JP6868541B2 - Modified nucleosides or modified nucleotides - Google Patents

Description

Background Some embodiments described herein relate to modified nucleotides or nucleosides containing 3'-hydroxy protecting groups, and the use of the nucleotides or nucleosides in polynucleotide sequencing methods. Some embodiments described herein relate to methods of preparing 3'-hydroxyprotected nucleotides or nucleosides.

Description of Related Techniques Advances in molecular research have been, in part, brought about by improvements in the techniques used to characterize molecules or their biological reactions. In particular, the study of nucleic acids DNA and RNA has benefited by developing techniques used in sequence analysis and in the study of hybrid formation events.

One example of a technique that has improved nucleic acid research is the development of an array of immobilized nucleic acids produced. These arrays typically consist of a dense matrix of polynucleotides immobilized on a solid support material. For example, Fodor et al., Trends Biotech. See 12: 19-26, 1994. This document is a nucleic acid using a chemically sensitized glass surface that is protected by a mask but exposed in a region defined to allow binding of properly modified nucleotide phosphoramidite. Describes how to assemble. The prepared array can also be produced by a technique of "pointing" known polynucleotides on a solid support at predetermined positions (eg, Simpson et al., Proc. Natl. Acad. Sci). .92: 6379-6383, 1995).

One method of determining the nucleotide sequence of nucleic acids bound to an array is called "synthetic sequencing" or "SBS". This technique for sequencing DNA ideally requires controlled (ie, one at a time) uptake of the correct complementary nucleotides opposite the nucleic acid being sequenced. This allows each nucleotide residue to be sequenced one at a time, allowing accurate sequencing by adding nucleotides in multiple cycles, thus preventing an uncontrolled sequence of uptake. To do. The incorporated nucleotide is read with a suitable label that binds to the nucleotide prior to removal of the labeled moiety and subsequent sequencing of the next round.

To ensure that only a single uptake occurs, structural modifications (“protecting groups”) are added to each labeled nucleotide added to the growing strand, and only one nucleotide is taken up. Make sure that. After the nucleotide with the protecting group is added, the protecting group is then removed under reaction conditions that do not interfere with the integrity of the DNA during sequencing. The sequencing cycle can then continue uptake of the next protected and labeled nucleotide.

To be useful in DNA sequencing, nucleotides, more generally nucleotide triphosphates, are generally replicated by the polymerase used to incorporate the nucleotide into the polynucleotide chain once the base on the nucleotide has been added. It requires a 3'-hydroxy protecting group that prevents it from continuing. There are many restrictions on the types of groups that can be added to nucleotides and may still be suitable. The protecting group should be easily removed from the sugar moiety without causing damage to the polynucleotide chain and at the same time prevent additional nucleotide molecules from being added to the polynucleotide chain. In addition, the modified nucleotide needs to be tolerated by the polymerase or other suitable enzyme used to incorporate the nucleotide into the polynucleotide chain. The ideal protecting group therefore exhibits long-term stability, is efficiently taken up by the polymerase enzyme, results in blockage of secondary or additional nucleotide uptake, and is mild without damage to the polynucleotide structure. It has the ability to be removed under conditions (preferably under aqueous conditions).
Reversible protecting groups have been previously described. For example, Metsker et al. (Nucleic Acids Research, 22 (20): 4259-4267, 1994) have synthesized and used eight 3'modified 2-deoxyribonucleoside 5'-triphosphates (3'modified dNTPs), as well as We disclose tests in two DNA template assays for uptake activity. WO2002/029003 describes a sequencing method that may include the use of an allyl protecting group to cap the 3'-OH group on the growing DNA strand in the polymerase reaction.
In addition, we develop some reversible protecting groups and deprotect them under DNA compatible conditions in WO 2004/018497, which is incorporated herein by reference in its entirety. I have already reported the method.

International Publication No. 2002/029003 International Publication No. 2004/018497

Fodor et al., Trends Biotechnology. 12: 19-26, 1994 Simpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995 Metsker et al. (Nucleic Acids Research, 22 (20): 4259-4267, 1994)

Abstract Some embodiments described herein are removable 3'-forming _{a structure-OC (R) 2} N ₃ covalently attached to a 3'-carbon atom with a purine or pyrimidine base. Here, with respect to a modified nucleotide molecule or a modified nucleoside molecule, including a sugar moiety of ribose or deoxyribose having a hydroxy protecting group.
R is hydrogen, -C (R ¹ ) _m (R ² ) _n , -C (= O) OR ³ , -C (= O) NR ⁴ R ⁵ , -C (R ⁶ ) ₂ O (CH ₂ ) Selected from the group consisting of _p NR ⁷ R ⁸ and -C (R ⁹ ) ₂ O-Ph-C (= O) NR ¹⁰ R ^11.
Each R ¹ and R ² is selected independently of hydrogen, optionally substituted alkyl, or halogen.
R ³ is selected from hydrogen or optionally substituted alkyl,
Each R ⁴ and R ⁵ is independent of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl. Selected
Each R ⁶ and R ⁹ is selected from hydrogen, optionally substituted alkyl, or halogen.
Each R ⁷ , R ⁸ , R ¹⁰ and R ¹¹ can be hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted. Selected independently of the Aryl Kills
m is an integer from 0 to 3 and n is an integer from 0 to 3, where the sum of m + n is equal to 3 and p is an integer from 0 to 6.
Neither R ¹ nor R ² can be halogen, and at least one R is not hydrogen.

Some embodiments described herein include a step of incorporating the modified nucleotide molecules described herein into a growing complementary polynucleotide with a target single-stranded polynucleotide in a sequencing reaction. With respect to methods of preparing complementary growing polynucleotides, the incorporation of modified nucleotides here prevents the introduction of any subsequent nucleotides into the growing complementary polynucleotide.

Some embodiments described herein are steps of monitoring the continuous uptake of complementary nucleotides, wherein at least one complementary nucleotide incorporated herein is described herein. It relates to a method for sequencing a target single-stranded polynucleotide, comprising the steps of being a modified nucleotide molecule and detecting the identity of the modified nucleotide molecule. In some embodiments, uptake of the modified nucleotide molecule is achieved by terminal transferase, terminal polymerase or reverse transcriptase.

Some embodiments described herein relate to kits comprising the plurality of modified nucleotide molecules or modified nucleoside molecules described herein, and packaging materials for them. In some embodiments, the identity of the modified nucleotide is determined by detecting a detectable label attached to a base. In some such embodiments, the 3'-hydroxy protecting group and the detectable label are removed prior to the introduction of the next complementary nucleotide. In some such embodiments, the 3'-hydroxy protecting group and the detectable label are removed in a single step chemical reaction.
The present invention provides, for example,:
(Item 1)
A purine base or a pyrimidine base and a sugar moiety of ribose or deoxyribose having a removable 3'-hydroxy protective group that forms _{a structure-OC (R) 2} N ₃ covalently bonded to a 3'-carbon atom. A modified nucleotide molecule or a modified nucleoside molecule containing, where R is hydrogen, -C (R ¹ ) _m (R ² ) _n , -C (= O) OR ³ , -C (= O). ) NR ⁴ R ⁵ , -C (R ⁶ ) ₂ O (CH ₂ ) _p NR ⁷ R ⁸ and -C (R ⁹ ) ₂ O-Ph-C (= O) NR ¹⁰ R ¹¹ selected from the group ,
Each R ¹ and R ² is selected independently of hydrogen, optionally substituted alkyl, or halogen.
R ³ is selected from hydrogen or optionally substituted alkyl,
Each R ⁴ and R ⁵ is independent of hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl. Selected
Each R ⁶ and R ⁹ is selected from hydrogen, optionally substituted alkyl, or halogen.
Each R ⁷ , R ⁸ , R ¹⁰ and R ¹¹ can be hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted. Selected independently of the Aryl Kills
m is an integer from 0 to 3 and
n is an integer from 0 to 3, where the sum of m + n is equal to 3 and p is an integer from 0 to 6.
Both R ¹ and R ² cannot be halogen, and at least one R is a modified nucleotide molecule or modified nucleoside molecule that is not hydrogen.
(Item 2)
The modified nucleotide molecule or modified nucleoside molecule of item 1, wherein one of R is hydrogen and the other R is -C (R ¹ ) _m (R ² ) _n.
(Item 3)
The modified nucleotide molecule or modification according to item 1 or 2, wherein -C (R ¹ ) _m (R ² ) _n is selected from -CHF ₂ , -CH ₂ F, -CHCl ₂ or -CH _{2 Cl.} Nucleoside molecule.
(Item 4)
The modified nucleotide molecule or modified nucleoside molecule according to any one of items 1-3, wherein −C (R ¹ ) _m (R ² ) _n is −CHF _2.
(Item 5)
The modified nucleotide molecule or modified nucleoside molecule according to any one of items 1-3, wherein -C (R ¹ ) _m (R ² ) _n is -CH _{2 F.}
(Item 6)
The modified nucleotide molecule or modified nucleoside molecule of item 1, wherein one of R is hydrogen and the other R is -C (= O) OR ^3.
(Item 7)
The modified nucleotide molecule or modified nucleoside molecule of item 6, wherein R ^{3 is hydrogen.}
(Item 8)
The modified nucleotide molecule or modified nucleoside molecule of item 1, wherein one of R is hydrogen and the other R is -C (= O) NR ⁴ R ^5.
(Item 9)
The modified nucleotide molecule or modified nucleoside molecule of item 1 or 8, wherein both R ⁴ and R ^{5 are hydrogen.}
(Item 10)
The modified nucleotide molecule or modified nucleoside molecule of item 1 or 8, wherein R ⁴ is hydrogen and R ⁵ is C _{1-6 alkyl.}
(Item 11)
The modified nucleotide molecule or modified nucleoside molecule of item 1 or 8, wherein both R ⁴ and R ⁵ are C _{1-6 alkyl.}
(Item 12)
The modified nucleotide molecule or modified nucleoside according to item 1, wherein one of R is hydrogen and the other R is -C (R ⁶ ) ₂ O (CH ₂ ) _p NR ⁷ R ^8. molecule. (Item 13)
R ⁶ are both hydrogen, modified nucleotide molecule or modified nucleosides molecule of claim 1 or 12.
(Item 14)
The modified nucleotide molecule or modified nucleoside molecule according to any one of items 1, 12 and 13, wherein both R ⁷ and R ^{8 are hydrogen.}
(Item 15)
The modified nucleotide molecule or modified nucleoside molecule according to any one of items 1 and 12-14, wherein p is 0.
(Item 16)
The modified nucleotide molecule or modified nucleoside molecule according to any one of items 1 and 12-14, wherein p is 6.
(Item 17)
The modified nucleotide molecule or modification according to item 1, wherein one of R is hydrogen and the other R is -C (R ⁹ ) ₂ O-Ph-C (= O) NR ¹⁰ R ^11. Nucleoside molecule.
(Item 18)
The modified nucleotide molecule or modified nucleoside molecule of item 1 or 17, wherein R ^{9 is both hydrogen.}
(Item 19)
The modified nucleotide molecule or modified nucleoside molecule according to any one of items 1, 17 and 18, wherein both R ¹⁰ and R ^{11 are hydrogen.}
(Item 20)
The modified nucleotide molecule or modified nucleoside molecule of any one of items 1, 17 and 18, wherein R ¹⁰ is hydrogen and R ^{11 is an amino-substituted alkyl.}
(Item 21)
The modified nucleotide molecule or modified nucleoside molecule according to any one of items 1 to 14, wherein the 3'-hydroxy protecting group is removed in a deprotection reaction using phosphine.
(Item 22)
The modified nucleotide molecule or modified nucleoside molecule of item 21, wherein the phosphine is tris (hydroxymethyl) phosphine (THP).
(Item 23)
The modified nucleotide molecule or modified nucleoside molecule of any one of items 1-22, wherein the base is attached to a detectable label via a cleavable or non-cleavable linker. ..
(Item 24)
The modified nucleotide molecule of any one of items 1-22, wherein the 3'-hydroxy protecting group is attached to a detectable label via a cleavable or non-cleavable linker. A modified nucleoside molecule.
(Item 25)
The modified nucleotide molecule or modified nucleoside molecule of item 23 or 24, wherein the linker is cleavable.
(Item 26)
The modified nucleotide molecule or modified nucleoside molecule of any one of items 23-25, wherein the detectable label is a fluorophore.
(Item 27)
The modified nucleotide molecule or modified nucleoside molecule of any one of items 23-26, wherein the linker is acid-labile, light-labile, or contains a disulfide bond. ..
(Item 28)
A method for preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, wherein the modified nucleotide molecule according to any one of items 1 to 27 is complemented during the growth. A method comprising the step of incorporating into a specific polynucleotide, wherein the incorporation of the modified nucleotide prevents the subsequent introduction of any subsequent nucleotide into the growing complementary polynucleotide.
(Item 29)
28. The method of item 28, wherein integration of the modified nucleotide molecule is achieved by terminal transferase, terminal polymerase or reverse transcriptase.
(Item 30)
A method for sequencing a target single-stranded polynucleotide,
A step of monitoring the continuous uptake of complementary nucleotides, wherein at least one complementary nucleotide incorporated is the modified nucleotide molecule of any one of items 23-27. A method comprising a step and a step of detecting the identity of the modified nucleotide molecule.
(Item 31)
30. The method of item 30, wherein the identity of the modified nucleotide is determined by detecting the detectable label attached to the base.
(Item 32)
30 or 31. The method of item 30 or 31, wherein the 3'-hydroxy protecting group and the detectable label are removed prior to introducing the next complementary nucleotide.
(Item 33)
32. The method of item 32, wherein the 3'-hydroxy protecting group and the detectable label are removed in a single step chemical reaction.
(Item 34)
A kit comprising a plurality of modified nucleotide molecules or modified nucleoside molecules according to any one of items 1-27, and a packaging material for the same.
(Item 35)
34. The kit of item 34, further comprising an enzyme and a buffer suitable for the action of the enzyme.

FIG. 1A illustrates various 3'-OH protecting groups.

FIG. 1B illustrates the thermal stability of various 3'-OH protecting groups.

FIG. 2A illustrates the deprotection rate curves of three different 3'-OH protecting groups.

FIG. 2B shows a chart of the deprotection half-lives of three different 3'-OH protecting groups.

FIG. 3 shows the phased and prephased values of various modified nucleotides with 3'-OH protecting groups and standard protecting groups for thermal stability in comparison.

FIG. 4A shows 2 × 400 bp sequencing data for the mono-F ffNs-A isomer in the uptake mixture (IMX).

FIG. 4B shows 2 × 400 bp sequencing data for the mono-F ffNs-B isomer in the uptake mixture (IMX).

Detailed Description One embodiment is a modified nucleotide or nucleoside containing a 3'-OH protecting group. In one embodiment, the 3'-OH protecting group is a monofluoromethyl-substituted azidomethyl protecting group. In another embodiment, the 3'-OH protecting group is a C-amide substituted azidomethyl protecting group. Yet another embodiment relates to a modified nucleotide having a difluoromethyl-substituted azidomethyl 3'-OH protecting group.
Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of other forms such as the terms "include" and "include", "includes" and "included" is not limiting. The use of the terms "having" and other forms such as "having", "has", and "had" is not limiting. .. As used herein, in both the transitional phase and the body of the claims, the terms "comprise (s)" and "comprising" are open ends. Should be interpreted as having the meaning of. That is, the above term should be construed as synonymous with the phrase "at least having at last" or the phrase "at least including at last". For example, when used in the context of a process, the term "comprising" means that the process includes at least the listed steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" includes, but additionally, the compound, composition, or device includes at least the listed features or components. Means that it may also contain various features or components.

As used herein, common organic abbreviations are defined as follows:
Ac Acetyl Ac ₂ O Acetic anhydride aq. Aqueous
Bn benzyl Bz benzoyl BOC or Boc tert-butoxycarbonyl Bun-butyl cat. Catalytic Cbz Carbobenzyloxy ° C. ° C. dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate ddNTP (s) Dideoxynucleotides (s)
DBU 1,8-diazabicyclo [5.4.0] Undec-7-ene DCA Dichloroacetate DCE 1,2-dichloroethane DCM Methylene chloride DIEA Diisopropylethylamine DMA dimethylacetonitrile DME dimethoxyethane DMF N, N'-dimethylformamide DMSO dimethylsulfoxide DPPA diphenylphosphoryl azide Et ethyl EtOAc Ethyl acetate ffN Fully functional nucleotide g ggram GPC gel permeation chromatography h or hr time iPr isopropyl KPi pH 7.0 10 mM potassium phosphate buffer KPS potassium persulfate IPA isopropyl alcohol IMX uptake mixture LCMS liquid Chromatography-mass spectrometry LDA diisopropylamide lithium m or min mCPBA meta-chloroperoxybenzoate MeOH methanol MeCN acetonitrile Mono-F-CH ₂ F
_{Mono-F ffN Modified nucleotide with -CH 2} F substituted at the methylene position of the azidomethyl 3'-OH protecting group mL milliliter MTBE methyl tert-butyl ether NaN ₃ sodium azide NHS N-hydroxysuccinimide PG protecting group Ph phenyl pt Precipitate rt Room temperature SBS synthesis sequencing TEA Triethylamine TEMPO (2,2,6,6-tetramethylpiperidin-1-yl) Oxyl TCDI 1,1'-thiocarbonyldiimidazole tert, t Tertiary TFA trifluoroacetic acid THF Tetrahydrofuran TEMPO tetramethylethylenediamine μL microliter

As used herein, the term "array" is a population of different probe molecules that are bound to one or more substrates, and the different probe molecules distinguish each other according to their relative position. Refers to a population of probe molecules that can be used. The array may contain different probe molecules, each located at a different addressable position on the substrate. Alternatively, or in addition, the array can include a separate substrate each carrying a different probe molecule, where the different probe molecule is the position of the substrate on the surface to which the substrate is attached, or in a liquid. It can be identified by the position of the substrate. Beads in wells, such as those described in US Pat. No. 6,355,431B1, US 2002/0102578 and PCT Publication WO 00/6437, as exemplary arrays in which the individual substrates are located on the surface. Includes, but is not limited to. An exemplary format that can be used in the present invention to distinguish beads in a liquid array (eg, using a microfluidic device such as a fluorescence activated cell sorter (FACS)) is, for example, a US patent. No. 6,524,793. Further examples of arrays that can be used in the present invention are US Pat. Nos. 5,429,807, 5,436,327, 5,561,071 and 5,583,211. 5,658,734, 5,837,858, 5,874,219, 5,919,523, 6,136,269, 6,287,768, 6,6 287,776, 6,288,220, 6,297,006, 6,291,193, 6,346,413, 6,416,949, 6,482 591, 6,514,751 and 6,610,482, and those described in WO93 / 17126, WO95 / 11995, WO95 / 35505, European Patent 742 287 and European Patent 799 897. However, the present invention is not limited to these.

As used herein, the term "covalently attached" or "covalently bounded" refers to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently bonded polymer coating refers to a polymer coating that forms a chemical bond with a functionalized surface of a substrate as compared to other means, such as bonding to a surface by adhesion or electrostatic interaction. It is recognized that the polymer covalently attached to the surface can also be attached by means in addition to the covalent bond.

As used herein, all "R" groups ^{(e.g., R 2, R 3, R} 4, R 5, R 6, R 7, and is a ^{R 8,} but not limited to) are shown Represents a substituent that can be attached to an atom. The R group may be substituted or unsubstituted. When two "R" groups are described as "integral", the R group and the atom to which the R group is attached can form a cycloalkyl, aryl, heteroaryl, or heterocycle. .. For example, if, but not limited to, R ² and R ³ , or R ² , R ³ , or R ⁴ and the atoms to which they bond are indicated to be "integral" or "bonded to each other." , They are covalently bonded to each other to form a ring, an example of which is shown below.

Whenever a group is described as being "substituted as needed", the group may be unsubstituted or substituted with one or more of the substituents shown. You may. Similarly, when a group is described as "unsubstituted or substituted", the substituent may be selected from one or more of the indicated substituents when substituted. If no substituent is indicated, the indicated "optionally substituted" or "substituted" groups are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, Heteroaricyclyl, aralkyl, heteroaralkyl, (heteroaricyclyl) alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl , O-thiocarbamyl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, Functional groups including, but not limited to, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, amino, monosubstituted amino groups, disubstituted amino groups, and protected derivatives thereof. It is meant that one or more groups selected individually and independently from the group may be substituted individually and independently.

As used herein, "alkyl" refers to a straight or branched chain hydrocarbon chain containing fully saturated (no double or triple bonds) hydrocarbon groups. In some embodiments, the alkyl group may have 1 to 20 carbon atoms (whenever this appears herein, a numerical range such as "1 to 20" is the end point. Refers to each integer in a given range including, for example, "1 to 20 carbon atoms" means that the alkyl group has the largest number of carbon atoms, such as 1 carbon atom, 2 carbon atoms, and 3 carbon atoms. It means that it may consist of 20 (and including 20) carbon atoms, but this definition also applies with the advent of the term "alkyl" for which no numerical range is specified). The alkyl group may also be a medium sized alkyl having about 7 to about 10 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group of the compounds may be specified in the "C ₁ -C ₄ alkyl" or similar designations. By way of example only, "C ₁ -C ₄ alkyl" is from 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is a methyl, ethyl, propyl, iso - propyl, n- butyl, Indicates that it is selected from iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl. The alkyl group may be substituted or unsubstituted.

As used herein, "alkenyl" refers to an alkyl group containing one or more double bonds in a straight or branched hydrocarbon chain. The alkenyl group may be unsubstituted or substituted.

As used herein, "alkynyl" refers to an alkyl group containing one or more triple bonds in a straight or branched hydrocarbon chain. The alkynyl group may be unsubstituted or substituted.

As used herein, "cycloalkyl" refers to a fully saturated (no double or triple bond) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings may be attached to each other in a condensed fashion. The cycloalkyl group can contain 3 to 10 atoms in the ring. In some embodiments, the cycloalkyl group can contain 3-8 atoms in the ring. The cycloalkyl group may be unsubstituted or substituted. Typical cycloalkyl groups include, but are by no means limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein, "aryl" is a carbocyclic (all carbon), monocyclic, or polycyclic having a fully delocalized π-electron system throughout at least one of its rings. A cyclic aromatic ring system (eg, a fused ring system, a bridged ring system or a spiro ring system (eg, with one or more aryl rings, one or more) in which two carbocyclic rings share a chemical bond. More aryl or non-aryl rings) can be mentioned). The number of carbon atoms in an aryl group can vary. For example, in some embodiments, the aryl group _may be a C 6 _{-C 14} aryl _group, C 6 _{-C 10} aryl group or a _{C 6} aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene, and azulene. The aryl group may be substituted or unsubstituted.

As used herein, "heterocyclyl" refers to a ring system containing at least one heteroatom (eg, O, N, S). Such systems can be unsaturated, can contain some unsaturated, or can contain some aromatic potions, or can be all aromatic. The heterocyclyl group may be unsubstituted or substituted.

As used herein, "heteroaryl" refers to one or more heteroatoms, ie, non-carbon elements including, but not limited to, nitrogen, oxygen, and sulfur, and at least one aromatic. Refers to a monocyclic or polycyclic aromatic ring system containing a ring (a ring system having at least one ring having a completely delocalized π-electron system). The number of atoms in the ring of heteroaryl groups can vary. For example, in some embodiments, the heteroaryl group contains 4 to 14 atoms in its ring, 5 to 10 atoms in its ring, or 5 to 6 atoms in its ring. Can be done. Further, the term "heteroaryl" is a fused ring system in which at least two rings (eg, at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings) share at least one chemical bond. including. Examples of heteroaryl rings include furan, flazan, thiophene, benzothiophene, phthalazine, pyrazole, oxazole, benzoxazole, 1,2,3-oxadiazol, 1,2,4-oxadiazole, thiazole, 1, 2,3-Thiazazole, 1,2,4-Thiazazole, benzothiazole, imidazole, benzimidazole, indol, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiazazole, tetrazole, Examples include, but are not limited to, pyridine, pyridazine, pyrimidin, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxalin, cinnoline, and triazine. The heteroaryl group may be substituted or unsubstituted.

As used herein, "heteroalicyclic" or "heteroaricyclyl" means 3 members, 4 members, 5 members, 6 members, 7 members, 8 members, 9 members, 10 members, up to 18 members. Refers to monocyclic, bicyclic, and tricyclic ring systems, where carbon atoms, together with 1 to 5 heteroatoms, constitute the ring system. However, the heterocycle optionally contains one or more unsaturated bonds placed in such a way that a completely delocalized π-electron system does not occur throughout the entire ring. May be good. Heteroatoms are selected independently of oxygen, sulfur, and nitrogen. Heterocycles further include one or more carbonyl or thiocarbonyl functional groups to include oxo and thio systems (eg, lactam, lactone, cyclic imide, cyclic thioimide, and cyclic carbamate) in the definition. It may be contained. When composed of two or more rings, the rings may be attached to each other in a condensed fashion. In addition, any nitrogen in the complex alicyclic may be quaternized. The heteroalicyclyl group or the complex alicyclic group may be unsubstituted or substituted. Examples of such "complex alicyclic" or "heteroaricyclyl" groups include 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-. Dioxolane, 1,4-dioxolane, 1,3-oxatian, 1,4-oxathione, 1,3-oxathiolane, 1,3-dithiol, 1,3-dithiolane, 1,4-oxatian, tetrahydro-1,4- Thiadine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazin, hydantin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline , Isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine, oxylane, piperazine N-oxide, piperazine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, 4H -Pyran, tetrahydropyran, thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, and benzo-condensation analogs thereof (eg, benzimidazolidinone, tetrahydroquinoline, 3,4-methylenedioxyphenyl). However, it is not limited to them.

As used herein, "aralkyl" and "aryl (alkyl)" refer to an aryl group attached as a substituent via a lower alkylene group. The lower alkylene and aryl groups of aralkyl may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

As used herein, "heteroaralkyl" and "heteroaryl (alkyl)" refer to heteroaryl groups attached as substituents via lower alkylene groups. The lower alkylene and heteroaryl groups of heteroaralkyl may be substituted or unsubstituted. Examples include 2-thienylalkyl, 3-thienylalkyl, frillalkyl, thienylalkyl, Helicobacter alkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, as well as benzo condensation analogs thereof. Not limited to.

As used herein, "alkoxy" refers to formula-OR, where R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, or cycloalkynyl, as defined above. A non-limiting list of alkoxys are methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy. Alkoxy may be substituted or unsubstituted.

As used herein, the "C-amide" group refers to the "-C (= O) N ( _Ra R _b )" group, in which _Ra and R _b are independent of each other. It can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroaricyclyl, aralkyl, or (heteroaricyclyl) alkyl. The C-amide may be substituted or unsubstituted.

As used herein, the "N-amide" group refers to the "RC (= O) N ( _Ra )-" group, in which R and _Ra are independently hydrogen, alkyl. , Alkyne, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroaricyclyl, aralkyl, or (heteroaricyclyl) alkyl. The N-amide may be substituted or unsubstituted.

The term "halogen atom", "halogen" or "halo" as used herein is any of the radiation stable atoms in the 7th column of the Periodic Table of the Elements, such as fluorine, chlorine, bromine, and iodine. Means one or one.

The term "amine" as used herein _{refers to two} -NH groups, in which one or more hydrogens can be optionally substituted with R groups. R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroaricyclyl, aralkyl, or (heteroaricyclyl) alkyl.

The term "aldehyde" as used herein _{refers to the -R c-} C (O) H group, where R _c may not be present or may be alkylene, alkenylene, alkynylene, cycloalkylene. , Cycloalkenylene, cycloalkynylene, arylene, heteroarylene, heteroaricyclylene, aralkylene, or (heteroaricyclyl) alkylene may be selected independently.

The term "amino" as used herein refers to _{two -NH groups.}

The term "hydroxy" as used herein refers to a -OH group.

The term "cyano" group, as used herein, refers to a "-CN" group.

The term "azide" as used herein refers to _{three -N groups.}

The term "thiol" as used herein refers to a -SH group.

The term "carboxylic acid" as used herein refers to -C (O) OH.

The term "thiocyanate" as used herein refers to the -SC≡N group.

The term "oxoamine" as used herein _{refers to two} -O-NH groups, where _{one or more hydrogens of -NH 2 in} the formula are optionally substituted with R groups. can do. R can independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroaricyclyl, aralkyl, or (heteroaricyclyl) alkyl.

As used herein, a "nucleotide" includes nitrogen-containing heterocyclic bases, sugars, and one or more phosphate groups. These are the monomeric units of the nucleic acid sequence. In RNA, this sugar is ribose, and in DNA, it is deoxyribose, a sugar that lacks the hydroxyl group present in ribose. The nitrogen-containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as modified derivatives or analogs thereof. The C-1 atom of deoxyribose is attached to N-1 of pyrimidine or N-9 of purine.

As used herein, a "nucleoside" is structurally similar to a nucleotide but has lost the phosphate moiety. An example of a nucleoside analog is one in which the label is attached to the base and the phosphate group attached to the sugar molecule is absent. The term "nucleoside" is used herein in the usual sense as understood by those skilled in the art. Examples include, but are not limited to, ribonucleosides containing ribose moieties and deoxyribonucleosides containing deoxyribose moieties. A modified pentose moiety is a pentose moiety in which the oxygen atom is replaced by a carbon and / or the carbon is replaced by a sulfur or oxygen atom. A "nucleoside" is a monomer that can have substituted base and / or sugar moieties. In addition, the nucleoside may be incorporated into larger DNA and / or RNA polymers and oligomers.

The term "purine base" is used herein in the usual sense as understood by those skilled in the art and includes tautomers thereof. Similarly, the term "pyrimidine base" is used herein in the usual sense as understood by those skilled in the art and includes tautomers thereof. A non-limiting list of optionally substituted purine bases includes purines, adenines, guanines, hypoxanthines, xanthines, alosanthins, 7-alkylguanines (eg, 7-methylguanines), theobromine, caffeine, uric acid. And isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (eg, 5-methylcytosine).

As used herein, "derivative" or "analog" means a synthetic nucleotide derivative or synthetic nucleoside derivative having a modified base moiety and / or a modified sugar moiety. Such derivatives and analogs are discussed, for example, in Sheet, Nucleoside Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90: 543-584, 1990. Nucleotide analogs can also include modified phosphodiester bonds, including phosphorothioates, phosphorodithioates, alkylphosphonates, phosphoranilides and phosphoramidate bonds. "Derivatives", "analogs" and "modified" may be used interchangeably as used herein and are encompassed by the terms "nucleotide" and "nucleoside" as defined herein. Will be done.

）を含む。本明細書で使用する場合、用語「モノホスフェート」、「ジホスフェート」、および「トリホスフェート」は、当業者によって理解されるとおりの通常の意味で使用され、プロトン化形態を含む。 As used herein, the term "phosphate" is used in the usual sense as understood by those skilled in the art and its protonated form (eg, eg).

)including. As used herein, the terms "monophosphate,""diphosphate," and "triphosphate" are used in the usual sense as understood by those skilled in the art and include protonated forms.

The terms "protecting group" and "protecting group" as used herein are molecules to prevent groups present in the molecule from undergoing unwanted chemical reactions. Refers to any molecule or group of atoms added to. The "protecting group" and the "blocking group" may be interchangeable.

As used herein, the prefix "photo" or "photo-" means light or electromagnetic radiation. The term includes, but is not limited to, one or more of the generally known ranges of radiation, microwave, infrared, visible, ultraviolet, X-ray or γ-ray parts of the spectrum. It can include all or part of the electromagnetic spectrum. Part of the spectrum can be blocked by metal regions on the surface, such as the metals shown herein. Alternatively, or in addition, a portion of the spectrum can pass through intervening regions of the surface (eg, regions made of glass, plastic, silica, or other materials shown herein). In certain embodiments, radiation that can pass through metal can be used. Alternatively, or in addition, radiation shielded by glass, plastic, silica, or other materials described herein can be used.

As used herein, the term "phased" refers to the incomplete removal of 3'terminating factors and fluorophores, as well as the incorporation of DNA strands into the cluster by polymerases in a given sequencing cycle. Refers to a phenomenon in SBS caused by failure to complete. Prephase is caused by the efforts of nucleotides without a valid 3'terminating factor, and this uptake event proceeds by one cycle. Due to phasing and prephase, the intensity extracted for a particular cycle will consist of the signal of the current cycle and the noise from the cycles before and after it. As the number of cycles increases, the proportion of sequences per cluster affected by phasing increases, interfering with the correct base identification. Prephase formation can occur due to the presence of trace amounts of unprotected or unblocked 3'-OH nucleotides during synthetic sequencing (SBS). Unprotected 3'-OH nucleotides can occur during the manufacturing process, or perhaps during the storage and reagent handling processes. Therefore, the discovery of nucleotide analogs that reduce the occurrence of prephase is surprising and offers superior advantages in SBS applications over existing nucleotide analogs. For example, the nucleotide analogs provided can result in shorter SBS cycle times, lower phased and prephased values, and longer sequencing read lengths.
3'-OH protecting group-C (R) ₂ N ₃

Some embodiments described herein relate to a modified nucleotide molecule or modified nucleoside molecule having a removable 3'-hydroxy protecting group-C (R) ₂ N _{3, where R is in the formula.} Hydrogen, -C (R ¹ ) _m (R ² ) _n , -C (= O) OR ³ , -C (= O) NR ⁴ R ⁵ , -C (R ⁶ ) ₂ O (CH ₂ ) _p NR ⁷ Selected from the group consisting of R ⁸ and -C (R ⁹ ) ₂ O-Ph-C (= O) NR ¹⁰ R ^{11 , where R 1} , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , m, n and p are defined above.

In some embodiments, one of R is hydrogen and the other R is -C (R ¹ ) _m (R ² ) _n . In some such embodiments, -C (R ¹ ) _m (R ² ) _n is selected from -CHF ₂ , -CH ₂ F, -C HCl ₂ or -CH ₂ Cl. In one embodiment, -C (R ¹ ) _m (R ² ) _n is -CHF ₂ . In another embodiment, -C (R ¹ ) _m (R ² ) _n is -CH ₂ F.

In some embodiments, one of R is hydrogen and the other R is -C (= O) OR ³ . In some such embodiments, R ³ is hydrogen.

In some embodiments, one of R is hydrogen and the other R is -C (= O) NR ⁴ R ⁵ . In some such embodiments, R ⁴ and R ⁵ are both hydrogen. In some other such embodiments, R ⁴ is hydrogen and R ⁵ is C _1-6 alkyl. In yet some other embodiments, ^{R 4} and ^{R 5} are both _{C 1-6} alkyl. In one embodiment, ^{R 5} is n- butyl. In another embodiment, R ⁴ and R ⁵ are both methyl.

In some embodiments, one of R is hydrogen and the other R is -C (R ⁶ ) ₂ O (CH ₂ ) _p NR ⁷ R ⁸ . In some such embodiments, R ⁶ are both hydrogen. In some such embodiments, R ⁷ and R ⁸ are both hydrogen. In some such embodiments, p is zero. In some other such embodiments, p is 6.

In some embodiments, one of R is hydrogen and the other R is -C (R ⁹ ) ₂ O-Ph-C (= O) NR ¹⁰ R ¹¹ . In some such embodiments, R ⁹ are both hydrogen. In some such embodiments, R ¹⁰ and R ¹¹ are both hydrogen. In some other such embodiments, R ¹⁰ is hydrogen and R ¹¹ is a substituted alkyl. In one embodiment, R ¹¹ is an amino-substituted alkyl.
Deprotection of 3'-OH protecting group

In some embodiments, the 3'-OH protecting group is removed in a deprotection reaction with phosphine. The azide group at -C (R) ₂ N ₃ can be converted to an amino group by contacting a modified nucleotide molecule or a modified nucleoside molecule with phosphine. Alternatively, the azide group at -C (R) ₂ N ₃ can be converted to an amino group by contacting such a molecule with a thiol, particularly a water-soluble thiol such as dithiothreitol (DTT). In one embodiment, the phosphine is tris (hydroxymethyl) phosphine (THP). Unless otherwise indicated, references to nucleotides are also intended to be applicable to nucleosides.
Detectable sign

Some embodiments described herein relate to the use of conventional detectable labels. Detection can be performed by any suitable method, including fluorescence spectroscopy or other optical means. A preferred label is a fluorophore, which emits a defined wavelength of radiation after energy absorption. Many suitable fluorescent labels are known. For example, Welch et al. (Chem. Eur. J. 5 (3): 951-960, 1999) disclose Dansyl-functionalized fluorescent moieties that can be used in the present invention. Zhu et al. (Cytometry 28: 206-211, 1997) also describe the use of fluorescent labels Cy3 and Cy5 that can be used in the present invention. Suitable labels for use are also Prober et al. (Science 238: 336-341, 1987), Connel et al. (BioTechniques 5 (4): 342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15 (11): 4593-4602, 1987) and Smith et al. (Nature 321: 674, 1986). Other commercially available fluorescent labels include, but are not limited to, fluorescein, rhodamine (including TMR, Texas Red and Rox), Alexa, body pie, acridine, coumarin, pyrene, benzanthracene and cyanine.

Multiple labels, such as dual fluorofore FRET cassettes (Tet. Let. 46: 8867-8871, 2000), can also be used in this application. A multiple fluorescence dendrimer system (J. Am. Chem. Soc. 123: 8101-8108, 2001) can also be used. Fluorescent labels are preferred, but other forms of detectable labels will be apparent to those skilled in the art. For example, quantum dots (Empodocles et al., Nature 399: 126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72: 6025-6029, 2000), and microbeads (Lacoste et al., Proc. Natl. Acad). All microparticles containing Sci USA 97 (17): 9461-9466, 2000) can be used.

Multi-component labels can also be used in this application. Multi-component labeling relies on interaction with additional compounds for detection. The most common multi-component label used in biology is the biotin-streptavidin system. Biotin is used as a label that binds to nucleotide bases. Streptavidin is then added individually to allow detection to occur. Other multi-component systems are available. For example, dinitrophenol has a commercially available fluorescent antibody that can be used for detection.

Unless otherwise indicated, references to nucleotides are also intended to be applicable to nucleosides. The application is also described further with reference to DNA, which is also applicable to RNA, PNA and other nucleic acids unless otherwise indicated.
Linker

In some embodiments described herein, the purine or pyrimidine base of a modified nucleotide molecule or modified nucleoside molecule can bind to a detectable label as described above. In some such embodiments, the linker used is cleaveable. The use of a cleavable linker ensures that the label can be removed after detection if desired and avoids any interference signal to any subsequently incorporated labeled nucleotide or nucleoside.

In some other embodiments, the linker used is non-cleavable. In each case where the labeled nucleotides of the invention are incorporated, the nucleotides do not need to be subsequently incorporated, so the labels do not need to be removed from the nucleotides.

Those of skill in the art are aware of the usefulness of dideoxynucleoside triphosphates in so-called Sanger sequencing methods and related protocols (Sanger type) that rely on random strand termination for specific types of nucleotides. An example of a Sanger-type sequencing protocol is the BASS method described by Metsker.

The Sanger method and the Sanger-type method generally work by performing experiments in which eight nucleotides are provided, four of which contain 3'-OH groups and of which the nucleotides. The four have no OH groups and are labeled differently from each other. The nucleotide used, without the 3'-OH group, is a dideoxynucleotide (ddNTP). As is known to those of skill in the art, ddNTPs are labeled differently, so the location of the incorporated terminal nucleotides can be determined and the information can be combined to determine the sequence of the target oligonucleotide.

The nucleotides of this application have been recognized to be useful in Sanger methods and related protocols, because the same effects achieved by using ddNTPs are the 3'-OH protecting groups described herein. Both can be achieved by use, preventing subsequent uptake of nucleotides.

Moreover, it is recognized that monitoring of the uptake of 3'-OH protected nucleotides can be determined by the use ^{of radioactive 32 P in the bound phosphate group.} These can be present either in the ddNTP itself or in the primers used for extension.

Cleaveable linkers are known in the art and conventional chemical reactions can be applied to attach the linker to nucleotide bases and labels. The linker can be cleaved by any suitable method including exposure to acids, bases, nucleophiles, electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes and the like. The linkers discussed herein may also be cleaved with the same catalyst used to cleave the 3'-O-protecting group bond. Suitable linkers can be adapted from standard chemical protecting groups as disclosed in Greene and Wuts, Protective Groups in Organic Synthesis, John Willey & Sons. More suitable cleavable linkers used in solid phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100: 2092-2157, 2000).

The use of the term "cleavable linker" is not intended to imply that the entire linker needs to be removed, eg, from nucleotide bases. If the detectable label is attached to a base, the nucleoside cleavage site can be in a position on the linker that ensures that part of the linker remains attached to the nucleotide base after cleavage.

If the detectable label is attached to a base, the linker may be attached at any position on the nucleotide base, provided that Watson-Crick base pairing can still be performed. In the context of purine bases, the linker is attached via the 7-position of the purine or preferred deazapurine analog, via the 8-modified purine, and via the N-6-modified adenosine or N-2-modified guanine. The case is preferable. For pyrimidines, the binding is preferably via the 5-position on cytosine, thymidine or uracil and the N-4 position on cytosine.
A. Electrophileally cleaved linker

Electrophilically cleaved linkers typically contain proton-cleaving and acid-sensitive cleavage. Suitable linkers include modified benzyl systems such as trityl, p-alkoxybenzyl ester and p-alkoxybenzylamide. Other suitable linkers include tert-butyloxycarbonyl (Boc) groups and acetal systems.

The use of thiophysic metals (such as nickel, silver or mercury) in the cleavage of thioacetals or other sulfur-containing protecting groups can also be considered for the preparation of suitable linker molecules.
B. Linker to be nucleophilically cleaved

Nucleophilic cleavage is also a well-recognized method in the preparation of linker molecules. Groups such as esters that are unstable in water (ie, can be simply cleaved at basic pH) and groups that are unstable to non-aqueous nucleophiles can be used. Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or t-butyldimethylsilane (TBDMS).
C. Light-cutting linker

Photocleavable linkers are widely used in hydrocarbon chemistry. It is preferred that the light required to activate cleavage does not affect other components of the modified nucleotide. For example, when a fluorophore is used as a label, it preferably absorbs light of a wavelength different from that required to cleave the linker molecule. Suitable linkers include those based on O-nitrobenzyl compounds and nitroveratril compounds. Linkers based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64: 3454-3460, 1999).
D. Cutting under reducing conditions

There are many linkers known to be susceptible to reductive cleavage. Catalytic hydrogenation with a palladium-based catalyst is used to cleave the benzyl and benzyloxycarbonyl groups. Reduction of disulfide bonds is also known in the art.
E. Cutting under oxidative conditions

Oxidation-based approaches are well known in the art. These include oxidation of the p-alkoxybenzyl group as well as oxidation of the sulfur and selenium linkers. The use of aqueous iodine solutions to cleave disulfides and other sulfur-based or selenium-based linkers is also within the scope of the invention.
F. Safety catch linker

The safety catch linker cuts in two steps. In a preferred system, the first step is the formation of reactive nucleophilic centers and the second step that follows involves intramolecular cyclization resulting in cleavage. For example, a levulinic acid ester bond can be treated with hydrazine or a photochemical reaction to release the active amine, which in turn cyclizes the ester elsewhere in the molecule. Can be cleaved (Burges et al., J. Org. Chem. 62: 5165-5168, 1997).
G. Cutting by detachment mechanism

Elimination reactions can also be used. For example, base-catalyzed elimination of groups such as Fmoc and cyanoethyl, and palladium-catalyzed reductive elimination of allyls can be used.

In some embodiments, the linker can include spacer units. The length of the linker is not important, provided that the label is retained at a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and the enzyme.

In some embodiments, the linker may consist of a functional group similar to the 3'-OH protecting group. This makes the deprotection and deprotection process more efficient, as only a single treatment is required to remove both the label and the protecting group. A particularly preferred linker is an azide-containing linker that can cleave phosphine.
Sequencing method

The modified nucleosides or nucleotides described herein can be used with a variety of sequencing techniques. In some embodiments, the process for determining the nucleotide sequence of a target nucleic acid can be an automated process.

The nucleotide analogs provided herein can be used in sequencing procedures such as synthetic sequencing (SBS) techniques. Briefly, SBS can be initiated by contacting the target nucleic acid with one or more labeled nucleotides, DNA polymerase, etc. Detectable labeled nucleotides are incorporated by the feature that the primer extends using the target nucleic acid as a template. If desired, the labeled nucleotide can further include a reversible termination property that terminates further primer extension once the nucleotide has been added to the primer. For example, a nucleotide analog with the moiety can be added to the primer so that subsequent elongation cannot occur until the deblocking agent has been delivered to remove the reversible termination factor moiety. Thus, for embodiments that use reversible termination, the deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washing can be performed during various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Illustrative SBS procedures, fluid systems and detection platforms that can be readily adapted for use with arrays produced by the methods of the present disclosure are described, for example, in Bentry et al., Nature 456: 53, each of which is incorporated herein by reference. ~ 59 (2008), WO04 / 018497, WO91 / 06678, WO07 / 123744, US Pat. Nos. 7,057,026, 7,329,492, 7,21,414, 7,315,019 No. 7,405,281, and US Patent Application Publication No. 2008/0108082 A1.

Other sequencing procedures that use cyclic reactions, such as pyrosequencing, can be used. Pyro sequencing detects the release of inorganic pyrophosphate (PPi) as a particular nucleotide is incorporated into a nascent nucleic acid chain (Ronaghi et al., Analytical Biochemistry 242 (1), each of which is incorporated herein by reference), 84-9 (1996), Ronagi, Genome Res. 11 (1), 3-11 (2001), Ronagi et al. Science 281 (5375), 363 (1998), US Pat. Nos. 6,210,891, No. 6, 258,568 and 6,274,320). In pyrosequencing, the released PPi can be detected by conversion to adenosine triphosphate (ATP) by ATP sulfylase, and the resulting ATP can be detected via luciferase-producing photons. Therefore, the sequencing reaction can be monitored via a luminescence detection system. The excitation radiation source used in the fluorescence-based detection system is not required for the pyrosequencing procedure. Useful fluid systems, detectors and procedures that can be used to apply pillow sequencing to the arrays of the present disclosure are described, for example, in WIPO Patent Application No. PCT / US11 / 57111, each of which is incorporated herein by reference. No., US Patent Application Publication No. 2005/0191698 A1, US Pat. No. 7,595,883, and US Pat. No. 7,244,559.

For example, those described in Science 309: 1728-1732 (2005), US Pat. No. 5,599,675, and US Pat. No. 5,750,341, each of which is incorporated herein by reference. Sequencing by including ligation reactions is also useful. As some embodiments, for example, Bains et al., Journal of Theoretical Biotechnology 135 (3), 303-7 (1988), Drmanac et al., Nature Biotechnology 16, 54-58 (1998), each of which is incorporated herein by reference. ), Fodor et al., Science 251 (4995), 767-773 (1995), and WO1989 / 10977 for sequencing procedures by hybrid formation. In both ligation-sequencing and hybridization-sequencing procedures, nucleic acids present in gel-containing wells (or other concave features) are subjected to repeated cycles of oligonucleotide delivery and detection. Fluid systems for SBS methods, such as those shown herein or in references cited herein, are readily adapted for delivery of reagents for sequencing procedures by ligation or hybridization. Can be done. Typically, oligonucleotides are fluorescently labeled and can be detected using a fluorescent detector similar to that described for the SBS procedure herein or in references cited herein.

For some embodiments, methods relating to real-time monitoring of DNA polymerase activity are available. For example, nucleotide uptake can be detected through a fluorescence resonance energy transfer (FRET) interaction between a fluorophore-supported polymerase and a γ-phosphate labeled nucleotide, or using a zero-mode waveguide. Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al. Science 299, 682-686 (2003), Lundquist et al. Opt. Lett. 33, 1026-1028 (2008), Koralach et al. Proc. Natl. Acad. Sci. USA 105, 1176 to 1181 (2008).

Some SGS embodiments include detection of protons released upon incorporation of nucleotides into extension products. For example, for sequencing based on the detection of released protons, electrical detectors and related technologies commercially available from Ion Patent (a subsidiary of Guilford, CT, Life Technologies), or each referred to herein. Sequencing methods and systems described in Incorporated US Patent Application Publication Nos. 2009/0026082 A1, 2009/0127589 A1, 2010/0137143 A1, or 2010/0282617 A1 may be used. it can.

Additional embodiments are disclosed in more detail in the examples below, which are by no means intended to limit the scope of the claims. The synthesis of various modified nucleotides with a protected 3'-hydroxy group is demonstrated in Examples 1-3.

スキーム１は、３’−ＯＨ保護基としてモノフルオロメチル置換アジドメチルを有する修飾されたヌクレオチドの調製のための合成経路を示す。化合物１ａ〜１ｆは、修飾されたチミン（Ｔ−ＰＡ）を塩基として使用する。使用することができる塩基の他の非限定的な例としては、Ｃｂｚ−ＰＡ、ＡＤＭＦ−ＰＡ、およびＧＰａｃ−ＰＡが挙げられ、それらの構造は、スキーム１において上記に示す。
実験手順

Scheme 1 shows a synthetic pathway for the preparation of modified nucleotides with a monofluoromethyl-substituted azidomethyl as a 3'-OH protecting group. Compounds 1a to 1f use modified thymine (T-PA) as a base. Other non-limiting examples of bases that can be used include Cbz-PA, ADMF-PA, and GPac-PA, the structures of which are shown above in Scheme 1.
Experimental procedure

2,6-Lutidine (0.87 mL, 7.5 mmol), (2-fluoroethyl) (4-methoxy) in a solution of starting nucleoside 1a (1.54 g, 2.5 mmol) in anhydrous CH _{3 CN (25 ml).} Phenyl) sulfan (MPSF) (3.26 g, 17.5 mmol) and then Bz ₂ O ₂ (purity 50%, 8.47 g, 17.5 mmol) were added at 4 ° C. The reaction mixture was gradually warmed to room temperature. The mixture was stirred for an additional 6 hours. Monitored by TLC to confirm complete consumption of the starting nucleoside (EtOAc: DCM = 2: 8v / v). The reaction was then concentrated under reduced pressure into an oily residue. Petroleum ether (500 ml) was added to this mixture and stirred vigorously for 10 minutes. The petroleum ether layer was decanted and the residue was repeatedly treated with petroleum ether (twice). The oily residue was _{fractionated between DCM and NaHCO 3} (1: 1) (300 mL). The organic layer was separated and the aqueous layer was further extracted into DCM (2 x 150 mL). The combined organic layers were dried on CVD ₄ and filtered to evaporate the volatiles under reduced pressure. The crude product 1c is purified by a Biotag silica gel column (50 g) using a gradient from petroleum ether to petroleum ether: EtOAc = 1: 1 (v / v), and 1.63 g of nucleoside 1b is pale yellow foam. It was obtained as a product (diasteromer, 82% yield).
^{1 1} H NMR (d ₆ DMSO, 400 MHz): δ, 0.95 (s, 9H, tBu), 2.16 − 2.28 (m, 2H, H-2'), 3.67 (s, OMe), 3.65 -3.85 (m, 2H, HH-5'), 3.77 (dd, J = 11.1, 4.5 Hz, 1H, HH-5'), 3.95-3.98 (m, 1H, H-4'), 4.04 (m, 2H, CH ₂ F ), 4.63-4.64 (m, 1H, H-3'), 5.01-5.32 (s, 1H, CH), 6.00 (m, 1H, H-1'), 6.72-6.87 (m, 3H, Ar), 7.35-7.44 (m, 7H, Ar), 7.55-7.60 (m, 4H, Ar), 7.88 (s, 1H, H-6), 9.95 (brt, 1H, NH), 11.70 (s, 1H, NH)

Cyclohexene (1.44 mL, 14 mmol) was added to a solution of starting nucleoside 1b (1.14 g, 1.4 mmol) in _{anhydrous CH 2} Cl ₂ (14 mL) containing molecular sieves (4 Å) under N _2. The mixture was cooled to −78 ° C. in a dry ice / acetone bath. DCM (14 ml) in a solution of sulfuryl chloride (580μL, 7.2mmol) was added slowly under _{N 2} for 90 minutes. After 20 minutes at that temperature, TLC (EtOAc: petroleum ether = 1: 1 v / v) showed complete consumption of the starting nucleoside. The volatiles were evaporated under reduced pressure (and at room temperature of 25 ° C.) and rapidly subjected to high vacuum for an additional 10 minutes until the oily residue foamed. The crude product _{was purged with N 2} and then dissolved in anhydrous DMF (5 mL) and NaN ₃ (470 mg, 7 mmol) was added all at once. The resulting suspension was stirred at room temperature for 2 hours or until TLC showed completion of the reaction and formation of 1c as the two isomers (a and b). The reaction mixture was _{fractionated between EtOAc and NaHCO 3} (1: 1) (200 mL). The organic layer was separated and the aqueous layer was further extracted into EtOAc (2 x 100 mL). The combined organic extracts were _{dried on volatiles 4} , filtered and the volatiles evaporated under reduced pressure. Two diastereoisomers (A and B) of 1c as pale yellow foam by Biotag silica gel column (25 g) using a gradient from petroleum ether to petroleum ether: EtOAc = 1: 1 (v / v). separated.

Isomer A (370 mg, yield: 38%).
^{1 1} H NMR (d ₆ DMSO, 400 MHz): δ 1.02 (s, 9H, tBu), 2.35 − 2.43 (m, 2H, H-2'), 3.76-3.80 (m, 1H, H-5'), 3.88 --3.92 (m, 1H, H-5'), 4.10 --4.12 (m, 1H, H-4'), 4.14 (d, J = 4.1 Hz 2H, NHCH ₂ ), 4.46-4.60 (m, 3H, H-3', CH2F), 5.05-5.09 (m, 1H, CHN ₃ ), 6.11 (t, J = 6.1 Hz, 1H, H-1'), 7.47 --7.51 (m, 6H, Ar), 7.64- 7.68 (m, 4H, Ar), 7.97 (s, 1H, H-6), 10.03 (bt, 1H, J = 10.0 Hz, NH), 11.76 (s, 1H, NH). ¹⁹ F NMR: -74.3 ( CF ₃ ), -230.2 (CH ₂ F)

Isomer B (253 mg, yield: 26%).
^{1 1} H NMR (d ₆ DMSO, 400 MHz): δ 1.01 (s, 9H, tBu), 2.38 − 2.42 (m, 2H, H-2'), 3.74-3.78 (m, 1H, H-5'), 3.86-3.90 (m, 1H, H-5'), 4.00-4.05 (m, 1H, H-4'), 4.12 (d, J = 4.1 Hz 2H, NHCH ₂ ), 4.45-4.60 (m, 3H, H-3', CH2F), 5.00-5.14 (m, 1H, CHN ₃ ), 6.09 (t, J = 6.1 Hz, 1H, H-1'), 7.41 --7.50 (m, 6H, Ar), 7.63- 7.66 (m, 4H, Ar), 7.95 (s, 1H, H-6), 10.01 (bs, 1H, NH), 11.74 (s, 1H, NH). ¹⁹ F NMR: -74.5 (CF3), -230.4 (CH2F)

Starting material 1c (isomer A) (500 mg, 0.71 mmol) was dissolved in THF (3 mL) and cooled to 4 ° C. in an ice bath. TBAF (1.0 M in THF, 5 wt% water, 1.07 mL, 1.07 mmol) was then added slowly over a period of 5 minutes. The reaction mixture was slowly warmed to room temperature. The progress of the reaction was monitored by TLC (petroleum ether: EtOAc = 3: 7 (v / v)). The reaction was stopped after 1 hour when the starting material was no longer visible by TLC. The reaction solution was dissolved in EtOAc (50 mL) and added to _{NaHCO 3 (60 mL).} The two layers were separated and the aqueous layer was extracted with additional DCM (50 mL x 2). The organic extracts were combined, dried (0054 ₄ ), filtered and evaporated to give a yellow oil. Crude product 1d (isomer A) on a Biotag silica gel column (10 g) using a gradient from petroleum ether: EtOAc = 8: 2 (v / v) to EtOAc, a white solid (183 mg, yield: It was purified as 56%).

Isomer A:
^{1 1} H NMR (400 MHz, d ₆ -DMSO): δ 2.24-2.35 (m, 2H, H-2'), 3.56-3.66 (m, 2H, H-5'), 3.96-4.00 (m, 1H, H-4'), 4.23 (s, 2H, CH ₂ NH), 4.33-4.37 (m, 1H, H-3'), 4.43-4.51 (m, CH2F), 5.12 (br.s, 1H, CHN ₃ ), 5.23 (br.s, 1H, 5'-OH), 6.07 (t, J = 6.7 Hz, 1H, H-1'), 8.26 (s, 1H, H-6), 10.11 (br s, 1H) , NH), 11.72 (br s, 1H, NH). ¹⁹ F NMR: -74.3 (CF3), -230.5 (CH2F)

The same reaction was carried out for 1c (isomer B) on a 360 mg scale to give the corresponding product 1d (isomer B, 150 mg, 63%).
^{1 1} H NMR (400 MHz, d ₆ -DMSO): δ 2.24-2.37 (m, 2H, H-2'), 3.57-3.70 (m, 2H, H-5'), 3.97-4.01 (m, 1H, H-4'), 4.23 (br.s, 2H, CH ₂ NH), 4.33-4.37 (m, 1H, H-3'), 4.44-4.53 (m, CH2F), 5.11-5.21 (br.s, 1H, CHN ₃ ), 5.23 (br.s, 1H, 5'-OH), 6.07 (t, J = 6.6 Hz, 1H, H-1'), 8.23 (s, 1H, H-6), 10.09 ( br s, 1H, NH), 11.70 (br s, 1H, NH). ¹⁹ F NMR: -74.1 (CF3), -230.1 (CH2F)

Preparation of the corresponding triphosphate 1e and further binding of the dye to the nucleobase to give rise to the fully functional nucleoside triphosphate (ffN) 1f have been reported in WO2004 / 0184997 and are generally known to those of skill in the art. is there.

スキーム２は、３’−ＯＨ保護基としてＣ−アミド置換アジドメチルを有する修飾されたヌクレオチドの調製のための合成経路を示す。化合物２ａ〜２ｉは、修飾されたチミン（Ｔ−ＰＡ）を塩基として使用する。使用することができる塩基の他の非限定的な例としては、Ｃｂｚ−ＰＡ、ＡＤＭＦ−ＰＡ、およびＧＰａｃ−ＰＡが挙げられ、これらの構造は、上記スキーム１に示されている。実験手順において、Ｎ，Ｎ−ジメチル−Ｃ（＝Ｏ）−置換アジドメチル保護基（Ｒ＝ＮＭｅ_２）を有する化合物２ｆおよびその後の反応を報告した。Ｎ−エチル−Ｃ（＝Ｏ）−（Ｒ＝ＮＨＥｔ）などの、他のＣ−アミド基を有する化合物も調製した。
実験手順

Scheme 2 shows a synthetic route for the preparation of modified nucleotides with a C-amide-substituted azidomethyl as a 3'-OH protecting group. Compounds 2a-2i use modified thymine (T-PA) as a base. Other non-limiting examples of bases that can be used include Cbz-PA, ADMF-PA, and GPac-PA, the structures of which are shown in Scheme 1 above. In the experimental procedure, compound 2f having an N, N-dimethyl-C (= O) -substituted azidomethyl protecting group (R = NMe ₂ ) and subsequent reactions were reported. Compounds with other C-amide groups, such as N-ethyl-C (= O)-(R = NHEt), were also prepared.
Experimental procedure

In a solution of starting nucleoside 2a (4.27 g, 6.9 mmol) in anhydrous CH ₃ CN (50 ml), 2,6-lutidine (2.4 mL, 20.7 mmol), S (CH ₂ CH ₂ OAc) ₂ ( 12.2 g, 69 mmol) and then Bz ₂ O ₂ (purity 50%, 33.4 g, 69 mmol) were added at 4 ° C. The reaction mixture was slowly warmed to room temperature. The mixture was stirred for an additional 12 hours. Monitored by TLC to confirm complete consumption of the starting nucleoside (EtOAc: DCM = 4: 6 v / v). The reaction was then concentrated under reduced pressure into an oily residue. Petroleum ether (800 ml) was added to this mixture and stirred vigorously for 10 minutes. The petroleum ether layer was decanted and the residue was repeatedly treated with petroleum ether (twice). The oily residue was then _{fractionated between DCM and NaHCO 3} (1: 1) (1000 mL). The organic layer was separated and the aqueous layer was further extracted into the DCM (2 x 500 mL). The combined organic layers were dried on CVD ₄ and filtered to evaporate the volatiles under reduced pressure. Crude product 2b on a Biotag silica gel column (100 g) using a gradient from petroleum ether to petroleum ether: EtOAc 2: 8 (v / v), pale yellow foam (4.17 g, yield: 74%). , Diastereoisomer).

Cyclohexene (5.62 mL, 56 mmol) was added to a solution of starting nucleoside 2b (4.54 g, 5.56 mmol) in _{anhydrous CH 2} Cl ₂ (56 mL) containing molecular sieves (4 Å) under N _2. The mixture was cooled to 4 ° C. in an ice bath. DCM (25 ml) solution of sulfuryl chloride (1.13 mL, 13.9 mmol) was added slowly under _{N 2} solution over 90 minutes a. After 30 minutes at that temperature, TLC (EtOAc: DCM = 4: 6v / v) showed that 10% of the starting nucleoside 2b remained. Additional sulfuryl chloride (0.1 mL) was added to the reaction mixture. TLC showed a complete conversion of 2b. The volatiles were evaporated under reduced pressure (and at room temperature of 25 ° C.) and rapidly subjected to high vacuum for an additional 10 minutes until the oily residue foamed. The crude product _{was purged with N 2} and then dissolved in anhydrous DMF (5 mL) and NaN ₃ (1.8 g, 27.8 mmol) was added all at once. The resulting suspension was stirred at room temperature for 2 hours, or at TLC until the reaction was completed and 2c was formed as the two isomers (A and B). The reaction mixture was _{fractionated between EtOAc and NaHCO 3} (1: 1) (1000 mL). The organic layer was separated and the aqueous layer was further extracted into EtOAc (2 x 300 mL). The combined organic extracts were then _{dried on Viyl 4} and filtered to evaporate the volatiles under reduced pressure. Two diastereoisomers 2c (isomers A and B) in a pale yellow foam with a Biotag silica gel column (100 g) using a gradient from petroleum ether to petroleum ether: EtOAc 1: 1 (v / v). Separated as. Isomer A: 1.68 g, yield: 40.7%. Isomer B: 1.79 g, yield: 43.2%.

Slowly add NaOH (1M aqueous solution) (2.2 mL, 2.2 mmol) to a solution of starting nucleoside 2c (isomer A) (1.63 g, 2.2 mmol) in MeOH / THF (1: 1) (20 mL). Was added to and stirred at 4 ° C. The progress of the reaction was monitored by TLC (EtOAc: DCM = 4: 6v / v). The reaction was stopped after 1 hour when the starting material was no longer visible by TLC. The reaction mixture was _{fractionated between DCM and NaHCO 3} (1: 1) (150 mL). The organic layer was separated and the aqueous layer was further extracted into DCM (2 x 70 mL). The combined organic extracts were _{dried on volatiles 4} , filtered and the volatiles evaporated under reduced pressure. The crude product 2d was added to a pale yellow foam (1.1 g, yield:) on a Biotag silica gel column (10 g) using a gradient from petroleum ether: EtOAc (8: 2) (v / v) to EtOAc. It was purified as 71%).

The same reaction was repeated for 2c (isomer B, 1.57 g) to give the corresponding product 2d (isomer B, 1.01 g, 69% yield).

A solution of the starting nucleoside 2d (isomer A) (700 mg, 1 mmol) in CH ₃ CN (10 mL) was treated with TEMPO (63 mg, 0.4 mmol) and BAIB (644 mg, 2 mmol) at room temperature. The progress of the reaction was monitored by TLC (EtOAc: DCM = 7: 3v / v). The reaction was stopped after 2 hours when the starting material was no longer visible by TLC. The reaction mixture was _{fractionated between DCM and Na 2} S ₂ O ₃ (1: 1) (100 mL). The organic layer was separated and the aqueous layer was further extracted into DCM (2 x 70 mL). The combined organic extracts were then washed with NaCl (saturated solution). For product is prevented from precipitating, in the dry without the over MgSO _4, the organic layer was evaporated under reduced pressure. The crude product 2e is pale yellow with a Biotag silica gel column (10 g) using a gradient from petroleum ether: EtOAc (1: 1) (v / v) to EtOAc and even MeOH: EtOAc (1: 9). Purified as a foam (isomer A, 482 mg, 68% yield).

The same reaction was carried out on 2d (isomer B, 700 mg) to give the corresponding product 2e (isomer B, 488 mg, 69% yield).

To _{a solution of the starting nucleoside 2e (isomer A) (233 mg, 0.33 mmol) in CH 3} CN (10 mL) was added Hunig base (173 μL, 1 mmol) and BOP (165 mg, 0.39 mmol) at room temperature. After stirring for 5 minutes, the solution was treated with Me2NH (2M THF solution) (0.41 ml, 0.82 mmol). The progress of the reaction was monitored by TLC (MeOH: DCM = 1: 9v / v). The reaction was stopped after 2 hours when the starting material was no longer visible by TLC. The reaction mixture was _{fractionated between DCM and NaHCO 3} (1: 1) (50 mL). The organic layer was separated and the aqueous layer was further extracted into DCM (2 x 30 mL). The combined organic extracts were _{dried on volatiles 4} , filtered and the volatiles evaporated under reduced pressure. Crude product 2f (R = NMe ₂ ) on a Biotag silica gel column (10 g) with a gradient from DCM: EtOAc (8: 2) (v / v) to EtOAc, a pale yellow foam (isomer). Purified as body A, 220 mg, 90% yield).

The same reaction was carried out on 2e (isomer B, 249 mg) to give the corresponding product 2f (isomer B, 240 mg, 92% yield).

Starting material 2f (mixture of isomers A and B) (455 mg, 0.61 mmol) was dissolved in THF (2 mL) and cooled to 4 ° C. in an ice bath. Next, TBAF (1.0 M THF solution, 5 wt% water, 1.0 mL, 1.0 mmol) was added slowly over a period of 5 minutes. The reaction mixture was slowly warmed to room temperature. The progress of the reaction was monitored by TLC (EtOAc). The reaction was stopped after 1 hour when the starting material was no longer visible by TLC. The reaction solution was dissolved in DCM (30 mL) and added to _{NaHCO 3 (30 mL).} The two layers were separated and the aqueous layer was extracted with additional DCM (30 mL x 2). The organic extracts were combined, dried (0054 ₄ ), filtered and evaporated to give a yellow oil. 2 g of crude product in a white solid (52% yield) with a Biotag silica gel column (10 g) using a gradient from DCM: EtOAc 8: 2 (v / v) to EtOAc and even MeOH: EtOAc (2: 8). Purified as a rate (160 mg).

Preparation of the corresponding triphosphate 2h to obtain a fully functional nucleoside triphosphate (ffN) 2i, and further binding of the dye to the nucleobase have been reported in WO2004 / 018497 and are generally available to those of skill in the art. It is known.

スキーム３は、ジフルオロメチル置換アジドメチル３’−ＯＨ保護基を有する修飾されたヌクレオチドの調製のための合成経路を示す。化合物３ａ〜３ｉは、修飾されたチミン（Ｔ−ＰＡ）を塩基として使用する。使用することができる塩基の他の非限定的な例としては、Ｃｂｚ−ＰＡ、ＡＤＭＦ−ＰＡ、およびＧＰａｃ−ＰＡが挙げられ、これらの構造は、前記スキーム１において示されている。３ｂ、３ｃおよび３ｄの合成のための手順は実施例２に記載した。
実験手順

Scheme 3 shows a synthetic pathway for the preparation of modified nucleotides with a difluoromethyl-substituted azidomethyl 3'-OH protecting group. Compounds 3a to 3i use modified thymine (T-PA) as a base. Other non-limiting examples of bases that can be used include Cbz-PA, ADMF-PA, and GPac-PA, the structures of which are shown in Scheme 1 above. Procedures for the synthesis of 3b, 3c and 3d are described in Example 2.
Experimental procedure

N-tert-Butylbenzenesulfine chloride in anhydrous DCM (2 ml) in a solution of starting nucleoside 3d (isomer A) (490 mg, 0.7 mmol) and DBU (209 μL, 1.4 mmol) in anhydrous DCM (5 mL). A solution of imidoyl (181 mg, 0.84 mmol) was added slowly at −78 ° C. The reaction mixture was stirred at −78 ° C. for 2 hours. The progress of the reaction was monitored by TLC (EtOAc: DCM = 4: 6v / v). The reaction was stopped after 2 hours with 10% starting material still remaining by TLC to prevent overreaction. The reaction mixture was _{fractionated between DCM and NaHCO 3} (1: 1) (50 mL). The aqueous layer was further extracted into DCM (2 x 30 mL). The organic extracts were combined, dried (0054 ₄ ), filtered and evaporated to give a yellow oil. Crude product 3e lightened by Biotag silica gel column (10 g) with a gradient from petroleum ether: EtOAc (8: 2) (v / v) to petroleum ether: EtOAc (2: 8) (v / v). Purified as a yellow foam (isomer A, 250 mg, 51% yield).

The same reaction was carried out on 3d (isomer B, 480 mg) to give the corresponding product 3e (isomer B, 240 mg, 50% yield).

A solution of the starting nucleoside 3e (isomer A) (342 mg, 0.49 mmol) in DCM (2.5 mL), EtOH (15 μL, 0.25 mmol) was added to DAST (181 mg, 0. It was added slowly to a solution of 84 mmol) at 4 ° C. (ice bath). The reaction mixture was stirred at 4 ° C. for 1 hour. The progress of the reaction was monitored by TLC (EtOAc: petroleum ether = 3: 7v / v). The reaction was stopped after 1 hour. The reaction mixture was _{fractionated between DCM and NaHCO 3} (1: 1) (50 mL). The aqueous layer was further extracted into DCM (2 x 30 mL). The organic extracts were combined, dried (0054 ₄ ), filtered and evaporated to give a yellow oil. The crude product 3f was subjected to a Biotag silica gel column (10 g) using a gradient from petroleum ether: EtOAc (9: 1) (v / v) to petroleum ether: EtOAc (2: 8) (v / v). Purified as a pale yellow foam (isomer A, 100 mg, 28%).

The same reaction was carried out on 3e (isomer B, 480 mg) to give the corresponding product 3f (isomer B, 240 mg, 50% yield).

Starting material 3f (isomer A) (124 mg, 0.17 mmol) was dissolved in THF (2 mL) and cooled to 4 ° C. in an ice bath. TBAF (1.0 M THF solution, 5 wt% water, 255 μL, 10.255 mmol) was then added slowly over a period of 5 minutes. The reaction mixture was slowly warmed to room temperature. The progress of the reaction was monitored by TLC (EtOAc). The reaction was stopped after 1 hour when the starting material was no longer visible by TLC. The reaction solution was dissolved in DCM (30 mL) and added to _{NaHCO 3 (30 mL).} The two layers were separated and the aqueous layer was extracted with additional DCM (30 mL x 2). The organic extracts were combined, dried (0054 ₄ ), filtered and evaporated to give a yellow oil. 3 g of crude product in pale yellow foam on a Biotag silica gel column (4 g) with a gradient from DCM: EtOAc = 8: 2 (v / v) to EtOAc and even MeOH: EtOAc (2: 8). Purified as a product (isomer A, 54% yield, 44 mg).

Isomer A:
^{1 1} H NMR (400 MHz, d ₆ -DMSO): δ 2.24-2.35 (m, 2H, H-2'), 3.56-3.66 (m, 2H, H-5'), 3.96-4.00 (m, 1H, H-4'), 4.23 (s, 2H, CH ₂ NH), 4.33-4.37 (m, 1H, H-3'), 4.85 (s, 2H, OCH ₂ N ₃ ), 5.23 (t, J = 5.1) Hz, 1H, 5'-OH), 6.07 (t, J = 6.7 Hz, 1H, H-1'), 8.19 (s, 1H, H-6), 10.09 (br s, 1H, NH), 11.70 ( br s, 1H, NH). ¹⁹ F NMR: -74.4 (CF3), -131.6 (CH2F).

The same reaction was carried out on 3f (isomer B, 133 mg) to give the corresponding product 3 g (isomer B, 48 mg, 54% yield).
^{1 1} H NMR (400 MHz, d ₆ -DMSO): δ 2.27-2.44 (m, 2H, H-2'), 3.58-3.67 (m, 2H, H-5'), 4.00-4.02 (m, 1H, H-4'), 4.24 (d, J = 4.1 Hz, 2H, CH ₂ NH), 4.57-4.58 (m, 1H, H-3'), 5.24-5.29 (m, 2H, 5'-OH, OCHN ₃ ), 6.07-6.34 (m, 2H, H-1', CHF ₂ ), 8.19 (s, 1H, H-6), 10.09 (br s, 1H, NH), 11.70 (br s, 1H, NH) . ¹⁹ F NMR: -74.2 (CF3), -131.4 (CH2F).

Preparation of the corresponding triphosphate 3h to obtain a fully functional nucleotide (ffN) 3i, and further binding of the dye to the nucleobase, have been reported in WO2004 / 018497 and are generally known to those of skill in the art. is there.
Example 4
Thermal stability test of 3'-OH protecting group

Various 3'-OH protecting groups were investigated for their thermal stability (Fig. 1A). Thermal stability, pH = 9 buffer and heated at (Tris _{-HCl50mM, NaCl50mM, tween0.05%, Mg} 2 SO 4 6mM) 60 ℃ respective 3'-OH protected nucleotides 0.1mM in Evaluated by. At various time points, HPLC was used to analyze the formation of unblocked material. Stability of -CH ₂ F and -C (O) NHBu a standard azidomethyl _(-CH 2 _{N 3)} was found to be about 2 times greater than the protective group. Stability of -CF ₂ H groups, was found to be about 10 times larger than the standard (Figure 1B).
Example 5
Deprotection of 3'-OH protecting group

The deprotection kinetics of some 3'-OH protecting groups were also tested. Deprotection speed standard azidomethyl protecting groups were compared with -CH ₂ F substituted azidomethyl and -C (O) NHBu-substituted azidomethyl. Both more thermostable 3'-OH blocking groups were observed to be removed more rapidly than standard azidomethyl protecting groups using phosphine (1 mM THP) as a deprotecting agent. See FIG. 2A. For example, -CH ₂ F and -C (O) half-life of NHBu, compared to the half-life of the azidomethyl of 20.4 minutes, 8.9 minutes, respectively, and 2.9 minutes (Figure 2B).
Example 6
Sequencing test

Modified nucleotides prepared having -CH ₂ F (mono-F) substituted azidomethyl 3'-OH protecting group was evaluated sequencing performance of the nucleotides on Miseq platform. Increased thermal stability of the 3'-OH protecting group was expected to result in higher quality nucleotides for sequencing chemistries, with fewer contaminating 3'unblocked nucleotides. The presence of 3'unblocked nucleotides in the SBS sequencing kit would therefore have resulted in a pre-phased event that is quantified as a pre-phased value.

A short 12-cycle sequencing experiment was first used to generate the phased and prephased values. Use ffN protected with mono-F substituted azidomethyl according to the following concentrations: ffA-dye 1 (2uM), ffT-dye 2 (10uM), ffC-dye 3 (2uM) and ffG-dye 4 (5uM). There was. The mono-F substituted azidomethyl group contains both isomers A and B. Two dyes, dye 2 and dye 5, as found in standard Miseq kits, were used to label the ffT. Table 1 shows the combinations of the A and B isomers of mono-F substituted azidomethyl with various nucleotides evaluated for their effects on phasing and prephase. In all cases, the pre-phased value was substantially lower than the control using the nucleotides of the standard V2 Miseq kit (Fig. 3).

(Sequencing quality inspection)
Sequencing of 2 x 400 bp was performed on Miseq to assess the ability of these nucleotides to improve the quality of sequencing. Sequencing runs were performed according to the manufacturer's instructions (Illumina, San Diego, CA). All mono-F blocked FFNs with standard uptake buffer (each individual dye label, ie ffA-dye 1 (2uM), ffT-dye 2 (1uM), ffC-dye 3 (2uM) and ffG -Replaced with uptake buffer containing dye 4 (5 uM)). The DNA library used was made from Bacillus genomic DNA according to the standard TruSeqHT protocol.

Very low prephased values were observed in both sequencing experiments (using mono-F blocks A and B isomers). In combination with low phased values, the application of these new nucleotides yielded excellent 2x400 bp sequencing data, with more than 80% bases above Q30 in both cases (Q of isomer A). See FIG. 4A for scores and FIG. 4B for isomer B Q score charts). These results demonstrate significant improvements compared to the Miseq version 2 kit (2 x 250 bp, 80% base> Q30 in typical R & D sequencing experiments, or 70% base> Q30 as specified). doing. As shown below, Table 2 summarizes the sequencing data for all mono-F ffN-A-isomers in IMX. Table 3 summarizes the sequencing data for all mono-F ffN-B-isomers in IMX.

Claims (22)

Modified to include a nucleobase and a sugar moiety of 2'-deoxyribose with a removable 3'-hydroxy protecting group that forms a structure-O-CH (R) N _{3 covalently attached to a 3'-carbon atom.} Nucleotide molecule, where R is selected from the group consisting of ^{-C (R 1} ) _m (R ² ) _n and -C (= O) NR ⁴ R ^5.
R ¹ is hydrogen,
R ² is a halogen,
R ⁴ is hydrogen,
R ⁵ is C _1-6 alkyl and is
m is an integer of 1 or 2
n is an integer of 1 or 2, provided that the sum of m + n is equal to 3, and the nucleobase flop phosphorus, deazapurine, cytosine, thymine, or saw including a U Rashiru, wherein the detectable The label is modified to bind to the purine or derazapurin via a linker at the 7-position of purine or derazapurin, or to the cytosine, thymine or uracil via a linker at the 5-position of cytosine, thymine or uracil. Nucleotide molecule. It is the nucleobase is de azapurines, modified nucleotide molecule of claim 1. The modified nucleotide molecule of claim 1 or 2, wherein R is -CHF ₂ or -CH _{2 F.} The modified nucleotide molecule of claim 1 or 2, wherein R is -C (= O) NR ⁴ R ⁵ , R ⁴ is hydrogen, and R ⁵ is C _{1-6 alkyl.} The modified nucleotide molecule according to any one of claims 1 to 4, wherein the nucleobase is attached to a detectable label via a cleavable linker. The modified nucleotide molecule of claim 5, wherein the cleaveable linker is acid-labile, light-labile, or contains a disulfide moiety. The modified nucleotide molecule of claim 6, wherein the cleaveable linker contains a disulfide moiety. The modified nucleotide molecule of claim 5, wherein the cleaveable linker comprises a moiety similar to the 3'-hydroxy protecting group. The modified nucleotide molecule of any one of claims 5-8, wherein the cleaving linker is cleaved under the same conditions as the 3'-hydroxy protecting group. The modified nucleotide molecule of any one of claims 5-9, wherein the detectable label is a fluorophore. An oligonucleotide comprising a nucleotide residue, wherein the nucleotide residue comprises the modified nucleotide according to any one of claims 1-10. The oligonucleotide according to claim 11, wherein the oligonucleotide is bound to a solid support. A method for preparing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, wherein the modified nucleotide molecule according to any one of claims 1 to 10 is being grown. A method comprising incorporating into a complementary polynucleotide, wherein the incorporation of the modified nucleotide molecule prevents the subsequent introduction of any subsequent nucleotide into the growing complementary polynucleotide. While incorporation of the modified nucleotide molecule, terminal transferase, is achieved by the end polymerase or reverse transcriptase, The method of claim 13. While incorporation of the modified nucleotide molecule is achieved by the end polymerase The method according to claim 13 or 14. A method for sequencing a target single-stranded polynucleotide,
A step of monitoring the continuous uptake of complementary nucleotide molecules , wherein at least one complementary nucleotide molecule incorporated is the modified nucleotide according to any one of claims 5-10. A method comprising the steps of being a molecule and detecting the identity of the modified nucleotide molecule. 16. The method of claim 16, wherein the identity of the modified nucleotide is determined by detecting the detectable label. The method of claim 16 or 17, wherein the 3'-hydroxy protecting group and the detectable label are removed prior to introducing the next complementary nucleotide molecule. 18. The method of claim 18, wherein the 3'-hydroxy protecting group and the detectable label are removed in a single step chemical reaction. A kit comprising a plurality of modified nucleotide molecules according to any one of claims 1 to 10, wherein each of the modified nucleotide molecules comprises a different detectable label. The kit of claim 20, comprising four modified nucleotide molecules. The kit according to claim 20 or 21, further comprising an enzyme and a buffer suitable for the action of the enzyme.

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