DE3407671A1 - A PREPARATION CONTAINING A NUCLEOTID COMPOUND AND METHOD FOR THE PRODUCTION THEREOF - Google Patents

Description

description

The invention relates to a protected nucleotide which is freeze-dried and a process for its preparation. The erfindungsgeraäße protected nucleotide is particularly suitable in that it can be used in a unit fraction, or as a "monomer", which is condensed in the synthesis of an oligonucleotide IQ Siert, in accordance with the phosphotriester method (which is developed by the applicant and in Nucleic Acids Research, 8, 5507, 1980 and other references).

An oligonucleotide that has a relatively small number of, for example, about 4 to 30 nucleotide units, the are bonded to each other to form a nucleotide chain or a nucleotide strand, is a Part of a molecular chain of a nucleic acid, such as Deoxyribonucleic acid (hereinafter referred to as "DNA") or ribonucleic acid (hereinafter referred to as "RNA") .

Because nucleic acids provide genetic information of the organism carry, yield oligonucleotides that form the molecular chain of nucleic acids form genes. As advances in recombinant genetic engineering have been used for synthesis of oligonucleotides that can be used for various methods have already been proposed. 30th

One of these processes, in which the production of an oligonucleotide with a predetermined nucleotide sequence takes place, is the solid phase synthesis process corresponding to the phosphotriester process. This procedure will described in the following references.

Tetrahedron Letters, 1979 , 3636 (1979)

Nucleic Acids Research, 8, 5473 (1980)

Nucleic Acids Research, 8, 5491 (1980)

Nucleic Acids Research, 8, 5507 (1980) Nucleic Acids Research Symposium Series, 7, 281 (1980) J. Am. Chem. Soc., 103 , 706 (1981)

Nucleic Acids Research, I _- O, 197 (1981).

According to this process, a polymeric carrier such as polystyrene is modified to make it functional Has groups. This polymer is reacted with a protected nucleoside, whereby one is attached to the polymer bound nucleoside is obtained, which is hereinafter referred to as "resin". Then the Protecting group removed from resin (usually there is a protecting group for the terminal 5'-hydroxyl group present), and the resulting resin is capped with a protected nucleotide block (usually the terminal one is 3'-phosphate group the phosphodiester triethylammonium form) reacted, whereby this nucleotide block is attached to the 5'-hydroxyl group of the resin and thereby joining the nucleotide chain. If a number of these process steps are repeated, the oligonucleotide can can be built up or compiled with the desired length sequence. With the power amplifier or at a stage close to the final stage, the oligonucleotide is isolated from the resin by cleavage. An essential feature of this method is the fact that it can be operated semi-automatically is.

Similar to DNA synthesis in general, i.e. the solid phase synthesis process, the most important one is point to be observed in order to obtain the desired product in high yield that the condensation reaction at absolutely anhydrous conditions must be carried out. This is because the bond is between the nucleotides takes place by a dehydration condensation reaction between phosphoric acid residues and hydroxyl groups.

Anhydrous conditions during the condensation reaction have been used according to the prior art, for example obtained by using a mixture of a protected nucleotide block and a resin of azeotropic distillation underwent with pyridine. As a result of a report [H.Ito, Y.Ike, S.Ikuta, K.Itakura; Nucleic Acids Research, 1-0, 1755 (1982)] and as a result of our own investigations it has been found that drying of the resin can be achieved by treating the resin with a volatile Solvent (e.g. anhydrous tetrahydrofuran) and then a stream of gas such as dry Nitrogen.

However, the protected nucleotide block to be condensed must be the fully protected nucleotide block [the compound described below [il]], in part deprotecting, if necessary, the 3'-terminal phosphodiester compound and further, the diester compound (the compound [I] described below) must be converted by repeated co-evaporation with pyridine can be transferred to an anhydrous state. Therefore, the phosphodiester compound [I] must be prepared in situ. Even if the phosphodiester salt compound [i]

is prepared beforehand (usually it is obtained as a powder, which is obtained by adding dropwise into pentane is stored), the yield will be poor if it is used directly as it is. To a high In order to obtain the yield, it must be rendered anhydrous at the point in time when it is necessary e.g. by azeotropic distillation with pyridine or another method.

The requirement that the phosphodiester compound [i] to the transferring the necessary point in time to an anhydrous state involves an extremely difficult process stage, and it is extremely difficult to carry out this stage mechanically in practice in the synthesis of DNA. This is also one of the reasons why one cannot produce a stable, received high yield.

The applicant has carried out extensive research and has finally succeeded in finding a phosphodiester compound [i] freeze-dry using a suitable solvent. It was found, that this compound can be stored as it is for long periods of time and a stable, high one Yield even when it is used directly for the condensation reaction.

The invention relates to a freeze-dried preparation a protected nucleotide compound represented by the following formula [i]

34U / ÖV1

OH-A

[I]

is shown in which

R denotes a hydrogen atom or a protected hydroxyl group,

R _{1 means} a 'chemical protecting group for the phosphate group,

R2 is a chemical protecting group for the hydroxyl group means,

B 'means a protected base selected from guanine, adenine, cytosine, uracil and thymine, A denotes a lower alkylamine and η denotes a natural number.

The inventive method for producing the freeze-dried Preparation from the protected nucleotide compound is characterized in that one Powdered form of a protected nucleotide compound of the general formula "[i], as it is listed below, together with pyridine in Dloxan and dissolve the solution subjected to freeze-drying.

OH-A

[I]

Therein mean:

R represents a hydrogen atom or a protected hydroxyl group;

R _{1 is} a chemical protecting group for the phosphate group;

Rp is a chemical protecting group for the hydroxyl group ;

B ^{1 is} a protected base selected from guanine, adenine, cytosine, uracil and thymine; A is a lower alkylamine group; and η is a natural number.

Another process for producing the freeze-dried preparation from the protected preparation according to the invention Nucleotide compound is characterized in that one has an oily form of a protected nucleotide represented by the general formula [i] shown below is dissolved in dioxane and the solution is subjected to freeze-drying.

B '

OH-A [I]

In it "mean:

R represents a hydrogen atom or a protected hydroxyl group;

R _{1 is} a chemical protecting group for the phosphate group;

R _{2 is} a chemical protecting group for the hydroxyl group;

B ^{1 is} a protected base selected from guanine, adenine, cytosine, uracil and thymine; A is a lower alkylamine; and η is a natural number.

In the above formula [i], when η is 2 or above, the radicals B ¹ , R and R .. occur several times and can therefore be identical or different.

The freeze-dried preparation according to the invention does not exist as a powder or in the form of dense particles or as a mass, but in the form that is used for freeze-dried Products is typical, namely as a light and flaky powder with good solubility. This preparation can be stored or transported in this state, with only the usual care spend to avoid moisture and contamination. The preparation dissolves easily in the Solvents normally used in the triester process used as in pyridine.

In the known phosphotriester method, the nucleotide reagent (of the formula [I]) was obtained by azeotropic distillation with pyridine immediately before use in the condensation reaction in an anhydrous state convicted. This process is unexpectedly complicated in actually performing the method, and in practice it is extremely difficult to simultaneously synthesize a number of oligonucleotides as above mentioned to perform. If the freeze-dried reagent according to the invention is used, the azeotropic Distillation that runs in parallel with the condensation reaction

is no longer required, and it is possible to carry out a series of condensation reactions at the same time. Low yields by Errors caused in the azeotropic distillation are avoided, and thereby a stable one is obtained Yield. It has been found that the yield per process stage is the same as or higher than in the case of azeotropic distillation. As a result of the fact that a stable, high yield can be obtained and that As the process steps are simplified, it has further become possible to increase the reaction scale to a large extent to reduce. It is a further advantage that the mechanical operation in the condensation reaction that occurs in mechanical operation was difficult to perform, also becomes possible.

In JA-OS 150700/1982 a product is described which is similar to the preparation according to the invention. However, the known freeze-dried preparation is that of a nucleotide compound used in the phosphite method [MD Mattucci, MH Caruthers: J. Am. Chem. Soc., 103 »3185 (1981)]. The freeze-dried preparation according to the invention differs from that of the known compound. With knowledge of the prior art, one could not assume that a freeze-dried sample could be produced in the form of a 1: 1 salt of a nucleotide and a lower alkyl amine by the method according to the invention. The salt is a salt with a lower alkylamine, ie a lower alkylamine with a boiling point of about 80 ^° C. or below at atmospheric pressure. It was therefore surprising that a 1: 1 salt can be produced in the freeze-drying step. In the azeotropic distillation with pyridine

3AU / 671

In contrast, excess quantities can always be removed.

The invention is explained in more detail with reference to the accompanying drawings; show it:

1 and 4 show chromatographies on Sephadex G-50 columns;

Figures 2 and 5 HPLC patterns of oligonucleotides with a dimethoxytrityl group; and

Figures 3 and 6 HPLC patterns of the target oligonucleotides.

Column: / U-Bondapak C 18 (Waters) Eluent: acetonitrile -0.02 M EDAA buffer (pH 7.8)

Concentration gradient: as indicated in the drawings

Flow rate: 2 ml / min Chart speed: 10 mm / rain Temperature: 50 ⁰ C.

The invention is explained in more detail below. Nucleotide compound [i]

The nucleotide compound that the freeze-dried of the invention Preparation represents corresponds to the aforementioned formula [i].

ι

In the above formula, the symbol - means, as is customary, the remainder of the ribose portion of a nucleotide from which the 2 ^f , 3 'and 5' hydroxyl groups and the base have been removed. More precisely, it has the following structure:

When R in the formula [ij is hydrogen, this is Ribose unit 2'-deoxyribosyl, and the compound of Formula [I] forms a DNA chain. When R is a hydroxyl group or a protected hydroxyl group, is this ribose unit ribosyl, and the compound of the formula [I] forms an EITA chain. If the degree of polymerization η = 2 or higher, two or more of the substituents R may be the same or different, and therefore the compound of the formula [i] may be a hybrid of DNA chain (s) and RNA chain (s).

B ¹ is a protected base. This generally includes the base adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U). When the compound of the formula [I] forms a DNA chain, the base is A, G, C or T, whereas when the compound forms an RNA chain, it is A, G, C or U. The protecting group is usually an acyl group and examples of protected bases are N -benzoyladenine, N-isobutyrylguanine, N -benzoylcytosine, thymine and uracil. As clearly follows from this, thymine and uracil are themselves examples of B ^! , and in general thymine and uracil need not be protected. They are themselves protected without a foreign protective group being present. Therefore, "protected thymine" and "protected uracil" are also included and are considered to be examples of those without a specific protecting group.

In this context it should be noted that the bases of the Formula [i] listed above are self-evident with groups with the exception of the acyl groups modified

34U7671

may be adorned, which fall under the term "protecting groups" as defined above, and the present invention also includes DMA and RNA derivatives in which changes that are generally customary for such bases have taken place, such as methylation or partial conversion of the amino groups in carbonyl groups when B is cytosine or adenine. Compounds with bases, which have been changed in this way are also the subject of the present invention.

The group R ^, the group Rp and the group R, when present as protecting groups for hydroxyl groups, are any protecting groups known as protecting groups for phosphate and hydroxyl groups in nucleic acid synthesis. Specific examples are those as described in the following reference, as well as the protecting groups explained in the above bases: Journal of the Society of Organic Synthetic Chemistry, Japan, Volume 36, No. 9 »Pages 723-731, (1978); Synthesis of Nucleosides and Nucleotides (Maruzen, Japan 1977); Organic Chemistry of Nucleic Acids (Kagaku Dojin, Japan 1979); Nucleic Acids (Asakura Shoten, Japan 1979); Tetrahedron, ^ 4, 3143 (1978); Journal of the Society of Synthetic Organic Chemistry, Japan, £ 4ι 723 (1978); Kagaku no Ryoiki (Domain of Chemistry), ^ 3 ,, 566 (197-9); and other summaries and textbooks. The protecting groups which are particularly preferred in the present invention are the o-chlorophenyl group and the p-chlorophenyl group as the group R ^; the monomethoxytrityl group and the diraethoxytrityl group as group R ₂ ; and hydrogen and the o-nitrophenyl protected hydroxyl group as group R.

η is any natural number. In general, η is about 1 to 10, preferably about 1 to 6 and particularly preferably about 1 to 3.

A is a lower alkyl amine. The term "base Alkyl "means an alkyl group having about 1 to 4 carbon atoms. Typical examples of lower alkyl amines are triethylamine, diisopropylamine, dimethylamine, t-butylamine, sec-butylamine, n-butylamine, n-propylamine and isopropylamines, with triethylamine being preferred.

The preparation of the compound [i] can be according to any one of suitable methods for the synthesis of nucleic acids are carried out as described in the above references or described in other publications. A typical example is explained below. First the compound of the formula [II] is synthesized by a method of the above literatures. She is also available in the stores.

B ¹
25th

R ₂ -4-0

^^ OR

-R O

-O P - OR., [II]

wherein

R, denotes a chemical protecting group for the phosphate group which can be cleaved off under the conditions at which other protecting groups are stable, usually a cyano ethyl group; mid R, R ₁ , B 'and η have the respective meanings as defined above.

3AU / 671

The compound of the formula [ill is assigned a low Alkylamine, preferably triethylamine, in pyridine or in pyridine-water to remove the protective group fW treated to form compound [i]. The compound [i] thus formed is obtained in an oily form by distillation of excess lower alkylamine, pyridine and water obtained from the reaction product.

This oily compound [i] is stored and so how it is used for DNA synthesis or as a powder, which is obtained by adding dropwise the oily form is obtained in hexane. The oily form of the compound [l] can be regarded as an oil, since it has compounds with Except for the compound [i], in particular pyridine, are contained in it.

Freeze drying Preparation of the solution:

The main solvent is dioxane. The compound [i] when it is in powder form is slowly dissolved in dioxane, and therefore it is preferably dissolved in a mixture of pyridine and dioxane. In this case, if the amount of pyridine is excessive, the freeze-drying of the solution cannot be carried out effectively. It is therefore preferred to use pyridine in an amount by volume of 2 to 10 % v / v based on dioxane. In preparing a solution of the compound [ij, pyridine may first be mixed with dioxane before the compound [I] is dissolved therein. Alternatively, the compound [i] can be dissolved in pyridine first and the resulting solution can then be dissolved in dioxane. The latter method may be preferred. The expression "dissolved in dioxane together with pyridine", as used in the present application, to

therefore summarizes both manufacturing processes described above.

When the compound [i] is in an oily form, can quite a high rate of dissolution can be obtained without the use of pyridine.

In both cases described above, the essential thing is Dioxane solvent. A small amount of another solvent or lower alkyl amine can also be used in conjunction therewith without departing from the subject matter of the present invention will. The scope of protection of the claims should also include this embodiment. Hence the use a small amount of pyridine when the compound [i] is in an oily form are also subject of the present invention Invention.

The concentration of the compound [ij in the solution can be can be appropriately determined depending on the viscosity at the freeze-drying step. Normally it is of the order of, for example, 10 to 100 mg / ml.

Freeze drying stage

Except for the fact that the compound in solution is compound [i] and the solvent is dioxane (and a small amount of pyridine), the Freeze drying stage not different from the known freeze drying stage.

Generally, this step involves freezing by cooling the solution (usually of the order of

3 4 O V b 7 Ί

of -30 C or below) and the sublimation of the frozen Solvent by applying a reduced pressure (usually on the order of several mmHg or below).

Regarding the other measures required the freeze-drying is based on textbooks such as Shin Jikken Kagaku KÜoza "New Course of Experimental Chemistry) 1 [1] 459 "'(Maruzen, Japan 1975), referenced.

Use of the freeze-dried preparation

The freeze-dried preparation of the invention Compound [l] not only has good solubility in pyridine, but also its reactivity in the condensation stage is equal to or better than that of the known preparations (dried by azeotropic distillation immediately before use).

The freeze-dried preparation according to the invention can therefore, as long as it is stored under anhydrous conditions, advantageously contribute as a nucleotide unit DNA synthesis can be used without azeotropic drying.

The solid phase method for the synthesis of an oligonucleotide using a freeze-dried nucleotide compound is exemplified below explained in more detail. However, the invention is not limited to this.

About 50 to 60 mg of a polystyrene resin with one on it bound nucleoside is placed in a reactor, which is equipped with a tightly fitting stopper and glass filter.

340767Ί

is equipped and well with an isopropanol-methylene chloride (i5: 85, V / V) solution washed. The resin is then treated with a 1 molar zinc bromide solution in an isopropanol-methylene chloride (15:85, V / v) solution four times during Treated for 5 min. After detritylation with zinc bromide solution the resin is treated with an isopropanol-methylene chloride (i5: 85, V / V) solution and then with pyridine, and the resin is dried with a vacuum pump. That The detritylation process can be replaced by a process using benzenesulfonic acid or trichloroacetic acid is used.

Alternatively, the resin can be dried by washing with a volatile organic solvent and then drying Drying can be carried out with a drying gas.

On the other hand, the nucleotide reagent used for the Condensation is used, can be prepared by adding-mesitylenesulfonyl-nitrotriazolide (hereinafter referred to as MSNT abbreviated) (about 30 mg) to the freeze-dried preparation of the nucleotide block (about 30 mg) according to Invention adds and the mixture dissolves in anhydrous pyridine (about 400 / ul) with stirring.

This reagent is quickly added to the dried resin and the reaction is carried out for 60 minutes. To After the reaction, the resin is washed with pyridine and the unreacted 5'-hydroxyl group is protected by acetylation. As an acetylating agent, 0.5 ml of an acetic anhydride-pyridine (1: 5) solution and 0.5 ml of one can be used Dimethylaminopyridine (hereinafter abbreviated as DMAP) -pyridine solution use together, and the reaction

34U / 671

22nd

follows for 5 min. After washing with pyridine, becomes initiates the following cycle. This procedure (steps 1 to 9 in Table 1) is repeated to to extend the chain length one by one.

Table 1 Reacts in front of, qänge

Level reagent or solution amount d. Time number of mean Solution (min) repetitions

by means of + (ml)

1 IsO-PrOH-CH ₂ Cl (15:85) 1 2 5 2 1.0 mol ZnBr2 / Iso-PrOH- 1 VJl CH ₂ Cl ₂ 3 IsO-PrOH-CH ₂ Cl ₂ (15:85) 1 1 3 4th dry pyridine 1 1 5 5 Vacuum drying - about 10 1 6th Nucleotide + MSNT / dry 0.5 60 1 nes pyridine 7th Pyridine 1 1 3 8th Ac20 pyridine (1: 5) 0.5 DMAP pyridine (200 mg / iOml) 0.5 9 Pyridine 1 1 3

25 based on 50 to 60 mg of resin

Experimental examples Example 1

About 30 mg of a compound [II] represented by the formula

DMTr -O-

-O P O-CE

I OR-,

where DMTr is a dimethoxytrityl group, R _{1 is} an o-chlorophenyl group, T Thyrain and CE is a cyanoethyl group, 1 ml of a pyridine-triethylamine-water (3: 1: 1, V / V) solution are treated with 1 ml of a pyridine-triethylamine-water (3: 1: 1, V / V) solution for 15 minutes at room temperature to remove the Treated cyanoethyl group. The solvent is evaporated and excess triethylamine and water are completely removed by evaporation using azeotropic distillation with pyridine. The resulting oily product is dissolved in 600 μl of dioxane and freeze-dried to obtain the freeze-dried preparation of the triethylamine salt of the aimed compound [i].

Example 2

About 30 mg of the compound [II] are mixed with 1 ml of diethylamine-pyridine (1: 9, V / V) treated for 30 min at room temperature. The solvent is removed and excess Diethylamine evaporated by azeotropic distillation with pyridine. The oily product obtained is in 600 / ul of dioxane dissolved and the freeze-dried preparation of the diethylamine salt of the compound [i] according to the procedure described in Example 1 obtained.

Similar results are obtained when diethylamine is replaced by n-butylamine or diisopropylamine.

Example 3

(1) Manufacture of freeze-dried preparations

Dimethoxytrityl-di-nucleotide of the formula 35

DMTr

• Ο-

^B i

-O

-ρ -ο-

^B 2

-0

I!

• ρ

OR

where DMTr is a dirnethoxytrityl group, IL an o-chloΓΙΟ phenyl group and B '^ and B ¹ ρ each a protected base selected from N-benzoyladenine (A), N-benzoylcytosine (C), N-isobutyrylguanine (G) and Thyrain ( T), are processed into freeze-dried preparations as follows.
15th

In the following examples these dinucleotides are referred to as AC or TG with reference to their bases.

35 mg each of (1) [CA], (2) [GG], (3) [CA], (4) [ta], (5) [TA], (6) [AA] and (7) [ TC) are dissolved in 50 / ul of anhydrous pyridine and frozen with addition of 800yul anhydrous dioxane at -50 ⁰ C. Drying is carried out by subliming the solvent at a reduced pressure of 3 mmHg. The freeze-dried dinucleotides

(1) to (7) are stored in a desiccator.

(2) Preparation of an oligonucleotide

60 mg (6.6 / µmol) dimethoxytrityladenosine resin are in treated a reactor according to the procedures listed in Table 1 above. That is, washing with isopropanol methylene chloride, detritylation with 1 molar zinc bromide, washing with isopropanol-methylene chloride, washing with pyridine and drying the resin

by means of a vacuum pump. 400 μl of a pyridine solution of the dinucleotide (1) is added to this dried resin [CA] and MSNT (30 mg) and carry out the reaction for 60 min. After the reaction, it is washed with pyridine, with acetic anhydride-pyridine-dimethylaminopyridine (1: 9: catalytic amount) acetylated and washed with pyridine, one cycle of condensation being completed.

IQ This procedure is used for the subsequent condensation of the dinucleotides (2) [GG], (3) [CA], (4) [TA], (5) [TA], (6) [AA] and (7) [TC ] repeated to synthesize a pentadecanucleotide d (TCAATATACAGGCAA). The average yield is 95% and the overall yield is 70 ^. This is a yield which is higher than that obtained by the known process, namely azeotropic distillation with pyridine.

The synthesized oligonucleotide is deprotected and purified in the usual way. More precisely, about 10 mg of the resin are mixed with 100 / ul of a solution of 0.5 M tetramethylguanidine-pyridine-2-aldoxime in dioxane-water (9: 1, v / v) overnight at room temperature and then overnight at 50 ⁰ C with the addition of 2.5 ml of concentrated ammonia water. The resin is filtered off, the filtrate concentrated and a crude purification on Sephadex C-50 (1.5c 120 cm), eluting with 0.05 M TEAB (triethyammonium bicarbonate) buffer (pH 7.5) (Fig. 1 ) subject.

The peak fraction is collected and the trityl derivative by high performance liquid chromatography (HPLC) Purified on a reverse phase column (Fig. 2). After the detritylation with 80% acetic acid, the cleaning takes place again by means of a reverse phase column, wherein

the desired pentadecanucleotide is obtained in high purity and high yield (FIG. 3).

Example 4

O) Production of freeze-dried preparations

35 mg each of the dinucleotide reagents (1) [CA],
(2) [TG], (3) [CA], (4) [TA], (5) [TA], (6) [AA) and (7) [TC] are dissolved in 50 / µl of anhydrous pyridine and freeze-dried with the addition of 800 μl of anhydrous dioxane.

(2) Preparation of an oliflonucleotide

60 mg (6.6 mmol) of dimethoxytrityladenosine resin are in given a reactor and successive condensations in the order of the nucleotide reagents (1) - (2) (3) - (4) - (5) - (6) - (7) for the synthesis of a pentadecane nucleoside d (TCAATATACATGCAA) carried out.

The average yield is £ 95 and the Ge overall yield 70%. Compared to the known method ren, the yield in the individual cycles is less uneven and very high.

The intended pentadecanucleotide is purified in a manner similar to the previous examples, and a product of high purity is obtained
Yield (Figures 4 to 6).

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Claims (13)

KRAUS & WElSERT PATENT LAWYERS AND APPROVED REPRESENTATIVES BEFORE THE EUROPEAN PATENT OFFICE DR. WALTER KRAUS DIPLOMCHEMIKER ■ D R.-l N Θ. ANN EKÄTE WEISERT DIPL.-ING. SPECIALIZATION CHEMICALS IRMGARDSTRASSE 15 · D-8OOO MUNICH 71 ■ TELEPHONE 089/79 70 77-79 70 78 · TELEX OS-212156 kpatd TELEGRAM CRAUSPATENT 4368 AW / My WAKUNAGA SEIYAKQ KABUSHIKI KAISHA, Kyoto, KABUSHIKI KAISHA, Osaka, Japan and Japan Preparation containing nucleotide compound and process for its production. Claims 1.) Freeze-dried preparation of a nucleotide mixture the general formula OH-A [I] wherein R is a hydrogen atom or a protected one Hydroxyl group means R _{1 is} a chemical protecting group for the phosphate group, R _{2 is} a chemical protecting group for the hydroxyl group, B ^{1 is} a protected base selected from guanine, adenine, cytosine, uracII and thymine, A denotes a lower alkylamine, and η denotes a natural number, where, when η is greater than 1, the substituents B ¹ , R and R ₁ can occur several times and can be identical or different. 2. The freeze-dried preparation of the nucleotide compound according to claim 1, wherein η is a natural number from 1 to 10 means. 3. The freeze-dried preparation of the nucleotide compound according to claim 1 or 2, wherein the lower alkyl amine is one in which each alkyl portion contains 1 to 4 carbon atoms. 4. The freeze-dried preparation of the nucleotide compound according to claim 1, 2 or 3, wherein the lower alkylamine is triethylamine. 5. Freeze-dried preparation of the nucleotide compound according to at least one of claims 1 to 4, wherein R is hydrogen. 6. Freeze-dried preparation of the nucleotide compound according to at least one of claims 1 to 4, wherein R is a protected hydroxyl group. 7. Freeze-dried preparation of the nucleotide compound according to at least one of claims 1 to 6, wherein B ^{1 is} selected from the group acylated guanine, acylated adenine, acylated cytosine, thymine and uracil. 8. Freeze-dried preparation of the nucleotide compound according to at least one of claims 1 to 7, wherein B 'is selected from the group consisting of benzoyladenine, isobutylguanine, benzoylcytosine, thymine and uracil. ' 9. A process for the manufacture of a freeze-dried preparation of a protected nucleotide compound, characterized by taking a powder form of a protected nucleotide compound represented by the general Formula [i] B " / Il \ OH-A [I] is shown in which R is hydrogen or a protected hydroxyl group, R _{1 is} a chemical protecting group for the phosphate group, R _{2 is} a chemical protecting group for the hydroxyl group means B ^{1 is} a protected base selected from guanine, adenine, cytosine, uracil and thymine, A is a lower alkylamine, and 35 η denotes a natural number, in which, if η is greater than 1, the substituents B ^f , R and R ₁ can occur several times and can be the same or different, dissolves in dioxane together with pyridine and subjects the solution to freeze-drying. 10. The method according to claim 9, characterized in that the powder in a mixture of pyridine and Dioxane is dissolved. 11. The method according to claim 9, characterized in that the powder is dissolved in pyridine and then the resulting Solution is dissolved in dioxane. 12. Process for the production of a freeze-dried preparation from a protected nucleotide compound, characterized in that one has an oily form of a protected nucleotide compound of the general Formula [ij -R II \ OH-A [I] wherein R is hydrogen or a protected hydroxyl group, R _{1 is} a chemical protecting group for the phosphate group, R _{2 is} a chemical protecting group for the hydroxyl group, B ^{1 is} a protected base selected from guanine, adenine, cytosine, uracil and. Thyrain, means, A means a lower alkylamine, and η denotes a natural number, where, if η is greater than 1, B ^f , R and R ₁ can occur several times and can be the same or different, dissolves in dioxane and throws the solution under freeze-drying! 13. The method according to claim 12, characterized in that the oily form of the protected nucleotide compound of the general formula [i] comprises an oily product which is formed by a protected Nucleotide compound represented by the following formula B ¹ 20th [II] with a lower alkylamine to remove the protecting group R, the terminal 3'-phosphate group wherein R, denotes a chemical protective group for the phosphate group and is split off under the conditions under which all other groups are stable, and R, R ^, R ₂ , B ¹ and η have the definitions given in claim 12,
implements.