CA1214735A - Nucleotide compound preparation and method for producing the same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A freeze-dried preparation of a protected nucleo-tide compound represented by the formula below, and a method for preparing the same, which comprises freeze drying a solution of an impure preparation whose solvent comprises dioxane [I] , wherein: R is hydrogen or a protected hydroxyl group;
R1 is a chemical protecting group for phos-phate group;
R2 is a chemical protecting group for hydroxyl group;
B' is a protected base selected from guanine, adenine, cytosine, uracil and thymine;
A is a lower alkylamine; and n is any desired natural number, wherein, when n is greater than 1, B', R and R1 in plural numbers may be either identical or different.

Description

~ 35 NUCLEOTIDE COMPOUND PREPARATION
A~D METHOD FOR PRODUCING THE SAME

BACKGROUND OF_TH~ INVENTION
This invention relates to a protected nucleotide which is freeze-dried and a method for producing the same. The protected nucleotide according -to this invention is particularly suitable for use as the unit fraction or "monomer" to be condensed in the synthesis of an oligonucleotide according to the phosphotriéster method (developed by us as disclosed in Nucleic Acids Research, 8, 5507, 1980, and others).
An oligonucleotide, comprising a relatively small number, for example, of about 4 to 30 of nucleotide units bonded to each other to form a nucleotide chain or strand is a constituent of the molecular chain of a nucleic acid such as deoxyribonucleic acid (herein-af~er called as DNA) or ribonucleic acid (hereinafter called as RNA).
Since nucleic acids bear genetic information of organisms, oligonucleotides forming the molecular chains of nucleic acids constitute genes. As progress in recombinant gene technique, various methods have already been proposed for synthesis of oligonucleotides -to be utilized therefor.
One of the methods ensuring production of an oligonucleotide of a predetermined nucleotide sequence - ~

~fæ~73~

is the solid phase synthesis according to the phos-photriester method we have developed. This method is disclosed in the references set forth below.
Tetrahedron Letters, 1979, 3636 (19791 Nucleic Acids Research, 8, 5473 (1980) Nueleic Acids Research, 8, 5491 (1980) Nucleic Acids Research, 8, 5507 (1980) Nucleic Acids Research Symposium Series t 7, 281 (1980) J. Am. Chem. Soc., 103 r 706 (1981) Nueleic Aeids Researeh, 10, 197 (1981) Aeeording to this method, a polymer support sueh as polystyrene is modified to have a funetional group, and this polymer is eaused to react with a protected nueleoside to make the nueleoside bonded polymer (hereinafter ealled the resin). Subsequently, the proteeting group is removed from the resin (ordinarily the proteeting group being for the terminal 5'~hydroxyl group), and the resultant resin is eaused to reaet with a proteeted nueleotide block (ordinarily the terminal 3'-phosphate group is phosphodiester tri-ethylammonium form), whereby this nueleotide bloek is bonded to the 5l-hydroxyl group on the resin, thus joining the nueleotide ehain. By repeating a series of these operations, the oligonucleotide with desired length sequenee ean be assembled.
In the final step or a step near the final step, the oligonucleo-tide is isolated by cleavage from the resin.
The salient feature of this method resides in that semi-automatic operations are possible.
Similarly as in the case of DNA synthesis in gene-ral, also in the solid phase synthetic method, the mostimportant point to be borne in mind for obtaining the desired product in a high yield is-that the condensa-tion reaction must be carried out under absolutely anhydrous conditions. This is because bonding between the nucleotides oecurs through dehydrating condensa-tion reaetion between phosphorie acid residues and hydroxyl groups.
Anhydrous conditions during condensation reaetion have been obtained in the prior art by, for example, subjecting a mixture of a protected nucleotide bloek and a resin to azeotropic distillation with pyridine.
As a result of a report [H. Ito, Y. Ike, S. Ikuta, K. Itakura; Nucleic Aeids Researeh, 10, 1755, (1982)]
and our investigations, drying of a resin can be aeeomplished b~; washing the resin with a volatile solvent (e.g , anhydrous tetrahydrofuran) and sub-sequently exposing the same to a stream of a gas such as dry nitrogen.
However, as for the protected nucleotide bloek to be condensed, the fully protected nucleo-tide block (the compound [II] as hereinafter described) 1;21l473~;

must be partially deprotected when necessary to convert the 3'-terminal phosphodiester compound, and moreover the diester compound (the compound [I]
as hereinafter described) must be rendered anhy-drous by repeated co-evaporation with pyridine.
Therefore, the phosphodiester compound [I] must be prepared in situ. Further, even when the phos?hodiester salt compound [I] is prepared beforehand (usually stored as powder formed by dropwise addition thereof into pentane), if it is used directly as it is, yield will be poor.
Thus, for obtaining a high yield, it must be rendered anhydrous at the time of necessity by way of azeotropic distillation with pyridine or by another method.
The operation to make anhydrous the phos-phodiester compound [I] at the time of necessity has been one of the most troublesome operations and has been considered difficult to practice mechanically in the synthesis of DNA. This has also been one of the causes of failure to obtain stable high yield in the preparation of an oligonucleotide.
SUMMARY OF THE INVENTION
We have made extensive studies and consequently sucseeded in freeze-drying a phosphodiester compound [I] by L4~3~

-the use of a suitable solvent. It was founcl -that this compound can be stored Eor a long period as it is, and can provide a stable high yield, even if it is used directly for the condensation reaction. This inven-tion is based on such discoveries.
More specifically, the present invention relates to a freeze-dried preparation of a protected nucleotide compound represented by the following formula [I].
B' ~ ~R O
t 1 LI]

wherein: R is hydrogen or a protected hydroxyl group;
Rl is a chemical protecting group for phos-phate group;
R2 is a chemieal protecting ~roup for hydroxyl group;
B' is a protected base selected fro~ guanine, adenine, cytosine, uracil and thy-mine;
A is a lower alkylamine; and n is a natural number.
The method for preparing the freeze-dried prepa-ration of the proteeted nucleotide eompound aecording to the present invention comprises dissolving a powder form 7~3~

of a protectecl nucleotide compound represented by the formula [I] shown below together with pyridine in dioxane and subjecting the solution to Ereeze-drying.
B' ~ ~R O
R2 t ~ - P - ~ O~-A [I]

wherein: R is hydrogen or a protected hydroxyl group;
Rl is a chemical protecting group for phos-phate group;
R2 is a chemical protecting group for hydroxyl group;
B' is a protected base selected from guanine, adenine, cytosine, uracil and thymine;
A is a lower alkylamine; and _ is a natural number.
Another method for preparing the freeze-dried preparation of the protected nucleotide compound according to the present invention comprises dis-solving an oily form of a protected nucleotide represented by the formula [I] shown below in dioxane and subjecting the solution to freeze-drying.

lZ~ 35 ~ ~-R O ~
R2 t ~ - P ~ OH-~ [I]

wherein: R is hydrogen or a protected hydroxyl group;
Rl is a chemical protecting group for phos-phate group;
R2 is a chemical protecting group for hydroxyl group;
L0 Bi is a protected base selected from guanine, adenine, cytosine, uracil and thymine;
A is a lower alkylamine; and n is a natural number.
In the above formula [I], when n is 2 or greater, B', R and Rl in plural numbers may be either identical or different.
The freeze-dried preparation according to the present invention does not exist as a powder of dense particles or mass but in the form typically seen in freeze-dried products, namely, light and 1uffy ~owder with good solubility. This pre-paration can be stored or transported in this state by exercising only ordinary care to remove humidity and to avoid contamination. The preparation can also readily dissolved in solvents conventionally used in the triester method such as pyridine.

~2~735 In the phosphotriester method of the prior art, the nucleotide reagent (the formula [I]) has been made anhydrous by azeotropic distillation with pyri-dine immediately before use for condensation reaction.
This operation in actual practice is unexpectedly complicated, and it has been practically difficult to perform concurrently syntheses of a number of oligo-nucleotides, as mentioned above. With the use of the - reagent freeze-dried according to the present invention, azeotropic distillation operation which has been conducted in parallel with the condensation reaction is no longer necessary, whereby it becomes possible to carry out a number of condensation reactions at the same time. Low yield due to erroneous opera-tions in azeotropic distillation was also found to beavoided to give a stable yield. The yield per one operation was also found to be equal to or higher than that in the case of the azeotropic dlstillation operation. As a result of affording such stable high yield and simplification of the operations, it has also become possible to reduce the reaction scale to a great extent. As another advantage, mechanical operation of the step of the condensation reaction, which has been considered to be difficult by mechanical operation, also becomes possible.
As a product similar to the preparation of the present invention, there is the preparation disclosed in Japanese Laid-Open Pa-tent Publication No.150700/
1982. However, the freeze-dried preparatlon describ-ed in this Publication is that of the nucleotide compound to be used ~or the phosphite method [M. D. Matteucci, M. H. Caruthers : J. Am.
Chem. Soc., 103, 3185 (1981)]. The freeze-dried preparation of the present invention is of course different as a compound from that of this prior in-vention. Furthermore, it can be stated that it could not be anticipated from this prior art that a freeze-dried specimen can be prepared by the present invention in the form of a l:l salt of a nucleotide and a lower alkylamine and also that it has become possible, due to the fact that the salt is one with a lower alkyl-amine, that is, that a lower alkylamine has a boiling point of about 80C or lower under atmospheric pressure, whereby excessive amount thereof can be easily removed in the pyridine azeotropi~ distillation step or in the freeze-drying step to prepare a l:l salt.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings:
FIG. l and FIG. 4 show chromatographs on Sephadex G-50 column;
FIGS. 2 and 5 show HPLC patterns of oligonucleo-tides having dimethoxytrityl group; and FIGS, 3 and 6 show HPLC patterns of the desired ~2~73S

oligonucleotides.
Column~ Bondapak C 18 (Waters) - Eluant: acetonitrile -0.02M EDAA buffer (pH 7.8) : Concentration gradient: as shown in the dxawings Flow rate: 2 ml/min.
Chart speed: 10 mm/minO
Temperature: 50C
DETAILED DESCRIPTION OF THE INVENTION
Nucleotide compound [I]
The nucleotide compound as the freeze-dried pre-paration according to the present invention is shown by the foregoing formula [I~.
In the above formula, the symbol ~ is conven-tionally used for showing a residue of the rebose portion of a nucleotide from which 2'-, 3'- and 5'-hydroxyl groups and the base are removed. More speci-fically, it has the followiny structure:

~

When R is hydrogen in the formula [I], this ribose unit is 2'-deoxyribosyl, and the compound of the formula [I] forms a DNA chain. When R is a hydroxyl group or a protected hydroxyl group, this ribose unit is ribosyl, and the compound of the .:

lZl 4735 formula [I] forms a RNA chain. When the degree of polymerization n is 2 or higher, the two or more of R may be the same or different, and therefore the compound of the formula [I] can be a hybrid of DNA
chain (s) and RNA chain (s).
B' is a protected base. This generally includes, as 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, while it is A, G, C or U when the eompound forms a RNA chain. The protecting group is usually an acyl group, and illustrative of the protected base are N6-benzoyladenine, N-isobutyryl-guanine, N6-benzoylcytosine, thymine and uracil. As clearly seen from thymine and uracil themselves as examples of s', thymine and uracil generally require no protection. They themselves stand protected with-out any foreign proteeting group. Thus, "the pro-tected thymine" and "the protected uracil" are inelusive also of those having no specific protecting group.
In this context, the bases in the formula [I], whieh are as specified above, may of course be modified with groups other than acyl groups falling within the "protecting groups" as deseribed above, and the present invention is clearly valid for DNA or RNA in which changes generally seen in this kind of bases, such as 73~

methylation or partial conversion of amino groups into carbonyl groups when B is cytosine or adenine have occurred. Accordingly, the compounds in which the bases are thus changed also belong to the cope of the present invention.
The group Rl, the group R2 and the group R
when showing a protected hydroxyl group may be those known as the protecting groups for phosphate group and hydroxyl group in the art of nucleic acid syn-thesis, specific examples together with those of the protecting groups for the above bases being dis-closed in "Journal of the Society of Organic Syn-thetic Chemistry, Japan", Vol. 36, No. 9, pages 723-731, (1978), "Synthesis of Nucleosides and Nucleo-tides" (Maruzen, Japan 1977), "Organic Chemistry of Nucleic Acids" (Kagaku Dojin, Japan 1979), "Nucleic Acids" (Asakura Shoten, Japan 1979), Tetrahedron, 34, 3143 (1978), Journal of the Society of Synthetic Organic Chemistry, Japan 34, 723 (1978), "Kagaku no Ryoiki" (Domain of Chemistry), 33, 566 (1979), and other review publications and textbooks. The protective groups particularly preferred in the present in~Jention are o-chlorophenyl group and p-chlorophenyl group as the group Rl, mono-methoxy-trityl group and di-m~thoxytrityl group as the group R2, and hydrogen and hydroxyl group protected with o-nitrophenyl as the group R.

:
_ is any desired natural number. Generally, is about l -to 10, preferably about l to 6, parti~
eularly about l to 3.
A is a lower alkylamine. The term "lower alkyl"
means an alkyl having about l to 4 carbon atoms.
Typieal examples of lower alkylamines are triethyl-amine, diisopropylamine, dimethylamine, t-butylamines, sec-butylamines, n-butylamines, n-propylamines, and isopropylamines, trlethylamine being preferable.
Production of the compound [I] can be carried out also aceording to any suitable method for syn-thesis o:E nucleic acids described in the above references or others. A typical example is as - described below. First, the compound of the formula [II] is synthesized aceording to the above references and is also commereially available.

~ ~R O ~

t ~ ORl [II]

wherein: R3 is a ehemical proteeting group for phos-phate group whieh ean be depro-teeted under the eonditions where other proteeting groups are all stable, usual-ly a eyanoethyl group; and R, Rl, B' and n have the same respeetive 7~i meanings as defined ahove.
The compound of the formula [II] is treated with a lower alkylamine, preferably triethylamine in pyridine or in pyridine-water to remove the protecting group R3 to form compound [I~. The compound [I] thus formed is obtained in an oily form by distilling off the excessive lower alkyla-mine, pyridine and water from the reaction product.
This oily compound ~I] is stored and used for the DNA synthesis as it is, or as a powder form pre-pared by dropwise addition of the oily form into hexane. The oily form of the compound ~I~ may be considered to be oily, because components other than the compound [I], particulariy pyri-dine, are contained.
Freeze-drying Preparation of Solution The essential solvent is dioxane. However, the compound [I], when it is a powder form, is dis-solved in dioxane at a slow rate, and therefore itis preferably disolved in a mixture of pyridine and dioxane. In this case, if the amount of pyridine is excessive, freeze-drying of the solution cannot be effectively carried out. Accordingly, it i lZ~4735 desirable to use pyridine in a volume of 2 to 10 v/v % oE dioxane. In prepariny a solution of the compound ~I], pyridine may be previously mixed with dioxane before dissolving the compound [I] therein, or alternatively the compound [I]
may first be dissolved in pyridine and the re-sultant solution further dissolved iII dioxane.
The latter procedure can en~oy better the ad-vantage thanks to use of tne pyridine. Thus, the expression l'dissolved in dioxane together - with pyridine" used in the present invention is inclusive of both of the procedures described above.
In the case where the compound [I] is in a oily form, an amply high dissol~ing rate can be obtained without using pyEidine in com~ination~
In either of the cases described above, the essential solvent is dioxane, but a small amount o another solvent or a lower alkylamine can be used in combination therewith without departing from the spirit of the invention, and the scope of claims should also be understood in this context.
Accordingly, the use of a small amount of pyri-dine in the case when the compound [I] is in a oily form is within the scope of the present invention.
The concen-tration of the compound [I] in the solution may be appropriately determined in view of 73~

its viscosity or convenience in the freeze-drying step. More specifically, it is ordinarily of the order of, for example, 10 to 100 mg/ml.
Freeze-drying Step Except for the point that the solute is the compound [I] and the solvent is dioxane (and a small amount of pyridine), the freeze drying operation is not different from the conventional one.
- Generally speaking, this step would comprise freezing by cooling the solution (ordinarily of the order of -30C or lower) and sublimation of the frozen solvent by application of a reduced pressure (ordi-narily of the order of several mmHg or lower).
With regard to other necessary information about freeze drying, reference may be made to text-books such as Shin Jikken Kagaku Koza "New Course of Experimental Chemistry) 1 [I] 459" (~laruzen, Japan 1975)-Utilization of FrPeze-Dried Preparation The freeze-dried preparation of the compound ~I] according to the present invention not oly has good solubility in pyridine but is also equal to or better than the preparations of -the prior art (dried by azeotropic dïstillation immediately before use) in reactivity at the condensation step.
Accordingly, the freeze-dried preparation, as ~16-~2~35 long as it is stored under moistureproof conditions, can be used advantageously as the nucleotide unit for the DNA synthesis withou~ azeotropic drying.
The solid phase method for synthesis of an oligonucleotide by the use of a freeze-dried nucleo-tide compound is speci~ically described by referring to an example as set forth below, by which the present invention is not limited.
About 50 to 60 mg of a polystyrene resin having a nucleoside bonded thereto is placed in a reactor equipped with a stop cock and glass ~ilter and washed thoroughly with an isopropanol-methylene chloride (15 : 85, y/v) solution.
The resin is then treated with 1 molar zinc bromide in isopropanol-methylene chloride (15 : 85, v/v solution for 5 minutes 4 times. After detrity-lation with ~inc bromide solution, the resin ;s washed with an isopropanol-methylene chloride (15 : 85, v/v) solution, then with pyridine, and the resin is dried by means of a vacuum pump.
Th2 detritylation method may be replaced by a method in WhiCh benzenesulfonic acid or trichloro-acetic acia is employed.
Alternatively, drying o~ ~he resin may ~e carried out by washing with a volatile organic sol-vent and subsequent drying with a dry gas.
On the other hand, the nucleotide reagent to be '73~i used for condensation is prepared by adding mesity-lenesulEonyl nitrotriazolide (hereinafter abbreviated as MSNT) (about 30 mg) to the freeze-dried prepara-tion of the nucleotide block (about 30 mg) of the present invention and dissolving the mixture in anhydrous pyridine (about 400 ~1) under stirring.
This reagent is added quickly to the dried resin, and the reaction is carried out for 60 minutes. After the reaction, the resin is washed with pyridine, and the unreacted 5'-hydroxyl group is protected through acetylation. As the acetylating agent, 0.5 ml of an acetic anhydride-pyridine ~1 :5) solution and 0.5 ml of dimethylaminopyridine (herein-after abbreviated as DMAP)-pyridine solution are added in combination, and the reaction is carried out for 5 minutes. After washing with pyridine, the following cycle is initiated. This operation (S-teps 1 - 9 in Table 1) is repeated to extend successively the chain length.

~Z~3S

Table 1 Reaction Operations Ouantity Time Number of Step Reagent or Solvent of solv- (min ) repeti-ent (ml) . tion _ _ 1 iso-PrOH-CH2C12 1 2 5 : 2 1.0 mol ZnBr2/ 1 5 4 iso-PrOH-CH2C12 : 3 iso-PrOH-CH2C12 1 1 3 4 Dry pyridine 1 1 5 Vacuum drying _ ca.l0 6 Nucleotide +MSNT/ 0.5 60 dry pyridine 7 Pyridine 1 1 3 8 Ac2O-pyridine(1:5) (0.5 5 1 DMAP-pyridine O.5 (200 mg/10 ml) 9 Pyridine 1 1 3 *) based on 50 to 60 mg of resin .

_xperimental Examples Example l _ About 30 mg of a compound [II] represented by the formula DMTr -O ~ -O - P - O-CE
ORl wherein: DMTr is a dimethoxytrityl group;
Rl is an o-chlorophenyl group;
T is thymine; and CE is a cyanoethyl group, was treated with l ml of a pyridine-triethylamine-water (3:1:1, v/v) solution at room temperature for 15 minutes to remove the cyanoethyl group. The solvent was evaporated, and excessive triethylamine and water were completely evaporated by pyridine azeotropic distillation. The resultant oily product was dissolved in 600 ~1 (microliters) of dioxane and freeze-dried to obtain the freeze-dried preparation of the triethylamine salt of the desired compound [I]-Example 2 About 30 mg of the compound [II] was treated with 1 ml of diethylamine-pyridine (1:9, v/v) at room temperature for 30 minutes. The solvent was removed, and excessive diethylamine was evaporated by pyridine ~2~4~

azeotropic distillation. The oily product obtained was d~ssolved in 600 ~1 of dioxane, and, following the procedure described in Example 1, the freeze-dried preparation of the diethylamine salt of the compound [I] was obtainedO
Similar results were also obtained when substi-tuting n-butylamine or diisopropylamine for diethyl-amine.
Example 3 1~ Production of freeze-dried preparations Dimethoxytrityl-di-nucleotides of the formula ` Bl B2 l 1l DMTr - O ~ -O - P - O ~ -O - P - OH-Et3N , \ ORl OR

wherein: DMTr is a dimethoxytrityl group;
Rl is an o-chlorophenyl group; and Bi and B2 are each a protected base selected from N-benzoyladenine (A), N-benzoylcytosine (C), N-isobutyryl-guanine (G) and thymine (T), were processed into freeze-dried preparations as des-cribed below.
In the following examples, these dinucleotides are called as AC or TG by referring to its bases.
35 mg of each of (1) [CA], (2) [GG], (3) [CA], ~L,fC~ L~L73~

(4) [TA], (5) [TA], (6) [AA], and (7) [TC] was dis-solved in 50 ~1 of anhydrous pyridine, respectively, and frozen with addition of 800 ~1 of anhydrous dioxane at -50C, and drying was carried out by sublimation of the solvent under a reduced pressure of 3 mmHg.
The freeze-dried dinucleotides (1) through (7) were stored in a dessicator.

2) Production of an oligonucleotide Dimethoxytrityladenosine resin (60 mg, 6.6 ~mol) was treated in a reactor according to the operations in Table 1 as shown above. That is to say, isopropanol-methylene chloride washing, detritylation with 1 molar zinc bromide, isopropanol-methylene chloride washing, pyridine washing and drying of the resin by means of a vacuum pump were conducted. To this dried resin was added a pyridine solution (400 ~1) of the dinucleotide (1) [CA]
and MSNT (30 mg), and reaction was carried out for 60 minutes. After the reaction, pyridine washing, acetylation with acetic anhydride-pyridine-dimethyl-aminopyridine (1: 9 :catalytic amount) and pyridinewashing were conducted to complete one cycle of con-densation. This procedure was repeated for successive condensation of the dinucleotides (2) [GG], (3) [CA], (4) [TA], (5) [TA], (6) [AA], and (7) [TC], thereby to synthesize a pentadecanucleotide d (TCAATATACAGGCAA).
The average yield was 95%, and the overall yield 70%.
This is a yield which is ~igher than that ~LZ~73~

of the prior art method according to pyridine azeo-tropic distillation.
The synthesized oligonucleotide was deprotected in a conventional manner and purified. More specifi-cally, about 10 mg of the resin was treated at roomtemperature overnight with 100 ~1 of a solution of 0.5 M tetramethylguanidine-pyridine-2-aldoxime in dioxane-water (9 : 1, v/v) and thexeafter treated at 50~C over-night with addition of 2.5 ml of conc. ammonia water.
The resin was filtered off, the filtrate was concentrat-ed and subjected to rough purification on Sephadex G-50 (1.5 x 120 cm~ with an eluent of 0.05 M TEAB
- (triethylammonium bicarbonate) buffer (pH 7.5) (FIG. 1).
The peak fraction was collected, and the trityl derivative was purified by high performance li~uid chromatography (HPLC) through a reversed phase column (FIG. 2). After detrityla~ion with 80~ acetic acid, purification was conducted again through a reversed phase column to obtain the desired pentadecanucleotide of high purity in a high yield (FIG. 3).
Example 4 1) Production of freeze-dried preparations 35 mg of each of the dinucleotide reagents (1) [CA], (2) [TG], (3) [CA], (4) [TA], (5) [TA], (6) [AA], and (7) [TC] was dissolved in anhydrous pyridine (50 ~1) and freeze-dried with addition of anhydrous dioxane (800 ~1).

, . . .

2) Production of an oligonucleotlde Dimethoxytrityladenosine resin (60 mg, 6.6 mmol) was placed in a reactor and condensations were suc-cessively conducted in the order of the nucleotide reagents (1) - (2) - (3) ~ (4) - (5) - (6) - (7) to synthesi~e a pentadecanucleoside d (TCAATATACATGCAA).
The average yield was 95%, and the overall yield 70%. AS compared with the method of the prior art, the yield was less deviantly scattered among the cycles and was hi~h.
The desired pentadecanucleotide was purified similarly as in the preceding Examples, whereupon a product of high purity was obtained in a high yield (FIG. 4 to FIG. 6).

Claims (13)

WHAT IS CLAIMED IS:

1. A freeze-dried preparation of a nucleotide compound represented by the formula [I] , wherein: R is hydrogen or a protected hydroxyl group;
R1 is a chemical protecting group for phos-phate group;
R2 is a chemical protecting group for hydroxyl group;
B' is a protected base selected from guanine, adenine, cytosine, uracil and thymine;
A is a lower alkylamine; and n is a natural number, wherein, when n is greater than 1, B', R and R1 in plural numbers may be either identical or dif-ferent.

2. A freeze-dried preparation of the nucleotide compound according to Claim 1, wherein n is a natural number of 1 to 10.

3. A freeze-dried preparation of the nucleotide compound according to Claim 1, wherein the lower alkylamine is one in which each alkyl moiety has 1 to 4 carbon atoms.

4. A freeze-dried preparation of the nucleotide compound according to Claim 3, wherein the lower alkyl-amine is triethylamine.

5. A freeze-dried preparation of the nucleotide compound according to Claim 1, 2 or 3 wherein R is hydrogen.

6. A freeze-dried preparation of the nucleotide compound according to Claim 1, 2 or 3 wherein R is pro-tected hydroxyl group.

7. A freeze-dried preparation of the nucleotide compound according to Claim 1, 2 or 3, wherein B' is selected from the group consisting of acylated guanine, acylated adenine, acylated cytosine, thymine and uracil.

8. A freeze-dried preparation of the nucleotide compound according to Claim 1, 2 or 3, wherein B' is selected from the group consisting of benzoyladenine, isobutyrylguanine, benzoyl cytosine, thymine and uracil.

9. A method for producing a freeze-dried prepa-ration of a protected nucleotide compound, which comprises dissolving a powder form of a protected nucleotide compound represented by the formula [I]
shown below together with pyridine in dioxane and subjecting the solution to freeze drying:

[I] , wherein: R is hydrogen or a protected hydroxyl group;
R1 is a chemical protecting group for phos-phate group;
R2 is a chemical protecting group for hydroxyl group;
B' is a protected base selected from guanine, adenine, cytosine, uracil and thymine;
A is a lower alkylamine; and n is a natural number, wherein, when n is greater than 1, B', R and R1 in plural numbers may be either identical or dif-ferent.

10. A method according to Claim 9, wherein said powder form is dissolved in a mixture of pyridine and dioxane.

11. A method according to Claim 9 , wherein said powder from is dissolved in pyridine and then the resultant solution is dissolved in dioxane.

12. A method for producing a freeze-dried prepa-ration of a protected nucleotide compound according to the present invention which comprises dissolving an oily form of a protected nucleotide compound represented by the formula [I] shown below in dioxane and subjecting the solution to freeze drying:

[I] , wherein: R is hydrogen or a protected hydroxyl group;
R1 is a chemical protecting group for phos-phate group;
R2 is a chemical protecting group for hydroxyl group;
B' is a protected base selected from guanine, adenine, cytosine, uracil and thymine;
A is a lower alkylamine; and n is a natural number, wherein, when n is greater than 1, B', R and R1 in plural numbers may be either identical or dif-ferent.

13. A method according to Claim 12, wherein the oily form of the protected nucleotide compound represented by the formula [I] comprises an oily product which is formed by causing a protected nucleotide compound represented by the following formula [II] to react with a lower alkylamine to remove the protecting group R3 of the terminal 3'-phosphate group [II] , wherein R3 is a chemical protecting group for the phosphate group and is deprotect-able under the conditions where other groups are all stable, and R, R1, R2, B' and n have the same meanings as defined in Claim 12.