JPH10500963A - 3 '-(4'-) non-radiolabeled nucleosides and nucleotides having an aminocarboxylic acid, peptide or carboxylic acid spacer - Google Patents

Abstract

(57) Abstract: 3 '-(4'-) modified nucleosides and nucleotides of general formula (1) are described, wherein R' is hydrogen, mono-, di- or triphosphate, alkyl phosphonate, dialkyl phosphinate Or is a phosphoramidite; X is oxygen, nitrogen, carbon or sulfur; Y is oxygen, nitrogen or sulfur; Z is a hydrogen, hydroxyl, amino or thiol group and R is non-radioactive detectable A spacer group to which a group or effector molecule is attached; the base is a purine or pyrimidine base or deazapurine or a derivative thereof; n is 0 or 1. Methods for making these compounds and their use in nucleic acid analysis and drug manufacture are also described. In particular, 3'-amino-3'-deoxynucleotide fluorescein, in which the fluorescein group is linked to the sugar via a mono-, di- or tetraglycine unit, has been found to be advantageous in sequencing.

Description

DETAILED DESCRIPTION OF THE INVENTION       Has aminocarboxylic acid, peptide or carboxylic acid spacer           3 '-(4'-) Non-radiolabeled nucleosides and nucleotides   The present invention provides a nucleoside in which the 3 ′ (4 ′) position is modified by a non-radiolabeled group, Nucleotides and oligonucleotides, methods for their production and their use It is about.   In the field of genetic engineering, labeled nucleotides are used in numerous applications. I have. The reason is that DNA digests obtained from natural genetic material by restriction enzyme digestion. It is more convenient to use lobes than to use them as hybridization probes Because it is easy.   Modified oligonucleotides used in the form of so-called "antisense" DNA oligonucleotides Ligonucleotides can intervene regulatoryly in cellular processes, thus For example, in vivo studies of protein expression and in cancer, virus and gene therapy Is becoming increasingly important. The mechanism of action in this case is DNA-DNA, DN A-RNA and RNA-RNA interactions, but with sufficient detail Ming is still needed.   Labeled oligonucleotides may be, for example, gene blots from a gene bank. To screen and identify pulls with the help of labeled oligonucleotides Therefore, they can be used in vitro to identify gene fragments contained in gene banks. Have been.   Apart from radiolabeling with suitable isotopes, for example, derivatized fluorescent colors Element is used as a more convenient and safer form of non-radioactive label. Have been.   Heretofore, this type of technique has been used for nonradioactive sequencing of DNA. Has been successful, but in that case, essentially the Sanger method [F. Sanger, S . Nicklen and S. Coulsen, Proc. Natl. Acad. Sci. USA 74,5463 (1977)] Based techniques were implemented.   In this method, the fluorescent label is the 5 'end of the nucleotide [L.E. Hood, L.M. Smith and C . Heiner, Nature 321, 674 (1986)] or a nucleobase [J.M. Prober, G.L. Tra inor and R.J. Dam, Science 238, 336 (1987); G.L. Trainor, Anal. Chem. 62 , 418 (1990)]. This method works only for specific polymerases. Not available for synthesis, triphosphate is poorly accepted by the polymerase And the disadvantage of requiring a large excess of substrate.   Also knows chemical sequencing by the Maxam-Gilbert method using fluorescent labels [H. Voss, C.E. Schwager, U.S. Wirkner, B .; Sproat, J .; Zimmermann, A .; Rosenthal, H .; Erfle, J .; Stegmann and W.S. Ansorge, Nucl. Acids. Res. 17, 2 517 (1989)].   In addition, fluorescent dyes are nucleosides, nucleotides or oligonucleotides. It can be linked directly by an amino or thiol group at the 3 'position, The synthesis of complementary strands in the presence of template strands and the transfer of genetic material in vivo and in vitro It is known that it can be used advantageously for detection (EP 0 490 281). However, The disadvantages of these compounds are that their quantum efficiency is relatively low and their synthesis is boring. It requires a long and long chromatographic purification step.   Thus, an object of the present invention is to provide a new non-ionic compound which does not have the disadvantages of the known compounds. Radioactively labeled nucleosides and nucleotides.   The purpose of this is:   (In the formula,   R ′ is hydrogen, mono-, di- or triphosphate, alkyl phosphonate, di Represents an alkyl phosphinate or phosphoramidite,   X represents oxygen, nitrogen, carbon or sulfur,   Y represents oxygen, nitrogen or sulfur,   Z represents a hydrogen, hydroxyl, amino or thiol group;   R is the spacer to which the non-radioactive detectable group or effector molecule is attached -Represents a group,   Bases are purine or pyrimidine bases or deazapurine or their derivatives Represents the body,   n is 0 or 1] By a modified nucleoside and nucleotide at the 3 'or 4' position having Achieved.   The spacer group is preferably a protected or unprotected 2-, 3- Or polyfunctional carboxylic acids, aminocarboxylic acids or peptides, or their Consists of the corresponding derivative or salt. All naturally occurring amino acids are preferred Corresponds to a spacer, for example, glycine or aspartic acid, Includes boronic acid or aminocarboxylic acid, but diamine, thiol and amino Alcohol is also suitable.   Dimers and tetramers of amino acid units are particularly suitable as spacer groups There was found. In particular, the length of the spacer group corresponding to a carbon chain having 2 to 12 atoms This is the case when Non-radioactive detectable groups, so-called reporter molecules Thus, all groups that can be detected chemically, physically or biologically are conceivable. This Such groups are known to those skilled in the art. In this case, directly or with a specific target molecule Chemical, physical or biological activation followed by chemical, physical or biological So-called effector molecules which can interact with Agents, π-based aromatic compounds, enzymes, etc.). As an example, acetylaminofluorene (Intercalation) into DNA / RNA by nuclease Cleavage of DNA / RNA, alkylation of DNA / RNA by alkylating agent or Partial or complete covalent bonding of aryl groups, or therapeutically effective electromagnetic radiation capture cross-sections Psoralens that produce a significant increase locally and for example metal clusters It is. In particular, peroxidase, a luminescent dye that emits light in the wavelength range of about 630 to 670 nm Enzymes such as lyase or alkaline phosphatase, biotin / iminobiotin or Haptens such as goxigenin are considered as reporter molecules. Related to this Thus, the phosphor is found to be particularly suitable for base sequencing by, for example, the Sanger method. won. Compounds substituted with effector groups are particularly effective for antisense therapy. You.   3 'and / or 4' of nucleoside, nucleotide or oligonucleotide The OH, amino or thiol group present at the position is Such as a reporter or effector molecule. To be combined. Then, the generated 3'- or 4'-hydroxyl-, amino- or Uses thiol-modified nucleosides, nucleotides and oligonucleotides To synthesize a complementary strand or to detect a specific sequence in the presence of a natural or synthetic template strand. Or place effector molecules in specific sequences of genetic material. Wear. In particular, they no longer have the modifying group at the 5 'end of the nucleotide, as in known labeling methods. And nucleobases.   The invention further relates to 3 ′ (4 ′)-modified nucleosides and nucleotides of general formula (1) And a method for producing the same. Compound of general formula (2): (In the formula,   The linker represents a selectively cleavable anchor group,   X represents oxygen, nitrogen, carbon or sulfur,   Y represents oxygen, nitrogen or sulfur,   Z represents a hydrogen, hydroxyl, amino or thiol group;   R represents hydrogen,   Bases are purine or pyrimidine bases or deazapurine or their derivatives Represents the body,   n is 0 or 1] Is bonded to a solid support via a linker group, and if necessary, a difunctional having a protecting group. Detectable by adding a functional or polyfunctional compound and then reactivating if necessary A nucleoside derivative from the carrier matrix, And, if desired, it is derivatized at the 5 'position.   In addition, a nucleoside linked to the solid support by a linker at the 5 '(6') position was prepared. The reaction can be accomplished using other methods known to those skilled in the art. Can be selectively disconnected Any functional anchor group can be used as a linker group [e.g., G.B.F. ields and R.L. Noble, Int. J. Peptide Prot. Res. 35 (1990), 165-171]. Warm Under mild conditions, for example photochemically, by hydrogenation or by acid or alkali Groups that can be cleaved in the sex range have been found to be particularly suitable. Preferred linker groups Is an optionally substituted trityl group. In this case, methoxy And halogen residues are suitable substituents; dimethoxy and o-chlorotrityl groups Was found to be particularly preferred. In addition, the linker group is attached to the 5 'or 6' position of the nucleoside. The basic method of attachment to a device is known to those skilled in the art [M.J. Gait: Oligonucleotide  synthesis; a practical approach; IRL Press, Oxford, Washington DC (1984) , P.12 ff].   Insoluble in the solvent used in each case, easy to filter as much as possible, Any inert solid materials are suitable as carrier materials in the present invention. Preferred Carrier material is inorganic based on polystyrene or polyethylene glycol It is a polymer material (resin).   Subsequently, the nucleoside bound to the solid support by this method is converted to an activating reagent, for example, For example, in the presence of a condensing agent such as DCC, DIC and HOBT, Mix with carboxylic acid or peptide derivative.   Aminocarboxylic acids, peptides, and nucleosides or their analogs bound to the solid phase Or after binding a carboxylic acid or a derivative or salt thereof, Combines several identical or different reporter / effector molecules, for example, Or amidation, sulfonamidation, sulfonic esterification, or Reaction with thiocyanate or N-hydroxysuccinimide ester gives side It is selectively introduced covalently to the chain or the C or N terminus. Appropriate methods are known to those skilled in the art. It is known.   Compounds and compounds considered as reporter or effector molecules Are those that can be covalently attached to a hydroxy, amino or thiol group (See above).   To cleave a nucleoside derivative from a linker group, a weakly acidic, weakly basic, It is preferably carried out under chemical or reducing conditions.   Nucleoside induction with one or several protecting groups or side chains on the spacer group The conductor can be manufactured in a corresponding manner.   The nucleoside derivatives produced in this way are subsequently prepared by methods known to those skilled in the art. Depending on the method, the corresponding 5 'or 6' mono-, di- or triphosphate (nucleo Ide).   The synthesis of 5 'and 6' modified nucleosides according to the invention is particularly advantageous. why Then, in contrast to known methods, use a large excess of the dye component without any problems Because they can be easily separated. In the case of the conventional method, Time-consuming and expensive chromatography after the actual production process to separate the corresponding dyes Rough purification steps had to be performed.   In the general formula (1), the compound of the present invention in which the group R ′ represents a triphosphate group includes Use primers and template strands in the presence of different nucleoside triphosphates Sometimes, it can be advantageously used as a substrate for DNA / RNA polymerase. So Suitable polymerases are T7 polymerase, Taq polymerase and DNA polymerase. Limerase I and reverse transcriptase. In addition, these compounds are DNA / RN It is also suitable as a terminator in A base sequencing. In this case, the synthetic Termination is achieved by using 3 ′-(4′-) modified A, C, G or T nucleotides of formula (1). Can be determined in each case. This is the synthesis of the complementary DNA strand in the presence of the template strand. (And therefore also for DNA strand sequencing). What is the use of modified nucleotides is a very base-specific This is because the termination is assured.   Synthesize RNA nucleosides and oligonucleotides in a similar manner Can be.   In addition, it has a spacer group at the 3 '(4') end and is amino- or thio-modified and That can be derivatized using a starting nucleotide immobilized on a polymeric material carrier It is also possible to synthesize gonucleotides.   Oligonucleotides can be any DNA or RNA nucleic acid produced by conventional methods. It is understood as a nucleotide, but is preferably 2 to 100 nucleotides, particularly preferably Having a length of 12 to 50 nucleotides (chemical synthesis) or the polymer used Having a length of up to about 3000 nucleotides depending on the efficiency of the enzyme (enzymatic synthesis) It is.   Oligonucleotide synthesis is performed in a conventional manner, ie, in the 3 'and 5' directions. Oligonucleotides having a defined sequence, performed starting from a nucleotide-carrier complex The synthesis of nucleotides is possible.   Immobilization of the starting nucleoside on a commercially available carrier involves a linking linker that can be cleaved after synthesis. Via a spacer (spacer), for example, with a succinic acid bond or urethane described in the literature. (Efimov et al., Nucl. Acids. Res. 11, 8369, 19). 83).   After completion of the synthesis, the oligonucleotide must be cleaved from the support using appropriate reagents. It is necessary. Thereafter, derivatization with the desired fluorescent dye can be performed directly.   Also, the starting nucleoside is linked to the linker group of the carrier material via its 5'OH group. Followed by proceeding the reaction according to conventional oligonucleotide synthesis methods. Alternatively, oligonucleotides can be synthesized in the opposite direction (5 '→ 3'). In that case, labeling of the oligonucleotide with the dye can be performed on a solid phase. You.   Thereafter, an oligonucleotide modified at the 3 ′ (4 ′) end and synthesized in this manner For example, for the detection of complementary oligonucleotides or nucleic acids and the corresponding derivatives Can be used.   However, the compounds according to the invention are useful for in vivo and in vitro sensors for analysis. It is also suitable as a gene or as a gene probe in gene therapy.   The present invention will be described in more detail with reference to the following examples.Example 1 2- Esterification of Fmoc-amino acid derivatives with chlorotrityl resin   1 g of resin loaded with 1.1 to 1.6 mmol of 2-chlorotrityl chloride linker [K . Barlos, O .; Chatzi, D. Gatos, G. Stauropoulos, Int. J. Peptide Prot. Re s. 37, (1991), 513-520] was shaken in anhydrous DCM for several minutes. 3 mmol / l DIEA After the addition, 1.2 mmol of Fmoc protected amino acid was added. The reaction time was 90 minutes. Additional After the addition of 3 mmol DIEA, the non-esterified linker groups were removed with 5 ml of anhydrous methanol. Esterified within about 30 minutes. Filter the coated resin, DMF, DCM, Washed several times with sopropanol and finally with diethyl ether. Oil pon After drying under reduced pressure in a pump, the quantitative ninhydrin test (Example 7b) or Fmoc cleavage Amino acid loading was measured by UV spectroscopy. By changing the amount of amino acids Amino acid loadings of 0.05-1.1 mmol / g were obtained.Example 2 Nα-binding of fluorescent dyes to resin-bound amino acids or peptides   For a dye having a carboxy group, the following procedure is performed.   3 equivalents per equivalent of resin-bound amino acid or peptide in a shake flask Add dye, 3.3 eq DIC and 4.5 eq HOBt and add DMF / DCM in dark room Shake at warm for 50 hours. Then, it is purified DMF, DCM, methanol, isopropano Wash several times with ethanol and ether, and remove the amino acid or peptide conjugate Separate from the solid support as described above.   3 equivalents per equivalent of resin-bound N-terminal protected peptide or amino acid to be labeled Fluorescent dye, 1.2 equivalent DIEA and catalytic DMAP (4,4'-dimethylaminopyridyl 48 hours in a solution of DMF / DCM (v / v 4: 1) in a shake flask with frit Reaction. Shaking was performed in a dark room to avoid the decomposition reaction of the dye. Reaction course Can be tracked by ninhydrin reaction or TLC. Free amino groups are still present If you want to_TwoAcetylation can be performed with O / DIEA (equivalent / equivalent 1: 2). Unreacted dye is removed by filtration, and each is diluted with 10 ml of DMF, DCM, DMSO, and MeOH. Washed twice and finally with diethyl ether. Dry under reduced pressure with an oil pump After that, the amino acid-peptide-dye conjugate can be stored in a refrigerator.   Depending on the orthogonality of the protecting groups, one or several identical or different fluorescent dyes It is also possible to introduce selectively at the chain or at the N-terminus. Side chain of trifunctional amino acid Fluorescent dyes can be introduced in the same manner as Nα-linkage, but the proportion of DMAP must be increased. It is necessary.Example 3 For producing fluorescently labeled amino acids or fluorescently labeled peptides2- Cleavage from chlorotrityl resin   Decomposition of the ester was performed under weakly acidic conditions. About 20 ml per g of peptide resin A mixture of glacial acetic acid / TFE / DCM (v / v / v 1: 2: 7) was used [K. Barlos, O .; Chatzi, D. Gat os, G. Stauropoulos, Int. J. Peptide Prot. Res. 37, (1991), 513-520]. Minute The solution time was usually about 90 minutes. His (N^imIf no (Trt) residue is present, DCM / TFE (v / v Can be cut using the 1: 1) mixture. Next, the resin is removed by filtration. The peptide solution was filled with about 100 ml of water. The added water leaves the volatile components To prevent the concentration of acetic acid when distilling off with a rotary evaporator . Hydrophobic amino acid and peptide derivatives are distilled on a rotary evaporator It was then lyophilized as it precipitated out.Example 4 2- Etherification of nucleosides via 5'-OH group with chlorotrityl resin   1 g of resin loaded with 1.1-1.6 mmol of 2-chlorotrityl chloride linker CHCl_ThreeShake for several minutes in a mixture of / DMSO (v / v 1: 2). After addition of 2.5 mmol DIEA , 0.52 mmol of nucleoside was added. The reaction time was about 120 minutes. Extra 3 mm After the addition of olDIEA, the non-etherified linker groups were removed with about 5 ml of anhydrous methanol. Etherified within minutes. Filter the coated resin, DMF, DCM, ISO Washed several times with propanol and finally with diethyl ether. Decompression with oil pump To After drying, in the case of 3'-aminonucleotides, a quantitative ninhydrin test (Example 7b) or after appropriate derivatization of the 3 'function of the nucleoside Alternatively, the resin load was measured by weighing the resin. Nucleo Varying the amount of sid resulted in a resin loading of 0.1-1 mmol / g.Example 5 Amino acids, peptides, carboxylic acids to carrier-bound nucleosidesAnd their derivatives   Binding was performed according to the activation method known from peptide chemistry (time: about 12 hours) . The use of base-labile protecting groups allows the use of amino acid chains or flags by known methods. It is possible to elongate the methyl condensate [M. Bodanszky Principles of Peptide Synth eses, 2nd Edition, Springer 1993, G.B. Fields & R.L. Noble, Int. J. Pept ide Prot. Res. 35, (1990), 161-214].Example 6 Nucleosides, nucleoside-amino acids from 2-chlorotrityl resin,Cleavage of nucleoside-peptides and their dye derivatives   Ether degradation was performed under acidic conditions. About 10 per gram of coated resin ml of dichloroacetic acid / DCM (v / v 3:97) solution was used. Decomposition time was about 1 minute . Thereafter, the resin was removed by filtration, and the product solution was treated with DIEA / DCM (v / v 63: 937). And immediately neutralized. The resin was washed several times with a small amount of ACN. Add about 10 ml of water Was. The product was placed in a freeze dryer and dried.Example 7 Ninhydrin test   The following three types of solutions are required to perform the ninhydrin test [I. Kaiser, R.L. Colescott, C.D. Bossinger, P.I. Cook, 3. Anal. Biochem. 34, (1970) , 595].   Solution 1: A solution containing 80 g of phenol in 20 ml of ethanol   Solution 2: 2% pyridine solution containing 33 mg of potassium cyanide KCN in 50 ml of water   Solution 3: A solution containing 500 mg of ninhydrin in 10 ml of ethanol a)Qualitative ninhydrin test   To determine whether the acylation has progressed quantitatively, In a dorf tube, apply 2-3 drops of the three solutions to a microspatula full of coaty. To react with the resin. The reaction mixture is heated in a water bath at 100 ° C. for about 5 minutes. If the acylation reaction is incomplete, the color of the solution will be dark blue, while quantitative In the case of a combined reaction, the reaction mixture remains yellow. b)Quantitative ninhydrin test   To determine the amino acid loading of the resin, the Fmoc-amino acid derivative was piperidine / D Deprotect with MF (v / v 40:60) for 40 minutes.   After washing with DMF, isopropanol and DCM, the resin is dried. About 3-5mg Samples were placed in a thermo-heating block for solution 1 (4 drops) and solution 2 (8 drops) And with solution 3 (4 drops) at 100 ° C. for 7 minutes. 60% of the reaction mixture Dilute immediately with ethanol and transfer to an appropriately sized measurement flask.   After calibrating the UV spectrometer with 60% ethanol, measure the absorbance and The resin load (μmol / g) is determined by applying an appropriate numerical value to the equation.   The compounds of Examples 8 to 23 described below were prepared according to the general conditions of Examples 1 to 6. Synthesized.Amino acid / peptide-dye conjugate Example 8 Fluorescein-5 (6) -aminothiono-N-glycine (FITC-Gly (OH))Three Preparation:   0.17 mmol (673 mg) resin-gly-NH_Two   0.5 mmol (193 mg) FITC   Yield (77.9 mg) 91%HPLC analysis:   Column: Nucleosil RP18, 5μm, 300A, 4 × 250 mm   Gradient: 2-100% ACN (0.1% TFA) 40 minutes   Flow rate: 1 ml / min, absorption at 235.0 nm and 225.0 nm   Retention time: 10.05 / 11.00 minutesExample 9 Fluorescein-5 (6) -aminothiono-N-glycyl-glycine (FITC-Gly_Two(OH))Four Preparation:   0.43 mmol resin-gly_Two-NH_Two   1.3 mmol (504 mg) FITC   Yield (220.4 mg) 89%HPLC analysis:   Column: Nucleosil RP18, 5μm, 300A, 4 × 250 mm   Gradient: 20-100% ACN (0.1% TFA) 40 minutes   Flow rate: 1 ml / min, absorption at 287.5 nm and 425.0 nm   Retention time: 8.00 minutesExample 10 Fluorescein-5 (6) -aminothiono-N-triglycyl-glycine (FITC-Gly_Four(OH))Five Preparation:   0.2 mmol resin-gly_Four-NH_Two   0.5 mmol (195 mg) FITC   Yield (77 mg) 74%HPLC analysis:   Column: Nucleosil RP18, 5μm, 300A, 4 × 250 mm   Gradient: 0-80% ACN (0.1% TFA) 40 minutes   Flow rate: 1 ml / min, absorption at 287.5 nm   Retention time: 17.5 minutesExample 11 Rhodamine MR 200-N-methylcarbonyl-N'-triglycyl-glycine Preparation:   44.4 mg resin-gly_Four-NH_Two(Equivalent to about 0.02 mmol)   30 mg dye MR 200, free carboxylic acid   8.0 mg HOBT   6.3 μl DICyield:   quantitativeHPLC analysis:   Column: Nucleosil RP18, 5μm, 300A, 4 × 250 mm   Gradient: 0-80% acetonitrile (0.1% TFA) in 40 minutes   Flow rate: 1ml / min   Detection: Absorption at 617 nm   Retention time: 19.5 minutesTLC :   Silica gel, CHCl_Three/ MeOH 1: 1, Rf 0.55mass:   Calculated value 943.7         Measured 943.5Spectral data:   λ_max.Ex615 nm, λ_max.Em642 nm (in MeOH)Example 12 Rhodamine JA 51-N-propylcarbonyl-N'-triglycyl-glycine Preparation:   0.047 mmol resin-gly_Four-NH_Two   0.141 mmol dye JA 51   0.2 mmol HOBT   0.2 mmol DICyield:   Almost quantitativeHPLC analysis:   Column: Nucleosil RP18, 5μm, 300A, 4 × 250 mm   Gradient: 0-80% acetonitrile (0.1% TFA) in 40 minutes   Flow rate: 1ml / min   Detection: absorbance at 631 nm   Retention time: 16.5 minutesmass:   Calculated 774.8         Found 774.6 Example 13 Digoxigenin 3-0-methylcarbonyl-triglycyl-glycine Retention time: 15 minutesPreparation:   0.2 mmol resin-gly_Four-NH_Two   0.5 mmol digoxigenin-3-0-acetic acid-N-hydroxysuccinimide ester   Yield (94 mg) 72%HPLC analysis:   Column: Nucleosil RP18, 5μm, 300A, 4 × 250 mm   Gradient: 0-80% acetonitrile (0.1% TFA) in 40 minutes   Flow rate: 1ml / min   Detection: Absorption at 215 nm   Retention time: 15 minutesNucleoside / nucleotide-dye conjugate Example 14 3'-O- [S-triphenylmethyl-Nα- (9-fluorenylmethoxycarbonyl) -system Inyl] -2'-deoxythymidine (thymidine-Cys (Trt) (Fmoc))6 Preparation:   0.25 mmol resin-thymidine   0.5 mmol (Fmoc) Cys (Trt) OH   Retention time: 28.5 minutes   Yield: cleavage performed on analytical scaleHPLC analysis of the crude product:   Column: Vydac RP 18, 5 μm, 300 A, 4 × 250 mm   Gradient: 0-60% ACN (0.1% TFA) 20 minutes, 60-100% 25 minutes, 100% 30 minutes,          0% 35 minutes   Flow rate: 1ml / min   Absorption at 265 nm   Retention time: 28.5 minutesExample 15 3'-O-N- [Nε-pentamethylchromyl-Nα- (9-fluorenylmethoxycarbonyl ) -Arginyl] -2'-deoxythymidine (thymidine-Arg (Pmc) (Fmoc))7 Preparation:   0.25 mmol resin-thymidine   0.5 mmol (Fmoc) Arg (Pmc) OH   Yield: cleavage performed on analytical scaleHPLC analysis of the crude product:   Column: Vydac RP 18, 5 μm, 300A, 4 × 250 mm   Gradient: 0-60% ACN (0.1% TFA) 20 minutes, 60-100% 25 minutes, 100% 30 minutes,         0% 35 minutes   Flow rate: 1ml / min   Absorption at 265 nm   Retention time: 26.7 minutesExample 16 3'-O- [Nα- (9-fluorenylmethoxycarbonyl) -leucinyl] -2'-deoxythi Midine (Thymidine-Leu (Fmoc))8 Preparation:   0.25 mmol resin-thymidine   0.5 mmol (Fmoc) LeuOH   Yield: cleavage performed on analytical scaleHPLC analysis of the crude product:   Column: Vydac RP 18, 5 μm, 300A, 4 × 250 mm   Gradient: 0-60% ACN (0.1% TFA) 20 minutes, 60-100% 25 minutes, 100% 30 minutes,          0% 35 minutes   Flow rate: 1ml / min   Absorption at 265 nm   Retention time: 25.5 minutesExample 17 Compound 9 of Example 17 was synthesized continuously on a support (1. 1． (Fmoc)-amino acid binding; 3. deprotection of amino acids; Pigmented Binding). 3'-O-glycyl- {N-thiono [5 (6) -amino-fluoresceinyl]}-2'-deoxy-  Thymidine (Thymidine-gly-FITC)9 Preparation:   0.5 mmol resin-thymidine   0.5 mmol (Fmoc) gly (OH)   1.0 mmol FITC   Yield: cleavage performed on analytical scale   Rf value (silica gel 60, F₂₅₄, Merck): 0.35; CHCl_Three/ MeOH (v / v 9: 1)   The following Examples 18-23 were also synthesized on the support, then cleaved from the resin, and To produce triphosphate. The synthesis of triphosphate is described in [Ludwig Eckstein, J .; Org. Chem. 54, 631-635, (1989)].Example 18 Fluorescein-5 (6) -amino-thiono- (N-glycyl)-[(3'-amino-2 ', 3'-dide Oxy) -thymidine-5'-triphosphate] (3'-amino-Gly-FITC, 3'-deoxy , 5'-Triphosphate-thymidine)Ten Preparation:   0.031 mmol resin-3'-amino-3'-deoxy-thymidine   0.047 mmol fluorescein-gly (OH)   Yield: 60.1% over all steps (17.4 mg)   Rf value (silica gel 60, F₂₅₄, Merck): 0.59; isopropanol / NH_FourOH / water                                                 (V / v / v 7: 1: 4)HPLC analysis of the crude product:   Column: Supersphere RP18 (e); 3 μm; 2 × 125 mm   Gradient: 0-2% B 20 minutes, 2-4% B 25 minutes, 4-20% B          A = TEAA, pH 7.0; B = ACN   Flow rate: 0.15 ml / min   Absorption at 265 nm   Retention time: 11.95 minutesExample 19 Fluorescein-5 (6) -amino-thiono- (N-diglycyl)-[(3'-amino-2 ', 3'-di Deoxy) -thymidine-5'-triphosphate] (3'-amino-Gly_Two-FITC, 3'-deoki (5,5-triphosphate-thymidine)11 Preparation:   0.031 mmol resin-3'-amino-3'-deoxy-thymidine   0.052 mmol fluorescein-gly_Two(OH)   Yield: 54% over all steps (19.9 mg)   Rf value (silica gel 60, F₂₅₄, Merck): 0.29; CHCl_Three/ MeOH (v / v 1: 1)HPLC analysis of the crude product:   Column: Supersphere RP18 (e); 3 μm; 2 × 125 mm   Gradient: 0-2% B 20 minutes, 2-4% B 25 minutes, 4-20% B           A = TEAA, pH 7.0; B = ACN   Flow rate: 0.15 ml / min   Absorption at 265 nm   Retention time: 12.8 minutesExample 20 Fluorescein-5 (6) -amino-thiono- (N-tetraglycyl)-[(3'-amino-2 ', 3' -Dideoxy) -thymidine-5'-triphosphate] (3'-amino-Gly_Four-FITC, 3'-de Oxy, 5'-triphosphate-thymidine)12 Preparation:   0.031 mmol resin-3'-amino-3'-deoxy-thymidine   0.045 mmol fluorescein-gly_Four(OH)   Yield: 64.2% for all steps (21.9 mg)   Rf value (silica gel 60, F₂₅₄, Merck): 0.53 isopropanol / NH_FourOH / water                                                 (V / v / v 7: 1: 4)   Rf value (silica gel 60, F₂₅₄, Merck): 0.26 CHCl_Three/ MeOH (v / v 1: 1)HPLC analysis of the crude product:   Column: Supersphere RP18 (e); 3 μm; 2 × 125 mm   Gradient: 0-2% B 20 minutes, 2-4% B 25 minutes, 4-20% B   A = TEAA, pH 7.0; B = ACN flow rate: 0.15 ml / min   Absorption at 265 nm   Retention time: 12.8 minutesExample 21 Rhodamine MR 200-N-methylcarbonyl- (N'-tetraglycyl)-[(3'-amino-2 ' , 3'-Dideoxy) -adenosine-5'-triphosphate] Preparation:   0.027 mmol resin-3'-amino-2 ', 3'-dideoxy-N⁶-Benzoyladenosine   0.050 mmol Gly_Four-MR200   0.1 mmol HOBT   0.1 mmol DIC After cleavage from the carrier resin and subsequent synthesis of the triphosphate, the benzoyl protecting group is Removed with concentrated ammonia solution. Yield over all steps: 49% of theoryHPLC analysis:   Column: Hypersil ODS, 5μm, 120A, 4 × 250 mm   Flow rate: 1ml / min   Gradient: 0-20% B 10 minutes            80% B 20 minutes           100% B 25 minutes   Solvent: A: 0.1 M TEAA aqueous solution          B: Acetonitrile   Detection: Absorption at 617 nm   Retention time: 18.3 minutesmass:   Calculated value 1411.9         Found 1411.5Example 22 Rhodamine MR 200-N-methylcarbonyl- (N'-tetraglycyl)-[(3'-amino-2 ', 3'-dideoxy) -guanosine-5'-triphosphate] Preparation:   0.027 mmol resin-3'-amino-2 ', 3'-dideoxy-N^Two-Isobutyrylguanosine   0.050 mmol Gly_Four-MR200   0.1 mmol HOBT   0.1 mmol DIC After cleavage from the carrier resin and subsequent triphosphate synthesis, the isobutyryl protecting group Was removed with a concentrated ammonia solution. Yield over all steps: 41% of theoryHPLC analysis:   Column: Hypersil ODS, 5μm, 120A, 4 × 250 mm   Flow rate: 1ml / min   Gradient: 0-20% B 10 minutes            80% B 20 minutes           100% B 25 minutes   Solvent: A: 0.1 M TEAA aqueous solution          B: Acetonitrile   Detection: Absorption at 617 nm   Retention time: 18.0 minutesmass:   Calculated value 1427.9         Actual value 1427.3 Example 23 Digoxigenin 3-0-methylcarbonyl- (N'-tetraglycyl)-[(3'-amino-2 ' , 3'-Dideoxy) -thymidine-5'-triphosphate] Preparation:   0.03 mmol resin-3'-amino-2 ', 3'-dideoxy-thymidine   0.06 mmol digoxigenin 3-O-methylcarbonyl-triglycyl-glycine   0.1 mmol HOBT   0.1 mmol DIC After completion of the condensation, cleavage from the carrier resin as in Examples 21 and 22, phosphorylation and 5 ' -Got the triphosphate. Yield over all steps: 58% of theoryHPLC analysis:   Column: Hypersil ODS, 5 μm, 120 A, 4 × 250 mm   Flow rate: 1ml / min   Gradient: 0-20% B 10 minutes            80% B 20 minutes           100% B 25 minutes   Solvent: A: 0.1 M TEAA aqueous solution          B: Acetonitrile   Detection: absorbance at 260 nm   Retention time: 15.2 minutesmass:   Calculated value 1139.9         Observed 1139.1Example 24 Use of 3'-modified nucleotides for enzymatic DNA sequencing   The following nucleotides prepared according to Examples 1-6 were used.   1 μg of M13mp18 single-stranded DNA (5 μl), 2 μl of fluorescein-labeled universal plasmid Immer (1 pmol, Pharmacia LKB), 2 μl of Mn buffer I (311 mM Tris-HCl, pH7. 5) 2 μl of Mn buffer II (177 mM DTT) and 2 μl of Mn buffer III (62 mM MnCl_Two, 460 mM sodium isocitrate), mixed together, heated to 70 ° C, Cooled to 5 ° C (about 40 minutes). Next, 2 μl of diluted T7 DNA polymerase (4 (Pharmacia) was added to the template and primer heated to 37 ° C. Meanwhile, 3μ l of the termination mixture (T mix 1: 150 μl / compound 1; T mix compound 2: 200 μM II; T mix compound 3: 300 μM compound 4; each mixture was further 1 mM d ATP, 1 mM dGTP, 1 mM dCTP, 1 mM dTTP, 50 mM NaCl, 40 mM Tris-HCl, pH Was added to the four reaction vessels and heated at about 37 ° C for at least 1 minute. Heated. Subsequently, 3.8 μl of the first mixture (annealing mixture) was preheated. Was immediately dispensed into the finished mixture. After incubation at 37 ° C for about 10 minutes, 4 μl Stop reagents (deionized formamide solution, dextran blue) were added to each reaction solution. Added. The reaction solution is heated at about 90 ° C for 2 minutes, and each slot of the 6% denaturing sequencing gel is (6 μl each). After that, individual DNA fragments are detected with a fluorescence detector. And identified.   FIG. 1 shows fluorescence emission F (λ) measured for Compounds 1 to 4 by RP-HPLC analysis._ex = 488 nm; λ_em= 520 nm) and absorbance A (λ_abs= 265 nm).   According to this, the fluorescence intensity of Compound 1 was the lowest. The strength of compounds 2 to 4 is 3 ' It increased remarkably with the length of the spacer group at position (4 ').   All four modified nucleotides were used as substrates by the DNA polymerase used. And had termination performance comparable to, for example, ddTTP. According to the invention Since there is a bulky residue at the 3 ′ (4 ′) position of the nucleotides of Compounds 2 to 4 These compounds are recognized and converted by DNA polymerase as a substrate. This is surprising because it could not be predicted.   Rhodamine and digoxigenin prepared as described in Examples 20 and 21 Can be used according to the above.                                   Abbreviation   Recommendations of the IUPAC-IUB Biochemical Nomenclature Committee (J. Biochem. 138, (1984), 9; J. Biol. Chem. 247, (1972), 977; Biochemistry 9, (1970), 3471). You. ACN acetonitrile AcOH glacial acetic acid AA amino acids CDCl_Three     Deuterochloroform CH_ThreeOH methanol CHCl_Three     Trichloromethane TLC thin layer chromatography DCA dichloroacetic acid DCC N-N'-dicyclohexylcarbodiimide DCM dichloromethane ddTTp 2 ', 3'-dideoxy, 5'-triphosphate DIC N-N'-diisopropylcarbodiimide DIEA diisopropylethylamine DMAP 4,4'-dimethylaminopyridine DMF N, N-dimethylformamide DMTr 4,4'-dimethoxytrityl DNA deoxynucleic acid Et_TwoO diethyl ether FITC fluorescein isothiocyanate (5-isomer) Fmoc 9-fluorenylmethoxycarbonyl HOBt 1-hydroxybenzotriazole HPLC high-performance liquid chromatography KCN potassium cyanide Linker Anchor group between AA and resin MeOH methanol MW molecular weight ml milliliter mM mmol Mn manganese nm nanometer NMR nuclear magnetic resonance Pmc pentamethylchroman Rf TLC relative sample path RNA ribonucleic acid RP reversed phase RT room temperature Rt retention time SPPS solid phase peptide synthesis T thymidine T7 E. coli phage T7 Taq Thermococcus aquaticus TBTU 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyl-           Uronium tetrafluoroborate TEAA Tetraethylammonium acetate TFA trifluoroacetic acid TFE trifluoroethanol Tris Tris (hydroxymethyl) aminomethane Trt triphenylmethyl (trityl) TTp 5'-triphosphate-thymidine UV UV VIS visible light μl microliter

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI C07H 19/16 8615-4C C07H 19/16 // C07D 311/82 9360-4C C07D 311/82 491/14 9271 -4C 491/14 (72) Inventor Wechselberger, Liner Dee-D 65779 Kelkheim 3 am Rosenwald 23 (72) Inventor Engels, Joahin D-61476 Kronberg Feldbergstrasse 1 Address (72) Inventor Griesinger, Christian Germany Dee 61440 Auber Azel Hans Roser Steg 52

Claims (1)

[Claims] 1. Compound of general formula (1): Wherein R ′ represents hydrogen, mono-, di- or triphosphate, alkylphosphonate, dialkylphosphinate or phosphoramidite, X represents oxygen, nitrogen, carbon or sulfur, and Y represents oxygen, nitrogen or sulfur. Represents Z represents a hydrogen, hydroxyl, amino or thiol group, R represents a non-radioactive detectable group or a spacer group to which an effector molecule is attached, and the base is a purine or pyrimidine base or deazapurine or a derivative thereof. And n is 0 or 1. 2. The compound according to claim 1, wherein the spacer group at residue R has 2 to 12 atoms. 3. The compound according to claim 1, wherein the residue R has a (aminoacyl) _2-6 group. 4. 3. The compound according to claim 1 or 2, wherein the non-radioactive detectable group is a hapten, fluorophore, metal chelate, luminophore, protein or intercalator. 5. 5. The compound according to claim 4, wherein the detectable group is digoxigenin or fluorescein. 6. 3'-amino- (glygly) _2-6- FITC-3'-deoxy-5'-triphosphate nucleotide. 7. Compound of general formula (2): (Wherein, linker represents a selectively cleavable anchor group, X represents oxygen, nitrogen, carbon or sulfur, Y represents oxygen, nitrogen or sulfur, and Z represents a hydrogen, hydroxyl, amino or thiol group. R represents hydrogen, and the base represents a purine or pyrimidine base or deazapurine or a derivative thereof, and n is 0 or 1.) is bonded to a solid support via a linker group, and if necessary, a protecting group is provided. Is added, followed by the addition of a reactivated detectable signal component, if necessary, to cleave the nucleoside derivative from the carrier matrix and optionally to derivatize it at the 5 'position. The method for producing a compound according to any one of claims 1 to 6, comprising: 8. The method according to claim 7, wherein the bifunctional or polyfunctional compound is one or more carboxylic acids, aminocarboxylic acids or peptide derivatives. 9. The method according to claim 7 or 8, wherein the amino acid component is added in a single or double equimolar amount to the compound (2), and the method is repeated once or several times as necessary. Ten. The method of claim 7, wherein the linker group is an unsubstituted methoxy- or halogen-substituted trityl group. 11． Use of the compound of the general formula (1) for determining the base sequence of RNA or DNA. 12． Use of a compound of general formula (1) for non-radioactive detection of oligonucleotides, polynucleotides or nucleic acids. 13. Use of a compound of general formula (1) as an antisense or gene therapy agent.