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WO2008039997A2 - Conjugués de nucléobase avec liants à ossature cationique - Google Patents

Conjugués de nucléobase avec liants à ossature cationique Download PDF

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WO2008039997A2
WO2008039997A2 PCT/US2007/079935 US2007079935W WO2008039997A2 WO 2008039997 A2 WO2008039997 A2 WO 2008039997A2 US 2007079935 W US2007079935 W US 2007079935W WO 2008039997 A2 WO2008039997 A2 WO 2008039997A2
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conjugate
polynucleotide
labeled
dye
polynucleotides
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PCT/US2007/079935
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WO2008039997A3 (fr
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Paul M. Kenney
Shaheer H. Khan
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Applera Corporation
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    • 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
    • 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
    • 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
    • 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
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present disclosure relates to, among other things, nucleoside, nucleotide, and polynucleotide compounds, compositions, kits and methods that may be useful for DNA sequencing, the formation anl29004d use of labeled polynucleotides, and/or other uses.
  • the analysis of complex mixtures of polynucleotides is important in many biological applications. In many situations, it is necessary to separate components of such mixtures to detect target polynucleotides of interest, to determine relative amounts of different components, and to obtain nucleotide sequence information, for example.
  • Electrophoresis provides a convenient tool for analyzing polynucleotides.
  • polynucleotides can be separated on the basis of length, due to differences in electrophoretic mobility.
  • polynucleotides typically migrate at rates that are inversely proportional to polynucleotide length, due to size-dependent obstruction by the crosslinked matrix.
  • polynucleotides tend to migrate at substantially the same rates because of their substantially identical mass-to-charge ratios, so that it is difficult to distinguish different polynucleotides based on size alone.
  • distinguishable electrophoretic mobilities can be obtained in free solution using polynucleotides that contain different charge/mass ratios, e.g., by attaching to the polynucleotides a polymer or other chemical entity having a charge/mass ratio that differs from that of the polynucleotides alone (e.g., see US Patent No. 5,470,705).
  • detection can usually be accomplished using a single detectable label, such as a radioisotope or fluorophore.
  • Electrophoresis of the labeled products generates ladders of fragments that can be detected on the basis of elution time or band position.
  • mixtures can be produced that contain the desired labeled polynucleotide product(s) plus residual labeled nucleotides. Electrophoresis of such mixtures can produce separation profiles in which the labeled nucleotides (if they are negatively charged) and breakdown products thereof (sometimes called "dye blobs) co-migrate with negatively charged labeled polynucleotides. Comigration of dye blobs can interfere with the identification or quantification of particular polynucleotide fragments and can cause incomplete or erroneous sequence determinations.
  • a factor that contributes to the presence of dye blobs is the need to use high concentrations of labeled nucleotides relative to unlabeled nucleotides (e.g., a ratio of 50:1) due to the weaker binding affinities of polymerase enzymes for labeled nucleotides.
  • Dye blobs can be removed or substantially reduced in amount by subjecting such reaction mixtures to purification by methods such as size-exclusion gel chromatography or ethanol precipitation.
  • purification methods can undesirably reduce the levels of smaller DNA fragments and also increase the overall time to obtain results.
  • the present disclosure provides labeled nucleotides that can be effectively incorporated into polynucleotides by polymerases or other enzymes.
  • the present disclosure provides labeled nucleotides that migrate more slowly in electrophoresis than the corresponding labeled forms of standard nucleotides such as ATP, CTP, GTP, and TTP, thereby allowing improved detection of polynucleotides that are obscured by comigrating labeled standard nucleotides.
  • the present disclosure provides labeled nucleotides that have a substantially net neutral or net positive charge under selected pH conditions so that they cannot co-migrate with negatively charged analytes such as ribo- or deoxyribopolynucleotides.
  • the present disclosure provides conjugates comprising a dye labeled nucleobase of the form: (1) B-L-D, wherein B is a nucleobase, L is a linker whose backbone comprises at least one imidazolium moiety, and D comprises at least one fluorescent dye, or (2) B-L1-D1-L2-D2, wherein B is a nucleobase, Ll and L2 are linkers such that at least one of Ll and L2 is a linker whose backbone comprises at least one imidazolium moiety, and Dl and D2 are members of an energy transfer pair, such that one of Dl and D2 is an energy donor capable of emitting energy at a first wavelength and the other of Dl and D2 is capable of absorbing the energy emitted from the donor and emitting energy at a second wavelength in response thereto.
  • the dye-labeled nucleobase is of the form B-L-D.
  • the backbone of L comprises a total of one, two, three, four, five, six, or seven imidazolium moieties.
  • the backbone of L comprises at least two, or at least three, or at least four, or at least five, or at least six, or at least seven imidazolium moieties.
  • L comprises 4 to 50 chain atoms.
  • D comprises at least one xanthene, rhodamine, fluorescein,
  • the labeled nucleobase is of the form: B-L1-D1-L2-D2.
  • the backbone of Ll comprises a total of one, two, three, four, five, six, or seven imidazolium moieties.
  • the backbone of Ll comprises at least two, or at least three, or at least four, or at least five, or at least six, or at least seven imidazolium moieties.
  • Ll comprises 4 to 50 chain atoms.
  • L2 comprises 4 to 50, or 4 to 30, or 4 to 20 chain atoms.
  • the backbone of L2 does not comprise an imidazolium moiety.
  • the backbone of L2 comprises at least one imidazolium moiety.
  • the backbones of both Ll and L2 each comprise at least one imidazolium moiety.
  • Ll and L2 taken together comprise a total of two, three, four, five, six, or seven imidazolium moieties.
  • Ll and L2 taken together comprise at least two, or at least three, or at least four, or at least five, or at least six, or at least seven imidazolium moieties.
  • At least one of Dl or D2 comprises a xanthene, rhodamine, fluorescein, [8,9]benzophenoxaz ⁇ ie, cyanine, phthalocyanine, or squaraine dye.
  • Dl is a donor dye and D2 is an acceptor dye.
  • B comprises adenine, 7-deazaadenine, 7-deaza-8- azaadenine, cytosine, guanine, 7-deazaguanine, 7-deaza-8-azaguanine, thymine, uracil, or inosine.
  • the conjugate has a net positive charge at a pH of 7 or greater, or has a net positive charge al a pH of 8 or greater.
  • labeled nucleoside triphosphates are provided comprising a conjugate that contains an imidazolium-containing linker moiety.
  • the labeled nucleoside triphosphate is not 3 '-extendable.
  • the labeled nucleoside triphosphate is a 2',3'- dideoxynucleotide or 3'-fluoro-2',3'-dideoxynucleotide.
  • the labeled nucleoside triphosphate contains a 3'-hydroxyl group.
  • polynucleotides comprising a conjugate that contains an imidazolium-containing linker moiety.
  • At least one said conjugate is contained in a 3 1 terminal nucleotide subunit.
  • the polynucleotide comprises a 3 1 terminal nucleotide subunit that is not 3 '-extendable, such as a 2',3'-dideoxynucleotide or 3'-fluoro-2',3'- dideoxynucleotide.
  • the 3' terminal nucleotide subunit contains a 3'-hydroxyl group.
  • At least one said conjugate is contained in a non-terminal nucleotide subunit.
  • mixtures comprising a plurality of different polynucleotides, wherein at least one polynucleotide contains a conjugate as described herein.
  • the mixture comprises a plurality of different polynucleotides that each comprises the same type of 3' terminal nucleotide subunit. [0039] In some embodiments, the mixture comprises four classes of polynucleotides, wherein the polynucleotides in each class terminate with a different type of 3' terminal nucleotide subunit that identifies the polynucleotides in that class, wherein at least one said polynucleotide comprises a conjugate as described herein.
  • mixtures comprising at least one labeled nucleoside triphosphate comprising a nucleobase conjugate that contains an imidazolium- containing linker moiety, and one or more of the following components: a 3'-extendable primer, a polymerase enzyme, one or more 3'-extendable nucleoside triphosphates that do not comprise a said conjugate, and/or a buffering agent.
  • kits comprising at least one labeled nucleoside triphosphate comprising a nucleobase conjugate that contains an imidazolium-containing linker moiety, and one or more of the following components: a 3'-extendable primer, a polymerase enzyme, one or more 3'-extendable nucleoside triphosphates that do not comprise a said conjugate, and/or a buffering agent.
  • At least one labeled nucleoside triphosphate is 3'- extendable.
  • At least one labeled nucleoside triphosphate is not 3'- extendable.
  • the kit or mixture comprises four different labeled nucleoside triphosphates that are complementary to A, C, T and G, such that at least one of the labeled nucleoside triphosphates comprises a nucleobase conjugate that contains an imidazolium-containing linker moiety.
  • the four different labeled nucleoside triphosphates each comprise the same donor.
  • the four different labeled nucleoside triphosphates are not 3 '-extendable.
  • the four different labeled nucleoside triphosphates are 3'- extendable ribonucleoside triphosphates.
  • At least one donor dye is an ortho-carboxyfluorescein.
  • the present disclosure provides a method of forming a labeled polynucleotide comprising reacting a first polynucleotide with a labeled nucleoside triphosphate comprising a conjugate as described herein in the presence of a primer extension reagent under conditions effective to form a modified polynucleotide containing at least one labeled nucleoside subunit from the labeled nucleoside triphosphate.
  • said reacting is performed in the presence of a template nucleic acid to which the first polynucleotide is complementary, and the primer extension reagent comprises a template-dependent polymerase.
  • the modified polynucleotide is subjected to electrophoresis.
  • the modified polynucleotide is subjected to electrophoresis without prior removal of residual labeled nucleoside triphosphate.
  • methods comprise forming one or more labeled different- sequence polynucleotides, wherein at least one different-sequence polynucleotide contains a unique conjugate of the type described herein.
  • one or more labeled different-sequence polynucleotides are separated by electrophoresis so as to separate different-sequence polynucleotides on the basis of size, and different-sequence polynucleotides are identified on the basis of electrophoretic mobility and, optionally, fluorescence signal,
  • methods comprise forming four classes of polynucleotides which are complementary to a target polynucleotide sequence, by template-dependent primer extension, wherein the polynucleotides in each class terminate with a different terminator subunit type, and at least one polynucleotide contains a conjugate as described herein, separating the polynucleotides of the four classes on the basis of size to obtain a mobility pattern, and determining the sequence of the target polynucleotide sequence from the mobility pattern.
  • terminator subunits are nonextendable.
  • terminator subunits contain a 3'-hydroxyl group.
  • At least one said conjugate has a net neutral or net positive charge at pH 7 or at pH 8.
  • detectable label refers to any moiety that, when attached to the compounds of the present teachings, renders such compounds detectable using known detection means.
  • detectable labels include but are not limited to fluorophores, chromophores, radioisotopes, spin-labels, enzyme labels, chemiluminescent labels that are detectable using a suitable detector or detection means, or a binding pair, for example, a ligand, such as an antigen or biotin, that can bind specifically with high affinity to a detectable anti-ligand, such as a labeled antibody or avidin.
  • the labels can be fluorescent dyes such as fluorescein, rhodamine, cyanine, or pyrene dyes, for example.
  • Enzymatically extendable or “3 1 extendable” means a nucleotide or polynucleotide that is capable of being appended to a nucleotide or polynucleotide by enzyme action.
  • a polynucleotide containing a 3' hydroxyl group is an example of an enzymatically extendable polynucleotide,
  • Enzymatically incorporatable means that a nucleotide is capable of being enzymatically incorporated onto the terminus, e.g. 3' terminus, of a polynucleotide chain, or internally through nick-translation of a polynucleotide chain, through action of a template- dependent or template-independent polymerase enzyme.
  • a nucleotide-5' -triphosphate is an example of an enzymatically incorporatable nucleotide.
  • Backbone-imidazolium linker moiety refers to a moiety that comprises one or more imidazolium moieties in the backbone chain of atoms of a linker.
  • Linker refers to a moiety that links a dye to a substrate such as an oligonucleotide, or links one dye to another dye (e.g., links a donor to an acceptor dye).
  • Nucleobase means a nitrogen-containing heterocyclic moiety capable of forming Watson-Crick type hydrogen bonds with a complementary nucleobase or nucleobase analog, e.g. a purine, a 7-deazapurine, or a pyrimidine.
  • nucleobases are the naturally occurring nucleobases adenine, guanine, cytosine, uracil, thymine, and analogs of naturally occurring nucleobases, e.g. 7-deazaadenine, 7-deaza-8-azaadenine, 7-deazaguanine, 7-deaza-8- azaguanine, inosine, nebularine, nitropyrrole, nitroindole, 2-amino-purine, 2,6-diamino- purine, hypoxanthine, pseudouridine, pseudocytidine, pseudoisocytidine, 5-propynyl- cytidine, isocytidine, isoguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4- thiouracil, (/-methylguanine, //-methyl-adenine, ⁇ y-methylthymine, 5,6-dihydrothymine
  • Nucleoside means a compound comprising a nucleobase linked to a C-Y carbon of a ribose sugar or sugar analog thereof.
  • the ribose or analog may be substituted or unsubstituted.
  • Substituted ribose sugars include, but are not limited to, those riboses in which one or more of the carbon atoms, preferably the 3 '-carbon atom, is substituted with one or more of the same or different substituents such as -R, -OR, -NRR or halogen (e.g., fluoro, chloro, bromo, or iodo), where each R group is independently -H, Ci-C ⁇ alkyl or C 3 -C 14 aryl.
  • substituents such as -R, -OR, -NRR or halogen (e.g., fluoro, chloro, bromo, or iodo)
  • riboses are ribose, 2'-deoxyribose, 2',3'-dideoxyribose > 3'-haloribose (such as 3'-fluororibose or 3'-chlororibose) and 3'-alkylribose.
  • the nucleobase is A or G
  • the ribose sugar is attached to the N 9 -position of the nucleobase.
  • the nucleobase is C, T or U
  • the pentose sugar is attached to the N '-position of the nucleobase (Kornberg and Baker, DNA Replication, 2 nd Ed., Freeman, San Francisco, CA, (1992)).
  • sugar analogs include, but are not limited to, substituted or unsubstituted furanoses having more or fewer than 5 ring atoms, e.g., erythroses and hexoses and substituted or unsubstituted 3-6 carbon acyclic sugars.
  • Typical substituted furanoses and acyclic sugars are those in which one or more of the carbon atoms are substituted with one or more of the same or different -R, -OR, -NRR or halogen groups, where each R is independently -H, (Ci-Ce) alkyl or (C 1 -C 14 ) aryl.
  • substituted furanoses having 5 ring atoms include but are not limited to 2-deoxyribose, 2'-(Ci-C 6 )alkylribose, 2'-(Ci- C 6 )alkoxyribose (e.g., 2'-O-methyl ribose), 2'-(C 5 -Ci 4 )aryloxyribose, 2',3'-dideoxyribose, 2',3 -didehydroribose, 2'-deoxy-3'-haloribose, 2'-deoxy-3'-fluororibose, 2'-deoxy-3'- chlororibose, 2'-deoxy-3'- amino-ribose, 2'-deoxy-3'-(Ci-C,s)alkylribose, 2'-deoxy-3'-(Ci- C 6 )alkoxyribose, 2'-deoxy-3 t -(Ci
  • sugar analogs include but are not limited to, locked nucleic acids such as
  • Nucleotide means a phosphate ester of a nucleoside, either as an independent monomer or as a subunit within a polynucleotide.
  • Nucleotide triphosphates also referred to herein as nucleoside triphosphates
  • NTP Nucleotide triphosphates
  • dNTP (2'-deoxypentose)
  • ddNTP (2 ⁇ 3'-dideoxypentose
  • Nucleotide 5'-triphosphate refers to a nucleotide with a triphosphate ester group at the 5' position.
  • the triphosphate ester group may include sulfur substitutions for one or more phosphate oxygen atoms, e.g. ⁇ -thionucleotide 5'-triphosphates.
  • Nonextendable or “3 1 nonextendable” refers to the fact that a terminator is incapable, or substantially incapable, of being extended in the 3' direction by a template- dependent DNA or RNA polymerase.
  • Nucleotide subunit or “polynucleotide subunit” refers to a single nucleotide or nucleotide analog within a polynucleotide or polynucleotide analog.
  • Polynucleotide refers to linear polymers of natural nucleotide monomers or analogs thereof, including for example, double- and single-stranded deoxyribonucleotides, ribonucleotides, ⁇ -anomeric forms thereof, and the like.
  • a polynucleotide may be composed entirely of deoxyribonucleotides, ribonucleotides, or analogs thereof, or may contain blocks or mixtures of two or more different monomer types.
  • nucleoside monomers are linked by phosphodiester linkages.
  • polynucleotides containing non-phosphodiester linkages are also contemplated.
  • Polynucleotide also encompasses polymers that contain one or more non-naturally occurring monomers and/or intersubunit linkages, such as peptide nucleic acids (PNAs, e.g., polymers comprising a backbone of amide-linked N-(2-aminoethyl)-glycine subunits to which nucleobases are attached via the non-amide backbone nitrogens.
  • PNAs peptide nucleic acids
  • Polynucleotides typically range in size from a few monomelic units, e.g. 8-40, to several thousand monomeric units.
  • a polynucleotide is represented by a sequence of letters, such as "ATGCCTG,” it will be understood that the nucleotides are in 5'->3' order from left to right and that "A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted.
  • Phosphate analog refers to an analog of phosphate wherein one or more of the oxygen atoms is replaced with a non-oxygen moiety.
  • Exemplary phosphate analogs including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoro- anilothioate, phosphotriester, phosphoranilidate, phosphoramidate, alkylphosphonates such as methylphosphonates, boronophosphates.
  • “Spectrally resolvable” means that two or more dyes have emission bands that are sufficiently distinct, i.e., sufficiently non-overlapping, that they can be distinguished on the basis of a unique fluorescent signal generated by each dye.
  • Temporal nucleic acid refers to any nucleic acid or polynucleotide that is capable of annealing with a primer polynucleotide.
  • Exemplary template nucleic acids include DNA, KNA, which DNA or RNA may be single stranded or double stranded. More particularly, template nucleic acid may be genomic DNA, messenger RNA, cDNA, DNA amplification products from a PCR reaction, and the like. Methods for preparation of template DNA may be found elsewhere (ABI PRISMTM Dye Primer Cycle Sequencing Core Kit).
  • Terminator means an enzymatically incorporatable nucleotide which prevents subsequent incorporation of nucleotides to the resulting polynucleotide chain and thereby halts polymerase-mediated extension.
  • Typical terminators lack a 3'-hydroxyl substituent and include 2 ⁇ 3'-dideoxyribose, 2',3'-didehydroribose, and 2 l ,3'-dideoxy-3'-haloribose, e.g. 3'-deoxy-3'- fluoro-ribose or 2 1 ,3'-dideoxy-3'-fluororibose, for example.
  • a ribofuranose analog can be used, such as 2',3'-dideoxy- ⁇ -D-ribofuranosyl, ⁇ -D-arabinofuranosyl, 3'- deoxy- ⁇ -D-arabinofuranosyl, 3'-amino ⁇ 2 ⁇ 3 > -dideoxy- ⁇ -D-ribofuranosyl, and 2',3'-dideoxy- 3'-fluoro- ⁇ -D-ribofuranosyl (see, for example, Chidgeavadze et al., Nucleic Acids Res., 12: 1671-1686 (1984), and Chidgeavadze et al. FEB. Lett, 183: 275-278 (1985)). Nucleotide terminators also include reversible nucleotide terminators (Metzker et al. Nucleic Acids Res., 22(20):42S9 (1994)).
  • SUBSTTTUTE SHEET (RULE 26) suitable counterfoil that balances the positive or negative charge.
  • exemplary positively charged counterions include, without limitation, H + , NH 4 + , Na + , K + , Mg 2+ , trialkylammonium (such as triethylammonium), tetraalkylammonium (such as tetraethyiammonium), and the like.
  • exemplary negatively charged counterions include, without limitation, carbonate, bicarbonate, acetate, chloride, and phosphate, for example. Also, although particular resonance structures may be shown herein, such structures are intended to include all other possible resonance structures.
  • compositions that comprise at least one dye-labeled nucleobase of the type described herein.
  • Such compositions include not only nucleobase-dye conjugates as independent molecules, but also as nucleosides, nucleotides and polynucleotides containing such conjugates, as well as mixtures, solids, or solutions containing any of the foregoing.
  • a dye-labeled nucleobase of the invention has the form B-L- D, wherein B is a nucleobase, L is a linker whose backbone comprises at least one imidazolium moiety, and D comprises at least one fluorescent dye.
  • Nucleobase B may be any moiety capable of forming Watson-Crick hydrogen bonds with a complementary nucleobase or nucleobase analog, as set forth above.
  • B is a nitrogen-containing heterocyclic moiety such as a 7-deazapurine, purine, or pyrimidine nucleotide base.
  • B is uracil, cytosine, 7-deazaadenine, or 7- deazaguanosine.
  • the linker is usually attached to the 8-position of the purine.
  • B is a 7-deazapurine
  • the linker to the dye is usually attached to the 7-position of the 7-deazapurine.
  • B is pyrimidine
  • the linker is usually attached to the 5-position of the pyrimidine.
  • Fluorescent dye D may be any fluorescent dye that is suitable for the purposes of the invention.
  • the fluorescent dye comprises a resonance-delocalized system or aromatic ring system that absorbs light at a first wavelength and emits fluorescent light at a second wavelength in response to the absorption event,
  • fluorescent dyes can be selected from any of a variety of classes of fluorescent compounds, such as xanthenes, rhodamines, fluoresceins, cyanines, phthalocyanines, squaraines, and bodipy dyes.
  • the dye comprises a xanthene-type dye, which contains a fused three-ring system of the form:
  • This parent xanthene ring may be unsubstituted (i.e., all substituents are H) or may be substituted with one or more of a variety of the same or different substituents, such as described below.
  • the dye contains a parent xanthene ring having the general structure:
  • a 1 is OH or NH 2 and A 2 is O or NH 2 + .
  • the parent xanthene ring is a fluorescein-type xanthene ring.
  • a 1 is NH 2 and A 2 is NH 2 +
  • the parent xanthene ring is a rhodamine-type xanthene ring.
  • a 1 is NH 2 and A 2 is O
  • the parent xanthene ring is a rhodol-type xanthene ring.
  • one or both nitrogens of A 1 and A 2 (when present) and/or one or more of the carbon atoms at positions C-I, C-2, C-4, C-5, C-I, C-8 and C-9 can be independently substituted with a wide variety of the same or different substituents.
  • typical substituents include, but are not limited to, -X, -R, -OR, -SR, - NRR, perhalp (Ci-C 6 ) alky!,-CX 3 , -CF 3 , -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO 2 , - N 3 , -S(O) 2 O " , -S(O) 2 OH, -S(O) 2 R, -C(O)R, -C(O)X, -C(S)R, -C(S)X, -C(O)OR, -C(O)O " , -C(S)OR, -C(O)SR, -C(S)SR, -C(O)NRR, -C(S)NRR and -C(NR)NRR, where each X is independently a halogen (preferably -F or Cl)
  • C-I and C-2 substituents and/or the C-I and C-8 substituents can be taken together to form substituted or unsubstituted buta[l,3]dieno or (Cs-C 2 o) aryleno bridges.
  • substituents which do not tend to quench the fluorescence of the parent xanthene ring are preferred, but in some embodiments quenching substituents may be desirable.
  • Substituents that tend to quench fluorescence of parent xanthene rings are electron- withdrawing groups, such as -NO2, -Br, and -I.
  • C-9 is unsubstituted.
  • C-9 is substituted with a phenyl group.
  • C-9 is substituted with a substituent other than phenyl.
  • a 1 is NH 2 and/or A 2 is NH 2 + , these nitrogens can be included in one or more bridges involving the same nitrogen atom or adjacent carbon atoms, e.g., (Ci-C 12 ) alkyldiyl,
  • each X is independently a halogen (preferably -F or -Cl) and each R' is independently hydrogen, (Ci-C ⁇ ) alkyl, 2-6 membered heteroalkyl, (Cs-C] 4 ) aryl or heteroaryl, carboxyl, acetyl, sulfonyl, sulfinyl, sulfone, phosphate, or phosphonate.
  • Exemplary parent xanthene rings include, but are not limited to, rhodamine-type parent xanthene rings and fiuorescein-type parent xanthene rings.
  • the dye contains a rhodamine-type xanthene dye that includes the following ring system:
  • one or both nitrogens and/or one or more of the carbons at positions C-I, C-2, C-4, C-5, C-7 or C-8 can be independently substituted with a wide variety of the same or different substituents, as described above for the parent xanthene rings, for example.
  • Exemplary rhodamine-type xanthene dyes include, but are not limited to, the xanthene rings of the rhodamine dyes described in US Patents 5,936,087, 5,750,409, 5,366,860, 5,231,191, 5,840,999, 5,847,162, and 6,080,852 (Lee et al.), PCT Publications WO 97/36960 and WO 99/27020, Sauer et al, J.
  • the dye comprises a fluorescein-type parent xanthene ring having the structure:
  • fluorescein-type parent xanthene ring depicted above one or more of the carbons at positions C-I, C-2, C-4, C-S, C-7, C-8 and C-9 can be independently substituted with a wide variety of the same or different substituents, as described above for the parent xanthene rings.
  • Exemplary fluorescein-type parent xanthene rings include, but are not limited to, the xanthene rings of the fluorescein dyes described in US Patents 4,439,356, 4,481,136, 5,188,934, 5,654,442, and 5,840,999, WO 99/16832, and EP 050684.
  • the dye comprises a rhodamine dye, which comprises a rhodamine-type xanthene ring in which the C-9 carbon atom is substituted with an orthocarboxy phenyl substituent (pendent phenyl group).
  • orthocarboxyfluoresceins Such compounds are also referred to herein as orthocarboxyfluoresceins.
  • a particularly preferred subset of rhodamine dyes are 4,7,-dichlororhodamines.
  • Typical rhodamine dyes include, but are not limited to, rhodamine B, 5-carboxyrhodamine, rhodamine X (ROX), 4,7-dichlororhodamine X (dROX), rhodamine 6G (R6G), 4,7-dichlororhodamine 6G, rhodamine 110 (RI lO), 4,7-dichlororhodamine 110 (dRHO), tetramethyl rhodamine (TAMRA) and 4,7-dichloro-tetramethylrhodamine (dTAMRA).
  • the dye is a 4,7-dichloro-orthocarboxyrhodamine.
  • the dye comprises a fluorescein dye, which comprises a fluorescein-type xanthene ring in which the C-9 carbon atom is substituted with an orthocarboxy phenyl substituent (pendent phenyl group).
  • fluorescein- type dyes are 4,7,-dichlorofluoresceins.
  • Typical fluorescein dyes include, but are not limited to, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM).
  • the dye is a 4,7-dichloro-orthocarboxyfluorescein.
  • the dye can be a cyanine, phthalocyanine, squaraine, or bodipy dye, such as described in the following references and references cited therein: Patent No. 5,863,727 (Lee et al.), 5,800,996 (Lee et al.), 5,945,526 (Lee et al.), 6,080,868 (Lee et al.), 5,436,134 (Haugland et al.), US 5,863,753 (Haugland et al.), 6,005,113 (Wu et al.), and WO 96/04405 (Glazer et al.).
  • Rhodamine dyes for use in connection with the present teachings can include, for example, a rhodamine dye having the structure:
  • R I -R O are each independently selected from -H, -F, -Cl, -Br, -I, -CN, -CO 2 H, -CO 2 X, - CO 2 R, -SO 3 H, -SO 3 X, -SO 3 R, halogen, C]-Ci 0 alkyl, Ci-Ci 0 alkenyl, Ci-Ci 0 alkynyl, Ci-C 10 alkoxy, Ci-Ci 0 alkylamine, Ci-Cio mercaptyl, Cj-Cio alkylsulfonate, C 3 -Ci 0 cycloalkyl, Gr Cio cycloalkenyl, C 3 -Ci 0 heterocyclic, C 3 -Ci O aromatic, Cs-C 6 heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsul
  • Rg-Rn are each independently selected from Ci-C 10 alkyl, Ci-Cio alkenyl, Q-Cio alkynyl, Ci-Ci 0 alkoxy, Ci-Cio alkylamine, Cj-Cio mercaptyl, Ci-Ci 0 alkylsulfonate, C 3 -Ci 0 cycloalkyl, C 4 -Ci O cycloalkenyl, C 3 -Ci O aromatic, benzyl, benzoyl, biphenyl where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alkylsulfonate, cycloalkyl, cycloalkenyl, aromatic, benzyl, benzoyl and biphenyl is optionally further substituted by F,
  • Ri taken together with R? forms a 5-7 membered ring that is saturated or unsaturated, and is optionally substituted by one or more C 1 -C 6 alkyl, Ci-C 6 alkylamine or
  • R 2 taken together with Rj 0 forms a 5-7 membered ring that is saturated or unsaturated, and is optionally substituted by one or more C)-C 6 alkyl, Ci-C 6 alkylamine or
  • R 3 taken together with R 4 forms a benzo or naphtha ring optionally substituted by one or more of -F, -Cl, -Br, -I, -CN, -CO 2 H, -CO 2 X, -CO 2 R, -SO 3 H, -SO 3 X, -SO 3 R, halogen,
  • R 5 taken together with R 6 forms a benzo or naphtha ring optionally substituted by one or more of -F, -Cl, -Br, -I, -CN, -CO 2 H, -CO 2 X, -CO 2 R, -SO 3 H, -SO 3 X, -SO 3 R, halogen,
  • R 3 taken together with Rn forms a 5- or 6- membered ring that is saturated or unsaturated, and is optionally substituted by one or more Ci-C 6 alkyl, Ci-C 6 alkylamine or
  • R 6 taken together with Re forms a 5- or 6- membered ring that is saturated or unsaturated, and is optionally substituted by one or more Cj-C 6 alkyl, Ci-C 6 alkylamine or
  • R 7 is selected from ⁇ H, -F, -CN, -CO 2 H, -CO 2 X, -CO 2 R, C r Cio alkyl, Ci-C 10 alkyl that is saturated or unsaturated and is optionally substituted by one or more -F, -Cl, -Br, - CO 2 H, -CO 2 X, -CO 2 R 5 -SO 3 H, -SO 3 X, -SO 3 R, where X is a counterion and R is Ci-C 6 alkyl, or R 7 is a radical of the formula:
  • R ⁇ 2 , R !3 , R 14 , R15 and Rj 6 are each independently selected from -H, -F, -Cl, -Br, -I, - CO 2 H, -CO 2 X, -CO 2 R, -SO3H, -SO 3 X, and -SO 3 R, where X is a counterion and R is C 1 -C 6 alkyl.
  • Exemplary rhodamine dyes useful labels in connection with the present teachings include, but are not limited to, tetramethylrhodamine (TAMRA), 4,7-dichlorotetramethyl rhodamine (DTAMRA), rhodamine X (ROX), 4,7-dichlororhodamine X (DROX), rhodamine 6G (R6G), rhodamine 110 (RIlO), 4,7-dichlororhodamine 110 (RI lO) and the like.
  • TAMRA tetramethylrhodamine
  • DTAMRA 4,7-dichlorotetramethyl rhodamine
  • ROX 4,7-dichlororhodamine X
  • R6G 4,7-dichlororhodamine 6G
  • rhodamine 110 RIlO
  • 4,7-dichlororhodamine 110 RI lO
  • the designation -1 or -2 is placed after an abbreviation of a particular dye, e.g., TAMRA-I.
  • the "-1" and “-2" designations indicate the particular 5 or 6 dye isomer being used.
  • the 1 and 2 isomers are defined by the elution order (the 1 isomer being the first to elute) of free dye in a reverse-phase chromatographic separation system utilizing a C-8 column and an elution gradient of 15% acetonitrile/85% 0.1 M triethylammonium acetate to 35% acetonitrile / 65% 0.1 M triethylammonium acetate.
  • Fluorescein dyes for use in connection with the present teachings can include, for example, any fluorescein dye having the structure:
  • Ri-R 6 are each independently selected from -H, -F, -Cl, -Br, -I, -CN, -CO 2 H, -CO 2 X, - CO 2 R, -SO 3 H, -SO 3 X 5 -SO 3 R, halogen, Ci-Ci 0 alkyl, C 1 -Cj 0 alkenyl, Ci-Ci 0 alkynyl, Ci-C 0 alkoxy, Ci-Ci 0 alkylamine, Ci-Ci 0 mercaptyl, Ci-Ci 0 alkylsulfoiiate, C 3 -Ci 0 cycloalkyl, C 4 - C 10 cycloalkenyl, C 3 -C 10 heterocyclic, C 3 -C 10 aromatic, Cs-C 6 heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alkylamine, mercaptyl, alky
  • R 3 taken together with R 4 forms a benzo or naphtha ring optionally substituted by - F, -Cl, -Br, -I, -CN, -CO 2 H, -CO 2 X, -CO 2 R, -SO 3 H, -SO 3 X, -SO 3 R, halogen, C]-C 10 alkyl, Cj-Cjo alkenyl, Ci-Cjo alkynyl, C 1 -C 1O alkoxy, Ci-Ci 0 alkylamine, Ci-Ci 0 mercaptyl, Cj-Cto alkylsulfonate, C 3 -Ci 0 cycloalkyl, C 4 -Cj 0 cycloalkenyl, C 3 -Ci 0 heterocyclic, C 3 -Cj 0 aromatic, Cs-C 6 heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, al
  • R 5 taken together with Rg forms a benzo or naphtha ring optionally substituted by - F, -Cl, -Br, -I, -CN, -CO 2 H, -CO 2 X, -CO 2 R, -SO 3 H, -SO 3 X, -SO 3 R, halogen, C r Ci 0 alkyl, Ci-C 10 alkenyl, C 1 -Ci 0 alkynyl, Cj-Cio alkoxy, Ci-Ci 0 alkylamine, Ci-C 10 mercaptyl, Ci-C 10 alkylsulfonate, C 3 -Ci 0 cycloalkyl, C 4 -C 10 cycloalkenyl, C 3 -Ci 0 heterocyclic, C 3 -Ci 0 aromatic, C 5 -C 6 heteroaromatic, where each alkyl, alkenyl, alkynyl, alkoxy, alky
  • R 7 is selected from -H, -F, -CN, -CO 2 H, -CO 2 X, -CO 2 R, Ci-C 10 alkyl, C r Ci 0 alkyl that is saturated or unsaturated and is optionally substituted by one or more -F, -Cl, -Br, - CO 2 H, -CO 2 X, -CO 2 R, -SO 3 H, -SO 3 X 5 -SO 3 R, where X is a counterion and R is C 1 -C 6 alkyl, or R 7 is a radical of the formula:
  • R i2 , Rn, Ri4, R15 and R i6 are each independently selected from -H, -F, -Cl, -Br, -I, - CO 2 H, -CO 2 X, -CO 2 R, -SO 3 H, -SO 3 X, and -SO 3 R, where X is a counterion and R is C 1 -C 6 alkyl.
  • Exemplary rhodamine dyes useful labels in connection with the present teachings include, but are not limited to, 6-carboxyfluorescein, 5-carboxyfluorescein, 5-carboxy- 4,7,2',7'-tetrachlorofluorescein, ⁇ -carboxy ⁇ J ⁇ ' ⁇ '-tetrachloro-fluorescein, 5-carboxy- 4,7,2' ,4 ' ,5 ' ,7 ' -hexachlorofluorescein, 6-carboxy-4,7,2 ' ,4 ' ,5 ' ,T -hexachlorofluorescein, 5- carboxy-4 ' ,5 ' -dichloro-2 ' ,T -dimethoxy-fluorescein, 6-carboxy-4 ' ,5 ' -dichloro-2 ' T -dimeth- oxyfluorescein and S-carboxy ⁇ ' ⁇ 'jS'J
  • L is a linker whose backbone comprises at least one imidazolium moiety in which the ring nitrogens in each imidazolium moiety are both substituted with non- hydrogen (e.g., a substituent other than hydrogen, such as alkyl) substituents so that the imidazolium ring has a permanent positive charge.
  • a linker backbone must pass through at least two ring atoms of the imidazolium moiety.
  • the imidazolium ring constitutes a divalent moiety when it is present in a linker backbone.
  • such substituents can be provided by B, D, or additional linker backbone structure.
  • N-substituents can be non-backbone moieties such as alkyl.
  • one or more non-bridging imidazolium ring atoms can be substituted, e.g., with Ci-Cg substituted or unsubstituted alkyl such as methyl or ethyl.
  • Ci-Cg substituted or unsubstituted alkyl such as methyl or ethyl.
  • the imidazolium ring nitrogens are linked covalently to B, D 5 or additional backbone structure of the linker chain.
  • this is illustrated by the bonds emanating from Nl and N3 whose ends terminate with squiggly lines.
  • an imidazolium moiety may be contained in a linker backbone via one or two of C2, C4 or C5 of the imidazolium moiety, in which case the ring nitrogen is substituted with a non- hydrogen substituent as noted above.
  • Formula II below illustrates embodiments in which an imidazolium moiety has backbone linkages emanating from N3 and from C5.
  • Nl is substituted with an R group such as C 1 -C 6 substituted or unsubstituted allcyl, such as methyl or ethyl, so that the imidazolium moiety is permanently positively charged.
  • R group such as C 1 -C 6 substituted or unsubstituted allcyl, such as methyl or ethyl, so that the imidazolium moiety is permanently positively charged.
  • each ring atom of an imidazolium moiety can be hydrogen.
  • a linker between B and D will have a linker chain length of from about 4 to about 50 linker chain atoms, or from about 4 to about 30 linker chain atoms, or from about 4 to about 20 linker chain atoms, although shorter and longer linkers may also be used.
  • linker chain length of from about 4 to about 50 linker chain atoms, or from about 4 to about 30 linker chain atoms, or from about 4 to about 20 linker chain atoms, although shorter and longer linkers may also be used.
  • linker chain length of from about 4 to about 50 linker chain atoms, or from about 4 to about 30 linker chain atoms, or from about 4 to about 20 linker chain atoms, although shorter and longer linkers may also be used.
  • linker chain length of from about 4 to about 50 linker chain atoms, or from about 4 to about 30 linker chain atoms, or from about 4 to about 20 linker chain atoms, although shorter and longer linkers may also be used.
  • linker chain length of from
  • the attachment site on the nucleobase is selected so as not to interfere with or eliminate the H-bonding capability of the nucleobase with respect to a complementary nucleobase.
  • the linker is usually attached to the N-8-position of the purine.
  • the linker is usually attached to the N-7-position of the 7-deazapurine.
  • B includes a pyrimidine base, the linkage is attached to the C-5-position of the pyrimidine.
  • a purine or 7-deazapurine is usually attached to a sugar moiety via the N-9-position of the purine or deazapurine
  • a pyrimidine is usually attached to a sugar moiety via the N-I -position of the pyrimidine.
  • other points of attachment can also be used.
  • the particular entity by which a linker is connected to a nucleobase can be any chemical group that is suitable for the purposes of the present invention.
  • suitable chemical groups are known.
  • the terminal chemical group in the linker that is covalently attached to the nucleobase can be an acetylene moiety (-CsC-), and often is a propargyl moiety (-C ⁇ CCHj-), since such linkage moieties tend to be particularly compatible with a variety of polymerase enzymes used for primer extension.
  • acetylene moiety acetylene moiety
  • -C ⁇ CCHj- propargyl moiety
  • linkage moieties tend to be particularly compatible with a variety of polymerase enzymes used for primer extension.
  • non- acetylenic chemical groups are also contemplated. Examples of suitable terminal groups for attachment to a nucleobase can be found in the following exemplary references:
  • linker L and dye moiety D can be located at any suitable position on the dye moiety, preferably so that the fluorescent properties of the dye are not adversely affected.
  • the linker can be joined to any available carbon atom, or to one of the nitrogen atoms in a rhodamine-type xanthene ring.
  • the substituent positions on the pendent phenyl ring are also available, particularly the positions which are para to C9 of the xanthene ring (5 position), or para to the ortho carboxyl group (6 position),
  • the particular chemical group by which a linker is connected to a nucleobase can be any chemical group that is suitable for the purposes of the present invention.
  • the linker is attached to the dye via a 5-carboxy ⁇ henyl (para to the xanthene C9 carbon atom) or 6-carboxyphenyl group (meta to the xanthene C9 carbon atom).
  • the linker is preferably attached to a 4-carbon atom or 5-carbon atom on the xanthene ring.
  • the linker is attached to the 3 or 6-nitrogen atom of the xanthene ring.
  • Dye-labeled nucleobase of the invention may also have the form B-L1-D1-L2-D2, wherein B is a nucleobase, Ll and L2 are linkers such that at least one of Ll and L2 comprises an imidazolium-containing linker moiety, and Dl and D2 are members of a fluorescent donor/acceptor pair.
  • Dl is a donor dye
  • D2 is an acceptor dye
  • D2 is a donor dye
  • Dl is an acceptor dye.
  • the donor dye and acceptor dye have different (non- identical) spectral properties.
  • the donor and acceptor may have the same type of aromatic ring structure (e.g., when both the donor and acceptor are fluorescein dyes, or both are rhodamine dyes), different spectral properties can arise for the donor and acceptor due to the nature of the substituents on each one.
  • the donor dye is effective to enhance the intensity of fluorescence emission of the acceptor dye relative to the intensity that would be observed in the absence of the donor dye under the same conditions.
  • Conjugates of this form may be referred to herein as "ET probes", "ET-labeled conjugates” or “ET-labeled nucleotides” because upon excitation of the donor dye, the conjugate can undergo energy transfer (by any of a variety of mechanisms, such as fluorescence resonance energy transfer) from the donor to the acceptor, such that the acceptor dye can then emit fluorescent light at a second wavelength in response thereto.
  • energy transfer by any of a variety of mechanisms, such as fluorescence resonance energy transfer
  • the donor dye and acceptor dye can be any fluorescent dye, and are each preferably fluorescent aromatic dyes.
  • the donor and acceptor dye, taken separately, can be a xanthene, rhodamine, dibenzorhodamine, fluorescein, [8,9]benzophenoxazine, cyanine, phthalocyanine, squaraine, or bodipy dye.
  • the donor and acceptor dyes can be linked together using any of a variety of attachment sites on each dye.
  • Dl is a fluorescein and D2 is a rhodamine (both of which contain pendent phenyl groups attached to C 9 of the xanthene rings)
  • Dl can be linked via its xanthene ring (preferably via C4)) to the pendent phenyl ring of D2 (e.g., via a 5- or 6- carboxy group on the pendent phenyl group).
  • This is referred to as a head to tail arrangement.
  • the positions of the connections can be reversed, such that D2 is linked via its xanthene ring to the pendent phenyl ring of Dl (another example of a head to tail arrangement).
  • Dl and D2 can be connected tail to tail, via their pendent phenyl rings, or head to head, via their xanthene rings, for example.
  • at least one of Ll and L2 comprises an iraidazolium-containing linker moiety linker. The properties of such linkers are generally as discussed above for linker L.
  • Ll comprises at least one imidazolium-containing linker moiety and L2 does not.
  • any of a variety of linkers can be used to connect Dl to D2.
  • D1-L2-D2 may comprise structure (a), (b) or (c) below:
  • R 2 ] is C]-Cs alkyldiyl
  • Zj is NH, S, or O
  • R 22 is an alkene, diene, alkyne, or a 5- or 6-membered ring having at least one unsaturated bond or a fused ring structure
  • R28 is a bond or spacer group. Details and examples of such inter-dye linkers can be found in U.S. Patent No. 5,800,996, for example.
  • R 22 is ethenediyl, ethynediyl, 1,3-butadienediyl, or 1,3-butadiynediyl.
  • L2 comprises at least one imidazolium-containing linker moiety and Ll does not.
  • any of a variety of linkers can be used to connect B to Dl. Descriptions of exemplary linkers can be found in the references in Table 1 above.
  • Ll can be or contain any of the following non-limiting examples:
  • left-hand ethene or ethyne moiety is linked to the nucleobase, and the right hand bond is typically linked directly to the dye or is linked indirectly to the dye through a carbonyl group.
  • Ll and L2 both comprise at least one imidazolium-containing linker moiety
  • the structures of Ll and L2 can be the same or different.
  • the present disclosure also includes nucleosides and nucleotides containing conjugates in accordance with the invention.
  • Exemplary nucleosides/tides of the present disclosure are illustrate by the following formula:
  • Wi is OH, H, F, Cl, NH 2 , N 3 , or OR, where R is C1-C6 alkyl (e.g., OCH 3 or OCH 2 CH 3 ); W 2 is OH or a group capable of blocking polymerase-mediated template-directed primer extension (such as H, F, Cl, NH 2 , N 3 , or OR, where R is C1-C6 alkyl (e.g., OCH 3 or OCH 2 CH 3 )); W 3 is OH, or mono-, di- or triphosphate or a phosphate analog thereof; and LB represents a dye-labeled nucleobase conjugate of the present disclosure.
  • R is C1-C6 alkyl (e.g., OCH 3 or OCH 2 CH 3 )
  • W 2 is OH or a group capable of blocking polymerase-mediated template-directed primer extension (such as H, F, Cl, NH 2 , N 3 , or OR, where R is C1-C6 alky
  • LB can comprise a conjugate of the form B-L-D or B-L1-D1-L2-D2, as described herein.
  • Wi is not OH.
  • W 2 is not OH, so that the compound is not 3' extendable.
  • Wi and W 2 are each separately selected from H, F, and NH 2 .
  • W 1 is F and W 2 is H, or W 1 is H and W 2 is F, or Wi and W 2 are each F, or W ( and W 2 are each H.
  • W 3 can be OH, monophosphate, diphosphate, or triphosphate.
  • exemplary nucleobases include adenine, 7-deazaadenine, 7-deaza-8-azaadenine, cytosine, guanine, 7-deazaguanine, 7-deaza-8- azaguanine, thymine, uracil, and inosine.
  • the present disclosure when W 3 is triphosphate, the present disclosure includes nucleotide triphosphates having the structure shown in the formula below:
  • the invention includes deoxynucleotide triphosphates having the structure shown in the formula below:
  • LB is defined as above.
  • Such compounds are examples of 3' extendable nucleotides.
  • Labeled 2'-deoxvnucleotides of this type find particular application as reagents for labeling polymerase extension products, e.g., in the polymerase chain reaction and nick- translation.
  • the invention includes ribonucleotide triphosphates having the structure shown in the formula below: o- flp P—— oO— 1P— O— i Pf— 0— CH 2 o.
  • Labeled nucleotides of this type find particular application as reagents for and in sequencing methods that utilize labile nucleotides having cleavable internucleotide linkages, as discussed for example in US Patent 5,939,292 (Gelfand et al.), Eckstein, Nucl. Acids Res. 16:9947-9959 (1988), U.S. Patent 6,887,690 (Fisher et al.), and Shaw, Nucl Acids Res. 23:4495 (1995).
  • nucleobase-linker-dye conjugates may be prepared by any suitable synthetic method.
  • conjugates are formed using a modular approach in which a nucleobase (which optionally may be provided in the form of a nucleoside or nucleotide .containing the nucleobase, for example), a first dye, a second dye (if present), and one or more linkers or linker precursors, are combined in serial and/or parallel steps to produce the desired labeled product.
  • the label can optionally be attached to a linker through a linkage formed by the reaction of a nucleophilic moiety of the linker with a complementary functionality located on the label.
  • the complementary functionality can be, for example, isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide (NHS) ester, sulfonyl chloride, aldehyde or glyoxal, epoxide, carbonate, aryl halide, imidoester, carbodiimide, 4,6- dichlorotriazinylamine, carboxylic acid anhydride, or other active carboxylate, see Hermanson, Bioconiugate Techniques. Academic Press, 1996.
  • the complementary functionality can optionally be an activated NHS ester that reacts with a nucleophilic moiety on the linker.
  • the activated NHS ester on the label can be formed by reacting a label, such as a carboxylate-complementary functionality, with dicyclohexylcarbodiimide and N-hydroxysuccinimide to form the NHS ester.
  • Table 1 shows a sampling of representative complementary functionalities and resulting linkages formed by reaction of the complementary functionality with an amine moiety on the linker.
  • the label can be any moiety that, when attached to the compounds of the present teachings, renders the compound to which the label is attached detectable using known detection means.
  • labels include but are not limited to fluorophores, chromophores, radioisotopes, spin- labels, enzyme labels, and chemiluminescent labels.
  • the label can optionally be, for example, a ligand, such as an antigen, or biotin, which can bind specifically with high affinity to a detectable anti-ligand, such as a labeled antibody or avidin,
  • detectable labels comprise fluorescent dyes such as fluorescein, rhodamine, rhodol or energy transfer dyes.
  • fluorescent dyes such as fluorescein, rhodamine, rhodol or energy transfer dyes.
  • various fluorescent dyes are described in U.S. Patent Application Publication US 2002/0102590 Al, which is incorporated herein by reference.
  • Examples 1 to 4 illustrate methods for forming linker synthons containing one, three, or four backbone imidazolium moieties.
  • Example 5 provides an alternative protocol to that of Example 2 in which 1,3- diiodopropane is used instead of 1,3-dibromopropane to alkylate protected aminoethyl imidazole A3, for further reaction with additional imidazole-containing synthons such as compound A6.
  • Examples 6 to 8 illustrate methods for forming linker synthons containing one, three, or four backbone imidazolium moieties using protection schemes that are different from those in Examples 1 to 4, and a convergent synthesis for forming a linker synthon containing six backbone imidazolium moieties.
  • Examples 9 to 12 illustrate methods for forming linker synthons containing one, three, or four backbone imidazolium moieties in which two imidazolium moieties are separated by an ethylene bridge instead of the propylene bridges shown in
  • Examples 13 to 15 illustrate methods for forming linker synthons containing one or two backbone imidazolium moieties using another protection scheme.
  • Examples 16 to 18C illustrate model reactions for conjugating an imidazolium linker moiety to an amino compound (represented by 3,4- dimethoxyphenethylamine) and a fluorescent dye label (FlO).
  • Examples 19 to 22 (Schemes 7 and 8) a modular, stepwise protocol for forming a hexaimidazolium linker labeled with an energy transfer dye.
  • Example 23 illustrates synthesis of a dye-labeled nucleoside triphosphate comprising a hexameric backbone-irm'dazolium moiety.
  • Example 24 illustrates synthesis of a dye-labeled nucleoside triphosphate comprising one backbone-imidazolium, built stepwise from a nucleoside triphosphate containing an aminoethoxypropargyl moiety by adding an imidazolium- containing synthon and then a fluorescent label. This contrasts with the approach shown in
  • Schemes 11 and 12 illustrate syntheses of dye-labeled nucleoside triphosphate comprising two and three backbone-imidazolium moieties, respectively, built stepwise from a nucleoside triphosphate.
  • the present disclosure also provides polynucleotides and mixtures of polynucleotides that contain one or more different nucleobase-linker-dye conjugates of the type discussed above.
  • Polynucleotides are useful in a number of important contexts, such as DNA sequencing, ligation assays, the polymerase chain reaction (PCR), probe hybridization assays, and various other sequence detection or quantitation methods.
  • Polynucleotide(s) may be formed by any appropriate method.
  • polynucleotides containing nucleobase linker dye conjugates may be synthesized enzymatically, e.g., using a DNA or RNA polymerase, nucleotidyl transferase, ligase, or other enzymes, e.g., Stryer, Biochemistry, Chapter 24, W.H.
  • Labels may be introduced during enzymatic synthesis utilizing labeled nucleotide triphosphate monomers as described above, or during chemical synthesis using labeled non-nucleoside or nucleoside phosphoramidites, or may be introduced subsequent to synthesis.
  • exemplary methods for forming labeled polynucleotides can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY (1989), US Patents 6,008,379 (Benson et al.), and references cited therein, for example.
  • a labeled polynucleotide is made using enzymatic synthesis, the following procedure may be used. An oligonucleotide primer is annealed to a complementary sequence in a template DNA strand.
  • a mixture of deoxynucleotide triphosphates (such as dGTP, dATP, dCTP, and dTTP) is added, where at least one of the deoxynucleotides contains a nucleobase-dye conjugate of the invention.
  • a dye- labeled polynucleotide is formed by incorporation of a labeled deoxynucleotide during polymerase-mediated strand synthesis.
  • the primer extension reagent includes a thermostable polymerase.
  • thermostable polymerases include but are not limited to rTth DNA polymerase, Bst DNA polymerase, Vent DNA polymerase, Pfu DNA polymerase, or Tag polymerase enzyme as described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., CSHL Press (1995).
  • the thermostable polymerase can be Tag DNA polymerase, or a mutant Tag polymerase enzyme having, for example, a mutation at the F667 position as described in, for example, Tabor and Richardson, EP 0 655506.
  • the mutation at the F667 position can be F667Y.
  • Tag polymerase enzyme can be a mutant that includes, in addition to the F667Y mutation, one or more mutations at the 660, 664, 665 and/or the 681 positions. See U.S. Patent 6,265,193.
  • representative mutations at the 660, 664, 665 and/or the 681 positions include, but are not limited to, R660D, R660E, R660C, R660S, R660P, and E681G.
  • the mutant Tag polymerase enzyme includes at least one of the mutations R660C or R660S, R660P and F667Y.
  • Labeled polynucleotides may be chemically synthesized using any suitable method, such as the phosphoramidite method Detailed descriptions of the chemistry used to form polynucleotides by the phosphoramidite method are provided elsewhere, e.g., Caruthers et al., U.S. Patents No. 4,458,066 and 4,415,732, Caruthers et al., Genetic Engineering 4: 1-17 (1982), Users Manual Model 392 and 394 DNA/RNA Synthesizers, pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (1991).
  • the phosphoramidite method is a preferred chemical method because of its efficient and rapid coupling and the stability of the starting materials.
  • the synthesis is performed with a growing polynucleotide chain attached to a solid support, so that excess reagents, which are in the liquid phase, can be easily removed by filtration, thereby eliminating the need for purification steps between synthesis cycles.
  • Nucleobase-dye conjugates of the present disclosure are suited for any method utilizing fluorescent detection, particularly methods requiring simultaneous detection of analytes which are not well separated by electrophoresis. Aspects of the present disclosure are particularly well suited for detecting classes of polynucleotides that have been subjected to a biochemical separation procedure, such as electrophoresis.
  • the present disclosure provides methods of identifying one or more polynucleotide(s).
  • the method utilizes one or more labeled different-sequence polynucleotides, which may have the same lengths or different lengths, wherein each different-sequence polynucleotide contains a unique nucleobase-dye conjugate.
  • the one or more labeled different-sequence polynucleotides are separated by electrophoresis to separate different-sequence polynucleotides on the basis of size.
  • Each different-sequence polynucleotide can then be identified on the basis of its electrophoretic mobility and, optionally, fluorescence signal.
  • each different polynucleotide is identifiable on the basis of a unique combination of electrophoretic mobility and fluorescence signal.
  • two different polynucleotides may contain identical dye moieties but may exhibit different electrophoretic mobilities.
  • two different polynucleotides can contain different dye moieties that produce distinct (spectrally resolvable) fluorescence signals but can exhibit the same electrophoretic mobilities.
  • different polynucleotides can differ in both their fluorescence signals and mobilities.
  • the method can be used in a multiplex format in which different labeled polynucleotides are formed by reaction with (i) a plurality of different target sequences and (ii) a plurality of different polynucleotides that are complementary to the target sequences.
  • the different polynucleotide can be designed to undergo a change in structure after hybridization to their complementary target sequences in a polynucleotide sample, e.g., due to modification by enzyme action, thereby producing different labeled polynucleotides having unique combinations of mobility and fluorescence to allow identification.
  • Such reactions can be performed simultaneously in a single reaction mixture or can be performed in separate reaction mixtures that can be combined prior to electrophoretic separation.
  • Sanger-type sequencing involves the synthesis of a DNA strand by a DNA polymerase in vitro using a single-stranded or double-stranded DNA template whose sequence is to be determined or confirmed. Synthesis is initiated at a defined site based on where an oligonucleotide primer anneals to the template. The synthesis reaction is terminated by incorporation of a nucleotide analog that will not support continued DNA elongation.
  • Exemplary chain-terminating nucleotide analogs include the 2',3'-dideoxynucleoside 5'- triphosphates (ddNTPs) which lack the 3'-OH group necessary for 5' to 3' DNA chain elongation.
  • dNTPs 2'-deoxynucleoside 5 -triphosphates
  • enzyme-catalyzed polymerization will be terminated in a fraction of the population of chains at each site where the ddNTP is incorporated.
  • a desired sequence read can be obtained by detection of the fluorescence signals of the terminated chains during or after separation by high-resolution electrophoresis.
  • dyes of the invention can be attached to either sequencing primers or terminator nucleotides.
  • labeled polynucleotide fragments can be generated through template-directed enzymatic synthesis using labeled primers or nucleotides, e.g., by polynucleotide ligation or polymerase-directed primer extension. The resultant fragments are then subjected to a size-dependent separation process, e.g.,
  • SUBSTTTUTE SHEET (RULE 26) electrophoresis or chromatography, and the separated fragments are detected, e.g., by laser- induced fluorescence.
  • multiple classes of polynucleotides are separated simultaneously and the different classes are distinguished by spectrally resolvable labels.
  • a fragment analysis method is based on amplified fragment length polymorphisms, i.e., restriction fragment length polymorphisms that are amplified by PCR. These amplified fragments of varying size serve as linked markers for following mutant genes in family lineages. The closer the amplified fragment is to the mutant gene on the chromosome, the higher the linkage correlation. Because genes for many genetic disorders have not been identified, these linkage markers serve to help evaluate disease risk or paternity.
  • the polynucleotides may be labeled by using a labeled polynucleotide PCR primer, or by utilizing labeled nucleotide triphosphates in the PCR.
  • nick translation another fragment analysis method, known as nick translation, one or more unlabeled nucleotide subunits in a double-stranded DNA molecule are replaced with labeled subunits. Free 3'-hydroxyl groups are created within the unlabeled DNA by "nicks" caused by treatment with deoxyribonuclease I (DNAase I). The DNA polymerase I then catalyzes the addition of one or more labeled nucleotides to the 3 -hydroxyl of the nick. At the same time, the 5' to 3'-exonuclease activity of this enzyme can remove one or more nucleotide subunits from the 5'-phosphoryl terminus of the nick.
  • DNAase I deoxyribonuclease I
  • a new nucleotide with a free 3'-OH group is incorporated at the position of the excised nucleotide, and the nick is shifted along by one nucleotide unit in the 3' direction. This 3' shift will result in the sequential addition of new labeled nucleotides to the DNA with the removal of existing unlabeled nucleotides.
  • the nick- translated polynucleotide is then analyzed using a separation process, e.g., electrophoresis.
  • Another exemplary fragment analysis method is based on the variable number of tandem repeats, or VNTRs.
  • VNTRs are regions of double-stranded DNA that contain adjacent multiple copies of a particular sequence, with the number of repeating units being variable among different members of a population (e.g., of humans). Examples of VNTR loci are pYNZ22, pMCT118, and Apo B. A subset of VNTR methods are based on the detection of microsatellite repeats, or short tandem repeats (STRs), i.e., tandem repeats of DNA characterized by a short (2-4 bases) repeated sequence.
  • STRs short tandem repeats
  • One of the most abundant interspersed repetitive DNA families in humans is the (dC-dA)n ⁇ (dG-dT)n dinucleotide repeat family (also called the (CA)n dinucleotide repeat family).
  • CA 50,000 to 100,000
  • label is introduced into the polynucleotide fragments by using a dye-labeled PCR primer.
  • oligonucleotide ligation assay two polynucleotides (probe pair) which are complementary to adjacent regions in a target sequence are hybridized to the target region of a polynucleotide, to create a nicked duplex structure in which the ends of the two polynucleotide abut each other.
  • the two probes can be joined by ligation, e.g., by treatment with ligase.
  • the ligated product is then detected, evidencing the presence of the target sequence.
  • the ligation product acts as a template for a second pair of polynucleotide probes which are complementary to the ligated product from the first pair.
  • the target sequence is amplified exponentially, allowing very small amounts of target sequence to be detected and/or amplified.
  • a fragment analysis method such as any of those discussed above can be performed in a multi-probe format, in which a sample is reacted with a plurality of different polynucleotide probes or probe sets which are each specific for a different target sequence, such as different alleles of a genetic locus and/or different loci.
  • the probes are designed to have a unique combination of mobility and fluorescence signal, to permit specific detection of the individual probes or probe products that are generated in the assay as a result of the presence of the different target sequences.
  • polynucleotides may be subjected to a size-dependent separation process.
  • the size-dependent separation process can comprise electrophoresis, chromatography, or hybridization to a set of polynucleotide probes that bind to the fragments in a sequence-dependent manner as described in, for example, Drmanac et al., Nature Biotechnology, 16: 54-58 (1998), Ramsay, Nature Biotechnology, 16: 40-44 (1998) and U.S. Patent No. 5,202,231.
  • the polynucleotides are detected, by, for example, laser-induced fluorescence.
  • multiple classes of polynucleotides can be separated or hybridized simultaneously and the different classes can be distinguished by a set of spectrally resolvable labels.
  • the electrophoretic matrix contains crosslinked or uncrosslinked polyacrylamide having a concentration (weight to volume) of between about 2-20 weight percent, and often about 4 to 8 percent.
  • the electrophoresis matrix usually includes a denaturing agent such as urea, formamide, or the like.
  • a denaturing agent such as urea, formamide, or the like.
  • Detailed exemplary procedures for forming such matrices are given by Maniatis et al., "Fractionation of Low Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98% Formamide or 7 M Urea," in Methods in Enzymology, 65: 299-305 (1980), Sambrook et al. (1989, supra), and ABI PRISMTM 377 DNA Sequencer User's Manual, Rev.
  • Optimal electrophoresis conditions e.g., polymer concentration, pH, temperature, voltage, concentration of denaturing agent, employed in a particular separation depends on many factors, including the size range of the nucleic acids to be separated, their base compositions, whether they are single stranded or double stranded, and the nature of the polynucleotides for which information is sought by electrophoresis. Accordingly application of the invention may require preliminary testing to optimize conditions for particular separations.
  • labeled polynucleotides can be detected or identified by recording fluorescence signals (or other detectable signals) and migration times (or migration distances) of the separated polynucleotides, or by constructing a chart of relative fluorescent and order of migration of the polynucleotides (e.g., as an electropherogram).
  • the labeled polynucleotides can be illuminated by standard means, e.g. a high intensity mercury vapor lamp, a laser, or the like.
  • the labeled polynucleotides are illuminated by laser light generated by a He-Ne gas laser or a solid-state diode laser.
  • the fluorescence signals can then be detected by a light-sensitive detector, e.g., a photomultiplier tube, a charged-coupled device, or the like.
  • a light-sensitive detector e.g., a photomultiplier tube, a charged-coupled device, or the like.
  • Exemplary electrophoresis detection systems are described elsewhere, e.g., U.S. Patent Nos. 5,543,026, 5,274,240, 4,879,012, 5,091,652 and 4,811,218.
  • labeled nucleoside triphosphates that comprise backbone- imazolium-containing linker moieties are designed to have a net neutral or net positive charge under selected conditions.
  • such nucleotide triphosphates have a net neutral or net positive charge at a pH of 7 or greater, or a pH of 8 or greater.
  • the triphosphate portion of a nucleoside triphosphate typically bears 3 to 4 negative charges, depending on the pH of the environment (3 negative charges if the pH is below about 6, and 4 negative charges if the pH is above about 6).
  • An simple fluorescein dye backbone typically contains about 2 negative charges under neutral or basic pH conditions.
  • a linker that comprises at least 6 positive charges should suffice to confer an overall net neutral or positive charge to the nucleotide triphosphate.
  • a simple rhodamine dye is present instead (typically having a net neutral charge)
  • a linker moiety containing at least 4 backbone-imidazolium moieties should be sufficient to confer an overall net neutral or positive charge to the nucleotide triphosphate.
  • Linkers containing fewer backbone-irnidazolium moieties can be used if additional positive charge are present elsewhere in the labeled nucleotide triphosphate.
  • nucleoside triphosphates that comprise backbone-imidazolium moieties and that have a net neutral or net positive charge can be used advantageously in primer extension or other reactions that are subsequently subjected to electrophoresis separation since such nucleoside triphosphates and their breakdown products will migrate away from, or only very slowly toward, the anode so that overlap with polynucleotides of interest can be reduced or avoided.
  • primer extension reaction mixtures are loaded into a capillary or other electrophoretic pathway without prior removal of residual triphosphates.
  • kits for performing the various methods of the invention comprises at least one labeled nucleoside triphosphate comprising a conjugate described herein.
  • the kit may also include one or more of the following components: a 3 '-extendable primer, a polymerase enzyme, one or more 3' extendable nucleotides which are not labeled with conjugate, and/or a buffering agent.
  • the kit includes at least one labeled nucleoside triphosphate that is nonextendable.
  • the kit comprises four different labeled nucleoside triphosphates which are complementary to A, C, T and G, and each of which contains a distinct conjugate as described herein.
  • the labeled nucleoside triphosphates are nonextendable.
  • the labeled nucleoside triphosphates are extendable ribonucleoside triphosphates.
  • the kit comprises at least one labeled, nonextendable nucleoside triphosphate comprising a conjugate described herein, and one or more of the following components: a 3 '-extendable primer, a polymerase enzyme, and/or a buffering agent.
  • the concentrated material was chromatographed on a 130 mm x 75 mm bed of silica gel 60 (Merck) packed in 20:1 dichloromethane:methanol (DCM:M) eluted with (1) 600 mL of 20:1 DCM:M, (2) 1 L of 10:1 DCM:M, then (3) 2 L of 5:1 DCM:M, during which fractions of 225 mL were collected.
  • TLC analyses of fractions were performed on silica eluted with 5:1 DCM:M, and product was visualized with iodine and ninhydrin. Product- containing fractions were collected and dried under reduced pressure, yielding 6.8 g (16.5 mmol) A5 as an opaque thick oil.
  • Example 3 t-Boc-Protected Linker Element Containing Three Imidazolium Moieties 3.3 g (8 mmol) of A5 and 10.7 g (60 mmol) A6 were dissolved in 30 mL DMF in a 100 mL flask and heated at 85°C using an oil bath. After NMR analysis of an aliquot indicated completion of the reaction (after about 16 h of heating), the reaction mixture was filtered through a coarse frit (which removed symmetrical side-product AT) and solvent was removed under high vacuum. The resultant product was dispersed in 35 mL of DCM, poured into 140 mL of THF, and stirred over night, yielding a gummy precipitate in a clear supernatant.
  • A3 (2.2 g, 10 mmol, see Example 1 for synthesis) and 1,3-diiodopropane AlO (H g, 37.3 mmol) were dissolved in 25 mL THF in a 50 ml flask equipped with a reflux condenser, and the mixture was refluxed in an oil bath at 65 0 C over night, forming a large precipitate. After refluxing, the flask was removed from the oil bath and allowed to cool to room temperature.
  • the solid was removed by filtration on a medium frit, chased with 5 mL THF, and then dried under high vacuum, yielding 1.2 g of solid that was identified as the adduct of 1,3-diiodopropane AlO with two molecules of A3. (This adduct, All, was not used further in this example.)
  • the THF filtrate was chromatographed on a 50 by 50 mm column of silica with 5:1 DCM/M. Product-containing fractions were combined, rotoevaporated and then rechromatographed on a 50 by 50 mm silica column as above with 10:1 DCM:M and then 5:1 DCM:M. Except for an early fraction that contained a substantial amount of residual AlO, product-containing fractions were pooled and evaporated, yielding 2.8 g (5.5 mmol) of gummy oil that solidified into a soft yellow solid.
  • N ⁇ -Bromopropyl-N ⁇ -Carboxypropyl Imidazole Ethyl Ester 4.4 g (24.2 mmol) of N-3-carboxypropyl imidazole ethyl ester Bl and 13 mL (24.4 g, 121 mmol) 1,3-dibromopropane (A4) were dissolved in 25 mL DMF in a 100 mL flask and heated over night at 80 0 C. The DMF was removed under reduced pressure, yielding 10 g of a yellow oil.
  • the oil was chromatographed on a 75 mm by 150 mm bed of silica gel 60 packed in 20:1 DCM:M, that was eluted with (I) I L of 20:1 DCM:M, (2) 1 L 10:1 DCM:M, then (3) 3 L of 4:1 DCM:M (225 mL fractions).
  • Thin layer chromatography (TLC) analyses of fractions were performed on silica eluted with 5:1 DCM:M, and product was visualized with iodine.
  • Product-containing fractions were collected and concentrated under reduced pressure, yielding 7.6 g (19.8 mmol) B2 as an opaque oil.
  • Example 7 r0050138-097.11 Ethyl Ester of Linker Element Containing Three Imidazolium Moieties
  • Compounds B2 (3.6 g) and A6 (14 g, prepared in accordance with Diez-Barra, E., et. al., Heterocvcles 34(7):1365-1373, 1992) were dissolved in DMF (15 mL) in a 250 mL flask, and the reaction was heated at 8O 0 C over night. Solvent was then removed under reduced pressure, and the resultant product was dispersed in 40 mL DCM followed by addition of 120 mL of THF.
  • Example 9 r0050138-033.11 N-trimethylsilylimidazole Dl (2.8 g, 20 mmol) and ethyl 4-bromobutanoate D2 (4 g, 20 mmol) were dissolved in DMF (8 mL) in a 25 mL flask and heated over night in a 7O 0 C oil bath. The reaction was poured into 100 mL of water and was saturated with solid sodium bicarbonate. The resulting liquid was decanted into a separatory tunnel, chased with 10 mL of water, and extracted three times with 100 mL portions of DCM.
  • D3 (1.82 g, 10 mmol) and A4 (10 g, 50 mmol) were mixed in DMF (10 mL) in a 50 mL flask and heated at 7O 0 C for 16 h.
  • the DMF was removed by rotoevaporation under high vacuum.
  • the product was chromatographed on silica (50 by 150 mm with 10:1 DCM/M). Product-containing fractions were analyzed by TLC (silica developed with 5:1 DCM:M), visualized with iodine, pooled and rotoevaporated, yielding 2.88 g (7.5 mmol) D4 of a cloudy oil.
  • reaction was then diluted with 300 mL of ethanol and filtered on #2 filter paper on a Buchner funnel. The solid was washed with 20 mL ethanol. The solid was dried under reduced pressure, dissolved in about 200 ml of 8:1 DCM/M, and chromatographed on a silica column, 75 by 180 mm, packed with 10:1 DCM/M. Elution was performed with 10:1 DCM/M. Fractions were analyzed by TLC with 5:1 DCM/M and visualized using iodine. Fractions that contained 1,2-diimidazole E3 were combined, dried, and rechromatographed through a 75 by 180 mm silica column packed with 10:1 DCM/M.
  • N-2-aminoethyl imidazole hydrobromide Al (2.29g, 10 mmol) was suspended in ethanol (30 mL), followed by addition of triethylamine (TEA, approx. 3 mL). The reaction mixture initially became clear and then formed a precipitate. To this mixture was added CF 3 CO 2 Et (Fl, approx. 2 mL), and the mixture was stirred at room temperature over night. The precipitate was removed by filtration, and the filtrate was dried by rotoevaporation and high vacuum. The solid residue was suspended in 30 mL THF, Undissolved solid was removed by filtration, and the filtrate was dried by rotoevaporation and high vacuum.
  • THF triethylamine
  • the mixture was then warmed gently by heat gun over about 30 min, then rotoevaporated at 4O 0 C to form a glass.
  • the glassy material was triturated with 45 mL of THF over night. Solid product was collected by filtration on a medium frit, yielding 2.8 g. This was triturated with 30 mL acetonitrile, warmed gently, then stirred in an ice bath, then warmed to room temperature over 2 h. Solid product was collected by filtration, yielding 1.4 g (2.1 mmol) of F6.
  • F4 (1.46 g, 3.5 mmol) was dissolved in about 12 mL of acetonitrile (ACN) with sonication and then was added to a stirred solution of F7 (990 mg, 5.46 mmol) in ACN (2 mL) and stirred at room temperature for about 2 h. Most of the acetonitrile was removed by rotoevaporation (leaving a volume of about 2 mL), and 50 mL ether was added and mixed. The opaque ether layer was decanted, and 20 mL of ether was added, mixed, and then decanted. The acetonitrile layer was dried under high vacuum, producing a sticky foam.
  • ACN acetonitrile
  • the sample was dissolved in aq HBr (10 drops HBr/L of water), applied to a Cl 8 reverse phase silica column (40 by 60 mm, BakerBond Octadecyl 40 Micron Prep LC packing material, PN 7025-01 from J.T. Baker Inc., USA) packed with aq HBr, and eluted with 400 mL of aq HBr, then 220 mL of 200:20 aq HBr/ACN, then 230 mL of 200:30 aq HBr/ACN, then 240 mL of 200:40 aq HBr/ACN.
  • Fractions were analyzed by silica TLC plates and visualized with ninhydrin and/or molybdic acid stain solution (12 g (NH 4 )SMo 7 O 24 "4H 2 O, 0.5 g cerric ammonium nitrate, 50 mL H 2 SO- J , and 450 mL water). Product fractions were combined and evaporated under high vacuum to produce F8 as a sticky foam (1.14 g, 2.24 mmol).
  • reaction was desalted on a small reverse phase column by loading the sample in 0.1% TFA (aq), washing with 10 column volumes of 0.1 % TFA, and eluting with 4:1 acetonitrile:0.1% TFA. After evaporation of solvent, mass spectrometric analysis confirmed that the large peak is product FIl (MW 814.73).
  • the dried material was redissolved in 300 mL 5% NaHCO 3 solution and washed two times with EA (100 mL portions), acidified with 6 N HCl, and extracted two times with EA (250 mL portions).
  • the combined EA layers were washed two times with brine (100 mL portions), then dried over Na 2 SO 4 , filtered, and rotoevaporated.
  • the collected product was then crystallized from 100 mL EA, yielding 6.0 g of crystalline 4- (trifiuoroacetyl)aminomethyl benzoic acid (first crop) and an additional 1.8 g in a second crop (total 7.8 g).
  • the dried EA solution was filtered and then rotoevaporated.
  • the residue was chromatographed on a silica column (25 by 80 mm) eluted with 10:1 DCM/MeOH containing 1% acetic acid (AA).
  • Product-containing fractions were combined and rotoevaporated and partitioned between EA (100 mL) and 1 N HCl (25 mL), washed with brine (25 mL), and dried over Na 2 SO 4 .
  • the EA layer was rotoevaporated and rechromatographed on silica (25 by 80 mm) with 15:1 DCM:M containing 1% AA.
  • the dried solution was filtered, rotoevaporated, reconstituted in EA, sonicated, followed by removal of precipitated DCU by filtration and rotoevaporation of the filtered reaction mixture.
  • the reaction mixture was then chromatographed on a silica column (25 by 80 mm) using 20:1 DCM/MeOH. Fractions were analyzed by TLC, and product-containing fractions were pooled and rotoevaporated, yielding 350 mg (0.47 mmol) product G5.
  • Poly-imidazole hexamer C2 (650 mg, 516 ⁇ mol, prepared supra) was dissolved in about 4 mL of formamide, then G5 (220 mg, 300 ⁇ mol), dissolved in about 5 mL of formamide, was added. The flask containing G5 was washed (chased) two times with one mL portions of formamide into the mixture of C2 and g5, then several drops of TEA were added until the reaction started to become orange.
  • the reaction mixture was then diluted to about 200 mL with 0.1% aqueous TFA and loaded on a reverse phase silica colun (20 x 60 mm) and eluted with 100:15 0.1% TFA:acetonitrile, then 100:25 , then 100:30, then 100:35 (-200 mL each).
  • the combined product fractions were diluted 2.5-fold with water and passed through a 10 mm by 15 mm pad of reverse phase Cl 8 silica (J.T. Baker, supra).
  • the trapped compound was washed with 1:10 acetonitrile/aqueous 0.5% TFA and then eluted with 100:1 MeOH/H 2 O containing about 0.3% TFA. During elution, the flow was stopped for 15 minutes between fractions.
  • the product fractions were concentrated under reduced pressure and then dissolved in 1 mL DMSO (dimethylsulfoxide) and precipitated with 14 mL of ether in a 15 mL Falcon tube. After 2 more precipitations from DMSO/ether, the sample was precipitated twice with DMSO-dg-ether. After removal of the ether by high vacuum, the sample was dissolved in DMSO-dg for NMR analysis, confirming that free acid G8 was obtained.
  • the G8 product in the NMR sample tube was treated with 10 mg of TSTU (O-(N- Succinimidyl)-l,l,3,3-tetramethyluroniurn tetrafluoroborate) and then with 5 microliters of triethylamine. After 1 hour, NMR analysis indicated that the desired NHS (N- hydroxysuccinimide) ester G9 had formed.
  • the sample was transferred to a 15 mL Falcon tube with the aid of a small amount of DMSO and precipitated with 13 mL of ethyl acetate. After decantation and vacuum concentration, the sample was dissolved in DMSOd 6 . NMR analysis showed a singlet at 2.5 ppm integrating as 4 protons, indicating formation of NHS ester ⁇ 2.- The sample was then precipitated with ether in portions in a 1.5 mL Eppendorf tube and vacuum dried.
  • nucleosides (nucleoside triphosphates in this case) containing dye-linker-nucleobase conjugates of the inventions by first attaching a backbone imidazolium linker moiety to a nucleobase of a nucleoside triphosphate and then attaching a dye moiety to the linker (see Scheme 10).
  • AE-HPLC anion-exchange high-performance chromatography
  • CE-HPLC Cation-exchange high-performance chromatography

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Abstract

La présente invention fournit des conjugués marqueur-nucléotide et des compositions associés dans lesquels un marqueur, tel qu'un colorant fluorescent, est lié directement ou indirectement à une nucléobase par un liant. Dans certains modes de réalisation, le liant possède une ossature comprenant une fraction d'imidazolium. De tels conjugués sont utiles, par exemple, dans des procédés d'extension d'amorce et dans des techniques associées ainsi que dans des procédés qui impliquent une électrophorèse des polynucléotides, telle que certaines techniques de séquençage d'ADN. L'invention décrit également des procédés, des kits et des compositions.
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