EP0973788A1 - Analogues a base tricyclique - Google Patents

Analogues a base tricyclique

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Publication number
EP0973788A1
EP0973788A1 EP98913963A EP98913963A EP0973788A1 EP 0973788 A1 EP0973788 A1 EP 0973788A1 EP 98913963 A EP98913963 A EP 98913963A EP 98913963 A EP98913963 A EP 98913963A EP 0973788 A1 EP0973788 A1 EP 0973788A1
Authority
EP
European Patent Office
Prior art keywords
analogue
moiety
nucleoside
triphosphate
reporter
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Withdrawn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
EP98913963A
Other languages
German (de)
English (en)
Inventor
Daniel Brown
David Loakes
David Williams
Fergal Hill
Shiv Kumar
Satyam Nampalli
Mark Mcdougall
Alan Hamilton
Clifford Smith
Adrian Christopher Simmonds
William Jonathan Cummins
Patrick Finn
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 UK Ltd
Original Assignee
Amersham Pharmacia Biotech UK Ltd
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Filing date
Publication date
Application filed by Amersham Pharmacia Biotech UK Ltd filed Critical Amersham Pharmacia Biotech UK Ltd
Priority to EP98913963A priority Critical patent/EP0973788A1/fr
Publication of EP0973788A1 publication Critical patent/EP0973788A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/16—Peri-condensed systems
    • 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
    • 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

  • This invention concerns base analogues which may be used to make nucleoside analogues and nucleotide analogues which may be incorporated into nucleic acids. Some of these analogues are base- specific and may be incorporated into DNA or RNA in the place of a single native base i.e. A, T, G, or C. Other analogues have the potential for base- pairing with more than one native base or base analogue.
  • the present invention provides a compound having the structured
  • W is an alkylene or alkenylene chain of 0 - 5 carbon atoms any of which may carry a substituent R 8
  • X is O or N or NR 12 or CR 10 ,
  • X' is O or S or N, provided that when X' is O or S, X is C, Y is CH or N,
  • R 6 is H or NH 2 or SMe or SO 2 Me or NHNH 2
  • each of R 7 and R 8 is independently H or F or alkyl or alkenyl or aryl or acyl or a reporter moiety
  • each of R 9 and R 12 is independently H or alkyl or alkenyl or aryl or acyl or a reporter moiety
  • Z is O, S, Se, SO, NR 9 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 a reporter moiety,
  • 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 a reporter moiety, 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
  • R 6 H or NH 2
  • R 7 H or reporter moiety.
  • Alkyl, alkenyl, aryl and acyl groups herein preferably contain 1-20 carbon atoms.
  • nucleotide is a naturally occurring compound comprising a base and a sugar backbone including a phosphate.
  • a nucleoside is a corresponding compound in which a backbone phosphate may or may not be present.
  • Nucleotide analogues and nucleoside analogues are analogous compounds having different bases and/or different backbones.
  • a nucleoside analogue is a compound which is capable of forming part of a nucleic acid (DNA or RNA) 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 two or three of the natural bases, e.g. with pyrimidines (T/C) or purines (A/G); or universal, by pairing with each of the natural bases without discrimination; or it may pair with another analogue or itself.
  • the base analogue is linked to a sugar moiety such as ribose or deoxyribose to form a nucleoside analogue.
  • a sugar moiety such as ribose or deoxyribose
  • the nucleoside triphosphate analogues of the invention are capable of being incorporated by enzymatic means into nucleic acid chains.
  • the nucleoside analogue or nucleotide analogue which contains a base analogue as defined is labelled with at least one reporter moiety.
  • a reporter moiety 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 an Iodine isotope. It may be an isotope detectable by mass spectrometry or NMR. It may be a signal moiety e.g.
  • the reporter moiety may comprise a signal moiety and a linker group joining it to the remainder of the molecule, which linker group may be a chain of up to 30 carbon, nitrogen, oxygen and sulphur atoms, rigid or flexible, unsaturated or saturated as well known in the field.
  • the reporter moiety may comprise a solid surface and a linker group joining it to the rest of the molecule.
  • Linkage to a solid surface enables the use of nucleic acid fragments containing nucleoside analogues to be used in assays including bead based probe assays or assays involving arrays of nucleic acid samples or oligonucleotides which are interrogated with e.g. oligonucleotide or nucleic acid probes.
  • the reporter moiety may consist of a linker group with a terminal or other reactive group, e.g. NH 2 , OH, COOH, CONH 2 or SH, by which a signal moiety and/or solid surface may be attached, before or after incorporation of the nucleoside analogue in a nucleic acid chain, before or after hybridisation.
  • Two (or more) reporter groups may be present, e.g. a signal group and a solid surface, or a hapten and a different signal group, or two fluorescent signal groups to act as donor and acceptor.
  • Various formats of these arrangements may be useful for separation purposes.
  • 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 ⁇ 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 addition to one or two present in the base.
  • R 5 is OH, SH, NH 2 or mono-, di- or tri-phosphate or - thiophosphate or corresponding boranophosphate.
  • one of R 2 and R 5 may be a phosphoramidite or H-phosphonate or methylphosphonate or phosphorothioate, 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.
  • phosphoramidites and related derivatives in synthesising oligonucleotides is well known and described in the literature.
  • At least one reporter moiety is preferably present in the base analogue or 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). Reporters in the form of isotopic labels may be introduced into phosphate groups by literature methods (Analytical Biochemistry, 214, 338-340, 1993; WO 95/15395).
  • Nucleoside analogues of this invention are useful for labelling DNA or RNA or for incorporating in oligonucleotides. Some have the possible advantage over conventional hapten labelled nucleotides such as fluorescein-dUTP of being able to replace more than one base.
  • 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. If a reporter moiety of the incorporated nucleoside analogue consists of a linker group, then 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 (as described in Example 13 of WO 97/28177).
  • the analogues when incorporated into oligonucleotides or nucleic acids may be acted upon by nucleic acid modification enzymes such as ligases or restriction endonucleases.
  • Primer walking sequencing a primer/template complex is extended with a polymerase and chain terminated to generate a nested set of fragments where the sequence is read after electrophoresis and detection (radioactive or fluorescent). A second primer is then synthesised using the sequence information near to the end of the sequence obtained from the first primer. This second ("walking") primer is then used for sequencing the same template. Primer walking sequencing is more efficient in terms of generating less redundant sequence information than the alternative "shot gun" approach.
  • the main disadvantage with primer walking is the need to synthesise a primer after each round of sequencing.
  • Cycle sequencing requires primers that have annealing temperatures near to the optimal temperature for the polymerase used for the cycle sequencing. Primers between 18 and 24 residues long are generally used for cycle sequencing. The size of a presynthesised walking primer set required has made primer walking cycle sequencing an impractical proposition.
  • base analogues that are degenerate or universal addresses this problem.
  • the use of such analogues that are also labelled, e.g. the nucleoside analogues of this invention will also help to overcome the problem.
  • Preferred reporters for this purpose are radioactive isotopes or fluorescent groups, such as are used in conventional cycle sequencing reactions. Where the nucleoside analogues are base specific chain terminators they may be used in chain terminating sequencing protocols.
  • the final analysis step in DNA sequencing involves the use of a denaturing polyacryiamide electrophoresis gel to separate the DNA molecules by size. Electrophoretic separation based solely on size requires the complete elimination of secondary structure from the DNA. For most DNA this is typically accomplished by using high concentrations of urea in the polyacryiamide matrix and running the gels at elevated temperatures. However certain sequences, for example those capable of forming "stem loop" structures retain secondary structure and, as a result, display compression artefacts under standard electrophoresis conditions. Here, adjacent bands of the sequence run at nearly the same position on the gel, "compressed" tightly together.
  • Such loops are typically formed when a number of GC pairs are able to interact since GC pairs can form 3 hydrogen bonds compared to the 2 hydrogen bonds of AT pairs.
  • a second form of compression artefact is seen when rhodamine-labelled terminators are used and there is a G residue close to the terminus. In these cases, anomalous mobility of the DNA strand in a gel is often seen, possibly due to an interaction between the dye and the G residue.
  • compression artefacts appear to be caused whenever stable secondary structures exist in the DNA under the conditions prevailing in the gel matrix during electrophoresis.
  • the folded structure runs faster through the gel matrix than an equivalent unfolded DNA.
  • gel compression artefacts are eliminated in one of two ways. One is to change to a stronger denaturing condition for the gel, for example 40% formamide with 7 M urea.
  • the other method is to incorporate a derivative of dGTP during the synthesis of DNA.
  • the first 7-deazadGTP
  • the second nucleotide dITP will remove all sequencing artefacts. It has reduced hydrogen bonding capabilities, so preventing secondary structure being a problem.
  • the downside of this analogue is that it is a very poor DNA polymerase substrate.
  • 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 polyacryiamide or agarose gel based methods or solution hybridization assays and other assays in microtitre plates or tubes or arrays 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.
  • nucleoside analogues of the present invention with the combination of molecular diversity and increased numbers of positions where reporter groups may be added can result in a series of improved enzyme substrates.
  • nucleoside analogue triphosphate into DNA by means of a polymerase but without a reporter label for the purpose of random mutagenesis. It has been shown by Zaccolo et al, 1996, J. Mol. Biol. 255, 589-603 that when nucleotide analogues with ambivalent base pairing potential are incorporated by the PCR into DNA products, they induce the formation of random mutations within the DNA products.
  • nucleotide analogue dPTP was shown to be incorporated into DNA by Taq polymerase in place of TTP and, with lower efficiency, dCTP.
  • RNA is an extremely versatile biological molecule.
  • RNA molecules have been proposed as drug candidates for the treatment of diseases like myasthenia gravis and several other auto-immune diseases.
  • RNA library to the protein or molecule of interest; washing to remove unbound RNA; then specifically eluting the RNA bound to the protein.
  • This eluted 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).
  • 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.
  • This sequence is optimised to produce a short oligonucleotide which shows improved specific binding which may then be used as a therapeutic, or member of a binding pair.
  • the base analogues described here when converted to the ribonucleoside triphosphate or ribonucleoside phosphoramidite, or to the deoxyribonucleoside triphosphate or deoxyribonucleoside 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 using the current building blocks.
  • the secondary structure of nucleic acids is also important when considering ribozyme function.
  • the base analogues of the present invention may cause the formation of secondary structures which would otherwise be unavailable using native bases or other modified nucleotide derivatives.
  • the hybridization binding properties of nucleic acids incorporating base analogues of the present invention may have particular application in the antisense or antigene field.
  • the base analogues of the present invention may have properties which are different to those of the native bases and therefore are particularly suited to other important applications.
  • the interaction of these base analogues with enzymes may be extremely important in vivo and may result in the development of new anti-viral therapeutics.
  • EXAMPLE 1 The synthesis of the novel tricyclic nucleoside analogue 9-(2'- deoxy- ⁇ -D-ribofuranosyl)-pyrrolo [4, 3, 2-de] pyrimido [4, 5 -c] dihydro- oxazepine referred to as dS (1.13) and its 5'-triphosphate referred to as dSTP (1.14) is described.
  • the nucleoside (1.6) (5.5g, ⁇ mmol) was suspended in methanol (50ml) and ammonium fluoride (3g, ⁇ Ommol) added and the solution heated at reflux for 3 hours. The solvent was evaporated and the product worked up as usual to give a foam which was chromatographed (CHCI 3 / 1% MeOH) to give the product as a white solid. Yield 3.98g, 83%.
  • the sulfanyl derivative (1.7) (3.9g, 6.5mmol) was suspended in ethanol (300ml) and to this was added magnesium monoperoxyphthalate (6.5g, 13mmol) in water (100ml) and the solution stirred at 50°C for 4 hours. The solution was then concentrated and worked up as usual to give a white foam which was chromatographed (CHCI 3 / 1% MeOH) to give a white foam. Yield 3.90g, 95%. M.p. 157-158°C.
  • H-NMR spectra were recorded at 250.13 MHz or at 300.13 MHz or on a on a Bruker WM 250 or AM300 spectrometer with tetramethylsilane as the external standard (J values in Hz). D2O was added to ⁇ H NMR samples for the identification of exchangeable protons.
  • UV spectra were obtained using a Perkin Elmer Lambda 2 spectrophotometer, all samples being dissolved in distilled water or analytical methanol (Aldrich). Mass spectra were recorded on a Kratos MS890 instrument. Melting points are uncorrected.
  • Precoated silica gel F254 plates for preparative (1 mm) or thin-layer chromatography (TLC) (Merck) were developed using one of the following solvent systems; A, ethyl acetate: cyclohexane (1 :2); B, MeOH:CHCl3 1 :9; C, MeOH:CHCl3 2:8; D, MeOH:CHCl3 15:85; E, MeOH:CHCl3 2;98; F, MeOH:CHCl3 5:95.
  • Tetrahydrofuranosyl derivatives were identified with anisaldehyde solution which contained anisaldehyde (9.2 ml), acetic acid (3.75 ml), cone. H2SO4 (1.25 ml) and 95% ethanol (388 ml).
  • the synthesis of the analogue of compound (1.7) containing an azido functional group (3.8) to allow introduction of a reporter moiety is described.
  • the azide group can then be reduced using triphenyl phosphine and water to provide the free primary amine (Mag et al, 1989, Nucleic Acids Research 17, 5973-5988).
  • This provides a means of incorporating a reactive group such as NH 2 .
  • the primary amine generated can either be protected with a suitable protecting group such as monomethoxy trityl or reacted with an active ester of a hapten containing molecule such as the N-hydroxysuccinimidyl ester of 2,4-dinitrophenyl acetic acid.
  • the secondary alcohol remaining can then be converted to its hydroxyphthalimide derivative according to the reaction conditions in example 1.9.
  • the formation of the corresponding tricyclic derivative can then be achieved by carrying out the subsequent synthetic steps illustrated in example 1.
  • the tricyclic derivative thus obtained can be hapten derivatised or have a reactive group attached as previously described in the term signal moiety. By having the amine protected the signal moiety can either be added at the triphosphate stage or after the nucleotide has been incorporated into an oligomer.
  • ninhydrin/EtOH solution (254nm), ninhydrin/EtOH solution (2% w/v), KMnO 4 /H 2 S0 4 /H 2 0 (10/1/100 w/v/v) or anisaldehyde solution (anisaldehyde/H 2 SO 4 /EtOH 5/1/50 v/v/v).
  • nucleoside analogue 1-(2- deoxyribofuranosyl)-pyrazolo[3,4-d]pyrimidine[2,3-c]dihydro-oxazepine (4.10) is described.
  • Manipulations to introduce a reactive group as described in example 3 can also be applied to compound (4.5) in this example.
  • Tributylammonium pyrophosphate in DMF 0.5M, 3ml
  • tri-n-butylamine 0.4ml
  • Triethylammonium bicarbonate (1 M, pH8.5, 20ml) was added and the reaction mixture stored at 4°C for 16h. then evaporated to dryness in vacuo.
  • the residue was dissolved in water (15ml) then the product purified by ion exchange chromatography (8 ⁇ M Hipex anion exchange column) eluting with 0-100% triethylammonium bicarbonate (0.3M) over 100min at a flow rate of
  • Each reaction contained 1 pmol of a 24-mer oligonucleotide: 5' GATCTGGTCATAGCTGTTTCCTGT annealed to 0.25pmol of a complementary 20-mer oligonucleotide 5' ACAGGAAACAGCTATGACCA which was labelled at the 5' end with 32 P, and 1 ⁇ l of lOxbuffer (for Taq polymerase: 500mM KCI, 100mM Tris pH 9 at room temperature, 1 % Triton-X 100, 15mM MgCI 2 ; for the other two enzymes: 50mM MgCI 2 , 100mM Tris pH 7.5 at room temperature, 75 mM DTT). Normal dNTPs, in various combinations, were added, as required, to 50 ⁇ M final concentration; dSTP was also used at 50 ⁇ M final concentration.
  • a single unit of the appropriate polymerase was added to start each of the reactions; these were incubated for 15 minutes (at 72°C under mineral oil for Taq, at 37°C for Pol I derivatives) before being terminated by the addition of 5 ⁇ l of termination mix (95% formamide, 20mM EDTA, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol FF).
  • the reactions were heated to 70°C for ten minutes before electrophoresis on a 20% polyacrylamide/7M urea gel, which was then exposed to autoradiography film.
  • dSTP as either dGTP or as dATP, that is opposite T or C in a template, but not opposite A or G, because no extension occurs in any of the assays opposite A or G unless TTP or dCTP are present.
  • a second primer extension assay was used to compare the triphosphate of the pyrrolo compound (1.14) (dSTP) with the triphosphate of the pyrazolo compound (4.11) as substrates for the 3' to 5' exonuclease- free version of Klenow fragment of DNA polymerase I (EFK).
  • the assay used a 33 P 5' end labelled 15mer primer hybridised to one of two different 24mer templates.
  • the sequences of the primer and templates are:
  • One picomole 33 P labelled primer was hybridised to 2 picomoles of template in x2 Klenow buffer. To this was added either 4 ⁇ M dNTP ⁇ S, 20 ⁇ M or 80 ⁇ M analogue triphosphate or a mixture of 4 ⁇ M dNTP ⁇ S and 20 or 80 ⁇ M analogue triphosphate. Two units EFK and 2mU inorganic pyrophosphatase were used per reaction. Primer alone, primer plus template, primer plus template plus enzyme controls were also carried out. The reactions were incubated at 37°C for 3 minutes. Reactions were then stopped by the addition of formamide EDTA stop solution.
  • Reaction products were separated on a 19% polyacryiamide 7M urea gel and the product fragments sized by comparison with a 33 P labelled 8 to 32 base oligonucleotide ladder after exposure to Kodak Biomax autoradiography film.
  • a 15mer primer (sequence: 5' TGC ATG TGC TGG AGA 3') and 8 to 32 base oligonucleotide markers were 5' end labelled with [ ⁇ 33 P] ATP and T4 polynucleotide kinase. Reactions were boiled for 5 minutes after labelling to remove any PNK activity. Four picomoles of the labelled primer, 25 U terminal deoxynucleotidyl transferase and 64 ⁇ M dNTP or analogue triphosphate were incubated in 25 ⁇ l 100mM cacodylate buffer pH7.2, 2mM CoCI 2 and 0.2mM 2-mercaptoethanol for 90 minutes at 37°C. The reactions were stopped by the addition of formamide stop solution and the reaction products run on a 19% polyacryiamide 7M urea gel with the labelled markers. Autoradiography using Biomax film was carried out on the dry gel.
  • dATP gave a tail estimated at greater than 100 bases long and dGTP produced shorter tails in the order of 50 bases.
  • the pyrrolo analogue (dSTP) produced tails intermediate in size between those of dATP and dGTP.
  • the pyrazolo analogue extended the primer predominantly by a single base although there were some +2 and +3 products as well as some unextended primer.
  • reaction master mix 16 ⁇ l Reaction buffer 80 ⁇ l DNA (M13mp18 0.2 ⁇ g/ ⁇ l
  • the dSTP "A" mix gave a sequence (>250 bases) with similar even band intensities.
  • the sequence was longer than that obtained with both dITP ( ⁇ 200 bases) and 8-oxo - dGTP.
  • the 8 - oxo dGTP sequence only extended 15-20 bases from the primer.
  • the dITP sequence had uneven band intensities especially in the G track.
  • dSTP is a very good polymerase substrate and that a G equivalent could be useful in removing compression artefacts from sequencing reactions by replacing the 7 - deaza - dGTP or dITP analogues currently used.

Abstract

L'invention a trait à des analogues de nucléosides ayant la structure (2) dans laquelle Q représente H ou une fraction glucidique ou un analogue glucidique ou un glucide modifié ou un squelette d'acide nucléique ou un analogue de ce squelette; W représente un alkilène ou une chaîne alcénylène contenant de 0 à 5 atomes de carbone, n'importe lequel d'entre eux pouvant porter un substituant R8; X représente O ou N ou NR?12 ou CR10¿; X' représente O ou S ou N, à condition que lorsque X' représente O ou S, X représente alors C; Y représente CH ou N; R6 représente H ou NH¿2? ou SMe ou SO2Me ou NHNH2; chaque R?7 et R8¿ représente indépendamment H ou F ou un alkyle ou un alcène ou un aryle ou un acyle ou une fraction marqueur; R12 représente indépendamment H ou un alkyle ou un alcényle ou un aryle ou un acyle ou une fraction marqueur; et R10 représente H ou =O ou F ou un alkyle ou un aryle ou une fraction marqueur.
EP98913963A 1997-04-02 1998-04-02 Analogues a base tricyclique Withdrawn EP0973788A1 (fr)

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EP97302265 1997-04-02
EP97302265 1997-04-02
EP98913963A EP0973788A1 (fr) 1997-04-02 1998-04-02 Analogues a base tricyclique
PCT/GB1998/000978 WO1998043991A1 (fr) 1997-04-02 1998-04-02 Analogues a base tricyclique

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