Preparation method of solution phase peptide nucleic acid oligomer

Abstract

The present invention provides a method for preparing a peptide nucleic acid oligomer. More specifically, the method comprises the step of preparing a peptide nucleic acid oligomer by reacting a peptide nucleic acid block with a peptide nucleic acid block in a solution phase, and the peptide nucleic acid oligomer can be prepared with surprisingly improved purity and yield through a simple solution process.

Description

Technical Field

[0001] The present invention relates to a method for preparing solution phase peptide nucleic acid (PNA) oligomers. Background Art

[0002] As is known to all, nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which are responsible for the genetic information of organisms.

[0003] In contrast, peptide nucleic acids are nucleic acids in which the sugar-phosphate backbone of nucleic acid is converted to an N-(2-aminoethyl)glycine backbone.

[0004] The sugar-phosphate backbone of DNA / RNA has a negative charge under neutral conditions, which results in electrostatic repulsion between complementary chains. However, the backbone structure of peptide nucleic acid does not have a charge, so there is no electrostatic repulsion.

[0005] That is, the main chain of peptide nucleic acid is substantially completely uncharged, which is a very important feature of peptide nucleic acid. Due to this feature, it can be used in many applications where oligonucleotides or oligonucleotide derivatives cannot be used.

[0006] Furthermore, peptide nucleic acids bind to deoxyribonucleic acid or ribonucleic acid with a higher affinity than oligonucleotides, and have the advantage of being very stable in serum compared to natural ribonucleic acid.

[0007] However, although the interest and research on the applications of PNAs are high and diverse, research on the synthesis methods of PNA oligomers has been slow.

[0008] Generally, the synthesis method of peptide nucleic acid oligomers utilizes solid-phase peptide synthesis. If the peptide nucleic acid monomer units are classified according to the backbone structure of the peptide nucleic acid, they can be divided into two types: Fmoc-type peptide nucleic acid monomer units and Boc-type peptide nucleic acid monomer units.

[0009] Although the synthesis method of Fmoc-type peptide nucleic acid monomer units has been established, it is not easy to produce in large quantities. In addition, there are problems with yield and purity.

[0010] For example, although WO2005-009998A1 discloses that a monomer for synthesizing peptide nucleic acid can be easily synthesized at a high yield, there is a problem that it is not economical in mass production due to the multiple steps.

[0011] Therefore, there is a need for a method for producing a solution-phase peptide nucleic acid oligomer that can efficiently produce a target peptide nucleic acid with high purity and yield through a simple process. Summary of the invention

[0012] Technical issues

[0013] The present invention provides a method for preparing a target peptide nucleic acid oligomer with surprisingly improved purity and yield through a simple process.

[0014] Technical Solution

[0015] The present invention provides a method for preparing peptide nucleic acid oligomers with extremely improved purity and yield through simple procedures and easy solution phase. The method for preparing peptide nucleic acid oligomers of the present invention is characterized in that it includes a step of preparing peptide nucleic acid oligomers by reacting peptide nucleic acid blocks with peptide nucleic acid blocks in a solution phase, wherein the peptide nucleic acid blocks are composed of two or more different or identical peptide nucleic acid monomers combined.

[0016] Preferably, the preparation method of the peptide nucleic acid oligomer of the present invention includes the step of mixing a first peptide nucleic acid dimer represented by the following Chemical Formula 1, a first peptide nucleic acid trimer represented by the following Chemical Formula 2, or a first peptide nucleic acid tetramer represented by the following Chemical Formula 3 with a second peptide nucleic acid dimer represented by the following Chemical Formula 4, a second peptide nucleic acid trimer represented by the following Chemical Formula 5, or a second peptide nucleic acid tetramer represented by the following Chemical Formula 6 in the presence of a coupling reagent and a solvent, and then adding an amine compound to prepare a peptide nucleic acid oligomer represented by the following Chemical Formulas 7 to 9.

[0017] Chemical formula 1

[0018]

[0019] Chemical formula 2

[0020]

[0021] Chemical formula 3

[0022]

[0023] Chemical formula 4

[0024]

[0025] Chemical formula 5

[0026]

[0027] Chemical formula 6

[0028]

[0029] Chemical formula 7

[0030]

[0031] Chemical formula 8

[0032]

[0033] Chemical formula 9

[0034]

[0035] In the above Chemical Formulae 1 to 9, A1 to A9 and A 11 To A 14 Each is independently a peptide nucleic acid monomer containing different or identical nucleic acid bases, P1 to P18 are independently hydrogen, or different or identical protecting groups, P1 and P2, P3 and P4, P5 and P6, P 11 With P 12 , P 13 With P 14 , P 15 With P 16 and P 17 With P 18 Except for the case where they are both hydrogen, a and b are each independently an integer of 1, and c and d are each independently an integer of 0 or 1.

[0036] Preferably, the preparation method of the peptide nucleic acid oligomer of one embodiment of the present invention comprises: step a), adding the second peptide nucleic acid dimer represented by the following chemical formula 4-1, the second peptide nucleic acid trimer represented by the following chemical formula 5-1 or the second peptide nucleic acid tetramer represented by the following chemical formula 6-1 to the first peptide nucleic acid dimer represented by the following chemical formula 1-1, the first peptide nucleic acid trimer represented by the following chemical formula 2-1 or the first peptide nucleic acid tetramer represented by the following chemical formula 3-1 in the presence of a coupling reagent and a solvent to obtain a mixed solution; and step b), adding an amine compound to the mixed solution of the above steps to prepare the compounds of the above chemical formulas 7 to 9.

[0037] Chemical formula 1-1

[0038]

[0039] Chemical formula 2-1

[0040]

[0041] Chemical formula 3-1

[0042]

[0043] Chemical formula 4-1

[0044]

[0045] Chemical formula 5-1

[0046]

[0047] Chemical formula 6-1

[0048]

[0049] In the above Chemical Formula 1-1, Chemical Formula 2-1, Chemical Formula 3-1, Chemical Formula 4-1, Chemical Formula 5-1 and Chemical Formula 6-1, A1 to A9 and A 11 To A 14 Each independently comprises a peptide nucleic acid monomer containing different or identical nucleic acid bases, P2, P4, P6, P7, P9 and P 11 Each is independently hydrogen or a different or identical protecting group, X1 to X3 are each independently an acid salt, a and b are each independently an integer of 1, and c and d are each independently an integer of 0 or 1.

[0050] Preferably, the amine compound according to an embodiment of the present invention may be N,N-diisopropylethylamine, but is not limited thereto.

[0051] Preferably, the method for preparing a peptide nucleic acid oligomer according to an embodiment of the present invention may further include repeatedly performing steps a) and b) using the product obtained in the above step b) as a starting material.

[0052] More preferably, the volume ratio of the starting material to the solvent in the mixed solution may be 1:10 or more.

[0053] The nucleic acid bases of one embodiment of the present invention may be adenine, cytosine, 5-methylcytosine, guanine, thymine, uracil, purine, 2,6-diaminopurine, N 4 N 4 -Ethylenediylcytosine, N 6 N 6 -Ethylene-2,6-diaminopurine, 5-(C3-C6)-alkynyluracil, 5-(C3-C6)-alkynyl-cytosine, 5-(1-propargylamino)uracil, 5-(1-propargylamino)cytosine, phenoxazine, 9-aminoethoxyphenoxazine, 5-fluorouracil, pseudoisocytosine, 5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil, dihydrouracil pyrimidine, 5-(C1-C6)-alkyluracil, 5-(C1-C6)-alkyl-cytosine, 5-(C2-C6)-alkenylcytosine, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil, 5-bromocytosine, 7-deazaadenine, 7-deazaguanine, 8-azapurine, 7-deaza-7-substituted purine, thiouracil or an artificial nucleic acid base.

[0054] The protecting group of one embodiment of the present invention can be fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, diphenylmethoxycarbonyl (Bhoc), acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, tosyl, trichloroethyl chloroformate, sulfonamides or isobutyryl.

[0055] In the preparation method of the peptide nucleic acid oligomer of the present invention, preferably, the first peptide nucleic acid dimer or the second peptide nucleic acid dimer of one embodiment of the present invention is represented by the following chemical formula 11, the above-mentioned first peptide nucleic acid trimer or the second peptide nucleic acid trimer is represented by the following chemical formula 12, and the above-mentioned first peptide nucleic acid tetramer or the second peptide nucleic acid tetramer is represented by the following chemical formula 13.

[0056] Chemical formula 11

[0057]

[0058] Chemical formula 12

[0059]

[0060] Chemical formula 13

[0061]

[0062] In the above Chemical Formulas 11 to 13, R1 to R 18 Each is independently hydrogen, an amino acid residue or an amino acid residue having a substituent, T1 to T3 are each independently an amine protecting group, Z1 to Z3 are each independently a hydroxyl protecting group, and B1 to B9 are each independently a nucleic acid base containing or not containing an amine protecting group.

[0063] In one embodiment of the present invention, relative to 1 equivalent of the first peptide nucleic acid dimer represented by the above Chemical Formula 1-1, the first peptide nucleic acid trimer represented by the above Chemical Formula 2-1, or the first peptide nucleic acid tetramer represented by the above Chemical Formula 3-1, 1 mol:0.8 mol to 1 mol:1.2 mol of the second peptide nucleic acid dimer represented by the above Chemical Formula 4-1, the second peptide nucleic acid trimer represented by the above Chemical Formula 5-1, or the second peptide nucleic acid tetramer represented by the above Chemical Formula 6-1 can be used.

[0064] Preferably, the coupling reagent of one embodiment of the present invention can be benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBop) or a mixture thereof, and step b) can be carried out at a temperature of -10°C to 5°C for 10 to 60 minutes.

[0065] The solvent of one embodiment of the present invention may be a mixed solvent of chlorinated (C1-C4) alkanes, N,N-dimethylformamide (DMF) and N,N-diisopropylethylamine (DIEA).

[0066] An embodiment of the present invention may further include the step of adding water to the compounds of Chemical Formulae 7 to 9 for separation and purification.

[0067] Effects of the Invention

[0068] The method for preparing a peptide nucleic acid oligomer of the present invention prepares a peptide nucleic acid oligomer by preparing a peptide nucleic acid block and then reacting the peptide nucleic acid blocks with each other, which epochally reduces the preparation steps of the peptide nucleic acid and can prepare the peptide nucleic acid oligomer very economically and with high purity.

[0069] Specifically, the method for preparing peptide nucleic acid oligomers of the present invention uses peptide nucleic acid dimers, peptide nucleic acid trimers or peptide nucleic acid tetramers for preparation. Compared with the existing method using peptide nucleic acid monomers, peptide nucleic acid oligomers can be prepared with simpler processes. Not only that, the target peptide nucleic acid oligomer can also be prepared more accurately.

[0070] Therefore, compared with the existing method using peptide nucleic acid monomers, the method for preparing peptide nucleic acid oligomers of the present invention can be prepared with a shorter process step and separation from by-products is very easy, so the yield and purity of peptide nucleic acid oligomers are very high.

[0071] Furthermore, all steps of the method for preparing a peptide nucleic acid oligomer of the present invention can be prepared in a solution phase, and mass production is possible, which is very advantageous for commercialization.

[0072] Furthermore, the method for preparing the peptide nucleic acid oligomer of the present invention uses peptide nucleic acid blocks, specifically, uses peptide nucleic acid dimers, peptide nucleic acid trimers or peptide nucleic acid tetramers for preparation. Compared with existing methods, very small amounts of peptide nucleic acid dimers, peptide nucleic acid trimers or peptide nucleic acid tetramers are used, so it is very economical and can easily prepare high-purity peptide nucleic acid oligomers in a mass production manner with high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 These are the results of high performance liquid chromatography (HPLC) measurement of the peptide nucleic acid oligomer prepared in Example 20 of the present invention.

[0074] Figure 2 The results of HPLC measurement of the peptide nucleic acid oligomer prepared in Comparative Example 1 of the present invention are shown. DETAILED DESCRIPTION

[0075] The following is a detailed description of the preparation method of the peptide nucleic acid oligomer of the present invention and the peptide nucleic acid oligomer prepared according to the method. In this case, unless otherwise defined, the technical terms and scientific terms used have the meanings generally understood by ordinary technicians in the technical field to which the present invention belongs. In the following description, well-known functions and structures that may unnecessarily confuse the gist of the present invention will be omitted.

[0076] The amino acids described in this specification are used in a broad sense, including not only natural amino acids such as serine (Ser), asparagine (Asn), valine (Val), leucine (Leu), isoleucine (Ile), alanine (Ala), tyrosine (Tyr), glycine (Gly), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), threonine (Thr), cysteine ​​(Cys), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro), but also non-natural amino acids such as amino acid variants and derivatives. It can be understood by those skilled in the art to which the present invention belongs that, considering the above broad definition, the amino acids in this specification can be chemically modified amino acids such as L-amino acids, D-amino acids, amino acid variants and derivatives; amino acids such as norleucine, β-alanine, and ornithine that are not used as constituent materials of proteins in organisms; and compounds synthesized by chemical methods that have amino acid properties and are well known in the art to which the present invention belongs. Examples of non-natural amino acids include, in addition to the threonine derivative A, α-methyl amino acids (α-methylalanine, etc.), D-amino acids, histidine-like amino acids (2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine, etc.), amino acids having an extra methylene group on the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced by a sulfonic acid group (cysteine, etc.).

[0077] The "amino acid residue having a substituent" recorded in the present specification means that the amino acid residue has a substituent, and may include, for example, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan or tyrosine substituted with an acetyl group, and may be a peptide bound to an amino acid.

[0078] The "protecting group" described in the present invention is a functional group used to protect a specific functional group in an organic reaction, such as an amine group or a hydroxyl group. Any functional group can be used as long as it is within the scope of knowledge of ordinary technicians in the field of organic synthesis. Specific examples of amine protecting groups include fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), diphenylmethoxycarbonyl (Bhoc), allyloxycarbonyl (Alloc), acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, toluenesulfonyl, trichloroethyl chloroformate, sulfonamide (Bts, nitrobenzenesulfonyl (Nosyl) and Nps), and isobutyryl.

[0079] The "chlorinated alkane" described in the present invention refers to an alkane in which one or more hydrogen atoms are replaced by chlorine. The alkane includes all forms of straight chain or branched chain. Unless otherwise specified, the alkane has 1 to 10 carbon atoms, preferably 1 to 7 carbon atoms, and more preferably 1 to 4 carbon atoms.

[0080] The terms "substituted", "having a substituent" and "substituents" in this specification, unless otherwise specified, may be substituted with halogen, hydroxyl, carboxylic acid, nitro, cyano, (lower) alkyl, haloalkyl, mono- or di-alkylamino, alkoxy, thioalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -NO2, -NR a1 R b1 、-NRa1C(=O)R b1 、-NR a1 C(=O)NR a1 R b1 、-NR a1 C(=O)OR b1 、-NR a1 S02R b1 、-OR a1 、-CN、-C(=O)R a1 、-C(=O)OR a1 、-C(=O)NR a1 R b1 、-OC(=O)R a1 、-OC(=O)OR a1 、-OC(=O)NR a1 R b1 、-NR a1 S02R b1 、-PO3R a1 、-PO(OR a1 )(OR b1 ),-SO2R a1 、-S(O)R a1、-SO(NR a1 )R b1 (e.g. sulfoximine), -S(NR a1 )R b1 (e.g. sulfilimine) and -SR a1 , where R a1 With R b1 may be the same or different and may each independently be hydrogen, halogen, amino, alkyl, alkoxyalkyl, haloalkyl, aryl and heterocyclic, or, like the nitrogen atom to which it is attached, R a1 With R b1 It can be in the form of a heterocyclic ring. a1 With R b1 There may be multiple alkyl groups. Preferably, the alkyl group may be C 1-6 Alkyl, cycloalkyl and heterocycloalkyl may be C 3-12 , the aryl group can be C 6-12 , heterocyclic and heteroaryl groups can be C 3-12 .

[0081] The peptide nucleic acid monomer described in the present invention comprises a basic peptide nucleic acid skeleton, specifically, a nucleic acid base having or not having an amine or hydroxyl protecting group at the N position of N-(2-aminoethyl)glycine) (Compound 5) or all Compounds 8.

[0082] Specifically, all the following structures having no hydroxyl group or no amine group and the like are also included.

[0083]

[0084] The peptide nucleic acid dimer described in the present invention is composed of two peptide nucleic acid monomers connected to the N-(2-aminoethyl)glycine (compound 5) or compound 8 which are bonded to the basic skeleton of the peptide nucleic acid and to which the nucleic acid bases are bonded. The nucleic acid bases contained in the peptide nucleic acid dimer can be different or the same, and specifically, can be represented by the above-mentioned chemical formula 11.

[0085] The peptide nucleic acid trimer described in the present invention is composed of three peptide nucleic acid monomers connected to the N-(2-aminoethyl)glycine (compound 5) or compound 8 which are bound to a nucleic acid base at the N position as the basic skeleton of the peptide nucleic acid. The nucleic acid bases contained in the peptide nucleic acid trimer can be different or the same, and specifically, can be represented by the above-mentioned chemical formula 12.

[0086] Furthermore, similar to the peptide nucleic acid trimer, the peptide nucleic acid tetramer described in the present invention refers to a structure formed by connecting four peptide nucleic acid monomers, and specifically, is represented by the above-mentioned Chemical Formula 13.

[0087] The "peptide nucleic acid block" described in the present specification means a combination of two or more peptide nucleic acid monomers, and refers to a peptide nucleic acid dimer, a peptide nucleic acid trimer, a peptide nucleic acid tetramer, and the like.

[0088] The present invention provides a method for preparing peptide nucleic acid oligomers with high purity and yield in a solution phase. The method for preparing peptide nucleic acid oligomers of the present invention is characterized in that it includes a step of preparing peptide nucleic acid oligomers by reacting peptide nucleic acid blocks with peptide nucleic acid blocks in a solution phase, wherein the peptide nucleic acid blocks are composed of two or more different or identical peptide nucleic acid monomers combined.

[0089] The method for preparing a peptide nucleic acid oligomer of the present invention is different from the existing method of connecting peptide nucleic acid monomers one by one on a support. After preparing a peptide nucleic acid block composed of two or more peptide nucleic acid monomers in a solution phase, these peptide nucleic acid blocks are reacted to prepare a peptide nucleic acid oligomer, thereby accurately preparing the target peptide nucleic acid oligomer through a short process step.

[0090] Furthermore, the method for preparing a peptide nucleic acid oligomer of the present invention can prepare a peptide nucleic acid oligomer with higher purity and yield using a peptide nucleic acid block, and all reactions are performed in a solution phase, so it has the advantages of being easier to prepare and capable of mass production.

[0091] Preferably, the preparation method of the peptide nucleic acid oligomer of the present invention includes the step of mixing a first peptide nucleic acid dimer represented by the following Chemical Formula 1, a first peptide nucleic acid trimer represented by the following Chemical Formula 2, or a first peptide nucleic acid tetramer represented by the following Chemical Formula 3 with a second peptide nucleic acid dimer represented by the following Chemical Formula 4, a second peptide nucleic acid trimer represented by the following Chemical Formula 5, or a second peptide nucleic acid tetramer represented by the following Chemical Formula 6 in the presence of a coupling reagent and a solvent, and then adding an amine compound to prepare a peptide nucleic acid oligomer represented by the following Chemical Formulas 7 to 9.

[0092] Chemical formula 1

[0093]

[0094] Chemical formula 2

[0095]

[0096] Chemical formula 3

[0097]

[0098] Chemical formula 4

[0099]

[0100] Chemical formula 5

[0101]

[0102] Chemical formula 6

[0103]

[0104] Chemical formula 7

[0105]

[0106] Chemical formula 8

[0107]

[0108] Chemical formula 9

[0109]

[0110] In the above Chemical Formulae 1 to 9, A1 to A9 and A 11 To A 14 Each independently comprises a peptide nucleic acid monomer containing different or identical nucleic acid bases, P1 to P 18 Each is independently hydrogen, or a different or identical protecting group, P1 and P2, P3 and P4, P5 and P6, P 11 With P 12 , P 13 With P 14 , P 15 With P 16 and P 17 With P 18 Except for the case where they are both hydrogen, a and b are each independently an integer of 1, and c and d are each independently an integer of 0 or 1.

[0111] Unlike the existing method of preparing peptide nucleic acid oligomers by binding peptide nucleic acid monomers on a support (resin), washing it and then binding peptide nucleic acid monomers again, the preparation method of peptide nucleic acid oligomers in one embodiment of the present invention utilizes peptide nucleic acid dimers, peptide nucleic acid trimers and peptide nucleic acid tetramers as nucleic acid block peptides. The synthesis steps are short and very economical. Compared with the preparation method in the solid phase, peptide nucleic acid oligomers can be prepared with high yield and purity.

[0112] Furthermore, the present invention utilizes peptide nucleic acid dimers, peptide nucleic acid trimers or peptide nucleic acid tetramers to prepare peptide nucleic acid oligomers, and the process steps are short and economical. An amine compound as a specific compound is added to a mixture of a starting material and a coupling reagent in a solution phase, and the generation of by-products is low, so that high-purity peptide nucleic acid oligomers can be prepared with a high yield.

[0113] That is, unlike the method of preparing peptide nucleic acid oligomers by mixing starting materials, coupling reagents and amine compounds, the method of preparing peptide nucleic acid oligomers of the present invention is to add amine compounds to the mixed solution of the mixed starting materials and coupling reagents, which significantly reduces the formation of by-products and can prepare peptide nucleic acid oligomers with high yield and purity.

[0114] In addition, the peptide nucleic acid oligomers prepared in this way can also be easily separated and purified.

[0115] Specifically, the preparation method of the peptide nucleic acid oligomer of one embodiment of the present invention utilizes a peptide nucleic acid dimer, a peptide nucleic acid trimer or a peptide nucleic acid tetramer that can be synthesized by a solution process. When preparing a peptide nucleic acid oligomer having n nucleic acid bases, a peptide nucleic acid oligomer having n-1 nucleic acid bases or a peptide nucleic acid oligomer having n-2 nucleic acid bases will not be prepared as a by-product. The peptide nucleic acid oligomer can be easily separated from other by-products and other impurities, so the purity of the separated peptide nucleic acid oligomer is very high.

[0116] More preferably, the preparation method of the peptide nucleic acid oligomer of one embodiment of the present invention includes: step a), adding the second peptide nucleic acid dimer represented by the following chemical formula 4-1, the second peptide nucleic acid trimer represented by the following chemical formula 5-1 or the second peptide nucleic acid tetramer represented by the following chemical formula 6-1 to the first peptide nucleic acid dimer represented by the following chemical formula 1-1, the first peptide nucleic acid trimer represented by the following chemical formula 2-1 or the first peptide nucleic acid tetramer represented by the following chemical formula 3-1 in the presence of a coupling reagent and a solvent to obtain a mixed solution; and step b), adding an amine compound to the mixed solution of the above steps to prepare the compounds of the above chemical formulas 7 to 9.

[0117] Chemical formula 1-1

[0118]

[0119] Chemical formula 2-1

[0120]

[0121] Chemical formula 3-1

[0122]

[0123] Chemical formula 4-1

[0124]

[0125] Chemical formula 5-1

[0126]

[0127] Chemical formula 6-1

[0128]

[0129] In the above Chemical Formula 1-1, Chemical Formula 2-1, Chemical Formula 3-1, Chemical Formula 4-1, Chemical Formula 5-1 and Chemical Formula 6-1, A1 to A9 and A 11 To A 14 Each independently comprises a peptide nucleic acid monomer containing different or identical nucleic acid bases, P2, P4, P6, P7, P9 and P 11 Each is independently hydrogen or a different or identical protecting group, X1 to X3 are each independently an acid salt, a and b are each independently an integer of 1, and c and d are each independently an integer of 0 or 1.

[0130] Preferably, in order to convert the ammonium salts of Chemical Formula 1-1, Chemical Formula 2-1 and Chemical Formula 3-1 into free amines while increasing the reactivity with the hydroxyl groups of Chemical Formula 4-1, Chemical Formula 5-1 and Chemical Formula 6-1, N,N-diisopropylethylamine is used as a specific amine compound, thereby enabling the preparation of peptide nucleic acid oligomers with a higher yield and purity.

[0131] Preferably, in one embodiment of the present invention, 5 to 40 moles of amine compounds can be used relative to 1 mole of the compounds of the above Chemical Formula 1-1, Chemical Formula 2-1 and Chemical Formula 3-1, and more preferably, 10 to 30 moles of amine compounds can be used.

[0132] Furthermore, the method for preparing a peptide nucleic acid oligomer according to an embodiment of the present invention may further include repeatedly performing steps a) and b) using the product obtained in the above step b) as a starting material.

[0133] The method for preparing a peptide nucleic acid oligomer according to one embodiment of the present invention utilizes a peptide nucleic acid dimer, a peptide nucleic acid trimer or a peptide nucleic acid tetramer. Unlike the existing methods, it does not require a capping step for protecting unreacted functional groups and has the advantage of being able to epochally shorten the preparation steps, thus enabling mass production.

[0134] The peptide nucleic acid oligomer of one embodiment of the present invention can be easily prepared as a peptide nucleic acid oligomer having a target number of nucleic acid bases, but preferably, it can contain more than 4 nucleic acid bases, and more preferably, it can contain 4 to 40 nucleic acid bases.

[0135] In one embodiment of the present invention, the volume ratio of the starting material to the solvent in the mixed solution may be greater than 1:10, preferably, may be 1:10 to 1:250, and more preferably, may be 1:10 to 1:100.

[0136] When the volume ratio of the starting material to the solvent in the mixed solution of one embodiment of the present invention is 1:10 or more, the peptide nucleic acid oligomer can be prepared with higher yield and purity.

[0137] The nucleic acid bases of one embodiment of the present invention may be adenine, cytosine, 5-methylcytosine, guanine, thymine, uracil, purine, 2,6-diaminopurine, N 4 N 4 -Ethylenediylcytosine, N 6 N 6 -Ethylene-2,6-diaminopurine, 5-(C3-C6)-alkynyluracil, 5-(C3-C6)-alkynyl-cytosine, 5-(1-propargylamino)uracil, 5-(1-propargylamino)cytosine, phenoxazine, 9-aminoethoxyphenoxazine, 5-fluorouracil, pseudoisocytosine, 5-(hydroxymethyl)uracil, 5-aminouracil, pseudouracil, dihydrouracil pyrimidine, 5-(C1-C6)-alkyluracil, 5-(C1-C6)-alkyl-cytosine, 5-(C2-C6)-alkenylcytosine, 5-fluorocytosine, 5-chlorouracil, 5-chlorocytosine, 5-bromouracil, 5-bromocytosine, 7-deazaadenine, 7-deazaguanine, 8-azapurine, 7-deaza-7-substituted purine, thiouracil or an artificial nucleic acid base.

[0138] The protecting group of one embodiment of the present invention can be fluorenylmethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, diphenylmethoxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, toluenesulfonyl, trichloroethyl chloroformate, sulfonamide or isobutyryl. Preferably, the amine protecting group can be tert-butoxycarbonyl, benzyloxycarbonyl, diphenylmethoxycarbonyl, acetyl, benzoyl, benzyl, and the protecting group of the hydroxyl group can be (C1-C4) alkyl.

[0139] Specifically, the first peptide nucleic acid dimer or the second peptide nucleic acid dimer of one embodiment of the present invention can be represented by the following chemical formula 11, the first peptide nucleic acid trimer or the second peptide nucleic acid trimer can be represented by the following chemical formula 12, and the first peptide nucleic acid tetramer or the second peptide nucleic acid tetramer can be represented by the following chemical formula 13.

[0140] Chemical formula 11

[0141]

[0142] Chemical formula 12

[0143]

[0144] Chemical formula 13

[0145]

[0146] In the above Chemical Formulas 11 to 13, R1 to R 18 Each is independently hydrogen, an amino acid residue or an amino acid residue having a substituent, T1 to T3 are each independently an amine protecting group, Z1 to Z3 are each independently a hydroxyl protecting group, and B1 to B9 are each independently a nucleic acid base containing or not containing an amine protecting group.

[0147] Relative to 1 mole of the first peptide nucleic acid dimer represented by the above Chemical Formula 1-1, the first peptide nucleic acid trimer represented by the above Chemical Formula 2-1, or the first peptide nucleic acid tetramer represented by the above Chemical Formula 3-1, 1 mole:0.8 mole to 1 mole:1.2 mole of the second peptide nucleic acid dimer represented by the above Chemical Formula 4-1, the second peptide nucleic acid trimer represented by the above Chemical Formula 5-1, or the second peptide nucleic acid tetramer represented by the above Chemical Formula 6-1 can be used. Preferably, 1 mole:0.9 mole to 1 mole:1.1 mole can be used.

[0148] The coupling reagent of one embodiment of the present invention can be benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop, (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate) or a mixture thereof, preferably, it can be benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate), but is not limited thereto.

[0149] Relative to 1 mole of the first peptide nucleic acid dimer represented by the above chemical formula 1-1, the first peptide nucleic acid trimer represented by the above chemical formula 2-1, or the first peptide nucleic acid tetramer represented by the above chemical formula 3-1, 1.2 moles to 2.5 moles of the coupling reagent can be used, preferably, 1.2 moles to 2.0 moles of the coupling reagent can be used, but it is not limited thereto.

[0150] Step b) of one embodiment of the present invention is a step of performing a coupling reaction, which can be performed at a temperature of -10°C to 5°C for 10 to 60 minutes, more preferably, at a temperature of -5°C to 5°C for 10 to 40 minutes.

[0151] The solvent of one embodiment of the present invention may be a mixed solvent of chlorinated (C1-C4) alkanes, N,N-dimethylformamide and N,N-diisopropylethylamine, preferably, may be N,N-dimethylformamide.

[0152] In this case, the solvent used can be a mixed solvent of chlorinated (C1-C4) alkanes, N,N-dimethylformamide and N,N-diisopropylethylamine (DIEA), and the chlorinated (C1-C4) alkanes can be one or more selected from chloroform, dichloromethane, chloroform, 1,1,2-trichloroethane, 1,1,1-trichloroethane, 1,2-dichloroethane, and 1,1-dichloroethane.

[0153] The solvent of one embodiment of the present invention uses N,N-dimethylformamide, N,N-diisopropylethylamine or chlorinated alkane. The solubility of the reactants is appropriately adjusted during the coupling reaction so that the product can be obtained with high yield and purity. From this aspect, preferably, the solvent can be N,N-dimethylformamide.

[0154] The peptide nucleic acid oligomers of Chemical Formula 7 to Chemical Formula 9 prepared according to one embodiment of the present invention can be separated and purified by various methods known to those skilled in the art, preferably, by adding water.

[0155] Furthermore, all steps of the method for preparing a peptide nucleic acid oligomer according to one embodiment of the present invention are carried out in a solution phase. Compared with the existing method of preparing a peptide nucleic acid oligomer from a peptide nucleic acid monomer in a solid phase using a support (resin), peptide nucleic acid dimers, peptide nucleic acid trimers or peptide nucleic acid tetramers can be used to prepare peptide nucleic acid oligomers with surprisingly improved yields and purity, which is a very effective and economical method.

[0156] That is, the preparation method of the peptide nucleic acid oligomer of the present invention can prepare a first peptide nucleic acid dimer, a first peptide nucleic acid trimer, a first peptide nucleic acid tetramer, a second peptide nucleic acid dimer, a second peptide nucleic acid trimer, a second peptide nucleic acid tetramer and a peptide nucleic acid oligomer in a solution phase, and can prepare a peptide nucleic acid oligomer with excellent purity and yield by a simple process, and has the advantage of being able to be mass-produced.

[0157] The results of high performance liquid chromatography (HPLC) analysis of the peptide nucleic acid oligomer of one embodiment of the present invention under the following conditions may show a purity of 70% or more, preferably 75% or more, and more preferably 80% or more.

[0158] Developing solvent: 0.1% trifluoroacetic acid aqueous solution (TFA 0.1% in Water), 0.1% trifluoroacetic acid acetonitrile solution (TFA 0.1% in MeCN), ultraviolet (UV) detector under gradient condition, chromatographic column 250 mm×4.6 mm.

[0159] Furthermore, the preparation method of the peptide nucleic acid oligomer of the present invention synthesizes the peptide nucleic acid oligomer in a manner without a covering process, and the purity of the prepared peptide nucleic acid oligomer is 70% or more, preferably 75% or more, and more preferably 80% or more according to the HPLC analysis results under the above conditions.

[0160] Hereinafter, the invention will be described in detail by way of examples, but the scope of the present invention is not limited to the following examples.

[0161] The substances and organic solvents used for the reaction were purchased from Novabiochem, Alfa Aesar, SA dichloromethane (MC), HUN CHEMICALS, Junsei chemicals co., Ltd., DUKSAN reagents chemical, etc., and were used without additional purification. 1H-nuclear magnetic resonance (1H-NMR) analysis of the synthesized compound was carried out at room temperature using a 400MHz or 500MHz nuclear magnetic resonance spectrometer of Bruker Company. HPLC (waters 1525 Binary hplc pump) used a solvent of acetonitrile (MeCN) containing 0.1% trifluoroacetic acid (TFA): water containing 0.1% trifluoroacetic acid in a ratio of 5:95 as a developing solvent of the high performance liquid chromatograph (waters 1525 Binary hplc pump). The ratio of the developing solvent was gradually changed so that the ratio of acetonitrile containing 0.1% trifluoroacetic acid: water containing 0.1% trifluoroacetic acid was changed to 20:80 within 20 minutes. Then, the analysis was carried out using a solvent of acetonitrile containing 0.1% trifluoroacetic acid: water containing 0.1% trifluoroacetic acid in a ratio of 95:5 within 10 minutes with a column heater at 60°C.

[0162] The nucleic acid bases or nucleic acid bases having an amine protecting group of the following compounds 1 to 4 were prepared in the same manner as in Korean Patent No. 10-0464261.

[0163]

[0164] Preparation Example 1. Preparation of Compound 5 (Boc-aeg-OEt)

[0165]

[0166] Place 11.0 g of ethylenediamine (183 mmol) in a 500 mL conical flask and dissolve in dichloromethane (MC). Dissolve 5.0 g of Boc2O (22.9 mmol) in dichloromethane and add dropwise, stirring at room temperature for 12 hours. After confirming the termination of the reaction by thin layer chromatography (TLC), add water to extract only the dichloromethane layer and wash with sat.NaCl. After treating with Na2SO4, filter and concentrate. Add dichloromethane to the concentrated solution and add 6.4 mL of triethylamine (45.8 mmol). Dissolve 2.4 mL of ethyl bromoacetate (22.0 mmol) in dichloromethane and slowly add dropwise, stirring at room temperature for 12 hours. After confirming the termination of the reaction by thin layer chromatography, add water to extract only the dichloromethane layer. After removing water with Na2SO4, the mixture was concentrated and purified by silica-column chromatography (eluent: EA:Hex=1:1 (v / v)) to obtain a transparent oily product, ie, compound 5 (3.79 g, 70%).

[0167] Preparation Example 2. Preparation of Compound 8 (Boc-Lys(Z)-OMe)

[0168] Preparation of compound 6

[0169]

[0170] In a 250 mL two-neck round-bottom flask under nitrogen atmosphere, 5.40 g of Boc-Lys(Z)-COOH (14.2 mmol) was placed in 100 mL of dry tetrahydrofuran (Dry THF). 9.21 g of 1,1'-Carbonyldiimidazole (56.8 mmol) was placed at once and stirred at room temperature for 10 minutes. When bubbles were no longer generated, 2.68 g of NaBH4 (71.0 mmol) was dissolved in 30 mL of distilled water at 0°C and slowly added, followed by stirring for 30 minutes. After confirming the termination of the reaction by thin layer chromatography, the solvent was concentrated. After placing 200 mL of EA, the mixture was transferred to a sorting funnel, washed with 1 M HCl, washed with saturated brine, and concentrated with sodium sulfate to remove water. The product, ie, Compound 6 (5.0 g, 96.2%), was obtained as a transparent yellow oil by purification using silica gel column chromatography (eluent: EA:Hex=1:1 (v / v)).

[0171] Preparation of compound 7

[0172]

[0173] Under nitrogen atmosphere, 8.71 mL of oxalyl chloride (17.4 mmol) was placed in a 250 mL double-necked round-bottom flask using a syringe, followed by 20 mL of dry dichloromethane. The temperature was lowered to -20°C within 5 minutes using NaCl, ice block, and methanol, and a solution of 2.47 mL of dry dimethyl sulfoxide (DMSO) (34.8 mmol) and 5 mL of dry dichloromethane was added dropwise to a 100 mL round-bottom flask. After 5 minutes, 5.80 g of Boc-Lys(Cbz)-OH (15.8 mmol) was dissolved in 20 mL of dry dichloromethane and added to the reaction solution. After 15 minutes, 15.2 mL of N,N-diisopropylethylamine (87.1 mmol) was added and stirred for 5 minutes. After removing the ice bath, thin layer chromatography (EA only, p-Anisaldehyde) was confirmed. After the reaction was terminated, the compound 7 (5.5 g, 95.3%) was obtained as a transparent oil after washing with NaHCO3 and concentrating the solvent.

[0174] Preparation of compound 8

[0175]

[0176] Compound 8 was prepared by the same method with reference to FILBERT TOTSINGAN et. Al. CHIRALITY, 2009, 21, 245-253, and the synthesis of compound 8 was confirmed by the above reference.

[0177] Into a 250 mL round-bottom flask, 5.50 g of Boc-Lys(Cbz)-CHO (15.1 mmol) was added, followed by 50.0 mL of methanol and 2.84 g of Gly-OMe (22.6 mmol). At 0°C, 1.30 mL of acetic acid (22.6 mmol) and 3.94 mL of N,N-diisopropylethylamine (22.6 mmol) were added, followed by 9.60 g of sodium triacetoxyborohydride (45.3 mmol), and the mixture was stirred at 0°C for 2 hours and then overnight. After confirming whether the starting material has reacted by thin layer chromatography (developing solvent: only EA, p-anisaldehyde), the solution was concentrated when the reaction was terminated, EA was added and washed with NaHCO3, and then concentrated. The product was purified by silica gel column chromatography (eluent: EA: Hex = 1: 1 (v / v)) to obtain the product in the form of a transparent oil (4.5 g, 68.2%).

[0178] Preparation Example 3. Preparation of Compound 11 (Boc-Glu(OcHex)-OMe)

[0179] Preparation of compound 9

[0180]

[0181] In a 500 mL one-neck round-bottom flask, 20 g of Boc-Glu(OcHex)-COOH (60.7 mmol) was placed and dissolved in 150 mL of dry N,N-dimethylformamide. 22.3 g of HBTU (O-(1H-Benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate)) (58.3 mmol) was placed. 23.3 mL of N,N-diisopropylethylamine (133.6 mmol) was added at 0°C, and then 7.11 g of N-methoxy-N-methylamine hydrochloride (72.9 mmol) was added, and the mixture was stirred overnight at room temperature. After confirming whether the starting material has reacted by thin layer chromatography (developing solution: 3% acetic acid (AcOH), 5% methanol (MeOH) / dichloromethane, ninhydrin), the solution was concentrated at the end of the reaction and 150 mL of ethyl acetate (EA) was added, followed by washing with 0.5 N HCl, saturated NaHCO3 and 1 N KHSO4. Sodium sulfate was added to the organic layer to remove water from the organic layer, and then concentrated to obtain the product in the form of a transparent colorless oil, i.e., compound 9 (21 g, 92.9%).

[0182] Preparation of compound 10

[0183]

[0184] In a 1L three-necked round-bottom flask under nitrogen, 21.0 g of compound 9 (56.4 mmol) was placed in 210 mL of dry tetrahydrofuran. At a temperature of -78°C, 26.2 g of 2.37 M lithium aluminum hydride (Litium Aluminium Hydride) (62.0 mmol) was added within 20 minutes and stirred for 1 hour. After confirming whether the starting material reacted by thin layer chromatography (developing solution: EA: Hex = 1: 1, ninhydrin), 600 mL of ethyl acetate was added at the end of the reaction, and 210 mL of 1 M KHSO4 was added within 10 minutes and stirred for 10 minutes. The organic layer was washed with 1 M KHSO4 and saturated NaHCO3, and the water was removed with sodium sulfate and concentrated to obtain the product in the form of a transparent yellow oil, i.e., compound 10 (18.5 g, 88.1%).

[0185] Preparation of compound 11

[0186]

[0187] After placing 18.5 g of compound 10 (59.0 mmol) in a 500 mL round-bottom flask, add 130 mL of dichloroethane and glycine methyl ester hydrochloride (HCl.Gly-OMe). After adding 3.38 mL of acetic acid (59.0 mmol) and 15.4 mL of N,N-diisopropylethylamine (88.5 mmol) at 0°C, add 18.9 g of sodium triacetoxyborohydride (88.5 mmol) and stir for 30 minutes. After confirming whether the starting material has reacted by thin layer chromatography (eluent: EA: Hex = 1:4, eluted twice, ninhydrin), when the reaction is terminated, add 130 mL of saturated NaHCO3 at 0°C and stir for 10 minutes. The organic layer was washed with saturated NaHCO3 and saturated brine, and the water was removed with sodium sulfate and concentrated. The product was purified by silica gel column chromatography (eluent: EA:Hex=1:1 (v / v)) to obtain a transparent oily product, i.e., compound 11 (Boc-Glu(OcHex)-OMe) (14 g, 61.4%).

[0188] Preparation Example 4. Preparation of Compound 12 (Boc-Lys(Z)-NH2)

[0189]

[0190] Preparation of compound 12

[0191] 20.0 g of Boc-Lys(Cbz)-COOH (52.6 mmol) was placed in a 1 L round bottom flask and dissolved in 200 mL of N,N-dimethylformamide. 29.9 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (78.9 mmol) and 5.62 g of NH4Cl (105.1 mmol) were added, and 36.6 mL of N,N-diisopropylethylamine (210.3 mmol) was added dropwise at 0°C. After stirring for about 30 minutes, dichloromethane (200 mL) was added after confirming the termination of the reaction by thin layer chromatography, and the mixture was washed once with 5% NaHCO3 and 0.5 M HCl using a separatory funnel. As the organic layer gradually became turbid and a solid was generated, it was filtered to obtain compound 12 (Boc-Lys(Z)-NH2) (19.9 g, 99.9%).

[0192] Example 1. Preparation of Peptide Nucleic Acid Monomers

[0193] Preparation of compound 13-3 (Boc-aeg-A(Z)-OEt)

[0194]

[0195] 20.0 g of compound 3 (61.1 mmol) was placed in a 3 L round bottom flask and dissolved in 1 L of N, N-dimethylformamide. 34.8 g of benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (122 mmol) was placed, and 15.1 g of compound 5 (61.1 mmol) was placed, and 31.9 mL of N, N-diisopropylethylamine (183 mmol) was added dropwise at 0°C and stirred for 30 minutes. After confirming the termination of the reaction by thin layer chromatography (developing solution: 5% methanol / dichloromethane), 1 L of dichloromethane (MC) was placed. The organic layer was washed with saturated NaHCO3 and 0.5 M HCl, and then concentrated by adding sodium sulfate to remove water. The residue was purified by silica gel column chromatography (eluent: 5% methanol / dichloromethane) to obtain a white solid product, ie, Compound 13 (Boc-A(Z)-OEt) (30.0 g, 88.4%).

[0196] Preparation of compound 14-3 (Boc-aeg-A(Z)-OH)

[0197]

[0198] 15.0 g of compound 13-3 (27.0 mmol) was placed in a 500 mL round-bottom flask, dissolved in 270 mL of 0.5 N LiOH (135 mmol), and stirred for about 39 minutes. The reaction was confirmed to be terminated by thin layer chromatography (eluent: 5% methanol / dichloromethane). After the pH was lowered to 2-3 using 0.5 M HCl at 0°C, the generated solid was filtered using a glass filter, washed several times with water, and dried using P2O5 to obtain the product in the form of a white solid, i.e., compound 14-3 (Boc-A(Z)-OH) (14.1 g, 99.0%).

[0199] Preparation of compound 15-1 (NH2-aeg-A(Z)-OEt)

[0200]

[0201] 15.0 g of compound 13-3 (27.0 mmol) was placed in a 250 mL round-bottom flask, dissolved in 135 mL of 50% trifluoroacetic acid / dichloromethane at 0°C and allowed to stand for 30 minutes. The reaction was confirmed to have terminated by thin layer chromatography (developing solution: 5% methanol / dichloromethane). The solution was added dropwise to an excess of diethyl ether to produce a precipitate, which was then filtered using a glass filter and washed several times with diethyl ether to obtain a product in the form of a white solid, i.e., compound 15-1 (NH2-A(Z)-OEt) (14.5 g, 94.3%).

[0202] Example 2. Preparation of Peptide Nucleic Acid Monomers

[0203] Peptide nucleic acid monomer 14-1 (Boc-aeg-T-OH) and peptide nucleic acid monomer 15-1 (NH2-aeg-T-OEt) were prepared in the same manner as in Example 1, except that compound 1 was used instead of compound 3 in the process of preparing compound 13-3 in Example 1.

[0204] Example 3. Preparation of Peptide Nucleic Acid Monomers

[0205] Peptide nucleic acid monomer 14-2 (Boc-aeg-C(Z)-OH), peptide nucleic acid monomer 14-4 (Boc-aeg-G(Z)-OH) and peptide nucleic acid monomer 15-4 (NH2-aeg-G(Z)-OEt) were prepared in the same manner as in Example 1, except that compound 2 or compound 4 was used instead of compound 3 in the process of preparing compound 13-3 in Example 1.

[0206] Example 4. Preparation of Peptide Nucleic Acid Monomers

[0207] Peptide nucleic acid monomer 14-5 (Boc-Lys(Z)-C(Z)-OH) and peptide nucleic acid monomer 15-6 (NH2-Lys(Z)-NH2) were prepared in the same manner as in Example 1, except that compound 8 was used instead of compound 5 and compound 2 was used instead of compound 3 in the process of preparing compound 13-3 in Example 1.

[0208] Example 5. Preparation of Peptide Nucleic Acid Monomers

[0209] Peptide nucleic acid monomer 15-3 (NH2-Glu(OcHex)-T-OMe) was prepared in the same manner as in Example 1, except that compound 11 was used instead of compound 5, and compound 1 was used instead of compound 3.

[0210] Example 6. Preparation of Peptide Nucleic Acid Monomers

[0211] Except that compound 2 or compound 4 was used instead of compound 1, peptide nucleic acid monomer 15-4 (NH2-Glu(OcHex)-C(Z)-OMe) and peptide nucleic acid monomer 15-5 (NH2-Glu(OcHex)-G(Z)-OMe) were prepared respectively by the same method as Example 5.

[0212] Example 7. Preparation of Peptide Nucleic Acid Dimer 17-1 (Boc-AA-OH)

[0213] Preparation of compound 16-1 (Boc-AA-OEt)

[0214]

[0215] In a 2L round-bottom flask, 13.0g of compound 14-3 (24.6mmol) was dissolved in 650mL of dry N,N-dimethylformamide, and then 14.0g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (37.0mmol) was added. After 14.0g of compound 15-1 (24.6mmol) was added, 21.5mL of N,N-diisopropylethylamine (123mmol) was added dropwise at 0°C and stirred for 30 minutes. After confirming the termination of the reaction by thin layer chromatography (eluent: 15% methanol / dichloromethane), 650mL of dichloromethane was added. After washing the organic layer with saturated NaHCO3 and 0.5M HCl, sodium sulfate was added to remove water and concentrate. The product Boc-AA-OEt (21.0 g, 87.9%) was obtained by purification by silica gel column chromatography (eluent: 10% methanol / dichloromethane).

[0216] Preparation of compound 17-1 (Boc-AA-OH)

[0217]

[0218] 21.0 g of compound 16-1 (21.7 mmol) was added to a 500 mL round-bottom flask, dissolved in 217 mL of 0.5 N LiOH (108 mmol) and stirred for 30 minutes at 0°C. The reaction was terminated by monitoring by thin layer chromatography (developing solution: 10% methanol / dichloromethane). After the pH was reduced to 2-3 using 1 M HCl at 0°C, the generated solid was filtered using a glass filter, washed several times with water, and then recrystallized using methanol / ether to obtain a white product, i.e., compound 17-1 (Boc-AA-OH) (19.7 g, 96.6%).

[0219] Example 8. Preparation of Peptide Nucleic Acid Dimers

[0220] Except that different peptide nucleic acid monomers were used to replace compound 14-3 and compound 15-3, peptide nucleic acid dimer 16-2 (Boc-C"A-OEt), peptide nucleic acid dimer 17-2 (Boc-CC^-OH), peptide nucleic acid dimer 17-3 (Boc-TT^-OH) and peptide nucleic acid dimer 17-4 (Boc-GG-OH) were prepared respectively by the same method as Example 7. In this case, base" is NH2-γLys(Z)-base-OMe, and base^ is NH2-γGlu(OcHex)-base-Ome.

[0221] Example 9. Preparation of Peptide Nucleic Acid Dimer 18-1 (NH2-C"A-OEt)

[0222]

[0223] 20.0 g of compound 16-2 (Boc-C"A-OEt) (17.4 mmol) was placed in a 250 mL round-bottom flask, dissolved in 180 mL of 50% trifluoroacetic acid / dichloromethane at 0°C and stirred for 30 minutes. The reaction was terminated by thin layer chromatography (eluent: 7% methanol / dichloromethane). After the solution was added dropwise to an excess of diethyl ether to produce a precipitate, it was filtered using a glass filter and washed with diethyl ether to obtain a white solid product NH2-C"A-OEt (19.2 g, 94.9%).

[0224] Example 10. Preparation of OK linker (Compound 18-2, NH2-OK-NH2)

[0225] Preparation of compound 16-6 (Boc-OK-NH2)

[0226]

[0227] In a 2L round-bottom flask, 13.0 g of 3,6,11-Trioxa-9-azatridecanoic acid, 12,12-dimethyl-10-oxo-97% (Boc-O-OH) (49.4 mmol) was dissolved in 650 mL of dry N,N-dimethylformamide, and then 22.5 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (59.3 mmol) was added. After 19.4 g of compound 15-6 (49.4 mmol) was added, 21.5 mL of N,N-diisopropylethylamine (123 mmol) was added dropwise at 0°C and stirred for 30 minutes. After confirming the termination of the reaction by thin layer chromatography (developing solution: 7% methanol / ethyl acetate), 650 mL of dichloromethane was added. After washing the organic layer with saturated NaHCO3 and 0.5 M HCl, sodium sulfate was added to remove water and concentrate. Boc-OK-NH2 (21.0 g, 81.1%) was obtained as a transparent colorless oil by purification by silica gel column chromatography (eluent: 7% methanol / ethyl acetate).

[0228] Preparation of compound 18-2 (NH2-OK-NH2)

[0229]

[0230] 21.0 g of compound 16-6 (Boc-OK-NH2) (40.0 mmol) was added to a 250 mL round-bottom flask and dissolved in 180 mL of 50% trifluoroacetic acid / dichloromethane at 0°C and stirred for 30 minutes. The reaction was terminated by thin layer chromatography (developing solution: 7% methanol / ethyl acetate). After the solution was added dropwise to an excess of diethyl ether to produce a precipitate, the oily product obtained by centrifugation was dissolved in dichloromethane and the solvent was concentrated to obtain a transparent oily product NH2-OK-NH2 (19.2 g, 89.1%).

[0231] Example 11. Preparation of Peptide Nucleic Acid Trimer 19-1 (Boc-AAG^-OMe)

[0232]

[0233] In a 2L round-bottom flask, 15.2g of compound 17-1 (16.2mmol) was dissolved in 750mL of dry N,N-dimethylformamide. After adding 12.6g of benzotriazole-1-yl-oxytripyrrolidinophosphate (PyBop), 11.7g of compound 15-5 was added. After adding 56.3mL of N,N-diisopropylethylamine at 0°C and stirring for about 30 minutes, the reaction was confirmed to be terminated by thin layer chromatography (developing solution: 20% methanol / dichloromethane). After adding 300mL of dichloromethane, the organic layer was washed with saturated NaHCO3 and 0.5M HCl solution, and the product was concentrated by removing water with sodium sulfate to obtain a transparent yellow oil. The product was recrystallized using methanol / diethyl ether to obtain a white solid compound, ie, compound 19-1 (Boc-AAG^-OMe) (21.3 g, 85.9%).

[0234] Example 12. Preparation of Peptide Nucleic Acid Trimer 19-2 (Boc-CC"A-OEt)

[0235] Peptide nucleic acid trimer 19-2 (Boc-CC"A-OEt) was prepared in the same manner as in Example 11, except that peptide nucleic acid monomer 14-2 and peptide nucleic acid dimer 18-1 were used instead of peptide nucleic acid dimer 17-1 and peptide nucleic acid monomer 15-5. In this case, Base" is NH2-γLys(Z)-base-OMe.

[0236] Example 13. Preparation of Peptide Nucleic Acid Trimer 20-1 (Boc-AAG^-OH)

[0237]

[0238] Into a 1L round-bottom flask, 20.3g of compound 19-1 (13.2mmol) was dissolved in 100mL of 1,4-dioxane (1,4-Dioxane). 132mL of 0.5M LiOH (66.1mmol) was added dropwise at 0°C and stirred for about 30 minutes. The reaction was confirmed to be terminated by thin layer chromatography (eluent: 20% methanol / dichloromethane). 0.5M HCl was used at 0°C to reduce the pH to 2-3, the generated solid was filtered using a glass filter, washed with water and then recrystallized using methanol / ether to obtain a white solid product, i.e., compound 20-1 (Boc-AAG^-OH) (18.3g, 90.8%).

[0239] Example 14. Preparation of Peptide Nucleic Acid Trimer 21-1 (NH2-CC"A-OEt)

[0240]

[0241] 25.3 g of compound 19-2 (16.5 mmol) was added to a 250 mL round-bottom flask and dissolved in 230 mL of 50% trifluoroacetic acid / dichloromethane at 0°C and stirred for 30 minutes. The reaction was confirmed to be terminated by thin layer chromatography (developing solution: 20% methanol / dichloromethane). After the solution was added dropwise to an excess of diethyl ether to generate a precipitate, it was filtered using a glass filter and washed with diethyl ether to generate a white solid product, i.e., compound 21-1 (NH2-CC"A-OEt) (23.8 g, 93.2%).

[0242] Example 15. Preparation of Peptide Nucleic Acid 23-1 (NH-2-GGOK-NH2)

[0243] Preparation of compound 22-1 (Boc-GGOK-NH2)

[0244]

[0245] In a 1L round-bottom flask, 13.8g of compound 17-4 (14.2mmol) was dissolved in 650mL of N,N-dimethylformamide. After adding 11.1g of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (21.4mmol), 7.67g of compound 18-2 (14.2mmol) was added. After adding 49.6mL of N,N-diisopropylethylamine (285mmol) at 0°C and stirring for about 30 minutes, the reaction was confirmed to be terminated by thin layer chromatography (eluent: 20% methanol / dichloromethane). After adding 650 mL of dichloromethane, the organic layer was washed with saturated NaHCO3 and 0.5 M HCl solution, and the water was removed by sodium sulfate to concentrate the mixture to obtain the product in the form of a transparent colorless oil. The product was recrystallized with methanol / ethyl ether to obtain a white solid compound, i.e., compound 22-1 (Boc-GGOK-NH2) (17.7 g, 90.4%).

[0246] Preparation of compound 23-1 (NH2-GGOK-NH2)

[0247]

[0248] 16.7 g of compound 22-1 (12.1 mmol) was placed in a 250 mL round-bottom flask and dissolved in 140 mL of 50% trifluoroacetic acid / dichloromethane at 0°C and stirred for 30 minutes, and then the reaction was terminated by thin layer chromatography (developing solution: 20% methanol / dichloromethane). After the solution was added dropwise to an excess of diethyl ether to produce a precipitate, it was filtered using a glass filter and washed with diethyl ether to obtain a white solid product, i.e., compound 23-1 (NH2-GGOK-NH2) (16.0 g, 94.8%).

[0249] Example 16. Preparation of Peptide Nucleic Acid Pentamer 25-1 (Boc-CC^CC"A-OH)

[0250] Preparation of compound 24-1 (Boc-CC^CC"A-OEt)

[0251]

[0252] 6.13 g of compound 17-2 (5.88 mmol) was added to a 1 L round bottom flask and dissolved in 300 mL of dry N,N-dimethylformamide. 4.59 g of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (8.82 mmol) was added, and then 9.08 g of compound 21-1 (5.88 mmol) was added, and then 20.5 mL of N,N-diisopropylethylamine was added dropwise at 0°C. After stirring for about 30 minutes, the reaction was confirmed to be terminated by thin layer chromatography (developing solution: 5% acetic acid, 10% methanol / dichloromethane), 300 mL of dichloromethane was added, and the organic layer was washed with saturated NaHCO3 and 0.5 M HCl. The organic layer was concentrated after removing water with sodium sulfate to obtain a product, which was recrystallized with methanol / diethyl ether to obtain a white solid product, namely, compound 24-1 (Boc-CC^CC"A-OEt) (13.2 g, 91.4%).

[0253] Preparation of compound 25-1 (Boc-CC^CC"A-OH)

[0254]

[0255] 13.0 g of compound 24-1 (5.29 mmol) was placed in a 500 mL round-bottom flask and dissolved in 71.5 mL of 1,4-dioxane. 52.9 mL of 0.5 N LiOH (26.5 mmol) was placed at 0°C and stirred for about 30 minutes. After confirming the termination of the reaction by thin layer chromatography (eluent: 5% acetic acid, 20% methanol / dichloromethane), 0.5 M HCl was used to reduce the pH to 2-3 to produce a solid, which was filtered through a glass filter and washed with water, and then recrystallized using methanol / ether. The generated solid was filtered through a glass filter, washed with diethyl ether, and dried to obtain a product in the form of a white solid, i.e., compound 25-1 (Boc-CC^CC"A-OH) (12.1 g, 94.2%).

[0256] Example 17. Preparation of Peptide Nucleic Acid Hexamer 27-1 (Boc-AAG^CC"A-OH)

[0257] Preparation of compound 26-1 (Boc-AAG^CC"A-OEt)

[0258]

[0259] In a 2L round-bottom flask, 11.7g of compound 20-1 (7.66mmol) was dissolved in 550mL of dry N,N-dimethylformamide. After adding 4.36g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.5mmol), 11.8g of compound 21-1 (7.66mmol) was added. 26.7mL of N,N-diisopropylethylamine (153mmol) was added at 0°C and stirred for 30 minutes. After confirming the termination of the reaction by thin layer chromatography (developing solution: 5% acetic acid, 15% methanol / dichloromethane), 550mL of dichloromethane was added and the organic layer was washed with saturated NaHCO3 and 0.5M HCl, and the water was removed with sodium sulfate and concentrated to obtain the product in the form of a transparent yellow oil. The compound was recrystallized using methanol / diethyl ether to obtain a solid compound, ie, compound 26-1 (Boc-AAG^CC"A-OEt) (yield: 18.2 g, 81.0%).

[0260] Preparation of compound 27-1 (Boc-AAG^CC"A-OH)

[0261]

[0262] 18.2 g (6.20 mmol) was placed in a 500 mL round-bottom flask and dissolved in 182 mL of 1,4-dioxane. 62.0 mL of 0.5 N LiOH was added dropwise at 0°C and stirred for 30 minutes. The reaction was terminated by monitoring by thin layer chromatography (developing solution: 5% acetic acid, 15% methanol / dichloromethane). 1 M HCl was used to reduce the pH to 2-3 at 0°C to generate a solid, which was filtered and washed with water, and then the dried solid was recrystallized with methanol / ether to obtain a white compound, i.e., compound 27-1 (15.1 g, 83.8%) (Prep purification was performed).

[0263] Example 18. Preparation of Peptide Nucleic Acid Tetramer Linker Compound 28-1 (TFA.NH2-TT^GGOK-NH2)

[0264] Preparation of compound 26-2 (Boc-TT^GGOK-NH2)

[0265] Compound 26-2 (Boc-TT^GGOK-NH2) was prepared in the same manner as Example 17 except that compound 17-3 and compound 23-1 were used instead of peptide nucleic acid trimer 20-1 and peptide nucleic acid trimer 21-1. In this case, Base^ was NH2-γGlu(OcHex)-base-OMe.

[0266] Preparation of compound 28-1 (TFA.NH2-TT^GGOK-NH2)

[0267]

[0268] 15.5 g of compound 26-2 (7.52 mmol) was placed in a 250 mL round-bottom flask and dissolved in 140 mL of 50% trifluoroacetic acid / dichloromethane at 0°C and stirred for 30 minutes. The reaction was terminated by monitoring by thin layer chromatography (developing solution: 20% methanol / dichloromethane). After the reaction was terminated, an excess of diethyl ether was added dropwise to the solution at 0°C to produce a precipitate. The product was filtered to obtain a white solid, i.e., compound 28-1 (TFA.NH2-TT^GGOK-NH2) (14.6 g, 94.3%).

[0269] Example 19. Preparation of Peptide Nucleic Acid Decamer Linker Compound 30-1 (NH2-CC^CC"AATT^GGOK-NH2)

[0270] Preparation of compound 29-1 (Boc-CC^CC"ATT^GGOK-NH2)

[0271] 5.76 g of compound 25-1 (2.37 mmol) was added to a 1 L round bottom flask and dissolved in 250 mL of dry N, N-dimethylformamide, and then 1.85 g of benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (3.56 mmol) was added. 4.92 g of compound 28-1 (2.37 mmol) was added, and 12.0 mL of N, N-diisopropylethylamine (68.8 mmol) was added dropwise at 0°C and stirred for 30 minutes. The reaction termination and crude purity of the prepared crude peptide nucleic acid oligomer were detected using a high performance liquid chromatograph (HPLC) (Agilent 1100 model, acetonitrile was increased from 10% to 70% within 20 minutes, and then reached 100% within 25 minutes (10% MeCN 70% for 20mins 100% for 25mins)). After adding 250 mL of dichloromethane, the organic layer was washed with saturated NaHCO3 and 0.5 M HCl, and then water was removed with sodium sulfate and concentrated to obtain the product in the form of a transparent yellow oil. The product was recrystallized with methanol / diethyl ether to obtain compound 29-1 (Boc-CC^CC"ATT^GGOK-NH2) (8.32 g, 80.3%) in the form of a white solid.

[0272] Preparation of compound 30-1 (TFA.NH2-CC^CC"ATT^GGOK-NH2)

[0273] 8.32 g of compound 30-1 (1.90 mmol) was placed in a 250 mL round-bottom flask and dissolved in 140 mL of 50% trifluoroacetic acid / dichloromethane at 0°C and stirred for 30 minutes. High performance liquid chromatograph (Agilent 1100, acetonitrile from 10% to 70% in 20 minutes, then 100% in 25 minutes) was used to detect the reaction termination and crude purity of the prepared crude peptide nucleic acid oligomer. After the reaction was terminated, the solution was added dropwise to an excess of diethyl ether at 0°C to produce a precipitate. The product was filtered to obtain a white solid, i.e., compound 30-1 (TFA.NH2-CC^CC"ATT^GGOK-NH2) (7.31 g, 87.6%) (Prep purification was performed).

[0274] Example 20. Preparation of Peptide Nucleic Acid Pentamer 31-1 (Boc-AAG^CC"ACC^CC"ATT^GGOK-NH2)

[0275] Into a 500 mL round-bottom flask, 3.54 g of compound 20-1 (1.22 mmol) was dissolved in 350 mL of dry N,N-dimethylformamide, and then 0.693 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.83 mmol) was added. After adding 5.34 g of compound 30-1 (1.22 mmol), 4.24 mL of N,N-diisopropylethylamine (24.4 mmol) was added at 0°C and stirred for 30 minutes. The crude purity of the prepared crude peptide nucleic acid oligomer was measured using a high performance liquid chromatograph (Agilent 1100, acetonitrile increased from 10% to 70% within 20 minutes, and then reached 100% within 25 minutes). The results are as follows: Figure 1 shown.

[0276] like Figure 1 As shown, the measured crude purity was 87%.

[0277] Comparative Example 1. Preparation of Compound 31-1 (Boc-AAG^CC"ACC^CC"ATT^GGOK-NH2)

[0278] In a 500 mL round-bottom flask, 1.00 g of compound 20-1 (0.344 mmol) was dissolved in 50 mL of dry N,N-dimethylformamide, and then 0.196 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.516 mmol) was added, and 0.6 mL of N,N-diisopropylethylamine (3.44 mmol) was stirred at room temperature for 10 minutes. 1.51 g of compound 30-1 (0.344 mmol) was dissolved in 50 mL of dry N,N-dimethylformamide, and then 0.6 mL of N,N-diisopropylethylamine (3.44 mmol) was added and stirred at 0°C for 10 minutes. After the prepared solutions were mixed and stirred at 0°C for 30 minutes, the crude purity of the prepared crude peptide nucleic acid oligomer was measured using a high performance liquid chromatograph (Agilent 1100, acetonitrile from 10% to 70% in 20 minutes, and then to 100% in 25 minutes). The results were as follows: Figure 2 shown.

[0279] like Figure 2 As shown, it can be seen that the crude purity varies significantly depending on the order of adding the amine compounds.

[0280] Example 21. Preparation of Peptide Nucleic Acid Oligomers

[0281] Peptide nucleic acid oligomer 31-1 (Boc-AAG^CC"ACC^CC"ATT^GGOK-NH2) was prepared in the same manner as in Example 20 except that 30 mL of dry N,N-dimethylformamide was used.

Claims

1. A method for preparing a peptide nucleic acid oligomer, characterized in that: The method comprises: a) obtaining a mixed solution by reacting reactants containing a first peptide nucleic acid block and a second peptide nucleic acid block in a solution phase in the presence of a coupling reagent and a solvent; and b) adding an amine compound to the mixed solution of step a); Wherein, the first peptide nucleic acid block and the second peptide nucleic acid block are formed by combining two or more different or identical peptide nucleic acid monomers; Wherein, the coupling reagent is benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate or a mixture thereof; The above amine compound is N,N-diisopropylethylamine.

2. The method for preparing a peptide nucleic acid oligomer according to claim 1, characterized in that: The above step a) comprises mixing a first peptide nucleic acid dimer represented by the following chemical formula 1, a first peptide nucleic acid trimer represented by the following chemical formula 2, or a first peptide nucleic acid tetramer represented by the following chemical formula 3 with a second peptide nucleic acid dimer represented by the following chemical formula 4, a second peptide nucleic acid trimer represented by the following chemical formula 5, or a second peptide nucleic acid tetramer represented by the following chemical formula 6, and then reacting Chemical formula 1: Chemical formula 2: Chemical formula 3: Chemical formula 4: Chemical formula 5: Chemical formula 6: In the above Chemical Formulas 1 to 6, A1 to A9 and A 11 To A 14 Each of P1, P3, P5, P7, P9 and P1 is independently a peptide nucleic acid monomer containing different or identical nucleic acid bases. 11 Each is independently hydrogen, or an amine protecting group that is different from or the same as each other; P2, P4, P6, P8, P 10 and P 12 Each is independently hydrogen, or a hydroxyl protecting group that is different from or the same as each other; But P1 and P2, P3 and P4, P5 and P6, P7 and P8, P9 and P 10 and P 11 With P 12 The exception is the case where they are both hydrogen.

3. The method for preparing a peptide nucleic acid oligomer according to claim 2, characterized in that: The above step a) comprises: The first peptide nucleic acid dimer represented by the following chemical formula 1-1, the first peptide nucleic acid trimer represented by the following chemical formula 2-1, or the first peptide nucleic acid tetramer represented by the following chemical formula 3-1 is mixed with the second peptide nucleic acid dimer represented by the following chemical formula 4-1, the second peptide nucleic acid trimer represented by the following chemical formula 5-1, or the second peptide nucleic acid tetramer represented by the following chemical formula 6-1, and then reacted Chemical formula 1-1: Chemical formula 2-1: Chemical formula 3-1: Chemical formula 4-1: Chemical formula 5-1: Chemical formula 6-1: In the above Chemical Formula 1-1, Chemical Formula 2-1, Chemical Formula 3-1, Chemical Formula 4-1, Chemical Formula 5-1 and Chemical Formula 6-1, A1 to A9 and A 11 To A 14 Each independently is a peptide nucleic acid monomer containing mutually different or identical nucleic acid bases, P2, P4 and P6 are each independently different or the same hydroxyl protecting group, P7, P9 and P 11 are each independently different or the same amine protecting group, X1 to X3 are each independently an acid salt.

4. The method for preparing a peptide nucleic acid oligomer according to claim 1, characterized in that: The method further comprises the step of repeatedly performing step a) and step b) by using the product obtained in the above step b) as a starting material.

5. The method for preparing a peptide nucleic acid oligomer according to claim 1, characterized in that: The volume ratio of the reaction material to the solvent in the mixed solution is 1:10 or more.

6. The method for preparing a peptide nucleic acid oligomer according to claim 2 or 3, characterized in that: The above-mentioned nucleic acid base is adenine, cytosine, guanine, thymine, uracil, or an artificial nucleic acid base.

7. The method for preparing a peptide nucleic acid oligomer according to claim 2 or 3, characterized in that: The above-mentioned amine protecting group and hydroxy protecting group are independently fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, diphenylmethoxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, toluenesulfonyl, trichloroethyl chloroformate, sulfonamide or isobutyryl.

8. The method for preparing a peptide nucleic acid oligomer according to claim 2, characterized in that: The first peptide nucleic acid dimer or the second peptide nucleic acid dimer is represented by the following chemical formula 11: The first peptide nucleic acid trimer or the second peptide nucleic acid trimer is represented by the following chemical formula 12: The first peptide nucleic acid tetramer or the second peptide nucleic acid tetramer is represented by the following chemical formula 13: Chemical formula 11: Chemical formula 12: Chemical formula 13: In the above Chemical Formulas 11 to 13, R1 to R 18 are each independently hydrogen, an amino acid residue or an amino acid residue having a substituent, T1 to T3 are each independently an amine protecting group, Z1 to Z3 are each independently a hydroxyl protecting group, B1 to B9 are each independently a nucleic acid base which may or may not contain an amine protecting group.

9. The method for preparing a peptide nucleic acid oligomer according to claim 3, characterized in that: Relative to 1 equivalent of the first peptide nucleic acid dimer represented by the above Chemical Formula 1-1, the first peptide nucleic acid trimer represented by the above Chemical Formula 2-1, or the first peptide nucleic acid tetramer represented by the above Chemical Formula 3-1, 1 mol:0.8 mol to 1 mol:1.2 mol of the second peptide nucleic acid dimer represented by the above Chemical Formula 4-1, the second peptide nucleic acid trimer represented by the above Chemical Formula 5-1, or the second peptide nucleic acid tetramer represented by the above Chemical Formula 6-1 is used.

10. The method for preparing a peptide nucleic acid oligomer according to claim 2 or 3, characterized in that: The above step b) is performed at a temperature of -10°C to 5°C for 10 to 60 minutes.

11. The method for preparing a peptide nucleic acid oligomer according to claim 2 or 3, characterized in that: The above solvent is chlorinated C1-C4 alkane or N,N-dimethylformamide.

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