671,816, which are incorporated by reference in their entireties. It can be enjoyed.

[0090] In some embodiments, for deprotection or removal of the 3'-OH blocking group The conditions are also compatible with the linearization process. The protection conditions include the use of Pd complexes and phosphines, e.g., Pd(OAc)2 and THP. In some embodiments, the Pd complex is compatible with a chemical linearization process comprising: It is a Pd(II) complex that generates Pd(0) in situ in the presence of phosphine.

[0091] Unless otherwise specified, references to nucleotides also apply to nucleosides. This is the intention.

[0092] (labeled nucleotides) According to one aspect of the present disclosure, the described 3'-OH blocked nucleotides also can be used for detection Such nucleotides also contain a possible label, and are called labeled nucleotides. For example, fluorescent dyes) are attached to the surface of the molecule by a variety of means, including hydrophobic attraction, ionic attraction, and covalent bonding. , can be attached via an optional linker. In some aspects, the dye is conjugated to the substrate by a covalent bond. More specifically, the covalent bond is In some cases, such labeled nucleotides are also called "modified nucleotides." Called.

[0093] Labeled nucleotides can be used in a variety of applications, including, but not limited to, PCR amplification, isothermal amplification, solid-phase amplification, polynucleotide amplification, and the like. Nucleotide sequencing (e.g., solid-phase sequencing), nick translation It is useful for labeling polynucleotides formed by enzymatic synthesis in reactions etc. be.

[0094] In some embodiments, the dye is attached to the oligonucleotide via the nucleotide base. or covalently attached to a nucleotide. For example, a labeled nucleotide or oligonucleotide The oxide is attached to the C5 position of the pyrimidine base or the 7-deazapurine salt via the linker moiety. The group can have a label attached to the C7 position.

[0095] Unless otherwise specified, references to nucleotides also apply to nucleosides This application will also be further described with reference to DNA, but unless otherwise specified, the description will be in accordance with the R It is also applicable to NA, PNA, and other nucleic acids.

[0096] (Linker) In some embodiments described herein, the nucleotides or nucleotide sequences described herein may be The purine or pyrimidine base of the riboside molecule can be attached to a detectable label as described above. In some such embodiments, the linker used is cleavable. The use of a cleavable linker ensures that the label can be removed after detection, if desired, and subsequently Interfering signals with incorporated labeled nucleotides or nucleosides can be avoided. In this embodiment, the cleavable linker may be an azide moiety, —O—C2-C6 alkenyl moieties (e.g., -O-allyl), disulfide moieties, acetal moieties (as described herein) 3' acetal blocking group), or thiocarbamate A moiety (the same as or similar to the 3' acetal blocking group described herein).

[0097] In some other embodiments, the linker used is non-cleavable. In each instance where a nucleotide is incorporated, no subsequent nucleotides need to be incorporated. Therefore, there is no need to remove the label from the nucleotide.

[0098] Cleavable linkers are known in the art and can be prepared by applying conventional chemistry to cleave the linker. The linker can be attached to a nucleotide base and a label. , including exposure to electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes, etc. Cleavage can be performed by any suitable method. Cleavage of the 3'-O-blocking group bond Suitable linkers are described in Greene & Wuts, Protein active Groups in Organic Synthesis, John These can be adapted from standard chemical protecting groups disclosed in the Wiley & Sons Further suitable cleavable linkers for use in solid phase synthesis are those described by Guillier et al. (Chem. Rev. 100:2092-2157, 2000) It will be disclosed.

[0099] If a detectable label is attached to the base, Watson-Crick base pairing still occurs. The linker can be attached at any position on the nucleotide base as long as it is feasible In the context of purine bases, the linker binds the 7-position of the purine or the preferred deazapurine analogue. via an N-8 modified purine, via an N-6 modified adenosine or an N-2 modified guanine In the case of pyrimidines, the bond is preferably between cytosine, thymidine or uracil. Preferably, via the 5-position of uracil and the N-4 position of cytosine.

[0100] In some embodiments, the linker can comprise a spacer unit. The label should be attached to the nuclease so as not to interfere with the interaction between the nucleotide and the enzyme, e.g., polymerase. The length of the linker is not important as long as it is kept sufficiently far from the oxidase.

[0101] In some embodiments, the linker comprises a functional group similar to the 3'-OH protecting group. This allows for the removal of both the label and the protecting group in a single step, facilitating the deprotection process. This makes the protection and deprotection process more efficient.

[0102] The use of the term "cleavable linker" means that the entire linker must be removed. The cleavage site is the site where a part of the linker becomes a dye and / or substrate part after cleavage. The cleavage site can be placed in a position on the linker that ensures that the cleavage site remains attached to the molecule. Possible linkers include, but are not limited to, electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, photocleavable linkers, and linkers that can be cleaved under reducing conditions (e.g., disulfide or aryl groups). dide-containing linker), cleavable under oxidative conditions and through the use of a safety catch linker It may be cleavable by a cleavage mechanism or by an exclusion mechanism. By using a linker to attach the dye compound to the substrate moiety, This allows for label removal after detection, avoiding interfering signals in downstream steps. can be avoided.

[0103] Useful linker groups are described in PCT Publication No. WO 2004 / 018493 (see and the like), examples of which include transition metals and at least Using water-soluble phosphines or water-soluble transition metal catalysts formed from partially water-soluble ligands In aqueous solution, the latter is at least partially soluble in water. The formation of soluble transition metal complexes, such as Pd(II) complexes and THP. The bases of the nucleotides can be linked to labels, such as the dyes described herein, using available linkers. can be connected to.

[0104] Particular linkers include those containing a moiety of the formula: The one disclosed in JP 2004 / 018493 (incorporated herein by reference) Included: [ka] wherein X is selected from the group consisting of O, S, NH and NQ, and Q is C 1-10 Replace or an unsubstituted alkyl group, and Y is selected from the group consisting of O, S, NH and N(allyl). and T is hydrogen or C1-C 10 is a substituted or unsubstituted alkyl group, and * indicates a moiety (This indicates where the residue is attached to the rest of the nucleotide or nucleoside.) In some embodiments, the linker connects the base of the nucleotide to a dye, e.g., a dye-containing dye as described herein. Connect to signs such as compounds.

[0105] Additional examples of linkers include those described in U.S. Publication No. 2004 / 012999, such as those containing a moiety of the formula: 16 / 0040225 (incorporated herein by reference). R: [ka] The linker moieties shown herein are linkers between the nucleotide / nucleoside and the label. - may include the entire structure or part.

[0106] Additional examples of linkers (“L”) include portions of the formula: [ka] where B is a nucleobase; Z is -N3 (azido), -O-C1-C6 alkyl, -O -C2-C6 alkenyl, or -O-C2-C6 alkynyl; F1 is an additional linker Those skilled in the art will recognize that the functional group (e.g., carboxyl) of the label can be linked to the ribonucleotide. The label is covalently attached to the linker by reacting with a functional group (e.g., amino) on the linker. I understand that it fits.

[0107] In particular embodiments, the linker between the fluorescent dye (fluorophore) and the guanine base The length can be varied, for example, by introducing a polyethylene glycol spacer group. and thereby linking the guanine base via other linkages known in the art. Exemplary linkers and their derivatives increase fluorescence intensity compared to the same fluorophore. These properties are described in PCT Publication No. WO 2007020457, which is incorporated herein by reference. The design of linkers, especially their increased length, allows for the synthesis of DNA, etc. When incorporated into a polynucleotide, it binds to the guanine base of the guanosine nucleotide Therefore, the brightness of the fluorophore can be improved by using a dye containing guanine. For use in analytical methods requiring detection of fluorescent dye labels attached to nucleotides, Carr, as described in WO 2007 / 020457, has the formula -((CH2) 2O) n - spacer groups of the formula -, where n is an integer between 2 and 50; , is advantageous.

[0108] Nucleosides and nucleotides can be labeled at sites on the sugar or the nucleobase. As is known in the art, a "nucleotide" is a molecule consisting of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA, the sugar is ribose, and in DNA, it is deoxyribose, i.e., ribosomal sugar. The nitrogenous base is a sugar that lacks the hydroxyl group present in saccharides. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T), or in the context of RNA, uracil (U). The C-1 atom of deoxyribose binds to the N-1 of a pyrimidine or the N-9 of a purine Nucleotides are also phosphate esters of nucleosides, with the sugar attached to C-3 or C-5. Esterification occurs at the attached hydroxy group. Nucleotides are usually mono-, di-, or tri- It is phosphate.

[0109] "Nucleosides" are structurally similar to nucleotides but lack the phosphate moiety. An example of a nucleoside analogue is one in which the label is attached to the base and the phosphate group is attached to the sugar molecule. It is something that has not been done before.

[0110] Although bases are commonly referred to as purines or pyrimidines, those skilled in the art may refer to them as nucleotides or nucleotides. Derivatives and derivatives that do not alter the ability of the thiol side to undergo Watson-Crick base pairing It will be understood that derivatives and analogs are available. The structure is the same as or very similar to the parent compound, but different, e.g. or additional side chains, such as derivatives that attach a nucleotide or nucleoside to another molecule. "A compound or molecule having chemical or physical modifications that allow it to be activated" means a compound or molecule having chemical or physical modifications that allow it to be activated. For example, the base may be a deazapurine. In particular embodiments, the derivative is Wat It should be possible to undergo son-Crick pairing. "Derivative" and "Analogs" also include those having, for example, modified base moieties and / or modified sugar moieties. Such derivatives and analogs include synthetic nucleotide or nucleoside derivatives. , e.g., Scheit, Nucleotide analogs (John Wiley y & Son, 1980) and Uhlman et al., Chemical Rev IEWS 90:543-584, 1990. Nucleotide analogs are also Also, phosphorothioates, phosphorodithioates, alkylphosphonates, phosphoranilines The amino acid may contain modified phosphodiester bonds, including phosphodiester, phosphoramidate, and the like.

[0111] The dye can be attached to any position on the nucleotide base, for example, via a linker. In particular embodiments, Watson-Crick base pairing can be used to Specific nucleobase labeling sites include pyrimidines, The C5 position of a 7-deazapurine base or the C7 position of a 7-deazapurine base is an example. A linker group may be used to covalently attach the dye to the nucleoside or nucleotide.

[0112] In particular embodiments, the labeled nucleoside or nucleotide is enzymatically incorporated. In rare cases, the linker moiety may be enzymatically extendible. The nucleotides are then inserted into the nucleotide chain so as not to significantly interfere with the overall binding and recognition of the nucleotide by the enzyme. The linker may be of sufficient length to connect the bond to the compound. A spacer can also include, for example, a nucleotide from the cleavage site or label. Release the base.

[0113] The dye-labeled nucleosides or nucleotides described herein have the formula: You may also: [ka] In the formula, Dye is a dye compound; B is, for example, uracil, thymine, cytosine, adenine, L is any nucleobase that may or may not be present, such as thiazolinone, ... R' is a linker group; R' is H, monophosphate, diphosphate, triphosphate, thiophosphate, phosphate ester Stereo analogs, -O- attached to a reactive phosphorus-containing group, or protected by a blocking group R''' may be H, OH, a phosphoramidite, or any of the groups defined herein. and R'' is H or OH. In the case of sulfamidites, R' is an acid-cleavable hydroxy protecting group, and This allows for subsequent monomer coupling under synthetic conditions.

[0114] In particular embodiments, the linker (between the dye and the nucleotide) and the blocking group are both In particular embodiments, the linker and blocking groups are both present and distinct moieties. Both of the blocking groups are cleavable under substantially similar conditions. Only one treatment is required to remove both the locking groups, so deprotection and deprotection are The blocking process may be more efficient. However, in some embodiments, The linker and blocking group do not have to be cleavable under similar conditions, but instead , which are individually cleavable under distinct conditions.

[0115] The present disclosure also encompasses polynucleotides incorporating dye compounds. Nucleotides are deoxyribonucleotides or ribonucleotides joined by phosphodiester bonds. The polynucleotide may be DNA or RNA, each composed of polynucleotides. The nucleotides may be naturally occurring nucleotides, non-natural nucleotides other than the labeled nucleotides described herein. or any combination thereof as used herein. In combination with at least one modified nucleotide (e.g., labeled with a dye compound) according to Polynucleotides according to the present disclosure may also include non-natural backbone linkages and / or It may also contain non-nucleotide chemical modifications. Chimeric structures consisting of mixtures of deoxyribonucleotides containing ribonucleotides are also contemplated. will be done.

[0116] Non-limiting exemplary labeled nucleotides described herein include: Can be: [ka] In the formula, L represents a linker, and R represents a sugar residue as described above, or a sugar residue having one, two, or three 5'-positions. represents a sugar residue substituted with a phosphate.

[0117] In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below: Can be: [ka] wherein PG represents a 3' hydroxy blocking group as described herein. In any embodiment of the labeled nucleotide, the nucleotide is a nucleotide triphosphate. do.

[0118] (kit) The present disclosure also provides one or more 3' blocked nucleosides and / or Nucleotides, such as 3'-blocking nucleotides of formula (I), (Ia), or (II) Such kits generally include at least one additional component. At least one 3' blocked nucleotide or nucleotide labeled with a dye is added to the Further components may be included as described herein or in the Examples below. The kit may be one or more components identified in the method described in the previous section. Some non-limiting examples of components that can be combined into a kit are listed below.

[0119] In certain embodiments, the kit comprises at least one labeled 3' blocked nucleotide or or nucleosides, along with labeled or unlabeled nucleotides or nucleosides. For example, dye-labeled nucleotides can be used in combination with unlabeled or natural nucleotides. , and / or in combination with fluorescently labeled nucleotides or any combination thereof The nucleotide combinations may be provided in separate individual components (e.g., containers or tubes). one nucleotide type per tube) or a mixture of nucleotides (e.g., in the same container) or two or more nucleotides mixed in a tube).

[0120] The kit may comprise a plurality of, in particular two or three, or more particularly, dye-labeled compounds. If the antibody contains four 3' blocked nucleotides, different nucleotides will bind to different dye compounds. One may be labeled with a dye compound, or one may be dark without a dye compound. When the peptides are labeled with different dye compounds, the dye compounds must be spectrally distinguishable. It is a feature of the kit that the dye is a spectrophotometric fluorescent dye. The term "tally distinguishable fluorescent dyes" is used when two or more such dyes are present. Then, use a fluorescence detection device (e.g., a commercially available capillary-based DNA sequencing platform) Refers to a fluorescent dye that emits fluorescent energy at wavelengths that can be distinguished by the Two nucleotides labeled with fluorescent dye compounds are provided in the kit. When provided, spectrally distinguishable fluorescent dyes can be simultaneously detected, e.g., by the same laser. It is a feature of some embodiments that the fluorescent dye compounds labeled with the fluorescent dye compounds can be excited at the same wavelength. The four selected 3'-blocking nucleotides (A, C, T, and G) are provided in the kit. In this case, two of the spectrally distinguishable fluorophores are both excited at one wavelength and The two spectrally distinct dyes can both be excited at different wavelengths. The wavelengths are 488nm and 532nm.

[0121] In one embodiment, the kit includes a first 3' blocked nucleotide labeled with a first dye. and a second nucleotide labeled with a second dye, the dye being at least 10 nm , especially with a difference in maximum absorbance between 20 nm and 50 nm. More specifically, the two dyes The compound has a Stokes shift of 15 to 40 nm, where "Stokes shift" means It is the distance between the peak absorption wavelength and the peak emission wavelength.

[0122] In an alternative embodiment, the kit of the present disclosure includes a method for labeling a nucleotide sequence in which the same base is labeled with two or more different dyes. The first nucleotide (e.g., a 3' blocking nucleotide) may include a 3' blocking nucleotide. 3'-blocked T nucleotide triphosphate or 3'-blocked G nucleotide triphosphate) The second nucleotide (e.g., a 3'-block C nucleotide) may be labeled with a first dye. The second dye, spectrally distinct from the first, e.g., 600 nm They can be labeled with a "green" dye that absorbs below 50 and a "blue" dye that absorbs below 50 0 nm, for example 400 nm to 500 nm, particularly 450 nm to 460 nm). The nucleotide triphosphate (e.g., 3'-block A nucleotide triphosphate) is a mixture of the first and second dyes. or the first, second, and third dyes and a fourth nucleotide (e.g., 3' block G ) can be labeled as a mixture of nucleotide triphosphates or 3'-block T nucleotides. The nucleoside triphosphates may be "dark" and contain no label. Channels 1-4 may be labeled "blue," "green," "blue / green," and dark. To further simplify the process, the four nucleotides are coupled to two dyes excited by a single laser. Therefore, the labels for nucleotides 1 to 4 are "Blue 1", "Blue 2", and so on. ", "Blue 1 / Blue 2", and Darkness.

[0123] In certain embodiments, the kit includes four labeled 3' blocked nucleotides (e.g., , A, C, T, G), where each type of nucleotide has the same 3' blocking group. The fluorescent label comprises a binding group and a fluorescent label, each of which has a distinct maximum fluorescence and each fluorescent The label can be distinguished from the other three labels. The kit is designed to detect when two or more fluorescent labels have similar absorbance maxima. Some other examples may have different Stokes shifts. In an embodiment, one type of nucleotide is unlabeled.

[0124] The present specification relates to a configuration having different nucleotides labeled with different dye compounds. Although the kit is exemplified with a dye compound, the kit may contain two, three, four or more dye compounds having the same dye compound. It will be understood that the sequence may contain more than one different nucleotide. In some cases, the kit also contains the enzyme and a buffer appropriate for the enzyme's action. In such embodiments, the enzyme is a polymerase, a terminal deoxynucleotidyl transferase, or In certain embodiments, the enzyme is a DNA polymerase. 812 (Pol 812) or DNA polymerase 1901 (Pol 1901), etc. DNA polymerase. Amino acids of Pol 812 and Pol 1901 polymerases. The amino acid sequence can be found, for example, in U.S. patent application Ser. No. 16 / 670, filed October 31, 2019. ,876 and 16 / 703,569 filed December 4, 2019. No. 6,239,133, which is incorporated herein by reference.

[0125] Other components that may be included in such a kit include buffers, etc. nucleotides, and any other nucleotide components, including mixtures of different nucleotides, should be diluted prior to use. It may be provided in the kit in a concentrated form that is to be diluted. In such an embodiment, an appropriate dilution buffer may be used. Again, one or more of the components identified in the methods described herein may be used in the present invention. It can be included in the disclosed kits.

[0126] (Sequencing method) A labeled nucleotide or nucleoside according to the present disclosure is a nucleotide or nucleoside. The method can be used in any analytical method, such as a method involving the detection of a fluorescent label attached to the electrode. In this context, the term "incorporated into a polynucleotide" refers to a polynucleotide in which the 5' phosphate is the 3' hydroxy-OH group of the second (modified or unmodified) nucleotide in an acid diester bond that is bound to a molecule and that itself forms part of a long polynucleotide chain. The 3' end of the nucleotides described herein may be further modified or unmodified. (Decorative) Whether or not bound to the 5' phosphate of a nucleotide by a phosphodiester bond Thus, in one non-limiting embodiment, the present disclosure provides: (a) a method for producing a medicament for the treatment of a medicament comprising: (b) incorporating at least one nucleotide into a polynucleotide; and detecting an incorporated nucleotide into a polynucleotide, The present invention provides a method for detecting a nucleotide by detecting a fluorescent signal from a dye compound attached to the nucleotide. The insertion into the polynucleotide is detected by detecting the insertion.

[0127] The method comprises incorporating one or more nucleotides according to the present disclosure into a polynucleotide. synthesis step (a), and detecting one or more nucleotides incorporated into the polynucleotide. The method may include a detection step (b) in which the oxidized nucleotides are detected or their fluorescence is quantified. Measure objectively.

[0128] Some embodiments of the present application are directed to a sequencing method that includes: (a) Incorporating at least one labeled nucleotide as described herein into a polynucleotide. (b) detecting a fluorescent signal from the new fluorescent dye attached to said nucleotide; The labeled nucleotide incorporated into the polynucleotide is detected by

[0129] Some embodiments of the present disclosure provide a method for determining the sequence of a target single-stranded polynucleotide, including: Regarding the method for determining: (a) a nucleoside containing a 3'-OH blocking group and a detectable label as described herein; The nucleotide is then transferred to a copy polynucleotide strand that is complementary to at least a portion of the target polynucleotide strand. Incorporating into; (b) detecting the identity of the nucleotide incorporated into the copy polynucleotide strand; and (c) Labeling and 3'-OH removal from the nucleotides incorporated into the copy polynucleotide strand Chemically removing the blocking group.

[0130] In some embodiments, the sequencing method further comprises (d) chemically removing the label. and washing the 3' blocking group from the copy polynucleotide strand. In some such embodiments, the 3' blocking group and the detectable label are It is removed before introducing the next complementary nucleotide. Thus, the 3' blocking group and detectable label are removed in a single chemical reaction step. In some embodiments, washing step (d) also removes unincorporated nucleosides. In some further embodiments, the palladium scavenger is also It is also used in the washing step after chemical cleavage of the label and 3' blocking group.

[0131] In some embodiments, steps (a) through (d) comprise: This is repeated until the sequence of a portion of the tide chain is determined. , Steps (a) to (d) are performed at least 50 times, at least 75 times, at least 100 times times, at least 150 times, at least 200 times, at least 250 times, or at least It is repeated 300 times.

[0132] In some embodiments, the label and the 3' blocking group are reacted in two separate chemical reactions. In some such embodiments, the fragments incorporated into the copy polynucleotide strand are removed. Removing the label from the incorporated nucleotide results in the removal of the copy containing the incorporated nucleotide. In some such embodiments, the method comprises contacting the first strand with a first cleavage solution. The cleavage solution in 1 contains a phosphine such as a trialkylphosphine. Non-limiting examples of phosphine include tris(hydroxypropyl)phosphine (THP), tris- (2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THMP), or tris(hydroxyethyl)phosphine (THEP) In one embodiment, the first cleavage solution comprises THP. In some such embodiments, In the method, a 3' blocking group is formed from the nucleotide incorporated into the copy polynucleotide strand. Removal of the incorporated nucleotide is accomplished by contacting the copy strand containing the incorporated nucleotide with a second cleavage solution. In some such embodiments, the second cleaving solution comprises palladium ( In some further embodiments, the Pd catalyst comprises a Pd(0) catalyst. In some such embodiments, Pd(0) is a Pd(II) complex [( It is prepared in situ by mixing PdCl(C3H5)2 with THP. The molar ratio of d(II) complex to THP was approximately 1:2, 1:3, 1:4, 1:5, 1:6, and 1: In one embodiment, the Pd:TH In some further embodiments, the molar ratio of ascorbic acid or P is 1:5. may contain one or more reducing agents, such as its salts (e.g., sodium ascorbate). In some embodiments, the second cleavage solution contains a primary amine, a secondary amine, or , a tertiary amine, a carbonate, a phosphate, or a borate, or a combination thereof. In some further embodiments, the buffering reagent may comprise one or more buffering reagents. Ethylaminoethanol (EA), tris(hydroxymethyl)aminomethane (Tris), glycine , Sodium carbonate, Sodium phosphate, Sodium borate, 2-dimethylaminomethanol DMEA, 2-diethylaminomethanol (DEEA), N,N,N',N'-tetramethylbenzyl ether Tetramethylethylenediamine (TEMED), or N,N,N',N'-tetraethyl ethylenediamine (TEEDA), or a combination thereof. In another embodiment, the buffering agent is a carbonate, phosphate, or borate. In one embodiment, the compound includes one or more inorganic salts, such as a carboxylic acid salt, ... In some other embodiments, the second cleavage solution is a Na In some further embodiments, 3 The blocking nucleotide contains an AOM group, and the second cleavage solution contains a palladium (Pd) catalyst. a solvent and one or more buffering agents as described herein (e.g., a tertiary amine such as DEEA), and has a pH of about 9.0 to about 10.0 (e.g., 9.6 or 9.8).

[0133] In some alternative embodiments, the label and the 3'-OH blocking group are removed in a single chemical reaction. In some such embodiments, the label is removed from the same site as the 3' blocking group. The nucleotide is attached via a cleavable linker comprising a moiety, e.g., a linker and a 3' Both of the blocking groups may be acetal moieties, as described herein. [ka] or thiocarbamate moiety [ka] In some such embodiments, the single chemical reaction may include the Pd catalyst described above. The cleavage is carried out in a solution containing a solvent.

[0134] In some further embodiments, the nucleoside used in the incorporation step (a) The triphosphates are fully functionalized A, C, T, and G nucleotide triphosphates, respectively. , comprising a 3' blocking group as described herein. The nucleotides herein are protected with standard 3'-O-azidomethyl blocking groups. provides superior stability in solution during sequencing runs compared to the same nucleotides used For example, the acetal or thiocarbamate blocking compounds disclosed herein The group was at least 100% protected compared to the 3'-OH protected with azidomethyl under the same conditions for the same period. Also 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, or 3000% improvement This may confer increased stability, thereby reducing prephase values and increasing sequence read length. In some embodiments, the stability is increased at ambient or sub-ambient temperatures. (e.g., 4-10°C). In other embodiments, stability is measured at 40°C, 45°C, Measurements are made at elevated temperatures such as 50°C, 55°C, 60°C or 65°C. In this form, stability is improved in basic pH environments, e.g., pH 9.0, 9.2, 9.4, 9.6, In some further embodiments, the method is performed in a solution of 9.8 or 10.0. The prephasing value using the 3' blocked nucleotides described in the specification is 50 SBS. , 100, or 150 or more cycles, the values were approximately 0.25, 0.24, 0.23, 0. .22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0 .14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0 In some further embodiments, the 3' block is less than 0.06, or less than 0.05. Phase values with nucleotides were obtained by performing SBS for 50, 100, or 150 cycles or more. After the test, the results were approximately 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, and 0.19. ,0.18,0.17,0.16,0.15,0.14,0.13,0.12,0.11 , 0.10, 0.09, 0.08, 0.07, 0.06, or less than 0.05. In an embodiment, each ffN comprises a 3'-AOM group.

[0135] In some embodiments, the 3' blocked nucleotides described herein are Compared to the same nucleotide protected with a 3'-O-azidomethyl blocking group, - Provides excellent deblocking rates in solution during the chemical cleavage step of a sequencing run For example, the acetals (e.g., AOM) or thiocarbamates disclosed herein The blocking group should be at least 5%, 10%, 20%, 30%, 40%, 50%, 60% ,70%,80%,90%,100%,150%,200%,300%,400%,50 0%, 600%, 700%, 800%, 900%, 1000%, 1500%, or 20 00% shows improved deblocking rate compared to azidomethyl-protected 3'-OH. Standard deblocking reagents (e.g., tris(hydroxypropyl)phosphine) can be used. This reduces the overall time for the sequencing cycle. Nucleotide deblocking times are approximately 5%, 10%, 20%, 30%, 40%, and 50%. or 60% shortening. For example, deprotection of 3'-AOM and 3'-O-azidomethyl The blocking times are approximately 4-5 seconds and 9-10 seconds, respectively, under certain chemical reaction conditions. In some embodiments, the half-life (t 1 / 2 ) is azidomethyl At least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times faster than the blocking group. In some such embodiments, the t 1 / 2 The time is about 1 minute, and the time of azidomethyl 1 / In some embodiments, the deblocking rate is about 11 minutes at ambient temperature or In another embodiment, the measurement is performed at a temperature lower than the deblocking temperature (e.g., 4 to 10°C). The conversion rate is measured at high temperatures such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, the deblocking rate is increased in a basic pH environment, e.g., It is measured in a solution of pH 9.0, 9.2, 9.4, 9.6, 9.8 or 10.0. In some such embodiments, the deblocking reagent and the substrate (i.e., the 3'-blocking The molar ratio of the nucleotides (or nucleosides) to the nucleotides is about 10:1, about 5:1, about 2:1, , about 1:1, about 1:2, about 1:5, or about 1:10. In one embodiment, each ffN is Contains a 3'-AOM group.

[0136] In any embodiment of the methods described herein, the labeled nucleotide is a nucleotide In any embodiment of the methods described herein, the target polynucleotide is a nucleotide triphosphate. The leutide chains are attached to a solid support, such as a flow cell.

[0137] In one embodiment, at least one nucleotide is cleaved by the action of a polymerase enzyme. In some such embodiments, In this study, the polymerase was either DNA polymerase Pol 812 or Pol 1901. However, other methods of joining nucleotides to polynucleotides, such as For example, chemical oligonucleotide synthesis or unlabeled oligonucleotides of labeled oligonucleotides. Ligation to a nucleotide can be used. Hence the term "incorporate." The term, when used in reference to nucleotides and polynucleotides, refers to the synthesis of nucleotides by chemical methods and Polynucleotide synthesis by enzymatic methods may be included.

[0138] In certain embodiments, a synthesis step is performed to prepare labeled 3' blocked nucleotides of the present disclosure. Incubating the template polynucleotide strand with a reaction mixture containing an oxidant and optionally, a polynucleotide strand annealed to the template polynucleotide strand. The phosphodiester bond between the free 3'-OH group on the back strand and the 5' phosphate group on the nucleotide The polymerase can also be provided under conditions that allow the formation of the bond. The steps are directed by complementary base pairing of nucleotides to the template strand. The present invention can include the formation of a polynucleotide chain.

[0139] In all embodiments of the method, the polynucleotide into which the labeled nucleotide is incorporated is during which the doped strand is annealed to the template strand, or after a denaturation step in which the two strands are separated. Between the synthesis and detection steps, further steps, e.g. For example, chemical or enzymatic reaction steps or purification steps may be included. The target strand incorporating the nucleotide is isolated or purified and further processed or used in subsequent analysis. For example, the nucleotides described herein can be used in the synthesis step. The labeled target polynucleotide is then used as a labeled probe or primer. In other embodiments, the products of the synthetic steps described herein may be further reacted. If necessary, the products of these subsequent steps can be purified or It may be isolated.

[0140] Suitable conditions for the synthetic steps are well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step comprises a nucleotide sequence comprising a nucleotide as described herein. Similar to a standard primer extension reaction using a protease precursor, the appropriate polymerase enzyme is used. In the presence of an enzyme, an extended target strand can be formed that is complementary to the template strand. In this embodiment, the synthesis step itself forms part of the amplification reaction, and the target and template polynucleotide strands are Generate a labeled double-stranded amplification product consisting of annealed complementary strands derived from the copies. Typical synthesis steps include nick translation, strand displacement polymerization, random primed D Polymerase enzymes particularly useful in the synthesis step include those described herein. Various natural or modified polymerases can catalyze the incorporation of nucleotides. For example, thermostable polymerases can be used using thermal cycling conditions. can be used for synthesis reactions carried out in a lab, but is thermostable for isothermal primer extension reactions. Polymerases that are not capable of incorporating nucleotides according to the present disclosure may be undesirable. Suitable thermostable polymerases include those described in WO2005 / 024010 or WO06 / 1 20433, each of which is incorporated herein by reference. In synthesis reactions carried out at low temperatures, such as 37°C, polymerase enzymes are not necessarily thermostable. Therefore, the choice of polymerase depends on the reaction temperature, pH, , strand displacement activity, and favorites.

[0141] In certain non-limiting embodiments, the present disclosure provides methods for nucleic acid sequencing, re-sequencing, Whole genome sequencing, methods for scoring single nucleotide polymorphisms, and Other applications include detection of labeled nucleotides or nucleosides as described herein when The present invention provides an advantage in the use of polynucleotides labeled with nucleotides containing fluorescent dyes. Any of a variety of other applications may be achieved by labeling nucleotides or nucleic acids with the dyes described herein. Creosides can be used.

[0142] In certain embodiments, the present disclosure provides a method for preparing synthetically labeled polynucleotides according to the present disclosure. The present invention provides a method for the preparation of a nucleic acid sequence for use in a nucleotide sequencing (SBS) reaction. Sequencing typically involves the use of polymerases or ligases to separate growing polynucleotides. A chain is sequentially inserted with one or more nucleotides or oligonucleotides in the 5' to 3' direction. Additionally, an extended polynucleotide strand is formed that is complementary to the template nucleic acid. The identity of the base present in one or more of the added nucleotides is used to detect or " The identity of the added base can be determined by the "imaging" step. The sequence of the template can then be determined after the step of incorporating the nucleotides. can be deduced using conventional Watson-Crick base pairing rules. The use of labeled nucleotides as described herein to determine the identity of a group can be achieved, for example, by Such single base sequences may be useful in scoring nucleotide polymorphisms. Extension reactions are within the scope of this disclosure.

[0143] In one embodiment of the present disclosure, the sequence of the template polynucleotide is The template to be sequenced is identified through the detection of fluorescent labels attached to the polynucleotides. one or more 3' blocked nucleotides described herein to the nascent strand complementary to the target polynucleotide The incorporation of a nucleotide into a template is determined by detecting the incorporation of the nucleotide into the template. Sequencing of polynucleotides is performed using appropriate primers (or as part of a hairpin) (prepared as a hairpin construct containing a primer) and can be primed with 3' The nascent chain is extended stepwise by the addition of nucleotides to the end. Amount of primer in the reaction.

[0144] In certain embodiments, those of different nucleotide triphosphates (A, T, G and C) Each can be labeled with a unique fluorophore and has a 3'-position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). The polymerase enzyme nucleotides the nascent strand that is complementary to the template polynucleotide. The nucleotide is incorporated and the blocking group prevents further incorporation of the nucleotide. Any unincorporated nucleotides can be washed away, and each incorporated nucleotide Fluorescence signals from charge-coupled detectors using laser excitation and appropriate emission filters The 3'-blocking factor can then be optically "read" by appropriate means such as a 3'-blocking factor. The blocking group and the fluorescent dye compound are removed (deprotected) simultaneously or sequentially to allow for the release of additional nucleotides. This allows the nascent strand to be exposed for nucleotide incorporation. The identity of the fragments may be determined after each incorporation step, although this is not strictly required. Similarly, U.S. Patent No. 5,302,509 (incorporated herein by reference). (1999) disclose a method for sequencing polynucleotides immobilized on a solid support.

[0145] This method uses fluorescently labeled 3'-blocked nucleotides A, G, as exemplified above. , C, and T in the presence of a DNA polymerase to form a polynucleotide complementary to the immobilized polynucleotide. The polymerase incorporates a salt sequence complementary to the target polynucleotide into the growing strand. group but further addition is prevented by a 3'-blocking group The label of the incorporated nucleotide is then determined and the protecting group is removed by chemical cleavage. Further polymerization can be performed. The nucleic acid template to be sequenced can be any polynucleotide for which sequencing is desired. The nucleic acid template for the sequencing reaction is typically a nucleic acid sequence that is It serves as a primer or starting point for the addition of additional nucleotides in the reaction. The template to be sequenced contains a double-stranded region with a free 3'-OH group. The region overhangs this free 3'-OH group on the complementary strand. The overhanging region of the template to be sequenced may be single-stranded, but The "nick" on the strand complementary to the base strand provides a free 3' site for initiation. It may be double-stranded, provided that it provides an -OH group. In certain embodiments, sequencing may proceed by strand displacement. A primer with an -OH group ligates to a single-stranded region of the template to be sequenced. They may be added as separate hybridizing components (eg, short oligonucleotides). Alternatively, the primer and template strand to be sequenced may be, for example, a hairpin loop. A portion of a partially self-complementary nucleic acid strand that can form an intramolecular duplex such as a loop structure. Hairpin polynucleotides and their attachment to a solid support can be formed, respectively. The attachment method is described in PCT Publication Nos. WO01 / 57248 and W2005 / 0473 01, each of which is incorporated herein by reference. Sequential additions to the growing primer synthesize a polynucleotide chain in the 5' to 3' direction The nature of the added base is determined specifically after each nucleotide addition, but not necessarily It does not have to be, and provides sequence information for the nucleic acid template. Nucleotides are attached to nucleic acids via the formation of a phosphodiester bond with the 5' phosphate group of the nucleotide. Nucleotides form nucleic acid chains (or polynucleotides) by binding to the free 3'-OH group of the chain. It will be incorporated into the Ochid.

[0146] The nucleic acid template to be sequenced can be DNA or RNA, or deoxyribonucleic acid. It may even be a hybrid molecule consisting of nucleotides and ribonucleotides. The rate is natural and non-natural as long as it does not interfere with the copying of the template in the sequencing reaction. and / or non-natural nucleotides and natural or non-natural backbone linkages.

[0147] In certain embodiments, the nucleic acid template to be sequenced is a nucleic acid sequence known in the art. The solid support may be attached by any suitable attachment method, for example, covalent attachment. In some embodiments, the template polynucleotide is attached to a solid support (e.g., a silica-based However, in other embodiments of the present disclosure, the solid support may be directly attached to the The body surface allows for direct covalent attachment of template polynucleotides, or Templated polynuclear molecules through hydrogel or polyelectrolyte multilayers, which themselves may be non-transformed. It can be modified in some way to immobilize the nucleotides - covalently attached to a solid support is doing.

[0148] Sequencing-by-Synthesis Embodiments and Alternatives Some embodiments include pyrosequencing technology. Pyrosequencing involves: The release of inorganic pyrophosphate (PPi) is detected when a specific nucleotide is incorporated into the nascent chain. (Ronaghi, M., Karamohamed, S., Pettersson , B., Uhlen, M. and Nyren, P. (1996) “Real-pylori Time-dependent DNA sequencing using detection of phosphate release.” Analytical Bio chemistry 242(1), 84-9; Ronaghi, M. (2001) Pyrosequencing sheds light on DNA sequencing. "GenomeRes. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren , P. (1998) "Real-time pyrophosphate-based sequencing methods." Scien ce 281(5375), 363; U.S. Patent No. 6,210,891; No. 6,258, The disclosures of US Pat. Nos. 5,568 and 6,274,320, which are incorporated herein by reference in their entireties. In pyrosequencing, the released PPi is converted to ATP sulfurylase. It can be detected by its immediate conversion to adenosine triphosphate (ATP) by enzymes. The level of ATP is detected via luciferase-generated photons. The nucleic acids to be detected can be attached to features in the array, and the array can detect nucleic acids attached to the features of the array. Imaging is performed to capture the chemiluminescent signal generated as the oxidase is incorporated. The array can be treated with a specific nucleotide type (A, T, C, G, etc.) and then Images obtained after the addition of each nucleotide type can be used to determine which features in the array These differences in the images are due to the sequence of features on the array. The relative position of each feature varies within the image. The images may be stored, processed, and analyzed using the methods described herein. For example, the results obtained after treating the array with each different nucleotide type can be The images shown show different detection channels for reversible terminator-based sequencing methods. Images obtained from the panel can be processed in the same manner as exemplified herein. do.

[0149] In another exemplary type of SBS, cycle sequencing is performed using the methods described in, for example, WO04 / 0 No. 18497 and U.S. Pat. No. 7,057,026, Stepwise addition of reversible terminator nucleotides containing cleavable or photobleachable dye labels This approach is achieved by adding are commercialized by Solexa (now Illumina), and each is incorporated herein by reference. Also described in WO91 / 06678 and WO07 / 123,744, which are incorporated herein by reference. Both ends can be reversed, resulting in fluorescently labeled terminators where the fluorescent label is cleaved. The availability of the ter- minator is based on an efficient cyclic reversible termination (CRT) sequence. Polymerases also incorporate these modified nucleotides efficiently. , and can be co-designed to extend from there.

[0150] Preferably, in reversible terminator-based sequencing embodiments, the label However, the detection label does not substantially inhibit extension under SBS reaction conditions. For example, the label can be removed by cleavage or degradation. After integration, images can be captured. In certain embodiments, each cycle , which involves the simultaneous delivery of four different nucleotide types to the array, with each nucleotide type have spectrally distinct labels. Then, selection is performed for one of four different labels. Four images can be acquired using different detection channels, respectively. Oxide types can be added sequentially and images of the array taken between each addition step In such an embodiment, each image may contain a particular type of nucleotide. The sequence content of each function will be different, so the Different features may or may not be present in different images, but the relative positions of the features may vary across images. The image obtained from such a reversible Terminator-SBS method is The image may be stored, processed, and analyzed as described herein. Following the labeling step, the label can be removed, allowing for subsequent addition and detection of nucleotides. The reversible terminator portion can be deleted for a particular cycle. After detection by NMR, removing the label before the next cycle reduces the background signal and The advantage of this method is that it reduces crosstalk between the labels. Examples of useful labels and removal methods are listed below. Shown below.

[0151] Some embodiments involve the use of four different nucleotides with fewer than four different labels. For example, SBS can be used for detection. Issue 32. As a first example, pairs of nucleotide types can be detected at the same wavelength, but based on the difference in strength between one member and the other, or one member of a pair are distinguished based on changes to the ), the apparent signal appears or disappears compared to the signal detected in the other member of the pair As a second example, three of the four different nucleotide types are The fourth nucleotide type is detectable under these conditions, but the fourth nucleotide type is detectable under these conditions. Not detected or minimally detected under these conditions (e.g., due to background fluorescence) The incorporation of the first three nucleotide types into nucleic acids is The presence of a fourth nucleotide tag to the nucleic acid can be determined based on the presence of each signal. Incorporation of the type can be determined based on the absence or minimal detection of any signal. As a third example, one nucleotide type can be detected in two different channels. Other nucleotide types can contain labels that are detected in one or fewer channels. The three exemplary configurations above are not to be considered mutually exclusive and various combinations are possible. An exemplary embodiment that combines all three examples is The first nucleotide type to be detected in the first channel (e.g., by the first excitation wavelength) A labeled dATP (which is detected in the first channel when excited) is introduced into the second channel. A second nucleotide type (e.g., when excited by a second excitation wavelength) is detected in the chromatogram. (dCTP with a label detected in the second channel), both the first and second channels A third nucleotide type (e.g., detected by the first and / or second excitation wavelengths) is detected in the dTT with at least one label that is detected in both channels when excited by P, and labeling that is not or minimally detected in either channel. A fluorescence-based SBS method using a fourth nucleotide type (e.g., label) .

[0152] Further, as described in the incorporated material of U.S. Publication No. 2013 / 0079232, As shown, sequence data can be acquired using a single channel. In the one-dye sequencing approach, the first nucleotide type is labeled. However, the label is removed after the first image is generated, and the second nucleotide type is The third nucleotide type is labeled only after the first image is generated. and the second image retains its label, and the fourth nucleotide type is It remains unlabeled.

[0153] Some embodiments may utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides. , identify the incorporation of such oligonucleotides. The oligonucleotides are typically The sequence of the oligonucleotide hybridizes to the specific nucleotide identity. As with other SBS methods, the sequencing reagents are labeled with different correlated labels. After processing a series of nucleic acid features, images can be acquired, each of which contains a specific type of label. The integrated nucleic acid features are displayed. The sequence content of each feature is different, so the different Different images may or may not have different features, but the relative positions of the features may change within the image. Images obtained from ligation-based sequencing methods are not included in the methods described herein. The method and method described herein can be used to store, process, and analyze the cells. Exemplary SBS systems and methods that can be utilized with the system are disclosed in U.S. Pat. Patents Nos. 6,969,488, 6,172,218, and 6,306,597 (the disclosures of which are incorporated herein by reference in their entireties).

[0154] Some embodiments may utilize nanopore sequencing (Deame r, DW & Akeson, M. "Nanopores and Nucleic Acids: Prospects for Ultrafast Sequencing". "Analysis of nucleic acids by nanopore analysis," Acc. Chem. Res. 35:817-825( 2002); Li, J., M. Gershow, D. Stein, E. Brandin , and J.A. Golovchenko, "DNA Molecular and Constructive Solid-State Nanopore Microscopy" Nat. Mater. 2:611-615 (2003), the disclosure of which is incorporated by reference in its entirety. In such embodiments, the target nucleic acid passes through the nanopore. The nanopore can be a synthetic pore or a biological membrane protein such as α-hemolysin. By measuring the change in electrical conductance of the nanopore as the target nucleic acid passes through it, By using the above method, each base pair can be identified. (U.S. Patent No. 7,001,792; So ni, G.V. & Meller, “A. Ultrafast DNA Sequencing Using Solid-State Nanopores” Progress Towards Pharmacology.” Clin. Chem. 53, 1996–2001 (2007); Healy, K. “Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481(2007); Cockroft, S.L., Chu, J., Amorin , M. & Ghadiri, M.R. "Single-molecule nanopore device for DNA separation with single nucleotides" Resolution to detect polymerase activity." J. Am. Chem. Soc. 130, 818-8 20 (2008), the disclosure of which is incorporated herein by reference in its entirety. Data obtained from pore sequencing may be stored and processed as described herein. In particular, the data can be analyzed using optical images and and can be treated as an image according to the exemplary processing of other images.

[0155] Some other embodiments of the sequencing method are described in U.S. Pat. No. 9,222,132. Nanoball sequencing techniques, such as those described herein, This includes the use of 3' blocked nucleotides, the disclosure of which is incorporated by reference. Through the process of repeating circle amplification (RCA), numerous individual DNA nanoballs are generated. The nanoball mixture can then be made to associate a single nanoball with each location. The DNA nanoparticles are dispensed onto a patterned slide surface containing features that allow for the To generate the balls, DNA is fragmented and ligated to the first of four adapter sequences. The template is amplified, circularized, and cleaved with a type II endonuclease. A second set of adapters is added, followed by amplification, circularization, and cleavage. This process is repeated for the remaining two adapters. A circular template with four adapters, each separated by a template sequence The library molecules undergo a rolling circle amplification step and are converted into DNA nanoballs. A large number of concatemers, called chromatin fragments, are generated and deposited in a flow cell. Goodwin et al. al., “Coming of age: ten years of next-g generation sequencing technologies,” Nat Rev Genet. 2016;17(6):333-51.

[0156] Some embodiments involve real-time monitoring of DNA polymerase activity. Nucleotide incorporation can be achieved by, for example, the method described in U.S. Pat. Nos. 7,492 and 7,211,414, both of which are incorporated herein by reference. Fluorophore-containing polymerase and γ-phosphate labeling were performed as described in (included). Detection is via fluorescence resonance energy transfer (FRET) interactions between nucleotides. Alternatively, nucleotide incorporation can be performed using methods such as those described in, for example, U.S. Patent No. 7,315,019. (incorporated herein by reference) See, for example, U.S. Patent No. 7,405,281 and U.S. Publication No. 2002 / 0022994. No. 008 / 0108082, both of which are incorporated herein by reference. Using fluorescent nucleotide analogs and engineered polymerases, as described Illumination allows the incorporation of fluorescently labeled nucleotides to be observed with low background light. Confinement to zeptoliter-scale volumes around surface-tethered polymerases (Levene, MJ et al. "Zero-mode wave" guides for single-molecule analysis at h Science 299, 682-68 6 (2003); Lundquist, PM et al. lel confocal detection of single molecule es in real time.” Opt. Lett. 33, 1026-10 28 (2008); Korlach, J. et al. aluminum passivation for targeted immob ilization of single DNA polymerase molec ules in zero-mode waveguide nano structure res.” Proc. Natl. Acad. Sci. USA 105, 11 76-1181 (2008), the disclosure of which is incorporated herein by reference in its entirety. .) Images obtained from such methods may be stored, processed, and can be analyzed.

[0157] Some SBS embodiments release nucleotides once they are incorporated into extension products. For example, sequencing based on the detection of released protons can be performed using Ion Torrent (Guilford, CT, Life Technologie) s subsidiary), or U.S. Publication No. 20 09 / 0026082;2009 / 0127589;2010 / 0137143;and No. 2010 / 0282617, all of which are incorporated herein by reference. The sequencing methods and systems described can be used. The method described herein for amplifying a target nucleic acid using a proton detector is More specifically, the method can be readily applied to substrates used for the The method was used to generate a clonal population of amplicons that were used to detect protons. It can be achieved.

[0158] The SBS method described above can be implemented in multiple formats so that multiple different target nucleic acids can be manipulated simultaneously. In certain embodiments, the different target nucleic acids can be advantageously performed in a single reaction. This allows for sequencing experiments to be performed in a reaction vessel or on the surface of a specific substrate. Multiplexed for convenient drug delivery, removal of unreacted reagents, and detection of uptake events In embodiments using surface-bound target nucleic acids, the target nucleic acids are in an array format. In an array format, the target nucleic acids are typically represented in a spatially distinguishable manner. The target nucleic acid can be attached to a surface by direct covalent bonding, attachment to beads or other particles, or or by binding to a polymerase or other molecule attached to a surface The array contains a single copy of a target nucleic acid (also called a feature) at each site. Alternatively, multiple copies of the same sequence can be present at each site or feature. Multiple copies can be generated by bridge amplification or emulsion amplification, as described in more detail below. It can be generated by amplification methods such as PCR.

[0159] The methods described herein can be used to detect, for example, at least about 10 features / cm 2 , 100 special Signs / cm 2 , 500 features / cm 2 , 1000 features / cm 2 , 5000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 functions / cm 2 , 1,000,000 features / cm 2 , 5,000,000 features / cm 2 Including Arrays having features of any of a variety of densities can be used.

[0160] An advantage of the methods described herein is that they allow for rapid and efficient detection of multiple target nucleic acids in parallel. Accordingly, the present disclosure provides a method for the preparation of a method for the preparation of a compound according to the present invention. The present invention provides an integrated system capable of preparing and detecting nucleic acids using techniques known in the art. Thus, the integrated system of the present disclosure provides amplification reagents and / or sequencing reagents. Fluidic components capable of delivering reagents to one or more immobilized DNA fragments The system may include components such as pumps, valves, reservoirs, fluid lines, etc. The flow cell may be configured and / or used in an integrated system for detecting a target nucleic acid. Exemplary flow cells can be used, for example, as described in U.S. Publication No. 2010 / 011. 1768 and U.S. Serial No. 13 / 273,666. Each is incorporated herein by reference. As exemplified for a flow cell One or more fluidic components of the integrated system may be used for amplification and detection methods. Taking the nucleic acid sequencing embodiment as an example, the integrated system One or more of the fluidic components may be used in conjunction with the amplification methods described herein and as exemplified above. It can be used to deliver sequencing reagents in such sequencing methods. Alternatively, an integrated system may comprise separate fluidic systems for carrying out the amplification method and for carrying out the detection method. The system can be used to generate amplified nucleic acids and to sequence the nucleic acids. An example of an integrated sequencing system that can Lumina, Inc., San Diego, California) and U.S. Serial Number No. 1 No. 3 / 273,666, which is incorporated herein by reference. .

[0161] Arrays in which polynucleotides are directly attached to silica-based supports are described, for example, in WO00 / 04999. / 06770 (incorporated herein by reference), The nucleotides are bonded to pendant epoxide groups on the glass and internal amino groups on the polynucleotide. Furthermore, polynucleotides can be prepared by, for example, the methods described in WO2005 / 047301 (the present invention is incorporated herein by reference). The nucleophilic coupling of a sulfur-based nucleophile with a solid support is described in The solid-supported template polymer can be attached to a solid support by a reaction. Further examples of oligonucleotides include those in which the template polynucleotide is a / 31148, WO01 / 01143, WO02 / 12566, WO03 / 014392 See U.S. Patents U.S. Pat. No. 6,465,178 and WO 00 / 53812. each of which is incorporated herein by reference.

[0162] A particular surface onto which the template polynucleotides can be immobilized is polyacrylamide gel. Polyacrylamide hydrogels are described in the references cited above and in WO2 No. 005 / 065814, which is incorporated herein by reference. Specific hydrogels that can be used include those described in WO 2005 / 065814 and US Pub. No. 20 14 / 0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-aminobenzoates)). Didoacetamidylpentyl)acrylamide-co-acrylamide).

[0163] DNA template molecules are described, for example, in U.S. Pat. No. 5,253,003. The hydroxybenzoates can be attached to beads or microparticles, such as those described in U.S. Patent No. 6,172,218. No. 5,239,623, incorporated herein by reference. Attachment to beads or microparticles is performed sequentially. Bead libraries are useful for sequencing applications, where each bead contains a different DNA sequence. Examples of libraries and how to create them can be found in Nature, 437, 376-380 (2005). Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Arrays of such beads using the nucleotides described herein are incorporated by reference. Sequencing of is within the scope of this disclosure.

[0164] The templates to be sequenced may form part of an "array" on a solid support. and the array can take any convenient form. The method can be used on any array, including single molecule arrays, clustered arrays, and bead arrays. The labeled nucleotides of the present disclosure can be applied to high density arrays of this type. essentially any nucleic acid molecule, including but not limited to those formed by immobilization of It can be used to sequence templates on any type of array.

[0165] However, the labeled nucleotides of the present disclosure may be used in the context of clustered array sequencing. In a clustered array, different regions (sites) on the array are or features) are formed on multiple polynucleotide template molecules Generally, multiple polynucleotide molecules are individually resolved by optical means. Instead, they are detected as a group. Depending on how the array is formed, Each site may be multiple copies of one individual polynucleotide molecule (e.g., the site may be a specific single-stranded or double-stranded nucleic acid species) or contain a small number of multiple copies Multiple copies of different polynucleotide molecules (e.g., two different nucleic acid species) Clustered arrays of nucleic acid molecules can be prepared using techniques commonly known in the art. For example, WO98 / 44151 and WO00 / 18957 ( each of which is incorporated herein by reference) to form an array of clusters. a method for amplifying nucleic acids in which both the template and the amplification product remain immobilized on a solid support; or "colonies" of immobilized nucleic acid molecules. The nucleic acid molecules present on the prepared clustered array are labeled with the dye compounds of the present disclosure. The nucleotides are suitable templates for sequencing.

[0166] The labeled nucleotides of the present disclosure can also be used for sequencing templates on single molecule arrays. As used herein, the term "single molecule array" or "SMA" refers to a It refers to a population of polynucleotide molecules dispersed (or arrayed) on a solid support, and individual polynucleotides The spacing between nucleotides and all other groups separates each individual polynucleotide molecule. Thus, the target nucleic acid molecule immobilized on the surface of the solid support can be In some embodiments, it can be resolved by optical means. One or more different signals, each representing a polynucleotide, may be used in the particular This means that it occurs within the resolvable area of the imaging device.

[0167] The spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more precisely at least 250 nm, even more particularly at least 300 nm, even more particularly Single molecule detection can be achieved at least 350 nm. The molecules can be individually resolved and detected as single molecule fluorescent dots, and the single molecule fluorescent dots Fluorescence from also shows single-step photobleaching.

[0168] In this document, the terms "individually decomposed" and "individually decomposed" are used to This specifies that one molecule on the array can be distinguished from its neighbors. The separation between the individual molecules above depends, in part, on the specific technique used to resolve the individual molecules. The general characteristics of single molecule arrays are described in published application WO 00 / 0 6770 and WO01 / 57248. Each of these is incorporated herein by reference. One use of the nucleotides of the present disclosure is Sequencing by synthesis, but the usefulness of nucleotides is limited to such methods In fact, the nucleotides are attached to nucleotides that are incorporated into the polynucleotide. It can be advantageously used in any sequencing method requiring detection of a fluorescent label. do.

[0169] In particular, the labeled nucleotides of the present disclosure are useful in automated fluorescent sequencing protocols, particularly sangina. and collaborators' fluorescent dye terminator cycle based chain termination sequencing method Such methods typically involve enzymes and cycle sequencing. Sequencing is used to catalyze the addition of fluorescently labeled dideoxynucleotides to primer extension sequences. The so-called Sanger sequencing method and related protocols ( Sanger-type (random chain termination with labeled dideoxynucleotides) Use the stop.

[0170] Thus, the present disclosure provides dideoxynucleotides that lack hydroxyl groups at both the 3' and 2' positions. Also encompassed are labeled nucleotides that are nucleotides, and such dideoxynucleotides are It is suitable for use in Angler-type sequencing methods.

[0171] The labeled nucleotides of the present disclosure incorporating a 3'-blocking group are useful in Sanger sequencing and related applications. It may also be useful in sequential protocols because dideoxynucleotides are used. The same effect as that achieved by blocking nucleotides with 3'-OH groups can be achieved by This can be achieved by using a nucleotide sequence: When the nucleotides according to the present disclosure are used in Sanger-type sequencing methods, The dye compound or detectable label attached to the nucleotide may be attached via a cleavable linker. It will be understood that the labeled nucleotides of the present disclosure do not need to be linked to the nucleotides. Each example shows how to remove the label from the nucleotide, since there is no need to incorporate the nucleotide later. There's no need to remove it.

[0172] In any embodiment of the methods described herein, in a sequencing application The nucleotides used may be 3' blocked nucleotides as described herein, such as those of the formula ( In any embodiment, the 3' nucleotide is (I), (Ia), or (II). The blocking nucleotides are nucleotide triphosphates. [Example]

[0173] Additional embodiments are described in the following examples, which are not intended to limit the scope of the claims in any way. This is disclosed in more detail in

[0174] Example 1. Preparation of 3'-acetal-blocked nucleosides In this example, various 3'-acetal protected T nucleosides were prepared according to Scheme 2. Ta. [ka]

[0175] Preparation of T1: In an oven-dried, nitrogen-purged 100 mL flask, add 5-iodine 1.0 g of 2'-deoxyuridine (5.0 g, 14.12 mmol) was added to 30 mL of this solution. The mixture was coevaporated with 25 mL of pyridine three times and then placed under nitrogen. Anhydrous pyridine (25 mL) was added and The reaction was stirred at room temperature until a homogeneous solution was obtained (approximately 15 min). Cool to 0 °C and add tert-butyldiphenylsilyl chloride (4.04 mL, 15.5 mmol). l) was added slowly dropwise with vigorous stirring (about 1 hour). The reaction was maintained at 0° C. for 8 hours until consumed by saturated aqueous ammonium chloride. (approximately 15 mL) was added and the reaction was allowed to warm to room temperature. The mixture was diluted with ethyl acetate (100 mL). The organic layer was separated and the aqueous layer was washed with saturated aqueous ammonium chloride (200 mL). The extract was extracted with ethyl acetate (4x50 mL). The organic layers were combined, dried (MgSO4), and After concentration in vacuo and removal of residual solvent under high vacuum, approximately 8 g of a clear yellow oil was obtained. Product T1 was isolated by flash column chromatography on silica as a white crystalline The solid was purified. The yield was 6.94 g (83%). Raynegative) 591.08 [MH]

[0176] Preparation of T2: In an oven-dried, nitrogen-purged, brown, 500 mL, three-neck flask, add T 1 (6.23 g, 10.5 mmol), copper(I) iodide (200 mg, 1.05 mmol) ) and bis(triphenylphosphine). Palladium(II) dichloride under nitrogen (369 mg, 0.526 mmol). Protect the flask from light and add anhydrous, degassed DMF (200 mL) was added to the solution. 4.74 g (31.6 mmol) of methyl-acetamide was added, followed by degassed triethyl ether. Dimethylamine (2.92 mL, 21.0 mmol) was added and the reaction was stirred under nitrogen at room temperature for 6 h. After stirring, no further starting material was observed by TLC analysis. The volatiles were removed in vacuo. (~15 min) and the DMF was removed under high vacuum (~1 h) to a brown residue. This was dissolved in ethyl acetate (200 mL) and diluted with 0.1M EDTA in water (2 x 200 mL). The aqueous layers were combined and further extracted with ethyl acetate (200 mL). The mixture was then dried (MgSO4), the volatiles removed in vacuo (~30 min), and the solution was further purified under high vacuum. The mixture was dried at rt (~1 h) to give approximately 8 g of a crude brown / yellow oil. Purified by column chromatography as an off-white solid. Yield: 6.0 g (85%). LC-MS (electrospray negative) 614.19 [MH].

[0177] Preparation of T3: Under nitrogen, 2.0 g of starting nucleoside T2 (2.0 g, 3.25 mmol) was added to the ozone layer. In an oven-dried, nitrogen-purged 100 mL flask, add anhydrous DMSO (6.9 mL, 97.5 mm ol) was added at once at room temperature and stirred until a homogeneous solution was formed. 1 mL, 195 mmol), followed by acetic anhydride (15.1 mL, 162.09 mmol). Both were added dropwise (approximately 5 min each). The mixture was warmed to 50° C. and analyzed by TLC (EtOAc / petroleum Stir until the starting nucleoside is completely consumed (approximately 5 hours) by ether (3:2). The reaction was then concentrated to half its volume and cooled to approximately 0.5°C in an ice bath. ) N Slowly add 45 mL of aHCO3 (aq, sat.) and stir until the mixture is fully dissolved. Workup was initiated until no more bubbles were observed (~15 min). The solution was allowed to warm to room temperature and then The water was extracted into EtOAc (3 x 100 mL). The combined organic layers were dried over MgSO4 and After filtration, the volatiles were evaporated under reduced pressure and then under high vacuum. The solid was purified by flash chromatography on silica gel. Yield: 1 0.79g (82%). LC-MS (electrospray negative) 674.20 [M- H] - .

[0178] Preparation of T4: Dissolve starting nucleoside T3 (1. A solution of 79 g, 2.649 mmol) of cyclohexene (1.34 mL, 13.2 mm The mixture was cooled to 0°C in an ice bath and distilled sulfuryl chloride (322 µL, 3. 97 mmol) was slowly added dropwise (about 20 minutes) under N2, and the mixture was stirred at that temperature for 20 minutes. Afterwards, TLC (EtOAc:petroleum ether) = 3:2 v / v) showed that the starting nucleoside was completely Next, as shown in Scheme 3, the corresponding freshly distilled The chloride intermediate was quenched by the direct dropwise addition of the unsaturated alcohol (5 equivalents). The solution was stirred at room temperature for 2 hours, followed by evaporation of the volatiles under reduced pressure. The organic layer was separated and the aqueous was further partitioned into Ac:brine (3:2) (125 mL). Extracted into EtOAc (2 x 50 mL). The combined organic extracts were dried over MgSO4 and filtered. The oily residue was extracted with EtOAc:brine (3:2) and the volatiles were evaporated under reduced pressure. (125 mL). The organic layer was separated and the aqueous was further diluted with EtOAc (2 x 50 mL The combined organic extracts were dried over MgSO4, filtered, and the volatiles were removed under reduced pressure. The crude product T4 was purified by flash chromatography on silica gel. The final product was obtained as a yellow oil. Yield: 1.20 g (69%) of AOM. 1.29g (71%) for M. 1.34g (71%) for DPrOM.

[0179] 3'-AOM: Yellow oil. LC-MS (electrospray negative) [MH ]684.24.

[0180] 3'-PrOM: yellow oil. LC-MS (electrospray negative) [M- H]682.22.

[0181] 3'-DPrOM: yellow oil. LC-MS (electrospray negative) [M -H]710.25. [ka] Scheme 3.

[0182] Preparation of T5: Add starting material T4 (1.04 g, 1.51 mL) to a 50 mL round-bottom flask under nitrogen. To the resulting solution (1.6 mmol), anhydrous THF (9 mL) was added at room temperature. Then, TBAF (1. 0M, 1.7 mL, 1.70 mmol) was added dropwise until all SM was consumed by TLC. The solution was stirred until the mixture was dissolved (approximately 2 hours). During the course of the reaction, the solution turned orange. The soluble material was removed in vacuo to give an orange residue, which was dissolved in EtOAc (100 mL). The mixture was dissolved in NaHCO3 (saturated aqueous solution) (60 mL) and separated. The two layers were separated and the aqueous layer was Extraction with EtOAc (60 mL) was performed. The organic layers were combined, dried (MgSO4), filtered, and Evaporation gave the crude product as a yellow oil. The crude product was purified by flash chromatography on silica gel. Purification by HPLC gave a clear yellow oil. Yield: 637 mg (94%) by AOM. %). 526 mg (78%) for PrOM. 617 mg (86%) for DPrOM. ).

[0183] 3'-AOM: Clear yellow oil. LC-MS (electrospray negative) MH]446.12.

[0184] 3'-PrOM: clear yellow oil (526 mg 78%). LC-MS (electrospray spectroscopy) Lenegative): [MH] 444.10.

[0185] 3'-DPrOM: clear yellow oil (617 mg 86%). LC-MS (electrolysis) Play Negative): [MH] 472.13.

[0186] In addition, two additional 3'-blocked T nucleosides (3'-eAOMT and 3'-i 3'-iAOMT) was prepared in the same manner as above. 3'-iAOMT: LC-MS (ES): (negative ion) m / z 325.5 (MH + ), (positive ion) 327.3 (M+H + ). 3 '-eAOMT:LC-MS(ES):(cation) m / z341.3(M+1H + ). [ka]

[0187] (Example 2. 3'-OH Blocking Group Stability Test) In this example, the stability test of the 5'-mP 3'-AOM T nucleotide was performed using the standard 5 Run side-by-side in uptake buffer containing '-mP3'-O-azidomethyl T nucleotide Ta. [ka]

[0188] Buffer Formulation 100 mM ethanolamine buffer (pH 9.8), 100 mM NaCl, and and 0.1 mM of each 5'-monophosphate 3'-protected T nucleotide in a solution of 2.5 mM EDTA. 1 mL of the solution was incubated at 65°C in a heating block for 2 weeks. An aliquot of 0 μL was taken and analyzed by HPLC to determine the remaining blocked nucleotides. The proportion of nucleotides and the final formation of unblocked nucleotides were determined.

[0189] AOM, PrOM, DPrOM acetal protecting groups and standard azidomethyl blocking The results of the stability test for 5'-monophosphate 3'-protected nucleotides bearing the 5'-monophosphate group are shown in Figure 1. 3'-blocked nucleotide monophosphates including AOM, PrOM, and DPrOM were observed. The blocking group improved the unblocking rate in solution by 30-50 times or more. This experiment demonstrated that the corresponding fully functionalized nucleotides (ffNs) were This mimics how the mix will behave when stored in the built-in mix on the cartridge of the device. The improved stability offered by these acetal protecting groups also facilitates the sequential This would lead to a lower prephasing speed in the execution. Improve the shelf life of the mixed reagent.

[0190] (Example 3.3'-AOM deblocking test) In this example, 5'-mP 3'-AOM T and the standard 5'-mP 3'-O-azide Methyl T nucleotide deblocking tests provide solutions specific to each blocking group. The conditions were designed to mimic Illumina's standard unblocking reagents as closely as possible. It was designed to mimic the same methodology as the active deblocking reagents, buffers, and nucleoside. The concentration of cid was kept the same in all tests, but the identity of each component was unique. Thus, the observed rate differences between individual deblocking chemistries may be due to differences in the concentrations of the formulations. It is not due to [ka]

[0191] (Standard azidomethyl deblocking conditions) Nucleotide: 5'-monophosphate 3'-O-azidomethyl T. Active deblocking reagent : Tris(hydroxypropyl)phosphine (THP) (1M in 18 mΩ water). (Optional) Additive: Sodium ascorbate (0.1 mM in 18 mΩ water) final concentration = 1 mM M. Buffer: Ethanolamine pH 9.8 (2M in 18 mΩ water). Quenching Reagent: H2O2.

[0192] (AOM unblocking conditions) Nucleotide: 5'-monophosphate 3'-O-azidomethyl T. Stock of 3'-AOMT The solution was diluted in 100 mM ethanolamine buffer (pH 9) in a glass vial under nitrogen. The sodium ascorbate additive stock solution was diluted to 0.1 mM with 0.8% NaCl. The solution was stirred for 5 minutes. Then, a deblocking reagent (Pd / THP=1 / 5; sodium ascorbate; ethanol The solution was stirred at room temperature and added to a final concentration of 1 mM THP. Take a µL aliquot and mix it in a 1:3 mixture of EDTA / HO (0.025:0.075 M). The mixture was quenched with 6 μL of the mixture. HPLC analysis revealed the initiation nucleoside peak, the 3'-OH peak, and the The areas of the nucleotide peaks and other nucleotide peaks that appear in the HPLC chromatogram were calculated. No other nucleotide-based by-products were observed. It was.

[0193] The comparative results are shown in Figure 2A. AOM, compared to the standard azidomethyl blocking group, It has been observed to provide a 10-fold speed improvement in unblocking rate in solution. The experiment shows how the corresponding ffN behaves in the sequence during the deblocking step. The significant improvement in unblocking speed allows for specific illumination Instead of the 10-20 second incubation time typically used on the scanning platform, This allows for a flush-through deblocking step. The conversion rate has a significant impact on the sequencing by synthesis (SBS) cycle time.

[0194] The same experimental conditions were used for the deblocking assay of 3'-eAOM T and 3'-iAOM T. As a single change, Pd was used to observe a clear difference in the deblocking rate. The catalyst to substrate ratio was reduced to 5:1. 3'-AOM T was used as a reference, and the results are shown in Figure 1. 2B These results show that the deblocking rates of eAOM and iAOM are consistent with this specific The Pd-catalyzed deblocking reagent was 2-3 times slower than AOM at a concentration of AO. The difference in deblocking rate between substituted and unsubstituted versions of the M blocking group is P d It can be expected that the higher the catalyst to substrate ratio, the smaller the

[0195] Example 4. Optimization of Palladium Cleavage Mixtures for Sequencing The Pd / THP catalyst used in the deblocking reaction described in Example 2 is very air sensitive. When exposed to air, it showed a substantial loss of activity. Developing a stress assay to evaluate the air sensitivity of different formulations of palladium cleavage mixtures did.

[0196] Dispense 0.5 mL of Pd cleave mix into a 5 mL glass vial and incubate at room temperature for 3 h. The residual activity of the oxidized cleavage mixture was determined by the 3'-A activity as follows: The cleavage of 3'-AOMT was assessed by measuring the cleavage of AOMT. Sodium ascorbate diluted to 0.1 mM in 100 mM cleavage mix buffer. Stock solution was added to a final concentration of 1 mM, followed by the oxidative cleavage mixture. The final dilution was 1 / 20. After 1 hour, 40 μL of the solution was diluted with 10 μL of EDTA / H2O2 (0 The reaction mixture was immediately quenched with a 1:1 mixture of 0.25M and 0.25M of ethanol and analyzed by HPLC. In the experiment, various buffer reagents were screened, including: primary amines (ethanoic acid, ethanol ... amine, tris, glycine, etc.). Tertiary amine (2-dimethylaminomethanol (( DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-tetramethylbenzyl methyl ether Methylethylenediamine (TEMED) or N,N,N',N'-tetraethylethylenediamine diamine (TEEDA), etc.); and various inorganic salts (borates, carbonates, phosphates, etc.) Inorganic buffer reagents (sodium borate, sodium carbonate, sodium phosphate, etc.) are the best. It was observed that the palladium complexes provided air stability and retained a high percentage of activity. Tertiary amines also significantly improved the stability of the Pd cleavage mixture compared to primary amines. .

[0197] Based on these findings, two palladium cleavage mixtures were prepared. In the first example, 2 Use a stock solution of 50 mM borate buffer (pH 9.6, 20 mL) with water (14 After dilution with 1 mL of THP (1 M in 100 mM Tris, pH 9, 5 mL, 5.0 mm ol) and allylpalladium(II) chloride dimer (183 mg, 0.5 mmol). The mixture was stirred vigorously at room temperature for several minutes, and then 1 M aqueous sodium ascorbate was added. (0.5 mL, 0.5 mmol), 5M NaCl aq. (10 mL) and 10% v / v Tween 20 (0.5 mL). In the second example, 2 M DEEA buffer solution Stock solution of TH (pH 9.6, 0.6 mL) was diluted with water (7.6 mL) and then P (1 M in 100 mM Tris, pH 9, 1.2 mL, 1.2 mmol) and solid chloride A stock solution of allylpalladium(II) dimer(II) was added: 43.9 mg The mixture was stirred vigorously at room temperature for several minutes, and then added to 1 M sodium ascorbate. Aqueous sodium solution (0.12 mL, 0.12 mmol), 5M NaCl aq. (2.4 mL) and 10% v / v Tween 20 (0.12 mL).

[0198] Example 5. Preparation of fully functionalized nucleotides and their use in sequencing applications ) In this example, various fully functionalized nucleotides with 3'-AOM blocking groups were These ffNs were prepared using the Illumina MiniSe It is also used for sequencing by synthesis applications on the q® platform. It was. Scheme 4. Synthesis of 3'-AOM-ffC-LN3-SO7181 [ka]

[0199] Synthesis of intermediate AOM C2: Nucleoside C1 (0.5 g, 0.64 mmol) was added to N2 The mixture was cooled to 0° C. Cyclohexene (0. 32 mL, 3.21 mmol) was added, followed by SO2Cl2 (1.0 M in DCM, 1.2 The reaction mixture was quickly transferred to a rotary evaporator. Add additional cyclohexene (0.32 mL) before removing all volatiles under reduced pressure. The solid residue was further dried under high vacuum for 10 minutes, and then The mixture was cooled to 0°C and dissolved in anhydrous DCM (5 mL) under N2. The reaction was stirred at 0°C for 2 hours and then quenched by the addition of sat. NaHCO3 (50 mL) and DCM (30 mL). The two phases were separated and The aqueous layer was extracted with EtOAc (2 x 50 mL). The organic layers were combined, dried over MgSO4, and Filtration and evaporation of volatiles under reduced pressure were carried out. The crude product was purified by elution with EtOAc / petroleum ether. AOM C2 was purified by flash chromatography on silica gel using Obtained as a white solid (264 mg, 52% yield). LC-MS (electrospray negative) tive):[MH]787, [M+Cl]823.

[0200] Synthesis of intermediate AOM C3: AOM C2 (246 mg, 0.31 mmol) was dissolved in 100 mL of HCl under N The mixture was cooled to 0° C. and acetic acid (0.054 mL) was added. , 0.94 mmol) was added, followed by TBAF (1.0 M in THF, 5 wt. % water, 0. The reaction was stirred at 0°C for 5 hours, after which EtOAc was added dropwise. The mixture was diluted with HCl (20 mL) and then poured into 0.05 M aqueous HCl (20 mL). The layers were separated and the aqueous layer was extracted with EtOAc (2 x 20 mL). The organic layers were combined and The crude product was dried over O, filtered and the volatiles were evaporated under reduced pressure. Purification was performed by flash chromatography on silica gel using AcOEt. C3 was obtained as a yellowish solid (114 mg, 66% yield). Trospray Negative): [MH] 549, [M+H2O-H] 567, [M+Cl ] 585, (electrospray positive): [M+H] 551, [M+H2O+H] 569.

[0201] Synthesis of intermediate AOMC4: AOM C3 (0.114 g, 0.21 mmol), fresh Activated 4Å molecular sieves, proton sponge (0.066 g, 0.31 mm l), place a magnetic stirrer under N2, and add trimethyl phosphate anhydride (1.0 mL). The reaction was cooled to -10 °C and freshly distilled POCl (23 μL, 0.25 The reaction was stirred at -10°C for 1 hour. Solution of pyrophosphate as the ammonium salt (0.5 M in DMF, 1.7 mL, 0.85 mL) mol) and anhydrous tri-n-butylamine (0.41 mL, 1.74 mmol) were premixed. A portion of the nucleoside solution was added to the ice-cold activated nucleoside solution. The mixture was stirred vigorously at room temperature for 5 minutes. The reaction mixture was poured into a separate flask containing a vigorously stirred solution of 2M TEAB in water. (~10 mL). The reaction flask was rinsed with a small amount of H2O and the washings were poured into 2 M TE The combined mixture was then stirred at room temperature for 4 hours, after which the solvent was removed under reduced pressure. The residue was dissolved in aqueous NH3 (35%, approximately 10 mL) at room temperature overnight. The reaction was concentrated in vacuo and purified by flash chromatography on DEAE-Sephadex. The product was further purified by preparative HPLC to give pure AO. M C4 (62 μmol, 30% yield, determined by UV-Vis spectroscopy, λ max =29 4nm, ε=8600M -1 cm -1 ) was obtained. Tib):[MH]589.

[0202] Synthesis of 3'-AOM-ffC-LN3-SO7181: LN3-SO7181(0.0 N,N-Diisopropyl methylcellulose (205 mmol) was dissolved in anhydrous DMA (4 mL) under N2. Ethylamine (28.6 μL, 0.164 mmol) was added, followed by TSTU (in DMA 0.1M, 234 μL, 0.0234 mmol) was added. The reaction was stirred at room temperature under N2 for 1 hour. During this time, an aqueous solution of AOM C4 (0.0101 mmol) was evaporated under reduced pressure. The mixture was evaporated to dryness and resuspended in 0.1 M TEAB (400 μL) and LN3-SO4. The reaction was stirred at room temperature for 17.5 hours, then added to the 7181 solution in 0.1M TEA. The crude product was quenched with aqueous solution of B (4 mL). The product was further purified by preparative HPLC. To obtain pure 3'-AOM-ffC-LN3-SO7181 (6.81 mol, 67% yield, Determined by UV-Vis spectroscopy, λ max = 644 nm, ε = 2,000,000 M -1 c m -1 ) was obtained. LC-MS (electrospray negative): [MH] 1561 , [M-2H]781, [M-3H]520. Scheme 5. Synthesis of 3'-AOM-ffA [ka] JPEG2025121979000037.jpg33115

[0203] Synthesis of intermediate AOM A2: Nucleoside A1 (716 mg, 0.95 mmol) was reacted with N Dissolve cyclohexene (481 μL, 4 0.75 mmol) was added and the solution was cooled to about -15°C. Sulfuryl chloride (distilled, 92µ L, 1.14 mmol) was added dropwise and the reaction was stirred for 20 min. All starting material was consumed. After the reaction, an extra portion of cyclohexene (481 μL, 4.75 mmol) was added and the reaction The material was evaporated to dryness under reduced pressure. The residue was quickly purged with nitrogen and then diluted with allyl alcohol ( 5 mL, approximately 100 mmol) was added with stirring at 0° C. The reaction was stirred at 0° C. for 1 hour. The mixture was then quenched with 50 mL of saturated aqueous NaHCO3. The combined organic phases were washed with 100 mL of water and 100 mL of brine. The residue was washed with water, then dried over MgSO4, filtered and evaporated to dryness. Purified by flash chromatography on silica gel using 1000 mg of ethyl acetate / Et0Ac. 60% yield (435 mg, 0.57 mmol). LC-MS (ES and CI): (positive ion) m / z 763 (M+H+); (negative ion) m / z 761 (M-H+).

[0204] Synthesis of intermediate AOM A3: Nucleoside AOM A2 (476 mg, 0.62 mmol) 1) was dissolved in dry THF (5 mL) under N2 atmosphere, and then THF (750 μL, 0.7 A solution of 1.0 M TBAF in 1.5 mmol) was added. The solution was stirred at room temperature for 1.5 hours. The solution was diluted with 50 mL of EtOAc and then washed with 100 mL of NaH2PO4 sat. The organic phase was dried over MgSO4 and filtered. The residue was purified by ?uorescence on silica gel using EtOAc / MeOH. The product was purified by flash chromatography in 90% yield (292 mg, 0.55 ml). LC-MS (ES and CI): (positive ion) m / z 525 (M+H+); (negative ion) ion) m / z 523 (M-H+).

[0205] Synthesis of intermediate AOM A4: Nucleoside AOM A3 (285 mg, 0.544 mm ol) was dried under reduced pressure over P2O5 for 18 hours. Triethyl phosphate anhydride (2 mL) and some freshly activated 4Å molecular sieves were added to it under nitrogen, and then the reaction The reaction flask was cooled to 0 °C in an ice bath. Freshly distilled POCl (61 μL, 0.65 mmol) was added dropwise, followed by ProtonSponge® (175 mg, 0. After the addition, the reaction was stirred for an additional 15 minutes at 0°C. Bis-tri-n-butylammonium salt (5.4 mL, 2.72 mmol) in water DMF A 0.5 M solution of pyrophosphate as a sachet was added quickly, followed immediately by tri-n-butylamine (540 μL, 2.3 mmol) was added. The reaction was kept in the ice-water bath for an additional 10 minutes. Then pour it into 1M aqueous triethylammonium bicarbonate solution (TEAB, 20 mL) The mixture was quenched with HCl and stirred at room temperature for 4 hours. All solvents were evaporated under reduced pressure. To the residue was added 35% aqueous ammonia (20 mL), and the mixture was stirred at room temperature for at least 5 minutes. The mixture was stirred for 1 hour. The solvent was then evaporated under reduced pressure. The crude product was first purified by DEAE-Self- The product was purified by ion exchange chromatography on Fadex A25 (100 g). The ram was eluted with a gradient of aqueous triethylammonium bicarbonate. Fractions containing triphosphate were The mixture was pooled and the solvent was evaporated to dryness under reduced pressure. The crude material was purified by YMC-Pack-Pro C1 Preparative scale H using 8 columns and eluting with 0.1M TEAB and acetonitrile Compound AOM A4 was obtained as the triethylammonium salt. 56% yield (306 μmol). LC-MS (ES and CI): (anion) (positive ion) m / z 614 (M+H+), 715 (M +Et3NH+).

[0206] General procedure for ffA synthesis: Dissolve the dye linker (0.020 mmol) in 2 mL of anhydrous N, Dissolved in N'-dimethylacetamide (DMA). Add 28.4 μL (0.163 mmol) of ethanol, followed by N,N,N',N'-tetramethylethanolamine. 0.1M anhydrous methyl-O-(N-succinimidyl)uronium tetrafluoroborate The reaction mixture was added as a DMA solution (TSTU, 232 μL). The mixture was stirred under nitrogen at room temperature for 1 hour, during which time AOM A4 triphosphate (0.01 mmol) The aqueous solution was evaporated to dryness under reduced pressure, and 200 μL of 0.1 M triethylammonium bicarbonate ( The activated dye linker solution was added to the triphosphate and the reaction mixture was stirred for 1 hour. The mixture was stirred at room temperature for 18 hours. The crude product was first separated by column chromatography on DEAE-Sephadex A25 ( The fraction containing the triphosphate was purified by ion exchange chromatography on a 25 g column. The crude material was collected by filtration and the solvent was evaporated to dryness under reduced pressure. Further purification was performed by preparative-scale RP-HPLC using 3'-AOM-f fA-LN3-NR7180A: 38% yield (3.8 μmol). LC-MS (ES) :(Anion) m / z1459(M-H+), 729(M-2H+), 486(M-3H +). 3'-AOM-ffA-LN3-BL-NR 5 50S0: 37% yield (3.7μ mol). LC-MS (ES): (negative ion) m / z 1771 (M-H+), 885 (M -2H+), 589(M-3H+). 3'-AOMffA-LN3-BL-NR 6 50C 5: 51% yield (51 μmol). LC-MS (ES): (negative ion) m / z 1917 (M-H+), 958(M-2H+), 645(M-3H+). Scheme 6. Synthesis of 3'-AOM-pppG [ka]

[0207] Synthesis of intermediate AOMG4: The known nucleoside dG3 (100 mg, 0.143 mmol) 1) was dissolved in 10 mL of anhydrous dichloromethane under a N2 atmosphere and cyclohexene (72 μ L, 0.714 mmol) was added and the solution was cooled to -12°C. (1M in DCM, 171 μL, 0.171 mmol) was added dropwise and the reaction was stirred for 10 min. An extra portion of cyclohexene (72 μL, 0.714 mmol) was added and the reaction The reaction was stirred for 30 minutes at −12° C. The reaction was evaporated to dryness under reduced pressure and the residue was purged with nitrogen. Ice-cold neat allyl alcohol (distilled, 0.8 mL, 12 mmol) was stirred at -12°C. The reaction was stirred at -12°C for 60 minutes and then quenched with 2 mL of saturated aqueous solution. The mixture was separated with ethyl acetate (2 mL) and the aqueous layer was washed with ethyl acetate. The combined organic phase was washed with 4 mL of water and 4 mL of brine, and The residue was dried, filtered, and evaporated to a crude oil. Purification by filtration gave AOMG4 as a clear oil in 36% yield (50.9 mg , 0.072 mmol). LC-MS (ES and CI): (positive ion) m / z 710 [ M+H]+; (negative ion) m / z 708 [M−H]−.

[0208] Synthesis of intermediate AOM G5: Nucleoside AOM-G4 (111 mg, 0.156 mm ol) was dissolved in dry THF (5 mL) under N2 atmosphere. Acetic acid (27 μL, 0.468 1.0 M TBAF in THF (296 μL, 0.296 mmol) was added, followed by mol) was added. The solution was stirred at room temperature for 5 hours. The solution was diluted with 10 mL of EtOAc. The aqueous phase was washed with 10 mL of 0.05 M aqueous HCl. The HCl and organics were separated. The combined organic phases were dried over MgSO4, filtered and evaporated to dryness. was purified by flash chromatography on silica gel to give AOMG5 as a white solid. 44% yield (32.4 mg, 0.068 mmol). LC-MS (ES and C I): (positive ion) m / z 472 [M+H]+; (negative ion) m / z 470 [MH]- .

[0209] Synthesis of 3'-AOM-pppG: Possessing a newly activated 4 Å molecular sieve Nucleoside AOM-G5 (79 mg, 0.168 mmol) was heated under reduced pressure on P2O5 It was dried for 18 hours. Proton Sponge® (175 mg, 0.816 mmol) ) and anhydrous triethyl phosphate (0.8 mL) were added under nitrogen and stirred at room temperature for 1 hour. The reaction flask was cooled to 0 °C in an ice bath and freshly distilled POCl (19 μL, 0.20 2 mmol) was added dropwise and the reaction was stirred at 0° C. for 15 min. Then, bis- Pyrroline as the tri-n-butylammonium salt (1.68 mL, 0.84 mmol) A 0.5 M solution of the acid was quickly added, followed immediately by tri-n-butylamine (168 μL, 0.70 The reaction was removed from the ice / water bath and stirred vigorously for 5 minutes, then The mixture was poured into 1M aqueous triethylammonium bicarbonate solution (TEAB, 6 mL). The mixture was cooled to 100°C and stirred at room temperature for 18 hours. All solvents were evaporated under reduced pressure. The residue was 35% The mixture was dissolved in aqueous ammonia (10 mL) and stirred at room temperature for at least 5 hours. The crude product was first purified by DEAE-Sepharose. The product was purified by ion exchange chromatography on Dex A25 (50 g). The triphosphate-containing fractions were collected and eluted with a linear gradient of aqueous triethylammonium chloride. The solvent was evaporated to dryness under reduced pressure. The crude material was purified using a YMC-Pack-Pro C18 column. The 3'-AOM-pppG was further purified by preparative-scale HPLC using a triethylamine. The compound was obtained as the methylammonium salt. Yield: 24% (39.7 μmol). LC-MS ( ES and CI): (negative ion) m / z 576 [MH]-; (positive ion) m / z 578 [M+H]+.

[0210] 3'-AOM-ffT-LN3-NR 5 50S0 contains 3'-AOMffA and ffC It was synthesized in a similar manner as described in the preparation of Scheme 7. 3'-AOM-ffT-LN3'-NR 5 Synthesis of 50S0 [ka]

[0211] Synthesis of intermediate T1: 5-iodo-2'-deoxyuridine (3 g, 8.4 mmol) and and palladium(II) acetate (1.6 g, 7.14 mmol) were dissolved in dry, degassed DMF. , and then dissolved in N-allyltrifluoroacetamide (6.4 mL, 42 mmol). The solution was placed under vacuum, then purged with nitrogen three times, and then degassed triethylene Dimethylamine (2.3 mL, 16.8 mmol) was added, and the solution was heated to 80° C. for 2 hours. The black mixture was cooled to room temperature and then diluted with 50 mL of methanol. Carbonated charcoal was added and the solution was filtered through Celite and then evaporated under reduced pressure to give a thick brown oil. The crude product was purified by chromatography on silica gel using EtOAc / MeOH. Purified by LC-MS (ES and CI): (negative ion) m / z 378 (M-H+).

[0212] Synthesis of intermediate T2: 5-[3-(2,2,2-trifluoroacetamido)-allyl] -2'-deoxyuridine (T1) (2.55 g, 6.72 mmol) was dissolved in dry DMF. Imidazole (1.37 g, 20.1 mmol) was added, followed by 4-(dimethylamino)- (amino)pyridine (410 mg, 3.36 mmol) was added. The reaction was cooled to 0° C. Then tert-butyl(chloro)diphenylsilane (1.92 mL, 7.39 mmol) was added slowly in three portions at 30 minute intervals. The reaction was stirred at 0° C. for 6 hours. The solvent was evaporated and the residue was resuspended in 200 mL of EtOAc and diluted with 2 x 200 mL of aqueous The organic phase was washed with saturated NaHCO3 and 200 mL of water, then 100 mL of brine. The crude product was purified using DCM / EtOAc. The product was purified by flash chromatography on silica gel in 68% yield (2.8%). 06g, 4.54mmol). LC-MS (ES and CI): (positive ion) m / z 61 8 (M+H+); (negative ion) m / z 616 (M-H+).

[0213] Synthesis of intermediate T3: 5'-O-(tert-butyldiphenylsilyl)-5-[3-( 2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T2) (2.8 g, 4.53 mmol) dissolved in 10 mL of anhydrous DMSO (136 mmol) Then glacial acetic acid (16 mL, 272 mmol) and acetic anhydride (16 mL, 158 mmol) The reaction was heated to 50°C for 6 hours and then quenched with 200 mL of aqueous After the solution stopped bubbling, extract with 2x150 mL of EtOAc. The organic phases were pooled and washed with 2x200 mL of saturated aqueous NaHCO3, 200 mL of water and 100 mL of brine. The organic phase was dried over MgSO4, filtered and evaporated to dryness. The crude product was purified by flash chromatography on silica using DCM / EtOAc. Purified by HPLC. 77% yield (2.375 g, 3.51 mmol). LC-MS S (ES and CI): (positive ion) m / z 678 (M+H+); (negative ion) m / z 6 76(M-H+).

[0214] Synthesis of intermediate T4: 5'-O-(tert-butyldiphenylsilyl)-3'-O-methyl Thiomethyl-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2 '-Deoxyuridine (T3) (310 mg, 0.45 mmol) was dissolved in 5 ml of water under a N2 atmosphere. Dissolve cyclohexene (228 μL, 2.25 mmol) in 1 L of anhydrous dichloromethane. The solution was cooled to approximately −15° C. Sulfuryl chloride (distilled, 55 μL, 0.675 mm ol) was added dropwise and the reaction was stirred for 20 min. After all the starting material was consumed, cyclohexane was added. An extra portion of hexene (228 μL, 2.25 mmol) was added and the reaction was evaporated under reduced pressure. The residue was quickly purged with nitrogen and then washed with ice-cold allyl alcohol (2.5 mL). was added with stirring at 0° C. The reaction was stirred at 0° C. for 35 minutes and then 25 mL of saturated aqueous The mixture was then quenched with 100 mL of saturated aqueous NaHCO3. aHCO3. The mixture was extracted with 2 x 50 mL of ethyl acetate. The pooled organic phases were washed with MgS The residue was purified by silica gel chromatography using DCM / EtOAc. Purification by flash chromatography on gel was performed in 69% yield (214 mg , 0.311 mmol). LC-MS (ES and CI): (positive ion) m / z 688 ( M+H+); (negative ion) m / z 686 (M-H+).

[0215] Synthesis of Intermediate T5: 5'-O-(tert-butyldiphenylsilyl)-3'-O-a Allyloxymethyl-5-[3-(2,2,2-trifluoroacetamido)-allyl]- 2'-Deoxyuridine (T4) (210 mg, 0.305 mmol) was added under a N2 atmosphere. Dissolved in dry THF (3 mL). A solution of 1.0 M T BAF in THF (367 μL, 0 0.367 mmol) was added. The solution was stirred at room temperature for 3 hours. The solution was diluted with 50 mL of EtOAc. c, then washed with 50 mL of NaH2PO4 sat. (pH = 3), and 50 mL of water was used. The organic phase was dried over MgSO4, filtered and evaporated to dryness. The product was purified by flash chromatography on silica gel using DCM / EtOAc. The product was purified by LC-MS (ES and and CI): (negative ion) m / z 448 (M-H+), 484 (M+Cl-).

[0216] Synthesis of Intermediate T6: 3'-O-allyloxymethyl-5-[3-(2,2,2-trimethylphenyl)-2-(2-methyl-2-propanol]-2-yl]methyl [Fluoroacetamido]-allyl]-2'-deoxyuridine (T5) (120 mg, 0. 267 mmol) was dried under reduced pressure. P2O5 for 18 hours. Triethyl phosphate anhydride (1 mL) and some freshly activated 4 Å molecular sieves were added to it under nitrogen. The reaction flask was then cooled to 0 °C. Freshly distilled POCl (30 μL, 0.3 2 mmol) was added dropwise, followed by ProtonSponge® (85 mg, 0. After the addition, the reaction was stirred for an additional 15 minutes at 0°C. Bis-tri-n-butylammonium salt (2.7 mL, 1.33 mmol) in DMF A 0.5 M solution of pyrophosphate was added quickly, followed immediately by tri-n-butylamine ( The reaction was kept in the ice-water bath for an additional 10 minutes, and then Then, pour it into 1M aqueous triethylammonium bicarbonate solution (TEAB, 10 mL) The mixture was quenched with HCl and stirred at room temperature for 4 hours. All solvents were evaporated under reduced pressure. To the residue was added 35% aqueous ammonia (10 mL), and the mixture was stirred at room temperature for 18 hours. The solvent was then evaporated under reduced pressure and the residue was resuspended in 10 mL of 0.1 M TEAB. The filtrate was first subjected to ion exchange with DEAE-Sephadex A25 (100 g). Purification was performed by chromatography. The column was filled with aqueous triethylammonium bicarbonate (T The triphosphate-containing fractions were pooled and the solvent was evaporated to dryness under reduced pressure. The crude material was purified by preparative scale HPLC using a YMC-Pack-ProC18 column. Compound T6 was obtained as a triethylammonium salt in 33% yield. (89 μmol). LC-MS (ES and CI): (negative ion) m / z 592 (MH +), 295(M-2H+).

[0217] 3'-AOM-ffT-LN3'-NR 5 Synthesis of 50S0: Dry known compound LN 3-NR 5 50SO (0.015 mmol) was dissolved in anhydrous DMA (2 mL) under N N,N-diisopropylethylamine (17 μL, 0.1 mmol) was added, followed by T STU (0.1 M in DMA, 180 μL, 0.018 mmol) was added. The reaction was purged with N The mixture was stirred at room temperature for 1 hour under reduced pressure. During this time, an aqueous solution of T6 (0.01 mmol) was evaporated under reduced pressure. The mixture was evaporated to dryness and resuspended in 0.1M TEAB (200 μL) and LN3-NR 5 The reaction was stirred at room temperature for 18 hours, then added to a 0.1M TEAB solution. The crude product was purified by flashing on DEAE-Sephadex. The product was further purified by preparative HPLC to give Pure 3'-AOM-ffT-LN3'-NR 5 50S0 was obtained in 67% yield (41· mol, measured by UV-Vis spectroscopy, λ max = 550 nm, ε = 125000 M-1c m-1). LC-MS (ES): (negative ion) m / z 1521 (M-H+), 761 (M -2H+), 507(M-3H+).

[0218] (Sequencing by synthesis experiment) The ffNs were then analyzed by sequencing using an Illumina MiniSeq® instrument. All standard market mixes were tested except for the new built-in mixes that contain these ffNs. Commercially available reagents were used. A standard 2x150 recipe was used. Standard synthetic sequences were used. In addition to the SBS protocol, a 5-second stimulation in a solution of palladium cleavage mixture was performed. Incubation (Pd:THP=1 / 5 with DEEA as described in Example 4) was added for 3 '-AOM unblocked.

[0219] In the first experiment, the following ffNs were used in the incorporation mix: 3'-AOM-ffT- LN3-NR 5 50S0, 3'-AOM-ffA-LN3-BL-NR 5 50S0, 3' -AOM-ffA-LN3-BL-NR 6 50C5, 3'-AOM-ffA-LN3-N R7180A, 3′-AOM-ffC-LN3-SO7181, and 3′-AOM-p ppG (dark G). The sequencing results for Read 1 are summarized below.

[0220] [Table 1]

[0221] In the second experiment, unlabeled 3'-AOM was used in the same manner as in the preparation of 3'-AOM-pppG described above. -pppT was synthesized (LC-MS (ES): (negative ion) m / z 551 (M-H+)) Commercially available green ffG-LN3-PEG12-ATTO532 (Illumina 4 channel) used in the system) and described in the first experiment above. The same ffA and ffC were used. The results are summarized below. A significant improvement in fading values was observed, and no signal attenuation was observed (Figure 3A). ,The error rates of both read 1 and read 2 also decreased.

[0222] [Table 2]

[0223] In another experiment, 3'-AOM-ffT-LN3'-NR 5 50S0, 3'-AOM-f fA-LN3-BL-NR 5 50S0, 3'-AOM-ffA-LN3-BL-NR 6 5 0C5, Incorporation Mix NR7180A containing 3'-AOM-ffA-LN3-, 3' -AOM-ffC-LN3-SO7181, and 3'-AOM-pppG (dark G) As in the previous work, a palladium catalyst (Pd / THP = 1:10; as described in Example 4) was used. A 5-second incubation with cleavage mixture containing 100 mM DEEA (cleaved) was used as a standard. The standard MiniSeq® DNA polymerase was used. The uptake time was doubled. No signal decay phenotype was observed (Figure 3). B). Furthermore, these sequencing results are consistent with those of f with a standard azidomethyl blocking group. compared to a commercial MiniSeq® run (average 3; N = 3) using fN The error rates were observed to be almost the same (Figure 3C). To summarize:

[0224] [Table 3]

[0225] Furthermore, the main sequence metrics of ffNs with 3'-AOM blocking groups were Standard MiniSeq® commercial kit containing DNA polymerase Pol 812 The results are shown in Figure 4A. Due to improved quality, very low prephasing was observed. However, the 2x uptake Even when a timed delay was used, the phase was still elevated.

[0226] In yet another experiment, the commercially available MiniSeq® kit (Pol 812) Instead of DNA polymerase, another DNA polymerase (Pol 1901) was used. Pol 1901 was used in the sequence at the standard 1x acquisition time instead of the double acquisition time mentioned above. Furthermore, incubation with the Pd cleavage mixture allowed for twice the uptake time. This reduces the total time required for the SBS chemistry cycle by half compared to the standard run. A 10% time savings was achieved. Sequencing metrics improved significantly, with 3'-O- This exceeded the values obtained from a standard commercial kit containing a dihydromethyl blocking group (Figure 4B ).

[0227] (3' Blocking Group Stability Test in Sequencing) To demonstrate the improved stability of ffN by 3'-AOM, 3'-O-azidomethyl The two sets of ffN were compared side-by-side with the standard MiniSeq® ffN. The standard incorporation mixture, excluding only the DNA polymerase, was incubated at 45°C for several days. At each time point, a new polynucleotide was added immediately before loading onto the MiniSeq®. The incorporation mix was completed by adding ribosomal enzyme. The previously described sequencing conditions were used. Prephase % is a direct indicator of the percentage of 3'OH-ffN present in the mixture Therefore, it directly correlates with the stability of the 3' block group. The rephasing values were recorded and plotted (Figure 5). At 45°C, the 3'-A containing ffN OM appears to be six times more stable than standard ffN containing a 3'-O-azidomethyl group. Sequencing metrics were observed during stability assays in solution. The predicted tendency was also confirmed. The 3'-AOM block was significantly more effective than the 3'-O-azidomethyl group. was stable.

[0228] Example 6. Preparation of 3'-O-thiocarbamate blocked nucleosides In this example, various 3'-O-thiocarbamate protected T nucleosides were skimmed. The solution was prepared according to the method described in Section 8. Scheme 8. Synthesis of 3'-O-dimethylthiocarbamate T nucleosides [ka]

[0229] Preparation of T-7: Add 5'-O- to an oven-dried, nitrogen-purged 100 mL flask. (4,4'-dimethoxytrityl)thymidine (1.0 g, 1.836 mmol) was added. This was co-evaporated with anhydrous DMF (3x20 mL) and placed under nitrogen. Anhydrous DCM (9. 2 mL) and 4-dimethylaminopyridine (224 mg, 0.184 mmol) were added. The mixture was stirred at room temperature until a homogeneous solution was formed. Add dazole (360 mg, 2.02 mmol) quickly under a stream of nitrogen and reseal the reaction. The reaction mixture was stirred at room temperature for 2 hours until all the starting material was consumed. Filter through a bed and wash the filter cake with EtOAc (10 mL). The material was removed in vacuo and the crude residue was used without further purification.

[0230] Preparation of T-8: Compound T-7 from the previous step was used immediately after drying in vacuo. The residue was placed under nitrogen in a 25 mL round-bottom flash tube and dimethylamine (2 in THF) was added. M, 7.3 mL, 14.6 mmol) was added and all starting material was consumed according to TLC. The reaction was stirred for 2 hours until all volatiles were removed in vacuo to give a clear crude residue. which was purified by flash column chromatography on silica gel to form T-8 was obtained as a white solid. Yield: 1.15 g (99%). LC-MS (electrochemical analysis) Spray Negative) 630.23 [MH].

[0231] Preparation of T-9: Starting nucleoside T-8 (320 mg, 0.504 mmol) was dissolved in air. Dissolve in minimal acetonitrile in a 50 mL round-bottom flask containing AcOH / H2O 5: Add the solution of 1 (12.5 mL:2.5 mL) in one portion and continue stirring until all the starting material is consumed. The reaction was stirred at room temperature for 2-4 hours. All volatiles were evaporated under vacuum. The residue was co-evaporated with toluene (2x60 mL) to give the crude product as an off-white solid. The crude product was purified by flash column chromatography to give T- 9 was obtained as a white solid. Yield: 123 mg (74%). LC-MS (electrospray -negative) [MH] 328.10.

[0232] The other two thiols were prepared following a similar synthetic procedure using the corresponding MeNH2 or NH3. Nucleosides bearing carbamate protecting groups were also prepared. The general reaction scheme is shown below. . [ka]

[0233] (Stability Test of 3'-O-Thiocarbamate Blocking Group) Stability testing of 5'-mP 3'-DMTC T nucleotides was performed using standard 5'-mP3' The sequences were performed in uptake buffer containing -O-azidomethyl T nucleotides. [ka]

[0234] Both 5'-mP 3'-DMTC T and 5'-mP 3'-O-azidomethyl T The final solution volume was 1 mL, and the final concentrations of the corresponding nucleotides were both 0.1 mM. Other components of the buffered aqueous solution include ethanolamine (EA), ethanolamine HCl, N The buffer contains 100 mM aCl, and 2.5 mM EDTA. 0.5MEA buffer, 0.5M NaCl, 0.01M EDTA.

[0235] (Stability test method) Place 200 μL of 10x buffer into a 1.7 mL polypropylene snap-lock microtube. The corresponding nucleoside was then added to the buffer and diluted with the appropriate amount of 18 mΩ water. Seal the vial and invert, gently agitate, or pump with a micropipette. A 40 μL aliquot was taken and analyzed by HPLC to determine the starting value (or t = 0). The vial was then placed in a preheated heating mantle set at 65°C. Place in a refrigerator, cover with a thick layer of aluminum foil, and heat for 1 month. Periodically, 40 μL aliquots were The samples were collected periodically (1st week: once a day, 2nd to 4th weeks: once every 2 days) and analyzed by HPLC. The percentage of starting material and the percentage of unblocked (3'-OH) nucleotides in the sample The area of the nucleotide peak and the 3'OH peak was measured by HPLC. These values were displayed graphically and the uptake buffer concentration between samples was Percentage of unblocked nucleotides used to compare stability was used to calculate the α-O-azidomethyl blocking group at 65°C. Three different thiocarbamate 3' blocking for blocked nucleotides The results show the stability comparison of nucleotides. 3'-OC(=S) N H2 or 3'- OC(=S) N HCH3-containing nucleotides are protected with the standard 3'-O-azidomethyl group Although less stable than nucleotides containing 3'-DMTC, nucleotides containing 3'-DMTC are an improvement. It was observed that DMTC was stable over the 9-day test period. It showed better stability than the didomethyl blocking group.

[0236] (Example 7. 3'-O-Thiocarbamate Blocking Group Deblocking Test) In this example, 5'-mP 3'-DMTC T and the standard 5'-mP 3'-O-A Deblocking or deblocking test of didemethyl T nucleotides was performed for each blocking Each condition was run separately in a group-specific solution. Active deblocking reagents have been formulated to mimic deblocking reagents as closely as possible and follow the same methodology. The buffer and nucleoside concentrations were kept the same for all tests, but the same amount of each component was used. Thus, the observed rate differences between individual deblocking chemicals were This is not due to differences in the concentration of the formulation. [ka]

[0237] (General methodology for unblocking tests) Each reaction component was individually formulated as a concentrated stock in 18 mΩ water and stored appropriately. The aliquots were combined in the specific order shown below: Preformulated Deblocking Reagent The reaction was initiated by the addition of final concentrations: nucleoside (0.1 mM), active deblocker Reagent (1 mM), additives (specific to the deblocking reagent), buffer (100 mM). Final volume Volume: 2000μL.

[0238] In a 3 mL glass vial, add the preformulated buffer solution followed by the preformulated excipient solution. This was diluted with the correct amount of 18 mΩ water and stirred for 10 minutes. An aliquot of the leutide solution was added and stirred for 5 minutes. Then, a 40 μL aliquot was taken. and quenching reagent is added to generate the reference (or t = 0 min) peak for HPLC analysis. The deblocking reagent was then added all at once to the stirring solution and timing was started. At 40 μL, aliquots were taken and immediately quenched with the appropriate quenching reagent. The unblocked nucleotides occurring at these specified time points were analyzed by LC. The results were plotted graphically to compare the efficiency and effectiveness of the unblocking. It is used for this purpose.

[0239] (DMTC deblocking) Nucleotide: 5'-mP 3'-DMTC T. Active deblocking reagent: NaI O4 (0.1M in 18mΩ water) or Oxone® (0.1M in 18mΩ water) M). Additives: None. Buffer for NaIO4: pH 6.75 phosphate buffer (18mM) 1M in water). Buffer for Oxone®: pH 8.65 phosphate buffer ( (1M in 18 mΩ water). Quenching reagent: sodium thiosulfate. 3'-O-azidomethyl deblocking agent. The cleavage conditions were the same as those described in Example 3.

[0240] HPLC analysis was performed by separating the nucleoside peak, the 3'-OH peak, and the HPLC chromatogram. This was performed by measuring the area of other nucleotide peaks that appear in the rAb. These values are used to calculate the percentage of initiating nucleotides and unblocked nucleotides. Calculate and graphically display unblocking rates, efficiency, and effectiveness across samples. The results are shown in Figure 7. The deblocking efficiency of DMTC by NaIO4 was However, when DMTC was cleaved with Oxone®, In this case, the proportion of remaining starting material, which is a nucleotide with a DMTC blocking group, is significantly lower. In summary, DMTC significantly improved the deblocking rate of the standard azidomethyl blocking group. It shows a better deblocking rate (using Oxone®) than

Claims

1. Covalently bonded to the 3'-carbon atom 【Chemical 1】 Ribose or deoxyribose having a removable 3'-OH blocking group forming A nucleoside or nucleotide containing: Each R １ａ and R １ｂ are independently H, C １ ~C ６ Alkyl, C １ ~C ６ Hello Aruki Lu, C １ ~C ６ Alkoxy, C １ ~C ６ Haloalkoxy, cyano, halogen, substituted optionally substituted phenyl, or optionally substituted aralkyl; Each R ２ａ and R ２ｂ are independently H, C １ ~C ６ Alkyl, C １ ~C ６ Hello Aruki aryl, cyano, or halogen; Or, R １ａ and R ２ａ together with the atoms to which they are attached, are replaced forming an optionally substituted 5- to 8-membered heterocyclyl group; R ３ is H, optionally substituted C ２ ~C ６ Alkenyl, optionally substituted C ３ ~C ７ Cycloalkenyl, optionally substituted C ２－ C ６ Alkynyl, or substituted (C １ ~C ６ alkylene)Si(R ４ ) ３ and Each R ４ are independently H, C １ ~C ６ Alkyl or optionally substituted C ６ ~C １０ aryl; provided that each R １ａ , R １ｂ , R ２ａ and R ２ｂ If is H, then R ３ is not H, Nucleosides or nucleotides.

2. R １ａ and R １ｂ 2. The nucleoside or or nucleotides.

3. Each R １ａ and R １ｂ The nucleoside or nucleotidin of claim 2, wherein Do.

4. R ２ａ and R ２ｂ each independently represents H, halogen, or C １ ~C ６ It is alkyl The nucleoside or nucleotide according to any one of claims 1 to 3.

5. Each R ２ａ and R ２ｂ The nucleoside or nucleotidin of claim 4, wherein Do.

6. Each R ２ａ and R ２ｂ But independently, C １ ~C ６ alkyl or halogen. Item 5. The nucleoside or nucleotide according to Item 4.

7. Each R ２ａ and R ２ｂ is methyl. Punch.

8. R ２ａ is H and R ２ｂ is halogen or C １ ~C ６ 4. The alkyl group of claim 3. A nucleoside or nucleotide as described above.

9. R ３ But halogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyls and combinations thereof C optionally substituted with one or more substituents independently selected from the group consisting of ２ ~C ６ The nucleoside or nucleotidin according to any one of claims 1 to 7, which is alkynyl. Do.

10. R ３ but, 【Chemistry 2】 10. The nucleoside or nucleotide of claim 9, wherein:

11. R ３ But halogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyls and their combinations C optionally substituted with one or more substituents independently selected from the group consisting of ２ ~C ６ The nucleoside or nucleotidin according to any one of claims 1 to 7, wherein the nucleoside or nucleotidin is alkenyl. Do.

12. R ３ but, 【Chemistry 3】 12. The nucleoside or nucleotide of claim 11, wherein:

13. R ３ may be substituted (C １ ~C ６ alkylene)Si(R ４ ) ３ and each R ４ But C １ ~C ６ 8. The nucleoside of any one of claims 1 to 7, which is alkyl. or nucleotides.

14. R ３ But -(CH ２ )-SiMe ３ The nucleoside or nucleoside of claim 13, Cleotide.

15. R １ａ and R ２ａ together with the atoms to which they are attached form a six-membered heterocycle. The nucleoside or nucleotide according to claim 1, which forms a ring.

16. The 6-membered heterocyclyl group has the structure 【Chemistry 4】 16. The nucleoside or nucleotide of claim 15, having the formula:

17. Each R １ｂ , R ２ｂ and R ３ is H. or nucleotide.

18. A 3'-OH blocking group is covalently attached to the 3'-carbon of ribose or deoxyribose. Combined: 【Chemistry 5】 The nucleoside or nucleotidin of claim 1, comprising a structure selected from the group consisting of: Do.

19. Covalently bonded to the 3'-carbon atom 【Chemistry 6】 Ribose or deoxyribose having a removable 3'-OH blocking group forming A nucleoside or nucleotide containing R ５ and R ６ each independently represents H, C １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl , C ２ ~C ６ Alkynyl, C １ ~C ６ Haloalkyl, C ２ ~C ８ Alkoxyalkyl, optional optionally substituted -(CH ２ ) ｍ -phenyl, optionally substituted -(CH ２ ) ｎ - (5 again is a 6-membered heteroaryl), optionally substituted —(CH ２ ) ｋ -C ３ ~C ７ Carbocycline or optionally substituted -(CH ２ ) ｐ -(3- to 7-membered heterocyclyl); - (CH ２ ) ｍ -, -(CH ２ ) ｎ -, -(CH ２ ) ｋ -, and -(CH ２ ) ｐ -of each is optionally substituted; and Each of m, n, k, and p is independently 0, 1, 2, 3, or 4; Nucleosides or nucleotides.

20. R ５ and R ６ At least one of is H or C １ ~C ６ 19. The alkyl group of claim 19. A nucleoside or nucleotide according to any one of claims 1 to 4.

21. Each R ５ and R ６ is H. 。

22. R ５ is H and R ６ But C １ ~C ６ Alkyl, C ２ ~C ６ Alkenyl, C ２ ~C ６ a alkynyl, optionally substituted -(CH ２ ) ｍ -phenyl or optionally substituted - (CH ２ ) ｎ -6-membered heteroaryl, and each of m and n is 0 or 1; be.

21. A nucleoside or nucleotide according to claim 20.

23. Each R ５ and R ６ But C １ ~C ６ 21. The nucleoside or nucleoside of claim 20, or nucleotides.

24. Each R ５ and R ６ is methyl. Chid.

25. The 3'-OH blocking group is: 【Chemistry 7】 20. The nucleoside or nucleoside of claim 19, comprising a structure selected from the group consisting of: Chid.

26. The nucleoside or nucleotide is optionally linked via a cleavable linker to a detectable 26. The nucleoside or nucleoside of any one of claims 1 to 25 covalently attached to a label. Cleotide.

27. The detectable label is attached to the nucleoside or nucleotide via a cleavable linker.

27. The nucleoside or nucleotide of claim 26, which is covalently linked to an acid base.

28. A detectable label is attached to the 3′ end of the nucleoside or nucleotide via a cleavable linker.

27. The nucleoside or nucleotide of claim 26, wherein the nucleoside or nucleotide is covalently linked to the '-oxygen.

29. The linker may comprise an azide moiety, an -O-allyl moiety, a disulfide moiety, an acetal moiety, or or a cleavable linker comprising a thiocarbamate moiety. The nucleosides or nucleotides described above.

30. The 3'-OH blocking group and the cleavable linker are removed under the same chemical reaction conditions.

30. The nucleoside or nucleotide according to any one of claims 27 to 29.

31. The nucleoside or nucleoside according to any one of claims 1 to 30, which contains 2' deoxyribose. or nucleotides.

32. 32. The nucleoside or nucleotide of claim 31, wherein the nucleotide is a nucleotide triphosphate. is a nucleotide.

33. An oligonucleotide comprising the nucleotide according to any one of claims 1 to 32.

34. A growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction A method for preparing a nucleotide according to any one of claims 1 to 32. into a growing complementary polynucleotide, The incorporation of a nucleotide results in the subsequent incorporation of a nucleotide into the growing complementary polynucleotide. Prevent the introduction of,.

35. Nucleotide incorporation occurs via polymerase, terminal deoxynucleotidyl transferase, 35. The method of claim 34, wherein the method is accomplished by a reverse transcriptase or a reverse transcriptase.

36. 1. A method for determining the sequence of a target single-stranded polynucleotide, comprising: (a) a nucleotide according to any one of claims 26 to 32, incorporating into a copy polynucleotide strand complementary to at least a portion of the strand; (b) detecting the identity of the nucleotide incorporated into the copy polynucleotide strand; and; and (c) labeling and 3' from the nucleotide incorporated into the copy polynucleotide strand. Chemically removing the blocking group A method comprising:

37. (d) Chemically remove the label and the 3' blocking group from the copied polynucleotide strand.

37. The method of claim 36, further comprising washing from

38. Steps (a) to (d) are performed until the sequence of said portion of the template polynucleotide strand is determined.

38. The method of claim 37, further comprising repeating until

39. 38. The method of claim 37, wherein steps (a) through (d) are repeated at least 50 times. 。

40. The label and 3' bromine from the nucleotides incorporated into the copy polynucleotide strand.

40. The method of claim 36, wherein the binding group is removed in a single chemical reaction. method.

41. Step (c) contacts the incorporated nucleotide with a cleavage solution containing a palladium catalyst.

41. The method of claim 40, comprising contacting.

42. The label and 3' bromine from the nucleotides incorporated into the copy polynucleotide strand.

40. The method of claim 36, wherein the binding group is removed in two separate chemical reactions. The method described below.

43. Step (c) reacting the incorporated nucleotide with a cleavage solution comprising phosphine, and 42. The method of claim 41, comprising contacting with a cleavage solution comprising a palladium catalyst.

44. Phosphines are tris(hydroxymethyl)phosphine and tris(hydroxyethyl)phosphine. 41 or 43, which is a sphingophosphine or tris(hydroxypropyl)phosphine. The method described below.

45. The cleavage solution containing the palladium catalyst is a primary amine, a secondary amine, a tertiary amine, a carbonate one or more buffers selected from the group consisting of salts, phosphates, borates, and combinations thereof; 45. The method of any one of claims 41, 43 or 44, further comprising a buffer reagent.

46. The buffering reagents are ethanolamine (EA), tris(hydroxymethyl)aminomethane ( Tris), glycine, carbonate, phosphate, borate, 2-dimethylaminomethanol ( DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-tetramethyl Methylethylenediamine (TEMED), N,N,N',N'-tetraethylethylenediamine amine (TEEDA), and combinations thereof.

45. The method according to claim 45.

47. A compound comprising one or more nucleosides or nucleotides according to any one of claims 1 to 32. Mu Kit.

48. 48. The kit of claim 47, further comprising an enzyme and a buffer suitable for enzyme action.

49. The enzyme is a polymerase, terminal deoxynucleotidyl transferase, or reverse transcription 49. The kit of claim 48, which is an enzyme.

50. 50. The kit of claim 49, wherein the polymerase is a DNA polymerase.

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