EP1208109A2 - Analogues de nucleoside - Google Patents

Analogues de nucleoside

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Publication number
EP1208109A2
EP1208109A2 EP00958770A EP00958770A EP1208109A2 EP 1208109 A2 EP1208109 A2 EP 1208109A2 EP 00958770 A EP00958770 A EP 00958770A EP 00958770 A EP00958770 A EP 00958770A EP 1208109 A2 EP1208109 A2 EP 1208109A2
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EP
European Patent Office
Prior art keywords
analogue
mmol
primer
nucleoside
compound
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Withdrawn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
EP00958770A
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German (de)
English (en)
Inventor
Clifford Smith
William Jonathan Cummins
Robert James Domett Nairne
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.)
GE Healthcare Ltd
Original Assignee
Nycomed Amersham PLC
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Priority to EP00958770A priority Critical patent/EP1208109A2/fr
Publication of EP1208109A2 publication Critical patent/EP1208109A2/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Withdrawn legal-status Critical Current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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
    • C07D249/10—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D249/12—Oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
    • C07D249/10—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D249/14—Nitrogen atoms
    • 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
    • C07H19/052—Imidazole radicals

Definitions

  • the present invention relates to compounds suitable for use as nucleoside analogues, and to polynucleotide chains comprising nucleoside analogues.
  • Nucleic acids are manipulated in vitro in a wide variety of research and diagnostic techniques.
  • the methods can involve the synthesis of nucleic acid probes by means of DNA or RNA polymerase, reverse transcriptase or terminal transferase enzymes for the purposes of labelling or determination of base sequence identity. Labelling often involves the incorporation of a nucleotide which is chemically labelled or which is of a particular chemical composition so as to make it detectable.
  • Nucleic acid probes made in this way can be used to determine the presence of a nucleic acid target which has a complementary sequence by means of hybridisation of the probe to the target.
  • WO 94/21658 T I Kalman describes novel nucleoside or nucleotide analogues having a 4-acetylimidazolin-2-one base and their use for inhibiting virally encoded reverse transcriptases.
  • T Fukuda et al describe the effect of incorporation of nucleoside analogues having an imidazolin-2-one base as both T and G in DNA duplexes.
  • Y is -CO-, -CONW-, -O-, -S-, -SO-, -SO 2 -, -NWCO-,-NW-, or -OCO-,
  • W is the same or different at different places in the molecule and each is H or alkyl or aryl or Rp or -Ln-Rp,
  • Ln is a linker group
  • Rp is a reporter moiety
  • Q is a sugar or a sugar analogue or a nucleic acid backbone or backbone analogue, provided that at least one reporter moiety Rp is present.
  • Z is O, S, Se, SO, NW or CH 2 ,
  • R 1 , R 2 , R 3 and R 4 are the same or different and each is H, OH, F, NH 2 , N 3 , O-hydrocarbyl or Rp or -Ln-Rp,
  • R 5 is OH, SH or NH 2 or mono-, di- or tri-phosphate or - thiophosphate, or corresponding boranophosphate, or one of R 2 and R 5 is a phosphoramidite or other group for incorporation in a polynucleotide chain, or Rp or -Ln-Rp, or Q consists of one of the following modified sugar structures
  • Q is a nucleic acid backbone consisting of sugar- phosphate repeats or modified sugar-phosphate repeats (e.g. LNA) (Koshkin et al, 1998, Tetrahedron 54, 3607-30) or a backbone analogue such as peptide or polyamide nucleic acid (PNA). (Nielsen et al, 1991 , Science 254, 1497 - 1500).
  • LNA sugar- phosphate repeats or modified sugar-phosphate repeats
  • PNA polyamide nucleic acid
  • the reporter moiety Rp with or without the linker group Ln, will be present in W or Y when Y contains W and will always have at least one atom between the reporter and the base ring.
  • Q is a sugar or sugar analogue or a modified sugar
  • these compounds are nucleotide analogues or nucleoside analogues.
  • Q is a nucleic acid backbone or a backbone analogue
  • these compounds are herein called nucleic acids or polynucleotides.
  • a nucleoside analogue is a molecule which is capable of being incorporated, either chemically or enzymatically, into an oligomeric or polymeric nucleic acid (DNA or RNA) chain, and when so incorporated of forming a base pair with a nucleotide residue in a complementary chain or base stacking in the appropriate nucleic acid chain.
  • a nucleotide is a naturally occurring compound comprising a heterocyclic base and a sugar moiety including a phosphate.
  • a nucleoside is a corresponding compound in which a phosphate is not present.
  • Nucleotide analogues and nucleoside analogues are analogous compounds having different bases and/or different sugar moieties.
  • a nucleoside analogue is a compound which is capable of forming part of a nucleic acid (DNA or RNA or PNA) chain, and is there capable of base-pairing with a base in a complementary chain or base stacking in the appropriate nucleic acid chain.
  • a nucleoside analogue may be specific, by pairing with only one complementary nucleotide; or degenerate, by base pairing with more than one of the natural bases, e.g. with pyrimidines (T/C) or purines (A/G); or universal, by pairing with each of the natural bases with little discrimination; or it may pair with another analogue or itself.
  • T/C pyrimidines
  • A/G purines
  • the base analogue is linked to a sugar moiety such as ribose, deoxyribose or dideoxyribose to form a nucleoside analogue.
  • a sugar moiety such as ribose, deoxyribose or dideoxyribose
  • the nucleoside triphosphate analogues of the invention are capable of being incorporated by enzymatic means into nucleic acid chains.
  • a reporter moiety Rp may be any one of various things. It may be a radioisotope by means of which the nucleoside analogue is rendered easily detectable, for example 32-P or 33-P or 35-S incorporated in a phosphate or thiophosphate or phosphoramidite or H-phosphonate group, or alternatively 3-H or 14-C or 125-1. It may be a stable isotope or a specific chemical moiety suitable for detection by mass spectrometry. (Or the compound as a whole may be suitable for detection by mass spectrometry.) It may be a signal moiety e.g. an enzyme, hapten, fluorophore, chemiluminescent group, Raman label or electrochemical label.
  • a signal moiety e.g. an enzyme, hapten, fluorophore, chemiluminescent group, Raman label or electrochemical label.
  • the reporter moiety may be a solid surface, to which the nucleoside analogue is attached and by means of which it may be distinguished from nucleoside analogues not so immobilised.
  • the reporter moiety may be a reactive group, either a nucleophilic group, e.g. NH 2 , OH, COOH, CONH 2 , ONH 2 , SH or a thiophosphate or a hydrazine or a hydrazide, or an electrophilic group e.g. an active ester or aldehyde or maleimide, by which a signal moiety and/or a solid surface may be attached, before or after incorporation of the nucleoside analogue in a nucleic acid chain.
  • a nucleophilic group e.g. NH 2 , OH, COOH, CONH 2 , ONH 2 , SH or a thiophosphate or a hydrazine or a hydrazide
  • a linker group Ln is a chain of 1 to 60 or more carbon, nitrogen, oxygen phosphorus and/or sulphur atoms, rigid or flexible, saturated or unsaturated, as well known in the field.
  • the linker group is joined to a 4-triazole ring (when X is N) or to a 4-imidazole ring (when X is CH) of the nucleoside analogue molecule by a group having an alpha carbonyl group, e.g. amide or an amine bond.
  • the linker group is joined to the reporter moiety by an amide bond.
  • a linker preferably has at least three chain atoms, e.g. -(CH 2 ) n - where n is at least 3.
  • Two (or more) reporter moieties may be present, e.g. a signal moiety and a solid surface, or a hapten and a different signal moiety, or two fluorescent signal groups to act as donor and acceptor.
  • Various formats of these arrangements may be useful for separation or detection purposes.
  • Purine and pyrimidine nucleoside derivatives labelled with reporter moieties are well known and well described in the literature. Labelled nucleoside derivatives have the advantage of being readily detectable during sequencing or other molecular biology techniques.
  • R 1 , R 2 , R 3 and R 4 may each be H, OH, F, NH 2 , N 3 , O-alkyl or a reporter moiety.
  • ribonucleosides, and deoxyribonucleosides and dideoxyribonucleosides are envisaged together with other nucleoside analogues.
  • These sugar substituents may contain a reporter moiety in place of or in addition to the one or two present in the base.
  • R 5 is OH or mono-, di- or tri-phosphate or -thiophosphate or corresponding boranophosphate. From nucleosides (R 5 is OH) it is readily possible to make the corresponding nucleotides (R 5 is triphosphate) by literature methods. Alternatively, one of R 2 and R 5 may be a phosphoramidite or H-phosphonate or methylphosphonate or phosphorothioate or amide, or an appropriate linkage to a solid surface e.g. hemisuccinate controlled pore glass, or other group for incorporation , generally by chemical means, in a polynucleotide chain. The use of phosphoramidites and related derivatives in synthesising oligonucleotides is well known and described in the literature.
  • At least one reporter moiety is present preferably in the base analogue and/or optionally in the sugar moiety or a phosphate group.
  • Reporter moieties may be introduced into the sugar moiety of a nucleoside analogue by literature methods (e.g. J. Chem. Soc. Chem. Commun. 1990, 1547-8; J. Med. Chem., 1988, 31 . 2040-8). Reporter moieties in the form of isotopic labels may be introduced into phosphate groups by literature methods (Analytical Biochemistry, 214, 338-340, 1993; WO 95/15395).
  • the nucleoside analogues are available for enzymatic incorporation in DNA or RNA.
  • the invention includes in another aspect the polynucleotide chain comprising at least one residue of the nucleoside analogue as defined.
  • Nucleoside analogues of this invention are useful for labelling DNA or RNA or for incorporating in oligonucleotides or PNA.
  • a reporter moiety is attached at a position where it does not have a significant detrimental effect on the physical or biochemical properties of the nucleoside analogue, in particular its ability to be incorporated in single stranded or double stranded nucleic acid.
  • a template containing the incorporated nucleoside analogue of this invention may be suitable for copying in nucleic acid synthesis.
  • a reporter moiety of the incorporated nucleoside analogue consists of a linker group
  • a signal moiety can be introduced into the incorporated nucleoside analogue by being attached through a terminal or other reactive group of the linker group.
  • a nucleoside analogue triphosphate of this invention may be incorporated by enzymes such as terminal transferase to extend the 3' end of nucleic acid chains in a non-template directed manner. Tails of the nucleoside analogue triphosphate produced in this way may be detected directly in the absence of any reporter label by use of antibodies directed against the nucleoside analogue.
  • the analogues when incorporated into oligonucleotides or nucleic acids may be acted upon by nucleic acid modification enzymes such as ligases or restriction endonucleases.
  • nucleoside analogues of this invention can also be used in any of the existing applications which use native nucleic acid probes labelled with haptens, fluorophores or other reporter groups, for example on Southern blots, dot blots and in polyacrylamide or agarose gel based methods or solution hybridisation assays and other assays in microtitre plates or tubes or assays of oligonucleotides or nucleic acids such as on microchips.
  • the probes may be detected with antibodies targeted either against haptens which are attached to the base analogues or against the base analogues themselves which would be advantageous in avoiding additional chemical modification.
  • Antibodies used in this way are normally labelled with a detectable group such as a fluorophore or an enzyme. Fluorescent detection may also be used if the base analogue itself is fluorescent or if there is a fluorophore attached to the nucleoside analogue.
  • RNA is an extremely versatile biological molecule. Experimental studies by several laboratories have shown that in vitro selection techniques can be employed to isolate short RNA molecules from RNA libraries that bind to proteins, not normally associated with RNA binding, including a few antibodies, with high affinity and specificity (Gold, Allen, Binkley, et al.1993, 497-510 in The RNA World, Cold Spring Harbor Press, Cold Spring Harbor N.Y., Gold, Polisky, Unlenbeck, and Yams, 1995, Annu.
  • RNA molecules have been proposed as drug candidates for the treatment of diseases like myasthenia gravis and several other auto-immune diseases.
  • the basic principle involves adding an RNA library to the protein or molecule of interest. Washing to remove unbound RNA. Then specifically eluting the RNA bound to the protein. The RNA is then reverse transcribed and amplified by PCR. The DNA is then transcribed using modified nucleotides (either 2' modifications to give nuclease resistance e.g. 2' F, 2' NH 2 , 2' OCH 3 and/or C5 modified pyrimidines and/or C8 modified purines). Those molecules that are found to bind the protein or other molecule of interest are cloned and sequenced to look for common sequences. The common sequence is taken and used to make a short oligonucleotide therapeutic.
  • modified nucleotides either 2' modifications to give nuclease resistance e.g. 2' F, 2' NH 2 , 2' OCH 3 and/or C5 modified pyrimidines and/or C8 modified purines.
  • the base analogues described here when converted to the nucleoside triphosphate or nucleoside phosphoramidite, will significantly increase the molecular diversity available for this selection process. This may lead to oligonucleotides with increased binding affinity to the target that is not available from the current building blocks.
  • triphosphate nucleotide analogues containing five membered heterocycles such as pyrrole have been demonstrated to act as substrates for enzymatic incorporation, (WO 97/28176).
  • the nucleotide base analogue pyrrole-3,4-dicarboxamide is a particularly good substrate.
  • the corresponding base analogue pyrrole-3-carboxamide is also a substrate but with a significant decrease in efficiency relative to the dicarboxamide. This illustrates that despite having the same groups being presented at the hydrogen bonding face subtle changes of structure can have significant effects that alter the analogue's ability to act as a substrate. These effects are not yet predictive.
  • Both the pyrrole mono and dicarboxamide analogues are also degenerate in that they will substitute for all the natural bases with varying degrees of efficiency e.g. the pyrrole-3,4-dicarboxamide will replace A and C and extension is then possible from there but it will also replace T and G and act as a terminator.
  • the nucleotide analogues of this invention have several advantages over those described above for enzymatic incorporation.
  • Direct enzymatic incorporation is but one aspect of enzymatic recognition and tolerance that has to be considered.
  • the attachment of a linker arm or the analogue itself can effect the ability of an enzyme to either extend from the analogue or read through the analogue and these properties to date are not predictive.
  • the base analogue difluorotoluene causes a pause on read through by an enzyme placing a base opposite in the growing complimentary strand (Proc. Natl. Acad. Sci. USA (1997), 94, 10506-1 1).
  • the universal base 5-nitroindole (WO 97/28176) when included in an oligo is readily read through by an enzyme and a base replaced opposite.
  • the 5-nitroindole is a universal base there is likely to be uncertainty as to the base placed opposite during the formation of the complimentary strand to the template.
  • the nucleotide analogues of this invention have advantages in that as phosphoramidites they can be selectively placed in a position within a DNA oligo via chemical synthesis. Once in that position the presence of a linker and reporter group has been demonstrated to permit an enzyme to read through it and place a base opposite the analogue, see examples 4A-4G. In a comparable experiment with a universal base such as 5-nitroindole the introduction of a linker and reporter was found to have detrimental effects on read through ability.
  • the amine salt (5) was dissolved in anhydrous DMSO and treated with N,N-diisopropyethylamine (5 ⁇ l) and 6-(fluorescein-5-(and 6-) carboxamidohexanoic acid NHS ester (3.6 mg). The mixture was allowed to stir for 20 hours and the mixture was purified by ion-exchange chromatography . ⁇ max 486 nm, 1 H nmr was consistent with expected structure.
  • a primer extension assay was used to evaluate compounds (4, 5 and 6) as a substrate for exonuclease free Klenow fragment DNA polymerase I (EFK).
  • the assay used a 33 P 5' end labelled 15mer primer hybridised to a 24mer template.
  • the sequences of the primer and template are:
  • N-(3-te/T-butoxycarbonylamino)propyl 4- trifluoroacetamidobutanamide (570 mg, 1.6 mmol) was treated with 4M hydrogen chloride in dioxan (5 ml) at room temperature for 1.5 hours. The solvent was removed in vacuo and the resulting oil dried in vacuo to give the desired amine salt as a thick gum that was used without further purification.
  • This sulfonamide was prepared in an analogous fashion to 14, using 4-(4-N,N-dimethylaminophenyl)azobenzenesulfonyl chloride (1.03 g, 3.2 mmol) and methyl 6-aminohexanoate hydrochloride salt (0.58 g, 3.2 mmol) giving the sulfonamide (810 mg, 59%) as an orange solid.
  • Methyl 4-(4-(4- dimethylaminophenyl)azobenzenesulfonamido)butanoate (1 g, 2.47 mmol) was dissolved in 1 ,3-diaminopropane (10ml) and the mixture was heated to 100°C under nitrogen for 3 hours. The solvent was removed in vacuo and the solid was partitioned between pH 6 citrate buffer and chloroform.
  • Methyl 6-(4-(4-N,N- dimethylaminophenyl)azobenzenesulfonamido)hexanoate (0.72 g, 1.7 mmol) was added to molten, stirred 1 ,6-diaminohexane (10.7 g, 92 mmol) at 50°C. The mixture was heated at 77°C for 6 hours and then the mixture was allowed to cool to room temperature. The mixture was then melted and poured into pH 6 citrate buffer and the solid extracted with chloroform, the extracts were washed with water (3 x 150 ml), then pH 6 citrate buffer, dried (MgSO4), filtered and evaporated.
  • Diphenylphosphoryl azide (300 mg) was added and the mixture stirred for a further 8 h. The solvent was removed in vacuo and the residue taken into ethyl acetate, washed with water, 2M hydrochloric acid and 2M sodium bicarbonate, dried (MgSO4), filtered and evaporated and then chromatographed in 5% methanol in dichloromethane to give the desired material as a pale yellow gum (440 mg, 48%).
  • This amide was prepared according to the same procedure as 23 using 1-(3,'5'-bis(fe/ ⁇ -butyldimethylsilyloxy)-2'-deoxyribos-1 '- yl)imidazolidin-2(3/- )-one-4-carboxylic acid (0.9 g, 1.9 mmol), N-(3- aminopropyl) 4-(4-(4-N,N- dimethylaminophenyl)azobenzenesulfonamido)butanamide (0.9 g, 2mmol), triethylamine (0.20 g, 2 mmol) and diphenylphosphoroyl azide (0.61 g, 2.2 mmol) in anhydrous N,N-dimethylformamide (10 ml).
  • This amide was prepared according to the same procedure as 23 using 1-(3,'5'-bis(te/ ⁇ -butyldimethylsilyloxy)-2'-deoxyribos-1 '- yl)imidazolidin-2(3/-/)-one-4-carboxylic acid (710 mg, 1.5 mmol), N-3- aminopropyl 4-(4-N,N-dimethylaminophenyl)azobenzenesulfonamide (361 mg, 1 mmol), triethylamine (101 mg, 1 mmol) and diphenylphosphoroyl azide (303 mg, 1.1 mmol) in anhydrous N,N-dimethylformamide (10 ml).
  • This amide was prepared according to the procedure used for the preparation of 23 using 1-(3,'5'-bis(te/ ⁇ -butyldimethylsilyloxy)-2'- deoxyribos-1 '-yl)imidazolidin-2(3/-/)-one-4-carboxylic acid (710 mg, 1.5 mmol), N-3-(4-(4-N,N-dimethylaminophenyl)azobenzenesulfonamido)propyl 4-aminobutanamide (446 mg, 1 mmol), triethylamine (101 mg, 1 mmol), and diphenylphosphoryl azide (303 mg, 1.1 mmol) in anhydrous N,N- dimethylformamide (10 ml).
  • the product was obtained after chromatography, eluting with 5 % methanol in dichloromethane to give the product as an orange oil. This was taken into dichloromethane (80 ml) and washed with water (2 x 100 ml), dried (MgSO ) and evaporated to give the product as an orange oil (500 mg, 55 %).
  • N-6-aminohexyl 6-(4-(4-N,N- dimethylaminophenyl)azobenzenesulfonamido)hexanamide (580 mg, 1.1 mmol) and 1 -(3,'5'-di(te/t-butyldimethylsilyloxy)-2'-deoxyribos-1 '- yl)imidazolidin-2(3 -/)-one-4-carboxylic acid (800 mg, 1.7 mmol) in anhydrous N,N-dimethylformamide (10 ml) were treated with bromotripyrrolidinephosphonium hexafluorophosphate (800 mg, 1.7 mmol) and triethylamine (222 mg, 2.2 mmol) at room temperature under an atmosphere of argon.
  • This nucleoside was prepared according to the same procedure as 24 using N-(3-(4-(4-(4-(4- dimethylaminophenyl)azobenzenesulfonamido)butanoyl)aminopropyl 1 - (3',5'-di(te/ ⁇ -butyldimethylsilyloxy)-2'-deoxyribos-1'-yl)imidazolidin-2(3/-/)- one-4-carboxamide (0.8 g, 0. 9 mmol) and ammonium fluoride (1.2 g, 32 mmol) in methanol (50 ml).
  • This nucleoside was prepared according to the same procedure as 24 using N-(4-(3-(4-(4-(4-N,N- dimethylaminophenyl)azobenzenesulfonamido)propylamino)-4-oxobutyl 1 - (3',5'-di(te/f-butyldimethylsilyloxy)-2'-deoxyribos-1 '-yl)imidazolidin-2(3/-/)- one-4-carboxamide (500 mg, 0.55 mmol) and ammonium fluoride (200 mg, 11.2 mmol) in methanol (20 ml).
  • N-(3-(4-trifluoroacetamidobutanoyl)aminopropyl) 1 -(2'- deoxyribos-1'-yl)imidazolidin-2(3 -/)-one-4-carboxamide (110 mg, 0.23 mmol) in dry pyridine (2 ml) was treated with 4,4'-dimethoxytrityl chloride (93 mg, 0.27 mmol) in dichloromethane (1 ml) and 4-N,N- dimethylaminopyridine (10 mg). The mixture was stirred at room temperature for 6 hours. Portions of dimethoxytrityl chloride were added until tic (9:1 dichloromethane:methanol) showed that no starting material remained.
  • This compound was prepared according to the procedure used for preparing 25 using N-3-(4-(4-N,N- dimethylaminophenyl)azobenzenesulfonamido)propyl 1 -(2'-deoxyribos-1 '- yl)imidazolidin-2(3H)-4-carboxamide (110 mg, 0.19 mmol) in anhydrous pyridine (2 ml) and 4,4'-dimethoxytrityl chloride (79 mg, 0.23 mmol) in dichloromethane (4 ml). Further aliquots of 4,4'-dimethoxytrityl chloride (20, 16,13 and 13 mg) were added.
  • N-(3-(4-trifluoroacetamidobutanoyl)aminopropyl) 1 -(2'-deoxy- 5'-(4,4'-dimethoxytrityloxy)ribos-r-yl)imidazolidin-2(3/-/)-one-4-carboxamide (180 mg, 0.18 mmol) was dissolved in anhydrous tetrahydrofuran (3 ml) under an atmosphere of argon.
  • This phosphoramidite was prepared according to the same procedure as 26 using N-(3-(4-(4-(4-(4- dimethylaminophenyl)azobenzenesulfonamido)-butanoyl)aminopropyl 1-(2'- deoxy- ⁇ '-(4,4'-dimethoxytrityloxy)ribos-1 '-yl)imidazolidin-2(3 -/)-one-4- carboxamide (138 mg, 0.14 mmol) and (2- cyanoethyl)diisopropylaminochlorophosphoramidite (50 mg, 0.21 mmol) and diisopropylethylamine (54 mg, 0.42 mmol).
  • This phosphoramidite was prepared according to the same procedure as 26 using N-3-(4-(4-N,N- dimethylaminophenyl)azobenzenesulfonamido)propyl 1-(2'-deoxy-5'-(4,4'- dimethoxytrityloxy)ribos-1 '-yl)imidazolidin-2(3/-/)-one-4-carboxamide (80 mg, 0.09 mmol), (2-cyanoethyl)diisopropylaminochlorophosphoramidite (43 mg, 0.18 mmol), and diisopropylethylamine (47 mg, 0.36 mmol).
  • oligonucleotides were prepared, each of the same sequence, differing only in the imidazolidin-2(3H)-one-4-carboxamide phosphoramidite used to synthesise them.
  • Template 4 synthesised using phosphoramidite 29
  • Template 5 synthesised using phosphoramidite 30
  • the phosphoramidites prepared as described above were dissolved in DNA reagent grade acetonitrile supplied by Cruachem to make 1 mmolar solutions.
  • the natural base phosphoramidites were obtained from Amersham Pharmacia Biotech and dissolved in DNA grade acetonitrile according to the manufacturer's instructions immediately before oligonucleotide synthesis.
  • the synthetic phosphoramidites were dissolved in DNA reagent grade acetonitrile to make 1 mmolar solutions with the exception of 29, which was dissolved in 1 :1 tetrahydrofuran:acetonitrile
  • the oligonucleotides were synthesised in three steps on an
  • the first 27 bases were synthesised using the preprogammed 0.2 ⁇ M CE cycle (DMT On) on a 1000A CPG A column from ABI.
  • the imidazolidin-2(3H)-one-4-carboxamide base was added using a manual cycle (DMT On) synthetic phosphoramidite (26) was reacted using this cycle with a coupling time of 6 minutes and the DABSYL labelled analogues (27 - 30) was reacted with a coupling time of 12 minutes.
  • DMT On manual cycle
  • synthetic phosphoramidite (26) was reacted using this cycle with a coupling time of 6 minutes and the DABSYL labelled analogues (27 - 30) was reacted with a coupling time of 12 minutes.
  • the oligonucleotides were cleaved from the CPG support using ammonia and the bases deprotected by heating the ammoniacal solution at 57°C for 18 hours.
  • the crude oligonucleotides were PAGE purified and desalted using a NAP-5 column using water as eluent. Read-through Experiments
  • oligonucleotides described above were used as templates and a 26 mer primer ( ⁇ '-FAM-TGC AGG TCG ACT CTA GAG GAT CCC C-3') (supplied by Oswell)
  • a hybridisation mixture consisting of primer (7 ⁇ l of a 46 ⁇ M solution in water), Template 1 (7 ⁇ l of a 100 ⁇ M solution in water), 5 X KGB buffer (260 mM Tris acetate, 17.5 mM magnesium acetate, 125 mM potassium glutamic acid, 10% glycerol, pH 7.9) (28 ⁇ l) and double distilled water (28 ⁇ l) was heated to 95°C for 10 minutes and cooled slowly to room temperature.
  • a premix of Thermosequenase IITM (20U, 5 ⁇ l), dNTP (5 ⁇ l of an 8 mM solution) and water (40 ⁇ l) was prepared.
  • the hybridisation mixture (50 ⁇ l) and premix were mixed and the reaction mixture incubated at 72°C and 10 ⁇ l samples were taken before incubation and after 30, 60, 90, 120, 150, 180, 210, 240, and 600 seconds.
  • the reaction samples were quenched with EDTA (2 ⁇ l of a 50 mM solution at pH 8).
  • Orange G in 80% formamide (5 ⁇ l) was added and the mixture heated to 95°C for 3 minutes.
  • Primer alone, primer plus template, primer plus template plus enzyme, primer plus enzyme plus dNTPs, template plus enzyme plus dNTPs, primer plus template plus dNTPs controls were also carried out, incubating at 72°C for 300 s.
  • the reactions were analysed on 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluorimager. This showed that full length extended primer was formed in the reaction and therefore that Thermosequenase IITM had read through the base analogue in the template oligonucleotide.
  • a hybridisation mixture consisting of primer (7 ⁇ l of a 46 ⁇ M solution in water), Template 2 (7 ⁇ l of a 100 ⁇ M solution in water), 5 X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate, 125 mM potassium glutamic acid, 10% glycerol, pH 7.9) (28 ⁇ l) and double distilled water (28 ⁇ l) was heated to 95°C for 10 minutes and cooled slowly to room temperature.
  • a premix of Thermosequenase IITM (20U, 5 ⁇ l), dNTP (5 ⁇ l of an 8 mM solution) and water (40 ⁇ l) was prepared.
  • the hybridisation mixture (50 ⁇ l) and premix were mixed and the reaction mixture incubated at 72°C and 10 ⁇ l samples were taken before incubation and after 30, 60, 90, 120, 150, 180, 210, 240, and 600 seconds.
  • the reaction samples were quenched with EDTA (2 ⁇ l of a 50 mM solution at pH 8).
  • Orange G in 80% formamide (5 ⁇ l) was added and the mixture heated to 95°C for 3 minutes.
  • Primer alone, primer plus template, primer plus template plus enzyme, primer plus enzyme plus dNTPs, template plus enzyme plus dNTPs, primer plus template plus dNTPs controls were also carried out, incubating at 72°C for 300 s.
  • the reactions were analysed on 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluorimager. This showed that full length extended primer was formed in the reaction and therefore that Thermosequenase IITM had read through the base analogue in the template oligonucleotide.
  • a hybridisation mixture consisting of primer (7 ⁇ l of a 46 DM solution in water), Template 3 (7 ⁇ l of a 100 ⁇ M solution in water), 5 X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate, 125 mM potassium glutamic acid, 10% glycerol, pH 7.9) (28 ⁇ l) and double distilled water (28 ⁇ l) was heated to 95°C for 10 minutes and cooled slowly to room temperature.
  • the hybridisation mixture (50 ⁇ l) and premix were mixed and the reaction mixture incubated at 72°C and 10 ⁇ l samples were taken before incubation and after 30, 60, 90, 120, 150, 180, 210, 240, and 600 seconds.
  • the reaction samples were quenched with EDTA (2 ⁇ l of a 50 mM solution at pH 8). Orange G in 80% formamide (5 ⁇ l) was added and the mixture heated to 95°C for 3 minutes.
  • Primer alone, primer plus template, primer plus template plus enzyme, primer plus enzyme plus dNTPs, template plus enzyme plus dNTPs, primer plus template plus dNTPs controls were also carried out, incubating at 72°C for 300 s.
  • the reactions were analysed on 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluorimager. This showed that full length extended primer was formed in the reaction and therefore that Thermosequenase IITM had read through the base analogue in the template oligonucleotide.
  • a hybridisation mixture consisting of primer (7 ⁇ l of a 46 ⁇ M solution in water), Template 4 (7 ⁇ l of a 100 ⁇ M solution in water), 5 X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate, 125 mM potassium glutamic acid, 10% glycerol, pH 7.9) (28 ⁇ l) and double distilled water (28 ⁇ l) was heated to 95°C for 10 minutes and cooled slowly to room temperature.
  • the hybridisation mixture (50 ⁇ l) and premix were mixed and the reaction mixture incubated at 72°C and 10 ⁇ l samples were taken before incubation and after 30, 60, 90, 120, 150, 180, 210, 240, and 600 seconds.
  • the reaction samples were quenched with EDTA (2 ⁇ l of a 50 mM solution at pH 8). Orange G in 80% formamide (5 ⁇ l) was added and the mixture heated to 95°C for 3 minutes.
  • Primer alone, primer plus template, primer plus template plus enzyme, primer plus enzyme plus dNTPs, template plus enzyme plus dNTPs, primer plus template plus dNTPs controls were also carried out, incubating at 72°C for 300 s.
  • the reactions were analysed on 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluorimager. This showed that full length extended primer was formed in the reaction and therefore that Thermosequenase IITM had read through the base analogue in the template oligonucleotide.
  • a hybridisation mixture consisting of primer (7 ⁇ l of a 46 ⁇ M solution in water), Template 5 (7 ⁇ l of a 100 ⁇ M solution in water), 5 X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate, 125 mM potassium glutamic acid, 10% glycerol, pH 7.9) (28 ⁇ l) and double distilled water (28 ⁇ l) was heated to 95°C for 10 minutes and cooled slowly to room temperature.
  • a premix of Thermosequenase IITM (20U, 5 ⁇ l), dNTP (5 ⁇ l of an 8 mM solution) and water (40 ⁇ l) was prepared.
  • the hybridisation mixture (50 ⁇ l) and premix were mixed and the reaction mixture incubated at 72°C and 10 ⁇ l samples were taken before incubation and after 30, 60, 90, 120, 150, 180, 210, 240, and 600.
  • the reaction samples were quenched with EDTA (2 ⁇ l of a 50 mM solution at pH 8).
  • Orange G in 80% formamide (5 ⁇ l) was added and the mixture heated to 95°C for 3 minutes.
  • Primer alone, primer plus template, primer plus template plus enzyme, primer plus enzyme plus dNTPs, template plus enzyme plus dNTPs, primer plus template plus dNTPs controls were also carried out, incubating at 72°C for 300 s.
  • the reactions were analysed on 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluorimager. This showed that full length extended primer was formed in the reaction and therefore that Thermosequenase IITM had read through the base analogue in the template oligonucleotide.
  • a hybridisation mixture consisting of primer (7 ⁇ l of a 46 DM solution in water), Template 2 (7 ⁇ l of a 100 DM solution in water), 10 x Thermopol buffer (Biolabs) (21 ⁇ l) and double distilled water (35 ⁇ l) was heated to 95°C for 10 minutes and cooled slowly to room temperature.
  • the hybridisation mixture (50 ⁇ l) and premix were mixed and the reaction mixture incubated at 72°C and 10 ⁇ l samples were taken before incubation and after 30, 60, 90, 120, 150, 180, 210, 240, and 600 seconds.
  • the reaction samples were quenched with EDTA (2 ⁇ l of a 50 mM solution at pH 8). Orange G in 80% formamide (5 ⁇ l) was added and the mixture heated to 95°C for 3 minutes.
  • Primer alone, primer plus template, primer plus template plus enzyme, primer plus enzyme plus dNTPs, template plus enzyme plus dNTPs, primer plus template plus dNTPs controls were also carried out, incubating at 72°C for 300 s.
  • a hybridisation mixture consisting of primer (7 ⁇ l of a 46 ⁇ M solution in water), Template 2 (7 ⁇ l of a 100 ⁇ M solution in water), 5 X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate, 125 mM potassium glutamic acid, 10% glycerol, pH 7.9) (28 ⁇ l) and double distilled water (28 ⁇ l) was heated to 95°C for 10 minutes and cooled slowly to room temperature.
  • a premix of TTS DNA polymerase ( as described in PCT Patent Application No. PCT/US96/20225 ) (20U, 5 ⁇ l), dNTP (5 ⁇ l of an 8 mM solution) and water (40 ⁇ l) was prepared.
  • the hybridisation mixture (50 ⁇ l) and premix were mixed and the reaction mixture incubated at 72°C and 10 ⁇ l samples were taken before incubation and after 30, 60, 90, 120, 150, 180, 210, 240, and 600 seconds.
  • the reaction samples were quenched with EDTA (2 ⁇ l of a 50 mM solution at pH 8).
  • Orange G in 80% formamide (5 ⁇ l) was added and the mixture heated to 95°C for 3 minutes.
  • Primer alone, primer plus template, primer plus template plus enzyme, primer plus enzyme plus dNTPs, template plus enzyme plus dNTPs, primer plus template plus dNTPs controls were also carried out, incubating at 72°C for 300 s.
  • the reactions were analysed on 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluorimager. This showed that full length extended primer was formed in the reaction and therefore that TTS had read through the base analogue in the template oligonucleotide.
  • reaction ii showed product due to the primer being extended by three bases, the remaining three reactions (i, iii, and iv) showed only the addition of two thymidine bases.
  • dATP is a base complement to the imidazolidin-2(3/-/)-one-4- carboxamide base analogue and therefore the analogue behaves as a thymidine analogue.
  • reaction ii showed product due to the primer being extended by three bases, the remaining three reactions (i, iii, and iv) showed only the addition of two thymidine bases.
  • dATP is a base complement to the imidazolidin-2(3H)-one-4- carboxamide base analogue and therefore the analogue behaves as a thymidine analogue.
  • T analogue using the 1 ,2,4-triazol-3-one-5- carboxamides may be achieved using scheme 1.
  • the preparation of compound 3 is known (T J Schwan and R L White, J Heterocycl. Chem., 1975, 771).
  • This compound may be glycosylated with the 1-D-chloro-3,5- ditoluoyl-2-deoxyribose. Removal of the toluoyl protecting groups and reprotection as the silyl ethers, followed by amidation with propylenediamine gives the extended amine (5).
  • the triphosphate may be prepared by protection of the amine as its tert- butyoxycarbamate, removal of the silyl protecting groups and phosphorylation.
  • the phosphoramidite may be prepared by extending the amine with TFA protected 4-aminobutyric acid NHS ester, removal of the silyl protecting groups, dimethoxytritylation and phosphitylation.

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Abstract

L'invention concerne des composés représentés par la structure (I) dans laquelle X est CH ou N, Y est CO-, -CONW-, -O-, -S-, -SO-, -SO2-, -NWCO-, -NW-, ou OCO-, W est identique ou différent à différents endroits de la molécule et chacun est H ou alkyle ou aryle ou Rp ou Ln-Rp, Ln est un groupe de liaison, Rp est un fragment rapporteur, et Q est un sucre ou un analogue de sucre, ou un squelette d'acide nucléique ou un analogue de squelette, à condition qu'au moins un fragment rapporteur soit présent. Ces composés constituent des nucléosides triphosphates qui sont de bons substrats enzymatiques.
EP00958770A 1999-08-31 2000-08-30 Analogues de nucleoside Withdrawn EP1208109A2 (fr)

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US7977108B2 (en) 2005-07-25 2011-07-12 Roche Molecular Systems, Inc. Method for detecting a mutation in a repetitive nucleic acid sequence

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