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

H-phosphonate oligonucleotide derivative and process for producing the derivative Download PDF

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Publication number
WO1998021226A1
WO1998021226A1 PCT/JP1997/004128 JP9704128W WO9821226A1 WO 1998021226 A1 WO1998021226 A1 WO 1998021226A1 JP 9704128 W JP9704128 W JP 9704128W WO 9821226 A1 WO9821226 A1 WO 9821226A1
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group
phosphonate
general formula
derivative
oligonucleotide
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PCT/JP1997/004128
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French (fr)
Japanese (ja)
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Mitsuo Sekine
Takeshi Wada
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Chugai Seiyaku Kabushiki Kaisha
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Priority to AU49648/97A priority Critical patent/AU4964897A/en
Publication of WO1998021226A1 publication Critical patent/WO1998021226A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07FACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02Phosphorus compounds
    • C07F9/06Phosphorus compounds without P—C bonds
    • C07F9/08Esters of oxyacids of phosphorus
    • C07F9/141Esters of phosphorous acids
    • C07F9/1411Esters of phosphorous acids with hydroxyalkyl compounds with further substituents on alkyl

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  • 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
  • HSV simple herpes virus
  • BPV pipapi lipovirus
  • cmyconcogene ELWickstrom et al, In Vitro Cell Develop.Biol., 25,297 (1989)
  • BCL-2 oncogene JC Reed et al, Proc. Natl. Acad. Sci.
  • VSV vesicular stomatitis virus
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the H-phosphone mononucleotide derivative of the present invention is represented by the following general formula (I).
  • 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.
  • 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.
  • 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,
  • H-phosphonate oligonucleotide derivative of the present invention can be synthesized, for example, by a method comprising the following steps.
  • R 3 represents a protecting group such as a trityl group, a monomethoxytrityl group, a dimethyloxytrityl group, a pixyl group, and the like;
  • R 5 represents a carrier such as aminopropyl CPG, long-chain alkylamino CPG, aminomethylpolystyrene, etc.
  • R 2 has the same meaning as described above.
  • R 6 represents the same protecting group as R 3 in formula (II);
  • 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). ).
  • 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.
  • ⁇ , ⁇ ,, ⁇ 1 2 ,, ⁇ , ⁇ 1 6 and n are as defined above] can be obtained oligomer represented by.
  • 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).
  • 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,
  • 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
  • R represents a hydrogen atom, a halogen atom or N0 2.
  • R represents a hydrogen atom or a N0 2.
  • R represents a hydrogen atom or a nitrophenyl group.
  • Azolide groups such as
  • i represents a hydrogen atom or a phenyl group.
  • R represents a halogen atom which may be different from each other, CF 3 or NO 2.
  • R 3 is a lower alkyl group having 1 to 6 carbon atoms such as a methyl group and an ethyl group], for example,
  • 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 ⁇ , ⁇ -bistrimethylsilyl trifluoroacetamide-provylamine-acetonitrinole (1: 2: 2, ⁇ / ⁇ / ⁇ ) 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, ⁇ / ⁇ ) or dioxane-water (1: 1, ⁇ / ⁇ ) or THF-water (1: 1 ⁇ / ⁇ ) at room temperature for 5 minutes to give the present invention.
  • the ⁇ -phosphonate oligonucleotide derivative represented by the general formula (I) can be obtained.
  • the ⁇ -phosphonate oligonucleotide derivative of the present invention obtained by the above reaction steps has the following features.
  • the ⁇ -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.
  • 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.
  • it since it has higher water solubility than methylphosphonate-type DNA, it can be administered to cells at a high concentration.
  • 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.
  • 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.
  • 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.
  • an oligonucleotide derivative represented by the general formula (I) having a length of about 4 to 30, preferably about 10 to 20.
  • 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. .
  • 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.
  • 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 ⁇ 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.
  • 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 aqueous phase and washings (washings) were combined, extracted 3 times with CHC1 3, was back extracted the organic phase with CHC1 3.
  • 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.
  • filters were performed (10 ⁇ X 50 ⁇ ) 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 ).
  • trimethylammonium 6- (dimethoxytrityloxy) hexylphosphonate (triethyla onium 6- (dime thoxytrityloxy) hexyl phosphonate) (3) is converted into monomer units (“2a”, “2b”). ”And“ 2c ”) (Fig. 5).
  • 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.
  • CAGT tetranucleotide H-phosphonate
  • 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 °.
  • Example 2 Chemical synthesis of H-phosphonate oligodeoxyribonucleotide Chemical synthesis of H-phosphonate oligodeoxyribonucleotide was performed under different reaction conditions from Example 1.
  • 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).
  • 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).
  • 5′-0-dimethoxytrityldeoxycytidine (5, -0-dimethoxytrityldeoxycytidine) (lc) (0.570 g, (0,708 g, 94%) as a colorless foam.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.

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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

H-ホスホネ一卜オリゴヌクレオチド誘導体、 及び該誘導体の製造方法 技術分野  TECHNICAL FIELD The present invention relates to an H-phosphonate oligonucleotide derivative and a method for producing the derivative.
本発明は、 新規な H-ホスホネ一卜ォリゴヌクレオチド誘導体、 及び該誘導体の 製造方法に関する。 詳しくは、 ホスホジエステラーゼ耐性で、 かつ細胞への取り 込み効率の高い H-ホスホネートオリゴヌクレオチド誘導体に関する。 背景技術  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
近年、 遺伝子発現を阻害するアンチセンス DNAとして数多くの DNA類似体が合成 されている。 中でも、 ホスホロチォエート型 DNAとメチルホスホネート型 DNAにつ いて広く研究されている。 例えば、 ホスホロチォェ一ト型 DNAに関しては、 ヒト免 疫不全ウィルス (HIV) の発現制御 (C.Cazenave et al, Nucleic Acids Res.,15, 10507(1987), W. J. Stec et al, J. Am.Chem. ,106,6077(1984)、 S.Agrawal et al,Pro c.Natl. Acad. Sci. USA, 85, 7079(1988), T.Vickers et al, Nucleic Acids Res. ,19, 3359(1991)) 、 単純へルぺスウィルス (HSV) の発現制御 (W.Gao et al,J.Biol. Chem., 2643, 11521(1989)) 、 ゥシパピ口一マウィルス (BPV) の発現の制御 (L.M .Cowsert et al, Antimicrobial Agents and Chemotherapy, 37, 171 ( 1993 ) ) 、 c-m yc oncogeneの発現制御 (E.L.Wickstrom et al, In Vitro Cell Develop. Biol. ,2 5,297(1989)) 、 BCL-2 oncogeneの発現制御 (J. C. Reed et al,Proc.Natl.Acad. S ci.USA,87, 3660(1990)) 、 I CAM- 1タンパク質の発現制御 (J.A.M.Maier et al,Sc ience., 249, 1570(1990)) や IL- 1?タンパク質の発現制御 (J.Manson et al,Lymp hokine Res. ,9,35(1990)) などが報告されている。 また、 メチルホスホネート型 MAに関しては、 単純へルぺスウィルス (HSV) の発現制御 (C.C.Smith et al,Pr oc. Natl. Acad. Sci.USA,83,2785( 1985), M.Koziolkiewicz et al,Chem. Scripta,26 ,251(1986)) 、 水疱性口内炎ウィルス (VSV) の発現制御 (C.H.Agris et al,Bio chemistry, 25, 6268(1986)) 、 SV40の発現制御 (P.S.Miller et al,Biochimie,67 ,769(1985)) 、 N-ras oncogeneの発現制御 (D.M.Tidd,Anti-Cancer Drug Design ,3,117(1988)) 、 JGlobinの発現制御 (K. R. Blake, Biochemistry,24,6132( 1985) ) やクロラムフエニコールァセチルトランスフェラ一ゼの発現制御 (C.J.Marcus -Sekura, Nucleic Acid Res. ,15,5749(1987)) などが報告されている。 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.
しかしながら、 ホスホロチォェ一ト型 DNAには、 その二重鎖の安定性が低く、 標 的とする m A以外のタンパク質とも結合し、 非選択的な阻害を惹起するという問 題点があった。 また、 メチルホスホネート型 DNAは、 標的 mMAに高い特異性を有し ているが、 親和性が低いために有効な生物活性を示すためには高濃度で使用しな ければならず、 さらに、 水に対する溶解度が低いため高濃度で細胞に投与できな いという問題点があった。  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.
このような問題点を改良すべくいくつかの提案がなされている。 例えば、 ヒド 口キシメチル基を導入することにより、 水溶性などを高めたヒドロキシメチルホ スホネ一ト型 DNAが提案されている(Bioorganic Chem.22, 128(1994)、 Tetrahedron LeH 8845(1995))。  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)).
なお、 構造上、 二重鎖の安定性が高く、 高い水溶性を有し、 さらに RNaseの基質 になりにくいと考えられる H-ホスホネ一卜オリゴヌクレオチド誘導体としては、 その有用性にも関わらず本発明者らの知る限りにおいては、わずかに 2量体のもの が報告されているのみである (J. Org. Chem., 49., 139(1984)) 、 Tetrahedron Let t., 29, 2911 (1989), Tetrahedron Lett. ,30,4713( 1989)s Tetrahedron Lett., l,4 145 (1980) ) 。 また、 固相担体に結合した状態の H-ホスホネートオリゴヌクレオ チドについての報告例もあるが (W090/1202(PCT/US90/01865)) 、 固相担体から切 り出すと該ヌクレオチドは直ちに加水分解されてしまう。 即ち、 長鎖の H-ホスホ ネ一卜オリゴヌクレオチドについては、 これまでに合成し、 単離された報告例は 皆無である。 発明の開示 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
本発明は、 新規な H-ホスホネ一卜オリゴヌクレオチド誘導体、 及び該誘導体の 合成方法を提供することを課題とする。  An object of the present invention is to provide a novel H-phosphonate oligonucleotide derivative and a method for synthesizing the derivative.
H -ホスホネートオリゴヌクレオチドは塩基性条件下において極めて不安定であ る。 このため通常の DNA合成における塩基部位のァシル型保護基の除去および固相 担体からのオリゴマーの切り出しを行う場合には、 用いられるアンモニァ水処理 の過程で完全に分解されてしまう。 この場合でも、 H-ホスホネートオリゴヌクレ ォチドの 3'および 5'水酸基に保護基が導入されてレ、れば無水塩基性条件下におレ、 ても比較的安定である。 しかし、 保護基を除去してしまうと、 H-ホスホネート DN Aの 3'末端に存在する遊離の水酸基が 2' -デォキシリボースを介して 5'側に隣接す る H-ホスホネ一トジエステルを分子内求核攻撃し、 これにより H-ホスホネ一ト DN Aがヌクレオシド 3', 5' -環状ホスホネートを形成しながら逐次 3,側から分解されて しまう。 H-ホスホネ一ト DNAの 5'末端に存在する遊離の水酸基も同様の反応機構に より 2' -デォキシリボ一スを介して 3'側に隣接する H-ホスホネ一トジエステルを分 子内求核攻撃し、 これにより H-ホスホネート DNAがヌクレオシド 3,,5,- 環状ホス ホネートを形成しながら逐次 5'側から分解されてしまう (図 1 A ) 。  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).
そこで、 本発明者らは、 合成過程における H-ホスホネートオリゴヌクレオチド の塩基性条件下における分解を抑制し、 高収率で H-ホスホネートオリゴヌクレオ チドを合成する方法について、 鋭意研究を行った。 この結果、 本発明者等は、 固 相法でォリゴマーを合成する際に、 ォリゴマーの 3'末端および 5'末端に適当な鎖 長のアルキレン基をもつアルコキシホスホン酸の導入を行うと、 オリゴマーの 3, および 5'水酸基とそれにより分子内求核攻撃を受けていた H-ホスホネートジエス テルとの分子間距離が離れるため、 該水酸基の H-ホスホネ一トジエステルへの攻 撃が不可能となり、 無水塩基性条件下で反応を行った場合でも、 H-ホスホネート オリゴヌクレオチドの分解が顕著に抑制され (図 1 B ) 、 これにより H-ホスホネ 一卜オリゴヌクレオチドが効率よく合成されることを見いだした。 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.
本発明の H-ホスホネ一トォリゴヌクレオチド誘導体は、 下記一般式(I )で表され るものである。  The H-phosphone mononucleotide derivative of the present invention is represented by the following general formula (I).
Figure imgf000006_0001
Figure imgf000006_0001
[式中、 Bは互いに異なっていてもよい、 チミン、 シトシン、 ゥラシル等のピリミ ジン塩基、 アデニン、 グァニン等のプリン塩基または 5-メチルシトシン、 5-フル ォロウラシル、 5—ヒドロキシメチルシトシン等のそれらの誘導体を表す。 R1は 水素原子、 メチル基、 ェチル基、 プロピル基、 ブチル基等のアルキル基、 ビニル 基、 ァリル基等のアルケニル基、 ヒドロキシ基、 メ トキシ基、 エトキシ基、 プロ ピルォキシ基、 ブチルォキシ基、 メ トキシェトキシ基等のアルコキシ基、 ァリル ォキシ基等のアルケニルォキシ基、 またはメチルカルボニル基、 ェチルカルボ二 ル基、 メ トキシカルボニル基、 エトキシカルボニル基等のァシル基、 好ましくは 、 水素原子、 ヒドロキシ基、 メ トキシ基を表す。 R2は炭素数 1〜; 10、 好ましくは、 炭素数 6〜8等の分岐していてもよいアルキレン基を表す。 また、 これらアルキレ ン基は 1以上の酸素原子を介していてもよい。 例えば、 [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,
- ( O C H 2 C H 2 O --(OCH 2 CH 2 O-
(式中 Xは、 1〜5の数を表す) 等が挙げられる。 Xは酸素原子、 硫黄原子、 セレン 原子、 好ましくは酸素原子等のへテロ原子を表す。 nは 1以上、 好ましくは、 10〜 30、 特に好ましくは、 12~15の数を表す。 ] 本発明の H-ホスホネートオリゴヌクレオチド誘導体は、 例えば、 以下の工程か らなる方法によって合成することができる。 (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.
<反応工程 1 >  <Reaction step 1>
この工程は、 アルキレン基を導入した固相にモノマーを結合させる工程である (図 5参照) 。 一般式(II)  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)
R3 - 0 - R2 - 0— R4 (II) R 3 - 0 - R 2 - 0- R 4 (II)
[式中、 R3は、 トリチル基、 モノメ トキシトリチル基、 ジメ 卜キシトリチル基、 ピキシル基等の保護基を表し、 Γは式 [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-R5 -COCONH-R 5
(式中、 R5はァミノプロピル CPG、 長鎖アルキルアミノ CPG、 アミノメチルポリス チレン等の担体を表す。 ) を表し、 R2は前記と同義を表す。 ] で表される化合物 の保護基 R3を、 通常、 約1%のトリフルォロ酢酸/ 112(;12、約2.5%のトリクロロ酢酸 /CH2CI2, 約 3¾のジクロ口酢酸/ CH2C12などによって除去後、 下記一般式(III) (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)
Figure imgf000007_0001
Figure imgf000007_0001
[式中、 、Β、および Xは前記と同義を表し、 R6は一般式(II)中の R3と同様の保護 基を表し、 [Wherein,, Β, and X have the same meanings as described above; R 6 represents the same protecting group as R 3 in formula (II);
+  +
X1 X 1
はトリェチルアンモニゥム、 1,8-ジァザビシクロ [5.4.0] ゥンデ力- 7-ェニゥム 、 トリプチルアンモニゥム等のアンモニゥム塩、 リチウム、 ナトリウム、 力リウ ム等の金属塩などのカチォニック塩を表す。 ] で表される塩基モノマー単位と、 該モノマー単位に対して、 通常、 1〜5当量の脱水縮合剤の存在下、 ビリジン、 あ るいはァセトニトリル—ピリジン(l : l、v/v)中、 室温で 1〜10分程度反応させる。 上記一般式(I I )で表される化合物および一般式(I I I )で表される塩基モノマ一単 位は、 後述の実施例に示すような方法によって得ることができる (図 2、 図 7参 昭) 。 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). ).
かくして一般式(IV)  Thus, the general formula (IV)
Figure imgf000008_0001
Figure imgf000008_0001
[式中、 Β、Χ、 、ίί2、Ι14および R6は前記と同義を表す. ] で表される化合物を得る ことができる。 Wherein, Β, Χ,, ίί 2 , Ι1 4 and R 6. Representing the same meaning] can be obtained a compound represented by the.
く反応工程 2 >  Reaction step 2>
この工程は、 反応工程 1において得た固相に結合したモノマーに、 さらにモノ マ一を結合させてヌクレオチド鎖を伸長させる工程である (図 5参照) 。  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).
反応工程 1で得られる一般式(IV)で表される化合物の保護基 R6を通常、 約 の トリフルォロ酢酸/ CH2C12などの条件下に除去し、 前記一般式(I I I )で表される塩 基モノマー単位を反応工程 1の方法と同様にして反応させる。 この操作を繰り返 し行うことによって式
Figure imgf000009_0001
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
Figure imgf000009_0001
[式中、 Β,Χ, ,ί12, ,Γ,Ι16および nは前記と同義を表す] で表されるオリゴマー を得ることができる。 Wherein, Β, Χ,, ί1 2 ,, Γ, Ι1 6 and n are as defined above] can be obtained oligomer represented by.
<反応工程 3 >  <Reaction step 3>
この工程は、 反応工程 2で合成したオリゴマーの 5'末端に、 縮合剤の存在下、 アルキレン基を導入する工程である (図 5参照) 。  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).
反応工程 2で得たォリゴマーと一般式(V)  Oligomers obtained in reaction step 2 and general formula (V)
X X
II - + II- +
R7— 0— R2—〇一 P— 0 . X2 (V) R 7 — 0— R 2 —〇-1 P— 0. X 2 (V)
I  I
H  H
[式中、 R7は一般式(II)中の R3と同様、 あるいはァクリジン等のインターカレー 夕一等の保護基または官能基を表し、 [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,
X2 X 2
は前記 X1と同様のカチォニック塩を表し、 R2および Xは前記と同義を表す。 ] とを 縮合剤の存在下、 ピリジンあるいはァセトニトリル—ピリジン(1:1, v/v)中、 室温 で 1~10分反応させる。 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.
ここで用いられる縮合剤としては、 例えば、 式  As the condensing agent used here, for example,
0  0
II II
RSPX3 で表されるようなリン酸クロリ ド、 式 RSPX 3 Phosphoric chloride as represented by the formula
0  0
II  II
R8CX3 で表されるようなァシルク口リ ド、式 R1 CX3, as shown by R 8 CX 3 , formula
0  0
II  II
(R8C) 20 で表されるような酸無水物、 式 An acid anhydride represented by (R 8 C) 20 ,
R PX3 - Y R PX 3 -Y
で表されるようなホスホニゥム塩などが挙げられる。 And the like.
[式中、 R8は互いに異なっていてもよく、 メチル基、 ェチル基、 イソプロピル基 、 t -プチル基等のアルキル基、 フエニル基等のァリール基、 メ トキシ基、 ェトキ シ基、 イソプロポキシ基、 t-ブトキシ基等のアルコキシ基、 フエノキシ基、 へキ サフルオロフエノキシ基等のァリールォキシ基、 N, N-ジメチルァミノ基、 N, N- ジェチルァミノ基、 N, N-メチルェチルァミノ基、 N, N-ジフエニルァミノ基等の N, N-ジアルキルアミノ基、 N, N-ジフエニルァミノ基等の N, N-ジァリールァミノ 基、 ピロリジニル基、 ビペリジニル基、 モノホニル基等の環状アミン残基等、 好 ましくは、 ビロリジニル基を表す。 X3は、 塩素原子、 臭素原子等のハロゲン原子
Figure imgf000010_0001
[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
Figure imgf000010_0001
(式中、 Rは水素原子、 ハロゲン原子または N02を表す。 )
Figure imgf000010_0002
(式中、 Rは水素原子または N02を表す。 )
(Wherein, R represents a hydrogen atom, a halogen atom or N0 2.)
Figure imgf000010_0002
(Wherein, R represents a hydrogen atom or a N0 2.)
N = N N = N
- ΝγΝ γ Ν
R  R
(式中、 Rは水素原子またはニトロフヱニル基を表す。 )
Figure imgf000011_0001
Figure imgf000011_0002
(In the formula, R represents a hydrogen atom or a nitrophenyl group.)
Figure imgf000011_0001
Figure imgf000011_0002
等のァゾリ ド基、
Figure imgf000011_0003
Azolide groups such as
Figure imgf000011_0003
(式中、 iま水素原子またはフヱニル基を表す。 )  (Wherein, i represents a hydrogen atom or a phenyl group.)
— 0-N ' — 0-N '
R R
(式中、 Rは互いに異なっていてもよいハロゲン原子、 CF3または N02を表す。 ) (In the formula, R represents a halogen atom which may be different from each other, CF 3 or NO 2. )
— 0-N' 、、N — 0-N ',, N
N 一。 n N one. n
(式中、 Rは互いに異なっていてもよい Cl、 Fまたは N02を表し、 mは 0~5の数を表 す。 ) 等の活性化エステル、 (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 Y
は塩素イオン、 PF 6イオン、 BF 4イオン、 C104イオン等のァニオンを表す。 ] 好ましくは、 Represents chlorine ion, PF 6 ion, BF 4 ion, an Anion such C10 4 ions. ] Preferably,
Figure imgf000012_0001
Figure imgf000012_0001
[式中、 R3はメチル基、 ェチル基等の炭素数 1〜 6の低級アルキル基]、 例えば、 [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
Figure imgf000012_0002
Figure imgf000012_0002
などが挙げられる。 And the like.
また、 本発明においては、 c - xd · γ (式中、 Γ、 X3および Further, in the present invention, c− xd · γ (Where Γ, X 3 and
Υ Υ
は前記と同義を表す) 、  Represents the same meaning as described above)
+ +
•X3 · Υ • X 3 · Υ
R R8 2 Ν' RR 8 2 Ν '
(式中、 、x3および (Where,, x3 and
Y Y
は前記と同義を表す) 、  Represents the same meaning as described above)
Figure imgf000013_0001
Figure imgf000013_0001
(式中、 Rs、 X3および Where R s , X 3 and
Y Y
は前記と同義を表す) 、 または Is as defined above), or
R
Figure imgf000013_0002
R
Figure imgf000013_0002
R ''  R ''
(式中、 Rs、 X3および Where R s , X 3 and
Y Y
は前記と同義を表す) で表される化合物などを縮合剤として使用することもでき る。 かくして一般式 (VI ) Represents the same meaning as described above), and the like, and the like. Thus, the general formula (VI)
Figure imgf000014_0001
で表される化合物を得ることができる。
Figure imgf000014_0001
Can be obtained.
く反応工程 4 >  Reaction step 4>
この工程は、 反応工程 3で固相合成された H-ホスホネートオリゴヌクレオチド 誘導体を塩基性条件下で固相から分離し、 さらに加水分解により脱保護化して、 H -ホスホネ一卜オリゴヌクレオチド誘導体を単離する工程である (図 6参照) 。 反応工程 3で得られる一般式 (VI )で表される化合物を、 Ν,Ο-ビストリメチルシ リルトリフルォロアセ トアミ ド-プロビルアミン-ァセ トニトリノレ (1 :2:2, ν/ν/ν ) 等の無水塩基性の条件下に、 通常、 室温で 30分〜 1時間反応させ、 次いで、 濾過 により固相担体を除去し、 濾液を減圧下留去した後に、 ァセトニトリル-水 (1 : 1 , ν/ν) またはジォキサン-水 (1 : 1 , ν/ν) または THF-水 (1 : 1 , ν/ν) で、 室温下 、 5分間加水分解処理することによって、 本発明の前記一般式 (I ) で表される Η -ホスホネートオリゴヌクレオチド誘導体を得ることができる。  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.
以上の反応工程により得られる本発明の Η-ホスホネートオリゴヌクレオチド誘 導体は以下のような特徴を有する。  The Η-phosphonate oligonucleotide derivative of the present invention obtained by the above reaction steps has the following features.
まず、 本発明の Η-ホスホネートオリゴヌクレオチド誘導体はリン酸部位の立体 障害が従来のものに比べて小さく、 アンチセンスとして用いた場合、 標的遺伝子 との二重鎖を形成し易く、 ホスホロチォェ一ト型 DNAやメチルホスホネ一ト型 DNA 、 さらには、 天然型 DNAよりも有利である。 また、 中性分子であるために、 二重鎖 を形成したときに相補鎖のリン酸負電荷との静電反発が起こらないため、 天然型 DNAよりも安定な二重鎖を形成できる点で有利である。 さらに、 DNAのリン酸部に 負電荷を持たないため、 RNAとの間に形成される二重鎖は、 DNAの負電荷を認識し て Aを分解する酵素である RNaseHの基質となりにくいが、 標的遺伝子に対するァ ンチセンス効果の選択性はホスホロチォェ一ト型 DNAより有利である。 さらに、 メ チルホスホネート型 DNAよりも高い水溶性を有するため、 高濃度で細胞に投与する ことが可能である。 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.
従って、 本発明の H-ホスホネートオリゴヌクレオチド誘導体は、 特に、 アンチ センスとして有用であり、 例えば、 HIV、 HCV、 HCMV、 HSV、 HTLV等のウィルスの増 殖に際して生成するメッセンジャー RNA、 通常、 対象とするウィルスの生存に必須 のタンパク質をコードするメッセンジャー RNA、 例えば、 HIVの 「TAR」 、 「U5」 、 「rev」 、 rtatj (O.Zelphati,et al. ,Antisense Res. Dev. ,3,323(1993), B.Bo rdier,et al., Nucleic Acids Res. ,20,5999(1992)) 、 HCVの 「5に UTR」 (M.Alt, et al.,Hepatology,22, 707(1995)) 、 HSVの 「IE110」 、 「UL30」 、 「UL37」 、 「 UL37j (A.Peyman,et al. ,Biol.Chem.Hoppe Seyler,376, 195(1995)、 J.A.Smith, et al., J. Virol, 69, 1925(1995)) 、 HTLV- 1の 「tax」 などに相補的な塩基配列を有 するウィルス増殖阻害剤として、 或いは急性および慢性白血病、 肝臓癌、 乳癌、 大腸癌等の腫瘍細胞の増殖に際して特異的に生成するメッセンジャー RNA、 通常、 対象とする腫瘍細胞の生存に必須のタンパク質をコードするメッセンジャー MA、 例えば、 急性白血病の 「AML- 1」 (C . Sakakura, et al . ,Proc . Natl .Acad. Sci . USA, 91,11723(1994)) 、 急性および慢性骨髄白血病に関する 「bcr- abl」 (A.M.Tarai ,et al., Blood, 84,601(1994)) 、 白血病、 その他の癌に関する 「c- myc」 、 「c - m ybj (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)) 、 大腸癌に関する 「APC」 、 乳癌に関する 「BCA - 1」 などに相補 14 的な塩基配列を有する腫瘍細胞増殖阻害剤等として使用されうる。 このアンチセ ンスは、 通常、 4〜30、 好ましくは、 10〜20程度の長さの前記一般式(I )で表され るオリゴヌクレオチド誘導体を有効成分としている。 また、 その製剤の成分や使 用割合などは特に制限はなく、 従来アンチセンス治療剤として知られている種々 の製剤と同様に使用することができる。 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.
なお、 本発明の H-ホスホネートオリゴヌクレオチド誘導体は合成化学的に、 塩 基部およびリン酸部に特に保護基を用いる必要がないため、 合成反応の工程が短 縮され、 大量合成にも適している。 図面の簡単な説明  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
図 1は、 H-ホスホネートヌクレオチドの塩基性条件下での挙動を示す図である 図 2は、 H-ホスホネートオリゴヌクレオチドの単位となるモノマーの合成法を 示す図である。  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.
図 3は、 5'末端のホスホネートの合成法を示す図である。  FIG. 3 is a diagram showing a method for synthesizing a 5 ′ terminal phosphonate.
図 4は、 固相へのリンカ一の導入方法を示す図である。  FIG. 4 is a diagram showing a method for introducing a linker into a solid phase.
図 5は、 H-ホスホネ一トオリゴヌクレオチドの固相合成の工程を示す図である 図 6は、 固相合成された H-ホスホネートオリゴヌクレオチドから保護基を除去 する工程を示す図である。  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.
図 7は、 H-ホスホネートオリゴヌクレオチドの単位となるモノマーの合成法を 示す図である。  FIG. 7 is a diagram showing a method for synthesizing a monomer that is a unit of an H-phosphonate oligonucleotide.
図 8は、 5,末端のホスホネートの合成法を示す図である。  FIG. 8 is a diagram showing a method for synthesizing 5, terminal phosphonate.
図 9は、 本発明において用いられる固相へのリンカーの導入方法を示す図であ る。 発明を実施するための最良の形態 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
以下に実施例を挙げてさらに本発明を具体的に説明するが、 下記の実施例は本 発明を制限するものではない。  Hereinafter, the present invention will be described more specifically with reference to Examples. However, the following Examples do not limit the present invention.
く実施例 1> H-ホスホネート DNAの化学合成  Example 1> Chemical synthesis of H-phosphonate DNA
( 1 ) 縮合剤、 2-ベンゾトリアゾール- 1 -ィルォキシ -1, 3-ジメチル- 2-ピロリ ジン- 1 -ィル- 1,3,2-ジァザホスホラニゥムへキサフルォロホスフェート (2 - B enzotriazol- 1 -yloxy-l,3-dimethyl-2-pyrrolydin- 1 -yl- 1,3,2-diazaphosp holanium hexaf luorophosphate) (BDPP)の合成  (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 -ジメチル- 2-ピロリジン- 1-ィル -1,3,2-ジァザホスホレン (1,3- Dimethyl- 2-pyrrolydin-l-yl-l,3,2-diazaphospholane) (F. Ramirez, A. V. Patwardham , H. J. Kugler, C. P. Smith, J. Am. Chem. Soc, 89, 6276 (1967)を参照) ( 4.59 g, 25廳 ol) をよく攪袢した無水 CH2C12 (50 mL)中の卜ヒドロキシベンゾト リアゾ一ル (1-hydroxybenzotriazol) (3.32g, 25誦 ol) 及び CC (4.8 mL, 5 O mmol)の懸濁液に、 アルゴン下、 - 78°Cで、 5分間かけて滴下した。 同じ温度で 2 時間攪拌後、 この混合液を室温まで徐々に暖め、 KPF6 (4.6 g, 25腿 ol)を含む水 (100 mL)で洗浄した。 その有機相を Na2S04で乾燥し、 濾過し、 半量まで濃縮した 。 エーテル(50 mL)を上記混合液に加え、 濾過により淡黄色の沈殿物を回収した。 その沈殿物をエーテルで洗浄し、 真空中(in vacuo), P205で乾燥し、 BDPP (8.56 g)を 73 の収率で得た。 この化合物は、 無色針晶として酢酸ェチルから再結晶さ せることができた。 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);  Mp 128-129 ° C (dec);
31P NMR (CDC )(541.52 (s, P+), -142.90 (7重線, J = 711.6 Hz, PF6- ); 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, ピロリジニ ル基の 3,4- CH2), 2.87 (6H, d, J = 11.2 Hz, N-CHa), 2.91 (2H, ddd, J: 8.9 Hz, 7.3 Hz, 5.6 Hz, ピロリジニル基の 2又は 5- CH2 ), 3.50(2H, ddd, J = 12.9 Hz, 9.1 Hz, 5.8 Hz, ピロリジニル基の 2又は 5- CH2 ), 3.64 (4H, ddd, J二 8.9 Hz, 4.3 Hz, 2.3 Hz, ジァザホスホレンの 4,5- CH2 ), 7.52 (1H, ddd, J = 8.3H z, 6.8 Hz, 1.3 Hz, ベンゾトリァゾリル基の 6- H), 7.72 (1H, dd, J = 8.6 Hz,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, ベンゾトリアゾリル基の 5- H), 7.78 (1H, d, J = 8.6 Hz, ベンゾトリ ァゾリル基の 4- H), 8.07 (1H, d, J 二 8.3 Hz, ベンゾトリアゾリル基の 7- H); 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);
13C NMR (CDCla) 526.45 (d, J = 9.8 Hz, N- CH3 ), 30.95 (d, J = 6.1 Hz,ピ ロリジニル基の 3,4-C), 45.78 (d, J二 15.8 Hz, ジァザホスホレンの 4,5- C), 4 8.68 (d, J = 4.9 Hz, ピロリジニル基の 2, 5- C) , 108.19, 120.63, 126.15,127. 58, 130.60, 142.89 (ベンヅトリアゾリル基); Anal. C14H22F6N60P2の計算値: C, 36.06; H, 4.76; N, 18.02. 実測値: C, 35.88; H, 4.59; N, 18.15. 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の
Figure imgf000018_0001
の計算値: 321.159.実測値: 321.158 (2 ) モノマー、 1,8-ジァザビシクロ [5.4.0]ゥンデク- 7-ェニゥム 5'- 0-ジメ トキシトリチルデォキシアデノシン- 3,-ィル ホスホネート (1,8- Diazabicyclo [5.4.0]undec-7-enium 5 -0-dimethoxytrityldeoxyadenosin-3' -yl phosphonat e) (以下、 「2a」 と称することがある)の合成
BDPP
Figure imgf000018_0001
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")
以下の方法 A及び方法 Bを用いてモノマーの合成を行った。  Monomers were synthesized using the following methods A and B.
方法 A (図 2) : 5,- 0-ジメ トキシトリチルデォキシアデノシン (5,_0- Dimeth ox tr i ty 1 deoxyadenos ine ) (以下、 「la」 と称することがある) (0.554 g, 1 m mol)を無水ピリジンを用いた繰り返し共脱水 (coevaporation) により乾燥し、 最 終的に無水ピリジン(5 mL)中に溶解した。 この溶液にホスホン酸ジフヱニル(1. 64 g,7 mmol)を加えた。 20分の攪拌の後、 この混合液を H20- Et3N (1: 1, v/v, 2 mL)で処理し、 さらに 20分室温で攪拌した。 この混合液を MeOH (5 mL)で希釈し、 1 Mのトリエチルアンモニゥムヒドロゲンカルボネ一卜 (triethyla画 onium hydr ogencarbonate) (10 mL)を加えた。 この混合液を Et20 (10 mL x 3)で 3回洗浄し、 その有機相を 1 Mの卜リエチルアンモニゥムヒドロゲンカルボネート (triethyla 誦 onium hydrogencarbonate) で逆抽出した。 この水相 (The aqueous layer) と 洗浄液 (washings) を混合し、 CHC -MeOH (2:1, v/v, 10 mL x 3)で 3回抽出し、 その水相を CHC13- MeOH (2:1, v/v)で数回逆抽出した。 その有機相と洗浄液 (wa shings) を混合し、 Na2S04を通じて乾燥し、 濾過し、 減圧下、 濃縮して乾固した 。 その残留物をシリカゲルカラム(30 gのシリカゲル)に通した。 クロマトグラフ ィ一は、 メタノール勾配(0-155 を適用して、 0.5%トリェチルァミンを含む CHC13 を用いて行った。 「2a」 を含む画分を混合し、 濃縮して乾固した。 生成物を CHC1 3-MeOH (2: 1, v/v, 10 mL)に溶解し、 0.2 Mの 1,8-ジァザビシクロ [5.4.0]ゥンデ ク- 7-ェニゥムヒドロゲンカルボネート (1,8- diazabicyclo[5.4.0]undec- 7- eniu m hydrogencarbonate) (10 mL)で洗浄した。 その水相を CHC13- MeOH (2:1)で数回 逆抽出し、 その有機相と洗浄液 (washings) を混合し、 Na2S04を通 じて乾燥させ 、 濾過し、 減圧下、 濃縮して乾固し、 無色の発泡体として 「2a」 (0.631 g,88%) を得た。 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.
方法 B (図 2) : 無水ピリジン(10 mL)中のリン酸(0.082 g, 10誦 ol, 無水ピ リジンを用いた繰り返し共脱水により乾燥したもの)溶液にビス (2-ォキソ -3-ォ キサゾリジニル) ホスフィニッククロライ ド (bis(2- OXO-3- oxazolidinyl)phosp hinic chloride ) (1.40 g, 5.5誦 ol)を加えた。 その反応混合液を室温で 20分間 攪拌した。 その混合液に 5'- 0-ジメチルトリルデォキシアデノシン (5'- 0- dimeth ox tr i ty 1 deoxyadenos ine ) (la) (0.554 g, 1 麵 ol)を添加した。 6時間の攪拌 後,H20 (1 mL)をその混合液に添加した。 それを CHC13- MeOH (2:1, v/v, 10 mいで 希釈し, 1 Mのトリェチルアンモニゥムヒドロゲンカルボネート (triethylammon ium hydrogencarbonate) (10 mL x 3)で 3回洗浄し,その水相を CHC13- MeOH (2: 1 , v/v)で数回逆抽出した。 その有機相と洗浄液 (washings) を混合し、 Na2S04を 通じて乾燥し、 濾過し、 濃縮して乾固した。 その粗生成物(トリェチルアンモニゥ ム塩 (triethyla腿 onium salt) )をシリカゲルカラムクロマトグラフィーで精製 し、 方法 Aで述べたようにして DBU塩に変換し、 無色の発泡体として 「2a」 (0.60 0 g,78%)を得た。 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.
31P墜 (CDC13)53.74 (dd, J 612.8 Hz, 8.5 Hz); Ή NMR (CDCL) (51.66 (2H, m, DBUの CH2 ), 1.73 (4H, m, DBUの CH2), 1.97 (2H, 5重線, J = 5.6 Hz, DBUの CH2 ),2.42-2.80 (2H, m, 2,,2"- H), 2.83 (2H, m, DBUの CH2), 3.36-3.44 (8H, m, 5,,5"-H, DBUの CH2 ),3.77 (6H, s, MTrの 0 CH3), 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, MTrの 3, 3,,5, 5, -H ) , 6.96 (1H, d, J = 612.3 Hz,P- H), 7.15-7.42 (9H, m, 3,3' ,5,5, - Hを除く DMT rの ArH), 7.99 (1H, s, 2- H), 8.26 (1H, s, 8 - H); 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), 84.1 7 ( - 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. 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.
「2a」 の Anal C4。H4
Figure imgf000020_0001
·5Η20の計算値: C,55.87;H,6.80;N,11.40.実測値: C, 55.83;H,6.72;N, 11.28.
Anal C 4 of "2a". H 4
Figure imgf000020_0001
· 5Η 2 0 Calculated:. C, 55.87; H, 6.80; N, 11.40 Found: C, 55.83; H, 6.72 ; N, 11.28.
「2a」
Figure imgf000020_0002
の計算値: 618.212.実測値: 618.212.
"2a"
Figure imgf000020_0002
Calculated for: 618.212. Found: 618.212.
( 3) モノマー、 1,8-ジァザビシクロ [5.4.0]ゥンデク -7-ェニゥム 5,- 0-ジメ トキシトリチルデォキシシチジン- 3,-ィル ホスホネート (l,8-Diazabicyclo[5 .4.0]undec-7-enium 5' -0-dimethoxytrityldeoxycytidin-3' -yl phosphonate) ( 以下、 「2b」 と称することがある)の合成 (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")
「2a」 の場合において述べたような方法 A及び Bを用いて、 5,- 0 -ジメトキシ 卜リナノレテオキシシナシン (5' -O-dimethoxytrityldeoxycytidine) (以下、 「1 b」 と称することがある) (0.530 g, 1 画 ol)から 「2b」 が無色の発泡体としてそ れぞれ 91% (0.679 g)と 74% (0.552 g)の収率で得られた。  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.
31P NMR (CDCh) 53.83 (dd, J = 612.8 Hz, 8.6 Hz); 3 1 P NMR (CDCh) 53.83 (dd, J = 612.8 Hz, 8.6 Hz);
Ή NMR(CDC13) 51.66 (2H, m, DBUの CH2), 1.71 (4H, m, DBUの CH2), 1.97 ( 2H, 5重線, J = 5.6 Hz, DBUの CH2), 2.22 (1H, m, 2,- H), 2.66 (1H, m, 2"- H ), 2.81 (2H, m, DBUの CH2), 3.35-3.51 (8H, m,DBl^5, ,5"-H, CH , 3.78 (6H , s, DMTrの 0CH3), 4.40 (1H, m, 4, - H), 5.05 (1H, m, 3, - H), 5.51(2H, d, J 二 7.3 Hz, 5-H), 5.91 (2H, bs, 4- NH2), 6.30 (1H, t, J = 6.1 Hz, l'-H), 6. 82 (4H, d, J = 8.9 Hz, DMTrの 3,3, ,5,5, -H), 6.92 (1H, d, J = 613.3 Hz, P- H), 7.17-7.41 (9H, m, 3, 3, ,5,5, -Hを除く DMTrの ArH), 7.80 (1H, d, J = 7.3 Hz, 6 - H); 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) ;
13C N R (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. 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.
「2b」 の Anal C39H4 eNsOaP, ·4Η20の計算値: 57.27;11,6.90; 8.56.実測値: C,57.38;H,7.06;N,8.82. 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」
Figure imgf000021_0001
の計算値: 594.201.実測値: 594.203.
"2b"
Figure imgf000021_0001
Calculated for: 594.201. Found: 594.203.
(4) モノマ一、 1,8-ジァザビシクロ [5.4.0]ゥンデク- 7-ェニゥム 5,- 0-ジメ トキシトリチルデォキシグアノシン- 3'-ィル ホスホネート (l,8-Diazabicyclo [5.4.0]undec-7-enium 5 -O-dimethoxytrityldeoxyguanosin-3' -yl pnosphonate ) (以下、 「2c」 と称することがある)の合成 (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")
「2a」 の場合において述べたような方法 A及び Bを用いて、 5'- 0-ジメトキシ 卜リチノレデォキシク、 'ソ7ノシン ( 5 ' -0-d imethoxy t r i t 1 deoxyguanos ι ne (以下、 「lc」 と称することがある) (0.570 g,. 1 mmol)から無色の発泡体として(2c)をそ れぞれ 94% (0.739 g)と 87% (0.684 g) の収率で得た。 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.
31P NMR (CDC13) 53.07 (dd, J = 618.9 Hz, 8.5 Hz); 3 1 P NMR (CDC1 3) 53.07 (dd, J = 618.9 Hz, 8.5 Hz);
Ή N R(CDC13) dl.58-1.78 (6H, m, DBUの CH , 1.95 (2H, 5重線, J : 5.6 Hz, DBUの CH2), 2.31-2.54(2H, m, 2, ,2" - H), 2.62-2.78 (2H, m, DBUの CH2), 3 .28-3.48 (8H, m, 5, ,5"-H, DBUの CH2), 3.78(6H, s, DMTrの 0CH3), 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-NH2), 6.82 (4H, d, J = 8.6 Hz, DMTrの 3,3,,5,5, - H), 7.02 ( 1H, d, J 二 619.3 Ηζ,Ρ- H), 7.16-7.44 (9H, m, 3,3,,5,5, - Hを除く DMTrの ArH), 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); 7.65 (1H, s, 8-H);
13C NMR (CDC13)19.89, 24.17, 26.97. 29.09, 33.26, 39.01, 41.33 (2,- C), 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. 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.
「2C」 の Anal
Figure imgf000022_0001
·6Η20の計算値 : 53.74;11,6.76;^10.97.実測値: C, 53.58;Η,6.54;Ν, 10.87.
Anal of "2C"
Figure imgf000022_0001
· 6Η 2 0 Calculated:. 53.74; 11,6.76; ^ 10.97 Found: C, 53.58; Η, 6.54 ; Ν, 10.87.
「2C」 の FAB+(M- DBU+H C H NsOeP,の計算値: 634.207.実測値: 634.208. Calculated value of FAB + (M-DBU + HCHNsOeP, for 2C): 634.207. Found: 634.208.
(5) 5,末端のホスホネ一ト、 トリエチルアンモニゥム 6- (ジメ トキシトリチ リレ才キシ)へキシノレ ホスホネ——卜 (Triethyla腿 onium 6— (dimethoxytrityloxy)h exyl phosphonate) (以下、 「3」 と称することがある)の合成 (図 3) (5) 5, terminal phosphonet, triethylammonium 6- (dimethoxytrityloxy) hexyl phosphonate (hereinafter referred to as “3”) (May be called) (Fig. 3)
1,6-へキサンジオール (1,6- Hexanediol) (0.104g, 1 舰 ol)を無水ピリジンを 用いた繰り返し共脱水により乾燥させ、 最終的に無水ピリジン(10 mL)中に溶解し た。 この溶液にジメ トキシトリチルクロライ ド (dimethoxytrityl chloride) (0 .373 g, 1.1 誦 ol)を添加し、 この混合液を室温で 3時間攪拌した。 この混合液を CHC13で希釈し、 5% NaHC03で 3回洗浄し、 その水相を CHC13で逆抽出した。 その有 機相と洗浄液 (washings) を混合し、 Na2S04を通じて乾燥し、 濾過し、 濃縮して 乾固し、 6- (ジメ トキシトリチルォキシ)へキサノ一ル (6- (dimethoxytrityloxy) hexanol) を粗混合液として得た。 他方、 無水ピリジン(10 mL)中のホスホン酸(0 .082 g, 10 mmol, 無水ピリジンを用いた繰り返し共脱水により乾燥したもの) の 溶液に、 ビス(2-ォキソ -3-ォキサゾリジニル)ホスフィニッククロライ ド (bis(2 -oxo-3-oxazolidinyl)Dhosphinic chloride) (1.40 g, 5.5 mmol)を添カ卩した。 2 0分間の攪拌の後、 この混合液を 6- ( dimethoxytr i ty 1 oxy ) hexano 1を含む混合液に 添加した。 20分間の攪拌後、 この混合液をピリジン(5 mL)で希釈し、 1 Mのトリエ チノレアンモニゥムヒ ドロゲンカノレボネ一卜 (triethylammonium hydrogencarbona te) (10 mL)を添加した。 この混合液を Et20で 5回洗浄し、 有機相を 1 Mのトリェチ Jレアンモニゥムヒ ド ンカリレホ不——卜 (triethylammonium hydrogencarbonate ) で逆抽出した。 水相と洗浄液 (washings) を混合し、 CHC13で 3回抽出し、 この 有機相を CHC13で逆抽出した。 有機相と洗浄液 (washings) を混合し、 Na2S04を通 じて乾燥し、 濾過し、 減圧下、 濃縮して乾固し、 「3」 (0.265 g, 63%)を淡黄色の 油状物として得た。 その残留物をシリカゲルカラム(30 gのシリカゲル)に通した 。 メタノールの勾配(0-4 )を適用して、 0.5%のトリチルァミン含む CHC13を用いて クロマトグラフィーを行った。 「3」 を含む画分を混合し、 濃縮して乾固した。 そ の生成物を CHCL- MeOH (2:1, v/v, 10 mL)に溶解し、 1 Mのトリェチルアンモニゥ ムヒドロゲンカノレボネー卜 (triethylammonium hydrogencarbonate) (10 mL)で洗 浄した。 水相を CHC13 (10 mL)で逆抽出し、 有機相と洗浄液 (washings) を混合し 、 Na2S04を通じて乾燥し、 濾過し、 減圧下、 濃縮して乾固し、 「3」 (0.230 g, 5 3%)を無色の油状物として得た。 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.
31P NMR (CDCla) 55.11 (dt, J = 607.9, 7.4 Hz); 31 P NMR (CDCla) 55.11 (dt, J = 607.9, 7.4 Hz);
NMR (CDCh) (51.19 (9H, t, J = 7.3 Hz, Et3Nの CH3), 1.28 (2H, m, CH2 ), 1.52 (4H, m, CH2), 2.77 (8H, q, J = 7.3 Hz, Et3Nの CH2, CH2), 2.94 (2H , t, J二 6.9 Hz, CH2), 3.67 (6H, s, DMTrの 0CH3), 3.74 (2H, t, J = 6.9 Hz , CH2), 6.72 (4H, d, J 二 8.9 Hz, DMTrの 3, 3,,5, 5, -H), 6.76 (1H, d, J = 6 08.1 Hz, P-H), 7.06-7.36 (9H, m, 3,3,,5,5, -Hを除く DMTrの ArH); 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);
13C NMR (CDC13)(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. 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) 固相 LCAA- CPGへのリンカ一の導入 (図 4) 1,6-へキサンジオール(0. 118 g, 1 mmol ) を無水ピリジンを用いた繰り返し共 脱水により乾燥させ、 最終的に無水ピリジン(10 mL )に溶解した。 この溶液にジメ トキシトリチルクロライ ド (dimethoxytrityl chloride) ( 0.373 g, 1. 1 腸 1 )を 添加し、 この混合液を室温で 3時間攪拌した。 この混合液を CHC13 (20 mL)で希釈し 、 5% NaHC03 (20 mL x 3)で 3回洗浄し、 その水相を CHC13 (50 mL)で逆抽出した。 そ の有機相と洗浄液 (washings) を混合し、 Na2 S04を通じて乾燥し、 濾過し、 濃縮 して乾固し、 6- (ジメ トキシトリチルォキシ)へキサノール (6- (dimethoxytrityl oxy)hexanol) を淡黄色の油状物として得た。 他方、 無水 CH3CN (4 mL)中のトリァ ゾール(0.173 g, 2.5 mmol, 無水ピリジンを用いた繰り返し共脱水により乾燥し たもの)の溶液にォキサリルクロライ ド (oxalyl chloride) (44 μ. , 0.5 mmol ) とピリジン(0.2 mL)を添加した。 5分間の放置後、 この混合液をピリジン- CH3CN( 2 : 1, v/v, 3 mL)中の 6- (ジメ 卜キシトリチルォキシ)へキサノール (6- (dimethox ytrityloxy)hexanol) の溶液に添加した。 1時間の放置後、 この混合液を反応容器 の中の LCAA-CPG ( 1 g)に添加した。 この混合液を 30分放置後、 CH3CN、 無水 MeOH 及び CH3CNで順次洗浄した。 CPGゲルを Ac20-ピリジン(1 : 9, v/v, 5 mL)で処理し た。 3時間の放置後、 CPGゲルをピリジン、 続いて CH3CNで洗浄し、 減圧下で乾燥さ せ、 LCAA-CPGに結合した 6- (ジメ トキシトリチルォキシ)へキシルォキザレート ( 6 - ( d imethoxy t r i ty 1 oxy ) hex 1 oxalate) を得た。 LCM- CPGに対する 6- (ジメ トキ シトリチルォキシ)へキシルォキザレート (6-(dimethoxytrityloxy)hexyl oxala te) の負荷量は、 DMTr解析により 29〃mol/g と評価された。 (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 ) オリゴマー、 H-ホスホネートオリゴデォキシリボヌクレオチドの合成 固相合成は、 「Appl ied Biosystems 380A synthesizers で、 又は 反応容器と して、 先端に制止装置をを有し底に栓を有するガラスフィルター(10 顏 X 50 誦 )を用いて手動で行った。 鎖の伸長の各サイクルは、 脱トリチル化 (detritylati on) (CH2C12中の 1% TFA ; 15秒)、 洗浄(CH2C12、 次いでピリジン)、 カップリン グ(ピリジン中において 0.1 Mのモノマ一( 「2a」、 「2b」、 「2c」 ) 及び 0.5 Mの BDPP; 2分)、 洗浄(ピリジン)、 キヤッビング (0· 1 M トリェチルアンモニゥムェ チルホスホネート, ピリジン中の 0.5 M BDPP; 2分)、 洗浄(ピリジン、 次いで CH2 Cl2 )から構成した。 合成の最終サイクルにおいて、 卜リメチルアンモニゥム 6- ( ジメ トキシトリチルォキシ) へキシルホスホネート (triethyla腿 onium 6- (dime thoxytrityloxy)hexyl phosphonate) (3) をモノマー単位( 「2a」 、 「2b」 、 「 2c」 )の代わりに用いた (図 5 ) 。 一般に、 1サイクル当たりの平均収率は、 DMTr 解析により 96- 99%と評価された。 鎖の伸長後、 DMTr群を CH2C12中の 1% TFAで 15秒 処理することによって除去し、 CH2C12、 次いで CHaCNで洗浄した。 CPGゲル上の H- ホスホネートオリゴマーを N, 0-ビス(トリメチルシリノレ)ァセトアミ ド- CH3CN (N , 0-bis(trimethylsi lyl )acetamide-CH3CN) ( 1 :2, v/v, 300 / L)で 20分間処理し 、 この混合液に n- PrNH2 (200〃L )を添加し 30分間放置した。 CPGゲルを濾過により 除去し、 濾液を減圧下濃縮した。 この粗精製物を 「01igo- Pak EX (Mi ll ipore社製 )」 カートリッジ上で精製した。 生成物を H20- CH3CN (3 :2, v/v)を用いて溶出させ 、 この溶出液を濃縮して乾固し、 3'及び 5'末端に 6- (ヒドロキシ) へキシルホスホ ネ一ト (6- (hydroxy)hexyl phosphonate) を有する H-ホスホネートオリゴデォキ シリボヌクレオチドを得た (図 6 ) 。 (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).
以 にテカチミ ンレート H-ホス小不一ト (Decathymidyiate H- phosphonate) (T10)とテトラヌクレオチド H-ホスホネート (Tetranucleotide H- phosphonate) (CAGT)の合成例を示す。  An example of the synthesis of decathyminate H-phosphonate (T10) and tetranucleotide H-phosphonate (CAGT) is shown below.
合成例 1 :化学式 Synthesis Example 1: Chemical formula
Figure imgf000026_0001
で示されるデカチミジレート H-ホスホネート (Decathymidylate H-phosphonate ) (T10 )の合成
Figure imgf000026_0001
Of decatimimidylate H-phosphonate (T10) shown by
差替 え 用紙 (規則 26) 24/1 - 上記した典型的な工程を用いて、 LCM- CPGに結合した l〃molの 6- (ジメ トキシ卜 リチノレ才キシ)へキシノレ才キザレー卜 (6— (dirmethoxytrityloxy)hexyl oxalate) からデカチミジレート H-ホスホネート (decathymidylate H-phosphonate) (61. 4 A260 units)を 82%の収率で得た。 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;  UV (CH3CN-H2O, 1: 1, v / vUmax: 262.8 run, λιηΐη 233.0 nm;
31P NMR (CD3CN- H20, 1: 1, v/v) (510.95- 11.49 (多重線, 平均値として J二 731.6 Hz); FAB M+D+C H SN O PHの計算値: 3165.29.実測値: 3165.25 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
差替え用紙 (規則 26) 25 合成例 2:化学式 Replacement form (Rule 26) 25 Synthesis example 2: Chemical formula
Figure imgf000028_0001
で示されるテトラヌクレオチド H-ホスホネート (Tetranucleotide H- phosphona te) (CAGT)の合成
Figure imgf000028_0001
Of tetranucleotide H-phosphonate (CAGT)
上記した典型的な工程を用いて、 LCM-CPGに結合した l〃molの 6- (ジメ トキシト リチルォキシ)へキシルォキザレート (6- (dirmethoxytrityloxy)hexyl oxalate) からテ卜ラヌクレオチド H-ホスホネ一卜 (tetranucleotide H - phosphonate ) (31.0 A2 60 units)を 84°の収率で得た。  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 (CH3CN-H20, 1:1, v/v)人 max 259.2 nm, 入 min 229.8 nm; UV (CH 3 CN-H 2 0, 1: 1, v / v) person max 259.2 nm, input min 229.8 nm;
31P丽 R(CD3CN- H20, 1:1, v/v) (510.97- 12.29 (多重線, 平均値として J二 7 28.8 Hz); FAB+(M+1)+C51H77N15025 P5の計算値: 1455.12.実測値: 1455.09. 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.
差替 え 用紙 (規則 26) 25/1 得られたテトラヌクレオチド H-ホスホネート (tetranucleotide H- phosphona te) (1.0 A26。 units)を濃 NH3-ピリジン(9: 1, v/v, 5 mL)で室温にて 10分間処理 し、 H-ホスホネートのジエステル結合を切断した。 この混合液を脱水して乾燥さ せ、 無水ピリジン(300〃L)中に溶解した。 この溶液に Et3N (42 zL)及び BSTFA (8 1〃L)を添加した。 室温で 2時間の放置後、 無水デカン(100 /L)中の 5.0 M t-Bu00 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)
差替え用紙 (規則 26) 26 Replacement form (Rule 26) 26
Hを添加し、 この混合液を室温で 10分間放置した。 この混合液を脱水して乾燥させ 、 濃 NH3-ピリジン(9: 1, v/v, 5 mL)中に溶解した。 5分間の放置後、 この混合液 を脱水して乾燥させ、 0.1M Tris- HC1緩衝液 (pH 8.0, 100 〃L)に溶解した。 この 溶液に、 子牛の腸のアルカリホスファタ一ゼ(10 units)を添加し、 37°Cで 3時間ィ ンキュベ一卜し、 dC, dA, dG,及び dTのほぼ 1:1:1:1の混合液を得た(逆相 解析 )。 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).
く実施例 2 > H-ホスホネートオリゴデォキシリボヌクレオチドの化学合成 実施例 1と異なる反応条件で H-ホスホネートオリゴデォキシリボヌクレオチド の化学合成を行った。  Example 2> Chemical synthesis of H-phosphonate oligodeoxyribonucleotide Chemical synthesis of H-phosphonate oligodeoxyribonucleotide was performed under different reaction conditions from Example 1.
( 1 ) モノマー、 トリメチルアンモニゥム 5, -0-ジメ トキシトリチルデォキシ ァテノシン一 3,ーィノレ ホスホ不一卜 (Triethylammonium 5,一 0— dimethoxytritylde oxyadenosin-3' -yl phosphonate) (2a)の合成  (1) Synthesis of the monomer, triethylammonium 5,10-dimethoxytrityldeoxyadenosin-3'-yl phosphonate (2a)
以下の方法 A及び方法 Bを用いてモノマ一の合成を行った。  Monomers were synthesized using Method A and Method B below.
方法 A (図 7) : 5,- 0-ジメ トキシトリチルデォキシアデノシン (5,- 0-Dimeth oxy tr i ty Ideoxyadenos ine ) (la) (0.554 g, 1 minol)を無水ピリジンを用いた繰り 返し共脱水により乾燥させ、 最終的に無水ピリジン(5 mL)中に溶解した。 この溶 液にジフエニルホスホネート (diphenyl phosphonate) (1.64 g, 7誦 ol)を添加 した。 20分の攪拌後、 この混合液を H20- Et3N (1:1, v/v, 2 mL)で処理し、 室温で さらに 20分攪拌した。 この混合液をピリジン(5 mL)で希釈し、 1 Mのトリエチルァ ンモニゥムヒドロゲンカノレボネ——卜 (triethyla腿 onium hydrogencarbonate) (1 O mL)を添加した。 この混合液を Et20で 5回洗浄し、 その有機相を 1 Mのトリェチル アンモニゥムヒ ドロゲン力ノレ不不——ト (triethylammonium hydrogencarbonate) で逆抽出した。 その水相と洗浄液を混合し、 CHC13で 3回抽出し、 この水相を CHC1 3で逆抽出した。 有機相と洗浄液を混合し、 Na2S04を通じて乾燥させ、 濾過し、 減 圧下、 濃縮して乾固した。 その残留物をシリカゲルカラム(30 gのシリカゲル)に 通した。 メタノールの勾配(0-15%)を適用して、 4%のトリチルァミン含む CHC13を 27 用いてクロマトグラフィーを行った。 「2a」 を含む画分を混合し、 濃縮して乾固 した。 その生成物を CHC13(10 mL)に溶解し、 1 Mのトリェチルアンモニゥムヒドロ ゲンカノレボネー卜 (triethyla腿 onium hydrogenca bonate) (10 mL)で洗净し、 シ リカゲルを除去した。 水相を CHC13 で逆抽出し、 有機相と洗浄液 (washings) を 混合し、 Na2S04を通じて乾燥し、 濾過し、 減圧下、 濃縮して乾固し、 「2a」 (0.5 89 g, 82%)を無色の発泡体として得た。 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.
方法 B (図 7) : 無水ピリジン(10 mL)中のホスホン酸(0.082 g, 10議 ol, 無 水ピリジンを用いた繰り返し共脱水により乾燥したもの) の溶液に、 ビス(2-ォキ ソ- 3-ォキサゾリジニル)ホスフィニッククロライ ド (bis(2- OXO- 3- oxazolidinyl )phosphinic chloride) (1.40 g, 5.5 腿 ol)を添加した。 反応混合液を室温で 20 分間攪拌した。 この混合液に 5'- 0-ジメ 卜キシトリチルデォキシアデノシン (5'- O-dimethoxytrityldeoxyadenosine) (la)(0.554 g, 1 腿 ol)を添加した。 6時間の 攪拌後、 混合液を CHC13で希釈し、 1 Mのトリェチルアンモニゥムヒドロゲンカル ボネ一ト (triethylammonium hydrogencarbonate) で 3回洗浄し、 その水相を CHC 13で逆抽出した。 有機相と洗浄液 (washings) を混合し、 Na2S04を通じて乾燥し 、 濾過し、 減圧下、 濃縮して乾固した。 その粗生成物を上記のようなシリカゲル カラムクロマトグラフィーにより精製し、 無色の発泡体として 「2a」 (0.563 g, 78%)を得た。 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.
31P NMR (CDCh) (53.88 (dd, J PH = 616.5 Hz, J PCH = 8.5 Hz); 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, Et3NH+の CH3), 2.70-2.83 (2H, 111, 2'-H and 2"- H), 2.92 (6H, q, J = 7.3 Hz, CH2Et3NH+), 3.32 (3H, m, 5, - H, and 5- H), 3.69 (6H, s, DMTrの 0CH3 ),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, DMTrの 3,3,,5,5,- H), 6.85 (1H, d, J PII = 617.0 Hz, P- H) , 7.14-7.34 (9H, m, 3,3,,5,5, - Hを除く DMTrの ArH), 7.91 (1H, s, 2- H), 8.20 (1H, s, 8 - H); 28 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
13C NMR (CDCls) 58.68 (Et3NH+の CH3 ), 39.70 (2,-C), 45.41 (Et3NH+の CH2 ), 55.11 (DMTrの 0CH3), 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の tert-C), 113.03 (DMTrの 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 8 (DMTr Ar-C). 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) モノマ一、 トリェチルアンモニゥム 5,- 0-ジメ トキシトリチルデォキシ シチジン一 3,ーィノレ 3、スホ不——卜 (Triethyla腿 onium 5,一 0— dimethoxytrityldeo xycytidin-3' -yl phosphonate) (2b)の合成  (2) Monomer, triethylammonium 5, -0-dimethoxytrityldeoxycytidine-1,3-inole3, triethylamonium 5,10-dimethoxytrityldeo xycytidin-3'-yl phosphonate) (2b)
「2a」 における場合に記載したような方法 A及び Bを用いて、 5'- 0-ジメトキシ トリチルデォキシシチジン (5'-0-dimethoxytrityldeoxycytidine) (lb)(0.530 g, 1 mmol)から 「2b」 を無色の発泡体として、 それぞれ収率 91% (0.635 g)及び 7 4¾ (0.518 g)で得た。  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.
31P NMR (CDCh) 53.67 (dd, JPH = 616.4 Hz, JPOCH = 8.5 Hz); 31 P NMR (CDCh) 53.67 (dd, J PH = 616.4 Hz, JPOCH = 8.5 Hz);
Ή NMR (CDC13)(51.21 (9H, t, J = 7.3 Hz, Et3NH+の CH3), 2.17 (1H, m, 2, - H), 2.59 (1H, m, 2"-H), 2.93 (6H,q, J = 7.3 Hz, Et3NH+の CH2), 3.32 (3H, m, 5, - H, and 5"- H), 3.69 (6H, s, DMTrの 0CH3 ),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 H z, 1,- H), 6.74 (4H, d, J = 8.9 Hz, DMTrの 3,3,,5, 5, -H), 6.81 (1H, d, JPH = 617.0 Hz, P-H), 7.11-7.34 (9H, m, 3,3,,5,5, - Hを除く MTrの ArH), 7.70 (1 H, d, J = 7.6 Hz, 6-H); 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);
,3C NMR(CDC13 ) (58.77 (Et3NH+の CH3 ), 40.61 (2,-C), 45.61 (Et3NH+の CH2) , 55.20 (MTrの 0CH3 ),62.82 (5,- C), 72.82 (3,- C), 84.91 (l'-C), 85.76 (4, - C), 86.67 (DMTrの tert- C), 94.68 (C-5), 113.21 (DMTrの 3, 3,,5, 5, -C), 141.2 4 (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の Ar- C). 29 グアノシン- 3,-ィル ホスホネ一ト (Triethyla画 onium 5, - 0- dimethoxytrityl , 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) モノマ一、 トリェチルアンモニゥム 5, -0-ジメ トキシトリチルデォキシ d eoxyguanosin-3' -yl phosphonate) (2c)の合成 (3) Monomer, synthesis of triethylammonium 5, -0-dimethoxytrityldeoxy deoxyguanosin-3'-yl phosphonate) (2c)
「2a」 の場合に記載したような方法 Aを用いて、 5'- 0-ジメ トキシトリチルデォ キシシチジン (5, - 0- dimethoxytrityldeoxycytidine) (lc)(0.570 g, 1 顏 ol)か ら 「2C」 (0,708 g, 94¾ )を無色の発泡体として得た。  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.
3 1P NMR (CDC ) 63.22 (dd, JPH 二 626.2 Hz, JPCH = 8.6 Hz); 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, Et3NH+の CH3 ),2.35 (1H, m, 2, - H), 2.58 (1H, in, 2"-H), 2.99 (6H, q, J = 7.3 Hz, Et3NH+の CH2), 3.26 (3H , m,5, - H, 及び 5"- H), 3.69 (6H, s, DMTrの 0CH3 ),4.29 (1H, m, 4,- H), 4.95 ( 1H, m, 3,- H). 6·10(1Η, dd, Jr , 2' /Jr , 2' ' = 4.6 Hz 及び 8.9 Hz, l'-H), 6. 74 (4H, d, J = 8.9 Hz, DMTrの 3,3,,5,5, - H),6.91 (1H, d, JPH 625.5 Hz, P - H), 7.11-7.35 (9H, m, 3,3,,5,5, - Hを除く DMTrの ArH),7.62 (1H, s, 8-H); )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);)
13C NMR (CDCla) 58.86 (Et3NH+の CH3 ), 40.81 (2,- C), 45.73 (Et3NH+の CH2 ), 55.19 (DMTrの 0CH3 ), 63.67 (5, - C), 74.81 (3,- C), 83.61 (1,-C), 85.14 ( 4,- C), 86.41(DMTrの tert- C), 113.21 (DMTrの 3,3,,5,5, - C), 117.10 (5-C), 13 5.60 (8 -ひ, 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 (DMTrの AT-C). 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) 5'末端ホスホン酸、 トリチルアンモニゥム 5- (ジメ トキシトリチルォキ シ)ペンチノレ ホスホネ一ト (Triethylammonium 5— (dimethoxytrityloxy)pentyl phosphonate) (3)の合成 (図 8)  (4) Synthesis of Triethylammonium 5- (dimethoxytrityloxy) pentyl phosphonate (3) (5) Phosphonic acid at the 5'-terminal, tritylammonium 5- (dimethoxytrityloxy) pentyl phosphonate
1,5-ペン夕ンジオール (1,5- Pentanediol) (0.104 g, 1 誦 ol)を無水ピリジン を用いた繰り返し共脱水により乾燥させ、 最終的に無水ピリジン(10 mL)中に溶解 した。 この溶液にジメ トキシトリチルクロライ ド (dimethoxytrityl chloride) (0.373 g, 1.1 醒 ol)を添加し、 この混合液を室温で 3時間攪拌した。 この混合液 を CHC13で希釈し、 5% NaHC03で 3回洗浄し、 その水相を CHC13で逆抽出した。 その 30 有機相と洗浄液 (washings) を混合し、 Na2S04を通じて乾燥し、 濾過し、 濃縮し て乾固し、 5- (ジメトキシトリチルォキシ)ペン夕ノール (5- (dimethoxytritylox y)pentanol) を粗混合液として得た。 他方、 無水ピリジン (10 mL)中のホスホン 酸(0.082 g, 10讓 ol, 無水ピリジンを用いた繰り返し共脱水により乾燥したもの ) の溶液に、 ビス(2-ォキソ -3-ォキサゾリジニル)ホスフィニッククロライ ド (b is(2-oxo-3-oxazolidinyl)phosphinic chloride) (1.40 g, 5.5 mmol)を添カロした 。 20分間の攪袢の後、 この混合液を 5- (ジメチルトリチルォキシ)ペン夕ノール ( 5 - ( d ime thoxy t r i ty 1 oxy ) pentano 1 ) を含む混合液に添加した。 20分間の攪拌後、 この混合液をピリジン(5 mL)で希釈し、 1 Mのトリエチルアンモニゥムヒドロゲン カノレポ'不一卜 (triethyla醒 onium hydrogencarbonate) (10 mL)を添カロした。 この 混合被を Et20で 5回洗浄し、 有機相を 1 Mのトリエチルアンモニゥムヒドロゲン力 レボネー卜 (triethylammonium hydrogencarbonate) で逆抽出した 0 水目と洗净 液 (washings) を混合し、 CHC13で 3回抽出し、 この有機相を CHC13で 逆抽出した 。 有機相と洗浄液 (washings) を混合し、 Na2S04を通じて乾燥し、 濾過し、 減圧 下、 濃縮して乾固し、 「3」 (0.265 g, 63%)を淡黄色の油状物として得た。 この物 質は、 これ以上精製しなくとも分析上純粋であった。 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.
31P NMR (CDCla) 54.99 (dt, JPH = 614.0 Hz, JPCH 二 7.3 Hz); 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 二 7.3 Hz, Et3NH+の CH3), 1.36 (2H, m, C H2), 1.50 (4H, m, 2xCH2), 2.87 (6H, q, J = 7.3 Hz, Et3NH+の CH2), 2.90 (2 H, dd, J = 6.9 Hz 及び 3.0 Hz, DMTrO-CH2), 3.63 (6H, s, DMTrの 0CH3), 3.74 (2H, dd, J = 6.9 Hz 及び 7.6 Hz, P-0CH2), 6.69 (4H, d, J = 8.6 Hz, DMTrの 3,3, ,5,5,- H), 6.73 (1H, d, JPH 613.7 Hz, P- H), 7.05-7.55 (9H, m, 3,3, , 5,5, - Hを除く DMTrの ArH); 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);
13C NMR (CDCla) (58.07 (Et3NH+の CH3 ), 22.32 (CH2), 29.35 (CH2), 30.32( JPOCC = 7.3 Hz, P-OCH2CH2), 44.93 (Et3NH+の CH2 ), 54.72 (DMTrの 0CH3), 62. 88 (CH2), 63.27 (JPOC = 4.9 Hz, P-0CH2), 85.18 (DMTrの tert-C), 112.53 (D 31 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
^1 の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 (DMTrの Ar- C). 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) 固相 LCAA- CPGへのリンカ一の導入 (図 9 )  (5) Introduction of linker to solid phase LCAA-CPG (Fig. 9)
1,5-ペン夕ンジオール (1,5- Pentanediol) (0.104 g, 1 腿 ol)を無水ピリジン を用いた繰り返し共脱水により乾燥させ、 最終的に無水ピリジン(10 mL)中に溶解 した。 この溶液にジメ トキシトリチルクロライ ド (dimethoxytrityl chloride) (0.373 g, 1.1 顏 ol)を添加し、 この混合液を室温で 3時間攪拌した。 この混合液 を CHC13で希釈し、 5% NaHC03で 3回洗浄し、 その水相を CHCLで逆抽出した。 その 有機相と洗浄液 (washings) を混合し、 Na2S04を通じて乾燥し、 濾過し、 濃縮し て乾固し、 粗 5- (ジメ トキシトリチルォキシ)ペン夕ノール (5- (dimethoxytrityl oxy)pentanol) を得た。 他方、 無水 CH3CN(4 mL)中の卜リアゾール(0.173 g, 2.5 腿 ol, 無水ピリジンを用いた繰り返し共脱水により乾燥したもの)の溶液に、 ォ キサリルクロライ ド (oxalyl chloride) (44 uL, 0.5 腿 ol)及びピリジン(0.2 mL)を添加した。 5分間の放置後、 この混合液をピリジン- CH3CN (2:1, v/v, 3 mL )中の 5- (ジメ トキシトリチルォキシ)ペンタノ一ル (5- (dimethoxytrityloxy)pen tanol) の溶液に添加した。 1時間の放置後、 この混合液を反応容器中の LCAA- CPG1,5-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)に添加した。 この混合液を 1時間放置し、 順次 CH3CN、 無水 Me0H、 及び CH3C Nで洗浄した。 CPGゲルを Ac20ピリジン(1:9, v/v, 5 mL)で処理した。 3時間の放置 後、 CPGゲルをビリジン、 次いで、 CH3CNで洗浄し、 減圧下乾燥させ、 LCAA- CPGに 結合した 5- (ジメ トキシトリチルォキシ)ペンチルォキザレ一ト (5- (dimethoxytr ityloxy;pentyl oxalate) を得た。 (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) H-ホスホネ一卜オリゴデォキシリボヌクレオチド (H- phosphonate olig odeoxyribionucleotide) の固相合  (6) Solid phase synthesis of H-phosphonate oligodeoxyribionucleotide
合成例 1.  Synthesis example 1.
実施例 1の合成例 1において使用したモノマーが、 方法 Aまたは方法 Bで DBUの 代わりに TEAを使用して得られたチミジンモノマーである以外は同様にして、 デカ 32 チミジレート H-ホスホネ一ト (Decathymidylate H-phosphonate) を合成した。 合成例 2. 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.
実施例 1の合成例 1において使用したモノマーが、 方法 Aまたは方法 Bで DBUの 代わりに TEAを使用して得られたチミジンモノマーであり、 縮合剤として BDPPの代 わりに PyFOPを使用した以外は同様にして、 デカチミジレート H-ホスホネート ( Decathymidylate H-phosphonate) を合成した。  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.
合成例 3.  Synthesis example 3.
実施例 1の合成例 2において使用したモノマーが、 方法 Aまたは方法 Bで DBUの 代わりに TEAを使用して得られたチミジンモノマーである以外は同様にして、 テト ラヌクレオチド H-ホスホネート (Tetranucleotide H- phosphonate) を合成した 以上の実施例の結果を表 1に示す。  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.
表 1 オリゴ モノマー 縮合剤 平均収率 (%) 脱保護前 脱保護後の 単位の塩 (計算値) の収率 (%) の収率 (%) 実施例 T 10 DBU BDP P 99 61. 4 OD 合成例 (82%) 実施例 2 T 10 TEA BDPP 96 65 43. 50D 合成例 1 (58%) 実施例 2 T 10 TEA PyFOP* 95 62 42. 0OD 合成例 2 (56%) 実施例 1 CAGT DBU BDPP 99 95 31. 0OD 合成例 2 (84%) 実施例 2 CAGT TEA BDPP 96 26. 40D 合成例 3 ( 71 %) 33 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 F O P : ( Ν - Ρ+— 0 * P y FOP: (Ν-Ρ + — 0
し/
Figure imgf000037_0001
I /
Figure imgf000037_0001
文献 (H.H. Jensen, C. E. Olsen, A. Holm, J. Org. Chem. , 59, 1257( 1994)) に 従って合成した。 く参考例 1 > テトラヌクレオチドメチルホスホネ一ト(tetranucleotide met hylphosphonate)の合成  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
ォキザリルリンカ一(oxalyl linker)を介して LCAA- CPGに結合したテトラヌクレ ォチド H-ホスホネート(tetranucleotide H-phosphonate )( 1 〃mol )を 12の 5 %メタ ノールーピリジン溶液 (9: 1, v/v)で室温で 1 0分間反応した後、 ピリジン、 ァセ トニトリルで順次洗浄した。 ついで、 CPGゲルを n- PrNH广ァセトニトリル (1 :4, v/v) 中で室温で 3 0分間処理した。 CPGゲルを濾過により除去し、 濾液を減圧下 濃縮した。 この粗精製物を逆相 HPLCにより精製することでテトラ pヌクレオチドメ ナリレホスホネ一卜 (tetranucleotide methylpnosphonate)を得た 0 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 1P NMR (CD3CN- H20, 1 : 1, v/v) ό" 0.05-1.16, 3 1 P NMR (CD 3 CN- H 2 0, 1: 1, v / v) ό "0.05-1.16,
FAB MS (FAB ) Calcd for CssH8SNi503。P5 : 1603, Found 1603. FAB MS (FAB) Calcd for C ss H 8S N i5 0 3. P 5 : 1603, Found 1603.
く参考例 2 > テトラヌクレオチドヒドロキシメチルホスホネート(tetranucl eotide hydroxymethylphosphonate )の合成  Reference Example 2> Synthesis of tetranucleotide hydroxymethylphosphonate
ォキザリルリンカー(oxalyl linker)を介して LCAA- CPGに結合したテトラヌクレ ォチド H-ホスホネート(tetranucleotide H- phosphate )( 1 〃mol )をジォキサン(d ioxane)中で 1 Mの N,0-ビス (トリメチルシリル) ァセトアミ ド(N,0- bis(trimet hylsilyl )acetamide )と室温で 2時間反応させ、 ついでホルムアルデヒドガス(ga seous formaldehyde )と 1 0分間反応した。 ジォキサンで洗浄した後、 n- PrNHr "メ 夕ノール (1 :4, v/v)中で室温で 3 0分間処理した。 CPGゲルを濾過により除去し 、 濾液を減圧下濃縮した。 この粗精製物を逆相 HPLCにより精製することでテトラ  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
差替 え 用紙(規則 26) 34 ヌクレオチドヒドロキシメチルホスホネート(tetranucleotide hydroxymethylph osphonatejを得た o Replacement form (Rule 26) O Obtained tetranucleotide hydroxymethylphosphonate
3 1P 腿 (CDaCN-aO, 1 : 1, ν/ν) δ 28.52-29.78, 3 1 P thigh (CDaCN-aO, 1: 1, ν / ν) δ 28.52-29.78,
FAB MS (FAB ) Calcd for C5tH86N1503。Ps: 1603, Found 1603. FAB MS (FAB) Calcd for C 5t H 86 N 15 0 3. P s : 1603, Found 1603.
く参考例 3 > テトラヌクレオチドボラノホスホネ一ト(tetranucleotide bor anophosphonate )の合成  Reference Example 3> Synthesis of tetranucleotide boranophosphonate
ォキザリルリンカ一(oxalyl l inker)を介して LCAA-CPGに結合したテトラヌクレ ォチド H-ホスホネート(tetranucleotide H- phosphate )( 1 〃mol )を 1 Mの N,0 -ビ ス (トリメチルシリル) ァセトアミ ド- Me2S · BH3 ( 5 : 2 , v/v)と室温で 1時間反 応させた。 メタノールで洗浄した後、 n-PrNH3-メタノール ( 1 : 4, v/v)中で室 温で 3 0分間処理した。 CPGゲルを濾過により除去し、 濾液を減圧下濃縮した。 こ の粗精製物を逆相 HPLCにより精製することでテトラヌクレオチドボラノホスホネ ―卜 (tetranucleotide boranophosphonate)を得た。 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 1P NMR (CD3CN-H¾0, 1 : 1, ν/ν) δ 92.41-94.89. 3 1 P NMR (CD 3 CN-H ¾ 0, 1: 1, ν / ν) δ 92.41-94.89.
く参考例 4 > テトラヌクレオチドホスホアミデート(tetranucleotide phosp hoamidate)の合成  Reference Example 4> Synthesis of tetranucleotide phosp hoamidate
テトラヌクレオチド H-ホスホネ一ト(tetranucleotide H- phosphonate )(CAGT)を 5 %の12を含む飽和アンモニアピリジン溶液で 0 °Cで 5分間反応した。 ついで、 過剰 の Lを NaHS03水溶液で処理し、 得られた粗精製物を逆相 HPLCにより精製することで テトラヌクレオナドホスホア 丁一ト (tetranucleotide phosphoamidate) ¾得た 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 1P NMR (CD3CN-H20, 1 : 1, v/v) δ 12.47-14.33, 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. 産業上の利用可能性  FAB MS (FAB) Calcd for CsHO P;: 1528, Found 1528. Industrial applicability
本発明により、 新規な H-ホスホネートオリゴヌクレオチド誘導体、 及び該誘導 35 体の合成方法が提供された。 本発明の H-ホスホネートオリゴヌクレオチド誘導体 は、 標的遺伝子との二重鎖を形成しやすく、 ホスホジエステラーゼ耐性で、 かつ 細胞への取り込み効率の高いなどの特性を有する。 本発明の H-ホスホネートオリ ゴヌクレオチド誘導体は、 特にアンチセンス核酸としての利用が期待される。 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 請求の範囲 36 Claims
1. 下記一般式 ( I ) で表される H-ホスホネートオリゴヌクレオチド誘導体 c 1. H-phosphonate oligonucleotide derivative c represented by the following general formula (I)
Figure imgf000040_0001
Figure imgf000040_0001
[式中、 Bは互いに異なっていてもよいピリミジン塩基、 プリン塩基またはそれら の誘導体を表し、 R1は水素原子、 アルキル基、 アルケニル基、 ヒドロキシ基、 ァ ルコキシ基、 アルケニルォキシ基またはァシル基を表し、 R2は分岐していてもよ いアルキレン基を表し、 該アルキレン基は酸素原子を介していてもよい。 Xはへテ 口原子を表し、 nは 1以上の数を表す。 ] [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) —般式(11)、  2. (a) — General formula (11),
R3-0-R2-0-R4 (II) R 3 -0-R 2 -0-R 4 (II)
[式中、 R3は保護基を表し、 は式 [Wherein, R 3 represents a protecting group;
-CO CONH-R5 -CO CONH-R 5
(式中、 は担体を表す。 ) を表し、 R2は前記と同義を表す。 ] で表される化合 物の保護基 R3を除去後、 (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
一般式(ΙΠ)、 37 General formula (ΙΠ), 37
Figure imgf000041_0001
Figure imgf000041_0001
[式中、 R Bおよび Xは前記と同義を表し、 R6は保護基を表し、 [Wherein, RB and X have the same meanings as described above; R 6 represents a protecting group;
X1 X 1
はカチォニック塩を表す。 ] で表される塩基モノマー単位と反応させて、一般式( IV), Represents a catonic salt. ] With the basic monomer unit represented by the general formula (IV),
(IV)
Figure imgf000041_0002
(IV)
Figure imgf000041_0002
[式中、 Β、Χ、 、Ι12、ίί4, および R6は前記と同義を表す。 ] で表される化合物を得 る工程、 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) 前記工程で得られる一般式(IV)で表される化合物の保護基 R6を除去後、 前 記一般式(III)で表される塩基モノマー単位と反応させる工程、 (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) 前記 (b) と同様の操作を繰り返し行うことによって得られる化合物と、  (c) a compound obtained by repeating the same operation as in (b),
X X
II - + II- +
R7— 0— R2— 0— P—〇 ■ X2 (V) R 7 — 0— R 2 — 0— P—〇 ■ X 2 (V)
I  I
H  H
[式中、 R7は保護基を表し、 38 Wherein R 7 represents a protecting group, 38
X2 X 2
は、 カチォニック塩を表し、 R2および Xは前記と同義を表す。 ] で表される化合物 とを縮合剤の存在下に反応させて、 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,
一般式 (VI)、 χρηπ  General formula (VI), χρηπ
Figure imgf000042_0001
Figure imgf000042_0001
[式中、 ΙΤ、Ι12、Ι14、 、Βおよび Xは前記と同義を表し、 ηは 1以上の数を表す。 ] で 表される化合物を得る工程、 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
(d) 前記工程 (c) で得られる一般式(VI)で表される化合物を無水塩基性条件 下に反応させ、 次いで、 加水分解処理することによって請求項 1記載の H ホスホ ネートオリゴヌクレオチド誘導体を得る工程、  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
から成ることを特徴とする H-ホスホネートオリゴヌクレオチド誘導体の製造方法 A method for producing an H-phosphonate oligonucleotide derivative, comprising:
3. 下記の一般式 (V) で表される H-ホスホネートオリゴヌクレオチドの 5, 末 端合成用試薬。 3. Reagent for 5, terminal synthesis of H-phosphonate oligonucleotide represented by the following general formula (V).
X X
II - + II- +
R7— 0— R2—〇— P— 0. X2 (V) R 7 — 0— R 2 —〇— P— 0. X 2 (V)
H 39 H 39
[式中、 R\ [Where R \
+  +
X2 X 2
、 R2および Xは前記と同義を表す。 ] , R 2 and X have the same meaning as described above. ]
4. 下記の一般式 (VII) で表される H-ホスホネートオリゴヌクレオチドの 5' 末端合成用試薬。  4. A reagent for synthesizing the 5 'end of an H-phosphonate oligonucleotide represented by the following general formula (VII).
X X
II - + II-+
R7— 0— R9— 0— P— 0 · X2 (VII) R 7 — 0— R 9 — 0— P— 0 · X 2 (VII)
I  I
H  H
[式中、 ITは炭素数 4以上の分岐をしていてもよいアルキレン基を表し、 該アル キレン基は酸素原子を介していてもよい。 Γ、  [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,
+  +
X2 X 2
、 Xは前記と同義を表す。 ]  And X have the same meaning as described above. ]
5. ウィルスの増殖に際して生成するメッセンジャー RNAに相補的な塩基配列 を有するオリゴヌクレオチド誘導体を有効成分とするウィルス増殖阻害剤であつ て、 該オリゴヌクレオチド誘導体が、 請求項 1記載の Η-ホスホネートオリゴヌク レオチド誘導体であることを特徴とするウィルス増殖阻害剤。  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. 腫瘍細胞の増殖に際して特異的に生成するメッセンジャー RNAに相補的な 塩基配列を有するオリゴヌクレオチド誘導体を有効成分とする腫瘍細胞増殖阻害 剤であって、 該ォリゴヌクレオチド誘導体が、 請求項 1記載の Η—ホスホネートォ リゴヌクレオチド誘導体であることを特徴とする腫瘍細胞増殖阻害剤。  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. 下記の一般式 (V) で表される化合物の Η-ホスホネートオリゴヌクレオチ ドの 5' 末端合成における試薬としての使用。  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 X
II - ÷ II-÷
R7— 0— R2— 0— Ρ— 0 · X2 (V) R 7 — 0— R 2 — 0— Ρ— 0 · X 2 (V)
I  I
Η 40 Η 40
[式中、 R\ [Where R \
+  +
X2 X 2
、 R2および Xは前記と同義を表す。 ] , R 2 and X have the same meaning as described above. ]
8. 下記の一般式 (VII) で表される化合物の H-ホスホネートオリゴヌクレオチ ドの 5' 末端合成における試薬としての使用。  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 X
II + II +
R7— 0— R9—〇一 P—〇 · X2 (VII) R 7 — 0— R 9 —〇 一 P—〇 · X 2 (VII)
I  I
H  H
[式中、 R9は炭素数 4以上の分岐をしていてもよいアルキレン基を表し、 該アル キレン基は酸素原子を介していてもよい。 R7[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 ,
X2 X 2
、 Xは前記と同義を表す。 ]  And X have the same meaning as described above. ]
PCT/JP1997/004128 1996-11-13 1997-11-12 H-phosphonate oligonucleotide derivative and process for producing the derivative WO1998021226A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023160A1 (en) * 1994-02-23 1995-08-31 Isis Pharmaceuticals, Inc. Novel phosphoramidate and phophorothioamidate oligomeric compounds
WO1996022297A1 (en) * 1995-01-18 1996-07-25 Pharmagenics, Inc. Non-nucleotide phosphorus ester oligomers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023160A1 (en) * 1994-02-23 1995-08-31 Isis Pharmaceuticals, Inc. Novel phosphoramidate and phophorothioamidate oligomeric compounds
WO1996022297A1 (en) * 1995-01-18 1996-07-25 Pharmagenics, Inc. Non-nucleotide phosphorus ester oligomers

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NUCLEIC ACIDS RESEARCH, Vol. 18, No. 8, (1990), A. WILK et al., "Backbone-Modified Oligonucleotides Containing a Butanediode-1,3 Moiety as a 'Vicarious Segment' for the Deoxyribosyl Moiety-Synthesis and Enzyme Studies", p. 2065-2068. *
TETRAHEDRON LETTERS, Vol. 36, No. 46, (1995), K. SCHUTZ et al., "Synthesis of Oligonucleotides Labeled with a Novel Type of Chemically Stable Acridine Dye", p. 8407-8410. *

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