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    NUCLEOSIDES OR MODIFIED NUCLEOTIDS Some of the embodiments described in this document refer to modified nucleotide and nucleoside molecules with innovative 3? - hydroxy protecting groups. Said 3'-hydroxy protecting groups form a? O-C (R) 2N3 structure covalently linked to the 3? -Carbon atom, wherein R is as defined in the Claims. Also provided herein are methods for making such modified nucleotide and nucleoside molecules and sequencing by synthesis processes using such modified nucleotide or nucleoside molecules.
    公开号:BR112015022448B1
    申请号:R112015022448-2
    申请日:2013-03-15
    公开日:2020-12-08
    发明作者:Xiaohai Liu；Xiaolin Wu；Geoffrey Paul Smith
    申请人:Illumina Cambridge Limited；
    IPC主号:
    专利说明:

    DESCRIPTIVE REPORT BACKGROUND Field of the Invention
    [0001] Some embodiments described in this document refer to modified nucleotides or nucleosides, comprising 3'-hydroxy protecting groups and their use in polynucleotide sequencing methods. Some of the achievements described in this document refer to a method for preparing nucleotides or nucleosides protected by 3'-hydroxy. Description of the Related Art
    [0002] Advances in the study of molecules have been driven, in part, by the improvement in technologies used to characterize molecules or their biological reactions. In particular, the study of DNA and RNA nucleic acids has benefited from the development of technologies used for sequence analysis and the study of hybridization events.
    [0003] An example of technologies that have improved the study of nucleic acids is the development of ordered assemblies made of immobilized nucleic acids. These ordered sets typically consist of a high density matrix of polynucleotides immobilized on a solid support material. See, for example, Fodor et al., Trends Biotech. 12: 19-26, 1994, which describes ways of assembling nucleic acids using a chemically sensitized glass surface protected by a mask, but exposed in defined areas to allow binding of appropriately modified nucleotide phosphoramidites. Fabricated ordered assemblies can also be manufactured using the "tagging" technique of known polynucleotides on a solid support at predetermined positions (for example, Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).
    [0004] One way of determining the nucleotide sequence of a nucleic acid linked to an ordered set is called "synthesis sequencing" or "SBS". This technique for determining the DNA sequence ideally requires controlled incorporation (that is, one at a time) of the correct complementary nucleotide opposite the nucleic acid being sequenced. This allows for precise sequencing through the addition of nucleotides in multiple cycles, since each nucleotide residue is sequenced one at a time, thus preventing the occurrence of an uncontrolled series of incorporations. The embedded nucleotide is read using an appropriate label attached to it prior to removal of the label segment and the next round of sequencing.
    [0005] In order to ensure that only a single incorporation occurs, a structural modification ("protecting group") is added to each labeled nucleotide that is added to the growing chain, to ensure that only one nucleotide is incorporated. After the nucleotide with the protecting group has been added, the protecting group is then removed, under reaction conditions that do not interfere with the integrity of the DNA being sequenced. The sequencing cycle can then continue with the next protected labeled donucleotide incorporation.
    [0006] To be useful in sequencing DNA, nucleotides, and more commonly nucleotide triphosphates, generally require a 3'-hydroxy protecting group, in order to prevent the polymerase used to incorporate into a polynucleotide chain from continuing to replicate once that the base on the nucleotide is added. There are many limitations on the types of groups that can be added to a nucleotide and still if appropriate. The protecting group must prevent additional nucleotide molecules from being added to the polynucleotide chain, while simultaneously being easily removable from the sugar segment, without causing damage to the polynucleotide chain. In addition, the modified nucleotide must be tolerated by the polymerase or other appropriate enzyme used to incorporate it into the polynucleotide chain. The ideal protection group, therefore, has long-term stability, is efficiently incorporated by the polymerase enzyme, causes secondary or additional nucleotide incorporation blocking and has the ability to be removed under moderate conditions, which do not cause damage to the polynucleotide structure, preferably under aqueous conditions.
    [0007] Reversible protection groups have been described previously. For example, Metzker et al., (Nucleic Acids Research, 22 (20): 4259-4267,1994) discloses the synthesis and use of eight 2'-deoxyribonucleoside 5'-triphosphates (3'-modified dNTPs) and testing in two model assays of DNA for incorporation activity. WO 2002/029003 describes a sequencing method that may include the use of an allyl protecting group to cap the 3'-OH group on a growing strand of DNA in a polymerase reaction.
    [0008] In addition, we have previously reported the development of a number of reversible protection groups and methods of their deprotection under compatible DNA conditions in International Application Publication No. WO 2004/018497, which is hereby incorporated by reference in its wholeness. SUMMARY
    [0009] Some embodiments described in this document refer to a modified nucleotide or nucleoside molecule, comprising a purine or pyrimidine base and a ribose or deoxyribose sugar segment having a removable 3'-hydroxy protecting group forming an -OC (R) structure 2N3 covalently linked to the 3'-carbon atom, where R is selected from the group consisting of hydrogen, - C (R1) m (R2) n, -C (= O) OR3, -C (= O) NR4R5, -C (R6) 2O (CH2) pNR7R8 and - C (R9) 2O-pH-C (= O) NR10R11; each R1 and R2 is independently selected from hydrogen, optionally substituted alkyl or halogen; R3 is selected from hydrogen or optionally substituted alkyl; each R4 and R5 is independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl; each R6 and R9 is selected from hydrogen, optionally substituted alkyl or halogen; each R7, R8, R10 and R11 is independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted aralkyl; m is an integer from 0 to 3; and n is an integer from 0 to 3; provided that the total of m + n is equal to 3; and p is an integer from 0 to 6; provided that R1 and R2 cannot both be halogen; and at least one R is not hydrogen.
    [0010] Some of the embodiments described in this document refer to a method of preparing a growing polynucleotide complementary to a single stranded polynucleotide in a sequencing reaction, comprising incorporating a modified nucleotide molecule described in this document into the growing complementary polynucleotide, in that the incorporation of the modified nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide.
    [0011] Some of the embodiments described in this document refer to a method for determining the sequence of a target single-stranded polynucleotide, comprising monitoring the sequential incorporation of complementary nucleotides, wherein at least one complementary nucleotide incorporated is a modified nucleotide molecule described in this document; and detecting the identity of the modified nucleotide molecule. In some embodiments, incorporation of the modified nucleotide molecule is achieved by a terminal transferase, a terminal polymerase or a reverse transcriptase.
    [0012] Some embodiments described in this document refer to a kit comprising a plurality of modified nucleotide or nucleoside molecules described in this document, and materials for packaging them. In some embodiments, the identity of the modified nucleotide is determined by detecting the detectable label attached to the 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 chemical reaction step. BRIEF DESCRIPTION OF THE DRAWINGS
    [0013] FIG. 1A illustrates a variety of 3'-OH protecting groups.
    [0014] FIG. 1B illustrated the thermal stability of several 3'-OH protecting groups.
    [0015] FIG. 2A illustrates the deprotection rate curve of three different 3'-OH protection groups.
    [0016] FIG. 2B shows a half-time graph of three different 3'-OH protection groups.
    [0017] FIG. 3 shows the phasing and pre-phasing values of several nucleotides modified with a thermally stable 3'-OH protecting group compared to the standard protecting group.
    [0018] FIG. 4A shows the 2x400bp deformation ffNs-A mono-F deisomer in incorporation mixture (IMX).
    [0019] FIG. 4B shows the sequencing data 2x400bp of mono-FfNs-B deisomer in incorporation mixture (IMX). DETAILED DESCRIPTION
    [0020] One embodiment is a modified nucleotide or nucleoside comprising a 3'-OH protecting group. In one embodiment, the 3'-OH protecting group is a substituted mono-fluoromethyl azidomethyl protecting group. In another embodiment, the 3'-OH protecting is a substituted C-starch azidomethyl protecting group. Yet another embodiment relates to modified nucleotides having substituted difluoromethyl 3'-OH azidomethyl protecting groups. Definitions
    [0021] Unless otherwise defined, all thermostats and scientific materials used in this document have the same meaning as is commonly understood by a person of ordinary skill in the art. The use of the term "including", as well as other forms, such as "include", "includes" and "included", not without limitation. The use of the term "having", as well as other forms, such as "having", "has" and "had" is non-limiting. As used in this specification, whether in a transitional phrase or in the body of the Claim, the terms "comprise (m)" and "comprising" are to be interpreted as having an open meaning. That is, the above terms should be interpreted synonymously with the phrases “having at least” or “including at least”. For example, when used in the context of a process, the term “comprising” means that the process includes at least the steps listed, but may include additional steps.When used in the context of a compound, composition or device, the term "comprising" means that the compound, composition, or device includes at least the listed features or components, but may also include features or additional components.
    [0022] As used herein, orgânicascomuns abbreviations are defined as follows: AcAcetila aq.Aquoso acetic Ac2OAnidrido BnBenzila BzBenzoila BOC or Bocterc Butóxicarbonila-Butyl Bun-cat.Catalítico CbzCarbobenzilóxi CTemperatura ° Centigrade in dATPDesoxiadenosina triphosphate triphosphate dCTPDesoxicitidina dGTPDesoxiguanosina triphosphate triphosphate ddNTP dTTPDesoxitimidina (s) Didesoxynucleotide (s) DBU1,8-Diazabicyclo [5.4.0] undec-7-en DCADichloroacetic acid DCE1,2-Dichloroethane DCMMethylene chloride DIEADiisopropylethylamine DMADimethylacetamide DMEDimethoxymethyl DMEmethylmethoxymethylamine functional GPGgram (s) GP permeation chromatography h or hrHour (s) iPrIsopropyl KPi 10 mM potassium phosphate buffer at pH 7.0 KPSPotassium sulphate IPAIsopropyl alcohol IMX Incorporation mixture LCMSSpectrometry of dough coupled with liquid chromatography LD chromatography LD chromatography lithium m or minMinute (s) mCPBAMetochloroperoxybenzoic acid MeOHMethanol MeCNAcetonitrile Mono-F-CH2F ffN Mono-FNucleotides modified with-CH2F substituted in methylene protection group 3'-OH azidomethylMethylmethyl Na sine MTNE3E NHSN-hydroxysuccinimide PGProtection group PhFenila pptPrecipitate rt Ambient temperature SBSSynthesis sequencing TEATriethylamine TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl) oxy TCDI1,1'-Tiocarbolina diimidazole Terc, tetriamide Tetra, tetrahydrofluoride
    [0023] As used in this document, the term "ordered set" refers to a population of different probe molecules that are linked to one or more substrates, such that different probe molecules can be differentiated from each other according to their relative location. An ordered set may include different probe molecules, which are each located in a different addressable location on a substrate. Alternatively or additionally, an ordered set includes different substrates, each carrying a different probe molecule, in which the different probe molecules they can be identified according to the locations of the substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid. Exemplary ordered sets in which separate substrates are located on a surface include, without limitation, those including granules in wells, as described, for example, in US Patent 6,355,431 B1, US 2002/0102578 and PCT Publication WO 00/63437. Exemplary formats that can be used in the invention to distinguish granules in an ordered liquid set, for example, using a microfluidic device, such as a fluorescence activated cell separator (FACS), are described, for example, in U.S. Pat. US 6,524,793. Additional examples of ordered sets that can be used in the invention include, without limitation, those described in U.S. Pat. US 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; 6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93/17126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799 897.
    [0024] As used in this document, the term "covalently attached" or "covalently attached" refers to the formation of a chemical bond that is characterized by the sharing of electron pairs between atoms. For example, a covalently bonded polymer coating refers to a polymer coating that forms chemical bonds with a functionalized surface of a substrate, compared to bonding to the surface by other means, for example, electrostatic adhesion or interaction. It will be appreciated that polymers that are covalently bonded to a surface can also be bonded via means in addition to the covalent bond.
    [0025] As used in this document, any (any) "R" group (s), such as, without limitation, R2, R3, R4, R5, R6, R7 and R8 represent substituents that can be attached to the indicated atom. Any group R can be substituted or substituted. If two "R" groups are described as "taken together", the R groups and the atoms, they are linked to form a cycloalkyl, aryl, heteroaryl or heterocycle. For example, without limitation, if R2 and R3, or R2, R3 or R4, and the atom to which it is attached, are indicated to be "taken together" or "joined", it means that they are covalently linked to each other to form a ring, an example of which is set out below:

    [0026] Whenever a group is described as being “optionally substituted”, that group can be replaced or substituted by one or more of the indicated substituents. Likewise, when a group is described as being "unsubstituted or substituted", if substituted, the substituent can be selected from one or more of the indicated substituents. If no substituent is indicated, it means that the “optionally substituted” or “substituted” group indicated can be individually and independently replaced by one or more group (s) individually and independently selected from a group of features including, but not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalkyl) alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, aryl, halo, aryl, halo, aryl, halo O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-starch, N-starch, S-sulfonamido, N-sulfonamido, C-carboxy, Protected C-carboxy, O-carboxy, isocyanate, thiocyanate, isothiocyanate , nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, monosubstituted amino group, disubstituted amino group and protected derivatives smos.
    [0027] As used in this document, "alkyl" refers to a straight or branched hydrocarbon chain, which comprises a fully saturated hydrocarbon group (without double or triple bonds). In some embodiments, the alkyl group can have 1 to 20 carbon atoms (whenever a numerical variation such as "1 to 20" appears in this document, it refers to each integer in the given variation, including end points; for For example, “1 to 20 carbon atoms” means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although this definition also covers the occurrence of the term "alkyl" where no numerical variation is designated). The alkyl group can also be a medium-sized alkyl having about 7 to about 10 carbon atoms. The alkyl group can also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group of the compounds can be referred to as "C1-C4 alkyl" or similar designations. By way of example only, "C1-C4 alkyl" indicates that there are one to four carbon atoms in the alkyl chain, that is, the alkyl chain is selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso -butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexis. The alkyl group can be substituted or unsubstituted.
    [0028] As used in this document, "alkenyl" refers to an alkyl group that contains in the linear or branched hydrocarbon chain one or more double bonds. An alkenyl group can be unsubstituted or substituted.
    [0029] As used in this document, "alkynyl" refers to an alkyl group that contains, in the linear or branched hydrocarbon chain, one or more triple bonds. An alkynyl group can be unsubstituted or replaced.
    [0030] As used in this document, "cycloalkyl" refers to a mono or multicyclic hydrocarbon ring system, fully saturated (without double or triple bonds). When composed of two or more rings, the rings can be fused together. Cycloalkyl groups can contain 3 to 10 atoms in the ring (s). In some embodiments, cycloalkyl groups may contain 3 to 8 ring atoms (rings). A cycloalkyl group can be unsubstituted or replaced. Typical cycloalkyl groups include, but are not limited in any way to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
    [0031] As used in this document, "aryl" refers to a monocyclic or multicyclic carbocyclic aromatic ring system (all carbon) (including, for example, fused, bridged, or spiro ring systems, where two carbocyclic rings share one chemical bond, for example, one or more aryl rings, with one or more aryl ring or non-aryl ring), which has a pi electron system fully relocated throughout at least one of the rings. The number of carbon atoms in an aryl group can vary. For example, in some embodiments, the aryl group can be a C6-C14 aryl group, a C6-C10 aryl group, or a C6 aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. An aryl group can be replaced or unsubstituted.
    [0032] As used in this document, "heterocyclyl" refers to ring systems including at least one heteroatom (for example, O, N, S). Such systems may be unsaturated, may include some unsaturation or may contain some aromatic part or aromatic sertodo. A heterocyclyl group may be unsubstituted or substituted.
    [0033] As used in this document, “heteroaryl” refers to a monocyclic or multicyclic aromatic ring system (a ring system having at least one ring with a fully displaced pi electron system), which contains (contains) one or more heteroatoms, that is, an element other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur, and at least one aromatic ring. The number of atoms in the ring (s) of a heteroaryl group can vary. For example, in some embodiments, a heteroaryl group may contain 4 to 14 ring atoms (rings), 5 to 10 ring (s) atoms or 5 to 6 ring atoms (rings). Furthermore, the term "heteroaryl" includes fused ring systems, where two rings, such as at least one aryl ring and at least one heteroaryl ring, or at least two heteroaryl rings, share at least one chemical bond. Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole, 1,2,3 - thiadiazole, 1,2,4-thiadiazole, benzothiazole. imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, quinoline, quinoline, quinoline, quinoline, quinoline, quinoline, quinoline, quinoline A heteroaryl group can be substituted or unsubstituted.
    [0034] As used in this document, “heteroalicyclic” or “heteroalicyclic” refers to a monocyclic, bicyclic and tricyclic ring system of three, four, five, six, seven, eight, nine, ten, up to 18 members, in which atoms of carbon, together with 1 to 5 heteroatoms, constitute this ring system. A heterocycle can optionally contain one or more unsaturated bonds, situated in such a way, however, that a fully displaced pi electron system does not occur across all rings. Heteroatoms are independently selected from oxygen, sulfur and nitrogen. A heterocycle can also contain one or more carbonyl or thiocarbonyl functionalities, in order to make the definition include oxo systems and thio systems, such as lactam, cyclic imides, cyclic thioimides and cyclic carbamates. When composed of two or more rings, the rings can be fused together. Additionally, any nitrogen in a heteroalicyclic can be quaternized. Heteroalicyclic or heteroalicyclic groups can be replaced or replaced. Examples of such "heteroalicyclic" or "heteroalicyclic" groups include, but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1, 4-dioxolane, 1,3-oxatian, 1,4-oxatiin, 1,3-oxathiolan, 1,3-dithiol, 1,3-dithiolane, 1,4-oxatian, tetrahydro-1,4-thiazine, 2H- 1,2- oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydanotoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazoline, thiazolidine, oxazoline, thiazoline, oxazoline, thiazolidine thiazolidine, morpholine, oxirane, piperidine N-oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine, tetrahydropyran, tetrahydramorhyroline, tetrahydroxytrorholine, tetrahydroxytron, , and its benzofused analogs (for example, benzimidazolidinone, tetrahydroquinoline, 3,4-methylenedioxyphenyl).
    [0035] As used in this document, "aralkyl" and "aryl (alkyl)" refer to a connected aryl group, as a substituent, by means of a lower alkylene group. The lower alkylene and aryl groups of an aralkyl can be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl and naphthylalkyl.
    [0036] As used in this document, "heteroaralkyl" and "heteroaryl (alkyl)" refer to a heteroaryl group connected, as a substituent, by means of a lower alkylene group. The lower alkylene and heteroaryl group of the heteroaralkyl can be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and their fused-analogs.
    [0037] As used in this document, "alkoxy" refers to the formula -OR, where R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or a cycloalkynyl, is defined above. A non-limiting list of alkoxies is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy. An alkoxy can be substituted or substituted.
    [0038] As used in this document, a “C-starch” group refers to a “-C (= O) N (RaRb)” group, where Ra and Rb can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl , cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl or (heteroalicyclyl) alkyl. A C-starch can be replaced or unsubstituted.
    [0039] As used in this document, an “N-starch” group refers to an “RC (= O) NRa) -” group, where R and Ra can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl , cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl or (heteroalicyclyl) alkyl. An N-starch can be replaced or unsubstituted.
    [0040] The term "halogen atom", "halogen" or "halo", as used in this document, means any of the radioestable atoms in column 7 of the Periodic Table of the Elements, such as fluorine, chlorine, bromine and iodine.
    [0041] The term "amine", as used in this document, refers to a -NH2 group, in which one or more hydrogens can be optionally substituted by an R group. R can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl , cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl or (heteroalicyclyl) alkyl.
    [0042] The term “aldehyde”, as used in this document, refers to a group -Rc-C (O) H, where Rc may be absent or independently selected from alkylene, alkenylene, alkylene, cycloalkylene, cycloalkenylene, cycloalkenylene , arylene, heteroarylene, heteroalicyclylene, aralkylene, or (heteroalicyclyl) alkylene.
    [0043] The term "amino", as used in this document, refers to a -NH2 group.
    [0044] The term "hydroxy", as used in this document, refers to an -OH group.
    [0045] The term "cyan" group, as used in this document, refers to a "-CN" group.
    [0046] The term "azido", as used in this document, refers to a group -N3.
    [0047] The term "thiol", as used in this document, refers to a -SH group.
    [0048] The term "carboxylic acid", as used in this document, refers to -C (O) OH.
    [0049] The term “thiocyanate”, as used in this document, refers to a group.
    [0050] The term "oxo-amine", as used in this document, refers to a group -O-NH2, in which one or more hydrogen of -NH2 can be optionally substituted by a group R. R can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl) alkyl.
    [0051] As used in this document, a "nucleotide" includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid sequence. In RNA, sugar is a ribose, and in DNA, a deoxyribose, that is, a sugar without a hydroxyl group, which is present in ribose. The nitrogen containing heterocyclic base can be either purine or pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified or analogous derivatives thereof. Pyrimidine bases include cytosine (C), thymine (T) and uracil (U), and modified or analogous derivatives thereof. The C-1 atom of deoxyribose is attached to either N-1 of a pyrimidine or N-9 of a purine.
    [0052] As used in this document, a "nucleoside" is structurally similar to a nucleotide, but the phosphate segments are missing. An example of a nucleoside analog would be one where the label is attached to the base and there is no phosphate group attached to the sugar molecule. The term "nucleoside" is used in this document in its common sense, as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose segment and a deoxyribonucleoside comprising a deoxyribose segment. A modified pentose segment is a pentose segment in which an oxygen atom has been replaced by a carbon and / or a carbon has been replaced by a sulfur or an oxygen atom. A "nucleoside" is a monomer that can have a substituted sugar base and / or segment. In addition, a nucleoside can be incorporated into larger DNA and / or RNA polymers and oligomers.
    [0053] The term "purine base" is used in this document in its common sense, as understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used in this document in its common sense, as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthin, 7-alkylguanine (e.g., 7-methylguanine), 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 (e.g., 5-methylcytosine).
    [0054] As used herein, "derivative" or "analog" means a synthetic nucleotide or nucleoside derivative having modified base segments and / or modified sugar segments. Such derivatives and analogs are discussed, for example, in Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman, et al., Chemical Reviews 90: 543-584,1990. Nucleotide analogs can also comprise phosphodiester chain bonds, including phosphorothioate, phosphorodithioate, alkyl phosphonate, phosphoranilidate and phosphoramidate chain bonds. "Derived", "analogous" and "modified", as used in this document, may be used interchangeably, and are encompassed by the terms "nucleotide" and "nucleoside" defined in this document.
    [0055] As used in this document, the term “phosphate” is used in its common sense, as understood by those skilled in the art and
    includes its protonated forms (for example, '' and ~). As used in this document, the terms "monophosphate", "diphosphate" and "triphosphate" are used in their common senses, as understood by those skilled in the art, and include protonated forms.
    [0056] The terms "protecting group" and "protecting groups", as used in this document, refer to any atom or group of atoms that is added to a molecule, in order to prevent existing groups in the molecule from undergoing chemical reactions. unwanted. Sometimes, “protection group” and “blocking group” can be used interchangeably.
    [0057] As used in this document, the prefixes “photo” or “photo-” mean in relation to light or electromagnetic radiation. The term may encompass all or part of the electromagnetic spectrum, including, but not limited to, one or more of the variety commonly known as radio, microwave, infrared, visible, ultraviolet, x-ray or gamma ray of the spectrum. The part of the spectrum can be one that is blocked by a metal region on a surface, such as those metals set forth in this document. Alternatively or additionally, the part of the spectrum may be one that passes through an interstitial region of a surface, such as a region made of glass, plastic, silica or other material set out in this document. In particular embodiments, radiation can be used, which is able to pass through a metal. Alternatively or additionally, radiation can be used, which is masked by glass, plastic, silica or other material set out in this document.
    [0058] As used in this document, the term “phasing” refers to phenomena in SBS, which are caused by incomplete removal of the 3 'and fluorophores terminators and failure to complete the incorporation of a part of DNA strands into clusters ) by polymerases in a given sequencing cycle. Pre-phasing is caused by the incorporation of nucleotides without effective 3 'terminators and the incorporation event follows 1 cycle ahead. The phasing and pre-phasing make the intensities extracted for a specific cycle consist of the current cycle signal, as well as noise from the preceding and subsequent cycles. As the number of cycles increases, the fraction of sequences per cluster affected by the phasing increases, making it difficult to identify the correct base. Pre-phasing can be caused by the presence of a small amount of unprotected or unblocked 3'-OH nucleotides during synthesis sequencing (SBS). The unprotected 3'-OH nucleotides could be generated during the manufacturing processes or possibly during the reagent storage and handling processes. Consequently, the discovery of nucleotide analogs that decrease the incidence of pre-phasing is surprising and provides a great advantage in SBS applications over existing nucleotide analogs. For example, the nucleotide analogs provided may result in faster SBS cycle time, shorter phasing and pre-phasing values, and longer sequencing reading length. Protection groups 3'-OH -C (RhN3
    [0059] Some embodiments described in this document refer to a modified nucleotide or nucleoside molecule, having a removable 3'-hydroxy protecting group -C (R) 2N3, where R is selected from the group consisting of hydrogen, -C ( R1) m (R2) n, -C (= O) OR3, - C (= O) NR4R5, -C (R6) 2O (CH2) pNR7R8 and -C (R9) 2O-Ph-C (= O) NR10R11 , where R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, m, nep are defined above.
    [0060] In some embodiments, one of R is hydrogen and the other R is -C (R1) m (R2) n. In some such embodiments, -C (R1) m (R2) n is selected from -CHF2, -CH2F, -CHCl2 or -CH2Cl. In one embodiment, -C (R1) m (R2) n is -CHF2. In another embodiment, -C (R1) m (R2) n is -CH2F.
    [0061] In some embodiments, one of R is hydrogen and the other R is -C (= O) OR3. In some such embodiment, R3 is hydrogen.
    [0062] In some embodiments, one of R is hydrogen and the other R is -C (= O) NR4R5. In some such embodiments, both R4 and R5 are hydrogen. In some other such embodiments, R4 is hydrogen and R5 is C1-6 alkyl. In still some other embodiments, both R4 and R5 are C1-6 alkyl. In one embodiment, R5 is n-butyl. In another embodiment, both R4 and R5 are methyl.
    [0063] In some embodiments, one of R is hydrogen and the other R is -C (R6) 2O (CH2) pNR7R8. In some such embodiments, both R6 are hydrogen. In some such embodiments, both R7 and R8 are hydrogen. In some such realization, p is 0. In some other such realization, p is 6.
    [0064] In some embodiments, one of R is hydrogen and the other R is -C (R9) 2O-Ph-C (= O) NR10R11. In some such embodiments, both R9 are hydrogen. In some such embodiments, both R10 and R11 are hydrogen. In some such embodiments, R10 is hydrogen and R11 is substituted alkyl. In one embodiment, R11 is substituted amino alkyl. Deprotection of 3'-OH Protection Groups
    [0065] In some embodiments, the 3'-OH protecting group is removed in an unprotected reaction with a phosphine. The azido group in -C (R) 2N3 can be converted to an amino group by contacting the modified nucleotide or nucleoside molecules with a phosphine. Alternatively, the azido group in -C (r) 2N3 can be converted to an amino group by contacting such molecules with thiols, in particular water-soluble thiols, such as dithiothreitol (DTT). In one embodiment, the phosphine is tris (hydroxymethyl) phosphine (THP). Unless otherwise indicated, the reference to nucleotides is also intended to be applicable to nucleosides. Detectable Labels
    [0066] Some achievements described in this document refer to the use of conventional detectable labels. Detection can be conducted by any appropriate method, including fluorescence spectroscopy or by other optical means. The preferred label is a fluorophore, which, after absorbing energy, emits radiation at a defined wavelength. Many appropriate fluorescent labels are known. For example, Welch et al. (Chem. Eur. J. 5 (3): 951-960,1999) discloses fluorescent segments functionalized by dansyl that can be used in the present invention. Zhu et al. (Cytometry 28: 206-211,1997) describes the use of the fluorescent labels Cy3 and Cy5, which can also be used in the present invention. Labels suitable for use are also disclosed in Prober et al. (Science 238: 336-341, 1987); Connell et al. (Biotechniques 5 (4): 342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15 (11): 4593-4602, 1987) and Smith et al. (Nature 321: 674,1986). Other commercially available fluorescent labels include, but are not limited to, fluorescein, rhodamine (including TMR, Texas red and Rox), alexa, bodipi, acridine, coumarin, pyrene, benzanthracene and cyanines.
    [0067] Multiple labels can also be used in the present application, for example, FRET fluorophore cassettes (Tet. Let. 46: 88678871,2000). Multifluorine dendrimeric systems (J. Am. Chem. Soc. 123: 8101-8108,2001) can also be used. Although fluorescent labels are preferred, other forms of detectable labels will appear to be useful to those of ordinary skill in the art. For example, microparticles, including quantum dots (Empodocles et al., Nature 399: 126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72: 6025-6029, 2000) and microspheres (Lacoste et al., Proc. Natl. Acad. Sci USA 97 (17): 9461-9466,2000) can be used.
    [0068] Multicomponent labels can also be used in the present application. A multicomponent label is one that is dependent on interaction with an additional compound for detection. The most common multicomponent label used in biology is the biotin-streptavidin system. Biotin is used as the label attached to the nucleotide base. Streptavidin is then added separately to allow detection to occur. Other multicomponent systems are available. For example, dinitrophenol has a commercially available fluorescent antibody, which can be used for detection.
    [0069] Unless otherwise indicated, reference to nucleotides is also intended to be applicable to nucleosides. The present application will also be further described with reference to DNA, although the description is also applicable to RNA, PNA and other nucleic acids, unless otherwise indicated. Binders
    [0070] In some embodiments described in this document, the purine or pyrimidine base of the modified nucleotide or nucleoside molecules can be linked to a detectable label, as described above. In some such embodiments, the binders used are cleavable. The use of a cleavable linker ensures that the label can, if required, be removed after detection, avoiding any sign of interference with any subsequently incorporated nucleotide or labeled nucleoside.
    [0071] In some other embodiments, the binders used are non-cleavable. Since in each case where a labeled nucleotide of the invention is incorporated, no nucleotide subsequently needs to be incorporated and thus the label does not need to be removed from the nucleotide.
    [0072] Those skilled in the art will be aware of the usefulness of didesoxynucleoside triphosphates in so-called Sanger sequencing methods, and related protocols (Sanger type), which depend on randomized chain termination on a particular type of nucleotide. An example of a Sanger-type sequencing protocol is the BASS method, described by Metzker.
    [0073] Sanger and Sanger-type methods generally operate by conducting an experiment in which eight types of nucleotides are provided, four of which contain a 3'-OH group; and four of which omit the OH group and which are labeled differently from each other. The nucleotides used, which omit the group 3'-OH - dideoxy nucleotides (ddNTPs). As known to a person skilled in the art, since ddNTPs are labeled differently, by determining the positions of the embedded terminal nucleotides, and combining this information, the sequence of the target oligonucleotide can be determined.
    [0074] The nucleotides of the present application, will be recognized, may be useful in Sanger methods and related protocols, since the same effect achieved through the use of ddNTPs can be achieved through the use of the 3'-OH protection groups described in this document: both prevent the incorporation of subsequent nucleotides.
    [0075] Furthermore, it will be appreciated that the monitoring of the incorporation of 3'-OH protected nucleotides can be determined by the use of radioactive 32P in the bound phosphate groups. These can be present both in the ddNTPs themselves, as well as in the primers used for the extension.
    [0076] Cleavable linkers are known in the art, and conventional chemistry can be applied to link a linker to a nucleotide base and label. The ligand can be cleaved by any appropriate method, including exposure to acids, bases, nucleophiles, electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes, etc. The linker, as discussed in this document, can also be cleaved with the same catalyst used to cleave the 3'-O protecting group. Appropriate binders can be adapted from standard chemical protection groups, as disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Additional suitable cleavable linkers used in solid phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100: 2092-2157, 2000).
    [0077] The use of the term "cleavable linker" is not intended to imply that the entire linker is required to be removed, for example, from the nucleotide base. Where the detectable label is attached to the base, the nucleoside cleavage site can be located on the linker, which ensures that part of the linker remains attached to the nucleotide base after cleavage.
    [0078] Where the detectable label is attached to the base, the linker can be attached at any position on the nucleotide base, provided that the Watson-Crick base pairing can still be conducted. In the context of purine bases, it is preferred if the linker is attached via the 7-position of the purine or the preferred deazapurine analogue, via an 8-modified purine, via a modified N-6 adenosine or N-2 guanine modified. For pyrimidines, binding is preferably via the 5-position in cytosine, thymidine, or uracil and the N-4 position in cytosine. A. Electrophilically cleaved binders
    [0079] Electrophilically cleaved ligands are typically cleaved by protons and include acid-sensitive cleavages. Suitable binders include modified benzyl systems, such as trityl, p-alkoxybenzyl esters and p-alkoxybenzyl amides. Other suitable binders include tert-butyloxycarbonyl (Boc) groups and the acetal system.
    [0080] The use of thiophilic metals, such as nickel, silver or mercury, in the cleavage of thioacetal or other sulfur-containing protecting groups can also be considered for the preparation of appropriate binder molecules. B. Nucleophilically cleaved ligands
    [0081] Nucleophilic cleavage is also a well-known method in the preparation of ligand molecules. Groups such as esters that are labile in water (i.e., can be cleaved simply at basic pH) and groups that are labile to non-aqueous nucleophiles can be used. Fluorine ions can be used to cleave silicon-oxygen bonds in groups such as triisopropyl silane (TIPS) or t-butyldimethyl silane (TBDMS). C. Photocleavable binders
    [0082] Photocleavable binders have been used widely in carbohydrate chemistry. It is preferable that the light required to activate the cleavage does not affect the other components of the modified nucleotides. For example, if a fluorophore is used as the label, it is preferable if it absorbs light of a wavelength different from that required to cleave the ligand molecule. Suitable binders include those based on O-nitrobenzyl compounds and nitroveratril compounds. Binders based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64: 3454-3460, 1999). D. Cleavage under reducing conditions
    [0083] There are many known ligands that are susceptible to reductive cleavage. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction is also known in the art. E. Cleavage under oxidative conditions
    [0084] Approaches based on oxidation are well known in the art. These include the oxidation of p-alkoxybenzyl groups and the oxidation of sulfur and selenium binders. The use of aqueous iodine to cleave disulfides and other sulfur and selenium-based binders is also within the scope of the invention. F. Security capture links
    [0085] Security capture binders are those that cleave in two stages. In a preferred system, the first step is the generation of a reactive nucleophilic center, followed by a second step involving an intramolecular cyclization that results in cleavage. For example, levulin ester chain linkages can be treated with hydrazine or photochemistry to release an active amine, which can then be cyclized to cleave an ester elsewhere in the molecule (Burgess et al., J. Org. Chem 62: 5165-5168, 1997). G. Cleavage by elimination mechanisms
    [0086] Elimination reactions can also be used. For example, catalyzed elimination based on groups such as Fmoc and cyanoethyl and palladium catalyzed reductive elimination from allyl systems can be used.
    [0087] In some embodiments, the binder may comprise a spacer unit. The length of the linker is not important, as long as the label is kept at a sufficient distance from the nucleotide, so as not to interfere with any interaction between the nucleotide and an enzyme.
    [0088] In some embodiments, the linker may consist of similar functionality as the 3'-OH protecting group. This will make the deprotection and deprotection process more efficient, since only a single treatment will be required to remove both the label and the protecting group. Particularly preferred binders are phosphine-cleavable azide containing binders. Sequencing methods
    [0089] The modified nucleosides or nucleotides described in this document can be used in conjunction 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.
    [0090] The nucleotide analogs presented in this document can be used in a sequencing procedure, such as a synthesis sequencing technique (SBS). Briefly, SBS can be initiated by contacting the target nucleic acids with one or more labeled nucleotides, DNA polymerase, etc. Those features where a primer is extended using the target nucleic acid as a template will incorporate a labeled nucleotide that can be detected. Optionally, the labeled nucleotides can further include a reversible termination property that terminates additional primer extension, once a nucleotide has been added to a primer. For example, a nucleotide analog having a reversible terminator segment can be added to a primer such that subsequent extension cannot occur until an unlocking agent is delivered to remove the segment. Thus, for embodiments that use reversible termination, an unlocking reagent can be delivered to the flow cell (before or after detection occurs). Washes can be conducted between the various dispensing steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluid systems and detection platform that can be easily adapted for use with an ordered set produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456: 53-59 (2008), WO 04/018497; WO 91/06678; WO 07/123744; US patent 7,057,026; 7,329,492; 7,211,414; 7,315,019; or 7,405,281 and US Patent Application Publication 2008/0108082 A1, each of which is incorporated herein by reference.
    [0091] Other sequencing procedures that use cyclic reactions can be used, such as pyro sequencing. Pyrosquencing detects the release of inorganic pyrophosphate (PPi), as particular nucleotides are incorporated into a nascent nucleic acid filament (Ronaghi, et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res 11 (1), 3-11 (2001); Ronaghi et al., Science 281 (5375), 365 (1998); US patents 6,210,891; 6,258,568 and 6,274,320, each of which is incorporated into this document by reference). In pyro sequencing, released PPi can be detected by being converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected by means of photons produced by luciferase. Thus, the sequencing reaction can be monitored by by means of a luminescence detection system. Excitation radiation sources used for fluorescence-based detection systems are not required for pyro-sequencing procedures. Fluidic systems, detectors and useful procedures that can be used for the application of pyrosequencing to ordered sets of the present disclosure are described, for example, in Patent Application Ser. WIPO PCT / US11 / 57111, US Patent Application Publication 2005 / 0191698 A1, US Patent 7,595,883, and US Patent 7,244,559, each of which is incorporated herein by reference.
    [0092] Link sequencing reactions are also useful, including, for example, those described in Shendure et al., Science 309: 1728-1732 (2005); US patent 5,599,675; and US Patent 5,750,341, each of which is incorporated herein by reference. Some achievements may include hybridization sequencing procedures, as described, for example, in Bains et al., Journal of Theoretical Biology 135 (3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251 (4995), 767773 (1995); and WO 1989/10977, each of which is incorporated herein by reference. In both sequencing by ligation and hybridization sequencing procedures, nucleic acids that are present in wells containing gel (or other concave characteristics) are subjected to repeated oligonucleotide distribution and detection cycles. Fluid systems for SBS methods are set out in this document, or in references cited in this document, can be easily adapted for the distribution of reagents for ligation sequencing or hybridization sequencing procedures. Typically, oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described in relation to SBS procedures in this document or in references cited in this document.
    [0093] Some achievements may use methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a polymerase bearing fluorophore and Y-phosphate labeled nucleotides, or with zero-mode waveguides. 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); Korlach et al., Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated into this document by reference.
    [0094] Some SBS achievements include detecting a proton released by incorporating a nucleotide into an extension product. For example, sequencing based on detecting protons released may use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies) or sequencing methods and systems described in US Patent Application 2009 / 0026082A1; 2009 / 0127589A1 ; 2010 / 0137143A1; or 2010/0282617 A1, each of which is incorporated herein by reference. EXAMPLES
    [0095] Additional achievements are revealed in greater detail in the following examples, which are in no way intended to limit the scope of the Claims. The synthesis of several nucleotides modified with the 3'-hydroxy protecting group is demonstrated in Examples 1-3. Example 1Synthesis of Nucleotides with the 3'-OH protecting group

    [0096] Scheme 1 illustrates a synthetic route for the preparation of modified nucleotides with azidomethyl monofluoromethyl substituted as 3'-OH protecting groups. Compounds 1a-1f employ a modified thymine (T-PA) as the base. Other non-limiting examples of the bases that can be used include Cbz-PA, ADMF-PA and GPac-PA, the structures of which are shown above in Scheme 1. Experimental Procedures
    [0097] To a solution of primary nucleoside 1a (1.54 g, 2.5 mmol) in anhydrous CH3CN (25 ml) was added 2,6-lutidine (0.87 ml, 7.5 mmol), (2- fluorethyl) (4-methoxyphenyl) sulfane (MPSF) (3.26 g, 17.5 mmol) and then BZ2O2 (50% pure, 8.47 g, 17.5 mmol) at 4 ° C. The reaction mixture was allowed to warm slowly to room temperature. The mixture was stirred for another 6 hours. Monitored TLC (EtOAc: DCM = 2: 8 v / v) to see complete consumption of the primary nucleoside. The reaction was then concentrated under reduced pressure to oily residue. To this mixture, petroleum ether (500 ml) was added and stirred vigorously for 10 min. The petroleum ether layer was decanted and the residue was repeated to treat with petroleum ether (x2). The oily residue was partitioned between DCM / NaHCO3 (1: 1) (300 ml). The organic layer was separated and the aqueous one was further extracted in DCM (2x150 ml). The combined organic layers were dried over MgSO4, filtered and the volatiles evaporated under reduced pressure. The crude product 1c was purified by a Biotag silica gel column (50g), using a gradient from petroleum ether to petroleum ether: EtOAc 1: 1 (v / v), to provide 1.63g of nucleoside 1b as a yellow foam pale (diastereomers, 82% yield). 1H NMR (d6 DMSO, 400 MHz): Ô, 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, CH2F), 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).
    [0098] To the primary nucleoside solution 1b (1.14g, 1.4mmol) in CH2Cl2 (14mL) with molecular sieve (4Å) under N2 was added cyclohexene (1.44mL, 14mmol). The mixture was cooled with an ice cream / acetone bath to -78 ° C. The solution of sulfuryl chloride (580 gL, 7.2 mmol) in DCM (14 ml) was slowly added over 90 minutes under N2. After 20 minutes at that TLC temperature (EtOAc: petroleum ether = 1: 1v / v), it indicated the total consumption of the primary nucleoside. The volatiles were evaporated under reduced pressure (and room temperature of 25 ° C) and the oily residue was quickly subjected to high vacuum for an additional 10 minutes until it foamed. The crude product was purged with N2 and then dissolved in anhydrous DMF (5 ml) and NaN3 (470 mg, 7 mmol) added at once. The resulting suspension was stirred at room temperature for 2 hours or until TLC indicated the completion of the reaction and the formation of 1c as two isomers (a and b). The reaction mixture was partitioned between EtOAc: NaHCO3 (1: 1) (200 ml). The organic layer was separated and the aqueous one was further extracted in EtOAc (2x100 ml). The combined organic extracts were dried over MgSO4, filtered and the volatiles evaporated under reduced pressure. The two diastereoisomers of 1c (A and B) were separated by a Biotag silica gel column (25 g), using a gradient from petroleum ether to petroleum ether: EtOAc 1: 1 (v / v), as a pale yellow foam .
    [0099] Isomer A (370 mg, yield: 38%). 1H NMR (d6 DMSO, 400 MHz): ô 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, NHCH2), 4.46 - 4.60 (m, 3H, H-3 ', CH2F), 5.05 - 5.09 (m, 1H, CHN3), 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). 19F NMR: -74.3 (CF3), -230.2 (CH2F).
    [00100] Isomer B (253 mg, 26% yield). 1H NMR (d6 DMSO, 400 MHz): ô 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, NHCH2), 4.45-4.60 (m, 3H, H-3 ', CH2F), 5.00-5.14 (m, 1H, CHN3), 609 (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). 19F NMR: -74.5 (CF3), -230.4 (CH2F).
    [00101] Primary material 1c (isomer A) (500 mg, 0.71 mmol) was dissolved in THF (3 mL) and cooled to 4 ° C in an ice bath. Then, TBAF (1.0 M in THF, 5% water, 1.07 mL, 1.07 mmol) was added slowly over a period of 5 mins. 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 no more primary material was visible by TLC. The reagent solution was dissolved in EtOAc (50 mL) and added to NaHCO3 (60 mL). The two layers were separated and the aqueous layer was extracted with additional DCM (50 mLx2). The organic extractions were combined, dried (MgSO4), filtered and evaporated to provide a yellow oil. The crude product 1d (isomer A) was purified by a Biotag silica gel column (10g), using a gradient of petroleum ether: EtOAc 8: 2 (v / v) to EtOAc as a white solid (183 mg, yield: 56 %).
    [00102] Isomer A: 1H NMR (400 MHz, d6-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, CH2NH), 4.33-4.37 (m, 1H, H-3 ') , 4.43-4.51 (m, CH2F), 5.12 (br.s, 1H, CHN3), 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) . 19F NMR: -74.3 (CF3), - 230.5 (CH2F)
    [00103] The same reaction was carried out for 1c (isomer B) at 360 mg scale and provided the corresponding product 1d (Isomer B, 150 mg, 63%). 1H NMR (400 MHz, d6-DMSO): δ 2.24-2.37 (m, 2H, H-2 '), 3.573.70 (m, 2H, H-5'), 3.97-4, 01 (m, 1H, H-4 '), 4.23 (br.s, 2H, CH2NH), 4.33-4.37 (m, 1H, H-3'), 4.44-4.53 (m, CH2F), 5.11-5.21 (br.s, 1H, CHN3), 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), 19F NMR: - 74.1 (CF3), - 230.1 (CH2F).
    [00104] The preparation of the corresponding triphosphates 1e and the additional dye binding to the nucleobase to provide the fully functional nucleoside triphosphate (ffN) 1f was reported in WO 2004/018497 and is generally known to a person skilled in the art. Example 2 Nucleotide Synthesis with 3'-OH protection group

    [00105] Scheme 2 illustrates a synthetic route for the preparation of nucleotides modified with substituted azidomethyl C-starch as 3'-OH protecting groups. Compounds 2a-2i employ a modified thymine (T-PA) as the base. Other non-limiting examples of the bases that can be used include Cbz-PA, ADMF-PA, and GPac-PA, the structures of which are shown above in Scheme 1. In the experimental procedure, compound 2f with an azidomethyl protecting group N, N-dimethyl-C (= O) - substituted (R = NMe2) and subsequent reactions were reported. Compounds with other C-starch groups were also prepared, such as N-ethyl-C (= O) - (R = NHEt). Experimental Procedures
    [00106] To a solution of primary nucleoside 2a (4.27 g, 6.9 mmol) in anhydrous CH3CN (50 ml) was added 2,6-lutidine (2.4 ml, 20.7 mmol), S (CH2CH2OAc ) 2 (12.2 g, 69 mmol) and then BZ2O2 (50% pure, 33.4 g, 69 mmol) at 4 ° C. The reaction mixture was allowed to warm slowly to room temperature. The mixture was stirred for another 12 hours. Monitored TLC (EtOAc: DCM = 4: 6 v / v) to see complete consumption of the primary nucleoside. The reaction was then concentrated under reduced pressure to an oily residue. To this mixture, petroleum ether (800 ml) was added and stirred vigorously for 10 min. The petroleum ether layer was decanted and the residue was repeatedly treated with petroleum ether (x2). The oily residue was partitioned between DCM / NaHCO3 (1: 1) (1000 mL). The organic layer was separated and the aqueous layer was further extracted in DCM (2x500 ml). The combined organic layers were dried over MgSO4, filtered and the volatiles evaporated under reduced pressure. The crude product 2b was purified by a column of silica gel Biotag (100g), using a gradient of petroleum ether to petroleum ether: EtOAc 2: 8 (v / v), as a pale yellow foam (4.17g, yield : 74%, diastereoisomers).
    [00107] To a solution of primary nucleoside 2b (4.54g, 5.56 mmol) in CH2 Cl2 (56 mL) with molecular sieve (4 µm) under N2 was added cyclohexene (5.62 mL, 56 mmol). The mixture was cooled with an ice bath to 4 ° C. The solution of sulfuryl chloride (1.13 mL, 13.9 mmol) in DCM (25 mL) was slowly added over 90 minutes under N2. After 30 minutes at that TLC temperature (EtOAc: DCM = 4: 6 v / v), it indicated that 10% of the primary nucleoside 2b had left. Additional sulfuryl chloride (0.1 mL) was added to the reaction mixture. TLC indicated complete conversion of 2b. The volatiles were evaporated under reduced pressure (and room temperature of 25 ° C) and the oily residue was quickly subjected to high vacuum for an additional 10 minutes until it foamed. The crude product was purged with N2 and then dissolved in anhydrous DMF (5 ml) and NaN3 (1.8 g, 27.8 mmol) added at once. The resulting suspension was stirred at room temperature for 2 hours or until TLC indicated the completion of the reaction and the formation of 2c as two isomers (A and B). The reaction mixture was partitioned between EtOAc: NaHCO3 (1: 1) (1000 ml). The organic layer was separated and the aqueous layer was further extracted in EtOAc (2x300 ml). The combined organic extracts were then dried over MgSO4, filtered and the volatiles evaporated under reduced pressure. The two diastereoisomers of 2c (isomer A and B) were separated by a column of silica gel Biotag (100 g), using a gradient from petroleum ether to petroleum ether: EtOAc 1: 1 (v / v), as a foam pale yellow. Isomer A: 1.68 g, yield: 40.7%. Isomer B: 1.79 g, yield: 43.2%.
    [00108] To a solution of primary nucleoside 2c (isomer A) (1.63 g, 2.2 mmol) in MeOH / THF (1: 1) (20 mL) was slowly added NaOH (1M in water) (2, 2 mL, 2.2 mmol) and stirred at 4 ° C. The progress of the reaction was monitored by TLC (EtOAc: DCM = 4: 6 v / v). The reaction was stopped after 1 hour when no more primary material was visible by TLC. The reaction mixture was partitioned between DCM: NaHCO3 (1: 1) (150 ml). The organic layer was separated and the aqueous layer was further extracted in DCM (2x70 ml). The combined organic extracts were dried over MgSO4, filtered and the volatiles evaporated under reduced pressure. The 2d crude product was purified by a Biotag silica gel column (10g), using a petroleum ether gradient: EtOAc (8: 2) (v / v) to EtOAc as a pale yellow foam (1.1g, yield: 71%).
    [00109] The same reaction was repeated for 2c (isomer B, 1.57g) and provided the corresponding product 2d (isomer B, 1.01 g, 69% yield).
    [00110] A solution of primary nucleoside 2d (isomer A) (700 mg, 1 mmol) in CH3CN (10 mL) was treated with TEMPO (63 mg, 0.4 mmol) and BAIB (644 mg, 2 mmol) at temperature environment. The progress of the reaction was monitored by TLC (EtOAc: DCM = 7: 3 v / v). The reaction was stopped after 2 hours, when no more primary material was visible by TLC. The reaction mixture was partitioned between DCM / Na2S2O3 (1: 1) (100 mL). The organic layer was separated and the aqueous layer was further extracted in DCM (2x70 mL). The combined organic extracts are then washed with NaCl (sat.). The organic layer was evaporated under reduced pressure, without drying over MgSO4, in order to prevent the product from precipitating out. The crude product 2e was purified by a Biotag silica gel column (10g), using a gradient of petroleum ether: EtOAc 1: 1 (v / v) to EtOAc to MeOH: EtOAc (1: 9) as a pale yellow foam ( isomer A, 482 mg, yield 68%).
    [00111] The same reaction was carried out for 2d (isomer B, 700 mg) and provided the corresponding product 2e (isomer B, 488 mg, 69% yield).
    [00112] To a solution of primary nucleoside 2e (isomer A) (233 mg, 0.33 mmol) in CH3CN (10 mL), Huning's base (173 gL, 1 mmol) and BOP (165 mg, 0, 39 mmol) at room temperature. After stirring for 5 minutes, the solution was treated with Me2 NH (2 M in THF) (0.41 ml, 0.82 mmol). The progress of the reaction was monitored by TLC (MeOH: DCM = 1: 9 v / v). The reaction was stopped after 2 hours, when no more primary material was visible by TLC. The reaction mixture was partitioned between DCM: NaHCO3 (1: 1) (50 ml). The organic layer was separated and the aqueous layer was further extracted in DCM (2x30 ml). The combined organic extracts were dried over MgSO4, filtered and the volatiles evaporated under reduced pressure. The 2f crude product (R = NMe2) was purified by a Biotag silica gel column (10 g), using a gradient from DCM: EtOAc (8: 2) (v / v) to EtOAc as a pale yellow foam (isomer A, 220 mg, 90% yield).
    [00113] The same reaction was performed for 2e (isomer B, 249, mg) and provided the corresponding product 2f (isomer B, 240 mg, 92% yield).
    [00114] The primary material 2f (mixture of isomer A and B) (455 mg, 0.61 mmol) was dissolved in THF (2 mL) and cooled to 4 ° C with an ice bath. Then, TBAF (1.0 M in THF, 5% water mass, 1.0 mL, 1.0 mmol) was added slowly over a period of 5 min. The reaction mixture was slowly warmed to room temperature. The reaction progress was monitored by TLC (EtOAc). The reaction was stopped after 1 hour, when no more primary material was visible by TLC. The reaction solution was dissolved in DCM (30 mL ) and added to NaHCO3 (30 mL). The two layers were separated and the aqueous layer was extracted with additional DCM (30 mLx2). The organic extractions were combined, dried (MgSO4), filtered and evaporated, to provide a yellow oil. The 2g crude product was purified by a Biotag silica gel column (10 g), using a gradient from DCM: EtOAc 8: 2 (v / v) to EtOAc to MeOH: EtOAc (2: 8) as a white solid (52 % yield, 160 mg).
    [00115] The preparation of the corresponding triphosphates 2h and the additional dye binding to the nucleobase, to provide the fully functional nucleoside triphosphate (ffN) 2i, have been reported in WO 2004/018497 and are generally known to a person skilled in the art. Example 3 Nucleotides with 3'-OH protection group

    [00116] Scheme 3 illustrates a synthetic route for the preparation of nucleotides modified with 3'-OH substituted difluormethyl azidomethyl protecting groups. Compounds 3a-3i employ a modified thymine (T-PA) as the base. Other non-limiting examples of the bases that can be used include Cbz-PA, ADMF-PA and GPac-PA, the structures of which are shown above in Scheme 1. The procedure for the synthesis of 3b, 3c and 3d was described in Example 2 . Experimental Procedures
    [00117] To a solution of primary nucleoside 3d (isomer A) (490 mg, 0.7 mmol) and DBU (209 gL, 1.4 mmol) in anhydrous DCM (5 mL), a N chloride solution was slowly added -tert-butyl benzene sulfinimidoyl (181 mg, 0.84 mmol) in anhydrous DCM (2 mL) at - 78 ° C. The progress of the reaction was monitored by TLC (EtOAc: DCM 4: 6 v / v). The reaction was stopped after 2 hours, when there was still 10% of primary material left by TLC, to prevent overreaction. The reaction mixture was partitioned between DCM: NaHCO3 (1: 1) (50 ml). The aqueous layer was further extracted in DCM (2x30 ml). The organic extractions were combined, dried (MgSO4), filtered, and evaporated to provide a yellow oil. The crude product 3e was purified by a Biotag silica gel column (10 g), using a gradient from petroleum ether: EtOAc (8: 2) (v / v) to petroleum ether: EtOAc (2: 8) (v / v) as a pale yellow foam (isomer A, 250 mg, 51% yield).
    [00118] The same reaction was performed for 3d (isomer B, 480 mg) and provided the corresponding product 3e (isomer B, 240 mg, 50% yield).
    [00119] To a solution of primary nucleoside 3e (isomer A) (342 mg, 0.49 mmol), EtOH (15 gL, 0.25 mmol) in DCM (2.5 mL) at 4 ° C (ice bath ). The reaction mixture was stirred for 1 h at 4 ° C. The progress of the reaction was monitored by TLC (EtOAc: petroleum ether = 3: 7 v / v). The reaction was stopped after 1 hour. The reaction mixture was partitioned between DCM: NaHCO3 (1: 1) (50 ml). The aqueous layer was further extracted in DCM (2x30 ml). The organic extractions were combined, dried (MgSO4), filtered and evaporated, to provide a yellow oil. The 3f crude product was purified by 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) as a pale yellow foam (isomer A, 100 mg, 28%).
    [00120] The same reaction was performed for 3e (B isomer, 480 mg) and provided the corresponding 3f product (B isomer, 240 mg, 50% yield).
    [00121] Primary material 3f (isomer A) (124 mg, 0.17 mmol) was dissolved in THF (2 ml) and cooled to 4 ° C with an ice bath. Then, TBAF (1.0 M in THF, 5% water mass, 255 gL, 10.255 mmol) was added slowly over a period of 5 min. The reaction mixture was slowly warmed to room temperature. The progression of the reaction was monitored by TLC (EtOAc). The reaction was stopped after 1 hour, when no more primary material was visible by TLC. The reaction solution was dissolved in DCM (30 mL) and added to NaHCO3 (30 mL) The two layers were separated and the aqueous layer was extracted with additional DCM (30 ml x2). The organic extractions were combined, dried (MgSO4), filtered, and evaporated to provide a yellow oil. The 3g crude product was purified by a Biotag silica gel column (4 g) using a gradient from DCM: EtOAc 8: 2 (v / v) to EtOAc to MeOH: EtOAc (2: 8) as a pale yellow foam (isomer A, 54% yield, 44 mg).
    [00122] Isomer A: 1H NMR (400 MHz, d6-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, CH2NH), 4.33-4.37 (m, 1H, H-3 ') , 4.85 (s, 2H, OCH2N3), 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). 19F NMR: -74.4 (CF3), -131.6 (CH2F).
    [00123] The same reaction was performed for 3f (isomer B, 133 mg) and provided the corresponding product 3g (isomer B, 48 mg, 54% yield). 1H NMR (400 MHz, d6-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, CH2NH), 4.57-4.58 (m, 1H, H-3' ), 5.24-5.29 (m, 2H, 5'-OH, OCHN3), 6.07-6.34 (m, 2H, H-1 ', CHF2), 8.19 (s, 1H, H-6), 10.09 (br s, 1H, NH), 11.70 (br s, 1H, NH). 19F NMR: -74.2 (CF3), -131.4 (CH2F).
    [00124] The preparation of the corresponding 3h triphosphates and the additional dye binding to the nucleobase to provide the fully functional nucleotide (ffN) 3i have been reported in WO 2004/018497 and are generally known to a person skilled in the art. Example 4 Thermal Stability Test of 3'-OH Protection Groups
    [00125] A variety of 3'-OH protecting groups have been investigated regarding their thermal stability (FIG. 1A). Thermal stability was assessed by heating 0.1 mM of each nucleotide protected by 3'-OH in a pH = 9 buffer (50 mM tis-HCl, 50 mM NaCl, 0.05% tween, 6mM Mg2SO4) at 60 ° C. Several time points were taken and HPLC was used to analyze the formation of unlocked materials. The stabilities of -CH2F and -C (O) NHBu were found to be about 2 times greater than the standard azidomethyl (-CH2N3) protecting group. The stability of the - CF2H group was found to be about 10 times greater than the standard (FIG. 1B). Example 5Protection of 3'-OH protection groups
    [00126] The deprotection reaction rates of several 3'-OH protection groups were also studied. The deprotection rate of the standard azidomethyl protecting group was compared with the substituted azidomethyl -CH2F and substituted azidomethyl -C (O) NHBu. It was observed that both the more thermally stable 3'-OH protecting groups were removed faster than the standard azidomethyl protecting group using phosphines (1mM THP) as the deprotecting agent. See FIG. 2A. For example, the half-life of -CH2F and -C (O) NHBu was 8.9 minutes and 2.9 minutes, respectively, compared to the 20.4 minutes of azidomethyl half life (FIG. 2B). Example 6 Sequencing Test
    [00127] Nucleotides modified with 3'-OH azidomethyl -CH2F (mono-F) substituted protecting group were prepared and their sequencing performance was evaluated on Miseq platforms. It was predicted that increased thermal stability of 3'-OH protecting groups would lead to a higher quality of nucleotides for sequencing chemistry with less contaminated 3'-unlocked nucleotides. The presence of 3'-unlocked nucleotides in the SBS sequencing kits would therefore result in pre-phasing events, which were numbered as pre-phasing values.
    [00128] 12 short cycle sequencing experiments were first used to generate phasing and pre-phasing values.ffNs protected by substituted mono-F azidomethyl were used according to the following concentrations: ffA-dye 1 (2uM); ffT-dye 2 (10uM), ffC-dye 3 (2uM) and ffG-dye 4 (5uM). The substituted mono-F azidomethyl group comprises both isomers A and B. Two dyes - dye 2 as in the standard Miseq kits and dye 5 were used to label ffT. Table 1 shows several combinations of nucleotides with isomers A and B of substituted mono-F azidomethyl, which were evaluated for phasing and pre-phasing impacts. In all cases, the pre-phasing values were substantially lower than the control that the standard V2 Miseq kit nucleotides used (FIG. 3). Table 1.

    Sequencing Quality Test
    [00129] 2x400bp sequencing was conducted in Miseq to assess the potential of these nucleotides for improving the quality of sequencing. The sequencing performed was performed according to the manufacturer's instructions (Illumina Inc., San Diego, CA). The standard incorporation buffer was replaced by an incorporation buffer containing all FFNs blocked by mono-F, each with a separate dye label: ffA-dye 1 (2uM), ffT-dye 2 (1uM), ffC-dye 3 (2 µM) and ffG-dye 4 (5 µM). The DNA library used was made following the standard TruSeq HT protocol of B cereus genomic DNA.
    [00130] In both sequencing experiments (with blocked A and B mono-F isomer), very low pre-phasing values were observed. Accompanied by low phasing values, the application of these new nucleotides generated superior 2x400bp sequencing data, with> 80% of bases above Q30 in both cases (see FIG. 4A for the Q score of isomer A and FIG. 4B for scoring chart Q of isomer B). These results demonstrate a great improvement compared to Miseq v2 kits (2x250bp, 80% of bases> Q30 in typical R&D sequencing experiments, or 70% of bases> Q30, as the specifications determined). As shown below, Table 2 summarizes the sequencing data when using all mono-F ffNs isomers in IMX. Table 3 summarizes the sequencing data using all mono-F ffNs B isomers in IMX.Table 2.
    Table 3.
    权利要求:
    Claims (15)
    [0001]
    1. Modified Nucleotide or Nucleoside molecule, which comprises a purine or pyrimidine base and a ribose or deoxyribose sugar group having a removable 3'-hydroxy protecting group forming a -OC (R) 2N3 structure covalently linked to 3'-carbon atom, characterized in that one of R is hydrogen and the other R is -CHF2, -CH2F, -CHCl2 or - CH2Cl; or one of R is hydrogen and the other R is -C (= O) NR4R5, where each R4 and R5 is selected from hydrogen or C1-6alkyl.
    [0002]
    2. Modified Nucleotide or Nucleoside molecule according to Claim 1, characterized in that one of R is hydrogen and the other R is -CHF2 or -CH2F.
    [0003]
    3. Modified Nucleotide or Nucleoside molecule according to Claim 1, characterized in that one of R is hydrogen and the other R is -C (= O) NR4R5 and where R4 is hydrogen and R5 is C1-6 alkyl.
    [0004]
    4. Modified Nucleotide or Nucleoside molecule according to Claim 3, characterized in that R5 is n-butyl.
    [0005]
    5. Modified Nucleotide or Nucleoside molecule according to any one of Claims 1 to 3, characterized in that the nucleotide or nucleoside molecule comprises a deoxyribose group.
    [0006]
    6. Modified Nucleotide or Nucleoside molecule according to any one of Claims 1 to 3, characterized in that the nucleotide or nucleoside molecule comprises a 7-deaza purine.
    [0007]
    7. Modified Nucleotide or Nucleoside molecule according to any one of Claims 1 to 6, characterized in that the 3'-hydroxy protecting group is removed in a deprotection reaction with a phosphine.
    [0008]
    8. Modified Nucleotide or Nucleoside molecule according to Claim 7, characterized in that the phosphine is tris (hydroxymethyl) phosphine (THP).
    [0009]
    9. Modified Nucleotide or Nucleoside molecule according to any one of Claims 1 to 8, characterized in that said base is linked to a detectable marker through a cleavable linker or a non-cleavable linker and wherein the detectable marker is a fluorophore.
    [0010]
    10. Modified Nucleotide or Nucleoside molecule according to any one of Claims 1 to 8, characterized in that said 3'-hydroxy protecting group is attached to a detectable marker via a cleavable linker or a non-cleavable linker and wherein the detectable marker is a fluorophore.
    [0011]
    11. Method for Preparing the Growth of Complementary Polynucleotide to a Single-Striped Target Polynucleotide in Sequencing Reaction, which comprises incorporating a modified nucleotide molecule as defined in any of Claims 1 to 10 in the complementary polynucleotide growth, characterized by that the incorporation of the modified nucleotide prevents the introduction of any subsequent nucleotide in the growth of the complementary polynucleotide.
    [0012]
    12. Method for Preparing Complementary Polynucleotide Growth on Single-Striped Target Polynucleotide in Sequencing Reaction according to Claim 11, characterized in that the incorporation of the modified nucleotide molecule is carried out by a terminal transferase, a terminal polymerase or a reverse transcriptase.
    [0013]
    13. Method for Determining the Single Strip Target Polynucleotide Sequence, characterized in that it comprises monitoring the sequential incorporation of complementary nucleotides, wherein at least one incorporated complementary nucleotide is a modified nucleotide molecule as defined in Claim 9 or 10; and detecting the identity of the modified nucleotide molecule.
    [0014]
    14. Method for Determining the Single-Striped Target Polynucleotide Sequence according to Claim 13, characterized in that the identity of the modified nucleotide is determined by detecting the detectable marker attached to the base.
    [0015]
    15. Kit, characterized in that it comprises a plurality of nucleotide or modified nucleoside molecules as defined in any one of Claims 1 to 10 and packaging materials therefor.
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    同族专利:
    公开号 | 公开日
    US9593373B2|2017-03-14|
    CA2903095C|2018-02-13|
    EP2970356A1|2016-01-20|
    KR101874680B1|2018-07-04|
    US10982277B2|2021-04-20|
    CN108864232A|2018-11-23|
    JP6333297B2|2018-05-30|
    KR20150132474A|2015-11-25|
    KR20180077296A|2018-07-06|
    HK1220463A1|2017-05-05|
    CN105377869A|2016-03-02|
    WO2014139596A1|2014-09-18|
    KR102230831B1|2021-03-22|
    US20170166961A1|2017-06-15|
    BR112015022448A2|2017-07-18|
    US20160002721A1|2016-01-07|
    EP3388442A1|2018-10-17|
    ES2685549T3|2018-10-09|
    AU2013382358B2|2017-01-12|
    CA2903095A1|2014-09-18|
    CN105377869B|2018-07-10|
    EP2970356B1|2018-05-30|
    US20200087725A1|2020-03-19|
    JP2016512206A|2016-04-25|
    CN108864232B|2022-01-04|
    DK2970356T3|2018-08-27|
    US10407721B2|2019-09-10|
    AU2013382358A1|2015-09-17|
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    法律状态:
    2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-10-15| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-05-05| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-09-08| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-12-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/03/2013, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    PCT/EP2013/055466|WO2014139596A1|2013-03-15|2013-03-15|Modified nucleosides or nucleotides|
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