JPS58135896A - Selective phosphorylating reagent in the 5'-position of nucleosides - Google Patents

Abstract

NEW MATERIAL:A compound of formulaI(X is halogen). EXAMPLE:Bis(5-chloro-8-quinolyl)-4-chlorophenyl phosphate. USE:A selective phosphorylating agent in the 5'-position of nucleosides, which are used in synthesis of artificial genes. In the molecule, it acts as a protecting group for phosphoric acid and contains eliminative groups. PREPARATION:The condensation reaction between 1-equivalent amount of 4- chlorophenyl phosphorodichloridate of formula II and 2-equivalent amount of 5-halogen-8-hydroxyquinoline is carried out in an organic solvent in the presence of a dehydrochlorinating reagent to give phosphoric triester of formula IV. Then, the triester is hydrolyzed to remove p-chlorophenyl group selectively, thus giving the objective compound of formulaI.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention is used for warping purposes. More specifically, the compound represented by the following formula (I) and its 01! Concerning methods and uses.

In recent years, advances in molecular biology have been remarkable, and deoxyribo1i
The structural functions of ribolium (凰Nmu) in al(DNA) were successively clarified, and at the same time, *S+O chemical synthesis also made great progress. It has become possible to synthesize 4IK artificial genes, and by performing genetic recombination operations using synthetic genes, we can produce notstatin, human in/nilin, human growth hormone, human interferon, etc., which are useful peptide hormones for batataeria. The technology is established
It's been coming.

Synthesis of oligogari leote F is essential for artificial gene synthesis, and various synthesis methods have been investigated, but there are still many problems with these synthesis methods, and there are many changes in synthesis/methods. Establishment of an excellent method is desired.

Ori containing 5'-terminal phosphoric acid among oligo-purchased Reote V:/! t-R)iA, which is naturally present in t-R) iA, is extremely important for the synthesis of rat N-mouligase and the synthesis of certain genes, and h9, one of its starting materials, 5'-terminal phosphorus at
A manufacturing method containing a reotide unit is desired.

The inventors! As a result of various studies on phosphorylating agents that selectively phosphorylate the 5' terminal hydroxyl group of the cleoside and contain a group in the molecule that acts as a phosphoric acid holding group and is easily released, we found that bis-(5-hen-8 Tomo ID discovered that -41noryl) phosphate is an extremely excellent phosphorylating agent and completed the present invention.

Nucleotides with a 5' terminal phosphate group polymerize to form tQ
- In the process of making 15t taleoted, it is necessary to preserve the phosphate group, and for that purpose, /d purchase 1v
A method of condensing a phosphoric acid monoester with an O/VK holding group or a method of condensation using deuterated cleotide and an alcohol t-dicyclohexylcarbodiimide such as 2-cyanoethanol, or a method of condensing a terminal phosphoric acid with 2.2. 2-
Diesterification such as trichloroethyl ester, ethylthioester, and phenylthioester, and amidation of phosphoric acid with aromatic amines were performed. but,
When these phosphate-retaining groups are used, the KFi-retaining group becomes unstable with respect to If2 and alkali, is difficult to eliminate, has an adverse effect on other retaining groups under degutation conditions, and furthermore esterifies KII.
There is no electrical point, such as the generation of side reactions.

However, when the compound of the present invention is used to phosphorylate a nucleon Vvr having a hydroxyl group at the 3' and 5' positions, only the 5' position is selectively phosphorylated, and the 3' position is not phosphorylated. - It is extremely convenient as there is no need to do so.

It is thought that the 31st position of the nucleoside is not phosphorylated probably because the 20S-dP noryl group contained in compound (I) is bulky.

This 0@- group is stable at 17 K in alkali and chloride.
It can be removed under more neutral conditions than K by working with aXa chloride steel (If). In addition, since it is fat-soluble, it dissolves well in organic solvents, and has the advantage of rapid reaction.It also emits fluorescence when irradiated with ultraviolet light, so it can be detected and preserved.
As a base, it has excellent features not found in conventional products.

The compounds of the present invention are produced by the following method.

(In the formula, X represents a halogen t'') That is, 4-chlorophenyl phosphorodichloride is subjected to a condensation reaction of 2 equivalents of 5-halogen-8-hydroxy-norrin in an organic solvent in the presence of a dehydrochlorination agent to obtain phosphoric acid. The phosphoric acid triester obtained by synthesizing the ester (1)
Shunyi Km can be prepared by hydrolysis to selectively eliminate Kp-tal phenyl groups. The organic solvent used in the condensation reaction may be any solvent as long as it is inert to the reaction, and examples thereof include ether, tetrahydrofuran, dioxane, and O-containing solvents such as acetone and methyl ethyl ketone. As the dehydrochlorination agent, not only organic bases such as trietherate, pyridine, and dimethylaminopyridine, but also alkali metal salts such as alkali carbonates can be used. Also, p- in the form of a metal salt of 5-halogen-8-oxy
R phenylphos can also react with rhodichloridate.

The reaction is carried out at low room temperature for 2 to 8 hours, preferably 4 to 6 hours or a
The reaction is completed by heating the reaction #i, and heating it slightly accelerates the reaction #i history. The elimination reaction of the p-chlorophenyl group from the produced triester (If) is carried out in the presence of a base, usually using a dilute alkali, aqueous ammonia or 2-pyridine aldosiloxane, but the reaction is prevented and the reaction is carried out selectively. Anmoeya water is a convenient source.

That is, [■] [object CI produced when mixed with an aqueous solution containing a base and stirred] becomes a solution and dissolves, so the progress of the reaction can be clearly seen. The reaction is carried out quantitatively, and is determined by obtaining the desired product in a yield of 9011 or higher even if it is purified.

Next, the compound of the present invention is useful as a selective phosphorylating agent at the 5'-position of nucleoside V in oligocleote V synthesis and to generate a protecting group at the 5'-position, and is further useful as a starting material unit for oligoiptate synthesis, for example, by the following formula: As shown in 4;
'rhp represents a tetrahydrofuranyl group) A compound in which the 5'-position is selectively phosphorylated when reacted with an allegoryoside [button] [Han] is obtained. [At Seki 5
-Hagen-B-The quinolyl group is stable in acids and alkalis, and if it needs to be removed, it can be easily eliminated using chlorinated steel [II] in zinc chloride under neutral conditions. Quiet is 3'
It is useful as a starting material for oligomers having a chain length on the side and a phosphoric acid group at the 5'-terminal position, and is used, for example, in the following reactions.

(In the formula, Todoroki represents uridine, enzoylated cytidine, enzoylated daanosine, or inzoylated adenosine, and tehp represents a tetrahydropyranyl group [, am represents a benzoyl group]. 'm meeting polymerization m
The desired oligonucleotide Vt can be generated by grinding the oat. As described above, the compounds of the present invention are extremely useful as selective phosphorylating agents for the terminal 5'-position of taleoside and as storage reagents for the 5'-position.

Next, the present invention will be explained with reference to Examples.
Of course, this is not something that can be determined.

II Example 1 Preparation of (5-rioo-49-4noryl)-4-chlorophenylphosphate 4-thalophenylphosphorodichloridate 548j1
15-Anhydrous Nato. 12. A solution of 5-talol-8- and 5.92 joules of shiquinoline dissolved in anhydrous tetrahydrofuran was added dropwise at 0°C. One
Dissolve 421 mtV ether in t-10ajo anhydrous tetrahydrofuran and add, and leave at room temperature for 6 hours! Mixed.

After adding cold water and mixing, the mixture was extracted with methylene chloride 20011, and the oil layer was washed with aqueous 5-sodium carbonate solution 9100, dehydrated over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography in a methylene chloride solvent system to give bis(5-chloro-a
-dFnoryl)-4-chlorophenylphosphate 6I
I (yield 76 capsules) was obtained. Melting point 118-120°C.

Elemental analysis (as CCtaHraC1sNtO*P):
Calculated value: C, 54, 21; kl, 2.6s;
N, 5.27%%-111: C, 5187;
H, 2,7+6; N, 5.281 Example 2 Preparation of bis(5-chloro-8-noryl)phosphate 6I bix (5-/o a -13-quinolyl) -
4-chlorophenylphosphate was added to tsooma ammonia water and stirred for 24 minutes at room temperature. Filter the fCO3 solution [reduced pressure a*t, and the residue found [recrystallized from water-acetonitrile, bis(5-chloro-8-quinolyl)phosphate] 3.1. A lE rate of 9011) was obtained. Melting point 171-175°C.

Elemental analysis (c,,n□C41N,O,P・5 horse 0) Two-point calculation value: C, 44,44; H, 575;
N, a7991 column value: C, 44, 58;
II, 570; N, 4081i Example S Preparation of 2'-.-Tetrahydropyranyluridine-5'-his(5-chloro-81'noryl)phosphate 2'-.-Tetrahydropyranyluridine 250 drops and bis(5-chloro-8-quinolyl)phosphate 5oo
Dissolve I1111 in pyridine 4-, add 750 Wt of 8-quinolinesulfonyltetoxol, and stir at room temperature for 2 hours. 1t reaction! After adding i to cold water 25- and stirring, extracting with methylene monochloride 100-, an oil layer tα5M T
The residue was purified by silica gel chromatography using a methylene chloride-methanol (98:2.V/V) solvent system, and the residue was purified by silica gel chromatography using a methylene chloride-methanol (98:2.V/V) solvent system. 500 mg (yield: 90) of -〇-tetra/1 idrovilaniru 5'-his(5-chloro-8-quinolyl phosphate) was obtained. Melting point: 99-101°C. Uv: λ 264 (# = 11800
).

-1 wealth 232aae; λ 246mm.

Elemental analysis (C,,H□C4,N,O,.P・2CH,
(as OH) 2 calculated values: C, 5145; H, 4
,66: N,7.191i actual value: C,52,
80; H, 4, 82; N, 491 Example 4 2'-〇-tetrano-1-hydrobyranyl-N4-indiylcytidine-5'-bis(5-chloro-8-quinolyl)
Synthesis of phosphates 2'-.-Tetrahydropyranyl-N4-benzoylcytidine 450q and bis(5-chloro-8-quinolyl) phosphate 459Iv? Dissolved in pyridine 5-8
- Added 979 drops of 1,000 norine sulfonyl tetrazolide and stirred for 2 hours, then carried out K161aYt in the same manner as in Example 5.
2'-・-tetra/-iF robi2-N4-benzoylcytidine-51-bis(5-chloro-8-quinolyl)
Phos7ate 66611 (yield 5 od) was obtained. Melting point 114-117°C.

Ma country UV, λ 262 (#=23000), 233
nm;λM@o&I245n,.

1m Elemental analysis (as CuHs@C1@Neo<*P): Calculated value: C, 54,42; II, 4.21;
N, 8.15 Am value: C, 54, d5; 11
, 4.46; N, 7.771! Okukoji Example 5 2'--Tetrahydropyranyl W proverb-<Nzoylquanosine-5'-bis(5-/Crow8-quinolyl)
Synthesis of phosphate 2'-.-Tetrahydropyranyl-Nl-,Hnzoyldaanosine 470 drops and bis(5-chloro-8=noryl) phosphate 459IIgf Dissolved in pyridine 5-, 8-? After adding 979 drops of norinsulfonyltetrazolide and reacting with stirring for 2 hours, post-treatment was carried out in the same manner as in Example 5 to obtain 2'-.-tetrahydropyranyl-Nm-inzoylguanosine-s'-biJ(s -Chloro-8-thousandoline) phosphate) 155Mg (yield 75%>k was obtained. Melting point 135-158°C. Uv: λ 290,264 (#=110
00).

Elemental analysis (C4°HstNyC40+. as P).

Calculated values: C, 54,93; H, M92; N
, 1121 Nut value: C, 55, 45; H, 4
, 45; Ru-t-benzoyladenosine 454~ and bis(5-chloro-8-quinolyl)
Dissolve 439 drops of phosphate in 5117 pyridine, 8
- 979m9'i of quinolinesulfonyltetrazolide was added, stirred for 2 hours, and treated with Kgk as in the case (Execution @ 5K). 5-chloro-8
-quinolyl) phosphate 609 drops (yield $71%) were obtained. One point 120256 t* wa * λ”@(M4
247no1 Hoi■ Elemental analysis (as C4゜HsacI*Nto@P):
Calculated value: C,55,? 5; H, 559; N
, 1t4211G actual column value: C, 5421; H,
&58; N, 1t4S9 过实儒7 Synthesis of diuridylyl (compound where in the formula (■) is a uridyl group) 2'-.-Tetrahydropyranyl uridine-5'-bis(5-terE!-8--IF noryl) phosphate) 5
48111F and 5-aa-9-quinolyl phosphate 187191 were dissolved in 3 ml of pyridine, 529 q/f of 8-quinolinesulfonyl chloride was added, and the mixture was stirred at room temperature for 1 hour, and then 10 d of cold water was added to the reaction solution for hydrolysis.
Extract with 100 methylene chloride. Oil layer t and 9α5MT
Wash with 1 m 50 m of water and 5 oaj of water, dehydrate with anhydrous sodium sulfate, and then reduce the pressure to II). The obtained phosphodiester was dissolved in pyridine 2.5114 and 2#
, 5e-,-dibenzoyluridine 1519 and 8-
? After adding Norinsulfoeltetrazolide 329M9 and stirring at room temperature for 2 hours, the reaction mixture was mixed with cold water and stirred, extracted with methylene chloride 10011j, and the oil layer t (15MTEA850-1, then washed with water 50-1,
After dehydrating with IJum (anhydrous sulfuric acid), it was concentrated under reduced pressure, and the residue was diluted with methylene chloride-methanol (97:3 v/v) I
The product was purified by silica gel chromatography using a medium I system to obtain 390 da of diuridylylic acid (yield: 85 min). Thin layer chromatography Rf = α56 (methylene chloride-methanol.

9:IV/V), UV: J”oH258s 25
0 avm *Agent Patent attorney 1) Parent Male

Claims (1)

[Scope of Claims] t A metal compound represented by the formula [1] (wherein X is a halogen t1!). 2. P-chlorophenyl phosphoric acid dichloride in the presence of a base with 5-halogen-8- and Shigyu Norin tffi
Triester obtained by fa [escape with alkali]
Method for producing compound (1) as rs mold. 5. The present sulforylating agent selected at the 5'-position shown in formula (1) 4.