DD287040A5 - NEW FLUORPHOR DERIVATIVES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE FOR THE PREPARATION OF NOVEL FLUORESCENCE-MARKED NUCLEOSIDES, NUCLEOTIDES AND OLIGONUCLEOTIDES - Google Patents

Abstract

The invention relates to a method for producing novel fluorescently labeled nucleosides, nucleotides and oligonucleotides which can be used in genetic engineering and molecular biology as probes and primers, in particular for manual and automatic sequence analysis. According to the invention, fluorophore derivatives of general formula I: FO (X) n R (I) are substituted with F fluorescent dye, X CH 2, n 1 to 18, R phosphate, substituted phosphate, phosphoramidite, substituted phosphoramidite, H-phosphonate, substituted phosphonate, thiophosphate thiophosphate; Phosphoramidate, substituted phosphoramidate under the usual conditions for the chain construction of oligonucleotides with nucleosides, nucleotides or oligonucleotides directly to compounds of general formula II, with the meaning explained for F, X and n, and YO, S, NH and R1 3- and 5- Oligonucleotide or nucleotide or nucleoside reacted. Formula III {fluorophore derivatives; fluorescein; Fluorescein-O-hydroxyalkyl ether; phosphorylation; Phosphitilierungsmittel; phosphonylating; fluorescently labeled nucleotides; Nucleosides and oligonucleotides}

Description

I - O - (Xi ₁₁ - Y - Γ - · - π - ίϊ _ν (II)

with the meaning denoting F, X, and η and for Y = O, S, NH and R ₁ = 3 'or 5' oligonucleotide or nucleoside or nucleotide. 2. The method according to claim 1, characterized in that Fluorophorphosphortriester, phosphoramidites, -H-phosphonates, -thiophosphite and -thiophosphate be used.

Field of application of the invention

The invention relates to a method for producing novel fluorescently labeled nucleosides, nucleotides and oligonucleotides which can be used in genetic engineering and molecular biology as probes and primers, in particular for manual and automatic sequence analysis.

Characteristic of the known state of the art

Previous methods for the chemical non-radioactive labeling of oligonucleotides with Ruorophoren require the introduction of special Ar.kergruppen, which are then reacted with well-known reactive derivatives of fluorophores> m following the actual Ollgonucleotldsynthese covalent binding of the dye. These anchor groups include amino groups at the 5'-end of the oligonucleotide (Smith, L.M., Fung, S., Hunkapiller, MW, Hunkapiller, T.J., and Hood, S., Nucl. Acids Res., 13 [1985 ] 2399; WO 88/00201, Smith, LM; Fung, S., and Kaiser, RJ Jr.) Describe various alkylamino groups which are linked via a phosphodiester bond (Tanaka, T. Sakata, T., Fujimoto, K., and Ikehara, M.W. Nucl. Acids Res. 15 [1987] 6209; DE-OS 3642939 Sproat, B.) or the heterocyclic base (Sarfati, SR; Pochet, S., Cureito, C; Nansame, A .; Hynh-Dinh, T , and Igolen, J .; Tetrahedron 43 (1987) 3491) into the oligonucleotide. Binding via sulfhydryl groups is also described (OE-OS 3642939, Sproat, B). Another variant for labeling oligonucleotides or natural DNA is the synthesis of fluorescent nucleoside triphosphates (Prober, JM, Trainer, GL, Dans, RJ, Hobbs, FW, Robertson, CW, Cagursky, RJ, Cocuzza, AJ, Jensen, MA and Baumeister, CW, Science 238 (1987) 336) and their enzymatic incorporation on a DNA template using an oligonucleotide primer. The listed methods for fluorescence labeling of oligonucleotides or DNA require in addition to the effort to synthesize these compounds, additional experimental work to introduce anchor groups or to represent the spacer when coupled via a Aminoalkylphosphorsäurediesterbindung. The synthesis of the fluorescent nucleoside triphosphates for enzymatic incorporation also requires considerable experimental effort. The disadvantage is w? ^! terhin that the oligonucleotide to be labeled must be gelelekfrophoretisch purified after the actual synthesis and after the conversion with the corresponding dye again on a Ausschlusschromatographie of unreacted dye and unlabeled oligonucleotide must be separated. This requires a lot of time.

Object of the invention

The object of the invention is to label nucleosides, nucleotides and oligonucleotides in good yields with fluorophores at the expense of goring, without the use of special anchor groups in the course of normal oligonucleotide synthesis.

Explanation of the essence of the invention The invention is based on the object, with the aid of novel synthesis building blocks, a process for the preparation of nonradioactive

to develop labeled nucleosides, nucleotides and oligonucleotides. The introduction of novel fluorophore derivatives is made in a straightforward way, without the use of special anchor groups or special protection group techniques in the course of

Oligonucleotldsynthese. It has been found that the fluorophore derivatives of general formula I

FO- (X) _n -R (I)

F = fluorescent dye

η = Ibis 18

R s = phosphate, substituted phosphate, phosphoric acid, substituted phosphoramidite, H-phosphonate, substituted

H-phosphonate, thiophosphate, substituted thiophosphate, phosphoramidate, substituted phosphoramidate under conditions customary for the chain construction of oligonucleotides with nucleosides nucleotides or oligonucleotides directly in the manual, semi-automatic and fully automatic synthesis regime to give compounds of general formula II

ci

Il

: · - i) iX) _t , - V-P-Ok ₁ dl)

II ...

with the meaning explained for F, X and η and for Y = 0, S, NH and R, = 3'- or 5'-oligonucleotide, nucleoside or nucleotide.

The compounds of the general formula I can be prepared by reacting fluorophores of the general formula III

FO- (X) _n -YH (ill)

with the meaning explained for F, X, η and Y with customary phosphorylation, phosphitylation or

Phosphonylating be obtained. Dio Preparation of the new fluorophore derivatives of general formula I is the subject of a separate patent application. The fluorescent dye in compound I is preferably fluorescein or fluorescoin O-hydroxyalkyl ether with a Alkyl spacers of 1 to 18 methylene groups, preferably 1 to 10 methylene groups, in particular 4 to 6 methylene groups. For the phosphylation of the fluorophores, preference is given to p-chlorophenylphosphoric acid ditriazolide, for phosphitylation

preferably N, N'-bis (diisopropylamino) cyanoethylphosphine or for H-phosphylation preferably

Salicoylchlorphosphit used. In structure, the dye derivative of general formula I corresponds to that used in oligonucleotide synthesis Synthesis intermediates and, like these compounds, analogously easy to handle. In the preferred use of the N, N'-bis-

(Diisopropylamino) -O-cyanoethylphosphines as R in the compound of general formula I, the reaction with the carrier-fixed or in solution oligonucleotide, nucleoside or nucleotide in the presence of weak acids, preferably tetrazole, in aprotic polar solvents, preferably acetonitrile.

If the novel fluorophore derivative by the Phosphotriesterverfahrens to be bound to the oligonucleotide, sets

the polymer-bound or in solution usually protected oligonucleotide with free 3 'or δ'-HydroKylgruppe with the compound of the general formula I with the meaning explained for F, X and η and R = substituted phosphate

Phosphate, preferably phosphoric acid p-chloro (or o-chloro) -plienyl ester in the presence of sulfonyl chlorides or

azolides, preferably triisopropylbenzenesulfonyl chloride in the presence of nucleophilic catalysts, preferably

N-methylimidazole, in aprotic polar solvents, preferably pyridine. When using the H-phosphonate method, the compound of the general formula I is explained with that for F, X and η Meaning and in which for R = phosphonate or substituted phosphonate, with the polymer bound or in solution

commonly protected oligonucleotide with free 3'- or 5'-hydroxyl function in the presence of Aktivierungsreagentien such as sulfonic acid or carboxylic acid chlorides, preferably pivaloyl chloride or adamantoyl chloride, in polar aprotic

Solvents or mixtures thereof, preferably a mixture of pyridine and acetonitrile, implemented. In the case of an automated synhesis strategy, that for normal nucleotide extension of the oligonucleotide chain

used synthesis cycle are used and repeated as often as desired to ensure the most quantitative possible labeling.

In the manner described, nucleosides and nucleotides can also be labeled according to the invention. After coupling of the fluorophore to the nucleoside, nucleotide or oligonucleotide and formation of the Internucleotidbindung, the synthesis product can be worked up in unlabeled oligonucleotides usually. To

The alkali-labile protecting groups are prepared by treatment with bases, preferably an aqueous concentrated solution of

Ammcniak, in a temperature range of 5 to 60 ° C, preferably at 5O ⁰ C, over a period of 12 to 60h, preferably in the Course of 15h, split off. The labeled oligonucleotides thus obtained can be detected both by gel electrophoresis Acrylamide as well as by high pressure liquid chromatography and in the for biological use

bring necessary form.

The inventive method is characterized in that starting from a synthesized by conventional methods Oligonucleotide, without additional anchor groups in the carbohydrate moiety, as well as in the heterocyclic evil, the introduction of

novel fluorophore derivatives of the invention directly in the course of oligonucleotide synthesis is possible.

The inventive method is further characterized by a mild reaction. The uncomplicated repeatability of the attachment ensures quantitative labeling of the oligonucleotide. The resulting products are available in high purity and in high yield. The bond between the fluorophore and the oligonucleotide is surprisingly stable to mild acid and alkaline conditions, thus allowing cleavage of protecting groups present on the oligonucleotide without affecting the binding of the fluorophore. The substances presented are novel and can be used as DNA probes or as primers in genetic engineering and molecular biology, in particular in the sequencing of genetic structures by chemical degradation method or the chain termination method are used. The fluorimetric determination of the compounds labeled according to the invention can be carried out with the hitherto customary technique for fluorescence measurement.

Exemplary embodiments The invention will be explained in more detail by means of examples without limiting them. example 1 Representation of the phosphoramidite

187 mg of F- (CHj) ₄ -OH (F = fluorescein methyl ester) are dissolved in 20 ml of absolute methylene chloride and 28 mg of diisopropylammonium tetrazolide and 0.12 ml of bis (diisopropylamino) methoxyphosphir) are added with stirring. One lets the

Stir reaction mixture for 1 h at room temperature. Then it is diluted to 50ml and saturated Sodium bicarbonate solution and water. After drying the organic phase over sodium sulfate concentrated

one and dries in an oil pump vacuum over phosphorus pentoxide. This gives 200 mg of the product (83% of theory).

Thin layer chromatography (chloroform / methanol 9/1 v / v) Ri = 0.65 Example 2 Representation of the phosphonate

100 mg of F- (CHj) ₁ -OH (F = fluorescein methyl ester) are codirected three times with 10 ml of absolute dioxane each time and in 12 ml

Dioxane added. To this solution is added 0.5 ml of lutidine and 1 ml of a one-molar solution of salicyl chlorophosphite

in absolute dioxane. After one hour, 1 ml of water is added and stirred for a further 30 min. The reaction solution becomes

Evaporated to dryness. The residue is taken up in chloroform and washed with saturated sodium bicarbonate solution,

one molar triethylammonium bicarbonate buffer (pH 8) and water in the order given. To

Drying of the organic phase with sodium sulfate, filtration and concentration, the phosphonate is in vacuo Phosphorus pentoxide dried. Thin layer chromatography (chloroform / methanol 9/1 v / v) Ri = 0.3 Example 3 Labeling of an oligonucleotide according to the phosphoramidite method (scale 1 pmol) The labeling takes place in the course of Oligonucleotidsynihese. For this purpose, a 0.1 molar solution of Example 1 prepared Depending on the type of DNA synthesizer, the compound in absolute acetonitrile is installed in the appropriate position and the Dye coupled according to the given synthesis cycle: Step Operation Time (s)

1 Wash with acetonitrile 60 2 Dry with argon 20 3 Dye derivative and 0.5 molar solution of tetraiol in acetonitrile in reaction vessel 10 4 reaction 30 5 Remove the reaction solution 5 6 Wash with acetonitrile 10 7 Remove the acetonitrile with argon 10 8th Oxidation with Ij / HjO / pyridine / TMF 40 9 Remove the oxidation solution 10 10 Wash with acetonitrile 30

Steps 2 to 6 can be repeated as often as required to complete the reaction. Example 4 Work-up of the labeled oligonucleotide The support with the fixed oligonucleotide is treated with concentrated ammonia for 60 h at room temperature and the

concentrated supernatant yellow fluorescent solution. The purification of the crude product thus obtained can over

Gel electrophoresis on polyacrylamide in the well-known, ι / rt manner, wherein labeled and unlabeled derivative

clearly distinguish in running behavior. The labeled oligonucleotide appears on viewing the gel

Transilluminator at a wavelength of 310nm as a weakly fluorescent band that excised and in for

Unlabeled oligonucleotides are usually eluted. (Desalting on RP18 silica gel or Sephadex)

Labeled and unlabeled oligonucleotides also differ by the retention times in the High pressure liquid chromatography and can be separated with their help.

Claims (1)

1. A process for preparing novel fluorescently labeled nucleosides, nucleotides and oligonucleotides, characterized in that fluorophor derivatives of the general formula (I) FO- (X) _n -R (I) F = fluorescent dye X = CH ₂ η = 1 to 18 R = phosphate, substituted phosphate, phosphoramidite, substituted phosphoramidite, H-phosphonate, substituted H-phosphonate, thiophosphonate, substituted thiophosphate, phosphorarnidate, substituted phosphoramidate under conditions customary for the chain construction of oligonucleotides with nucleosides nucleotides or oligonucleotides directly to compounds of the general formula (II)