JPH0684396B2 - Deoxynucleoside phosphorous sulfide compound - Google Patents

Description

TECHNICAL FIELD The present invention relates to an intermediate compound for producing an oligodeoxynucleotide, more specifically, a deoxynucleoside-3′-phosphite compound for producing an oligodeoxynucleotide by a phosphite triester method. It is about.

2. Description of the Related Art Currently, the phosphate triester method and the phosphite triester method are widely used as methods for producing oligodeoxynucleotides. In the phosphoric acid triester method, a deoxynucleoside-3′-phosphate compound [II] as shown below is generally used, and in the phosphite triester method, a deoxynucleoside-containing compound is used.
3'-phosphoryl chloride compound [III] and deoxynucleoside-3'-phosphoramidite compound [IV]
Are used as intermediates for the production thereof (for the production method of compound [II] and its use, see CBRees
e, Tetrahedron, 34 , 3143 (1978), for compound [III], see RL Letsinger et al., J. Am. Chem. Soc., 97 , 3278.
(1975), and for compound [IV], see MHCaruther.
S. et al., Tetra-hedron Lett., 22 , 1859 (1981). ) (In the formula, R ¹ and R ² represent a protecting group, B represents a base residue which may have a protecting group, and R ⁴ represents an alkyl group.) Problems to be Solved by the Invention Compounds as intermediate compounds An advantage of producing an oligodeoxynucleotide using [II] is that the compound [II] is stable to oxygen and water, so that the compound [II] can be easily produced and handled. Therefore, the method using the compound [II] as an intermediate compound (phosphoric acid triester method) has dominated the oligodeoxynucleotide production method. However, in this method, the compound [II] and 5'-O-deoxynucleoside or 5'-
Since the condensation reaction with —O-deoxynucleotide, so-called internucleotide formation reaction, is not so fast,
There is a drawback that it takes time to produce oligos.

On the other hand, the phosphite triester method using the compound [III] or the compound [IV] is performed by using the compounds [III] and [I].
V] is 5'-O-deoxynucleoside or 5'-
Since the reactivity of O-deoxynucleotide with the alcoholic hydroxyl group is large, the internucleotide forming reaction is fast, and therefore, the desired oligodeoxynucleotide can be obtained in a short time. In particular, these methods are effective for producing oligodeoxynucleotides by the solid phase method. However, these compounds [II
I] and [IV], especially compound [III], are not easy to prepare and handle, and because they are unstable to oxygen and water, care must be taken not to decompose them during storage. There are drawbacks such as In fact, compound [III] is generally such a compound that it is difficult to isolate it from the productive system, and compound [IV] easily cleaves the PN bond even in the presence of a weak acid. It is known to be unstable.

MEANS TO SOLVE THE PROBLEMS, ACTIONS AND EFFECTS The present inventors have taken into consideration the situation of the prior art as described above, and have found an intermediate compound which is easy and stable to produce and which can promptly produce an oligodeoxynucleotide. As a result of searching, it was found that a deoxynucleoside phosphoryl sulfide compound represented by the following general formula [I] was created and this object can be advantageously achieved by using the compound.

(In the formula, R ¹ and R ² represent a protecting group, R ³ represents an aryl group, and B represents a base residue which may have a protecting group.) Accordingly, the present invention is as described above. The present invention relates to a novel intermediate compound for producing an oligodeoxynucleotide, that is, a novel deoxynucleoside compound represented by the general formula [I] which is easy to produce and provides a prompt internucleotide bond-forming reaction. The novel compound [I] will be specifically described.

As the protecting group R ¹ of the phosphoryl sulfide compound represented by the general formula [I], basically all hydroxy protecting groups are possible. Specifically, in the production of oligonucleotides, hydroxy-protecting groups which have been found to be useful, for example, triphenylmethyl group, 4-methoxytriphenylmethyl group, tria such as 4,4'-dimethoxytriphenylmethyl group and the like. Lille methyl group, pixyl group (ie 9-
(A phenylxanthen-9-yl group), an alkoxycarbonyl group, an aryloxycarbonyl group, an arylthioalkyloxycarbonyl group, and a trialkylsilyl group such as a t-butyldimethylsilyl group. There is no limitation as to which R ¹ of the compound [I] is selected from these hydroxy-protecting groups, but as far as the triarylmethyl group is concerned, it is easy to introduce and deprotect it. 5'-O used as a starting material in the production of compound [I] as described above.
Of the-, N-protected deoxynucleosides, 5'-O-
Some triarylmethyl-N-protected deoxynucleosides are commercially available. Considering their easy availability, they are preferably used.

As the organic group R ² of the compound [I], basically all phosphate protecting groups are possible. Specifically, a protecting group researched and developed for producing an oligonucleotide,
For example, a lower alkyl group having up to 5 carbon atoms, an allyl group,
Examples thereof include a cyanoalkyl group, a haloalkyl group, an arylsulfonylalkyl group, an aryl group and a haloaryl group. Of these protecting groups, the compound [I]
There is no limitation as to which R ² to choose,
Methyl group, which has been widely studied as a phosphate protecting group for oligonucleotide production and its usefulness has been confirmed,
An allyl group, β-cyanoethyl group, 2-chlorophenyl group and the like are preferably used.

Next, the organic group R ³ of the compound [I] will be described. It may be an aryl group, specifically, a phenyl group,
2-methylphenyl group, 4-methylphenyl group, 2,4-
Dimethylphenyl group, 2,6-dimethylphenyl group, 2,4,6
-Trimethylphenyl group, 2-chlorophenyl group, 4-
Examples thereof include a chlorophenyl group, a 3,4-dichlorophenyl group and an α-naphthyl group. Which of these aryl groups is selected as R ³ cannot be limited because it depends on the kind of the protecting group R ^{2 in} the compound [I], but the ease and stability of the production of the compound [I] are not limited. In consideration of the above, generally, an aryl group having a substituent at the α-position such as a 2-methylphenyl group, a 2,6-dimethylphenyl group and a 2-chlorophenyl group, which gives it easy and good stability, is preferred. Used.

Next, the base residue B in the compound [I] will be described. As a typical B, a thymine residue which may have a protecting group at the 3-position represented by the following general formula [V], a general formula [VI] A cytosine residue which may have a protecting group at the 4-position amino group, an adenine residue which may have a protecting group at the 6-position amino group represented by the general formula [VII], and a general formula [VIII] And a guanine residue which may have a protecting group at the 1,2,6-position amide group, amino group and keto group.

Examples of the protecting group for these bases include, but are not limited to, triarylmethyl groups, trialkylsilylalkyl groups, arylthioalkyl groups, phthaloyl groups, aryloxycarbonyl groups, and alkoxy groups that have been developed to date for producing oligonucleotides. Examples thereof include a carphonyl group, a diarylcarbamoyl group, an arylcarbonyl group, an alkylcarbonyl group, and a 1,2-dialkylcarbonyloxyethylene group (which is protected by a base residue represented by the general formula [V]-[VIII]). The use of groups has already been shown in many reports and reviews (see, for example, Akira Hatsuji et al., Journal of Synthetic Organic Chemistry, 42 , 429 (1984)).

The deoxynucleoside phosphoryl sulfide compound of the general formula [I] according to the present invention has the following formula (1): a deoxynucleoside of the general formula [IX] and a compound of the general formula [X] It can be produced by a reaction with a 2,4-triazolylphosphine compound.

(In the formula, R ¹ , R ² , R ³ and B have the same meanings as described above.) For example, 2-chlorophenyloxy-2-methylphenylthio-1 in an equimolar amount to 5′-O-dimethoxylytylthymidine. After reacting with 2,2,4-triazolylphosphine in deuterated chloroform at room temperature for 1.5 hours, ¹ HNMR of the reaction solution was measured. As a result, 3'of 5'-O-dimethoxytritylthymidine as a starting material -OH group proton signal (δ3.45ppm), 2-chlorophenyloxy-2-methylphenylthio-1,2,4-triazolylphosphine methyl group (δ2.31ppm) and 1,2 The absorption of the 4,4-triazolyl group (δ 8.12 and 8.44 ppm) disappeared, and the newly appeared absorption was 5'-O-dimethoxytritylthymidine-3 '-(2-chlorophenyloxy-2-methylphenylthio). ) Phosphine [of the main organic groups Gunaru is,
δ 1.46 (5-methyl group of thymine residue), 2.28 and 2.38
(Methyl group of 2-methylphenylthio group), 3.69 and
3.70 (methoxy group of dimethoxytrityl group) ppm] and 1H
It was shown that -1,2,4-triazole [δ 8.16 (hydrogens at 3 and 5 positions), 12.8 (hydrogen at 1st position) ppm] was produced almost quantitatively. Further, this solution was washed with saturated saline solution, dried and concentrated, and then added dropwise to n-pentane to give fine powder of 5'-O-dimethoxytritylthymidine-3 '-(2-chlorophenyloxy- 2-Methylphenylthio) phosphine was obtained almost quantitatively.

As described above, the deoxynucleoside phosphoryl sulfide compound represented by the general formula [I] can be obtained in a high yield by the reaction (1), but the deoxynucleoside represented by the general formula [IX], which is the starting material, is already known. Method (for example, Akira Hatsuji et al., Journal of Synthetic Organic Chemistry, 42 , 429 (198
The products produced in 4) and the references cited therein) or commercially available products can be used, but it is better to dry them sufficiently by an appropriate means before use.

The 1,2,4-triazolylphosphine compound [X] is
Krihildorf's method (Angew.Chem.Int.Ed.Engl. 15 ,
305 (1976)) (reaction formula (2)).

(In the formula, R ² and R ³ have the same meanings as described above.) That is, an alkoxy or aryloxyarylthiochlorophosphine represented by the general formula [XI] and trimethylsilyl-1,2,4-triazole [XII] And are reacted in an organic solvent such as chloroform, methylene chloride, benzene, and toluene at a molar ratio of 1: 1 to 1.5 at room temperature for about 5 minutes, and then the organic solvent and by-produced trimethylchlorosilane [XIII] are depressurized. The compound [X] is obtained as a residue by distilling off underneath. The compound [X] is not stable to heat and water so that it can be purified by distillation or chromatography, but the compound [X] obtained by the above method
Is almost pure, which is readily demonstrated by measuring the ¹ H NMR of the above residue. An organic solvent for carrying out the reaction (1) is added to the compound [X] thus obtained and used in the reaction with the deoxynucleoside [IX].

Next, the alkoxy or aryloxyarylthiochlorophosphine compound [XI] and trimethylsilyl-1,2,4-triazole [XII], which are the starting materials for the reaction (2), will be explained. That is, it can be easily produced by the following reactions (3) and (4) (for the production method of the compound [XI], see, for example, GM Kosolapoff et al., “Organic Phosphor”).
us Compounds ", Vol.5, John Wiley & Sons (New Yor
k), 1973 and its references, and for compound [III], see L. Birkofer et al., Chem. Ber., 93 , 2804 (1960)).

(In the formula, R ² and R ³ have the same meanings as described above, and the base represents a base such as triethylamine.) Reaction (1) according to the present invention proceeds more smoothly in an organic solvent, and examples of such a solvent include chloroform, methylene chloride, 1,2-dichloroethane, benzene, tetrahydrofuran, p-dioxane and the like. These solvents are preferably used after sufficiently dehydrated with an appropriate desiccant and then purified by distillation or the like. In the reaction (1) according to the present invention, the molar ratio of the compound [IX] and the compound [X] is 1: 1.
Although it may be in the range of -10, it is better not to use the compound [X] in a large excess in view of economy, and the range of 1: 1 to 2 is preferable. Further, in actually carrying out the reaction (1), it is desirable to gently add the organic solvent solution of the compound [X] to the organic solvent solution or suspension of the compound [IX]. The reaction is preferably carried out in the range of 0 ° to 35 ° C. The reaction time depends on the molar ratio of the compound [IX] and the compound [X], and the protective group R ¹ of the compound [IX].
It is generally in the range of 10 minutes to 2 hours, though it varies depending on the kinds of the base residue B, the protecting group R ² and the organic group R ³ of the compound [X]. However, when actually carrying out the reaction (1), it is better to confirm the termination of the reaction by thin layer chromatography (TLC), ¹ H NMR, or the like.

The deoxynucleoside phosphoryl sulfide compound [I] produced by the reaction (1) according to the present invention is a so-called reaction solution of the compound [IX] and the compound [X] as it is,
Although it may be used for the subsequent reaction in situ, the one isolated by the following method is usually used for the subsequent reaction and the like. After confirming the termination of reaction (1) by the above method,
After the reaction solution is washed with saturated saline, the organic layer is dried over anhydrous sodium sulfate and the organic solvent is mostly distilled off under reduced pressure. The solution containing the concentrated compound [I] is dropped into stirred n-pentane or n-hexane. The precipitated fine powdery compound [I] is separated and dried under reduced pressure. In some cases, the solution containing the above-mentioned concentrated compound [I] is subjected to column chromatography on silica gel and developed with chloroform, methylene chloride, ethyl acetate or the like as a solvent to obtain a distillate containing the compound [I]. The liquid can also be concentrated to dryness under reduced pressure and isolated as a foamy solid.

The deoxynucleoside phosphoryl sulfide compound [I] thus obtained is stable, and is, for example, 5'-
O-dimethoxytritylthymidine-3 '-(2-chlorophenyloxy-2-methylphenylthio) phosphine can be stably stored at -20 ° C for 5 months. Further, when a compound [I] is used to synthesize an oligodeoxynucleotide, a dideoxynucleotide is obtained. For example, in the presence of tri-n-butylstannyl-1,2,4-triazole, with 5'-O-dimethoxytritylthymidine-3 '-(2-chlorophenyloxy-2-methylphenylthio) phosphine After reacting 3'-0-t-butyldimethylsilylthymidine in methylene chloride,
Oxidation with iodine gives 5'-O-dimethoxytritylthymidine (3 '→ 5')-3'-t-butyldimethylsilylthymidine-3'-0- (P1-2-chlorophenyl) phosphate in good yield. .

The meanings of the abbreviations used in this specification and the structure of each base residue are as follows.

Me; methyl group, MMTr; monomethoxytrityl group, DMTr; dimethoxytrityl group, EXAMPLES The present invention will be specifically described with reference to examples below, but the present invention is not limited to these examples.

The 1,2,4-triazolylphosphine compound [X] used in each Example was synthesized according to Reference Examples 1 to 3 below.

Reference Example 1 As a chlorophosphine compound [XI], 2 ml of a toluene solution containing 0.3 mmol (0.095 g) of 2-chlorophenyloxy-2-methylphenylthiochlorophosphine was added to trimethylsilyl-1,2,4-triazole [ XII] was added to the toluene solution containing 0.33 mmol (0.046 g) at 0 ° C., the mixture was stirred at this temperature for 10 minutes, and the low boiling fraction was reduced under reduced pressure at −78 ° C.
I was trapped. 1,2,4-triazolylphosphine compound [X] as the residue, namely 2-chlorophenyloxy-2-methylphenylthio-1,2,4-triazolylphosphine, was also trapped. Fractional fractionation of trimethylchlorosilane [XIII] was obtained. The results are as shown in Table 2 below.

Reference Example 2 As chlorophosphine compound [XI], methoxy-2-
Methoxy-2 was used in the same manner as in Reference Example 1 except that 30 ml of a toluene solution containing 5 mmol of methylphenylthiochlorophosphine and 20 ml of a toluene solution containing 5 mmol of trimethylsilyl-1,2,4-triazole [XII] were used. The results of synthesizing -methylphenylthio-1,2,4-triazolylphosphine [X] are shown in Table 2 below.

Reference Example 3 As shown in Table 2 below, by the same method as in Reference Example 2 except that 2-chlorophenyloxy-2,6-dimethylphenylthiochlorophosphine (5 mmol) was used as the chlorophosphine compound [XI]. 2-chlorophenyl-2,6-
Dimethylphenylthio-1,2,4-triazolylphosphine [X] was obtained.

Example 1 5'-O-dimethoxytritylthymidine [IX] (0.163
2-chlorophenyloxy-2 synthesized in Reference Example 1 in a suspension of deuterated chloroform (0.5 ml) (g, 0.3 mmol).
A solution of -methylphenylthio-1,2,4-triazolylphosphine [X] in deuterated chloroform (1 ml) was slowly added at 0 ° C. After returning to room temperature and stirring at that temperature for 1.5 hours, ¹ H NMR of the solution was measured. ¹ HNMR (CDCl ₃ , TMS) δ; 1.46 (s, 3H, 5-CH ₃ ), 2.20-2.
85 (m, 5H, 2.28 and 2.38ppm include singlet. 2'and CH
₃ C ₆ H ₄ S-), 3.35-3.65 (m, 2H, 5 '), 3.69 (s, 3H, CH ₃ O
C ₆ H ₄ −), 3.70 (s, 3H, CH ₃ OC ₆ H ₄ −), 4.25−4.50 (m, 1H,
4 '), 5.45-5.80 (m, 1H, 3'), 6.49 (t, 1H, J = 7.0Hz,
1 '), 6.65-6.90 (m, 4H, ph), 6.90-7.75 (m, 18H, ph and 6), 8.16 (s, 2H, HNC ₂ H ₂ N ₂ ), 10.3 (s, 1H, NH) , 12.
8 (s, 1H, HNC ₂ H ₂ N ₂ ).

Then, chloroform (50 ml) was added to this solution, the chloroform solution was washed with saturated saline (30 ml), and dried over anhydrous sodium sulfate. Chloroform was mostly distilled off under reduced pressure, and the residue (about 2 ml) was added dropwise to stirred n-pentane (200 ml) at 0 ° C. to immediately precipitate a white powder. Stir for 30 minutes, separate the powder, and dry under reduced pressure at room temperature for 8 hours to obtain 0.235 g (95%) of 5'-O-dimethoxytritylthymidine-3 '-(2-chlorophenyloxy-2-methyl). Phenylthio) phosphine [I]
Got Rf (CHCl ₃ : MeOH = 80: 1) = 0.30.

Examples 2 to 8 The deoxynucleoside [IX] described in Table 2 and the 1,2,4-triazolylphosphine compound [X] synthesized in Reference Examples 1 to 3 are shown in Table 2 in deuterated chloroform. After reacting at the indicated molar ratio for a predetermined time, the same operation as in Example 1 was performed to obtain the deoxynucleoside phosphoryl sulfide compound [I] shown in Table 2.

Claims (1)

[Claims] 1. A deoxynucleoside phosphoryl sulfide compound represented by the general formula [I]. (In the formula, R ¹ and R ² represent a protecting group, R ³ represents an aryl group, and B represents a base residue which may have a protecting group.)