WO2006074351A2

WO2006074351A2 - Reversible nucleotide terminators and uses thereof

Info

Publication number: WO2006074351A2
Application number: PCT/US2006/000432
Authority: WO
Inventors: Douglas Smith; Kevin Mckernan; Alan Blanchard
Original assignee: Agencourt Personal Genomics
Priority date: 2005-01-05
Filing date: 2006-01-05
Publication date: 2006-07-13
Also published as: EP1879906A2; EP1879906A4; WO2006074351A3; JP2008526877A

Abstract

The present invention provides nucleotide analogs which are useful in one or more DNA sequencing techniques.

Description

REVERSIBLE NUCLEOTIDE TERMINATORS AND USES THEREOF

BACKGROUND OF THE INVENTION

[0001] Various methods are known for sequencing nucleic acids, including dideoxy sequencing methods, chemical degradation methods, and sequencing by synthesis (i.e., multiple iterations of the minisequencing method), among others. Some of these methods are well adapted to automation. Such automated sequencers are typically based on the chain termination method utilizing fluorescent detection of product formation. These chain termination methods are based upon the ability of an enzyme to add specific nucleotides onto the 3' hydroxyl end of a primer annealed to a template. In these systems, primers, to which deoxynucleotides and dideoxynucleotides are added, are dye labeled. Alternatively, the added dideoxynucleotides are dye labeled. In addition, dye labeled deoxynucleotides can be used in conjunction with unlabeled dideoxynucleotides. In each case, the base pairing property of nucleic acids determines the specificity of nucleotide addition. The resulting dye labeled products are then separated electrophoretically on a polyacrylamide gel and detected by a method approriate for the label used. [0002] Although both the chemical degradation method and the dideoxy chain termination method are in widespread use, there are many associated disadvantages. For example, the methods require gel-electrophoretic separation. Furthermore, it is typical that only 400-800 base pairs can be sequenced from a single clone. As a result, the systems are both time- and labor-intensive and methods avoiding gel separation have been developed in attempts to increase the sequencing throughput.

[0003] Sequencing by hybridization (SBH) methods have been proposed by Crkvenjakov (Drmanac et al., Genomics 4:114 (1989); Strezoska et al., (Proc. Natl. Acad. Sci. USA 88:10089 (1991)), Bains and Smith (Bains and Smith, J. Theoretical Biol. 135:303 (1988)) and in U.S. Pat. No. 5,202,231. This type of system utilizes the information obtained from multiple hybridizations of the polynucleotide of interest, using short oligonucleotides to determine the nucleic acid sequence. These methods can potentially increase the sequence throughput beacuse multiple hybridization reactions are performed simultaneously. To reconstruct the sequence, however, an extensive computer search algorithm is required to determine the most likely order of all fragments obtained from the multiple hybridizations. [0004] The SBH methods are problematic in several respects. For example, the hybridization is dependent upon the sequence composition of the duplex of the oligonucleotide and the polynucleotide of interest, so that GC-rich regions are more stable than AT-rich regions. As a result, false positives and false negatives during hybridization detection are frequently present and complicate sequence determination. Furthermore, the sequence of the polynucleotide is not determined directly, but is inferred from the sequence of the known probe, which increases the possibility for error. [0005] An alternative sequencing method that uses reversibly blocked nucleotides is known as Sequencing by Synthesis (SBS). SBS determines the DNA sequence by incorporating nucleotides and detecting the sequence one base at a time. To effectively sequence long stretches of a nucleic acid using SBS, it is advantageous to be able to perform multiple iterations of the single nucleotide incorporation. Accordingly, SBS- based methods typically employ methods which prevent the extention of the growing nucleic acid strand. For example, the use of groups which sterically hinder the incorporation of additional nucleotides have been used as well as 3'-OH protecting groups that are removable under conditions that do not disrupt the primer and target DNA interactions. As such, there remains a need for nucleotide analogs which are useful in one or more DNA sequencing techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0007] Figure 1 A depicts the ABI 373 OXL DNA analysis of primer only.

[0008] Figure IB depicts the ABI 373 OXL DNA analysis of primer extended by T nucleotide on a template containing 5A's.

[0009] Figure 2A depicts the ABI 3730XL DNA analysis of an incomplete termination

[0010] Figure 2B depicts the ABI 3730XL DNA analysis of an incomplete extension.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE

INVENTION

1. General Description of the Invention:

[0011] The present invention provides nucleotide analogs useful in a method for sequencing a nucleic acid. One of ordinary skill in the art would appreciate that the present compounds are useful in a variety of sequencing methods. Such methods are readily apparent to one of ordinary skill in the art and include the Sanger method, chemical degradation methods, and the Sequencing by Synthesis (SBS) method, to name but a few. In certain embodiments, the present compounds are used in the SBS method. Thus, according to one aspect, the present invention provides a method for sequencing a nucleic acid by detecting the identity of a nucleotide analog after that nucleotide analog is incorporated into a growing nucleic acid strand e.g. a DNA strand.

[0012] According to another embodiment, a nucleotide analog of the invention is first incorporated into a growing nucleic acid strand e.g. a DNA strand, wherein the incorporation of said nucleotide analog terminates the growth of the nucleic acid strand e.g. a DNA strand. The nucleotide analog thus incorporated is then detected by methods appropriate for the specific detectable moiety present in that nucleotide analog. [0013] In some embodiments, the growing nucleic acid strand e.g. a DNA strand is synthesized by a polymerase-catalyzed reaction. [0014] The present invention provides a compound of formula I:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R² is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂;

R is a nucleobase or a nucleobase mimetic, wherein R is optionally substituted with L-

R⁴;

R⁵ is hydrogen or a thiol protecting group and is optionally substituted with L-R⁴; each L is independently a cleavable linker group; and each K^* is independently a detectable moiety; provided that:

(a) R⁵ is other than -S-pyridin-2-yl when R¹ is -OTBS;

(b) R⁵ is other than hydrogen when R² is TBDPSi; and

(c) at least one of R³ or R⁵ is substituted with L-R⁴.

[0015] The present invention also provides a compound of formula II:

wherein:

R^la is hydrogen or a thiol protecting group and is optionally substituted with L^a-R^4a;

R^2a is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂;

R^3a is a nucleobase or a nucleobase mimetic, wherein R^3a is optionally substituted with L^a-

R^4a;

R^5a is hydrogen or a suitable hydroxyl protecting group; each L^a is independently a cleavable linker group; and each R^4a is independently a detectable moiety; provided that:

(a) at least one of R^3a or R^la is substituted with L^a-R^4a; and

(b) R^la is other than trityl when R^5a is either acetyl or benzyl; and said compound is other than

[0016] According to another embodiment, the present invention provides a compound of formula III:

wherein:

R^lb is OH, a suitably protected hydroxyl group, or hydrogen;

Q is oxygen or sulfur;

R⁶ is a suitable hydroxyl or thiol protecting group and is optionally substituted with L^b-

R^4b;

R^3b is a nucleobase or a nucleobase mimetic, wherein R^3b is optionally substituted with L^b- R^4b;

R^5b is hydrogen or a suitable hydroxyl protecting group; each L^b is independently a cleavable linker group; and each R^4b is independently a detectable moiety; provided that:

(a) R⁶ is other than allyl when R^5b is TBS;

(b) R⁶ is other than p-nitrobenzyl when Q is sulfur; and

(c) at least one of R^3b or R⁶ is substituted with L^b-R^4b.

2. Definitions

[0017] Compounds of this invention include those described generally above, and are further illustrated by the embodiments, sub-embodiments, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5^th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference. [0018] As described herein, compounds of the invention may optionally be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the invention. It will be appreciated that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." In general, the term "substituted", whether preceded by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. [0019] The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and preferably their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 4O⁰C or less, in the absence of moisture or other chemically reactive conditions, for at least a week. [0020] As used herein, the term "detectable moiety" is used interchangeably with the term "label" and relates to any moiety capable of being detected, e.g., primary labels and secondary labels. Primary labels, such as radioisotopes (e.g., ³²P, ³³P, ³⁵S, or ¹⁴C), mass- tags, and fluorescent moieties are signal generating reporter groups which can be detected without further modifications.

[0021] The term "secondary label" as used herein refers to moieties such as biotin and various protein antigens that require the presence of a second intermediate for production of a detectable signal. For biotin, the secondary intermediate may include streptavidin- enzyme conjugates. For antigen labels, secondary intermediates may include antibody- enzyme conjugates. Some fluorescent groups act as secondary labels because they transfer energy to another group in the process of nonradiative fluorescent resonance energy transfer (FRET), and the second group produces the detected signal. [0022] The terms "fluorescent label", "fluorescent dye", and "fluorophore" as used herein refer to moieties that absorb light energy at a defined excitation wavelength and emit light energy at a different wavelength. Examples of fluorescence labels include, but are not limited to: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), CAL dyes, Carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin, 4',5'- Dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Oyster dyes, Pacific Blue, PyMPO, Pyrene, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-Tetra-bromosulfone- fluorescein, Tetramethyl-rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

[0023] The term "quencher" used herein includes any moiety that is capable of absorbing the energy of an excited fluorescent label when located in close proximity and of dissipating that energy without the emission of visible light. Examples of quenchers include, but are not limited to DABCYL ( 4-(4'-dimethylaminophenylazo) benzoic acid) succinimidyl ester, diarylrhodamine carboxylic acid, succinimidyl ester (QSY-7), and 4',5'-dinitrofluorescein carboxylic acid, succinimidyl ester (QSY-33) (all available from Molecular Probes), quencher 1 (Ql; available from Epoch), or "Black hole quenchers" BHQ-I, BHQ-2, and BHQ-3 (available form BioSearch, Inc.).

[0024] The term "mass-tag" as used herein refers to any moiety that is capable of being uniquely detected by virtue of its mass using mass spectrometry (MS) detection techniques. Examples of mass-tags include electrophore release tags such as N-[3-[4'-[(p- Methoxytetrafluorobenzy^oxyJpheny^-S-methylglyceronylJisonipecotic Acid, 4'-[2,3,5,6- Tetrafluoro-4-(pentafluorophenoxyl)]methyl acetophenone, and their derivatives. The synthesis and utility of these mass-tags is described in US patents: No. 4,650,750, 4,709,016, 5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, 5,650,270, and in US Provisional patent No. 60/209,415. Other examples of mass-tags include, but are not limited to, nucleotides, dideoxynucleotides, oligonucleotides of varying length and base composition, oligopeptides, oligosaccharides, and other synthetic polymers of varying length and monomer composition. A large variety of organic molecules, both neutral and charged (biomolecules or synthetic compounds) of an appropriate mass range (100-2000 Daltons) may also be used as mass-tags.

[0025] The terms "electrophoretic-tag", or "e-tag" as used herein refer to any moiety that is capable of being uniquely detected by virtue of its charge-to-mass ratio using electrophoretic separation techniques. Such electrophoretic separation techniques include capillary electrophoresis and separation in polymer- or gel-filled microchannels in manufactured "chips" or devices made of silica, glass, plastic, or other materials (eg., "sequencing chips"). Examples of e-tags include charged molecules of the type described in PCT application WO066607A1, and may be attached to DNA primers by means of the labeling methods described herein.

[0026] The term "substrate", as used herein, refers to any material or macromolecular complex to which a nucleic acid can be attached either directly or via covalent or noncovalent attachment means to another moiety that is attached to the substrate. Substrates can typically be separated from an aqueous solution by virtue of their solidity or insolubility under an appropriate condition, but polymeric substrates that lack this property, gels for axample, may also be used. Examples of commonly used substrates include, but are not limited to, glass surfaces, silica surfaces, plastic surfaces, metal surfaces, surfaces containing a metallic or chemical coating, membranes (eg., nylon, polysulfone, silica), micro-beads (eg., latex, polystyrene, or other polymer or resin, including magnetic beads), porous polymer matrices (eg., polyacrylamide gel, polysaccharide, polymethacrylate, thermo-reversible polymers), macromolecular complexes (eg., protein, polysaccharide).

[0027] The term "aliphatic" or "aliphatic group", as used herein, means a straight- chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "carbocycle" "cycloaliphatic" or "cycloalkyl"), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in yet other embodiments aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C₃-Cs hydrocarbon or bicyclic C₈-Ci₂ hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule wherein any individual ring in said bicyclic ring system has 3-7 members. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0028] The term "heterocycle", "heterocyclyl", "heterocycloaliphatic", or

"heterocyclic" as used herein means non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring members is an independently selected heteroatom. In some embodiments, the "heterocycle", "heterocyclyl", "heterocycloaliphatic", or

"heterocyclic" group has three to fourteen ring members in which one or more ring members is a heteroatom independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.

[0029] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl)

OrNR⁺ (as in N-substituted pyrrolidinyl)).

[0030] The term "unsaturated", as used herein, means that a moiety has one or more units of unsaturation.

[0031] The term "alkoxy", or "thioalkyl", as used herein, refers to an alkyl group, as previously defined, attached to the principal carbon chain through an oxygen ("alkoxy") or sulfur ("thioalkyl") atom.

[0032] The terms "haloalkyl", "haloalkenyl" and "haloalkoxy" means alkyl, alkenyl or alkoxy, as the case may be, substituted with one or more halogen atoms. The term

"halogen" means F, Cl, Br, or I.

[0033] The term "aryl" used alone or as part of a larger moiety as in "aralkyl",

"aralkoxy", or "aryloxyalkyl", refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term

"aryl" may be used interchangeably with the term "aryl ring". The term "aryl" also refers to heteroaryl ring systems as defined hereinbelow.

[0034] The term "heteroaryl", used alone or as part of a larger moiety as in

"heteroaralkyl" or "heteroarylalkoxy", refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members. The term "heteroaryl" may be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic". [0035] An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) or heteroaryl (including heteroaralkyl and heteroarylalkoxy and the like) group may contain one or more substituents. Suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl group are selected from halogen; N₃, CN, R°; OR°; SR°; 1,2-methylene-dioxy; 1,2-ethylenedioxy; phenyl (Ph) optionally substituted with R°; -0(Ph) optionally substituted with R⁰; (CH₂)_I-2(Ph), optionally substituted with R⁰; CH=CH(Ph), optionally substituted with R⁰; NO₂; CN; N(R°)₂; NR⁰C(O)R⁰; NR°C(O)N(R°)₂; NR⁰CO₂R⁰; -NR⁰NR⁰C(O)R⁰; NR°NR°C(0)N(R°)₂; NR⁰NR⁰CO₂R⁰; C(O)C(O)R⁰; C(O)CH₂C(O)R⁰; CO₂R⁰; C(O)R⁰; C(O)N(R°)₂; OC(O)N(R°)₂; S(O)₂R⁰; SO₂N(R°)₂; S(O)R⁰; NR°SO₂N(R°)₂; NR⁰SO₂R⁰; C(=S)N(R°)₂; C(=NH)-N(R°)₂; or (CH₂)_0-2NHC(O)R^o wherein each independent occurrence of R⁰ is selected from hydrogen, optionally substituted Ci_-6 aliphatic, an unsubstituted 5-6 membered heteroaryl or heterocyclic ring, phenyl, O(Ph), or CH₂(Ph), or, notwithstanding the definition above, two independent occurrences of R⁰, on the same substituent or different substituents, taken together with the atom(s) to which each R⁰ group is bound, form a 3-8 membered cycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Optional substituents on the aliphatic group of R° are selected from N₃, CN, NH₂, NH(Ci_-4aliphatic), N(Ci_-4aliphatic)₂, halogen, Ci_-4aliphatic, OH, 0(C_1- ₄aliphatic), NO₂, CN, CO₂H, CO₂(Cj ^aliphatic), O(haloCi₄ aliphatic), or haloCμ ₄aliphatic, wherein each of the foregoing Ci^aliphatic groups of R° is unsubstituted. [0036] An aliphatic or heteroaliphatic group or a non-aromatic heterocyclic ring may contain one or more substituents. Suitable substituents on the saturated carbon of an aliphatic or heteroaliphatic group, or of a non-aromatic heterocyclic ring are selected from those listed above for the unsaturated carbon of an aryl or heteroaryl group and additionally include the following: =0, =S, =NNHR*, =NN(R*)₂, =NNHC(0)R^*, =NNHCO₂(alkyl), =NNHSO₂(alkyl), or =NR^*, where each R* is independently selected from hydrogen or an optionally substituted Ci_-6 aliphatic. Optional substituents on the aliphatic group of R^* are selected from NH₂, NH(Ci_-4 aliphatic), N(Ci_-4 aliphatic)₂, halogen, Ci_-4 aliphatic, OH, 0(Q_-4 aliphatic), NO₂, CN, CO₂H, CO₂(Ci_-4 aliphatic), O(halo Ci_-4 aliphatic), or ImIo(Ci_-4 aliphatic), wherein each of the foregoing Ci^aliphatic groups of R* is unsubstituted.

[0037] Optional substituents on the nitrogen of a non-aromatic heterocyclic ring are selected from R⁺, N(R⁺K C(O)R⁺, CO₂R⁺, C(O)C(O)R⁺, C(O)CH₂C(O)R⁺, SO₂R⁺, SO₂N(R⁺)₂, C(=S)N(R⁺)₂, CC=NH)-N(RO₂, or NR⁺SO₂R⁺; wherein R⁺ is hydrogen, an optionally substituted C_1-6 aliphatic, optionally substituted phenyl, optionally substituted 0(Ph), optionally substituted CH₂(Ph), optionally substituted (CH₂)i_-2(Ph); optionally substituted CH=CH(Ph); or an unsubstituted 5-6 membered heteroaryl or heterocyclic ring having one to four heteroatoms independently selected from oxygen, nitrogen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R⁺, on the same substituent or different substituents, taken together with the atom(s) to which each R⁺ group is bound, form a 3-8-membered cycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Optional substituents on the aliphatic group or the phenyl ring of R⁺ are selected from NH₂, NH(Ci_-4 aliphatic), N(C_1-4 aliphatic^, halogen, C_1-4 aliphatic, OH, 0(Ci_-4 aliphatic), NO₂, CN, CO₂H, CO₂(C_1-4 aliphatic), O(halo Ci_-4 aliphatic), or halo(Ci-₄ aliphatic), wherein each of the foregoing Ci^aliphatic groups of R⁺ is unsubstituted.

[0038] As detailed above, in some embodiments, two independent occurrences of R⁰ (or R⁺, or any other variable similarly defined herein), are taken together with the atom(s) to which each variable is bound to form a 3-8-membered cycloalkyl, heterocyclyl, aryl, or heteroaryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Exemplary rings that are formed when two independent occurrences of R° (or R , or any other variable similarly defined herein) are taken together with the atom(s) to which each variable is bound include, but are not limited to the following: a) two independent occurrences of R° (or R⁺, or any other variable similarly defined herein) that are bound to the same atom and are taken together with that atom to form a ring, for example, N(R°)₂, where both occurrences of R° are taken together with the nitrogen atom to form a piperidin-1-yl, piρerazin-1-yl, or morpholin-4-yl group; and b) two independent occurrences of R° (or R⁺, or any other variable similarly defined herein) that are bound to different atoms and are taken together with both of those atoms to form a ring, for example

where a phenyl group is substituted with two occurrences of OR° \ , these two occurrences of R° are taken together with the oxygen atoms to which they are bound

to form a fused 6-membered oxygen containing ring:

\ *-* . It will be appreciated that a variety of other rings can be formed when two independent occurrences of R° (or R⁺, or any other variable similarly defined herein) are taken together with the atom(s) to which each variable is bound and that the examples detailed above are not intended to be limiting.

[0039] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays. [0040] As used herein, the term "nucleotide" consists of a nitrogenous base ("nucleobase"), a sugar, and one or more phosphate groups. It is understood that in RNA, the sugar is a ribose, and in DNA is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose. A nucleotide is also a phosphate ester of a nucleoside, with esterification occurring on the hydroxyl group attached to C-5 of the sugar. Nucleotides are usually mono, di- or triphosphates.

[0041] As used herein, the term "nucleoside" is structurally similar to a nucleotide, but is missing the phosphate moieties.

[0042] As used herein, the term "nucleobase", without the modifier "mimetic" or "universal", refers to the natural or "normal" nucleobases that form the base pairs of naturally occuring nucleic acids, e.g. DNA or RNA. These are derivatives of purine or pyrimidine. The purines are adenosine (A) and guanidine (G), and the pyrimidines are cytidine (C) and thymidine (T), or in the context of RNA, uracil (U). Also included as nucleobases are inosine, xanthine, hypoxanthine, and 2-aminopurine. Typically, the C-I atom of deoxyribose is bonded to N-I of a pyrimidine or N-9 of a purine. [0043] As used herein, the term "nucleobase mimetic" refers to nucleobases, not including the natural nucleobases, which form the appropriate hydrogen bonds, i.e. base pairs, with each of the natural nucleobases in the Watson-Crick mode. See Seela and Debelak, Nucleic Acids Research 28:17, 3224-3232 (2000). Alternatively, such nucleobases may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases. See Berger, et al, Nucleic Acids Research 28:15, 2911-2914 (2000). Nucleobase mimetics include those nucleobase analogs that specifically pair with one or more of the natural nucleobases. In certain embodiments, the nucleobase mimetic pairs specifically with C. In other embodiments, the nucleobase mimetic pairs specifically with T. In yet other embodiments, the nucleobase mimetic pairs specifically with A. In still other embodiments, the nucleobase mimetic pairs specifically with G.

3. Description of Exemplary Embodiments

[0044] As described generally above, the present invention provides a compound of formula I:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R³ is a nucleobase or a nucleobase mimetic, wherein R³ is optionally substituted with L-

R⁴;

R⁵ is hydrogen or a suitable thiol protecting group and is optionally substituted with L-R⁴; each L is independently a cleavable linker group; and each R⁴ is independently a detectable moiety; provided that at least one of R³ and R⁵ is substituted with L-R⁴.

[0045] In certain embodiments, the R³ group of formula I is substituted with L-R⁴. In other embodiments, R³ is substituted with L-R⁴ and R^s is a thiol protecting group which is removable under the same conditions used to cleave L-R⁴.

[0046] In certain embodiments, the R³ group of formula I is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine. [0047] In other embodiments, the R^J group of formula I is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[0048] As described generally above, the L group of formula I is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R³ is substituted with L-R⁴, the L-R⁴ group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[0049] In certain embodiments, L has a functional moiety at the terminal end for coupling to the detectable R⁴ group. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R⁴ detectable group utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [0050] Thiol protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable thiol protecting groups of the R⁵ moiety of formula I include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, trichloroethoxycarbonyl, to name but a few. In certain embodiments, the thiol protecting group of the R⁵ moiety of formula I is -S-pyridin-2-yl.

[0051] According to one aspect of the present invention, the R⁵ moiety of formula I is a thiol protecting group that is removable under neutral conditions e.g. with AgNO₃, HgCl₂, and the like. Other neutral conditions include reduction using a suitable reducing agent. Suitable reducing agents include dithiothreitol (DTT), mercaptoethanol, dithionite, reduced glutathione, reduced glutaredoxin, reduced thioredoxin, substituted phosphines such as tris carboxyethyl phosphine (TCEP) and any other peptide or organic based reducing agent, or other reagents known to those of ordinary skill in the art. According to another aspect of the present invention, the R⁵ moiety of formula I is a thiol protecting group that is cleaved under conditions where the pH is from about 4 to about 9. According to yet another aspect of the present invention, the R⁵ moiety of formula I is a thiol protecting group that is "photocleavable". Such suitable thiol protecting groups are known in the art and include, but are not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, a trityl group, -CH₂SCH₃ (MTM), dimethylmethoxymethyl, or -CH₂-S-S-pyridin-2-yl. One of ordinary skill in the art would recognize that many of the suitable hydroxyl protecting groups, as described herein, are also suitable as thiol protecting groups.

[0052] In certain embodiments, R² is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trjmethylacetyl), crotonate, 4-methoxy-crotonate, benzoate, p-benylbenzoate, 2,4,6- trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2- trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p- nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t- butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [0053] In other embodiments, the R² of formula I is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH)₂.

[0054] In certain embodiments, the R¹ of formula I is hydrogen. [0055] In other embodiments, the R¹ of formula I is -OH. [0056] According to yet other embodiments, the R¹ of formula I is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R² group.

[0057] In certain embodiments, the present invention provides a compound of formula

Ia:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R² is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂; R³ is a nucleobase or a nucleobase mimetic; L is independently a cleavable linker group; and R⁴ is a detectable moiety.

[0058] In certain embodiments, the R³ group of formula Ia is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[0059] In other embodiments, the R³ group of formula Ia is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[0060] As described generally above, the L group of formula Ia is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R³ is substituted with L-R⁴, the L-R⁴ group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out. [0061] In certain embodiments, L has a functional moiety at the terminal end for coupling to the detectable R⁴ group. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R⁴ detectable group utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [0062] In certain embodiments, the R² group of formula Ia is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy-crotonate, benzoate, p-benylbenzoate, 2,4,6-trimethylbenzoate, carbonates such as methyl, 9- fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2- (phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p- methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [0063] In other embodiments, the R² of formula Ia is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH)₂.

[0064] In certain embodiments, the R¹ of formula Ia is hydrogen. [0065] In other embodiments, the R¹ of formula Ia is -OH.

[0066] According to yet other embodiments, the R¹ of formula Ia is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R² group. [0067] In other embodiments, the present invention provides a compound of formula Ib:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R² is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂; R³ is a nucleobase or a nucleobase mimetic; T is oxygen, sulfur, NH, or optionally substituted phenyl; L is independently a cleavable linker group; and R⁴ is a detectable moiety.

[0068] In certain embodiments, the R³ group of formula Ib is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[0069] In other embodiments, the R³ group of formula Ib is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[0070] As described generally above, the L group of formula Ib is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R³ is substituted with L-R⁴, the L-R⁴ group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out. [0071] In certain embodiments, L has a functional moiety at the terminal end for coupling to the detectable R⁴ group. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R⁴ detectable group utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [0072] In certain embodiments, R² is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy-crotonate, benzoate, p-benylbenzoate, 2,4,6- trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2- trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p- nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t- butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [0073] In other embodiments, the R² of formula Ib is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH)₂.

[0074] In certain embodiments, the R¹ of formula Ib is hydrogen. [0075] In other embodiments, the R¹ of formula Ib is -OH.

[0076] According to yet other embodiments, the R¹ of formula Ib is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R² group. [0077] In other embodiments, the present invention provides a compound of formula Ic:

wherein:

W is 0(C_1-4 aliphatic) or S(Ci_-4 aliphatic); W is Ci_-4 aliphatic;

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R² is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)_2, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂; R³ is a nucleobase or a nucleobase mimetic; L is a cleavable linker group; and R⁴ is a detectable moiety.

[0078] According to one aspect, the present invention provides a compound of formula Ic, wherein W is -OCH₃ or -SCH₃. According to another aspect, the present invention provides a compound of formula Ic, wherein W is -CH3, CH₂CH3, and the like. [0079] In certain embodiments, the R³ group of formula Ic is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[0080] In other embodiments, the R³ group of formula Ic is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[0081] As described generally above, the L group of formula Ic is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R³ is substituted with L-R⁴, the L-R⁴ group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[0082] In certain embodiments, L has a functional moiety at the terminal end for coupling to the detectable R⁴ group. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R⁴ detectable group utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [0083] In certain embodiments, the R² group of formula Ic is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [0084] In other embodiments, the R² of formula Ic is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH)₂. [0085] In certain embodiments, the R¹ of formula Ic is hydrogen. [0086] In other embodiments, the R¹ of formula Ic is -OH.

[0087] According to yet other embodiments, the R¹ of formula Ic is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R² group.

[0088] Yet another embodiment of the present invention provides a compound of formula Id:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R⁴; each L is independently a cleavable linker group; and each R⁴ is independently a detectable moiety.

[0089] In certain embodiments, the R³ group of formula Id is substituted with L-R⁴.

[0090] In certain embodiments, the R³ group of formula Id is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[0091] In other embodiments, the R³ group of formula Id is a nucleobase mimetic.

Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[0092] As described generally above, the L group of formula Id, when present, is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application

2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^J is substituted with L-R⁴, the L-R⁴ group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[0093] In certain embodiments, L has a functional moiety at the terminal end for coupling to the detectable R⁴ group. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R⁴ detectable group utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxy! group, a carboxylic acid group, or a thiol group. [0094] In certain embodiments, the R² group of formula Id is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [0095] In other embodiments, the R² of formula Id is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH)₂.

[0096] In certain embodiments, the R¹ of formula Id is hydrogen. [0097] In other embodiments, the R¹ of formula Id is -OH. [0098] According to yet other embodiments, the R¹ of formula Id is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R² group.

[0099] As described generally above, the present invention also provides a compound of formula II:

wherein:

R^4a;

R^Sa is hydrogen or a suitable hydroxyl protecting group; each L^a is independently a cleavable linker group; and each R^4a is independently a detectable moiety; provided that at least one of R^la or R^3a is substituted with L^a-R^4a.

[00100] In certain embodiments, the R^3a group of formula II is substituted with L^a-R^4a.

In other embodiments, the R^3a group of formula II is substituted with L^a-R^4a and R^5a is a thiol protecting group which is removable under the same conditions used to cleave L^a-

R^4a.

[00101] In certain embodiments, the R^3a group of formula II is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[00102] In other embodiments, the R^3a group of formula II is a nucleobase mimetic.

Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases. [00103] As described generally above, the L^a group of formula II is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3a is substituted with L^a-R^4a, the L^a-R^4a group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[00104] In certain embodiments, L^a has a functional moiety at the terminal end for coupling to the detectable R^4a moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4a detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [00105] Thiol protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^vd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable thiol protecting groups of the R^la moiety of formula II include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, trichloroethoxycarbonyl, to name but a few. In certain embodiments, the thiol protecting groups of the R^la moiety of formula II is -S-S-pyridin-2-yl.

[00106] According to one aspect of the present invention, the R^la moiety of formula II is a thiol protecting group that is removable under neutral conditions e.g. with AgNO₃, HgCl₂, and the like. Other neutral conditions include reduction using a suitable reducing agent. Suitable reducing agents include dithiothreitol (DTT), mercaptoethanol, dithionite, reduced glutathione, reduced glutaredoxin, reduced thioredoxin, substituted phosphines such as tris carboxyethyl phosphine (TCEP), and any other peptide or organic based reducing agent, or other reagents known to those of ordinary skill in the art. According to another aspect of the present invention, the R^la moiety of formula II is a thiol protecting group that is cleaved under conditions where the pH is from about 4 to about 9. According to yet another aspect of the present invention, the R^la moiety of formula II is a thiol protecting group that is "photocleavable". Such suitable thiol protecting groups are known in the art and include, but are not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, a trityl group, -CH₂SCH₃ (MTM), dimethylmethoxymethyl, or -CH₂-S-S-pyridin-2-yl. One of ordinary skill in the art would recognize that many of the suitable hydroxyl protecting groups, as described herein, are also suitable as thiol protecting groups.

[00107] In certain embodiments, the R²⁸ group of formula II is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00108] In other embodiments, the R^2a group of formula II is -P(O)(OH)-O-P(O)(OH)- 0-P(O)(OH)₂.

[00109] In certain embodiments, the R^5a group of formula II is hydrogen. [00110] According to yet other embodiments, the R^5a group of formula II is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R^2a group.

[00111] In certain embodiments, the present invention provides a compound of formula Ha:

wherein:

R^2a is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂; R^3a is a nucleobase or a nucleobase mimetic, wherein R^3a is optionally substituted with L^a- R^4a;

R^5a is hydrogen or a suitable hydroxyl protecting group; L^a is a cleavable linker group; and R^4a is a detectable moiety.

[00112] In certain embodiments, the R^3a group of formula Ha is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminoρurine.

[00113] In other embodiments, the R^3a group of formula Ha is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[00114] As described generally above, the L^a group of formula Ha is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3a is substituted with L^a-R^4a, the L^a-R^4a group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out. In the context of purine bases, it is preferred if the linker is attached via the 7 position of the purine or the preferred deazapurine analogue, via an 8-modified purine, via an N-6 modified adenosine or an N-2 modified guanine. For pyrimidines, attachment is preferably via the 5 position on cytidine, thymidine or uracil and the N-4 position on cytosine. [00115] In certain embodiments, L^a has a functional moiety at the terminal end for coupling to the detectable R^4a moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4a detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [00116] In certain embodiments, the R^2a group of formula Ha is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00117] In other embodiments, the R^2a group of formula Ha is -P(O)(OH)-O- P(O)(OH)-O-P(O)(OH)₂.

[00118] In certain embodiments, the R^5a group of formula Ha is hydrogen. [00119] According to yet other embodiments, the R^Sa group of formula Ha is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R^2a group. [00120] In certain embodiments, the present invention provides a compound of formula lib:

wherein:

R^5a is hydrogen or a suitable hydroxyl protecting group; T^a is oxygen, sulfur, NH, or optionally substituted phenyl; each L^a is independently a cleavable linker group; and each R^4a is independently a detectable moiety.

[00121] In certain embodiments, the R^3a group of formula lib is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[00122] In other embodiments, the R^3a group of formula lib is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[00123] As described generally above, the L^a group of formula Hb is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3a is substituted with L^a-R^4a, the L^a-R^4a group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out. [00124] In certain embodiments, L^a has a functional moiety at the terminal end for coupling to the detectable R^4a moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4a detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [00125] In certain embodiments, the R^2a group of formula lib is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-ρhenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00126] In other embodiments, the R^2a group of formula lib is -P(O)(OH)-O- P(O)(OH)-O-P(O)(OH)₂.

[00127] In certain embodiments, the R^5a group of formula lib is hydrogen. [00128] According to yet another embodiment, the R^5a group of formula lib is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R^2a group. [00129] The present invention also provides a compound of formula Hc:

wherein:

W^a is 0(Ci_-4 aliphatic) or S(Ci_-4 aliphatic); W^b is Ci_-4 aliphatic;

R^5a is hydrogen or a suitable hydroxyl protecting group; each L^a is independently a cleavable linker group; and each R^4a is independently a detectable moiety.

[00130] In certain embodiments, the W^a group of formula lie is -OCH₃ or -SCH₃. In other embodiments, the W^b group of formula Hc is -CH₃, -CH₂CH₃ and the like. [00131] In certain embodiments, the R^3a group of formula Hc is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[00132] In other embodiments, the R^3a group of formula Hc is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[00133] As described generally above, the L^a group of formula He is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3a is substituted with L^a-R^4a, the L^a-R^4a group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[00134] In certain embodiments, If has a functional moiety at the terminal end for coupling to the detectable R^4a moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4a detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [00135] In certain embodiments, the R^2a group of formula Hc is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00136] In other embodiments, the R^2a of formula Hc is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH)₂. [00137] In certain embodiments, the R^5a group of formula lie is hydrogen. [00138] According to yet other embodiments, the R^ia group of formula Hc is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R^2a group.

[00139] In certain embodiments, the present invention provides a compound of formula

Hd:

wherein:

R^2a is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂; R^3a is a nucleobase or a nucleobase mimetic, wherein R^3a is substituted with L^a-R ^a; R^5a is hydrogen or a suitable hydroxyl protecting group; L^a is a cleavable linker group; and R^4a is a detectable moiety.

[00140] In certain embodiments, the R^3a group of formula Hd is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[00141] In other embodiments, the R^3a group of formula lid is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[00142] As described generally above, the L^a group of formula Hd, when present, is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3a is substituted with L^a-R^4a, the L^a-R^4a group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out. [00143] In certain embodiments, L^a has a functional moiety at the terminal end for coupling to the detectable R^4a moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4a detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [00144] In certain embodiments, .the R^2a group of formula Hd is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R² moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxy alkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00145] In other embodiments, the R^2a group of formula lid is -P(O)(OH)-O- P(O)(OH)-O-P(O)(OH)₂.

[00146] In certain embodiments, the R^5a group of formula ITd is hydrogen. [00147] According to yet other embodiments, the R^5a group of formula Hd is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R^2a group. [00148] As described generally above, the present invention provides a compound of formula III:

wherein:

R^lb is OH, a suitably protected hydroxyl group, or hydrogen;

Q is oxygen or sulfur;

R⁶ is a suitable hydroxyl protecting group when Q is O, or a suitable thiol protecting group when Q is S, and is optionally substituted with L^b-R^4b;

R^3b is a nucleobase or a nucleobase mimetic, wherein R^3b is optionally substitute with L^b- R^4b;

R^5b is hydrogen or a suitable hydroxyl protecting group; each L^b is independently a cleavable linker group; and each R^4b is independently a detectable moiety; provided that at least one of R⁶ or R^3b is substituted with L^b-R^4b.

[00149] In certain embodiments, the R^3b group of formula in is substituted with L^b-R^4b. In other embodiments, R^3b is substituted with L^b-R^4b and R⁶ is a hydroxyl or thiol protecting group which is removable under the same conditions used to cleave L -R^4b. [00150] According to one aspect of the present invention, the Q group of formula III is O.

[00151] According to another aspect of the present invention, the Q group of formula III is S.

[00152] In certain embodiments, the R^3b group of formula HI is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[00153] In other embodiments, the R^3b group of formula HI is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases. [00154] As described generally above, the V group of formula III is a cleavable linker group. Such groups are generally known in the art and include those described in IJS 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3b is substituted with L^b-R^4b, the L^b-R^4b group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[00155] In certain embodiments, L^b has a functional moiety at the terminal end for coupling to the detectable R^4b moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4b detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxy 1 group, a carboxylic acid group, or a thiol group. [00156] Thiol protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable thiol protecting groups of the R⁶ moiety of formula III include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, trichloroethoxycarbonyl, to name but a few. In certain embodiments, the thiol protecting groups of the R⁶ moiety of formula III is -S-S-pyridin-2-yl.

[00157] According to one aspect of the present invention, the R⁶ moiety of formula III is a thiol protecting group that is removable under neutral conditions e.g. with AgNO₃, HgCl₂, and the like. Other neutral conditions include reduction using a suitable reducing agent. Suitable reducing agents include dithiothreitol (DTT), mercaptoethanol, dithionite, reduced glutathione, reduced glutaredoxin, reduced thioredoxin, substituted phosphines such as tris carboxyethyl phosphine (TCEP) and any other peptide or organic based reducing agent, or other reagents known to those of ordinary skill in the art. According to another aspect of the present invention, the R⁶ moiety of formula III is a thiol protecting group that is cleaved under conditions where the pH is from about 4 to about 9. According to yet another aspect of the present invention, the R⁶ moiety of formula III is a thiol protecting group that is "photocleavable". Such suitable thiol protecting groups are known in the art and include, but are not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, a trityl group, -CH₂SCH₃ (MTM), dimethylmethoxymethyl, or -CH₂-S-S-pyridin~2-yl. One of ordinary skill in the art would recognize that many of the suitable hydroxyl protecting groups, as described herein, are also suitable as thiol protecting groups.

[00158] In certain embodiments, the R⁶ group of formula III is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R⁶ moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifiuoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylρropionate, 4-oxopentanoate, 4,4-

(ethylenedithio)ρentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy-crotonate, benzoate, p-benylbenzoate, 2,4,6-trimethylbenzoate, carbonates such as methyl, 9- fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl, 2- (phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p- methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00159] In certain embodiments, the R^lb group of formula III is hydrogen. [00160] In other embodiments, the R^lb group of formula in is -OH. [00161] According to yet other embodiments, the R^lb of formula III is a suitably protected hydroxyl group. Such suitable hydroxyl protecting groups include those described above for the R⁶ group.

[00162] In certain embodiments, the R^5b group of formula III is hydrogen. [00163] In other embodiments, the R^5b group of formula III is a suitable hydroxyl protecting group. Such suitable hydroxyl protecting groups include those described above for the R⁶ group. [00164] According to another embodiment, the present invention provides a compound of formula Ilia:

wherein:

R^lb is OH, a suitably protected hydroxyl group, or hydrogen;

R^3b is a nucleobase or a nucleobase mimetic, wherein R^3b is optionally substituted with L^b-

R^4b;

R^5b is hydrogen or a suitable hydroxyl protecting group; each L^b is independently a cleavable linker group; and each R^4b is independently a detectable moiety.

[00165] In certain embodiments, the R^3b group of formula Ilia is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[00166] In other embodiments, the R^3b group of formula Ilia is a nucleobase mimetic.

[00167] As described generally above, the L^b group of formula IHa is a cleavable linker group. Such groups are generally known in the art and include those described in

US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO

04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3b is substituted with L^b-R^4b, the L^b-R^4b group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[00168] In certain embodiments, L^b has a functional moiety at the terminal end for coupling to the detectable R^4b moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4b detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [00169] In certain embodiments, the R^5b group of formula Ilia is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R^5b group further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxy acetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00170] In certain embodiments, the R^lb group of formula Ilia is hydrogen. [00171] In other embodiments, the R^lb group of formula Ilia is -OH. [00172] According to yet other embodiments, the R^lb of formula IQa is a suitably protected hydroxyl group. Such suitable hydroxyl protecting groups include those described above for the R^5b group.

[00173] According to another embodiment, the present invention provides a compound of formula IHb:

wherein:

R^lb is OH, a suitably protected hydroxyl group, or hydrogen;

R^4b;

[00174] In certain embodiments, the R^3b group of formula HIb is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[00175] In other embodiments, the R^3b group of formula IHb is a nucleobase mimetic.

[00176] As described generally above, the L^b group of formula IHb is a cleavable linker group. Such groups are generally known in the art and include those described in

[00177] In certain embodiments, L^b has a functional moiety at the terminal end for coupling to the detectable R^4b moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4b detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [00178] In certain embodiments, the R^SB group of formula IIIb is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R^5b group further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00179] In certain embodiments, the R^lb group of formula IIIb is hydrogen. [00180] In other embodiments, the R^lb group of formula IIIb is -OH. [00181] According to yet other embodiments, the R^lb of formula IIIb is a suitably protected hydroxyl group. Such suitable hydroxyl protecting groups include those described above for the R^sb group.

[00182] According to another embodiment, the present invention provides a compound of formula IIIc or IHc':

wherein: each R^lb is independently OH, a suitably protected hydroxyl group, or hydrogen; each R^3b is independently a nucleobase or a nucleobase mimetic, wherein each R^3b is optionally substitute with L^b-R^4b; each T^b is independently O, S, NH, or optionally substituted phenyl; each R^5b is independently hydrogen or a suitable hydroxyl protecting group; each L^b is independently a cleavable linker group; and each R^4b is independently a detectable moiety.

[00183] In certain embodiments, the R^3b group of formulae πic and IIIc' is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine. [00184] In other embodiments, the R^3b group of formulae IIIc and HIc' is a nucleobase mimetic. Such nucleobase mimetics are known to one of ordinary skill in the art and include those which form the appropriate hydrogen bonds, i.e. base pairs, with the natural nucleobases in the Watson-Crick mode. Alternatively, such nucleobase mimetics may not form any hydrogen bonds at all yet still pack efficiently in duplex DNA with nearly equal efficiency with that of the natural nucleobases.

[00185] As described generally above, the L^b group of formulae HIc and IIIc' is a cleavable linker group. Such groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3b is substituted with L^b-R^4b, the L^b-R^4b group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[00186] In certain embodiments, L^b has a functional moiety at the terminal end for coupling to the detectable R^4b moiety. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4b detectable moiety utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. [00187] In certain embodiments, the R^5b group of formulae IIIc and HIc' is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R^5b group further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-

(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00188] In certain embodiments, the R^lb group of formulae IIIc and IIIc' is hydrogen. [00189] In other embodiments, the R^lb group of formulae IIIc and IIIc' is -OH. [00190] According to yet other embodiments, the R^lb of formulae IIIc and IIIc' is a suitably protected hydroxyl group. Such suitable hydroxyl protecting groups include those described above for the R^sb group.

[00191] Catalytic editing is a process in which a substituent attached to the 3' OH of a nucleoside (in an ester, amide, or thiourea linkage) is removed by a DNA polymerase in the presence of the (correct) incoming dNTP complementary to the next base position (n+1) on the template. See Canard, et al, Proc. Natl. Acd. Sci. USA, "Catalytic editing properties of DNA polymerases" 92, 10859-10863 (1995). Unfortunately, this property limits the utility of certain types of teminators in conjunction with enzymes that are capable of catalytic editing. In particular, it is not possible to use a mixture of all four dNTP analogs in a reaction with an enzyme capable of catalytic editing because editing will occur at each position when the next nucleotide analog binds to the enzyme-template- primer complex. However, such terminators can still be used with enzymes that do not catalyze the editing reaction, such as AMV reverse transcriptase.

[00192] To overcome the possibility of catalytic editing, new reversible terminators are provided that are resistant to such catalytic editing. These nucleotide analogs share the property that they include a universal nitrogenous base moiety attached to the cleavable linker in such a way as to promote base stacking and/or pairing of the universal base at the 3' position of the primer in the n+1 position of the primer-template complex. Without wishing to be bound by any particular theory, it is believed that this will block the next correct nucleoside triphosphate from occupying that position, hence preventing catalytic editing from occuring. Reversal of the termination is achieved by cleavage of a linker that releases the universal nucleoside mimic with attached label, and regenerates a 3' OH, or, alternatively, a 3' SH in the case where a 3' S- compound has been used. Accordingly, another embodiment of the present invention relates to a compound of formula IV:

IV wherein:

R^lc is OH, SH, a suitably protected hydroxyl group, a suitably protected thiol group, or hydrogen;

Q is oxygen or sulfur;

U is a universal nucleoside analog;

R^2c is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂;

R^3c is a nucleobase or a nucleobase mimetic, wherein R^3c is optionally substituted with L^c-

R^4c; each L^c is independently a cleavable linker group; L^c is a non-cleavable linker group; and each R^4c is independently a detectable moiety.

[00193] According to one aspect of the present invention, the Q group of formula IV is oxygen.

[00194] According to another aspect of the present invention, the Q group of formula

IV is sulfur.

[00195] In certain embodiments, the R^3c group of formula IV is substituted with L^c-R^4c.

[00196] In certain embodiments, the R^3c group of formula IV is a nucleobase. Such nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.

[00197] In other embodiments, the R^3c group of formula IV is a nucleobase mimetic.

[00198] As described generally above, the L^c group of formula IV is a cleavable linker group. In certain embodiments, the L^c group of formula IV is attached to the universal nucleoside, U, at the 5 '-position of U. Such L^c groups are generally known in the art and include those described in US 6,664,079, US 6,664,079, US 6,511,803, US Published

Application 2003104437, WO 04/18497 and WO 03/48387, the entirety of which are hereby incorporated herein by reference. When R^3c is substituted with L^c-R^4c, the L^c-R^4c group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.

[00199] In certain embodiments, L^c has a functional moiety at the terminal end for coupling to the detectable R^4c group. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R^4c detectable group utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group.

[00200] In other embodiments, the L^c group of formula IV is of such a length that it is possible for the molecule to assume a conformation in the active site of the enzyme that allows pairing of the universal base analog with the n+1 position in the DNA helix. Many linker configurations are possible, as long as they are capable of folding in a configuration that fits within the space normally occupied by the sugar-phosphate linkage in the DNA backbone.

[00201] As described generally above, the L^0' group of formula IV is a non-cleavable linker group. According to one aspect of the present invention, the L^cl group of formula IV is attached to U at the 3 '-position of U. In certain embodiments, L^c> has a functional moiety at the terminal end for coupling to the detectable R^4c group. Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R⁴° detectable group utilized. Examples of such functional moieties include, but are not limited to, amino groups, an aldehyde group, a hydroxyl group, a carboxylic acid group, or a thiol group. Such linker groups include a Ci_-8 alkylidene chain wherein 0-2 methylene units of the chain are optionally and independently replaced by -O-, -S-, -NH-, - C(O)-, -C(O)NH-, -NHC(O)-, -SO-, -SO₂-, -NHSO₂-, -SO₂NH-, -C(O)O-, or -OC(O)-. In certain embodiments, the L^c' group of formula IV is a Cj_-6 alkylidene chain wherein the methylene unit adjacent to R⁴° is replaced by -NHC(O)-.

[00202] Thiol protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable thiol protecting groups of the R^lc moiety of formula IV include, but are not limited to, disulfides, thioethers, silyl thioethers, thioesters, thiocarbonates, thiocarbamates, and the like. Examples of such groups include, but are not limited to, alkyl thioethers, benzyl and substituted benzyl thioethers, triphenylmethyl thioethers, trichloroethoxycarbonyl, to name but a few. In certain embodiments, the thiol protecting group of the R^lc moiety of formula IV is -S-pyridin-2-yl.

[00203] According to one aspect of the present invention, the R^lc moiety of formula IV is a thiol protecting group that is removable under neutral conditions e.g. with AgNO₃, HgCl₂, and the like. Other neutral conditions include reduction using a suitable reducing agent. Suitable reducing agents include dithiothreitol (DTT), mercaptoethanol, dithionite, reduced glutathione, reduced glutaredoxin, reduced thioredoxin, substituted phosphines such as tris carboxyethyl phosphine (TCEP) and any other peptide or organic based reducing agent, or other reagents known to those of ordinary skill in the art. According to another aspect of the present invention, the R^lc moiety of formula IV is a thiol protecting group that is cleaved under conditions where the pH is from about 4 to about 9. According to yet another aspect of the present invention, the R^lc moiety of formula IV is a thiol protecting group that is "photocleavable". Such suitable thiol protecting groups are known in the art and include, but are not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, a trityl group, -CH₂SCH₃ (MTM), dimethylmethoxymethyl, or -CH₂-S-S-pyridin-2-yl. One of ordinary skill in the art would recognize that many of the suitable hydroxyl protecting groups, as described herein, are also suitable as thiol protecting groups.

[00204] In certain embodiments, R^2c is a suitable hydroxyl protecting group. Hydroxyl protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Examples of suitable hydroxyl protecting groups of the R^2c moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples of such esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3- phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetyl), crotonate, 4-methoxy-crotonate, benzoate, p-benylbenzoate, 2,4,6- trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2- trichloroethyl, 2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, and p- nitrobenzyl. Examples of such silyl ethers include trimethylsilyl, triethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t- butyl, allyl, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl, benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of arylalkyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p- nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. [00205] In other embodiments, the R^2c of formula I is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH)₂.

[00206] In certain embodiments, the R^lc of formula I is hydrogen. [00207] In other embodiments, the R^{1 c} of formula I is -OH.

[00208] According to yet other embodiments, the R^Ic of formula I is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R^2c group. [00209] As described generally above, the U group of formula IV is a universal nucleoside analog. In certain embodiments, U comprises a universal base, capable of stacking and/or base pairing with any of the four canonical nucleobases in a DNA helix. [00210] In certain embodiments, the present invention provides a compound of formula IVa:

4. Uses, Methods, and Compositions

[00211] As discussed above, the present invention provides nucleotide analogs that are reversibly blocked and optionally labeled. These nucleotide analogs have a detectable moiety that may be linked via a cleavable linker group to the nucleobase or nucleobase mimetic. Alternatively, these nucleotide analogs have a detectable moiety that may be linked via a cleavable linker group to the 3'-SH or 2'-SH protecting group. Yet another aspect provides nucleotide analogs having a detectable moiety that may be linked via a cleavable linker group to the cc-phosphate. As such, these compounds are useful as reagents in almost any method for sequencing a nucleic acid molecule. In addition, the compounds may be used generally for polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridization assays, single nucleotide polymorphism studies, and other techniques using enzymes such as polymerases, reverse transcriptases, terminal transferases, or other nucleic acid modifying enzymes. The invention is especially useful in techniques that use labelled dNTPs, such as sequencing, nick translation, random primer labeling, end-labeling (e.g., with terminal deoxynucleotidyltransferase), reverse transcription, or nucleic acid amplification. For example, one aspect of the present invention provides a method for sequencing a nucleic acid by detecting the identity of a nucleotide analog after that nucleotide analog is incorporated into a growing strand of DNA.

[00212] In another aspect, the present invention provides a method for determining the sequence of a target single-stranded polynucleotide, where the method comprises the steps of:

(a) monitoring the sequential incorporation of complementary nucleotide analogs of the present invention, where the identity of each nucleotide incorporated is determined by detection of the label linked to the base, and

(b) removal of the label.

[00213] In certain embodiments, detection of the label is performed prior to removal of the label.

[00214] In certain embodiments, the present invention provides a method for determining the sequence of a target single-stranded polynucleotide, where the method comprises the steps of:

(a) providing at least one compound of formula I, II, III, or IV, where the detectable label of said nucleotide can be distinguished upon detection from the detectable label used for other nucleotides;

(b) incorporating the nucleotide of step (a) into the complement of the target single stranded polynucleotide;

(c) detecting the label of the nucleotide of (b), thereby determining the type of nucleotide incorporated;

(d) removing the label of the nucleotide of (b); and

(e) optionally repeating steps (b)-(d) one or more times; thereby determining the sequence of a target single-stranded polynucleotide.

[00215] In other embodiments, at step (a) above, the method comprises providing a set of nucleotides wherein each nucleotide is a compound of formula I, II, III, or IV. Yet another embodiment provides a set of nucleotides wherein each nucleobase present in the set is associated with a distinct detectable label such that the nucleobase is identifiable upon detection of that label.

[00216] During a sequencing reaction, the primer and target nucleic acid sequences may be combined so that the primer anneals or hybridizes to the target nucleic acid in a sequence specific manner. A polymerase enzyme, e.g. a DNA polymerase, is then used to incorporate additional nucleotides onto the primer in a sequence specific or template- dependent manner such that the nucleotides added to the primer are complementary to the target nucleic acid. For example, a nucleotide analog or a mixture of nucleotide analogs is added to the sequencing reaction at a sufficient concentration so that the DNA polymerase incorporates onto the primer a single nucleotide analog that is complementary to the target sequence. The incorporation of the nucleotide analog can be detected by any known method that is appropriate for the type of label used.

[00217] A second or subsequent round of incorporation for the nucleotide analog can occur after deprotecting the 3' protecting group. Further, each round of incorporation can be completed without disrupting the hybridization between primer and target sequence. After deprotection, the 3'-OH or 3'-SH becomes unblocked and ready to accept another round of nucleotide incorporation. Alternatively, the α-phosphate becomes unblocked and the resulting strand ready to accept another round of nucleotide incorporation. The incorporation and deprotection steps can be repeated as needed to complete the sequencing of the target sequence. In this way, it may be advantageous to differentially label the individual nucleotides so that incorporation of different nucleotides can be detected. Such a method can be used in single sequencing reactions, automated sequencing reactions, and array based sequencing reactions.

[00218] The compounds of the present invention can also be used for synthesizing oligonucleotides. In this process, the 2'-OH or 2'-SH is protected during synthesis. A method for synthesizing an oligoribonucleotide using the present compounds can proceed as follows. A first nucleoside is linked to a solid support using known methods. See, e.g., Pon, R T, "Chapter 19 Solid-phase Supports for Oligonucleotide Synthesis," Methods in Molecular Biology Vol. 20 Protocols for Oligonucleotides and Analogs, 465-497, Ed. S. Agrawal, Humana Press Inc., Towata, NJ. (1993). The 2'-OH or 2'-SH moiety can be protected with a suitable protecting group as described herein. It is to be understood that the 5'-OH and the 3'-OH/SH are also protected as needed using known methods or the methods described herein. After the initial nucleoside is tethered to the solid support, additional compounds of the present invention are added to the growing oligoribonucleotide using any of the existing strategies for internucleotide bond formation. The completed oligoribonucleotide is then deprotected at all positions by using means appropriate for the specific protecting groups used. Such deptrotection means are known to one of skill in the art.

[00219] In certain embodiments, sequencing methods of the present invention are carried out with the target polynucleotide attached to a substrate. Multiple target polynucleotides can be immobilised on the substrate through linker molecules, or can be attached to particles, e.g., microspheres, which can also be attached to a substrate material. [00220] The polynucleotides can be attached to the substrate by a number of means, including the use of biotin-avidin interactions. Methods for immobilizing polynucleotides on a substrate are well known in the art, and include lithographic techniques and "spotting" individual polynucleotides in defined positions on a solid support. In certain embodiments, the substrate is a solid support. Such solid supports are known in the art, and include glass slides and beads, ceramic and silicon surfaces and plastic materials. The support is usually a flat surface although microscopic beads (microspheres) can also be used and can in turn be attached to another solid support by known means. The microspheres can be of any suitable size, typically in the range of 100 nm to 10 μm in diameter. In another embodiment, the polynucleotides are attached directly onto a planar surface, such as a planar glass surface. Attachment may be by means of a covalent linkage. In other embodiments, the arrays that are used are single molecule arrays that comprise polynucleotides in distinct optically resolvable areas, e.g., as disclosed in International App. No. WO 00/06770.

[00221] The sequencing methods of the present invention can be carried out on both single polynucleotide molecule and multi-polynucleotide molecule arrays, i.e., arrays of distinct individual polynucleotide molecules and arrays of distinct regions comprising multiple copies of one individual polynucleotide molecule. Single molecule arrays allow each individual polynucleotide to be resolved separately.

[00222] Chemical synthesis of an oligonucleotide can be done by attaching a first nucleoside monomer to a solid support. Any known solid support can be used including non-porous and porous solid supports and organic and inorganic solid supports. Useful solid supports include polystyrenes, cross-linked polystyrenes, polypropylene, polyethylene, teflon, polysaccharides, cross-linked polysaccharides, silica, and various glasses. Conventional linkers and methods for attaching monomers or oligonucleotides to a solid support are known. See Beaucage & Iyer, Tetrahedron, 48(12):2223-2311 (1992). [00223] In certain embodiments, the present invention provides a kit, where the kit includes: (a) individual nucleotide analogs of formula I, II, III, or IV; and (b) a DNA polymerase, (c) reaction buffer, (d) natural 2'deoxynucleoside triphosphates, and optionally, (e) a cleavage reagent. Kits may also be formulated to include additional reagents to support sequencing by synthesis experiments. Such additional reagents may include one or more of the following items (a) substrate with one or more attached oligonucleotide primers, (c) reagents for clonal amplification of DNA fragments onto the substrate, (d) reagents for creating an array of microbeads with clonally amplified DNA fragments in the case where the substrate is plurality of microbeads, (e) reagents for creating libraries of genomic DNA fragments with attached oligonucleotide primers.

EXAMPLES

[00224] The compounds of this invention may be prepared or isolated in general by synthetic and/or pseudo-synthetic methods known to those skilled in the art for analogous compounds and as illustrated by the general schemes and the preparative examples that follow.

[00225] Certain organic and inorganic starting materials were obtained from Aldrich

Chemical, Alfa Aesar, or Acros Organics and were used without further purification. The

NHS ester of AlexaFluor 430 was obtained from Molecular Probes, Inc., of Eugene, OR,

All solvents were HPLC grade, or ultra-dry grade from Fisher Scientific.

[00226] NMR spectra were recorded on a Bruker Avant System operating at 200 MHz, or on a Bruker Avant System operating at 250 MHz.

[00227] Melting points were obtained on a Mel-Temp capillary system and are uncorrected.

[00228] HPLC data were obtained using a Hewlett-Packard 1100 equipped with a

Zorbax SB-C₈ column (50 X 4.6 mm) using a gradient of 15% acetonitrile: 85% water to

95% acetonitrile:0.5% water in 5 minutes (both solvents contain 0.1% TFA), at a flow rate of 2mL/min.

[00229] TLC data were obtained using Analtech F₂₅₀ 0.25mm silica plates.

[00230] Compounds were purified using bench top flash chromatographic silica from

Silicycle. All compounds gave satisfactory analytical data. Example 1

J. Org. Chem., 65, 249 (20Q0)

Synthesis of 3'-Deoxythymidine-3'-yl Methyl Disulfide 5'-Triphosphate: A solution of 3'-deoxythymidine-3'-yl methyl disulfide (145 mg), prepared in five steps according to the procedure of Chambert, et al. (J. Org. Chem., 65, 249 (2000)), was co-evaporated from anhydrous pyridine (5 mL) and dissolved in anhydrous dioxane (1.35 mL). To this solution was added, under an argon atmosphere, a stock solution of 2-chloro-4H- 1,2,3 - dioxaphosphori-4-one (524 μl, IM in anhydrous dioxane). After stirring for 10 minutes, it was treated with bis (tri-n-butylammonium)-pyrophosphate (1.4 mL of a 0.5M solution in DMF) and tri-n-butylamine (470 μl). After stirring for 10 minutes, the reaction was treated with iodine (10 μl, 1% in 2% aqueous pyridine) and allowed to stir for 15 minutes. The excess iodine was quenched by treatment with NaHSO₃ (700 μl, 5%)and evaporated to dryness. The residue was treated with water (10 mL) and stirred for 30 minutes, whereupon it was treated with concentrated aqueous ammonia (20 mL), stirred for one hour and evaporated to dryness. The residue was dissolved in water and applied to a column packed with DEAE Sephadex A25 resin in 0.05 M triethylammonium bicarbonate and eluted with a linear gradient up to IM TEAB. Pooled product fractions were lyophilized to afford the title compound (65 mg).

Example 2 Synthesis of PEG Linker with Fluorescent Label

Part A:

FmocOSucc

DCM Reaction J

TsCI- Pyridine Reaction 2

Reaction 4

Reaction 1: Synthesis of [2-(2-hydroxyethoxy)- ethylj-carbamic acid 9H-fluoren-9- ylmethyl ester. A 250 mL round bottom flask was charged with a solution of 2- aminoethoxyethanol (10 gm, 0.095 mol; Acros Chemical) in CH₂Cl₂ (100 mL). To this was added N-(9-fluorenylmethoxycarbonyloxy)-succinimide (33 gm, 0.1 mol) and the solution stirred at room temperature for five hours. The solution was stripped to dryness and taken up in CH₂Cl₂, extracted with bicarbonate solution, then brine. The organic layer was dried over sodium sulfate, filtered and evaporated in vacuo to afford [2-(2- hydroxyethoxy)- ethyl]-carbamic acid 9H~fluoren-9-ylmethyl ester as a crystalline solid (28 gm, 90%) HPLC: 97%.

Reaction 2: Synthesis of Toluene-4-sulfonic acid 2-[2-(3-methyl-2-vinyl-lH-inden- 1- ylmethoxycarbonylamino)-ethoxy]-ethyl ester. A 1000 mL flask was charged with [2- (2-hydroxyethoxy)- ethyl] -carbamic acid 9H-fluoren-9-ylmethyl ester (33 gm, O.lmol) and the flask flushed with argon. The solid was dissolved in dry pyridine (300 mL) and the resulting solution stirred under argon in an ice bath at O⁰C. The solution was treated with /7-toluenesulfonyl chloride (21.1 gm, 0.112mol) and the resulting solution stirred stored under argon at 0⁰C overnight. The solvent was removed in vacuo and the residue taken up in ethyl acetate, washed with water (2X), 10% citric acid (2X), and saturated sodium chloride. The organic phase was dried over sodium sulfate, filtered and evaporated to afford toluene-4-sulfonic acid 2-[2-(3-methyl-2-vinyl-lH-inden-l- ylmethoxy-carbonylamino)-ethoxy]-ethyl ester. This was purified on 500 gm silica, using EtOAc:Hex, starting at a ratio of 1:4 and then the product eluted at 1:1. The pooled fractions containing product λvere evaporated to afford 28 gm pure product, assayed at 98% by HPLC.

Reaction 3: Synthesis of [2-(2-Bromoethoxy)-ethyI]-carbamic acid 9H-fiuoren-9- ylmethyl ester A 500 mL round bottom flask was equipped with an oil bath, a reflux condenser, a magnetic stir bar and a source of dry nitrogen. The flask was flushed with nitrogen and charged with toluene-4-sulfonic acid 2-[2-(3-methyl-2-vinyl-lH-inden-l- ylmethoxycarbonylamino)-ethoxy]-ethyl ester(25 gm, 52 mmol) and this was dissolved in anhydrous THF (250 mL). The clear solution was treated with tetra-«~butylammonium bromide (65 gm, 202 mmol). The mixture was heated to 70°C.and stirred for one hour. The heating bath was removed and the reaction allowed to stir overnight under nitrogen. The solution was concentrated in vacuo and the residue dissolved in EtOAc: Hexane (20OmL, 10:1). The resulting solid was removed by filtration. The filtrate was washed with water (2x300mL), followed by saturated NaCl (300 mL). The organic phase was dried over sodium sulfate, filtered and evaporated in vacuo to afford [2-(2-bromoethoxy)- ethyl]-carbamic acid 9H-fluoren-9-ylmethyl ester as cream colored solid (20 gm). Reaction 4: Synthesis of [2-(2-TrityIsuIfanyl-ethoxy)-ethyI]-carbamic acid 9H- fluoren-9-yImethyl ester. A solution of sodium hydroxide (3.08 gm, 77 mmol) in water (3 mL) and absolute ethanol (300 mL) was treated with triphenylmethylsulfide (21.3 gm) and the resulting mixture stirred for 15 minutes. The cloudy mixture was added to the solid from the previous step and stirred for 15 minutes. It was then heated to 70⁰C with an oil bath. After two hours the reaction was cooled to room temperature and stored in the freezer overnight. The resulting mixture was dissolved in ethyl acetate (500 mL), filtered, and concentrated in vacuo to afford a yellow oil (38 gm). This was purified on 450 mL silica beginning with 6: 1 hexane:ethyl acetate, and then 2:1. The fractions containing the product were pooled and evaporated to dryness to afford [2-(2~tritylsulfanyl-ethoxy)- ethyl]-carbamic acid 9H-fluoren-9-ylmethyl ester as an oil (10.5 gm, 35%). Reaction 5: Synthesis of 2-(2-Tritylsulfanyl-ethoxy)-ethy-amine. To a stirred solution of [2-(2-tritylsulfanyl-ethoxy)-ethyl]-carbamic acid 9H-fluoren-9-ylmethyl ester (9.5 gm, 16.2 mmol) in anhydrous THF (100 mL), under nitrogen, is treated with piperidine (940 mL). The solution was stirred for 90 minutes and concentrated in vacuo to give an oily solid. This was washed in hexane, and the solution decanted, concentrated to dryness and the residue purified on silica gel using an eluant of 5% methanol in CH₂Cl₂ containing 1% triethylamine to afford 2-(2-tritylsulfanyl-ethoxy)-ethylamine (1.6 gm, 29%). Reaction 6: Synthesis of Thiol-terminated PEG Dye.

Step (A): A solution of NHS AlexaFluor 430 (5 mg) in anhydrous methanol (500μl) was treated with a solution of 2-(2-tritylsulfanyl-ethoxy)-ethylamine (3.5 mg) in anhydrous methanol (250μl) and the stirred solution monitored by HPLC. Upon completion, the solvent was removed in vacuo and the residue dissolved in 1 mL 1 :1 methanol: water. This was loaded onto a SepPak-C₁₈ cartridge and eluted with 10 mL each of 1:1 methanol: water, 3:1 methanol :water, and 100% methanol. Product fractions were pooled and evaporated in vacuo to afford the trityl protected thiol-terminated PEG dye in 90% yield.

Step (B): The trityl group was removed by treating the protected dye linker with 2 mL 50% trifluoroacetic acid in CH₂Cl₂ in the presence of one equivalent triethylsilane, using the procedure reported by Pearson, et al. {Tetrahedron Letters, 30, 2739 (1989)). Reaction 7: Synthesis of Activated Disulfide. The mixed disulfide was prepared by treating the free thiol of Reaction 6 with 2,2'-pyridinedisulfide, following the procedure of Sun, et al, RNA, 3, 1352 (1997).

Part B:

Thymidine 6 Steps AcO 3% TFA/ CH₃CI;

— ► Pyridine fltaw,j.o«M..7.4oi i!9%i Reaction 9

Reaction 8

Reaction 8: Synthesis of 5'-Acetyl-5'-(dimethoxytrityl)-5-[S-(2,4-dinitrophenyI)- thiol]-2'-deoxyuridine. A solution of 5'-(dimethoxytrityl)-5-[S-(2,4-dinitrophenyl)-thiol]- 2'-deoxyuridine (7.1 gm, prepared by the procedure of Meyer and Hanna, Bioconjugate Chemistry, 7, 401-412 (1996)) was dissolved in anhydrous pyridine (30 mL) and treated with acetic anhydride (0.92 mL). After being allowed to stir at room temperature for 20 hours, the solution was evaporated to dryness and the residue taken up in ethyl acetate. This was washed with 10% aqueous citric acid and brine. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness to afford a solid. This was purified on silica using an eluting solvent of 6:4 ethyl acetate: hexane, containing 2% triethylamine. Fractions containing the product were pooled and evaporate in vacuo to afford 5'-acetyl-5'-(dimethoxytrityl)-5-[S-(2,4-dinitrophenyl)-thiol]-2'-deoxyuridine (2.5 gm, 33%). Reaction 9: Synthesis of 5'-AcetyI-5-[S-(2,4-dinitrophenyI)-thiol]-2'-deoxyuridine. A sample of 5'-acetyl-5'-(dimethoxytrityl)-5-[S-(2,4-dinitrophenyl)-thiol]-2'-deoxyuridine (1 gm) was treated with of a solution of 3% TFA in CH₂Cl₂ (15 mL ) and the resulting pale orange solution allowed to stir at room temperature for 30 minutes. The solvent was evaporated and the residue dissolved in fresh CH₂Cl₂. This was washed with saturated sodium bicarbonate, dried over sodium sulfate, filtered and evaporated to a solid. This was purified on silica using 6:4 ethyl acetate; hexane to afford 5'-acetyl-5-[S-(2,4- dinitrophenyl)-thiol]-2'-deoxyuridine (250 mg, 41%).

Reaction 10: Synthesis of 5-[S-(2,4-dinitrophenyl)-thiol]-2'-deoxyuridine-5'- triphosphate. This conversion was carried according to the procedure of Ludwig and Eckstein ( J, Org. Chem., 54, 631-635 (1989)). Ion exchange chromatography was performed with DEAE Sephadex A25 resin, using a gradient of 0.05 M to 1 M triethylammonium bromide. Fractions showing a positive sugar and phosphate test were pooled and lyophilized to afford the nucleotide as its tetra-triethylamine salt. Reaction 11: Synthesis of 5-Thiol-2'-deoxyuridine-5'-triphosphate. The free thiol was generated by removal of the 2,4-dinitrophenyl group using. -mercaptoethanol in TEAB buffer (pH 8), using the procedure reported by Shaltiel (Niochem. Biophys. Res. Commun., 29, 178 (1967).

Reaction 12: Synthesis of Fluorescent Nucleotide. The activated thiol synthesized in Reaction 7 was reacted with the thiol product of Reaction 11 to afford the mixed disulfide fluorescent nucleotide with cleavable linker, following the procedure of Futaki and Kitigawa (Tetrahedron Letteres, 35, 1267-1270 (1994).

Example 3 Scheme III: Synthesis of 3'-O-(tert-Butyldimethylsilyl)thymidine 5 '-triphosphate

tBDMSlCl/pyπdine TFA/DCM / water

3^5'-bis(O-te/Y-butyIdimethylsiIyl)thymidine: Following the procedure described by Tronchet, et al, Nucleotides and Nucleosides., 20, 1927 (2001), a 1250 mL round bottom flask was equipped with an argon source, a stir bar and a heating mantle. The flask was charged with thymidine (10 gm). Under argon, the thymidine was suspended in pyridine (100 mL) and treated with tert-butyldimethylchlorosilane (15.6 gm). The mixture was stirred for one hour at room temperature and then warmed to ~65°C and stirred for seven hours. The reaction was cooled to room temperature and allowed to stand overnight. The resulting pale yellow solution (with some solid in it) was treated with water (4.1 mL) and stirred for 15 minutes. The solvent was evaporated and the residue co-evaporated with toluene (2 x 5OmL). The residue was taken up in cold EtOAc (200 mL) and washed with cold IN HCl, followed by water and saturated sodium bicarbonate, then dried over Na₂SO₄, filtered and evaporated to dryness to give 20 gm white solid. This was recrystallized from 3OmL 10:1 hexane: EtOAc to afford 3',5'-bis(O-tert- butyldimethylsilyl)thymidine, 15.5 gm (80%). 3'-O-tø/^butyIdimethylsilyl)thymidine: A 250 mL round bottom flask was equipped with a stir bar and the flask charged with 15.5 gm 3',5'-bis(O-tert- butyldimethylsilyl)thymidine. This was dissolved in DCM (178 mL) and treated with trifluoroacetic acid : water (17.9 mL, 10:1 v/v) and the mixture stirred at room temperature. Contrary to the reference, the reaction proceeded very slowly and the product over-desilylated to generate a substantial amount of thymidine. The reaction was stopped by cooling to O⁰C and treating with saturated sodium bicarbonate. The solution was washed with saturated NaCl, dried over sodium sulfate, filtered and evaporated to give 15 gm of an oil. This was purified by flash chromatography on 150 gm silica with a step-gradient Of CH₂Cl₂ and acetone in ratios of 6: 1, then 4:1, respectively to give 2.4 gm 3'-O-te7*/-butyldimethylsilyl)thymidine.

3'-O-(tert-Butyldimethylsilyl)thymidine 5'-triphosphate: A 50 mL flask was equipped with a stir bar and a source of dry argon. The flask was charged with 3'-0-tert- butyldimethylsilyl)thymidine (107 mg). This was dissolved in anhydrous pyridine (6 mL) and the solution evaporated to dryness. The flask was flushed with argon and the residue treated with anhydrous pyridine (300 μl) and anhydrous dioxane (900 μl), followed by IM 2-chloro-4H-l,2,3-dioxaphosphori-4-one in anhydrous dioxane (330 μl) and the reaction mixture stirred for ten minutes at room temperature. The reaction mixture was treated with a well-stirred mixture of 0.5M solution of bis(tri-n-butylamrnoniurn)pyrophosphate in anhydrous dimethylformamide (900 μl) and tri-n-butylamine (300 μl) and the mixture stirred ten minutes at room temperature. This was treated with of a solution of 1% iodine in a mixture of 98:2 pyridine : water (6 μl) and stirred 15 minutes and the excess iodine was quenched by treatment with 5% NaHSO₃ (700 μl) and the solution evaporated to dryness. The residue was treated with water (30 mL) and stirred for 30 minutes, whereupon it was treated with concentrated aqueous ammonia (60 mL), stirred for one hour and evaporated to dryness. The residue was dissolved in water and applied to a column packed with DEAE Sephadex A25 resin in 0.05 M triethylammonium bicarbonate and eluted with a linear gradient up to IM TEAB. Pooled product fractions were lyophilized to afford the title compound (54 mg). Example 4 Thymidine-S'-O-d-CS-Csubstitutedithiotriphosphate.

Pyridine

HCl Methanol

Reaction 1: Synthesis of 3'-O-Acetyl- 5'-0-(ter£-ButyIdimethylsilyI)thymidine: A

25OmL round bottom flask was equipped with a stir bar, a source of dry argon and an ice bath. The flask was charged with thymidine (24.2 gm) and the flask flushed with argon. The thymidine was suspended in pyridine (6OmL) and the mixture cooled to O⁰C. Next, fert-butyldimethylsilylchloride (16.6 gm) was added at once. The mixture was stirred for 3 hours and allowed to warm to room temperature. The turbid mixture was treated with acetic anhydride (12.25 gm) and the reaction stirred at room temperature for 14 hours. The turbid mixture was precipitated by pouring, with stirring, into water (50OmL). After being allowed to sit for a few minutes, the solid was isolated by filtration and washed with water. The solid was dissolved in about acetone (200 mL) and the resulting clear solution was re-precipitated into water (600 mL). The resulting precipitate was collected by filtration, rinsed with water and dried in vacuo at ~40°C. to afford 3'-O-acetyl- 5'-0-(tert- butyldimethylsilyl)thymidine 32 gm (93%). Reaction 2: Synthesis of 3'-O-AcetyI thymidine: A 2 L round bottom flask was equipped with an overhead stirrer and an ice bath. The flask was charged with 3'-O- acetyl- 5'-O-(tert-butyldimethylsilyl)thymidine (200 gm) and the solid treated with methanol (1.5 L). The mixture was cooled on an ice bath and treated with acetyl chloride (5.9 gm) and the reaction stirred. The ice bath was removed after 15 minutes and the reaction stirred at room temperature and monitored by HPLC. After three hours, it was 97% complete and a solution. This was treated with solid sodium bicarbonate until neutral to pH paper (approximately 30 gm), stirred briefly, filtered and the solid washed with 50 mL methanol. The solvent was concentrated in vacuo until a white solid appeared. About two-thirds of the solvent was removed, the resulting solid collected by filtration, dried in vacuo to afford 3'-O-acetyl thymidine, 71.4 gm (50%).

Reaction 3: Synthesis of Thymidine~5'-0-(l-thiotriphosphate): The title compound was prepared, in the following manner, by methods substantially similar to those of Arabshashi and Frey (Biochem. Biophys. Res. Commun., 204, 150 (1994). 3'-O-Acetyl thymidine (570 mg), previously evaporated from pyridine (5 mL), was dissolved in triethylphosphate (7.5 mL) under argon, treated with tri-«-butylamine (525 μl), and the reaction cooled to 0⁰C. This was treated with thiophosphoryl chloride (204 μl) and allowed to stir at 0⁰C for one hour. A solution of tr-m-butylammonium pyrophosphate (1.21 gm) dissolved in triethylphosphate (19 mL) was added and the resulting mixture allowed to stir at room temperature for 30 minutes. Triethylamine (10.4 mL) was added to precipitate inorganic phosphate. The mixture was filtered and the crude product taken up in water (125 mL) and treated with concentrated ammonia (25 mL). This was allowed to stand for 1 hour at room temperature and the ammonia removed in vacuo. The resulting concentrate was purified by ion exchange chromatography with DEAE Sephadex A25 resin, using a gradient of 0.05 M to 1 M triethylammonium bromide. Fractions showing a positive sugar and phosphate test were pooled and lyophilized to afford the nucleotide as its tetra-triethylamine salt 3'-0-acetylthymidine-5'-0-(l-thiotriphosphate). Reaction 4: Synthesis of Thymidine-5'-O-(l-(S-(2-pyridyIthio)thiotriphosphate): The thymidine-5'-O-(l-thiotriphosphate) from the previous reaction was dissolved in a mixture of 1:1 methanol/water and treated with Aldrithiol 2. The product was purified by IEX chromatography as above to give Product 1 (See Scheme). Additional groups can be attached as disulfides by reaction of Product 1 with any number of free thiols. Example 5

Evaluation of Reversible Terminators

[00231] The reversible terminators of the present invention were tested for efficiency of polymerase incorporation and their termination efficiency and reversibility. To that end, a Capillary Electrophoresis based strand shift assay was used. Similar to gel shift assays, the goal was to observe an extended primer based on the difference in mobility in the matrix of a DNA fragment which has been extended by one nucleotide in comparison to a fragment that has not been extended.

[00232] The assay was developed on an ABI 373 OXL DNA Analyzer. To test the efficiency of incorporation, a dye labeled primer was annealed to a template containing a 5' biotin attached to magnetic beads coated with Streptavidin. The template contains a stretch of complementary bases to the reversibly terminating nucleotide. DNA polymerase was added in the presence of a nucleoside triphosphate of interest, here a reversible terminator, and appropriate buffers. The reaction was incubated at an appropriate temperature (usually 37°C) for 4 minutes, stopped with EDTA, washed 3X, resuspended in deionized water, heated to 95°C to denature the primer-template structure, placed on a magnet to concentrate the beads, and the supernatant removed. The supernatant contained the primer, which was then analyzed on the 3730XL against fragments of known size to determine if the primer had been extended. Efficiency of incorporation was estimated from the amount of unextended primer versus the amount of extended primer. Efficiency of termination can be estimated from the amount of next base addition because the template contains a stretch of complementary bases to the terminating nucleotide being interrogated, a nucleotide that does not terminate efficiently will allow the primer to be extended by more than one nucleotide. How reversible the terminating nucleotide can be assessed by reversing the termination and attempting an additional incorporation. The protocol is as follows.

[00233] 1 micron Dynal Streptavidin beads (MyOne) (100 μl) at approximately 10 Million beads/μl were washed 3X in 1OmM Tris-Cl, 1 mM EDTA, pH 8 (TE), then resuspended in 200ul of TE containing 300 pmol of 5' biotinylated Template oligonucleotide (oligo). The Biotinylated template oligo contained a region complementary to a sequencing primer followed by a stretch of 5 Adenosines and then by a mix of various bases. The bead/oligo mixture was incubated on a rotator at room temperature for 30 minutes, applied to a magnet, the supernatant was removed, and the beads washed 3X with 200 μl TE. The beads were resuspended in 100 μl of TE. 10 μl of resuspended beads are added to 90 μl of TE containing 100 pmol of sequencing primer. The sequencing primer was complementary to a stretch of the 5'Biotinylated Template Oligo allowing sequencing of the 5 consecutive Adenosines. The sequencing primer was labeled with a 5' FAM dye. The beads with attached template oligo in the primer solution were incubated at 65°C for 2 minutes then cooled to 40⁰C for 2 minutes followed by a 4⁰C incubation of 2 minutes to anneal the sequencing primer. Excess primer was washed away and the beads resuspended at an appropriate concentration of beads. [00234] Two million beads with template oligo attached and sequencing primer annealed were added to a sequencing reaction containing 10 U of Klenow exo-, for example, an appropriate amount of Thymidine (typically around 1 μM final concentration for unmodified Thymidine) in a reaction volume of 10 μl. The reaction is then incubated at 37⁰C for 4 minutes and stopped by adding EDTA to a final concentration of 50 mM. The beads were applied to a magnet, washed 3X with TE buffer, once with deionized water and then resuspended in 20 μl of deionized water. The resuspended beads were heated to 95°C to denature the primeπtemplate, releasing the primer into solution. The beads were concentrated with a magnet and the supernatant removed to a fresh tube. 0.5 μl of supernatant was loaded on the ABI 3730XL with 9 μl of HiDi Formamide and 0.5 μl of the ABI Liz Genescan size marker. Typical results are shown in Figures IA, IB, 2A, and 2B.

Example 6

Sequencing by Synthesis

[00235] Four reversibly terminating nucleotides were used, each capable of being incorporated specifically at a position complementary to adenosine, guanosine, cytosine or thymidine on a DNA template. Each reversibly terminating nucleotide was labeled with a different fluorescent dye that permitted its unique detection and identification at a particular wavelength of light. The DNA templates to be sequenced were dispersed and immobilized onto an array such that a fluorescent signal corresponding each template could be detected and recorded. In this example, the templates were immobilized onto 1 micron diameter magnetic beads. Each bead contained approximately 150,000 copies of a DNA template that was derived by clonal amplification from a single DNA molecule. The amplification was accomplished by emulsion PCR, as described by Dressman, et al., 2003, Proc. Natl. Acad. Sci. USA. 100: 8817-22. As a consequence of the template construction and amplification process, each immobilized DNA fragment on each bead contained a sequence complementary to a universal sequencing primer immediately juxtaposed to the unknown DNA that was to be sequenced. The beads were imbedded in a thin layer of polyacrylamide on the surface a glass microscope slide. To accomplish this, template- bound beads were mixed with the ingredients necessary to produce a 5% polyacrylamide gel and poured onto a masked, bind-silane treated microscope slide, and covered with a cover slip as described by Mitra, et al., (2002) Anal Biochem. 320:55-65. After polymerization the template-containing beads were trapped in a single focal plane and at a specific x, y location on the glass slide, allowing visualization and identification of each bead according to its location.

[00236] The universal primer, labeled with a fluorescent dye (in this case, FAM) was hybridized to the beads by adding sufficient primer (in a TE solution) to achieve a concentration approximately 10 times the molarity of template bound to the beads (i.e., if a total of lOpmol of template was present on the combined beads in the gel, lOOpmol of primer was added). The slide was incubated at 65°C for 2 minutes, then 40⁰C for 2 minutes and 4°C for 2 minutes to allow the primer to anneal. Excess primer was washed away by immersing the slide three times in TE for 3 minutes each, refreshing the TE each time. The slide was imaged on a microscope with the appropriate light source to allow visualization of the FAM labeled primer in the plurality of primer-template complexes attached to each bead. The location of each bead was recorded so that the fluorescent history of each bead could be determined after stepwise addition of labeled reversible terminators. In an alternate protocol for initial detection of all of the beads, the sequencing primer was designed in such a way that one base of the universal primer-binding sequence adjacent to the DNA to be sequenced was added in the first nucleotide addition step (thus, the same nucleotide was incorporated on all of the primer-template complexes, allowing detection of all bead location on the array).

[00237] Each addition cycle was accomplished as follows. The primers were extended on distinct beads by adding a solution containing a DNA Polymerase capable of incorporating the reversible terminators, such as exonuclease deficient mutant of E. coli polymerase I Klenow fragment, in an appropriate buffer with the four fluorescently labeled reversible terminators at an appropriate concentration (typically 2-50 micromolar) to effect >95% complete addition of a single nucleotide at the 3' end of each primer- template complex when incubated at 37°C (or another temperature more appropriate for the particular polymerase used). Excess reversible terminators were then removed by immersing the slide in TE three times for 3 minutes, refreshing the TE buffer each time. The slide was then imaged on a microscope using appropriate excitation and emission wavelengths to allow visualization of one of the fluorescently labeled reversible terminators. This process was repeated at appropriate excitation and emission wavelengths to allow specific detection of each of the three remaining fluorescently labeled terminators. Each bead that contained an extended primer-template complex fluoresced in one of the four detection steps at a wavelength corresponding to the particular fluorescently labeled terminator that was incorporated. The nature of the added nucleotide for each bead was recorded. In an alternate protocol, in which the fluorescent labels attached to the reversible terminators were all excited at the same wavelength but the emission from each of the four detected at a different wavelength, a single detection step was used with simultaneous data collection at each of the four emission wavelengths, using a color CCD camera. After detection, the slide was washed with a solution containing 500 mM DTT for 5 minutes, and then washed 3 times with TE for 3 minutes to cleave the linker by which the fluorescent label was attached to the incorporated nucleotide and wash away the released molecules. The conditions used for cleavage maintain the integrity of the hybridized primer-template complexes (no denaturation is allowed to occur). Depending on the nature of the terminator, cleavage of the linker also generates an extendable 3' terminus on the incorporated nucleotide. If the reversible terminator was of a type in which two linkers with different cleavage chemistries were used (1) to attach the fluorescent label and (2) to prevent addition to the 3' terminus, a second cleavage step is added to effect cleavage of the second linker. Once an extendable terminus has been generated at the 3' end of the extended primer in each primer-template complex, the addition cycle was repeated with a fresh aliquot of reversible terminators. The process of reversible terminator addition, detection, and cleavage to regenerate an extendable 3' terminus on the incorporated nucleotide is repeated for multiple cycles in order to read out the sequence of the template attached to each bead. [00238] While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

Claims

CLAIMSWe claim:

1. A compound of formula I:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R⁴;

R⁵ is hydrogen or a thiol protecting group and is optionally substituted with L-R⁴; each L is independently a cleavable linker group; and each R⁴ is independently a detectable moiety; provided that:

(a) R⁵ is other than -S-pyridin-2-yl when R¹ is -OTBS;

(b) R⁵ is other than hydrogen when R² is TBDPSi; and

(c) at least one of R³ or R⁵ is substituted with L-R⁴.

2. The compound according to claim 1, wherein R³ is a nucleobase selected from adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or 2- aminopurine.

3. The compound according to claim 1, wherein R⁵ is selected from a disulfide, a thioether, a silyl thioether, a thioester, a thiocarbonate, or athiocarbamate.

4. The compound according to claim 3, wherein R⁵ is -S-pyridin-2-yl.

5. The compound according to claim 1, wherein R² is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH)₂.

6. The compound according to claim 5, wherein R¹ is OH or hydrogen.

7. The compound according to claim 1, wherein said compound is of formula Ia:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R² is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)_2, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂;

R³ is a nucleobase or a nucleobase mimetic;

L is independently a cleavable linker group; and

R⁴ is a detectable moiety.

8. The compound according to claim 1, wherein said compound is of formula Ib:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R² is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂; R^J is a nucleobase or a nucleobase mimetic;

T is oxygen, sulfur, NH, or optionally substituted phenyl;

L is independently a cleavable linker group; and

R⁴ is a detectable moiety.

9. The compound according to claim 1, wherein said compound is of formula Ic:

wherein:

W is 0(Ci_-4 aliphatic) or S(Ci_-4 aliphatic);

W is Ci-₄ aliphatic;

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R³ is a nucleobase or a nucleobase mimetic;

L is a cleavable linker group; and

R⁴ is a detectable moiety.

10. The compound according to claim 1, wherein said compound is of formula Id:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R² is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂; R is a nucleobase or a nucleobase mimetic, wherein R is optionally substituted with L-

11. A compound of formula II:

wherein:

R^4a;

(a) at least one of R^3a or R^la is substituted with L^a-R^4a; and

(b) R^la is other than trityl when R^5a is either acetyl or benzyl; and said compound is

other than

12. The compound according to claim 11, wherein R^3a is a nucleobase selected from adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or 2- aminopurine.

13. The compound according to claim 11, wherein R^la is selected from a disulfide, a thioether, a silyl thioether, a thioester, a thiocarbonate, or a thiocarbamate.

14. The compound according to claim 13, wherein R⁵ is -S-pyridin-2-yl.

15. The compound according to claim 11, wherein R² is -P(O)(OH)-O-P(O)(OH)- 0-P(O)(OH)₂.

16. The compound according to claim 11, wherein R^5a is hydrogen.

17. The compound according to claim 11, wherein said compound is of formula

Ha:

wherein:

R^2a is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)_2, -P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-S- P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O- P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-P(O)(OH)₂;

R^4a;

R^Sa is hydrogen or a suitable hydroxyl protecting group;

L^a is a cleavable linker group; and

R^4a is a detectable moiety.

18. The compound according to claim 11, wherein said compound is of formula Hb:

wherein:

R^4a;

R^5a is hydrogen or a suitable hydroxyl protecting group;

T^a is oxygen, sulfur, NH, or optionally substituted phenyl; each L^a is independently a cleavable linker group; and each R^4a is independently a detectable moiety.

19. The compound according to claim 11, wherein said compound is of formula Hc:

wherein:

W^a is O(C_]-4 aliphatic) or S(Ci_-4 aliphatic);

W^b is Ci_-4 aliphatic;

R^4a;

R^Sa is hydrogen or a suitable hydroxyl protecting group; each L^a is independently a cleavable linker group; and each R^4a is independently a detectable moiety.

20. The compound according to claim 11, wherein said compound is of formula IW:

wherein:

R^3a is a nucleobase or a nucleobase mimetic, wherein R^3a is substituted with L^a-R^4a;

R^5a is hydrogen or a suitable hydroxyl protecting group;

L^a is a cleavable linker group; and

R^4a is a detectable moiety.

21. A compound of formula III:

wherein:

R^lb is OH, a suitably protected hydroxyl group, or hydrogen;

Q is oxygen or sulfur;

R^4b;

R^sb is hydrogen or a suitable hydroxyl protecting group; each L^b is independently a cleavable linker group; and each R^4b is independently a detectable moiety; provided that: (a) R° is other than allyl when R^5b is TBS;

(b) R⁶ is other than p-nitrobenzyl when Q is sulfur; and

(c) at least one of R^3b or R⁶ is substituted with L^b-R^4b.

22. The compound according to claim 21, wherein Q is oxygen.

23. The compound according to claim 22, wherein R⁶ is a suitable hydroxyl protecting group selected from an ester, an allyl ether, an ether, a silyl ether, an alkyl ether, an arylalkyl ether, or an alkoxyalkyl ether.

24. The compound according to claim 23, wherein R⁶ is substituted with L^b-R^4b.

25. The compound according to claim 21, wherein Q is sulfur.

26. The compound according to claim 25, wherein R⁶ is a suitable thiol protecting group selected from a disulfide, a thioether, a silyl thioether, a thioester, a thiocarbonate, or athiocarbamate.

27. The compound according to claim 21, wherein R^Ib is hydrogen or OH.

28. The compound according to claim 27, wherein R^5b is hydrogen.

29. The compound according to claim 21, wherein said compound is of formula

Ilia:

wherein:

R^lb is OH, a suitably protected hydroxyl group, or hydrogen;

R^4b; R^5b is hydrogen or a suitable hydroxyl protecting group; each L^b is independently a cleavable linker group; and each R^4b is independently a detectable moiety.

30. The compound according to claim 21, wherein said compound is of formula

HIb:

wherein:

R^lb is OH, a suitably protected hydroxyl group, or hydrogen;

R^4b;

31. The compound according to claim 21, wherein said compound is of formula

Illc or IIIc':

32. A compound of formula IV:

wherein:

Q is oxygen or sulfur;

U is a universal nucleoside analog;

R^3c is a nucleobase or a nucleobase mimetic, wherein R^3c is optionally substituted with L^c~

R^4c; each L^c is independently a cleavable linker group;

L^c is a non-cleavable linker group; and each R^4c is independently a detectable moiety.

33. A method for determining the sequence of a target single-stranded polynucleotide, said method comprising the steps of: (a) providing at least one compound of formula I:

wherein:

R¹ is OH, a suitably protected hydroxyl group, or hydrogen;

R² is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂,

-P(O)(OH)-S-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂-

P(O)(OH)₂;

R³ is a nucleobase or a nucleobase mimetic, wherein R³ is optionally substituted with L-R⁴;

R⁵ is hydrogen or a thiol protecting group and is optionally substituted with L-R⁴; each L is independently a cleavable linker group; and each R⁴ is independently a detectable moiety; provided that at least one of R³ or R⁵ is substituted with L-R⁴, a compound of formula II:

wherein:

R^la is hydrogen or a thiol protecting group and is optionally substituted with L^a- R^4a;

R^2a is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂,

-P(O)(OH)-S-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂- P(O)(OH)₂;

R^3a is a nucleobase or a nucleobase mimetic, wherein R^3a is optionally substituted with L^a-R^4a;

R^5a is hydrogen or a suitable hydroxyl protecting group; each L^a is independently a cleavable linker group; and each R^4a is independently a detectable moiety; provided that at least one of R^3a or R^la is substituted with L^a-R^4a, a compound of formula III:

wherein:

R^lb is OH, a suitably protected hydroxyl group, or hydrogen; Q is oxygen or sulfur;

R⁶ is a suitable hydroxyl or thiol protecting group and is optionally substituted with

L^b-R^4b;

R^3b is a nucleobase or a nucleobase mimetic, wherein R^3b is optionally substituted with L^b-R^4b;

R^5b is hydrogen or a suitable hydroxyl protecting group; each L^b is independently a cleavable linker group; and each R^4b is independently a detectable moiety; provided that at least one of R^3b or R⁶ is substituted with L^b-R^4b, or a compound of formula IV:

IV wherein:

R^lc is OH, SH, a suitably protected hydroxyl group, a suitably protected thiol group, or hydrogen; Q is oxygen or sulfur; U is a universal nucleoside analog;

R^2e is a suitable hydroxyl protecting group, -P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-S-P(O)(OH)₂,

-P(O)(OH)-S-P(O)(OH)-O-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-S-P(O)(OH)₂, -P(O)(OH)-O-P(O)(OH)-NH-P(O)(OH)₂, or -P(O)(OH)-O-P(O)(OH)-CH₂- P(O)(OH)₂; R^3c is a nucleobase or a nucleobase mimetic, wherein R^3c is optionally substituted with L^c-R^4c; each L^c is independently a cleavable linker group; L^c is a non-cleavable linker group; and each R^4c is independently a detectable moiety, wherein the detectable label of said nucleotide can be distinguished upon detection from the detectable label used for other nucleotides;

(b) incorporating the nucleotide of step (a) into the complement of the target single stranded polynucleotide; (c) detecting the label of the nucleotide of (b), thereby determining the type of nucleotide incorporated;

(d) removing the label of the nucleotide of (b); and

34. A kit, wherein the kit includes: (a) individual nucleotide analogs of formula I, II, III, or IV; and (b) a DNA polymerase, (c) reaction buffer, (d) natural 2'deoxynucleoside triphosphates, and optionally, (e) a cleavage reagent. Kits may also be formulated to include additional reagents to support sequencing by synthesis experiments. Such additional reagents may include one or more of the following items (a) substrate with one or more attached oligonucleotide primers, (c) reagents for clonal amplification of DNA fragments onto the substrate, (d) reagents for creating an array of microbeads with clonally amplified DNA fragments in the case where the substrate is plurality of microbeads, (e) reagents for creating libraries of genomic DNA fragments with attached oligonucleotide primers.