JP2003219874A - Method for using peptide nucleic acid selectively binding to rna as antisense oligonucleotide, and method for producing the peptide nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for selectively linking an oligonucleotide analogue to an RNA rather than an DNA. <P>SOLUTION: This method for linking a peptide nucleic acid represented by formula I [wherein, R<SP>1</SP>is a hydrogen atom or a protective group of an amino acid; R<SP>2</SP>is a hydroxy group or a protective group of a carboxy group; R<SP>3</SP>is a direct bond, a methylene group or an ethylene group; B is a base or a derivative thereof, and may be the same or different; and m is an integer of 1-60] to an RNA having a sequence complementary to the peptide nucleic acid is provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method of using a peptide nucleic acid that selectively binds to RNA as an antisense oligonucleotide, a method of producing the peptide nucleic acid, and a composition containing the peptide nucleic acid.

[0002]

Various structural modifications of oligonucleotides have been studied in order to design new drugs that modify the expression of genetic information by antisense and antigene oligonucleotides. Peptide nucleic acid (PNA) developed by Nielsen et al. Is an oligonucleotide mimetic in which the sugar-phosphate backbone of the oligonucleotide is replaced by a peptide chain or a pseudopeptide chain (Nielsen, P.
E .; Egholm, M .; Berg, RM; Buchardt, O. Science
1991 , 254, 1497 .; Egholm, M .; Buchardt, O .; Nie
lsen, PE; Berg, RHJ Am. Chem. Soc. 1992 ,
114, 1895 .; Egholm, M.; Nielsen, PE; Bucha
rdt, O .; Berg, RHJ Am. Chem. Soc. 1992 , 114,
9677. ； Egholm, M.; Buchardt, O.; Christensen,
L.; Behrens, C.; Freier, SM; Driver, DA
; Berg, RH; Kim, SK; Norden, B.; Nielse
n, PE Nature 1993 , 365, 566 .; Hyrup, B.; Egh
olm, M.; Nielsen, PE; Witlung, P.; Norden, B.
Buchardt, OJ Am. Chem. Soc. 1994 , 116, 7964.
Hyrup, B .; Nielsen, PE Bioorg. Med. Chem.
1996 , 4, 5.). PNAs can bind to complementary DNA, RNA or PNAs, and in some cases,
It is also known to form triple chains by hydrogen bonding (Nielsen, PE et al., Bioconjugate Chem., 1994 , 5, 3-).
7, and M. Egholm et al., Nature, 365, 566-568, 199.
3 ). When PNA binds to the DNA double strand, transcription is inhibited, and when PNA binds to RNA, translation is inhibited.

Generally, PNA-DNA or PNA-RN
The bond of A is stronger than the usual bond of DNA-DNA or DNA-RNA and is excellent in thermal stability, and has the property of bonding even if some base pairs are mismatched. Furthermore, peptide nucleic acids are biologically and chemically stable in various biological fluids, including extracts of human and eukaryotic cells, as well as DNase and RNa.
Since it is not decomposed by se, it exists stably in vivo. Also, PNAs can be manufactured on a large scale at a relatively low cost. From the above characteristics,
Peptide nucleic acids are expected to be applied as effective unsense oligonucleotides and primers.

Various peptide nucleic acids have been synthesized so far for the purpose of improving the solubility of peptide nucleic acids having low solubility, structural flexibility and nucleic acid recognition ability. As such an attempt, for example, introduction of a side chain (Stammers, T.
A.; Burk, MJ Tetrahedron Lett. 1999 , 40, 332
5.; Falkiewicz, B.; Kowalska, K.; Kolodziejczy
k, AS; Winsniewski, K .; Lankiewicz, L. Nucleo
sides Nucleotides, 1999 , 18, 353. ； Sforza, S.;
Corradini, R.; Ghirardi, S.; Dossena, A.; Marche
lli, R. Eur. J. Org. Chem. 2000 , 2905.) and introduction of double bond (Roberts, CD; Schutz, R .; Leumann,
CJ Synlett, 1999 , 819.), Conversion of carbonyl group to sulfonyl group (Liu, Y .; Hudson, RHE Synlet
t, 2001 , 1626.), construction of ethereal peptide skeleton and / or cyclic peptide skeleton (Kuwahara, M .; Arimitsu, M.
; Sisido, MJ Am. Chem. Soc. 1999, 121, 256.
D'Costa, M.; Kumar, V.; Ganesh, KN Org. Let
t. 1999 , 1, 1513 .; Altmann, K.; Husken, D.; Cu
enoud, B .; Garcia-Echeverria, C. Bioorg. Med. Ch
em. Lett. 2000 , 10, 929. ； Shigeyuki, M.; Kuwaha
ra, M.; Sisido, M.; Ishikawa, T. Chem. Lett. 200
1 , 634.) and so on.

However, conventionally synthesized peptide nucleic acid (P
NA) was synthesized as a substance mimicking DNA or RNA, and its affinity for RNA is higher than its affinity for DNA, but the difference in its affinity is only slight, and it has a high degree of selectivity. Peptide nucleic acids that bind preferentially to RNA have not been developed.

[0006]

The object of the present invention is to provide a method for selectively binding an oligonucleotide mimetic to RNA complementary thereto.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that non-genetic 2 ′,
5'-linked oligonucleotide (2 ', 5'-isoDN
By using a peptide nucleic acid having a skeleton structure similar to the skeleton structure of A), it was found that the above problems can be solved, and the present invention has been completed.

That is, the present invention includes the following inventions. (1) A method of binding a peptide nucleic acid having a skeletal structure similar to that of 2 ', 5'-isoDNA and having a sequence complementary to the target RNA to the RNA.

(2) The structure of the peptide portion of the peptide nucleic acid is glycine and alanine:

[Chemical 7] The method according to (1), wherein [where n = 0, 1 or 2] are alternately bonded.

(3) The peptide nucleic acid has the formula I:

[Chemical 8] [Wherein R ¹ is a hydrogen atom or an amino group protecting group, R ² is a hydroxyl group or a carboxyl group protecting group, R ³ is a direct bond, or a methylene group or an ethylene group, and B is Are bases or derivatives thereof, which may be the same or different, and m is an integer of 1 to 60], The method according to (1).

(4) The method according to any one of (1) to (3), wherein the peptide nucleic acid has a higher affinity for complementary RNA than that for complementary DNA.

(5) A base or a derivative thereof is introduced into aspartic acid or a derivative thereof to obtain a compound of the formula II:

[Chemical 9] [Wherein R ¹ is a hydrogen atom or an amino group-protecting group] or a derivative thereof to give a compound of formula III:

[Chemical 10] [Wherein R ¹ is a hydrogen atom or an amino group protecting group, R ² is a hydroxyl group or a carboxyl group protecting group, and B is a base or a derivative thereof] A method of manufacturing.

(6) Introducing a base or a derivative thereof into serine or a derivative thereof, and formula II:

[Chemical 11] [Wherein R ¹ is a hydrogen atom or an amino group-protecting group] to give a compound of the formula IV:

[Chemical 12] [Wherein R ¹ is a hydrogen atom or an amino group protecting group, R ² is a hydroxyl group or a carboxyl group protecting group, and B is a base or a derivative thereof] A method of manufacturing.

(7) The method according to (6), wherein tosylation and / or halogenation is carried out before the step of introducing the base or its derivative.

(8) A composition for regulating the expression of a gene in an organism, which comprises the peptide nucleic acid according to any one of (1) to (4).

(9) A composition for treating a condition associated with unwanted protein production in an organism, which comprises the peptide nucleic acid according to any one of (1) to (4).

(10) A composition for inducing the degradation of RNA in cells of an organism, which comprises the peptide nucleic acid according to any one of (1) to (4).

(11) A composition for killing a cell or a virus, which comprises the peptide nucleic acid according to any one of (1) to (4).

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, a peptide nucleic acid means a peptide skeleton or a pseudo peptide skeleton, for example, an aminoethylglycine main chain, instead of a skeleton composed of deoxyribose or ribose of nucleic acids such as DNA and RNA And other similar backbone nucleic acid mimetics, including polyamides, polythioamides, polysulfinamides and polysulfonamides, meaning compounds having a nucleobase attached to the backbone.

In the present specification, the peptide nucleic acid monomer means one unit of the peptide nucleic acid which constitutes the peptide nucleic acid of the present invention, and the peptide nucleic acid monomer is obtained by a method commonly used in the art, such as solid phase synthesis. By sequentially binding, the peptide nucleic acid of the present invention can be produced.

In the present specification, alanines are represented by the following formula:

[Chemical 13] It means a compound represented by the formula [wherein n = 0, 1 or 2]. In the above formula, n = 0 means Ala (naturally occurring alanine), and n = 1 means Ala1, n
= 2 is referred to as Ala2.

In the present specification, aspartic acid and serine derivatives include amino groups of these amino acids and / or
Alternatively, those having a protected carboxyl group are included. In the present specification, 2 ', 5'-isoDNA means natural D
In NA, each nucleoside has an ester bond at the 3rd and 5th positions of the sugar via a phosphate, while it means an isomer of DNA having an ester bond at the 2nd and 5th positions of the sugar. A structural comparison of native DNA and 2 ', 5'-isoDNA is shown in Figure 1.

In the present specification, the substituent B in the formulas I, III and IV represents a base or a derivative thereof. As the base, as naturally occurring, adenine, guanine,
Nucleic acid bases such as cytosine, thymine or uracil,
Or hypoxanthine, xanthine and the like, but not limited thereto, DNA or R
Any base or a derivative thereof that can form a double chain or a triple chain by hydrogen bonding with NA or the like may be used. Derivatives of bases include
Also included are modified bases and amino-protected bases. Typical modified bases include 6-methylaminopurine, 7
-Methylguanine and 5-methylcytosine.

In the present specification, the protecting group for the amino group in R ¹ of the formulas I to IV is a protecting group used in ordinary peptide synthesis, and can be eliminated if desired, and a free There is no particular limitation as long as the amino group can be regenerated. Such a protecting group has 1 to 20 carbon atoms, preferably 1 to 15 carbon atoms, and more preferably 1 to 1 carbon atoms.
A straight-chain or branched alkyl group having 0 to 7 carbon atoms,
Preferably 7 to 15 monocyclic, polycyclic or condensed ring aralkyl groups, alkylcarbonyl groups consisting of the above alkyl groups, 6 to 20 carbon atoms, preferably 6 to 15 monocyclic or polycyclic groups. Or an arylcarbonyl group consisting of a condensed cyclic aryl group, 1 to 5, preferably 1 to 5 5 to 7 membered rings having 1 to 4 nitrogen atoms, oxygen atoms or sulfur atoms in the ring A heterocyclic carbonyl group consisting of a monocyclic, polycyclic or condensed cyclic saturated or unsaturated heterocyclic group,
Heterocyclic sulfenyl group, alkoxycarbonyl group consisting of the above alkyl group, aryloxycarbonyl group consisting of the above aryl group, aralkyloxycarbonyl group consisting of the above aralkyl group, heterocyclic group consisting of the above heterocyclic group Examples thereof include an oxycarbonyl group.

Preferred R ¹ is Fmoc (9-fluorenylmethoxycarbonyl) group, Boc (t-butoxycarbonyl) group, Z (benzyloxycarbonyl)
Group, Npys (3-nitro-2-pyridylsulfenyl) group, Z (OMe) (p-methoxybenzyloxycarbonyl) group and the like.

Examples of the protecting group for the carboxyl group represented by R ² in the formulas I, III and IV include groups capable of forming carboxylic acid derivatives such as esters, acid halides and acid anhydrides. Examples of the group capable of forming an ester include an alkoxy group including the above alkyl group, an aralkyloxy group including the above aralkyl group, and an aryloxy group including the above aryl group. Examples of preferable R ² include an OBn (benzyloxy) group and a t-butoxy group in addition to the hydroxyl group. As R ² in formula IV, it is preferable to use an OBn group.

As the derivative of the compound represented by the formula II,
Examples thereof include carboxylic acid derivatives such as esters, acid halides and acid anhydrides. Examples of the group capable of forming an ester include an alkoxy group including the above alkyl group, an aralkyloxy group including the above aralkyl group, and an aryloxy group including the above aryl group. The method for synthesizing the peptide nucleic acid monomer of the present invention will be described below.

Of the Gly-Ala1 peptide nucleic acid monomer
Synthesis The R ¹ -Gly-Ala1 peptide nucleic acid monomer represented by formula III of the present invention can be produced by various methods. For example, it can be produced by using D-aspartic acid or L-aspartic acid as a starting material by a synthetic route represented by the following formula. However, it is not limited to the following synthetic route.

[0029]

[Chemical 14]

By methods commonly used in the art,
When the carboxyl group of D-aspartic acid or L-aspartic acid is protected with a Bn group or the like and the amino group is protected with a Boc group or the like, an aspartic acid derivative (Compound 3) is obtained. This was made into an acid anhydride with ethyl chloroformate, and Na was added.
When it is reduced with a reducing agent such as BH _4, a D-form and an L-form of hydroxymethyl-β-alanine (compound 4) having protected amino groups and carboxyl groups are obtained. Then, the base is introduced into the above hydroxymethyl-β-alanine derivative by reacting with the target base or its derivative. In order to prevent a side reaction from occurring in the base, it is preferable to protect the amino group with a Bz (benzoyl) group or the like before the reaction. The protecting group for the base is not particularly limited, but may be Bz group, Z (benzyloxycarbonyl) group,
It is preferable to use a Bhoc (Ph ₂ CHOC═O) group or the like. Subsequently, after deprotecting the base, glycine is bound in the presence of the peptide condensation reagent. Regarding the binding of glycine, it is preferable to bind glycine having an amino group protected in order to prevent side reactions. The peptide condensation reagent is not particularly limited, but for example, TBTU (O-
(Benzotriazol-1-yl) -N, N, N ',
N'-tetramethyluronium tetrafluoroborate), HBTU (O-benzotriazole-N, N,
N ', N'-tetramethyluronium-hexafluoro-phosphate), HATU (O- (7-azabenzotriazol-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate , DCC
Etc., and it is preferable to use HATU. Subsequently, when the carboxyl group of the condensation product is deprotected, R ¹ -Gly-Ala containing the target base or its derivative is obtained.
One peptide nucleic acid monomer is obtained.

In the above-mentioned method for synthesizing a peptide nucleic acid monomer, the D-form of a peptide nucleic acid monomer is obtained when the D-form of aspartic acid is used as a starting material, and the L-form of the peptide nucleic acid monomer is obtained when the L-form of aspartic acid is used. can get. The protecting groups for the amino acid and the carboxyl group used here are not particularly limited as long as they are commonly used in the art. It is preferable to use a Boc group as the amino-protecting group and a Bn group as the carboxyl-protecting group.

Combination of Gly-Ala Peptide Nucleic Acid Monomers
The R ¹ -Gly-Ala peptide nucleic acid monomer represented by formula IV of the present invention can be produced by various methods.
For example, it can be produced using D-serine or L-serine as a starting material by a synthetic route represented by the following formula. However, it is not limited to the following synthetic route.

[0033]

[Chemical 15]

By methods commonly used in the art,
When the amino group of D-serine or L-serine is protected with a protecting group such as Boc group and the carboxyl group is protected with a protecting group such as Bn group, a serine derivative (Compound 8) is obtained.
The free hydroxyl group is put into a state where it can be easily removed by a method such as tosylation (Compound 9). The tosyl group is then replaced with halogen using a halogenating reagent (compound 10). The halogenating reagent is not particularly limited, but examples thereof include sodium iodide, sodium bromide, and the like, and sodium iodide is preferably used because of its reactivity in coupling with a base. Compound 8 is converted to, for example, I ₂ , P
Compound 10 can also be produced by direct iodination by reacting in the presence of h ₃ P and imidazole. Then, the base is introduced by reacting with the target base or its derivative. In order to prevent the side reaction from occurring in the base, it is preferable to protect the base or its derivative with a Bz group or the like before the reaction. As the base protecting group, the same protecting groups as described above can be used. Chromatography of the reaction product followed by recrystallization gives compound 11a with the desired base or its derivative and compound 11b with the deprotected base.
When compound 11a is treated in the presence of a basic catalyst, it is converted to compound 11b in high yield. As a basic catalyst,
Is not particularly limited, for _{example, K 2 CO 3, Na 2} CO 3 and the like, preferably used K ₂ CO _3. Compound 1
After deprotecting the amino group of 1b, glycine is attached in the presence of a peptide condensation reagent. Regarding the binding of glycine, it is preferable to bind glycine having an amino group protected in order to prevent side reactions. Subsequently, the carboxyl group of the condensation product is deprotected to obtain the R ¹ -Gly-Ala peptide nucleic acid monomer having the target base or its derivative. Examples of the respective protecting groups for amino acid and base that can be used here include the same ones as described above.

In the synthesis of the above-mentioned Gly-Ala peptide nucleic acid monomer, when a base or its derivative is introduced into the serine derivative of compound 8, the free hydroxyl group is eliminated by a method such as tosylation rather than direct introduction. From the viewpoint of the progress of the reaction is preferably in an easy state,
Furthermore, it is preferable to halogenate the tosylated group before introducing the base or its derivative. In the above method for synthesizing a peptide nucleic acid monomer, the D-form of serine is used as a starting material to obtain the D-form of the peptide nucleic acid monomer, and the L-form of serine is used to obtain the L-form of the peptide nucleic acid monomer.

The solvent that can be used for synthesizing the peptide nucleic acid monomer represented by the formula III or formula IV of the present invention is not particularly limited as long as it is a solvent usually used in the art. Alcohol solvents such as methanol and ethanol, ether, methyl butyl ether, TH
Ether solvents such as F and dioxane, ester solvents such as ethyl acetate, ketone solvents such as acetone and cyclohexanone, amide solvents such as DMF (N, N-dimethylformamide), dimethylacetamide and HMPT, dichloromethane, chloroform and the like. Halogen-based solvent, n-
Examples thereof include aliphatic hydrocarbon solvents such as hexane and aromatic hydrocarbon solvents such as benzene and toluene.

The peptide nucleic acid monomer represented by the above formula III or IV is sequentially acid amidated by a method usually used in peptide synthesis or the like, whereby two or more peptide nucleic acid monomers have an acid amide bond. Peptide nucleic acids can be produced.

Examples of the peptide synthesis method that can be used here include a solid phase method and a liquid phase method, but the solid phase method is preferably used. The outline of the method for synthesizing a peptide nucleic acid when the solid phase synthesis method is used will be described below. First, a resin protected by a protective group such as an Fmoc group is washed with a solvent such as DMF and swollen overnight. The amino group is deprotected to a free amino group and a primary amino acid such as Fmoc-lysine (Boc) -OH is attached to the free amino group.

After coupling the first amino acid, the desired peptide nucleic acid chain is systematically synthesized. This synthesis is carried out by repeating deprotection and coupling. Temporary protecting groups such as Boc or Fmoc on the last attached amino acid may be treated with a suitable treatment to liberate the N-terminal amino group, eg acid hydrolysis such as using trifluoroacetic acid in the case of Boc. Quantitatively removed by degradation or by base treatment such as with piperidine in the case of Fmoc. The desired peptide nucleic acid monomer of formula III or IV is then attached to the N-terminus of the last attached monomer. The linking of the N-terminus of the last linked amino acid to the C-terminus of the amino acid can be done in several ways. For example, 2,4,5-trichlorophenyl ester (Pless et al., Helv. Chim. Acta, 1963 , 46,1
609), phthalimido ester (Nefkens et al., J. Am. Che
m.Soc., 1961 , 83,1263), pentachlorophenyl ester (Kupryszewski, Rocz.Chem., 1961 , 35,595), pentafluorophenyl ester (Kovacs et al., J. Am.Che.
m.Soc., 1963 , 85,183), o-nitrophenyl ester (Bodanzsky, Nature, 1955 , 175,685), imidazole ester (Li et al., J. Am. Chem. Soc., 1970 , 92,7608).
And 3-hydroxy-4-oxo-3,4-dihydroquinazoline (Dhbt-OH) ester (Koning et al., Che
m.Ber., 1973 , 103, 2024 and 2034) or the initial formation of active ester derivatives or symmetrical anhydrides (Wieland et al., An.
gew.Chem., Int.Ed.Engl., 1971 , 10,336), etc.
It can be carried out by supplying a peptide nucleic acid monomer having an activated carboxyl group. Alternatively, the carboxyl group of the supplied peptide nucleic acid monomer is, for example, dicyclohexylcarbodiimide (Sheeha
n et al., J. Am. Chem. Soc., 1955 , 77, 1067) or a derivative thereof, and a condensation reagent such as HATU, TBTU, HBTU directly reacts with the N-terminal of the peptide nucleic acid monomer finally bound. You can also In the synthesis of the peptide nucleic acid according to the present invention, only the monomer of formula III or formula IV
It is preferable to bond only the monomers of.

The condensation reaction between the peptide nucleic acid chain bound to the resin and the peptide nucleic acid monomer can be confirmed by the Kaiser test. Kaiser test, 1-5mg resin
Transfer to a test tube, a) 5% ninhydrin in ethanol, b) 80% phenol in ethanol, c) 0.
It is carried out by adding 5 drops of 2 mM potassium cyanide solution in pyridine and heating in boiling water for 5 minutes.
By this operation, the condensation reaction is continued when the solution is blue, and when the solution becomes yellow, the amino group is deprotected and the condensation reaction with the next peptide nucleic acid monomer is performed. By repeating this cycle as many times as necessary, a peptide nucleic acid having a sequence complementary to the target RNA and having a desired length can be obtained.

The final deprotection of the peptide nucleic acid side chain and release of the peptide nucleic acid molecule from the solid support is accomplished by using anhydrous HF (Sa
kakibara et, Bull.Chem.Soc.Jpn., 1965, strong acid such 38,4921), trifluoroacetic acid, boron tris (trifluoroacetate) (Pless, et al., Helv.Chim.Acta, 1973, 46,160
9), sulfonic acids such as trifluoromethanesulfonic acid and methanesulfonic acid (Yajima et al., J Chem. Soc., Chem.
Comm., 1974 , 107), and a mixed solution of the above compound and cresol.

The length of the peptide nucleic acid of the present invention depends on the desired R
There is no particular limitation as long as it can bind to NA or the like,
In the formula I, m = 3 to 24 is preferable, and m = 9
The thing of -18 is more preferable.

Further, in the method of the present invention, since the D-form or L-form of the peptide nucleic acid monomer is respectively synthesized, it is possible to synthesize the peptide nucleic acid by binding only the D-form or L-form. From the structural point of view, among the above, a peptide nucleic acid monomer derived from D-aspartic acid (D-form of the peptide nucleic acid monomer represented by formula III)
It is preferable to synthesize only the peptide nucleic acid monomer derived from L-serine (L-form of the peptide nucleic acid monomer represented by the formula IV) to synthesize the peptide nucleic acid.

DN having PNA and a sequence complementary thereto
The affinity between the peptide nucleic acid of the present invention and DNA or RNA was tested by measuring the melting temperature with A or RNA. The melting temperature is a mixture of PNA and DNA or PNA.
It was measured by observing the absorbance at 260 nm while changing the temperature of the mixed solution of RNA with RNA. As a result, PN
It was observed that the melting temperatures of A and RNA were higher than the melting temperatures of PNA and DNA. That is, it was revealed that the peptide nucleic acid of the present invention has higher affinity for complementary RNA than complementary DNA and selectively binds to RNA.

2 ', 5'-isoDNA has a higher affinity for the RNA complementary strand than the DNA complementary strand, and the A-type double helix structure of 2', 5'-isoDNA and RNA Have reported that the distance between C2′-O5 ′, which is an index of the distance between bases, is closer than the distance between C3′-O5 ′ of natural DNA (9th Antisense Symposium). Lecture summary p.46). When bound to RNA, the sites corresponding to C2 ′ and O5 ′ in the peptide backbone of the peptide nucleic acid of the present invention retain the same distance as between C2′-O5 ′ of 2 ′, 5′-isoDNA. As a result, the distance between the bases in the peptide nucleic acid of the present invention when bound to RNA and the distance between the bases in 2 ', 5'-isoDNA become almost equal. That is,
In the present invention, a skeleton structure similar to the skeleton structure of 2 ', 5'-isoDNA means a skeleton structure in which the distance between bases when bound to RNA is almost equal to that of 2', 5'-isoDNA. means.

Peptide nucleic acid developed by imitating conventional natural type DNA has a 2 ',
RNA binding selectivity is low because it cannot form the same three-dimensional structure as 5'-isoDNA, whereas the peptide nucleic acid of the present invention has a backbone structure of 2 ', 5'-isoDNA. It is considered to bind selectively to complementary RNA because it has a similar structure and can form a three-dimensional structure similar to 2 ', 5'-isoDNA when bound to RNA.

As described above, the peptide nucleic acid of the present invention is RN
Since it selectively binds to A, the amount bound to DNA is reduced as a result, and even a small amount preferentially binds to RNA, and translation of RNA can be efficiently inhibited.
Therefore, it is expected to be useful in the antisense method. Currently in clinical trials as an antisense drug S
-Oligo (phosphorothioate type) antisense agents are
This is an improvement in the natural antisense agent's deficiency in that it is easily decomposed by a nucleolytic enzyme and improved stability against that enzyme. However, it is inferior in terms of binding affinity with RNA as compared with the natural type. So R
If the peptide nucleic acid of the present invention that strongly binds to NA is used instead of S-oligo, a better antisense effect is expected. In addition, if it is an antisense that has a strong binding affinity only for RNA, various RNA (mRNA, tR
It is considered to be very useful as an effective biological tool for elucidating the biological mechanism of NA, rRNA, snRNA, etc.).

The present invention is also directed to the use of peptide nucleic acids in therapy or prophylaxis. Potential therapeutic or prophylactic targets include herpes simplex virus (HSV), human papilloma virus (HPV), human immunodeficiency virus (H
IV), Candida albicans, influenza virus, cytomegalovirus (CMV), intracellular adhesion molecule (ICAM), 5-lipoxygenase (5-LO),
Phospholipase A ₂ (PLA ₂ ), protein kinase C
(PKC), and the RAN oncogene. Such a targeting method can be applied to: For example,
Eye, labial, genital and generalized herpes simplex I and II
Infections; genital warts (warts, warts); cervical cancer; vulgaris vulgaris; Kaposi's sarcoma; AIDS; cutaneous and systemic fungal infections; influenza; pneumonia; retinitis and immunosuppressed patients Pneumonia; mononucleosis; eye, skin and systemic inflammation; cardiovascular disease; cancer; asthma; psoriasis; cardiovascular collapse; myocardial infarction; gastrointestinal disease; kidney disease; osteoarthritis; osteoarthritis; acute pancreatitis; septicemia Shock; and Crohn's disease.

For therapeutic or prophylactic treatment, the peptide nucleic acids of the invention can be incorporated into pharmaceutical compositions, which include carriers, thickeners, diluents, buffers, preservatives, interfaces. An activator or the like may be contained. The pharmaceutical composition comprises one or more active ingredients such as antimicrobial agents,
An anti-inflammatory drug, an anesthetic, etc. may be contained in addition to the peptide nucleic acid.

The pharmaceutical composition may be intended for either local or systemic application or may be administered in a number of ways depending on the area to be treated. Administration should be local (including eye, uterus, rectum, intranasal), orally, by inhalation, or parenterally, for example by intravenous drip or by subcutaneous, intraperitoneal or intramuscular injection. You can

Formulations for topical administration include ointments, lotions,
Includes creams, gels, drops, suppositories, sprays, solutions and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like are necessary or desirable. Coated condoms would also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, fragrances, diluents, emulsifiers, dispersion aids or binders are desirable. Formulations for parenteral administration include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

The dosage depends on the degree and responsiveness of the condition to be treated but is usually once or more than once daily,
The treatment period lasts from a few days to several months, or until a cure or reduction of the disease state is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.

This type of treatment is applicable to a wide variety of organisms, ranging from unicellular prokaryotes and eukaryotes to multicellular eukaryotes. Any organism that utilizes DNA-RNA transcription or RNA-protein translation as a fundamental part of its genetic, metabolic or cellular regulation can be subjected to therapeutic and / or prophylactic treatment according to the present invention. It is believed that a wide variety of organisms can be treated, including bacteria, yeasts, protozoa, algae, all plants, and all higher animal forms, including warm-blooded animals. Furthermore, each cell of multicellular eukaryotes has DN as a component of their cellular activity.
It includes both A-RNA transcription and RNA-protein translation so that they can be treated. In addition, many organelles of eukaryotic cells (eg mitochondria and chloroplasts) also contain transcription and translation mechanisms. Thus, single cells, cell populations or organelles can also be treated with therapeutic or diagnostic peptide nucleic acids. Treatment as used herein is meant to include eliminating the disease state by killing the organism or by controlling erroneous or harmful cell growth or expression. The present invention will be explained by the following examples, but the present invention is not limited to the scope of the examples.

[0055]

EXAMPLES Example 1. Fmoc-Gly-Ala1
Min) Synthesis of peptide nucleic acid monomer (D-form) Boc-Ala1 (benzoylthymine) -OBn
Synthesis of (compound 5a; D form)

[Chemical 16]

0.309 g (1 mmol) Boc-hydroxymethyl-β-alanine-OBn (compound 4a), 0.230 g (1 mmol) 3-benzoylthymine and 0.294 g (1.1 mmol) triphenylphosphine. To, add 10 ml of dry tetrahydrofuran,
0.182 ml (1.1 mm
ol) DEAD was added dropwise. After completion of the dropping, the temperature was returned to room temperature and stirring was continued. After 24 hours, the transparent solution was subjected to solvent removal under reduced pressure and dried. The residue was purified by silica gel column chromatography using a 50: 1 mixed solution of dichloromethane and acetone as an eluent to give 0.225 g of Bo.
c-Ala1 (benzoylthymine) -OBn (Compound 5
a) was obtained (yield: 43%). δ _H (300 MHz, CDCl ₃ ) : 1.36 (9H, s, (CH ₃ ) ₃ ), 2.01 (3
H, s, CH ₃ ), 2.50-2.70 (2H, m, CH ₂ ), 4.10-4.45 (3H,
m, CH and CH ₂ ), 5.10 (2H, s, CH ₂ ), 7.30 (5H, s, Ph
of benzyl), 7.50-7.70 (5H, m, Ph of benzoyl), 7.85
(1H, s, CH = ofthymine)

2. Synthesis of Fmoc-Gly-Ala1 (thymine) -OBn (Compound 6a; D-form)

[Chemical 17]

3N of 3N hydrochloric acid ethyl acetate solution was added to 0.10
Add 4 g (0.2 mmol) of compound 5a and add 3 at room temperature.
Stir for 0-45 minutes. The reaction was monitored by TLC, and after the disappearance of the raw materials, excess hydrochloric acid was removed under reduced pressure to obtain an oily hydrochloride, which was dried under reduced pressure in a potassium hydroxide desiccator for 2 hours. The hydrochloride was adjusted to pH 7 with a 5% aqueous sodium hydrogen carbonate solution, extracted with ethyl acetate, washed with saturated saline and distilled water, and the solvent was removed under reduced pressure to obtain a de-Boc form of compound 5a as 0.05 g of white crystals. (Yield 79%).

0.743 g (2.5 mmol) of Fmo
c-glycine and 0.825 g (2.6 mmol) de-Bo
20 ml of acetonitrile was added to the c-form and 0.275 g (2.5 mmol) of triethylamine, and this was added to 0 ° C.
At 0.835 g (2.6 mmol) of TBTU was added. After stirring the mixture at 0 ° C for 1 hour, the temperature was returned to room temperature and stirring was continued. After 6 hours, 70 ml of saturated saline was added to 50 ml.
It was extracted 3 times with ethyl acetate. The organic phase was washed with 5 ml of 2N hydrochloric acid, 5 ml of water, 5 ml of 5% aqueous solution of sodium hydrogen carbonate, washed again with 5 ml of water, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure to remove the remaining crystals. Further, when recrystallized from a mixed solvent of ethyl acetate-hexane, Fmoc-Gly-Ala1 (thymine) -OBn
1 g of crystals of (compound 6a) was obtained (yield 64.5%).
The physicochemical data of the product obtained are shown below: Melting point : 128-129 ° C ν _max / cm ^-1 : 3329 (NH) 1724, 1645 (C = O) δ _H (300 MHz, DMSO-d ₆ ) : 1.74 (3H, s, CH ₃ ), 2.56 (2
H, m, CH ₂ ), 3.5 (2H, m, CH ₂ ), 3.9 (2H, m, CH ₂ ), 4.
2 (3H, m, CH ₂ , CH), 4.5 (1H, m, CH), 5 (2H, s, C
H ₂ ), 7.3 (5H, s, CH ₂ Ph ), 7.2-7.5 (6H, m, Fmoc prot
on, thymine proton, NH), 7.33 (2H, d, J 7.7, Ar-H),
7.87 (2H, d, J 7.5, Ar-H), 10.85 (1H, br, NH of t
hymine) FAB-MAS : m / z 597 (m + 1)

3. Synthesis of Fmoc-Gly-Ala1 (thymine) peptide nucleic acid monomer (compound 7a; D form)

[Chemical 18]

To 0.5065 g (0.85 mmol) of compound 6a was added 1.013 g of 10% palladium on carbon and 10 ml of ethanol, and 2.35 ml (25 mmol) of 1,4-while stirring under an argon atmosphere. Cyclohexadiene was added and the stirring was continued overnight.
Next day, the mixture was filtered through Celite, and Celite was washed with hot ethanol three times. The filtrate and the washing solution were combined and the solvent was removed under reduced pressure. To the residue was added 10 ml of 5% aqueous sodium hydrogen carbonate solution, and the mixture was shaken with ethyl acetate to remove by-products. 1 until the pH of the remaining aqueous solution becomes 2-3
A 0% aqueous solution of potassium hydrogen sulfate was added, and the mixture was diluted with ethyl acetate to 20%.
It was extracted 3 times by ml. The organic phase was washed with saturated brine and distilled water, dried over anhydrous magnesium sulfate, the solvent was removed under reduced pressure, and the remaining crystals were recrystallized from ethanol. 0.266 g of Fmoc-Gly-Ala1 (thymine)
A peptide nucleic acid monomer (compound 7a) was obtained (yield 6
1.8%). The physicochemical data of the obtained product are shown below: Melting point : 144 ° C Experimental value : C, 61.21; H, 5.19; N, 10.94. Theoretical value (C ₂₆ H ₂₆ N ₄ O ₇ ) : C, 61.65 H, 5.17; N, 11.06 ν _max / cm ^-1 : 1714, 1638 (C = O) δ _H (300 MHz, DMSO-d ₆ ) : 1.75 (3H, s, CH ₃ ), 2.35-2.
46 (2H, m, CH ₂ ), 3.30 (1H, br, NH), 3.39-3.62 (2H,
m, CH ₂ ), 3.83-4.01 (2H, m, CH ₂ ), 4.21-4.27 (3H, m,
CH and CH ₂ ), 4.40-4.56 (1H, m, CH), 7.23 (1H, s, C
H-6 of T), 7.32 (2H, dd, J 7.5 and 7.5, Ar-H), 7.4
0 (2H, dd, J 7.5 and 7.5, Ar-H), 7.71 (2H, d, J 7.
5, Ar-H), 7.87 (2H, d, J 7.5, Ar-H), 10.81 (1H, br
s, NH), 12.14 (1H, br, COOH) δ _C (75 MHz, DMSO-d ₆ ) : 12.48 (CH ₃ ), 37.03 (CH ₂ ), 4
2.72 (CH ₂ ), 43.39 (CH ₂ ), 44.55 (CH), 46.63 (CH), 6
5.74 (CH ₂ ), 107.13 (C), 120.06 (CH), 125.27 (CH),
127.06 (CH), 127.60 (CH), 136.28 (CH), 140.69 (C),
143.85 (C), 151.54 (C), 156.30 (C), 164.14 (C), 1
68.48 (C), 171.94 (C) FAB-MAS : m / z 507 (M + 1).

Example 2. Fmoc-Gly-Ala1
Synthesis of (thymine) peptide nucleic acid monomer (L-form) Boc-Ala1 (benzoylthymine) -OBn
Synthesis of (Compound 5b; L-form)

[Chemical 19]

0.309 g of compound 4b (1 mmol)
To this, 10 ml of dry tetrahydrofuran was added, 0.23 g (1 mmol) of 3-benzoylthymine and 0.294 g (1.1 mmol) of triphenylphosphine were further added, and 182 μl was added while stirring at −15 ° C.
(1.1 mmol) DEAD was added dropwise. Then, after returning to room temperature and stirring for another 24 hours, the transparent solution was subjected to solvent removal under reduced pressure and dried. The residue was purified by silica gel column chromatography using a 50: 1 mixed solution of dichloromethane and acetone as an eluent to obtain 0.21 g of compound 5b (yield 40%). The physicochemical data of the product obtained are shown below: δ _H (300 MHz, CDCl ₃ -d ₆ ) : 1.4 (9H, s, (CH ₃ ) ₃ ), 2.02
(3H, s, CH ₃ ), 2.5-2.7 (2H, m, CH ₂ ), 4.1-4.45 (3H,
m, CH and CH ₂ ), 5.1 (2H, s, CH ₂ ), 7.3 (5H, s, Ph),
7.5-7.7 (5H, m, benzoyl protons), 7.85 (1H, m, CH =
of thymine).

2. Synthesis of Fmoc-Gly-Ala1 (thymine) -OBn (Compound 6b; L-form)

[Chemical 20]

3 ml of 3N hydrochloric acid ethyl acetate solution was added to 0.10
Add 4 g (0.2 mmol) of compound 5b and add 3 at room temperature.
Stir for 0-45 minutes. The reaction was monitored by TLC, and after the disappearance of the raw materials, excess hydrochloric acid was removed under reduced pressure to obtain an oily hydrochloride, which was dried under reduced pressure in a potassium hydroxide desiccator for 2 hours. The hydrochloride was adjusted to pH 7 with a 5% aqueous sodium hydrogen carbonate solution, extracted with ethyl acetate, washed with saturated brine and distilled water, and the solvent was removed under reduced pressure to obtain the de-Boc form of compound 5b as white crystals (0.0475 g). (Yield 75
%).

0.743 g (2.5 mmol) of Fmo
c-glycine and 0.825 g (2.6 mmol) de-Bo
To the c-form, 20 ml of acetonitrile was added, and further 0.
275 g (2.5 mmol) triethylamine was added. To this was added 0.835 g (2.6 mmol) TBTU at 0 ° C. The mixture was stirred at 0 ° C. for 1 hour, then returned to room temperature and further stirred, and after 6 hours, saturated saline solution was added to 70%.
ml was added, and the mixture was extracted 3 times with 50 ml of ethyl acetate. The organic phase was washed with 5 ml of 2N hydrochloric acid, 5 ml of water, 5 ml of 5% aqueous solution of sodium hydrogen carbonate, washed again with 5 ml of water, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure to remove the remaining crystals. Further, when recrystallized from a mixed solvent of ethyl acetate-hexane, Fmoc-G was obtained as white crystals.
1.01 g of ly-Ala1 (thymine) -OBn (compound 6b) was obtained (yield 65%). The physicochemical data of the product obtained are shown below: Melting point : 130-131 ^° C Experimental value : C, 66.15; H, 5.54; N, 9.51. Theoretical value (C ₃₃ H ₃₂ N ₄ O ₇ ) : C, 66.43; H, 5.41; N, 9.39) ν _max / cm ^-1 : 3330 (NH); 1668 (C = O), δ _H (300MHz, DMSO-d ₆ ) : 1.73 (3H, s, CH ₃ ), 2.53 (2
H, m, CH ₂ ), 3.49 (2H, m, CH ₂ ), 3.9 (2H, m, CH ₂ ),
4.2 (3H, m, CH ₂ , CH), 4.5 (1H, m, CH), 5.0 (2H, s, C
H ₂ ), 7.2 (5H, s, CH ₂ Ph ), 7.2-7.5 (6H, m, arom of F
moc, CH = of thymine and NH), 7.698 (2H, d, J 7.14
6, arom of Fmoc), 7.875 (2H, d, J 7.3, arom of Fmo
c), 10.8 (1H, br, NH of thymine). FAB-MAS : m / z 597 (m + 1). Synthesis of Fmoc-Gly-Ala1 (thymine) peptide nucleic acid monomer (Compound 7b; L-form)

[0067]

[Chemical 21]

To 0.5065 g (0.85 mmol) of compound 6b 1.013 g of 10% palladium on carbon and 1
0 ml of ethanol was added, 2.35 ml (25 mmol) of 1,4-cyclohexadiene was added with stirring under an argon atmosphere, and stirring was continued overnight. The next day, filter through Celite and the Celite is washed with hot ethanol.
After washing twice, the filtrate and the washing solution were combined and the solvent was removed under reduced pressure and dried. To the residue was added 10 ml of 5% aqueous sodium hydrogen carbonate solution, and the mixture was shaken with ethyl acetate to remove by-products. 10 until the pH of the remaining aqueous solution becomes 2-3
% Potassium hydrogensulfate aqueous solution, 20m with ethyl acetate
It was extracted 3 times, l times. The organic phase was washed with saturated saline and distilled water, dried over anhydrous magnesium sulfate, the solvent was removed under reduced pressure, and the obtained precipitate was recrystallized from ethanol. 0.266 g of Fmoc-Gly-Ala1 (thymine)
A peptide nucleic acid monomer (compound 7b; L-form) was obtained (yield 60%). The physicochemical data of the obtained product are shown below: Melting point : 144 ° C Experimental value : C, 61.29; H, 5.01; N, 11.28 Theoretical value (C ₂₆ H ₂₆ N ₄ O ₇ ) : C, 61.65; H, 5.17; N, 11.06 ν _max / cm ^-1 : 1714, 1638 (C = O) δ _H (300 MHz, DMSO-d ₆ ) : 1.75 (3H, s, CH ₃ ), 2.35-2.
46 (2H, m, CH ₂ ), 3.30 (1H, br, NH), 3.39-3.62 (2H,
m, CH ₂ ), 3.83-4.01 (2H, m, CH ₂ ), 4.21-4.27 (3H, m,
H and CH ₂ ), 4.40-4.56 (1H, m, CH), 7.23 (1H, s, CH
-6 of T), 7.32 (2H, dd, J 7.5 and 7.5, Ar-H), 7.40
(2H, dd, J 7.5 and 7.5, Ar-H), 7.71 (2H, d, J 7.5,
Ar-H), 7.87 (2H, d, J 7.5, Ar-H), 10.81 (1H, brs, N
H), 12.14 (1H, br, COOH) δ _C (75 MHz, DMSO-d ₆ ) : 12.48 (CH ₃ ), 37.03 (CH ₂ ), 4
2.72 (CH ₂ ), 43.39 (CH ₂ ), 44.55 (CH), 46.63 (CH), 6
5.74 (CH ₂ ), 107.13 (C), 120.06 (CH), 125.27 (CH),
127.06 (CH), 127.60 (CH), 136.28 (CH), 140.69 (C),
143.85 (C), 151.54 (C), 156.30 (C), 164.14 (C), 1
68.48 (C), 171.94 (C); FAB-MAS : m / z 507 (M + 1).

Example 3. Fmoc-Gly-Ala1
Synthesis of (adenine) peptide nucleic acid monomer Boc-Ala1 (benzoyladenine) -OBn
Synthesis of (compound 5c; D form)

[Chemical formula 22]

150 ml of dry tetrahydrofuran was added to 5.57 g (18.0 mmol) of compound 4a, 5.19 g (19.8 mmol) of triphenylphosphine and 8.6 g (36 mmol) of 6-benzoyladenine, and the mixture was added at 0 ° C. 2.83 ml (18 mmol) with stirring
Of DEAD was dropped. After completion of the dropping, the mixture was further stirred at 0 ° C. for 2 hours and further at room temperature for 24 hours, and then filtered,
The insoluble matter was removed. The residue obtained by distilling off the solvent under reduced pressure was purified by silica gel column chromatography using a 4: 1 mixed solution of dichloromethane and acetone and later a 1: 1 mixed solution as an eluent to give Boc crystals as Boc crystals.
-Ala1 (benzoyl adenine) -OBn (compound 5
4.3 g of c) was obtained (yield: 45%). The physicochemical data of the obtained product are shown below: Melting point : 127-129 ^o C Experimental value : C, 63.05; H, 5.71; N, 15.36. Theoretical value (C ₂₆ H ₃₀ N ₆ O ₅ ) : C, 63.38; H, 5.70; N, 15.84 ν _max / cm ^-1 : 3386 (NH), 1680 (C = O), δ _H (300 MHz, DMSO-d ₆ ) : 1.22 (9H, s, C ( CH ₃ ) ₃ ), 2.5
(2H, m, CH ₂ ), 4.25-4.40 (3H, m, CH ₂ and CH), 5.06
(2H, s, CH ₂ ), 7.02 (1H, d, J 7.8, NH), 7.34 (5H,
s, CH ₂ Ph ), 7.53-7.62 (3H, m, benzoyl), 8.00-8.03
(2H, m, benzoyl), 8.27 (1H, s, CH =), 8.60 (1H, s,
CH =), 11.09 (1H, s, NH). FAB-MAS : m / z 531 (M + 1)

2. Synthesis of Fmoc-Gly-Ala1 (adenine) -OBn (Compound 6c; D-form)

[Chemical formula 23]

A mixture of dichloromethane (8 ml) and trifluoroacetic acid (8 ml) was added with 1.06 g of compound 5c (2
mmol) was added, and the mixture was stirred at room temperature until the raw material disappeared on TLC. The solvent was evaporated under reduced pressure, 4% aqueous sodium hydrogen carbonate solution (100 ml) was added to the obtained residue,
It was extracted 3 times with ml of ethyl acetate. The residue obtained by concentrating the organic phase under reduced pressure was dried and used as it was in the next reaction. D
Obtained residue, 0.59 g of Fm in MF (30 ml)
oc glycine (2 mmol), 0.31 g HOBt (2
mmol), 0.64 g of TBTU (2 mmol), 0.
697 ml DIEA (diisopropylethylamine)
(4 mmol) was sequentially added, and the mixture was stirred at room temperature for 24 hours.
After distilling off the solvent under reduced pressure, ethyl acetate (200 ml) was added to the residue, purified water (100 ml), 5% potassium hydrogen sulfate aqueous solution (100 ml × 3), 4% sodium hydrogen carbonate aqueous solution (100 ml × 3), purified water ( It was washed with 100 ml).
The organic phase was dried over magnesium sulfate and then evaporated under reduced pressure. Fmoc-Gly-Ala1 was obtained by recrystallizing the obtained crude crystal in a mixed solvent of ethyl acetate and methanol.
1.05 g of (adenine) -OBn (compound 6c) was obtained (yield 73%). The physicochemical data of the obtained product are shown below: Melting point : 133-135 ° C Experimental value : C, 67.11; H, 5.14; N, 13.49. Theoretical value (C ₄₀ H ₃₅ N ₇ O ₆ ) : C , 67.69; H, 4.97; N, 13.81 ν _max / cm ^-1 : 3296 (NH), 1726 (C = O), δ _H (300 MHz, DMSO-d ₆ ) : 2.55-2.78 (2H, m, CH ₂ ),
3.50 (2H, d, J 5.9, CH ₂ ) 4.2-4.3 (3H, m, CH ₂ CH),
4.36-4.50 (2H, m, CH ₂ ), 4.50-4.62 (1H, m, CH), 5.06
(2H, s, CH ₂ ), 7.25-7.35 (4H, m, arom of Fmoc), 7.5
5-7.70 (3H, m, benzoyl), 7.70 (2H, d, J 7.5, arom
of Fmoc), 7.86 (2H, d, J 7.509, arom ofFmoc) 8.02
-8.04 (2H, m, benzoyl), 8.33 (1H, s, CH =), 8.69 (1
H, s, CH =), 11.12 (1H, s, NH). FAB-MAS : 710 (M + 1) 3. Synthesis of Fmoc-Gly-Ala1 (adenine) peptide nucleic acid monomer (compound 7c; D form)

[0073]

[Chemical formula 24]

0.19 g of compound 6c (0.27 mmo
10% after dissolving l) in methanol (100 ml)
Palladium carbon (200 mg) was added, and the mixture was stirred at room temperature in a hydrogen gas atmosphere until the raw material disappeared on TLC. After the palladium carbon was distilled off, the solvent was concentrated and the resulting residue was recrystallized from methanol to obtain Fmoc-Gly-A.
la1 (adenine) peptide nucleic acid monomer (compound 7
0.10 g of c) was obtained (60% yield). The physicochemical data of the obtained product are shown below: Melting point : 213-215 ° C Experimental value : C, 63.87; H, 4.81; N, 15.61. Theoretical value (C ₃₃ H ₂₉ N ₇ O ₆ ) : C , 63.97; H, 4.72; N, 15.82 ν _max / cm ^-1 : 1697, 1646 (C = O) δ _H (300 MHz, DMSO-d ₆ ) : 2.32-2.78 (2H, m, CH ₂ ), 3 .
49 (2H, d, J 5.2, CH ₂ ), 4.13-4.56 (6H, m, CH ₂ x 2
and CH x 2), 7.10 (1H, brs, NH), 7.30 (2H, dd, J
7.5 and 7.5, Ar-H), 7.39 (2H, dd, J 7.5 and 7.5, A
rH), 7.53-7.63 (3H, m, Ph), 7.70 (2H, d, J 7.5, A
rH), 7.87 (2H, d, J 7.5, Ar-H), 7.95-8.10 (2H,
m, Ph), 8.32 (1H, s, Ar-H), 8.71 (1H, s, Ar-H), 1
1.13 (1H, brs, NH) δ _C (75 MHz, DMSO-d ₆ ) : 36.45 (CH ₂ ), 43.4 (CH ₂ ), 4
5.81 (CH ₂ ), 46.13 (CH), 46.58 (CH), 65.73 (CH), 12
0.02 (CH), 125.05 (C), 125.21 (CH), 127.01 (CH), 1
27.56 (CH), 128.37 (CH), 132.28 (C), 133.52 (CH),
140.66 (C), 143.80 (C), 144.96 (CH), 149.94 (C), 1
51.24 (CH), 152.77 (C), 156.40 (C), 165.58 (C), 16
8.89 (C), 171.73 (C) FAB-MAS : m / z 620 (M + 1).

Example 4 Fmoc-Gly-Ala
Min) Synthesis of peptide nucleic acid monomer (Compound 13) Tosylation of Boc-L-serine-OBn (Compound 8)

[Chemical 25]

Boc-L-serine-OBn (18.0
g, 0.061 mol) in dry pyridine solution (120 m
l) was cooled to -10 ° C and stirred with TosCl
(13.92 g, 0.073 mol) was added, and -10
The mixture was stirred at -15 ° C for 24 hours. The reaction solution was poured into ice water (300 ml), and the precipitated crystals were separated by filtration. The obtained crude crystals were recrystallized from ethanol to give a tosylated product (Compound 9).
1 g was obtained (yield 62%). The physicochemical data of the product obtained are shown below: δ _H (300 MHz, CDCl ₃ ) : 1.41 (9H, s, CH ₃ x 3), 2.43
(3H, s, CH ₃ ), 4.30 (1H, dd, J 10.0 and 4.0, CHH),
4.42 (1H, dd, J 10.0 and 3.0, CHH), 4.50 (1H, br
s, CH), 5.08 (1H, d, J 11.8, CHHPh), 5.17 (1H, d,
J 11.8, CHHPh), 5.30 (1H, br, NH), 7.27 (2H, d, J
9, arom), 7.34 (5H, s, Ph), 7.71 (2H, d, J 9, arom) 2. Iodination of Boc-L-serine (OTos) -OBn (Compound 9)

[0077]

[Chemical formula 26]

Boc-L-serine (OTos) -OBn
(12.6 g, 0.028 mol) of dry acetone (4
0 ml) solution at room temperature under stirring with sodium iodide (6.28 g, 0.042 mol) in dry acetone (4
0 ml) solution was added dropwise. After stirring for 24 hours, the reaction solvent was evaporated under reduced pressure, and the residue was diluted with dichloromethane (250 ml).
And washed with purified water (150 ml × 3), 1M sodium thiosulfate (200 ml), and purified water (150 ml × 3). The dichloromethane phase was dried over anhydrous magnesium sulfate and evaporated under reduced pressure, and the obtained crude crystals were recrystallized from ethanol to obtain 8.17 g of an iodinated compound (Compound 10) (yield 72%). The physicochemical data of the product obtained are shown below: δ _H (300 MHz, CDCl ₃ ) : 1.45 (9H, s, CH ₃ x 3), 3.52
-3.60 (2H, m, CH ₂ ), 4.50-4.61 (1H, m, CH), 5.18 (1
H, d, J 11.8, CHHPh), 5.23 (1H, br, NH), 5.37 (1
H, br, NH), 7.38 (5H, s, Ph).

3. Boc-Ala (thymine) -OBn
Synthesis of (Compound 11b)

[Chemical 27]

To DMF (20 ml) was added 1.76 g of compound 10 (4.34 mmol), 3.00 g of 3-benzoylthymine (13.2 mmol) and 1.80 g of anhydrous potassium carbonate (13.03 mmol) and argon. The mixture was stirred under a stream of air at room temperature for 2 days. After distilling off the reaction solvent under reduced pressure, silica gel column chromatography (ethyl acetate: n-
0.75 g of compound 11a with hexane 7: 3)
(Yield 34%) and 0.52 g of compound 11b (Yield 30%) were obtained. Compound 11a (0.203 g, 0.4
0.25 M potassium carbonate aqueous solution (5 ml) was added to a dioxane solution (5 ml) of (mmol) and compound 11 was analyzed by TLC.
Stir at room temperature until a disappears. The reaction solvent was evaporated under reduced pressure, purified water (10 ml) was added to the residue, and the precipitated crystals were collected by filtration. Compound 11b was obtained by recrystallizing the obtained crude crystal in a mixed solvent of ethanol-n-hexane (yield 0.129 g, 80%). The physicochemical data of the obtained product are shown below: Compound 11a Melting point : 133-134 ° C Experimental value : C, 63.98; H, 5.73; N, 8.30. Theoretical value (C ₂₇ H ₂₉ N ₃ O ₇ ). : C, 63.89; H, 5.76; N, 8.28 ν _max / cm ^-1 : 1752, 1712, 1658, 1640 (C = O) δ _H (300 MHz, CDCl ₃ ) : 1.43 (9H, s, CH ₃ x 3), 1.90
(3H, s, CH ₃ ), 3.70-3.85 (1H, m, C H H), 4.34-4.42 (1
H, m, CH H ), 4.59-4.70 (1H, m, CH), 5.07 (1H, d, J 1
2.0, PhC H H), 5.19 (1H, d, J 12.0, PhCH H ), 5.47 (1
H, d, J 6.6, NH), 7.02 (1H, s, CH-6 of T), 7.33 (5
H, s, Ph), 7.48 (2H, dd, J 7.8 and 7.8, PhC = O), 7.
63 (1H, dd, J 7.8 and 7.8, PhC = O), 8.04 (2H, d, J
7.8, PhC = O) δ _C (75 MHz, CDCl ₃ ) : 12.30 (CH ₃ ), 28.21 (CH ₃ ), 50.
47 (CH ₂ ), 52.26 (CH), 68.06 (CH ₂ ), 80.68 (C), 110.4
1 (C), 128.53 (CH), 128.63 (CH), 129.01 (CH), 130.
68 (CH), 131.59 (C), 134.68 (C), 134.89 (CH), 140.
46 (CH), 150.06 (C), 155.22 (C), 163.04 (C), 168.86
(C), 169.39 (C) FAB-MAS : m / z 508 (M + 1). Compound 11b Melting point : 191-192 ° C Experimental value : C, 59.65; H, 5.95; N, 10.23. Theoretical value (C ₂₇ H ₂₉ N ₃ O ₇ ) : C, 59.54; H, 6.25; N, 10.42) ν _max / cm ^-1 : 1748, 1715, 1683 (C = O); δ _H (300 MHz, CDCl ₃ ) : 1.41 ( 9H, s, CH ₃ x 3), 1.85
(3H, s, CH ₃ ), 4.02 (1H, dd, J 14.2 and 6.8, C H H),
4.20 (1H, dd, J 14.2 and 5.6, CH H ), 4.52 (1H, dd,
J 6.8 and 5.6, CH), 5.16 (1H, d, J 12.0, PhC H H),
5.22 (1H, d, J 12.0, PhCH H ), 5.46 (1H, br, NH), 6.
92 (1H, s, CH-6 of T), 7.34 (5H, s, Ph), 8.25 (1H,
brs, NH) δ _C (75 MHz, DMSO-d ₆ ) : 11.95 (CH ₃ ), 27.98 (CH ₃ ), 4
8.12 (CH ₂ ), 51.70 (CH), 66.38 (CH ₂ ), 78.72 (C), 10
8.03 (C), 127.89 (CH), 128.11 (CH), 128.39 (CH), 1
35.58 (C), 141.80 (CH), 150.87 (C), 155.26 (C), 16
4.20 (C), 169.76 (C) FAB-MAS : m / z 404 (M + 1).

4. Fmoc-Gly-Ala (thymine)
Synthesis of -OBn (Compound 12)

[Chemical 28]

0.55 g of compound 11b (1.36 mm
ol) with dichloromethane (5 ml) and trifluoroacetic acid
It was added to the mixed solvent of (5 ml) and stirred at 0 ° C. for 2 hours. Anti
After evaporating the solvent under reduced pressure, the residue was washed with 4% aqueous sodium hydrogen carbonate.
Add the solution (100 ml) and add dichloromethane (70 ml).
It was extracted in × 3). Dichloromethane phase is magnesium sulfate
Compound 11b was removed by evaporation under reduced pressure.
Crude crystals of Boc body were obtained. After drying this, DMF (10
ml) and 0.45 g of Fmoc glycy in the solution.
(1.36 mmol), 0.29 g of HOBt (1.
36 mmol), 0.438 g TBTU (1.36 m
mol), 0.475 ml DIEA (2.73 mmo
1) were sequentially added, and the mixture was stirred at room temperature for 3 hours. Reduce reaction solvent
After distilling off under pressure, ethyl acetate (100 ml) was added to the residue and purified.
Water (100 ml), 5% potassium hydrogen sulfate aqueous solution (1
00 ml × 3), 4% aqueous sodium hydrogen carbonate solution (10
It was washed with 0 ml × 3) and purified water (100 ml). Organic phase
Was dried over magnesium sulfate and evaporated under reduced pressure. Got
The crude crystals were recrystallized with a mixed solvent of ethyl acetate and methanol.
570 mg of Fmoc-Gly-Al by crystallization
a (thymine) -OBn (compound 12) was obtained (yield 72
%). The physicochemical data of the obtained product are shown below.
You:Melting point : 194-195 ℃ν _max / cm ^-1 : 1681 and 1735 (C = O)δ _H (300 MHz, DMSO-d ₆ ) : 1.69 (3H, s, CH₃), 3.56-3.
65 (2H, m, CH₂), 3.77 (1H, dd, J 13.8 and 8.7, CH
H), 4.14-4.29 (2H, m, CH and CHH), 4.27 (2H, s, C
H₂), 4.60-4.66 (1H, m, CH), 5.11 (2H, d, J 2.1, CH
₂Ph), 7.27-7.43 (5H, m, arom and CH = of T), 7.38
(5H, s, Ph), 7.58 (1H, t, J 7.3, NH), 7.70 (2H, d,
 J 7.2, arom), 7.88 (2H, d, J 7.2, arom), 8.46 (1
H, d, J 8.1, NH), 11.25 (1H, br, NH)δ _C (75 MHz, DMSO-d ₆ ) : 11.83 (CH₃), 40.33 (CH₂), Four
6.60 (CH), 47.82 (CH₂), 50.62 (CH), 65.73 (CH₂), 6
6.51 (CH₂), 108.42 (C), 120.01 (CH), 125.21 (CH), 1
27.01 (CH), 127.61 (CH), 127.86 (CH), 128.08 (CH),
 128.40 (CH), 135.50 (C), 140.69 (C), 141.63 (CH),
143.81 (C), 150.94 (C), 156.43 (C), 164.23 (C), 16
9.36 (C), 169.64 (C)FAB-MS : M / z 583 (M + 1).

5. Fmoc-Gly-Ala (thymine)
Synthesis of peptide nucleic acid monomer (compound 13)

[Chemical 29]

0.30 g of compound 12 (0.51 mmo
l) was added to methanol (180 ml) and dichloromethane (5
After dissolving it in a mixed solvent (0 ml), 10% palladium carbon (0.30 g) was added, and the mixture was stirred under a hydrogen gas atmosphere until the spot of compound 12 disappeared on TLC. After the palladium carbon was filtered off, the reaction solvent was distilled off under reduced pressure, and the resulting residue was recrystallized with purified water to obtain 0.188 g of Fmoc-Gly-Ala (thymine) peptide nucleic acid monomer (Compound 13). (Yield 74%). The physicochemical data of the obtained product are shown below: Melting point : 260-262 ° C ν _max / cm ^-1 : 1684 (C = O) δ _H (300 MHz, DMSO-d ₆ ) : 1.65 (3H, s, CH ₃ ), 3.33-3.
56 (3H, m, CH ₂ and C H H), 4.17-4.28 (5H, m, CH x 2a
nd CH ₂ and CH H ), 7.25 (1H, s, CH-6 of T), 7.27 (2
H, dd, J 7.5 and 7.5, Ar-H), 7.36 (2H, dd, J 7.5 a
nd 7.5, Ar-H), 7.58 (1H, br, NH), 7.66 (2H, d, J
7.5, Ar-H), 7.83 (2H, d, J 7.5, Ar-H), 10.85 (1H,
br, NH) δ _C (75 MHz, DMSO-d ₆ ) : 11.86 (CH ₃ ), 43.54 (CH ₂ ), 4
6.61 (CH), 49.29 (CH ₂ ), 51.87 (CH), 65.77 (CH ₂ ), 1
07.75 (C), 120.09 (CH), 125.27 (CH), 127.10 (CH), 1
27.62 (CH), 140.69 (C), 141.93 (CH), 143.81 (C), 1
50.96 (C), 156.41 (C), 164.41 (C), 168.91 (C), 17
1.22 (C) FAB-MS : m / z 493 (M + 1).

Example 5 Synthesis of peptide nucleic acid by solid phase synthesis
Synthesis 13.75 mg (2.2 μmol) Fmoc-PAL
-30% PEG resin (Applied Biosystems)
It was washed 3 times with 0 to 400 μl of DMF for peptide synthesis (hereinafter, all DMF is for peptide synthesis). Same DMF40
Swelled in 0 μl overnight. The next day, DMF was removed and 400 μl of a 20% piperidine DMF solution was added, and the mixture was slowly stirred. After 30 minutes, the solution was removed and 400 μl of DM
Wash 5 times with F. The procedure was repeated until the Kaiser test was positive. When the Kaiser test showed positive, 10 equivalents of lysine with amino group protected by Fmoc and hydroxyl group protected by Boc was added, and 10 equivalents of HATU, 10 equivalents of HOBt and 31 equivalents of DIEA were n-methylated. Equivalent mixture of pyrrolidone (NMP) and DMF 300-400μ
The solution dissolved in 1 was added and stirred slowly. 4-5
After the time, the plate was washed 5 times with 400 μl of DMF. ~ Was repeated until the Kaiser test was negative. 400 μl of 20% DMF solution of piperidine was added and stirred slowly. After 30 minutes, remove the solution and add 5 μl of 400 μl DMF.
Washed twice. The procedure was repeated until the Kaiser test was positive. Equivalent mixture of NMP and DMF 300-400 μl of 5 equivalents of peptide nucleic acid monomer, 4.5 equivalents of HATU, 5 equivalents of DIEA and 7.5 equivalents of lutidine.
It was added to the solution dissolved in and stirred slowly. 4-
After 5 hours, the plate was washed 5 times with 400 μl of DMF. The procedure was repeated until the Kaiser test was negative. When the Kaiser test showed a negative result, the procedure was repeated 12 times until returning to.

The resin was transferred to Millipore Ultra Free MC with methylene chloride, 200 μl of a 9: 1 solution of trifluoroacetic acid and m-cresol was added, the mixture was allowed to stand at room temperature for 90 minutes, and then centrifuged at 200 rpm for 5 minutes. After 800 μl of ether was added to the filtrate while cooling with ice and mixed well, the mixture was transferred to another Millipore Ultra Free MC and the speed was increased to 5 at 200 rpm.
The supernatant was removed by centrifuging for minutes to obtain crude crystals of peptide nucleic acid. The crude crystal was freeze-dried by taking the target peak portion by HPLC. HPLC purification conditions: Column: Wakopak Navi C18-5, 4.
6 mmφ × 150 mm, flow rate: 1.5 ml / min, 35 ° C. Detection: 260 nm Injection: 10 μl Eluent: A: Purified water (containing 0.1% trifluoroacetic acid), B: Acetonitrile (0.1% trifluoroacetic acid) Included). A composition of A 90% and B 10% was first flowed for 5 minutes, and then a gradient was applied to A 65% and B 35% after 30 minutes.

Example 6 Measurement of melting temperature (1) Measuring apparatus: V-530 manufactured by JASCO Corporation
Type UV-visible spectrophotometer, E manufactured by JASCO
TC-505 type Peltier constant temperature prismatic cell holder, double-sided transparent cell for spectrophotometer 10 x 2 x 45 mm (optical path width 2 mm) with PTFE cap

(2) Preparation of PNA and DNA aqueous solution: P
S so that the absorbance of NA and DNA solutions is 0.25
SC aqueous solution (0.15M sodium chloride-0.015M
The concentration was adjusted with sodium citrate).

(3) Measurement 700 μl of the measurement sample was put in the measurement cell, and the sample was kept at 90 ° C. for 20 minutes.
After holding for a minute and confirming that the absorbance was almost constant, the temperature was lowered from 90 ° C to 1 ° C at 0.5 ° C / min.
When the temperature reached 1 ° C, the measurement was terminated and the temperature was kept at 1 ° C. The change in absorbance was measured, and after 10 minutes, the temperature was increased from 1 ° C. to 90 ° C. at 0.5 ° C./min and the temperature was measured.

(4) Analysis: The melting temperature (Tm) was determined from the melting curves of the temperature decrease and the temperature increase respectively. To calculate Tm from melting curve, Spect as spectrum analysis software
raManagerfor Windows 95 / NT
(Version 1.52.00 [Build4])
Was used.

(5) Conventional DN whose bases are all thymine
The peptide nucleic acid imitating A is PNA1. Similarly, PNA2 is the Gly-Ala type peptide nucleic acid of the present invention whose bases are all thymine. Peptide nucleic acid 8mer simulating conventional DNA
And a mixed peptide nucleic acid of Gly-Ala type peptide nucleic acid 4mer as PNA3. A conventional mixed peptide nucleic acid consisting of a peptide nucleic acid 4mer simulating DNA and a Gly-Ala type peptide nucleic acid 8mer is designated as PNA4. The Tm of 12-mer DNA and RNA having all adenine bases and the peptide nucleic acids PNA1 to 4 were measured. The results are shown in Table 1.

[0092]

[Table 1] From the above results, it can be seen that the peptide nucleic acid of the present invention has a very high binding selectivity for RNA as compared with conventional peptide nucleic acids.

[0093]

INDUSTRIAL APPLICABILITY The peptide nucleic acid of the present invention binds to RNA with a high degree of selectivity, can bind efficiently to RNA even in a small amount, and is biologically and chemically stable. , Can be used as a very useful antisense oligonucleotide mimetic.

[Brief description of drawings]

FIG. 1 2 ′, 5′-isoDNA and 3 ′, 5′-DNA
It is a figure showing the structure of.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) A61P 1/04 A61P 9/00 1/18 9/10 9/00 103 9/10 11/00 103 11 / 06 11/00 13/12 11/06 15/00 13/12 17/00 15/00 19/02 17/00 27/02 19/02 29/00 27/02 31/04 29/00 31/10 31 / 04 31/16 31/10 31/18 31/16 31/20 31/18 31/22 31/20 33/14 31/22 35/00 33/14 37/04 35/00 C07K 1/06 37 / 04 5/023 C07K 1/06 5/027 5/023 5/062 5/027 C12N 15/00 A 5/062 A61K 37/02 (72) Inventor Masami Otsuka 1-4 Koto, Kumamoto City, Kumamoto Prefecture 3 Koto Housing 3 -303 F term (reference) 4B024 AA01 CA05 CA10 HA17 4C084 AA01 AA02 AA07 AA13 BA09 BA10 BA14 BA15 BA16 BA17 BA18 BA19 BA20 BA23 BA35 CA59 MA01 NA01 NA14 ZA331 ZA361 ZA401 ZA961 ZB111ZB011B011B011B011ZB1 ZB1 ZB1 ZB091 ZB091 ZB091 ZB091 BB09 4H04 5 AA10 AA20 AA30 BA11 BA54 EA22 EA28 FA34

Claims (11)

[Claims] 1. A method of binding a peptide nucleic acid having a skeletal structure similar to that of 2 ′, 5′-isoDNA and having a sequence complementary to the RNA of interest to the RNA. 2. The structure of a peptide portion of a peptide nucleic acid is
Glycine and alanines: The method according to claim 1, wherein [where n = 0, 1 or 2] are alternately combined. 3. The peptide nucleic acid has the formula I: [In the formula, R ¹ is a hydrogen atom or an amino group protecting group, R ² is a hydroxyl group or a carboxyl group protecting group, R ³ is a direct bond, or a methylene group or an ethylene group, and B is Is a base or a derivative thereof, which may be the same or different, and m is an integer of 1 to 60]. 4. The method according to claim 1, wherein the peptide nucleic acid has a higher affinity for complementary RNA than that for complementary DNA. 5. A base or a derivative thereof is introduced into aspartic acid or a derivative thereof to obtain a compound represented by the formula II: [Wherein R ¹ is a hydrogen atom or an amino group-protecting group] or a derivative thereof to give a compound of formula III: [Wherein R ¹ is a hydrogen atom or an amino group protecting group, R ² is a hydroxyl group or a carboxyl group protecting group, and B is a base or a derivative thereof] A method of manufacturing. 6. A base or a derivative thereof is introduced into serine or a derivative thereof to obtain a compound of formula II: [Wherein R ¹ is a hydrogen atom or an amino-group-protecting group] to give a compound of the formula IV: [Wherein R ¹ is a hydrogen atom or an amino group protecting group, R ² is a hydroxyl group or a carboxyl group protecting group, and B is a base or a derivative thereof] A method of manufacturing. 7. The method according to claim 6, wherein tosylation and / or halogenation is carried out before the step of introducing the base or its derivative. 8. A composition for regulating the expression of a gene in an organism, which comprises the peptide nucleic acid according to any one of claims 1 to 4. 9. A composition for treating a condition associated with undesired protein production in an organism, comprising the peptide nucleic acid according to any one of claims 1 to 4. 10. A composition for inducing degradation of RNA in cells of an organism, which comprises the peptide nucleic acid according to any one of claims 1 to 4. 11. A composition for killing a cell or virus, which comprises the peptide nucleic acid according to any one of claims 1 to 4.