DE4110085A1 - New 2'O-alkyl-oligo-ribonucleotide(s) with 8-35 nucleotide units - useful as anti-sense oligo-nucleotide(s), primers and probes - Google Patents

Abstract

The following are claimed: (A) Prepn. of an oligoribonucleotide (A), based on 8-35 nucleotides of formula (II) (or a 2'-OH or 2'-deoxy deriv.) comprises: (a) linking the corresp. ribonucleotide building blocks in the presence of a coupling agent; and (b) opt. incorporating labelled gps. and/or lipophilic gps. in (A) and opt. cleaving protecting gps.. In (II), B = cytosine, uracil, adenine, guanine or inosine; R = 1-30C alkyl; E1 = a phosphate diester-, methyl phosphonate, phosphoramidate- or phosphorthioate-residue; Provided that (i) at least one R is 2-30C alkyl and (ii) the terminal 3' and 5' positions are an OH gp. (opt. modified by a labelled or lipophilic gp.) or a phosphite ester-, H-phosphonate- or phosphate ester-residue. (B) Prepn. of an oligoribonucleotide (A'), based on 8-35 nucleotides; (C) (A) are new (A'') when the terminal 3' and 5' positions are OH (opt. modified by labelled or lipophilic gps.) or a phosphate ester residue; (D) Prepn. of nucleosides of formula (III) where B'' = opt. protected cytosine, uracil, adenine, guanine or inosine; R'' = 2-30C alkyl when B is opt. protected adenine, otherwise 1-30C. (E) (III) are new (i) when B'' is guanine protected in 6-position by nitrophenylethyl and R'' is methyl, and (ii) when R'' is 2-30C alkyl and B'' is opt. protected cytosine, inosine, uracil or guanine. (F) Alkylated ribonucleotide building blocks are new; and (G) Liposomes, in which are incorporated (A'') modified by 3'cholesterol or 3'-thiocholesterol, are new. USE - (A'') are useful as antisense oligonucleotides, probes, primers or primer sections. They may be used to inhibit gene expression or virus replication.

Description

The present invention relates to 2'-O-alkyl oligoribonucleotides, the building blocks from which they are built up, processes for their manufacture as well their use as antisense oligonucleotides, probes, Primers or primer sections and furthermore to 2′-O-alkyl oligoribonucleotides, the at the positions on 3'- and 5'-end are modified.

Oligonucleotides bind through specific Hydrogen bonds from base pairs complementary sequences of genomic DNA or RNA, already relatively short oligomers of <20 bases hybridize specifically to and onto DNA or RNA Manner of gene expression or viral replication can deliberately impair.

The following are as antisense substances Defined oligonucleotides due to Base sequence specific interaction a can inhibit certain genes. These antisense oligonucleotides can by Base pairing complementary single-stranded Nucleic acid sequences (DNA or RNA) or also double stranded nucleic acid sequences (DNA) from Binding gene sections, whereby from this binding a Inhibition of the corresponding gene results. Here the antisense oligonucleotide can be complementary to that RNA strand ("antisense"), or in the given case also have the same sequence ("sense"); the latter is thus complementary to the corresponding DNA strand of the Sequence.

The use of synthetic oligonucleotides as specific inhibitors of gene expression or of Virus replication, so-called "antisense oligonucleotides"  has become increasingly interesting in recent years. A major limitation in the use of this Strategy, however, is that short single-stranded nucleic acid molecules according to their Entering the cell are biologically unstable. Around To counter this disadvantage are already a few Attempts have been made not in nature to synthesize occurring nucleic acid analogs that with increased biostability, the inhibitory activity of have natural nucleic acids. Results of this Attempts that so far have mainly focused on Have concentrated oligodeoxyribonucleotide analogs, are z. B. phosphorothioates, methylphosphonates and Phosphoramidates.

Because the RNA-RNA duplex is more stable than that DNA-RNA hybrid, the present invention was the Task focused on RNA-RNA hybrids, at which have the advantage of greater hybrid stability greater susceptibility of the RNA to nuclease degradation faces.

There are relatively few examples of oligoribonucleotide analogs known who do not have this disadvantage or less than natural ones Have ribonucleotides (this is partly due to it attributed to technical difficulties in the RNA synthesis passed).

One of these known examples is modification the ribose by adding a 2′-O-methyl group, from which has been found to reduce the effects of RNA and DNA-specific nucleases (Sproat et al., Nucleic Acids Research, 1 (1989) 3373-3386). A another example of 2'-O-modified oligoribonucleotides  are 2'-O-allyl oligoribonucleotides, from who were shown to have the nuclease resistance of the 2'-O-methyl-oligoribonucleotides and an additional one increased ability to bind to a complementary Have RNA section. From the same authors 2'-O-Dimethylallyl-oligoribonucleotides described, the stability against nucleases, but relative low binding affinity for the target sequences (Iribarren et al., Proc. Natl. Acad. Sci. USA, 87 (1990) 7747-7751).

It is known to make oligoribonucleotides Ribonucleotide building blocks using the solid phase method to manufacture. According to Sproat et al., Nucleic Acids Research 17, (9), (1989) 3373-3386, for example 2′-O-methyloligoribonucleotides after the Solid phase method from 5'-O-dimethoxytrityl-2'-O-methyl ribonucleoside-3′-O- (2-cyanoethyl N, N-diisopropyl- phosphoramidites using 5- (4-nitrophenyl) -1H-tetrazole be produced as an activator. At This procedure involves post-attachment alkylation appropriate protection groups in a later stage of the overall process with usually bad Yields carried out, so that in particular by the separation required in the reaction formed 3'-O-methyl isomers in a later stage large losses in yield must be accepted.

Conventional processes for the production of methylated Ribonucleotide building blocks are based primarily on the Alkylation with diazomethane. Because of the unfavorable Properties, especially the explosiveness of this Alkylating agent is the production of larger quantities hardly any alkylated compounds in this way possible. As a very special further disadvantage of the  Procedure results in the fact that a variation of the alkyl radical is hardly possible. Methods of Production of 2'-O-methyl nucleosides under Use of methyl iodide are also known. To the Example is in EP 2 69 574 the production of 2'-O-methyladenosine using methyl iodide and silver oxide. The production of certain 2'-O-methyl nucleosides using Methyl iodide and silver oxide and / or the steric hindered strong organic base 2-tert.- Butylimino-2-diethylamino-1,3-dimethylperhydro- 1,3,2-diazaphosphorin (BDDDP) is published by Brian S. Sproat et al., 1990, Nucleic Acids Research 18, 41-49 described. These procedures need for that Methylation a large number of reaction stages what very disadvantageous for the production of larger quantities is. J. Yano, L.S. Kan and P.O.P. TS'O, Biochim. Biophys. Acta, 629 (1980), 178-183 describes the Production of 2'-O-methyladenosine by methyl iodide in an anhydrous alkaline medium. This Process is based on the manufacture of others 2′-O-alkyl nucleosides are not readily transferable.

The present invention was based on the object new, 2′-O-modified oligoribonucleotides provide that as antisense oligonucleotides are usable and an improved method for Production of 2'-O-modified oligoribonucleotides and of 2′-O-modified ribonucleotide building blocks.

The present invention relates to new ones Oligoribonucleotides made up of 8 to 35 nucleotides the general formula II,

wherein
B means cytosine, uracil, adenine, guanine or inosine,
R is alkyl having 1 to 30 carbon atoms, and
E¹ is a phosphate diester, methylphosphonate, phosphoramidate or phosphorothioate residue,
or their 2′-OH or 2′-deoxy derivative, where in at least one of the nucleotides R is alkyl having 2 to 30 C atoms, and in which the terminal groups in positions 3 ′ and 5 ′ independently of one another are a hydroxyl group or a phosphate ester residue, or a hydroxyl group modified by a labeling group or lipophilic group. Oligoribonucleotides are preferred in which unprotected adenine, cytosine, uracil or guanine is in the nucleotide of the formula II B, in particular those in which R is alkyl having 2 to 8 (in particular 2 to 4) C atoms, preferably ethyl.

The aforementioned 2'-O-alkyl oligoribonucleotides according to the invention are made up of nucleotides, their sugars can be identical or different from one another and their nucleobases (as that generally known Oligoribonucleotides is common) are different.  

Preferred are oligoribonucleotides that only consist of Nucleotides of formula II are constructed as well Oligoribonucleotides consisting predominantly of nucleotides of the Formula II are built and between these building blocks Contain blocks that are in position 2 'OH or H exhibit. The length of the invention 2'-O-alkyl oligoribonucleotides depends on the Purpose. Parameters for the length of the Oligoribonucleotides is the ability to bind to the Target sequence under the respective application conditions, e.g. B. when used as antisense oligonucleotides physiological conditions or when used as Primer under the specific test conditions. in the When used as an antisense oligonucleotide these oligonucleotides preferably consist of at least 8 and at most approx. 30-35 nucleotides, in particular 8 up to 19 nucleotides.

The oligoribonucleotides according to the invention have a much more inhibitory than conventional ones Antisense connections and are regarding their inhibiting antisense properties of the 2′-O-methyloligoribonucleotides at least equal. Compared with the known 2'-O-methyloligoribonucleotides show the oligoribonucleotides according to the invention increased lipophilicity and increased stability versus Nucleases within the cell compared to the they show known 2′-O-allyl oligoribonucleotides lower chemical reactivity in biological Systems. The mentioned increased lipophilicity causes the application of the oligoribonucleotides according to the invention as an antisense oligonucleotide improved cell uptake.  

The present invention preferably relates to new ones 2'-O-alkyl oligoribonucleotides in which alkyl ethyl and their use as antisense oligonucleotides, in particular to inhibit gene expression or Virus replication.

The oligoribonucleotides according to the invention can furthermore, optionally marked, also as probes for Detection of specific DNA or RNA can be used. The oligoribonucleotides according to the invention can also as primer or primer sections in the enzymatic DNA amplification can be used.

In addition to the 2'-modification, the 2'-modified oligoribonucleotides according to the invention Have modifications at other positions, preferably at the 3'- and / or at the 5'-end. Possible Modifications are known per se; these include fluorescent groups, biotin or others prosthetic groups as well - with regard to a higher affinity for membranes - lipophilic groupings such as thiocholesterol or cholesterol, liposomes into which such lipophilic oligoribonucleotides are incorporated, are also the subject of the present invention!

The 2'-O-alkyl oligoribonucleotides according to the invention can be prepared by methods known per se will.

A method of making a Oligoribonucleotides made up of 8 to 35 Nucleotides of the general formula II,

wherein
B means cytosine, uracil, adenine, guanine or inosine,
R is alkyl having 1 to 30 carbon atoms, and
E¹ is a phosphate diester, methylphosphonate, phosphoramidate or phosphorothioate residue,
or their 2′-OH or 2′-deoxy derivatives,
where in at least one of the nucleotides R is alkyl with 2 to 30 C atoms,
and in which the terminal groups in positions 3 'and 5' independently of one another represent a hydroxyl group, a phosphite ester, H-phosphonate or phosphate ester residue, or a hydroxyl group modified by a labeling group or lipophilic group, characterized in that the corresponding ribonucleotide building blocks linked in the presence of a coupling reagent and, if desired, introducing groups and / or lipophilic groups into the oligoribonucleotide thus obtained and, if desired, splitting off protective groups.

Another aspect of the present invention relates an improved method of making a Oligoribonucleotides made up of 8 to 35 Nucleotides of the general formula II

wherein
B means cytosine, uracil, adenine, guanine or inosine,
R is alkyl having 1 to 30 carbon atoms, and
E¹ is a phosphate ester, phosphate diester, methylphosphonate, phosphoramidate or phosphorothioate residue,
or their 2′-OH or 2′-deoxy derivatives,
and in which the terminal groups in positions 3 'and 5' independently of one another represent a hydroxyl group, or a phosphate ester residue, or a hydroxyl group modified by a labeling group or lipophilic group, which is characterized in that the corresponding ribonucleotide building blocks are prepared by the following multistage process

• a) alkylating a nucleoside of the general formula IV, wherein B is protected or unprotected adenine, cytosine or inosine or O⁶-protected guanine or N³-protected uracil and, if desired, the hydroxy group in position 5 'is protected, in the presence of a deprotonating base with an alkylating agent of the general formula V, RZ (V) wherein R is alkyl with 1 to 30 C atoms and Z represents a nucleophilic leaving group (eg halogen, tosyl, mesyl, ethyl sulfate residue), preferably iodine, is reacted;
• b) introduction of protective groups in the bases and the Position 5 '(preferably mono- or Dimethoxytrityl);
• c) phosphorylating the from the reaction mixture isolated 2'-O-alkyl nucleosides by reaction with a phosphorylating agent, preferably Phosphoramidite;
• d) isolating the 2'-O-alkyl nucleoside phosphoramidite by standard methods or preferably by Addition of a lipophilic alcohol, preferably sec-butanol or iso-propanol and separating the desired 2'-O-alkyl nucleoside phosphoramidites through extraction and precipitation; and
• e) the ribonucleotide building blocks obtained in this way (if desired together with the above 2′-OH and / or 2′-H derivatives) are linked by standard methods, the protective groups are separated off and, if desired, introduced into the oligonucleotide-marking groups and / or lipophilic groups thus obtained.
  A nucleoside (IV) in which B is O⁶- [2- (4-nitrophenyl) ethyl] guanine is preferably used for the production of guanine-containing end products and, if appropriate, a nucleoside (IV) in which B Is N³-cyanoethyluracil.
  According to the invention, the alkylation is preferably carried out in the presence of sodium hydride as the deprotonating base, using R-iodide as the alkylating agent (V) at temperatures up to at most room temperature.
  Further information on these processes can be found in the examples.

The inventive method for the production of 2'-O-alkyl oligoribonucleotides compared to conventional method for the production of oligoribonucleotides and 2'-O-methyl oligoribonucleotides a simplified procedure and better Yields on. The last stage of the process, the Structure of the oligoribonucleotides from the Nucleotide building blocks are made according to conventional ones Coupling methods. An essential aspect of The inventive method is that the Alkylation of the nucleoside to the required 2'-O-alkyl nucleoside in contrast to the conventional Procedure in an earlier stage of the procedure he follows. The alkylation is within the Overall, the most lossy step, what before all through the formation of the unwanted By-products (base alkylated products, 2 ', 3'-O-dialkyl nucleosides and in particular 3′-O-alkyl nucleoside) is conditional. Since according to the invention this stage at the beginning of the overall process, one results substantial improvement in the overall yield of the Procedure. Through the execution of the invention Alkylation process will form the By-products (particularly advantageous of the by-product 3'-O-alkyl nucleoside) kept relatively low. (In  Several studies have found that the Share of 3'-O-alkyl nucleosides only 7-20% of Total amount of monoalkylated (i.e. 2'-O- and 3′-O-alkyl nucleosides) products.) In addition, at the process according to the invention the working up of Reaction mixture by chromatography or Crystallization possible, which is essential Represents simplification. (In particular, the Refurbishment due to the low proportion of 3′-O-alkyl nucleoside due.)

The use of methyl iodide for the production of 2'-O-methyladenosine without using a catalyst such as silver oxide is known (Yano, J., Biochim. Biophys. Acta 629 (1980), 178-183). The application of this method for the production of other 2'-O-methyl nucleosides and 2′-O-alkyl nucleosides (especially 2′-O-ethyl nucleosides) is however not easy possible. (So is the reactivity of methyl iodide much higher than that of the other alkyl iodides. The other nucleosides differ in their Reactivity essential of adenosine.) According to the Invention is the alkylation of adenosine and cytidine preferably on the unmodified (none Connections containing protecting groups executed. Thus, the alkylation represents the first stage of the Overall process. The alkylation of guanosine and Uridine is preferably carried out on O⁶-protected Guanosine or N³-protected uridine. (The preferred protecting groups are O⁶-nitrophenylethyl or N³-cyanoethyl.) Thus for Guanosine and uridine make the alkylation the second stage the manufacturing process. Generally are in themselves known protecting groups suitable, such as. B. are described in Welch, Ch. J. et al., Acta Chem. Scand. B37 (1983), 147-150.  

As has already been described, this is done alkylation process according to the invention Alkyl halide (preferably alkyl bromide and especially alkyl iodide) in the presence of Sodium hydride. By alkylating at low temperatures you can reduce the selectivity of the alkylation the 3'-O-alkylation) significantly increase, which the Yield of the desired product increased and the Workability of the reaction mixture is essential is improved. On the other hand, the Reaction temperature the reactivity of the Reaction components. As shown in examples, is the ethylation of cytidine and adenosine preferably starting from 0 ° C. of the reaction mixture up to room temperature. However, the Ethylation of guanosine preferably starting from -60 ° C to 0 ° C and the ethylation of uridine preferably starting from -40 ° C to room temperature.

Generally it can be stated that the alkylation according to the invention at the lowest possible temperature is carried out to selectivity of the Increase 2'-O-alkylation.

After the alkylation and the introduction of the necessary protective groups, the nucleosides are phosphorylated using known methods (e.g. using H-phosphonate, phosphoramidite, etc.). If the phosphorylation is carried out with a phosphoramidite (preferably with (2-cyanoethoxy) -N, N-diisopropylmonochlorophosphoramidite) which is used in excess, a lipophilic alcohol (e.g. sec-butanol or iso -Propanol) added, whereby the excess phosphorylating agent is converted to a lipophilic phosphoramidite. After extractive workup, the nucleoside phosphoramidite can then be isolated by precipitation;
the lipophilic by-product, which would significantly interfere in an oligonucleotide synthesis, remains in solution and is thereby completely separated. The method has the advantage that chromatographic purification is not necessary. In the specific case of the building blocks with base-labile phenoxyacetyl protective groups (especially guanosine), this work-up is particularly advantageous, since the chromatographic separation is only possible with difficulty or leads to products with low coupling efficiency in oligonucleotide synthesis.

Of those used in the process described Interconnections are new and the following connections also subject of the present invention:

Alkylated ribonucleotide building block of the general Formula I,

wherein
B means unprotected or protected cytosine, uracil, adenine, guanine or inosine,
R denotes alkyl with 2 to 30 C atoms,
E is a phosphite ester, H-phosphonate, phosphate ester, phosphate diester or phosphoramidate residue and
S¹ represents a protective group customary in oligonucleotide synthesis.

Ribonucleotide building blocks of the formula I are preferred, in which B is N⁶-benzoyladenine or N⁶-phenoxyacetyladenine, N⁴-benzoyl-cytosine, uracil or N³-cyanoethyl-uracil, N²-benzoyl-guanine, N²-phenoxyacetyl guanine or N²-phenoxyacetyl-O⁶-nitrophenylethyl guanine is, especially those Ribonucleotide building blocks, wherein R is alkyl with 2 to 8 C atoms, preferably ethyl or n-butyl and / or E N, N-Di (1-methylethyl) methoxyphosphoramidite or Is N, N-di (1-methylethyl) cyanoethoxy phosphoramidite and / or S¹ dimethoxytrityl or monomethoxytrityl is.

Nucleoside of the general formula III,

wherein
B in position 6 guanine protected by nitrophenylethyl and
R is methyl,
or
B is unprotected or protected cytosine, inosine, uracil or guanine and
R alkyl with 2 to 30 carbon atoms.

These nucleosides can be made in a manner known per se Processes are made.  

Preferred nucleosides are those in which B N⁴-benzoyl-cytosine, N³-cyanoethyl-uracil, N²-phenoxyacetyl guanine or N²-phenoxyacetyl-O⁶-nitrophenylethyl guanine and / or R is alkyl with 2 is up to 8 carbon atoms, preferably ethyl or n-butyl.

To demonstrate the biological inhibitory activity of 2'-O-ethyl oligoribonucleotides according to the invention and of those produced by the process according to the invention 2′-O-methyl-oligoribonucleotides became a system used in the inhibition of histone RNA biosynthesis can be tracked:

One of the essential checkpoints in the complex The histone RNA biosynthesis is the synthetic route Interaction of the "U7 small nuclear ribonucleoprotein" (snRNP) with the histone precursor mRNA (histone pre-mRNA). This interaction is required to control the pre-mRNA to cleave endonucleolytically. This split leads to Development of the mature mRNA, which is then ins Cytoplasm is transported (Birnstiel, M. L., et al. (1985 Cell 41, 349-359). The 15-20 nucleotides are close the 5'-end of the U7 RNA contained for Histone pre-mRNA sequence complementary sequences (Strub, K., et al., (1984) EMBO J. 3, 2801-2807; Mowry, K., et al., (1987) Science 238, 1682-1687; Cotten, M., et al., (1988) EMBO J. 7, 801-808; Soldati, D. et al., (1988) Mol. Cell, Biol. 8, 1518-1524). These Complementarities are essential for processing (Mowry, K., et al., (1987) Science 238, 1682-1687; Cotten, M., et al., (1988) EMBO J. 7, 801-808; Soldati, D. et al., (1988) Mol. Cell. Biol. 8, 1518-1524; Schaufele, F., et al. (1986) Nature 323, 777-781; Cotten, M., et al. (1989) Mol. Cell. Biol. 9, 4479-4487). It has also been shown that during the Cell cycle the accessibility of these 5'-nucleotides the U7 snRNP from the cell along with the  DNA synthesis is modulated (Hoffmann, I. et al. (1990) Nature 346, 665-668). Since the processing of the histone pre-mRNA through U7 snRNP an important role in the Cell cycle control of histone biosynthesis has been done an in vitro system designed for this processing to reproduce (Gick, O., et al., (1986) EMBO J. 5, 1319-1326). This system uses as a source for the processing factors, primarily U7 snRNP (Gick, O., et al., (1987) Proc. Natl. Acad. Sci. USA 84, 8937-8940; Vasserot, A., et al., (1989) Proc. Natl. Acad. Sci. USA 86, 4345-4349), a core extract rapidly dividing mammalian cells. When a radioactively labeled RNA molecule that contains the processing signals contains a histone pre-mRNA, this extract is added, a specific split takes place pre-mRNA takes place, forming a molecule that is identical to the product cleaved in vivo (Gick, O., et al., (1986) EMBO J. 5, 1319-1326). There this cleavage event is over base pairing ongoing interaction between U7 snRNP and the pre-mRNA (Schaufele, F., et al. (1986) Nature 323, 777-781) requires the sequence of the U7 RNA is known could use oligonucleotides Complementarity to pre-mRNA or U7 snRNP designed who, after being bound to their target sequence, the Impair processing (Cotten, M., et al. (1989) Mol. Cell. Biol. 9, 4479-4487). This test system was recently used the inhibitory effect various natural RNA and DNA antisense molecules and ribozymes directed against the U7 sequence to compare.

In the context of the present invention was with the help this test system examines whether the after Process according to the invention 2′-O-methyl-oligoribonucleotides and the 2'-O-ethyl-oligoribonucleotides according to the invention  are suitable for specifically inhibiting genes. It was found that both the 2'-O-methyl and the 2'-O-ethyl oligoribonucleotides the unmodified Antisense RNA molecules in inhibiting RNA processing are about 5 times superior, whereby for the inhibition only a small excess (less than 5-fold) the inhibitor against the target RNA is required to increase the processing response to 80% inhibit. Under conditions that are inhibited by a natural antisense RNA molecule make is the inhibition by the modified Antisense oligonucleotides practically irreversible without that this is accompanied by a modification of the target RNA, as recently for adenosine deaminase activity (Bass, B.L., et al. (1987) Cell 48, 607-613; Bass, B. L, et al. (1988) Cell 55, 1089-1098; Rebagliati, M.R., et al. (1987) Cell 48, 599-605). A possible explanation for irreversibility could be found in reduced degradation of the non-hybridized 2′-O-methyl-RNA be located in the extract. The presumed high Melting temperature of the inhibitor / U7 hybrid (due to the longer length became a higher melting temperature than that of Inoue, H., et al. (1987) Nucl. Acids Res. 15, 6131-6148, described) and the Difficulty, sufficient complement to the inhibitor add to inhibitor / U7 dissociation too effect, make it almost impossible to bind undo under physiological conditions. This irreversibility suggests that this class of inhibitors is suitable for in vivo applications.

The experiments in the context of the present invention were carried out to determine the effectiveness of various To investigate inhibitors of U7 function. (It was found that the 20 nucleotides at the 5 'end of the active U7 snRNP accessible for digestion  Micrococcal nuclease are and that the complexation of these 5'-nucleotides with a complementary one RNA nucleotide or its removal using a coupled deoxyoligo binding / RNase H cleavage Processing blocks (Cotten, M., et al. (1989) Mol. Cell. Biol. 9, 4479-4487; Hoffmann, I. et al. (1990) Nature 346, 665-668; Gilmartin, G., et al. (1988) Mol. Cell. Biol. 8, 1076-1084). In preliminary experiments for the present invention, it was found that the - on a molar basis - strongest inhibitor of the reaction an antisense RNA molecule complementary to 61 of the 63 nucleotides of the U7 sequence and that the Limitation of the function of the antisense inhibitors mainly in their rapid degradation in the extracts rich in nucleases located in the Attempts have been used. It turned out that a 19 obtained by the method according to the invention Nucleotides of 2'-O-methyl-oligoribonucleotides 61mer RNA is superior. It was also shown that the 2′-O-methyl-19mer in a phosphorothioate-DNA-19mer in the Considerably superior inhibition of processing response is from which it was concluded that the Nuclease resistance for the effectiveness of Inhibition is not the only determining factor is.

Example 1 Inhibitory effect of a natural 63mer antisense RNA in the Comparison with a 2′-O-methyl-7U-19mer antisense RNA

The response to inhibit the processing response in this and the following examples performed, as with Cotten, M., Schaffner, G. and Birnstiel, M. L ,. (1989) Mol. Cell. Biol. 9, 4479-4487,  described: A 15 @ l or 7.5 @ l aliquot of core extract (in buffer D: 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 20% glycerol, 20 mM HEPES, pH 7.4) from one Mouse hybridoma cell line, containing approx. 10 or 20 fmol U7 RNA (Dignam, J., Lebovitz, R. and Roeder, R. (1983) Nucl. Acids Res. 11, 1475-1489; Gick, O. Kramer, A., Keller, W. and Birnstiel, M. L. (1986) EMBO J. 5, 1319-1326), was with the respective Test oligonucleotide in the presence of 5 mM MgCl₂ (in a volume of 15 @ l) 30 min on ice, 30 min at Pre-incubated room temperature and 30 min at 30 ° C. After that 15 @ l of a reaction mixture containing tRNA, RNasin, EDTA (final concentrations 0.17 mg / ml, 400 Units / ml or 20 mM) and approx. 10 fmol (10,000 CPM) a 32 P-labeled histone pre-mRNA of the sample added. The mixture was at 30 ° C for 2 hours reacted, then Proteinase K (on 0.5 mg / ml) and SDS (0.5%) were added to the samples incubated at 37 ° C for 30 min. The RNA from the sample was then treated with phenol / chloroform, with ethanol like, in 80% formamide / 0.5 × TBE plus 0.025% Bromophenol blue & 0.025% xylene cyanole dissolved and on a preheated 10.7% acrylamide / 8.3 M Urea / TBE gel separated. The received radioactive pattern was obtained by exposing one X-ray film made visible at -70 ° C.

The sequences used in the experiments are shown in FIG. 1. The U7 sequence and the mouse histone H4 pre-mRNA are described in Cotten, M., Schaffner, G. and Birnstiel, ML (1989) Mol. Cell. Biol. 9, 4479-4487 (the U7 RNA probably functions due to a base pairing interaction with the purine-rich element in the pre-mRNA, which is identified in Fig. 1). Furthermore, the sequences of 1 7U 19mer (as 2'-O-methyl and 2'-O-ethyl-RNA) and the DNA 18-mer are shown in Fig., The check-2'-O-methyl oligonucleotide NS19mer ( without complementarity to U7) and the antisense U7 RNA 63mer (the additional vector sequences are boxed) (antisense U7 RNA (7U) was obtained by in vitro T7 polymerase transcription of a derivative of pTZ19 (Pharmacia) with a mouse U7 insert obtained from synthetic DNA oligonucleotides, synthesized).

To investigate whether a 2′-O-methyl-RNA molecule has inhibitory properties, the inhibition of processing by a 19 nucleotide-2′-O-methyl-RNA molecule (hereinafter referred to as 2′-O- Methyl-7U-19mer)) compared with a naturally synthesized RNA molecule containing the sequence complementary to 61 nucleotides of the U7-RNA (this complementary sequence is called 7U-RNA). (Preliminary experiments had shown that the 63mer antisense RNA was the strongest inhibitor of in vitro processing, this molecule completely blocking the reaction with a 30-fold molar excess over the U7 RNA (Cotten, M., Schaffner, G. and Birnstiel, ML (1989) Mol. Cell. Biol. 9, 4479-4487.) Accordingly, the inhibition test, the results of which are shown in FIG blocked and that 30 fmol resulted in a partial reduction in processing activity (lanes 4 and 5) In comparison, the 2′-O-methyl-7U-19mer gave complete inhibition at 300 fmol and inhibition of approx. 80% (lanes 8 and 9). As a control, the 2′-O-methyl-7U-19mer was prehybridized with a 10-fold excess of U7-RNA before it was added to the core extract. This procedure resulted in a total blocking of the inhibition (lane 12), which with 300 fmol of 2′-O-methyl-7U-19mer alone was complete (lane 9). These results show that the 2'-O-methyl inhibitor works via hybridization with the target RNA via base pairing and that the inhibition is not due to unspecific effects. (If the complement to the inhibitor is added after preincubation of the inhibitor with the extract, the inhibition caused by antisense RNA can be reversed; Cotten, M., Schaffner, G. and Birnstiel, ML (1989) Mol. Cell. Biol 9, 4479-4487 Carrying out the same experiment with the 2′-O-methyl inhibitor showed that only a small proportion of the inhibition could be reversed (lane 13).

Fig. 3:

Lane 1: unreacted substrate
Lane 2: control processing
Lanes 3 to 6:
Processing after preincubation with 3, 30, 300, 3000 fmol 7U-63merRNA
Lanes 7 to 11:
Processing after preincubation with 3, 30, 300, 3000 or 30 000 fmol 2'-O-methyl-7U-19mer
Lane 12:
300 fmol of 2′-O-methyl-7U-19mer were preincubated with 3 pmol of U7-RNA (in buffer D) before addition to the core extract
Lane 13:
The core extract was preincubated with 300 fmol 2'-O-methyl-7U-19mer. Then 3 pmol of U7 RNA was added to the reaction
Lane 14:
Control processing (the addition of 3 pmol U7-RNA has no influence on the processing reaction)
Tracks M:
Molecular weight standards: HpaII-cut pBr322 ( ^{32 p} -labeled using the Klenow fragment of polymerase I and @ -32 P-dCTP), the size of some of the fragments being shown to the left of the figure. The abbreviations "sub." and "proc." indicate the positions of the pre-mRNA substrate and the processed product.

Example 2 Inhibitory effect of a phosphorothioate antisense DNA 19mer in Comparison with a 2′-O-methyl-7U antisense RNA 19mer

The following consideration was assumed in the implementation of this example: If the only determinant for the increased inhibition by the 2′-O-methyl-RNA molecule is the stability against nucleases, then the use of a phosphorothioate derivative of the 7U-19mer- Oligonucleotide also show an increased ability to inhibit (however, the increased nuclease resistance is accompanied by a reduced binding affinity for the complementary sequence (Zon, 1988, Pharm. Res. 5, 539-549)). The experiment was carried out as indicated in Example 1; the results are shown in Fig. 4. With regard to the 2′-O-methyl-7U-19mer, identical results were found as in Example 1 (columns 4-7). A nonsense-2′-O-methyl oligonucleotide, a 19mer without sequences complementary to U7, was used as a control. This oligonucleotide (NS19mer) has no effect on processing when 3 pmol is added to the reaction (lane 8). If the phosphorothioate DNA 7U-19mer is tested in this system, it is found that there is a slight inhibition of the reaction at 300 fmol and approximately 30% inhibition at 3 pmol (lanes 9-13).

Fig. 4:

Lane 1: unreacted substrate
Lane 2: control processing
Lane 3: Processing after pretreatment with magnesium
Lanes 4-7:
Processing after preincubation with 3, 30, 300 or 3000 fmol 2′-O-methyl-7U-19mer
Lane 8:
Processing after preincubation with 3 pmol of a control 2′-O-methyl-19mer
Lanes 9-13:
Processing after preincubation with 0.3, 3, 30, 300, 3000 fmol phosphorothioate DNA 7U-19mer
Lane 14: control processing
Lane M: molecular weight standards as in Fig. 3

Example 3 Inhibitory effect of a 2'-O-ethyl-7U-19mer in comparison with a nitrophenylethyl-G-blocked 2'-O-ethyl-7U-19mer

The experiments carried out in the context of this example were based on the following consideration: Guanine groups modified with nitrophenylethyl (NPE) have lost the ability to form GC base pairs. Since in the 7U-19mer the G residues are distributed over the sequence ( FIG. 1), the presence of NPE-modified G residues should block the hybridization of the inhibitor to the U7 target sequence and thus the inhibition. NPE-G blocked 2′-O-ethyl-7U-19mer was therefore used as a control for the specificity of the inhibition. (The NPE-G-blocked 2′-O-ethyl-7U-19mer obtained as an intermediate in an example described below before the removal of the NPE protective groups was used.) The result of the experiments is shown in FIG. 5: the 2′-O-ethyl-7U-19mer inhibited the processing reaction completely at 300 fmol and <95% at 30 fmol. This inhibitory activity is similar to that observed for 2′-O-methyl compounds (see FIG. 3). In contrast, the NPE-G blocked 2′-O-ethyl-7U-19mer did not show inhibitory activity even at the highest concentrations examined (3 pmol; Fig. 5). Hence the importance of guanosine base pairing for inhibition.

Fig. 5:

Lane 1: unreacted substrate
Lane 2: control processing
Lane 3: Processing after pretreatment with magnesium
Lanes 4-7:
Processing after preincubation with 3, 30, 300 or 3000 fmol 2′-O-ethyl-7U-19mer
Lanes 8-11:
Processing after preincubation with 3, 30, 300 or 3000 fmol ^NPE G-2′-O-ethyl-7U-19mer
Tracks M:
Molecular weight standards as in Fig. 3

Example 4 Inhibitory effect of a 2′-O-methyl-7U-19mer in comparison with a 2'-O-ethyl-7U-19mer

In this experiment, the core extract concentration was reduced to 7.5 @ l in order to be able to carry out a more sensitive analysis of the processing response. The result of the tests carried out is shown in FIG. 6. When the two inhibitors were examined at concentrations of 3, 9, 30, 90 and 300 fmol, practically identical inhibitory activity was found (lanes 3-12). To test the inhibitory activity in the absence of magnesium, the test was modified slightly. If the two inhibitors were added directly to the extract in the absence of the divalent cation and the processing substrate RNA was then added rapidly in the presence of 20 mM EDTA, a slightly higher activity of the 2′-O-ethyl inhibitor was found (cf. lanes 13 and 14). Both inhibitors showed an increase in their activity of 30 fmol when they were preincubated with the extract plus magnesium (see lanes 5 and 13 for 2'-O-methyl and lanes 10 and 14 for 2'-O-ethyl).

Fig. 6:

Lane 1: control processing
Track 2: Processing after pretreatment with magnesium
Lanes 3-7:
Processing after preincubation with 3, 9, 30, 90 or 300 fmol 2′-O-methyl-7U-19mer
Lanes 8-12:
Processing after preincubation with 3, 9, 30, 90 or 300 fmol 2′-O-ethyl-7U-19mer
Lane 13:
Processing after adding 30 fmol of 2′-O-methyl-7U-19mer without pretreatment with magnesium
Lane 14:
Processing after adding 30 fmol 2′-O-ethyl-7U-19mer without magnesium pretreatment
Lane 15: control processing
Tracks M:
Molecular weight standards as in Fig. 3

Example 5 Determination of U7 RNA concentrations in nuclear extracts before and after treatment with antisense inhibitor

There is a possibility that in the presence of magnesium both the 2'-O-ethyl and the 2'-O-methyl oligonucleotides trigger a modification (or destruction) of the U7 target sequence which contributes to the inhibitory effect, which if necessary for the irreversibility of 2'-O-methyl inhibition ( Fig. 3) plays a role. Although the RNA-RNA-like duplex in the 2′-O-methyl or ethyl-U7 hybrid was not expected to be a substrate for the enzymatic degradation of U7 (like the DNA-RNA hybrid for RNase H), it was found that preincubation of the two inhibitors with core extracts in the presence of 5 mM Mg ²⁺ leads to increased inhibition and, in the case of 2′-O-methyl-RNA, to irreversibility ( FIGS. 3 and 6). A sensitive RNase protection assay was used to investigate whether there has been a modification to the U7 RNA that affects its ability to form base pair interactions. This determined the amount of U7 and its ability to hybridize before and after treatment with the various oligonucleotide inhibitors. After incubation with the various inhibitors, the EDTA concentration was increased to 20 mM in the presence of 5 mM Mg ²⁺ or without magnesium.

The individual procedure was as follows: The core extract samples (7.5 @ 1) were in buffer D 7.5 / 11.5 (buffer D, diluted by a factor 7.5 / 11.5), containing those in FIG. 7 specified amounts of the oligoribonucleotides ssDNA-7U-18mer, 2'-O-ethyl or 2'-O-methyl-7U-19mer or 2'-O-methyl-NS-19mer, incubated. The samples labeled "Mg ²⁺ " (4, 10-14, 16) contained 5 mM MgCl₂ and were incubated for 30 min on ice, 30 min at room temperature and 30 min at 30 ° C, followed by 20 mM EDTA have been added. For the remaining samples (3, 5-9, 15), the sample was incubated in buffer D 7.5 / 11.5 (containing 0.13 mM EDTA and without divalent cations) for 20 min on ice, followed by 20 mM EDTA have been added. Mapping was performed with an antisense mouse U7 RNA as described by Cotten, M., Schaffner, G. and Birnstiel, ML (1989) Mol. Cell. Biol. 9, 4479-4487. The protein in the sample was destroyed by Proteinase K digestion, the RNA was harvested and hybridized with radioactive 7U-RNA. The sample was then treated with RNase A plus RNase T1 to destroy all non-double-stranded RNA, and the protected RNA was separated by gel electrophoresis ( FIG. 7): The control, untreated core extract samples, gave the band expected for intact U7 RNA (Lanes 3 and 17). From the measurement of the radioactivity of this band, it could be concluded that 7.5 @ l of this core extract contained about 10-15 fmol U7-RNA. Incubation of the extract with Mg ²⁺ alone caused a slight reduction in the amount of U7 and the appearance of a small amount of a U7 subfragment ( FIG. 7, lane 4). Minor U7 destruction occurs with the DNA-7U-19mer in the absence of Mg ²⁺ (lane 5). The destruction of U7 is complete when the sample is incubated with 5 mM Mg ²⁺ (lane 10). However, in the absence and in the presence of Mg ²⁺ , neither 2′-O-methyl-7U-19mer nor 2′-O-ethyl-7U-19mer of the control 2′-O-methyl-NS19mer was detectable Change in the amount of U7 RNA detected. The conditions used were the same that caused complete inhibition of histone processing for the 7U-19mer oligonucleotides ( Figure 6). The slight decrease in U7 concentrations and the appearance of the U7 subfragment in lanes 11-14 are no more pronounced than the changes observed with Mg ²⁺ incubation in the absence of oligonucleotide (lane 4). Based on the results of this experiment, it was concluded that the strong inhibitory activity of the 2′-O-methyl and ethyl oligoribonucleotides was not accompanied by an enzymatic change in the target RNA.

Fig. 7:

Track 1: 7U probe
Lane 2: 7U, hybridized with 20 @g tRNA
Lanes 3 and 17: Control: extract, treated without oligonucleotide, without Mg ²⁺
Lane 4: extract, treated with Mg ²⁺
Lane 5: extract, treated with 30 pmol ssDNA-7U-19mer
Lanes 5 and 7: extract treated with 30 and 300 fmol 2′-O-methyl-7U-19mer
Lanes 8 and 9: extract treated with 30 and 300 fmol 2′-O-ethyl-7U-19mer
Lane 10: extract treated with 30 pmol ssDNA-7U-19mer plus Mg ²⁺
Lanes 11 and 12: extract treated with 30 and 300 fmol 2'-O-methyl-7U-19mer plus Mg ²⁺
Lanes 13 and 14: extract treated with 30 and 300 fmol 2′-O-ethyl-7U-19mer plus Mg ²⁺
Lane 15: extract, treated with 3 pmol of control 2′-O-methyl NS-19mer
Lane 16: extract, treated with 3 pmol of control 2′-O-methyl NS-19mer plus Mg ²⁺
Lanes M: molecular weight standards as in Fig. 3.

The truncated U7 species caused by NA oligonucleotide / RNase H cleavage were formed are given.

The following examples illustrate another Subject of the present invention.

The synthesis of the oligodeoxynucleotides can be carried out according to Standard procedure on the ABI 380 B DNA synthesizer (Applied Biosystems, 380 B DNA synthesizer, users manual, version 1.0, July 1985). The Oligodeoxynucleotide is caused by ethanol precipitation and denaturing polyacrylamide gel electrophoresis or Reverse phase HPLC cleaned.

The 19mer 2'-methoxyoligonucleotides can with a ABI 380 B DNA synthesizers can be synthesized, whereby CPG solid phase (controlles-pore glass) with a 3′-deoxynucleoside is used as a starter.

Preferably 2'-O-methyl nucleoside (2-cyanoethyl) - (N, N-diisopropylamino) phosphoramidite with the following Protection groups used:

Adenosine:
N⁶-phenoxyacetyl 5′-O-DMTr (dimethoxytrityl)
Cytidine:
N⁴-benzoyl 5′-O-MMTr (monomethoxytrityl)
Guanosine: N²-phenoxyacetyl 5′-O-DMTr (dimethoxytrityl)
Uridine:
5′-O-MMTr (monoethoxytrityl)

The preparation of these compounds is described in the examples. The standard DNA method is used with an extended coupling time (5 minutes instead of 3 minutes). After the solid phase synthesis, the oligonucleotide is freed from the protective groups by treatment with 25% aqueous NH₄OH solution (for example 15 hours at 55 ° C.) and separated from the solid support. The NPE group on guanosine (if present) is split off using the DBU. After the final detritylation, the crude oligonucleotide is purified by denaturing polyacrylamide gel electrophoresis. For U7 inhibition, the material is further purified by RP-HPLC (reverse phase HPLC) ( Fig. 2).

The synthesis of phosphorothioatoligodeoxynucleotides can be achieved using the H-phosphonate method with an AB 380 B DNA synthesizer, using the recommended procedures respectively. (Applied Biosystems, User Bulletin DNA synthesizer model 380, issue no. 44, Dec. 1987. Froehler, B.C. and Matteucci, M.D. (1986) Tetrahedron Lett. 27, 469-72 and (1986) Nucl. Acids Res. 14, 5399-5407. The sulfurization can be carried out with sulfur (5% in Carbon disulfide, pyridine, triethylamine 12: 12: 1) the solid support material, the 5'-end group is protected. After disconnecting from that solid support and removing the Protecting groups of the bases, the 5'-trityl phosphorothioate oligonucleotide by RP-HPLC (reverse  phase HPLC). The material of the main peak is detritylated and again by RP-HPLC (reverse phase HPLC).

The synthesis of phosphorothioate-2'-O-alkyl oligoribonucleotides can also through the Phosphoramidite method analogous to the standard method (Applied Biosystem, 380B DNA Synthesizer, users manual, version 1.0, July 1985) with 2′-O-alkyl nucleosides take place with the modification that in Oxidation step instead of iodine a 0.2 M solution of 3H-1,2-benzodithiol-3-one, 1,1-dioxide in acetonitrile is used, as in IYER, R. P. J. Am. Chem. Soc. 112: 1253-1254 (1990).

The synthesis of the 2'-O-ethyl oligoribonucleotides can e.g. B. on a Pharmacia Gene Assembler, the with 0.2 µmol solid carrier material (5 µm Polymer beads based on polystyrene) with deoxythymidine is loaded as a 3'-starter nucleotide.

Preferably 2'-O-ethyl nucleoside (2-cyanoethyl) - (N, N-diisopropylamino) phosphoramidite with the following Protection groups used:

Adenosine:
N⁶-phenoxyacetyl 5′-O-DMTr (dimethoxytrityl)
Cytidine:
N⁴-benzoyl 5′-O-DMTr (dimethoxytrityl)
Guanosine: N²-phenoxyacetyl
O⁶-NPE (NPE: 2- (4-nitrophenyl) ethyl)
5′-O-DMTr (dimethoxytrityl)
Uridine:
5′-O-DMTr (dimethoxytrityl)

The conditions correspond to the standard of DNA synthesis, except for the coupling time, which is expediently increased to 5 minutes. (Procedure: removing the protective groups: 0.4 min, 3% trichloroacetic acid in dichloroethane, coupling: 5.0 min, 25 equivalents of amidite, 500 equivalents of tetranol in acetonitrile; capping: 0.4 minutes, 10% acetic anhydride, 3% 4- (N, N-Dimethylamino) pyridine (DMAP), 15% collidine in acetonitrile; oxidation: 0.1 min, 0.01 MI₂, 5% collidine in acetonitrile). These measures are necessary to counteract the reduced coupling efficiency, which is due to the steric hindrance caused by the bulky 3′-ethoxy group. The average coupling efficiency is approximately 98.3% (calculated from the amount of trityl cations released when the protective groups are removed). Removal of the protective groups of the base and purification: after the last de-tritylation, the bound oligonucleotide is treated with 25% strength aqueous ammonia for 2 hours at 55 ° C. in order to separate it from the support and to remove all protective groups (except the O⁶-2- ( 4-nitrophenyl) ethyl group (NPE) in guanosine). The solution is evaporated to dryness in vacuo and a portion of the crude oligonucleotide (or NPE-protected guanosine) is purified by RP-HPLC (reverse phase-HPLC) ( Fig. 2) to be used as a negative control in the inhibition studies. In the preparation of guanosine compounds, in order to remove the NPE group, the oligonucleotide for 24 hours with 500 μl of 1 M DBU (1,8-diazabicyclo [5,4,0] undec-7-ene) in pyridine at 55 ° C treated. (Mag, M. and Engels, JW (1988) Nucl. Acids Res. 16, 3525-3543). After neutralizing the DBU with 1.1 equivalents of acetic acid and partial evaporation in vacuo, the yellow solution is subjected to gel filtration and the product is further purified by preparative polyacrylamide gel electrophoresis. The yield of purified 19mer: 240 ug (36 pmol, 18% based on the solid support material, RP-HPLC profile, Fig. 2).

The other oligonucleotides of the invention can can be produced analogously to the instructions above.

Examples of the production of nucleosides N⁴-Benzoyl-5'-O-dimethoxytrityl-2'-O-methyl-cytidine 3a

40 g (0.164 mol) of cytidine were dried under high vacuum at 80 ° C. for 20 hours and then dissolved in 650 ml of anhydrous dimethylformamide with heating. The solution was cooled to 0 ° C. and 4.33 g (0.18 mol) of washed sodium hydride were added and the mixture was stirred at 0 ° C. for 45 minutes. Then a total of 36.9 g (0.26 mol) of methyl iodide (20% in dimethylformamide) were added dropwise in portions over a period of 4 hours and while warming to room temperature. The reaction was monitored by thin layer chromatography and stopped at a time when the proportion of dimethylated product in the reaction mixture rose sharply. Precipitated sodium iodide was separated off during the work-up and the reaction mixture was evaporated under a high vacuum. The residue was purified by flash chromatography (substance / silica gel = 1/10, eluent: chloroform / methanol = 5/1). Crude yield: 22.2 g (mixture of the 2′- and 3′-O-methyl isomers, contaminated with sodium iodide; TLC: chloroform / methanol = 3/2. R _f = 0.3 for both isomers). 20.8 g of the crude product were towed several times with benzene / pyridine and dissolved in 250 ml of anhydrous pyridine. Then 34.9 g (0.32 mol) of trimethylchlorosilane were added dropwise at room temperature and the mixture was stirred for 60 minutes. Then 13.5 g (0.096 mol) of benzoyl chloride were added and the mixture was left overnight at room temperature. The reaction solution was poured onto 300 ml of water, diluted with 300 ml of methanol and mixed with 250 ml of concentrated ammonia. After 20 minutes, the mixture was poured onto dichloromethane and the aqueous phase was extracted ten times with dichloromethane. The collected organic phases were combined, dried with sodium sulfate, filtered and evaporated. 32 g of crude product 2a / 5a = 4.26 / 1 were obtained (TLC: dichloromethane / methanol = 10/1. R _f = 0.2, both regioisomers). The mixture 2a / 5a was towed three times with pyridine / benzene, 33 g (0.0976 mol) of dimethoxytrityl chloride were added in anhydrous pyridine and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and partitioned between dichloromethane and sodium hydrogen carbonate solution. The aqueous phase was extracted several times with dichloromethane. The collected organic phases were dried with sodium sulfate and evaporated. The crude product obtained was purified by means of flash chromatography (1200 g of silica gel, eluent: dichloromethane then dichloromethane / acetone = 8/1)). 25.3 g (25%) 3a and 5.1 g (5%) 6a were obtained as yellow solid oils (starting from cytidine). TLC: dichloromethane / acetone = 3.5 / 1.5. R _f = 0.28 for 3a, R _f = 0.24 for 6a.

1 H-NMR (CDCl₃, 90 MHz): δ = 8.58 (d, J = 7.2 Hz, 1 H; 6-H), 7.98-7.18 (m, 16H; DMTr, Bz, NH, 5-H), 6.89 (d, J = 7.5 Hz, 4H; DMTr), 6.02 (s, 1H; 1′-H), 4.65-3.92 (m, 4H; 2′-H, 3′-H, 4′-H, 3′-OH), 3.82 (s, 6H; DMTr OCH₃), 3.7 (s, 3H); 2'-OCH₃), 3.6 (m, 2H; 5'-CH₂).

13 C NMR (CDCl 3, 50 MHz): δ = 166.22 (s, C = O), 162.36 (s, 4-C *), 158.25 (s, aroma, C-OCH₃), 154.37 (s, 2-C), 144.58 (d, 6-C), 143.73 / 135.29 (2 s, DMTr), 134.98 / 132.7 / 132.54 (aroma, Bz), 129.74 / 129.62 / 128.38 / 127.90 / 127.64 / 127.39 / 126.754 (arom.), 112.94 (d, DMTr), 96.46 (d, 5-C), 87.99 (d, 1′-C), 86.62 (s, C- (Ph) 3), 83.51 (d, 2′-C), 82.46 (d, 4′-C), 67.42 (d, 3'-C), 60.58 (t, 5'-CH₂), 58.22 (q, OCH₃), 54.78 (q, DMTr OCH₃).

N⁴-Benzoyl-5'-O-dimethoxytrityl-3'-O-methylcytidine 6a

1 H-NMR (CDCl₃, 200 MHz): δ = 8.43 (d, J = 7.7 Hz, 1H; 6-H), 7.91 (d, J = 7.7, 2H, Bz), 7.65-7.2 (m, 13H; DMTr, Bz, 5-H), 6.9 (d, J = 9.7, 4H; DMTr), 5.98 (s, 1H; 1'-H), 4.47 (m, 1H; 2'-H), 4.31 (m, 1H; 4'-H), 4.02 (m, 1H; 3'-H), 3.82 (s, 6H; DMTr OCH₃), 3.6 (d, J = 10.3, 1H; 5'-H), 3.44 (s, 3H; 3'-OCH₃), 3.5-3.35 (m, 1H; 5'-H).  

13 C-NMR (CDCl 3, 50 MHz): δ = 166.5 (s, Ar-NH-C = O), 162.51 (s, 4-C), 158.62 (s, arom., C-OCH₃), 155.34 (s, 2-C), 144.78 (d, 6-C), 144.01 / 135.49 / 135.24 / 132.98 / 130.0 / 129.88 / 128.83 / 128.08 / 127.96 / 127.65 / 127.08 / 113.26 (aroma), 96.89 (d, 5-C), 92.24 (d, 1'-C), 86.96 (s, C- (Ph) 3), 81.5 (d, 4'-C), 78.01 (d, 3'-C), 73.92 (d, 2'-C) 61.64 (t, 5'-CH₂), 58.27 (q, OCH₃), 55.14 (q, DMTr OCH₃).

N⁴-Benzoyl-2'-O-methylcytidine 2a (was characterized as the product obtained by detritylation of 3a): 430 mg (0.65 mmol) 3a was stirred for 5 min in 16 ml of 2% trichloroacetic acid solution in dichloromethane. The reaction mixture was extracted with water, the combined aqueous phases were evaporated and purified by flash chromatography (9 g of silica gel, eluent: dichloromethane / acetone = 4/1 then 3/2): 220 mg (94%) 2a, white foam, TLC: dichloromethane / Methanol = 10/1. R _f = 0.2.

1 H-NMR (DMSO, 200 MHz): δ = 8.9 (m, 1H; 6-H), 8.69-7.42 (m, 3H; 3'-OH, 5'-OH, NH), 8.15 (d, J = 7.7 Hz, 2H; Bz), 7.78-7.42 (m, 4H; Bz, 5-H), 5.83 (s, 1H; 1'-H), 4.12 (m, 1H; 3'-H), 3.98 (m, 1H; 4'-H), 3.87 (m, 2H; 2′-H, 5′-H), 3.67 (d, J = 12.7 Hz, 1H; 5′-H), 3.48 (s, 3H; 2'-OCH₃).

13 C-NMR (DMSO, 50 MHz): δ = 166.69 (s, Ar-NH-C = O), 160.09 (s, 4-C), 149.22 (s, 2-C / d, 6-C), 133.87 (d, aroma), 131.71 / 128.86 / 128.79 (aroma), 95.34 (d, 5-C), 88.78 (d, 1′-C), 84.55 (d, 4′-C), 83.38 (d, 2′-C), 67.11 (d, 3′-C), 58.96 (t, 5′-CH₂), 58.09 (q, OCH₃).  

N⁴-Benzoyl-3'-O-methylcytidine 5a (obtained by detritylation of 6a): 400 mg (0.60 mmol) 6a were dissolved in 10 ml of anhydrous dichloromethane and cooled to -5 ° C. The solution was mixed with 0.2 ml of a 10% ethereal hydrochloric acid solution. The reaction mixture immediately turned red and a precipitate formed. The reaction mixture was evaporated and the residue was purified by flash chromatography (8 g of silica gel, eluent: dichloromethane / acetone = 2/1.) And precipitated from ethyl acetate / methanol. 194 mg (89.5%) 5a were obtained as a white foam. TLC: dichloromethane / methanol = 10/1. R _f = 0.4.

1 H-NMR (DMSO, 200 MHz): δ = 8.75 (d, J = 7.7 Hz, 1H; 6-H), 8.1 (d, J = 7.7 Hz, 2H; Bz) 7.74-7.37 (m, 4H; Bz, 5-H), 6.9-5.5 (m, 2H; 3'-OH; 5'-OH), 5.76 (s, 1H; 1'-H), 4.33 (m, 1H; 2'-H), 4.08 (m, 1H; 4'-H), 3.9-3.7 (m, 2H; 3′-H, 5′-H), 3.63 (d, J = 12.7 Hz, 1H; 5′-H), 3.34 (s, 3H; 3'-OCH₃).

13 C-NMR (DMSO, 50 MHz): δ = 166.95 (s, Ar-NH-C = O), 160.83 (s, 4-C), 150.54 (s, 2-C), 148.14 (d, 6-C), 133.53 / 132.15 / 128.75 / 128.66 (aroma), 95.40 (d, 5-C), 91.31 (d, 1′-C), 82.54 (d, 4′-C), 77.01 (d, 3′-C), 72.24 (d, 2′-C), 59.46 (t, 5′-CH₂), 57.38 (q, OCH₃).  

N⁴-Benzoyl-2'-O-ethylcytidine 2b

45 g (0.185 mol) of cytidine were described as for 3a dissolved and cooled, with 7.4 g (0.185 mol) Sodium hydride (60% in paraffin, washed in anhydrous n-hexane) was added and the mixture was stirred at 0 ° C. for 1 h. Analogously to 3a, 40.52 g (3.825 mol) of ethyl iodide admitted. Cleaning by SC (fine silica gel, Eluens dichloromethane / THF = 7/1) purified and gave 25.5 g of colorless foam with 32.8 g (0.301 mol) Trimethylchlorosilane and 19.8 g (0.140 mol) Benzoyl chloride was added. After working up taken up in ethyl acetate and for crystallization brought. After drying, 16.6 g (24%) of pure product 2b, melting point 193-194 °.

1 H-NMR (CDCl₃ / DMSO, 200 MHz): δ = 10.5 (s br, 1H; NH), 8.62 (d, J = 8 Hz, 1H; 6-H), 8.03 (d, 2H; Bz), 7.70-7.30 (m, 4H; Bz, 5-H), 5.87 (s, 1H; 1′-H), 5.18 * (s br, 1H; 5′-OH), 4.75 * (s br, 1H; 3′-OH), 4.30-3.50 (m, 7H; 2'-H, 3'-H, 4'-H, O-CH₂-CH₃, 5'-CH₂), 1.21 (t, J = 7 Hz, 3H; O-CH₂-CH₃).

13 C NMR (CDCl 3 / DMSO): δ = 166.25 (s, Ar-NH-C = O) ,. 161.98 (s, 4-C), 153.48 (s, 2-C), 143.70 (d, 6-C), 132.01 (d, aroma), 131.22 / 127.01 (aroma), 95.18 (d, 5-C), 87.56 (d, 1′-C), 82.88 (d, 4′-C), 80.97 (d, 2'-C), 65.89 (d, 3'-C), 64.55 (t, O-CH₂-CH₃), 57.95 (t, 5'-CH₂), 13.98 (q, O-CH₂-CH₃).  

N⁴-Benzoyl-5'-O-dimethoxytrityl-2'-O-ethylcytidine 3b

7.30 g (19.4 mmol) 2b were mixed with 9.88 g (29.2 mmol) Dimethoxytrityl chloride implemented and analogous to 3a worked up. Cleaning by SC (finest silica gel, Eluens dichloromethane / isopropanol = 30: 1) gave 7.43 g (56.6%) pure product 3b.

1 H-NMR (CD₃CN, 250 MHz): δ = 9.36 (s br, 1H, NH), 8.50 (d, J = 8 Hz, 1H; 6-H), 7.95 (d, J = 8 Hz, 2H; Bz 2,6-H), 7.70-7.20 (m, 12H; DMTr, Bz 3,4,5-H), 7.13 (d, J = 8Hz, 1H; 5-H), 6.91 (m, 4H; DMTr), 5.84 (s, 1H; 1'-H), 4.42 (s br, 1H; 3'-OH), 4.20-3.10 (m, 13H; 2'-H, 3'-H, 4'-H, O-CH₂-CH₃, 5'-CH₂, [with 3.80 (s, 6H; DMTr OCH₃]), 1.22 (t, 3H; O-CH₂-CH₃).

13 C-NMR (CD₃CN, 62.5 MHz) δ = 168.05 (s, Ar-NH-C = O), 163.64 (s, DMTr, C-OCH₃), 155.49 (s, 2-C), 145.81 (d, 6-C), 145.32 / 136.81 (aroma, DMTr), 136.43 / 134.30 / 133.73 (arom.-C, C-benzoyl), 130.98 / 130.81 / 129.50 / 129.06 / 128.92 / 128.88 / 127.93 (arom.-C), 114.06 (arom., DMTr), 96.92 (d, 5-C), 90.06 (d, 1'-C), 87.50 (s, C- (Ph) 3), 83.49 (d, 4'-C), 82.74 (d, 2'-C), 68.66 (d, 3'-C), 67.00 (t, O-CH₂-CH₃), 62.01 (t, 5′-CH₂), 55.78 (q, DMTr OCH₃), 15.45 (q, O-CH₂-CH₃).

5'-O-Dimethoxytrityl-2'-O-methyl-N⁶- (phenoxyacetyl) adenosine 9a

50 g (0.187 mol) of adenosine were dried under high vacuum at 80 ° C. for 20 hours and then dissolved in 700 ml of anhydrous dimethylformamide with heating. The solution was cooled to 0 ° C., 5.34 g (0.224 mol) of washed sodium hydride was added and the mixture was stirred at 0 ° C. for 45 minutes. A total of 42.2 g (0.3 mol) of methyl iodide (20% in dimethylformamide) were then added dropwise in portions over a period of 4 hours and while warming to room temperature. The reaction was monitored by thin layer chromatography and stopped at a time when the proportion of dimethylated product in the reaction mixture rose sharply. During working up, undissolved sodium iodide was separated off and the reaction mixture was evaporated under a high vacuum. The residue was purified by means of flash chromatography (substance / silica gel = 1/10, eluent: gradient of 5-20% methanol in chloroform). The 42 g of crude product obtained (mixture of the 2′- and 3′-O-methyl isomers, contaminated with sodium iodide; TLC: chloroform / ethanol = 3/1, R _f = 0.3 for both isomers) were extracted several times with a mixture Towed benzene and pyridine and dissolved in 500 ml of anhydrous pyridine. 64.8 g (0.597 mol) of trimethylchlorosilane were then added dropwise at room temperature, and after a further 2 hours, 51.2 g (0.179 mol) of phenoxyacetic anhydride were added and the mixture was stirred at room temperature overnight. The reaction solution was poured onto 1 liter of water, diluted with 400 ml of methanol, so that a clear solution was obtained. After 40 minutes, sodium bicarbonate was added and the mixture was stirred for a further 10 minutes and then extracted several times with dichloromethane. The collected organic phases were combined, dried with sodium sulfate, filtered and evaporated. The crude product obtained (45 g) with 8a / 11a = 4.6 / l was towed three times with pyridine / benzene and 53.2 g (0.157 mol) of dimethoxytrityl chloride were added in anhydrous pyridine and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and partitioned between dichloromethane and sodium hydrogen carbonate solution. The aqueous phase was extracted several times with dichloromethane. The collected organic phases were dried with sodium sulfate, filtered and evaporated. The 112 g of crude product thus obtained were purified by means of flash chromatography (1200 g of silica gel, eluent: dichloromethane then dichloromethane / acetone = 8/1). 32.8 g of 9a (24.5% based on adenosine) and 3.3 g of 12a were obtained as a colorless solid. TLC: dichloromethane / acetone = 3.5 / 1.5 9a: R _f = 0.52, 12a: R _f = 0.48.

1 H-NMR (CDCl₃, 90 MHz): δ = 9.5 (s br, 1H; NH), 8.72 (s, 1H; 2-H), 8.28 (s, 1H; 8-H), 7.5-6.7 (m, 18H; DMTr, PhOAc), 6.2 (d, J = 3 Hz, 1H; 1'-H), 4.87 (s, 2H; PhOAc CH₂), 4.65-4.4 (m, 2H; 2'-H, 3'-H), 4.25 (m, 1H; 4'-H), 3.77 (s, 6H; DMTr OCH₃), 3.56 (s, 3H; 2'-OCH₃), 3.52 (m, 2H; 5'-H), 2.82 (s, 1H; 3'-OH).

13 C-NMR (CDCl 3, 22.5 MHz): δ = 167.03 (s, C = O), 158.09 (s, aroma, C-OCH₃), 156.9 (s, aroma, C-O-CH₂), 151.9 (d, 2-C), 150.89 (s, 6-C), 148.02 (s, 4-C), 144.17 (arom., DMTr), 141.95 (d, 8-C), 135.24 (aroma, DMTr), 129.6 (d, aroma, PhOAc), 129.22 / 128.68 / 127.70 / 127.38 / 126.46 (aroma, DMTr), 122.45 (s, 5-C), 121.63 / 114.48 (2d, aromatic, PhOAc), 112.75 (arom., DMTr), 86.41 (d, 1′-C), 86.15 (s, C- (Ph) 3), 83.75 (d, 4′-C), 82.68 (d, 2′-C), 69.35 (d, 3'-C), 68.0 (t, Ph-O-CH₂), 62.69 (t, 5'-CH₂), 58.24 (q, OCH₃), 54.67 (q, DMTr OCH₃).  

Goodbye to 5'-O-dimethoxytrityl-3'-O-methyl-N⁶- (phenoxyacetyl) nosin 12a

1 H-NMR (CDCl₃, 200 MHz): δ = 9.43 (s br, 1H; NH), 8.73 (s, 1H; 2-H), 8.22 (s, 1H; 8-H), 7.46-6.99 (m, 14H; DMTr, PhOAc), 6.81 (d, J = 9.7 Hz, 4H; DMTr), 6.04 (d, J = 6.5 Hz, 1H; 1'-H), 5.01-4.8 (m, 1H; 2'-H), 4.89 (s, 2H; PhOAc CH₂), 4.4-4.27 (m, 1H; 4′-H *), 4.18-4.06 (m, 1H; 3′-H *), 3.79 (s, 6H; DMTr OCH₃), 3.57-3.26 (m, 2H; 5'-CH₂), 3.5 (s, 3H; 3'-OCH₃).

13 C NMR (CDCl 3, 50 MHz): δ = 166.74 (s, Ar-NH-C = O), 158.47 (s, aroma, C-OCH₃), 156.98 (s, aroma, C-O-CH₂), 152.29 (d, 2-C), 151.40 (s, 6-C), 148.25 (s, 4-C), 144.34 (arom., DMTr), 142.11 (d, 8-C), 135.43 (aroma, DMTr), 129.87 (d, aroma, PhOAc), 129.67 / 127.94 / 127.78 / 126.84 (4d, DMTr), 122.85 (s, 5-C), 122.21 / 114.83 (d, aroma, PhOAc), 113.09 (d, arom., DMTr), 89.27 (d, 1′-C), 86.52 (s, C- (Ph) 3), 81.89 (d, 4′-C), 79.97 (d, 3′-C), 73.59 (d, 2′-C), 68.06 (t, Ph-O-CH₂), 63.12 (t, 5′-CH₂), 58.13 (q, OCH₃), 55.09 (q, DMTr OCH₃).

2'-O-Methyl-N⁶- (phenoxyacetyl) adenosine 8a: The product mixture 8a / 11a obtained from an analogous approach was purified by flash chromatography (400 g silica gel, eluent: dichloromethane / acetone = 4/1). After single chromatography, 5.5 g (7.05%) 8a, with the analytical NMR data given below, and 14.1 g (18.1%) 8a / 11a were obtained. TLC: dichloromethane / methanol = 4.5 / 0.5. 8a: R _f = 0.50; 11a: R _f = 0.48.

1 H-NMR (CDCl₃, 90 MHz): δ = 9.70 (s br, 1H; NH), 8.7 (s, 1H; 2-H), 8.1 (s, 1H; 8-H), 7.4-6.8 (m, 5H; PhOAc arom.), 5.95 (d, J = 6 Hz, 1H; 1′-H), 5.7 (s br, 1H; 5'-OH), 4.88 (s, 2H; PhOAc CH₂), 4.7-4.5 (m, 2H; 2′-H, 3′-H), 4.3 (s, 1H; 4′-H), 4.05-3.6 (m, 2H; 5'-CH₂), 3.45 (s, 3H; 2'-OCH₃), 3.12 (s, 1H; 3′-OH).

13 C NMR (CDCl 3, 22.5 MHz): δ = 167.5 (s, Ar-NH-C = O), 157.01 (s, arom., C-O-CH₂), 151.6 (d, 2-C), 150.41 (s, 6-C), 148.67 (s, 4-C), 143.41 (d, 8-C), 129.38 (d, aroma), 123.26 (s, 5-C), 121.85 / 114.73 (d, aroma), 88.53 (d, 1′-C), 87.40 (d, 4′-C), 82.8 (d, 2′-C), 69.73 (d, 3′-C), 68.21 (t, Ph-O-CH₂), 62.3 (t, 5'-CH₂), 58.3 (q, 2'-OCH₃).

3′-O-Methyl-N⁶- (phenoxyacetyl) adenosine 11a was obtained as a pure product by detritylation of 12a: 1 g (1.39 mmol) of 12a was dissolved in 20 ml of anhydrous dichloromethane and cooled to -5 ° C. The solution was mixed with 0.5 ml of a 10% ethereal hydrochloric acid solution. The reaction mixture immediately turned red and a precipitate precipitated out. After one minute, the reaction solution was diluted with aqueous sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases were dried with sodium sulfate, filtered and evaporated. The residue was purified by means of flash chromatography (12 g of silica gel, eluent: dichloromethane / acetone = 3.5 / 1). 530 mg (92%) 11a were obtained. TLC: dichloromethane / methanol = 9/1; R _f = 0.48, white foam.

1 H-NMR (CDCl₃, 200 MHz): δ = 9.58 (s br, 1H; NH), 8.72 (s, 1H; 2-H), 8.08 (s, 1H; 8-H), 7.42-7.26 (m, 2H; PhOAc aroma.), 7.13-7.0 (m, 3H; PhOAc aroma.), 5.82 (d, J = 7.7 Hz, 1H; 1′-H), 5.02 (dd, J₁ = 5.5, J₂ = 7.7, 1H; 2'-H), 4.88 (s, 2H; PhOAc CH₂), 4.36 * (s, 1H; 4′-H), 4.1 * (d, J = 5.5 Hz, 1H; 3′-H), 4.02 / 3.74 (2d, J = 12.8 Hz, 2H; 5'-CH₂), 3.54 (s, 3H; 3'-OCH₃).

13 C NMR (CDCl 3, 22.5 MHz): δ = 167.0 (s, Ar-NH-C = O), 156.95 (s, arom., C-O-CH₂), 151.79 (d, 2-C), 150.38 (s, 6-C), 148.77 (s, 4-C), 143.5 (d, 8-C), 129.71 (d, aroma), 123.59 (s, 5-C), 122.28 / 114.82 (d, aroma), 91.8 (d, 1′-C), 84.57 (d, 4′-C), 80.79 (d, 3′-C), 73.52 (d, 2′-C), 68.06 (t, Ph-O-CH₂), 63.34 (t, 5'-CH₂), 58.0 (q, 3'-OCH₃).

5'-O-Dimethoxytrithyl-2'-O-ethyl-N⁶- (phenoxyacetyl) ad enosin 9b

50 g (0.187 mol) adenosine were as for 9a dissolved, cooled and with 8.98 g (0.225 mol) Sodium hydride suspension (60% in paraffin, washed with anhydrous n-hexane). 34.8 g (0.225 mol) Ethyl iodide in 40 ml of anhydrous DMF added in portions. After stirring for 2 h at 0 ° C 46.5 g (0.300 mol) of ethyl iodide in 50 ml of anhydrous DMF added and stirred at 0 ° C for a further 2 h. The Working up took place in analogy to 8a and gave 30.0 g of colorless foam. This was like for 9a described with 44.1 g (0.406 mol) of trimethylchlorosilane and 58.4 g (0.203 mol) of phenoxyacetic anhydride implemented and revised. SC cleaning (silica gel finest, eluent dichloromethane / THF = 1/1) gave 19.7 g  (25%) crude product 8b / 11b (ratio 14: 1) as colorless foam. 19.6 g of the crude product were dried, then in 100 ml of anhydrous pyridine dissolved and with 23.2 g (0.069 mol) of dimethoxytrityl chloride transferred. The implementation was analogous to 8a / 11a. SC cleaning (finest silica gel, eluent Dichloromethane / THF = 12/1 to 6/1) gave 4.8 g 9b (3.5% Th. Starting from adenosine) as a colorless foam.

1 H-NMR (CD₃CN, 250 MHz): d = 9.5 (s, 1H; NH), 8.58 (s, 1H; 2-H), 8.30 (s, 1H; 8-H), 7.5-6.7 (m, 18H; DMTr, PhOAc), 6.15 (d, J = 3 Hz, 1H; 1'-H), 4.99 (s, 2H; PhOAc CH₂), 4.64-4.44 (m, 2H; 2'-H, 3'-H), 4.11 (m, 1H; 4'-H), 3.76 (s, 6H; DMTr OCH₃), 3.7-3.3 (m, 5H; O-CH₂-CH₃, 5'-CH₂, 3'-OH), 1.15 (t, 3H; O-CH₂-CH₃).

13 C-NMR (CD₃CN, 62.5 MHz): δ = 168.17 (s, Ar-NH-C = O), 159.52 (s, aroma, C-OCH₃), 158.54 (s, aroma ., C-OCH₂), 152.76 (d, 2-C), 152.28 (s, 6-C), 149.51 (s, 4-C), 145.86 (aroma; DMTr), 143.49 (d, 8-C), 136.67 (aroma, DMTr), 130.90 (d, aroma PhOAc), 130.86 / 130.54 / 128.86 / 128.68 / 127, 71 (aroma, DMTr), 123.86 (s, 5-C), 122.56 / 115.56 (d, aroma, PhOAc), 113.88 (aroma; DMTr), 87.99 (d , 1′-C), 87.02 (s, C- (Ph) 3), 84.81 (d, 4′-C), 81.76 (d, 2′-C), 70.47 (d , 3'-C), 68.92 (t, Ph-O-CH₂), 67.20 (t, O-CH₂-CH₃), 64.03 (t, 5'-CH₂), 55.76 (q , DMTr OCH₃), 15.38 (q, O-CH₂-CH₃).
2′-O-ethyl- (phenoxyacetyl) adenosine (8b): NMR data obtained from 8b / 11b mixture (14: 1).
1 H-NMR (CDCl₃, 90 MHz): δ = 9.93 (s br, 1H; NH), 8.96 (s, 1H; 2-H), 8.36 (s, 1H; 8-H), 7.6-6.9 (m, 5H; PhOAc arom.), 6.10 (d, J = 6 Hz, 1H; 1′-H), 5.8-5.6 (m, 1H; 5 ′ -OH), 4.92-4.55 (m, 4H; PhOAc CH₂, 2'-H, 3'-H), 4.40 (s, 1H; 4'-H), 4.05-3, 41 (m, 5H; 5'-CH₂, O-CH₂-CH₃, 3'-OH), 1.09 (t, 3H; O-CH₂-CH₃). - Characteristic position of 11b in the mixture: d = 5.9 (d, J = 6 Hz, 1'-H).
13 C-NMR (CDCl₃, 22.5 MHz): δ 167.50 (s, Ar-NH-C = O), 157.05 (s, aroma, CO-CH₂), 151.67 (d, 2- C), 150.45 (s, 6-C), 148.73 (s, 4-C), 143.40 (d, 8-C), 129.42 (d, aromatic), 123.35 ( s, 5-C), 121.91 / 114.73 (d, aroma), 88.60 d, 1′-C), 87.44 (d, 4′-C), 82.81 (d, 2'-C), 69.75 (d, 3'-C), 68, 31 (t, Ph-O-CH₂), 67.60 (t, O-CH₂-CH₃); 63.01 (t, 5'-CH₂), 15.45 (q, O-CH₂-CH₃).

2′-O-methyl-O⁶- [2- (4-nitrophenyl) ethyl] guanosine 14a

8.1 g (18.7 mmol) of 13 (2) were towed several times with dimethylformamide / benzene, dissolved in 130 ml of anhydrous dimethylformamide and 0.72 g (30 mmol) of washed sodium hydride were added at -60 ° C. and the mixture was stirred for 45 minutes -50 ° C stirred. Then 21.3 g (0.15 mol) of methyl iodide were added dropwise within 5 hours and the reaction mixture was warmed to -15 ° C. The reaction was followed by thin layer chromatography. For working up, the solution was mixed with ammonium chloride and evaporated to dryness. The oily residue was purified by means of flash chromatography (220 g of silica gel, eluent: chloroform / methanol = 80/1). 4.0 g (48%) of isomer mixture 14a / 19a = 86/14 were obtained. Recrystallization from benzene / chloroform three times gave 1.8 g (22%) 14a (<1% 19a) with the analytical data given below. The mother liquors were evaporated and the residue, 2.2 g (26.5% 19a) was purified by column chromatography (250 g silica gel, eluent: dichloromethane / isopropanol = 30/1), 1.7 g (20.4%) white crystals 14a / 19a = 82/18 were obtained. TLC: dichloromethane / isopropanol = 5/1, R _f = 0.57 (14a), R _f = 0.61 (19a).
14a: colorless crystals, m.p .: 109-111 ° C.
1 H-NMR (CDCl₃, 200 MHz): δ = 8.17 (d, J = 9 Hz, 2H; NPE), 7.65 (s, 1H; 8-H), 7.48 (d, J = 9 Hz, 2H; NPE), 6.68 (d, J = 12 Hz, 1H; 5′-OH), 5.75 (d, J = 8 Hz, 1H; 1′-H), 4.98 (see br, 2H; NH₂,), 4.73 (t, J = 7.3 Hz, 2H; NPE O-CH₂), 4.65 (dd, J₁ = 8 Hz, J₂ = 4.6 Hz, 1H, 2 ′ -H), 4.55 (d, J = 4.6 Hz, 1H; 3′-H), 4.32 (s, 1H; 4′-H), 3.95 / 3.74 (AB, J _AB = 13.3 Hz, J _BX = 12 Hz, 2H; 5'-CH₂), 3.35 (s, 3H; 2'-OCH₃), 3.28 (t, J = 7.3 Hz, 2H ; NPE CH₂-Ph), 2.87 (s, 1H; 3'-OH).
13 C-NMR (CDCl₃, 22.5 MHz): δ = 159.1 (s, 6-C), 157.07 * (s, 2-C), 150.96 * (s, CH₂-C (Ph) ), 145.24 * (s, 4-C), 143.93 * (s, O2N-C (Ph)), 138.02 (d, 8-C), 128.69 / 122.25 (d, arom., NPE), 115.55 (s, 5-C), 88.42 (d, 1′-C), 87.1 (d, 4′-C), 82.11 (d, 2′- C), 70.36 (d, 3'-C), 66.29 (t, O-CH₂NPE), 63.0 (t, 5'-CH₂), 58.54 (q, 2'-OCH₃) , 35.54 (t, CH₂-Ph NPE).

2′-O-ethyl-O⁶- [2- (4-nitrophenyl) ethyl] guanosine 14b

3.15 g (7.29 mmol) 13 were dried as described for 14a, dissolved in 30 ml DMF, cooled to -60 ° C. and with 0.263 g (6.6 mmol) 60% sodium hydride suspension (washed with anhydrous n-hexane) added and stirred at -15 ° C for 1 h. Then 4.31 g (29.2 mmol) of ethyl iodide, after 60 min 2.15 g (14.6 mmol) and after another 60 min again 2.15 g (14.6 mmol) of ethyl iodide were added and the temperature in during this time slowly increased to 0 ° C. 3 h after the last addition, the mixture was worked up and purified using SC (finest silica gel, eluent: dichloromethane / isopropanol = 16: 1). 0.440 g (13%) of pure product 14b were obtained as a colorless foam.
1 H-NMR (CDCl₃, 200 MHz): δ = 8.17 (d, J = 9.5 Hz, 2H; NPE), 7.65 (s, 1H; 8-H), 7.48 (d, J = 9.5 Hz, 2H; NPE), 6.68 (d, J = 12.5 Hz, 1H; 5′-OH), 5.75 (d, J = 7.6 Hz, 1H; 1′- H), 4.98 (s, 2H; NH₂), 4.83-4.62 (m, 2H; NPE O-CH₂), 4.49 (d, J = 4.5 Hz, 1H; 3′- H), 4.33 (s, 1H; 4'-H), 4.12-3.88 (m, 2H; 5'-CH₂), 3.85 (m, 2H; O-CH₂-CH₃), 3.28 (t, J = 6.4 Hz, 2H; NPE CH₂-Ph), 2.85 (s, 1H; 3′-OH), 1.10 (t, J = 6.4 Hz, O- CH₂-CH₃).
13 C-NMR (CDCl₃, 22.5 MHz): δ = 159.1 (s, 6-C), 157.07 (s, 2-C), 150.96 * (s, 4-C), 145, 24 * (s, CH₂-C (Ph)), 143.93 (s, O2N-C (Ph)), 138.02 (d, 8-C), 128.65 / 122.25 (d, aroma. NPE), 115.55 (s, 5-C), 88, 42 (d, 1′-C), 87.1 (d, 4′-C), 82.11 (d, 2′-C), 70.36 (d, 3'-C), 66.29 (t, O-CH₂NPE), 65.38 (t, O-CH₂-CH₃), 63.01 (t, 5'-CH₂), 35 , 54 (t, CH₂-Ph NPE), 15.04 (q, O-CH₂-CH₃).

2'-O-Methyl-O⁶- [2- (4-nitrophenyl) ethyl] -N²- (phenoxyacetyl) guanosine 15a

a) 1.5 g (3.3 mmol) 14a were three times with Towed pyridine / benzene and in 150 ml pyridine solved. Within 30 minutes Room temperature 1.78 g (16.5 mmol) trimethylchlorosilane dripped. After 2 hours, 1.32 g (4.6 mmol) Phenoxyacetic anhydride added and at Room temperature stirred overnight. The The reaction mixture was treated with 250 ml of water / methanol = 1/1 and with 10 ml conc. aqueous ammonia added after The aqueous phase was seven times with 10 minutes Extracted dichloromethane. The united organic Phases were dried over sodium sulfate, filtered and evaporated. 1.85 g (95%) 15a, colorless, firm foam with those given below get analytical data.

b) The same experimental conditions were chosen, but 1.7 g (3.81 mmol) 14a / 19 / a = 82/18 were used. 1.8 g (81%) 15a / 20a were obtained. TLC: chloroform / methanol = 5 / 0.3, R _f = 0.36 for 15a, R _f = 0.38 for 20a.
1 H-NMR (CDCl₃, 200 MHz): δ = 8.98 (s br, 1H; NH), 8.15 (d, J = 8.5 Hz, 2H; NPE) 8.00 (s, 1H; 8 -H), 7.52 (d, J = 8.5 Hz, 2H; NPE), 7.42-7.26 (m, 2H; PhOAc aroma), 7.14-6.93 (m, 3H ; PhOAc arom.), 5.92 (d, J = 6.2 Hz, 1H; 1'-H), 5.05 (s br, 1H; OH), 4.85 (t, J = 6.8 Hz, 2H; NPE O-CH₂), 4.69 (s, 2H; PhOAc CH₂), 4.65 (m, 2H; 2'-H, 3'-H), 4.3 (s, 1H; 4 ′ -H), 4.02 (d, J = 12.5 Hz, 1H; 5′-H), 3.84 (d, J = 12.5 Hz, 1H; 5′-H), 3.39 (s, 3H; 2'-OCH₃), 3.35 (t, J = 6.8 Hz, 2H; NPE CH₂-Ph).
13 C-NMR (CDCl₃, 50 MHz): δ = 165.95 (s, NH-C = O), 160.8 (s, 6-C), 156.8 (s, CH₂-OC (Ph)), 151.68 * (s, 2-C), 150.61 * (s, CH₂-C (Ph) NPE), 146.76 * (s, 4-C), 145.40 * (s, O₂N-C (Ph)), 142.18 (d, 8-C), 129.91 / 129.75 (d, aroma, phenoxyac.), 123.62 (d, aroma, NPE), 122.41 (aroma .), 119.81 (s, 5-C), 114.77 (aroma), 88.54 (d, 1′-C), 87.11 (d, 4′-C), 82.44 ( d, 2'-C), 69.85 (d, 3'-C), 67.56 (t, O-CH₂NPE), 67.17 (t, CH₂-O-Ph), 62.47 (t , 5'-CH₂), 58.63 (q, OCH₃), 34.87 (t, CH₂-Ph NPE).

2′-O-ethyl-O⁶- [2- (4-nitrophenyl) ethyl] -N²- (phenoxyacetyl) guanosine 15b

0.440 g (0.96 mmol) 14b was treated analogously to 15a with 0.521 g (4.8 mmol) trimethylchlorosilane and 0.440 g (1.54 mmol) phenoxyacetic anhydride. After the same reaction conditions and analogous work-up, 0.470 g (82%) of pure product 15b was obtained as a colorless foam by SC purification (finest silica gel, eluent: dichloromethane / isopropanol = 10: 1).
1 H-NMR (CDCl₃, 200 MHz): δ = 8.98 (s br, 1H; NH), 8.62 (m, 1H; OH), 8.15 (d, J = 8.4 Hz, 2H; NPE aroma.), 7.91 (s, 1H; 8-H), 7.56 (d, J = 8.4 Hz; NPE aroma.), 7.44-7.25 (m, 2H; PhOAc aroma .), 7.15-6.95 (m, 3H; PhOAc arom.), 5.90 (d, J = 7.3 Hz, 1H; 1'-H), 4.88 (d, J = 7 , 3 Hz, 1H; 2'-H), 4.85 (t, J = 7.3 Hz, 2H; NPE O-CH₂), 4.68 (s, 2H; PhOAc CH₂), 4.65 (m , 1H; 3′-H), 4.30 (s, 1H; 4′-H), 4.02 / 3.84 (2d, J = 12.5 Hz, 2H; 5′-CH₂), 3, 44-3.27 (m, 2H; O-CH₂-CH₃), 3.35 (t, J = 7.3 Hz, 2H; NPE CH₂-Ph), 1.15 (t, 3H; O-CH₂- CH₃).
13 C-NMR (CDCl₃, 50 MHz): δ = 165.95 (s, NH-C = O), 160.80 (s, 6-C), 156.50 (s, CH₂O-C (Ph)), 151.70 (s, 2-C), 150.65 * (s, 4-C), 146.75 * (s, CH₂-C (Ph) NPE), 145.40 (s, O2N-C (Ph )), 142.20 (d, 8-C), 129.90 / 129.75 (d, aroma, PhOAc), 123.62 (d, aroma NPE), 122.40 (aroma), 119 , 85 (s, 5-C), 114.76 (aroma), 88.55 (d, 1′-C), 87.13 (d, 4′-C), 82.43 (d, 2 ′ -C), 69.87 (d, 3'-C), 67.56 (t, O-CH₂NPE), 67.16 (t, CH₂-O-Ph), 67.61 (t, O-CH₂ -CH₃), 62.42 (t, 5'-CH₂), 34.83 (t, CH₂-Ph NPE), 15.18 (q, O-CH₂-CH₃).

5′-O-dimethoxynitrile-2′-O-methyl-O⁶- [2- (4- nitrophenyl) ethyl] -N²- (phenoxyacetyl) guanosine 16a

3.0 g (5.18 mmol) of 15a as a mixture with 0.3 g of regioisomer 20a were towed three times with pyridine / benzene, dissolved in 350 ml of anhydrous pyridine and with 2.13 g (6.3 mmol) of dimethoxytrityl chloride for 2 hours Room temperature stirred. The reaction mixture was concentrated and partitioned between dichloromethane and aqueous sodium bicarbonate solution. The aqueous phase was shaken out with dichloromethane. The combined organic phases were dried with sodium sulfate, filtered and evaporated. The foam obtained was purified by column chromatography (500 g of silica gel, eluent: toluene / isopropanol = 30/1). 4.1 g 16a (81%) and 0.3 g 21a (6%) were obtained as yellowish solid oils. TLC: toluene / isopropanol = 9/1, R _f = 0.24 for 16a, R _f = 0.28 for 21a.
1 H-NMR (CDCl₃, 200 MHz): δ = 8.75 (s, 1H; NH), 8.18 (d, J = 10 Hz, 2H; NPE arom.), 8.09 (s, 1H; 8 -H), 7.55 (d, J = 10 Hz, 2H; NPE aroma), 7.47-6.93 (m, 14H; DMTr, PhOAc), 6.80 (d, J = 9.6 Hz, 4H; DMTr), 6.05 (d, J = 2.6 Hz, 1H; 1'-H), 4.86 (t, J = 6.7 Hz, 2H; NPE O-CH₂), 4 , 69 (s, 2H; PhOAc CH₂), 4.57 (dd, J₁ = 10, J₂ = 6.7, 1H; 3′-H), 4.42-4.32 (m, 1H; 2′- H), 4.24-4.13 (s, 1H; 4'-H), 3.78 (s, 6H; DMTr OCH₃), 3.63 (s, 3H; 2'-OCH₃), 3.48 (s, 2H; 5'-CH₂), 3.34 (t, 2H; NPE CH₂-Ph), 2.65 (d, J = 6.7 Hz, 1H; 3'-OH).
13 C-NMR (CDCl₃, 50 MHz): δ = 165.77 (s, C = O), 160.23 (s, 6-C), 158.15 (s, CH₃O-C (Ph), DMTr), 156.67 (s, CH₂-OC (Ph)), 152.00 * (s, 2-C), 150.65 * (s, CH₂-C (Ph) NPE), 146.48 * (s, 4th -C), 145.23 * (s, O2N-C (Ph)), 144.13 (s, arom., DMTr), 140.02 (d, 8-C), 135.3 / 135.21 / 129.68 / 129.62 / 129.46 / 127.76 / 127.48 / 126.54 / 123.34 / 122.04 (aroma) 118.53 (s, 5-C), 114.48 ( arom., PhOAc), 112.78 (arom., DMTr), 86.19 (s, C (Ph) 3), 86.19 (d, 1′-C), 83.57 (d, 4′- C), 83.07 (d, 2'-C), 69.35 (d, 3'-C), 67.53 (t, O-CH₂NPE), 66.75 (t, CH₂-O-Ph ), 62.79 (t, 5'-CH₂), 58.5 (q, OCH₃), 54.78 (q, DMTr OCH₃), 34.87 (t, CH₂-Ph NPE).

5′-O-dimethoxytrityl-3′-O-methyl-O⁶- [2- (4-nitrophenyl) ethyl] -N²- (phenoxyacetyl) guanosine 21a

1 H-NMR (CDCl₃, 200 MHz): δ = 8.83 (s, 1H; NH), 8.07 (d, J = 9.3 Hz, 2H; NPE arom.), 8.02 (s, 1H ; 8-H), 7.4 (d, J = 9.3 Hz, 2H; NPE aroma), 7.33-6.9 (m, 14H; DMTr, PhOAc), 6.6 (d, J = 9 Hz, 4H; DMTr), 5.87 (d, J = 6.3 Hz, 1H; 1′-H), 5.64 (s, 1H; 2′-OH), 4.94 (dd, J₁ = 6.3, J₂ = 5.3, 1H; 2'-H), 4.75 (t, J = 6.7 Hz, 2H; NPE O-CH₂), 4.57 (s, 2H; PhOAc CH₂), 4.29 * (m, 1H; 3'-H), 4.03 * (m, 1H; 4'-H), 3.67 (s, 6H; DMTr OCH₃), 3.5 (s , 3H; 3'-OCH₃, 3.33-3.08 (m, 4H; 5'-CH₂, NPE CH₂-Ph).
13 C-NMR (CDCl₃, 50 MHz): δ = 165.76 (s, C = O), 160.3 (s, 6-C), 158.08 (s, CH₃O-C (Ph), DMTr), 156.54 (s, CH₂-OC (Ph)), 151.55 * (s, 2-C), 150.18 * (s, NPE), 146.47 * (s, 4-C), 145, 17 * (s, NPE), 144.03 (arom., DMTr), 140.63 (d, 8-C), 135.15 / 135.06 / 129.53 / 127.57 / 127.37 / 126 , 42 / 123.34 / 122.18 (aroma), 118.82 (s, 5-C), 114.51 (aroma., PhOAc), 112.68 (aroma., DMTr), 90.40 ( d, 1'-C), 86.15 (s, C (Ph) ₃), 83.47 (d, 4'-C), 81.21 (d, 3'-C), 75.29 (d , 2′-C), 67.40 (t, O-CH₂NPE), 66.82 (t, CH₂-O-Ph), 63.51 (t, 5′-CH₂), 58.45 (q, 3'-OCH₃), 54.73 (q, DMTr OCH₃), 34.65 (t, CH₂-Ph NPE).

5′-O-dimethoxytrityl-2′-O-ethyl-O⁶- [2- (4-nitrophenyl) ethyl) -N²- (phenoxyacetyl) guanosine 16b

0.470 g (0.79 mmol) 15b were dried analogously to 16a, dissolved, mixed with 0.321 g (0.95 mmol) dimethoxytrityl chloride and worked up in the same way. SC (silica gel finest, eluent dichloromethane / acetone = 15/1) gave 483 mg (67.6%) of pure product 16b as a yellow powder.
1 H-NMR (CDCl₃, 250 MHz): δ = 8.88 (s, 1H; NH), 8.10 (d, J = 9.2 Hz, 2H; NPE arom.), 8.04 (s, 1H ; 8-H), 7.56 (d, J = 9.2 Hz, 2H; NPE aroma), 7.44-6.60 (m, 18H; DMTr, PhOAc), 6.00 (d, J = 4.5 Hz, 1H; 1′-H), 4.88 (s, 2H; PhOAcCH₂), 4.83 (t, J = 6.9 Hz, 2H; NPE O-CH₂), 4.66- 4.48 (m, 2H; 2'-H, 3'-H), 4.18-3.94 (m, 1H); 4'-H), 3.72 (s, 6H; DMTr OCH₃), 3.68-3.51 (m, 2H; O-CH₂-CH₃), 3.50-3.15 (m, 5H; 5 '-CH₂, NPE CH₂-Ph), 3'-OH), 1.13 (t, 3H; O-CH₂-CH₃).
13 C-NMR (CDCl₃, 50 MHz): δ = 165.77 (s, NH-C = O), 160.59 (s, 6-C), 158.50 (s, CH₃O-C (Ph)), 157.02 (s, CH₂-OC (Ph)), 152.38 (s, 2-C), 150.98 * (s, 4-C), 146.86 * (s, CH₂-C (Ph, NPE)), 145.56 (s, O₂N-C (Ph)), 144.48 (s, arom., DMTr), 140.39 (d, 8-C), 135.65 / 135.56 / 130 , 01 / 129.81 / 128.10 / 127.82 / 126.88 / 123.71 / 122.40 (aroma), 118.89 (s, 5-C), 114.82 (aroma, PhOAc ), 113.12 (arom., DMTr), 86.95 (d, 1′-C), 86.53 (s, C (Ph) 3), 84.01 (d, 4′-C), 81 , 54 (d, 3'-C), 76.36 (d, 2'-C), 67.87 (t, O-CH₂NPE), 67.10 (t, CH₂-O-Ph), 66, 91 (t, O-CH₂-CH₃), 63.22 (t, 5'-CH₂), 55.13 (q, DMTr OCH₃), 35.03 (t, CH₂-Ph NPE), 15.20 (q , O-CH₂-CH₃).

5'-O-dimethoxytrityl-2'-O-methyl-N²- (phenoxyacetyl) - guanosine 18a

150 mg (0.17 mmol) 16a were stirred in 5 ml of a 0.5 molar solution of 1,8-diazabicyclo (5.4.0) undec-7-ene (DBU) in pyridine for 2 hours at room temperature. 1.5 ml of 1M aqueous acetic acid were then added dropwise, the mixture was diluted with water and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried with sodium sulfate, filtered and evaporated. The oily residue was purified by means of flash chromatography (4 g of silica gel, eluent: dichloromethane / acetone / triethylamine = 100/10/2). 95 mg (76%) of colorless solid oil 18a were obtained. TLC: chloroform / methanol = 4.8 / 0.3, R _f = 0.38.
1 H-NMR (CDCl₃, 200 MHz): δ = 9.1 (s br, 1H; NH), 7.87 (s, 1H; 8-H), 7.49-6.75 (m, 18H; DMTr ), 6.02 (d, J = 5.3 Hz, 1H; 1'-H); 4.65 (s, 2H; PhOAc CH₂), 4.48 (m, 1H; 3'-H), 4.33-4.15 (m, 2H; 2'-H, 4'-H), 3 , 78 (s, 6H; DMTr OCH₃), 3.49 (s, 3H; 2′-OCH₃), 3.44 (s, 2H; 5′-CH₂), 2.78 (s br, 1H; 3 ′ -OH).
13 C-NMR (CDCl₃, 50 MHz): δ = 169.5 (s, NH-C = O), 158.61 (s, CH₃O-C (Ph), DMTr), 156.3 (s, CH₂-OC (Ph)), 155.26 (s, 6-C), 147.78 (s, 2-C), (146.29 (s, 4-C), 144.33 (s, arom., DMTr) , 137.17 (d, 8-C), 135.47 / 135.37 (aroma, DMTr) 129.98 (aroma-C, PhOAc), 128.09 / 127.91 / 127.03 (aroma ., DMTr), 123.01 (s, 5-C), 122.05 / 114.83 (aroma., PhOAc), 113.2 (aroma., DMTr), 86.74 (s, C (Ph3) , 85.47 (d, 1′-C), 84.15 (d, 4′-C), 83.85 (d, 2′-C), 69.96 (d, 3′-C), 66 , 88 (t, CH₂-O-Ph), 63.3 (t, 5'-CH₂), 58.85 (q, 2'-OCH₃), 55.2 (q, DMTr OCH₃).

N³-cyanoethyl-5'-O-monomethoxytrityluridine 24a

7 g (13.6 mmol) 23a (manufactured according to Schaller, H., et al. (1963), J. Am. Chem. Soc., 3821-27) towed three times with dimethylformamide / benzene and dissolved in 100 ml of anhydrous dimethylformamide Cooled 0 ° C and washed with 325 mg (13.6 mmol) Sodium hydride added and one hour at 0 ° C. touched. Then 14.4 g were added within 12 hours (0.272 mol) acrylonitrile added and that Reaction mixture heated to 80 ° C. The reaction mixture a 44013 00070 552 001000280000000200012000285914390200040 0002004110085 00004 43894 was emptied onto water / dichloromethane and the aqueous one Phase was shaken out with dichloromethane. The combined organic phases were with Dried sodium sulfate, filtered and evaporated.  

The crude product was purified by means of flash chromatography (200 g silica gel, eluent: dichloromethane / acetone = 30/1). 3.5 g (45%) of white foam 24a were obtained. TLC: dichloromethane / acetone = 33/2, R _f = 0.42.
1 H-NMR (CDCl₃, 90 MHz): δ = 7.95 (d, J = 8.1 Hz, 1H; 6-H), 7.45-7.1 (m, 12H; MMTr), 6.83 (d, J = 9 Hz, 2H; MMTr), 5.92 (d, J = 3 Hz, 1H; 1′-H), 5.44 (d, J = 8.1 Hz, 1H; 5-H ), 4.6-4.0 (m, 5H; 2'-H, 3'-H, N-CH₂, 4'-H), 3.78 (s, 3H; MMTr OCH₃), 3.51 ( m, 2H; 5'-H), 2.53 (t, J = 7.2 Hz, 2H; CH₂CN).
13 C-NMR (CDCl₃, 22.5 MHz): δ = 161.89 (s, 4-C), 158.64 (s, aroma, C-OCH₃), 150.99 (s, 2-C), 143.68 / 143.17 (aroma), 138.59 (d, 6-C), 134.48 / 132.25 / 128.19 / 127.81 / 127.11 (aroma), 117.52 (s, CN), 113.18 (arom.), 101.37 (d, 5-C), 90.43 (d, 1′-C), 87.18 (s, C- (Ph) 3) , 83.66 (d, 4'-C), 75.26 (d, 2'-C), 69.84 (d, 3'-C), 62.09 (t, 5'-CH₂), 55 , 05 (q, OCH₃), 36.46 (t, N-CH₂) 15.98 (t, CH₂CN).

N³-cyanoethyl-5'-O-dimethoxytrityluridine 24b

40 g (0.076 mol) 23b (manufactured according to Schaller, H., et al. (1963) J. Am. Chem. Soc., 3821-27) were treated analogously to the above with 2.9 g (0.076 mol) of sodium hydride suspension ( 60% suspension, washed with anhydrous n-hexane). 1.52 mol of acrylonitrile were added within 12 h and the reaction mixture was heated to 110.degree. Working up as described above and purification by SC (finest silica gel, eluent dichloromethane / acetone = 30: 1) gave 24.0 g (55%) of 24b as a white foam.
1 H-NMR (CDCl₃, 90 MHz): δ = 7.95 (d, J = 8 Hz; 1H; 6-H), 7.45-7.10 (m, 9H; DMTr), 6.83 (d , J = 9 Hz, 4H; DMTr), 5.92 (d, J = 3 Hz, 1H; 1′-H), 5.45 (d, J = 8 Hz, 1H; 5-H), 4, 60-4.00 (m, 5H; 2'-H, 3'-H, N-CH₂, 4'-H), 3.78 (s, 6H; DMTr OCH₃), 3.50 (m, 2H; 5'-H), 2.53 (t, J = 7.2 Hz, 2H; CH₂CN).
13 C-NMR (CDCl₃, 22.5 MHz): δ = 161.89 (s, 4-C), 158.64 (s, aroma, C-OCH₃), 150.96 (s, 2-C) 143 , 65 (aroma), 138.54 (d, 6-C), 134.46 / 132.23 / 128.18 / 127.80 (aroma), 117.50 (s, CN), 113.18 (aroma) 101.36 (d, 5-C), 90.41 (d, 1′-C), 87.18 (s, C- (Ph) 3), 83.66 (d, 4′- C), 75.24 (d, 2'-C), 69.82 (d, 3'-C), 62.09 (t, 5'-CH₂), 55.05 (q, DMTr OCH₃), 36 , 46 (t, N-CH₂), 15.95 (t, CH₂CN).

N³-cyanoethyl-2'-O-methyl-5'-O-monomethoxytrityluridine 25a

a) 600 mg (1.05 mmol) 24a were dissolved in 10 ml dimethylformamide and cooled to -40 ° C. After the addition of 25 mg (1.04 mmol) of washed sodium hydride, the mixture was stirred for a further 45 minutes at this temperature and then 284 mg (2 mmol) of methyl iodide were injected and warmed to room temperature within 3 hours. For working up, ammonium chloride was added, the reaction mixture was evaporated to dryness and partitioned between water and dichloromethane. The aqueous phase was extracted with dichloromethane. The collected organic phases were dried with sodium sulfate, filtered and evaporated. The product mixture of 2′-methylated 25a, 3′-methylated 28a and 2 ′, 3′-dimethylated 29a was separated by column chromatography (90 g of silica gel, eluent: diethyl ether). 200 mg (33%) 25a, 48 mg (8%) 28a and 80 mg (13%) 29a were obtained as colorless solid oils. TLC: dichloromethane / acetone = 3.2 / 1.8, R _f = 0.80 for 25a, R _f = 0.74 for 28a, R _f = 0.87 for 29a.
1 H-NMR (CDCl₃, 90 MHz): δ = 8.11 (d, J = 8.1 Hz, 1H; 6-H), 7.65-7.3 (m, 12H; MMTr), 6.88 (d, J = 9 Hz, 2H; MMTr), 6.0 (d, J = 1.5 Hz, 1H; 1′-H), 5.35 (d, J = 8.1 Hz, 1H; 5 -H), 4.48 (m, 1H; 3'-H), 4.29 (t, J = 6 Hz, 2H; N-CH₂), 4.08 (m, 1H; 4'-H), 3.95 (m, 1H; 2′-H), 3.82 (s, 3H; MMTr OCH₃, 3.75 (s, 3H; 2′-OCH₃), 3.60 (m, 2H; 5′- CH₂), 2.82 (t, J = 6 Hz, 2H; CH₂CN).
13 C-NMR (CDCl₃, 22.5 MHz): δ = 161.73 (s, 4-C), 158.64 (s, aroma, C-OCH₃), 150.13 (s, 2-C), 143.69 / 143.41 (2s, aroma), 138.32 (d, 6-C), 134.42 / 130.25 / 128.24 / 127.81 / 127.05 (aroma), 116 , 81 (s, CN), 113.13 (arom.) 101.15 (d, 5-C), 87.61 (s, C- (Ph) ₃), 87.18 (d, 1'-C ), 83.82 d, 2'-C), 83.01 (d, 4'-C), 68.11 (d, 3'-C), 61.06 (t, 5'-CH₂), 58 , 52 (q, 2'-OCH₃), 55.05 (q, MMTr OCH₃), 36.03 (t, N-CH₂), 15.07 (t, CH₂CN).

N³-cyanoethyl-3'-O-methyl-5'-O-monomethoxytrityluridine 28a

1 H-NMR (CDCl₃, 200 MHz): δ = 7.86 (d, J = 8.4 Hz, 1H; 6-H), 7.54-7.2 (m, 12H; MMTr), 6.86 (d, J = 9 Hz, 2H; MMTr), 5.91 (d, J = 3.6 Hz, 1H; 1′-H), 5.48 (d, J = 8.4 Hz, 1H; 5 -H), 4.4-4.15 (m, 4H; 2'-H, 4'-H, N-CH₂), 4.03 (m, 1H; 3'-H), 3.80 (s , 3H; MMTr OCH₃), 3.64-3.39 (m, 2H; 5'-H), 3.44 (s, 3H; 3'-OCH₃), 2.72 (t, J = 7.2 Hz, 2H; CH₂CN), 2.6 (s, 1H; OH).
13 C-NMR (CDCl₃, 50 MHz): δ = 161.61 (s, 4-C), 158.44 (s, aroma, C-OCH₃), 150.31 (s, 2-C), 143, 40 / 143.20 (aroma), 138.30 (d, 6-C), 134.19 / 129.99 / 127.92 127.66 / 126.93 (aroma), 116.87 (s, CN), 112.94 (aroma), 101.21 (d, 5-C), 90.13 (d, 1'-C), 86.96 (s, C (Ph) ₃), 80.70 (d, 4'-C), 78.27 (d, 3'-C), 73.35 (d, 2'-C), 61.98 (t, 5'-CH₂), 57.96 (q , 3'-OCH₃), 54.88 (q, MMTr OCH₃), 35.99 (t, N-CH₂), 15.62 (t, CH₂CN).

N³-cyanoethyl-2 ', 3'-O-dimethyl-5'-O-monomethoxytrityluridine 29a

1 H-NMR (CDCl₃, 200 MHz): δ = 8.12 (d, J = 7.7 Hz, 1H; 6-H), 7.45-7.21 (m, 12H; MMTr), 6.87 (d, J = 9.3 Hz, 2H; MMTr), 5.95 (s, 1H; 1'-H), 5.35 (d, J = 7.7 Hz, 1H; 5-H), 4 , 32-3.88 (m, 5H; 2'-H 3'-H 4'-H, N-CH₂), 3.8 (s, 3H; MMTr OCH₃), 3.66 (s, 3H; OCH₃ ), 3.6-3.39 (m, 2H; 5'-CH₂), 3.46 (s, 3H; OCH₃), 2.78 (t, J = 7 Hz, 2H; CH₂CN).
13 C-NMR (CDCl₃, 50 MHz): δ = 161.83 (s, 4-C), 158.60 (s, aroma, C-OCH₃), 150.07 (s, 2-C), 143, 67 / 143.36 (aroma), 138.40 (d, 6-C), 134.32 / 130.2 / 128.15 / 128.10 127.82 / 127.07 (aroma), 116, 95 (s, CN), 113.1 (arom.), 101.07 (d, 5-C), 88.25 d, 1′-C), 87.1 (s, C (Ph) 3), 81.75 d, 4'-C), 80.69 (d, 2'-C), 76.36 (d, 3'-C), 60.83 (t, 5'-CH₂), 58.31 / 58.07 (2q, 2 ′ - / 3′-OCH₃), 55.02 (q, MMTr OCH₃), 35.95 (t, N-CH₂), 15.86 (t, CH₂CN).

b) 500 mg (0.88 mmol) 24a were in 7 ml Dimethylformamide dissolved and at room temperature with added a total of 10 ml of diazomethane solution. The Reaction was by thin layer chromatography followed and after 4 hours between dichloromethane and Sodium bicarbonate solution distributed. The United organic phases were dried, filtered and evaporated. After purification by column chromatography (60 g silica gel, eluent: diethyl ether) became 330 mg (64%) 25a and 100 mg (19%) 28a.

2'-O-methyl-5'-O-monomethoxytrityluridine 26a

38 mg (0.34 mmol) of t-potassium butoxide were added to 100 mg (0.17 mmol) of 25a in dichloromethane. After 30 minutes at room temperature, the reaction mixture was partitioned between dichloromethane and sodium bicarbonate solution, the collected organic phases were dried with sodium sulfate, filtered and evaporated. The crude product was purified by means of flash chromatography (4 g of silica gel, eluent: dichloromethane / acetone = 9/1). 80 mg (89%) of 26a, white foam were obtained. TLC: dichloromethane / acetone = 3/2, R _f = 0.41.
1 H-NMR (CDCl₃, 200 MHz): δ = 9.2 (s br, 1H; NH), 8.02 (d, J = 8.1 Hz, 1H; 6-H), 7.4-7, 25 (m, 12H; MMTr), 6.85 (d, J = 8.9 Hz, 2H; MMTr), 5.97 (s, 1H; 1′-H), 5.27 (d, J = 8 , 1 Hz, 1H; 5-H), 4.5 * (dd, J₁ = 5.3 Hz, J₂ = 9.3 Hz, 1H; 3′-H), 4.0 * (d, J = 8 Hz, 1H; 4'-H, 3.8 (s, 4H; MMTr OCH₃, 2'-H), 3.65 (s, 3H; 2'-OCH₃), 3.56 (d, J = 2 Hz , 2H; 5'-CH₂).
13 C-NMR (CDCl₃, 50 MHz): δ = 163.76 (s, 4-C), 158.55 (s, aroma, C-OCH₃), 150.19 (s, 2-C), 143, 75 / 143.42 (aroma) 139.85 (d, 6-C), 134.4 / 130.25 / 128.22 / 128.12 / 127.78 / 127.05 / 113.08 (aroma. ), 101.87 (d, 5-C), 87.06 (s, C- (Ph) 3), 86.88 (d, 1′-C), 83.8 (d, 2′-C) , 82.82 (d, 4'-C, 68.15 (d, 3'-C), 61.07 (t, 5'-CH₂), 58.43 (q, OCH₃), 54.98 (q , MMTr OCH₃).

5'-O-Dimethoxytrityl-2'-O-ethyluridine 26b

24 g (0.042 mol) 24b were treated analogously to 25a (methyl iodide alkylation approach) with 1.6 g (42 mmol) 60% sodium hydride suspension (washed with anhydrous n-hexane) and stirred at -45 ° C. for 1 hour. 10.53 g (0.068 mol) of ethyl iodide were then added and the procedure followed as in 25a. SC (finest silica gel, eluent dichloromethane / acetone = 12/1) gave 6.0 g of 25b (contaminated with regioisomers) as a white foam. This was dried as in 26a, dissolved in 60 ml of anhydrous dichloromethane, mixed with 4.2 g (18.2 mmol) of potassium t-butoxide and worked up in the same way. The crude product was purified by SC (finest silica gel, eluent: dichloromethane / acetone = 9/1), 4.1 g (17%) 26b being obtained as a gray-brown foam.
1 H-NMR (CDCl₃, 250 MHz): δ = 9.30 (s br, 1H; NH), 7.79 (d, J = 8.1 Hz, 1H; 6-H), 7.5-7, 08 (m, 9H; DMTr), 6.89 (d, J = 8.9 Hz, 4H; DMTr), 5.80 (s, 1H 1′-H), 5.24 (d, J = 8, 1 Hz, 1H; 5-H), 4.30 * (m, 1H; 2′-H), 3.94 * (m, 2H; 3′-H, 4′-H), 3.75 (s , 6H; DMTr OCH₃), 3.45-3.20 (m, 4H; O-CH₂-CH₃, 5'-CH₂), 1.21 (t, J = 7.4 Hz, 3H; O-CH₂- CH₃).
13 C-NMR (CDCl₃, 62.5 MHz): δ = 163.95 (s, 4-C), 159.63 (s, aroma), 159.61 (s, C-OCH₃), 151.97 (s, 2-C), 145.69 (aroma), 141.10 (d, 6-C), 136.52 / 136.26 / 130.97 / 130.95 / 128.91 / 128.82 / 127, 86 (arom.), 102.29 (d, 5-C), 88.52 * (s, C- (Ph) 3), 87.39 * (d, 1′-C), 83.87 (d , 4'-C), 82.44 (d, 2'-C), 69.50 (d, 3'-C), 67.01 (t, O-CH₂-CH₃), 62.96 (t, 5'-CH₂), 55.81 (q, DMTr OCH₃), 15.41 (q, O-CH₂-CH₃).

2′-O-methyluridine 30a

8 g (14.3 mmol) of 26a were dissolved in 100 ml of methanol and 5 ml of a 10% methanolic hydrochloric acid were added at room temperature. The reaction mixture was evaporated and the residue was partitioned between diethyl ether and water, the organic phase was extracted twice with water, the combined aqueous phases were evaporated and the product was towed several times with benzene and pyridine. 3.27 g (93%) 30a were obtained as a white foam (TLC: dichloromethane / methanol = 3/1, R _f = 0.37).
1 H-NMR (DMSO, 90 MHz): δ = 8.00 (d, J = 8.1 Hz, 1H; 6-H), 6.0 (d, J = 5.4 Hz, 1H; 1'- H), 5.78 (d, J = 8.1 Hz, 1H; 5-H), 5.08 (s br, 3H; 5′-OH), 3′-OH, NH), 4.25 ( ABX system, J _AB = 4.5 Hz, J _AX = 5.4 Hz, 1H; 2′-H), 4.1-3.85 (m, 2H; 3′-H, 4′-H) , 3.74 (m, 2H; 5'-H), 3.30 (s, 3H; 2'-OCH₃).
13 C-NMR (DMSO, 22.5 MHz): δ = 163.19 (s, 4-C), 150.62 (s, 2-C), 140.55 (d, 6-C), 101.91 (d, 5-C), 86.15 (d, 1′-C), 85.33 (d, 4′-C), 82.9 (d, 2′-C), 68.43 (d, 3'-C), 60.57 (t, 5'-CH₂), 57.60 (q, OCH₃).

2'-O-n-butyl-5'-O-MMT-N-cyanoethyluridine

Approach:
2 g (3.51 mmol) 5'-O-MMT-N-cyanoethyl uridine
150 mg (1.1 equivalents) NaH
0.67 ml (1.8 equivalents) of n-butyl bromide

2 g (3.51 mmol) of 5'-O-MMT-N-cyanoethyl-uridine was towed three times with benzene, dissolved in absolute DMF and cooled to minus 60 ° C. Then 150 mg of a 60% NaH suspension (1.1 equivalents) were added and the mixture was stirred at -50 ° C. for 30 minutes. Now 0.52 ml (1.4 equivalents) of butyl bromide was slowly added dropwise to the reaction solution. After 20 minutes the temperature was slowly raised to 10 ° C (within three hours). The reaction was followed by TLC chromatography. 0.15 ml (0.4 equivalents) of butyl bromide were added and the mixture was stirred at room temperature for one hour before the reaction was stopped. About 1 g of NH₄Cl was added to the solution and the mixture was evaporated under a high vacuum. The residue was partitioned between dichloromethane and sodium hydrogen carbonate, dried with sodium sulfate and evaporated. The raw product was cleaned:
Column: 100 g silica gel, eluent: dichloromethane
Yield: 300 mg pure (15% of theory)
TLC solvent: dichloromethane: acetone 9: 1)
R _f = 0.65 (2'-O-butyl), R _f = 0.44 (3'-O-butyl).
1 H NMR (CDCl₂): 8.05 (d, H6); 7.5-7.2 (m, MMT); 6.9-6.7 (m, MMT); 5.95 (d, H1 ′); 5.3 (d, H5), 4.6-3.6 (m, H2 ′, H3 ′, H4 ′, OCH₂, NCH₂); 3.8 (S, OCH₃, MMT); 3.55 (d, C5 ′), 2.7 (t, CH₂CN), 1.8-1.2 (m, butyl CH₂CH₂ 4H); 0.9 (t, CH₃-butyl).
C-NMR (CDCl₃): 161 = (C4); 158.8 (MMT); 150.3 (C2); 143.9 (MMT); 143.5 (MMT); 138.5 (C6); 134.6, 130.4, 128.4, 127.8, 127.3 (MMT), 117.0 (CN); 113.3 (MMT), 101.3 (C5); 88.1 (MMT); 87.4 (C1 ′); 83.3 (C4 ′); 62.4 (C2 ′); 70.8 (2'-OCH₂); 68.2 (C3 ′); 61.1 (C5 ′); 55.2 (O-CH₃-MMT); 36.2 (N-CH₂); 31.6 (butyl-β-CH₂); 19.2 (butyl CH₂); 16.1 (CH₂CH); 13.8 (CH₃ butyl). Chemical shift in ppm.

Examples of phosphorylation

After drying by towing with pyridine, Toluene and THF are 1-3 mmol of the protected Nucleosides 3a, 3a ′, 3b, 9a, 9b, 16a, 16b, 18a, 26a, 26b in 25 ml THF (in the case of 3a, 16a in dry DCM) solved. The solution is added under nitrogen 2-3.6 moleq. N, N-diisopropylethylamine (Hünig's base) and then within 5 minutes with 1.2 to 2.4 moleq. 2-cyanoethoxy-N, N-diisopropylmonochlorophosphoramidite at room temperature. The reaction mixture is then stirred for 2 hours and by TLC observed (the phosphoramidite gives Ninhydrin reagent a yellow color). If always raw material is still available additional 0.3 moleq. Phosphorylating agents added. The excess reagent is then through the addition of 3-5 moleq. Isopropanol, sec-butanol or quenched methanol and the reaction mixture is stirred for another hour. By Evaporation in vacuo becomes the reaction mixture concentrated and then with degassed ethyl acetate (50 ml) transferred. The solution is degassed twice one molar aqueous sodium bicarbonate solution (100 ml) extracted under argon at 0 ° C. The organic phase is dried over sodium sulfate and evaporated. The residue is towed twice with toluene and in 3-5 ml degassed toluene (in the case of pyrimidines) or ethyl acetate (in the case of purines). The Solution becomes dropwise dry petroleum ether (200 ml, boiling range 40-60 ° C) and the colorless precipitate is filtered off under Nitrogen separated. After careful washing with Hexane, the phosphoramidite is dried in vacuo and  in dry acetonitrile to a 100 mM solution solved that directly for oligonucleotide synthesis is used. The content of precipitated Cyanoethyl-N, N-diisopropyl-H-phosphonate 31 is replaced by 1 H and 31 p NMR detected, but interferes with Coupling efficiency not (see table below).

Table: Phosphorylation of protected 2'-methyl and 2'-ethyl nucleosides

N⁴-benzoyl-5'-O-dimethoxytrityl-2'-O-methylcytidine 3'-O - [(2-cyanomethyl) -N, N-diisopropyl phosphoramidite] 4a

1 H-NMR (CDCl₃, 90 MHz): δ = 8.6, 8.5 (2d, J = 7.5 Hz, 1H; 6-H, 2 diast.), 7.93-6.65 (m, 19H; NH, DMTr), 7.03, 6.95 (2d, J = 7.5 Hz, 1H; 5-H, 2 diast.), 6.05, 6.01 (2d, 1H; 1′-H, 2 diast.), 4.72-4.03 (m, 2H; 2′-H, 3'-H), 3.94 (m, 1H; 4'-H), 3.94-3.3 (m, 4H; P-O-CH₂, 2 × (CH (CH₃) ₂), 3.78 (s, 6H; DMTr OCH₃), 3.65 (s, 3H; 2'-OCH₃), 3.53 (m, 2H; 5'-CH₂), 2.68, 2.37 (2t, J = 7.3 Hz, 2H; CH₂CN, 2 diast.), 1.4-1.0 (12H; 2 × CH (CH₃) ₂).  

N⁴-benzoyl-2'-O-methyl-5'-O-monomethoxytritylcytidine 3′-O - [(2-cyanoethyl) -N, N-diisopropyl- phosphoramidite] 4a ′

TLC: 2 diastereomers, R _f = 0.69, 0.55 (ethyl acetate)
1 H-NMR (250 MHz, CD₃CN; 1: 1 mixture of 2 diastereomers, the amidite contains 3% 31 [d, 6.87 ppm, J = 640 Hz, PH; t, 2.75 ppm, J = 6 Hz , -CH₂CN]): 9.2 (br s, 1H; NH), 8.51, 8.40 (2d, J = 8 Hz, 1H; 6-H, 2 diast.), 7.93 (m, 2H; Bz), 7.7-7.15 (m, 15H; MMTr, Bz), 7.06, 7.04 (2d, J = 8 Hz, 1H; 5-H, 2 diast.), 6, 9 (m, 2H; MMTr 3,5-H), 5.92 (d, J = 5 Hz, 1H; 1′-H), 4.60, 4.46 (2m, 1H; 3′-H, 2 diast.), 4.18 (m, 2H; 4′-H), 3.96 (2d, 1H; 2′-H), 3.8-3.3 (m, 12H; 5′-CH₂, O-CH₂-CH₂CN, 2 × CH- (CH₃) ₂, MMTr OCH₃ [3.79; s, 3H], 2′-OCH₃ [3.57; s, 3H]), 2.64, 2.51 ( 2t, J = 6 Hz; O-CH₂-CH₂CN 2 diast.), 1.3-0.8 (m, 12H; i-Pr CH₃, 2 diast.)

31 P NMR (101.26 MHz, CD₃CN): 149.75, 149.05 (2nd diast.); 31: 13.71

N⁴-benzoyl-5'-O-dimethoxytrityl-2'-O-ethylcytidine-3'- O - [(2-cyanoethyl) -N, N-diisopropyl phosphoramidite] 4b

TLC: 2 diastereomers R _f = 0.51, 0.61 (DCM / ethyl acetate 1: 1)
1H-NMR (250 MHz, CD₃CN; 1: 1.1 mixture of 2 diastereomers, the amidite contains 8% hexane [m, 0.86 ppm] and 7% 31 [d, 6.87 ppm, J = 635 Hz, PH; t, 2.76 ppm, J = 6 Hz, -CH₂CN]): 9.25 (s, br, 1H; 4-NH-Bz), 8.54, 8.44 (2d, J = 8 Hz, 1H ; 6-H) 2 diast.), 7.95 (d, J = 8 Hz, 2H; Bz 2,6-H), 7.7-7.2 (m, 12H; DMTr, Bz 3.4, 5-H), 7.06, 7.03 (2d, J = 8 Hz, 1H; 5-H, 2 diast.), 6.90 (m 4H; DMTr 3.5-H), 5.89 ( d, J = 4 Hz, 1H; 1'-H), 4.61, 4.47 (2m, 1H; 3'-H, 2 diast.), 4.19 * (m, 1H; 4'-H ), 4.06 * (m 1H; 2'-H), 4.0-3.4 (m, 14H; DMTr 2 × -OCH₃ [3.79 s, 6H, 2'-OCH₂-, 2 × N -CH, PO-CH₂-, 5'-CH₂), 2.64, 2.52 (2t, J = 6 Hz, 2H; -CH₂CN, 2 diast.), 1.3-1.0 (m, 15H ; 2'-OCH₂CH₃, 2 × CH (CH₃) ₂)
31 P NMR (CD₃CN, 101.26 MHz): 149.64, 148.77 (2 diast.); 31: 13.72

5'-O-Dimethoxytrityl-2'-O-methyl-N⁶- (phenoxyacetyl) - adenosine-3'-O - [(2-cyanoethyl) -N, N-diisopropyl- phosphoramidite] 10a

TLC: 2 diastereomers R _f = 0.63, 0.56 (DCM / ethyl acetate 1: 1)
1 H-NMR (250 MHz, CD₃CN; 1: 1 mixture of 2 diastereomers, the amidite contains 1% 31 [d, 6.87 ppm, J = 640 Hz, PH; t, 2.75 ppm, J = 6 Hz , -CH₂CN]): 9.3 (br s, 1H; NH), 8.59, 8.58 (2s, 1H; 2-H, 2 diast.), 8.31, 8.29 (2s, 1H ; 8-H, 2 diast.), 7.45-7.15 (m, 11H, MMTr, PhOAc 2,6-H), 7.03 (m, 3H; PhOAc 3,4,5-H), 6.82 (m, 4H; DMTr 3,5-H), 6.11 (d, J = 5 Hz, 1H; 1′-H), 4.98 (s, 2H; PhOAc-CH₂), 4, 80 (m, 1H; 2′-H), 4.69 (m, 1H; 3′-H), 4.30 (m, 1H; 4′-H), 3.8-3.3 (m, 15H; 5'-CH₂, O-CH₂-CH₂CN, 2 × CH- (CH₃) ₂, 2 × DMTr OCH₃ [3.74; s, 6H], 2'-OCH₃ [3.46, 3.44; 2s, 3H , 2 diast.]), 2.66, 2.51 (2t, J = 6 Hz; O-CH₂-CH₂CN, 2 diast.), 1.3-0.8 (m, 12H; i-Pr CH₃, 2 diast.)
31 P NMR (101.26 MHz, CD₃CN): 149.88, 149.57 (2 diast.); 31: 13.71

5′-O-dimethoxytrityl-2′-O-ethyl-N⁶- (phenoxyacetyl) - adenosine-3'-O - [(2-cyanoethyl) -N, N-diisopropyl- phosphoramidite] 10b

TLC: 2 diastereomers R _f = 0.74, 0.81 (DCM / ethyl acetate 1: 1)
1 H-NMR (250 MHz, CD₃CN; 1: 1 mixture of 2 diastereomers, the amidite contains 1.5% 31 [d, 6.87 ppm, J = 635 Hz, PH; t, 2.75 ppm, J = 6 Hz, -CH₂CN]: 9.3 (s br, N6-NH), 8.59, 8.57 (2s, 1H; 2-H, 2 diast.), 8.30, 8.28 (2s, 1H; 8-H, 2 diast.), 7.5-7.2 (m, 11H; DMTr, PhOAc 2,6-H), 7.03 (m, 3H, PhOAc 3,4,5-H) , 6.81 (m, 4H; DMTr, 3,5-H), 6.10 (d, J = 4.5 Hz, 1H; 1′-H), 4.97 (s, 2H; PhOAc CH₂) , 4.78 (m, 1H; 2'-H), 4.71 (m, 1H; 3'-H), 4.30 (m, 1H; 4'-H), 3.9-3.5 (m, 12H; DMTr 2 × -OCH₃ [3.75 s, 6H], 2′-OCH₂-, 2 × N-CH, PO-CH₂-), 3.38 (m, 2H; 5′-CH₂) , 2.67, 2.48 (2t, J = 6 Hz, 2H; -CH₂CN, 2 diast.), 1.18, 1.16 (2d, J = 7 Hz, 12H; 2 × CH (CH₃) ₂ ), 1.10 (t, J = 7 Hz, 3H; 2′-OCH₂CH₃)
31 P NMR (CD₃CN, 101.26 MHz): 149.71, 149.32 (2 diast.); 31: 13.72

5'-O-Dimethoxytrityl-2'-O-methyl-O⁶- [2- (4-nitrophenyl) - ethyl] -N²- (phenoxyacetyl) guanosine-3'-O - [(2- cyanomethyl) -N, N-diisopropyl phosphoramidite] 17a

1 H-NMR (CDCl₃, 90 MHz): δ = 8.5 (s br, 1H; NH), 8.12 (d, J = 9 Hz, 2H; NPE arom.), 8.02, 7.96 (2s, 1H; 8-H, 2 diast.), 7.5 (d, J = 9 Hz, 2H; NPE arom.), 7.5-6.6 (m, 18H; DMTr, PhOAc), 6.1 (m, 1H; 1′-H), 4.85 (t, J = 6.4 Hz, 2H; NPE O-CH₂), 4.68 (s, 2H; PhOAc CH₂), 4.7-4.0 (m, 3H; 2'-H, 3'-H, 4'-H), 4.0-3.18 (m, 8H; 5′-CH₂, NPE CH₂-Ph, 2 × CHCCH₃) ₂), P-O-CH₂), 3.76 (s, 6H; DMTr OCH₃), 3.5 (s, 3H; 2′-OCH₃), 2.62, 2.45 (2t, J = 6.3 Hz, CH₂CN, 2 diast.), 1.17, 1.04 (2d, J = 7.2 Hz, 12H; 2 × CH (CH₃) ₂, 2 diast.)

5′-O-dimethoxytrityl-2′-O-ethyl-O⁶- [2- (4-nitrophenyl) - ethyl] -N²- (phenoxyacetyl) guanosine-3'-O- [(2-cyanoethyl) -N, N-diisopropyl phosphoramidite] 17b

TLC: R _f = 0.87 (DCM / ethyl acetate 1: 1)
1 H-NMR (250 MHz, CD₃CN; 1: 1 mixture of 2 diastereomers, the amidite contains 2% hexane [m, 0.86 ppm] and 2% 31 [d, 6.87 ppm, J = 635 Hz, PH ; t, 2.75 ppm, J = 6 Hz, -CH₂CN]: 8.79, 8.77 (2s, 1H; 2-NH, 2 diast.), 8.14 (d, J = 9 Hz, 2H ; PhNO₂ 3,5-H), 8.07, 8.04 (2s, 1H; 8-H, 2 diast.), 7.57 (d, J = 9 Hz, 2H; PhNO₂ 2,6-H) , 7.45-7.15 (m, 11H; DMTr, PhOAc, 2,6-H), 6.99 (m, 3H, PhOAc 3,4,5-H), 6.78 (m, 4H; DMTr 3,5-H), 6.02 (d, J = 5 Hz, 1H; 1'-H), 4.91 (s, 2H; PhOAc CH₂), 4.84 (t, J = 7 Hz, 2H; O⁶-CH₂), 4.73 (m, 1H; 2'-H), 4.58 (m, 1H; 3'-H), 4.28 (m, 1H; 4'-H), 3 , 8-3.25 (m, 16H; DMTr 2 × -OCH₃ [3.73 s, 6H], 2′-OCH₂-, 2 × N-CH, PO-CH₂-, 5′-CH₂, NPE CH₂Ph [ 3.32 t, J = 7 Hz, 2H], 2.63, 2.46 (2t, J = 6 Hz, 2H; -CH₂CN 2 diast.), 1.2-1.0 (m, 15H; 2 '-OCH₂CH₃, 2 × CH (CH₃) ₂
31 P NMR (CD₃CN, 101.26 MHz): 149.71, 149.49 (2 diast.); 31: 13.72

5'-O-dimethoxytrityl-2'-O-methyl-N²- (phenoxyacetyl) - guanosine-3'-O - [(2-cyanoethyl) -N, N-diisopropyl- phosphoramidite] 18a

TLC: R _f = 0.5 (acetone / DCM 3: 7)
1 H-NMR (250 MHz), CD₃CN; 1: 1 mixture of 2 diastereomers, the amidite contains 2% O-2-cyanoethyl-N, N-diisopropylamino-H-phosphonate 31 [d, 6.87 ppm, J = 640 Hz, PH; t, 2.75 ppm, J = 6 Hz, -CH₂CN]): 7.89, 7.86 (2s, 1H; 8-H, 2 diast.), 7.5-7.1 (m, 11H; DMTr, PhOAc 2,6-H), 7.1-7.0 (m, 3H; PhOAc 3,4,5-H), 6.85-8.7 (m, 4H; DMTr 3.5-H) ), 5.94 (m, 1H; 1′-H), 4.74 (s, 2H; PhOAc-CH₂), 4.55-4.45 (m, 2H; 2 ′, 3′-H), 4.28 (m, 1H; 4'-H), 3.8-3.3 (m, 15H; 2 × DMTr OCH₃ [3.73; s, 6H], 5'-CH₂, O-CH₂-CH₂CN , 2 × CH- (CH₃) ₂, 2′-OCH₃ [2 diast., 3.44, 3.42; 2s, 3H]), 2.66, 2.47 (2t, J = 6 Hz; O- CH₂-CH₂CN, 2 diast.), 1.3-1.0 (m, 12H; i-Pr CH₃, 2 diast.)
31 P NMR (101.26 MHz, CD₃CN): 149.97, 149.86 (2 diast.); O- (2-cyanoethyl) -N, N-diisopropylamino-H-phosphonate 31: 13.71

2′-O-methyl-5′-O-monomethoxytrityluridine-3′-O - [(2- cyanoethyl) -N, N-diisopropyl phosphoramidite] 27a

TLC: 2 diastereomers R _f = 0.76, 0.70 (ethyl acetate / DCM 1: 1)
1 H-NMR (250 MHz, CD₃CN; 1: 1 mixture of 2 diastereomers, the amidite contains 3% 31 [d, 6.87 ppm, J = 638 Hz, PH; t, 2.75 ppm, J = 6 Hz , -CH₂CN]): 8.9 (br s, 1H; NH), 7.82, 7.69 (2d, J = 8 Hz, 1H; 6-H, 2 diast.), 7.5-7, 2 (m, 12H, MMTr), 6.9 (m, 2H; MMTr), 5.87 (d, J = 5 Hz, 1H; 1′-H), 5.21, 5.18 (2d, J = 8 Hz, 1H; 5-H, 2 diast.), 4.52 4.42 (2m, 1H; 3′-H, 2 diast.), 4.12 (m, 1H; 4′-H), 3.93 (m, 1H; 2'-H), 3.85-3.3 (m, 12H; 5'-CH₂, O-CH₂-CH₂CN, 2 × CH- (CH₃) ₂, MMTr OCH₃ [3rd , 77; s, 3H], 2′-OCH₃ [3.50, 3.48; 2s, 3H, 2 diast.]), 2.67, 2.51 (2t, J = 6 Hz; O-CH₂- CH₂CN 2 diast.), 1.3-1.0 (m, 12H; i-Pr CH₃, 2 diast.)
31 P NMR (101.26 MHz, CD₃CN): 149.75, 149.41 (2 diast.); 31: 13.70

5′-O-dimethoxytrityl-2′-O-ethyluridine-3′-O - [(2- cyanoethyl) -N, N-diisopropyl phosphoramidite] 27b

TLC: R _f = 0.83 (ethyl acetate)
1 H-NMR (250 MHz, CD₃CN; 1: 1.1 mixture of 2 diastereomers, the amidite contains 5% hexane [m, 0.86 ppm] and 1.6% 31 [d, 6.86 ppm, J = 635 Hz, PH; t, 2.75 ppm, J = 6 Hz, -CH₂CN]: 9.0 (s, br, 1H; 3-NH), 7.82, 7.73 (2d, J = 8 Hz, 1H; 6-H, 2 diast.), 7.5-7.2 (m, 9H; DMTr), 6.88 (m, 4H; DMTr 3,5-H), 5.85, 5.83 (2d, J = 4 Hz, 2 diast.), 4.50, 4.40 (2m, 1H; 3′-H, 2 diast.), 4.14 * (m, 1H; 4′-H), 4.05 * (m, 1H; 2'-H), 3.8-3.5 (m, 12H; DMTr 2 × -OCH₃ [3.77 s, 6H], 2′-OCH₂-, 2 × N-CH, PO-CH₂-), 3.41 (m, 2H; 5'-CH₂), 2.67, 2.52 (2t, J = 6 Hz, 2H; -CH₂CN, 2 diast.), 1.25-1 , 0 (m, 15H; 2′-OCH₂CH₃, 2 × CH (CH₃) ₂)
31 P NMR (CD₃CN, 101.26 MHz): 149.57, 149.18 (2 diast.); 31: 13.72

Examples of modified oligoribonucleotides 3'-amino-modified 2'-O-methyloligoribonucleotides

The at the 3'-end with (3-amino-2-hydroxypropyl) phosphate modified 19mer 2'-O-methylated Oligoribonucleotides can be used with an ABI 380 B DNA synthesizer (Applied Biosystems) synthesized a modified LCAA-CPG (controlled pore glass) solid carrier is used, the 1-O-DMTr, 3-N- [fluorenyl (methoxycarbonyl)] aminopropanediol groups (3'-amine-ON CPG, Clontech) carries. 2′-O- Methyl nucleoside- (2-cyanoethyl) -N, N-diisopropyl- phosphoramidites with the following protective groups used:

A:
N⁶-phenoxyacetyl
5′-O-DMTr
C:
N⁴-benzoyl
5′-O-MMTr
G:
N²-phenoxyacetyl
5′-O-DMTr
U:
5′-O-MMTr

Standard DNA synthesis methods with increased Coupling time (5 minutes instead of 3 minutes) is used. The oligonucleotide is treated with 25% aqueous NH₄OH solution (15 h at 55 ° C) of Protective groups released and separated from the carrier. To the final de-tritylation becomes the raw Oligonucleotide purified by ethanol precipitation.

Quantitative determination:
The oligonucleotides are quantified by the UV absorption at 260 nm, if necessary the value is corrected by subtracting the corresponding UV absorption of the FITC, dithiopyridine or buffer at 260 nm. The value of the dithiopyridine linkers in modified oligonucleotides is determined after reduction of a partial amount with dithiotreitol by measuring the absorption of released pyridine-2-thione at 343 nm (molar extinction coefficient 8.08 × 10³M⁻cm⁻¹).

The fluorescein content is determined spectrophotometrically Absorbance determined at 495 nm. The PMDBD content (Heinzerbase, Fluka) is affected by UV absorption Determined 219 nm; 1 µmol (as hydrochloride, in water) corresponds to approximately 16.3 AU₂₁₉.

3′-fluorescein-modified 2′-O-methyloligoribonucleotides

Labeling with FITC can be carried out similarly to the method described for 5′-amino-modified oligodeoxyribonucleotides (Agrawal, S., Christodoulon, C., and Gait, MJ, (1986) Nucleic Acids Res. 14, 6227-45). A solution of 10.4 AU₂₆₀ (approx. 350 µg; 52 nmol) 2′-O-methyloligoribonucleotide 19mer with the sequence 5′-GAG CAC ACU UCA UGC AGUG-3′-modified with (3-amino-2-hydroxypropyl) Phosphate at the 3'-end in 100 µl water is treated with a solution of 2.8 mg (7.2 µmol, 150 equivalents) fluorescein isothiocyanate (Sigma) in 300 µl 1M aqueous sodium bicarbonate solution (pH 8.5) for 4 h at room temperature . The mixture is subjected to gel filtration (Sephadex G 25-PD 10, Pharmacia) with water as the eluent. The first eluate, which contains oligoribonucleotide fractions, is evaporated in vacuo (possibly with Speedvac, Savant), the residue is taken up in 200 μl of a 100 mM triethylammonium acetate buffer (pH 7). The separation is carried out by RP-HPLC (Nucleosil RP-18, 250 × 4 mm;
Buffer A: 100 mM triethylammonium acetate, pH 7
Buffer B: acetonitrile, gradient elution: 0-50 min., 0-50% B, detection by UV absorption at 260 nm and fluorescence detection at 490 nm excitation, 520 nm emission) does not result in 3.3 AU₂₆₀ (32%) oligoribonucleotide FITC-modified form at a gradient concentration of approximately 18-19% acetonitrile, and 4.1 AU₂₆₀ (also 1.1 AU₄₉₆; the AU₂₆₀ value corresponds to approximately 3.75 AU₂₆₀ unlabeled oligoribonucleotide, 36%) of fluorescein-modified oligoribonucleotide at a gradient concentration of 19-20.5% acetonitrile.

Synthesis of thiocholesterol modified Oligoribonucleotides

a) Modification with dithiopyridine groups:
A solution of 14.1 AU₂₆₀ (about 450 µg; 67 nmol) of 2-O-methyloligoribonucleotide 19mer with the sequence 5'-CUA AAA GAG CUG UAA CACU-3 'modified with (3-amino-2-hydroxypropyl) phosphate on 3'-end, in 0.75 ml 50 mM HEPES buffer (pH 7.9) with a solution of 4.2 mg (13.4 µmol, 200 equivalents) of N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP, Pharmacia) treated in 300 ul ethanol for 4 hours at room temperature. The mixture is subjected to gel filtration (Sephadex G25-PD10, Pharmacia) with 20 mM HEPES buffer (pH 7.3) as an eluent. The first eluate, which contains fractions containing oligoribonucleotide, is evaporated in vacuo (Speedvac, Savant), dissolved in 200 .mu.l of water, and further purified by reverse phase HPLC. (Nucleosil RP-18, 250-4 mm;
Buffer A: 100 mM triethylammonium acetate, pH 6.5;
Buffer B: acetonitrile.
Gradient elution: 0-40 min., 0-40% B) gives 12.2 AU₂₆₀ oligoribonucleotide (corresponds to approximately 11.9 AU₂₆₀ (57 nmol) of the starting compound, 85%) modified with approximately 52 nmol dithiopyridine groups (after UV determination for Pyridine (1H) 2-thione released from a subset of a pattern by dithiothreitol treatment and AU value at 343 nm).

b) PMDBD salt formation: an aqueous solution of 8.1 AU₂₆₀ (about 255 µg, 37 nmol) from 2′-O-methylated 19mer oligoribonucleotide, which on 3'-end is modified with pyridyldithio groups (about 35 nmol after UV determination) in 250 µl Water is mixed with 250 µl of a solution of 4 mg PMDBD carbonate (PMDBD): 3,3,6,9,9-pentamethyl 2,10-diazabicyclo [4.4.0] dec-1-ene, Heinzer base, Fluka, Heinzer, F., Soukup, M., and Eschenmoser, A. (1978) Helv. Chim. Acta 61, 2851-74) in 50% treated aqueous methanol, then with water diluted a volume of 3 ml and freeze-dried. The lyophilisate is in 1 ml Methanol / water (1: 4) dissolved and one Subjected to gel filtration (Sephadex G25 PD10: Elution with methanol / water 1: 4). The Fraction containing oligoribonucleotide (7.1 AU₂₆₀, according to absorption of the nucleotide) 15 AU₂₁₉ (predominantly according to absorption of about 0.65 µmol BMDBD; 88%) is partially in a vacuum concentrated (Speedvac) to the methanol remove, freeze-dried and then in 1.0 ml Methanol / dichloromethane (1: 2) dissolved.  

c) Conjugation with thiocholesterol:
Half of the above solution (0.5 ml, which corresponds to approximately 16 nmol of the oligoribonucleotide) is mixed with 200 ul methanol, 30 ul 180 mM methanolic PMDBD trifluoroacetate buffer (pH 9) and 600 ug (1.5 µmol) thiocholesterol (Sigma) in 300 µl dichloromethane. The reaction mixture is kept under argon for 20 hours at room temperature. The solution is evaporated in vacuo (Speedvac), the residue is dissolved in 400 μl methanol / chloroform (1: 1) and extracted (first with 4 × 100 μl water, then further with 4 × 100 μl water. The two aqueous extracts (the 1.6 AU₂₆₀ and 0.7 AU₂₆₀ containing oligoribonucleotide) are combined and concentrated to remove the methanol in vacuo (Speedvac) to a volume of approximately 500 µl. Fractionation by reverse phase HPLC (nucleosil RP-18, 250 × 4 mm; buffer A: 100 mM triethylammonium acetate pH 7, buffer B: acetonitrile; gradient elution: 0-50 min., 0-50% B; 50-70 min., 50-100% B) gives 0.84 AU₂₆₀ (24% ) Oligoribonucleotide in non-cholesterol-modified form at a gradient concentration of about 25% acetonitrile, and 1.5 AU₂₆₀ (42%) of thiocholesterol-modified oligoribonucleotide at a gradient concentration of 55-68% acetonitrile.

d) In an analogous manner, a 2'-O-methylated Oligoribonucleotide 19mer, the one contains amino-modified 3'-end with the Sequence 5'-GAG CAC ACU UCA UGC AGVG-3 ' Thiocholesterol bound. That through Pyridyl dithiopropionate modified  Oligoribonucleotide (12 Au₂₆₀; about 60 nmol) as PMDBD salt is the raw product of the implementation of the amino modified oligoribonucleotide with 200 Equivalents of SPDP and conversion to that Preserved PMDBD salt. According to the reverse phase HPLC analysis the raw product contains about 75% dithiopyridine modified product (eluted at 20% Acetonitrile), not contaminated with 25% dithiopyridine-modified material (eluted at about 19% acetonitrile). The raw product comes with Treated 100 equivalents of thiocholesterol (Implementation in a total volume of 2 ml Methanol / dichloromethane (2: 3), the 5 mM PMDBD trifluoroacetate contains, pH 8.5). Refurbish and HPLC purification gives 4.7 AU₂₆₀ (about 160 µg; 39%) of oligoribonucleotide in non-thiocholesterol modified form (eluted at about 18% acetonitrile), and 5.7 AU₂₆₀ (about 190 µg; 48%) of the thiocholesterol modified form (eluted at 55-69% acetonitrile).

Fig. A1 and A2

Reducing division of the thiocholesterol-modified oligoribonucleotide: A solution of 0.1 AU₂₆₀ (46 nmol) thiocholesterol modified oligoribonucleotide (prepared according to point (d) above) in 900 µl 25 mM HEPES buffer (pH 7.3) is applied to a Concentration of 10 mM dithiothreitol (by Addition of 1.4 mg DTT) brought and under argon kept at 37 ° C for 18 hours. The analysis by reverse phase HPLC (under conditions as above has been described) shows complete  Cleavage of the entire oligoribonucleotide. A similar reducing cleavage with β-mercaptoethanol (BME) as a reducing agent gives the same cleavage product. The peak of the new product, presumably the split off thiocholesterol-free oligoribonucleotide, eluted at a gradient concentration of around 18% Acetonitrile. The remaining previous elution peaks are caused by the reducing agent.

Incorporation of thiocholesterol modified 2′-O-methyl oligoribonucleotides in liposomes Label the oligoribonucleotides

Thicholesterol-modified 2′-O-methyloligoribonucleotide and an unmodified one 2'-O-methyl oligoribonucleotide as a control 5′-³²-P-labeled, using an incubation buffer with a minimal amount of reducing agent starts. 1 pmol of oligoribonucleotide is incubated in 10 µl buffer [50 nM Tris · HCl pH = 7.5, 10 mM MgCl₂, 100 nM NaCl, 1 mM dithioerythritol (DTE)] with 10 T4 polynucleotide kinase units (Boehringer Mannheim) and 2 pmol g-32 P-ATP for 30 minutes at 37 ° C. The labeled oligoribonucleotide is then through Gel filtration cleaned with the addition of 10 pmol unlabeled oligoribonucleotide to the non-specific To minimize absorption (Sephadex G-25 (Pharmacia), 1.8 ml bed volume, 50 mM HEPES pH 7.3; the first radioactive peak that appears in the flow will collected).  

Production of liposomes

The liposomes are replaced by REV (reverse phase evaporation). Aqueous phase: 175 µl 50 mM HEPES (pH 7.3), 1 mM Calcein, 1 µM 2′-O-methyl-RNA (thiocholesterol modified or unmodified as a control), provided with a trace amount of 5′-³²-P-marked Material. Organic phase: solution of 5 µmol L-α-lecithin (from egg yolk, predominantly palmitoyloleoyl phosphatidylcholine; Avanti Polar Lipids) in 600 µl Diethyl ether (extracted with 50 mM HEPES buffer). The Both phases are carefully in a glass tube mixed by swirling and 5 min. at 0 ° C in one public address system similar to a bath. The resulting stable emulsion is slowly in one rotating evaporator with occasional whirling evaporated the mixture. After the full Remove the diethyl ether (about 30 minutes at 200 mbar) the liposome solution is again 20 min. sonicated at 0 ° C. The solution obtained is with Diluted 175 µl 50 mM HEPES pH 7.3 and at 2 ° C stored.

Gel filtration of the liposomes

The liposomes (a subset of 200 µl) are from unincorporated material by gel filtration [Sephadex G-75 superfine (Pharmacia), 10 ml Bed volume, 50 mM HEPES pH 7.3] separated. Fractions of 600 µl are collected and UV analysis and Subject to scintillation counting. The calcein Absorption (493 nm) is used for the non-specific Absorbance of the liposomes measured at 600 nm. As Control becomes a subset of the highlighted Oligoribonucleotide that is not due to thiocholesterol is modified, chromatographed analogously.  

Encapsulation efficiency is calculated from the calcein absorption associated with the liposome fractions (in fractions 4-7) and the non-incorporated calcein eluted with low molecular weight fractions (10-25). The incorporation of the oligoribonucleotide is determined by comparing the radioactivity of the liposome fractions (4-7) with the total eluted radioactivity in the case of unmodified oligoribonucleotides and the radioactivity in the oligonucleotide fractions (8-16, see Figs. 13 and 14) in the case of the thiocholesterol-modified oligoribonucleotides . (The level of radioactivity associated with the liposomes is corrected for the level of non-specifically incorporated 32 P-phosphates or nucleotides derived from the degradation of labeled oligoribonucleotides). The distribution of radioactivity is as follows: unmodified oligoribonucleotides: liposomes associated 8%, oligoribonucleotides 31%, phosphates 61%; thiocholesterol-modified oligoribonucleotides: liposomes associated 46%; Oligoribonucleotide 19%; Phosphate 34%.

Fig. A3: Gel filtration of the liposome preparation with thiocholesterol-modified 2'-O-methyl-RNA.

Fig. A4: Gel filtration of the liposome preparation with unmodified 2'-O-methyl-RNA. As a control, the 5'-labeled oligoribonucleotide (with broken line) is shown with a maximum in fraction 14. The second peak of radioactivity (in fractions 16-24, low molecular weight) that occurs in the liposome preparation (filled triangles in the drawing) is probably due to nuclease degradation or damage from sonication during liposome production.

The efficiency of incorporating the Oligoribonucleotides in liposome preparation are used in the summarized in the following table:

Degradation through alkaline phosphatase

A subset (400 µl) of the combined Liposome fractions that modified the thiocholesterol Contain oligoribonucleotides (Sephadex G 75, fractions 5 + 6, in 50 mM HEPES pH 7.3) is with 5 units (from calf intestine, Boehringer Mannheim) treated at 37 ° C for 2 hours.

As a control, the same amount of liposomes with 5 Units of alkali phosphatase in the presence of 1 µl Triton X-100 treated at 37 ° C for 1 hour. The  Degradation products are analyzed by gel filtration (Sephadex G-25 PD-10 columns) and the ones obtained Fractions (800 µl) are described as above characterized.

The results of the gel filtration of the alkali phosphatase degradation products are summarized in FIG. 5:

The 5'-labeled of about half of the oligoribonucleotide associated with the membrane is removed (³²PO₄ ^2- , fractions 7-12). This corresponds to the fact that half of the oligoribonucleotide is associated on the outside of the liposome membrane.

The data of the gel filtration of the alkali phosphatase degradation in the presence of Triton X-100, which causes the breakdown of the liposome, are summarized in Fig. A6: all oligoribonucleotides are vulnerable and are degraded by phosphatase (all radioactivity is in the phosphate fractions 7-12). The calcein also exits the liposomes and elutes as a broad peak in fractions 7-17, which show low molecular weights.

These examples clearly show that the degree of Incorporation of the thiocholesterol-modified Oligoribonucleotide is significantly higher than that of the unmodified.

Analogous to the examples above, several cholesterol- or thiocholesterol-modified 2'-O-alkyl oligoribonucleotides incorporated in liposomes will.

Claims (23)

1. oligoribonucleotides composed of 8 to 35 nucleotides of the general formula II, wherein
B means cytosine, uracil, adenine, guanine or inosine,
R is alkyl having 1 to 30 carbon atoms, and
E¹ is a phosphate diester, methylphosphonate, phosphoramidate or phosphorothioate residue,
or their 2′-OH or 2′-deoxy derivative,
wherein in at least one of the nucleotides R is alkyl having 2 to 30 C atoms, and in which the terminal groups in positions 3 'and 5' independently of one another represent a hydroxyl group or a phosphate ester residue, or a hydroxyl group modified by a labeling group or lipophilic group . 2. The oligoribonucleotide according to claim 1, wherein in the Nucleotide of formula II B unprotected adenine, Is cytosine, uracil or guanine.   3. Oligoribonucleotide according to claim 1 or 2, wherein R Is alkyl with 2 to 8 carbon atoms, preferably ethyl. 4. Oligoribonucleotide according to one of claims 1 to 3, in position 3 'by fluorescein or Thiocholesterol is modified. 5. Liposome, in the 3'-cholesterol or 3'-thiocholesterol modified Oligoribonucleotide as defined in one of claims 1 until 3 is defined, is incorporated. 6. alkylated ribonucleotide building block of the general formula I, wherein
B means unprotected or protected cytosine, uracil, adenine, guanine or inosine,
R is alkyl with 2 to 30 carbon atoms, E is a phosphite ester, H-phosphonate, phosphate ester, phosphate diester or phosphoramidate residue and
S¹ represents a protective group customary in oligonucleotide synthesis. 7. The ribonucleotide building block according to claim 6, wherein B N⁶-benzoyladenine or N⁶-phenoxyacetyl-adenine, N⁴-benzoyl-cytosine, uracil or N³-cyanoethyl-uracil, N²-benzoyl-guanine, N²-phenoxyacetyl guanine or N²-phenoxyacetyl-O⁶-nitrophenylethyl guanine is. 8. The ribonucleotide building block according to claim 6 or 7, wherein R alkyl with 2 to 8 carbon atoms, preferably ethyl or is n-butyl. 9. Ribonucleotide building block according to one of claims 6 to 8, where E N, N-Di (1-methylethyl) methoxyphosphoramidite or Is N, N-di (1-methylethyl) cyanoethoxy phosphoramidite. 10. Ribonucleotide building block according to one of claims 6 to 9, wherein S¹ is dimethoxytrityl or Is monoethoxytrityl. 11. nucleoside of the general formula III, wherein
B in position 6 guanine protected by nitrophenylethyl and
R is methyl, or
B is unprotected or protected cytosine, inosine, uracil or guanine and
R alkyl with 2 to 30 carbon atoms. 12. A nucleoside according to claim 11, wherein B N⁴-benzoyl-cytosine, N³-cyanoethyl-uracil, N²-phenoxyacetyl guanine or N²-phenoxyacetyl-O⁶-nitrophenylethyl guanine and / or R is alkyl with 2 to 8 carbon atoms, preferably ethyl or is n-butyl. 13. Process for the preparation of an oligoribonucleotide composed of 8 to 35 nucleotides of the general formula II, wherein
B means cytosine, uracil, adenine, guanine or inosine,
R is alkyl having 1 to 30 carbon atoms, and
E¹ is a phosphate diester, methylphosphonate, phosphoramidate or phosphorothioate residue,
or their 2′-OH or 2′-deoxy derivative,
wherein in at least one of the nucleotides R is alkyl having 2 to 30 carbon atoms, and
wherein the terminal groups in positions 3 'and 5' independently of one another represent a hydroxyl group, a phosphite ester, H-phosphonate or phosphate ester residue, or a hydroxyl group modified by a labeling group or lipophilic group, characterized in that the corresponding ribonucleotide building blocks in Linked presence of a coupling reagent and, if desired, introduces groups and / or lipophilic groups into the oligoribonucleotide thus obtained and, if desired, cleaves off protective groups. 14. Method for producing an oligoribonucleotide, built up from 8 to 35 nucleotides of the general formula II wherein
B means cytosine, uracil, adenine, guanine or inosine,
R is alkyl having 1 to 30 carbon atoms, and
E¹ is a phosphate ester, phosphate diester, methylphosphonate, phosphoramidate or phosphorothioate residue,
or their 2'-OH or 2'-deoxy derivative, at least one of the nucleotides being a 2'-OR nucleotide (II), and
wherein the terminal groups in positions 3 'and 5' independently of one another represent a hydroxyl group, or a phosphate ester residue, or a hydroxyl group modified by a labeling group or lipophilic group, which is characterized in that the corresponding ribonucleotide building blocks are prepared by the following multistage process

• a) alkylating a nucleoside of the general formula IV, wherein B is protected or unprotected adenine, cytosine or inosine or O⁶-protected guanine or N³-protected uracil and, if desired, the hydroxy group in position 5 'is protected, in the presence of a deprotonating base with an alkylating agent of the general formula V, RZ (V) wherein R is alkyl with 1 to 30 C atoms and Z represents a nucleophilic leaving group (eg halogen, tosyl, mesyl, ethyl sulfate residue), preferably iodine, is reacted;
• b) introduction of protective groups in the bases and position 5 '(preferably mono- or dimethoxytrityl);
• c) phosphorylating the 2′-O-alkyl nucleoside isolated from the reaction mixture by reaction with a phosphorylating agent, preferably phosphoramidite;
• d) isolating the 2'-O-alkyl nucleoside phosphoramidite by standard methods or preferably by adding a lipophilic alcohol, preferably sec-butanol or iso-propanol and separating the desired 2'-O-alkyl nucleoside phosphoramidite by extraction and precipitation; and
• e) the ribonucleotide building blocks obtained in this way (if desired together with the above 2′-OH and / or 2′-H derivatives) are linked by standard methods, the protective groups are separated off and, if desired, introduced into the oligonucleotide-marking groups and / or lipophilic groups thus obtained.

15. The method according to claim 14, wherein for the preparation of guanine containing end products a nucleoside (IV) is used in which B Is O⁶- [2- (4-nitrophenyl) ethyl] guanine.   16. The method according to claim 14 or 15, characterized characterized in that the alkylation in the presence of sodium hydride as a deprotonating base, under Use of R-iodide as alkylating agent (V) at temperatures up to a maximum of room temperature is performed. 17. Process for the preparation of the liposomes according to Claim 4, by mixing and sonicating the two Phases, one of which is the liposome and the other the cholesterol and / or thiocholesterol modified oligoribonucleotides contains. 18. Process for producing a Ribonucleotide building block according to one of claims 6 to 10 by implementing the appropriate Nucleosides with a phosphorylating agent and Isolate any protective groups containing nucleotides. 19. The method according to claim 18, characterized in that phosphoramidide as a phosphorylating agent is used and isolating the 2'-O-alkyl nucleoside phosphoramidites by Standard procedures or preferably by addition a lipophilic alcohol, preferably sec-butanol or iso-propanol, extraction and precipitation he follows.   20. Process for the preparation of a nucleoside of the general formula III, where B is protected or unprotected cytosine, uracil, adenine, guanine or inosine and R is alkyl with 1-30 C atoms if B is protected or unprotected cytosine, uracil, guanine or inosine and is alkyl with 2-30 C atoms, when B is protected or unprotected adenine, characterized in that a nucleoside of the general formula IV wherein B is protected or unprotected adenine, cytosine or inosine or O⁶-protected guanine or N³-protected uracil and, if desired, the hydroxy group in position 5 'is protected, in the presence of a deprotonating base with an alkylating agent of the general formula V, RZ (V) wherein R is alkyl with 1 to 30 C atoms and Z stands for a nucleophilic leaving group, is reacted and, if desired, protective groups are removed. 21. The method according to claim 20, characterized in that an alkylating agent (V) is used, wherein Z is bromine or preferably iodine. 22. The method according to claim 20 or 21, characterized characterized in that an alkylating agent used in which R is alkyl with 2 to 8 carbon atoms, is preferably ethyl. 23. Use of an oligonucleotide according to one of the Claims 1 to 4 as an antisense oligonucleotide, Probe, primer or section of primer.