WO1998021226A1

WO1998021226A1 - H-phosphonate oligonucleotide derivative and process for producing the derivative

Info

Publication number: WO1998021226A1
Application number: PCT/JP1997/004128
Authority: WO
Inventors: Mitsuo Sekine; Takeshi Wada
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1996-11-13
Filing date: 1997-11-12
Publication date: 1998-05-22
Also published as: AU4964897A

Abstract

A novel H-phosphonate oligonucleotide derivative. A process for synthesizing the derivative which comprises synthesizing by the solid-phase method an oligomer having an alkoxyphosphonic acid having an alkylene group with a moderate chain length at each of the 3' and 5' ends, in order to synthesize H-phosphonate oligonucleotide in a high yield while preventing decomposition thereof under basic conditions during synthesis. The derivative is apt to form a double strand together with the target gene, has resistance to phosphodiesterases, and is incorporated into cells efficiently. The derivative is expected to be utilized especially as an antisense.

Description

TECHNICAL FIELD The present invention relates to an H-phosphonate oligonucleotide derivative and a method for producing the derivative.

The present invention relates to a novel H-phosphonucleotide nucleotide derivative and a method for producing the derivative. More specifically, the present invention relates to an H-phosphonate oligonucleotide derivative that is resistant to phosphodiesterase and has high uptake efficiency into cells. Background art

In recent years, many DNA analogs have been synthesized as antisense DNA that inhibits gene expression. In particular, phosphorothioate-type DNA and methylphosphonate-type DNA have been widely studied. For example, regarding phosphorothioate-type DNA, expression control of human immunodeficiency virus (HIV) (C. Cazenave et al, Nucleic Acids Res., 15, 10507 (1987), WJ Stec et al, J. Am. Chem. , 106, 6077 (1984), S. Agrawal et al, Proc. Natl. Acad. Sci. USA, 85, 7079 (1988), T. Vickers et al, Nucleic Acids Res., 19, 3359 (1991). ), Expression control of simple herpes virus (HSV) (W. Gao et al., J. Biol. Chem., 2643, 11521 (1989)), control of expression of pipapi lipovirus (BPV) (LM. Cowsert et al, Antimicrobial Agents and Chemotherapy, 37, 171 (1993)), expression control of cmyconcogene (ELWickstrom et al, In Vitro Cell Develop.Biol., 25,297 (1989)), expression control of BCL-2 oncogene (JC Reed et al, Proc. Natl. Acad. Sci. USA, 87, 3660 (1990)), ICAM-1 protein expression control (JAMMaier et al, Science., 249, 1570 (1990)) IL-1? Regulation of protein expression (J. Manson et al, Lymp hokine Res., 9, 35 (1990)) and the like have been reported. For methylphosphonate-type MA, expression control of simple herpes virus (HSV) (CC Smith et al, Proc. Natl. Acad. Sci. USA, 83, 2785 (1985), M. Koziolkiewicz et al. Chem. Scripta, 26 , 251 (1986)), Regulation of vesicular stomatitis virus (VSV) expression (CHAgris et al, Biochemistry, 25, 6268 (1986)), Regulation of SV40 expression (PSMiller et al, Biochimie, 67, 769 (1985)) ), N-ras oncogene expression control (DMTidd, Anti-Cancer Drug Design, 3,117 (1988)), JGlobin expression control (KR Blake, Biochemistry, 24, 6132 (1985)), chloramphenicol acetyl chloride transfer Regulation of the expression of Ise (CJMarcus-Sekura, Nucleic Acid Res., 15, 5749 (1987)) and the like have been reported.

However, the phosphorothioate-type DNA has a problem that the stability of the duplex is low, and it binds to proteins other than the target mA and causes non-selective inhibition. Methylphosphonate-type DNA has high specificity for the target mMA, but must be used at a high concentration in order to exhibit effective biological activity due to low affinity. However, it has a problem that it cannot be administered to cells at a high concentration due to its low solubility.

Several proposals have been made to improve such problems. For example, a hydroxymethylphosphonate-type DNA whose water solubility is improved by introducing a hydroxymethyl group has been proposed (Bioorganic Chem. 22, 128 (1994), Tetrahedron LeH 8845 (1995)).

Despite its usefulness, this H-phosphonate oligonucleotide derivative, which is structurally highly stable in double strands, has high water solubility, and is considered to be unlikely to be an RNase substrate, To the inventors' knowledge, only dimers have been reported (J. Org.Chem., 49., 139 (1984)), Tetrahedron Let t., 29, 2911 ( 1989), Tetrahedron Lett., 30, 4713 (1989) _s Tetrahedron Lett., L, 4145 (1980)). There are also reports of H-phosphonate oligonucleotides bound to a solid support (W090 / 1202 (PCT / US90 / 01865)), but the nucleotides are immediately hydrolyzed when cut from the solid support. Will be done. That is, there have been no reports on the synthesis and isolation of long-chain H-phosphonate oligonucleotides. Disclosure of the invention

An object of the present invention is to provide a novel H-phosphonate oligonucleotide derivative and a method for synthesizing the derivative.

H-phosphonate oligonucleotides are extremely unstable under basic conditions. For this reason, when removing the acyl-type protecting group at the base site and excising the oligomer from the solid support in the ordinary DNA synthesis, it is completely decomposed in the process of using ammonia water. Even in this case, if a protecting group is introduced into the 3 ′ and 5 ′ hydroxyl groups of the H-phosphonate oligonucleotide, it is relatively stable even under anhydrous basic conditions. However, when the protecting group is removed, the free hydroxyl group at the 3 'end of the H-phosphonate DNA is converted to an H-phosphonate diester adjacent to the 5' side via 2'-deoxyribose. Nuclear attack, which causes the H-phosphonate DNA to be sequentially degraded from the third side while forming the nucleoside 3 ', 5'-cyclic phosphonate. The free hydroxyl group at the 5 'end of H-phosphonate DNA also undergoes intramolecular nucleophilic attack on the 3'-adjacent H-phosphonate diester via 2'-deoxyribose by the same reaction mechanism. As a result, H-phosphonate DNA is sequentially degraded from the 5 'side while forming nucleoside 3,5, -cyclic phosphonate (Fig. 1A).

Thus, the present inventors have conducted intensive studies on a method for synthesizing H-phosphonate oligonucleotides in a high yield by suppressing the decomposition of H-phosphonate oligonucleotides under basic conditions in the synthesis process. As a result, when synthesizing oligomers by the solid-phase method, the present inventors introduce an alkoxyphosphonic acid having an alkylene group having an appropriate chain length at the 3 ′ end and the 5 ′ end of the oligomer, thereby obtaining oligomers. Since the intermolecular distance between the 3, and 5 'hydroxyl groups and the H-phosphonate diester which has been subjected to intramolecular nucleophilic attack is large, it is impossible to attack the H-phosphonate diester of the hydroxyl group, Even when the reaction was carried out under anhydrous basic conditions, degradation of the H-phosphonate oligonucleotide was significantly suppressed (Fig. It was found that a single oligonucleotide was efficiently synthesized.

The H-phosphone mononucleotide derivative of the present invention is represented by the following general formula (I).

[Wherein B may be different from each other; pyrimidine bases such as thymine, cytosine and peracyl; purine bases such as adenine and guanine; and those such as 5-methylcytosine, 5-fluorouracil and 5-hydroxymethylcytosine Represents a derivative of R1 is a hydrogen atom, an alkyl group such as a methyl group, an ethyl group, a propyl group or a butyl group, an alkenyl group such as a vinyl group or an aryl group, a hydroxy group, a methoxy group, an ethoxy group, a propyloxy group, a butyloxy group, or a methoxyxetoxy group. An alkoxy group such as a group, an alkenyl group such as an aryloxy group, or an acyl group such as a methylcarbonyl group, an ethylcarbonyl group, a methoxycarbonyl group or an ethoxycarbonyl group, preferably a hydrogen atom, a hydroxy group, a methoxy group, or the like. Represents a group. R ² represents an optionally branched alkylene group having 1 to 10 carbon atoms, preferably 6 to 8 carbon atoms. Further, these alkylene groups may be via one or more oxygen atoms. For example,

-(OCH ₂ CH ₂ O-

(Wherein X represents a number from 1 to 5). X represents an oxygen atom, a sulfur atom, a selenium atom, preferably a hetero atom such as an oxygen atom. n represents 1 or more, preferably 10 to 30, particularly preferably 12 to 15. ] The H-phosphonate oligonucleotide derivative of the present invention can be synthesized, for example, by a method comprising the following steps.

In this step, the monomer is bonded to the solid phase into which the alkylene group has been introduced (see FIG. 5). General formula (II)

^{^{R 3 - 0 - R 2 -}} 0- R 4 (II)

[Wherein, R ³ represents a protecting group such as a trityl group, a monomethoxytrityl group, a dimethyloxytrityl group, a pixyl group, and the like;

-COCONH-R ⁵

(Wherein, R ⁵ represents a carrier such as aminopropyl CPG, long-chain alkylamino CPG, aminomethylpolystyrene, etc.), and R ² has the same meaning as described above. The protecting group R ³ of the compound represented by, typically, about 1% Torifuruoro acetate / 11 ₂ (; 1 _2, about 2.5% trichloroacetic acid / CH2CI2, about 3¾ of dichloroethane port acetate / CH ₂ C1 _2, etc. After removal by the following general formula (III)

[Wherein,, Β, and X have the same meanings as described above; R ⁶ represents the same protecting group as R ³ in formula (II);

+

X ¹

Is a salt of ammonium, such as triethylammonium, 1,8-diazabicyclo [5.4.0] indene-7-ene, triptylammonium, or a metal salt such as lithium, sodium, or potassium. Represent. ] And a base monomer unit represented by The monomer unit is usually reacted in the presence of 1 to 5 equivalents of a dehydration condensing agent in pyridine or acetonitrile-pyridine (l: l, v / v) at room temperature for about 1 to 10 minutes. The compound represented by the general formula (II) and the base monomer represented by the general formula (III) can be obtained by a method shown in Examples described later (see FIGS. 2 and 7). ).

Thus, the general formula (IV)

^{Wherein, Β, Χ,, ίί 2} , Ι1 4 and R ^6. Representing the same meaning] can be obtained a compound represented by the.

Reaction step 2>

In this step, a monomer is further bound to the monomer bound to the solid phase obtained in reaction step 1 to extend the nucleotide chain (see FIG. 5).

The protecting group R ⁶ of the compound represented by the general formula obtained in the reaction step 1 (IV) typically removed under conditions such as Torifuruoro acetate / CH ₂ C1 ₂ to about, represented by the general formula (III) The basic monomer units are reacted in the same manner as in the reaction step 1. By repeating this operation, the expression

^{Wherein, Β, Χ,, ί1 2} ,, Γ, Ι1 6 and n are as defined above] can be obtained oligomer represented by.

In this step, an alkylene group is introduced into the 5 ′ end of the oligomer synthesized in Reaction Step 2 in the presence of a condensing agent (see FIG. 5).

Oligomers obtained in reaction step 2 and general formula (V)

X

II- ₊

R ⁷ — 0— R ² —〇-1 P— 0. X ² (V)

I

H

[Wherein, R ⁷ is the same as R ³ in the general formula (II), or represents a protecting group or a functional group such as intercalation such as acridine,

X ²

Represents the same Kachionikku salt and said X ^1, R ² and X represent the same meaning. Is reacted in pyridine or acetonitrile-pyridine (1: 1, v / v) at room temperature for 1 to 10 minutes in the presence of a condensing agent.

As the condensing agent used here, for example,

0

II

RSPX ³ Phosphoric chloride as represented by the formula

0

II

R1 CX3, as shown by R ⁸ CX ³ , formula

0

II

An acid anhydride represented by (R ⁸ C) ₂₀ ,

R PX ³ -Y

And the like.

[In the formula, R ⁸ may be different from each other, and may be an alkyl group such as a methyl group, an ethyl group, an isopropyl group or a t-butyl group, an aryl group such as a phenyl group, a methoxy group, an ethoxy group, or an isopropoxy group. , Alkoxy groups such as t-butoxy group, phenoxy group, aryloxy groups such as hexafluorophenoxy group, N, N-dimethylamino group, N, N-methylethylamino group, N, N-methylethylamino group And N, N-dialkylamino groups such as N, N-diphenylamino groups, N, N-diarylamino groups such as N, N-diphenylamino groups, pyrrolidinyl groups, biperidinyl groups, and cyclic amine residues such as monophonyl groups. Represents a virolidinyl group. X ³ is a halogen atom such as a chlorine atom or a bromine atom

(Wherein, R represents a hydrogen atom, a halogen atom or N0 _2.)

(Wherein, R represents a hydrogen atom or a N0 _2.)

N = N

^-Ν γ ^Ν

R

(In the formula, R represents a hydrogen atom or a nitrophenyl group.)

Azolide groups such as

(Wherein, i represents a hydrogen atom or a phenyl group.)

— 0-N '

R

(In the formula, R represents a halogen atom which may be different from each other, CF ₃ or NO _2. )

— 0-N ',, N

N one. n

(Wherein, R represents an optionally different from each other represents Cl, F or N0 _2, the number of tables to the m is 0-5.) Activated esters, such as,

Y

Represents chlorine ion, PF ₆ ion, BF ₄ ion, an Anion such C10 ₄ ions. ] Preferably,

[Wherein, R ³ is a lower alkyl group having 1 to 6 carbon atoms such as a methyl group and an ethyl group], for example,

one

And the like.

Further, in the present invention, c− ^xd · γ (Where Γ, X ³ and

Υ

Represents the same meaning as described above)

+

• X ³ · Υ

RR ⁸ ₂ Ν '

(Where,, ^x3 and

Y

Represents the same meaning as described above)

^Where R ^s , X ³ and

Y

Is as defined above), or

R

R ''

^Where R ^s , X ³ and

Y

Represents the same meaning as described above), and the like, and the like. Thus, the general formula (VI)

Can be obtained.

Reaction step 4>

In this step, the H-phosphonate oligonucleotide derivative synthesized in the solid phase in the reaction step 3 is separated from the solid phase under basic conditions, deprotected by hydrolysis, and the H-phosphonate oligonucleotide derivative is simply synthesized. This is the separation step (see Fig. 6). The compound represented by the general formula (VI) obtained in the reaction step 3 was converted into Ν, Ο-bistrimethylsilyl trifluoroacetamide-provylamine-acetonitrinole (1: 2: 2, ν / ν / ν) or the like, usually at room temperature for 30 minutes to 1 hour, then the solid phase carrier is removed by filtration, the filtrate is evaporated under reduced pressure, and then acetonitrile-water (1 : 1, ν / ν) or dioxane-water (1: 1, ν / ν) or THF-water (1: 1 ν / ν) at room temperature for 5 minutes to give the present invention. The Η-phosphonate oligonucleotide derivative represented by the general formula (I) can be obtained.

The Η-phosphonate oligonucleotide derivative of the present invention obtained by the above reaction steps has the following features.

First, the Η-phosphonate oligonucleotide derivative of the present invention has less steric hindrance at the phosphate site than conventional ones, and when used as an antisense, easily forms a double strand with the target gene, and has a phosphorothioate type. DNA and methylphosphonate DNA Further, it is more advantageous than natural DNA. Also, since it is a neutral molecule, it does not generate electrostatic repulsion with the negative charge of phosphate of the complementary strand when forming a double strand, so it can form a more stable duplex than natural DNA. It is advantageous. Furthermore, since the phosphate moiety of DNA does not have a negative charge, the double strand formed with RNA is unlikely to be a substrate for RNaseH, an enzyme that recognizes the negative charge of DNA and degrades A. The selectivity of the antisense effect on the target gene is advantageous over phosphorothioate-type DNA. Furthermore, since it has higher water solubility than methylphosphonate-type DNA, it can be administered to cells at a high concentration.

Therefore, the H-phosphonate oligonucleotide derivative of the present invention is particularly useful as an antisense, for example, a messenger RNA produced upon propagation of a virus such as HIV, HCV, HCMV, HSV, HTLV, etc. Messenger RNAs encoding proteins essential for the survival of the virus, for example, HIV TAR, U5, rev, rtatj (O. Zelphati, et al., Antisense Res. Dev., 3, 323 (1993), B. Bordier, et al., Nucleic Acids Res., 20,5999 (1992)), HCV "5 to UTR" (M. Alt, et al., Hepatology, 22, 707 (1995)), HSV "IE110", "UL30", "UL37", "UL37j (A. Peyman, et al., Biol. Chem. Hoppe Seyler, 376, 195 (1995), JASmith, et al., J. Virol, 69, 1925 (1995)), as a virus growth inhibitor having a nucleotide sequence complementary to the "tax" of HTLV-1, etc., or acute and chronic leukemia, liver cancer, breast cancer, colorectal cancer, etc. Messenger RNA specifically generated during the growth of tumor cells, usually messenger MA encoding a protein essential for the survival of the target tumor cells, such as AML-1 in acute leukemia (C. Sakakura, et al. Natl. Acad. Sci. USA, 91,11723 (1994)), "bcr-abl" for acute and chronic myeloid leukemia (AMTarai, et al., Blood, 84, 601 (1994)), leukemia, and other cancers "C-myc", "c-mybj (S.Kimura, et al., Cancer Res., 55, 1379 (1995), M.Ratajczak, Proc.Natl. A cad. Sci. USA, 89, 11823 _{.. (1992) N Y.Mizutani,} J. Immunother Emphasis Tumor Immun ol, 17,78 (1995)), "APC" for colon cancer, about breast "BCA - 1", etc. to a complementary It can be used as a tumor cell growth inhibitor having a unique base sequence. This antisense comprises, as an active ingredient, usually an oligonucleotide derivative represented by the general formula (I) having a length of about 4 to 30, preferably about 10 to 20. There is no particular limitation on the components of the preparation or the proportion used, and it can be used in the same manner as various preparations conventionally known as antisense therapeutics.

Since the H-phosphonate oligonucleotide derivative of the present invention does not require the use of a protective group particularly in the base and phosphate groups due to its synthetic chemistry, the synthesis reaction process is shortened and is suitable for mass synthesis. . BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the behavior of an H-phosphonate nucleotide under basic conditions. FIG. 2 is a diagram showing a method for synthesizing a monomer as a unit of an H-phosphonate oligonucleotide.

FIG. 3 is a diagram showing a method for synthesizing a 5 ′ terminal phosphonate.

FIG. 4 is a diagram showing a method for introducing a linker into a solid phase.

FIG. 5 is a diagram showing a step of solid-phase synthesis of an H-phosphonate oligonucleotide. FIG. 6 is a view showing a step of removing a protecting group from a solid-phase synthesized H-phosphonate oligonucleotide.

FIG. 7 is a diagram showing a method for synthesizing a monomer that is a unit of an H-phosphonate oligonucleotide.

FIG. 8 is a diagram showing a method for synthesizing 5, terminal phosphonate.

FIG. 9 is a diagram showing a method for introducing a linker into a solid phase used in the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to Examples. However, the following Examples do not limit the present invention.

Example 1> Chemical synthesis of H-phosphonate DNA

(1) Condensing agent, 2-benzotriazole-1-yloxy-1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphosphoranidium hexafluorophosphate Synthesis of (2-Benzotriazol-1-yloxy-l, 3-dimethyl-2-pyrrolydin-1-1-yl-1,3,2-diazaphosp holanium hexaf luorophosphate) (BDPP)

1,3-Dimethyl-2-pyrrolidine-1-yl-1,3,2-diazaphospholane (1,3-Dimethyl-2-pyrrolydin-l-yl-l, 3,2-diazaphospholane) (F. Ramirez, AV Patwardham, HJ Kugler, CP Smith , J. Am. Chem. Soc, 89, 6276 (1967) a reference) (4.59 g, 25 Hall ol) well攪袢anh CH _₂ C1 ₂ (50 mL) solution of To a suspension of 1-hydroxybenzotriazol (3.32 g, 25 ol) and CC (4.8 mL, 5 O mmol) in argon at -78 ° C over 5 minutes did. After stirring at the same temperature for 2 hours, the mixture was gradually warmed to room temperature, and washed with water (100 mL) containing KPF ₆ (4.6 g, 25 mol). The organic phase was dried over Na ₂ S0 _4, filtered and concentrated to half volume. Ether (50 mL) was added to the above mixture, and a pale yellow precipitate was collected by filtration. Washing the precipitate with ether, in vacuo (in vacuo), and dried over P ₂ 0 _5, to give BDPP a (8.56 g) in 73 yield. This compound could be recrystallized from ethyl acetate as colorless needles.

Mp 128-129 ° C (dec);

³¹ P NMR (CDC) (541.52 (s, P +), -142.90 (7-fold, J = 711.6 Hz, PF _6- );

Ή NMR (CDCla) 52.14 (4H, ddd, J = 7.7 Hz, 5.3 Hz, 1.3 Hz, 3,4-CH ₂ of pyrrolidinyl group), 2.87 (6H, d, J = 11.2 Hz, N-CHa), 2.91 (2H, ddd, J: 8.9 Hz, 7.3 Hz, 5.6 Hz, 2 or 5-CH ₂ of pyrrolidinyl group), 3.50 (2H, ddd, J = 12.9 Hz, 9.1 Hz, 5.8 Hz, 2 or 5 of pyrrolidinyl group) 5-CH ₂ ), 3.64 (4H, ddd, J2 8.9 Hz, 4.3 Hz, 2.3 Hz, 4,5-CH _{2 of} diazaphospholene), 7.52 (1H, ddd, J = 8.3 Hz, 6.8 Hz, 1.3 Hz, 6-H of benzotriazolyl group), 7.72 (1H , dd, J = 8.6 Hz,

6.8 Hz, 5-H of benzotriazolyl group, 7.78 (1H, d, J = 8.6 Hz, 4-H of benzotriazolyl group), 8.07 (1H, d, J-8.3 Hz, benzotriazolyl group 7-H);

¹³ C NMR (CDCla) 526.45 (d, J = 9.8 Hz, N-CH ₃ ), 30.95 (d, J = 6.1 Hz, 3,4-C of pyrrolidinyl group), 45.78 (d, J 2 15.8 Hz, 4,5-C for diazaphospholene, 4 8.68 (d, J = 4.9 Hz, 2,5-C for pyrrolidinyl group), 108.19, 120.63, 126.15, 127.58, 130.60, 142.89 (benzyltriazolyl group) Anal. Calculated for C ₁₄ H ₂₂ F ₆ N ₆ 0P ₂ : C, 36.06; H, 4.76; N, 18.02. Found: C, 35.88; H, 4.59; N, 18.15.

BDPP

Calculated value: 321.159. Observed value: 321.158. (2) Monomer, 1,8-diazabicyclo [5.4.0] index-7-enidum 5'-0-dimethoxytrityldeoxyadenosine-3, -ylphosphonate ( Synthesis of 1,8-Diazabicyclo [5.4.0] undec-7-enium 5 -0-dimethoxytrityldeoxyadenosin-3'-yl phosphonat e) (hereinafter sometimes referred to as "2a")

Monomers were synthesized using the following methods A and B.

Method A (Fig. 2): 5, -0-Dimethoxtrityldeoxyadenosine (5, _0-Dimethoxtrityl 1 deoxyadenosine) (hereinafter sometimes referred to as "la") (0.554 g, 1 mmol) was dried by repeated coevaporation using anhydrous pyridine, and finally dissolved in anhydrous pyridine (5 mL). To this solution was added diphenyl phosphonate (1.64 g, 7 mmol). After stirring for 20 minutes, the mixture _{_{H 2 0- Et 3 N (1}} : 1, v / v, 2 mL) and stirred an additional 20 minutes at room temperature. This mixture was diluted with MeOH (5 mL), and 1 M of triethylammonium hydrogen carbonate (10 mL) was added. The mixture was washed 3 times with _{Et 2 0 (10 mL x 3} ), and back extracted the organic phase with 1 M of Bok Li ethyl ammonium Niu arm hydrogencarbonate carbonate (Triethyla誦onium hydrogencarbonate). This aqueous layer (The aqueous layer) and the washings (washings) were mixed and extracted three times with CHC-MeOH (2: 1, v / v, 10 mL x 3). The aqueous phase _{CHC1 3 - MeOH (2: 1} , v / v) several times were back extracted with. The organic phase and washings (wa shings) were combined, dried over Na ₂ S0 _4, filtered under reduced pressure to dryness and concentrated. The residue was passed through a silica gel column (30 g silica gel). Chromatograph I one applies a methanol gradient (0-155 was performed using CHC1 ₃ containing 0.5% Toryechiruamin. The fractions containing "2a", dryness and concentrated. The product Was dissolved in CHC1 3-MeOH (2: 1, v / v, 10 mL), and 0.2 M 1,8-diazabicyclo [5.4.0] indene-7-enidium hydrogen carbonate (1,8 - diazabicyclo [5.4.0] undec- 7- eniu m hydrogencarbonate) ( washed with 10 mL) the aqueous phase CHC1 ₃ - MeOH (2:. several times back extracted with 1), the organic phase and the washing liquid (Washings ) Were dried over Na ₂ SO ₄ , filtered, concentrated under reduced pressure to dryness to give “2a” (0.631 g, 88%) as a colorless foam.

Method B (Figure 2): A solution of phosphoric acid (0.082 g, 10 ol, dried by repeated co-dehydration with anhydrous pyridine) in anhydrous pyridine (10 mL) was added with bis (2-oxo-3-o). Oxazolidinyl) phosphinic chloride (bis (2-OXO-3-oxazolidinyl) phosp hinic chloride) (1.40 g, 5.5 recited ol) was added. The reaction mixture was stirred at room temperature for 20 minutes. To the mixture was added 5'-0-dimethyltolyldoxyadenosine (5'-0-dimethylmethod 1 deoxyadenosine) (la) (0.554 g, 1 mol). After stirring for 6 hours, it was added H ₂ 0 to (1 mL) to the mixture. It _{CHC1 3 - MeOH (2: 1} , v / v, 10 m Ide diluted, washed 3 times with 1 M tri E chill ammonium Niu arm hydrogencarbonate carbonate of (triethylammon ium hydrogencarbonate) (10 mL x 3), the aqueous phase _{CHC1 3 - MeOH. (2:} 1, v / v) was several times back extracted with the organic phase and washings (washings) were mixed, dried through Na ₂ S0 _4, filtered, concentrated The crude product (triethylammonium salt) was purified by silica gel column chromatography and converted to the DBU salt as described in Method A to give a colorless foam. “2a” (0.60 g, 78%) was obtained as a body.

³¹ P墜_{(CDC1 3) 53.74 (dd,} J 612.8 Hz, 8.5 Hz); Ή NMR (CDCL) (51.66 (2H, m, DBU CH ₂ ), 1.73 (4H, m, DBU CH ₂ ), 1.97 (2H, quintuple, J = 5.6 Hz, DBU CH ₂ ), 2.42 -2.80 (2H, m, 2, , 2 "- H), 2.83 (2H, m, CH of _{DBU 2), 3.36-3.44 (8H,} m, 5,, 5" -H, the DBU CH _2), 3.77 (6H, s, 0 CH 3 of MTr), 4.22 (1H, m , 4, - H), 4.91 (1H, m, 3, - H), 5.96 (2H, bs, 6-NH2), 6.52 ( 1H, t, J = 6.8 Hz, 1, -H), 6.78 (4H, d, J = 8.9 Hz, 3, 3, 5, 5, -H of MTr), 6.96 (1H, d, J = 612.3) Hz, PH), 7.15-7.42 (ArH of DMT r excluding 9H, m, 3, 3 ', 5, 5, -H), 7.99 (1H, s, 2-H), 8.26 (1H, s , 8-H);

¹ C NMR (CDC) δ 19.50, 23.99, 26.85, 28.99, 32.11, 37.90, 39.86 (2, -C), 48.56, 54.22, 55.15, 63.65 (5, -C), 73.67 (d, J = 3.7 Hz, 3, -C), 84.17 (-C), 85.61 (d, J = 7.3 Hz, 4, -C), 86.38, 113.08,119.84 (5-C), 126.76, 127.75, 128.18, 130.06, 135.67, 135.74 , 138.78 (8- C), 144.55, 149.72 (2-0,152.81 (4-C), 155.40 (6- C), 158.40, 166.11.

Anal C ₄ of "2a". H ₄

· 5Η ₂ 0 Calculated:. C, 55.87; H, 6.80; N, 11.40 Found: C, 55.83; H, 6.72 ; N, 11.28.

"2a"

Calculated for: 618.212. Found: 618.212.

(3) Monomer, 1,8-diazabicyclo [5.4.0] dex-7-enedium 5, -0-dimethoxytrityl deoxycytidine-3, -yl phosphonate (l, 8-Diazabicyclo [5.4.0] Synthesis of undec-7-enium 5'-0-dimethoxytrityldeoxycytidin-3'-yl phosphonate) (hereinafter sometimes referred to as "2b")

Using methods A and B as described in the case of "2a", 5, -5-O-dimethoxytrinanoleteoxycinosine (5'-O-dimethoxytrityldeoxycytidine) (hereinafter sometimes referred to as "1b" ) (0.530 g, 1 fraction ol) gave “2b” as colorless foams in 91% (0.679 g) and 74% (0.552 g) yields, respectively.

3 ¹ P NMR (CDCh) 53.83 (dd, J = 612.8 Hz, 8.6 Hz);

_{Ή NMR (CDC1 3) 51.66 (} 2H, m, CH of _{DBU 2), 1.71 (4H,} m, CH 2 of DBU), 1.97 (2H, 5 quintet, J = 5.6 Hz, CH of DBU _2), 2.22 (1H, m, 2, -H), 2.66 (1H, m, 2 "-H ), 2.81 (2H, m, CH 2) of DBU, 3.35-3.51 (8H, m, DBl ^ 5,, 5 "-H, CH, 3.78 (6H, s, 0CH 3 of DMTr), 4.40 (1H, m, 4, - H), 5.05 (1H, m, 3, - H), 5.51 (2H, d, J two 7.3 Hz, 5-H), 5.91 (2H, bs, 4- NH 2), 6.30 ( 1H, t, J = 6.1 Hz, l'-H), 6.82 (4H, d, J = 8.9 Hz, 3, 3, 5, 5, 5, -H of DMTr), 6.92 (1H, d, J = 613.3 Hz, P-H), 7.17-7.41 (9H, m, 3, 3, 5, 5, 5, excluding -H of DMTr), 7.80 (1H, d, J = 7.3 Hz, 6-H) ;

¹³ CNR (CDC) (519.32, 23.87, 26.62, 30.44,32.04, 37.83, 40.42 (2 C), 48.41, 54.11, 55.13, 62.97 (5, -C), 72.60 (3, -C), 84.82 (l ') -C), 85.59 (4, -C), 86.47, 94.74 (5-C), 113.08, 126.77, 127.76, 128.01, 129.97, 135.38, 135.47, 140.56 (6-C), 144.46, 155.89 (2- C), 158.38, 165.48 (4-C), 165.89.

_{_{Anal C 39 H 4 eNsOaP, ·}} 4Η 2 0 Calculated the "2b":. 57.27; 11,6.90; 8.56 Found: C, 57.38; H, 7.06 ; N, 8.82.

"2b"

Calculated for: 594.201. Found: 594.203.

(4) Monomer, 1,8-Diazabicyclo [5.4.0] index-7-enidum 5, -0-Dimethoxytrityldeoxyguanosine-3'-yl phosphonate (l, 8-Diazabicyclo [5.4.0 ] undec-7-enium 5 -O-dimethoxytrityldeoxyguanosin-3'-yl pnosphonate) (hereinafter sometimes referred to as "2c")

Using method A and B as described in the case of "2a", 5'-0-dimethoxy Bok Li Chino Rede O carboxymethyl click, 'source ⁷ Noshin (5' -0-d imethoxy trit 1 deoxyguanos ι ne ( Hereinafter, it may be referred to as “lc” (0.570 g, 1 mmol) to give (2c) as colorless foam in 94% (0.739 g) and 87% (0.684 g) yields, respectively. Obtained.

^{_{3 1 P NMR (CDC1 3)}} 53.07 (dd, J = 618.9 Hz, 8.5 Hz);

_{Ή NR (CDC1 3) dl.58-1.78 (} 6H, m, DBU in CH, 1.95 (2H, 5 quint, J: 5.6 Hz, CH ₂ of DBU), 2.31-2.54 (2H, m , 2,, 2 "- H), 2.62-2.78 ( 2H, m, CH 2 of DBU), 3 .28-3.48 (8H, m, 5,, 5" -H, the _{DBU CH 2), 3.78 (6H} , s, DMTr 0CH ₃ ), 4.37 (1H, m, 4, -H), 4.99 (1H, m, 3, -H), 6.17 (1H, dd, J = 9.6 Hz, 4.3 Hz, l'-H), 6.48 (2H, bs, 2- NH ₂ ), 6.82 (4H, d, J = 8.6 Hz, 3, 3, 5, 5, 5, -H of DMTr), 7.02 (1H, d, J 2 619.3 Ηζ, Ρ-H), 7.16-7.44 ( 9H, m, 3, 3, 5, 5, 5, -H except DMTr)

7.65 (1H, s, 8-H);

^{_{1 3 C NMR (CDC1 3)}} 19.89, 24.17, 26.97 29.09, 33.26, 39.01, 41.33. (2, - C),

48.55, 54.13, 55.20. 63.90 (5, -C), 74.90 (3, -C), 83,70 (l'-C), 85.16 (4, -C), 86.43, 113.19, 117.29 (5-C) , 126.82, 127.81, 128.12, 130.03, 130.0 6, 133.69, 135.67 (8-C), 135.70, 144.56, 150.71 (4-C), 155.58 (2-C), 158.49 (6-C), 165.57.

Anal of "2C"

· 6Η ₂ 0 Calculated:. 53.74; 11,6.76; ^ 10.97 Found: C, 53.58; Η, 6.54 ; Ν, 10.87.

Calculated value of FAB + (M-DBU + HCHNsOeP, for 2C): 634.207. Found: 634.208.

(5) 5, terminal phosphonet, triethylammonium 6- (dimethoxytrityloxy) hexyl phosphonate (hereinafter referred to as “3”) (May be called) (Fig. 3)

1,6-Hexanediol (0.104 g, 1 olol) was dried by repeated co-dehydration using anhydrous pyridine, and finally dissolved in anhydrous pyridine (10 mL). Dimethoxytrityl chloride (0.373 g, 1.1 ol) was added to the solution, and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with CHC1 _3, washed three times with 5% NaHCO _3, and back extracted the aqueous phase with CHC1 _3. The organic phase and the washings are mixed, dried over Na ₂ SO ₄ , filtered, concentrated to dryness, and 6- (dimethoxytrityloxy) hexanol (6- (dimethoxytrityloxy)) hexanol) was obtained as a crude mixture. On the other hand, a solution of phosphonic acid (0.082 g, 10 mmol, dried by repeated co-dehydration using anhydrous pyridine) in anhydrous pyridine (10 mL) was added to a solution of bis (2-oxo-3-oxazolidinyl) phosphine. Nick chloride (bis (2-oxo-3-oxazolidinyl) Dhosphinic chloride) (1.40 g, 5.5 mmol) was added to the mixture. Two After stirring for 0 minutes, the mixture was added to a mixture containing 6- (dimethoxytrityl1oxy) hexano1. After stirring for 20 minutes, the mixture was diluted with pyridine (5 mL), and 1 M triethylammonium hydrogencarbonate (10 mL) was added. The mixture was washed 5 times with Et ₂ 0, the organic phase of 1 M Toryechi J Reanmoniumuhi de Nkarireho not the - was back extracted with Bok (triethylammonium hydrogencarbonate). The aqueous phase and washings (washings) were combined, extracted 3 times with CHC1 _3, was back extracted the organic phase with CHC1 _3. Combine the organic phase with the washings, dry over Na ₂ SO ₄ , filter, concentrate under reduced pressure to dryness, and add 3 (0.265 g, 63%) to a pale yellow oil Obtained as a product. The residue was passed through a silica gel column (30 g silica gel). By applying a gradient of methanol (0-4) was subjected to chromatography using a CHC1 ₃ containing 0.5% Torichiruamin. The fractions containing "3" were combined, concentrated and dried. The product was dissolved in CHCL-MeOH (2: 1, v / v, 10 mL), and washed with 1 M triethylammonium hydrogencarbonate (10 mL). . The aqueous phase was back-extracted with CHC1 ₃ (10 mL), and the organic phases combined and washes (Washings), dried through Na ₂ S0 _4, filtered under reduced pressure, concentrated to dryness, "3" ( (0.230 g, 53%) as a colorless oil.

³¹ P NMR (CDCla) 55.11 (dt, J = 607.9, 7.4 Hz);

NMR (CDCh) (51.19 (9H , t, J = 7.3 Hz, Et 3 N CH 3 of), 1.28 (2H, m, CH2), 1.52 (4H, m, CH 2), 2.77 (8H, q, J = 7.3 Hz, Et ₃ N CH ₂ , CH ₂ ), 2.94 (2H, t, J 2 6.9 Hz, CH ₂ ), 3.67 (6H, s, 0Tr _{3 of} DMTr), 3.74 (2H, t, J = _{6.9 Hz, CH 2), 6.72} (4H, d, J two 8.9 Hz, 3 of DMTr, 3,, 5, 5 , -H), 6.76 (1H, d, J = 6 08.1 Hz, PH), 7.06- 7.36 (9H, m, 3,3, 5,5, excluding -H of DMTr);

^{_{13 C NMR (CDC1 3) (}} 5 9.20, 25.55, 25.79, 29.80, 30.57, 45.32, 63.1, 63.31, 76.53, 85.30, 112.63, 126.20, 127.35, 127.85, 129.67, 136.41, 145.16, 157.97.

(6) Introduction of linker to solid phase LCAA-CPG (Fig. 4) 1,6-Hexanediol (0.118 g, 1 mmol) was dried by repeated co-dehydration using anhydrous pyridine, and finally dissolved in anhydrous pyridine (10 mL). To this solution was added dimethoxytrityl chloride (0.373 g, 1.1 intestine 1), and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with CHC1 ₃ (20 mL), washed 3 times with _{5% NaHC0 3 (20 mL x} 3), and back extracted the aqueous phase CHC1 in ₃ (50 mL). The organic phase and the washings are mixed, dried over Na ₂ SO ₄ , filtered, concentrated to dryness and 6- (dimethoxytrityloxy) hexanol ) Was obtained as a pale yellow oil. On the other hand, a solution of triazole (0.173 g, 2.5 mmol, dried by repeated co-dehydration with anhydrous pyridine) in anhydrous CH ₃ CN (4 mL) was added to oxalyl chloride (44 , 0.5 mmol) and pyridine (0.2 mL) were added. After standing for 5 minutes, the mixture was treated with 6- (dimethyloxytrityloxy) hexanol in pyridine-CH ₃ CN (2: 1, v / v, 3 mL). Was added to the solution. After standing for 1 hour, the mixture was added to LCAA-CPG (1 g) in the reaction vessel. After leaving this mixture for 30 minutes, it was washed sequentially with CH ₃ CN, anhydrous MeOH and CH ₃ CN. The CPG gel Ac ₂ 0- pyridine (1: 9, v / v , 5 mL) was treated with. After standing for 3 hours, the CPG gel was washed with pyridine, followed by CH ₃ CN, dried under reduced pressure, and 6- (dimethoxytrityloxy) hexyl oxalate (6-) bound to LCAA-CPG. (dimethoxytrity1oxy) hex1oxalate). The loading of 6- (dimethoxytrityloxy) hexyl oxalate on LCM-CPG was estimated to be 29 mol / g by DMTr analysis.

(7) Synthesis of oligomers and H-phosphonate oligodeoxyribonucleotides Solid-phase synthesis is performed using `` Applied Biosystems 380A synthesizers '' or as a reaction vessel, a glass with a stopper at the top and a stopper at the bottom. . filters were performed (10顏X 50誦) manually with each cycle of chain extension are detritylation _{(detritylati on) (CH 2 C1} 1% TFA in _2; 15 seconds), washed (CH ₂ C1 _2, then pyridine), (0.1 M monomers in one pyridine ( "2a", "2b", "2c") cup-ring and 0.5 M of BDPP; 2 min), washing (pyridine), tubing (0.1 M triethylammonium methylphosphonate, 0.5 M BDPP in pyridine; 2 min), washing (pyridine, then CH ₂ Cl ₂ ). In the final cycle of the synthesis, trimethylammonium 6- (dimethoxytrityloxy) hexylphosphonate (triethyla onium 6- (dime thoxytrityloxy) hexyl phosphonate) (3) is converted into monomer units (“2a”, “2b”). ”And“ 2c ”) (Fig. 5). In general, the average yield per cycle was estimated to be 96-99% by DMTr analysis. After chain extension, it was removed by treating 15 seconds DMTr group in 1% TFA in CH ₂ C1 in _2, CH ₂ C1 _2, then washed with CHaCN. On CPG gel H- phosphonate oligomer N, 0- bis (trimethyl silicon Honoré) Asetoami de _{- CH 3 CN (N, 0} -bis (trimethylsi lyl) acetamide-CH3CN) (1: 2, v / v, 300 / was treated with L) 20 minutes, allowed to stand the mixture in n- PrNH ₂ (200〃L) was added for 30 minutes. The CPG gel was removed by filtration, and the filtrate was concentrated under reduced pressure. This crude product was purified on a “01igo-Pak EX (manufactured by Millipore)” cartridge. The product was eluted with H ₂ 0-CH ₃ CN (3: 2, v / v), the eluate was concentrated to dryness, and 6- (hydroxy) hexylphospho was added at the 3 ′ and 5 ′ ends. An H-phosphonate oligodeoxyribonucleotide having a net (6- (hydroxy) hexyl phosphonate) was obtained (FIG. 6).

An example of the synthesis of decathyminate H-phosphonate (T10) and tetranucleotide H-phosphonate (CAGT) is shown below.

Synthesis Example 1: Chemical formula

Of decatimimidylate H-phosphonate (T10) shown by

Replacement form (Rule 26) 24/1-Decatimidylate from l〃mol of 6- (dimethoxytrityloxy) hexyl oxalate bound to LCM-CPG using the typical process described above H-phosphonate (decathymidylate H-phosphonate) (61.4 A260 units) was obtained with a yield of 82%.

UV (CH3CN-H2O, 1: 1, v / vUmax: 262.8 run, λιηΐη 233.0 nm;

^{_{31 P NMR (CD 3 CN- H}} 2 0, 1: 1, v / v) (510.95- 11.49 ( multiplet, J two 731.6 Hz as a mean value); calcd FAB M + D + CH SN O PH: 3165.29. Found: 3165.25

Replacement form (Rule 26) 25 Synthesis example 2: Chemical formula

Of tetranucleotide H-phosphonate (CAGT)

Using the typical process described above, tetranucleotide H-phosphoneone is obtained from l〃mol of 6- (dirmethoxytrityloxy) hexyl oxalate bound to LCM-CPG. (31.0 A2 60 units) was obtained at a yield of 84 °.

UV (CH ₃ CN-H ₂ 0, 1: 1, v / v) person max 259.2 nm, input min 229.8 nm;

³¹ P 丽 R (CD ₃ CN-H ₂ 0, 1: 1, v / v) (510.97-12.29 (multiple line, J 2 7 28.8 Hz as average); FAB ⁺ (M + 1) ⁺ C ₅₁ H Calculated for ₇₇ N ₁₅ 0 ₂₅ P ₅ : 1455.12. Found: 1455.09.

Replacement form (Rule 26) 25/1 Treat the obtained tetranucleotide H-phosphonate (1.0 A _26. Units) with concentrated NH ₃ -pyridine (9: 1, v / v, 5 mL) for 10 minutes at room temperature Then, the diester bond of the H-phosphonate was cleaved. The mixture was dehydrated, dried, and dissolved in anhydrous pyridine (300 L). It was added Et ₃ N (42 zL) and BSTFA (8 1〃L) to the solution. After standing at room temperature for 2 hours, 5.0 M t-Bu00 in anhydrous decane (100 / L)

Replacement form (Rule 26) 26

H was added and the mixture was left at room temperature for 10 minutes. The mixture was dehydrated dried, concentrated NH ₃ - pyridine (9: 1, v / v , 5 mL) was dissolved in. After standing for 5 minutes, the mixture was dehydrated, dried, and dissolved in 0.1 M Tris-HCl buffer (pH 8.0, 100 μL). To this solution, add calf intestinal alkaline phosphatase (10 units) and incubate at 37 ° C for 3 hours to obtain approximately 1: 1: 1 of dC, dA, dG, and dT. A mixture of 1 was obtained (reverse phase analysis).

Example 2> Chemical synthesis of H-phosphonate oligodeoxyribonucleotide Chemical synthesis of H-phosphonate oligodeoxyribonucleotide was performed under different reaction conditions from Example 1.

(1) Synthesis of the monomer, triethylammonium 5,10-dimethoxytrityldeoxyadenosin-3'-yl phosphonate (2a)

Monomers were synthesized using Method A and Method B below.

Method A (Fig. 7): 5,0-Dimethoxytrityldeoxyadenosine (5, -0-Dimethoxytrityl Ideoxyadenosine) (la) (0.554 g, 1 minol) was repeated using anhydrous pyridine. It was dried by return co-dehydration and finally dissolved in anhydrous pyridine (5 mL). To this solution, diphenyl phosphonate (1.64 g, 7 ol) was added. After stirring for 20 minutes, the mixture _{_{H 2 0- Et 3 N (1}} : 1, v / v, 2 mL) and stirred an additional 20 minutes at room temperature. The mixture was diluted with pyridine (5 mL) and 1 M triethylammonium hydrogen canolebonate (1 O mL) was added. The mixture was washed 5 times with Et ₂ 0, the organic phase 1 M Toryechiru Anmoniumuhi Dorogen force Honoré non non of - back-extracted with preparative (triethylammonium hydrogencarbonate). The aqueous phase and washings were mixed and extracted three times with CHC1 _3, was back extracted the aqueous phase with CHC1 _3. The organic phase and washings were combined, dried through Na ₂ S0 _4, filtered and reduced pressure to dryness and concentrated. The residue was passed through a silica gel column (30 g silica gel). By applying a gradient of methanol (0-15%), the CHC1 ₃ containing 4% Torichiruamin 27 was used for chromatography. The fractions containing "2a" were combined, concentrated and dried. The product was dissolved in CHC1 ₃ (10 mL), and lavage with 1 M tri E chill ammonium Niu arm hydro Genkanorebone Bok of (Triethyla thigh onium hydrogenca bonate) (10 mL) , to remove the sheet Rikageru. The aqueous phase was back extracted with CHC1 _3, mixing an organic phase and washings (washings), dried through Na ₂ S0 _4, filtered under reduced pressure, concentrated to dryness, "2a" (0.5 89 g, 82%) as a colorless foam.

Method B (Figure 7): A solution of phosphonic acid (0.082 g, 10 ml, dried by repeated co-dehydration with anhydrous pyridine) in anhydrous pyridine (10 mL) was added to bis (2-oxo). -(3-oxazolidinyl) phosphinic chloride (bis (2-OXO-3-oxazolidinyl) phosphinic chloride) (1.40 g, 5.5 t ol) was added. The reaction mixture was stirred at room temperature for 20 minutes. To this mixture was added 5'-0-dimethyloxytrityldeoxyadenosine (la) (la) (0.554 g, 1 t ol). After stirring for 6 hours, the mixture was diluted with CHC1 _3, washed 3 times with 1 M bird E chill ammonium Niu arm hydrogencarbonate Cal Bonnet Ichito (triethylammonium hydrogencarbonate), and back extracted the aqueous phase with CHC 1 ₃ . The organic phase and the washings were combined, dried over Na ₂ SO ₄ , filtered, and concentrated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography as described above to obtain “2a” (0.563 g, 78%) as a colorless foam.

³¹ P NMR (CDCh) (53.88 (dd, J _PH = 616.5 Hz, J _P. _CH = 8.5 Hz);

Ή NMR (CDCh) dl. 21 (9H, t, J = 7.3 Hz, Et ₃ NH + CH ₃ ), 2.70-2.83 (2H, 111, 2'-H and 2 "-H), 2.92 (6H, q , J = 7.3 Hz, CH ₂ Et ₃ NH +), 3.32 (3H, m, 5, -H, and 5-H), 3.69 (6H, s, 0CH _{3 of} DMTr), 4.33 (1H, m, 4, -H), 4.98 (1H, m, 3, -H), 5.93 (2H, bs, 6-NH2), 6.45 (1H, t, Jr, _2- = 5.9 Hz, l'-H), 6.70 (4 H, d, J = 8.6 Hz, 3, 3, 5, 5, 5, -H of DMTr, 6.85 (1H, d, J _PII = 617.0 Hz, PH), 7.14-7.34 (9H, m, 3 , 3,5,5, -H except DMTr, 7.91 (1H, s, 2-H), 8.20 (1H, s, 8-H); 28

¹³ C NMR (CDCls) 58.68 (CH _{3 in} Et ₃ NH +), 39.70 (2, -C), 45.41 (CH _{2 in} Et ₃ NH +), 55.11 (0CH _{3 in} DMTr), 63.60 (5, -C), 73.84 (JPOC = 4.9 Hz, 3, -C), 84.13 (l'-C), 85.50 (JPOCC = 7.3 Hz, 4, -C), 86.34 (MTr tert-C), 113.03 (DMTr 3,3 ,, 5, 5, -C), 119.87 (5-C), 138.67 (8-C), 149.70 (C-2), 152.83 (C-4), 15 5.42 (C-6), 126,74, 127.73, 128.12, 130.03, 135.58, 135.63, 144.47, 158.3 (DMTr Ar-C).

(2) Monomer, triethylammonium 5, -0-dimethoxytrityldeoxycytidine-1,3-inole3, triethylamonium 5,10-dimethoxytrityldeo xycytidin-3'-yl phosphonate) (2b)

Using methods A and B as described in the case of `` 2a '', 5′-0-dimethoxytrityldeoxycytidine (lb) (0.530 g, 1 mmol) was converted to `` 2b Was obtained as a colorless foam in 91% (0.635 g) yield and 74% (0.518 g), respectively.

³¹ P NMR (CDCh) 53.67 (dd, J _PH = 616.4 Hz, JPOCH = 8.5 Hz);

_{Ή NMR (CDC1 3) (51.21} (9H, t, J = 7.3 Hz, Et 3 NH + of _{CH 3), 2.17 (1H,} m, 2, - H), 2.59 (1H, m, 2 "-H), 2.93 (6H, q, J = 7.3 Hz, Et 3 NH + of _{CH 2), 3.32 (3H,} m, 5, - H, and 5 "- H), 3.69 (6H, s, 0CH 3 of DMTr), 4 .15 (1H, m, 4, -H), 4.83 (1H, m, 3, -H), 5.57 (1H, d, J = 7.6 Hz, 5-H), 6.20 (1H, t, J, ₂ '= 5.9 Hz, 1, -H), 6.74 (4H, d, J = 8.9 Hz, 3,3, 5, 5, -H of DMTr), 6.81 (1H, d, J _PH = 617.0 Hz, PH), 7.11-7.34 (9H, m, 3, 3, 5, 5,-ArH of MTr excluding -H), 7.70 (1 H, d, J = 7.6 Hz, 6-H);

^{_{, 3 C NMR (CDC1 3)}} (58.77 (Et 3 NH + of _{CH 3), 40.61 (2,} -C), 45.61 (Et 3 NH + of CH _2), 55.20 (0CH ₃ of MTr), 62.82 (5, - C), 72.82 (3, -C), 84.91 (l'-C), 85.76 (4, -C), 86.67 (tert-C of DMTr), 94.68 (C-5), 113.21 (DMTr 3, 3 ,, 5, 5, -C), 141.24 (6-C), 154.47 (2-C), 163.97 (C-4), 123.68, 126,93,127.91, 128.12, 130.10, 135.38, 135.45, 144.46, 158.53 (DMTr Ar-C). 29 Guanosine-3, -yl phosphonet (Triethyla fraction onium 5,-0-dimethoxytrityl

(3) Monomer, synthesis of triethylammonium 5, -0-dimethoxytrityldeoxy deoxyguanosin-3'-yl phosphonate) (2c)

Using method A as described in the case of “2a”, 5′-0-dimethoxytrityldeoxycytidine (5, -0-dimethoxytrityldeoxycytidine) (lc) (0.570 g, (0,708 g, 94%) as a colorless foam.

³¹ P NMR (CDC) 63.22 (dd, JPH-626.2 Hz, J _P. _CH = 8.6 Hz);

Ή NMR (CDCla) (51.18 (9H, t, J = 7.3 Hz, Et ₃ NH + CH ₃ ), 2.35 (1H, m, 2, -H), 2.58 (1H, in, 2 "-H), 2.99 (6H, q, J = 7.3 Hz, Et 3 NH + of _{CH 2), 3.26 (3H,} m, 5, - H, and 5 "- H), 3.69 ( 6H, s, 0CH 3 of DMTr), 4.29 ( 1H, m, 4, -H), 4.95 (1H, m, 3, -H). 6 · 10 (1Η, dd, Jr, ₂ '/ Jr, ₂ ''= 4.6 Hz and 8.9 Hz, l'- H), 6.74 (4H, d, J = 8.9 Hz, 3,3, 5, 5, 5, -H of DMTr), 6.91 (1H, d, JPH 625.5 Hz, P-H), 7.11-7.35 ( 9H, m, 3,3,5,5, -H except DMH), 7.62 (1H, s, 8-H);)

¹³ C NMR (CDCla) 58.86 (CH _{3 of} Et ₃ NH +), 40.81 (2, -C), 45.73 (CH _{2 of} Et ₃ NH +), 55.19 (0CH _{3 of} DMTr), 63.67 (5, -C), 74.81 (3, -C), 83.61 (1, -C), 85.14 (4, -C), 86.41 (tert-C of DMTr), 113.21 (3,3,5,5, -C of DMTr), 117.10 (5-C), 13 5.60 (8-hi, 150.75 (4-C), 154.66 (C-2), 158.13 (6-C), 126,84, 127.85, 12 8.09, 130.02, 133.80, 144.51, 158.47 (AT-C for DMTr).

(4) Synthesis of Triethylammonium 5- (dimethoxytrityloxy) pentyl phosphonate (3) (5) Phosphonic acid at the 5'-terminal, tritylammonium 5- (dimethoxytrityloxy) pentyl phosphonate

1,5-Pentanediol (0.104 g, 1 ol) was dried by repeated co-dehydration using anhydrous pyridine, and finally dissolved in anhydrous pyridine (10 mL). To this solution was added dimethoxytrityl chloride (0.373 g, 1.1 ol) and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with CHC1 _3, washed three times with 5% NaHCO _3, and back extracted the aqueous phase with CHC1 _3. That 30 Combine the organic phase and the washings, dry over Na ₂ SO ₄ , filter, concentrate and dry to dryness, and dry 5- (dimethoxytrityloxyl) pentanol. Was obtained as a crude mixture. On the other hand, bis (2-oxo-3-oxazolidinyl) phosphinic acid was added to a solution of phosphonic acid (0.082 g, 10 mL of ethanol, dried by repeated co-dehydration using anhydrous pyridine) in anhydrous pyridine (10 mL). Chloride (bis (2-oxo-3-oxazolidinyl) phosphinic chloride) (1.40 g, 5.5 mmol) was added to the mixture. After stirring for 20 minutes, the mixture was added to a mixture containing 5- (dimethyltrityloxy) pentanol (5- (dimethyl thoxy trity 1oxy) pentano 1). After stirring for 20 minutes, the mixture was diluted with pyridine (5 mL), and added with 1 M triethylammonium hydrogen canolepo 'citrate (triethyla onium hydrogencarbonate) (10 mL). The mixture to be washed five times with Et ₂ 0, mixed with the organic phase of 1 M triethylammonium ammonium Niu arm hydrogencarbonate force Rebone Bok of (triethylammonium hydrogencarbonate) in reverse extracted ₀ water eyes and lavage solution (washings), extracted CHC1 ₃ three times and reverse extracted organic phase with CHC1 _3. Combine the organic phase with the washings, dry over Na ₂ SO ₄ , filter, concentrate under reduced pressure to dryness to give “3” (0.265 g, 63%) as a pale yellow oil. Was. This material was analytically pure without further purification.

³¹ P NMR (CDCla) 54.99 (dt, J _PH = 614.0 Hz, J _P. _CH2 7.3 Hz);

Ή NMR (CDC) (51.15 (9H, t, J 2 7.3 Hz, Et ₃ NH + CH ₃ ), 1.36 (2H, m, CH ₂ ), 1.50 (4H, m, 2xCH ₂ ), 2.87 (6H, q , J = 7.3 Hz, Et ₃ NH + CH ₂ ), 2.90 (2 H, dd, J = 6.9 Hz and 3.0 Hz, DMTrO-CH ₂ ), 3.63 (6H, s, DMTr 0CH ₃ ), 3.74 (2H , Dd, J = 6.9 Hz and 7.6 Hz, P-0CH ₂ ), 6.69 (4H, d, J = 8.6 Hz, DMTr 3, 3, 5, 5, 5, -H), 6.73 (1H, d, J _PH 613.7 Hz, P-H), 7.05-7.55 (9H, m, 3,3,, 5,5, -H except DMTr);

¹³ C NMR (CDCla) (58.07 (CH _{3 in} Et ₃ NH +), 22.32 (CH ₂ ), 29.35 (CH ₂ ), 30.32 (JPOCC = 7.3 Hz, P-OCH2CH2), 44.93 (CH _{2 in} Et ₃ NH +) , 54.72 (0CH _{3 of} DMTr), 62.88 (CH2), 63.27 (JPOC = 4.9 Hz, P-0CH ₂ ), 85.18 (tert-C of DMTr), 112.53 (D 31

3,3,, 5,5, -0,123.40,126.09,127.24,127.71,129.56.135.78,136.2 1,145.01,149.07,157.84 of ^ 1 (Ar-C of DMTr).

(5) Introduction of linker to solid phase LCAA-CPG (Fig. 9)

1,5-Pentanediol (0.104 g, 1 mol) was dried by repeated co-dehydration using anhydrous pyridine, and finally dissolved in anhydrous pyridine (10 mL). To this solution was added dimethoxytrityl chloride (0.373 g, 1.1 mmol), and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with CHC1 _3, washed three times with 5% NaHCO _3, and back extracted the aqueous phase with CHCl. Mixing the organic phase and wash solution (Washings), dried through Na ₂ S0 _4, filtered, dryness and concentrated to give the crude 5- (dimethyl butoxy trityl O carboxymethyl) Pen evening Nord (5- (Dimethoxytrityl the oxy) pentanol). On the other hand, a solution of triazole (0.173 g, 2.5 t, dried by repeated co-dehydration using anhydrous pyridine) in anhydrous CH ₃ CN (4 mL) was added to oxalyl chloride (44 uL, 0.5 l) and pyridine (0.2 mL) were added. After standing for 5 minutes, the mixture was treated with 5- (dimethoxytrityloxy) pentanol in pyridine-CH ₃ CN (2: 1, v / v, 3 mL). Was added to the solution. After standing for 1 hour, mix this mixture with the LCAA-CPG

(1 g). The mixture was allowed to stand for 1 hour and washed sequentially with CH ₃ CN, anhydrous Me0H, and CH ₃ CN. The CPG gel Ac ₂ 0 pyridine (1: 9, v / v , 5 mL) was treated with. After standing for 3 hours, the CPG gel was washed with pyridine and then with CH ₃ CN, dried under reduced pressure, and conjugated with 5- (dimethoxytrityloxy) pentyloxalate bound to LCAA-CPG (5- (dimethoxytrityloxy; pentyl oxalate).

(6) Solid phase synthesis of H-phosphonate oligodeoxyribionucleotide

Synthesis example 1.

The procedure was similar except that the monomer used in Synthesis Example 1 of Example 1 was a thymidine monomer obtained in Method A or Method B using TEA instead of DBU. 32 Thymidylate H-phosphonate was synthesized. Synthesis example 2.

Same as in Synthesis Example 1 of Example 1 except that the monomer used in Method A or Method B was thymidine monomer obtained by using TEA instead of DBU, and PyFOP was used instead of BDPP as a condensing agent Thus, Decathymidylate H-phosphonate was synthesized.

Synthesis example 3.

In the same manner as in Synthesis Example 2 of Example 1 except that the monomer used in Method A or Method B was a thymidine monomer obtained by using TEA instead of DBU, tetranucleotide H-phosphonate (Tetranucleotide H Table 1 shows the results of the above examples in which phosphonate was synthesized.

Table 1 Oligomonomer Condensing agent Average yield (%) Before deprotection After deprotection Unit salt (calculated) yield (%) yield (%) Example T 10 DBU BDP P 99 61.4 OD Synthesis Example (82%) Example 2 T10 TEA BDPP 96 65 43.50D Synthesis Example 1 (58%) Example 2 T 10 TEA PyFOP * 95 62 42.0OD Synthesis Example 2 (56%) Example 1 CAGT DBU BDPP 99 95 31.0OD Synthesis Example 2 (84%) Example 2 CAGT TEA BDPP 96 26.40D Synthesis Example 3 (71%) 33

* P y FOP: (Ν-Ρ ⁺ — 0

I /

Synthesized according to the literature (H.H. Jensen, CE Olsen, A. Holm, J. Org. Chem., 59, 1257 (1994)). Reference Example 1> Synthesis of tetranucleotide methylphosphonate

Okizarirurinka one (oxalyl linker) Tetoranukure Ochido H- phosphonate bonded to LCAA- CPG via a (tetranucleotide H-phosphonate) (1 〃Mol) 1 ₂ 5% methanol over pyridine solution (9: 1, v / v ) At room temperature for 10 minutes, and then washed successively with pyridine and acetonitrile. The CPG gel was then treated for 30 minutes at room temperature in n-PrNH broad acetonitrile (1: 4, v / v). The CPG gel was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was obtained tetra p nucleotide main Narirehosuhone one Bok by purified by reverse phase HPLC (tetranucleotide methylpnosphonate) ₀

^{_{3 1 P NMR (CD 3 CN-}} H 2 0, 1: 1, v / v) ό "0.05-1.16,

FAB MS (FAB) Calcd for C ss H 8S N i5 0 3. P ₅ : 1603, Found 1603.

Reference Example 2> Synthesis of tetranucleotide hydroxymethylphosphonate

Tetranucleotide H-phosphonate (1 mol) bound to LCAA-CPG via oxalyl linker is converted to 1 M N, 0-bis (dioxane) in dioxane. The reaction was carried out at room temperature for 2 hours with trimethylsilyl) acetoamide (N, 0-bis (trimethyl hylsilyl) acetamide) and then for 10 minutes with gaseous formaldehyde. After washing with dioxane, the mixture was treated in n-PrNHr "ethanol (1: 4, v / v) at room temperature for 30 minutes. The CPG gel was removed by filtration, and the filtrate was concentrated under reduced pressure. Purification of the product by reverse phase HPLC

Replacement form (Rule 26) O Obtained tetranucleotide hydroxymethylphosphonate

^{3 1} P thigh (CDaCN-aO, 1: 1, ν / ν) δ 28.52-29.78,

FAB MS (FAB) Calcd for C 5t H 86 N 15 0 3. P _s : 1603, Found 1603.

Reference Example 3> Synthesis of tetranucleotide boranophosphonate

Tetranucleotide H-phosphonate (1 mol) bound to LCAA-CPG via oxalyl linker is converted to 1 M N, 0-bis (trimethylsilyl) acetamide-Me ₂ The reaction was performed with S · BH ₃ (5: 2, v / v) at room temperature for 1 hour. After washing with methanol, it was treated in n-PrNH ₃ -methanol (1: 4, v / v) at room temperature for 30 minutes. The CPG gel was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was purified by reverse phase HPLC to obtain tetranucleotide boranophosphonate.

^{3 1} P NMR (CD ₃ CN-H _¾ 0, 1: 1, ν / ν) δ 92.41-94.89.

Reference Example 4> Synthesis of tetranucleotide phosp hoamidate

It was reacted for 5 minutes at 0 ° C with saturated ammonium pyridine solution containing tetranucleotide H- phosphonate Ichito the (tetranucleotide H- phosphonate) (CAGT) 5% 1 _2. Then, it processes the excess L in NaHS0 ₃ solution, tetra resulting crude product by purification by reverse phase HPLC quinuclidine Leona de phosphoamidinato Ding one preparative (tetranucleotide phosphoamidate) to give ¾

^{3 1} P NMR (CD ₃ CN-H ₂ 0, 1: 1, v / v) δ 12.47-14.33,

FAB MS (FAB) Calcd for CsHO P;: 1528, Found 1528. Industrial applicability

According to the present invention, a novel H-phosphonate oligonucleotide derivative, and its derivative A method for synthesizing 35 bodies was provided. The H-phosphonate oligonucleotide derivative of the present invention has properties such as easy formation of a double strand with a target gene, resistance to phosphodiesterase, and high efficiency of cell uptake. The H-phosphonate oligonucleotide derivative of the present invention is expected to be used particularly as an antisense nucleic acid.

Claims

36 Claims

1. H-phosphonate oligonucleotide derivative _c represented by the following general formula (I)

[Wherein, B represents a pyrimidine base, a purine base or a derivative thereof which may be different from each other, and R ¹ represents a hydrogen atom, an alkyl group, an alkenyl group, a hydroxy group, an alkoxy group, an alkenyloxy group or an acyl group. R ² represents an alkylene group which may be branched, and the alkylene group may be via an oxygen atom. X represents a heteroatom atom, and n represents a number of 1 or more. ]

2. (a) — General formula (11),

R ³ -0-R ² -0-R ⁴ (II)

[Wherein, R ³ represents a protecting group;

-CO CONH-R ⁵

(Wherein, represents a carrier.) Wherein R ² has the same meaning as described above. After removing the protecting group R ³ of the compound represented by

General formula (ΙΠ), 37

[Wherein, RB and X have the same meanings as described above; R ⁶ represents a protecting group;

X ¹

Represents a catonic salt. ] With the basic monomer unit represented by the general formula (IV),

(IV)

^{Wherein, Β, Χ,, Ι1 2} , ίί 4, and R ⁶ represents the same meaning. A step of obtaining a compound represented by the formula:

(b) removing the protecting group R ⁶ of the compound represented by the general formula (IV) obtained in the above step, and then reacting the compound with the basic monomer unit represented by the general formula (III);

(c) a compound obtained by repeating the same operation as in (b),

X

II- ₊

R ⁷ — 0— R ² — 0— P—〇 ■ X ² (V)

I

H

Wherein R ⁷ represents a protecting group, 38

X ²

Represents a cationic salt, and R ² and X have the same meanings as described above. Is reacted in the presence of a condensing agent,

General formula (VI), χρηπ

^{^{Wherein, ΙΤ, Ι1 2, Ι1 4}} ,, Β and X represents the same meaning, eta represents a number of 1 or more. ] Obtaining a compound represented by

The H phosphonate oligonucleotide derivative according to claim 1, wherein (d) reacting the compound represented by the general formula (VI) obtained in the step (c) under anhydrous basic conditions, followed by hydrolysis. The step of obtaining

A method for producing an H-phosphonate oligonucleotide derivative, comprising:

3. Reagent for 5, terminal synthesis of H-phosphonate oligonucleotide represented by the following general formula (V).

X

II- ₊

R ⁷ — 0— R ² —〇— P— 0. X ² (V)

H 39

[Where R \

+

X ²

, R ² and X have the same meaning as described above. ]

4. A reagent for synthesizing the 5 'end of an H-phosphonate oligonucleotide represented by the following general formula (VII).

X

II-+

R ⁷ — 0— R ⁹ — 0— P— 0 · X ² (VII)

I

H

[In the formula, IT represents an alkylene group having 4 or more carbon atoms which may be branched, and the alkylene group may be via an oxygen atom. Γ,

+

X ²

And X have the same meaning as described above. ]

5. A virus growth inhibitor comprising, as an active ingredient, an oligonucleotide derivative having a base sequence complementary to messenger RNA generated during virus growth, wherein the oligonucleotide derivative is the Η-phosphonate oligonucleotide according to claim 1. A virus growth inhibitor, which is a reotide derivative.

6. A tumor cell growth inhibitor comprising, as an active ingredient, an oligonucleotide derivative having a nucleotide sequence complementary to a messenger RNA specifically generated during the growth of tumor cells, wherein the oligonucleotide derivative is selected from the group consisting of: A tumor cell growth inhibitor, which is a ホスホ -phosphonate oligonucleotide derivative.

7. Use of a compound represented by the following general formula (V) as a reagent in the synthesis of 5'-terminal of Η-phosphonate oligonucleotide.

X

II-÷

R ⁷ — 0— R ² — 0— Ρ— 0 · X ² (V)

I

Η 40

[Where R \

+

X ²

, R ² and X have the same meaning as described above. ]

8. Use of a compound represented by the following general formula (VII) as a reagent in the synthesis of the 5 'end of H-phosphonate oligonucleotide.

X

II ₊

R ⁷ — 0— R ⁹ —〇一 P—〇 · X ² (VII)

I

H

[In the formula, R ⁹ represents an alkylene group having 4 or more carbon atoms which may be branched, and the alkylene group may be via an oxygen atom. R ⁷ ,

X ²

And X have the same meaning as described above. ]