WO2006095740A1

WO2006095740A1 - Method for synthesizing nucleic acid by using substituted carbamoyl group as protecting group

Info

Publication number: WO2006095740A1
Application number: PCT/JP2006/304400
Authority: WO
Inventors: Mitsuo Sekine; Kenichi Miyata; Ryuji Tamamushi; Kohji Seio
Original assignee: Tokyo Institute Of Technology
Priority date: 2005-03-09
Filing date: 2006-03-07
Publication date: 2006-09-14
Also published as: JP2006248931A

Abstract

Disclosed is a method for synthesizing a nucleic acid which is characterized by comprising a step wherein a compound represented by the general formula (III) below is obtained by reacting a compound represented by the general formula (I) below with an isocyanate derivative represented by the general formula (II) below, and another step wherein the compound represented by the general formula (I) below is obtained by heating the compound represented by the general formula (III) below or irradiating it with microwaves. (In the formula (I), X represents a portion other than an amino group in a nucleic acid base or the like that has an amino group.) (In the formula (II), Y represents an electron-withdrawing group.) (In the formula (III), X and Y are as defined above.)

Description

Specification

Method for synthesizing nucleic acid using substitutional ruberamoyl group as protecting group

Technical field

[0001] The present invention relates to a method for synthesizing a nucleic acid using a substitutional power ruberamoyl group as a protecting group, and a method for deprotecting the protecting group.

Background art

[0002] The amino group of the nucleobase portion requires a protecting group when chemically modifying other nucleic acid sites such as a highly reactive sugar moiety or when synthesizing DNA or RNA oligomers. Commonly used protecting groups include the trityl skeleton, the acyl skeleton, the force rubamate skeleton, the amidine skeleton (Strength Protocols in Nucleic Acid Chemistry, Chapter 2, Unit 2.1 John Wiley and Sons). And a protecting group. However, both protecting groups require acidic or basic conditions for deprotection, and the development of new protecting groups that can be deprotected under neutral conditions is required.

[0003] Nucleobase amino protecting groups for the synthesis of DNA and RNA oligomers are commonly used having an acyl skeleton and an amidine skeleton. All protecting groups must be deprotected using ammonia or methylamine, and oligomers containing molecules that are unstable under basic conditions cannot be synthesized. As a method of overcoming this problem, a method using a carboxy group that can be deprotected under neutral conditions as a protecting group for the nucleobase amino group (The Journal of Organic Chemistry 51 卷 P2400—2402 1986) Although it has been developed, there is a problem because it is necessary to use a transition metal for deprotection. On the other hand, the base-part unprotected phosphoramidite method using no protecting group in the base part (Journal of the American Chemi- nox Society 12684 P10884-10896 2004) has been developed by the present inventors.

[0004] Modified nucleic acid with N-strength rubamoyl group as a modified base of adenine base amino group (Journal of the American Chemical Society 125 卷 P 8086-8087 2003), modification of cytosine base amino group Modified nucleic acid with N-strength rubermoyl group as the base (Nuclideic Acids Research Supplement 2 P161—162 2002) has been reported. Furthermore, as a modified nucleoside, 4-N- (N aryl rubamoyl) deoxycytidine (Non-patent Document 1) has been reported by the present inventors.

[0005] Non-Patent Document 1: Tetrahedron Letters 45 卷 P9365— 9368 2004

Disclosure of the invention

Problems to be solved by the invention

[0006] As described above, there are very few protecting groups for nucleobase amino groups that can be deprotected under neutral conditions, and transition metals must also be used for deprotecting protecting groups. It was difficult. The present invention has been made under the above technical background, and an object of the present invention is to provide a novel means for protecting an amino group of a nucleobase.

Means for solving the problem

[0007] As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that the aryl group ruber moyl group contained in 4N— (N allylcarbamoyl) deoxycytidine (Non-patent Document 1) or the like is Based on this finding, the present invention has been completed based on the finding that it can be eliminated under sexual conditions and has suitable properties as a protecting group for the amino group of a nucleobase.

[0008] That is, the present invention provides the following (1) to (7).

[0009] (1)-General formula (1):

[0010] [Chemical 1]

NH ₂ — X (I)

[In the formula, X represents a moiety other than the amino group of a nucleoside or a similar compound containing a nucleobase having an amino group. ]

A compound represented by the general formula (Π):

[0011] [Chemical 2]

0 = C = N-Y (I I)

[Wherein Y represents an electron-withdrawing group. ] Is reacted with an isocyanate derivative represented by the general formula (ΠΙ):

[0012] [Chemical 3]

[Wherein, X and γ are as defined above. ]

And a step of obtaining a compound represented by the general formula (I) by heating or microwave irradiation of the compound represented by the general formula (in). Nucleic acid synthesis method.

(2) Y in the general formulas (II) and (III) may have a substituent! /, May have an aryl group or a substituent, or may have an aryl group. The method for synthesizing a nucleic acid according to (1), which is a group.

[0014] (3) —Y-force in the general formulas (Π) and (III) 4 -trophenyl group, 3 -trophenyl group, 2

-The method for synthesizing a nuclear acid according to (1), which is a trope group or a phenol sulfol group.

[0015] (4) General formula (III):

[0016] [Chemical 4]

[Wherein X represents a moiety other than the amino group of a nucleoside containing a nucleobase having an amino group or an analogous compound, and Y represents an electron-withdrawing group. ]

The compound represented by general formula (I):

[0017] [Chemical 5]

[Wherein X is as defined above. ] A method for deprotecting an amino group in a nucleobase, which comprises converting to a compound represented by the formula:

[0018] (5) Y in the general formula (III) may have a substituent! /, May have an aryl group or a substituent, and may have an aryl group. (4) The method for deprotecting an amino group in a nucleobase according to (4).

[0019] (6) —Y force in general formula (III) is characterized by being 4-trophenyl group, 3-trophenyl group, 2-trophenyl group, or phenylsulfol group ( 4) A method for deprotecting an amino group in the nucleobase.

[0020] (7) —General formula (m,):

[0021] [Chemical 6]

0

To _{^{HN Ν ζ Υ, (H I}} ')

[In the formula, X represents a moiety other than an amino group of a nucleoside containing a nucleobase having an amino group or an analogous compound, and Y, represents a 4-trophenyl group, a 3-trophenyl group, or a 2-trophenyl group. Or a phenylsulfol group. ]

A compound represented by

The invention's effect

[0022] The present invention provides a novel method for synthesizing a nucleic acid using a substitutional ruberamoyl group as a protecting group for an amino group of a nucleobase. The substitution force rubamoyl group can be removed by heating or the like under neutral conditions, and can be easily introduced by an isocyanate derivative, and has very excellent properties as a protecting group. Yes.

BEST MODE FOR CARRYING OUT THE INVENTION

[0023] Hereinafter, the present invention will be described in detail.

[0024] The nucleic acid synthesis method of the present invention is characterized by including at least the following protecting group introduction step and deprotection step.

[0025] The protective group introduction step is represented by the general formula (I)

[0026] [Chemical 7] The compound represented by NH ₂ — X is represented by the general formula (II)

[0027] [Chemical 8]

0 = C = N-Y (I I) is reacted with an isocyanate derivative represented by the general formula (III)

[0028] [Chemical 9]

(I I I)

Is a step of obtaining a compound represented by the formula:

[0029] X in the general formulas (I) and (III) represents a nucleoside containing a nucleobase having an amino group, or a moiety other than the amino group of an analogous compound.

[0030] Here, "nucleoside containing a nucleobase having an amino group" refers to all substances in which ribose 1-yl or 2-deoxyribose 1-yl group and any heteroaromatic ring having an amino group are bonded. Examples thereof include, but are not limited to, 2′-deoxyadenosine, 2′-deoxyguanosine, 2 ′ deoxycytidine, adenosine, guanosine, cytidine, and the like.

[0031] "Similar nucleoside compound" refers to a naturally-occurring or artificially synthesized compound containing a nucleobase in its molecule as in the case of a nucleoside. At least a nucleoside derivative, a nucleoside phosphoramidite Monomers of compounds, oligo DNAs, oligo RNAs, PNAs, and PNAs are included in “analogues of nucleosides”.

[0032] "Nucleoside derivative" refers to a compound in which a part of a nucleoside molecule is replaced with another atom or group, for example, a part of a hydroxyl group present at a sugar site in a nucleoside. In addition, all compounds protected by appropriate protecting groups include compounds in which the hydrogen atom at the 2-position of deoxyribose is substituted with a functional group other than a hydroxyl group. [0033] "Nucleoside phosphoramidite complex" refers to a compound in which the hydroxyl group at the 5-position of ribose or deoxyribose is protected with an appropriate protecting group and the compound at the 3-position is a free hydroxyl group. Is a compound having a group represented by the formula (IV) bonded thereto.

[0034] [Chemical 10]

R ¹

, _P ^N , R2 (, V)

\

⁰ , R3 wherein R ² is the same or different alkyl group, or R ¹ and R ² may be bonded to each other to contain a hetero atom! R ³ represents a phosphate group, and R ³ represents a phosphate group Represents a protecting group. Examples of R ² include an isopropyl group, and examples of R ³ include a 2-cyanoethyl group, but are not limited thereto.

[0035] Oligo DNA and oligo RNA include those that have undergone chemical modification.

[0036] Y in the general formulas (II) and (III) represents an electron-withdrawing group. Examples of the electron-withdrawing group may include a substituent, may be an aryl group, may have a substituent! /, May have a arylsulfonyl group, and the like, more specifically. Examples thereof include 4--trophenyl group, 3--trophenyl group, 2--phenol group, and phenylsulfol group. Among these, a 2-trophenyl group and a phenolsulfol group can be mentioned as particularly suitable groups. In addition, when Y is 4—trophenyl group, 3—trophenyl group, 2—trophenyl group, or fullsulfol group, the compound represented by the general formula (III) is novel. It is a compound.

[0037] The reaction of the compound represented by the general formula (I) with the isocyanate derivative represented by the general formula (II) is carried out by a known method (for example, Tetrahedron Letters 45 卷 P9365-9368 2004). Described method). For example, the compound represented by the general formula (I) and the isocyanate derivative represented by the general formula (Π) should not inhibit the reaction! ヽ Organic solvent (a plurality of organic solvents can be mixed at any volume ratio). The compound represented by the general formula (III) can be obtained by mixing in a mixed organic solvent in step 2) and stirring for a certain period of time.

[0038] The deprotection step involves heating or microwave irradiation of the compound represented by the general formula (III). And a step of obtaining a compound represented by the general formula (I).

[0039] The heating temperature is not particularly limited as long as it can be deprotected, but it is preferably 40 to 100 ° C, more preferably 50 to 90 ° C. Heating time is a force that varies depending on the type of substituent of the force rubamoyl group.For example, if the substituent is 2--phenol group, the caloric heat time is preferably 1 to 30 hours. 3 to 20 hours Is more preferable.

[0040] The frequency of the microwave to be irradiated is not limited as long as it can be deprotected, but it is preferably 1000 to 10,000 MHz, and more preferably 1000 to 5000 MHz. The irradiation time of the microwave required for deprotection varies depending on the type of substituent of the force rubamoyl group. For example, if the substituent is a 2-trophenyl group, the irradiation time is preferably 1 to 30 hours. More preferably, it is 20 hours.

[0041] The above-described heat treatment or the like is usually performed in a solvent. As the solvent, a solvent obtained by mixing an organic solvent that does not inhibit the reaction and an aqueous solvent at an arbitrary volume ratio of 0: 100-100: 0 can be used. As the organic solvent, the deprotection reaction is not inhibited. A mixed organic solvent obtained by mixing a plurality of organic solvents at an arbitrary volume ratio may be used. Further, as the aqueous solvent, not only water but also any buffer solution can be used, and a mixed buffer solution obtained by mixing a plurality of buffer solutions at an arbitrary concentration may be used. Additives that do not inhibit the reaction include, for example, ammonia, primary amine, secondary amine, tertiary amine, amidine compound, guanidine compound, tetra (n-butyl) ammonium fluoride, carbonate , Carboxylates, metal alkoxides, metal hydroxides, metal hydrides and the like can be used in combination.

[0042] The nucleic acid synthesis method of the present invention can be carried out in the same manner as a general nucleic acid synthesis method (for example, phosphoramidite method) except for the protecting group introduction step and the deprotection step described above. An example of a synthesis method using the phosphoramidite method is shown below. First, a nucleoside phosphoramidite complex containing a nucleobase having an amino group is synthesized. A protecting group is introduced into the amino group of this nucleoside phosphoroamidaitoi compound according to the protecting group introduction step described above. A nucleoside phosphoramidite complex having a protecting group introduced is sequentially ligated to synthesize an oligonucleic acid containing a protecting group. The oligonucleic acid containing this protecting group is removed according to the deprotection step described above to obtain an oligonucleic acid.

[0043] It should be noted that the nucleic acid to be synthesized in the synthesis method of the present invention includes a non-natural PNA or the like. Types of nucleic acids are also included.

Example

[0044] [Example 1] 4-N— [N— (4-Tropheyl) carno

Completion

[0045] [Chemical 11]

Deoxycytidine hydrochloride (264 mg, 1.0 mmol) was suspended in dimethylformamide (10 ml), and triethylamine (141 n 1.0 mmol) was stirred for 5 minutes. Subsequently, 4-trophenyl isocyanate (164 mg, 1.0 mmol) was added and stirred for 30 minutes, and the solvent was distilled off under reduced pressure. The precipitate formed by adding isopropyl alcohol was dried to obtain the title compound (334 mg, yield 88%).

[0046] IH NMR (DMSO— d6) δ 2.01— 2.09 (1H, m), 2.27— 2.33 (1H, m), 3.50— 3.64 (1H, m), 3.84— 3.90 (1H, m), 4.19—4.25 (1H, m), 5. 07 (1H, br), 5. 18 (1H, br), 6.08 (IH, dd, J = 6. OHz, J = 5.9Hz), 6.37 (IH, d, J = 7.6Hz), 7.74—7.77 (2H, m), 7.92 (1H, d, J = 7.6Hz), 8.12 —8.23 (3H, m)

Example 2 Synthesis of 4 N— [N— (3-Trophele) Force Rubamoyl] Deoxycytidine

[0047] [Chemical 12]

z Hydrochloride (264 mg, 1.0 mmol) was suspended in dimethylformamide (10 ml), and triethylamine (141 n 1.0 mmol) was stirred for 5 minutes. Subsequently, 3 -trophenyl isocyanate (164 mg, 1.0 mmol) was added and stirred for 30 minutes, and the solvent was distilled off under reduced pressure. Isopropyl alcohol was added and the resulting precipitate was dried to obtain the title compound (352 mg, yield 90%).

[0048] IH NMR (DMSO— d6) δ 1.98— 2.10 (1H, m), 2.25— 2.35 (1H, m), 3.50— 3.64 (1H, m), 3.84— 3.90 (1H, m), 4.19—4.25 (1H, m), 5. 08 (IH, br), 5.25 (IH, br), 6.08 (IH, dd, J = 6.3Hz, J = 5.9Hz), 6.36 (IH, d, J = 7.2Hz) , 7.55 (IH, m), 7.79 (1H, m), 7.89 (IH, m), 8.18 (1 H, d, J = 7.2Hz), 8.56 (IH, m)

[Example 3] 4— N— [N— (2—-trophele) cal

Completion

[0049] [Chemical 13]

z Hydrochloride (264 mg, 1.0 mmol) was suspended in dimethylformamide (10 ml), and triethylamine (141 n 1.0 mmol) was stirred for 5 minutes. Subsequently, 2-tro-phenol isocyanate (164 mg, 1.0 mmol) was added and stirred for 30 minutes, and the solvent was distilled off under reduced pressure. The product was purified by column chromatography using 60N silica gel (black mouth form-methanol) to obtain the title compound (352 mg, yield 90%).

[0050] IH NMR (DMSO— d6) δ 1.97— 2.08 (1H, m), 2.25— 2.34 (1H, m), 3.50— 3.64 (1H, m), 3.84— 3.89 (1H, m), 4.16—4.21 (IH, m), 5. 05 (IH, br), 5.21 (IH, br), 6.07 (1H, dd, J = 6.3Hz, J = 6.2Hz), 6.32 (IH, d, J = 7.2Hz) , 7.32 (1H, m), 7.67 (1H, m), 7.91 (IH, m), 8.02 (1 H, m), 8.19 (IH, m, J = 7.2Hz)

[Example 4] 3, 5, — O-bis- (tert-butyldimethylsilyl) — 4— N— (N-hue [0051] [Chem. 14]

2.0 mmol) was suspended in methylene chloride (20 mL), and phenol sulfone isocyanate (271 μ2.0 mmol) was added and stirred for 5 minutes. Dilute with black mouth form (20 mL) and wash with aqueous sodium chloride followed by saturated aqueous sodium bicarbonate. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Purification by column chromatography using hexane silica gel (C200 silica gel) gave the title compound (1.1 g, yield 87%).

[0052] 1H NMR (CDC13) δ 0.08— 0.11 (12H, m), 0.90— 0.92 (18H, m), 2.07-2.17 (1H, m), 2.59— 2.67 (1H, m), 3.76— 3.95 (2H, m), 3.95 —4.03 (1H, m), 4.34—4.42 (1H, m), 6.26 (1H, dd, J = 5.9Hz, J = 5.6 Hz), 7.42-7.75 (4H, m), 8.09— 8.13 (2H, m), 8.32 (1H, d, J = 7.6H z)

Example 5 Synthesis of 6-N- [N- (2-Trophele) force rubermoyl] deoxyadenosine

[0053] [Chemical 15]

Deoxyadenosine (502 mg, 2.0 mmol) was suspended in pyridine (20 mL), and trimethinoresilinorechloride (758 L, 6.0 mmol) was calored and stirred for 30 minutes. Then, 2-nitrophenol isocyanate (361 mg, 2.2 mmol) was stirred for 90 minutes. After diluting with pyridine (20 mL), concentrated aqueous ammonia (20 mL) was added and stirred for 2 hours. Chloroform (50 mL) and aqueous sodium chloride solution (50 mL) were added, and the mixture was extracted 3 times with black mouth form-pyridine (1: 1, v / v, 30 mL). The organic layer was recovered, the solvent was distilled off under reduced pressure, and water was added. The resulting precipitate was collected and dried to obtain the title compound (740 mg, yield 89%).

[0054] 1H NMR (DMSO— d6) δ 2.31— 2.42 (1H, m), 2.71— 2.81 (1Η, m), 3.50— 3.69 (2H, m), 3.88— 8.93 (1H, m), 4.41—4.57 (1H, m), 5. 03 (1H, t, J = 5.6Hz), 5.36 (1H, d, J = 4.0Hz) 6.45 (1H, dd, J = 6, 6Hz, J = 6.9Hz), 7.22 -7.33 (1H, m), 7.71— 7.80 (1H, m), 8.10— 8.15 (2H, m), 8.43-8.50 (1H, m), 8.65 (1H, s), 8.69 (1H, s), 10.63 (1H, br), 13.09 (1H, br)

[Example 6] 3 ,, 5, O-Bis- (tert-butyldimethylsilyl) -6-N- (N-Fe [0055] [Chemical 16]

1.0 mmol) was suspended in methylene chloride (20 mL), and phenol sulfone isocyanate (136 μ1.0 mmol) was added and stirred for 5 minutes. The mixture was diluted with black mouth form (20 mL) and washed with an aqueous sodium chloride solution and then with a saturated aqueous sodium hydrogen carbonate solution. The organic layer was collected, dried over anhydrous sodium sulfate, and the solvent was lifted under reduced pressure. Purification by column chromatography (hexane monochloroform) using C200 silica gel gave the title compound (613 mg, 92% yield).

[0056] 1H NMR (CDC13) δ 0.05— 0.12 (12H, m), 0.82— 0.97 (18H, m), 2.42-2.52 (1 Η, m), 2.61— 2.73 (1 Η, m), 3.73— 3.91 (2Η, m), 4.02 -4.07 (1H, m), 4.60-4.67 (1H, m), 6.46 (1H, dd, J = 6, 6Hz, J = 6.3 Hz), 7.51-7.67 (3H, m) , 8.16— 8.21 (2H, m), 8.41 (1H, s), 8.61 (1 H, br), 8.65 (1H, s), 13.12 (1H, br)

[Example 7] 3 ,, 5, O-bis- (tert-butyldimethylsilyl) -2-N- (N-Hye [0057] [Chemical 17]

1.0 mmol) was suspended in pyridine (20 mL), and trimethylsilyl chloride (631 μ5.0 mL) was stirred for 30 minutes. Ferrosulfol isocyanate (136 L, 1.0 mmol) was added and stirred for 20 minutes. The solvent was distilled off under reduced pressure, and azeotroped three times with toluene and three times with chloroform. The precipitate resulting from the formation of ethyl acetate was collected and dried to obtain the title compound (1.24 g, yield 91%).

[0058] 1H NMR (CDC13) δ 0.04— 0.11 (12H, m), 0.83— 0.96 (18H, m), 2.26-2.57 (2H, m), 3.70— 3.80 (2H, m), 3.91— 3.94 (1H, m), 4.49 —4.58 (1H, m), 6.62 (1H, dd, J = 6, 6Hz, J = 6.3Hz), 7.35—7.60 (3H, m), 7.94 (1H, s), 7.90 — 8.01 (2H, m), 8.17 (1H, br), 12.46 (1H, s), 13.12 (1H, br)

Example 8 Deoxycytidine deprotection

Deoxycytidine protected with various substitution force rubamoyl groups was heat-treated, and the time until deprotection was measured.

[0059] The deprotection reaction was carried out in D OZDMSO-d6, and the heating temperature was 80 ° C.

2

[0060] Time to deprotect half of protected doxycytidine (T) and all protection

1/2

Table 1 shows the time (T) until deoxycytidine is deprotected.

comp

[0061] [Table 1] Protecting group τ _1/2 τ 丄 com ϋ

N phenylcarbamoyl group ^a 1 1 hour nd ^d

N (Naphtho 1 Gil) Powerful Rubamoyl group "2.5 hours 20 hours

N (Quinole 5 yl) Powerful Rubamoyl group ^a 1.5 hours 10 hours

N— (2-Futaguchi Phenolic) Powerful Rubamoyl group ^b 30 minutes 3 hours

N— (3-Nitrophenol) Forced rubermoyl group ^b 3 hours 25 hours

N— (4 twelve trophenols) force rubermoyl group ^b 2.5 hours 20 hours

The amount of N-phenylalanine sulfonylcarbamoyl group nd ^d less than 10 minutes a protected nucleoside is a 2 Omm, the volume ratio of D ₂ 0 and DMSO-d 6 was 1: 1. b The amount of protected nucleoside was 1 OmM, and the volume ratio of D ₂ 0 to DMSO d 6 was 1: 3. The amount of c-protected nucleoside was 7 mM, and the volume ratio of D ₂₀ to DMS O—d 6 was 1: 5. dnot determined As shown in Table 1, groups with high electron-withdrawing properties such as N- (2--trophenol) force rubamoyl group and N-phenylsulfodicarbamoyl group required a short time for deprotection.

This specification includes the contents described in the specification and Z or drawings of the Japanese patent application (Japanese Patent Application No. 2005-64892) which is the basis of the priority of the present application. In addition, all publications, patents and patent applications cited in the present invention are incorporated herein by reference as they are.

Claims

Claims [1] General formula (I):

[Chemical 1]

A compound represented by the general formula (Π):

[Chemical 2]

0 = C = N-Y (I I)

[Wherein Y represents an electron-withdrawing group. ]

Is reacted with an isocyanate derivative represented by the general formula (ΠΙ):

[Chemical 3]

[Wherein, X and γ are as defined above. ]

[2] γ in the general formulas (II) and (in) is an aryl group which may have a substituent or a substituent, and may be an arylsulfonyl group The method for synthesizing a nuclear acid according to claim 1.

[3] Y-force in general formulas (Π) and (ΙΠ) 4 2-trophenyl group, 3-trophenyl group, 2

2. The method for synthesizing a nucleic acid according to claim 1, wherein the method is a trifluoro group or a phenyl sulfonyl group. General formula (m)

[Five]

[6]

[7]

[In the formula, X represents a moiety other than an amino group of a nucleoside containing a nucleobase having an amino group or an analogous compound, and Υ, is a 4--trophenyl group, 3--trophenyl group, 2-- Represents a tropenyl group or a phenylsulfol group. ] A compound represented by