JPH0269191A - Deacylation of nucleosides - Google Patents

Abstract

PURPOSE:To selectively and easily produce a deacylated nucleoside with simple process by reacting nucleosides with lipase or protease. CONSTITUTION:(A) A nucleoside of formula I (B is substituted or unsubstituted nucleic acid base such as uracil; R1 is H or -OCOR; R is alkyl or substituted or unsubstituted phenyl) is dissolved in an aqueous mixed solvent containing 10-50wt.% of a polar solvent (e.g., pyridine) to obtain (B) a solvent system. The component B is added with (C) a lipase originated from microbial strain of genus Pseudomonas, etc., or (D) a protease originated from microbial strain of genus Rhizopus, etc., in an amount of 20-300g per 1mol of the component A and the components are made to react with each other in the presence of a buffering agent at pH6.5-7.5 and 10-60 deg.C for 0.5hr-2 days to obtain (E) a reaction product. The component E is extracted and purified to obtain a deacylated compound such as a compound of formula II (R2 is H or OH) as a reaction product of the enzyme C or a compound of formula III, etc., as a reaction product of the enzyme D.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for selectively deacylating sugar acyl groups of nucleosides using lipase and protease. Useful as a manufacturing intermediate.

(Prior art) Acyl groups are widely used as general protecting groups for the hydroxyl groups of sugar moieties of nucleosides, but their selective protection and deprotection require multi-step reaction steps, and at that time, acid and No satisfactory solutions have been found in terms of manufacturing methods, such as using alkali. Furthermore, there have been no examples in the field of nucleoside substrates in which primary or secondary esters of sugar hydroxyl groups are recognized regioselectively and deesterified.

(Problems to be Solved by the Invention) An object of the present invention is to provide a method for selectively, simple and easy deacylation of sugar moiety acyl groups of nucleosides using an enzyme.

(Means for Solving the Problems) The present invention relates to the general formula (wherein, B represents a substituted or unsubstituted nucleobase, R represents a hydrogen atom or -0COR, and R represents a fulkyl group or a substituted or unsubstituted nucleobase). A nucleoside represented by ) representing a phenyl group is reacted with lipase or protease to produce a compound represented by the general formula (wherein R2 represents a hydrogen atom or a hydroxyl group, and B and R have the same meanings as above). or a method for deacylating nucleosides, which is characterized by leading to a compound represented by the general formula (wherein B, R, and R have the same meanings as above).

More specifically, the compound represented by the general formula (]) is
When lipase is used as the sylation M, the sugar moiety 2
The acyl group -COR substituted at the `` or 3'' position is selectively eliminated, and a compound represented by general formula (2) is produced. On the other hand, when protease is used as a deacylating agent, the 7-syl group -COR substituted at the 5'-position of the sugar moiety is selectively eliminated, producing a compound represented by general formula (3). . In this way, by selectively using lipase and protease as deacylating agents, it is possible to control the position at which the sugar acyl group is eliminated.

In general formulas (1), (2) and (3), substituted or non-fi12 substituted nucleic acid represented by B (such as uracil, 5-
Fluorouracil, 5-trifluoromethyluracil,
5-r! such as 5-chlorouracil, 5-bromouracil, etc.
! biriminone bases such as uracil, thymine, and cytosine,
Purine bases such as atenine, guanine, xanthine, hypoxanthine, etc. are represented by R as an alkyl group,
Methyl, ethyl, propyl, 1-propyl, butyl, t
- C1-C18 alkyl groups such as butyl, hexyl, octyl, decyl, dodecyl, tetradecyl, octadecyl, substituted phenyl groups include chlorine, fluorine, alkyl-substituted phenyl, specifically 0-chlorophenyl,
p-chlorophenyl, 0-fluorophenyl, p-fluorophenyl, 0-methylphenyl, methylphenyl, p-ethylphenyl, p-butylphenyl.

Furthermore, as the lipase for this reaction, lipases derived from microorganisms, animals, and plants can be used.

Specific microorganisms include Enterobacter, Arthrobacter, Brevibacterium, Pseudomonas, alkaline earth metals, Microfuccus, Chromobacterium, Corynebacterium, Bacillus,
Lactobacillus metal, Trifderma sp., Candida sp.
Satucharomyces, Rhodotorgue, Cryptococcus, Torlobus, Vichia, Penicillium, Aspergillus, Rhizopus, Mucor, Aureobasidihum, Actinomucor, Mecardia, Streptomyces, Hansenula Examples include microorganisms belonging to the genus Achromobacter. These lipases are commercially available, and representative examples include porcine pancreatic lipase (Hanka Kagaku), Pseudomonas lipase P (Ohno Pharmaceutical),
Aspergillus lipase AP (Ohno Pharmaceutical Co., Ltd.), lipase B (Wako Pure Chemical Industries), Kyanzig.
(Meito Sangyo), Arthrobacter lipase joint BS
L (Godo Shusei), Chromobacterium lipase (Toyo Jozo), Candida (candida cylin)
lipase of the genus Rhizopus (Tanabe Seiyaku), lipase of the genus Rhizopus (Osaka Atomic Bacteria Research Institute), and lipase of the genus Rhizopus (Osaka Atomic Research Institute); )
? An example is P. Particularly, lipase P derived from Neudomonas fluorescens (Ohno Pharmaceutical Co., Ltd.) was suitable.

In addition, proteases used in this reaction include proteases from microbial Central Rice, animal origin, and plant white rice.
Commercially available proteases include Rhizopus niveus neurase F (Ohno Pharmaceutical), Aspergillus oryzae protease M (Ohno Pharmaceutical), Pacilus subtilis protease N (Ohno Pharmaceutical), Aspergillus meleus protease P, and animal pancreas. Vancreatin F (Ohno Pharmaceutical), Papain W-4 of Caripa Papain
An example is Protease N (manufactured by Ohno Pharmaceutical Co., Ltd.), and Protease N is preferred. This reaction is usually carried out in a mixed solvent system of polar solvent and water. As the polar solvent, trimethylformamide, pyridine, acetonitrile, trimethylsulfoxide, acetone, etc. can be used. The above mixed solvent system usually has a pH of
A buffer is added to keep it constant. The optimum pH for this reaction is usually 6.5 to 7.5, preferably 6.8 to 7.
．． 2, and in the reaction in which a buffer within these pH ranges could be used, a pH of 7.0 gave the best results. A single polar solvent or a mixture of a plurality of polar solvents can be used. The mixing ratio of water and polar solvent is appropriately selected depending on the solubility of the compound to be subjected to the deacylation reaction, and is usually preferably 10 to 50%.

The reaction temperature was 10-60°C. The optimum temperature for enzyme activation is 25-35°C. The reaction time is 0.
It takes 5 hours to 2 days, and the desired parent acylated nucleoside can be obtained in good yield. The ratio of lipase and protease used is nucleoside! (1) 20 to 300g per mole, preferably 100 to 200F
It is K.

The compound obtained in this reaction can be isolated and purified by conventional separation means, extraction, separation, concentration, reconsolidation, column chromatography, etc.

(Example) Examples of the present invention are shown below.

Example 1 3',5'-Nohexamyl-2'-deoxyuridine 142u6 (0,1ml Iole) was mixed with 20% trimethylformamide-0,IM'J in RH buffer solution (pH 7,0
) 1 nl of FJ suspension 11t and then Lipase P (Ohno Pharmaceutical) 40B were added and stirred at room temperature.・After hours,
After confirming by thin layer chromatography that the spots of the raw material had disappeared, 20 ml of ethyl acetate was added for extraction.

The lfi layer was diluted with saturated sodium bicarbonate solution (5k I
X 2 ) and saturated saline (50+al X 2 ), and then dried with Glauber's salt. After concentrating the organic layer under reduced pressure,
Isolation and purification by silica sol column chromatography gave 30 mg (yield 94%) of the target product, 5'-hexamyl-2'-deoxyuridine.

H' NMR (DMSO-di) 0.95 (3H,3,-CH-) 1.00-1.70 (6H2I11.(CH2)3
)2.00-2.50 (4H, m, I
i-2°, -CH2CO-)3.80~4
．． 40(4) (, cocoon, H-3', H-4'H-5") 5.35 (I H, d, 3'-0H) 5.6
5 (I H, d, H-5) 6.15 (I
H, t, H-1°) 7.82 (I H, d,
H-6) 11.40 (I H,s, N 3
H) Implementation-Examples 2 to 7 The following compounds were obtained in the same manner as in Example 1.

5°-Butyryl-2°-deoxyuridine Yield 90%) 1'-NMR (DMSO-d,) 0.90 (3+4, s, CH3)
1.05-1.88 (2H, m, -CH2-
)2, OO~2.40 (4H, II, T-1-2°
, -C82Co-)4,OO~4.20 (3H,
theory, ) (-3', t''-5') 4.30 (I +
(,lj, H-4')5.40<IH,d,3'-
0 to I) 5.60 (I H, d, T(-5) 6.15
(I H, L, H-1') 7.60 <1 1
4. d, H-6) 11.30 (I H, S
, N3-H) 5'-octa/yl-2'-deoxyturinone Yield 8296 II''-NMR (DMSO-d6) 0.90 (3H,!i, -C11,) 1.03-1
．． 95 (IOH, button, -(CH2)5-)2.00-
2.60 (489m, H2', CH2C0-)3
．． 85~4.35 (4H, rumor, H-3', H-4゜H
-5") 5.40 (I H, d, 3°-0H) 5.60 (
I H+ d, H-5) 6.20 (I H, L, H-1') 7.60 (I
H,d, H-(i)11.20 (L H,s,
N3-H) 5'-hexamyl-2'-deoxyurinone yield 84%! 1'-NMR(r)MS〇-dS)0.9
0 (3I(,s, -CI-13)1.80 (3H
, d, -CI()) 1.05-1.60 (G H,
+s, (CI-12)) )2.00~2.40 (
4H, Ifi, H-2', C)-(□Co-13
, 90 (I HIL II 4') 4.05-4.
25 (3H, m, t1-3',
H-5') 5.39 (I H, d, 3'-0H
)6.12 (1f-i, L, H-1')7.40
(l H, d, H-6) 11.40 (I H, S, N 3 H) 5'-hexaphyll-2'-deoxy-5-bromocrinone Yield 67% H'-NMR (DMSO-d, ) 0.95 (3H, stC+-13) 1.10-1.70 (6H2m, (CH2)-)1.
95-2.50 (4H, J l-(-2°, -C
1(, C0-) 3.85-4.25 (4H, ua,
H-3', H4'H-5°) 5.45 (I II, d, 3'-〇l-
()6.15 (I II, L, H-1')
7.60 (1tl, d, H-6) 11.4
5 (I H, st N 3-H) 5'-hexamyl-2'-deoxy-5-fluorouridine Yield 92% H'-NMR (DMSO-d,) 0.92 (3H, m, CHd l ,00~1.70 (GI(,J-(CH2)p
-)2.05~2.40 (4H, 1m, H-2',
-C82CO) 4.00-4.20 (3H, Chin, H
-3', H-5°) 3.90 (I H, theory, H-61
) 5.40 (I H, b, 3'-OH) 6.10
(I H, L, H-1') 7.80 (I H, d,
HG) 11.70 (I H, bs+ N 3+ () 5°-hexamethyl-2°-deoxyazo7sine yield 72% H'-NMR (DMSO-d,) 0.98 (3H, s, -CH )) 1.00~1
．． 70 (GH, n-(CH2)) )2.05
~2.45 (4H, +II, H2°, -CH2C0
-) 3.90 to 4,40 (41-1, In, H-3
°, ) (-, 1'H-5') 5.28 (I H, d, 3'-〇H) 6.1
0 (I H, t, +-1-1')7.30
(2H, s, N H2) 8.12 (I H, s, c-8) 8.31
(I H2St C2) Example 8 142 mg (0,1m+6ole) of 3°, 5°-nohex/yl-2′-deoxyurinone was added to 20% trimethylformamide-Oj, M phosphate buffer (pH 7,0 ) 1
ml, and then 40 mg of Protease N (Ohno Pharmaceutical Co., Ltd.) was added and stirred at room temperature.・After confirming by thin layer chromatography that the spot of the raw material had disappeared, enal acetate 20+al was added for extraction. There are 8! 1) Rum solution (5 kl x 2
), washed with saturated saline (50 [ΩI x 2), and dried with Glauber's salt. After concentrating the a layer under reduced pressure, the target product 3'-hexamyl-2'-deokyneuridine 31+n was isolated and purified by silica sol column chromatography.
H (yield 96%) was obtained.

+1'-NMR (DMSO-d,) 0.92 (3H, III, CH,) 1.001,70 (G II, m,
(CH2)3)2.00~28. iQ
(4I-1, jet, )l-2', -C
H2CO-)3.50-3.65 (3t(, IIl,
H4°, H-5°) 5.35 (IH, bs, H-
3') 5.20 (I H9bs, 5' Ol-1
) 5.65 (1t f, d, H-5) 6.1
2 (I H, L, Il -1') 7.85 (J
H, d, H-6) Il, 30 (I H, S, N 3-1-1) Examples 9 to 12 The following compounds were produced in the same manner as in Example 8.

3°-hexaphyll-2′-deoxythyminone yield 86
% H'-NMR (DMSO-c16) 0.90 (3H, s, CI+z) 1.00-1.68 (GH, In, -(CH□)
3- )1.82 (3+(,d, -CI-(,)2
．． 05~2.60(4F(, w, H-2°, -C
I-1, CO-) 3.60-3.70 (31-(, plow, H-4', H-5') 5.30 (I H, bS,
H-3')5.10 (I H, L, 5'-0H)6
．． 15 (1)(,t, H-1')7.60 (I
H, d, H-6) 11.40 (1, Hls, N 3 ) ()3゛-
Hexa/yl-2'-deoxy-5-fluorourinone Yield 88% H'-NMR (DMSO-d,) 0.85 (314, s, CH3) 1.00-1.80 (6H, recited, -(CH,),-)
2.052.60 (4H9m, H2', CH2
C0) 3.80-4.10 (3H, J H4',
l-15')5.10 (1)(, b, H-
3°) 5.15 (111,t, 5'-
0H) 6.10 (I H, L, H-1') 7
．． 77 (l H, d, H-6) 11.6
5 (I I-1, bs, N 3-14) 2',
3'-Nohexamyl-5-fluorourinone yield 40
% 1('-NMR(DMSOda) 0.82.0.85 (6t-1, S, CI-(3
)1.00~1.80 (12H1I11. (CH
2),)2,OO~ 2.40 (41-1, Mouse,!
(-2°, -CH2CO-)3.60 (2H,
b, H-5') 4.05 (I H2b+ H4') 5.20-5.50 (3H, m, H-2°, H-3
'5'O)+) 5.70 (I H, d, H-5) 6.00 (I H, d, H-1') 7.85 (I
H, d, H-61 11,28 (I H, s, N 3 H) 3°-hexa/yl-2°-deoxyazo/syn Yield 82% H'-NMR (DMSO-d6) 0.90 ( 3H, s, CHz) 1.03 to 1.75 (6H, In, (c+-12
)) )2.00~2.45 (4H+ u+, H2
', Cl-12co) 3.70-3.85 (3H,
Castle, H-4", H-5') 5.28 (I H, b,
H-3') 5.20 (5'-OH) 6.15 (I H, t, H-1') 7.3], (
2H= s, N H2) 8.14 (I H, s, l
-[-8) 8.32 (IH, s, H-2) (and above) Applicant Taiho Pharmaceutical Co., Ltd. Agent Patent attorney 1) Iwao Mura procedural amendment dated October 6, 1989, 1988 Patent Application No. 222059 Name of the invention Method for deacylation of nucleosides 3 Relationship with the case by the person making the amendment Patent applicant Taiho Pharmaceutical Co., Ltd. 7 Contents of the amendment (1) Specification Nakaon page 9 line 13 Correct "4 hours later" to "24 hours later".

(2) 3rd line from right below 14 on the statement Correct "4 hours later" to "24 hours later".

(3) In the specification, page 18, line 9 [5, 20 (5°-0H) J is corrected to "5, 20 (I H, L, 5'-0H) J.

4, generation implantation Address: 1-2-8 Sonezaki, Kita-ku, Osaka-shi, Osaka 530 (hereinafter referred to as Up)

Claims (1)

[Claims] (1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, B is a substituted or unsubstituted nucleobase, R_1 is a hydrogen atom or -OCOR, R is an alkyl group, or a substituted or unsubstituted phenyl The nucleosides represented by (representing a group) are reacted with lipase or protease, and the general formula ▲ has a mathematical formula, chemical formula, table, etc. ▼ (where R_2 is a hydrogen atom or a hydroxyl group, and B and R are the same as above). Compounds or general formulas (indicating meanings) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formulas, B, R_1 and R have the same meanings as above)
A method for deacylating nucleosides, which leads to a compound represented by: