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EP1879906A4 - Terminateurs nucleotidiques reversibles et leurs utilisations - Google Patents

Terminateurs nucleotidiques reversibles et leurs utilisations

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Publication number
EP1879906A4
EP1879906A4 EP06733635A EP06733635A EP1879906A4 EP 1879906 A4 EP1879906 A4 EP 1879906A4 EP 06733635 A EP06733635 A EP 06733635A EP 06733635 A EP06733635 A EP 06733635A EP 1879906 A4 EP1879906 A4 EP 1879906A4
Authority
EP
European Patent Office
Prior art keywords
group
nucleobase
hydrogen
independently
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06733635A
Other languages
German (de)
English (en)
Other versions
EP1879906A2 (fr
Inventor
Douglas Smith
Kevin J Mckernan
Alan Blanchard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AB Advanced Genetic Analysis Corp
Original Assignee
AB Advanced Genetic Analysis Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AB Advanced Genetic Analysis Corp filed Critical AB Advanced Genetic Analysis Corp
Publication of EP1879906A2 publication Critical patent/EP1879906A2/fr
Publication of EP1879906A4 publication Critical patent/EP1879906A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • 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.
  • dye labeled deoxynucleotides can be used in conjunction with unlabeled dideoxynucleotides.
  • 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.
  • the methods require gel-electrophoretic separation.
  • the systems are both time- and labor-intensive and methods avoiding gel separation have been developed in attempts to increase the sequencing throughput.
  • the SBH methods are problematic in several respects.
  • 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.
  • false positives and false negatives during hybridization detection are frequently present and complicate sequence determination.
  • sequence of the polynucleotide is not determined directly, but is inferred from the sequence of the known probe, which increases the possibility for error.
  • SBS Sequencing by Synthesis
  • Figure 1 A depicts the ABI 373 OXL DNA analysis of primer only.
  • Figure IB depicts the ABI 373 OXL DNA analysis of primer extended by T nucleotide on a template containing 5A's.
  • Figure 2A depicts the ABI 3730XL DNA analysis of an incomplete termination
  • Figure 2B depicts the ABI 3730XL DNA analysis of an incomplete extension.
  • 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.
  • the present compounds are used in the SBS method.
  • 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.
  • 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.
  • the growing nucleic acid strand e.g. a DNA strand is synthesized by a polymerase-catalyzed reaction.
  • the present invention provides a compound of formula I:
  • R 1 is OH, a suitably protected hydroxyl group, or hydrogen
  • R 2 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)-CH 2 -P(O)(OH) 2 ;
  • R is a nucleobase or a nucleobase mimetic, wherein R is optionally substituted with L-
  • R 5 is hydrogen or a thiol protecting group and is optionally substituted with L-R 4 ; each L is independently a cleavable linker group; and each K * is independently a detectable moiety; provided that:
  • R 5 is other than -S-pyridin-2-yl when R 1 is -OTBS;
  • R 5 is other than hydrogen when R 2 is TBDPSi;
  • the present invention also provides a compound of formula II:
  • 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)-CH 2 -P(O)(OH) 2 ;
  • R 3a is a nucleobase or a nucleobase mimetic, wherein R 3a is optionally substituted with L a -
  • 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:
  • R la is other than trityl when R 5a is either acetyl or benzyl; and said compound is other than
  • 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
  • R 6 is a suitable hydroxyl or thiol protecting group and is optionally substituted with L b -
  • 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:
  • R 6 is other than allyl when R 5b is TBS;
  • R 6 is other than p-nitrobenzyl when Q is sulfur
  • 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.
  • 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.
  • stable 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.
  • a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 4O 0 C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
  • 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., 32 P, 33 P, 35 S, or 14 C), mass- tags, and fluorescent moieties are signal generating reporter groups which can be detected without further modifications.
  • secondary label refers to moieties such as biotin and various protein antigens that require the presence of a second intermediate for production of a detectable signal.
  • the secondary intermediate may include streptavidin- enzyme conjugates.
  • 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.
  • FRET nonradiative fluorescent resonance energy transfer
  • fluorescent label fluorescent dye
  • fluorophore refer to moieties that absorb light energy at a defined excitation wavelength and emit light energy at a different wavelength.
  • 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, Dial
  • 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.
  • 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.).
  • mass-tag refers to any moiety that is capable of being uniquely detected by virtue of its mass using mass spectrometry (MS) detection techniques.
  • 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.
  • 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.
  • electrophore release tags such as N-[3-[4'-[(p
  • 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.
  • electrophoretic-tag refers to any moiety that is capable of being uniquely detected by virtue of its charge-to-mass ratio using electrophoretic separation techniques.
  • 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”).
  • 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.
  • substrate 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.
  • 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).
  • 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, polysacc
  • 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.
  • aliphatic groups contain 1-20 aliphatic carbon atoms.
  • 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.
  • cycloaliphatic refers to a monocyclic C 3 -Cs hydrocarbon or bicyclic C 8 -Ci 2 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.
  • heterocycle means “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.
  • heterocycle means non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring members is an independently selected heteroatom.
  • heterocyclyl means non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring members is an independently selected heteroatom.
  • 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.
  • 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)
  • alkoxy refers to an alkyl group, as previously defined, attached to the principal carbon chain through an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.
  • haloalkyl means alkyl, alkenyl or alkoxy, as the case may be, substituted with one or more halogen atoms.
  • halogen means F, Cl, Br, or I.
  • 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.
  • aryl may be used interchangeably with the term “aryl ring”.
  • aryl also refers to heteroaryl ring systems as defined hereinbelow.
  • heteroaryl used alone or as part of a larger moiety as in
  • heteroarylkyl 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.
  • heteroaryl may be used interchangeably with the term “heteroaryl ring” or the term “heteroaromatic".
  • 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.
  • Optional substituents on the aliphatic group of R° are selected from N 3 , CN, NH 2 , NH(Ci -4 aliphatic), N(Ci -4 aliphatic) 2 , halogen, Ci -4 aliphatic, OH, 0(C 1- 4 aliphatic), NO 2 , CN, CO 2 H, CO 2 (Cj ⁇ aliphatic), O(haloCi 4 aliphatic), or haloC ⁇ 4 aliphatic, wherein each of the foregoing Ci ⁇ aliphatic groups of R° is unsubstituted.
  • An aliphatic or heteroaliphatic group or a non-aromatic heterocyclic ring may contain one or more substituents.
  • Optional substituents on the aliphatic group of R * are selected from NH 2 , NH(Ci -4 aliphatic), N(Ci -4 aliphatic) 2 , halogen, Ci -4 aliphatic, OH, 0(Q -4 aliphatic), NO 2 , CN, CO 2 H, CO 2 (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.
  • Optional substituents on the aliphatic group or the phenyl ring of R + are selected from NH 2 , NH(Ci -4 aliphatic), N(C 1-4 aliphatic ⁇ , halogen, C 1-4 aliphatic, OH, 0(Ci -4 aliphatic), NO 2 , CN, CO 2 H, CO 2 (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.
  • R 0 or R + , or any other variable similarly defined herein
  • R 0 is 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, pi ⁇ erazin-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
  • 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.
  • nucleotide consists of a nitrogenous base (“nucleobase”), a sugar, and one or more phosphate groups.
  • RNA the sugar is a ribose
  • 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.
  • nucleoside is structurally similar to a nucleotide, but is missing the phosphate moieties.
  • nucleobase 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).
  • nucleobases are inosine, xanthine, hypoxanthine, and 2-aminopurine.
  • 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.
  • Nucleobase mimetics include those nucleobase analogs that specifically pair with one or more of the natural nucleobases.
  • the nucleobase mimetic pairs specifically with C.
  • the nucleobase mimetic pairs specifically with T.
  • the nucleobase mimetic pairs specifically with A.
  • the nucleobase mimetic pairs specifically with G.
  • R 1 is OH, a suitably protected hydroxyl group, or hydrogen
  • R 2 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)-CH 2 -P(O)(OH) 2 ;
  • R 3 is a nucleobase or a nucleobase mimetic, wherein R 3 is optionally substituted with L-
  • R 5 is hydrogen or a suitable thiol protecting group and is optionally substituted with L-R 4 ; each L is independently a cleavable linker group; and each R 4 is independently a detectable moiety; provided that at least one of R 3 and R 5 is substituted with L-R 4 .
  • the R 3 group of formula I is substituted with L-R 4 .
  • R 3 is substituted with L-R 4 and R s is a thiol protecting group which is removable under the same conditions used to cleave L-R 4 .
  • the R 3 group of formula I is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • 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.
  • 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.
  • R 3 is substituted with L-R 4
  • the L-R 4 group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.
  • L has a functional moiety at the terminal end for coupling to the detectable R 4 group.
  • Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R 4 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.
  • 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 5 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.
  • the thiol protecting group of the R 5 moiety of formula I is -S-pyridin-2-yl.
  • the R 5 moiety of formula I is a thiol protecting group that is removable under neutral conditions e.g. with AgNO 3 , HgCl 2 , 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.
  • the R 5 moiety of formula I is a thiol protecting group that is cleaved under conditions where the pH is from about 4 to about 9.
  • the R 5 moiety of formula I is a thiol protecting group that is "photocleavable".
  • suitable thiol protecting groups include, but are not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, a trityl group, -CH 2 SCH 3 (MTM), dimethylmethoxymethyl, or -CH 2 -S-S-pyridin-2-yl.
  • THP tetrahydropyranyl
  • MTM trityl group
  • dimethylmethoxymethyl or -CH 2 -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.
  • R 2 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.
  • suitable hydroxyl protecting groups of the R 2 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.
  • silyl ethers examples 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.
  • 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.
  • the R 2 of formula I is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH) 2 .
  • the R 1 of formula I is hydrogen. [0055] In other embodiments, the R 1 of formula I is -OH. [0056] According to yet other embodiments, the R 1 of formula I is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R 2 group.
  • the present invention provides a compound of formula
  • R 1 is OH, a suitably protected hydroxyl group, or hydrogen
  • the R 3 group of formula Ia is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3 group of formula Ia is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • R 3 is substituted with L-R 4
  • the L-R 4 group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.
  • L has a functional moiety at the terminal end for coupling to the detectable R 4 group.
  • R 4 detectable group Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R 4 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.
  • the R 2 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.
  • Suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • 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-
  • silyl ethers examples 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-
  • R 2 of formula Ia is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH) 2 .
  • the R 1 of formula Ia is hydrogen. [0065] In other embodiments, the R 1 of formula Ia is -OH.
  • the R 1 of formula Ia is a suitably protected hydroxyl group.
  • suitable hydroxyl groups include those described above for the R 2 group.
  • the present invention provides a compound of formula Ib:
  • R 1 is OH, a suitably protected hydroxyl group, or hydrogen
  • the R 3 group of formula Ib is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3 group of formula Ib is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • R 3 is substituted with L-R 4
  • the L-R 4 group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.
  • L has a functional moiety at the terminal end for coupling to the detectable R 4 group.
  • R 2 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.
  • Suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • 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.
  • silyl ethers examples 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.
  • 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.
  • the R 2 of formula Ib is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH) 2 .
  • the R 1 of formula Ib is hydrogen. [0075] In other embodiments, the R 1 of formula Ib is -OH.
  • the R 1 of formula Ib is a suitably protected hydroxyl group.
  • suitable hydroxyl groups include those described above for the R 2 group.
  • the present invention provides a compound of formula Ic:
  • W is 0(C 1-4 aliphatic) or S(Ci -4 aliphatic); W is Ci -4 aliphatic;
  • R 1 is OH, a suitably protected hydroxyl group, or hydrogen
  • the present invention provides a compound of formula Ic, wherein W is -OCH 3 or -SCH 3 .
  • the present invention provides a compound of formula Ic, wherein W is -CH3, CH 2 CH3, and the like.
  • the R 3 group of formula Ic is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3 group of formula Ic is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • R 3 is substituted with L-R 4
  • the L-R 4 group can be attached at any position on the nucleobase provided that Watson-Crick base pairing can still be carried out.
  • L has a functional moiety at the terminal end for coupling to the detectable R 4 group.
  • Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R 4 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.
  • the R 2 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.
  • suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • esters include formates, acetates, carbonates, and sulfonates.
  • formate benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-
  • silyl ethers examples 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-
  • R 2 of formula Ic is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH) 2 .
  • R 1 of formula Ic is hydrogen.
  • the R 1 of formula Ic is -OH.
  • the R 1 of formula Ic is a suitably protected hydroxyl group.
  • suitable hydroxyl groups include those described above for the R 2 group.
  • R 1 is OH, a suitably protected hydroxyl group, or hydrogen
  • R 2 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)-CH 2 -P(O)(OH) 2 ;
  • R 3 is a nucleobase or a nucleobase mimetic, wherein R 3 is optionally substituted with L-
  • each L is independently a cleavable linker group; and each R 4 is independently a detectable moiety.
  • the R 3 group of formula Id is substituted with L-R 4 .
  • the R 3 group of formula Id is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3 group of formula Id is a nucleobase mimetic.
  • 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.
  • 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
  • L has a functional moiety at the terminal end for coupling to the detectable R 4 group.
  • Such functional moieties will be apparent to one of ordinary skill in the art and will be appropriate for the particular R 4 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.
  • the R 2 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.
  • suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • esters include formates, acetates, carbonates, and sulfonates.
  • formate benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-
  • silyl ethers examples 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-
  • R 2 of formula Id is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH) 2 .
  • the R 1 of formula Id is hydrogen. [0097] In other embodiments, the R 1 of formula Id is -OH. [0098] According to yet other embodiments, the R 1 of formula Id is a suitably protected hydroxyl group. Such suitable hydroxyl groups include those described above for the R 2 group.
  • the present invention also provides a compound of formula II:
  • 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)-CH 2 -P(O)(OH) 2 ;
  • R 3a is a nucleobase or a nucleobase mimetic, wherein R 3a is optionally substituted with L a -
  • 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 .
  • the R 3a group of formula II is substituted with L a -R 4a .
  • 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 -
  • the R 3a group of formula II is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3a group of formula II is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the thiol protecting groups of the R la moiety of formula II is -S-S-pyridin-2-yl.
  • the R la moiety of formula II is a thiol protecting group that is removable under neutral conditions e.g. with AgNO 3 , HgCl 2 , 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.
  • 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.
  • the R la moiety of formula II is a thiol protecting group that is "photocleavable".
  • suitable thiol protecting groups include, but are not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, a trityl group, -CH 2 SCH 3 (MTM), dimethylmethoxymethyl, or -CH 2 -S-S-pyridin-2-yl.
  • THP tetrahydropyranyl
  • MTM trityl group
  • dimethylmethoxymethyl or -CH 2 -S-S-pyridin-2-yl.
  • the R 28 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.
  • suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • 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-
  • silyl ethers examples 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-
  • R 2a group of formula II is -P(O)(OH)-O-P(O)(OH)- 0-P(O)(OH) 2 .
  • the R 5a group of formula II is hydrogen.
  • the R 5a group of formula II is a suitably protected hydroxyl group.
  • suitable hydroxyl groups include those described above for the R 2a group.
  • the present invention provides a compound of formula Ha: wherein:
  • R 5a is hydrogen or a suitable hydroxyl protecting group
  • L a is a cleavable linker group
  • R 4a is a detectable moiety.
  • the R 3a group of formula Ha is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-amino ⁇ urine.
  • the R 3a group of formula Ha is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • 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-
  • silyl ethers examples 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-
  • R 2a group of formula Ha is -P(O)(OH)-O- P(O)(OH)-O-P(O)(OH) 2 .
  • the R 5a group of formula Ha is hydrogen.
  • 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.
  • the present invention provides a compound of formula lib:
  • 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
  • each R 4a is independently a detectable moiety.
  • the R 3a group of formula lib is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3a group of formula lib is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • 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.
  • L a has a functional moiety at the terminal end for coupling to the detectable R 4a moiety.
  • R 4a detectable 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.
  • 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.
  • Suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3- ⁇ henylpropionate, 4-oxopentanoate, 4,4-
  • silyl ethers examples 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-
  • R 2a group of formula lib is -P(O)(OH)-O- P(O)(OH)-O-P(O)(OH) 2 .
  • the R 5a group of formula lib is hydrogen.
  • the R 5a group of formula lib is a suitably protected hydroxyl group.
  • suitable hydroxyl groups include those described above for the R 2a group.
  • 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.
  • the W a group of formula lie is -OCH 3 or -SCH 3 .
  • the W b group of formula Hc is -CH 3 , -CH 2 CH 3 and the like.
  • the R 3a group of formula Hc is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3a group of formula Hc is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • 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.
  • R 4a 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.
  • 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.
  • suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • esters include formates, acetates, carbonates, and sulfonates.
  • formate benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-
  • silyl ethers examples 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-
  • R 2a of formula Hc is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH) 2 .
  • R 5a group of formula lie is hydrogen.
  • 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.
  • the present invention provides a compound of formula
  • the R 3a group of formula Hd is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3a group of formula lid is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • 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.
  • L a has a functional moiety at the terminal end for coupling to the detectable R 4a moiety.
  • R 4a detectable 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.
  • 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.
  • Suitable hydroxyl protecting groups of the R 2 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxy alkyl ethers.
  • 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-
  • silyl ethers examples 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-
  • R 2a group of formula lid is -P(O)(OH)-O- P(O)(OH)-O-P(O)(OH) 2 .
  • the R 5a group of formula ITd is hydrogen.
  • the R 5a group of formula Hd is a suitably protected hydroxyl group.
  • suitable hydroxyl groups include those described above for the R 2a group.
  • R lb is OH, a suitably protected hydroxyl group, or hydrogen
  • Q is oxygen or sulfur
  • R 6 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 R 6 or R 3b is substituted with L b -R 4b .
  • the R 3b group of formula in is substituted with L b -R 4b .
  • R 3b is substituted with L b -R 4b and R 6 is a hydroxyl or thiol protecting group which is removable under the same conditions used to cleave L -R 4b .
  • the Q group of formula III is O.
  • the Q group of formula III is S.
  • the R 3b group of formula HI is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3b group of formula HI is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Suitable thiol protecting groups of the R 6 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.
  • the thiol protecting groups of the R 6 moiety of formula III is -S-S-pyridin-2-yl.
  • the R 6 moiety of formula III is a thiol protecting group that is removable under neutral conditions e.g. with AgNO 3 , HgCl 2 , 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.
  • the R 6 moiety of formula III is a thiol protecting group that is cleaved under conditions where the pH is from about 4 to about 9.
  • the R 6 moiety of formula III is a thiol protecting group that is "photocleavable".
  • suitable thiol protecting groups include, but are not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, a trityl group, -CH 2 SCH 3 (MTM), dimethylmethoxymethyl, or -CH 2 -S-S-pyridin ⁇ 2-yl.
  • THP tetrahydropyranyl
  • MTM trityl group
  • dimethylmethoxymethyl or -CH 2 -S-S-pyridin ⁇ 2-yl.
  • the R 6 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.
  • suitable hydroxyl protecting groups of the R 6 moiety further include, but are not limited to, esters, allyl ethers, ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.
  • esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifiuoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenyl ⁇ ropionate, 4-oxopentanoate, 4,4-
  • silyl ethers examples 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-
  • R lb group of formula III is hydrogen.
  • R lb group of formula in is -OH.
  • R lb of formula III is a suitably protected hydroxyl group.
  • suitable hydroxyl protecting groups include those described above for the R 6 group.
  • the R 5b group of formula III is hydrogen.
  • the R 5b group of formula III is a suitable hydroxyl protecting group.
  • suitable hydroxyl protecting groups include those described above for the R 6 group.
  • the present invention provides a compound of formula Ilia:
  • 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 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.
  • the R 3b group of formula Ilia is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3b group of formula Ilia is a nucleobase mimetic.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • esters include formates, acetates, carbonates, and sulfonates.
  • silyl ethers examples 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-
  • R lb group of formula Ilia is hydrogen.
  • R lb group of formula Ilia is -OH.
  • 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.
  • the present invention provides a compound of formula IHb: 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 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.
  • the R 3b group of formula HIb is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3b group of formula IHb is a nucleobase mimetic.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • esters include formates, acetates, carbonates, and sulfonates.
  • formate benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p- chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-
  • silyl ethers examples 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-
  • R lb group of formula IIIb is hydrogen.
  • R lb group of formula IIIb is -OH.
  • 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.
  • 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.
  • the R 3b group of formulae ⁇ ic and IIIc' is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil. Such nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3b group of formulae IIIc and HIc' is a nucleobase mimetic.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • esters include formates, acetates, carbonates, and sulfonates.
  • formate benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4-
  • silyl ethers examples 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-
  • R lb group of formulae IIIc and IIIc' is hydrogen.
  • R lb group of formulae IIIc and IIIc' is -OH.
  • the R lb of formulae IIIc and IIIc' is a suitably protected hydroxyl group.
  • suitable hydroxyl protecting groups include those described above for the R sb group.
  • 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.
  • a substituent attached to the 3' OH of a nucleoside in an ester, amide, or thiourea linkage
  • a DNA polymerase in the presence of the (correct) incoming dNTP complementary to the next base position (n+1) on the template.
  • 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.
  • 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)-CH 2 -P(O)(OH) 2 ;
  • R 3c is a nucleobase or a nucleobase mimetic, wherein R 3c is optionally substituted with L c -
  • 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.
  • the Q group of formula IV is oxygen
  • IV is sulfur
  • the R 3c group of formula IV is substituted with L c -R 4c .
  • the R 3c group of formula IV is a nucleobase.
  • nucleobases include adenine, guanine, cytosine, thymine, and uracil.
  • nucleobases also include inosine, xanthine, hypoxanthine, or 2-aminopurine.
  • the R 3c group of formula IV is a nucleobase mimetic.
  • 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.
  • the L c group of formula IV is a cleavable linker group.
  • 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
  • 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.
  • 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.
  • the L 0' group of formula IV is a non-cleavable linker group.
  • the L cl group of formula IV is attached to U at the 3 '-position of U.
  • 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 4 ° 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-, -SO 2 -, -NHSO 2 -, -SO 2 NH-, -C(O)O-, or -OC(O)-.
  • the L c' group of formula IV is a Cj -6 alkylidene chain wherein the methylene unit adjacent to R 4 ° is replaced by -NHC(O)-.
  • 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.
  • the thiol protecting group of the R lc moiety of formula IV is -S-pyridin-2-yl.
  • the R lc moiety of formula IV is a thiol protecting group that is removable under neutral conditions e.g. with AgNO 3 , HgCl 2 , and the like.
  • 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.
  • 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.
  • the R lc moiety of formula IV is a thiol protecting group that is "photocleavable".
  • suitable thiol protecting groups include, but are not limited to, a nitrobenzyl group, a tetrahydropyranyl (THP) group, a trityl group, -CH 2 SCH 3 (MTM), dimethylmethoxymethyl, or -CH 2 -S-S-pyridin-2-yl.
  • THP tetrahydropyranyl
  • MTM trityl group
  • dimethylmethoxymethyl or -CH 2 -S-S-pyridin-2-yl.
  • 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.
  • 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.
  • silyl ethers examples 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.
  • 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.
  • the R 2c of formula I is -P(O)(OH)-O-P(O)(OH)-O- P(O)(OH) 2 .
  • the R lc of formula I is hydrogen.
  • the R 1 c of formula I is -OH.
  • the R Ic of formula I is a suitably protected hydroxyl group.
  • suitable hydroxyl groups include those described above for the R 2c group.
  • the U group of formula IV is a universal nucleoside analog.
  • U comprises a universal base, capable of stacking and/or base pairing with any of the four canonical nucleobases in a DNA helix.
  • the present invention provides a compound of formula IVa:
  • 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.
  • these compounds are useful as reagents in almost any method for sequencing a nucleic acid molecule.
  • 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.
  • 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.
  • the present invention provides a method for determining the sequence of a target single-stranded polynucleotide, where the method comprises the steps of:
  • detection of the label is performed prior to removal of the label.
  • the present invention provides a method for determining the sequence of a target single-stranded polynucleotide, where the method comprises the steps of:
  • step (b) incorporating the nucleotide of step (a) into the complement of the target single stranded polynucleotide;
  • steps (e) optionally repeating steps (b)-(d) one or more times; thereby determining the sequence of a target single-stranded polynucleotide.
  • the method comprises providing a set of nucleotides wherein each nucleotide is a compound of formula I, II, III, or IV.
  • each nucleobase present in the set is associated with a distinct detectable label such that the nucleobase is identifiable upon detection of that label.
  • 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
  • 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.
  • 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 ⁇ -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.
  • the compounds of the present invention can also be used for synthesizing oligonucleotides.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m in diameter.
  • the polynucleotides are attached directly onto a planar surface, such as a planar glass surface. Attachment may be by means of a covalent linkage.
  • 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.
  • 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.
  • Chemical synthesis of an oligonucleotide can be done by attaching a first nucleoside monomer to a solid support.
  • 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).
  • 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.
  • 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.
  • NHS ester of AlexaFluor 430 was obtained from Molecular Probes, Inc., of Eugene, OR,
  • NMR spectra were recorded on a Bruker Avant System operating at 200 MHz, or on a Bruker Avant System operating at 250 MHz.
  • HPLC data were obtained using a Hewlett-Packard 1100 equipped with a
  • TLC data were obtained using Analtech F 250 0.25mm silica plates.
  • 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 CH 2 Cl 2 (100 mL).
  • 2- aminoethoxyethanol 10 gm, 0.095 mol; Acros Chemical
  • CH 2 Cl 2 100 mL
  • N-(9-fluorenylmethoxycarbonyloxy)-succinimide 33 gm, 0.1 mol
  • 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 O 0 C.
  • 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).
  • 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).
  • Thymidine 6 Steps AcO 3% TFA/ CH 3 CI;
  • 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).
  • 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).
  • 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 proceeded very slowly and the product over-desilylated to generate a substantial amount of thymidine.
  • the reaction was stopped by cooling to O 0 C 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 CH 2 Cl 2 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 ⁇ l) and anhydrous dioxane (900 ⁇ l), followed by IM 2-chloro-4H-l,2,3-dioxaphosphori-4-one in anhydrous dioxane (330 ⁇ l) 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 ⁇ l) and tri-n-butylamine (300 ⁇ l) and the mixture stirred ten minutes at room temperature.
  • Reaction 1 Synthesis of 3'-O-Acetyl- 5'-0-(ter£-ButyIdimethylsilyI)thymidine: A
  • 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 ⁇ l), and the reaction cooled to 0 0 C. This was treated with thiophosphoryl chloride (204 ⁇ l) and allowed to stir at 0 0 C for one hour.
  • the reversible terminators of the present invention were tested for efficiency of polymerase incorporation and their termination efficiency and reversibility.
  • 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.
  • 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.
  • 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 ⁇ l TE.
  • the beads were resuspended in 100 ⁇ l of TE. 10 ⁇ l of resuspended beads are added to 90 ⁇ l 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 40 0 C for 2 minutes followed by a 4 0 C 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 ⁇ M final concentration for unmodified Thymidine) in a reaction volume of 10 ⁇ l.
  • Thymidine typically around 1 ⁇ M final concentration for unmodified Thymidine
  • the reaction is then incubated at 37 0 C 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 ⁇ l of deionized water.
  • the resuspended beads were heated to 95°C to denature the prime ⁇ template, releasing the primer into solution.
  • the beads were concentrated with a magnet and the supernatant removed to a fresh tube. 0.5 ⁇ l of supernatant was loaded on the ABI 3730XL with 9 ⁇ l of HiDi Formamide and 0.5 ⁇ l of the ABI Liz Genescan size marker. Typical results are shown in Figures IA, IB, 2A, and 2B.
  • 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.
  • 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.
  • 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.
  • 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 40 0 C 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.
  • 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).
  • 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.
  • 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).
  • 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.

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Abstract

La présente invention a trait à des analogues nucléotidiques qui sont utiles dans une ou des techniques de séquençage d'ADN.
EP06733635A 2005-01-05 2006-01-05 Terminateurs nucleotidiques reversibles et leurs utilisations Withdrawn EP1879906A4 (fr)

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