Method of obtaining 3'-(2')-amino- or thiolo-modified nucleosides, nucleotides and oligonucleotides coupled with a fluorescent dye

The present invention relates to a process for producing 3'-amino or thiolomodified fluorescent dye-linked nucleosides, nucleotides and oligonucleotides.

custom many uses in gene technology,

Marked oligonucleotides have been found because manipulation is easier than manipulation of DNA probes traditionally used as hybridization assays, which are produced by restriction digestion from native gene material.

Labeled oligonucleotides, which are used in the form of so-called DNS antisense oligonucleotides, can regulate cellular action and therefore become increasingly important, e.g. for in vivo testing of protein expression. The mechanism is presently recognized by the interaction of DNA-DNA, DNA-RNA and RNA-RNA, but it is not explained in detail yet.

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The tagged oligonucleotides are used in vitro, e.g., to identify gene bank gene fragments, whereby a tagged oligonucleotide is probed and blotted gene bank gene samples are identified.

To be able to perform such in vitro or in vivo tests, oligonucleotides must be labeled as already mentioned. In addition to radioactive labeling by appropriate isotopes, derivatives of fluorescent dyes are already used as a form of non-radioactive labeling , which make it easier and safer to manipulate.

Until now, this technique has also been used successfully for non-radioactive DNA sequencing. Extensions based essentially on the Sanger method were carried out here (F. Sanger, S. Nicklen and S. Coulson, Proc. Natl. Acad. Sci. USA 74, 5463 (1977)).

The fluorescent label is located either at the 5 'end of the oligonucleotide (LE Hood, L.

M. Smith and C. Heiner. Nature 321, 674 (1986)), or at the nucleobase (JM Prober, GL Trainor and RJ Dam, Science 238, 336 (1987)) (GL Trainor, Anal. Chem. 62, 418 (1990)). The great disadvantage of the method cited at the end is that the fluorescent label is introduced during synthesis, i.e. during polymerization and here especially during enzymatic polymerization. The consequence of this process step is that only some polymerases can be used for synthesis, that the acceptance of triphosphates by polymerases decreases and that a strong excess of substrate is also needed.

The introduction of a fluorescent tag, however, is not limited to Sanger sequencing. Chemical sequencing by Maxam-Gilbegt fluorescence label is also known (H. Voss, C. Schwager, U. Wirkner, B. Sproat J. Zimmermann, A. Rosenthal, H. Erfle, J. Stegemann and W. Ansorge , Humming. Acids

Res. 17, 2517 (1989)).

Similarly to this, the transfer of restriction fragments by means of fluorescent detectors is also described (AV Carrano, J. Lamerdin, LK Ashworth, B. Watkins, E. Branscomb, T. Slezak, M. Raff, PJ de Jong, D. Keith, L. McBride, S. Meister, M. Kronic, Genomics, 129 (1989) and S. Brenner and KJ Livak, Proc. Natl. Acad. Sci, USA 86.8902 (1989)).

It has now been found that a fluorescent dye can be attached via an amino or thiol group at the 3 'position (at the α or β site) of a nucleoside, nucleotide or oligonucleotide and this compound can be advantageously used for the synthesis of opposing strands in the presence of a matrix or oligonucleotide strand and for detection genetic material in vivo and in vitro.

The subject of the invention is therefore a process for the preparation of a compound of formula 1, wherein R1 is a purine or pyrimidine base, R³ is an fluorescent dye attached to the amino or thiol group in the a or (3, R position)^from is a hydrogen atom, a hydroxyl group, a protected hydroxyl or methoxy group in the a or β position, n is the number> 0, R is a 5 'protecting group or phosphate, pyrophosphate, triphosphate, R⁵ is oxygen, fluoromethylene, difluoromethylene or methylene, R⁶ is a hydroxyl or methoxy group or a hydrogen atom in the a or β position, wherein R \ R5 and R6 may each have the same or different meanings, Y and X are oxygen, sulfur, NH or methylene, whereby X and Y may be identical or different, which method is characterized in that the 3 'OH group of the nucleoside, nucleotide or oligonucleotide is converted to an amine or thiol derivative and then coupled to a fluorescent dye.

The invention is described below in detail, especially in its preferred ones. embodiments, the invention is further defined by the content of the claims.

Compounds of general formula I are synthesized essentially according to methods known in the literature (M. Gait, Oligonucleotide Synthesis, JRL-Press, Oxford 1984).

To attach the dye to the 3 'position, you start with a compound of formula II, especially a nucleoside. A compound of formula II in which R is used for attachment¹ to r6, X, Y and n have the meanings given, the substituents being optionally the same or different and R⁷ or R⁸ represents a hydrogen atom, a hydroxyl group, a protected hydroxyl group or methoxy group. Attachment of the dye is carried out by introducing the amine or thiol through the 3 'hydroxyl group.

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To introduce the azido group into the 3 'position, a leaving group is introduced. The leaving group is usually triflate (trifluoromethanesulfonic acid), mesylate or tosylate. By nucleophilic action with azide, usually lithium azide, the introduction of azide azide occurs. Nucleophilic attack of thiolate or S-protected thiolate results in thiol or protected thiol.

The desired stereochemistry at the sugar portion of the nucleoside can best be obtained by SN substitutions

The amino group can be obtained straight through the azido group and then reduced to the amine (lit .: WS Mungall and RL Letsinger, J. Org. Chem., Vol. 40, No. 11, 1659 (1975). At this stage, the Staudinger reaction stands out. with triphenylphosphine and water (liters: M. May and JW Engels, Nuci. Acids Res. 17, 15, 5973 (1989)).

All compounds regarding their structure and configuration were determined by NMR, elemental analysis, UV, IR etc.

At the end of polycondensation, the free 3'-amino or thiol group of the sugar portion at the 3 'end of DNA can react with the reactive fluorescent dye according to methods known in the literature (Kunkapiller, Nuci. Acids, Res. 13, 2399, 1985; Rodges RR et al. Biochemistry, Vol. 28, 261 (1989).

Alternatively, the amino group is also obtained by the Mitsunobu reaction (Synthesie, vol. 11981, page 1) and suitable for the reaction described by Yamamoto and colleagues (J. Chem. Soc. Perk. Trans. I. 1, 306, 1980).

Attachment of a fluorescent dye via a thiol group occurs in an analogous manner and by an analogous route. Instead of the azido group, however, the thiol group is introduced in protected or unprotected form.

Attachment of a fluorescent dye to a free amino group or thiol function of a nucleoside, nucleotide or oligonucleotide may alternatively only take place after their application. For example, it is possible to proceed in such a way that the fluorescent dye reaction is carried out after the termination step of the DNA or RNA sequencing reaction, the entire extender being labeled with a fluorescent dye.

As fluorescent dyes, essentially all commercially available dyes that react with an amino or thiol group, usually fluorescein, rhodamine, Texas red, NBD (4-fluoro-7-nitrobenzofurazan Sigma), coumarin, fluorescamin, succinylfluorescin and dansyl (1- (dimethylamino) -naphthalene-5-sulfonyl).

The derivatized reaction mixture can be separated by gel electrophoresis and detected by photometry or laser spectroscopy (H. Swerdlow and R. Gesterland, Nuci. Acids Res. 18. page 1415 (1990)). Capillary electrophoresis (AS Cohen et al., PNAS US 85, 9660 (1988)), e.g. filled with acrylamide gels, has also proved to be well suited for use. Detection then usually occurs at the capillary outlet (H. Swerdlow, S. Wu, H. Harke, N. Dovichi, Chromatography 516, 61 (1990)).

Starting from the compound of formula 1, which has a triphosphate at the 5 'position, using any primer and strand-matrix, by means of a polymerase, i.e. an enzyme which in the presence of suitable substrates synthesizes a faithful, complementary copy of the sequence, can be synthesized in the presence of four nucleoside triphosphates double-stranded DNA, usually with T7-or Taqpolymerase, DNA-polymerase I and reverse transcriptase. Triphenylmethyl, methoxy- or dimethoxytriphenyl-pipetines serve as protecting groups in the 5 'position! (Gait, Oligonucleotide Synthesis, JRL-Press, Oxford 1984).

Synthesis termination can be specifically determined each time by using the 3'-aminomodified nucleotide A, C, G, T of formula 1. This is of particular importance for the synthesis of opposing DNA strands in the presence of the template strand, and thus also for DNA strand sequencing because the use of modified nucleotide provides very specific alkaline reaction termination.

The synthesis of RNA-nucleosides, nucleotides and oligonucleotides occurs in an analogous manner and by analogous route.

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In addition, it is possible to synthesize derivatizable oligonucleotides using a primer nucleotide that has been modified with an amino or thio at the 3 'end and fixed on an oligomeric support.

As oligonucleotides, all DNA and RNA nucleotides produced in the traditional sense are important, but usually 2 to 100, particularly preferably 12 to 50 nucleotides (chemical synthesis) or up to about 3000 nucleotides (enzymatic synthesis), depending on the effectiveness of the polymerase used.

Oligonucleotide synthesis occurs starting from the primer-carrier nucleotide complex in the traditional sense, i.e. in the 3 'and 5' direction, and allows the oligonucleotide to be synthesized with a specific sequence.

Fixation of the start nucleoside on commercially available carriers occurs via the compound arm (Spacer), which can be cleaved after synthesis; for example, by a combination of succinic acid known from the literature or by combination with urethane (Efimov et al., Nuci. acids. Res. 11, 8369.1983).

After synthesis, the oligonucleotide must be cleaved from the support with appropriate reagents. Then followed directly derivatization with any fluorescent dye.

The 3'-oligonucleotides so synthesized and modified at the end can then be used to detect e.g. complementary oligonucleotides.

The method according to the invention combines two advantages, namely firstly, when producing a marked opposite strand, the strand is terminated at the same time by a 3'-amino terminus or a thiol-modified nucleotide. Safe labeling is also possible by labeling with its fluorescent dye in the 3 'position. The second advantage is that in the production of precisely defined oligonucleotides by labeling the attached to the carrier and also the 3'-amino- or thiolomodifiable primer nucleotide, accurate oligonucleotide synthesis combined with the possibility of fluorescent labeling and thus detection is possible.

The examples below serve to further explain the invention.

Examples

1. Anomeric 3'-amino- or -azido-nucleoside-5'-triphosphates

Example 1 Preparation of 5'-triphosphate-3'-amino-3'-deoxyribosidothymidine according to the scheme 1,5, 5'-monophosphate-3'-azido-3'-deoxyribosidothymidine.

160 mg (0.6 mmol) 3'azidothymidine (Sigma) is dissolved in 10 ml triethyl phosphate while stirring. 0.2 ml of POCl3 in 6 ml of triethyl phosphate are added to the solution through a dropping funnel at 4 ° C. After 24 hours, it is neutralized with 50 ml saturated NaHCO3 solution and shaken twice with 60 ml toluene and 100 ml ter. The aqueous phase is diluted to 500 ml with distilled water and applied to an anionite column (®Sephadex A-25; 50 x 2.5 cm) which by passing 200 ml of 0.2 M TBK buffer (triethylammonium bicarbonate buffer; pH 7.5 is adjusted itself) is charged with HCO3 as a counterion. It is then purified by passing 300 ml of distilled water and eluted first with 200 ml 0.1 M TBK buffer and then with a linear gradient of 0.1 - 0.2 M TBK buffer (total gradient volume 1350 ml). The peak, product peak is shown in the chromatogram. The product eluted at a buffer concentration of 0.12 to 0.15 M TBK buffer. 20 ml DC positive fractions are combined and concentrated by lyophilization. Triethylammonium bicarbonate is removed by repeated addition of ethanol. The product appears as a white, crystalline trletylammonium salt. Yield: 220 mg (56.2%); molecular weight: 651.81; Rf (ammonia: isopropanol: water = 10:70:20): 0.40 300 MHz -'H-NMR (D₂O): 1.36 (t, 3H, CH 3); 1.90 (s, 3H, CH 3); 2.50 (m, 4H, 2 ', 2 "-H); 3.18-3.30 (dd, 2H, CH2); 4.10 (m, 1H, 4'-H); 4.3 (m, 1H, 3'-H); 6.31 (t, 1H, 1'-H); 7.7 (s, 1H, 6-H); 11.50 (s, 1H, NH) 300MHz-3¹P-NMR (85% H₃P04extem, D₂0): 1.23 (s, 1P).

'- triphosphate-3' -azido-3 '-deoxyribosidimothymidine

130 mg (0.2 mmol) 5'-monophosphate-3'-azido-3 ', 2'-deoxyribosidothymine as the triethylammonium salt is dissolved in 6 ml of absolute DMF (dimethylformamide) and 162.15 mg is added to the solution at 25 ° C (1 mmol) N ', N'-carbonyldiimidazole in 3 ml absolute6

168 874 ne DMF while mixing. After a reaction time of 2 hours, 1 ml of absolute methanol is added, stirred for a further 15 minutes and the methanol is distilled off. The residue is mixed with 5 ml of a 0.2 M solution of tri-n-butylammonium pyrophosphate in DMF and stirred overnight at room temperature. The precipitate formed from imidazole pyrophosphate is filtered off, washed with 20 ml DMF and the filtrate is concentrated on a rotary evaporator. The residue is dissolved in 250 ml of 0.1 M TBK buffer and the solution is subjected to an HCO3 'loaded Sephadex A-25 anion exchange column. The product is purified by passing 200 ml distilled water and eluting with a linear gradient of 0 to 0.5 M TBK buffer (total gradient volume 2000 ml). Two peaks appear in the chromatogram, the first of which is produced by monophosphate and the second by the product. The product eluted at a buffer concentration of 0.30 to 0.35 M TBK buffer. The positive 20 ml fractions are combined and concentrated by lyophilization. Triethylammonium bicarbonate is removed by repeated addition of ethanol. The product appears as a white, crystalline triethylammonium salt. Yield: 92 mg (50.4%): molecular weight: 911.95; Rf (ammonia: isopropanol: water = 10:70:20): 0.14 300 MHz-T-NMR (85% H3PO4 extem, D20): -10.74 (d,²Jpp = 22.1 Hz, 1P, alpha-P-atom): -11.45 (d, 2jpp = 21.9 Hz, 1P, gamma-P-atom); -22.99 (t,³Jpp = 20.3 Hz, 1P, beta-P-atom).

3'-triphosphate-3 '-amino-3' -deoxyriboside-thymidine mg (0.075 mmol) 5'-triphosphate-3'-azido-3 ', 2'-deoxyribosidothymine is dissolved in 5 ml of dioxane-distilled water (2: 1 ) and 200 mg (0.75 mmol) triphenylphosphine are added under stirring at room temperature. The reaction mixture is stirred at 25 ° C for 30 hours and then the solvent is removed. The residue is dissolved in 30 ml distilled water and extracted three times with 30 ml ether each. The aqueous phase is diluted to 150 ml and the solution is applied to a Sephadex A-25 anion exchange column loaded with HCO3 '. The product is purified by passing 200 ml distilled water and eluting with a linear gradient of 0 to 0.5 M TBK buffer (gradient volume 2000 ml). The chromatogram shows three peaks, the first of which produces monophosphate compound 6, the second the product peak, and the third shows the education peak 7. The product eluted at a buffer concentration of 0.40 to 0.42 M TBK buffer. Positive 25 ml fractions are combined and concentrated by lyophilization. Triethylammonium bicarbonate is removed by adding ethanol. The product appears as a white-amorphous triethylammonium salt. Yield: 57 mg (85.7%); molecular weight: 885.90; Rf (ammonia: isopropanol: water = 10:70:20): 0.08; 300 MHz¹H-NMR (D2O): 1.30 (t, 3H, CH3); 1.92 (s, 3H, CH 3); 2.64 (m, 4H, 2 ', 2 "-H); 3.19-3.21 (dd, 2H, CH2); 4.26 (m, 1H, 4'-H): 4.41 (m, 1H, 3'-H); 6.34 (t, 3j_hh= 6.74 Hz, 1H, 1'-H); 7.69 (s, 1H, 6-H); 11.50 (s, 1H, NH). 300 MHz ^ P-NMR (85% H3PO4 extem, D20); -10.75 (d, 2jpp = 22 Hz, 1P; alpha-P-atom); -11.45 (d, 2jpp = 21.9 Hz, 1P, gamma-P-atom); -22.05 (t, 3jpp = 20.4 Hz, 1P, beta-P-atom).

Example II Preparation of 1- (3'-amine-2 ', 3'-dideoxy-5'-triphosphate-3-D-treopentofuranosyl) -thymine according to scheme 2- 1- (3'-azido-2', 3'-dideoxy -5 '0-dimetoksytrifenylometyIo-3-D-treopentofuranozylo) -tymina

To a solution of 5.98 g (11 mmol) of 5'-dimethoxytriphenylmethylthymidine (for preparation see M. Gait, Oligonucleotide synthesis, IRL Press (1984), page 27), triphenylphosphine (3.076 g, 11.73 mmol) and 3.1 g ( 55 mmol) lithium azide in 37 ml dry DMF are added with 4.10 g (11.91 mmol) of carbon tetrabromide while stirring. The mixture is stirred for 56 hours at room temperature and then 15 ml of methanol are added. The product is then precipitated in 800 ml ice-cold distilled water. The precipitate is taken up in chloroform and purified by flash chromatography (ethyl acetate: n-hexane = 3: 1).

Yield: 4.95 g (80%); Rf (chloroform: methanol = 9: 1): 0.52 IR (cm '¹): 2100 azide.

- (3 '-amino-2', 3'-dideoxy-5'-0-dimethoxytriphenylmethyl-eD-treopentofuranoxyl) -thymine

To a stirred solution of 0.5 g (0.8 mmol) 1- (3'-azido-2 ', 3-dideoxy-5'-O-dimethoxytriphenylmethyl-eD-tropentofuranosyl) -thymine is added 0.15 g triphenylphosphine (1 , 3 g, 4.94 mmol) and leave to react for 4 hours. To hydrolyze phosphinoimine, 3 ml of distilled water are added and mixed for a further 3 hours. The oil remaining after removal of the solvent on the rotary evaporator is purified by flash chromatography (chloroform: methanol = 99: 1 + 1% triethylamine). Rt value in the same eluent = 0.27. Through

168 874 treatment with ninhydrin in thin layer chromatography (DC) can be violet colored. Yield: 0.42 g (87.5%). The product shows an appropriate 'H-NMR spectrum.

Reaction to 1- (3 '-amino-2', 3 '-dideoxy-5'-triphosphate-3-D-treopentofuranosyl) -thymine

For further reaction to 5-triphosphate, analogous to that of M. Gait and Oligonuclideide synthesis, IRL Press, page 49 (1984), the dimethoxytriphenylmethyl group is removed and the triphosphate attachment to the 5 'position is carried out, as mentioned in the example AND.

Example III. Preparation of 3'-amino-2 ', 3'-dideoxy-5'-adenosine triphosphate according to scheme 4.

N-benzoyl ^ - ^ - ^ O-benzoyl-deoxy-pD-treopentofuranozylohadenina.

920 mg (2 mmol) suspension N.⁶, 5'-0-dibenzoyl-2'-deoxyadenosine (prepared by the method described by Nishino et al., Nucleosides & Nucleotides 1986, 5, 159) in 30 ml absolute dichloromethane (containing 2 ml pyridine) is cooled to -30 ° C and 5 ml of a solution of trifluoromethanesulfonic anhydride in dichloromethane (10% by volume) are slowly added dropwise. After removing the cooling bath, the solution is allowed to warm and 1 ml of water is added to it. After 3 hours, a further 5 ml of water are added and the organic phase is washed. After removal of the solvent, the residue obtained is dissolved in 50 ml of methanol and 100 mg of sodium bicarbonate are added. The mixture is stirred for a further 2 hours at room temperature, neutralized with 10% acetic acid, the solvent is distilled off and purified by flash chromatography on silica gel (chloroform: methanol = 97: 3).

Yield: 710 mg (1.48 mmol) 75%.

Rf value (chloroform: methanol = 97: 3): 0.49.

MS = 459 '-azido-2', 3 '-dideoxyadenosine

920 mg (2 mmol) solution N⁶-benzoyl-9- (5-0-bfenzoyl-2-deoxy-β-D-tteopentofuranosyl) -adenine in 20 ml of absolute dichloromethane (and 2 ml of pyridine) is cooled to -30 ° C. Then 5 ml (3 mmol) of a solution of trifluoromethanesulfonic anhydride in absolute dichloromethane are added dropwise. The cooling bath is removed, stirred for a further 20 minutes and 980 mg (20 mmol) lithium azide in 20 ml DMF is added to the solution. After stirring for a further 2 hours at room temperature, 50 ml of water and 150 ml of chloroform are added, the organic phase is shaken and washed with distilled water. The solvent is removed on a rotary evaporator (®Rotyvapor) and treated with ammonia methanol overnight to remove the basic protecting group. After again removal of the solvent, purification is carried out by flash chromatography (chloroform: methanol = 95: 5) on silica gel.

Yield: 430 mg (1.6 mmol, 79%) of a white crystalline powder.

IR: 2100 cm¹ azido group '-amino-2', 3 '-dideoxy-5'-adenosine triphosphate

Preparation of the 5'-triphosphate is carried out in analogy to Example 1. Then the azido group is reduced to an amino group as in Example II.

Example IV Preparation of 3'-amino-2 ', 3'-dideoxy-5'-triphosphate guanosine according to scheme 5.

The production of 3'-amino-2 ', 3'-dideoxy-5'-triphosphate-guanosine is carried out starting from N²-isobutyty-5'-O-bfenzoyl-2'-deoxy guanosine, which can be produced by the method of Nishino et al. (Nucleosides & Nucleotides 5, 159, 1986). The reaction of the nucleoside to the 3'-azide occurs analogously to the production given in the example m. It should be noted that treatment with an ammoniacal methanol solution does not take place, since the removal of the isobutyryl protecting group occurs only after the introduction of the triphosphate group and before the reduction of azide into the amine. The reaction to triphosphate also occurs in the case of guanosine according to the method mentioned in Example 1. The reduction to amine is described in Example II.

2. Preparation of 3'-amino-oligomers and subsequent 3'-fluorescent labeling.

Example V. Synthesis of oligomer from 20 nucleotides 3'-H2N-TTTTTTTTTTTTTTTTTTTT-5 'occurs starting from 3'amino-3'-deoxy-thymidine protected with 5'-dimethoxytriphenylmethyl. The preparation of this compound proceeds according to the description of Example I.

168 874 from 5'-dimethoxytriphenylmethyl protected AZT (azido-3'-deoxyribosidothymidine), which can be prepared from AZT and dimethoxytriphenylmethyl chloride according to the M. Gait method (Oligonucleotide Synthesis, IRL Press, page 27).

The compound is then reduced, as described in Example 1, with triphenylphosphine. The next step is to make the carrier. To this end, 200 mg of 5'-0- (4,4'-dimethoxytriphenylmethyl) -3'amino-3-deoxy-thymidine is placed in 700 (il of absolute pyridine. To this solution is added 45 mg of DMAP (dimethylaminopyridine) and then 40 mg succinic anhydride After standing overnight, the remaining succinic anhydride is hydrolysed by adding 10 g of water. It is co-evaporated three times with toluene and the residue is taken up in 12 ml of methylene chloride, washed with 4 ml of cold citric acid (10%) and twice with 4 ml of water. The organic phase is dried over sodium sulfate, the solvent volume is concentrated and the substance is dissolved in 1 ml of methylene chloride. This solution is slowly added dropwise to 30 ml of n-hexane while stirring. The precipitate is filtered off with suction and dried at 40 ° C under reduced pressure using an oil pump. Further production of the CPG-based carrier follows standardized protocols, and a standard cycle (ABI 380A User Bulletin, Issue No. 36, July 1986) on the AB1 380 A DNA synthesizer according to the phosphoramidate method is used to produce the 12 nuccotide oligomer. After removal of the protecting group and cleavage of the 3'-aminooligonucleotide from the support according to the standard, it is purified and characterized. Better results can be obtained by more base-friendly coupling methods for attachment to the support. Substitution of the cleavable acid amide by urethane function is preferred, as described in Efimov (Nucleic Acid Res. 11, 8369, 1983).

Example VI. Synthesis of oligomer from 23 nucleotides with a 3 '-amino 3'-H2N-ACACCCAATTCTGAAAATGGAT-5' end is carried out as described in Example 1. Purification and characterization is carried out by standard methods.

Example VII. The subsequent formation of oligomer derivatives at the 3'-amino terminal occurs using fluorescein isothiocyanate. In an Eppendorf 50 vessel, nmol of the 3'-aminooligonucleotide is dissolved in 25 g of a 500 mM sodium bicarbonate solution. 20 g of a 300 mM FITC solution (Sigma) in DMSO is added. After a reaction time of 6 hours at room temperature, the reaction mixture is purified by passing in a 20 mM ammonium acetate solution through a Sephadex G-25.3'-FITC-oligomer column, analyzed by analytical HPLC by both fluorescein detection and UV detection. HPLC results were also verified by analyzes on capillary gel electrophoresis (Dionex company). Fluorescein isothiocyanate is represented by the formula 3.

Example VIII. Both the 3'-aminooligomers of Examples 1 and 2 are reacted by the method described in Example 3 with a rhodamine isothiocyanate of formula 4 and tetramethylorodamine of formula 5. 3'-fluorescence labeled oligomers are analyzed by analytical HPLC by both fluorescein detection and detection UV. HPLC results were also verified by analyzes on capillary gel electrophoresis (Dionex company).

Example IX. Both the 3'-aminooligomers of Examples 1 and 2 are reacted by the method described in Example 3 with a reconstituted coumarin derivative. The 3'-fluorescently labeled oligomer is analyzed by analytical HPLC by both fluorescein detection and UV detection. HPLC results were also verified by analyzes on capillary gel electrophoresis (Dionex company). The coumarin dye molecule is represented by the formula 6.

3. Structure of modified nucleotides by DNA polymerases and analysis of sequencing experiments.

To re-examine the acceptance of aminotriphosphates by available sequencing enzymes, appropriate termination solutions were prepared and polymerases tested (Klenow, Boehringer; Taq, Amearsham; T7, Pharmacia; Sequenaste®, USB). The stages of construction and termination properties of triphosphates were analyzed both classic and fluorescent dyeing.

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Example X. Prepared according to Example 1, 5'-triphosphate-3'-amino-3'-deoxyriboside-thymidine was replaced with 2 ', 3-dideoxy-5'-triphosphate-thymidine. This occurred with termination solutions for all the polymerases listed above. Each one in% / y> enterprise / in the second half of the mill / p H71 Amprinkrnn in 7Q and 7th

X * »> - *> Background V VT JVV» V »» <* A «K * Λ- \ -f» ▼ Λ Λ VAV ·· S ··. ** «Αηρ Α —-Α. ** ΑΑΑΑ V. >_Λ ΑΑΑΆΜ V Α »Α VW Λ. V Λ. V ΑΧΑ kŁAXV AA AAAA «^ J WMW dNTP relations / terminator. Sequencing was carried out according to standardized protocols. In these studies, detection occurred autoradiographically by embedding alpha35S-dATP. It turns out that a) the 3'-amino-nuldeotide terminates the enzymes used, b) the construction steps in the case of T7, Taq and sequencase are identical to ordinary dideoxy terminators and c) finally, the aminonucleotide can be sequenced without any problems.

Example XI. The 5'-triphosphate-3'-amino-3'-deoxyriboside-thymidine compound was reacted with fluorescein isothiocyanate to 5'-triphosphate-3'-amino-3'-deoxyribosidate-3'-N-fluoresceinisothiocyanato-thymidine.

2.5 mg (2.8 gmol) 5'-triphosphate-3'-amino-3 ', 2'-deoxyribosidothymine is dissolved in an Eppendorf vessel in 200 g of distilled water and 200 μ1 1 M Na2CC ^ 3 / NaHCC ^ buffers are added 3 (pH 9). The reaction mixture is protected from daylight by an aluminum foil sheath and 80 ml of fluorescein isothiocyanate solution (10 mg in 1 ml DMF) are added using a 100 μΐ pipette. After a reaction time of 10 minutes at room temperature, a quantitative reaction of the educate is found according to DC. Purification is carried out by gel chromatography. The Sephadex G-10 column is protected from light by means of an aluminum foil cover, then the entire reaction mixture is applied to the column material and eluted with a water jet of 1 ml / minute. The chromatogram shows two peaks. The first peak is generated by the product and the second by unreacted dye. The fraction size is 5 ml, in total 24 fractions are obtained. Based on the chromatogram, 4 fractions turn out to be positive. This confirms DC. The solvent is removed by lyophilization and the substance is stored at -80 ° C.

Yield: 3.32 mg (93%); molecular weight: 1,274,32;

Rf value (ammonia: isopropanol: water = 10:70:60): 0.62;

Fluorescent emission spectrum: the wavelength of the radiated light is 420 nm. The maximum emission of the compound is 514.8 nm. A 2.5 mM solution in distilled water was measured. The maximum emission of the 2.5 mM dye solution not derivatized in distilled water is at a wavelength of 519.4 nm.

The resulting 5'-triphosphorane-3'-amino-3'-deoxyriboside-3'-Ń-fluoresceinoisothiocyanato-thymide was replaced with 2 ', 3'-dideoxoxy-5'-triphosphate-thymidine. This occurred in termination solutions of all the polymerases listed above. Each time equimolar, ten times larger and ten times smaller dNTP / terminator ratios were tried. Sequencing was carried out according to standardized protocols. In these studies, the detection was done audioradiographically by embedding alpha ^ S-dATP. It turns out that a) a 3'-fluorescently-labeled nucleotide terminates the enzymes used, b) stages of construction in the case of T7, Taq and sequenase on the basis of a sterically demanding dye residue at ten times higher concentrations are identical to ordinary dideoxy-terminators and c) 3'-fluorescently labeled the terminator can be used for trouble-free sequencing.

Example XII. The resulting 5 '-triUosUoranf-3' -amino-3 'deoxyribosidium-3'-N-fluoresceinisothiocyanato-thymidine was exchanged for 2', 3'-dideoxy-5'-triUosUorar ^ o -> midine. This occurred with termination solutions of T7 and Taq polymerases. The basis was dNTP / terminator ten times higher. Sequencing was carried out according to standardized protocols. For these studies, alpha ^ S-dATP was discontinued and sequence analysis was successfully performed with a 3'-fluorescent labeled terminator on a commercially available DNA sequencing device.

Example XIII. Preparation of 5'-triphosphate-3'-thio-3'-deoxyribosidothymidine according to scheme 6.

5'-0-dimetoksytrifenylometylo-3'-S-benzoyl-tiotymidyna

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The introduction of sulfur to the 3 'position takes place by substitution of the starting group (messyate) with sodium thiobenzoate.

The preparation of sodium thiobenzoate is carried out by adding 10 M NaOH to an ice-cold solution of thiobenzoic acid (10 g) in 15 ml water until the solution becomes alkaline. The pH is then adjusted to 7 with aqueous thiobenzoic acid, cooled to -5 ° C and the solution filtered to remove solid residues. After removing the water on a rotary evaporator with ethanol, the yellow salt is dried under reduced pressure over phosphorus pentoxide.

A solution of 5 '-0-dimethoxytriphenylmethyl-3' -0-methanesulfonyl-2'-desoxyxyl-thymidine (8.45 mmol) (synthesis of mesylate: Miller, Fox (1964) J. Org. Chem. 29, 1772) and 33 mmol Sodium thiobenzoate in 30 ml DMF is stirred for 4 hours at 100 ° C. It is shaken with dichloromethane and the organic phase is washed with saturated NaHCO3 solution and saturated NaCL solution. After drying the organic phase over sodium sulfate, it is co-evaporated twice with toluene and the oily product is purified by flash chromatography (silica gel 60 H, chloroform / methanol 0-10%).

Performance; 60%; the product has the expected 1H-NMR spectrum.

The 5'-dimethoxytriphenylmethyl protecting group at the 5 'position for phosphorylation is cleaved by methods known in the literature (M. Gait, Oligonucleotide Synthesis, JRL-Press, Oxford 1984). Then, by Eckstein and Ludwig's method (J. Org. Chem., 1989, vol. 54, No. 3, 631) 5'-triphosphate with salicylphosphoric chloride (Aldrich) and by pyrophosphate treatment is prepared. Thiol is released by reaction with 10 M NaOH in argon saturated ethanol (R. Cosstick, Nucleic Acids Research 18.4, 829).

3'-thio-5'-triphosphate-thymidine is coupled according to the method of Example 8 with iodoacetamidofluorescein (molecular trials) and fluorescein-labeled thionucleotide is obtained in 80% yield.

Example XIV. Preparation of 1- (3'-thio-2 ', 3'-dideoxy-5'-triphosphate-PD-treopentofuranosyl) -thymine according to scheme 7.

Starting with 5'-DMTr-O-, 3'-O-mesyl-thymidine, as described in Example 13, it is substituted with sodium thiobenzoate. This is followed by cleavage of the 5'-protecting group, introduction of 5'-triphosphate, release of thiol and attachment of iodoacetamidofluorescein analogously to the method of Example 13.

Example XV Preparation of the oligomer from 10 nucleotides 3'-3-HzN-T'TT TTT TTT T-5 '.

The 3'-P-H2N-TTT TTT TTT TT-T 'oligomer is produced starting from 1- (3'-amino-2', 3'-dideoxy-5'-DMTr-3-treopentofuranosyl) -thimine, which according to the method described in Example V is reacted to the support for the phosphoramidite process. Subsequent labeling of the fluorescein isothiocyanate 3'-3-amino-oligonucleotide according to example VII.

<img file="PL168874B1_D0001.tif" />

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PATTERN 2

168 874

<img file="PL168874B1_D0003.tif" />

<img file="PL168874B1_D0004.tif" />

PATTERN 3 PATTERN 5

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MODEL 4

MODEL 6

168 874

<img file="PL168874B1_D0007.tif" />

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168 874

<img file="PL168874B1_D0009.tif" />

OH

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°9^P3 ~ V ° 4^AT

OH N₃

DIAGRAM 3

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DIAGRAM 4

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<img file="PL168874B1_D0014.tif" />

DMTrO-_koj

IN

<img file="PL168874B1_D0015.tif" />

DIAGRAM 7

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