CA2190982A1 - 3'-(4'-) nonradioactively tagged nucleosides and nucleotides with aminocarboxylic acid, peptide or carboxylic acid spacer - Google Patents

Abstract

Nucleosides or nucleotides of the general formula (1) modified at the 3'(2') position

Description

g~2 3'-(4'-)non-radioactively labelled nucleosides and nucleotides with aminocarboxylic acid, peptide or carboxylic acid spacer~

The invention concerns nucleosides, nucleotides and oligonucleotides modified in the 3'(4') position with non-radioactively labelled groups, a process for their production as well as their use.

Extremely many uses have been found for labelled nucleotides in genetic engineering since it is easier to use them than the DNA probes conventionally used as hybridization probes which are produced from native gene material by restriction digestion.

Modified oligonucleotides which are used in the form of so-called "antisense" DNA oligonucleotides can intervene in a regulatory manner in cellular processes and are thus becoming increasingly important for example for the in vivo investigation of the expression of proteins as well as in cancer, viral and gene therapy. The mechanism in this case involves DNA-DNA, DNA-RNA and RNA-RNA
interactions but still requires a complete detailed elucidation.

Labelled oligonucleotides serve in vitro for example to identify gene fragments within a gene bank by screening and identifying blotted gene samples of the gene bank with the aid of a labelled oligonucleotide.

Apart from radioactive labelling by means of suitable - 213~2 isotopes, derivatised fluorescent dyes have for example been used as a form of non-radioactive labelling which enable easier and safer handling.

Previously such a technique has been used successfully for the non-radioactive sequencing of DNA. In this case approaches which are essentially based on the method of Sanger [F. Sanger, S. Nicklen and S. Coulsen, Proc.
Natl. Acad. Sci. USA 74, 5463 (1977)] have been carried out.

In this method the fluorescent label was either attached to the 5' end of the nucleotide [L.E. Hood, L.M. Smith and C. Heiner, Nature 321, 674 (1986)] or to 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 is associated with the disadvantages that only particular polymerases can be used for the synthesis, the triphosphates are accepted less by the polymerases and that in addition a large substrate excess is necessary.

Chemical sequencing according to Maxam-Gilbert using fluorescent labels 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)].

In addition it is known that a fluorescent dye can be directly coupled via an amino or thiol group in the 3' position of a nucleoside, nucleotide or oligonucleotide and that this compound can be used advantageously for the synthesis of complementary strands in the presence of a template strand as well as for the detection of - 21g~g2 genetic material in vivo and in vitro (EP O 490 281).
However, a disadvantage of these compounds is that the quantum yield is relatively low and the synthesis of the compounds involves tedious and time-consuming chromatographic purification steps.

Therefore the object of the present invention was to provide new non-radioactively labelled nucleosides and nucleotides which do not have the disadvantages of the previously known compounds.

This object is achieved by nucleosides and nucleotides modified at the 3' or 4' position having the following general formula:

Base R'-X- ~

~Z
~ (1) \ R

in which R': represents hydrogen, mono-, di- or triphosphate, alkylphosphonate, dialkyl-phosphinate or phosphoramidite X: oxygen, nitrogen, carbon or sulphur Y: oxygen, nitrogen or sulphur Z: a hydrogen, hydroxyl, amino or thiol group R: denotes a spacer group to which a non-radioactive, detectable group or an effector molecule is bound base: represents a purine or pyrimidine base or a deazapurine or derivatives thereof n: is o or 1.

- ~l9~g82 The spacer group is preferably composed of a protected or unprotected bi-, tri- or polyfunctional carboxylic acid, aminocarboxylic acid or a peptide or corresponding derivatives or salts thereof. All naturally occurring amino acids represent particularly preferred spacers such as for example glycine or aspartic acid, carboxylic acids or aminocarboxylic acids with 2 to 20 atoms, but also diamines, thiols and aminoalcohols.

Di- and tetramers of amino acid units have proven to be especially suitable for spacer groups; particularly when the spacer group has a length which corresponds to a carbon chain of 2 to 12 atoms. All chemically, physically or biologically detectable groups come into consideration as non-radioative, detectable groups, so-called reporter molecules. Such groups are known to a person skilled in the art. In this case so-called effector molecules are also suitable which are capable of chemical, physical or biological interactions with particular target molecules either directly or after chemical, physical or biological activation (such as alkylation agents, ~ systems of aromatic compounds, enzymes etc.). The intercalation with DNA/RNA by acetyl-aminofluorenes, cleavage of DNAtRNA by nucleases or the partial or complete covalent binding of alkyl or aryl groups to DNA/RNA by alkylation agents or psoralens and for example metal clusters which lead locally to a strong increase in the capture cross-section of therapeutically effective electromagnetic radiation are mentioned here as examples. Luminescent dyes which emit in the wavelength range of ca. 630 to 670 nm, enzymes such as peroxidase or alkaline phosphatase and haptens such as for example biotin/iminobiotin or digoxigenin come into particular consideration as reporter molecules. In this connection fluorophores have proven 21~0~82 to be particularly suitable for example for sequencing according to Sanger. Compounds substituted with effector groups are of particular value especially for antisense therapy.

The OH group, amino or thiol group located at the 3' and/or 4' position of a nucleoside, nucleotide or oligonucleotide is coupled to a spacer group such as for example an aminocarboxylic acid, carboxylic acid or a peptide and this is subsequently linked to a reporter or effector molecule. The 3'- or 4'- hydroxyl-, amino- or thiol-modified nucleosides, nucleotides and oligonucleotides that are formed can then be used for the synthesis of complementary strands in the presence of a natural or synthetic template strand as well as for the detection of particular sequences or to localize an effector molecule at particular sequences in the genetic material. They have in particular the advantage that the modification is no longer attached to the 5' end of the nucleotide or to the nucleobase as in previously known labelling techniques.

The invention in addition concerns a process for the production of the 3'(4'-)modified nucleosides and nucleotides according to the general formula (1).
Compounds of the general formula (2) B~e carrier - ~ker)-X - l - '/0~~

~ (2) 21gO~82 in which: linker: represents a selectively cleavable anchor group X: represents oxygen, nitrogen, carbon or sulphur Y: represents oxygen, nitrogen or sulphur Z: represents a hydrogen, hydroxyl, amino or thiol group R: represents hydrogen base: represents purine or pyrimidine base or a deazapurine or derivatives thereof n: is O or 1.

are bound via the linker group to a solid carrier a bifunctional or polyfunctional compound provided if necessary with protecting groups is added, subsequently a detectable signal component which is reactivated if necessary is added, the nucleoside derivative is cleaved from the carrier matrix and, if desired, it is derivatized in the 5' position.

The reaction to form nucleosides bound via a linker in the 5'(6') position to a solid carrier can in addition be achieved by other methods known to a person skilled in the art. All selectively cleavable anchor groups can be used as the linker group [e.g. G.B. Fields and R.L.
Noble, Int. J. Peptide Prot. Res. 35 (1990), 165 - 171].
Groups which can be cleaved under mild conditions such as for example photochemically, by hydrogenation or in the acid or alkaline range have proven to be particularly suitable. Preferred linker groups are trityl groups which can be substituted if desired.
Methoxy and halogen residues are suitable substituents in this case. Dimethoxy and o-chlorotrityl groups have proven to be particularly suitable in this case. In 2 ~ 8 2 addition the basic procedure for linking the linker group to the 5' or 6' position of nucleosides is well-known to a person skilled in the art (M.J. 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 the respective case and which should be as easy to filter as possible is suitable according to the invention as the carrier material.
Preferred carrier materials are inorganic polymeric materials (resins) based on polystyrene or polyethylene glycol.

Nucleosides bound in this manner to a solid carrier are subsequently admixed with an appropriate carboxylic acid, aminocarboxylic acid or peptide derivative in the presence of an activation reagent such as for example condensation agents such as DCC, DIC or HOBT.

After linking the aminocarboxylic acid, peptide or carboxylic acid or their derivatives or salts to the nucleoside or analogue thereof bound to the solid phase, one or several, identical or different reporter/effector molecules are introduced covalently and selectively into the side chain or C or N terminus, for example by means of esterification, amidation, sulfonamidation, sulfonic acid esterification or by reaction with isothiocyanates or N-hydroxysuccinimide esters. Appropriate methods are known to a person skilled in the art.

Compounds and classes of compounds which come into consideration as reporter molecules or effector molecules are those which can be covalently linked to a 219~g~2 hydroxy, amino or thiol group (see above).

The cleavage of the nucleoside derivatives from the linker group is preferably carried out under weakly acidic, basic, photochemical or reductive conditions.

Nucleoside derivatives which have one or several protecting groups or side chains on the spacer group can be produced in a corresponding manner.

The nucleoside derivatives produced in this manner can be subsequently converted into the corresponding 5' or 6' mono-, di- or triphosphates (nucleotides), alkyl-phosphonates, dialkylphosphinates or phosphoramidite according to methods known to a person skilled in the art.

The synthesis according to the invention of the 5' and 6' modified nucleosides is particularly advantageous because - in contrast to the well-known processes - a large excess of the dye components can be used without problems and easily separated. In the case of the previously known processes tedious and expensive chromatographic purification steps follow the actual production process in order to separate the non-reacted dye.

The compounds according to the invention having the general formula (1) in which residue R' represents a triphosphate group can be used advantageously as substrates for DNA/RNA polymerases with the aid of a primer and a template strand and in the presence of the four nucleoside triphosphates. Suitable polymerases in this case are T7 polymerase, Taq polymerase, DNA

g polymerase I and reverse transcriptases. In addition the compounds are suitable as terminators in enzymatic DNA/RNA sequencing. In this case termination of the synthesis can be specifically determined in each case by the use of a 3'-(4'-)modified A, C, G or T nucleotide of formula (1). This is of particular importance for the synthesis of DNA complementary 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 is achieved in an analogous manner.

In addition it is also possible to synthesize derivatizable oligonucleotides with the aid of a start nucleotide which carries a spacer group at its 3'(4') end and which has been amino-modified or thio-modified and immobilized on a polymeric carrier.

Oligonucleotides are understood as all DNA and RNA
nucleotides produced in a conventional manner preferably, however, having a length of 2 to 100, particularly preferably of 12 - 50 nucleotides (chemical synthesis) or having a length of up to ca. 3000 nucleotides (enzymatic synthesis) depending on the efficiency of the polymerase used.

The oligonucleotide synthesis is carried out starting with the start nucleotide-carrier complex in the conventional sense i.e. in the 3' and 5' direction and enables the synthesis of an oligonucleotide with a defined sequence.

-- 21~82 The immobilization of the start nucleoside to commercial carriers is achieved via a connecting arm (spacer) which can be cleaved after the synthesis; for example by means of the succinic acid linkage known in the literature or by means of a linkage with urethane (Efimov et al., Nucl. Acids. Res. 11, 8369, 1983).

After the synthesis is completed the oligonucleotide must be cleaved from the carrier using suitable reagents. Derivatization with any desired fluorescent dye is carried out directly afterwards.

It is also possible to synthesize oligonucleotides in the opposite direction (5'~ 3') by linking the start nucleoside via the 5' OH group to the linker group of the carrier material and then proceeding according to conventional oligonucleotide synthesis. In this case the labelling of the oligonucleotide with dye can be achieved on the solid phase.

The oligonucleotides modified at their 3'(4') end and synthesized in this manner can then be used to detect for example complementary oligonucleotides or nucleic acids and corresponding derivatives.

The compounds according to the invention are, however, also suitable as in vivo and in vitro sensors for analysis and as gene probes in gene therapy.

The invention is elucidated further by the following examples:

- 11- 21~82 ExamPle 1:

Esterification of Fmoc-amino acid derivatives with 2-chlorotrityl resin 1 gram resin which is loaded with 1.1-1.6 mmol 2-chloro-trityl chloride linker [K. Barlos, O. Chatzi, D. Gatos, G. Stauropoulos, Int. J. Peptide Prot. Res. 37, (lg91), 513 - 520] is shaken for several minutes in absolute DCM. After addition of 3 mmol/l DIEA, 1.2 mmol Fmoc-protected amino acid is added. The reaction period is 90 minutes. After addition of a further 3 mmol DIEA, the unesterified linker function is etherified with 5 ml absolute methanol within approximately 30 minutes. The coated resin is filtered 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 a quantitative ninhydrin test (example 7b) or by using W spectrometric determination of the Fmoc cleavage. Amino acid loadings between 0.05 and 1.1 mmol/g is obtained by varying the amount of amino acid.

Example 2:

Na-linking of fluorescent dyes to resin-bound amino acids or peptides For dyes with carboxy groups the following applies:

3 equivalents dye, 3.3 equivalents DIC and 4.5 equivalents HOBt are added per equivalent of resin-bound amino acid or peptide in a shaking flask and shaken for - 2~ gV9~2 50 hours at room temperature in DMF/DCM in the dark.
Subsequently it is washed several times with pure DMF, DCM, methanol, isopropanol and ether and the amino acid or peptide conjugate is cleaved from the solid carrier as described below.

3 equivalents of the fluorescent dye, 1.2 equivalents DIEA and a catalytic amount of DMAP (4,4'-dimethylamino pyridine) are reacted per equivalent of the resin-bound, N-terminally-deprotected peptide or amino acid to be labelled in a solution of DMF/DCM (v/v 4:1) in a shaking flask with a frit for 48 hours. In order to avoid decomposition reactions of the dyes, it is shaken in the dark. The reaction course can be monitored using the ninhydrin reaction or TLC. If free amino functions are still present, then it can be acetylated with Ac20/DIEA
(equivalents/equivalents 1:2). Non-reacted dye is removed by filtration and it is washed several times with 10 ml in each case 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 a refrigerator.

Orthogonality of the protecting groups also allows one or several identical or different fluorescent dyes to be introduced selectively into the side chain or N-terminus. The introduction of fluorescent dyes into the side chain of trifunctional amino acids is carried out analogously to the N~-linking, but the proportion of DMAP has to be increased.

- 21gV~82 Example 3 Cleavage from 2-chlorotrityl resin to prepare fluorescent-labelled amino acids or fluorescent-labelled peptides The ester cleavage is carried out under weakly acidic conditions. Approximately 20 ml of a mixture of glacial acetic acid/TFE/DCM in a ratio of (v/v/v 1:2:7) is used per 1 g peptide resin [K. Barlos, O. Chatzi, D. Gatos, G. Stauropoulos, Int. J. Peptide Prot. Res. 37, (1991), 513 - 520]. The cleavage period is usually approximately 90 minutes. If no His(Nim Trt) residue is present, then it can be cleaved with a mixture of DCM/TFE (v/v 1:1).
Subsequently the resin is removed by filtration and the peptide solution is filled up with approximately 100 ml water. The added water prevents the concentration of acetic acid in the subsequent removal of the readily volatile components by distillation on a rotary evaporator. The hydrophobic amino acid and peptide derivatives are precipitated during the rotary evaporation and are then dried in a freeze dry apparatus.

Example 4:

Etherification of nucleosides via the 5'-OH group using the 2-chlorotrityl resin 1 gram resin which is loaded with 1.1-1.6 mmol 2-chloro-trityl chloride linker is shaken for several minutes in a mixture of CHCl3/DMSO (v/v 1:2). After addition of 2.5 mmol DIEA, 0.52 mmol nucleoside is added. The reaction period is ca. 120 minutes. After addition of a further 21g~

3 mmol DIEA the non-etherified linker function is etherified with methanol within 30 minutes using 5 ml absolute methanol. The coated resin is filtered and washed several times with DMF, DCM, isopropanol and finally with diethyl ether. After drying in an oil-pump vacuum, the loading of the resin is determined using the quantitative ninhydrin test, in the case of the 3'-amino nucleotides (example 7b) by W spectrometric determination after suitable derivatization of the 3' function of the nucleoside or by weighing the resin. By varying the amount of nucleoside, resin loadings between 0.1 and 1 mmol/g is obtained.

Example 5:

Coupling of amino acids, peptides and carboxylic acids and their derivatives to carrier-bound nucleosides The coupling is carried out according to the activation methods known from peptide chemistry (period: ca. 12 hours). The use of base-labile protecting groups enables the extension of the amino acid chains or fragment condensations according to the well-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].

,~ .

21gO98~

Exam~le 6:

Cleavage of nucleosides, nucleoside-amino acids, nucleoside-peptides and their dye derivatives from 2-chlorotrityl resin The ether cleavage is carried out under acidic conditions. Approximately 10 ml of a solution of dichloroacetic acid/DCM in a ratio of (v/v 3:97) is used per l g coated resin. The cleavage period is ca. 1 minute. Subsequently the resin is removed by filtration and the product solution is neutralized immediately with an equimolar solution of DIEA/DCM in a ratio of (v/v 63:937). The resin is washed several times with a small amount of ACN. Ca. 10 ml water is added. The product is now dried in a freeze drying apparatus.

Example 7:

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

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.

21g~82 a) Qualitative ninhydrin test In order to test whether an acylation has proceeded quantitatively, two to three drops of the three solutions are reacted in each case with a microspatula-tip full of the coated resin in an Eppendorf tube.
The reaction mixture is heated for about 5 minutes in a water-bath to 100C. If the acylation reaction is not completed then the solution has a deep dark-blue colour whereas in the case of quantitative coupling reactions the reaction mixture retains its yellow colour.

b) Quantitative ninhydrin test In order to quantitatively determine the loading of the resin with amino acid, the Fmoc-amino acid derivatives are deprotected for 40 minutes with piperidine/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 to 100C with solutions 1 (4 drops), 2 (8 drops) and 3 (4 drops) in a thermo-heating block for 7 minutes. The reaction mixture is diluted immediately with 60 ~ ethanol and transferred to a measuring flask of suitable size.

After calibrating the W spectrometer with 60 ~ ethanol, the absorbance is determined ~2lsa~s2 -and the resin loading in ~mol/g is determined by entering the appropriate values in the following equation.

[absorbance x dilution (ml)]
Loading ~mol/g] = x 106 extinction coefficient x weighed mass (mg) The compounds of examples 8-23 set forth in the following were synthesized according to the general conditions of examples 1 - 6.

Amino acid/peptide-dye conjugates 2190~8~

Example 8:

Fluorescein-5(6)-aminothiono-N-glycine (FlTC-Gly(OH))3 HO~N\~S

~ OOH

O ~ OH

Preparation:
0.17 mmol (673 mg) resin-gly-NH2 0.5 mmol (193 mg) FITC
Yield (77.9 mg) 91 %

HPLC ana lYS i S:
Column Nucleosil RP18, 5 ~m, 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 minutes.

'~lg~gB~

ExamPle 9:

Fluorescein-5(6)-aminothiono-N-glycyl-glycine (FlTC-Gly2(0H))4 HO~ N~S x = 2 H~ `

~ OOH

O ~ OH

Preparation:
0.43 mmol resin-gly2-NH2 1.3 mmol (504 mg) FITC
Yield (220.4 mg) 89 %

HPLC analYsis:
Column Nucleosil RP18, 5 ~m, 300A, 4 x 250 mm Gradient 20 - 100 % ACN (0.1 % TFA) 40' Flow rate 1 ml/min. Abs. at 287.5 and 425.0 nm.
Retention time: 8.00 minutes.

Z190$8Z

Exam~le 10:

Fluorescein-5(6)-aminothiono-N-triglycyl-glycine (FlTC-Gly4(0H))5 ~ OOH

O ~ OH

Preparation:
0.2 mmol resin-gly4-NH2 0.5 mmol (195 mg) FITC
Yield (77 mg) 74 %

HPLC ana 1YS i S:
Column Nucleosil RP18, 5 ~m, 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 minutes.

-Exam~le 11:

Rhodamine MR 200-N methylcarbonyl-N'-triglycyl-glycine o o HotC~,NH~ IC2H5 =~<

Cl~COOH
CI~CI
Cl Preparation:
44.4 mg resin-gly4-NH2 (corresponding to ca. 0.02 mmol) 30 mg dye MR 200, free carboxylic acid 8.0 mg HOBT
6.3 ~l DIC

Yield: quantitative HPLC ana lYs i s:
Column: Nucleosil RP18, 5 ~m, 300 A, 4 x 250 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 TLC: silica gel, CHCl3/MeOH l:l:Rf 0.55 Mass: calculated 943.7 found 943.5 s~ectral data: ~maX,EX615 nm, ~aX,Em642 nm (in MeOH) 219~2 Example 12:

Rhodamine JA 51-N-propylcarbonyl-N'-triglycyl-glycine O o HOtC NHtC~ Cl 02CH3 4 (CH2)3 (CH2)3 ,t Pre~aration:
0.047 mmol resin-gly4-NH2 0.141 mmol dye JA 51 0.2 mmol HOBT
0.2 mmol DIC

Yield: nearly quantitative HPLC analysis:
Column: Nucleosil RP18, 5 ~m, 300 A, 4 x 250 mm Gradient: 0-80 % acetonitrile (0.1% TFA) in 40 min Flow: 1 mllmin Detection: Abs. at 631 nm Retention time: 16.5 min Mass: calculated 774.8 found 774.6 219~
_ Example 13:

Digoxigenin 3-0-methylcarbonyl-triglycyl-glycine o ~o OH
1 C~/
CH
O
HO ~ ~ ~ OH

Retention time: 15 minutes Preparation:
0.2 mmol resin-gly4-NH2 0.5 mmol digoxigenin-3-O-acetic acid-N-hydroxy-succinimide ester Yield (94 mg) 72 %

HPLC analysis:
Column: Nucleosil RP 18, 5 ~m, 300 A, 4 x 250 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 - 21g~982 Example 14:

3'-0-[S-triphenylmethyl-Na-(9-fluorenylmethoxycarbonyl)-cysteinyl]-2'-deoxy thymidine (thymidine-Cys(Trt)(Fmoc)) 6 ~ CH3 o~N~
HO -O
(Trt) ~S ~CH2 -f 6 ~moc) Preparation:
0.25 mmol resin-thymidine 0.5 mmol (Fmoc)Cys(Trt)OH
Retention time: 28.5 min Yield: Cleavage was achieved on an analytical scale HPLC analysis of the crude product:
Column Vydac RP 18, 5 ~m, 300 A, 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 - 2~g~82 ExamPle 15:

3'-0-N-[N-pentamethylchromyl-Na-(9-fluorenylmethoxycarbonyl)-arginyl]-2'-deoxy thymidine (thymidine-Arg(Pmc)(Fmoc)) 7 ~ H3 o~NJ
HO~

~mc) H
EDN- C -~nH - (CH~)3 -~nH NH
I

~moc) Preparation:
0.25 mmol resin-thymidine 0.5 mmol (Fmoc)Arg(Pmc)OH
Yield: Cleavage was achieved on an analytical scale HPLC analysis of the crude product:
Column Vydac RP 18, 5 ~m, 300 A, 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 - 2~98~

Example 16:

3'-0-lNa-(9-fluorenylmethoxycarbonyl)-leucinyl]-2'-deoxy-thymidine (thymidine-Leu(Fmoc)) 8 ~CH3 ol~NJ
HO

O
(CH3)2 --CH--CH2 --Cl ~

~moc) Preparation:
0.25 mmol resin-thymidine O.5 mmol (Fmoc)LeuOH
Yield: Cleavage was achieved on an analytical scale HPLC analysis of the crude product:
Column Vydac RP 18, 5 ~m, 300 A, 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 - 2~9~g~

Example 17:

Example 17, compound 9 was successively synthesized on a carrier (1. Coating of the resin with thymidine, 2.
Coupling a ~Fmoc)-amino acid, 3. Deprotecting the amino acid, 4. Coupling the dye).

3'-0-glycyl~N-thiono-[5(6)-amino-fluoresceinyl]}-2'-deoxy-thymidine (thymidine-gly-FlTC) 9 ~ H3 HO o~N

O ~S

r OOH

O ~ OH

Preparation:
0.5 mmol resin-thymidine 0.5 mmol (Fmoc)gly(OH) 1.0 mmol FITC

Yield: Cleavage was achieved on an analytical scale Rf value (silica gel 60, F254, Merck): 0.35; CHC13/MeOH
(v/v 9: 1) .
The following examples 18-23 were also synthesized on a carrier, subsequently cleaved from the resin and further ~ 19~

reacted in solution to form 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-FlTC, 3'-deoxy, 5'-triphosphate-thymidine) 1 0 O
~ _,CH3 H~
(4 ) 09P30~

HN~N~S

H ~

~ OOH

O ~ OH

Preparation:
0.031 mmol resin-3'amino-3'-deoxy-thymidine 0.041 mmol fluorescein-gly(OH) Yield: over all steps (17.4 mg) 60.1 %
Rf value (silica gel 60, F254 Merck): 0.59; isoprop./
NH40H/water (v/v/v 7:1:4) HPLC analysis of the crude Product:
Column Supersphere RP18(e); 3 ~m; 2 x 125 mm Gradient 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.

Z~9~98~

ExamPle 19:

Fluorescein-5(6)-amino-thiono-(N-diglycyl)-1(3'-amino-2',3'-dideoxy)-thymidine-5'-triphosphate] (3'-amino-Gly2-FlTC,3'-deoxy,5'-triphosphate-thymidine) 1 1 ~ _,CH3 (¢)OgP3O ~ '~

H~N ~ ~ S x=2 ~`~
`r OOH

O~OH

Preparation:
0.031 mmol resin-3'amino-3'-deoxy-thymidine 0.052 mmol fluorescein-gly2(OH) Yield: over all steps (19.9 mg) 54 %
Rf value (silica gel 60, F254 Merck): 0.29; CHCl3/MeOH
(v/v 1: 1) HPLC analysis of the crude Product:
Column Supersphere RP18 (e); 3 ~m; 2 x 125 mm Gradient 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: 12.8 min.

- ~g~98~

Example 20:

Fluorescein-5(6)-amino-thiono-(N-tetraglycyl)-[(3'-amino-2',3'-dideoxy)-t]-thymidine-5'-triphosphate]
(3'-amino-Gly4-FlTC,3'-deoxy,5'-triphosphate-thymidine) 1 2 (¢) gP30~

~HN~bS X = 4 H~ , ~COOH

O~OH
Preparation:
0.031 mmol resin-3'amino-3'-deoxy-thymidine 0.045 mmol fluorescein-gly4(OH) Yield: over all steps (21.9 mg) 64.2 %
Rf value (silica gel 60, F254 Merck): 0.53 isoprop./
NH40H/water (v/v/v 7:1:4) Rf value (silica gel 60, F254 Merck): 0.26 CHCl3/MeOH
(v/v 1: 1) HPLC analYsis of the crude product:
Column Supersphere RP18 (e); 3 ,um; 2 x 125 mm Gradient O - 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: 12.8 min.

9 ~

Example 21:

Rhodamine MR 200-N-methylcarbonyl-(N'-tetraglycyl)-1(3'-amino-2',3'-dideoxy)-adenosine-5'-triphosphate]

. ~NJ
09PJO~

HN~C NH~
~CH2 C2H~
~' C~COOH
C~CI
Preparation:
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 was removed with concentrated ammonia solution.

Yield over all steps: 49 % of theory ` ~l9~g82 HPLC ana lYs i s:
Column: Hypersil ODS, 5 ~m, 120 A, 4x2S0 mm Flow: 1 ml/min Gradient: 0-20 % B in 10 min 80 % B in 20 min 100 % B in 25 min Solvent: A: 0.1 M TEAA in water B: acetonitrile Detection: Abs. 617 nm Retention time: 18.3 min Mass: calculated 1411.9 found 1411.5 Example 22:

Rhodamine MR 200-N-methylcarbonyl-(N'-tetraglycyl)-1(3'-amino-2'.3'-dideoxy)-guanosine-5'-triphosphate] l ~ NH2 gP30~o \J
O O
HI~C NHt4C~ C2H5 Cl~COOH
C~CI
Cl Preparation:
0.027 mmol resin-3'-amino-2',3'-dideoxy-N2-isobutyryl-2 1 ~
-guanosine 0.050 mmol Gly4-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 concentrated ammonia solution.

Yield over all steps: 41 % of theory HPLC analysis:
Column: Hypersil ODS, 5 ~m, 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: 0.1 M TEAA in water B: acetonitrile Detection: Abs. 617 nm Retention time: 18.0 min Mass: calculated 1427.9 found 1427.3 ~19~98~

Example 23:

Digoxigenin 3-0-methylcarbonyl-(N'-tetraglycyl)-~(3'-amino-2',3'-dideoxy)-thymidine-5'-triphosphate]

0~ ~0 ~ CH ~
O ~
H ~ ~ O

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 the condensation was completed it was cleaved from the carrier resin as in examples 21 and 22 and the 5'-triphosphate was obtained after the phosphorylation.

Yield over all steps: 58 % of theory 21g~g~2 HPLC analysis:
Column: Hypersil ODS, 5 ~m, 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: 0.1 M 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 nucleotideQ for enzymatic DNA
sequencing The following nucleotides were used that had been prepared according to examples 1 to 6:
H3C a ~r ~
~N~_ 4-o9p30_1 o - J U
OOC ~C) ~\NH3'n NH

l:n=O (\ ~
2:n=l ~o 3:n=2 4:n=4 Z1~0~8Z

1 ~g M13mpl8 single-stranded DNA (5 ~1), 2 ~1 fluorescein-labelled universal primer (1 pmol, Pharmacia LKB), 2 ~1 Mn buffer I (311 mM Tris HCl, pH 7.5), 2 ~1 Mn buffer II (177 mM DTT) and 2 ~1 Mn buffer III (62 mM
MnC12, 460 mM sodium isocitrate) are mixed together and heated to 70C and then cooled to 25C (ca. 40 min).
2 ~1 of a diluted T7 DNA polymerase (4 units, Pharmacia) are then added to the template and primer heated to 37C. In the meantime 3 ~1 of a termination mixture (T-mix 1: 150 ~l/compound 1; T-mix compound 2: 200 ~M II;
T-mix compound 3: 300 ~M compound 4; each mixture in addition contains: 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 1 mM
dTTP, 50 mM NaCl, 40 mM Tris HCl, pH 7.5) is pipetted into four reaction vessels and heated for at least 1 minute to ca. 37C. Subsequently 3.8 ~1 of the first mixture (annealing mixture) is immediately pipetted into the preheated termination mixtures. After an incubation of ca. 10 minutes at 37C, 4 ~1 of a stop reagent (deionised formamide solution, Dextran blue) is added to each reaction solution. The reaction solutions are heated for 2 minutes at ca. 90C and placed in the individual slots (6 ~1 of each) of a 6 % denaturing sequencing gel. Subsequently the individual DNA
fragments were detected and identified using a fluorescence detection instrument.

Figure 1 shows the quotient of fluorescence emission F (~ex = 488 nm; ~em = 520 nm) and absorbance A (~abs =
265 nm) which was determined for the compounds 1 - 4 during the RP-HPLC analysis.

According to this the fluorescence intensity was lowest for compound 1. The intensity for compounds 2 to 4 significantly increases with the length of the spacer function at the 3'(4') position.

Z1 9~2 All four modified nucleotides are accepted as a substrate by the DNA polymerase used and they have a termination quality that is comparable for example to ddTTP. This is remarkable since, due to the bulky residues at the 3'(4') position of the nucleotides of compounds 2 to 4 according to the invention the compounds would not have been expected to be recognized and converted as substrates by DNA polymerases.

The triphosphates of rhodamine and digoxigenin prepared as described in examples 20 and 21 are used accordingly.

~9~2 Abbreviation~

According 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 AA amino acid CDCl3 deuterochloroform CH30H methanol CHCl3 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 Et20 diethyl ether FITC fluoroescein isothiocyanate (5-isomer) Fmoc 9-fluorenylmethoxycarbonyl HOBt 1-hydroxybenzotriazole HPLC high performance liquid chromatography KCN potassium cyanide linker anchor group between AA and the resin MeOH methanol MW molecular weight ml millilitre mM millimolar 219~g8~

Mn manganese nm nanometre NMR nuclear magnetic resonance Pmc pentamethylchromane Rf relative migration path of the sample in TLC
RNA ribonucleic 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-(lH-benzotriazol-lyl)-1,1,3,3-tetramethyl-uroniumtetrafluoroborate TEAA tetraethylammonium acetate TFA trifluoroacetic acid TFE trifluoroethanol Tris Tris(hydroxymethyl)aminomethane Trt triphenylmethyl(trityl) TTp 5'-triphosphate-thymidine W ultraviolet VIS visible ~1 microlitre

Claims (13)

Claims

1. Compounds of the general formula (1) (1) in which R': represents hydrogen, mono-, di- or triphosphate, alkylphosphonate, dialkylphosphinate or phosphoramidite X: represents oxygen, nitrogen, carbon or sulphur Y: represents nitrogen or sulphur Z: represents a hydrogen, hydroxyl, amino or thiol group R: represents a spacer group to which a non-radioactive, detectable group or an effector molecule is bound base: represents a purine or pyrimidine base or a deazapurine or derivatives thereof n: is 0 or 1.

2. Compound as claimed in claim 1, wherein the spacer group of residue R has 2 to 12 atoms.

3. Compounds as claimed in claim 1 or 2, wherein the residue R has an (aminoacyl)2-6 group.

4. Compounds as claimed in one of the claims 1 or 2, wherein the non-radioactive, detectable group is a hapten, fluorophore, metal chelate, lumiphore, protein or an intercalator.

5. Compounds as claimed in claim 4, wherein the detectable group is digoxigenin or fluorescein.

6. 3'-amino-(glygly)2-6-FITC-3'-deoxy-5'-triphosphate nucleotides.

7. Process for the production of compounds as claimed in one of the claims 1 to 6, wherein compounds of the general formula (2) (2) in which linker: represents a selectively cleavable anchor group X: represents oxygen, nitrogen, carbon or sulphur Y: represents oxygen, nitrogen or sulphur Z: represents a hydrogen, hydroxyl, amino or thiol group R: represents hydrogen base: represents a purine or pyrimidine base or a deazapurine or derivatives thereof n: is 0 or 1 are bound via the linker group to a solid carrier a bifunctional or polyfunctional compound provided if necessary with protecting groups is added, subsequently a detectable signal component which is reactivated if necessary is added, the nucleoside derivative is cleaved from the carrier matrix and, if desired, it is derivatized in the 5' position.

8. Process as claimed in claim 7, wherein the bifunctional or polyfunctional compound is one or several carboxylic acid, amino carboxylic acid or peptide derivatives.

9. Process as claimed in claim 7 or 8, wherein the amino acid component is added to compound (2) in a single or double equimolar amount and this process is repeated once or several times if desired.

10. Process as claimed in claim 7, wherein the linker group is an unsubstituted methoxy or halogen-substituted trityl residue.

11. Use of compounds of the general formula (1) for sequencing RNA or DNA sequences.

12. Use of compounds of the general formula (1) for the non-radioactive detection of oligonucleotides, polynucleotides or nucleic acids.

13. Use of compounds of the general formula (1) as antisense or gene therapeutic agents.