JPS6133195A - Oligonucleotide derivative and preparation thereof - Google Patents

Abstract

NEW MATERIAL:An oligonucleotide derivative expressed by formula I (m and n respectively represents 0 or a natural number; R<1> represents divalent hydrocarbon residue; B represents base constituting the nucleotide). USE:An oligonucleotide used as a non-radioactive hybridization probe or for immobilization in genetic engineering, transformable to a natural type oligonucleotide, recoverable after use, and easily synthesized. PREPARATION:Protecting groups R<2> and COR<4>, and protecting groups of both basic parts and phosphoric acid groups in a compound expressed by formula II(R<0> is a protecting group of phosphoric acid part; R<2> is a protecting group of amino group; COR<4> is a protecting group of 3'-terminal hydroxyl group in nuclotide; B' is a base constituting the nucleotide which may be protected if necessary) are eliminated by suitable methods.

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION TECHNICAL FIELD The present invention generally relates to novel oligonucleotide derivatives convertible to naturally occurring oligonucleotides. More specifically, the present invention relates to an oligonucleotide derivative in which a primary amino group is introduced onto the 5'-terminal phosphate amide group of a nucleotide via a spacer of appropriate length. The invention also relates to a method for producing such oligonucleotide derivatives. PRIOR ART In recent years, chemical synthesis of nucleic acids has progressed dramatically through the introduction of new protecting groups and the development of new condensation methods such as the triester method and the phosphite method. Additionally, with the rapid progress of genetic engineering, chemical synthesis of nucleic acids has come to have important significance in this field. For example, useful substances are being produced by synthesizing artificial genes and using genetic recombination (interface x
o7: Nature, 281.544
(1979), Leukocyte-derived interferon: Natu
re, 287.411 (1980)), and
Examples as probes for hybrid methods (Nucl
, Ac1dsRes, 9.879 (1981)
), or mltNA or - Complementary to the template DNA required when synthesizing double-stranded DNA from woody DNA using reverse transcriptase or DNA polymerase.
Example of use as a DNA fragment (primer) (Nucl
, Ac1ds Res, 8.4057 (1980
)), and other application examples. Among these, DNA probes in particular have been used not only as tools for identifying and detecting unknown genes in the field of molecular biology, but also for the analysis of genetic diseases, viral infections, food testing, etc. It is becoming increasingly important. By the way, affinity probes for non-radioactive nucleic acids are more difficult to handle, risk of exposure to radiation, price, and more expensive than radioactive probes.
Due to its advantages in waste disposal and preservation, interest in its use has increased over the past few years, and various methods of use have already been proposed. For example, an adenosine triphosphate (ATP) derivative to which 2,4-dinitrobenzene is attached is incorporated into the 31-terminus of DNA using terminal transferase to generate an antibody that recognizes the 2,4-dinitrophenyl group as an antigen. Using rabbit antiserum and peroxidase-conjugated sheep antiserum (which recognizes rabbit immunoglobulin type G as an antigen)2,4
-Method for detecting DNA labeled with dinitrophenyl group (Nucl, Ac1ds Res, 1997, 6787)
-6796, (1982) ``l,'r4 Using RNA ligase, the substrate derivative labeled with biotin etc.
A method for detecting a target RNA by incorporating it into the 31-terminus of NA [Nucl, Ac1ds R
es,, ■, 6167-6184 (1983)),
A method of detecting the target RNA by modifying cytosine in RNA q with ethylenediamine through a transamination reaction and introducing a marking substance [Nu
cl Ac1dsRes,, 12.989-1002
(1984)). However, all of the non-radioactive nucleic acid affinity probes used above have the following disadvantages. B. Probe preparation is complicated and troublesome. However, it is difficult to synthesize probes with arbitrary and defined base sequences. . C. Since the base portion is modified, the Tm value may change. Therefore, the present inventors proposed a new oligonucleotide derivative to overcome the disadvantages of the above-mentioned compounds (Japanese Patent Application Laid-Open No. 59-27900, Japanese Patent Application No. 58-22
516, specifications of Japanese Patent Application No. 58-75878, JP-A-59-93098, JP-A-59-93099, JP-A-59-93100, each publication 1 Japanese Patent Application No. 59-22474
(Japanese Patent Application No. 59-22475). However, not only the affinity probes for non-radioactive nucleic acids mentioned above but also the oligonucleotide probes previously developed by the present inventors, once used in combination with a labeling substance or a carrier, cannot be used for binding with a marker or a carrier. Currently, the scope of its application is limited in that it cannot be recovered as the original oligonucleotide (ie, natural oligonucleotide). SUMMARY OF THE INVENTION The present invention aims to provide a solution to the above-mentioned problems, and as a result of extensive research aimed at synthesizing oligonucleotide derivatives that can be converted into natural oligonucleotides, phosphoalkylamidates are converted into hydrochloric acid derivatives. We found that water decomposition is faster in acetic acid than in acetic acid, and debridement is slower in acetic acid.Based on this fact, we developed an oligonucleotide derivative that can be converted into a natural oligonucleotide and a method for producing it. The invention was completed. That is, the present invention achieves the above object by using an oligonucleotide derivative having a phosphoric acid amide group at the 51-terminus of a nucleotide and introducing a primary amino group into the extension thereof via a spacer of an appropriate length. It is what we are trying to achieve. Therefore, the oligonucleotide derivative according to the present invention is characterized by being represented by the following formula [V]. Furthermore, the method for producing the oligonucleotide derivative represented by the following formula (V) according to the present invention includes a protecting group R2,3- of the amino group on the 51-terminal extension of the compound represented by the following formula
It is characterized by removing all the terminal COR' groups, base moieties, and phosphoric acid moiety protecting groups. [However, m and n are each 0 or any natural number, Ro is a phosphate group protecting group, R1 is a divalent straight chain or branched hydrocarbon residue, and R2 is an amino group The COR4 group is a protecting group, and the COR4 group is a protecting group.
A protecting group for the terminal hydroxyl group, B1 is a base constituting the nucleotide and is protected as necessary,
B is a base constituting a nucleotide (Bl or B
and when there are multiple Ro's, they may be the same or different). [Effect] Since the 5I-aminoalkyl-oligodeoxyribonucleotide according to the present invention has a phosphoric acid amide bond in its structure, it can be easily converted into a natural oligonucleotide by being cleaved at this part under weakly acidic conditions. I can do it. Further, according to the present invention, the following effects can also be obtained. (1) Immobilized oligonucleotides for affinity chromatography and non-radioactive hybridization probes having any base sequence can be produced. (2) This oligotide derivative is extremely simple to synthesize and can be synthesized in large quantities. (3) This oligonucleotide derivative has a primary amino group that is more reactive than other functional groups (hydroxyl group, phosphate group, amino group in the base moiety, etc.) present in it, so depending on the settings of the reaction conditions, etc. Since it is possible to convert other compounds into gonucleotides that selectively bind to amine group moieties, the range of applications is expanded as immobilized oligonucleotides and non-radioactive polymerization probes as described above. That is, in the past, immobilized oligonucleotides could only be used as affinity columns and non-radioactive hybridization globes could only be used as probes, but using oligonucleotide derivatives with phosphate amide bonds like the compounds of the present invention If the above-mentioned probe etc. are not used, the compound can be converted into a natural oligonucleotide. They can be recovered as oligonucleotides and used for other purposes, such as linkers and primers (template DNA fragments necessary for synthesizing double-stranded I), or for genetic recombination. Furthermore, when used as these primers and linkers, the recovered oligonucleotides have the advantage that they do not require phosphorylation because they are natural, whereas conventional synthetic primers and linkers require phosphorylation. . Definition The oligonucleotide derivative convertible into a natural oligonucleotide according to the present invention is represented by the above formula [V] (hereinafter, the deoxyliponucleoside residues of this oligonucleotide excluding the 31- and 51-hydroxyl groups are referred to as The substituent B represents a base forming a nucleotide, usually adenine, thymine, cytosine or guanine.Compound [V ), when there are multiple B's, they may be the same or higher. The degree of polymerization of compound [V] is expressed as m+n because
In the preferred production method of the present invention, the degree of polymerization is m and n, respectively.
This is due to the condensation of the fractals (details will be described later). In that case, m is practically 0 to 6, particularly 1 to 4. Practically, n is θ˜40, particularly θ˜20. The group R1 is a divalent straight-chain or branched hydrocarbon residue that connects the 51-terminal phosphoric acid amide group of the nucleotide moiety of the compound [■] and the primary amino group moiety. This is preferably a linear or branched alkylene group having about 2 to 20 carbon atoms. Preferably R1 is an alkylene group having 2 to 6 carbon atoms. Since the compound [V] of the present invention has a phosphoric acid amide bond, the conversion of the compound [V] of the present invention into a natural oligonucleotide can be carried out using the usual acid treatment procedure for oligonucleotide synthesis [o, IN hydrochloric acid treatment fm (Tetrahedron 1etters
, 22.1463-1466 (1981), go
% acetic acid treatment (dimethoxytrityl group elimination conditions), etc.]
This can be easily done by However, since deoxyadenosine is unstable under such acidic conditions (cleavage of the glycosidic bond of adenosine (debrine Vvan) occurs), the various effects described above are not limited (for example, deoxyadenosine in the oligonucleotide sequence is (If adenosine is present, acid treatment will cause debridement, making it impossible to recover the oligonucleotide in its intact form.) To achieve this, conditions are required in which depurination is unlikely to occur and the phosphate amide bonds are rapidly thawed. The present inventors investigated various conditions and found that 80% acetic acid treatment was the most efficient. Therefore, the compound of the present invention can be efficiently converted into a natural oligonucleotide (depurination is unlikely to occur and the phosphoric acid amide bond is quickly thawed) by treatment with 80% acetic acid, and the compound is treated under these conditions. You can definitely get the above effects from Kototrap. here,
80elb acetic acid means that the acetic acid to be used for hydrolysis is an aqueous solution with a concentration of 80 parts. In addition, selective hydrolysis of the compound [V] of the present invention is generally observed when an acetic acid aqueous solution having a concentration of 30 to 90% is used. Synthesis of Compound [V] General Description Compound [V], the oligonucleotide derivative according to the present invention, can be synthesized by any convenient method. One preferred method is to introduce a protected primary amino group via the group R1 into the 5' to terminal phosphate amide group of the oligonucleotide derivative of the formula [IV], that is, the oligodeoxynucleotide, and to form a ring group of the nucleotide. and the phosphoric acid group are protected, bonded to the 31-terminus, and the hydrogen atom of the hydroxyl group is replaced with a COR' group, without removing all the protective groups. On the other hand, the compound of formula (IV) (hereinafter referred to as compound [IV]) introduces a protected primary amine group on the 51-terminal phosphoric acid amide extension of an oligonucleotide in which other functional group moieties are attached. It can be synthesized using the following method. FIG. 1 is a flowchart illustrating one example of this preferred synthesis method. The symbols in the flowchart have the following meanings. A substituent that protects the ROynic acid group, usually an or)chlorophenyl group. R1 is a divalent straight or branched hydrogen port group. R2 is a protecting group for the amino group, and a dimethoxytrityl group is usually used. B3 is a substituent from which all other protecting groups can be easily removed under stable conditions to give a phosphorus-free diester, and a cyanoethyl group is usually used. ■R4 3 used in normal oligonucleotide synthesis method
It is a 1-hydroxyl retention group. Specifically, R4 is a lower argyl group, an aryl group (particularly a phenyl group or a methoxy-substituted phenyl group), or a support having an appropriate spacer used in solid phase synthesis (polystyrene resin,
Some are made of polyamide resin). R551-Terminal hydroxyl group, which is usually a methoxytrityl group. m O or any natural number. n o or any natural number. B indicates a base. B1 indicates an optionally protected base, but is usually selected from R6-benzoyladenine, N-isobutyrylguanine, -1pensoylcytosine and yohithymine (ie, no protection required). Synthesis of Compound -] The compound represented by formula (IV) can be obtained by bonding the 5'-hydroxyl group of an oligonucleotide whose other functional group is protected and one amino group of diaminoalkylene via phosphoric acid. can be synthesized by any convenient method. The method for synthesizing compound [IV] in one embodiment (FIG. 1) is as follows. In Figure 1, 5
′-hydroxyl compound

A diaminoalkylene compound [I] in which [0] is oxidized by the action of a phosphorylating agent (for example, phosphoditriazolide, phosphodichloride, or phosphobenzotria 7zolide), and then either one of the amino groups is protected. (This compound can be obtained by protecting either one of the amino groups of diaminoalkylene (NH2-R'-NH2) with R2) to obtain compound [II]. In addition, the compound

[0] is an oligonucleotide and can be produced by a normal oligonucleotide synthesis method. Synthesis methods include the triester method, the phosphite method, and their respective solid phase and liquid phase methods. On the other hand, a conventional oligonucleotide synthesis method, preferably the solid phase synthesis method of the present inventors (TetrahθdronLet
tθrθ1979.3635 (1979), Nucle
ic AcjdsResearch 8.5473(
1980), Nucleic Ac1ds Re
ee-arch 8.5491 (1980), Nucl
eic Ac1ds meeearch8.5507(
1980), Nucleic Ac1ds Resea
Compound [IV] can be obtained by condensing the previously synthesized compound [II] with a condensing agent. As the condensing agent, menthylenesulfonyltetrazolide and mesitylenesulfonylnitrotriazolide are preferred. For details such as reaction conditions, please refer to the experimental examples described later. Compound [V] can be obtained by removing all the protecting groups from the above compound [1]. The protecting group COR' group, the orthochlorophenyl group in the phosphoric triester, and the afur group in the base moiety were protected by dioxane-water (9:1 (v/v) of 0.5M tetramethylguanidine-pyridine-2-carbaladoxime. )) After treatment with a solution, it is removed by performing an alkali treatment (concentrated ammonia water). When R2 is a trifluoroacetyl group,
Although it is sufficiently eliminated by treatment with ammonia, if it is an orthonitrophenylsulfenyl group, treatment with mercaptoethanol is necessary. When another protecting group is used as R2, it is also possible to add another treatment under conditions where the oligonucleotide moiety is stable. Various methods for synthesizing deoxyoligoliponnucleotides are already known, and details other than those mentioned above regarding the protection group 8%4, their introduction or removal, condensation, and other matters are detailed in the bottom book on the chemical synthesis of nucleic acids. For example, in the review article “Nucleoside
``Synthesis of Nucleotides'' (Maruzen 1977), ``Nucleic Acid Framework Chemistry'' (Kagaku Doujin 1979), ``Nucleic Acids'' (Breakfast Shoten 1)
979), Tetrahedron, 34.31
0978), conjugation, 34.723 self---■----
---■■■■■■-1-ρ (1978) and Chemistry Region, 2, 566 (1979). Application of the compound [V] of the present invention The compound of the present invention can be used to create non-radioactive affinity probes and immobilized oligonucleotides by binding a labeling substance or carrier via the primary amino group introduced on the 51-terminal extension. can do. It goes without saying that such compounds can be converted into natural oligonucleotides and have the various effects described above. The general formula of such a compound is as follows. [However, [Q] is a labeling substance or a carrier, and the definitions of other groups are the same as those for compound [V]] Synthesis of compound [VI) (when [÷] is a labeling substance) Compound [ The label can be obtained by any purposeful method capable of bonding a labeling substance to the primary amino group on the 5'-terminal phosphoric acid amide group extension of the compound [V]. Examples of labeling substances include biotin, 2,4-dinitohairsen, 'Ml photon' fH (rhodamine, fluorescein, etc.), enzymes (horseradish peroxidase, alkaline phosphatase, β-galactosidase, etc.), and metalloproteins (). , Eritin, etc.). In one embodiment, binding of biotin may be carried out according to the method described in Japanese Patent Application No. 58-22516, which was previously proposed by the present inventors. That is, the bonding between the two can be carried out by any method that can realize the formation of an amide bond by dehydration between the carboxyl group of biotin and the amino group of compound [V]. When compound [V] contains an amino group or a hydroxyl group capable of reacting with the carboxyl group of biotin, this reaction can be carried out with these groups suitably protected. on the other hand,
Biotin may also be in its functional conductor form. Specific examples of functional conductors of biotin are its biotin halides or its active esters. One preferred method for bonding an amino group and biotin in this sense is by reaction between an amino group and a biotin active ester. In general, biotin active esters are preferred because they do not react with the amino group of the base portion of the oligonucleotide.
This is because it selectively reacts only with the primary amino group on the terminal extension of the phosphoric acid amide group, and the reaction operation is simple. "Biotin active ester" means a biotin derivative with an ester bond that easily reacts with other functional groups (usually amine groups), specifically succinimide, varanitrophenyl, pencitriazolide-12,4° Examples include 5-trichlorophenyl ester. The first two are preferred. Another preferred method for binding an amino group to biotin is to perform the binding of both in the presence of a condensing agent. Examples of suitable condensing agents include dicyclohex7carbodiimide, carbonylimidazole, and Woodward's reagent 1 to 6. Synchhexylcarbodiimide is preferred. In either method, the reaction method may be any suitable method. Specific reaction methods for a given reaction system can be found in the aforementioned patent application specifications, later published experimental examples, and various base materials, such as "Peptide Synthesis" (Maruzen 1975).
) and 'Kunno (Liquid Chemistry IVJ (1977))
You can determine it appropriately by referring to . On the other hand, when the substance to be bound is 2,4-dinitrobenzene, the q″r application previously proposed by the present inventors
Please refer to the specification ψ of No. 878. That is, the bond between the two is the 1-position of 2,4-dinitrobenzene and the compound e.
It can be carried out by any method capable of realizing the formation of an O-N bond between +[V] and the amino group. The connection between the two is generally one conductor and two conductors, i.e. DNP.
-X (DNP is 29.4-dinitrophenyl group,
is usually due to deH-X condensation between the 1-substituent) and the amino group. -X is preferably /.rogen. Preferred derivatives in which This is because it selectively reacts only with primary amino groups, and the reaction operation is simple. In particular, 1-fluoroTh-2,4-dinitrobenzene is commercially available and easily obtained, and the reaction with the amino group of compound [VI] proceeds under mild reaction conditions. l-halogen-2,s-dinitrobenzene compound [■
] in a homogeneous solution (the solvent is, for example, a hydrous alcohol) or in a heterogeneous solution (the solvent is, for example, water), with a hydrogen scavenger (for example, sodium bicarbonate, triethylamine, hydroxide). potassium, etc.)
It can be carried out at a temperature of about 10 to 50°C in the presence of. The desired product may be recovered, for example, by extraction. Regarding NP conversion, in addition to the above patent application specifications, you may refer to appropriate reviews such as "Experimental Chemistry Course ■, Chemistry of Proteins ■," p. 118 J (1976 (published by Maruzen Co., Ltd.)). Even when the substance to be bound is a carrier, the target product can be obtained by binding to the primary amino group on the 5'-terminal phosphoric acid amide group extension of compound [V] as in the case of the labeling substance described above. Possible carriers include natural insoluble carriers such as agarose, dextran, cellulose, and glass particles, and synthetic polymers such as polyacrylamide. Preferred. In the case of agarose, several types of Sepharose derivatives (Pharmacia) are commercially available as its derivatives. For example, if the carrier is a Sepharose derivative, the carboxyl group in the derivative and the amino acid of the compound [■] It can be carried out by any method that can realize an amide bond by dehydration between groups.Compound [
When there is an amino group or hydroxyl group in V] that can react with the carboxyl group of the Sepharose derivative, this reaction can be carried out with these groups appropriately preserved. On the other hand, the carrier may also be in the form of a functional derivative thereof. When the carrier is Sepharose, specific examples of functional derivatives of Sepharose include cyanogen bromide-activated suprapharose (@), activated OH Sepharose (■), and epoxidized Sepharose (■). Other derivatives of Sepharose include OH-Sepharose (■) and AH-Sepharose (■) (in this case, a condensing agent such as dicyclohexylcarbodiimide (DOO
) )is necessary). Activated C
α-Sepharose is most preferred (see below for details of these). The condensation reaction is arbitrary for any purpose, and the specific reaction method for a given reaction system can be found in the specification of Japanese Patent Application No. 59-27900, which was previously proposed by the present inventors, as well as in publications and literature. (Affinity chromatography, Elsevier
8cientific Pub, Co, Amst
erdam (1g7B), Agric, Biol,
Ohem, 37.465 (1973), ibl 1
1191 (1973), iMd, 80.409 (
1976), Proteins, Nucleic Acids, and Enzymes (separate volume) No. 22
(1980) ) and the experimental examples below. ■ Sepharose-〇-0H2-OH-OH2-0-O
H (OH2) 4-O-OH2-OH-OH. ■ Sepharose-kJH (C1L2), -C0O
H ■ Sepharose-Nu (0H2) lI-Nli. The hydrolysis of phosphoalkylamidates under acidic conditions and the stability of deoxyadenosine under the same conditions were investigated. To examine the water decomposition of phosphoalkylamidate, 3
1-Leproylnucleoside compound [J', Am,
Ohem, 5oc0.97.1614, (1975
) A compound in which a phosphoalkylamidate is introduced into ),
Compound (2) was used, and in order to examine the stability of deoxyadenosine, compound (2) was obtained by synthesizing an oligonucleotide using a conventional method and then deprotecting it. In addition, as acidic conditions, 0.01 N@acid(1]H2
) [Deprotection conditions in oligonucleotide synthesis], 8
゜Thiacetic acid [conditions for removing dimethoxytrityl group in oligonucleotide synthesis] and 0. IN hydrochloric acid (pH1)
Each solution was used. (1) Synthesis of compound [■] Compound [■] (31-lebroyl nucleoside compound,
After making the structural formula (below) anhydrous by pyridine azeotropy, compound [@] (1.0 mmol, 340 mg) K
Orthochlorophenylphosphorobenzotriasolide solution (1.5 mmol, 9 ml) was added, and reaction (phosphorylation) was carried out for 1 hour. After confirming the progress of the reaction by thin layer chromatography, n-butylamine (3 mmO1,2
20 mg) and l-methyl-imidazole (3 mmo
1.240! 11g) was added and the reaction was carried out for 2 hours. After concentrating the reaction solution, it was dissolved in chloroform (40 ml), and then dissolved in water (2 times with 40 ml), O, aM IJ acid (2 times with 40 ml), and saturated sodium bicarbonate (2 times with 40 ml). After washing with 5% sodium chloride (2 times with 40 ml) and drying with anhydrous sodium sulfate. After concentrating the chloroform layer, silica gel short column (Silica gel 50Crn3, φ4
Compound (2) was obtained as an oil (yield f5y 480 mg s, recovery rate 81%). Compound (2) (structure is The following (approximately 20 mg) K Virigi 7 (0.5m+1) and concentrated ammonia (2.5m
After adding 1), sealing the container, and reacting at 50°C overnight,
This was concentrated, dissolved in 1 ml of 50n]M triethylammonium picarbonate buffer (pH 7,5), washed three times with ether (1 ml), and the aqueous layer was concentrated to obtain compound (2). . (2) Examination of ice-melting properties of phosphoalkylamidates under acidic conditions Compound (2) synthesized above was mixed with 80% thiacetic acid and 0.0% thiacetic acid. IN
After dissolving in hydrochloric acid and 0.01N hydrochloric acid solution, the ice-melting properties of the phosphoalkylamidate in each solution were examined. The water-dissolving properties of phosphoalkylamidates vary over time for compound (2) in each acid solution. An example of the HPLO elution pattern (in 80 thiacetic acid) is shown in FIG. In the figure, the numbers in [ ] indicate the elapsed time (hours) in 80% acetic acid. The change over time of compound ■] is the residual amount [related]
I calculated it. That is, the residual amount of compound (4) at each time was determined from the area ratio of compound (total) and its decomposition products in the elution pattern (chromatogram) when subjected to HPLO. They also calculated the half-life of the compound. ``The results at that time were as shown in Table 1. Table 1 - From the above results, the decomposition rate of phosphoric acid amide bond (phosphoalkylamidate) in acid solution was the highest among 80% acetic acid. It can be seen that it is fast and slowest in 0.01N hydrochloric acid. (3) Oligonucleotide stability under acidic conditions (
Under the same acidic conditions as in (2) above, oligonucleotides were synthesized using the following compound l1ol (obtained by synthesizing the oligonucleotide using a conventional synthesis method and then deprotecting it). We investigated the stability of The stability of the oligonucleotide is determined by the stability of the compound (2) in each acid solution as in (2) above.
) was analyzed by HPLO analysis. The change over time of Compound (2) was also investigated by calculating the residual amount [H] and half-life from the chromatogram results in the same manner as for Compound 0]. The results obtained are as shown in Table 2. From this result, depurination was slowest in 80% acetic acid, and 0.5% in acetic acid. It was found that it was the fastest among IN hydrochloric acid. From the above experimental results, it is clear that phosphoalkylamidates are more rapidly decomposed in acetic acid than in hydrochloric acid (when hydrolyzed), and conversely, depurination is slower in acetic acid, i.e., phosphoric acid amidates do not undergo depurination in hydrochloric acid. It has been found that while it is difficult to hydrolyse the bond, the phosphoric acid amide bond can be hydrolyzed in acetic acid after treatment for 5 to 6 hours with almost no depuration.Therefore, the desired oligonucleotide If deoxyadenosine is present in the base sequence of A compound (Compound ■ in the same figure) was produced. In Figure 3, the symbols have the following meanings: B1 Thymidine B Thymidine 1 & iTr Dimethoxytrityl country 2-Ratham 22- Ro Orthochlorophenyl 5t Ethyl OR-Cyanoethyl [ About labeling substance or carrier ml n 唖 6 (2) Production experiment of compound (compound [V]) 1-1 Dimethoxytritylthymidine/resin [■] (The resin is only a carrier, but the target compound supported on the resin Since the appearance is essentially the same as the resin itself, the compound supported on the resin will be simply referred to as the resin below.)3
0mg (3μm01) of methidine chloride (hereinafter 0H2ON)
After washing 5 times in 1 ml of 3-trichloroacetic acid-containing methylene chloride (hereinafter referred to as 3-TOA 10H2012), it was reacted 6 times (detritylation) for 20 seconds each with 1 ml of 3-trichloroacetic acid-containing methylene chloride (hereinafter referred to as 3-TOA 10H2012) to obtain a resin [■]. . resin (to)] to 0H2(
Washed 5 times with J121 ml, and further washed with anhydrous pyridine tm.
After washing with x 5 times, the resin was dried. This is dinucleotide■] (thymidine dimer 25mg
20μm01) Oyohimesitylenesulfonyl nitrotriazolide (hereinafter referred to as M8NT) 30mg (100
1'm01) and 400 μm of anhydrous pyridine.
Reaction condensation) was carried out for 00 minutes. After the reaction, the reaction was washed five times with 1 ml of pyridine, and acetic anhydride-pyridine (l
:9, (v/v)) solution tmx was added and reacted for 5 minutes to acetylate and protect the unreacted 5-hydroxyl group, and this was washed with 1 ml of pyridine to prepare the nucleotide resin [@
(n-2) was obtained. The above operation was repeated three times to obtain nucleotide resin C@(n-6). On the other hand, Compound Garden was synthesized by reacting 51-hydroxynucleotide (phosphorylated compound [of]) with monotrifluoroacetyldiaminoalkylene (compound ■). That is, first, compound (1) was synthesized according to the following procedure. Hexamethylenediamine (2.6g, 22.4
After dissolving 50 ml of dimethylformamide (hereinafter referred to as DM'F), trifluoroacetic anhydride (4.2 g, 20 mmol) was added while stirring under water cooling.
) was added dropwise, and 30 minutes later, IN hydrochloric acid (50 ml) was added, and the mixture was concentrated to dryness under reduced pressure. Salt the residue IN (
After washing three times with ether (50 ml) (this operation removes the di-IJ fluoroacetyl compound), the aqueous layer was concentrated to dryness, and the aqueous layer was dissolved in isopropanol (100 ml).
ml) extraction was performed. The isopropanol layer obtained here was concentrated to dryness and then extracted with 100ml of acetone.
After distilling off the acetone, the precipitated crystals were washed with ether and dried to obtain the compound [Shun's hydrochloride (1.8 g, yield 36%)]. Next, 5'-hydroxy Thymidine compound [■] (1 mmoyu, 4 somg
) (synthesized by the usual nucleonate synthesis method) was made anhydrous by pyridine azeotropy, and then an ortho-chlorophenylphosphorodibenzotriazolide solution (1.4 mm n1
．． 8.4 ml) was added and the reaction was carried out for 1 hour (phosphorylation). To this was added hydrochloric acid of the above compound [[of]] which had been made anhydrous, and 1-methylimidazole (2.8 mmol, 22
0 mg) was added thereto, and the reaction was carried out for 2 hours. After the reaction was completed, the solvent was distilled off and the residue was dissolved in chloroform.
Water, 0.5M sodium dihydrogen phosphate aqueous solution, saturated hydrogen carbonate)
Each was washed with an aqueous solution of sodium chloride and dried over anhydrous sodium sulfate. After concentrating the chloroform layer, it was purified with a silica gel column (using chloroform containing 0 to 4 ml of methanol as the eluent), and the eluate was concentrated and dropped into pentane to obtain a powdery compound [C]. (330 mg, yield 38%). To the resin [(9) (n = 6) detritylated in the same manner as described above (6)], 30 mg of the compound (to)] was added (
38 μmob) to triethylamine-pyridine-water (1
:3+1,''/v) solution was added to the treated (decyanoethylated) compound [0, MSNT 30mg (
100 pmol) and 400 μl of anhydrous pyridine were added, and reaction condensation was performed for 500 minutes. After the reaction is complete,
After washing with pyridine and methanol and drying, a completely protected oligonucleotide derivative (g) was obtained. This was mixed with 0.5M tetramethylguanidine-pyridine-
2-aldoxime pyridine-water (9:1 (v/v)
)) After treating the solution overnight at room temperature with a solution of 300 μm, concentrated ammonia water (2.5 ml) was added, the mixture was tightly stoppered, and the mixture was allowed to stand at 50° C. overnight (deprotection). Next, the resin was separated and the aqueous layer was concentrated, followed by a 50111M) ethyl ammonium bicarbonate (hereinafter referred to as TFIAB) buffer (pFi7.
5) Dissolved in 2 ml and extracted with ether. After concentrating the aqueous layer, Sephadex G-50 (diameter 1.5×
The length was 1000 m, and the eluate was desalted and purified with a 0.05 M triethylammonium bicarbonate buffer (pH 7.5) to obtain an octatimidylic acid derivative (9). In addition, experiments 1-2.1-3 and 1-4 were performed in the same manner.
An oligonucleotide derivative of was obtained. Table 3 shows the base sequences of the compounds of Experimental Examples 1-1 to 1-4. Table 3 However, in the above table, A represents adenine, T represents thymine, G represents guanine, and C represents cytotone. The elution pattern results for experiments 1-2, ta and 1-4 when applied to a Sephadex G-50 column are shown in Figures 4.6 and 8, and the elution pattern results when HBLO was performed are shown in Figures 4.6 and 8. are respectively 5-(a) and 7-0)
and 8-pi) as shown in the figure. These results show that compound [V] was produced. In addition,
The numbers above the peaks in Figures 5.7 and 8 indicate the retention time (minutes). (1) Production experiment 2-1 of piotine oligonucleotide derivative Octatimidylic acid derivative synthesized in the above experiment 1-1]
Approximately 1. QOD was dissolved in 0.1M sodium bicarbonate aqueous solution (p
H8, 3) was dissolved in 10 μm, and a 10 μm solution of biotin succinimide ester in dimethylformamide (corresponding to several hundred times excess) was added, and the reaction was carried out at 4° C. overnight to synthesize biotin-octatimidylic acid. The reaction was confirmed by HPLO and 20% polyacrylamide toge/L-electrophoresis. Experiments 2-2 to 2-4 The same operations as in Experiment 2-1 were performed for the compounds synthesized in Experiments 1-2, 1-3 and 1-4, to produce compounds for each of them. When the compound [OD] obtained at that time was subjected to HPLC, the results were as shown in Figures 5-(b), 7-(b) and 9-(b). In the figure, the numerical value above the peak indicates the retention time. In Figures 5.7 and 9, (a) is a chemical compound.

It shows the elution pattern when [0] was subjected to HPI, O, and (b) shows the elution pattern when compound (to)] was subjected to HPI, O.
This shows the elution pattern when the sample is applied to the sample, and in each figure, it is noted that (b) and (b) are single peaks, and that (c) has a longer retention time than (c). From the fact revealed in the specification of Japanese Patent Application No. 58-22516 (--class amino group selectively binds to biotin), it can be seen that biotin oligonucleotide derivatives are certainly produced. Experiment 3- 1 (Conversion to natural oligonucleotide) Compound [ol (Experiment 1-1) and compound [■] (Experiment 2-1) synthesized above were converted to natural oligonucleotide according to the following procedure. , since the operations for conversion of all compounds are the same, only those for compound (9) will be described. In the case of compound (8) (approximately 1.0 OD) [compound (to)], approximately 2 Acid hydrolysis was carried out by adding 100μ of 880% acetic acid to . As shown. In the figure, A is a compound

The peak [0] is shown, and B shows the peak when the compound [2] is converted into a natural oligonucleotide. Also,

[0] to [4]
] The numerical value in parentheses indicates the time (C time) from the start of hydrolysis. Looking at Figure 10, we can see that as time passes, the peak A
It can be seen that as the peak B decreases, peak B appears, and then peak A disappears and only peak B remains. This shows that Compound (1) was almost converted into a natural oligonucleotide after 4 hours of hydrolysis. Furthermore, when Compound (1) was subjected to acid hydrolysis in the same manner as Compound (1), the change over time was as shown in FIG. 11. Numerical values and symbols in the figure have the same meanings as in the case of the compound [(e). Therefore, it can be seen that Compound (9)] was also converted to a natural oligonucleotide in about 6 hours. Experiments 3-2 to 3-4 Experiments 1-2 to 1-4 and Experiments 1-1-■ to 1-1-■
The compound was also converted into a natural oligonucleotide in the same manner as above. The structure of the natural oligonucleotide (g) obtained by the above procedure is summarized in Table K below. Experiment 4-i (reproduction of 2*4-jetrophenyl oligonucleotide derivative) Octatimidylic acid derivative synthesized in Experiment 1-1 above
Approximately 1. OOD was dissolved in 0.1M aqueous sodium bicarbonate solution (p
H8,3) Dissolved in 10 μm, 1-fluoro-2,4
- Ethanol solution of dinitrobenzene (50 mg/ml
) 5 μm (large excess) was added and reacted at 37° C. for 2 hours, then 30 μm of water was added and extraction was performed four times with 150 μl of ether to obtain 2,4-dinitrophenyl-octatimidylic acid [σ]. The reaction was confirmed by HPLO. The same operations as in Experiment 2-1 were performed on the compounds [O] synthesized in Experiments 1-2.1-3 and 1-4, and the compounds for each were

[0] was manufactured. Experiment 5-1 Activated OH Sephas 4B (40mg) at 1mM
Wash with hydrochloric acid, and then add 0.5M sodium chloride-0.1
After washing with M sodium bicarbonate buffer (pH 8,3),
Compound [V] (Real r!iA1-1! 4.00Dll
) (1) 200 μm of 0.5 M sodium chloride-0.1 M sodium bicarbonate buffer (pHs, 3) was added, and the reaction was carried out overnight at room temperature with shaking. After the reaction, the resin was washed with 10mM) Liss-HCl buffer (pH 7,5) and 0.5m NaCl (IQrnM) and Liss-HCl buffer ('I)H7,5), and the resin [σ(
However, X=Sepharose) was obtained. In addition, natural oligonucleotides were recovered by performing acid hydrolysis for about 6 hours in the same manner as in Experiment 3-1.

[Brief explanation of the drawing]

FIG. 1 is a flowchart showing an example of a method for synthesizing compounds [V] and [VI] of the present invention. FIG. 2 is a reproduction of a chromatogram obtained by HPLO analysis of the change over time of compound (4) in a 0.1N sulfuric acid solution. Figure 3 shows the actual underframe [V”+ and [
■] is a flowchart of synthesis. Chapter 4.6 and Compound [V] (each experiment)-2
, 1-3 and 1-4) were applied to Sephadex G-50K. Figures 5-(a), 7-vi) and 9-(a) show the chromatography of compound [V] (compounds of experiments 1-2.1-3 and 1-4, respectively) when subjected to I (PLO). Figures 5-(b), 7-(b), and 9-(b) show the compound [■] (compounds of Experiments 2-2.2-3 and 2-4, respectively). This is a reproduction of the chromatogram obtained when the compound [V] (Experimental Example 1-1) was subjected to HPL (j).
80 thiacetic acid), the time course of compound [V] is shown on HP
This is a reproduction of a chromatogram obtained when analyzed by LC. Figure 11 shows compound [■] (Experimental Example 2-1) treated with acid (
80% acetic acid) HPL of compound [■] over time
This is a reproduction of the chromatogram when analyzed with O. Applicant's agent Ino Mata Holds 2 diagrams between Elt (8) and 4th diagram fractional number confusion.

Claims (1)

[Scope of Claims] 1. An oligonucleotide derivative represented by the following formula [V]. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [V] [However, m and n are each 0 or a natural number, R^1 is a divalent linear or branched hydrocarbon residue, and B is a nucleotide is a base constituting (when multiple Bs exist, they may be the same or different)
. 2. The oligonucleotide derivative according to claim 1, wherein base B is selected from the group consisting of adenine, thymine, cytosine and guanine. 3. The oligonucleotide derivative according to claim 1 or 2, wherein R^1 is a linear or branched alkylene group having 2 to 20 carbon atoms. 4. The oligonucleotide derivative according to any one of claims 1 to 3, wherein m is a natural number of 0 or up to 6, and n is a natural number of 0 or up to 40. 5. Protecting group R^2 for the amino group on the 5'-end extension of the compound represented by the following formula [IV], COR^4 group at the 3'-terminus,
It is characterized by removing all protective groups on the base moiety and phosphoric acid moiety. A method for producing an oligonucleotide derivative represented by the following formula [V]. ▲There are mathematical formulas, chemical formulas, tables, etc.▼[IV] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[V] [However, m and n are each 0 or natural,
R^0 is a protecting group for the phosphate group, R^1 is a divalent straight-chain or branched hydrocarbon residue, R^2 is a protecting group for the amino group, and COR^4 is a 3'- of the nucleotide
It is a protecting group for the terminal hydroxyl group, and B' is a base constituting the nucleotide and is protected as necessary,
B is a base constituting a nucleotide (B' or B
and when there are multiple R^0s, they may be the same or different). ]