WO2011061115A1

WO2011061115A1 - Solid phase-bound nucleosides

Info

Publication number: WO2011061115A1
Application number: PCT/EP2010/067320
Authority: WO
Inventors: Sabine MÜLLER; Janczyk Matthäus
Original assignee: Ernst-Moritz-Arndt-Universität Greifswald
Priority date: 2009-11-23
Filing date: 2010-11-11
Publication date: 2011-05-26
Also published as: DE102009046982A1

Abstract

The invention relates to solid phase-bound nucleosides, in which the nucleoside is covalently bound to a solid phase by a 3'-O-disulfide bridge, and to a method for the production and use thereof.

Description

Solid-phase bound nucleosides

All previous methods for the synthesis of trinucleotide synthons proceed in solution by the Posphortriester- method or the Phosphoramiditverfahren. The solution chemistry has the advantage that the scale of the synthesis is Prinzi ^¬ Piell selected as desired, including greater amounts of trinucleotides can be produced in one batch. However, the multistage process is costly, since after each synthesis step, an isolation and purification of the Pro ^¬ domestic product is necessary. Particularly in the production of exemplary meh ^¬ trinucleotides time is a critical factor, since such a synthesis can partially take several weeks.

By solid phase chemistry, this very time wändigen steps can be circumvented, since only one down washing the by-products, reagents and unreacted starting materials ^¬ from the carrier is required while the product remains on the solid phase. In addition, the construction ^¬ blocks for the solid phase synthesis of trinucleotides which - 3 ^λ -O-phosphoramidites of the N-acyl-5 ^λ -O-DMT-protected nucleosides are commercially available, and so permit fast ^¬ le synthesis of trinucleotides in a few hours , This is a decisive advantage over the solution chemistry, which requires at least several days through the above purification steps.

In the preparation of RNA or DNA strands on the solid phase, solid-phase-bound nucleosides are nowadays used. which are bound by means of an O-succinate linker to the solid ^¬ phase material.

The solid-phase synthesis of protected trinucleotides has hitherto failed due to the fact that cleavage of the trinucleotides from the commercial support materials was accompanied by a loss of the protective groups. The starting nucleoside is usually bound to the solid phase by an ester linkage to the linker molecule. Such binding is übli ^¬ cherweise cleaved by reaction with suitable nucleophiles, for example, aqueous or methanolic ammonia.

However, nucleophiles also attack the acyl protecting groups on the nucleobases as well as the usual phosphate protecting groups, leading to their cleavage, i. almost all

Protective groups are lost along the way. After removal of almost all protecting groups, a resulting trinucleotide as an unprotected polyanion no longer has any significance from a synthetic point of view. Due to numerous reactive groups (hydroxyl groups, amino groups), coupling reactions can no longer be specifically carried out. In addition, requires the high polarity of the molecule polar, mög ^¬ lichst protic solvents which are with the usual loading of the solid phase synthesis conditions incompatible.

It was therefore an object of the present invention to provide solid-phase-bound nucleosides or a process for the solid-phase synthesis of nucleosides, which overcome or overcome the abovementioned disadvantages. In particular, was of interest to the release of the tri-nucleotide from the solid phase under conditions to Errei ^¬ chen that make all other protecting groups intact.

This object is achieved by solid phase-bound nucleo ^¬ side according to claim 1 and by a manufacturing method of solid phase-bound nucleosides according to claim 8. width ^¬ re preferred embodiments result from the dependent claims.

In other words, the object is achieved by Festphasengebunde ^¬ ne nucleosides of the general formula 1, wherein the radical R is hydrogen, a protective group PG or more, bound via phosphate nucleosides, wherein the free OH groups of the phosphates themselves have protecting groups PG 'and wherein the nucleoside has a base B from the Grup ^¬ pe of the purine and pyrimidine bases, wherein in the case of free NH ₂ ~ groups these are acylated and wherein in the case of several nucleosides, the bases B are the same or different, it being characteristic that the nucleo ^¬ sid is covalently bound via a 3'-0- disulfide bridge to a solid phase ^¬

formula 1 wherein n is 0 or 1 and X, when n = 1 a - (CR R) _m - (NH) _p -C (= Y) group, wherein Rl and R ² independently of one ^¬ other hydrogen or branched or unbranched Al - are kylgruppen each having 1 to 10 carbon atoms and wherein m is a number between 1 and 10, wherein the sum of all C atoms is not greater than 10 and wherein p is 0 or 1 and Y is oxygen or sulfur.

The term "phosphate" as used herein generally a phosphoric acid-di- or tri-ester. With "phosphite" are called gene ^¬ rell phosphorous.

Preferably, a solid phase-bound nucleoside of the general formula 2, which is characterized in that it comprises two additional nucleosides (ie that the radical R of formula 1, two additional nucleosides means) wel ^¬ che each bonded 3 5 'phosphate groups, wherein the phosphate groups and the terminal 5'-OH group have protecting groups PC and PG

Formula 2

In a preferred embodiment, n = 1 and X is a - (CHR ¹ ) _m - (NH) p C (= Y) group, wherein R ^{1 is} hydrogen or a branched or unbranched alkyl group each having 1 to 10 C atoms and wherein m is a number between 1 and 10, wherein the sum of all C atoms is not greater than 10 and wherein p is 0 or 1 and Y is oxygen or sulfur. Most preferably, n = 1 and X is a - (CHCH3) -C (= O) group.

The protecting groups PC of the phosphate groups are preferably selected from methyl groups and substituted or unsubstituted ethyl groups, preferably β-cyanoethyl groups (CE). The protecting groups PG of the 5'-OH group are preferential ^¬ as dimethoxytrityl group (DMT). For methyl groups or Unsubstituted ethyl groups later in the synthesis still another cleavage step with a soft nucleophile, preferably a thiolate, is required.

The solid phase material can be liquid-phase polymers, gela ^¬ tineartige polymers, macroporous polymers, non-porous Po ^¬ mers and grafted polymers, preferably polystyrene or controlled pore glass (CPG), particularly preferably CPG, is used, said covalently bonded functional groups, preferably thiol groups or amino groups , are present at the solid phase. Also usable are (paper) filter discs, membranes or sintered blocks.

Liquid phase polymers are polymers with a high Molekularge ^¬ weight, which can be dissolved completely in the space required for the synthesis Lö ^¬ solvents. After completion of the coupling reaction, they can be reprecipitated by adding solvents in which they are insoluble. Liquid phase polymers are in particular PEG polymers with average molecular weights of 5,000 to 2,000, cellulose acetates and poly (N-acryloylmorpholine). Gelatinous polymers are, in particular, polystyrene-divinylbenzene polymers with a low degree of cross-linking (1-5%) and polyacrylamide supports. Grafted polymers have long polymer chains grafted onto a solid support. In particular, polystyrene-tetrafluoroethylene (PS-PTFE) or polyethylene glycol-polystyrene (PEG-PS) grafted copolymers may be mentioned here. Not ^¬ porous support materials include silica beads, which have no pore.

Macroporous solid phase materials include silica gels and porous glasses (CPG), as well as polymers such as polystyrene or polycarbonate. lymethacrylate or their copolymers with other monomers such as polymethacrylate-vinyl alcohol copolymers. In this case, variable degrees of crosslinking are used, ie polymers with high, but also low degrees of crosslinking can be used.

Macroporous polymers are preferably used as Festphasenmateri ^¬ al. The solid phase is preferably selected from polystyrene or porous glass (CPG), preferably CPG, where covalently bonded functional groups, preferably thiol groups or amino groups, are present on the solid phase.

In the case of solid-phase-bonded nucleosides according to formula 1 or 2 in which n = 0, in a preferred embodiment, the solid phase used is polystyrene with thiol groups bound to the solid phase as carrier material.

The method for producing a solid phase-bound nucleoside according to formula 1, wherein R stands for water ^¬ cloth, a protective group PG, or more, attached via phosphate ^¬ groups nucleosides, wherein the free OH self-protecting groups PG include groups of the phosphates' and wherein the Nucleoside has a base B from the group of Pu ^¬ rin and pyrimidine bases, wherein in the case of free NH ₂ ~ groups present acylated and wherein in the case of several nucleosides, the bases B are the same or different, and wherein the nucleoside on a 3 '-O disulfide bridge is covalently bonded to a solid phase

formula 1

where n is 0 or 1 and X is n = 1

- (CR ¹ ^2!) _M - (NH) p C (= Y) group, wherein Rl and ^R2 indepen ^¬ pendently hydrogen or branched or unbranched alkyl groups each having 1 to 10 carbon atoms and m is a number from 1 to 10, wherein the sum of all C atoms is not greater than 10, and wherein p is 0 or 1, and Y is oxygen or sulfur, comprising the following steps:

Reacting a nucleoside of the general formula

3, which has one with a protecting group PG ge ^¬ protected 5'-OH group and a free 3'-OH group, wherein the radical B has the abovementioned Be ^¬ significance,

Formula 3

with dimethyl sulfoxide (DMSO) to the 3 '-O-methylthio methyl derivative of the general formula 4

Formula 4

and

• subsequent reaction of the 3'-O-methylthio methyl derivative with a solid phase material which has covalently bonded thiol or amino groups, resulting in a solid phase-bound nucleoside according to formula 1 with R = PG.

A preferred embodiment is characterized in that in solid phases with covalently bonded amino groups, the 3 '-O-Methylthiomethylderivat the general formula 4 is reacted at the Methylthiomethylgruppe first with a bifunktiona len reporter molecule, which is a Thiolgrup pe and an implementable with amino groups electrophilic Grup ^¬ pe, preferably a carboxyl, an isothiocyanate or isocyanate group, wherein between the thiol group and the group which can be reacted with amino groups, a (CR ¹ R ² ) _m - group is present, wherein R ¹ and R ^{2 are} independently hydrogen or branched or unbranched alkyl groups each having 1 to 10 carbon atoms, and m is a number from 1 to 10, wherein the sum of all carbon atoms is not RESIZE ^¬ SSER 10, and subsequent reaction with the solid phase material, which amino groups covalently bound comprising wherein when a carboxyl group on the reporter molecule this is first activated, resulting in a festphasenge Linked nucleoside according to formula 1 with R = PG and n = l results. The (CR R) _m group is preferably a (CHR) m group, wherein R ^{1 is} hydrogen or a branched or unbranched alkyl group each having 1 to 10 C atoms and m is a number between 1 and 10, wherein the Sum of all C atoms is not greater than 10 and wherein p is 0 or 1 and Y is oxygen or sulfur. Preferably, the reporter molecule is 2-mercaptopropanoic acid.

In further steps of the manufacturing process is eliminated these Schutzgrup ^¬ pe PG of a nucleoside according to formula 1 with R = PG, the solid phase-bound nucleoside at ^¬ closing with a nucleoside-3 '-O-phosphoramidite of all ^¬ common formula 5, wherein the phosphate group and from the ^¬ closing 5'-OH group protecting groups PG 'and PG aufwei ^¬ sen,

PG '

Formula 5 wherein B has the meaning given above, implemented. An ^¬ closing the 5 '-OH protective group is cleaved and there ^¬ after carried out a new reaction with another nucleoside 3' -O-phosphoramidite of the general formula 5, wherein PG, PG 'and B have the abovementioned meaning.

The protective groups PG 'of the phosphate groups are preferably selected from methyl groups and substituted or unsubstituted ethyl groups, preferably β-cyanoethyl groups (CE). The protective group PG of the 5'-OH group is preferred a dimethoxytrityl group (DMT). The peculiarities of methyl and unsubstituted ethyl groups have already been explained above.

Scheme 1 gives an overview of the synthesis method for solid phase-bound nucleosides according to formula 1 or 2, in which n = 0. In this case, the solid phase used is preferably polystyrene with thiol groups bound to the solid phase as carrier material. The respective radicals / protective groups in Scheme 1 have the meanings already mentioned, with a DMT group being exemplified here for the protective group PG. For reasons of clarity, the solid phase is symbolized by a circle.

Scheme 1 First, by reacting the N-acyl-5 ^λ- O-DMT protected nucleoside with acetic anhydride, DMSO and acetic acid, a 3 ^λ- O-methylthiomethyl-functionalized nucleoside Herge ^¬ provides. This is covalently bonded to a thiol-modified solid phase, for example mercaptoethylpolystyrene, by forming a disulfide bond in a subsequent reaction. Subsequently, the trinucleotide is synthesized on the solid phase by coupling two N-acyl-5 ^λ - (DMT) -3 ^λ- O-nucleoside phosphoramidites according to the standard phosphoramidite method. The cleavage of the protected trinucleotide from the solid phase is carried out by treatment with dithiothreitol (DTT) in a mixture of DMF and water (1: 1) at 45 ° C. for 10 hours. The Disulfidbrü- is first divided blocks, and generates a 3 ^λ -O-Methylenthiolgruppe on nucleo ^¬ sid. This mixed hemiacetal is subject to a rapid succession reaction with release of methanthial and generation of the free 3 ^λ -OH group.

For solid-phase-bound nucleosides according to formula 1 or 2, in which n = 1, the solid phase is preferably CPG with amino groups bound to the solid phase. Various chemically modified CPG carriers with different functional groups are commercially available, all of which are useful for this synthesis. The surface as well as the maximum load depend on the pore size, so that larger pores mean a higher loading capacity.

Most preferably, a CPG support is used which carries a long chain alkyl amine (LCAA, LCAA-CPG).

Alternatively, in a second process, the 3 ^λ- O-methylthiomethyl-functionalized nucleoside is converted via a reaction porter molecule linked to the solid phase, with preferential ^¬ manner as described above 2-mercapto propionic acid is used. But can also be any other difunctional molecule, which is equipped on the one hand with a thiol group and on the other hand ^¬ with an electrophilic moiety that can react with amines, preferably Carbonsäurederiva ^¬ te, isocyanates or isothiocyanates.

Scheme 2 gives an overview of the procedure for solid-phase-bound nucleosides according to Formula 1 or 2, wherein wel ^¬ chen n = l, and 2-mercapto propionic acid is used as a reporter molecule. The respective residues / protection groups have already mentioned meaning, being here for protection ^¬ group PG example a DMT group is shown. For clarity, the solid phase is symbolized by ei ^¬ nen circle.

Scheme 2

The reaction of the 3-O-methylthiomethyl-functionalized nucleoside with 2-mercaptopropanoic acid after activation with sulfuryl chloride and potassium thiotosylate leads to the formation of a disulfide bridge and thus to the covalent linkage of the nucleoside with the reporter molecule. The carboxyl group of mercaptopropionic acid is then set after activation, preferably with TSTU, with amino-functionalized CPG by ^¬, and so linked the nucleoside to the solid phase. Other activators besides TSTU are HBTU, HATU and TBTU. The synthesis of the trinucleotide was carried out according to the phosphoramidite method as described above, and the release of the protected trinucleotides was also carried out under identical conditions. conditions, preferably by treatment with DTT in a DMF-water mixture in the ratio 1: 1. Here, a 3 '-O-thiomethyl derivative formed as an intermediate which supplies ^¬ closing to release methanthial the free 3'-OH group.

By HPLC and MALDI-MS analysis, the Identi ty ^¬ the synthesized trinucleotides and the receipt of all protecting groups (see Fig. 1) has been clearly demonstrated. In addition to the acyl protecting groups on the nucleobases and the 5 ^λ- O-DMT groups, both methyl groups and β-cyanoethyl groups on the phosphates proved to be stable under these conditions.

The second method using CPG as the solid phase proved advantageous over the use of mercaptoethylpolystyrene, since the amino-functionalized CPG used is better adapted to the conditions of the oligonucleotide synthesis, for example, does not swell and egg ^¬ nen greater distance between the polymer phase and Nucleoside guaranteed.

Solid phase-bound nucleosides according to the general formula 2 are used for the preparation of a trinucleotide 5'-O-PG-3 '-0-phosphoramidite of the general formula 6 in which PG, PG' and B have the abovementioned meaning,

Formula 6 wherein a cleavage of the solid phase, in which all protecting groups are retained on the trinucleotide, and a subsequent phosphitylation of the free 3'-OH group takes place.

For cleavage from the solid phase thereby a non basi ^¬ ULTRASONIC soft nucleophile dissolved in an organic solvent, optionally with the addition of water at ei ^¬ NEM pH of 6.5 to 7.5 is used. For cleavage from the solid phase, dithiothreitol (DTT) dissolved in an organic solvent is preferably used, preferably dimethylformamide (DMF).

The phosphitylation of the free 3'-OH group of the trinucleotides is preferably carried out by reaction with 2-cyanoethyl-N, N, N ^λ , N ^x -tetraisopropylphosphoramidite, whereby a 3 ^λ -0-phosphoramidite is obtained. Description of the figure

Fig.l The figure shows three different trinucleotides synthesized on a CPG solid phase. The corresponding MALDI mass spectra show that the synthesis was successful. The cleavage from the solid phase resulted in all cases in the release of the respective trinucleotide to obtain all the necessary protective groups, with a 3'-O-thiomethyl group being formed as an intermediate. Shown are the recorded at different times mass spectra. After three hours, a beginning loss of the thiomethyl group can be recognized. After 8-10 hours almost complete loss of the thiomethyl group can be detected to generate a free 3'-OH group (spectra II, IV and VI).

The invention will be explained below with reference to examples.

Examples

Example 1: IV acylation of 2 "-deoxyadenosine / 2'-deoxycytidine / 2" -deoxyguanosine

1

Approach:

1. 500 ml of pyridine

2. 50 mmol 2'-desoxynucleoside (13.45 g dA hydrate, 13.35 g dG, 13.15 g dC hydrochloride)

3. 5 eq. Trimethylchlorosilane (32.5 ml)

4. 5 eq. Acylating agent (42.5 ml of isobutyric anhydride or 30 ml of benzoyl chloride)

In a 1000 ml one-necked flask, 50 mmol of 2'-deoxyadenosine / 2'-deoxycytidine / 2'-deoxyguanosine were coevaporated twice with 100 ml each of dry pyridine and finally suspended in 500 ml of dry pyridine. While cooling, 5 equivalents of trimethylchlorosilane (32.5 ml) were added dropwise to the solution over 10 minutes, then the solution was further stirred for 15 minutes. 5 equivalents of Iso ^¬ buttersäureanhydrid (deoxyadenosine, deoxyguanosine, 42.5 ml) and benzoyl chloride (deoxycytidine, 30 ml) added dropwise within 10 minutes to the further cooled solution, the ice bath then removed. After 20 minutes, the solution was again cooled in an ice bath and the reaction was stopped by the addition of 200 ml of cold water, after a further 15 minutes 100 ml of a 30% ammonia solution were added. ben. After stirring for 30 minutes, all solvents were removed, the residue was dissolved in as little water (about 50 ml) completely. The aqueous phase was then washed with 100 ml of diethyl ether and transferred to an Erlenmeyer flask. Crystallization started sometimes already after a few seconds, and could, if necessary, by cooling or by removing a portion of the water accelerates or be made ^possible. The Erlenmeyer flask was stored overnight in the refrigerator, the crystallized product was aspirated the next day, washed with water and dried in a desiccator at reduced pressure overnight. As a product color and odorless crystals of wat ^¬ teartiger consistency was obtained. The mother liquor was stored for any post-precipitation, the crystallization repeated under the conditions mentioned above.

In absence of crystallization, the product was concentrated to dryness and purified by column chromatography (CH ₂ Cl _2: methanol 80:20) ge of the organic salts ^¬ separates. After removal of the dichloromethane / methanol mixture, a colorless solid was obtained.

R _f value (CH ₂ Cl ₂ : MeOH 8: 2): 0.5

Yields:

• dA (N-acyl): 94%

• dC (N-acyl): 90%

• dG (N-acyl): 96% Example 2: 5 "O-tritylation

2

Approach:

1. 100 ml pyridine (dry)

2. 7 mmol W-acyl-nucleoside 1

3. 2 ml of triethylamine (dry)

4. 0.05 eq. DMAP (0.043 g)

5. 1.3 eq. DMTC1 (3.1 g)

7 mmol of the W-acylated deoxynucleoside 1 or thymidine were coevaporated twice in a 250 ml one-neck flask with 30 ml of dry pyridine and finally suspended in 100 ml of dry pyridine and 1.5 ml of dry triethylamine. 0.05 equivalents of DMAP (43 mg) and then 1.3 equivalents of dimethoxytrityl chloride (3.1 g) were added to the suspension. The formation of the tritylated product was checked by thin layer chromatography. When not fully implemented, further DMTC1 was given to ^¬. The reaction was stopped after adding 100 ml of water. The product was extracted with 100 ml of diethyl ether until no more product was present in the aqueous phase (about 10 ×). After the solution be ^¬ medium were completely removed under reduced pressure on a rotary evaporator ^¬, the product could säulenchro- matographisch chromatography (CH ₂ Cl _2: MeOH = 99: 1 _→ 95: 5). R _f value (CH ₂ Cl ₂ : MeOH 9: 1): 0.5

Yields:

• 5 ^{'-O-trityl-dA} (N-acyl): 92%

• 5'-O-trityl-dA (N-acyl): 91%

• 5 ^{'-O-trityl-dA} (N-acyl): 86%

• 5'-O-trityl-T: 97%

The tritylated nucleosides were detected by means of MALDI as sodium signals.

• 5'-O-trityl-dA (N-acyl): 646.3 m / z (sodium peak)

• 5'-O-trityl-dA (N-acyl): 656.1 m / z (sodium peak)

• 5'-O-trityl-dA (N-acyl): 663.0 m / z (sodium peak)

• 5'-O-Trityl-T: 566 m / z (sodium peak)

Example 3: Synthesis of a 3 "-0-MTM Functionalized Nucleoside

3

Approach:

1. 50 ml of DMSO

2. 50 ml of acetic anhydride

3. 40 ml of acetic acid

4. 10 mmol nucleoside 2

In a 250 ml one-necked flask filled with 10 mmol of a 5'-O-DMT-protected deoxynucleoside 2, 50 ml of DMSO, 50 ml of acetic anhydride and 40 ml of acetic acid added. The solution was stirred until the starting material had reacted completely (5-24 h). After complete reaction, the solution was poured into 200 ml of saturated sodium bicarbonate solution, the product was extracted by adding a further 200 ml of dichloromethane. If product was still present in the aqueous phase, the extraction was repeated with a further 100 ml of dichloromethane.

The solvents were removed by distillation under reduced pressure, the product was then purified by column chromatography.

R _f value (CH ₂ Cl ₂ : MeOH 95: 5): 0.7

Yields:

• 3 ^'-0-MTM-dA (N-acyl): 71%

• 3 ^'-0-MTM-dC (N-acyl): 80%

• 3 ^'-0-MTM-dG (N-acyl): 66%

• 3'-0-MTM-T: 75%

The following nucleosides were detected by MALDI:

dA: calculated: 684 g / mol; measured: 685 m / z

(no sodium peak)

dC: calculated: 694 g / mol; measured: 717 m / z

dG: calculated: 700 g / mol; measured: 701 m / z

(no sodium peak)

T: calculated: 604 g / mol; measured: 627 m / z Example 4 Synthesis of a Nucleoside Bonded to a Solid Phase via a Disulfide Bridge

3'-O-disulfide acid

4

3'-0-MTM functionalized nucleoside 3 was reacted with 2-mercaptopropanoic acid to obtain the desired product.

Rf value (CH ₂ Cl ₂ : MeOH 95: 5): 0.2

Yields:

• dA: 62%

• dC 55%

• T: 60%

The following nucleosides were detected by MALDI as sodium signals:

dA: calculated: 794 g / mol; measured: 774 m / z

dC: calculated: 783 g / mol; measured: 786 m / z

T: calculated: 694 g / mol; measured: 694 m / z Coupling with an amino-CPG carrier

5

In a 2 ml Eppendorf tube 0.1 mmol of 3'-0-disulfidic acid 4 were dissolved in 1.5 ml of a 1: 1 mixture of DMF and water, there were added 50 .mu.l triethylamine and 0.2 mmol TSTU, whereupon the Solution browned. Finally, 1 g of CPG-support was; added, the Eppendorf vessel was shaken overnight at Raumtem ^¬ temperature (105 ymol / g 1 eq.). The next day the Eppendorfge ^¬ fäß was anzentrifugiert, then carefully removed the supernatant from ^¬ and discarded. The support was then in 1 ml of a 1: 1 mixture of water was added and ethanol, shaken briefly, again anzentrifugiert, the supernatant ver ^¬ worfen. The process was repeated five times, the CPG carrier then dried in the open Eppendorf tube in the air. Example 5: Synthesis of a trinucleotide on the solid phase

The preparation of the trinucleotide on the solid phase (CPG) was carried out according to the standard phosphoramidite method on a 1 μιηοΐ scale using a DNA synthesizer (Geneasser, Pharmacia). Commercially available N-acyl-5 '- DMT protected 3' -O-phosphoramidite of the four nucleosides A, C, G and T were (CPG bound with starting nucleoside by a disulfide bond) to the solid phase according to the desired sequence ge ^¬ coupled. The syntheses were done in "trityl off" mode. Average coupling yields were 99.4%. Example 6: Cleavage of the trinucleotide from the solid phase

Approach:

1. 100 mg of CPG support (500 A; 105 ymol / g; ca. 1 ymol ge ^¬ bundenes trinucleotide)

2. 15 mg DTT (154 g / mol, 100 equivalents)

3. 1 ml of dry DMF

The CPG support obtained after solid-phase synthesis was washed five times with 1 ml of a 1: 1 mixture of water and acetonitrile. After removal of the solvents, the carrier was slurried in 1 ml of DMF in an Eppendorf tube, a 100-fold excess of DTT was added. The Eppendorf vessel was sealed with parafilm and incubated at 40 ° C. At 1 h intervals, the supernatant was examined for completeness of the cleavage reaction (DC, MALDI). Extracts of the MALDI mass spectra for cleavage are given in FIG. After complete cleavage of the 3'-O-thiomethyl group (compounds 8), the solvent was The product was taken up in 100 μl acetonitrile / water (1: 1), filtered and purified by preparative RP-HPLC.

Solvent system:

Buffer A: 5% acetonitrile in water

Buffer B. 70% acetonitrile in water

Example 7: Synthesis of trinucleotide 3'-O-phosphoramidite

SSN-acyl

9

Approach:

1. 5 ml dichloromethane (dry)

2. 0.5 mmol trinucleotide 1_

3. 0.55 mmol 2-cyanoethyl-W, A / ^' , A / ^'' , ΛΓ-tetraisopropylphosphoramidite

4. 0.6 mmol BMT (0.12 g) In a 10 ml one-necked flask, 0.5 mmol of the purified trinucleotide 7 was dissolved in 5 ml of dry dichloromethane, to which 0.55 mmol of 2-cyanoethyl-N _/ .N _/ .N ', N'-tetraisopropylphosphoramidite and 0.6 mmol of BMT were added. The reaction solution was stirred for about two hours until the starting material had completely disappeared. After complete reaction, the solvent was removed, the phosphoramidite coevaporated twice with dry dichloromethane and dried overnight on oil pump vacuum. An aqueous Aufarbei ^¬ processing and cleaning did not take place. The phosphoramidite was used immediately for solid phase synthesis.

Rf value (CH ₂ Cl ₂ : MeOH 9: 1): 0.7

Yield: 50-70%

Claims

claims

1. A solid-phase-bound nucleoside of the general formula 1, wherein the radical R is hydrogen, a protective group PG or more, bound via phosphate groups nucleosides, wherein the free OH groups of the phosphates themselves have protective groups PG ', and wherein the

Nucleoside has a base B from the group of purine and pyrimidine bases, wherein in the case of free NH ₂ groups are present acylated and wherein in the case of several nucleosides, the bases B are the same or different, characterized in that the

Nucleoside is covalently bound to a solid phase via a 3'-O-disulfide bridge

Formula 1 wherein n is 0 or 1 and X at n = 1 is a - (CRV) ,, - (NH) p-C (= Y) group, wherein

R 1 and R ² independently of one another are hydrogen or branched or unbranched alkyl groups each having 1 to 10 C atoms and in which m is a number between 1 and 10, the sum of all C atoms not being greater 10 is and

wherein p is 0 or 1 and Y is oxygen or

Sulfur is.

Solid phase-bound nucleoside according to claim 1 according to general formula 2, characterized in that it comprises two further nucleosides which are each bound via 3 5 '-phosphate groups, the phosphate groups and the terminal 5'-OH group protecting groups PC n

Formula 2

3. A solid-phase bound nucleoside according to claim 1 or 2, characterized in that n = 1 and X is a - (CHR ¹ ^ - (NH) _p -C (= Y) group, wherein R ^{1 is} hydrogen or a branched or unbranched alkyl group each with 1 to 10 C atoms and wherein m is a number between 1 and 10, wherein the sum of all C atoms is not greater than 10 and wherein p is 0 or 1 and Y is oxygen or sulfur.

4. Solid-phase-bound nucleoside according to one of Ansprü ^¬ che 1 to 3, characterized in that n = 1 and X is a - (CHCH ₃ ) -C (= 0) group.

5. solid phase-bound nucleoside according to one of claims 1 to 4, characterized in that the protective groups are selected of the phosphate groups PC pen from Methylgrup ^¬ groups and substituted or unsubstituted Ethylgrup-, preferably ß-cyanoethyl (CE) and the protecting groups PG of the 5'- OH group dimethoxytrityl groups (DMT) are.

6. solid phase-bound nucleoside according to one of Ansprü ^¬ che 1 to 5, characterized in that the Festpha ^¬ se is selected from liquid phase polymers gelati ^¬ near term polymers, macroporous polymers, non-porous polymers and grafted polymers, preferably polystyrene or controlled pore glass (CPG), particularly preferred CPG, wherein covalently bonded functional groups, preferably thiol groups or amino groups, are present on the solid phase.

7. solid phase-bound nucleoside according to one of claims 1 to 6, characterized in that when n = 0 olgruppen the hard ^¬ phase polystyrene bound to the solid phase Thi ^¬ and wherein n = l, the solid phase CPG with bound to the solid phase amino groups. Process for producing a solid phase bound

Nucleosides according to formula 1, where the radical R is hydrogen, a protective group PG or further phosphate groups bound by nucleosides, wherein the free OH groups of the phosphates themselves have protecting groups PG 'and wherein the nucleoside is a base B from the group of purines and pyrimidine bases, wherein in the case of free NH ₂ ~ groups, these are acylated and wherein in the case of several nucleosides, the bases B are the same or different, and wherein the nucleoside is covalently bound to a solid phase via a 3 '-O-disulfide bridge is

formula 1

where n is 0 or 1 and X is n = 1

- (CRV) ,, - (NH) p-C (= Y) group, in which

R 1 and R ^{2 are} independently hydrogen or branched or unbranched alkyl groups each having 1 to 10 carbon atoms and m is a number from 1 to 10, wherein the sum of all C atoms is not greater than 10 and wherein p is 0 or 1 and Y is oxygen or, the method comprising the steps of: Reacting a nucleoside of the general formula 3 which has a 5'-OH group protected by a protective group PG and a free 3'-OH group, the radical B having the abovementioned meaning,

Formula 3

Formula 4

and subsequent reaction of the 3'-O-methylthio- methyl derivative with a solid phase material having covalently bonded thiol or amino groups, resulting in a solid phase-bound nucleoside according to formula 1 with R = PG.

9. A process for the preparation of a solid phase-bound nucleoside according to claim 8, characterized in that in solid phases with covalently bound Aminogrup ^¬ pen the 3 '-O-Methylthiomethylderivat the general formula 4 is reacted at the Methylthiomethylgruppe first with egg ^¬ nem bifunctional reporter molecule which is a thiol group and with amino groups to electrophilic ^¬ settable group, preferably a carbonyl Xyl, has an isothiocyanate or isocyanate group ^¬, wherein between the thiol group and the group with Ami ^¬ nogruppen reactable group is a (CR ¹ R ²⁾ _m in front- is in which

R ¹ and R ^{2 are} independently hydrogen or branched or unbranched alkyl groups each having 1 to 10 C atoms and m is a number from 1 to 10, wherein the sum of all C atoms is not greater than 10, and subsequent reaction with the solid phase material , which has covalently bound amino groups, where ^¬ at a carboxyl group on the reporter molecule this is first activated, resulting in a solid phase-bound nucleoside according to formula 1 with R = PG and n = l results.

A process for producing a solid-phase-bound nucleoside according to claim 8 or 9, characterized in that the (CR ¹ R ² ) _m group is a (CHR ¹ ) m group, wherein R ^{1 is} hydrogen or a branched or unbranched alkyl group, respectively 1 to 10 carbon atoms and wherein m is a number between 1 and 10, wherein the sum of all C atoms is not greater than 10 and wherein p is 0 or 1 and Y is oxygen or sulfur.

11. A process for preparing a solid phase-bound nucleoside according to any one of claims 8 to 10, characterized in that the reporter molecule is 2-mercaptopropanoic acid.

12. A process for preparing a solid phase-bound nucleoside according to any one of claims 8 to 11, characterized in that in further steps from a nucleoside according to formula 1 with R = PG this protective group PG is cleaved, the solid-phase-bound nucleoside with a nucleoside -3 ' -O-phosphoramidite of the general formula 5, wherein the phosphate group and the final 5'-OH group has protecting groups PC and PG, is reacted,

PG '

Formula 5 wherein B has the meaning given above,

Subsequently, the 5 '-OH protective group is cleaved off and then a new reaction with another nucleoside 5' -O-dimethoxytrityl-3 '-O-phosphoramidites of the general formula 5, wherein PG, PG' and B above ge ^¬ named Have meaning takes place.

13. A method for producing a solid phase-bound nucleoside according to one of claims 8 to 12, characterized in that the protecting groups PG 'of Phos ^¬ phatgruppen are selected from methyl groups and sub ^{¬-substituted} or unsubstituted ethyl groups, preferably beta-cyanoethyl (CE) and the Schutzgrup ^¬ PG of the 5'-OH group Dimethoxytrityl groups (DMT) are.

14. Use of a solid phase-bound nucleoside of the general formula 2 for the preparation of a tri- nucleotide-5 '-O-PG-3' -O-phosphoramidite of general formula 6, wherein PG, PG 'and B have the above significance ^¬ tung .

15. Use according to claim 14, wherein for cleavage from the solid phase, a non-basic soft nucleophile dissolved in an organic solvent, optionally with the addition of water at a pH of 6.5 to 7.5 is used.

16. Use according to claim 14 or 15, wherein for cleavage of the solid phase dithiothreitol (DTT), ge ^¬ triggers in an organic solvent, preferably dimethylformamide (DMF) is used.