WO1995032985A1 - 3'-(4'-) nonradioactively tagged nucleosides and nucleotides with aminocarboxylic acid, peptide or carboxylic acid spacer - Google Patents

Abstract

Disclosed are 3'-(4'-) modified nucleosides and nucleotides of general formula (1), wherein R' is hydrogen, mono-, di- or triphosphate, alkyl phosphonate, dialkyl phosphinate or phosphoramidite; X is oxygen, nitrogen, carbon or sulfur; Y is oxygen, nitrogen or sulfur; Z is hydrogen, an hydroxyl, amino or thiol group, R is a spacer group to which is bonded a nonradioactive detectable group or an effector molecule; Base is purine, pyrimidine base or deazapurines or their derivatives; n is 0 or 1. Disclosed also is a method for preparing the compounds and their use in nucleic acid analysis and the manufacture of drugs. In particular, 3'-amino-3'-deoxynucleotide fluoresceins in which the fluorescein groups are bonded to the sugar via mono-, di- or tetraglycine units have proved advantageous in sequencing.

Description

3 '- (4'-) non-radioactive labeled nucleosides and nucleotides with aminocarboxylic acid, peptide or carboxylic acid spacers

The invention relates to nucleosides, nucleotides and oligonucleotides modified in the 3 '- (4'-) position with non-radioactively labeled groups, a process for their preparation and their use.

Labeled nucleotides have found an extraordinarily large number of applications in genetic engineering, since their use is easier than that of the DNA probes conventionally used as hybridization samples, which are produced by restriction digestion from native genetic material.

Modified oligonucleotides, which are used in the form of so-called "antisense" DNA oligonucleotides, can intervene in a regulating manner in the cell process and thus gain z. B. for the in vivo investigation of the expression of proteins and in cancer, virus and gene therapy is becoming increasingly important. The mechanism works via DNA-DNA, DNA-RNA and RNA-RNA interactions, but still requires detailed clarification.

Labeled oligonucleotides, e.g. B. the identification of gene fragments within a gene bank by probing and identifying blotted gene samples from the gene bank with the aid of a labeled oligonucleotide.

In addition to the radioactive labeling by means of suitable isotopes, derivatized fluorescent dyes, for example, are used as a form of non-radioactive labeling, which offer the possibility of easier and safer handling.

So far, such a technique has been successfully used for non-radioactive sequencing of DNA. Here are essentially based on the Sanger method [F. Sanger, S. Nicklen and S. Coulsen, Proc. Natl. Acad. Be. USA 74, 5463 (1977)] based approaches.

The fluorescence label was either at the 5 'end of the nucleotide [L. E. Hood, L. M. Smith and C. Heiner, Nature 321, 674 (1986)] or on the nucleobase [J. M. Prober, G.L. Trainor and R.J. Dam, Science 238, 336 (1987)] [G. L. Trainor, Anal. Chem. 62, 418 (1990)]. This has the disadvantages that only certain polymerases can be used for the synthesis, the acceptance of the triphosphates by the polymerases decreases and that a large excess of substrate is also necessary.

Chemical sequencing according to Maxam-Gilbert with a fluorescence label is also known [H. Voss, C. Schwager, U. Wirkner, B. Sproat, J. Zimmermann,

A. Rosenthal, H. Erfle, J. Stegmann and W. Ansorge, Nucl. Acids. Res. 17, 2517

(1989)].

It is also known that a fluorescent dye is coupled directly via an amino or thiol group in the 3 'position of a nucleoside, nucleotide or oligonucleotide, and this compound is advantageous for the synthesis of counter-strands in the presence of a template strand and for the detection of genetic strands Material in vivo and in vitro can be used (EP 0 490 281). However, these compounds are associated with the disadvantage that the quantum yield is relatively low and the synthesis of the compounds is associated with lengthy, complex chromatographic purification steps.

The object of the present invention was therefore to make available new non-radioactive labeled nucleosides or nucleotides which do not have the disadvantages of the known compounds. The problem is solved by nucleosides or nucleotides of the following general formula modified at the 3 'or 4' position:

base

where: R ': hydrogen, mono-, di- or triphosphate, alkylphosphonate, dialkylphosphinate or phosphoramidite

X: oxygen, nitrogen, carbon or sulfur Y: oxygen, nitrogen or sulfur Z: hydrogen, hydroxyl, amino or thiol group R: spacer group, followed by a non-radioactive, detectable

Group or an effector molecule is bound

Base: purine, pyrimidine base or desazapurins or their derivatives, n: 0 or 1.

The spacer grouping preferably consists of a protected or unprotected bi-, tri- or polyfunctional carboxylic acid, aminocarboxylic acid or a peptide or corresponding derivatives or salts. Particularly preferred spacers are all naturally occurring amino acids, such as, for example, glycine or aspartic acid, carboxylic acids or aminocarboxylic acids having 2 to 20 atoms, but also diamines, thiols and amino alcohols.

Dimers or tetramers of amino acid units have proven to be very particularly suitable for spacer groups; furthermore if the spacer group has a length which corresponds to a carbon lette of 2 to 12 atoms. All chemically, physically or biologically detectable groups come into consideration as non-radioactive, detectable groups, so-called reporter molecules. Such groupings are known to the person skilled in the art. So-called effector molecules are also suitable here, which are capable, directly or after chemical, physical or biological activation, of interactions of a chemical, physical or biological nature with certain target molecules (such as alkylating agents, π systems of aromatic compounds, enzymes, etc.). Examples include the intercalation on DNA / RNA by acetylaminofluorenes, cleavage of DNA / RNA by nucleases or the partial or complete covalent binding of alkyl or alyl groups to DNA / RNA by alkylating agents or psoralenes, and z. B. metal clusters that locally lead to a sharp increase in the cross-section of therapeutically effective electromagnetic radiation. In particular, luminescent dyes which emit in the wavelength range from approximately 630 to 670 μm, enzymes such as peroxidase or alkaline phosphatase and haptens, such as, for example, biotin, come as reporter molecules. Iminobiotin or digoxxigenin. In particular, fluorophers have proven to be suitable here, e.g. B. for Sanger sequencing. The compounds substituted with effector groups are of particular value in particular for antisense therapy.

The OH group, amino or thiol group of a nucleoside, nucleotide or oligonucleotide located in the 3 'and / or 4' position is coupled to a spacer group, such as an aminocarboxylic acid, carboxylic acid or a peptide, and then a reporter or effector molecule attached. The resulting 3'- or 4'-hydroxyl-, amino- or TTιiol-modified nucleosides, nucleotides and oligonucleotides can then be used for the synthesis of counter-strands in the presence of a natural or synthetic template strand as well as for the detection of certain sequences or the localization of a Effector molecule to be used at certain sequences in genetic material. In particular, they offer the advantage that the modification is no longer applied to the 5 'end of the nucleotide or to the nucleobase, as in previously known labeling techniques. Another object of the invention is a method for producing the 3 * - (4 '-) modified nucleosides or nucleotides according to the general formula (1). Compounds of the general formula (2)

Base Carrier I —— ((LLiirnker) -X

where: Left: a selectively cleavable anchor group

X: oxygen, nitrogen, carbon or sulfur

Y: oxygen, nitrogen or sulfur

Z: hydrogen, hydroxyl, amino or thiol group

R: hydrogen

Base: purine, pyrimidine base or desazapurins or their derivatives, n: 0 or 1

are bound to a solid support via the linker grouping, a bifunctional or polyfunctional compound optionally provided with protective groups, then an optionally reactivated, detectable signal component is added, the nucleoside derivative is cleaved from the support matrix and, if appropriate, the 5'- Position.

The conversion to nucleosides bound to a solid support via a linker in the 5 '- (6' -) position can moreover be carried out by other methods known to the person skilled in the art. All selectively cleavable anchor groups can be used as the linker group [z. BGB Fields and RL Noble, Int. J. Peptide Prot. Res. 35: 165-171 (1990)]. Groupings which can be cleaved under mild conditions, such as, for example, photochemically, by hydrogenation or in the acidic or alkaline range, have proven particularly suitable proven. Preferred linker groups are trityl groups, which may be substituted. Methoxy and halogen radicals are suitable substituents here. Dimethoxy and o-chlorotrityl groups have proven to be particularly suitable here. In addition, the basic procedure for linking the linker grapple with the 5 'or 6' position of nucleosides is known to the person skilled in the art (MJ Gait: "Oligonucleotide synthesis; a practical approach"; IRL Press, Oxford, Washington DC (1984 ), P. 12 ff).

Any solid, chemically inert material which is insoluble in the solvent used in each case and which should be as easy to filter as possible is suitable as the support material according to the invention. Preferred carrier materials are inorganic, polymeric materials (resins) based on polystyrene or polyethylene glycol.

In this way, nucleosides bound to solid supports are then mixed with a corresponding carboxylic acid, aminocarboxylic acid or peptide derivative in the presence of an activation reagent, such as, for example, condensing agents such as DCC, DIC or HOBT.

After the aminocarboxylic acid, the peptide or the carboxylic acid or their derivatives or salts have been linked to the nucleoside or its analogue bound to the solid phase, one or more identical or different reporter Zeffector molecules are introduced covalently selectively in the side chain or C or N-teπninal, for example via esterification, amidation, sulfonamidation, sulfonic acid esterification or by reaction with isothiocyanates or N-hydroxysuccinimide esters. Appropriate methods are known to the person skilled in the art.

Compounds or classes of compounds which can be covalently linked to a hydroxyl, amino or thiol group can be considered as reporter molecules or effector molecules (see above).

The nucleoside derivatives are preferably cleaved from the linker group under weakly acidic, basic, photochemical or reductive conditions.

Nucleoside derivatives which have one or more protective groups or side chains on the spacer group can be prepared accordingly. The nucleoside derivatives prepared in this way can then be converted into the corresponding 5'- or 6'-mono-, di- or triphosphates (nucleotides), alkylphosphonates, dialkylphosphinates or phosphoramidite by methods known to those skilled in the art.

The synthesis of the 5'- or 6'-modified nucleosides according to the invention is particularly advantageous because, unlike in known processes, a large excess of the dye component can be used without problems and can be easily separated off. For the previously known processes, lengthy and expensive chromatographic purification steps follow the actual production process for separating the unreacted dye.

The compounds of the general formula (1) according to the invention, where radical R 'represents a triphosphate group, can advantageously be used as substrates for DNA / RNA polymerases by means of a primer and a template strand and in the presence of the four nucleoside triphosphates. Suitable polymerases here are T7, Taq polymerase, DNA polymerase I and reverse transcriptases. The compounds are also suitable as terminators in enzymatic DNA RNA sequencing. The termination of the synthesis can in each case be determined specifically by using a 3 '- (4' -) modified A, C, G, T nucleotide of the formula (1). This is of particular importance for the synthesis of DNA counter-strands in the presence of a template strand (and thus also for the sequencing of DNA strands), since the use of a modified nucleotide ensures a very base-specific termination of the reaction.

The synthesis of RNA nucleosides and oligonucleotides takes place in an analogous manner.

In addition, the derivatizable oligonucleotides can be synthesized using a start nucleotide which has a spacer group at the 3 '- (4') end and has been amino- or thiovariated and fixed to a polymeric support.

All DNA and RNA nucleotides produced in the conventional sense are considered oligonucleotides, but preferably in a length of 2 to 100, particularly preferably 12 - 50 nucleotides (chemical synthesis) or in a length of up to approx. 3,000 nucleotides (enzymatic synthesis), depending on the efficiency of the used

Polymerase.

The oligonucleotide synthesis takes place, starting from the starting nucleotide-carrier complex, in the conventional sense, i. H. in the 3 'and 5' direction and enables the synthesis of an oligonucleotide of a defined sequence.

The start nucleoside is fixed to commercially available carriers via a connecting arm (spacer) which can be cleaved after synthesis; for example via the succinic acid linkage known from the literature or else via a linkage with urethane (Efimov et al, Nucl. Acids. Res. 11, 8369, 1983).

After synthesis has taken place, the oligonucleotide must be cleaved from the support using suitable reagents. The derivatization with any fluorescent dye takes place immediately afterwards.

The reverse synthesis direction (5 ¹ -. 3 ') of oligonucleotides is also possible in that the starting nucleoside is linked to the linker grouping of the support material via the 5'-OH group and the procedure is analogous to conventional oligonucleotide synthesis. The dye labeling of the oligonucleotide can take place on the solid phase.

The oligonucleotides thus synthesized and modified at the 3 '(4') end can then be used for the detection of e.g. B. complementary oligonucleotides or nucleic acids and corresponding derivatives can be used.

However, the compounds according to the invention are also suitable as in vivo and in vitro sensors in analysis and as gene probes in gene therapy and for related analytical purposes.

The following examples further illustrate the invention: Example 1:

Esterification of Fmoc-amino acid derivatives with the 2-chlorotrityl resin

1 gram of resin mixed with 1, 11, 6mmol 2-chlororritylchloride linker [K. Barlos, O. Chatzi, D. Gatos, G. Stauropoulos, Int. J. Peptide Prot. Res. 37, (1991), 513-520] is loaded, several minutes in abs. DCM shaken. After adding 3 mmol DIEA, 1.2 mmol Fmoc-protected amino acid are added. The reaction time is 90 min. After the addition of a further 3 mmol DIEA with 5 ml abs. Methanol etherified the unesterified linker functionality for about 30 min. The coated resin is filtered off and washed several times with DMF, DCM, isopropanol and finally with diethyl ether. After drying in an oil pump vacuum, the amino acid loading is determined using the quantitative ninhydrin test (Example 7b) or by UV spectrometric determination of the Fmoc elimination. By varying the amount of amino acid, amino acid loads between 0.05 and 1.1 mmol / g are obtained.

Example 2:

Nα coupling of fluorescent dyes to resin-bound amino acids or peptides

The following applies to dyes with carboxy groups:

For each equivalent of resin-bound amino acid or peptide, 3 equivalents of dye, 3.3 equivalents of DIC and 4.5 equivalents of HOBt are placed in a shaker and shaken for 50 hours at room temperature in DMF DCM with exclusion of light. The mixture is then washed several times with pure DMF, DCM, methanol, isopropanol and ether and the amino acid or peptide conjugate is split off from the solid support, as described below.

For each equivalent of the resin-bound, N-terminally deprotecting peptide or the amino acid to be labeled, 3 equivalents of the fluorescent dye, 1.2 equivalents of DEEA and a catalytic amount of DMAP (4,4'-dimethylaminopyridine) in a solution of DMF / DCM (v / v 4: 1) reacted in a pouring barrel with frit for 48 h. In order to avoid decomposition reactions of the dyes, it is shaken in the absence of light. The course of the reaction can be followed with the ninhydrin reaction or with TLC. If free amino functionalities are still available, acetylation can be carried out using Ac ₂ O / DIEA (equivalents / equivalents 1: 2). Unreacted dye is filtered off and washed several times with 10 ml of DMF, DCM, DMSO, MeOH and finally with diethyl ether. After drying in an oil pump vacuum, the amino acid-peptide-dye conjugates can be stored in the refrigerator.

Orthogonality of the protective groups also allows the introduction of one or more identical or different fluorescent dyes selectively into the side chain or N-terminal. The introduction of fluorescent dyes into the side chain of trift-functional amino acids proceeds analogously to the Nα coupling, but the proportion of DMAP must be increased. Example 3:

Cleavage from 2-chlorotrityl resin to display fluorescence-labeled amino acids or fluorescence-labeled peptides

The ester cleavage is carried out under weakly acidic conditions. Approx. 20 ml of a mixture of glacial acetic acid / TFE / DCM in a ratio of (v / v / v 1: 2: 7) [K. Barlos, O. Chatzi, D. Gatos, G. Stauropoulos, Int. J. Peptide Prot. Res. 37, (1991), 513-520]. The cleavage time is usually approximately 90 minutes. If no His (N ™ ¹ Trt) residue is contained, it can be split off with a mixture of DCM / TFE (v / v 1: 1). The resin is then filtered off and the peptide solution is made up with about 100 ml of water. The added water prevents the acetic acid from concentrating when the volatile components are subsequently distilled off on a rotary evaporator. The hydrophobic amino acid and peptide derivatives are precipitated on spinning in and are now dried in the freeze dryer.

Example 4:

Etherification of nucleosides via the 5'-OH group with the 2-chlorotrityl resin

1 gram of resin loaded with 1.1-1.6 mmol of 2-chlorotrityl chloride linker is shaken for several minutes in a mixture of CHCl ₃ / DMSO (v / v 1: 2). After adding 2.5 mmol DIEA, 0.52 mmol nucleoside are added. The reaction time is approx. 120 min. After the addition of a further 3 mmol DIEA with 5 ml abs. Methanol etherified the unetherified linker functionality with methanol for 30 min. The coated resin is filtered off and washed several times with DMF, DCM, isopropanol and finally with diethyl ether. After drying in an oil pump vacuum, the resin loading is carried out with the quantitative ninhydrin test, in the case of the 3'-amino nucleotides (example 7b), by UV spectrometric determination after suitable derivatization of the 3'-functionality of the nucleoside or by weighing the resin certainly. By varying the amount of nucleosides, resin loads between 0.1 and 1 mmol g are obtained. Example 5:

Coupling of amino acids, peptides and carboxylic acids and their derivatives to carrier-bound nucleosides

The coupling takes place according to the activation methods known from peptide chemistry (duration: approx. 12 hours). The use of base-labile protective groups allows the amino acid chain extension or fragment condensation according to the known methods [M. Bodanszky Principles of Peptide Syntheses, 2nd Edition, Springer 1993, G.B. Fields & R.L. Noble, Int. J. Peptide Prot. Res. 35, (1990), 161-214]

Example 6:

Cleavage of nucleosides, nucleoside amino acids, nucleoside peptides and their dye derivatives from the 2-chlorotrityl resin

The ether cleavage is carried out under acidic conditions. About 1 ml of a solution of dichloroacetic acid / DCM in a ratio of (v / v 3:97) is used per 1 g of loaded resin. The split-off time is approx. 1 min. The resin is then filtered off and the product solution is immediately neutralized with an equimolar solution of DIEA DCM in the ratio (v / v 63: 937). The resin is washed several times with a little ACN. About 10 ml of water are added. The product is now dried in the freeze dryer.

Example 7:

Ninhydrin test

To carry out the ninhydrin test [I. Kaiser, R.L. Colescott, C.D. Bossinger, P.I. Cook, 3. Anal. Biochem, 34, (1970), 595] the following three solutions are required:

Solution 1: A solution of 80 g phenol in 20 ml ethanol.

Solution 2: A 2% solution of 33 mg potassium cyanide KCN in 50 ml water in pyridine. Solution 3: A solution of 500 mg ninhydrin in 10 ml ethanol

a) Qualitative ninhydrin test

To demonstrate whether an acylation has proceeded quantitatively, two to three drops of the three solutions are reacted with a micro spatula tip of the coated resin in an Eppendorf tube. The reaction mixture is heated to 100 ° C. for about 5 minutes in a water bath. If the acylation reaction is not complete, the solution acquires a deep dark blue color, while in the case of quantitative coupling reactions the reaction mixture retains its yellow color.

b) Quantitative ninhydrin test

For quantitative determination of the resin loading with amino acid, the Fmoc-amino acid derivatives are deprotected for 40 min with Piperidin / DMF (v / v 40:60).

After washing with DMF, isopropanol and DCM, the resin is dried. A sample of about 3-5 mg is heated with solutions 1 (4 drops), 2 (8 drops) and 3 (4 drops) in the thermal heating block at 100 ° C. for 7 minutes. The reaction mixture is immediately diluted with 60% ethanol and transferred to a measuring flask of a suitable size. After the calibration of the UV spectrometer with 60% ethanol, the extinction is determined and the resin loading in μmol / g is determined by inserting the corresponding values into the following equation.

[Extinction x dilution (ml,

Load [μmol / g] = _{extinction coefficient x} weighed mass ₍ mg) ^{x 10}

The compounds of Examples 8-23 listed below were synthesized according to the general conditions of Examples 1-6:

Amino acid / peptide dye conjugates

Example 8:

Fluorescein - 5 (6) aminothiono-N-glycine (FITC-Gly (OH)) 3

Approach:

0.17 mmol (673 mg) resin-Gly-NH ₂ 0.5 mmol (193 mg) FITC yield (77.9 mg) 91%

HPLC analysis:

Nucleosil RP18 column, 5 μm, 300A, 4 x 250 mm

Gradient 20-100% ACN (0.1% TFA) 40 'flow rate 1 ml / min. Abs at 235.0 nm and 225.0 nm.

Retention time: 10.05 / 11.00 min.

Example 9:

Fluorescein - 5 (6) aminothiono-N-glycyl-glycine (FITC-Gly ₂ (OH)) 4

Approach:

0.43 mmol resin-Gly ₂ -NH2 1.3 mmol (504 mg) FITC yield (220.4 mg) 89%

HPLC analysis:

Nucleosil RP18 column, 5 μm, 300A, 4 x 250 mm

Gradient 20-100% ACN (0.1% TFA) 40 'flow rate 1 ml / min.

287.5 and 425.0 nm

Retention time: 8.00 min.

Example 10:

Fluoresscein - 5 (6) aminothiono-N-triglycylglycine (FITC-Gly ₄ (OH)) 5

Approach:

0.2 mmol resin-Gly ₄ -NH2 0.5 mmol (195 mg) FITC yield (77 mg) 74%

HPLC analysis:

Nucleosil RP18 column, 5 μm, 300A, 4 x 250 mm

Gradient 0 - 80% ACN (0.1% TFA) 40 'flow rate 1 ml / min. Abs at 287.5 nm.

Retention time: 17.5 min.

Example 11

Rhodamine MR 200-N-methylcarbonyl-N'-triglycyl-glycine

Approach:

44.4 mg resin-Gly4-NH2 (corresponding to approx.0.02 mmol)

30 mg dye MR 200, free carboxylic acid

8.0 mg HOBT

6.3 ul DIC

Yield: quantitative

HPLC analysis

Column: Nucleosil RP 18, 5 μm, 300 A, 4x250 mm

Gradient: 0-80% acetonitrile (0.1% TFA) in 40 min.

Flow: 1 ml / min.

Detection: Abs. At 617 nm

Retention time: 19.5 min.

PC: silica gel, CHCl ₃ / MeOH 1: 1: Rf 0.55

Mass: calculated 943.7 found 943.5

Spectral data: λ _m aχjϊχ 615 nm, λ _max _ß _m 642 nm (in MeOH) Example 12:

Rhodamine JA 51-N-propylcarbonyI-N'-triglycyl-glycine

Approach:

0.047 mmol resin-Gly4-NH2

0.141 mmol dye JA 51

0.2 mmol HOBT

0.2 mmol DIC

Yield: almost quantitative

HPLC analysis:

Column: Nucleosil RP18, 5 μm, 300 A, 4x250 mm

Gradient: 0-80% acetonitrile (0.1% TFA) in 40 min.

Flow: 1 ml min.

Detection: Abs. At 631 nm

Retention time: 16.5 min.

Mass: calculated 774.8 found 774.6

Example 13:

Digoxigenin 3-O-methylcarbonyl-triglycyl-glycine

Retention time: 15 min.

Approach:

0.2 mmol resin-Gly4-NH2

0.5 mmol digoxigenin-3-O-acetic acid N-hydroxysuccinimide ester

Yield (94 mg) 72%

HPLC analysis: column: Nucleosil RP18, 5 μm, 300 A, 4x250 mm gradient: 0-80% acetonitrile (0.1% TFA) in 40 min. Flow: 1 ml / min.

Detection: Abs. At 215 nm

Retention time: 15 min.

Nucleoside / nucleotide dye conjugates

Example 14

3'-0- [S-Trippphenylmethyl-N ^α - (9- ^fl u ° « ^* enylmethoxycarbonyl) -cysteinyl] -2'- deoxymethymidine (Thymidine-Cys (Trt) (Fmoc)) 6

NH

I.

(Fmoc)

Approach:

0.25 mmol resin thymidine

0.5 mmol (Fmoc) Cys (Trt) OH

Retention time: 28.5 min.

Yield: The cleavage was carried out on an analytical scale

HPLC analysis of the raw product:

Vydac RP18 column, 5 μm, 300A, 4 x 250 mm

Gradient 0-60% ACN (0.1% TFA) 20 '. 60 - 100% 25 * 100% 30 '. 0% 35 '

Flow rate 1 ml / min

Abs at 265 nm

Retention time: 28.5 min Example 15:

3 ^' -0-N- [N ^ε -pentamethylchromyl-N ^α - (9-fluorenylmethoxycarbonyl) -arginyl] - 2' -deoxy-thymidine (thymidine-Arg (Pmc) (Fmoc)) 7

(Fmoc)

(Fmoc)

Approach:

0.25 mmol resin thymidine

0.5 mmol (Fmoc) Arg (Pmc) OH

Yield: The cleavage was carried out on an analytical scale

HPLC analysis of the raw product:

Vydac RP18 column, 5 μm, 300A, 4 x 250 mm

Gradient 0 - 60% ACN (0.1% TFA) 20 ', 60 - 100% 25' 100% 30 ', 0% 35'

Flow rate 1 ml / min

Abs at 265 nm

Retention time 26.7 min. Example 16:

3'-0- [N ^α - (9-fluorenylmethoxycarbonyl) leucinyl] -2 'deoxy-thymidine (Thymidine-Leu (Fmoc)) 8

NH

I.

(Fmoc)

Approach:

0.25 mmol resin thymidine

0.5 mmol (Fmoc) LeuOH

Yield: The cleavage was carried out on an analytical scale

HPLC analysis of the raw product:

Vydac RP18 column, 5 μm, 300A, 4 x 250 mm

Gradient 0 - 60% ACN (0.1% TFA) 20 ', 60 - 100% 25' 100% 30 ', 0% 35'

Flow rate 1 ml / min

Abs at 265 nm

Retention time: 25.5 min. Example 17:

Example 17, compound 9 was built up successively on the support (1. coating of the resin with thymidine, 2. coupling of a (Fmoc) amino acid, 3. deprotection of the amino acid, 4. coupling of the dye).

3 '-O-Glycyl- {N-thiono- [5 (6) -amino-fluoresceinyl]} -2' -deoxy-thymidine (Thymidine-Gly-FITC) 9

Approach:

0.5 mmol resin-thymidine 0.5 mmol (Fmoc) Gly (OH) 1.0 mmol FITC

Yield: The cleavage was carried out on an analytical scale

R _f value (silica gel 60, F ₂₅₄ , Merck): 0.35; CHCl ₃ / MeOH (v / v 9: 1)

The following examples 18-23 were also built up on the support, then cleaved from the resin and further reacted in solution to the triphosphates.

Triphosphate synthesis was carried out according to [Ludwig, Eckstein, J. Org. Chem. 54,

631-635, (1989)]. Example 18:

Fluorescein-5 (6) -amino-thiono- (N-glycyl) - [(3'-amino-2 ^' , 3 ^' -dideoxy) -thymidine- 5'-triphosphate] (3 * -amino-Gly-FITC, 3'-deoxy, 5'-triphosphate

Thymidine) 10

Approach:

0.031 mmol resin-3'amino-3'-deoxy-thymidine

0.047 mmol fluorescein-Gly (OH)

Yield: over all stages (17.4 mg) 60.1%

I ^ value (Kieselgel 60, F ₂₅₄ , Merck): 0.59; Isoprop./NH ₄ OH / water (v / v / v 7: 1: 4)

HPLC analysis of the raw product:

Column Supersphere RP18 (e); 3 µm; 2 x 125 mm degrees. 0-2% B 20 ';

2-4% B 25 '; 4 - 20% B

A = TEAA, pH 7.0; B = ACN flow rate 0.15 ml min. Abs at 265 mn

Retention time: 11.95 min. Example 19:

Fluorescein-5 (6) -amino-thiono- (N-dig.ycyI) - [ ^' -amino -2 \ 3'-dideoxy) -thymidine-

5 ^{'triphosphate]} 3 ^* -amino-Gly ₂ -FITC, 3'-deoxy, 5-triphosphate ^τ T hymidin 11

Approach:

0.031 mmol Haιτ: -3'.Amino-3'-deoxy-Thyπιidin

0.052 mmol fluorescein-Gly ₂ (OH)

Yield over all stages (19.9 mg) 54%

R ^ value (Kieselgel 60, F ₂₅₄ , Merck): 0.29; CHCl ₃ / MeOH (v / v 1: 1)

HPLC analysis of the raw product:

Column Supersphere RP18 (e); 3 µm; 2 x 125 mm degrees. 0-2% B 20 ';

2 - 4% B 25 *; 4 - 20% B

A = TEAA, pH 7.0; B = ACN flow rate 0.15 ml / min. Abs at 265 nm

Retention time: 12.8 min Example 20:

Fluorescein -5 (6) -amino-thiono- (N-tetraglycyl) - [(3 '-amino-2', 3 '-dideoxy) -tl thymidine-5 ^' -triphosphate] (3 ^* -Amino-Gly ₄ - FITC, 3 * deoxy, 5 * triphosphate thymidine) 12

Approach:

0.031 mmol Haι -3'amino-3'-deoxy-thymidine

0.045 mmol fluorescein-Gly ₄ (OH)

Yield over all stages (21.9 mg) 64.2%

R value (silica gel 60, F _2J4 , Merck): 0.53 isoprop./NH ₄ OH water (v / v / v 7: 1: 4)

R ^ value (silica gel 60, F ₂₅₄ , Merck): 0.26 CHCl ₃ / MeOH (v / v 1: 1)

HPLC analysis of the raw product:

Column Supersphere RP18 (e); 3 µm; 2 x 125 mm degrees. 0-2% B 20 ";

2-4% B 25 '; 4 - 20% B

A = TEAA, pH 7.0; B = ACN flow rate 0.15 ml / min. Abs at 265 nm

Retention time: 12.8 min. Example 21:

Rhodamine MR 200-N-methylcarboπyl- (N'-tetraglycyl) - [(3'-amino-2 ', 3'-dideoxy) adenosine-5'-triphosphate]

Approach:

0.027 mmol resin-3 ^* -amino-2 ', 3 ^* -dideoxy-N6-benzoyl-adenosine

0.050 mmol Gly4 -MR200

0.1 mmol HOBT

0.1 mmol DIC

After cleavage from the carrier resin and subsequent triphosphate synthesis, the benzoyl protecting group with conc. Removed ammonia solution.

Yield over all stages: 49% of theory Th.

HPLC analysis:

Column: Hypersil ODS, 5 μm, 120 A, 4x250 mm flow: 1 ml / min gradient: 0-20% B in 10 min 80% B in 20 min 100% B in 25 min Solvent: A: 0.1M TEAA in water

B: Acetonitrile detection: Abs. 617 nm retention time: 18.3 min.

Mass: calculated 1411.9 found 1411.5

Example 22:

Rhodamine 200 -methylcarbonyl- MR ^(N, -tetraglycyl) - ^{[(3 -amino-2,} 3'-dideoxy) -guanosine-5'-triphosphate]

Approach:

0.027 mmol resin-3 ^* -amino-2 ', 3'-dideoxy-N2-isobutyryl-guanosine

0.050 mmol Gly4-MR200

0.1 mmol HOBT

0.1 mmol DIC

After separation from the carrier resin and subsequent triphosphate synthesis, the isobutyryl protective cap was concentrated. Removed ammonia solution.

Yield over all stages: 41% of theory Th.

HPLC analysis:

Column: Hypersil ODS, 5 μm, 120 A, 4x250 mm flow: 1 ml / min gradient: 0-20% B in 10 min 80% B in 20 min 100% B in 25 min Solvent: A: 0.1M TEAA in water

B: Acetonitrile detection: Abs. 617 nm retention time: 18.0 min.

Mass: calculated 1427.9 found 1427.3

Example 23:

Digoxigenin 3-0-methylcarbonyI- (N-tetraglycyI) - [(3'-amino-2 \ 3'-dideoxy) - thymidine-5'-triphosphate]

Approach:

0.03 mmol resin-3'-amino-2 ', 3'-dideoxothymidine

0.06 mmol digoxigenin 3-0-methylcarbonyl-triglycyl-glycine

0.1 mmol HOBT

0.1 mmol DIC

As in Examples 21 and 22, after the condensation had taken place, the carrier resin was cleaved off and the 5'-triphosphate was obtained after phosphorylation.

Yield over all stages: 58% of theory Th.

HPLC analysis:

Column: Hypersil ODS, 5 μm, 120 A, 4x250 mm flow: 1 ml / min gradient: 0-20% B in 10 min 80% B in 20 min 100% B in 25 min

Solvent: A: 0.1M TEAA in water B: acetonitrile

Detection: Abs. 260 nm retention time: 15.2 min.

Mass: calculated 1139.9 found 1139.1 Example 24:

Use of 3'-modified nucleotides for the enzymatic DNA sequencing

The following nucleotides, prepared according to Examples 1 to 6, were used:

3: n = 2 4: n = 4

3: n = 2 4: n = 4

1 μg M13mpl8 single-stranded DNA (5 μl), 2 μl fluorescein-labeled universal primer (1 pmol, Pharmacia LKB), 2 μl Mn buffer I (311 mM Tris • HC1, pH 7.5),

2 μl Mn buffer II (177 mM DTT) and 2 μl Mn buffer Iü (62 mM MnCl ₂ , 460 mM sodium isocitrate) are mixed together and heated to 70 ° C. and then cooled to 25 ° C. (approx. 40 min) . 2 μl of a diluted T7 DNA polymerase (4 units, Pharmacia) are then added to the template and primer heated to 37 ° C. In the meantime, 3 μl of a termination mix (T-mix 1:

150 ul / compound 1; T-mix compound 2: 200 μM II; T-mix compound 3: 300 μM compound 4; each mix also contains: 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 1 mM dTTP, 50 mM NaCl, 40 mM Tris • HC1, pH 7.5) pipetted into four reaction vessels and at least 1 minute at approx. 37 ° C warmed up. Then 3.8 μl of the first mix (annealing mix) are pipetted into the preheated termination mixes. After an incubation of approximately 10 minutes at 37 ° C., 4 μl of a stop reagent (deionized formamide solution, dextran blue) are added to each reaction solution. The reaction solutions are heated for 2 minutes at about 90 ° C and to a 6% denaturing Sequence gel added to the individual pockets (a 6 μl). The individual DNA fragments were then detected and identified using a fluorescence detection device.

FIG. 1 shows the quotient of fluorescence emission F (λ _ex = 488 nm; λ _m = 520 mm) and adsorption A (λ ^ = 265 nm) for compounds 1 - 4 determined during RP-HPLC analysis.

The lowest fluorescence intensity was then obtained for compound 1. The intensity for compounds 2 to 4 increases significantly with the length of the spacer function in the 3 '- (4' -) position.

All four modified nucleotides are accepted as a substrate by the DNA polymerase and show comparable termination quality, such as. B. ddTTP. This is noteworthy since, owing to the bulky residues in the 3 '- (4' -) position of the nucleotides of the compounds 2 to 4 according to the invention, it would have been expected that the compounds would not be recognized and converted by DNA polymerases as substrates.

The triphosphates of rhodamine and digoxigenin prepared according to Examples 20 and 21 are used accordingly.

Abbreviations

correspond to the proposals of the IUPAC-IUB Commission for Biochemical Nomenclature (J. Biochem. 138, (1984), 9; J.Biol. Chem. 247, (1972), 977; Biochemistry 9, (1970), 3471).

ACN acetonitrile AcOH glacial acetic acid

AS amino acid

CDC1 ₃ Deuterochloroform

CH3OH methanol

CHCI3 trichloroethane

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 ₂ 0 dieethyl ether

FITC fluorescein isothiocyanate (5-isomer)

Fmoc 9-fluorenylmethoxycarbonyl

HOBt 1-hydroxybenzotriazole

HPLC high performance liquid chromatography

KCN potassium cyanide

Left anchor group between AS and the Harz

MeOH methanol

MG Molecular Weight ml Müliüter mM Millimolar

Mn manganese nm nanometers

NMR Nuclear Magnetic Resonance

Pmc pentamethylchroman

Rf Relative migration distance of the sample at the DC

RNA ribunucleic acid

RP reversed phase

RT room temperature

Rt retention time

SPPS solid phase peptide synthesis

T thymidine

T7 Escherichia coli Phage T7

Taq Thermococcus aquaticus

TBTU 2- (1H-B enzotriazolyl) - 1, 1, 3, 3 -tetramethyl uronium tetrafluoroborate

TEAA tetraethylammonium ac etat

TFA trifluoroacetic acid

TFE trifluoroethanol

Tris tris (hydroxymethyl) aminomethane

Trt triphenylmethyl (trityl)

TTp 5'-triphosphate thymidine

UV ultraviolet

VIS Visible μl microliters

Claims

Claims 1. Compounds of the general formula (1) base

where: R 'is hydrogen, mono-, di- or triphosphate, Alkylphosphonate, dialkylphosphinate or phosphoramidite X oxygen, nitrogen, carbon or sulfur Y oxygen, nitrogen or sulfur Z is hydrogen, hydroxyl, amino or thiol group R spacer group, to which a non-radioactive, detectable group or an effector molecule is bound, base purine, pyrimidine base or desazapurine, their derivatives, n is 0 or 1. 2. A compound according to claim 1, characterized in that the spacer grouping of the radical R has 2 to 12 atoms. 3. Compounds according to claim 1 or 2, characterized in that radical R has an (aminoacyl) 2-6-G ^ru PPi ^erun g. 4. Compounds according to one of claims 1 or 2, characterized in that the non-radioactive, detectable group is a hapten, fluorophore, metal chelate, lumiphor, protein or an intercalator. 5. Compounds according to claim 4, characterized in that the detectable group is digoxigenin or fluorescein. 6. 3'-Amino- (glygly) 2-6-FITC-3'-deoxy-5'-triphosphate nucleotides. 7. A process for the preparation of the compounds of one of claims 1 to 6, characterized in that compounds of the general formula (2) base Carrier | - (Linker) -X

where: Left is a selectively cleavable anchor group X oxygen, nitrogen, carbon or sulfur Y oxygen, nitrogen or sulfur Z is hydrogen, hydroxyl, amino or thiol group R is hydrogen Base represent purine, pyrimidine base or desazapurine or their derivatives, n is 0 or 1. are bound to a solid support via the linker grouping, a bi- or polyfunctional compound optionally provided with protective groups, then an optionally reactivated, detectable signal component is added, the nucleoside derivative is cleaved from the support matrix and, if appropriate, the 5'- Position. A method according to claim 7, characterized in that the bi- or poly-functional compound is one or more carboxylic acid, aminocarboxylic acid or peptide derivatives. 9. The method according to claim 7 or 8, characterized in that the amino acid component is added in one or two equimolar amounts of the compound (2) and this process is optionally repeated one or more times. 10. The method according to claim 7, characterized in that the linker group is an unsubstituted methoxy or Halogen¬ substituted trityl radical. 11. Use of the compounds of general formula (1) for the sequencing of RNA or DNA sequences. 12. Use of the compounds of general formula (1) for the non-radioactive detection of oligo-, polynucleotides or nucleic acids. 13. Use of the compounds / the general formula (1) as antisense or gene therapeutics.