JPH0374238B2 - - Google Patents

Description

[Detailed Description of the Invention] As a method for producing oligodeoxynucleotides, a method of forming internucleotide bonds using a dehydration condensing agent is generally used.
The so-called triester method, in which the bond is in the form of a phosphotriester, is currently the mainstream method because the reaction product is stable and has high fat solubility, making it easy to separate and purify by chromatography. is doing. As the condensing agent, nitrogen-containing heterocyclic compounds of arenesulfonic acids such as 2,4,6-triisopropylbenzenesulfonyltriazole, 2,4,6-triisopropylbenzenesulfonyltetrazole, and 8-quinolinesulfonyltetrazole are used. .
These condensing agents have a strong dehydration effect and form internucleotide bonds in a short time, but
As a side reaction, it induces a sulfonation reaction to the 5' hydroxyl group and guanosine base moiety. Tetrazole condensing agents also have the disadvantage of converting the guanosine base moiety into tetrazole. Therefore, it is desired to develop an internucleotide bond forming reaction that suppresses these side reactions. The present invention provides a method for producing oligodeoxynucleotides that can suppress the side reactions described above and form internucleotide bonds in a short period of time and in high yield. For example, oligodeoxynucleotides can be simply and easily synthesized in high yield by a method that shortens the synthesis steps by at least two steps compared to the conventional triester method. First, to explain the deoxynucleotide represented by the above general formula [], which is the raw material compound of the method of the present invention, the protective group R ¹ of the 5'-position hydroxyl group includes a trityl group, a methoxytrityl group, a dimethoxytrityl group, etc. However, methoxytrityl group and dimethoxytrityl group, which can be easily and selectively eliminated by acid or Lewis acid, are suitable. Protecting groups R ² include phenyl, 2-chlorophenyl, 4-chlorophenyl, etc., but 2 can be easily deprotected with oxamate.
-Chlorophenyl group is most suitable. X is a leaving group, and a nitrogen-containing five-membered ring such as an imidazolyl group, a triazolyl group, or a benzotriazolyl group is suitable, but a triazolyl group is the most preferable group in consideration of the reactions described below. The deoxynucleotide represented by the general formula [] (hereinafter referred to as compound []) is a combination of a deoxynucleoside represented by the following general formula [] and a phosphorus-nitrogen-containing five-membered ring compound represented by the following general formula []. It can be easily produced in good yield by reacting in an inert solvent. (In the formula, R ¹ and B have the same meanings as above.) (In the formula, R ² and Methylene chloride is the most suitable inert solvent for the synthesis. There are various reasons for this, including that it is inexpensive, that dehydration and purification is easy, that the reaction yield is good, and that the deoxynucleotide represented by the general formula [], which is the target product of the reaction, can be isolated. In addition to being able to be used in the production of the oligodeoxynucleotide according to the present invention as a methylene chloride solution without using methylene chloride, the compound represented by the above general formula [] can also be advantageously produced using methylene chloride as a solvent, and can be directly used as is. It has various advantages such as being usable as a methylene chloride solution for the synthesis of deoxynucleotides of the general formula []. Furthermore, as is already known, the deoxynucleoside represented by the general formula [] can be easily produced by reacting the deoxynucleoside represented by the following general formula [] with trityl chloride, methoxytrityl chloride or dimethoxytrityl chloride. However, those of the general formula [] in which R ¹ is a dimethoxytrityl group are commercially available, so it is convenient to use them. (In the formula, B has the same meaning as above.) Phosphorus-nitrogen-containing 5 represented by the above general formula []
The membered ring compound is an arene phosphorodichloridate and imidazole represented by the following general formula [],
It can be easily produced by reacting triazole or benzotriazole in methylene chloride in the presence of a base such as triethylamine. (In the formula, R ² has the same meaning as above.) The reaction product, the phosphorus-nitrogen-containing five-membered ring compound represented by the general formula [ ], is synthesized in the present invention as a methylene chloride solution without isolation. It can be used as a raw material for the production of compound [], and since this solution can be stored in a dry state at -20°C for about 3 weeks, it is useful as a phosphorylating agent for deoxynucleosides. Next, the other synthetic raw material used in the present invention will be explained. The above general formula []
Deoxynucleoside or its amine salt represented by, where n is 0 (hereinafter referred to as compound [.])
is a new compound, in which an organotinamide compound represented by the general formula [] and a deoxynucleoside represented by the general formula [] are mixed in a molar ratio of preferably 1.0 to 1.2 ([]/[]) in methylene chloride at room temperature. The compound produced in this way can be easily produced by mixing and stirring for about 15 minutes. ] is used for the production of the next oligodeoxynucleotide without isolation. (In the formula, R ³ is the same as above. Either R ⁵ or R ⁶ is an alkyl group or an aryl group, and the remaining R ⁶ or R ⁵ is a hydrogen atom, an alkyl group, or an aryl group.) (In the formula, R ⁴ and B are the same as above.) The organic tin amide compound represented by the general formula [] and the deoxynucleoside represented by the general formula [] can be produced by a known method and used. However, since the organic tinamide compound represented by the general formula [] is unstable in water,
Preferably stored under dry nitrogen. The object of the method of the present invention, the general formula []
The dideoxynucleotide in which n is 0 (hereinafter referred to as compound [ ₀ ]) can be obtained by reacting the compound [] that can be produced by the method described above with the compound [ ₀ ] in methylene chloride at a reaction temperature of 0 to 35°C. This allows for easy production with high yield. Reaction time is compound [] and compound [ ₀ ]
and the substituent X in compound []
The end of the reaction can be easily determined by thin layer chromatography, etc., although this varies depending on the type of reaction.
For example, if the protecting group R ¹ of compound [] is a dimethoxytrityl group, dimethoxytrityl can be detected by slowly heating (300 to 400°C) a spot on the thin layer chromatogram from the back side of the thin layer chromatogram plate. Since the group develops a brown color, it is convenient for observing the progress of the reaction. Conventionally, it has been known to identify dimethoxytrityl groups by coloring them with acids such as sulfuric acid, but this method has the disadvantage that the coloring is sparse and detailed identification is difficult. On the other hand, the heat-induced coloration described above is uniform, so it has the advantage of being easy to identify. After completion of the reaction, the target compound [ ₀ ] can be purified by column chromatography and isolated with a high yield of around 95%. The trideoxynucleotide with n=1 in the general formula [], which is another object of the method of the present invention, is a compound with n=1 in the general formula [] (hereinafter referred to as compound [ ₁ ]) and the compound []. , can be easily obtained in high yield by reacting in methylene chloride at a reaction temperature of 0 to 35°C. Since the reaction time varies depending on the molar ratio of compound [] and compound [ ₁ ] and the type of substituent X in compound [], it is preferable to check the progress of the reaction by thin layer chromatography and confirm the completion of the reaction. . Compound [ ₁ ] is obtained by selectively removing the protecting group R ¹ from compound [ ₀ ] by a known method to obtain a deoxynucleotide represented by the following general formula [], and then converted to a deoxynucleotide represented by the following general formula []. It can be produced by reacting the organic tin amide compound shown in methylene chloride under the same conditions as when compound [ ₀ ] was synthesized. (In the formula, R ² , R ⁴ and B have the same meanings as above.) Still another object of the method of the present invention is that in the general formula [], n=N (however, N is an integer of 2 or more). Oligodeoxynucleotides are a compound in which n=N in the general formula [] (hereinafter referred to as compound [ _N ]) and a compound [],
It can be produced by reacting in the same manner as above. Also, the synthesis of compound [ _N ] is similar to the method described above, where n=N-1 in the general formula []
selectively removing the protecting group R ¹ of the compound,
This may be carried out by reacting an organic tin amide compound represented by the above general formula [] with this. In this manner, according to the present invention, a long-chain oligodeoxynucleotide having a base residue, in which n in the general formula is 10 or more, can be produced easily and with high yield in a small number of steps. It is. The structural formula of each residue described in this specification is as follows. Thymine residue N ⁴ -benzoylcytosine residue ^N2 -isobutyrylguanine residue N ⁶ - enzoyladenine residue The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to the Examples. Example 1 Dideoxynucleotides were produced by the following reaction. (In the formula, DMTr represents a dimethoxytrityl group, T
represents a thymine residue, TBDMS represents a t-butyldimethylsilyl group, and Et represents an ethyl group. ) 5'-0-dimethoxytrityl-cymidine (131
Pyridine (0.02 ml) and a methylene chloride solution of 2-chlorophosphoditriazolide (0.8 M, 0.36 ml, 0.288 mmol) were added to
After reacting for a minute, a solution of pyridine in water (1M,
methylene chloride of 5'-0-dimethoxytrityl-thymidine-3 ¹ -0-(2-chlorophenyl)phosphotriazolide ['] prepared by adding 0.012 ml, 0.012 mmol) and stirring for 15 minutes at room temperature. The solution was combined with (dimethylamino)triethylstannane (58 mg, 0.05 ml, 0.21 mmol) and 3′-0-t
-butyldimethylsilyl-thymidine (71 mg, 0.2
mmol) in methylene chloride (0.2 ml) for 15 minutes.
The mixture was added to diethylamine salt of 3'-0-t-butyldimethylsilyl-thymidine ['] at 20°C. After reacting at that temperature for 30 minutes and confirming completion of the reaction by thin layer chromatography (hereinafter abbreviated as TLC), a 50% aqueous pyridine solution (0.5 ml) was added.
Extract the organic layer with methylene chloride (10 ml);
0.5M potassium phosphate aqueous solution (10ml)
0.1M triethylamine bicarbonate (PH7.5, 10
ml), and methylene chloride was distilled off under reduced pressure. The residue was passed through silica gel column chromatography and eluted with chloroform-methanol (40:1). The eluate was concentrated, a small amount of the residue was dissolved in methylene chloride, and the solution was added dropwise to vigorously stirring n-hexane. The white crystals formed were separated and dried to give 5'-0-dimethoxytrityl-thymidyl ( 206 mg of 3'-0-t-butyldimethylsilyl-thymidine-3'-0-(P1-2-chlorophenyl)phosphate ['] was obtained.
The yield was 96% (vs. 5'-0-triethylstannyl-
3'-0-t-butyldimethyl-thymidine), and its physical properties were as follows. Thin layer chromatography (TLC); R ^f =0.67
( _CHCl3 :MeOH=9:1). ¹ HNMR ( _CDCl3 ,
TMS)〓, 0.08 (s, 6H, Si( CH ₃ ) ₂ ), 0.90 (s,
9H, SiC( CH3 ) ₃ ), _1.41 ( _s ,3H,5- CH3 ),
1.85 (s, 3H, 5- CH ₃ ), 2.0-2.80 (m, 4H,
2′ and 2′), 3.20−3.60 (m, 2H, 5′), 3.80 (
s,
6H, OC H ₃ and OC H ₃ ), 3.90−4.70 (m,
5H3′, 4′, 4′ and 5′), 5.30−5.50 (m, 1H,
3'), 6.29 (t, 1H, J=7.0Hz, 1'), 6.50 (t,
1H, J = 7.0Hz, 1′) 6.80−7.00 (m, 4H, ph),
7.10−7.80 (m, 15H, 6, 6 and ph) and
9.72 (s, 2H, NH and NH ) ppm. In addition, the 5′-
The physical properties of the diethylamine salt of 0-triethylstannyl-3'-0-t-butyldimethylsilyl-thymidine are as follows. ¹ HNMR (CD ₂ Cl ₂ , TMS) = 0.10 (S.6H, Si(C
H ₃ ) ₂ ), 0.92 (S, 9H, SiC( CH ₃ ) ₃ ), 1.09 (t
,
6H _, T=7.0Hz , N( _CH2CH3 ) ₂ ), 1.28(S,
15H, SnEt ₃ ), 1.85 (S, 3H, 5), 2.12−2.28
(m, 2H, 2′), 2.62(q, 4H, J=7.0Hz, N(C
_H2CH3 ) ₂ ), 3.60-3.96 (m, 3H, 4' _and 5'),
4.28 (S, 2H, 3 and H NEt ₂ ), 4.36−4.60 (m,
1H, 3′), 6.24 (t, 1H, J=7.0Hz, 1′) and
7.63 (S, 1H, 6) ppm. ¹³ CNMR (CD ₂ Cl ₂ , TMS) = −4.7, −4.6 (Si
( _CH3 ) ₂ ) , 7.6(Sn( _CH2CH3 ) _{3 )} , 10.3(Sn( C
H ₂ CH ₃ ) ₃ ), 13.1 (5th position C H ₃ ), 15.2 (N(CH ₂ C
_H3 ) ₂ ) _, 18.3( SiCMe3 ), 26.0(SiC( CH3 _{) 3} ), 41
.Four
(2′), 44.0(N( CH ₂ CH ₃ ) ₂ , 62.1(5′), 72.7(
3′),
85.9 (1′), 88.6 (4′), 110.1 (5′), 136.7 (6
), 154.2
(4) and 168.6 (2) ppm. Examples 2 to 9 Deoxynucleotides and deoxynucleosides or deoxynucleotides listed in Table 1
Using the amine salt of 5'-0-triorganostannyl derivative, the same operation as in Example 1 was carried out to obtain the first
Deoxynucleotides and trideoxynucleotides listed in the table were obtained. The meanings of the abbreviations in Table 1 are as follows. Et; ethyl group, n-Bu; n-butyl group,
DMTr: dimethoxytrityl group, Bz: benzoyl group, TBDMS: t-butyldimethylsilyl group,
T; thymine residue, C ^bz ; N ⁴ -benzoylcytosine residue, A ^bz ; N ⁶ -benzoyl adenine residue,
^Gib ; ^N2 -isobutylguanine residue. The amine salt of the 5'-0-triorganostannyl derivative of deoxynucleoside and deoxynucleotide used as a raw material was 5'-0-triethylstannyl-3'-0- in Example 1.
It was synthesized in the same manner as the diethylamine salt of t-butyldimethylsilyl-thymidine. In addition, the signal of the ¹ HNMR spectrum of the amine salt of the 5′-0-triorganostannyl derivative of these deoxynucleosides and dideoxynucleotides [ ¹ HNMR (CD ₂ Cl ₂ , TMS);
(ppm)] is as follows. Examples 2, 3 and 4 1.09 (t, 6H, J=7.0Hz, N(CH ₂ C H ₃ ) ₂ ),
1.30 (s, 15H, Sn(C ₂ H ₅ ) ₃ ), 1.89 (s, 3H, 5
-C H ₃ ), 2.36-2.82 (m, 6H, including quartet at 2.63, J = 7.0 Hz, 2' and N(C H ₂ CH ₃ ) ₂ ), 3.80
−4.10 (m, 2H, 5′), 4.15−4.40 (m, 3H, 4.28
4′, H NEt ₂ and N H )), 5.50
−5.65 (m, 1H, 3′), 6.44 (t, 1H, J=7.0Hz,
1'), 7.30−7.70 (m, 3H, ph), 7.78 (s, 1H,
6) and 7.95-8.10 (m, 2H, ph). Example 5 0.10 (s, 6H, Si( CH ₃ ) ₂ ), 0.80−1.20 (m,
24H, with singlet at 0.92 and triplet at _1.05 , J=7.0Hz _, SiC( CH3 ) ₃ , _Sn ( _{CH2CH2CH2C
H3} ) ₃ , N (CH2CH3 _{) 2} ) _, 1.20-1.80(m, 18H,
Sn ₍ CH2CH2CH2CH3 _{) 3 )} , _2.05-2.70 ( m , 6H,
2.60 with quartet, J = 7.0Hz, 2' and N(C
_H2CH3 ) ₂ ), 3.60-4.20 (m, 3H, 4' _and 5'),
4.40−4.70 (m, 3H, including singlet at 4.48, 3′, H
NEt ₂ and NH), 6.22 (t, 1H, J = 6.0Hz, 1′)
7.14 (d, 1H, J=7.0Hz, 5), 7.25−7.65 (m,
3H, ph), 7.90−8.10 (m, 2H, ph) and 8.66
(d, 1H, J=7.0Hz, 6). Example 6 0.80−1.40 (m, 21H, including triple line at 1.02, J
=7.0Hz, N(CH ₂ C H ₃ ) ₂ and Sn(C ₂ H ₅ ) ₃ , 2.30
−3.00 (m, 6H, 2.58 includes quartet, J=7.0
Hz, 2′ and N( CH ₂ CH ₃ ) ₂ , 3.36(s, 2H, H
NEt ₂ and N H ), 3.80−4.10 (m, 2H, 5′),
4.10−4.35 (m, 1H, 4′), 5.50−5.70 (m, 1H,
3'), 6.40-6.60 (m, 1H, 1'), 6.90-7.65 (m,
8H, ph), 7.80−8.00 (m, 2H, ph), 8.20−8.50
(contains singlet at m, 2H, 8.44, 2 and 8). Example 7 0.10 (s, 6H, Si(C H ₃ ) ₂ ), 0.92 (s, 9H,
SiC( CH3 ) ₃ ) _, 1.05(t, 6H, J=7.0Hz, N
( _CH2CH3 ) ₂ , 1.08-1.60(m _, 15H, Sn ( _{C2H5 )
3} ), 2.04-2.72 (m, 6H, including quartet at 2.60,
J=7.0Hz _, 2′ and N( CH2CH3 ) ₂ ) _, 3.60−4.20
(m, 3H, 4′ and 5′), 4.40−4.68 (m, 3H,
4.47 with singlet, 3′, H NEt ₂ and N H ),
6.22 (t, 1H, J = 6.0Hz, 1'), 7.14 (s, 1H, J
=7.0Hz, 5), 7.26-7.64 (m, 3H, ph), 7.88-
8.10 (m, 2H, ph) and 8.66 (d, 1H, J = 7.0
Hz, 6). Example 8 1.00−1.50 (m, 27H, including singlet at 1.21, N
(CH ₂ C H ₃ ) ₂ , CH(CH 3 ) ₂ and Sn ₍ C ₂ H ₅ ) ₃ ,
2.40−3.20 (m, 7H, including quartet at 2.70, J=
7.0Hz _, CH ( _CH3 ) _2,2 ' and _N ( CH2CH3 ) ₂ ),
3.80−4.00 (m, 2H, 5′), 4.20−4.35 (m, 1H,
4'), 5.55-5.70 (m, 1H, 3'), 6.32 (t, 1H, J
=7.0Hz, 1′), 7.01 (s, 3H, H NEt ₂ , NH and NH ), 7.15−7.60 (m, 3H, ph), 7.80−8.00
(m, 2H, ph) and 8.08 (s, 1H, 8). Example 9 0.10(s, 6H, Si( CH ₃ ) ₂ , 0.92(s, 9H, SiC
( CH ₃ ) ₃ ), 1.10(t, 6H, J=7.0Hz, N(CH ₂ C
H ₃ ) ₂ ), 1.18 (s, 15H, Sn (C ₂ H ₅ ) ₃ ), 1.86 (s
,
6H, 5- CH3 and ₅ - CH3 ), _2.10-2.80
(m, 8H, 2.66 includes quartet, J=7.0Hz, 2′,
2' and N( CH2CH3 ) ₂ ), 3.60-4.80( _{m ,} 7H,
5′, 5′, 4′, 4′, and 3′), 5.04(s, 3H, H
NEt ₂ , NH and NH ), 5.25−5.50 (m, 1H,
3'), 6.38 (t, 1H, J=7.0Hz, 1'), 6.44 (t,
1H, J = 7.0Hz, 1′), 7.10−7.70 (m, 5H, 6 and ph) and 7.76 (s, 1H, 6). [Table] [Table] [Table] [Table]

Claims (1)

[Scope of Claims] 1. An oligonucleotide represented by the general formula [], which is characterized by reacting a deoxynucleotide represented by the general formula [] with a deoxynucleoside or deoxynucleotide or an amine salt thereof represented by the general formula []. Method for producing deoxynucleotides. (In the formula, R ¹ and R ² represent a protecting group, B represents a basic residue that may have a protective group, and X represents a nitrogen-containing 5-membered ring.) (In the formula, R ³ is an alkyl group or an aryl group, R ²
and R ⁴ represents a protecting group, B represents a base residue that may have a protective group, and n represents 0 or a positive integer. ) (In the formula, the meanings of R ¹ , R ² , R ⁴ , B and n are the same as above.)