RU2818762C2 - Nucleosides and nucleotides with 3'-hydroxy blocking groups and their use in methods of sequencing polynucleotides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to methods for sequencing polynucleotides. Disclosed is a nucleotide containing ribose or deoxyribose, having removable 3'-OH blocking group covalently attached to 3'-carbon atom. Also disclosed is an oligonucleotide, a method of producing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction, method of determining the sequence of a target single-stranded polynucleotide, a kit for use in multiplex sequencing by synthesis, use of a nucleotide in multiplex sequencing by synthesis.

EFFECT: group of inventions provides a wider range of products for a specific purpose.

54 cl, 7 dwg, 7 ex

Description

The present disclosure generally relates to nucleotides, nucleosides or oligonucleotides containing 3'-hydroxy protecting groups, and their use in polynucleotide sequencing methods. Methods for preparing 3'-hydroxy protected nucleotides, nucleosides or oligonucleotides are also disclosed.

Description of related area

Advances in molecular research have been driven in part by improvements in the technologies used to characterize molecules or their biological reactions. In particular, the study of nucleic acids—DNA and RNA—has benefited from the development of technologies used for sequence analysis and studies of hybridization events.

An example of technologies that have improved nucleic acid research is the development of ready-to-use immobilized nucleic acid chips. These chips typically consist of a high-density array of polynucleotides immobilized on a solid support material. See, for example, Fodor et al., Trends Biotech. 12: 19-26, 1994, which describes ways to assemble nucleic acids using a chemically sensitized glass surface, protected by a mask, but exposed in certain areas to allow the attachment of suitably modified nucleotide phosphoramidites. Ready-to-use chips can also be made by "spotting" known polynucleotides onto a solid support at predetermined positions (eg, Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).

One method of determining the nucleotide sequence of a nucleic acid associated with a chip is called sequencing-by-synthesis or SBS. This DNA sequencing technique ideally requires the controlled (i.e., sequential) incorporation of the correct complementary nucleotide opposite the nucleic acid being sequenced. This ensures accurate sequencing by adding nucleotides in multiple rounds as each nucleotide residue is sequenced in turn, thus preventing uncontrolled series of additions from occurring. The included nucleotide is read using a suitable tag attached to it, before removing the tag cluster and subsequent further round of sequencing.

To ensure that only one incorporation occurs, a structural modification ("protecting group" or "blocking group") is included in each labeled nucleotide that is added to the growing strand to ensure that only one nucleotide is incorporated. After adding a nucleotide with a protecting group, 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 inclusion of the next protected labeled nucleotide.

To be useful in DNA sequencing, nucleotides, which are typically nucleotide triphosphates, typically require a 3'-hydroxy protecting group to prevent the polymerase used to be incorporated into the polynucleotide chain from continuing replication once a base on the nucleotide is added. There are many restrictions on the types of groups that can be added to a nucleotide and still be suitable. The protecting group should prevent the addition of additional nucleotide molecules to the polynucleotide chain, while at the same time being easily removed from the sugar moiety without causing damage to the polynucleotide chain. In addition, the modified nucleotide must be compatible with the polymerase or other suitable enzyme used to incorporate it into the polynucleotide chain. An ideal protecting group should therefore exhibit long-term stability, be efficiently incorporated by the polymerase enzyme, cause blocking of secondary or further incorporation of nucleotides, and be removable under mild conditions that do not cause damage to the polynucleotide structure, preferably under aqueous conditions.

Reversible protecting 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 3'-modified 2-deoxyribonucleoside 5'-triphosphates (3'-modified dNTP) and testing in two DNA template assays for incorporation activity. WO 2002/029003 describes a sequencing method that may involve the use of an allyl protecting group to cap a 3'-OH group on a growing DNA strand in a polymerase reaction.

In addition, the development of a variety of reversible protecting groups and methods for deprotecting them under DNA-compatible conditions has previously been described in International Application Publications No. WO 2004/018497 and WO 2014/139596, each of which is hereby incorporated by reference in its entirety .

SUMMARY OF THE INVENTION

Some embodiments of the present disclosure provide a nucleotide or nucleoside containing ribose or deoxyribose having a 3'-OH leaving protecting or blocking group forming the structure covalently attached to the 3' carbon atom, in which:

each R ^1a and R ^1b independently represents H, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy, cyano, halogen, optionally substituted phenyl or optionally substituted aralkyl ;

each R ^2a and R ^2b independently represents H, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, cyano or halogen;

alternatively, R ^1a and R ^2a , along with the atoms to which they are attached, form an optionally substituted five to eight membered heterocyclyl group;

R ³ is H, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₃ -C ₇ cycloalkenyl, optionally substituted C ₂ -C ₆ alkynyl or optionally substituted (C ₁ -C ₆ alkylene)Si(R ⁴ ) ₃ ; And

each R ⁴ independently represents H, C ₁ -C ₆ alkyl or optionally substituted C ₆ -C ₁₀ aryl. In some embodiments, when R ^1a and R ^1b are each H or C ₁ -C ₆ alkyl, both R ^2a and R ^2b are H, then R ³ is substituted C ₂ -C ₆ alkenyl, optionally substituted C ₃ - C ₇ cycloalkenyl, optionally substituted C ₂ -C ₆ alkynyl or optionally substituted (C ₁ -C ₆ alkylene)Si(R ⁴ ) ₃ . In some embodiments, when ^R1a , ^R1b , ^R2a , and ^R2b are each H, then ^R3 is not H.

Some embodiments of the present disclosure provide a nucleoside or nucleotide containing ribose or deoxyribose having a removable 3'-OH blocking group forming the structure covalently attached to the 3' carbon atom, in which:

each of R ⁵ and R ⁶ independently represents H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₂ -C ₈ alkoxyalkyl, optionally substituted - (CH ₂ ) _m -phenyl, possibly substituted

-(SN₂)_n-(5 or 6 membered heteroaryl), possibly substituted-(CH₂)_k-WITH₃-WITH₇ carbocyclyl or possibly substituted -(CH₂)_R-(3-7 membered heterocyclyl);

each of -(CH ₂ ) _m -, -(CH ₂ ) _n - -(CH ₂ ) _k - and -(CH ₂ ) _p - is optionally substituted; And

m, n, k, and p are each independently 0, 1, 2, 3, or 4.

Some embodiments of the present disclosure relate to an oligonucleotide or polynucleotide containing a 3'-OH-blocked nucleotide molecule described herein.

Some embodiments of the present disclosure relate to a method of producing a growing polynucleotide complementary to a target single-stranded polynucleotide in a sequencing reaction comprising incorporating a nucleotide molecule described herein into a growing complementary polynucleotide, wherein inclusion of the nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide. In some embodiments, the incorporation of a given nucleotide is accomplished by a polymerase, terminal deoxynucleotidyl transferase (TdT), or reverse transcriptase. In one embodiment, the incorporation is accomplished by a polymerase (eg, DNA polymerase).

Certain other embodiments of the present disclosure provide a method for determining the sequence of a target single-stranded polynucleotide, comprising:

(a) incorporating a nucleotide containing a 3'-OH blocking group and a detectable tag as described herein into a copy of the polynucleotide chain complementary to at least a portion of the target polynucleotide chain;

(b) identifying the identity of a nucleotide included in a copy of the polynucleotide chain; And

(c) chemical removal of the tag and 3'-OH blocking group from a nucleotide included in a copy of the polynucleotide chain.

In some embodiments, the sequencing method further comprises (d) washing the tag and 3' blocking group chemically removed from a copy of the polynucleotide chain. In some embodiments, this washing step also removes unincorporated nucleotides. In some such embodiments, the 3' blocking group and detectable tag of the included nucleotide are removed before the introduction of the next complementary nucleotide. In some other embodiments, the 3' blocking group and the detectable tag are removed in a single chemical reaction step. In some embodiments, the sequential actuation described herein is performed at least 50 times, at least 100 times, at least 150 times, at least 200 times, or at least 250 times.

Certain other embodiments of the present disclosure provide kits containing a variety of nucleotide or nucleoside molecules described herein and packaging materials therefor. The nucleotides, nucleosides, oligonucleotides or kits that are set forth herein can be used to detect, measure or identify a biological system (including, for example, its processes and components). Typical techniques in which nucleotides, oligonucleotides or kits may be used include sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis, cellular analysis (eg, cell binding or cell function assay), or protein analysis (eg, protein binding assay or protein activity assay). This application can be carried out on an automatic instrument for performing a specific technique, such as an automatic sequencer. The sequencer may contain two or more than two lasers operating at different wavelengths to distinguish between different detectable marks.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1 is a graph illustrating the stability of various 3' blocked nucleotides as a function of time in buffer solution at 65°C.

FIG. 2A is a graph illustrating the percentage (%) of nucleotide (starting material) remaining as a function of time when comparing the rate of unblocking of a nucleotide with a 3'-AOM blocking group with a nucleotide with a 3'-O-azidomethyl (-CH ₂ N ₃ ) blocking group in solution.

FIG. 2B is a graph illustrating the percentage (%) of 3' unblocked nucleotides as a function of time when comparing the rate of unblocking of 3' blocked nucleotides with different acetal blocking groups in solution.

FIG. 3A and 3B illustrate sequencing results on an Illumina MiniSeq® instrument using fully functionalized nucleotides (ffN) with a 3'-AOM blocking group in the inclusion mixture.

FIG. 3C illustrates the sequencing error rate using fully functionalized nucleotides (ffN) with a 3'-AOM blocking group in the inclusion mixture compared to standard ffNs with a 3'-O-azidomethyl blocking group.

FIG. 4A and 4B each illustrate a comparison of raw sequencing metrics including phasing, prephasing, and error rates using fully functionalized nucleotides with 3'-AOM and 3'-O-azidomethyl blocking groups using two different DNA polymerases (Pol 812 and Pol 1901 ) respectively.

FIG. 5 is a graph illustrating the sequencing stability of fully functionalized nucleotides with 3'-AOM and 3'-O-azidomethyl blocking groups as a function of time in buffer solution at 45°C.

FIG. 6 is a graph illustrating the stability of nucleosides with different 3' blocking groups as a function of time in buffer solution at 65°C.

FIG. 7 is a graph illustrating the percentage (%) of 3' blocked nucleotide remaining as a function of time when comparing the rate of cleavage (deblocking) of the dimethylthiocarbamate (DMTC) thiocarbamate 3' blocking group under two different conditions (Oxone® or NaIO ₄ ) with the rate of cleavage 3 '-O-azidomethyl (3'-O-CH ₂ N ₃ ) blocking group.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present disclosure relate to nucleosides and nucleotides with 3'-OH acetal or thiocarbamate blocking groups for sequencing applications, such as sequencing by synthesis (SBS). These blocking groups provide better stability in solution compared to blocking groups known in the art. In particular, 3'-OH blocking groups have improved stability during the synthesis of fully functionalized nucleotides (ffN) and also greater stability in solution during formulation, storage, and operation on sequencers. Additionally, with the 3'-OH blocking groups described herein, low prephasing, less signal decay for improved data quality can also be achieved, enabling longer reads from sequencing applications.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the meaning that is within the meaning of ordinary skill in the art. The use of the term “including,” as well as other forms such as “include,” “includes,” and “included,” is not intended to be limiting. The use of the term “having”, as well as other forms such as “have”, “has” and “had”, is not limiting. The terms “comprise” and “comprising” as used in this specification, whether in a transitional phrase or a distinctive portion of the claims, are to be interpreted as having a non-limiting 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 method, the term “comprising” means that the method 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” means that the compound, composition, or device includes at least the listed features or components, but may also include additional features or components.

As used herein, the usual abbreviations for organic compounds are defined as follows:

The term "chip" as used herein refers to a population of different probe molecules that are attached to one or more than one substrate, such that the different probe molecules can be differentiated from each other according to relative position. The chip may include different probe molecules, each positioned at a distinct addressable location on the substrate. Alternatively or additionally, the chip may include separate substrates, each carrying a different probe molecule, where the different probe molecules can be identified according to the positions of the substrates on the surface to which the substrates are attached, or according to the positions of the substrates in the liquid. Typical chips in which individual substrates are disposed on a surface include, but are not limited to, chips comprising beads in wells, as described, for example, in US Pat. No. 6,355,431 B1, US 2002/0102578 and PCT Publication No. WO 00/63437. Typical formats that can be used in this invention to distinguish beads in a liquid chip, for example, using a microfluidic device such as a fluorescence activated cell sorter (FACS), are described, for example, in US patent No. 6524793. Other examples of chips that can be used in this invention include, but are not limited to, those described in US Pat. No. 5,429,807; 5436327; 5561071; 5583211; 5658734; 5837858; 5874219; 5919523; 6136269; 6287768; 6287776; 6288220; 6297006; 6291193; 6346413; 6416949; 6482591; 6514751 and 6610482; and WO 93/17126; WO 95/11995; WO 95/35505; EP 742287; and EP 799897.

The term "covalently attached" or "covalently bonded" as used herein refers to the formation of a chemical bond that is characterized by the sharing of pairs of electrons between atoms. For example, a covalently attached polymer coating refers to a polymer coating that forms chemical bonds with the functionalized surface of a substrate, as opposed to attaching to the surface through other means, such as adhesion or electrostatic interaction. It will be appreciated that polymers that are covalently attached to a surface may also be linked by means other than covalent attachment.

Any "R" group(s) as the term is used herein represents substituents that can be attached to the indicated atom. The R group may be substituted or unsubstituted. If two "R" groups are described as "together with the atoms to which they are attached" forming a ring or ring system, this means that the aggregate block of atoms located between the bonds and two R groups is the described ring. For example, given the following substructure:

both R ¹ and R ² are defined as being selected from the group consisting of hydrogen and alkyl, or R ¹ and R ² together with the atoms to which they are added form aryl or carbocyclyl, which means that R ¹ and R ² can be selected from hydrogen or alkyl, or alternatively, the given substructure has the structure:

where A represents an aryl ring or a carbocyclyl containing said double bond.

It should be understood that certain radical naming conventions may include either a monoradical or a diradical, depending on the context. For example, in the case where a substituent requires two points of attachment to the rest of the molecule, it is clear that the substituent is a diradical. For example, a substituent identified as alkyl that requires two points of attachment includes diradicals such as -CH ₂ - -CH ₂ CH ₂ -, -CH ₂ CH(CH ₃ )CH ₂ - and the like. Other radical naming conventions clearly indicate that the radical is a diradical such as "alkylene" or "alkenylene".

The term “halogen” or “halo-” as used herein means any of the radioactively stable atoms in column 7 of the Periodic Table of the Elements, such as fluorine, chlorine, bromine or iodine, with fluorine and chlorine being preferred.

The term "C _a to C _b ", in which "a" and "b" are integers, as used herein, refers to the number of carbon atoms in an alkyl, alkenyl or alkynyl group, or to the number of ring atoms of the cycloalkyl or aryl group. That is, a given alkyl, alkenyl, alkynyl, cycloalkyl ring and aryl ring may contain from “a” to “b”, inclusive, carbon atoms. For example, "C ₁ -C ₄ alkyl" group refers to all alkyl groups having from 1 to 4 carbons, that is, CH ₃ -, CH ₃ CH ₂ -, CH ₃ CH ₂ CH ₂ -, (CH ₃ ) ₂ CH-, CH ₃ CH ₂ CH ₂ CH ₂ -, CH ₃ CH ₂ CH(CH ₃ )- and (CH ₃ ) ₃ C-; The C ₃ -C ₄ cycloalkyl group refers to all cycloalkyl groups having from 3 to 4 carbon atoms, that is, cyclopropyl and cyclobutyl. Likewise, a “4-6 membered heterocyclyl” group refers to all heterocyclyl groups with 4-6 common ring atoms, for example, azetidine, oxetane, oxazoline, pyrrolidine, piperidine, piperazine, morpholine and the like. Unless "a" and "b" are indicated with respect to an alkyl, alkenyl, alkynyl, cycloalkyl or aryl group, the widest range described in these definitions should be assumed. The term "C ₁ -C ₆ " as used herein includes C ₁ , C ₂ , C ₃ , C ₄ , C ₅ and C ₆ , and the interval defined by any two of these numbers. For example, C ₁ -C ₆ alkyl includes C ₁ , C ₂ , C ₃ , C ₄ , C ₅ and C ₆ alkyl, C ₂ -C ₆ alkyl, C ₁ -C ₃ alkyl, etc. Likewise, C ₂ -C ₆ alkenyl includes C ₂ , C ₃ , C ₄ , C ₅ and C ₆ alkenyl, C ₂ -C ₅ alkenyl, C ₃ -C ₄ alkenyl, etc.; and C ₂ -C ₆ alkynyl includes C ₂ , C ₃ , C ₄ , C ₅ and C ₆ alkynyl, C ₂ -C ₅ alkynyl, C ₃ -C ₄ alkynyl, etc. Each C ₃ -C ₈ cycloalkyl includes a hydrocarbon ring containing 3, 4, 5, 6, 7 and 8 carbon atoms, or a range defined by any two numbers, such as C ₃ -C ₇ cycloalkyl or C ₅ -C ₆ cycloalkyl.

The term "alkyl" as used herein refers to a straight or branched hydrocarbon chain that is fully saturated (ie, does not contain double or triple bonds). An alkyl group may have from 1 to 20 carbon atoms (a numerical value such as "1 to 20" whenever it appears herein refers to each integer in the range, e.g. "1 to 20 carbon atoms " means that an alkyl group may 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 designation is given interval). The alkyl group may also be a medium-sized alkyl having from 1 to 9 carbon atoms. The alkyl group could also be lower alkyl having from 1 to 6 carbon atoms. An alkyl group may be designated "C ₁ -C ₄ alkyl" or similar designations. By way of example only, "C ₁ -C ₆ alkyl" indicates that the alkyl chain has from one to six carbon atoms, i.e. the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are not in any way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

The term "alkoxy" as used herein refers to the formula -OR in which R is alkyl as defined above, such as "C ₁ -C ₉ alkoxy", including methoxy, ethoxy , n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy and tert-butoxy, and the like, but not limited to.

The term "alkenyl" as used herein refers to a straight or branched hydrocarbon chain containing one or more double bonds. A given alkenyl group may have from 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term "alkenyl" where no numerical range is designated. The alkenyl group may also be a medium sized alkenyl group having from 2 to 9 carbon atoms. The alkenyl group could also be lower alkenyl, having from 2 to 6 carbon atoms. An alkenyl group may be designated "C ₂ -C ₆ alkenyl" or similar designations. By way of example only, “C ₂ -C ₆ alkenyl” indicates that the alkenyl chain has from two to six carbon atoms, i.e. the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4 -yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl , buta-1,2-dienyl and buta-1,2-dien-4-yl.

Typical alkenyl groups include, but are in no way limited to ethenyl, propenyl, butenyl, pentenyl and hexenyl, and the like.

The term "alkynyl" as used herein refers to a straight or branched hydrocarbon chain containing one or more triple bonds. A given alkynyl group may have from 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term "alkynyl" where no numerical range is indicated. The alkynyl group may also be a medium sized alkynyl group having from 2 to 9 carbon atoms. The alkynyl group could also be a lower alkynyl group having from 2 to 6 carbon atoms. An alkynyl group may be designated "C ₂ -C ₆ alkynyl" or similar designations. By way of example only, "C ₂ -C ₆ alkynyl" indicates that the alkynyl chain has from two to six carbon atoms, i.e. the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl and 2-butynyl. Typical alkynyl groups include, but are by no means limited to, ethynyl, propynyl, butynyl, pentynyl and hexynyl, and the like.

The term "heteroalkyl" as used herein refers to a straight or branched hydrocarbon chain containing one or more than one heteroatom, that is, a non-carbon element in the backbone of the chain including nitrogen, oxygen and sulfur, but not limited to them. A heteroalkyl group may have from 1 to 20 carbon atoms, although the present definition also covers the occurrence of the term "heteroalkyl" where no numerical range is designated. The heteroalkyl group may also be a medium-sized heteroalkyl having from 1 to 9 carbon atoms. This heteroalkyl group could also be a lower heteroalkyl having from 1 to 6 carbon atoms. This heteroalkyl group may be designated "C ₁ -C ₆ heteroalkyl" or similar designations. A given heteroalkyl group may contain one or more than one heteroatom. By way of example only, "C ₄ -C ₆ heteroalkyl" indicates that there are four to six carbon atoms in the heteroalkyl chain and additionally one or more than one heteroatom in the backbone of the chain.

The term "aromatic" refers to a ring or ring system having a system of conjugated pi electrons, and it includes both carbocyclic aromatic (eg phenyl) and heterocyclic aromatic groups (eg pyridine). This term includes a monocyclic group or a polycyclic group with a fused ring (i.e., rings that share adjacent pairs of atoms), provided that the entire ring system is aromatic.

The term "aryl" as used herein refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) containing only the carbon at the core of the ring. When aryl is a ring system, each ring in the system is aromatic. An aryl group may have from 6 to 18 carbon atoms, although the present definition also covers the occurrence of the term "aryl" where no numerical range is indicated. In some embodiments, the aryl group has from 6 to 10 carbon atoms. This aryl group may be designated "C ₆ -C ₁₀ aryl", "C ₆ or C ₁₀ aryl" or similar designations. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl.

"Aralkyl" or "arylalkyl" is an aryl group attached, as a substituent, through an alkylene group, such as "C _7-14 aralkyl" and the like, including benzyl, 2-phenylethyl, 3-phenylpropyl and naphthylalkyl, but not limited to them. In some cases, the alkylene group is a lower alkylene group (ie, a C ₁ -C ₆ alkylene group).

The term "heteroaryl" as used herein refers to an aromatic ring or ring system (i.e., two or more fused rings that have two adjacent atoms) which contains ( zhat) one or more than one heteroatom, that is, to an element other than carbon in the backbone of the ring, including, but not limited to, nitrogen, oxygen and sulfur. When heteroaryl is a ring system, each ring in the system is aromatic. A heteroaryl group may have 5-18 ring members (ie, the number of atoms constituting the backbone of the ring, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heteroaryl” where no numerical range is indicated. In some embodiments, the heteroaryl group has from 5 to 10 ring members or from 5 to 7 ring members. A heteroaryl group may be designated "5-7 membered heteroaryl", "5-10 membered heteroaryl" or similar designations. Examples of heteroaryl rings include furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl and benzothienyl, but are not limited to them.

"Heteroaralkyl" or "heteroarylalkyl" is a heteroaryl group linked, as a substituent, through an alkylene group. Examples include 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (ie, a C ₁ -C ₆ alkylene group).

The term "carbocyclyl" as used herein means a non-aromatic cyclic ring or ring system containing only carbon atoms in the backbone of the ring system. When the carbocyclyl is a ring system, two or more than two rings may be linked to each other in a fused, bridged or spiro-linked manner. Carbocyclyls can have any degree of saturation, as long as at least one ring in the ring system is non-aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls and cycloalkynyls. A carbocyclyl group may have from 3 to 20 carbon atoms, although the present definition also covers the occurrence of the term "carbocyclyl" where no numerical range is indicated. The carbocyclyl group may also be a medium sized carbocyclyl having from 3 to 10 carbon atoms. The carbocyclyl group may also be a carbocyclyl group having from 3 to 6 carbon atoms. A carbocyclyl group may be designated "C ₃ -C ₆ carbocyclyl" or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycl[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

The term "cycloalkyl" as used herein means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term “heterocyclyl” as used herein means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the core of the ring. Heterocyclyls can be linked to each other in a fused, bridged or spiro-linked manner. Heterocyclyls can have any degree of saturation, as long as at least one ring in the ring system is non-aromatic. The heteroatom(s) may be present on either a non-aromatic or an aromatic ring in a ring system. A heterocyclic group may have from 3 to 20 ring members (ie, the number of atoms that make up the core of the ring, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heterocyclyl” where no numerical range is indicated. The heterocyclyl group may also be a medium-sized heterocyclyl group having from 3 to 10 ring members. This heterocyclyl group could also be a heterocyclyl having 3 to 6 ring members. This heterocyclyl group may be designated "3-6 membered heterocyclyl" or similar designations. In preferred six-membered monocyclic heterocyclyls, the heteroatom(s) are selected from one to three O, N or S, and in preferred five-membered monocyclic heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O, N or S Examples of heterocyclyl rings include azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, tiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3 -dioxynyl, 1,3-dioxanyl, 1,4-dioxynyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro -1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3 -oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl and tetrahydroquinoline.

The "O-carboxy" group refers to the group "-OC(=O)R", in which R is selected from hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ - C ₇ carbocyclyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl as defined herein.

The "C-carboxy" group refers to the group "-C(=O)OR", in which R is selected from the group consisting of hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl , C ₃ -C ₇ carbocyclyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl, as defined herein. A non-limiting example includes carboxyl (ie -C(=O)OH).

A "sulfonyl" group refers to the group "-SO ₂ R" in which R is selected from hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ carbocyclyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl as defined herein.

The “sulfino” group refers to the “-S(=O)OH” group.

The "S-sulfonamido" group refers to the group "-SO ₂ NR _A R _B ", in which each of R _A and _RB is independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ - C ₆ alkynyl, C ₃ -C ₇ carbocyclyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl, as defined herein.

The "N-sulfonamido" group refers to the group "-N( _RA )SO ₂ R _b ", in which each of _RA and _RB is independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ carbocyclyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl, as defined herein.

The "C-amido" group refers to the group "-C(=O)NR _A R _B ", in which each of R _A and R _B is independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ carbocyclyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl, as defined herein.

An "N-amido" group refers to the group "-N(R _A )C(=O)R _B ", in which each of R _A and R _B is independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ carbocyclyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl, as defined herein.

"Amino group" refers to the group "-NR _A R _B ", in which each of R _A and R _B is independently selected from hydrogen, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ carbocyclyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl and 3-10 membered heterocyclyl, as defined herein. A non-limiting example includes free amino (ie -NH ₂ ).

An "aminoalkyl" group refers to an amino group attached via an alkylene group.

An "alkoxyalkyl" group refers to an alkoxy group attached through an alkylene group, such as "C ₂ -C ₈ alkoxyalkyl" and the like.

A substituted group, as the term is used herein, is derived from an unsubstituted parent group in which one or more hydrogen atoms have been replaced by another atom or group. Unless otherwise indicated, when a group is considered "substituted", it is meant that the group is substituted with one or more substituents independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, C ₁ -C ₆ heteroalkyl, C ₃ -C ₇ carbocyclyl (possibly substituted with halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy), C ₃ -C ₇ carbocyclyl-C ₁ -C ₆ -alkyl (possibly substituted with halogen-, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy), 3- 10-membered heterocyclyl (possibly substituted with halogen-, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy), 3-10-membered heterocyclyl-C ₁ - C ₆ -alkyl (possibly substituted with halogen-, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy), aryl (possibly substituted with halogen-, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy), aryl(C ₁ -C ₆ )alkyl (possibly substituted with halogen, C ₁ -C ₆ alkyl, C ₁ - C ₆ alkoxy, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy), 5-10 membered heteroaryl (possibly substituted with halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy), 5-10-membered heteroaryl (C ₁ -C ₆ )alkyl (possibly substituted with halogen-, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkyl and C ₁ -C ₆ haloalkoxy), halogen-, -CN, hydroxy, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkoxy (C ₁ -C ₆ )alkyl (i.e. ether), aryloxy, sulfhydryl (mercapto), halogen(C ₁ -C ₆ )alkyl (for example, -CF ₃ ), halogenC ₁ -C ₆ )alkoxy (for example, -OCF ₃ ), C ₁ -C ₆ alkylthio, arylthio, amino, amino(C ₁ -C ₆ )alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C- carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, -SO ₃ H, sulfin, -OSO ₂ C1-4alkyl and oxo (=O). In all cases, if a group is described as "optionally substituted", the group may be substituted with the substituents listed above.

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

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

The term "azido" as used herein refers to the -N ₃ group.

The term "nucleotide" as used herein 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, the sugar is ribose, and in DNA it is deoxyribose, i.e. a sugar lacking the hydroxyl group found in ribose. The nitrogen-containing heterocyclic base may be a purine or pyrimidine base. Purine bases include adenine (A) and guanine (G), and their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and their modified derivatives or analogs. The C-1 atom of deoxyribose binds to N-1 of pyrimidine or N-9 of purine.

A "nucleoside" as the term is used herein is structurally similar to a nucleotide but lacks the phosphate moieties. An example of a nucleoside analog would be one in which a tag is bound to the base and there is no phosphate group attached to the sugar molecule. The term "nucleoside" is used herein in its original sense, as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside containing a ribose moiety and a deoxyribonucleoside containing a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which the oxygen atom has been replaced by carbon, and/or the carbon has been replaced by sulfur or an oxygen atom. A "nucleoside" is a monomer that has a substituted base and/or sugar moiety. Additionally, the nucleoside may be included in large DNA and/or RNA polymers and oligomers.

The term “purine base” is used herein in its original sense, as understood by those skilled in the art, and includes its tautomers. Likewise, the term “pyrimidine base” is used herein in its original sense, as understood by those skilled in the art, and includes tautomers thereof. A non-limiting list of possibly substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (eg, 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 (eg, 5-methylcytosine).

When an oligonucleotide or polynucleotide, as these terms are used herein, is described as “containing” a nucleoside or nucleotide described herein, it means that the nucleoside or nucleotide described herein forms a covalent bond oligonucleotide or polynucleotide. Likewise, when describing a nucleoside or nucleotide as part of an oligonucleotide or polynucleotide, such as “incorporated into” an oligonucleotide or polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. In some such embodiments, a covalent bond is formed between the 3'-hydroxy group of the oligonucleotide or polynucleotide with the 5'-phosphate group of the nucleotide described herein, in the form of a phosphodiester bond between the 3' carbon atom of the oligonucleotide or polynucleotide and the 5' carbon atom of the nucleotide.

The term “derivative” or “analogue” as used herein means a synthetic nucleotide or nucleoside derivative having modified base moieties and/or modified sugar moieties. 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 may also contain modified phosphodiester bonds, including phosphorothioate, phosphorodithioate , alkyl phosphonate,

phosphoranidate and phosphoramidate bonds. The terms “derivative,” “analog,” and “modified,” as used herein, may be used interchangeably and are encompassed by the terms “nucleotide” and “nucleoside” as defined herein.

The term “phosphate” as used herein is used in its ordinary sense as understood by those skilled in the art and includes its protonated forms (e.g. ). The terms "monophosphate", "diphosphate" and "triphosphate" as used herein are used in their ordinary sense as understood by those skilled in the art and include the protonated forms.

The terms “protecting group” and “protecting groups” as used herein refer to any atom or group of atoms that is added to a molecule to prevent unwanted chemical reactions of existing groups in the molecule. Sometimes the terms "protecting group" and "blocking group" may be used interchangeably.

The prefixes “photo” or “photo-” as used herein refer to light or electromagnetic radiation. The term may cover all or part of the electromagnetic spectrum, including, but not limited to, one or more than one range commonly known as the radio, microwave, infrared, visible, ultraviolet, x-ray or gamma ray portions of the spectrum. This portion of the spectrum may be a portion of the spectrum that is blocked by a metallic region of the surface, such as those metals disclosed herein. Alternatively or additionally, this portion of the spectrum may be the portion of the spectrum that passes through an intermediate surface region, such as a region made of glass, plastic, silica, or other substance set forth herein. In particular embodiments, radiation that is capable of passing through metal may be used. Alternatively or additionally, radiation may be used that is masked by glass, plastic, silica, or other substance set forth herein.

The term "phasing" as used herein refers to a phenomenon in SBS that is caused by incomplete removal of 3' terminators and fluorophores, and failure to complete the incorporation of a portion of DNA strands into clusters by polymerases in a given sequencing run. Prephasing is caused by the incorporation of nucleotides without effective 3' terminators, where the incorporation event occurs 1 cycle earlier due to termination failure. Phasing and prephasing cause the measured signal intensities for a particular cycle to consist of the signal from the current cycle as well as noise from the preceding and subsequent cycles. As the number of cycles increases, the proportion of sequences per cluster that undergo phasing and prephasing increases, making identification of the correct base difficult. Prephasing can be caused by the presence of trace amounts of exposed or unblocked 3'-OH nucleotides during sequencing by synthesis (SBS). Unprotected 3'-OH nucleotides could be generated during manufacturing processes or possibly during storage and reagent handling processes. Accordingly, the discovery of nucleotide analogs that reduce the frequency of prephasing is unexpected and provides a major advantage in SBS applications over existing nucleotide analogs. For example, the provided nucleotide analogues can result in shorter SBS cycle times, lower phasing and prephasing values, and longer sequencing read lengths.

3'-Hydroxyacetal blocking groups

Some embodiments of the present disclosure provide a nucleotide or nucleoside molecule containing ribose or deoxyribose having a 3'-OH leaving protecting or blocking group forming the structure covalently attached to the 3' carbon atom, in which: each R ^1a and R ^1b independently represents H, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy , cyano, halogen, optionally substituted phenyl or optionally substituted aralkyl;

each R ^2a and R ^2b independently represents H, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, cyano or halogen;

alternatively, R ^1a and R ^2a , along with the atoms to which they are attached, form an optionally substituted five to eight membered heterocyclyl group;

R ³ is H, optionally substituted C ₂ -C ₆ alkenyl, optionally substituted C ₃ -C ₇ cycloalkenyl, optionally substituted C ₂ -C ₆ alkynyl or optionally substituted (C ₁ -C ₆ alkylene)Si(R ⁴ ) ₃ ; And

each R ⁴ independently represents H, C ₁ -C ₆ alkyl or optionally substituted C ₆ -C ₁₀ aryl with the proviso that when R ^1a , R ^1b , R ^2a and R ^2b are each H, then R ³ is not H .

Some other embodiments of the present disclosure relate to a compound having the structure of Formula (I):

wherein R' is H, monophosphate, diphosphate, triphosphate, thiophosphate, phosphate ester analogue, -O- attached to a reactive phosphorus-containing group, or -O- protected by a protecting group; R" represents H or OH; B represents a nucleic base; each of R ^1a , R ^1b , R ^2a , R ^2b and R ³ is defined above.

In some other embodiment, B represents is In some other embodiments, the nucleic base is covalently linked to a detectable label (eg, fluorescent dye (Dye)), optionally via a linker, for example, B is In some such embodiments, R' is a triphosphate. In some such embodiment, R" is H.

In some embodiments of the acetal blocking group described herein, at least one of R ^1a and R ^1b is H. In some such embodiments, R ^1a and R ^1b are each H. In some other embodiments, at least one of R ^1a and R ^1b is C ₁ -C ₆ alkyl, for example methyl, ethyl, isopropyl or tert-butyl. In some embodiments, R ^2a and R ^2b are each independently H, halogen, or C ₁ -C ₆ alkyl. In some embodiments, at least one of R ^2a and R ^2b is H or C ₁ -C ₆ alkyl. In some such embodiment, R ^2a and R ^2b are each H. In some such embodiment, R ^2a and R ^2b are each C ₁ -C ₆ alkyl, for example, methyl, ethyl, isopropyl or tert-butyl. In one embodiment, R ^2a and R ^2b are each methyl. In certain such embodiments, R ^2a and R ^2b are each independently C ₁ -C ₆ alkyl or halogen. In some such embodiments, R ^2a is H and R ^2b is halogen or C ₁ -C ₆ alkyl.

In some embodiments of the acetal blocking group described herein, R ³ is optionally substituted with C ₂ -C ₆ alkenyl. In some such embodiments, R ³ is C ₂ -C ₆ alkenyl (eg, vinyl, propenyl), optionally substituted with one or more substituents independently selected from the group consisting of halogen, C ₁ -C ₆ alkyl, C ₁ - With ₆ haloalkyl and their combinations. In some other embodiments, R ³ is

In some other embodiments, R ³ is optionally substituted with C ₂ -C ₆ alkynyl. In certain such embodiments, R ³ is C ₂ -C ₆ alkynyl (eg, ethynyl, propynyl), optionally substituted with one or more substituents independently selected from the group consisting of halogen, C ₁ -C ₆ alkyl, C ₁ - With ₆ haloalkyl and their combinations. In one embodiment, R ³ is optionally substituted with ethynyl In some other embodiments, R ³ is optionally substituted with (C ₁ -C ₆ alkylene)Si(R ⁴ ) ₃ . In some such embodiments, at least one of R ⁴ is C _1-4 alkyl. In some other embodiments, each one of R ⁴ is C ₁ -C ₄ alkyl, for example, methyl, ethyl, isopropyl or tert-butyl. In one embodiment, R ³ is -(CH ₂ )-SiMe ₃ . In some alternative embodiments, R ³ represents C ₁ -C ₆ alkyl.

In some alternative embodiments, R ^1a and R ^2a together with the atoms to which they are attached form a five- to seven-membered heterocyclyl. In some such embodiments, R ^1a and R ^2a together with the atoms to which they are attached form a six-membered heterocyclyl. In some such embodiments, the six-membered heterocyclyl group has the structure In certain other embodiments, at least one of each R ^1b , R ^2b , and R ³ is H. In certain other embodiments, at least one of each R ^1b , R ^2b , and R ³ is C ₁ -C ₆ alkyl. In one embodiment, R ^1b , R ^2b and R ³ are each H.

In certain other embodiments, the compound of Formula (I) is also represented by Formula (Ia):

wherein each R ^2c and R ^2d independently represents H, halogen (eg fluoro-, chloro-), C ₁ -C ₆ alkyl (eg methyl, ethyl or isopropyl) or C ₁ -C ₆ haloalkyl (eg -CHF ₂ , -CH ₂ F or -CF ₃ ). In some such embodiments, one of R ^1a and R ^1b is H. In some such embodiments, each of R ^1a and R ^1b is H. In some other embodiments, at least one of R ^1a and R ^1b is C ₁ -C ₆ alkyl, for example methyl, ethyl, isopropyl or tert-butyl. In some embodiments, R ^2a and R ^2b are each independently H, halogen, or C ₁ -C ₆ alkyl. In some such embodiment, R ^2a and R ^2b are each H. In some such embodiments, R ^2c and R ^2d are each independently H, halogen, or C ₁ -C ₆ alkyl. In some such embodiments, R ^2c and R ^2d are each C ₁ -C ₆ alkyl, for example, methyl, ethyl, isopropyl or tert-butyl. In one embodiment, R ^2c and R ^2d are each methyl. In some such embodiments, R ^2c and R ^2d are each independently halogen. In some such embodiments, R ^2c is H, and R ^2d is H, halogen (fluoro-, chloro-) or C ₁ -C ₆ alkyl (eg, methyl, ethyl, isopropyl or tert-butyl). In other embodiments, R ^1a and R ^1b are each H; R ^2a represents H; R ^2b is H, halogen or methyl; R ^2c represents H; and R ^2d is H, halogen, methyl, ethyl, isopropyl or tert-butyl.

Non-limiting embodiments of the blocking groups described herein include groups having a structure selected from the group consisting of: covalently attached to the 3' carbon of ribose or deoxyribose.

3'-Hydroxythiocarbamate blocking groups

Some further embodiments of the present disclosure relate to a nucleoside or nucleotide containing ribose or deoxyribose having a 3'-OH leaving blocking group forming the structure covalently attached to the 3' carbon atom, in which:

each of R ⁵ and R ⁶ independently represents H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₂ -C ₈ alkoxyalkyl, optionally substituted - (CH ₂ ) _m -phenyl, possibly substituted

-(CH ₂ ) _n -(5 or 6 membered heteroaryl), optionally substituted-(CH ₂ ) _k -C ₃ -C ₇ carbocyclyl or optionally substituted-(CH ₂ ) _p -(3-7 membered heterocyclyl);

alternatively, R ⁵ and R ⁶ , together with the atoms to which they are attached, form an optionally substituted five- to seven-membered heterocyclyl;

each of -(CH ₂ ) _m -, -(CH ₂ ) _n -, -(CH ₂ ) _k - and -(CH ₂ ) _p - is optionally substituted; And

each of m, n, k and p is independently equal to 0, 1, 2, 3 or 4.

Some additional embodiments relate to the compound of Formula (II):

wherein R' is H, monophosphate, diphosphate, triphosphate, thiophosphate, phosphate ester analogue, -O- attached to a reactive phosphorus-containing group, or -O- protected by a protecting group; R" is H or OH; B is a nucleic acid base; ^R5 and ^R6 are each as defined above. In some other embodiment, B is

In some other embodiments, the nucleic base is covalently linked to a detectable label (eg, a fluorescent dye), possibly through a linker, for example, B is In some such embodiments, R' is a triphosphate. In some such embodiment, R" is H.

In some embodiments of the thiocarbamate blocking group described herein, at least one of R ⁵ and R ⁶ is H. In some such embodiments, R ⁵ and R ⁶ are each H. In some such embodiments, R ⁵ is H. and R ⁶ is C ₁ -C ₆ alkyl, for example methyl, ethyl, isopropyl or tert-butyl. In some such embodiments, R ⁵ is H and R ⁶ is C ₂ -C ₆ alkenyl (eg, vinyl or allyl) or C ₂ -C ₆ alkynyl (eg, ethynyl or propynyl). In some such embodiments, R ⁵ is H, and R ² is optionally substituted -(CH ₂ ) _m -phenyl, optionally substituted -(CH ₂ ) _n -(5- or 6-membered heteroaryl), optionally substituted -(CH ₂ ) _k -C ₃ -C ₇ carbocyclyl or possibly substituted -(CH ₂ ) _p -(3-7-membered heterocyclyl). In some other embodiment, the C ₃ -C ₇ carbocyclyl group may be C ₃ -C ₇ cycloalkyl or C ₃ -C ₇ cycloalkenyl. A 3-7 membered heterocyclyl group may contain zero or one double bond in the ring structure. In other embodiments, R ⁵ is H and R ⁶ is optionally substituted (CH ₂ ) _m -phenyl, optionally substituted -(CH ₂ ) _n -6-membered heteroaryl, optionally substituted -(CH ₂ ) _k -C ₅ or C ₆ carbocyclyl, or possibly substituted -(CH ₂ ) _p -(5- or 6-membered heterocyclyl). In some embodiments, m, n, k, or p is 0. In other embodiments, m, n, k, or p is 1 or 2. In some other embodiments, at least one of R ⁵ and R ⁶ is C ₁ -C ₆ alkyl eg methyl, ethyl, isopropyl or tert-butyl. In some other embodiments, both R ⁵ and R ⁶ are C ₁ -C ₆ alkyl. In one embodiment, both R ⁵ and R ⁶ are methyl.

In some alternative embodiments, R ⁵ and R ⁶ together with the atoms to which they are attached form an optionally substituted five- to seven-membered heterocyclyl. In some such embodiments, R ⁵ and R ⁶ together with the atoms to which they are attached form an optionally substituted piperidinyl.

Non-limiting embodiments of the 3'-O-thiocarbamate blocking groups described herein, including embodiments having a structure selected from the group consisting of covalently attached to the 3' carbon of ribose or deoxyribose.

Additional embodiments of the present disclosure relate to an oligonucleotide or polynucleotide containing a nucleoside or nucleotide described herein.

In any of the embodiments of the blocking groups described herein, when a group is described as “optionally substituted,” it may be either unsubstituted or substituted.

In any embodiments of nucleotides or nucleosides with a 3' hydroxy blocking group described herein, the nucleoside or nucleotide can be covalently attached to a detectable label (eg, a fluorophore), possibly through a linker. This linker may be cleavable or non-cleavable. In some such embodiments, a detectable label (eg, a fluorophore) is covalently attached to the nucleic acid base of the nucleoside or nucleotide through a cleavable linker. In some other embodiments, a detectable label (eg, a fluorophore) is covalently attached to the 3' oxygen of the nucleoside or nucleotide through a cleavable linker. In some other embodiments, such a cleavable linker may contain an azide moiety or a disulfide moiety, an acetal moiety, or a thiocarbamate moiety. In some embodiments, the 3' hydroxy blocking group and the cleavable linker (and attached tag) can be removed under the same or substantially the same chemical reaction conditions, for example, the blocking group and the detectable tag can be removed in a single chemical reaction. In other embodiments, the blocking group and the detectable label are removed in two separate steps.

In some embodiments, the nucleotides or nucleosides described herein contain 2'-deoxyribose. In some other aspects, 2'-deoxyribose contains one, two, or three phosphate groups at the 5' position of the sugar ring. In another aspect, the nucleotides described herein are nucleotide triphosphate.

In some embodiments, the 3' blocked nucleotides or nucleosides described herein provide superior stability in solution during storage or reagent handling during sequencing applications compared to the same nucleotides or nucleosides protected by the standard 3'-OH blocking group disclosed in the prior art, for example, a 3'-O-azidomethyl protecting group. For example, acetal or thiocarbamate blocking groups disclosed herein can impart at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 %, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500% or 3000% improved stability compared to azidomethyl protected 3'- OH under the same conditions for the same period of time, thereby reducing prephasing values and leading to longer read lengths during sequencing. In some embodiments, stability is measured at ambient temperature or at a temperature below ambient temperature (such as 4-10°C). In other embodiments, stability is measured at elevated temperature, such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, stability is measured in solution at a basic pH environment, for example, pH 9.0; 9.2; 9.4; 9.6; 9.8 or 10.0. In some such embodiments, stability is measured with or without the presence of an enzyme, such as a polymerase (eg, DNA polymerase), terminal deoxynucleotidyl transferase, or reverse transcriptase.

In some embodiments, the 3' blocked nucleotides or nucleosides described herein provide superior release rates in solution during the chemical digestion step of sequencing applications compared to the same nucleotides or nucleosides protected by the standard 3'-OH blocking group disclosed in the preceding prior art, for example, a 3'-O-azidomethyl protecting group. For example, acetal or thiocarbamate blocking groups disclosed herein can impart at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 %, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500% or 2000% improved release rate compared to azidomethyl protected 3'-OH with using a standard deblocking reagent (such as tris(hydroxypropyl)phosphine), thereby reducing the overall time for a sequencing run. In some embodiments, the release rate is measured at ambient or sub-ambient temperature (such as 4-10°C). In other embodiments, the release rate is measured at an elevated temperature, such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, the deblocking rate is measured in solution in a basic pH environment, for example, pH 9.0; 9.2; 9.4; 9.6; 9.8 or 10.0. In some such embodiments, the molar ratio of deblocking reagent to substrate (ie, 3' blocked nucleoside or nucleotide) is about 10:1, about 5:1, about 2:1, or about 1:1.

In some embodiments, a palladium deblocking reagent (eg, Pd(0) is used to remove 3' acetal blocking groups (eg, an AOM blocking group)). Pd can form a chelated complex with the two oxygen atoms of the AOM group as well as the double bond of the allyl group, allowing removal of the deblocking reagent in close proximity to the functional group, and can result in an increased rate of deblocking.

Deprotection of 3'-OH blocking groups

The 3'-acetal blocking groups described herein can be removed or cleaved under a variety of chemical conditions. For acetal blocking groups which contain a vinyl or alkenyl moiety, the non-limiting cleavage condition includes a Pd(II) complex, such as Pd(OAc) ₂ or an allylPd(II) chloride dimer, in the presence of a phosphine ligand, such as tris(hydroxymethyl)phosphine (THMP) or tris( hydroxypropyl)phosphine (THP or THPP). For those blocking groups that contain an alkynyl group (eg ethynyl), they can also be removed by a Pd(II) complex (eg Pd(OAc) ₂ or allylPd(II) chloride dimer) in the presence of a phosphine ligand (eg THP or TNMR).

Palladium decomposition reagents

In some embodiments, the acetal blocking group described herein can be cleaved with a palladium catalyst. In some such embodiments, the Pd catalyst is water soluble. In some such embodiments, it is a Pd(0) complex (e.g., tris(3,3',3"-phosphinidintris(benzenesulfonato)palladium(0) nonasodium salt). In some cases, the Pd(0) complex can be generated in in situ due to the reduction of the Pd(II) complex by reagents such as alkenes, alcohols, amines, phosphines or metal hydrides. Suitable sources of palladium include Na ₂ PdCl ₄ , Pd(CH ₃ CN) ₂ Cl _2: (PdCl(C ₃ H ₅ )) ₂ , [Pd(C ₃ H ₅ )(THP)]Cl, [Pd(C ₃ H ₅ )(THP) ₂ ]Cl, Pd(OAc) ₂ , Pd(Ph ₃ ) ₄ , Pd( dba) ₂ , Pd(Acac) ₂ , PdCl ₂ (COD), and Pd(TFA) ₂ . In one such embodiment, the Pd(0) complex is generated in situ from Na ₂ PdCl ₄ . In another embodiment, the palladium source is an allyl palladium chloride dimer. (II) [(PdCl(C ₃ H ₅ )) ₂ ] In some embodiments, the Pd(0) complex is generated in aqueous solution by mixing the Pd(II) complex with a phosphine. Suitable phosphines include water-soluble phosphines such as tris(hydroxypropyl) phosphine (THP), tris(hydroxymethyl)phosphine (THMP), 1,3,5-triaza-7-phosphadamantane (PTA), bis(p-sulfonatophenyl)phenylphosphine potassium salt, tris(carboxyethyl)phosphine (TCEP) and trisodium salt triphenylphosphine-3,3',3"-trisulfonic acid.

In some embodiments, Pd(0) is prepared by mixing the Pd(II) complex [(PdCI(C ₃ H ₅ )) ₂ ] with THP in situ. The molar ratio of Pd(II) complex to THP can be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In some other embodiments, one or more reducing agents, such as ascorbic acid or a salt thereof (eg, sodium ascorbate), may be added. In some embodiments, the cleavage mixture may contain additional buffering reagents, such as a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In some other embodiments, the buffering reagent contains ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylaminomethanol (DMEA), 2-diethylaminomethanol (DEEA), N,N, N',N'-tetramethylethylenediamine (TEMED) or N,N,N',N'-tetraethylethylenediamine (TEEDA), or combinations thereof. In one embodiment, the buffering reagent is DEEA. In another embodiment, the buffering reagent contains one or more inorganic salts, such as a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In one embodiment, the inorganic salt is a sodium salt.

Alternatively, an alkynyl moiety containing blocking groups can also be cleaved in the presence of (NH ₄ ) ₂ MoS ₄ . Another non-limiting cleavage condition for the alkynyl moiety involves complexing Cu(II) with the ligand THPTA (tris(3-hydroxypropyltriazolylmethyl)amine), and ascorbate. Non-limiting cleavage conditions for blocking groups containing a six-membered heterocycle (eg, tetrahydropyran) include cyclodextrin or Ln(OTf) ₃ (lanthanide triflate). A non-limiting cleavage condition for blocking groups containing an alkylsilane group (eg, -CH ₂ SiMe ₃ ) includes LiBF ₄ (lithium tetrafluoroborate). Other acetal blocking groups, such as -O(CH ₂ )OC ₁ -C ₆ alkyl, can be removed by LiBF ₄ or Bi(OTf) ₃ (bismuth triflate). Non-limiting typical conditions for the cleavage of the various blocking groups described are illustrated in Scheme 1 below.

Scheme 1. Illustration of 3'-deblocking conditions (Dye + Linker - dye + linker)

The 3'-thiocarbamate blocking groups described herein can be removed or cleaved under a variety of chemical conditions. Non-limiting typical conditions for the cleavage of thiocarbamate blocking groups described herein include NaIO ₄ and Oxone® (potassium peroxymonosulfate).

In addition, the azido group in -CH ₂ N ₃ can be converted to an amino group via phosphine. Alternatively, the azido group in -CH ₂ N ₃ 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 THP.

Linearization compatible

In order to maximize the throughput of nucleic acid sequencing reactions, the ability to sequence multiple template molecules in parallel is useful. Parallel processing of many templates can be achieved using nucleic acid chip technology. These chips typically consist of a high-density array of polynucleotides immobilized on a solid support material.

Both WO 98/44151 and WO 00/18957 describe nucleic acid amplification methods that immobilize the amplification products on a solid support to form chips consisting of clusters or “colonies” formed from multiple identical immobilized polynucleotide chains and multiple identical immobilized complementary chains. This type of chip is referred to in this document as “cluster chips.” Nucleic acid molecules present in DNA colonies on cluster chips obtained according to these methods can provide templates for sequencing reactions, for example, as described in WO 98/44151. The products of solid phase amplification reactions, such as those described in WO 98/44151 and WO 00/18957, are so-called “bridge” structures formed by annealing pairs of immobilized polynucleotide chains and immobilized complementary strands, both types of chains being attached to a solid support on 5' end. In order to provide more suitable templates for nucleic acid sequencing, it is preferred to remove substantially all or at least a portion of one of the immobilized strands in the bridge structure in order to obtain a template that is at least partially single-stranded. The portion of the template that is single-stranded will thus be available for hybridization with the sequencing primer. The process of removing all or part of one immobilized strand in a bridged double-stranded nucleic acid structure is called “linearization.” There are various linearization methods including, but not limited to, enzymatic digestion, photochemical digestion, or chemical digestion. Non-limiting examples of linearization methods are disclosed in PCT Publication No. WO 2007/010251, US Patent Publication No. 2009/0088327, US Patent Publication No. 2009/0118128, and US Patent Application No. 62/671816, which are incorporated by reference in their entirety.

In some embodiments, the condition for deprotection or removal of 3'-OH blocking groups is also compatible with linearization processes. In some other embodiments, the deprotection condition is compatible with a chemical linearization process that involves the use of a complex of Pd and a phosphine, for example, Pd(OAc) ₂ and THP. In some embodiments, the Pd complex is a Pd(II) complex that generates Pd(0) in situ in the presence of phosphine.

Unless otherwise stated, reference to nucleotides is also intended to apply to nucleosides.

Labeled nucleotides

According to one aspect of this disclosure, the described 3'-OH blocked nucleotide also contains a detectable label, and such nucleotide is called a labeled nucleotide. A label (eg, a fluorescent dye) can be conjugated through a possible linker through a variety of methods, including hydrophobic attraction, ionic attraction, and covalent attachment. In some aspects, dyes are conjugated to a substrate through covalent attachment. More specifically, covalent attachment is accomplished through a linker group. In some cases, such labeled nucleotides are also called "modified nucleotides".

Labeled nucleosides and nucleotides are useful for labeling polynucleotides formed by enzymatic synthesis, such as, by way of non-limiting example, in PCR amplification, isothermal amplification, solid-phase amplification, polynucleotide sequencing (e.g., solid-phase sequencing), nick-translation reactions, and the like .

In some embodiments, the dye may be covalently attached to oligonucleotides or nucleotides via a nucleotide base. For example, a labeled nucleotide or oligonucleotide may have a label attached to the C5 position of a pyrimidine base or to the C7 position of a 7-deazapurine base via a linker moiety.

Unless otherwise indicated, reference to nucleotides is also intended to apply to nucleosides. The present application will also be further described with reference to DNA, although this description will also apply to RNA, PNA (peptide nucleic acid) and other nucleic acids unless otherwise noted.

Linkers

In some embodiments described herein, the purine or pyrimidine base of the nucleotide or nucleoside molecules described herein may be linked to a detectable label as described above. In some such embodiments, the linkers used are cleavable. The use of cleavable linkers ensures that the label can, if desired, be removed after detection, avoiding any interfering signal with any labeled nucleotide or nucleoside included below. In some embodiments, the cleavable linker contains an azide moiety, an -O-C ₂ -C ₆ alkenyl moiety (e.g., -O-allyl), a disulfide moiety, an acetal moiety (same or similar to the 3'-acetal blocking group described herein) or a thiocarbamate moiety (same or similar to the 3'-acetal blocking group described herein).

In some other embodiments, the linkers used are non-cleavable. Because in each case where a labeled nucleotide of the invention is included, no nucleotides then need to be included, and thus no label needs to be removed from the given nucleotide.

Cleavable linkers are known in the art, and conventional chemistry can be used to attach the linker to the nucleotide base and tag. This linker can be cleaved by any suitable means, including exposure to acids, bases, nucleophiles, electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes, etc. The linker, as discussed herein, can also be cleaved by the same catalyst used to cleave the bond of the 3'-O-blocking group. Suitable linkers can be adapted from standard chemical protecting groups as disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Other suitable cleavable linkers used in solid phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100:2092-2157, 2000).

When a detectable tag is attached to a base, the linker can attach at any position on the nucleotide base as long as Watson-Crick base pairing can still occur. In the context of purine bases, it is preferred if the linker is attached via the 7-position of a purine or a preferred deazapurine analogue, via an 8-modified purine, via an N-6 modified adenosine or an N-2 modified guanine. For pyrimidines, the attachment is preferably via the 5-position on cytosine, thymidine or uracil and the N-4 position on cytosine.

In some embodiments, the linker may contain a spacer unit. The length of a given 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, such as a polymerase.

In some embodiments, the linker may consist of a similar functional group, such as a 3'-OH protecting group. This will make the deprotection and deprotection process more efficient since only one processing will be required to remove both the label and the protecting group.

The use of the term "cleavable linker" is not intended to imply that removal of the entire linker is required. The cleavage site may be located at a position on the linker that ensures that a portion of the linker remains attached to the dye and/or substrate moiety after cleavage. Cleavable linkers, by way of non-limiting example, can be electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavable under reducing conditions (e.g., disulfide or azide containing linkers), oxidative conditions, cleavable through the use of latch linkers, or cleavable through mechanisms elimination. The use of a cleavable linker to attach the dye compound to the substrate moiety ensures that the label, if required, can be removed after detection, avoiding any interfering signal in subsequent steps.

Useful linker groups can be found in PCT Publication No. WO 2004/018493 (incorporated herein by reference), examples of which include linkers that can be cleaved using water-soluble phosphines or water-soluble transition metal catalysts formed from a transition metal and at least partially water-soluble ligands, for example, the Pd(II) complex and THP. In aqueous solution, the latter form at least partially water-soluble transition metal complexes. Such cleavable linkers can be used to connect nucleotide bases to labels, such as the dyes set forth herein.

Specific linkers include those disclosed in PCT Publication No. WO 2004/018493 (incorporated herein by reference), such as linkers that include portions of the following formulas:

(where X is selected from the group containing O, S, NH and NQ, where Q is a C _1-10 substituted or unsubstituted alkyl group, Y is selected from the group containing O, S, NH and N(allyl), T is hydrogen or a C _1-10 substituted or unsubstituted alkyl group, and * indicates where the fragment is connected to the rest of the nucleotide or nucleoside). In some aspects, linkers connect nucleotide bases to tags, such as, for example, dye compounds described herein.

Additional examples of linkers include those disclosed in US Publication No. 2016/0040225 (incorporated herein by reference), such as linkers comprising portions of the following formulas:

The linker moieties illustrated herein may contain full or partial linker structure between nucleotides/nucleosides and tags.

Additional examples of linkers (“L”) include portions of the following formulas:

where B represents a nucleic base; Z represents -N ₃ (azido), -O-C ₁ -C ₆ alkyl, -O-C ₂ -C ₆ alkenyl or -O-C ₂ -C ₆ alkynyl; and FI contains a fluorescent label, which may contain an additional linker structure. One of ordinary skill in the art will appreciate that a label is covalently linked to a linker by reacting a functional group of the label (eg, carboxyl) with a functional group of the linker (eg, amino).

In specific embodiments, the length of the linker between the fluorescent dye (fluorophore) and the guanine base can be changed, for example, by introducing a polyethylene glycol spacer group, thereby increasing the fluorescence intensity compared to the same fluorophore attached to the guanine base through other linkages known in the art. areas. Typical linkers and their properties are set forth in PCT Publication No. WO 2007020457 (incorporated herein by reference). The design of the linkers, and especially their increased length, can provide improvements in the brightness of fluorophores attached to the guanine bases of guanosine nucleotides when incorporated into polynucleotides such as DNA. Thus, when the dye is intended for use in any assay that requires detection of a fluorescent dye tag attached to a guanine-containing nucleotide, it is useful if the linker contains a spacer group of the formula -((CH ₂ ) ₂ O) _n - where n is an integer from 2 to 50, as described in WO 2007/020457.

Nucleosides and nucleotides can be labeled at sites on the sugar or nucleic acid base. As is known in the art, a "nucleotide" consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA the sugar is ribose, and in DNA it is deoxyribose, i.e. a sugar that does not have a hydroxyl group, which is present in ribose. The nitrogenous base is a purine or pyrimidine derivative. Purines are adenine (A) and guanine (G), and pyrimidines are cytosine (C) and thymine (T) or, in the context of RNA, uracil (U). The C-1 atom of deoxyribose binds to N-1 of pyrimidine or N-9 of purine. A nucleotide is also a phosphate ester of a nucleoside with esterification occurring on the hydroxyl group attached to the C-3 or C-5 of the sugar. Nucleotides are usually mono-, di- or triphosphates.

A "nucleoside" is structurally similar to a nucleotide, but lacks the phosphate moieties. An example of a nucleoside analog would be one in which the label binds to the base and there is no phosphate group attached to the sugar molecule.

Although the base is commonly referred to as a purine or pyrimidine, one skilled in the art will appreciate that derivatives and analogs are available that do not alter the Watson-Crick base pairing ability of the nucleotide or nucleoside. "Derivative" or "analogue" means a compound or molecule whose core structure is the same or closely similar to the structure of the parent compound, but which has a chemical or physical modification, such as, for example, a different or additional side group that allows the nucleotide to be derived or nucleoside to bind to another molecule. For example, the base may be deazapurine. In specific embodiments, these derivatives must be capable of forming Watson-Crick pairs. "Derivative" and "analog" also include, for example, a synthetic derivative of a nucleotide or nucleoside having modified base moieties and/or modified sugar moieties. 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 may also contain modified phosphodiester linkages, including phosphorothioate, phosphorodithioate , alkylphosphonate, phosphoranilidate, phosphoramidate bonds and the like.

The dye can be attached at any position on the nucleotide base, for example through a linker. In certain embodiments, Watson-Crick base pairing can still be performed for the resulting analogue. Specific nucleobase labeling sites include the C5 position of the pyrimidine base or the C7 position of the 7-deazapurine base. As described above, a linker group can be used to covalently attach a dye to a nucleoside or nucleotide.

In specific embodiments, the labeled nucleoside or nucleotide may be enzymatically incorporated and enzymatically extended. Accordingly, the linker fragment may be of sufficient length to connect a nucleotide to a compound such that the connection does not significantly interfere with the overall binding and recognition of the nucleotide by the nucleic acid replication enzyme. Thus, this linker may also contain a spacer element. This spacer, for example, places a nucleotide base at a distance from the cleavage site or tag.

Nucleosides or nucleotides labeled with the dyes described herein may have the following formula:

wherein Dye is a dye compound, B is a nucleic acid base such as, for example, uracil, thymine, cytosine, adenine, guanine and the like; L represents a possible linker group which may or may not be present; R' may be H, monophosphate, diphosphate, triphosphate, thiophosphate, phosphate ester analog, -O- attached to a reactive phosphorus-containing group, or -O- protected by a blocking group; R'' may be H, OH, phosphoramidite, or a 3'-OH blocking group as described herein, and R" is H or OH. When R''' is phosphoramidite, R' is an acid-cleavable hydroxy a protecting group that ensures subsequent binding of the monomer under automatic synthesis conditions.

In a particular embodiment, both a linker (between the dye and the nucleotide) and a blocking group are present and are separate moieties. In certain embodiments, both the linker and the blocking group are cleavable under substantially similar conditions. Thus, the deprotection and deblocking methods may be more efficient since only one treatment will be required to remove both the dye compound and the blocking group. However, in some embodiments, the linker and blocking group do not need to be cleavable under similar conditions, but instead are individually cleavable under different conditions.

This disclosure also covers polynucleotides including dye compounds. Such polynucleotides may be DNA or RNA containing, respectively, deoxyribonucleotides or ribonucleotides linked in a phosphodiester bond. The polynucleotides may contain naturally occurring nucleotides, non-naturally occurring (or modified) nucleotides other than the labeled nucleotides described herein, or any combination thereof in combination with at least one modified nucleotide (e.g., a labeled dye compound) , as set forth in this document. The polynucleotides of this disclosure may also include non-natural backbone linkages and/or non-nucleotide chemical modifications. Chimeric structures consisting of mixtures of ribonucleotides and deoxyribonucleotides containing at least one labeled nucleotide are also contemplated.

Non-limiting exemplary labeled nucleotides as described herein include the following:

where L represents a linker and R represents a sugar residue as described above, or a sugar residue with the 5' position substituted by one, two or three phosphates.

In some embodiments, non-limiting typical fluorescent dye conjugates are shown below:

where PG refers to the 3' hydroxy blocking groups described herein. In any embodiments of the labeled nucleotide described herein, the nucleotide is a nucleotide triphosphate.

Sets

The present disclosure also provides kits comprising one or more 3' blocked nucleosides and/or nucleotides described herein, for example, a 3' blocked nucleotide of Formula (I), (Ia) or (II). Such kits will typically include at least one 3' blocked nucleotide or dye-labeled nucleoside, along with at least one additional component. The additional component(s) may be one or more than one component identified in the method set forth herein or in the Examples section below. Some non-limiting examples of components that may be combined in the present disclosure are set forth below.

In a particular embodiment, the kit may include at least one labeled 3' blocked nucleotide or nucleoside together with labeled or unlabeled nucleotides or nucleosides. For example, dye-labeled nucleotides may be provided in combination with unlabeled or naturally occurring nucleotides and/or fluorescently labeled nucleotides or any combination thereof. Combinations of nucleotides may be provided as separate individual components (eg, one type of nucleotide per vial or tube) or as mixtures of nucleotides (eg, two or more than two nucleotides mixed in the same vial or tube).

When the arrays contain multiple, in particular two or three or more, in particular four 3' blocked nucleotides labeled with a dye compound, different nucleotides may be labeled with different dye compounds, or one may be dark without dye compounds. When different nucleotides are labeled with different dye compounds, the characteristic of these kits is that the dye compounds are spectrally distinguishable fluorescent dyes. The term "spectrally detectable fluorescent dyes" as used herein refers to fluorescent dyes that emit fluorescent energy at wavelengths that can be detected by fluorescence detection equipment (e.g., a commercially available DNA sequencing platform on capillary base) in the presence of two or more than two such dyes in one sample. When two nucleotides labeled with fluorescent dye compounds are supplied as a set, it is a feature of some embodiments that the spectrally distinct fluorescent dyes can be excited at the same wavelength, such as by the same laser. When four 3' blocked nucleotides (A, C, T and G) labeled with fluorescent dye compounds are supplied as a set, a characteristic of some embodiments is that two of the spectrally distinguishable fluorescent dyes can both be excited at the same wavelength, and the other two spectrally distinct dyes can both be excited at a different wavelength. The specific excitation wavelengths are 488 nm and 532 nm.

In one embodiment, the kit includes a first 3' blocked nucleotide labeled with a first dye and a second nucleotide labeled with a second dye, wherein the dyes have a difference in absorbance maximum of at least 10 nm, in particular from 20 nm to 50 nm. More specifically, the two dye compounds have Stokes shifts of 15 to 40 nm, where the “Stokes shift” is the distance between the wavelengths of the absorption peak and the emission peak.

In an alternative embodiment, the kits of this disclosure may contain 3'-blocked nucleotides, where the same base is labeled with two or more different dyes. A first nucleotide (eg, 3' blocked nucleotide triphosphate T or 3' blocked nucleotide triphosphate G) may be labeled with a first dye. The second nucleotide (e.g., 3' blocked nucleotide triphosphate C) may be labeled with a second dye that is spectrally different from the first dye, for example, a “green” dye that absorbs at less than 600 nm and a “blue” dye that absorbs at less than 500 nm , for example, at 400-500 nm, in particular 450-460 nm). The third nucleotide (e.g., 3' blocked nucleotide triphosphate A) may be labeled as a mixture of the first and second dyes or a mixture of the first, second and third dyes, and the fourth nucleotide (e.g., 3' blocked nucleotide triphosphate G or 3' blocked nucleotide triphosphate T) may be " dark" and does not contain a label. In one example, nucleotides 1-4 may be labeled blue, green, blue-green, and dark. To further simplify the equipment, four nucleotides can be labeled with two dyes excited by a single laser, and thus the labeling of nucleotides 1-4 can be blue 1, blue 2, blue 1/blue 2, and dark.

In specific embodiments, these kits may contain four labeled 3' blocked nucleotides (e.g., A, C, T, G), where each type of nucleotide contains the same 3' blocking group and a fluorescent label, and where each fluorescent label has a different fluorescence maximum, and each of the fluorescent marks is different from the other three marks. These arrays may be such that two or more than two fluorescent labels have a similar absorption maximum but a different Stokes shift. In some other embodiments, one type of nucleotide is unlabeled.

Although the sets are exemplified herein with respect to configurations having different nucleotides that are labeled with different dye compounds, it will be appreciated that the sets may include 2, 3, 4 or more different nucleotides that have the same dye compound. In some embodiments, the kit also includes an enzyme and a buffer suitable for the action of the enzyme. In some such embodiments, the enzyme is a polymerase, terminal deoxynucleotidyl transferase, or reverse transcriptase. In specific embodiments, the enzyme is a DNA polymerase, such as DNA polymerase 812 (Pol 812) or DNA polymerase 1901 (Pol 1901). The amino acid sequences of the polymerases Pol 812 and Pol 1901 are described, for example, in US patent applications No. 16/670876, filed October 31, 2019, and 16/703569, filed December 4, 2019, both of which are incorporated herein by reference.

Other components to be included in such kits may include buffers and the like. The nucleotides of the present disclosure and any other nucleotide components, including mixtures of different nucleotides, may be provided in this kit in concentrated form for dilution before use. In such embodiments, a suitable dilution buffer may also be included. Again, one or more than one components identified in the method set forth herein may be included in the kit of the present disclosure.

Sequencing methods

The labeled nucleotides or nucleosides of the present disclosure can be used in any assay method, such as a method that involves detecting a fluorescent label attached to the nucleotide or nucleoside, whether alone or included in or associated with a larger molecular structure or conjugate. As used herein, the term "embedded in a polynucleotide" may mean that the 5'-phosphate is linked in a phosphodiester bond to the 3'-OH group of a second (modified or unmodified) nucleotide, which itself may form part of a longer polynucleotide chain. The 3' end of a nucleotide set forth herein may or may not be linked in a phosphodiester bond to the 5'-phosphate of another (modified or unmodified) nucleotide. Thus, in one non-limiting embodiment according to this disclosure, there is provided a method for detecting a nucleotide included in a polynucleotide, which includes: (a) including at least one nucleotide of the disclosure in the polynucleotide and (b) identifying the nucleotide(s) included in a given polynucleotide by detecting a fluorescent signal from a dye compound attached to said nucleotide(s).

The method may include: a synthesis step (a), in which one or more than one nucleotide according to this disclosure is included in a polynucleotide, and a detection step (b), in which one or more than one nucleotide included in the polynucleotide is detected by detection or quantitative measurement of their fluorescence.

Some embodiments of the present application are directed to sequencing methods comprising: (a) incorporating at least one labeled nucleotide as described herein into a polynucleotide and (b) identifying the labeled nucleotide(s) included in the polynucleotide. polynucleotide, by detecting a fluorescent signal from a new fluorescent dye attached to the specified polynucleotide(s).

Some embodiments of the present disclosure provide a method for determining the sequence of a target single-stranded polynucleotide, comprising:

(a) incorporating a nucleotide containing a 3'-OH blocking group and a detectable tag as described herein into a copy of the polynucleotide chain complementary to at least a portion of the target polynucleotide chain;

(b) identifying the identity of a nucleotide included in a copy of the polynucleotide chain; And

(c) effecting chemical removal of the tag and the 3'-OH blocking group from a nucleotide included in a copy of the polynucleotide chain.

In some embodiments, the sequencing method further comprises (d) washing the chemically removed tag and 3' blocking group from a copy of the polynucleotide chain. In some such embodiments, the 3' blocking group and detectable tag are removed before introducing the next complementary nucleotide. In some other embodiments, the 3' blocking group and detectable tag are removed in a single chemical reaction step. In some embodiment, washing step (d) also removes unincorporated nucleotides. In some other embodiments, a palladium radical scavenger is also used in the washing step after chemical cleavage of the label and 3' blocking group.

In some embodiments, steps (a)-(d) are repeated until the sequence of a portion of the template polynucleotide chain is determined. In some embodiments, steps (a)-(d) are repeated at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, or at least at least 300 times.

In some embodiments, the tag and the 3' blocking group are removed in two separate chemical reactions. In some such embodiments, removing the label from a nucleotide included in a copy of the polynucleotide strand includes contacting the copy of the strand including the included nucleotide with a first digestion solution. In some such embodiment, the first cleavage solution contains a phosphine, such as trial kylphosphine. Non-limiting examples of trialkylphosphines include

tris(hydroxypropyl)phosphine (THP), tris-(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THMP) or tris(hydroxyethyl)phosphine (THEP). In one embodiment, the first cleavage solution contains THP. In some such embodiments, removing a 3' blocking group from a nucleotide included in a copy of the polynucleotide strand involves contacting the copy of the strand including the included nucleotide with a second cleavage solution. In some such embodiments, the second cleavage solution contains a palladium (Pd) catalyst. In some other embodiments, the Pd catalyst is a Pd(0) catalyst. In some such embodiments, Pd(0) is prepared by mixing the Pd(II) complex [(PdCl(C ₃ H ₅ )) ₂ ] with THP in situ. The molar ratio of Pd(II) complex and THP can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In one embodiment, the Pd:THP molar ratio is 1:5. In some other embodiments, one or more reducing agents, such as ascorbic acid or a salt thereof (eg, sodium ascorbate), can be added. In some embodiments, the second cleavage solution may contain one or more buffering reagents, such as a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, or a borate salt, or combinations thereof. In some other embodiments, the buffering reagent contains ethanolamine (EA),

tris(hydroxymethyl)aminomethane (Tris), glycine, sodium carbonate, sodium phosphate, sodium borate, 2-dimethylaminomethanol (DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED) or N ,N,N',N'-tetraethylethylenediamine (TEEDA), or combinations thereof. In one embodiment, the buffering reagent is DEEA. In another embodiment, the buffering reagent contains one or more inorganic salts, such as a carbonate salt, a phosphate salt or a borate salt, or combinations thereof. In one embodiment, the inorganic salt is a sodium salt. In some other embodiments, the 3' blocked nucleotide contains an AOM group, and the second cleavage solution contains a palladium (Pd) catalyst and one or more than one buffering reagent described herein (e.g., a tertiary amine such as DEEA), and has a pH of about 9.0 to about 10.0 (for example, 9.6 or 9.8).

In some alternative embodiments, the tag and the 3'-OH blocking group are removed in a single chemical reaction. In some such embodiments, the label is attached to the nucleotide through a cleavable linker containing the same moiety as the 3' blocking group, e.g., both the linker and the 3' blocking group may contain an acetal moiety or thiocarbamate fragment as described in this document. In some such embodiment, one chemical reaction is carried out in a cleavage solution containing the Pd catalyst described above.

In certain other embodiments, the nucleotides used in incorporation step (a) are fully functionalized nucleotide triphosphates A, C, T, and G, each containing a 3' blocking group as described herein. In some such embodiments, the nucleotides described herein provide superior stability in solution during sequencing runs compared to the same nucleotides protected by a standard 3'-O-azidomethyl blocking group. For example, acetal or thiocarbamate blocking groups disclosed herein can impart at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 %, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500% or 3000% improved stability compared to azidomethyl protected 3'- OH under the same conditions for the same period of time, thereby reducing prephasing values and resulting in longer read lengths when sequencing. In some embodiments, stability is measured at ambient temperature or at a temperature below ambient temperature (such as 4-10°C). In other embodiments, stability is measured at elevated temperature, such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, stability is measured in solution at the basic pH of the environment, for example, pH 9.0; 9.2; 9.4; 9.6; 9.8 or 10.0. In some other embodiments, the prephasing value with the 3' blocked nucleotide described herein is less than about 0.25; 0.24; 0.23; 0.22; 0.21; 0.20; 0.19; 0.18; 0.17; 0.16; 0.15; 0.14; 0.13; 0.12; 0.11; 0.10; 0.09; 0.08; 0.07; 0.06 or 0.05 after more than 50, 100 or 150 SBS cycles. In some other embodiments, the 3' blocked nucleotide prephasing value is less than about 0.25; 0.24; 0.23; 0.22; 0.21; 0.20; 0.19; 0.18; 0.17; 0.16; 0.15; 0.14; 0.13; 0.12; 0.11; 0.10; 0.09; 0.08; 0.07; 0.06 or 0.05 after more than 50, 100 or 150 SBS cycles. In one embodiment, each ffN contains a 3'-AOM group.

In some embodiments, the 3' blocked nucleotides described herein provide superior release rates in solution after the chemical cleavage step of the sequencing run, compared to the same nucleotides protected by a standard 3'-O-azidomethyl blocking group. For example, acetal (eg, AOM) or thiocarbamate blocking groups disclosed herein can impart at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500% or 2000% improved release speed compared to 3' -OH protected with azidomethyl using a standard deblocking reagent (such as tris(hydroxypropyl)phosphine), thereby reducing the overall sequencing run time. In some embodiments, the unblocking time for each nucleotide is reduced by about 5%, 10%, 20%, 30%, 40%, 50%, or 60%. For example, the release time for 3'-AOM and 3'-O-azidomethyl is about 4-5 seconds and about 9-10 seconds, respectively, under certain chemical reaction conditions. In some embodiments, the half-life (t _1/2 ) of the AOM blocking group is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times less than the azidomethyl blocking group. In some such embodiment, the t1 _/2 of AOM is about 1 minute, while the t1 _/2 of azidomethyl is about 11 minutes. In some embodiments, the release rate is measured at ambient temperature or at a temperature below ambient temperature (such as 4-10°C). In other embodiments, the release rate is measured at an elevated temperature, such as 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some such embodiments, the deblocking rate is measured in solution in a basic pH environment, for example, pH 9.0; 9.2; 9.4; 9.6; 9.8 or 10.0. In some such embodiments, the molar ratio of deblocking reagent to substrate (i.e., 3' blocked nucleoside or nucleotide) is about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1 :5 or approximately 1:10. In one embodiment, each ffN contains a 3'-AOM group.

In any embodiments of the methods described herein, the labeled nucleotide is a nucleotide triphosphate. In any embodiments of the method described herein, a strand of the target polynucleotide is attached to a solid support, such as a flow cell.

In one embodiment, at least one nucleotide is incorporated into a polynucleotide during synthesis through the action of a polymerase enzyme. In some such embodiments, the polymerase may be DNA polymerase Pol 812 or Pol 1901. However, other methods of joining nucleotides to polynucleotides can be used, such as chemical synthesis of oligonucleotides or ligation of labeled oligonucleotides to unlabeled oligonucleotides. Therefore, the term "incorporation" when used in relation to a nucleotide and a polynucleotide can cover the synthesis of a polynucleotide by chemical methods as well as enzymatic methods.

In a particular embodiment, the synthesis step is carried out and may optionally include incubating the template polynucleotide strand with a reaction mixture containing labeled 3' blocked nucleotides according to this disclosure. The polymerase may also be provided under conditions that allow formation of a phosphodiester bond between the free 3'-OH group on the polynucleotide chain annealed to the template polynucleotide chain and the 5' phosphate group on the nucleotide. Thus, the synthesis step may involve the formation of a polynucleotide strand as driven by complementary nucleotide pairing with the template strand.

In all embodiments of these methods, the detection step may be carried out while the polynucleotide strand into which the labeled nucleotides are included is annealed to the template strand or after a denaturation step in which the two strands are separated. Other steps, for example chemical or enzymatic reaction steps, or purification steps, may be included between the synthesis step and the detection step. In particular, the target strand including the labeled nucleotide(s) can be isolated or purified and then further processed or used in subsequent analysis. As an example, target polynucleotides labeled with nucleotide(s) as described herein in the synthesis step can then be used as labeled probes or primers. In other embodiments, the product of the synthesis step set forth herein may be subjected to further reaction steps and, if desired, the product of these subsequent steps is purified or isolated.

Suitable conditions for the synthesis step will be well known to those familiar with standard molecular biology techniques. In one embodiment, the synthesis step may be similar to a standard primer extension reaction using precursor nucleotides comprising nucleotides as described herein to form an extended target strand complementary to the template strand, in the presence of a suitable polymerase enzyme. In other embodiments, the synthesis step may itself form part of an amplification reaction producing a labeled double-stranded amplification product consisting of annealed complementary strands resulting from copying of the target and template polynucleotide chains. Other typical synthesis steps include nick translation, strand displacement polymerization, random DNA labeling with primers, etc. A particularly useful polymerase enzyme for the synthesis step is one that is capable of catalyzing the incorporation of nucleotides, as set forth herein. A variety of naturally occurring or modified polymerases can be used. As an example, for a synthetic reaction that is carried out using thermocycling conditions, a thermostable polymerase may be used, whereas a thermostable polymerase may not be desirable for isothermal primer extension reactions. Suitable thermostable polymerases that are capable of incorporating nucleotides according to this disclosure include the polymerases described in WO 2005/024010 or WO 06/120433, each of which is incorporated herein by reference. In synthetic reactions that are carried out at lower temperatures, such as 37°C, polymerase enzymes do not necessarily need to be thermostable polymerases, therefore the choice of polymerase will depend on a number of factors such as reaction temperature, pH, chain displacement activity and the like .

In specific non-limiting embodiments, this disclosure covers methods for nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism counting, any other application involving the detection of a labeled nucleotide or nucleoside as set forth herein when incorporated into a polynucleotide. Any of the many other applications that benefit from the use of polynucleotides labeled with nucleotides containing fluorescent dyes can use the labeled nucleotides or nuceosides with dyes set forth herein.

In a specific embodiment, this disclosure provides the use of labeled nucleotides according to this disclosure in a polynucleotide sequencing by synthesis (SBS) reaction. Sequencing by synthesis typically involves the sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase or ligase to form an extended polynucleotide chain complementary to the template nucleic acid to be sequenced. The identity of the base present in one or more than one added nucleotide can be determined during the detection or "visualization" step. The identity of the added base can be determined after each nucleotide incorporation step. The template sequence can then be deduced using traditional Watson-Crick base pairing rules. The use of labeled nucleotides set forth herein to determine single base identity may be useful, for example, in the counting of single nucleotide polymorphisms, and such single base extension reactions are within the scope of this disclosure.

In one embodiment of the present disclosure, the sequence of a template polynucleotide is determined by detecting the inclusion of one or more 3' blocked nucleotides described herein in a growing strand complementary to the template polynucleotide to be sequenced by detecting fluorescent tag(s) , attached to the included nucleotide(s). Sequencing of the template polynucleotide can be started using a suitable primer (or it can be produced as a hairpin construct that will contain the primer as part of the hairpin), and the growing strand is completed stepwise by adding nucleotides to the 3' end of the primer in a polymerase-catalyzed reaction.

In specific embodiments, each of the different nucleotide triphosphates (A, T, G and C) may be labeled with a unique fluorophore and also contains a blocking group at the 3' position to prevent uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). The polymerase enzyme incorporates a nucleotide into a growing strand complementary to the template polynucleotide, and the blocking group prevents further incorporation of nucleotides. Any unincorporated nucleotides can be washed away, and the fluorescent signal from each incorporated nucleotide can be "read" optically by suitable means, such as a charge-coupled device using laser excitation and suitable emission filters. The 3' blocking group and fluorescent dye compounds can then be removed (deprotection) simultaneously or sequentially to expose the growing chain for further incorporation of nucleotides. Typically the identity of the incorporated nucleotide will be determined after each incorporation step, but this is not strictly essential. Similarly, US Pat. No. 5,302,509 (which is incorporated herein by reference) discloses a method for sequencing polynucleotides immobilized on a solid support.

This method, as illustrated by the examples above, uses the incorporation of fluorescently labeled, 3'-blocked nucleotides A, G, C and T into a growing strand complementary to the immobilized polynucleotide in the presence of DNA polymerase. This polymerase includes a base complementary to the target polynucleotide, but further addition is prevented by a 3' blocking group. The label of the included nucleotide can then be determined and the blocking group is removed by chemical cleavage to allow further polymerization. The template nucleic acid to be sequenced in a sequencing reaction by synthesis may be any polynucleotide that is desired to be sequenced. A template nucleic acid for a sequencing reaction will typically comprise a double-stranded region having a free 3'-OH group that serves as a primer or initiation point for adding further nucleotides to the sequencing reactions. The region of the template to be sequenced will form a sticky end with this free 3'-OH group on the complementary strand. The sticky end region of the template to be sequenced may be single-stranded, but may be double-stranded, provided that a "single-strand break is present" on a strand complementary to the template strand to be sequenced, providing a free 3'-OH group to initiate the sequencing reaction. In such embodiments, sequencing may proceed through strand displacement. In some embodiments, a primer carrying a free 3'-OH group (eg, a short oligonucleotide) that hybridizes to the single-stranded region of the template to be sequenced may be added as a separate component. Alternatively, the primer and template strand to be sequenced may each form part of a partially complementary nucleic acid strand capable of forming an intramolecular duplex, such as, for example, a hairpin loop structure. Hairpin polynucleotides and methods by which they can be attached to solid supports are disclosed in PCT Publication No. WO 01/57248 and WO 2005/047301, each of which is incorporated herein by reference. Nucleotides can be added to the growing primer sequentially, resulting in the synthesis of a polynucleotide chain in the 5' to 3' direction. The nature of the base that has been added may be determined, particularly but not necessarily after the addition of each nucleotide, thereby providing sequence information for the nucleic acid template. Thus, a nucleotide is incorporated into a nucleic acid chain (or polynucleotide) by combining the nucleotide with a free 3'-OH group of the nucleic acid chain through the formation of a phosphodiester bond with the 5' phosphate group of the nucleotide.

The template nucleic acid to be sequenced may be DNA or RNA, or even a hybrid molecule consisting of deoxynucleotides and ribonucleotides. The template nucleic acid may contain naturally occurring and/or non-naturally occurring nucleotides and natural or non-natural backbone linkages, provided they do not prevent copying of the template in the sequencing reaction.

In some embodiments, the template nucleic acid to be sequenced can be attached to a solid support by any suitable coupling method known in the art, such as covalent attachment. In some embodiments, the template polynucleotides may be attached directly to a solid support (eg, a silica support). However, in other embodiments of this disclosure, the surface of the solid support may be modified in some manner to permit either direct covalent attachment of the template polynucleotides or to immobilize the template polynucleotides through a hydrogel or polyelectrolyte multilayer, which itself may be non-covalently attached to the solid support.

Implementations and alternatives of sequencing by synthesis

Some embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic phosphate (PPi) as specific nucleotides are incorporated into the growing chain (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real- time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing." Genome Res. 11(1), 3-11; Ronaghi , M., Uhlen, M. and Nyren, P. (1998) "A sequencing method based on real-time pyrophosphate." Science 281(5375), 363; US patents 6210891 and 6274320, the disclosures of which are included herein. document by reference in their entirety). In pyrosequencing, released PPi can be detected by immediate conversion to adenosine triphosphate (ATP) by ATP sulfurase, and the level of ATP generated is detected by photons produced by luciferase. Nucleic acids to be sequenced can be attached to elements on a chip, and the chip can be imaged to capture the chemiluminescent signals that are produced due to the incorporation of nucleotides into elements of the chip. An image can be obtained after treating the chip with a specific type of nucleotide (for example, A, T, C or G). The images obtained after adding each type of nucleotide will differ in what features on the chip are detected. These differences in the image reflect the different content of the sequence of elements on the chip. However, the relative positions of each element will remain the same in the images. The image data can be stored, processed and analyzed using the methods described herein. For example, images obtained after processing the chip with each other type of nucleotide can be manipulated in the same manner, as illustrated herein by examples for images obtained from different detection channels for reversible terminator-based sequencing methods.

In another exemplary type of SBS, cycle sequencing is accomplished by stepwise addition of nucleotides with a reversible terminator containing, for example, a cleavable or photobleachable dye tag, as described, for example, in WO 04/018497 and US Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference. . This approach is being marketed by Solexa (now Illumina, Inc.) and is also described in WO 91/06678 and WO 07/123744, each of which is incorporated herein by reference. The availability of fluorescently labeled terminators, in which both termination can be reversed and the fluorescent tag cleaved, facilitates efficient cycle sequencing based on reversible termination (CRT). Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

Preferably, in embodiments of reversible terminator sequencing, the tags do not substantially inhibit extension under SBS reaction conditions. However, the detected marks may be removable, for example, by cleavage or degradation. Images can be captured by incorporating tags into ordered nucleic acid elements. In specific embodiments, each cycle involves the simultaneous delivery of four different types of nucleotides to the chip, and each type of nucleotide has a spectrally distinct label. Four images can then be acquired, each using a detection channel that is selective for one of four different marks. Alternatively, different types of nucleotides can be added sequentially, and an image of the chip can be obtained between each addition step. In such embodiments, each image will show nucleic acid elements that include a particular type of nucleotide. Different elements will be present or absent in different images due to the different sequence content of each element. However, the relative position of these elements will remain unchanged in the images. Images obtained from such reversible terminator SBS methods can be stored, processed and analyzed as described herein. After the image fixation step, tags can be removed and reversible terminator fragments can be removed for subsequent rounds of addition and nucleotide detection. Removing cues after they have been identified in a particular run and before a subsequent run may have the benefit of reducing background signal and overlap between runs. Examples of useful tags and removal methods are outlined below.

In some embodiments, detection of four different nucleotides using less than four different labels may be used. For example, SBS can be performed using the methods and systems described in the included materials of US Publication No. 2013/0079232. As a first example, a pair of nucleotide types may be detected at the same wavelength but distinguished based on a difference in intensity for one member of the pair compared to the other or based on a change in one member of the pair (e.g., through chemical modification, photochemical modification, or physical modification ), which causes the appearance or disappearance of a distinguishable signal compared to the signal detected for the other member of the pair. As a second example, three of four different types of nucleotides can be detected under particular conditions, whereas a fourth type of nucleotide has no label that is detected under given conditions, or is only minimally detectable under given conditions (e.g., minimal detection due to background fluorescence and etc.). The inclusion of the first three types of nucleotides in a nucleic acid can be determined based on the presence of their respective signals, and the inclusion of a fourth type of nucleotide in a nucleic acid can be determined based on the absence or minimal detection of any signal. As a third example, one type of nucleotide may include tag(s) that are detected in two different channels, while other types of nucleotides are detected in at most one of the channels. The above three typical configurations are not considered mutually exclusive and can be used in different combinations. A typical embodiment that combines all three examples is a fluorescence-based SBS method that uses a first type of nucleotide that is detected in a first channel (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength) , a second type of nucleotide that is detected in the second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third type of nucleotide that is detected in both the first and second channels (e.g. dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelength), and a fourth type of nucleotide that is unlabeled, that is, not detected or minimally detected in either channel (e.g. , unlabeled dGTP).

In addition, as described in the included materials of US Publication No. 2013/0079232, sequencing data can be obtained using a single channel. In these so-called single-dye sequencing approaches, the first type of nucleotide is labeled, but the label is removed after the first image is acquired, and the second type of nucleotide is labeled only after the first image is acquired. The third type of nucleotide retains its label in both the first and second images, and the fourth type of nucleotide remains unlabeled in both images.

In some embodiments, sequencing via ligation techniques may be used. Such techniques use DNA ligase to incorporate oligonucleotides and identify the inclusion of such oligonucleotides. These oligonucleotides typically have different labels that correlate with the identity of the particular nucleotide in the sequence to which the oligonucleotide hybridizes. As with other SBS methods, images can be obtained after processing the nucleic acid element chip using labeled sequencing reagents. Each image will show elements with nucleic acids that have included a specific type of tag. Different elements will be present or absent in different images due to the different sequence content of each element, but the relative position of these elements will remain the same in the images. Images obtained from ligation-based sequencing methods can be stored, processed and analyzed as described herein. Exemplary SBS systems and methods that can be used with the methods and systems described herein are described in US Pat. Nos. 6,969,488, 6,172,218, and 6,306,597, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, nanopore-based sequencing may be used (Deamer, D.W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton , "Characterization of nucleic acids by nanopore analysis", Ace, Chem, Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J.A. and configurations in a solid-state nanopore microscope" Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference in their entirety). In such embodiments, the target nucleic acid passes through the nanopore. The nanopore may be a synthetic pore or a biological membrane protein such as α-chemolysin. As the target nucleic acid passes through the nanopore, each base pair can be identified by measuring fluctuations in the electrical conductivity of that pore (U.S. Patent No. 7001792; Soni, G.V. & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); & Ghadiri, M.R. "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution." J. Am. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference. their completeness). Data obtained from nanopore-based sequencing can be stored, processed and analyzed as described herein. In particular, the data may be processed in image form according to typical processing of optical images and other images as set forth herein.

Some other embodiments of the sequencing method include the use of the 3' blocked nucleotide described herein in a nanobead-based sequencing technique, such as the techniques described in US Pat. No. 9,222,132, the disclosure of which is incorporated by reference. Through the rolling circle amplification (RCA) method, a large number of discrete DNA nanobeads can be obtained. The mixture of nanobeads is then distributed on a structured glass slide surface containing elements that ensure the association of a single nanobead with each position. When producing DNA nanobeads, the DNA is fragmented and ligated to the first of four adapter sequences. The template is amplified, circularized, and cleaved by type II endonuclease. A second set of adapters is added, followed by amplification, circularization and cleavage. This method is repeated for the remaining two adapters. The final product is a ring matrix with four adapters, each separated by a matrix sequence. The library molecules undergo a rolling ring amplification step, generating a large mass of concatemers called DNA nanobeads, which are then applied to the flow cell. Goodwin et al., "Coming of age: ten years of next-generation sequencing technologies," Nat Rev Genet. 2016; 17(6):333-51.

In some embodiments, methods may be used that include monitoring DNA polymerase activity in real time. Nucleotide inclusions can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-carrying polymerase and γ-phosphate-labeled nucleotides, as described, for example, in US Pat. Nos. 7,329,492 and 7,211,414, both of which are incorporated herein by reference, or Nucleotide inclusions can be detected using zero-mode waveguides, as described, for example, in US Pat. No. 7,315,019, which is incorporated herein by reference, and using fluorescent nucleotide analogues and modified polymerases, as described, for example, in US Pat. No. 7,405,281 and US Publication No. 2008/0108082, both of which are incorporated herein by reference. Illumination can be limited to the zeptoliter-scale volume around the surface-bound polymerase such that the incorporation of fluorescently labeled nucleotides can be observed with little background (Levene, M.J. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations." Science 299, 682-686 (2003); Lundquist, P. M. et al. "Parallel confocal detection of single molecules in real time." Opt. 33, 1026-1028 (2008); aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entirety) . Images obtained from such methods can be stored, processed and analyzed as described herein.

Some embodiments of SBS involve detecting a proton released upon incorporation of a nucleotide into an extension product. For example, released proton sequencing may use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a division of Life Technologies), or the sequencing methods and systems described in US Publication No. 2009/0026082; 2009/0127589; 2010/0137143 and 2010/0282617, all of which are incorporated herein by reference. The methods outlined herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for proton detection. More specifically, the methods set forth herein can be used to generate clonal ampicon populations that are used for proton detection.

The above SBS methods can advantageously be carried out in multiplex formats such that many different target nucleic acids are manipulated simultaneously. In specific embodiments, different target nucleic acids can be processed in a common reaction vessel or on the surface of a specific substrate. This enables convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of inclusion events in a multiplex manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids may be in a chip format. In a chip format, target nucleic acids can typically bind to a surface in a spatially distinct manner. Target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle, or binding to a polymerase or other molecule that is attached to a surface. A given chip may include one copy of the target nucleic acid at each site (also called an element), or many copies having the same sequence may be present at each site or element. Many copies can be produced by amplification methods such as bridge amplification or emulsion PCR (polymerase chain reaction), as described in further detail below.

The methods set forth herein may use chips having elements in any of a range of densities including, for example, at least about 10 elements/cm ² , 100 elements/cm ² , 500 elements/cm ² , 1000 elements/cm 2 ^cm2 , 5000 elements/ ^cm2 , 10000 elements/ ^cm2 , 50000 elements/ ^cm2 , 100000 elements/ ^cm2 , 1000000 elements/ ^cm2 , 5000000 elements/ ^cm2 or more.

An advantage of the methods described herein is that they provide rapid and efficient parallel detection of large quantities of target nucleic acid. Accordingly, the present disclosure provides integrated systems capable of obtaining and detecting nucleic acids using techniques known in the art, such as the systems exemplified above. Thus, the integrated system of the present disclosure may include fluid components capable of delivering amplification reagents and/or sequencing reagents to one or more immobilized DNA fragments, a system containing components such as pumps, valves, reservoirs, fluid lines, and the like. similar. In an integrated system, a flow cell may be configured and/or used to detect target nucleic acids. Exemplary flow cells are described, for example, in US Publication No. 2010/0111768 and US Publication Serial No. 13/273666, each of which is incorporated herein by reference. As illustrated by the flow cell examples, one or more fluid components of the integrated system can be used for the amplification method and for the detection method. Taking an embodiment of nucleic acid sequencing as an example, one or more liquid components of an integrated system can be used for the amplification method set forth herein and for delivering sequencing reagents in a sequencing method such as the methods illustrated in the examples above. Alternatively, the integrated system may include separate fluid systems for carrying out the amplification methods and for carrying out the detection methods. Examples of integrated sequencing systems that are capable of generating amplified nucleic acids and also determining the sequence of these nucleic acids include, but are not limited to, the MiSeq™ platform (Illumina, Inc., San Diego, CA) and devices described in US Publication Serial No. 13/ 273666, which are incorporated herein by reference.

Chips in which polynucleotides have been directly attached to silica substrates are chips, such as those disclosed in WO 00/06770 (incorporated herein by reference), where the polynucleotides are immobilized on a glass substrate by reaction between a pendant epoxy group on the glass with an internal amino group on the polynucleotide. In addition, polynucleotides can be attached to a solid support by reacting a sulfur-based nucleophile with the solid support, for example, as described in WO 2005/047301 (incorporated herein by reference). Yet another example of template polynucleotides on a solid support is where template polynucleotides are attached to a hydrogel support on silica or other solid supports, for example as described in WO 00/31148, WO 01/01143, WO 02/12566, WO 03/014392, US Patent No. 6465178 and WO 00/53812, each of which is incorporated herein by reference.

A particular surface on which template polynucleotides can be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the references cited above and in WO 2005/065814, which is incorporated herein by reference. Specific hydrogels that can be used include those described in WO 2005/065814 and US Publication No. 2014/0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl)acrylamide-coacrylamide)).

Template DNA molecules can be attached to beads or microparticles, for example, as described in US Pat. No. 6,172,218 (which is incorporated herein by reference). Attachment to beads or microparticles may be useful for sequencing applications. Bead libraries can be generated where each bead contains a different DNA sequence. Typical libraries and methods for their construction are described in Nature, 437, 376-380 (2005); Science, 309, 5741, 1728-1732 (2005), each of which is incorporated herein by reference. Sequencing chips of such beads using the nucleotides set forth herein is within the scope of this disclosure.

The templates to be sequenced can form part of a “chip” on a solid support, and in this case the chip can take any convenient shape. Thus, the method of this disclosure is applicable to all types of high-density chips, including single-molecule chips, cluster chips, and ball chips. The labeled nucleotides of the present disclosure can be used for sequencing templates on essentially any type of chip, including, but not limited to, chips formed by immobilizing nucleic acid molecules on a solid support.

However, the labeled nucleotides of this disclosure are particularly useful in the context of cluster chip sequencing. In cluster chips, distinct regions on the chip (often called sites or elements) contain many template polynucleotide molecules. In general, these multiple polynucleotide molecules are not individually resolved by optical means and are instead detected as a unit. Depending on how the chip is formed, each site on the chip may contain many copies of one individual polynucleotide molecule (e.g., a given site is homogeneous for a particular type of single- or double-stranded nucleic acid) or even many copies of a small number of different polynucleotide molecules (e.g. , many copies of two different types of nucleic acids). Cluster chips of nucleic acid molecules can be prepared using techniques generally known in the art. By way of example, WO 98/44151 and WO 00/18957, each of which are incorporated herein, describe methods for amplifying nucleic acids where both the template and the amplification products remain immobilized on a solid support to form chips consisting of clusters or “colonies” of immobilized nucleic acid molecules. The nucleic acid molecules present on cluster chips prepared according to these methods are suitable templates for sequencing using nucleotides labeled with the dye compounds of this disclosure.

The labeled nucleotides of the present disclosure are also useful in sequencing templates on single-molecule chips. The term "single molecule chip" or "SMA" as used herein refers to a population of polynucleotide molecules distributed (or arrayed) over a solid support, where the distance of any individual polynucleotide from all others in the population is such that it is possible to individually resolve individual polynucleotide molecules. Target nucleic acid molecules immobilized on the surface of a solid support may, in some embodiments, thus be capable of resolution by optical means. This means that one or more distinct signals, each representing a single polynucleotide, will appear within the resolution region of the particular imaging device being used.

Detection of single molecules can be achieved where the distance between adjacent polynucleotide molecules on the chip is at least 100 nm, more particularly at least 250 nm, even more particularly at least 300 nm, even more particularly at least 350 nm. Thus, each molecule is individually resolvable and detectable as a single-molecule fluorescent dot, and the fluorescence from said single-molecule fluorescent dot also exhibits one-step photobleaching.

The terms "individually resolvable" and "individually resolved" are used herein to specifically define that, when imaged, it is possible to distinguish one molecule on a chip from its neighboring molecules. The separation between individual molecules on a chip will be determined, in part, by the specific technique used to resolve individual molecules. The general characteristics of single-molecule chips will be understood by reference to published applications WO 00/06770 and WO 01/57248, each of which is incorporated herein by reference. Although one use of the nucleotides of this disclosure is in sequencing reactions by synthesis, the usefulness of these nucleotides is not limited to such methods. In fact, these nucleotides can be usefully used in any sequencing methodology that requires the detection of fluorescent tags attached to nucleotides included in a polynucleotide.

In particular, the labeled nucleotides of this disclosure can be used in automated fluorescent sequencing protocols, particularly fluorescent dye terminator cycle sequencing, based on the chain termination sequencing method of Sanger and co-workers. Such methods typically use enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides into the primer extension sequencing reaction. So-called Sanger sequencing methods and related (Sanger-type) protocols use randomized chain termination with labeled dideoxynucleoids.

Thus, the present disclosure also covers labeled nucleotides that are dideoxynucleoids lacking hydroxyl groups at both the 3' and 2' positions, such dideoxynucleoids being suitable for use in Sanger-type sequencing methods and the like.

It will be understood that the labeled nucleotides of the present disclosure, including 3' blocking groups, can also be useful with Sanger and related protocols, since the same effect achieved by using dideoxynucleotides can be achieved using nucleotides having 3'- OH blocking groups: both prevent the incorporation of subsequent nucleotides. When nucleotides of the present disclosure having a 3' blocking group are to be used in Sanger sequencing methods, it will be appreciated that the dye compounds or detectable tags attached to the nucleotides should not be attached via cleavable linkers, since in each case, where a labeled nucleotide of this disclosure is included, no further nucleotides should be included, and thus the label should not be removed from the nucleotide.

In any embodiments of the methods described herein, the nucleotide used in the sequencing application is a 3' blocked nucleotide described herein, for example, a nucleotide of Formula (I), (Ia) or (II). In any embodiments, the 3' blocked nucleotide is a nucleotide triphosphate.

EXAMPLES

Additional embodiments are disclosed in further detail with the following examples, which are in no way intended to limit the scope of the claims.

Example 1. Preparation of 3'-acetal-capped nucleosides

In this example, various 3'-acetal protected nucleosides T were prepared according to Scheme 2.

Preparation T1: 5-iodo-2'-deoxyuridine (5.0 g, 14.12 mmol) was added to an oven-dried, nitrogen-purged 100 mL flask. It was co-evaporated 3 times with 30 ml of pyridine, then placed under nitrogen. Anhydrous pyridine (25 ml) was added and the reaction mixture was stirred at room temperature until a homogeneous solution was obtained (~15 minutes). This mixture was cooled to 0°C in an ice-water bath, and tert-butyldiphenylsilyl chloride (4.04 mL, 15.5 mmol) was added slowly dropwise with vigorous stirring (~1 hour). This reaction mixture was maintained at 0°C for 8 hours until all SM was consumed as determined by TLC (thin layer chromatography). Saturated aqueous ammonium chloride (~15 mL) was added and the reaction mixture was allowed to warm to room temperature. This mixture was diluted with ethyl acetate (100 ml) and washed with saturated aqueous ammonium chloride (200 ml). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (4×50 ml). The organic layers were combined, dried (MgSO ₄ ) and concentrated in vacuo to give ~8 g of a clear yellow oil after removal of residual solvent under high vacuum. The crude product T1 was purified by flash silica column chromatography as a white crystalline solid. The yield is 6.94 g (83%). LC-MS (liquid chromatography-mass spectrometry) (electrospray, negative ion mode) 591.08 [M-N].

Preparation of T2: T1 (6.23 g, 10.5 mmol), copper(I) iodide (200 mg, 1.05 mmol) and bis(triphenylphosphine)palladium( II) dichloride (369 mg, 0.526 mmol) under nitrogen. The flask was protected from light and anhydrous degassed DMF (200 ml) was added. To this solution was added 2,2,2-trifluoro-N-prop-2-ynyl-acetamide (4.74 g, 31.6 mmol), followed by the addition of degassed triethylamine (2.92 mL, 21.0 mmol). This reaction mixture was stirred at room temperature under nitrogen for 6 hours until no additional starting material was observed by TLC analysis. Volatiles were removed under vacuum (~15 min) and DMF was removed under high vacuum (~1 hour) to a brown precipitate. It was dissolved in ethyl acetate (200 ml) and extracted with 0.1 M EDTA (ethylenediaminetetraacetic acid) in water (2 x 200 ml). The aqueous layers were combined and further extracted with ethyl acetate (200 ml). The organic phases were combined, dried (MgSO ₄ ), volatiles were removed in vacuo (~30 min) and further dried under high vacuum (~1 hour) to obtain approximately 8 g of a crude brown/yellow oil. This mixture was purified by flash column chromatography on silica gel as an off-white solid. Yield: 6.0 g (85%). LC-MS (electrospray, negative ion mode) 614.19 [M-H].

Preparation of T3: To an oven-dried, nitrogen-purged 100 mL flask containing the starting nucleoside T2 (2.0 g, 3.25 mmol), under nitrogen, anhydrous DMSO (6.9 mL, 97.5 mmol) was added in one portion ) at room temperature and stirred until a homogeneous solution was formed. Acetic acid (11.1 mL, 195 mmol) was added dropwise, followed by acetic anhydride (15.1 mL, 162.09 mmol) dropwise (~5 minutes each). This mixture was heated to 50°C and stirred until complete consumption of the starting nucleoside (~5 hours) as determined by TLC (EtOAc/petroleum ether 3:2). This reaction mixture was then concentrated to half volume and cooled using an ice bath to approximately 0.5°C. The treatment was started by slowly adding cold (~0.5°C) NaHCO ₃ (aq, sat) (45 ml) and continued stirring until no more fizzing was observed (~15 min). This solution was allowed to warm to room temperature, then the aqueous phase was extracted into EtOAc (3×100 ml). The combined organic layers were dried over MgSO ₄ , filtered, and the volatiles were evaporated under reduced pressure and additionally under high vacuum. The crude product T3 was purified by flash silica gel chromatography as an off-white solid. Yield: 1.79 g (82%). LC-MS (electrospray, negative ion mode) 674.20 [M-H] ^- .

Preparation of T4: cyclohexene (1.34 ml, 13.2 mmol) was added to a solution of the starting nucleoside T3 (1.79 g, 2.649 mmol) in anhydrous CH ₂ Cl ₂ (50 ml) under N ₂ . This mixture was cooled in an ice bath to 0°C, and distilled sulfuryl chloride (322 μl, 3.97 mmol) was slowly added dropwise (~20 min) under N ₂ . After stirring for 20 minutes at this temperature, TLC (EtOAc:petroleum ether - 3:2 (v/v)) showed complete consumption of the starting nucleoside. The chloride intermediate was then quenched by direct dropwise addition of freshly distilled corresponding unsaturated alcohol (5 eq.) as shown in Scheme 3. The resulting solution was stirred at room temperature for 2 hours, followed by evaporation of the volatiles under reduced pressure. The oily residue was partitioned between EtOAc:brine (3:2) (125 ml). The organic layer was separated and the aqueous layer was further extracted into EtOAc (2×50 ml). The combined organic extracts were dried over MgSO ₄ , filtered and the volatiles were evaporated under reduced pressure. The oily residue was partitioned between EtOAc:brine (3:2) (125 ml). The organic layer was separated and the aqueous layer was further extracted into EtOAc (2×50 ml). The combined organic extracts were dried over MgSO ₄ , filtered and the volatiles were evaporated under reduced pressure. The crude T4 products were purified by flash silica gel chromatography to give the final products as a yellow oil. Yield: 1.20 g (69%) for AOM; 1.29 g (71%) for PrOM; 1.34 g (71%) for DPrOM.

3'-AOM: yellow oil. LC-MS (electrospray, negative ion mode) [M-H] 684.24.

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

3'-DprOM: yellow oil. LC-MS (electrospray, negative ion mode) [M-H] 710.25.

Preparation of T5: Anhydrous THF (9 mL) was added to the starting material T4 (1.04 g, 1.516 mmol) in a 50 mL round bottom flask under nitrogen at room temperature. TBAF (1.0 M in THF, 1.7 mL, 1.70 mmol) was then added dropwise and the solution was stirred until all SM was absorbed as determined by TLC (~2 hours). This solution turned orange as the reaction progressed. Volatiles were removed in vacuo to leave an orange residue, which was dissolved in EtOAc (100 ml) and partitioned with NaHCO ₃ (sat. aq.) (60 ml). The two layers were separated and the aqueous layer was extracted with EtOAc (60 ml). The organic layers were combined, dried (MgSO ₄ ), filtered and evaporated to give the crude product as a yellow oil. This crude product was purified by flash chromatography on silica gel to give a clear yellow oil. Yield: 637 mg (94%) for AOM; 526 mg (78%) for PrOM; 617 mg (86%) for DPrOM.

3'-AOM: clear yellow oil. LC-MS (electrospray, negative ion mode): [M-H] 446.12.

3'-PrOM: clear yellow oil (526 mg, 78%). LC-MS (electrospray, negative ion mode): [M-H] 444.10.

3'-DPrOM: clear yellow oil (617 mg, 86%). LC-MS (electrospray, negative ion mode): [M-H] 472.13.

In addition, two additional 3' blocked T nucleosides (3'eAOM T and 3'iAOM T) were prepared following a similar method as described above. 3'-iAOM T: LC-MS (ER): (negative ion) m/z 325.5 (M-H ⁺ ), (positive ion) 327.3 (M + H ⁺ ). 3'-eAOM T: LC-MS (ER): (positive ion) m/z 341.3 (M+1H ⁺ ).

Example 2 Stability Analysis of 3'-OH Blocking Group

In this example, stability assays for the nucleotide 5'-mP 3'-AOM were carried out in parallel in an inclusion buffer solution with a standard nucleotide 5'-mP 3'-O-azidomethyl T.

Preparation of bubeer solution

1 ml of 0.1 mM each 5'-monophosphate 3'-protected nucleotide T in a solution of 100 mM ethanolamine buffer (pH 9.8), 100 mM NaCl and 2.5 mM EDTA was incubated at 65°C in a heating block for 2 weeks At specified time points, 40 μl aliquots were removed and analyzed by HPLC (high performance liquid chromatography) to determine the percentage of blocked nucleotide remaining and the final formation of unblocked nucleotide.

The results of the stability analysis related to 5'-monophosphate 3'-protected nucleotides with acetal protecting groups AOM, PrOM, DPrOM and a standard azidomethyl blocking group are illustrated in FIG. 1. It was observed that 3' blocked nucleotide monophosphate with blocking groups AOM, PrOM and DPrOM gave more than 30-50-fold improvement in reducing the deblocking rate in solution. This experiment simulates how the corresponding fully functionalized nucleotides (ffN) would behave when stored in a mixture for inclusion on a sequencing device cartridge. The improved stability provided by these acetal protecting groups would also result in less prephasing in sequencing runs. Finally, it improves the shelf life of the reagent mixture to be included.

Example 3: 3'-AOM Deblocking Assay

In this example, unblocking assays for 5'-mP 3'-AOM T and the standard nucleotide 5'-mP 3'-O-azidomethyl T were performed individually in a solution unique to each blocking group. Conditions were set to mimic the standard Illumina release reagent as closely as possible and followed the same methodology. The same concentrations of active deblocking reagent, buffer, and nucleoside were maintained in all assays, but the identity of each component was unique. With this method, the observed differences in rates between individual deblocking chemistries cannot be due to differences in drug concentration.

Standard conditions for azidomethyl deprotection

Nucleotide: 5'-monophosphate 3'-O-azidomethyl T. Active deblocking reagent: tris(hydroxypropyl)phosphine (THP) (1 M in 18 mΩ water). (Possible) additive: sodium ascorbate (0.1 mM in 18 mΩ water), final concentration 1 mM. Buffer: ethanolamine, pH 9.8 (2 M in 18 mΩ water). Quenching reagent: H ₂ O ₂ .

Conditions for releasing A OM

Nucleotide: 3'-O-azidomethyl T 5'-monophosphate. The stock solution of 3'-AOM T was diluted to 0.1 mM in 100 mM ethanolamine buffer (pH 9.8) in a glass vessel under nitrogen. A stock solution of the additive, sodium ascorbate, was added to a final concentration of 0.1 mM, and this solution was stirred for 5 minutes. To begin this assay, deblocking reagents (Pd/THP - 1/5; sodium ascorbate; ethanolamine) were added to a stirred solution at room temperature to a final concentration of 1 mM THP. At certain time points, 40 μl aliquots were removed and quenched with 6 μl of a mixture of 1:3 EDTA/H ₂ O ₂ (0.025:0.075 M). HPLC analysis was performed by measuring the area of the parent nucleoside peak, the 3'-OH peak, and any other nucleotide peaks that appeared in the HPLC chromatogram. No other nucleotide-based byproducts were observed.

The comparative result is shown in FIG. 2A. It was observed that AOM provided a 10-fold improvement in terms of deblocking rate in solution compared to the standard azidomethyl blocking group. This experiment serves the purpose of simulating how the corresponding ffNs would behave when sequenced during the unblocking step. A significant improvement in deblocking speed would provide a deblocking wash step instead of the 10-20 second incubation times typically used in certain Illumina sequencing platforms. As a result, the release rate will have a significant impact on the sequencing-by-synthesis (SBS) turnaround time.

Similar experimental conditions were used for deblocking assays for 3'-eAOM T and 3'-iAOM T. As a single change, the Pd catalyst to substrate ratio was reduced to 5:1 in order to observe less distinct differences in deblocking rates. 3'-AOM T was used as a control and the results are illustrated in FIG. 2B. These results showed that the deblocking rate of eAOM and iAOM is 2-3 times slower than AOM at a given specific concentration of the deblocking reagent with a Pd catalyst. It can be expected that this difference in deblocking rate between the substituted version and the unsubstituted version of the AOM blocking groups would be smaller at a higher Pd catalyst to substrate ratio.

Example 4: Optimization of Palladium Digestion Mixture for Sequencing

The Pd/THP catalyst used in the deprotection reaction described in Example 2 is very air sensitive. When exposed to air, it showed a significant loss of activity. In this example, an oxidative stress assay was developed to evaluate the air sensitivity of different palladium cleavage mixture preparations.

0.5 ml of Pd digestion mixture was aliquoted into a 5 ml glass vial and left open to air for 3 hours at room temperature. The residual activity of the oxidized cleavage mixture was assessed by measuring 3'-AOM cleavage as follows. The 3'-AOM T stock solution was diluted to 0.1 mM in 100 mM digestion buffer. Sodium ascorbate stock solution was added to a final concentration of 1 mM, followed by an oxidized digestion mixture to a final dilution of 1/20. After 1 hour, 40 μl of the solution was immediately quenched with 10 μl of a mixture of 1:1 EDTA/H ₂ 0 ₂ (0.25:0.25 M) and analyzed by HPLC. In this experiment, various buffer reagents were screened, including: primary amines (such as ethanolamine, Tris, and glycine); tertiary amines (such as 2-dimethylaminomethanol (DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED) or N,N,N',N'-tetraethylethylenediamine (TEEDA)); and various inorganic salts (such as borate salt, carbonate salt, phosphate salt). It was observed that inorganic buffer reagents (such as sodium borate, sodium carbonate, sodium phosphate) provided the best stability in air, and the palladium complex retained a high % of activities. In addition, tertiary amines also significantly improved the stability of the Pd cleavage mixture compared to primary amines.

Based on these data, two palladium fission mixtures were prepared. In the first example, the stock solution is 250 mM aq. borate buffer (pH 9.6, 20 mL) was diluted with water (14 mL) before adding THP stock solution (1 M in 100 mM Tris, pH 9. 5 mL, 5.0 mmol) and allyl palladium(II) chloride dimer (183 mg, 0.5 mmol). This mixture was stirred vigorously for several minutes at room temperature before adding 1 M aq. sodium ascorbate (0.5 ml, 0.5 mmol), 5 M aq. NaCl (10 ml) and 10% v/v. Tween 20 (0.5 ml). In the second example, the stock solution is 2 M aq. DEEA buffer (pH 9.6, 0.6 mL) was diluted with water (7.6 mL) before adding THP stock solution (1 M in 100 mM Tris, pH 9, 1.2 mL, 1.2 mmol) and solid dimer allylpalladium(II) chloride (43.9 mg, 0.12 mmol). This mixture was stirred vigorously for several minutes at room temperature before adding 1 M aq. sodium ascorbate (0.12 ml, 0.12 mmol), 5 M aq. NaCl (2.4 ml) and 10% v/v. Tween 20 (0.12 ml).

Example 5: Preparation of Fully Functionalized Nucleotides and Use for Sequencing Applications

This example details the preparation of various fully functionalized nucleotides (ffN) with a 3'-AOM blocking group. The ffN data were also used in a sequencing-by-synthesis application on the Illumina MiniSeq® platform.

Synthesis of AOM intermediate C2: Nucleoside C1 (0.5 g, 0.64 mmol) was dissolved in anhydrous DCM (12 ml) under N ₂ and the mixture was cooled to 0°C. Cyclohexene (0.32 mL, 3.21 mmol) was added, followed by dropwise addition of SO ₂ Cl ₂ (1.0 M in DCM, 1.27 mL, 1.27 mmol). Additional cyclohexene (0.32 mL, 3.21 mmol) was added before quickly transferring this reaction mixture to a rotary evaporator to remove all volatiles under reduced pressure. The solid residue was further dried under high vacuum for 10 min before dissolving in anhydrous DCM (5 ml) under N ₂ . This mixture was cooled to 0°C and ice-cold allylic alcohol (5 ml) was added dropwise. This reaction mixture was stirred at 0°C for 2 hours before quenching by adding sat. aq. NaHCO ₃ (50 ml) and DCM (30 ml). The two phases were separated and the aqueous layer was extracted with EtOAc (2×50 ml). The organic layers were combined, dried over MgSO ₄ , filtered, and the volatiles were evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel using EtOAc/petroleum ether to give AOM C2 as a white solid (264 mg, 52% yield). LC-MS (electrospray, negative ion mode): [M-H] 787, [M+Cl] 823.

Synthesis of intermediate compound AOM C3: AOM C2 (246 mg, 0.31 mmol) was dissolved in anhydrous THF (9.5 ml) under N ₂ and the mixture was cooled to 0°C. Acetic acid (0.054 mL, 0.94 mmol) was added, followed by dropwise addition of TBAF (1.0 M in THF, 5% water by weight, 0.99 mL, 0.94 mmol). This reaction mixture was stirred at 0°C for 5 hours before diluting with EtOAc (20 ml) and then poured into 0.05 M HCl aq. (20 ml). The two layers were separated and the aqueous layer was extracted with EtOAc (2×20 ml). The organic layers were combined, dried over MgSO ₄ , filtered, and the volatiles were evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel using DCM/EtOAc to give AOM C3 as a yellowish solid (114 mg, 66% yield). LC-MS (electrospray, negative ion mode): [M-H] 549, [M+H ₂ O-H] 567, [M+Cl] 585, (electrospray, positive ion mode): [M +H] 551, [M+H ₂ O+H] 569.

Synthesis of intermediate compound AOM C4: AOM C3 (0.114 g, 0.21 mmol), freshly activated 4A molecular sieves, proton sponge (0.066 g, 0.31 mmol) and a magnetic stirrer were placed under N ₂ , and anhydrous trimethylphosphate (1.0) was added ml). This reaction mixture was cooled at ~10°C, and freshly distilled POCl ₃ (23 μl, 0.25 mmol) was added dropwise. This reaction mixture was stirred at ~10°C for 1 hour. A solution of pyrophosphate as bis-tri-n-butylammonium salt (0.5 M in DMF, 1.7 ml, 0.85 mmol) and anhydrous tri-n-butylamine (0.41 ml, 1.74 mmol) was premixed and added to an ice-cold solution of activated nucleosides in one portion. This mixture was stirred vigorously for 5 minutes at room temperature. This reaction mixture was poured into a separate flask containing vigorously stirred aq. solution of 2 M TEAB (~10 ml). This reaction flask was washed with a small amount of H ₂ O, and the washes were added to a 2 M TEAB solution. The combined mixture was then stirred at room temperature for 4 hours, after which the solvent was evaporated under reduced pressure. The residue was dissolved in aq. NH ₃ (35%, ~10 ml) and stirred at room temperature overnight. This reaction mixture was concentrated in vacuo and purified by flash chromatography on DEAE-Sephadex. The product was further purified by preparative HPLC to obtain pure AOM C4 (62 μmol, 30% yield, determined by UV-vis spectrometry, λ _max equal to 294 nm, ε equal to 8600 M ^-1 cm ^-1 ). LC-MS (electrospray, negative ion mode): [M-N] 589.

Synthesis of 3'-AOM-ffC-LN3-SO7181: LN3-SO7181 (0.0205 mmol) was dissolved in anhydrous DMA (4 ml) under N ₂ . N,N-Diisopropylethylamine (28.6 μL, 0.164 mmol) was added, followed by TSTU (0.1 M in DMA, 234 μL, 0.0234 mmol). The reaction mixture was stirred under N ₂ at room temperature for 1 hour. Meanwhile, an aqueous solution of AOM C4 (0.0101 mmol) was evaporated to dryness under reduced pressure, resuspended in 0.1 M aq. TEAB (400 μL) and added to the LN3-SO7181 solution. This reaction mixture was stirred at RT (room temperature) for 17.5 hours and then quenched with 0.1 M aq. TEAB (4 ml). The crude product was purified by flash chromatography on DEAE-Sephadex. This product was further purified by preparative HPLC to obtain pure 3'-AOM-ffC-LN3-SO7181 (6.81 µmol, 67% yield, determined by UV-visible spectrometry, λ _max = 644 nm, ε = 200,000 M ^-1 cm ^-1 ). LC-MS (electrospray, negative ion mode): [M-H] 1561, [M-2H] 781, [M-3H] 520.

Synthesis of AOM intermediate A2: nucleoside A1 (716 mg, 0.95 mmol) was dissolved in 10 ml of anhydrous dichloromethane under N ₂ atmosphere, cyclohexene (481 μl, 4.75 mmol) was added and this solution was cooled to approximately -15°C . Sulfuryl chloride (distilled, 92 µl, 1.14 mmol) was added dropwise and the reaction mixture was stirred for 20 minutes. After all of the starting material had been consumed, additional cyclohexene (481 μL, 4.75 mmol) was added and the reaction mixture was evaporated to dryness under reduced pressure. The residue was quickly purged with nitrogen, then allyl alcohol (5 ml, ~100 mmol) was added with stirring at 0°C. This reaction mixture was stirred at 0°C for 1 hour, then quenched with 50 ml of saturated aq. _NaHCO3 . This reaction mixture was extracted with 2×100 ml ethyl acetate. The combined organic phases were washed with 100 ml water and 100 ml brine, then dried over MgSO ₄ , filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel using petroleum ether/EtOAc. Yield 60% (435 mg, 0.57 mmol). LC-MS (ES (electrospray ionization) and CI (chemical ionization): (positive ion) m/z 763 (M+H ⁺ ); (negative ion) m/z 761 (M-H ⁺ ).

Synthesis of intermediate AOM A3: AOM A2 nucleoside (476 mg, 0.62 mmol) was dissolved in dry THF (5 ml) under N ₂ atmosphere, then a solution of 1.0 M TBAF in THF (750 μl, 0.75 mmol) was added. . This solution was stirred at room temperature for 1.5 hours. This solution was diluted with 50 ml EtOAc, then washed with 100 ml sat. NaH ₂ PO ₄ (pH 3) and 100 ml of brine. The organic phase was dried over MgSO ₄ , filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel using EtOAc/MeOH. Yield 90% (292 mg, 0.55 mmol). LC-MS (ER and CI): (positive ion) m/z 525 (M+H ⁺ ); (negative ion) m/z 523 (M-H ⁺ ).

Synthesis of intermediate AOM A4: AOM A3 nucleoside (285 mg, 0.544 mmol) was dried under reduced pressure over P ₂ O ₅ for 18 hours. Anhydrous triethyl phosphate (2 ml) and some freshly activated 4 were added to it under nitrogen. molecular sieves, then this reaction flask was cooled to 0°C in an ice bath. Freshly distilled POCl ₃ (61 µl, 0.65 mmol) was added dropwise, followed by Proton Sponge® (175 mg, 0.816 mmol). After addition, this reaction mixture was further stirred at 0°C for 15 minutes. A 0.5 M solution of bis-tri-n-butylammonium pyrophosphate (5.4 mL, 2.72 mmol) in anhydrous DMF was then quickly added, followed immediately by tri-n-butylamine (540 μL, 2.3 mmol). This reaction mixture was kept in an ice-water bath for an additional 10 minutes, then quenched by pouring it into 1 M aqueous triethylammonium bicarbonate (TEAB, 20 ml) and stirred at room temperature for 4 hours. All solvents were evaporated under reduced pressure. 35% aqueous ammonium solution (20 ml) was added to the above residue and the mixture was stirred at room temperature for at least 5 hours. The solvents were then evaporated under reduced pressure. The crude product was first purified by ion exchange chromatography on DEAE-Sephadex A25 (100 g). The column was eluted with a gradient of aqueous triethylammonium bicarbonate. The fractions containing the triphosphate were combined and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by preparative scale HPLC using a YMC-Pack-Pro C18 column, eluting with 0.1 M TEAB and acetonitrile. LC-MS (ER and CI): (negative ion) m/z 612 (M-H ⁺ ); (positive ion) m/z 614 (M+H ⁺ ), 715 (M+Et ₃ NH ⁺ ).

General procedure for the synthesis of ffA: linker dye (0.020 mmol) was dissolved in 2 ml of anhydrous N,N'-dimethylacetamide (DMA). N,N'-diisopropylethylamine (28.4 µl, 0.163 mmol) was added, followed by N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate as a 0.1 M solution in anhydrous DMA (TSTU, 232 µl, 0.023 mmol). This reaction mixture was stirred under nitrogen at room temperature for 1 hour. Meanwhile, an aqueous solution of AOM A4 triphosphate (0.01 mmol) was evaporated to dryness under reduced pressure and resuspended in 200 μl of a 0.1 M solution of triethylammonium bicarbonate (TEAB) in water. An activated dye-linker solution was added to this triphosphate, and the reaction mixture was stirred at room temperature for 18 hours. The crude product was first purified by ion exchange chromatography on DEAE-Sephadex A25 (25 g). The fractions containing the triphosphate were combined and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by RP-HPLC (reverse phase high performance liquid chromatography) on a preparative scale using a YMC-Pack-Pro C18 column. 3'-AOM-ffA-LN3-NR7180A: yield 38% (3.8 µmol). LC-MS (ES): (negative ion) m/z 1459 (M-H ⁺ ), 729 (M-2H ⁺ ), 486 (M-3H ⁴ ). 3'-AOM-ffA-LN3-BL-NR550S0: yield 37% (3.7 µmol). LC-MS (ES): (negative ion) m/z 1771 (M-H ⁺ ), 885 (M-2H ⁺ ), 589 (M-3H ⁺ ). 3'-AOM ffA-LN3-BL-NR650C5: yield 51% (51 µmol). LC-MS (ES): (negative ion) m/z 1917 (M-H ⁺ ), 958 (M-2H ⁺ ), 645 (M-3H ⁺ ).

Synthesis of intermediate compound AOM G4: the known nucleoside dG3 (100 mg, 0.143 mmol) was dissolved in 10 ml of anhydrous dichloromethane under an atmosphere of N ₂ , cyclohexene (72 μl, 0.714 mmol) was added and this solution was cooled to -12°C. Sulfuryl chloride (distilled, (1 M in DCM), 171 µl, 0.171 mmol) was added dropwise and the reaction mixture was stirred for 10 minutes. An additional portion of cyclohexene (72 μl, 0.714 mmol) was added and the reaction mixture was stirred for 30 minutes at -12°C. This reaction mixture was evaporated to dryness under reduced pressure, the residue was purged with nitrogen, and ice-cold pure allylic alcohol (distilled, 0.8 mL, 12 mmol) was added with stirring at -12°C. This reaction mixture was stirred at -12°C for 60 minutes, then quenched with 2 ml of saturated aq. _NaHCO3 . This mixture was separated with ethyl acetate (2 ml), the aqueous layer was extracted with ethyl acetate. The combined organic phases were washed with 4 ml water and 4 ml brine, dried over MgSO ₄ , filtered and evaporated to a crude oil. The residue was purified by flash chromatography on silica gel to give AOM G4 as a clear oil. Yield 36% (50.9 mg, 0.072 mmol). LC-MS (ER and CI): (positive ion) m/z 710 [M+H] ⁺ ; (negative ion) m/z 708 [M-H] ^- .

Synthesis of AOM G5 intermediate: AOM-G4 nucleoside (111 mg, 0.156 mmol) was dissolved in dry THF (5 ml) under N ₂ atmosphere. Acetic acid (27 μL, 0.468 mmol) was added, followed by the addition of a solution of 1.0 M TBAF in THF (296 μL, 0.296 mmol). This solution was stirred at room temperature for 5 hours. This solution was diluted with 10 ml of EtOAc, washed with 10 ml of 0.05 M aq. HCl, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over MgSO ₄ , filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel to obtain AOM G5 as a white solid. Yield 44% (32.4 mg, 0.068 mmol). LC-MS (ER and CI): (positive ion) m/z 472 [M+H] ⁺ ; (negative ion) m/z 470 [M-H] ^- .

Synthesis of 3'-AOM-pppG: nucleoside AOM-G5 (79 mg, 0.168 mmol) with freshly activated 4 molecular sieves were dried under reduced pressure over P ₂ O ₅ for 18 hours. Proton Sponge® (175 mg, 0.816 mmol) and anhydrous triethyl phosphate (0.8 ml) were added under nitrogen and stirred at room temperature for 1 hour. The reaction flask was cooled to 0°C in an ice bath, freshly distilled POCl ₃ (19 μL, 0.202 mmol) was added dropwise, and the reaction mixture was stirred at 0°C for 15 minutes. A 0.5 M solution of pyrophosphate as bis-tri-n-butylammonium salt (1.68 ml, 0.84 mmol) in anhydrous DMF was then quickly added, followed by the immediate addition of tri-n-butylamine (168 µl, 0.705 mmol) . This reaction mixture was removed from the ice/water bath and stirred vigorously for 5 minutes, then quenched by pouring it into 1 M aqueous triethylammonium bicarbonate (TEAB, 6 ml) and stirred at room temperature for 18 hours. All solvents were evaporated under reduced pressure. The residue was dissolved in 35% aqueous ammonia (10 ml) and stirred at room temperature for at least 5 hours. The solvents were then evaporated under reduced pressure and further co-evaporated with water. The crude product was first purified by ion exchange chromatography on DEAE-Sephadex A25 (50 g). The column was eluted with a linear gradient of aqueous triethylammonium. Fractions containing triphosphate were collected and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by HPLC on a preparative scale using a YMC-Pack-Pro C18 column. 3'-AOM-pppG was obtained as triethylammonium salt. Yield 24% (39.7 µmol). LC-MS (ER and CI): (negatively charged ion) m/z 576 [M-H] ^- ; (positively charged ion) m/z 578 [M+H] ⁺ .

3′-AOM-ffT-LN3-NR550S0 was synthesized in the same manner as described for the preparation of 3′-AOM ffA and ffC.

Synthesis of intermediate T1: 5-iodo-2'-deoxyuridine (3 g, 8.4 mmol) and palladium(II) acetate (1.6 g, 7.14 mmol) were dissolved in dry degassed DMF, then N-allyltrifluoroacetamide was added (6.4 ml, 42 mmol). The solution was placed under vacuum, then purged with nitrogen 3 times, then degassed triethylamine (2.3 mL, 16.8 mmol) was added. This solution was heated to 80°C for 2 hours. The black mixture was cooled to room temperature, then diluted with 50 ml of methanol. Approximately 0.5 g of activated carbon was added and the solution was filtered on Celite, then evaporated under reduced pressure to obtain a brown thick oil. This crude material was purified by silica gel chromatography using EtOAc/MeOH. Yield: (2.27 g, 5.99 mmol). LC-MS (ER and CI): (negatively charged ion) m/z 378 (M-H ⁺ ).

Synthesis of intermediate T2: 5-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T1) (2.55 g, 6.72 mmol) was dissolved in dry DMF. Imidazole (1.37 g, 20.1 mmol) was added, followed by 4-(dimethylamino)pyridine (410 mg, 3.36 mmol). This reaction mixture was cooled to 0°C, then tert-butyl(chloro)diphenylsilane (1.92 mL, 7.39 mmol) was slowly added in 3 portions at 30 minute intervals. This reaction mixture was stirred at 0°C for 6 hours. The solvent was then evaporated, the residue was resuspended in 200 ml EtOAc and washed with 2× 200 ml aq. saturated NaHCO ₃ and 200 ml of water, then 100 ml of brine. The organic phase was dried over MgSO ₄ , filtered and evaporated to dryness. The crude product was purified by flash silica chromatography using DCM/EtOAc. Yield 68% (2.806 g, 4.54 mmol). LC-MS (ER and CI): (positively charged ion) m/z 618 (M+H ⁺ ); (negatively charged ion) m/z 616 (M-H ⁺ ).

Synthesis of intermediate T3: 5'-O-(tert-butyldiphenylsilyl)-5-[3-(2,2,2-trifluoroacetamido)-allyl]-2'-deoxyuridine (T2) (2.8 g, 4.53 mmol) was dissolved in 10 ml of anhydrous DMSO (136 mmol), then glacial acetic acid (16 ml, 272 mmol) and acetic anhydride (16 ml, 158 mmol) were added. This reaction mixture was heated to 50°C for 6 hours, then quenched with 200 ml of aq. saturated NaHCO ₃ . Once the solution stopped forming bubbles, it was extracted with 2 x 150 ml EtOAc. The organic phases were combined and washed with 2×200 ml aq. saturated NaHCO ₃ , 200 ml of water and 100 ml of brine. The organic phase was dried over MgSO ₄ , filtered and evaporated to dryness. The crude product was purified by flash silica chromatography using DCM/EtOAc. Yield 77% (2.375 g, 3.51 mmol). LC-MS (ER and CI): (positive ion) m/z 678 (M+H ⁺ ); (negative ion) m/z 676 (M-H ⁴ ).

Synthesis of intermediate T4: 5'-O-(tert-butyldiphenylsilyl)-3'-O-methylthiomethyl-5-[3-(2,2,2-trifluoroacetamido)allyl]-2'-deoxyuridine (T3) (310 mg, 0.45 mmol) was dissolved in 5 ml of anhydrous dichloromethane under N ₂ atmosphere, cyclohexene (228 μl, 2.25 mmol) was added and this solution was cooled to approximately -15°C. Sulfuryl chloride (distilled, 55 μl, 0.675 mmol) was added dropwise and the reaction mixture was stirred for 20 minutes. After all the starting material had been consumed, an additional portion of cyclohexene (228 μL, 2.25 mmol) was added and the reaction mixture was evaporated to dryness under reduced pressure.

The residue was quickly purged with nitrogen, then ice-cold allylic alcohol (2.5 ml) was added with stirring at 0°C. This reaction mixture was stirred at 0°C for 35 minutes, then quenched with 25 ml of saturated aq. NaHCO ₃ , then further diluted with 100 ml saturated aq. _NaHCO3 . This mixture was extracted with 2×50 ml ethyl acetate. The combined organic phases were dried over MgSO ₄ , filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel using DCM/EtOAc. Yield 69% (214 mg, 0.311 mmol). LC-MS (ER and CI): (positively charged ion) m/z 688 (M+H ⁺ ); (negatively charged ion) m/z 686 (M-H ⁺ ).

Synthesis of intermediate T5: 5'-O-(tert-butyldiphenylsilyl)-3'-O-allyloxymethyl-5-[3-(2,2,2-trifluoroacetamido)allyl]-2'-deoxyuridine (T4) (210 mg, 0.305 mmol) was dissolved in dry THF (3 ml) under N ₂ atmosphere. A solution of 1.0 M TBAF in THF (367 μL, 0.367 mmol) was added. This solution was stirred at room temperature for 3 hours. The solution was diluted with 50 ml EtOAc, then washed with 50 ml sat. NaH ₂ PO ₄ (pH 3) and 50 ml of water. The organic phase was dried over MgSO ₄ , filtered and evaporated to dryness. The residue was purified by flash chromatography on silica gel using DCM/EtOAc. Yield 95% (130 mg, 0.289 mmol). LC-MS (ER and CI): (negatively charged ion) m/z 448 (M-H ⁺ ), 484 (M+Cl ^- ).

Synthesis of intermediate T6: 3'-O-allyloxymethyl-5-[3-(2,2,2-trifluoroacetamido)allyl]-2'-deoxyuridine (T5) (120 mg, 0.267 mmol) dried under reduced pressure over P ₂ O ₅ for 18 hours. Anhydrous triethyl phosphate (1 ml) and a certain amount of freshly activated 4 were added to it under nitrogen. molecular sieves, then the flask with the reaction mixture was cooled to 0°C. Freshly distilled POCl ₃ (30 µl, 0.32 mmol) was added dropwise, followed by Proton Sponge® (85 mg, 0.40 mmol). After addition, this reaction mixture was further stirred at 0°C for 15 minutes. A 0.5 M solution of bis-tri-n-butylammonium pyrophosphate (2.7 mL, 1.33 mmol) in anhydrous DMF was then quickly added, followed immediately by tri-n-butylamine (270 μL, 1.2 mmol). This reaction mixture was kept in an ice-water bath for an additional 10 minutes, then quenched by pouring it into 1 M aqueous triethylammonium bicarbonate (TEAB, 10 ml) and stirred at room temperature for 4 hours. All solvents were evaporated under reduced pressure. A 35% aqueous ammonia solution (10 ml) was added to the above residue, and the mixture was stirred at room temperature for 18 hours. The solvents were then evaporated under reduced pressure and the residue was resuspended in 10 ml of 0.1 M TEAB and filtered. The filtrate was first purified by ion exchange chromatography on DEAE-Sephadex A25 (100 g). The column was eluted with aqueous triethylammonium bicarbonate (TEAB). The fractions containing the triphosphate were combined and the solvent was evaporated to dryness under reduced pressure. The crude material was further purified by HPLC on a preparative scale using a YMC-Pack-Pro C18 column. Compound T6 was obtained as triethylammonium salt. Yield 33% (89 µmol). LC-MS (ER and CI): (negatively charged ion) m/z 592 (M-H ⁺ ), 295 (M-2H ⁺ ).

Synthesis of S'-AOM-ffT-LN3'-NRSSOSO: The dried known compound LN3-NR550S0 (0.015 mmol) was dissolved in anhydrous DMA (2 ml) under N ₂ . N,N-Diisopropylethylamine (17 μL, 0.1 mmol) was added, followed by TSTU (0.1 M in DMA, 180 μL, 0.018 mmol). This reaction mixture was stirred under N ₂ at room temperature for 1 hour. Meanwhile, an aqueous solution of T6 (0.01 mmol) was evaporated to dryness under reduced pressure, resuspended in 0.1 M aq. TEAB (200 μl) and added to the LN3-NR550S0 solution. This reaction mixture was stirred at RT for 18 hours and then quenched with 0.1 M aq. TEAB (4 ml). The crude product was purified by flash chromatography on DEAE-Sephadex. The product was further purified by preparative HPLC to obtain pure 3'-AOM-ffT-LN3'-NR550S0. Yield 67% (41 µmol, as determined by spectrometry in the UV-visible region, λ _max equal to 550 nm, e equal to 125000 M ^-1 cm ^-1 ). LC-MS (ES): (negatively charged ion) m/z 1521 (M-H ⁺ ), 761 (M-2H ⁴ ), 507 (M-3H ⁺ ).

Sequencing-by-synthesis experiments

ffN was then analyzed by sequencing using an Illumina MiniSeq® instrument. With the exception of the new inclusion mixture including these ffN, all standard commercially available reagents were used. We used the standard 2×150 recipe. In addition to standard sequencing-by-synthesis (SBS) protocols, a 5-second incubation in a palladium digest solution (Pd:THP equal to 1/5 in DEEA as described in Example 4) was added to unblock the 3'-AOM.

In the first experiment, the following ffNs were used in the inclusion mixture: 3'-AOM-ffT-LN3-NR550S0, 3'-AOM-ffA-LN3-BL-NR550S0, 3'-AOM-ffA-LN3-BL-NR650C5, 3' -AOM-ffA-LN3-NR7180A, 3′-AOM-ffC-LN3-SO7181 and 3′-AOM-pppG (dark G), and the sequencing result for read 1 is summarized below.

In the second experiment, unlabeled 3'-AOM-pppT was synthesized similarly to the preparation of 3'-AOM-pppG described above (LC-MS (ES): (negative ion) m/z 551 (M-H ⁴ )). It was used in the presence of the commercially available green ffG-LN3-PEG12-ATT0532 (used on 4-channel Illumina systems) in sequencing, and the same ffA and ffC were used as the ffA and ffC described in the first experiment above. The results are summarized below. There were significant improvements in phasing and prephasing values, and no signal attenuation was observed (FIG. 3A). In addition, the error rates for both read fragment 1 and read fragment 2 also decreased.

In another experiment, an inclusion mixture containing 3'-AOM-ffT-LN3'-NR550S0, 3'-AOM-ffA-LN3-BL-NR550S0, 3'-AOM-ffA-LN3-BL-NR650C5, 3'- AOM-ffA-LN3-NR7180A, 3′-AOM-ffC-LN3-S07181, and 3′-AOM-pppG (dark G). Similar to previous runs, a 5-second incubation with a digestion mixture containing palladium catalyst (Pd/THP equal to 1:10; 100 mM DEEA, as described in Example 4) was added to the standard SBS run. Standard MiniSeq® DNA polymerase was used, but with a 2-fold turn-on time. No signal attenuation phenotype was observed (FIG. 3B). Additionally, these sequencing results were compared to commercially available MiniSeq® runs (average of 3; N = 3) using ffN with a standard azidomethyl blocking group. It was observed that the error rates were almost identical (FIG. 3C). The sequencing results are summarized below.

In addition, raw sequencing metrics for ffN with 3'-AOM blocking groups were compared to raw sequencing metrics generated by a standard commercially available MiniSeq® kit including Pol 812 DNA polymerase and FIG. 4A shows comparable results. Very low prephasing was observed due to the improved stability of 3′-AOM-ffN. However, phasing was still elevated even when 2x the on time was used.

In yet another experiment, a different DNA polymerase (Pol 1901) was used instead of the DNA polymerase in the commercially available MiniSeq® kits (Pol 812). Pol 1901 provided the standard 1× turn-on time in sequencing instead of the 2× turn-on time described above. In addition, the Pd incubation of the digestion mixture was reduced by half compared to the standard run. This provided a 10% time saving for the complete SBS chemical cycle. Sequencing performance was significantly improved and exceeded that obtained from standard commercially available kits containing a 3'-O-azidomethyl blocking group (FIG. 4 B).

Analysis of the stability of the 3' blocking group during sequencing

To demonstrate the improved stability of ffNs with a 3'-AOM, they were compared in parallel to standard MiniSeq® ffNs with a 3'-O-azidomethyl group. Two sets of ffN were incubated at 45°C for several days in standard inclusion mixture preparations, excluding only DNA polymerase. For each time point, fresh polymerase was added to complete the inclusion mixture immediately before loading onto the MiniSeq®. Sequencing conditions described previously were used. % prephasing is a direct indicator of the percentage of 3'OH-ffN present in the mixture, therefore it directly correlates with the stability of the 3' blocking group. Prephasing values for both sets of ffN were recorded and plotted (FIG. 5). At 45°, it was observed that ffNs containing 3'-AOM were 6 times more stable than standard ffNs with a 3'-O-azidomethyl group. Sequencing performance also confirmed the trend observed during solution stability analysis—the 3'-AOM block was significantly more stable than the 3'-O-azidomethyl group.

Example 6. Preparation of 3'-O-thiocarbamate-protected nucleosides In this example, various 3'-O-thiocarbamate-protected nucleosides T were prepared according to Scheme 8.

Preparation of T-7: 5'-O-(4,4'-dimethoxytrityl)thymidine (1.0 g, 1.836 mmol) was added to an oven-dried, nitrogen-purged 100 mL flask. This mixture was co-evaporated with anhydrous DMF (3×20 ml) and placed under nitrogen. Anhydrous DCM (9.2 mL) and 4-dimethylaminopyridine (224 mg, 0.184 mmol) were added and stirred at room temperature until a homogeneous solution was formed. 1,1'-thiocarbonyldiimidazole (360 mg, 2.02 mmol) was then quickly added under a stream of nitrogen and the reaction mixture was resealed and stirred at room temperature for 2 hours until all starting material was consumed. This reaction mixture is filtered through a pad of silica gel and the filter cake is washed with EtOAc (10 ml). Volatiles were removed in vacuo and the crude residue was used without further purification.

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

Preparation of T-9: the starting nucleoside T-8 (320 mg, 0.504 mmol) was dissolved in a minimal amount of acetonitrile in a 50 ml round-bottom flask in air. A 5:1 AcOH/H ₂ O solution (12.5 ml: 2.5 ml) was added at one time and the reaction mixture was stirred at room temperature until all the starting material was consumed (2-4 hours). Evaporation of all volatiles under vacuum and co-evaporation of the residue in toluene (2 x 60 ml) gave the crude product as a whitish solid. This crude product was purified by flash column chromatography to give T-9 as a white solid. Yield: 123 mg (74%). LC-MS (electrospray, negative ion mode) [M-H] 328.10.

Following a similar synthetic procedure using the appropriate MeNH ₂ or NH ₃ , nucleosides with two thiocarbamate protecting groups were also prepared. The general reaction scheme is shown below:

Analysis of the stability of 3'-O-thiocarbamate blocking groups

Stability assays for the nucleotide 5'-mP 3'-DMTC T were performed in parallel in inclusion buffer solution with the standard nucleotide 5'-mP 3'-O-azidomethyl T.

For both 5'-mP 3'-DMTC T and 5'-mP 3'-O-azidomethyl T, the final solution volume was 1 ml, and the final concentrations of the corresponding nucleotides were 0.1 mM for both. Other components of the aqueous buffer solution include ethanolamine (EA), ethanolamine HCl, NaCl (100 mM) and EDTA (2.5 mM). This buffer solution has the following concentration: 0.5 M buffer EA, 0.5 M NaCl, 0.01 M EDTA.

Stability Analysis Methodology

200 μL of 10× buffer solution was added to a 1.7 mL polypropylene microtube with a snap cap and diluted with the correct volume of 18 mΩ water. The appropriate nucleoside was then added, the vial was capped, and mixing was accomplished by inversion, gentle stirring, or micropipette suction. A 40 μl aliquot was taken and analyzed by HPLC to act as the input value (or t equal to 0). These vials were then placed in a heating mantle preheated to 65°C, covered with a thick layer of aluminum foil and allowed to heat for one month. 40 μl aliquots were taken periodically (week 1: once daily. Weeks 2-4: 1 times every 2 days) and analyzed by HPLC to determine the percentage of starting material and the percentage of deblocked (3'-OH) nucleotide in these samples. HPLC analysis was performed by measuring the peak area of the parent nucleotide and the 3'OH peak. These values were used to graphically calculate the percentage of unblocked nucleotide that was present and used to compare stability in inclusion buffer between samples. In FIG. 6 illustrates comparative stability results of three different 3' thiocarbamate-blocked nucleotides with 3'-O-azidomethyl blocking group-blocked nucleotides at 65°C. It was observed that while a nucleotide with 3'-OC(=S)NH ₂ or 3'-OC(=S)NHCH ₃ was less stable than a nucleotide protected by a standard 3'-O-azidomethyl group, a nucleotide with 3'-DMTC provided improved stability during the 9-day assay period. DMTC, as such, exhibited superior stability over the standard azidomethyl blocking group.

Example 7 Deblocking Assay for 3'-O-thiocarbamate Blocking Group

In this example, deblocking or deblocking assays for 5'-mP 3'-DMTC T and standard 5'-mP 3'-O-azidomethyl T were performed individually in a unique solution for each blocking group. Conditions were set to mimic the standard Illumina release reagent as closely as possible and following the same methodology. The same concentrations of active deblocking reagent, buffer, and nucleoside were maintained in all assays, but the identity of each component was unique. In this manner, the observed differences in rates between individual deblocking chemistries cannot be due to differences in drug concentration.

General methodology for release analysis

Each reaction component was prepared individually as a concentrated stock solution in 18 mΩ water, stored appropriately, and aliquots were combined in the specific order given below. The reaction was started by adding a previously prepared deblocking reagent. Final concentrations: nucleoside (0.1 mM), active deblocking reagent (1 mM), additive (specific to the deblocking reagent), buffer (100 mM). Final volume: 2000 µl.

A pre-prepared buffer solution was added to a 3 ml glass vial, followed by the addition of a pre-prepared additive. This mixture was diluted with the correct volume of 18 mΩ water and stirred for 10 minutes. An aliquot of the nucleotide solution was then added and mixed for 5 minutes. A 40 μL aliquot was then removed, a quenching reagent was added, and analyzed by HPLC as a reference peak (or t = 0 min). Then the deblocking reagent was added to the stirred solution in one step, and the countdown began. At specific time points, 40 μl aliquots were removed and immediately quenched with a suitable quenching reagent, then analyzed by HPLC to determine the amount of released nucleotide that appeared at those specific time points. The results were plotted graphically and used to compare the effectiveness and efficiency of release.

DMTC release

Nucleotide: 5'-mP 3'-DMTC T. Active deblocking reagent: NaIO ₄ (0.1 M in 18 mΩ water) or Oxone® (0.1 M in 18 mΩ water). Additive: no. Buffer for NaIO ₄ : phosphate buffer with pH 6.75 (1 M in 18 mΩ water). Buffer for Oxone®: phosphate buffer pH 8.65 (1 M in 18 mΩ water). Quenching reagent: sodium thiosulfate. The conditions for deprotection of 3'-O-azidomethyl are the same as those described in Example 3.

HPLC analysis was performed by measuring the area of the parent nucleoside peak, the 3'-OH peak, and any other nucleotide peaks that appear in the HPLC chromatogram. These values were used to calculate the percentage of original nucleotide and unblocked nucleotide, which was presented graphically and used to compare the rate, efficiency and effectiveness of unblocking between samples. The comparative result is shown in FIG. 7. It was observed that deblocking of DMTC using NaIO ₄ was not effective. However, the % of starting material remaining was significantly less for the nucleotide with the DMTC blocking group when cleaving DMTC using Oxone®. In conclusion, DMTC demonstrated superior deprotection rate (with Oxone®) over that of the standard azidomethyl blocking group.

Claims (65)

1. A nucleotide containing ribose or deoxyribose having a 3'-OH leaving blocking group covalently attached to the 3' carbon atom. 2. Nucleotide according to claim 1, containing 2’-deoxyribose. 3. A nucleotide according to claim 1 or 2, wherein said nucleotide contains a triphosphate at the 5' position of ribose or deoxyribose. 4. Nucleotide according to any one of paragraphs. 1-3, where the specified nucleotide is covalently attached to the detectable label, possibly through a cleavable linker. 5. The nucleotide according to claim 4, in which the detectable label is covalently attached to the nitrogenous base of the nucleotide through a cleavable linker. 6. The nucleotide of claim 5, wherein said cleavable linker contains an azide moiety, an -O-allyl moiety, a disulfide moiety, an acetal moiety, or a thiocarbamate moiety. 7. The nucleotide according to claim 5, where the linker contains a fragment selected from the group consisting of: where X is selected from the group consisting of O, S, NH and NQ, where Q is a substituted or unsubstituted C _1-10 -alkyl group, Y is selected from the group consisting of O, S, NH and N(allyl), T is represents hydrogen or a substituted or unsubstituted C ₁ -C ₁₀ alkyl group, and the symbol “*” indicates where the fragment is connected to a nucleotide residue. 8. The nucleotide according to claim 5, where the linker is selected from the group consisting of: where B represents a nitrogenous base; Z represents -N ₃ (azido), -OC ₁ -C ₆ alkyl, -O-C ₂ -C ₆ alkenyl or -O-C ₂ -C ₆ alkynyl; and FI contains a fluorescent label, which may contain an additional linker structure. 9. Nucleotide according to any one of paragraphs. 1-3 containing a nitrogenous base selected from the group consisting of 10. Nucleotide according to any one of paragraphs. 1-8 containing a nitrogenous base selected from the group consisting of where L is a linker and Dye is a fluorescent dye. 11. Nucleotide according to any one of paragraphs. 1-8, having a structure selected from the group consisting of where L is a linker, Dye is a fluorescent dye, and R is ribose or deoxyribose having a 3'-OH leaving a blocking group covalently attached to the 3' carbon atom. 12. An oligonucleotide comprising a nucleotide according to any one of claims. 1-11, wherein the phosphodiester bond is formed between the 3' carbon atom of the oligonucleotide and the 5' carbon atom of the specified nucleotide. 13. A method for producing a growing polynucleotide complementary to the target single-stranded polynucleotide in a sequencing reaction, including the inclusion of a nucleotide according to any one of claims. 1-11 into a growing complementary polynucleotide, wherein inclusion of said nucleotide prevents the introduction of any subsequent nucleotide into the growing complementary polynucleotide. 14. The method according to claim 13, in which the inclusion of a nucleotide according to any one of claims is carried out. 3-11 into growing complementary polynucleotide. 15. The method according to claim 13 or 14, in which the incorporation of the nucleotide is carried out by a polymerase. 16. A method for determining the sequence of a target single-stranded polynucleotide, comprising: (a) including a nucleotide according to any one of claims. 4-11 to a copy of a polynucleotide chain complementary to at least part of the target polynucleotide chain; (b) identifying the identity of a nucleotide included in a copy of the polynucleotide chain; And (c) chemical removal of the tag and 3'-OH blocking group from a nucleotide included in a copy of the polynucleotide chain. 17. The method of claim 16, wherein the nucleotide included in step (a) is a nucleotide 5'-triphosphate. 18. The method according to claim 16 or 17, further comprising (d) washing a copy of the polynucleotide chain from chemically removing the label and 3'-OH blocking group. 19. The method according to claim 18, in which in step (d) a palladium radical scavenger is used. 20. The method of claim 18 or 19, further comprising repeating steps (a)-(d) until the sequence of said portion of the template polynucleotide chain is determined. 21. Method according to any one of paragraphs. 18-20, in which steps (a)-(d) are repeated at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times or at least 300 times. 22. Method according to any one of paragraphs. 16-21, where the label and the 3'-OH blocking group are removed from a nucleotide included in a copy of the polynucleotide chain in a single chemical reaction. 23. The method of claim 22, wherein step (c) comprises contacting the incorporated nucleotide with a cleavage solution containing a palladium catalyst. 24. Method according to any one of paragraphs. 16-21, in which the label and 3'-OH blocking group are removed from a nucleotide included in a copy of the polynucleotide chain in two separate chemical reactions. 25. The method of claim 24, wherein step (c) comprises contacting the incorporated nucleotide with a cleavage solution containing phosphine and a palladium catalyst. 26. The method of claim 25, wherein the phosphine is tris(hydroxymethyl)phosphine, tris(hydroxyethyl)phosphine or tris(hydroxypropyl)phosphine. 27. Method according to any one of paragraphs. 23, 25, or 26, wherein the cleavage solution containing the palladium catalyst further contains one or more buffering reagents selected from the group consisting of a primary amine, a secondary amine, a tertiary amine, a carbonate salt, a phosphate salt, a borate salt, and combinations. 28. The method of claim 27, wherein the buffering reagents are selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonate salt, phosphate salt, borate salt, 2-dimethylaminoethanol (DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED) and N,N,N',N'-tetraethylethylenediamine (TEEDA) and combinations thereof. 29. Method according to any one of paragraphs. 23 and 25-28, where the splitting solution additionally contains ascorbic acid or its salt. 30. Method according to any one of paragraphs. 16-29, carried out in multiplex format. 31. Method according to any one of paragraphs. 16-30, carried out in a chip format using surface-bound target nucleic acids, wherein the target nucleic acids are bound to the surface in a spatially distinguishable manner. 32. The method of claim 31, wherein the chip includes multiple copies having the same target nucleic acid sequence at each site. 33. The method according to claim 32, wherein multiple copies of the target nucleic acids are formed as a result of bridging amplification. 34. The method of claim 32, wherein multiple copies of the target nucleic acids are present in the concatemers. 35. Method according to any one of paragraphs. 31-34, where the chip has a density of at least about 10 elements/cm ² , 100 elements/cm ² , 500 elements/cm ² , 1000 elements/cm ² , 5000 elements/cm ² , 10,000 elements/cm ² , 50,000 elements /cm ² , 100,000 elements/cm ² , 1,000,000 elements/cm ² or 5,000,000 elements/cm ² . 36. Method according to any one of paragraphs. 16-35, characterized in that the method uses the first type of nucleotides - which is detected in the first channel, the second type of nucleotides - which is detected in the second channel, the third type of nucleotides - which is detected in both the first and second channels, and the fourth type nucleotides - which lacks a label and is not detected or minimally detected in both channels. 37. Method according to any one of paragraphs. 16-35, characterized in that the method uses four different types of nucleotides, each type of nucleotide having a spectrally different label. 38. Method according to any one of paragraphs. 16-37, where the incorporation of the nucleotide is carried out using DNA polymerase. 39. A kit for use in multiplex sequencing by synthesis, containing one or more than one nucleotide according to any one of paragraphs. 1-11. 40. The set of claim 39, wherein at least one nucleotide is a nucleotide 5'-triphosphate having a 3'-OH blocking group structure covalently attached to the 3' carbon of the 2' deoxyribose. 41. The kit according to claim 39 or 40, further containing an enzyme and a buffer suitable for the action of the enzyme. 42. The kit according to claim 41, wherein the enzyme is a polymerase. 43. The kit according to claim 42, wherein the polymerase is a DNA polymerase. 44. Set according to any one of paragraphs. 39-43, containing at least one nucleotide labeled with a detectable label according to any one of paragraphs. 4-11. 45. Set according to any one of paragraphs. 39-43, containing two, three or four types of nucleotides labeled with a detectable label, according to any one of paragraphs. 4-11. 46. The set according to claim 45, additionally containing a “dark” nucleotide that does not have a detectable label. 47. The kit of claim 45 or 46, wherein the different types of labeled nucleotides are labeled with spectrally different dyes. 48. Set according to any one of paragraphs. 45-47, wherein at least one type of nucleotide is labeled at the nitrogenous base with two or more different dyes. 49. Set according to any one of paragraphs. 45-48, where the first type of nucleotides is labeled with a first dye, the second type of nucleotides is labeled with a second dye spectrally different from the first dye, the third type of nucleotides is labeled as a mixture of the first and second dyes or a mixture of the first, second and third dyes, and the fourth the nucleotide does not contain a label. 50. The kit of claim 45, comprising four types of nucleotides labeled with a detectable label, wherein the nucleotides labeled with the detectable label are A, C, T and G, wherein each type of nucleotide has a fluorescent label with a noticeable fluorescence maximum, and each of the fluorescent tags is different from the other three fluorescent tags. 51. Set according to any one of paragraphs. 39-50, further containing a cleavage solution including tris(hydroxypropyl)phosphine, palladium (Pd) catalyst, ascorbic acid or a salt thereof, and a buffering reagent selected from the group consisting of ethanolamine (EA), tris(hydroxymethyl)aminomethane (Tris), glycine, carbonate salt, phosphate salt, borate salt, 2-dimethylaminoethanol (DMEA), 2-diethylaminomethanol (DEEA), N,N,N',N'-tetramethylethylenediamine (TEMED) and N,N,N' ,N'-tetraethylethylenediamine (TEEDA) and combinations thereof. 52. Set according to any one of paragraphs. 39-51, additionally containing a washing solution containing a palladium radical scavenger. 53. Use of a nucleotide according to any one of paragraphs. 1-11 in multiplex sequencing by synthesis. 54. Use according to claim 53, where the sequencing steps are repeated at least 50 times, at least 75 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times or at least at least 300 times.