JP2000001478A - New delta-amino acid bearing nucleic acid base on side chain and derivative thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound useful as a raw material for peptide nucleic acids each bindable to a specific sequence in a DNA or RNA contributing to disease and thereby capable of inhibiting the onset of relevant disease. SOLUTION: This new compound is shown by formula I [R1 is H or an amino-protecting group; R2 is OH or a group representing a carboxyl derivative; Bs is a nucleic acid base (derivative)], e.g. 2-[(4-adenyl-(S)-2-(9- fluorenylmethoxycarbonyl)-aminobutyl)oxy]acetic acid. The compound of formula I is obtained, for example, by the following procedure : the amino group of L-homoserine of formula II is protected with butoxycarbonyl group or the like followed by converting the L-homoserine to the corresponding ester form by the action of ethyl bromide or the like, the γ-hydroxyl group of the ester form is then protected by etherifying it with tetrapyranyl group or the like followed by reducing the ester group to form the corresponding hydroxyl form, which is then reacted with an α-haloacetic ester to deprotect the hydroxyl group to form the corresponding ester form of formula III, a base is subsequently introduced into the above ester form to afford a compound with the base protected, followed by deprotecting the base.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel amino acid which is a raw material of a peptide nucleic acid (PNA) capable of binding to a specific sequence of DNA or RNA causing a disease and suppressing the onset of the disease.

[0002]

2. Description of the Related Art Nucleic acids such as DNA and RNA are composed of nucleobases (bas
e), the development of nucleic acids using a peptide backbone instead of the backbone composed of deoxyribose or ribose is under way. Nucleic acids having these peptide skeletons are referred to as “peptide nucleic acids” (PNA (Peptiden
ucleic acid)). Although this name is not chemically accurate, it is named because it has both a nucleic acid chain and a peptide (or pseudopeptide) bond (P, E, Nielsen et al., Bioconjugate Chemistry, No. 5). Vol., Pp. 3-7 (19
1994) (PENielsen, et al., Bioconjugate Chem.,
1994, 5, 3-7)).

[0003] These "peptide nucleic acids" (PNA)
The base position is designed to be distance-homologous to the backbone composed of phosphate bonds of deoxyribose and ribose such as DNA and RNA, and can hybridize with ordinary DNA and RNA. In some cases, it is known to form three kinds of complexes by hydrogen bonding, such as PNA-DNA-PNA (Id., And M., Egholm et al., Nature, 365-
566-568 (1993) (M. Egholm, et a.
l., Nature, 365, 566-568 (1993)).

In general, PNA-DNA or PNA-
RNA binding can be performed using ordinary DNA-DNA or DNA-RN.
It has better thermal stability than the bond of A,
It has the property of hybridizing no matter how many base pairs are mismatched. For this reason, PNA is expected to be applied as an unsense or primer. However, the conventional PNA has a major drawback of being poorly soluble in water, and has been a major obstacle in practical use.

[0005]

DISCLOSURE OF THE INVENTION The present invention is directed to a novel peptide nucleic acid having a flexible and easily hydrated polyamide ether main chain structure and a 2- [4-base-2 as a raw material thereof, which has improved the above-mentioned disadvantages. [Aminobutyroxy] acetic acid and derivatives thereof.

[0006]

The present invention provides a compound represented by the general formula (I):

Embedded image (Wherein, R ₁ represents a protecting group for a hydrogen atom or an amino group, R ₂ represents a group that becomes a derivative of a hydroxyl group or a carboxyl group, and Bs represents a base of a nucleic acid or a derivative thereof). The present invention relates to a δ-amino acid derivative or a salt thereof, a peptide nucleic acid derivative comprising two or more δ-amino acid derivatives linked by an acid amide bond, and a method for producing the same.

The substituent Bs of the general formula (I) of the present invention is preferably a base of a nucleic acid such as DNA or RNA such as adenine, guanine, cytosine, thymine or uracil, but is not limited thereto. Any base or derivative thereof that can form a hydrogen bond capable of forming a double chain or triple chain with the compound is acceptable. As such a base or a derivative thereof, the following formula:

[0008]

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[0009]

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[0010]

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[0011]

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Or

[0013]

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(Wherein, R ₃ represents a hydrogen atom or an amino-protecting group).

The amino-protecting group for the group R _{1 in} the general formula (I) and the group R ₃ in the base or the derivative thereof may be a protecting group used in ordinary peptide synthesis. There is no particular limitation as long as it can be eliminated as required and can regenerate a free amino group. Such protective groups include those having 1 to 2 carbon atoms.
0, preferably 1 to 15, more preferably 1 to 10 linear or branched alkyl groups, 7 to 20 carbon atoms, preferably 7 to 15 monocyclic, polycyclic or condensed cyclic aralkyl An alkylcarbonyl group comprising the above-mentioned alkyl group; an arylcarbonyl group comprising a monocyclic, polycyclic or condensed cyclic aryl group having 6 to 20 carbon atoms, preferably 6 to 15 carbon atoms; 1 to 5, preferably 1 to 5 5 to 7 membered rings having nitrogen, oxygen or sulfur atoms
Heterocyclic carbonyl group consisting of a monocyclic, polycyclic or condensed saturated or unsaturated heterocyclic group having three, an alkoxycarbonyl group consisting of the above-mentioned alkyl group, aryloxy consisting of the above-mentioned aryl group Examples include a carbonyl group, an aralkyloxycarbonyl group comprising the above-mentioned aralkyl group, and a heterocyclic oxycarbonyl group comprising the above-mentioned heterocyclic group.

Preferred amino-protecting groups include benzoyl, isobutyroyl, benzyloxycarbonyl, Fmoc and tert-butoxycarbonyl.

In the group R ₂ of the formula (I), examples of the group that becomes a derivative of a carboxyl group include groups that can form a carboxylic acid derivative such as an ester, an acid halide, and an acid anhydride. Can be The group R ₂ capable of forming an ester, an alkoxy group consisting of alkyl groups described above, the above-mentioned aralkyloxy group consisting of an aralkyl group, an aryloxy group consisting of aryl groups mentioned above. Examples of preferred radicals R ₂ are Besides a hydroxyl group, a methoxy group, an ethoxy group, a tert-butoxy group, a benzyloxy group and the like can be mentioned.

The δ-amino acid derivative represented by the general formula (I) of the present invention can be produced by various methods. For example, it can be produced by using a synthesis route represented by the following formula using L-homoserine as a starting material.

[0019]

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The amino group of L-homoserine (1) is protected with a butoxycarbonyl group or the like to give an N-protected form (2), which is converted into an ester form (3) with ethyl bromide or the like. Next, after protecting the γ-hydroxyl group by etherification with a tetrapyranyl group or the like, the ester group is reduced to obtain a hydrolyzate (5), to which an α-haloacetic acid ester such as α-bromoacetic acid tert-butyl ester is added. To give an amino acid derivative ester (6).
By removing the hydroxyl-protecting group, the desired ester (7) can be produced.

The resulting ester (7) can be converted to the δ-amino acid derivative of the present invention by the reaction represented by the following formula.

[0022]

Embedded image

First, the free hydroxyl group of the ester (7) is
A compound having a protecting group attached to the base by introducing a base by reacting with a target base or by directly introducing a base from the ester (7) by a method such as tosylation, 9) is obtained, the free carboxylic acid (10) can be obtained by removing the protecting group of the base and, if necessary, removing or converting the protecting group of the amino group. The free carboxyl group can be esterified, halogenated, or the like, if necessary.

The present invention relates to a peptide nucleic acid derivative in which two or more δ-amino acid derivatives of the general formula (I) are linked by an acid amide bond. The peptide nucleic acid derivative of the present invention can be produced by sequentially acid-amidating the δ-amino acid derivative represented by the general formula (I) by a method commonly used in peptide synthesis. By sequentially coupling δ-amino acid derivatives having a desired base, a peptide nucleic acid derivative having a target base sequence can be obtained.

FIG. 1 shows a production example of the peptide nucleic acid of the present invention. In this example, an example of a solid phase synthesis method using a polyethylene glycol resin is shown.
Examples of the production of polyadenine are shown.

The δ-amino acid derivative of the present invention having an adenine base protected by a benzoyl group is immobilized on a polyethylene glycol resin, and the D derivative of 20% piperidine (PIP)
After reacting in an MF solution for 7 minutes to remove the protecting group of the amino group to form a free amino group and performing a Kaiser test, 3 equivalents of monomeric amino acid in dimethylacetamide (DMAA) and 3 equivalents of benzotriazole -1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBop), 3 equivalents of N-
Hydroxy-benzotriazole (HOBt), and
Reaction with 6 equivalents of N-methylmorpholine (NMM) can produce one additional δ-amino acid derivative.

By performing this cycle a required number of times,
A peptide nucleic acid of a desired length can be obtained. For example, in the case of producing an 8-mer or 12-mer polyadenine chain, the target product can be obtained by performing an 8-cycle or 12-cycle reaction using an amino acid derivative having adenine in each cycle.

The peptide nucleic acid having the desired length is then deprotected with a protecting group such as a benzoyl group in a base using an ethanol solution of ethylenediamine (1: 1),
m-cresol and trifluoroacetic acid (TFA) (1:
The target peptide nucleic acid can be obtained by releasing from the stationary phase using the method 4).

FIG. 2 shows the results of HPLC analysis of polymerdenine having a length of 12 mer obtained by the above method. The measurement conditions were as follows: Column: C18 column Developing solvent: CH ₃ CN / 0.1 M NH ₄ OAc (pH 4.5) 0 → 100%, 0 → 50 minutes Flow rate: 0.6 ml / min Retention time: about 21 minutes Was.

FIG. 3 shows the ¹ H of 12 mer polyadenine.
1 shows an NMR chart.

The peptide nucleic acid derivative of the present invention can be hybridized with DNA or RNA by a conventional method.
It is useful as an antisense or primer.

[0032]

The present invention will be described below with reference to specific examples.
The present invention is not limited to these specific examples.

Production Example 1 Boc-L-homoserine; Synthesis of compound (2)

Embedded image L-homoserine (4.0 g; 33.58 mmol) was transferred to an eggplant flask and dissolved well in distilled water (18 ml). (Boc) ₂ O (8.79 g; 1.2 eq) was dissolved in dioxane (18 ml) and added to the eggplant flask.
The mixture was stirred under ice-cooling for 3 minutes, and then added with sodium hydrogen carbonate (7.06
g; 2.5 eq), and the mixture was stirred at room temperature overnight. Dioxane was distilled off under reduced pressure using an evaporator, dissolved in distilled water, and washed with ether. Ethyl acetate was added, and the mixture was extracted to pH 2 with a 5% aqueous potassium hydrogen sulfate solution, and washed twice with a saturated saline solution. It was dried over anhydrous magnesium sulfate and filtered. Dicyclohexylamine (7 ml; le
q) was added, and the mixture was precipitated and distilled off under reduced pressure using an evaporator. Further, dicyclohexylamine (14 ml; 2e
q) was added and precipitation was performed overnight in a refrigerator. The solution was subjected to suction filtration and dried in a desiccator. Yield 11.15 g,
The yield was 82.9%.

Production Example 2 Boc-L-homoserine ethyl ester; synthesis of compound (3)

Embedded image Compound (2) obtained in Production Example 1 (11.15 g; 2
7.9 mmol) in dimethylformamide (80 ml)
And bromoethane (4.57 g; 1.5 eq).
Was added. Stir at room temperature for 21 hours. Dimethylformamide was distilled off under reduced pressure using an evaporator. It was dissolved in distilled water, extracted with ethyl acetate, and washed three times with saturated saline.
It was dried over anhydrous magnesium sulfate and filtered. The solvent was distilled off under reduced pressure with a vaporizer and purified by a silica gel column (silica gel column solvent; ethyl acetate: hexane = 1: 1,
f. 12-36, Rf value 0.50). Yield 5.34 g,
The yield was 77.6%. NM of compound (3) obtained
The R chart is shown in FIG.

Production Example 3 Boc-O-tetrahydropyranyl-L-homoserine ethyl ester; synthesis of compound (4)

Embedded image Compound (3) obtained in Production Example 2 (5.28 g; 21.
38 mmol) was completely dissolved in dichloromethane (50 ml), and pyridinium paratoluenesulfonate (0.52 g; 0.1 eq) dissolved in a small amount of dichloromethane.
And 3,4-dihydro-2H-pyran (2.1
0 g; 1.2 eq) was added dropwise. The mixture was stirred at room temperature for 24 hours. Dichloromethane was distilled off under reduced pressure with a mechanical evaporator (at this time, dichloromethane is a halogen-based solvent, so be careful of waste water. The same applies hereinafter), extracted with ethyl acetate, and washed twice with saturated saline. It was dried over anhydrous magnesium sulfate and filtered. The solvent was distilled off under reduced pressure using an evaporator, and the residue was purified twice using a silica gel column (silica gel column solvent; ethyl acetate: hexane = 1: 3, f.
f. 15-35, Rf value 0.47). Yield 5.68 g,
The yield was 80.3%. NM of compound (4) obtained
The R chart is shown in FIG.

Production Example 4 Boc-O-tetrahydropyranyl-L-homoselinol; synthesis of compound (5)

Embedded image Compound (4) obtained in Production Example 3 (5.68 g; 17.
16 mmol) was completely dissolved in ethanol, NaBH ₄ dissolved in ethanol was added, and the mixture was stirred at room temperature for 4 hours. After confirming the progress of the reaction by TLC, the reaction was stopped by adjusting the pH to 7 with a 5% aqueous solution of potassium hydrogen sulfate under ice-cooling. The reaction solvent was distilled off under reduced pressure using an evaporator, dissolved in distilled water, and extracted with ethyl acetate. The extract was washed with saturated saline and dried over anhydrous magnesium sulfate. After filtration, the filtrate was distilled off under reduced pressure using a vaporizer. Purification was performed using a silica gel column (silica gel column solvent; ethyl acetate: hexane = 13: 7 f.11-50, Rf value 0.44). The yield was 4.59 g and the yield was 92.9%. FIG. 6 shows an NMR chart of the obtained compound (5).

Production Example 5 t-butyl 12-[(4-tetrahydropyranyloxy- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester; synthesis of compound (6)

Embedded image 13. Compound (5) obtained in Production Example 4 (4.28 g;
86 mmol) was added with tetrahydrofuran (30 ml), and NaH / 60% (1.19 g; 2 eq) was added.
N ₂ atmosphere, under ice-cold BrCH ₂ COOBu ^t (8.4
6 g; 3 eq) and gradually warmed to room temperature, N
_The mixture was stirred under _two atmospheres. The reaction solution was transferred to a centrifuge tube, centrifuged (3000 rpm, 5 min), and the supernatant was transferred to an eggplant flask. The reactor was washed with ethyl acetate, the washing solution was transferred to a centrifuge tube, and centrifuged in the same manner. This was repeated five times, and the solvent was distilled off under reduced pressure using an evaporator. The mixture was extracted with ethyl acetate and adjusted to pH 7 with a 5% aqueous potassium hydrogen sulfate solution under ice-cooling. The extract was washed with saturated saline and dried over anhydrous magnesium sulfate. After filtration, ethyl acetate was distilled off under reduced pressure using an evaporator. This was purified with a silica gel column (silica gel column solvent; ethyl acetate: hexane = 1: 4f.14-33, Rf value 0.23). The yield was 2.50 g and the yield was 43.1%. FIG. 7 shows an NMR chart of the obtained compound (6).

Production Example 6 Synthesis of compound (7) t-butyl 12-[(4-hydroxy- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester

Embedded image Compound (6) obtained in Production Example 5 (2.50 g; 6.4)
1 mmol) in ethanol (50 ml), and pyridinium paratoluenesulfonate (0.12 g; 0.1 eq) dissolved in a small amount of ethanol was added.
Stir for 4 hours. The reaction solvent was distilled off under reduced pressure using an evaporator. Extract with ethyl acetate, wash twice with saturated saline,
Washed once with distilled water. It was dried over magnesium sulfate, filtered, and ethyl acetate was distilled off under reduced pressure using an evaporator.
Purified by silica gel column (silica gel column solvent;
Ethyl acetate: hexane = 1: lf. 22-59, Rf value 0.57). The yield was 1.52 g, and the yield was 77.6%. FIG. 8 shows an NMR chart of the obtained compound (7).

Production Example 7 Synthesis of compound (8) t-butyl 12-[(4-tosyloxy- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester

Embedded image Compound (7) obtained in Production Example 6 (1.11 g; 3.6)
To 3 mmol) was added pyridine (3.07 ml). Ts-Cl (0.8
3g; 1.2eq) at -10 ° C for 1 hour at -10 ° C
With stirring. Thereafter, the mixture was stirred at 0 ° C. for 7 hours. After confirming the completion of the reaction by TLC, the mixture was extracted with ether. Washed 4 times with 25% citric acid (50 ml). Dried over magnesium and filtered. After ether was distilled off under reduced pressure with an evaporator, the residue was purified with a silica gel column (silica gel column solvent; ethyl acetate: hexane = 2:
3f. 11 to 24, Rf value 0.54). Yield 0.91
g, yield was 54.2%. FIG. 9 shows an NMR chart of the obtained compound (8).

Embodiment 1 t-butyl 12-[(4-benzoyladenyl- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester; compound (9
Synthesis of a)

Embedded image Compound (8) obtained in Production Example 7 (0.81 g; 1.7)
5 mmol) and DMF (10 ml) was added to suspend the mixture.
Benzoyl adenine (1.05 g; 2.5 eq), anhydrous potassium carbonate (0.61 g; 2.5 eq) and 18-crown-6-ether (0.175 g) were added, and the mixture was stirred at room temperature for 7 hours. The reaction solvent was distilled off under reduced pressure using an evaporator. Extracted with dichloromethane and washed once with distilled water. After dehydration with anhydrous magnesium sulfate and filtration, dichloromethane was distilled off under reduced pressure using an evaporator. Purified three times with a silica gel column (silica gel column solvent; ethyl acetate: hexane = 1: 1f.19, 54-94, 30-9).
3, Rf value 0.39). Yield 0.70 g, yield 81.4
%Met. The NMR chart of the obtained compound (9a) is shown in FIG.

Embodiment 2 FIG. t-butyl 12-[(4-benzoylthyminyl- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester; compound (9
Synthesis of b)

Embedded image Compound (8) obtained in Production Example 7 (0.91 g; 1.9)
7 mmol) and added with DMF (10 ml) to suspend the mixture.
Benzoylthymine (1.13 g; 2.5 eq), anhydrous potassium carbonate (0.68 g; 2.5 eq) and 18-crown-6-ether (0.197 g) were added, and the mixture was added at room temperature for 7 hours.
Stirred for hours. The reaction solvent was distilled off under reduced pressure using an evaporator. Extracted with dichloromethane and washed once with distilled water.
After dehydration with anhydrous magnesium sulfate and filtration, dichloromethane was distilled off under reduced pressure using a vaporizer. Purified three times with a silica gel column (silica gel column solvent; ethyl acetate:
Hexane = 1: 1 f. 19, 54-94, 30-93,
Rf value 0.39). The yield was 0.70 g and the yield was 81.4%. FIG. 11 shows an NMR chart of the obtained compound (9b).

Embodiment 3 FIG. t-butyl 12-[(4-benzoylcytosyl- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester; compound (9
Synthesis of c)

Embedded image Compound (8) obtained in Production Example 7 (0.22 g; 0.4
8 mmol), add DMF (5 ml) and suspend,
Benzoylcytosine (0.18 g; 1.7 eq), anhydrous potassium carbonate (0.33 g; 5.0 eq), 18-crown-6-ether (0.048 g) were added under N ₂ atmosphere, and the mixture was added at 50 ° C. overnight. Stirred. The reaction solvent was distilled off under reduced pressure using an evaporator. Extracted with dichloromethane and washed 4 times with distilled water. After dehydration with anhydrous magnesium sulfate and filtration, the filtrate was evaporated under reduced pressure using an evaporator. Purified by silica gel column (silica gel column solvent; ethyl acetate: hexane = 1: 1 f.23-37, Rf value 0.3)
4). The yield was 5 mg, and the yield was 0.001%. FIG. 12 shows an NMR chart of the obtained compound (9c).

Embodiment 4 FIG. t-butyl 12-[(4-benzoyladenyl- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester; compound (9
Synthesis of a): Mitsunobu Reaction
action)

Embedded image Compound (7) obtained in Production Example 6 (1.0 g; 3.27)
ml) was suspended in tetrahydrofuran (40 ml), and benzoyladenine (2.346 g; 3 eq) was added.
Triphenylphosphine (0.960 g; 1.1e
q), the mixture was cooled to -15 ° C with methanol-dry ice under an argon atmosphere, diethylazodicarbonate (540 µl; 1.1 eq) was added using an injection needle, and the mixture was stirred overnight. The reaction solvent was distilled off under reduced pressure using an evaporator.
Extracted with ether. It was washed with distilled water and dehydrated with anhydrous magnesium sulfate. After filtration, the filtrate was distilled off under reduced pressure by an evaporator and purified by a silica gel column three times (silica gel column solvent; ethyl acetate 100%, f.
68-100 and 200 ml, 42-79, Rf value 0.1
9). The yield was 0.57 g and the yield was 33.0%.

Example 5.2 2-[(4-Adenyl- (S)
-2- (9-Fluorenylmethoxycarbonyl) -aminobutyl) oxy] acetic acid; synthesis of compound (10a)

Embedded image Compound (9a) obtained in Example 1 (0.970 g;
1.83 mol) in a fume hood with HBr / acetic acid (15 m
l) was added and the mixture was stirred at room temperature for 30 minutes. The reaction solvent was distilled off under reduced pressure using an evaporator. A 5% aqueous sodium hydrogen carbonate solution / acetonitrile (10/8 ml) was added, and 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide (Fmo) dissolved in acetonitrile (2 ml) was added.
(c-OSu) (0.68 g; 1.1 eq) was added and the mixture was stirred at room temperature overnight. The reaction solution was distilled off under reduced pressure using an evaporator, and distilled water was added thereto.
Washed with ether to remove c-OSu. Extract with ethyl acetate and adjust to pH 2 with 5% aqueous potassium hydrogen sulfate solution.
Then, the target substance was transferred to an ethyl acetate layer. It was separated and purified by HPLC. FIG. 13 shows an NMR chart of the obtained compound (10a).

Example 6.2-[(4-thyminyl- (S)
-2- (9-Fluorenylmethoxycarbonyl) -aminobutyl) oxy] acetic acid; synthesis of compound (10b)

Embedded image Compound (9b) obtained in Example 2 (0.970 g;
1.83 mol) in a fume hood with HBr / acetic acid (15 m
l) was added and the mixture was stirred at room temperature for 30 minutes. The reaction solvent was distilled off under reduced pressure using an evaporator. A 5% aqueous sodium hydrogen carbonate solution / acetonitrile (10/8 ml) was added, and 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide (Fmo) dissolved in acetonitrile (2 ml) was added.
(c-OSu) (0.68 g; 1.1 eq) was added and the mixture was stirred at room temperature overnight. The reaction solution was distilled off under reduced pressure using an evaporator, and distilled water was added thereto.
Washed with ether to remove c-OSu. Extract with ethyl acetate and adjust to pH 2 with 5% aqueous potassium hydrogen sulfate solution.
Then, the target substance was transferred to an ethyl acetate layer. It was separated and purified by HPLC. FIG. 14 shows an NMR chart of the obtained compound (10b).

Example 7.2 2-[(4-cytosyl- (S)
-2- (9-Fluorenylmethoxycarbonyl) -aminobutyl) oxy] acetic acid; synthesis of compound (10c)

Embedded image Compound (9c) obtained in Example 3 (5 mg; 9.5 µ)
HBr / acetic acid (15 ml) was added to the mol in a fume hood, and the mixture was stirred at room temperature for 30 minutes. The reaction solvent was distilled off under reduced pressure using an evaporator. A 5% aqueous solution of sodium bicarbonate / acetonitrile (10/8 ml) was added, and 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide (Fmoc-OS) dissolved in acetonitrile (2 ml) was added.
u) (0.68 g; 1.1 eq) and stirred at room temperature overnight. The reaction solution was distilled off under reduced pressure using an evaporator, and distilled water was added thereto.
Washed with ether to remove u. Extract with ethyl acetate, bring to pH 2 with 5% aqueous potassium hydrogen sulfate,
The target substance was transferred to the ethyl acetate layer. It was separated and purified by HPLC. FIG. 15 shows an NMR chart of the obtained compound (10c).

Embodiment 8 FIG. H- [NH-CH (CH ₂ CH _{2
-A) -CH 2 -O-CH 2} -CO] 12 -Lys-OH
Production of (OPNA (A ₁₂ )) Super Acid Labail polyethylene glycol resin (Fmoc-NH-SAL) using benzotriazol-1-yl-oxy-trispirolidinephosphonium hexafluorophosphate (PyBop) / HOBt as a coupling agent The condensation of an Fmoc-protected δ-amino acid derivative having N ⁶ -benzoyladenine was carried out by a solid phase method using (-PEG) (manufactured by Watanabe Chemical) (see FIG. 1). First, Nε-Z-lysine was immobilized on the resin. Next, the coupling reaction of the Fmoc-δ-amino acid derivative was repeated 12 times. After the coupling reaction, the benzoyl group was removed using ethylenediamine. Next, the generated oligopeptide was removed from the resin using trifluoroacetic acid (TFA) containing 20% m-cresol, and purified as a single peak on HPLC (ODS column) (see FIG. 2). ¹ H-NMR
(See FIG. 3) and MALDI-TOF MS (m / z
(M + H) ^<+ > 3292.7 (calculated), 3295.7
(Actual value)) indicated that it was the target compound.

Comparative Example A Nielsen-type peptide nucleic acid (PNA (A ₁₂ )) consisting of 12 adenyl groups was produced by a solid phase method. MALDI-TOF MS data of the obtained PNA (A ₁₂ ) (m / z (M + Na) ⁺ 3469.4 (calculated value), 3
465.3 (actual value) showed that it was the desired product. The peptide nucleic acid (OPN) obtained in Example 8 of the present invention
A (A ₁₂ )), a Nielsen-type peptide nucleic acid (PNA)
(A ₁₂ )) and various heats in hybridization between DNA (A ₁₂ ) and DNA (T ₁₂ ) which is a complementary strand thereof or DNA (T ₆ CT ₅ ) in which the seventh base is not complementary. Mechanical parameters were measured. The results are shown in Table 1 below. Table 1. Thermodynamic parameters for hybridization of DNA (T ₁₂ ) or DNA (T ₆ CT ₅ ) with DNA (A ₁₂ ), OPNA (A ₁₂ ), and PNA (A ₁₂ ) ─────────────────────────── Component Tm ΔH ΔS (℃) (kcal mol ^-1 ) (cal mol ^-1 K ^-1 ) ─────────────────────────────────── Fully matching DNA (A ₁₂ ) -DNA (T ₁₂ ) 30- _{83.7 -251 OPNA (A 12) -DNA} (T 12) 43 -112 -329 PNA (A 12) -DNA (T 12) 55 -47.7 -120 -79.5 -217 Single mismatch DNA ( _{A 12) -DNA (T 6 CT} 5) 12 -70.5 -222 OPNA (A 12) -DNA (T 6 CT 5) 30 -97.7 -29 _{7 PNA (A 12) -DNA (} T 6 CT 5) 42 -59.0 -161 -77.3 -220 ─────────────────────── ───────────── In addition, these hybridization temperatures and 26
The UV absorption at 0 nm is shown in FIG. (A) in FIG.
Is a mixture of DNA (A ₁₂ ) and DNA (T ₁₂ ), (b)
Is a mixture of the OPNA (A ₁₂ ) of the present invention and DNA (T ₁₂ ), and (c) is a Nielsen-type PNA (A ₁₂ ) and DNA
(T ₁₂ ), 150 mM NaCl, 10 m
M NaH ₂ PO ₄ , 0.1 mM EDTA (pH 7.
0) shows the case where the concentration is 5.9 μM. The diagram inserted in FIG. 16 shows these association constants (associatio
n constant) is shown. FIG. 17 shows a DNA in which the seventh base is not complementary to DNA.
A (T ₆ CT ₅ ) and UV of 260 nm
It is a measurement of absorption. From these results, it was found that the peptide nucleic acid of the present invention was composed of Nielsen-type PNA and normal DN.
It has an intermediate Tm with A and makes a very sharp transition (see FIGS. 16 and 17). Therefore,
The peptide nucleic acid of the present invention is particularly useful as a medical antisense strand.

[Brief description of the drawings]

FIG. 1 shows an example of a process for producing a peptide nucleic acid of the present invention.

FIG. 2 shows a HPLC chart of a 12-mer peptide nucleic acid obtained by the method of the present invention.

FIG. 3 shows an NMR chart of a 12-mer peptide nucleic acid obtained by the method of the present invention.

FIG. 4 shows an NMR chart of Boc-L-homoserine ethyl ester.

FIG. 5 shows Boc-O-tetrahydropyranyl-
3 shows an NMR chart of L-homoserine ethyl ester.

FIG. 6 shows Boc-O-tetrahydropyranyl-
1 shows an NMR chart of L-homoselinol.

FIG. 7 shows an NMR chart of t-butyl 12-[(4-tetrahydropyranyloxy- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester.

FIG. 8 shows an NMR chart of t-butyl 12-[(4-hydroxy- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester.

FIG. 9 shows an NMR chart of t-butyl 12-[(4-tosyloxy- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester.

FIG. 10 shows an NMR chart of t-butyl 12-[(4-benzoyladenyl- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester.

FIG. 11 shows an NMR chart of t-butyl 12-[(4-benzoylthyminyl- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester.

FIG. 12 shows an NMR chart of t-butyl 12-[(4-benzoylcytosyl- (S) -2-t-butoxycarbonylaminobutyl) oxy] acetic acid ester.

FIG. 13 shows 2-[(4-adenyl- (S)-
2 shows an NMR chart of 2- (9-fluorenylmethoxycarbonyl) -aminobutyl) oxy] acetic acid.

FIG. 14 shows 2-[(4-thyminyl- (S)-
2 shows an NMR chart of 2- (9-fluorenylmethoxycarbonyl) -aminobutyl) oxy] acetic acid.

FIG. 15 shows 2-[(4-cytosyl- (S)-
2 shows an NMR chart of 2- (9-fluorenylmethoxycarbonyl) -aminobutyl) oxy] acetic acid.

FIG. 16 shows a chart of UV absorption at 260 nm of temperature-dependent hybridization with DNA (T ₁₂ ).

FIG. 17 shows a chart of UV absorption at 260 nm of temperature-dependent hybridization with DNA (T ₆ CT ₅ ).

Claims (9)

[Claims] 1. A compound of the general formula (I) (Wherein, R ₁ represents a protecting group for a hydrogen atom or an amino group, R ₂ represents a group that becomes a derivative of a hydroxyl group or a carboxyl group, and Bs represents a base of a nucleic acid or a derivative thereof). Or a salt thereof. 2. The group Bs has the following formula: Embedded image Embedded image Embedded image Or The δ-amino acid derivative or a salt thereof according to claim 1, wherein R ₃ is a group represented by a protecting group for a hydrogen atom or an amino group. 3. The δ-amino acid derivative or a salt thereof according to claim 2, wherein the group R ₃ is a hydrogen atom, a benzoyl group, an isobutyroyl group, or a benzyloxycarbonyl group. 4. The group R ₁ is a hydrogen atom, Fmoc or
The δ-amino acid derivative or a salt thereof according to any one of claims 1 to 3, which is a tert-butoxycarbonyl group. 5. The δ-amino acid derivative or a salt thereof according to claim 1, wherein the group R ₂ is a hydroxyl group, a methoxy group, a benzyloxy group, or a tert-butoxy group. 6. A peptide nucleic acid derivative comprising two or more δ-amino acid derivatives according to any one of claims 1 to 5 linked by an acid amide bond. 7. The peptide nucleic acid derivative according to claim 6, wherein the terminal of the carboxyl group is esterified. 8. The peptide nucleic acid derivative according to claim 6, which is capable of hybridizing with DNA or RNA. 9. A method for producing a peptide nucleic acid comprising two or more δ-amino acid derivatives by acid-amidating the δ-amino acid derivatives according to claim 1 with each other.