JPS60199899A - Nucleoside 3'-phosphotriester derivative - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula I [B is N<6>,N<6>-dibenzoyladenine, N<2>-isobutylguanine, N<4>-benzoylcytosine, or thymine, X is halogen or nitro; Tr is group shown by the formula II (R1-R2 are H, or methoxyl)]. EXAMPLE:5'-O-( 4,4'-Dimethoxytrityl )-N<6>, N<6>-dibenzoyldeoxyadenosine 3'-( 2, 6-dichlorophenyl 5-chloro-8-guinolyl)phosphate. USE:A raw material for synthesizing oligonucleotide. PREPARATION:2,6-Dichlorophenyl phosphorodichloridate is reacted with 2,6-dichlorophenyl 5-substituted-8-quinoline in a solvent (e.g., THF, etc.) to give 2,6- dichlorophenyl 5-substituted-8-quinolyl-phosphorochloridate, which is reacted with 5'-substituted nucleoside in the presence of a condensation activator (e.g., 1- methylimidazole, etc.) to give a compound shown by the formula I .

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to nucleoside 3'-phosphotriester derivatives. More specifically, the general formula [5] useful for the production of oligonucleotides (in the formula B, N6, N6-dibencyyladenine, N
2-Inbuterylguanine N4-benzoylcytosine, thymine, X is a halogen atom or a nitro group or a methoxy group).

In recent years, DNA-? The structure and mechanism of action of RNA have been gradually elucidated. These advances in molecular biology are largely due to the development of nucleic acid synthetic chemistry. Applying this synthesis technology, artificial genes were synthesized, and through genetic manipulation, matostatin, human incillin,
BACKGROUND OF THE INVENTION Trace components found in living organisms that are extremely useful as pharmaceuticals, such as human growth hormone and human interferon, are now being produced in large quantities. Oligonucleotides are required for the synthesis of artificial genes. Although various methods have been developed for synthesizing oligonucleotides, the unit method is the most commonly used. That is, after synthesizing a nucleoside 3'-phosphotriester derivative in which a phosphoric acid group ester-bonded with a protecting group that can be removed under specific reaction conditions is introduced into the hydroxyl group at the 3'-position, this protecting group is In this method, oligonucleotides are synthesized from the resulting intermediates by treating triesters under specific reaction conditions, including the elimination of oligonucleotides. Therefore, in order to advantageously synthesize oligonucleotides, it is important to synthesize nucleoside 3'-phosphotriesters containing excellent protecting groups.

Conventionally, the following groups have been proposed as protecting groups for this purpose, but all of them have their own problems. For example, the leanoether group (K, Itakura
, etal, 1Can, J, Chem, +
51 +3649 (1973)) is unstable to bases, so it is partially eliminated even during reactions requiring the function of a protecting group, affecting the yield. More careful reaction handling is required. 2,2.2-trichloroethyl group (J, H,
It must be carried out under combined acidic conditions. Therefore, the protecting group for another purpose is removed, and on the other hand, the 2,2,2-trichloroethyl group is not removed sufficiently, resulting in low yield and purity of the target product. 4-rioo phenyl group (H, Takaku, et al., J, O
rg.

Chem, 46-2589 (1981))
Because it is relatively stable to bases, the removal conditions must be strong, causing reactions other than the intended one, reducing the yield of the target product, and complicating the operation to separate the target product from reagents and by-products. Make it. 2.5-dichlorophenyl group (E, Vh
lmann et al., Tetrahedro
n Lett, +21 +1181 (1980))
is unstable to silica gel, and secondary changes occur during product separation F*membrane production, making the purification of the target product complicated@ Therefore, the protecting groups required for oligonucleotide synthesis are It has properties such as being stable to some extent, being easily desorbed in a short time under commonly used basic conditions, easy to remove the reagent after desorption, and being stable under acidic conditions. It is desirable to have one.

The inventors have found that the 2,6-dichlorophenyl group is useful as a protecting group with the properties described above. Furthermore, the 5-substituted-8-quinolyl group (H, Tak
aku, etal, , Tetrahedron
2,6-dichlorophenyl 5-ft-substituted-8-quinolyl phosphorochloridate represented by the following formula [3], which is obtained in combination with Lett., 3857 (1979)), is an excellent phosphorylating agent for nucleosides. I found that. Furthermore, this compound [3] and nucleoside [
It has been found that the compound of the following formula [5] obtained by the reaction with [4] is an extremely useful starting material for oligonucleotide synthesis by the unit method.

Compound [5] is synthesized as follows.

[1] [2) [3] [5] (In the above formula, B, X, and Tr are the same as shown above) In the presence of a base, react with 5-substituted 18-hydroxyquinoline [2] to give 2.6
-dichlorophenyl-5-11-substituted-8-quinolylphosphorochloridate [3]. 5′-substituted nucleoside [4] in the presence of a condensation activator without isolating [3].
], nucleoside 3'-phosphotriester [5] can be easily obtained. The solvent used in this reaction may be any inert solvent, and examples include ether, tetrahydrofuran (THF'), acetone, mether ether lactone (MEK) dioxane, and dichloromethane.

Triethylamine, pyridine, and dimethylaminopyridine are used as the base, and the reaction temperature and pressure are in the range of -15° to +5°. When the reaction to obtain compound [3] is carried out at a temperature close to room temperature, bis(5-substituted-8-quinolyl) 2.6
-Dichlorophenyl phosphate is produced as a by-product. 2,6-
(1) and 5
The reaction with -substituted-8-hydroxyquinoline quantitatively produces 2,6-dichlorophenyl 5-substituted-8-quinolylphosphorochloridate [3] under the above conditions. Therefore, the reaction between [3] and 5'-substituted nucleoside [4] can be carried out continuously in the same vessel without isolating [3]. 2,6- combined with 8-quinolyl group
This is an advantage of using dichlorophenyl 5-substituted-8-quinolyl phosphorochloridate [3] as a phosphorylating agent.◎ The reaction between the compound of formula [3] and the compound of formula [4] produces 1-methylimidazole and triethylamine. , dimethylaminobi IJ, ? The process proceeds easily at room temperature in the presence of a condensation activator such as phosphorus, and provides the nucleoside 3'-phosphotriester [5] in good yield. The compound of formula [5] can be isolated and purified by silica gel column chromatography.

The compound of formula [5] thus obtained serves as a starting material for oligonucleotide synthesis. First, when the compound of formula [5] is hydrolyzed in the presence of a base, it can be converted in good yield to nucleoside 3'-phosphonoester [6] from which the 2,6-dichlorophenyl group is selectively removed. . Effective solvent systems used in this reaction include pyridine-water-tert-butylamine, acetonitrile-water-tert-butylamine, or systems using triethylamine or isopropylamine in place of tert-butylamine. In particular, pyridine-water-tert-butylamine (8:1:1 v/v) yarn gave the best results. The 2,6-dichlorophenol produced by the elimination can be easily removed by extracting the reaction system with diether or reprecipitating it in n-hexane.Next, the compound of formula [5] is added to 3-tidolychloroacetic acid. When treated with, nucleoside 3'-phosphotriester [7] from which the 5'-1 lythyl group has been removed is obtained. Under this condition 2
, 6-dichlorophenyl group is stable. When the compound of formula [5] and the compound of formula [6] obtained in this manner are reacted with the compound of formula [7] according to a conventional method in the presence of a condensing agent, dinucleotide [8] is obtained. The dinucleotide of formula [8] is further 3'-5'
By extending the linkage, oligonucleotides can be introduced.

B of the compound of general formula [5] of the present invention is H6,
H6-dibencyyladenine N2-imbutylguanine H4-benzoylcytosine or thymine,
The method for producing a compound in which X is chlorine is as follows: Production Examples 1 to 4
Shown below.

Production example 1 5'-0-(4,4'-dimethoxytrityl) -H6
, H6-shihenzoyldeoxyadenosine 3'-(2,
6-cyclophenylene/l15-chloro-8-quinolyl)
Phosphate i) 2.6-dichlorophenylphosphorodichloridate 3.35 g 'ff: 1JF=water THF 20 m
A solution of 2.58 g of 5-chloro-8-hydroxyquinoline dissolved in 20 ml of completely anhydrous THF was added dropwise to the mixture at -15°C. Next, triethylamine 2.
A solution of Omlf dissolved in 5 ml of anhydrous THF is added dropwise. When the reaction solution was warmed to room temperature and stirred for about 60 minutes, 2,6-dichlorophenyl 5-chloro-8-quinolyl phosphorochloridate [3] was obtained at constant volume.

11) Completely anhydrous 5'-0-(4,4'-dimethoxytrithiAI)-H by repeated concentration with anhydrous pyridine
6,H6-Dibensyyldeoxyadenosine At 3.72 pm, add the reaction solution containing [3] obtained in 1) above, further add 1.7 TrLl'i of 1-methylimidazole, and stir at room temperature for 45 minutes. . After adding 5 rnl of cold water to the reaction solution and stirring, it was concentrated, and the residue was extracted with cH2ct2.

The extract was washed with water, dried, concentrated, and the residue was dissolved in CH2Ct2.
When purified by silica gel column chromatography using -MeOH (98:2 v/v) solvent, 5'-0-(
dimethoxytrityl) -H6,H6-dipencyyldeoxyadenosine 3'-(2,6-dichlorophenyl 5
-chloro-8-quinolyl)phosphate) 4.33 g
(98%) was obtained. Melting point 114-117℃'H-N
MR (cDcz3): δ9.00-6.35 (m,
33H.

C2-H, C8-H, Ar)-, 6,10(d, IH,
J, /2/==6Hz, C1'-1(), 3.
75 (s, 6H, 0CH3).

3.27-3-95 (m, 2H*C2'-H
).

Elemental analysis (C6oH46N601oPC23) calculated value:
C, 62,75: H, 404: N, 7.32 disaster measurement value: C, 62,33; H, 3,89; N, 7.
47Rf = 0.75 (CH2Cl!2-MeOH
, 9: 1) UV: λ”0H277, 232nm
;λFOH254nmax Production example 2 5'-〇-(4,4'-dimethoxytrityl) -N2
-Imbutyryldeoxyguanosine 3'-(2,6-dichlorophenyl 5-chloro-8-quinolyl)phosphate In the same manner as in Production Example 1, 1.67 g of 2,6-dichlorophenylphosphorodichloridate, 5- Chloro-8-hydroxyquinoline 1.211. ) Liethylamide 71. O
1m(!, 5'-0-(4,4'-dimethoxytrityl) -N2-inbutyryldeoxyguanosine 1.61
When 1.141 nt of i1-methylimidazole is reacted, 5'-0-(dimethoxytrityl)-N2-imbutyryldeoxyguanosine 3'-(2,6-dichlorophenyl5-lyao-(3-quinolyl)phosphate-hl) ,
65.9 (81%) was obtained. Melting point 131-134℃
.

Rf 0.60 (CH2CCH2C1l2-.9:
1) UV: λ”0H282, 260, 234nm;
λ -269+255nm0eOH max man'H-NMR (cDcz5): δ11.80 (br
, , IH, NH).

9.70-8.45 (m, 3H, NH, Ar),
7.82 (d.

IH, Ca-H), 7.55-6.60 (m
, 19K, -Ar).

6, 40 (m, IH, C1'-H), 3.7
0 (s, 6H, 0CHs).

2, 30-1.70 (m, 2H, C2'-H).

Elemental analysis (049H44N6°10PC13) calculated value I
C, 58,03; H, 4,37; N, 8.29
Actual value: C, 58, 18; H, 4, 29; N,
7.98 Production Example 3 5'-0-(4,4'-dimethoxytrityl) -H4
-Pensoylcytidine 3'-(2,6-dichlorophenyl 5-chloro-8-quinolyl)phosphate In the same manner as in Production Example 1, 2,6-dichlorophenylphosphorocyclo+jr-to 4.1si, 5- Chloro-8-hydroxyquinoline 3.23,9. Triethylamine 2.52-1
5'-0-(4,4'-dimethoxytrityl)-H4-
5'-0-(4,
4'-dimethoxytrityl)-H4-benzoylcytidine 3'-(2,6-dichlorophenyl 5-chloro-8-
quinolyl)phosphate) 4.311 (84) were obtained. Melting point: 109-111°C.

Rf O,66(CH2(J2-MeOH, 9:1
).

IJv: λM@OH260+ 213 nm; λ
M80H252nm0max m1n 1H-NMR (CDC13) δ8.80-6.65 (
m, 29H, Cs-H.

C6-H, NH, Ar) 16.30 (t, IH,
J112/=6Hz.

C1”H), 3.73 (a, 6H, 0CHs)
, 2.70 to 2-20 (br, , 2H, C2'-H
).

Elemental analysis (052H42N4010P013) calculated value:
C, 61,22; H, 4,12; N・5.49 Actual value: C, 61,39; H, 4,10; N, 5
．． 59 Production Example 4 5'-0-(4,4'-dimethoxytrityl)thymidine 3'-(2,6-dichlorophenyl 5-chloro-8-
2.6-dichlorophenylphosphorodichloridate 2.51. p,5-chloro-8-hydroxyquinoline 1.93. ! i', h 1.51 m rietheramine, 2.14 g 5'-0-(4,4'-dimethoxytrityl)thymidine, 1-methylimidazo-A/
l, 71 Ml f reacted cell and 5'-0-(4
,4'-dimethoxytrityl)thymidine3'-(2,6
2.72 g (80 g) of -dichlorophenyl 5-chloro-8-quinolyl) phosphate were obtained. Melting point 115~
118°C, RfO,48(CH2Cl2-MeOH
, 9: 1).

UV: λMeOH, 268, 232 nm; λm251
nm.

aX 1HNMR (CDC13) δ9.85 (br, I
H, NH), 8.70-842 (m, 2H1Ar
), 7.70-665 (m.

19 Hr Ar ), 6-50 (t + IH, J
"-6Hz, C1'2-H), 3.74 (s, 6H, 0CR5),
2.70 (m, 2H.

C2'-H) + 1.45 (s + 3 H, C
Hs).

Elemental analysis (C46H6, N601oPC43・C6H4
4) Calculated values: C, 60, 88; H, 5, 39; N,
4.28 Actual value: C, 60,85; H, 5,29;
N, 4.04 An example of a method for producing the oligonucleotide represented by formula [8] using the compound of the present invention is shown below.

5'-0-(4,4'-dimethoxytrityl) -H6
-H6-dipencyyldeoxyadenylyl (3' -5
)N'-benzoyldeoxycytenone 3'-(2,
6-dichlorophenyl 5-chloro-8-quinolyl)phosphate i) 5'-0-(4,4'-dimethoxytrityl)-H6,H6-dipensyyldeoxyatenosine 3'-(2 ,6-dichlorophenyl5-chloro-
8-quinolyl) phosphate 9.11'! Dissolve in 20 g of digin-water-tert-butylamine (8:1:1),
After standing at room temperature for 1.5 hours, the reaction solution is concentrated. The residue was dissolved in CH2Cl2 and diluted with hexane-ether (9:
1 v/v), white crystals precipitate. When all P is lost, 6.42 g of 5'-o-(4,r=nidimethoxytrityl-H6,H6-dibensyyldeoxyadenosine 3'-(5-chloro-8-quinolyl)phos7ate) is obtained.

11) 5'-0-(4,4'-dimethoxytrityl)-H4-penzoyldeoxycintidine 3 obtained in Production Example 3
'-(2,6-dichlorophenyl5chloro-8-quinolyl)phosphate-) 2.04 g'e CH3No2-MeOH (95:5) mixture containing 3% trichloroacetic acid 6
Dissolve at 0d and leave at 0°C for 5 minutes. Neutralize the reaction solution with phosphoric acid buffer (pH-8,0). The entire organic layer was washed with water, dried and concentrated, and the resulting residue was washed with hexane to give N4-benzoyldeoxycytidine 3'-(2
, 6-dichlorophenyl 5-chloro-8-quinolyl)phosphate) 1.94& was obtained.

1ii) Phosphodiester obtained in i) 2.51. li
+ and phosphotriester 1.29 obtained in 11), ? Dissolve in anhydrous pyridine, concentrate, and repeat this process several times to dry. Dissolve this in 9 ml of pyridine, add 1.84.9 ml of 8-quinolinesulfonyl chloride and 1.29 ml f of 1-methylimidazole, and stir at room temperature for 1.5 hours. Add 2 liters of cold water to the reaction solution for hydrolysis, and extract with CH2Cl240 rrtl. Extract 'c 0. After washing with 101 rLl of laethyl ammonium vicar and 10 ml of water,
Concentrate and purify the residue by silica gel column chromatography. Elution with CH2Cl2-MeOH (95:5) yielded 5'-0-(4,4'-dimethoxytrityl)-N',N'-dibenzoyldeoxyadenylyl (
3'-5') N'-benzoyldeoxycytidine 3'-(2,6-dichlorophenyl 5-chloro-8-
Quinolyl) phosphate 2.51 F was obtained. Rf
= 0.59 (CH2Cl2-MeOH, 9:1
).

UV: λMeOH26o, 232 nm, λ, 24
0nm.

, MeOH maX mln Patent applicant: Fujirebio Co., Ltd. Patent applicant Hisahiro Taka

Claims (1)

[Claims] General formula (wherein B is N6, N6-dipencyyladenine, N
A nucleoside 3'-phosphotriester derivative having 2-inbutylguanine N4-penzoylcytosyl or thymine;