KR20030009903A - A preparation method of p-Nitrophenylsulfonylethoxycarbonyl protected PNA monomer as the amino protected group of 2-aminoethylglycine backbone - Google Patents

Abstract

PURPOSE: Provided is a preparation method of a p-nitrophenylsulfonylethoxycarbonyl protected PNA monomer with the amino protected group of 2-aminoethylglycine backbone, thereby easily producing a PNA oligomer by effectively deprotecting the PNA monomer under mild condition. CONSTITUTION: The peptide nucleic acid(PNA) monomer is represented by the formula(1) or (2). In the formula(1), B is adenine, guanine and cytosine and R1 is a 4-methoxyphenyldiphenylmethyl group, and in the formula(2) B is adenine, guanine, cytosine, R1 is a 4-methoxyphenyldiphenylmethyl group and R' is a C1-C6 alkyl group. The preparation method of the PNA monomer employs an intermediate represented by the formula(3), wherein R' is a methyl group or an ethyl group.

Description

A preparation method of p-nitrophenylsulfonylethoxycarbonyl protected PNA monomer as the amino protected group of 2-aminoethylglycine backbone }

The present invention relates to a method for preparing a peptide nucleic acid monomer (Peptide Nucleic Acid; hereinafter referred to as 'PNA') in which the primary amino group of the main chain 2-aminoethylglycine is protected with p-nitrophenylsulfonylethoxycarbonyl group. Preferably 2-aminoethylglycine and p-nitrophenylthioethoxycarbonyl group protected by p-nitrophenylthioethoxycarbonyl group prepared by protecting the primary amino group in the backbone of the backbone 2-aminoethylglycine group P-nitrophenylthioethoxycarbonyl group / 4 by reacting a nucleobase group protected by a 4-methoxyphenyldiphenylmethyl group prepared by protecting a primary amino group of a cleobase group with a 4-methoxyphenyldiphenylmethyl group After preparing a PNA monomer protected with -methoxyphenyldiphenylmethyl group, the p-nitrophenylthioethoxycarbonyl group was finally p-nitrophenylsulfonylethoxycar by oxidation reaction. P-nitrophenyl, which is a base-sensitive main chain protecting group in the preparation of a conventional PNA monomer, by preparing a PNA monomer protected by p-nitrophenylsulfonylethoxycarbonyl group / 4-methoxyphenyldiphenylmethyl group by conversion to a carbonyl group By solving the problem of sulfonylethoxycarbonyl group being removed in the hydrolysis step and reintroducing the protecting group reagent, it is economical, easy to introduce the PNA monomer protecting group, and it is possible to prepare PNA monomer with high yield. The present invention relates to a method for preparing a PNA monomer, which effectively deprotects the protecting group of the PNA monomer under mild conditions, thereby facilitating preparation of a PNA oligomer useful for a diagnostic probe.

Solid phase peptide synthesis is widely used as an active substance in the manufacture of biologically active peptides used in medical and biological research and in pharmacy, veterinary medicine and diagnostics. The nature of solid phase peptide synthesis is summarized by the stepwise elongation of the chain by repetition of the chemical reaction, starting from the C-terminal monomer attached to the solid support. During the synthesis, all targets of the reaction remain bound to the support, while the reactants and by-products are removed by filtration and washing of the support.

The amino groups of the backbone and nucleobase moieties of the PNA monomers used to perform solid phase synthesis of such peptides should be protected with appropriate protecting groups. The amino protecting group of the monomer backbone attached to the solid support is deprotected step by step to form a free amine to bond with the carboxylic acid terminal of the monomer backbone in the next step, and the amino group of the nucleobase part is permanently protected. Finally, the desired PNA oligomer must be synthesized and then deprotected upon cleavage from the solid support.

PNA oligomer is a synthetic polyamide in which a base such as adenine, guanine, cytosine, or thymine, which is a DNA base, is connected to a repeating unit of 2-aminoethylglycine as a main chain by a methylenecarbonyl group. Patent Publication No. WO92 / 20702. PNA oligomers recognize DNA or RNA fragments to be compensated and are stable DNA / PNA duplexes and PNA / DNA / PNA triplex [J. Org. Chem. 1994, 59, 5767]. Such conjugates are more stable than oligonucleotide-only conjugates, and studies are actively underway to replace oligonucleotides as antisense therapeutics.

The most common method of preparing a PNA oligomer is a benzyloxycarbonyl group having a backbone protected with a tertiary butyloxycarbonyl group (hereinafter referred to as 't-Boc group') and the primary amino group present in the heterocycle of the base (carbamate type). It is prepared from PNA monomer protected with 'Cbz group') (Science 1991, 254, 1497). PNA monomers protected with t-Boc / Cbz groups are commercially available and sold, but very strong acidic conditions are required to remove these protecting groups (Perseptive Biosystems). Generally, to completely remove the Cbz group, the PNA oligomer should be treated with liquid hydrogen fluoride or trifluoromethanesulfonic acid for at least 1 hour. However, these conditions can often lead to the breakdown of nucleic acids and sugars, which are acid-sensitive moieties that can be attached to PNA oligomers, resulting in highly reactive cationic intermediates that can cause alkylation and acylation of specific portions of the PNA chain. This side reaction can also be partially reduced by using scavengers such as anisole, phenol, dimethylsulfide and mercaptoethanol. In addition, the extremely dangerous nature of liquid hydrogen fluoride or trifluoromethanesulfonic acid can corrode PNA oligomer synthesis equipment and therefore requires appropriate safety measures in handling.

The t-Boc / Cbz protecting group strategy of PNA monomers is the differential principle. This strategy protects groups essentially in the same form of reagents and conditions, but relies on the difference in relative velocity in the deprotection reaction of one protecting group to another protecting group. That is, in the t-Boc / Cbz protecting group strategy, both protecting groups are deprotected under acidic conditions, and stronger acidic conditions are required for complete removal of Cbz groups. Therefore, when the t-Boc group, which is more sensitive to acidic conditions, is deprotected with acid, some Cbz groups may be deprotected at the same time. PNA oligomer preparation, on the other hand, is a stepwise extension of the polymer chain by a chemical reaction in which only t-Boc groups are cleaved from the amino groups of the main chain in each synthesis cycle, and the free amino groups of the main chain are bonded to the free carboxylic acids of the next monomer backbone. The t-Boc group, the primary amino protecting group of the main chain, is a medium-strength acidic reagent such as trifluoroacetic acid and its chlorinated hydrocarbon solution, hydrogen chloride solution in organic solvents, borontrifluoride / diethylether complexes and some other By the action of acids it can be segmented by the formation of isobutylene and carbon dioxide. While the t-Boc group is deprotected and the PNA oligomer chain is extended, the Cbz group, the primary amino protecting group of the base heterocycle, should not be segmented. Trifluoroacetic acid, however, can also fragment some Cbz groups at the same time, resulting in polymers that are branched as byproducts, reducing the total yield of the desired product.

The orthogonal principle, on the other hand, fragments protecting groups under different conditions. In other words, one protecting group is segmented under acidic conditions and the other protecting group is segmented under basic conditions. As a PNA monomer backbone primary amino protecting group, a 9-fluorenylmethyloxycarbonyl group which is segmented at basic conditions instead of t-Boc group (hereinafter, 'Fmoc term') (Tetrahedron 1995, 51, 6179). Although this strategy (Fmoc / Cbz) excludes the possibility of fragmentation of the Cbz group, the primary amine protecting group of the nucleobases, upon deprotection of the Fmoc group in the stepwise extension of the polymer chain, finally the liquid hydrogen fluoride or tree during the Cbz phase fragmentation. Strong acids such as fluoromethanesulfonic acid must be used. Perseptive Biosystems recently used Fmoc as a backbone protecting group, and instead of Cbz, a benzhydryloxycarbonyl group (hereinafter referred to as 'Bhoc group'), which can be easily fragmented under carbamate form or under mild acidic conditions, is a primary amine of a nucleobase. PNA monomers introduced as protecting groups have been developed (WO 96/40685). Such PNA monomers protected with Fmoc / Bhoc groups have been commercialized and widely used in synthesizing PNA oligomers. However, in the preparation of PNA monomers, 20-30 moles of the Bhoc group-protected base moiety and the primary amino terminus are protected by Fmoc group and the carboxylic acid terminus is part of the methyl chain in the final hydrolysis reaction. There is a problem of having to reinject% Fmoc-succinimide. In addition, Fmoc groups must be particularly careful when used because of their extreme base sensitivity and instability in neutral aprotic solvents [WO96 / 25394], and besides, the high production costs for the production of large quantities of PNA oligomers also limit the use of Fmoc groups.

Another problem in the preparation of PNA monomers relates to the introduction of protecting groups of primary amino groups present in the heterocycle of the base. Generally, amino groups of bases such as adenine, guanine and cytosine are protected in carbamate form by reaction with chloroformate. However, the chloroformate compound is unstable and the nucleophilic amino group of the nucleobase is weak, making the reaction difficult. Carbamate preparation by imidazolide [J. Org. Chem., 1982, 47, 4471-4477] and alkylimidazolium salts [J. Am. Chem. Soc., 1982, 104, 5702-5708, etc., have been reported using activated carbonates. This method can overcome the difficulties associated with carbamate formation, but few examples are widely used for nucleobases. Meanwhile, a 4-methoxyphenyldiphenylmethyl group (hereinafter referred to as 'MMT group'), which is not a carbamate form, has been reported as a primary amino protecting group of the nucleobase of the PNA monomer [Bioorg. & Med. Chem. Lett. 1996, 6, 665.

As described above, there is an urgent need for a PNA monomer having a primary amino protecting group combination of a main chain and a nucleobase, which is easy to be prepared as a PNA monomer, can be easily segmented under selective conditions, and is also industrially applicable. .

In order to improve the above problems, the inventors of the present invention, Korean Patent Application No. 2000-30712, have developed a PNA monomer protected with an alkyl or arylsulfonylethoxycarbonyl group / 4-methoxyphenyldiphenylmethyl group. The present invention provides a method for protecting the primary amino group of the main chain 2-aminoglycine skeleton portion with p-nitrophenylsulfonylethoxycarbonyl group as an improved invention of the above invention, wherein the primary amino group of the main chain 2-aminoglycine skeleton portion is first The p-nitrophenylthioethoxycarbonyl group, which is stable to the hydrolysis reaction, is protected and finally oxidized to convert p-nitrophenylthioethoxycarbonyl group to p-nitrophenylsulfonylcarbonyl group to convert p-nitrophenylsulfonylethoxy It is an object to provide a method for producing a PNA monomer protected with a carbonyl group / 4-methoxyphenyldiphenylmethyl group.

The present invention provides a method for producing a peptide nucleic acid,

1) reacting 2-aminoethylglycine with p-nitrophenylthioethylsuccinimidyl carbonate to prepare 2-aminoethylglycine protected with p-nitrophenylthioethoxycarbonyl group;

2) preparing an alkyl ester hydrochloride thereof by reacting 2-aminoethylglycine protected by p-nitrophenylthioethoxycarbonyl group prepared in step 1 with cionyl chloride in an alcohol solvent;

3) reacting the nucleobase group with 4-methoxyphenyldiphenylmethylchloride to prepare a nucleobase group protected with 4-methoxyphenyldiphenylmethyl group;

4) 2-aminoethylglycinate hydrochloride protected with p-nitrophenylthioethoxycarbonyl group prepared in step 2 and the nucleobase group protected with 4-methoxyphenyldiphenylmethyl group prepared in step 3 above Reacted with -ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter referred to as 'EDC') to form a primary amino group of the main chain portion represented by the following formula (2): p-nitrophenylthioethoxycarbonyl group Preparing an alkyl ester of a PNA monomer which is protected with and wherein the primary amino group of the nucleobase moiety is protected with a 4-methoxyphenyldiphenylmethyl group; And

5) Alkyl of the PNA monomer in which the primary amino group of the main chain portion prepared in step 4 is protected with p-nitrophenylthioethoxycarbonyl group and the primary amino group of nucleobase portion is protected with 4-methoxyphenyldiphenylmethyl group. The ester is hydrolyzed using an alkali metal hydroxide, and then oxidized with hydrogen peroxide under a sodium molybdate catalyst to form p-nitrophenylthioethoxycarbonyl as a main chain protecting group with p-nitrophenylsulfonylethoxycarbonyl group. Converting to prepare a peptide nucleic acid monomer represented by Formula 1;

Characterized in that the method for producing a peptide nucleic acid monomer consisting of.

In Chemical Formula 1; B represents adenine, guanine and cytosine, and R ₁ represents a 4-methoxyphenyldiphenylmethyl group.

In Chemical Formula 2; B represents adenine, guanine and cytosine, R ₁ represents a 4-methoxyphenyldiphenylmethyl group, and R 'represents an alkyl group of C ₁ to C ₆ .

The present invention includes an intermediate represented by the following formula (3) for preparing a peptide nucleic acid monomer represented by the formula (1).

In Chemical Formula 3; R 'represents an alkyl group of C ₁ ~ C _6.

Referring to the present invention in more detail as follows.

In the present invention, the primary amino group of the main chain 2-aminoethylglycine skeleton portion is protected by p-nitrophenylsulfonylethoxycarbonyl group which is easily removed under weak base condition, and the primary amino group of nucleobase is easily desorbed under mild acidic condition. A method for producing a PNA monomer protected by a protected MMT group, wherein the primary amino group in the backbone of the main chain 2-aminoethylglycine is first protected with a p-nitrophenylthioethoxycarbonyl group that is stable to hydrolysis reaction and then nucleo P-nitrophenylthioethoxycarbonyl group / 4- reacted with a nucleobase protected by a 4-methoxyphenyldiphenylmethyl group prepared by protecting the primary amino group of the base with a 4-methoxyphenyldiphenylmethyl group After preparing PNA monomer protected by methoxyphenyldiphenylmethyl group in high yield, the p-nitrophenylthioethoxycarbonyl group was finally converted to p-nitro by oxidation reaction. Was converted to the ethoxycarbonyl group in nilseol sulfonyl relates to a method for producing a ethoxycarbonyl group / 4-methoxyphenyl de-protected PNA monomer to the phenyl group in the p- nitrophenyl sulfonyl.

The p-nitrophenylsulfonylethoxycarbonyl group is easily segmented under weak base conditions and is more suitable as a backbone protecting group than conventional Fmoc groups, which are expensive to produce in large quantities of PNA oligomers.

Meanwhile, conventional Cbz / Bhoc groups, such as nucleobase protecting groups, are introduced by reacting with amino groups of nucleobases in the form of chloroformates. Chloroformate compounds are unstable, and the nucleophilic amino groups of nucleobases have weak reactions. In order to facilitate the introduction, an additional reaction is required first of activating a base primary amino group with imidazolide and then reacting with alcohol. However, the MMT group described in the present invention can be easily introduced into a protecting group by reacting 4-methoxyphenyldiphenylmethyl chloride with a primary amino group of nucleobase in the presence of triethylamine or pyridine and is easily deprotected under weak acid conditions. . That is, the p-nitrophenylsulfonylethoxycarbonyl group is easily removed in weak base conditions, and the MMT group is easily deprotected in weak acidic conditions, thereby reducing side reactions in preparing PNA oligomers, which is easy to use (orthogonal theory).

The present invention relates to a method for producing the above-described PNA monomer, which will be described in detail below.

First, 2-aminoethylglycine is reacted with p-nitrophenylthioethylsuccinimidyl carbonate to prepare 2-aminoethylglycine protected by p-nitrophenylthioethoxycarbonyl group, and more specifically, backbone 2 Introduction of a new protecting group, p-nitrophenylthioethoxycarbonyl group, to the primary amino group of -aminoethylglycine was made by reacting 2-aminoethylglycine with chlorotrimethylsilane in a mixed solvent of dimethylformamide and dichloromethane. After preparing -trimethylsilyl-2-aminoethylglycine, a protecting group is introduced by reaction of p-nitrophenylthioethyl rucinimidyl carbonate which is a protecting group reagent with N-methylmorpholine.

In addition, step 2 is a step of reacting 2-aminoethylglycine protected by p-nitrophenylthioethoxycarbonyl group prepared by step 1 with the zonyl chloride to prepare an alkyl ester thereof, more specifically, a hydrochloride salt. After introduction of the protecting group, the carboxylic acid terminal of the 2-aminoethylglycine skeleton is added dropwise at 0 ° C. under an alcohol solvent to reflux, and the main chain portion of the amino acid group represented by the formula (3) is added to p-nitrophenylthio. 2-aminoethylglycinate hydrochloride protected with a oxycarbonyl group is prepared. The alcohol may be used methanol or ethanol.

The next three step is to prepare a nucleobase protected by a 4-methoxyphenyldiphenylmethyl group by reacting a nucleobase group and 4-methoxyphenyldiphenylmethylchloride, more specifically, a nucleobase for the production of PNA monomer The cleobase moiety is prepared by known methods of carboxymethyl-nucleobases in which the primary amino group present in the heterocycle in the case of adenine, guanine, cytosine except thymine is protected by MMT groups [Bioorg. & Med. Chem. Lett. 1996, 6, 665.

In step 4, 2-aminoethylglycinate hydrochloride protected by p-nitrophenylthioethoxycarbonyl group prepared in step 2 was activated with triethylamine in a dichloromethane solvent, which was then prepared in step 3- The primary amino group of the main chain portion represented by the formula (2) is protected with p-nitrophenylthioethoxycarbonyl group by reacting with a nucleobase protected with methoxyphenyldiphenylmethyl group and EDC and the primary amino group of the nucleobase portion. Alkyl esters of PNA monomers, protected with a 4-methoxyphenyldiphenylmethyl group, are prepared in high yield.

Finally, step 5 is a PNA monomer in which the primary amino group of the main chain portion of step 4 is protected with p-nitrophenylthioethoxycarbonyl group and the primary amino group of nucleobase portion is protected with 4-methoxyphenyldiphenylmethyl group. The carboxyl terminus of the alkyl ester of is converted to the free carboxylic acid by hydrolysis using an alkali metal hydroxide [see Scheme 1], and then oxidized with hydrogen peroxide under a sodium molybdate catalyst to form p as a main chain protecting group. The final peptide nucleic acid monomer represented by Chemical Formula 1 is prepared by converting the -nitrophenylthioethoxycarbonyl group to the p-nitrophenylsulfonylethoxycarbonyl group in high yield. The alkali metal hydroxide used in the hydrolysis reaction may be lithium hydroxide or sodium hydroxide.

In particular, the p-nitrophenylthioethoxycarbonyl group, which is the primary amino protecting group of the main chain in step 5, is stable in hydrolysis reaction, and thus, some of the base-sensitive main chain protecting groups are removed in the conventional PNA monomer production method, and thus 20 to 30 mol% of protecting groups The problem of having to reload the reagent can be solved.

In addition, the present invention includes an intermediate represented by the formula (3) for producing a peptide nucleic acid monomer represented by the formula (1). In the intermediate represented by Formula 3, R 'is preferably a methyl or ethyl group.

On the other hand, the method for producing a PNA oligomer using the PNA monomer prepared by the above method is as follows. PNA oligomers can be applied to a variety of synthetic methods known in the literature for the preparation of peptides, e.g. in the solid phase synthesis, the first PNA monomer is combined and the next step is to extend the desired PNA chain, which is a repeated cycle of deprotection / bonding. It includes.

A method of preparing a PNA oligomer using the PNA monomer according to the present invention will be briefly described as follows. First, p-nitrophenylsulfonylethoxycarbonyl group, a temporary backbone protecting group of the PNA monomer, is easily quantitatively divided under weak base conditions to form an N-terminal amine group. In the N-terminal amine group bonded to the PNA chain, the C-terminus of the next PNA monomer is bound in the presence of a binding reagent such as dicyclohexylcarbodiimide (hereinafter referred to as 'DCC'). After the PNA chain production is complete, the weak acid is used to remove the MMT group, a nucleobase protecting group, to obtain a synthesized PNA oligomer from the solid phase.

As such, the PNA monomer according to the present invention is a p-nitrophenylsulfonylethoxycarbonyl group, which is a skeletal protecting group in a chain extension step in preparing a PNA oligomer, in ammonia, morpholine, piperidine, piperazine and their aprotic organic solvents. Easily deprotected under weak base conditions using solutions of PNA oligomer, MMT group, which is the primary amino protecting group of nucleobase, is co-segmented at the final segment from solid support to facilitate PNA oligomer preparation. In particular, p-nitrophenylsulfonylethoxycarbonyl group, which is more economical than the existing Fmoc group, is introduced as a main chain protecting group, and is industrially useful.

In addition, the method for preparing a PNA monomer according to the present invention removes p-nitrophenylsulfonylethoxycarbonyl group, which is a part of the main chain protecting group, which is sensitive to the base during the final hydrolysis reaction in the preparation of the conventional PNA monomer, thereby removing 20 to 30 mol% of a protecting group reagent. This can solve the problem of having to re-inject the.

Although this invention is demonstrated in detail based on an Example, this invention is not limited to an Example.

Example 1 Preparation of N- [p-nitrophenylthioethoxycarbonyl- (2-aminoethyl)] glycine

134 ml (1 mol) of chlorotrimethylsilane was added to a suspension of 50 g (0.4 mol) of 2-aminoethylglycine contained in 850 ml of dichloromethane and 50 ml of dimethylformamide mixed solvent. The mixture was stirred for 40 minutes and then cooled to 5 ° C. 136 g (0.4 mol) of p-nitrophenylthioethylsuccinimidyl carbonate was added thereto, and 186 ml (1.7 mol) of N-methylmorpholine was added dropwise over 30 minutes. After stirring for 2 hours, 100 ml of methanol was added thereto, followed by stirring for 20 minutes, and then 1 L of ethyl acetate was added thereto. The suspension was filtered and the solid obtained was washed with ethyl acetate. The solid was poured into 1.5 L of water and stirred vigorously for 1 hour, and the remaining white solid was filtered under reduced pressure, washed with water and placed in 1 L of methanol, and the suspension was stirred vigorously. The product was obtained by filtration under reduced pressure, washed with methanol and dried in vacuo to give 82 g (yield 60%) of N- [p-nitrophenylthioethoxycarbonyl- (2-aminoethyl)] glycine.

¹ H-NMR (DMSO): δ 8.1 (d, 2H), 7.5 (d, 2H), 4.2 (t, 2H), 3.6 (m, 2H), 3.4 (t, 2H), 3.1 (m, 2H) , 3.0 (t, 2H)

Example 2 Preparation of Methyl N- [p-nitrophenylthioethoxycarbonyl- (2-aminoethyl)] glycinate hydrochloride

After cooling 100 g of methanol to which 11 g (0.032 mol) of N- [p-nitrophenylthioethoxycarbonyl- (2-aminoethyl)] glycine prepared in Example 1 was added was cooled to 0 ° C. 71 mL of nichloride was added dropwise under nitrogen. The reaction mixture was stirred at 0 ° C. for 15 minutes and then refluxed for 11 hours. The solvent was removed under reduced pressure and the concentrated portion was dissolved in an ether / methanol (9: 1) mixed solvent, cooled to 0 ° C. and stirred for 1 hour. The precipitated white solid was filtered under reduced pressure, washed with ether and dried in vacuo to afford 11.3 g (yield 90%) of methyl N- [p-nitrophenylthioethoxycarbonyl- (2-aminoethyl)] glycinate hydrochloride. .

¹ H-NMR (DMSO): δ 9.0 (br s, 1H), 8.2 (d, 2H), 7.6 (d, 2H), 4.2 (t, 2H), 4.0 (m, 2H), 3.8 (s, 3H ), 3.4 (t, 2H), 3.0 (t, 2H)

Example 3: Preparation of 2- [N'4-4-methoxyphenyldiphenylmethyl (1'-cytosyl)] acetic acid

1 g (0.009 mol) of cytosine, 4.1 g (0.013 mol) of methoxyphenyldiphenylmethyl chloride, and 0.9 g (0.009 mol) of triethylamine were added to a 100 ml round flask containing 50 ml of anhydrous pyridine and stirred at room temperature for 10 hours. . Excess pyridine was removed by concentration under reduced pressure, water was added thereto, stirred for 2 hours, filtered under reduced pressure, and the obtained solid was purified by silica gel chromatography to obtain 3.6 g of N'4-4-methoxyphenyldiphenylmethylcytosine (yield 100). %) Was obtained. The obtained solid 3.5g (0.009 mol) was put into a 100 ml round flask containing 50 ml of anhydrous dimethylformamide, cooled to 0 degreeC, 0.44 g (0.011 mol) of sodium hydrides were added, and it stirred for 1 hour. 1.1 ml (0.01 mol) of ethyl bromoacetate was added dropwise, followed by stirring at room temperature for 10 hours to complete the reaction. 15 ml of methanol was added thereto, stirred for 1 hour, and concentrated under reduced pressure. Dichloromethane was added, washed with water, and purified by silica gel chromatography to obtain 3.9 g (91% yield) of a white solid of 2- [N'4-4-methoxyphenyldiphenylmethyl (1'-cytosyl)] acetic acid ethyl ester. Got it. 1.62 g of the solid obtained was placed in a 250 ml round flask containing 20 ml of acetonitrile, 20 ml of methanol, and 10 ml of water, and then 13.8 ml of 2.5 normal lithium hydroxide aqueous solution was stirred for 30 minutes, followed by 20% aqueous citric acid solution. 60 ml was added and acidified. Filtration under reduced pressure afforded 1.4 g of 2- [N'4-4-methoxyphenyldiphenylmethyl (1'-cytosyl)] acetic acid.

¹ H-NMR (DMSO): δ 8.3 (br s, 1H), 7.5-6.8 (m, 16H), 4.2 (m, 2H), 3.7 (s, 3H)

Example 4: Methyl N- [N ''-p-nitrophenylthioethoxycarbonyl- (2 ''-aminoethyl)]-N- [2- [N'4-4-methoxyphenyldiphenylmethyl (1'-cytosyl)] acetyl] glycine preparation

1.0 g (2.6 mmol) of methyl N- [p-nitrophenylthioethoxycarbonyl- (2-aminoethyl)] glycinate hydrochloride prepared in Example 2 was dissolved in 40 ml of dichloromethane and cooled to 0 ° C. Then, 0.39 ml of triethylamine was added dropwise, stirred for 1 hour, activated with free amine, and then filtrated under reduced pressure, 2- [N'4-4-methoxyphenyldiphenylmethyl (prepared in Example 3). 1'-cytosyl)] acetic acid was added sequentially, followed by 1.2 g (2.6 mmol) of EDC 0.5 g (2.6 mmol). The reaction mixture was stirred at room temperature for 10 hours and then the solvent was removed under reduced pressure. Ethyl acetate was added to the concentrated portion, and the mixture was washed with NaHCO 3 solution, and then the organic layer was concentrated to give methyl N- [N ''-p-nitrophenylthioethoxycarbonyl- (2 ''-aminoethyl)]-N- [2 2.0 g (yield 97%) of-[N'4-4-methoxyphenyldiphenylmethyl (1'-cytosyl)] acetyl] glycinate were prepared.

¹ H-NMR (DMSO): δ 8.3 (br s, 1H), 8.1 (d, 2H), 7.5 (d, 2H), 7.3 to 6.8 (m, 16H), 4.6 (s, 2H), 4.3 to 3.7 (m, 6H), 3.71 (s, 3H), 3.67 (s, 3H), 3.3 to 3.0 (m, 4H)

Example 5: N- [N ''-p-nitrophenylsulfonylethoxycarbonyl- (2 ''-aminoethyl)]-N- [2- [N'4-4-methoxyphenyldiphenylmethyl (1'-cytosyl)] acetyl] glycine

Methyl N- [N ''-p-nitrophenylthioethoxycarbonyl- (2 ''-aminoethyl)]-N- [2- [N'4-4-methoxy prepared by Example 4 above 1.6 g (2.0 mmol) of phenyldiphenylmethyl (1′-cytosyl)] acetyl] glycinate was dissolved in 25 ml of acetonitrile: acetone: water in a 1: 1: 1 ratio, cooled to 0 ° C. and 2.5 normal lithium 4.2 ml of aqueous hydroxide solution was added, and the mixture was stirred for 5 minutes to cause a hydrolysis reaction. After acidifying with 20% citric acid, ethyl acetate and water were added, the ethyl acetate layer was taken, concentrated under reduced pressure, dissolved in dichloromethane, and diethyl ether was added dropwise while stirring to precipitate the product. N- [N ''-p-nitro Phenylthioethoxycarbonyl- (2 ''-aminoethyl)]-N- [2- [N'4-4-methoxyphenyldiphenylmethyl (1'-cytosyl)] acetyl] glycine 1.3 g (yield) 84%). 1.1 g (1.4 mmol) of the obtained solid was dissolved in 20 ml of methanol, cooled to 0 ° C., 1.1 ml of 0.3 M aqueous sodium molybdate solution and 2.5 ml of hydrogen peroxide were added thereto, followed by stirring for 10 hours. Ethyl acetate and water were added, the ethyl acetate layer was taken, water was removed with anhydrous sodium sulfate, and concentrated under reduced pressure. 1.1 g (yield 87%) of N- [N ''-p-nitrophenylsulfonylethoxycarbonyl- (2 '' -Aminoethyl)]-N- [2- [N'4-4-methoxyphenyldiphenylmethyl (1'-cytosyl)] acetyl] glycine was obtained.

¹ H-NMR (DMSO): δ 8.4 (d, 2H), 8.2 (d, 2H), 7.4 to 6.8 (m, 16H), 4.5 (s, 2H), 4.3 to 3.9 (m, 6H), 3.8 ( s, 3H), 3.1 (m, 2H), 2.9 (m, 2H)

As shown in Example 5, in the conventional hydrolysis reaction, since some of the base-sensitive backbone protecting groups are removed, there has been a problem of replenishing the 20-30 mol% protecting group reagent to replenish the removed backbone protecting groups. In the present invention, this problem is solved by using a p-nitrophenylthioethoxycarbonyl group stable to the hydrolysis reaction.

Experimental Example 1: Preparation of PNA Oligomer GATGACCGGCACACT

The PNA oligomer was prepared in the following synthesis cycle using the PNA monomer protected by the p-nitrophenylsulfonylethoxycarbonyl group / MMT group prepared in Example 5 and the Fmoc-Pal-Peg-PS solid support. The whole reaction and washing of the resin were carried out in a small reaction vessel, and the solutions were removed by filtration under reduced pressure through frit for the designated time.

[Synthesis cycle]

1) Primary amino group deprotection of 2-aminoethylglycine skeletal moiety by dimethylformamide solution with 20% piperidine dissolved for 10 minutes

2) three times washing with dimethylformamide and two times washing with dichloromethane

3) bound with 3 equivalents of PNA monomer (0.1M dimethylformamide solution):

The reaction was activated by addition of 3 equivalents of HATU (O-7-azabenzotriazolyl-1,1,3,3-tetramethyluranium hexafluorophosphate) and 3 equivalents of diisopropylethylamine, followed by addition to the resin. The bond was completed in 30 minutes.

4) After washing three times with dimethylformamide and washing twice with dichloromethane PNA oligomer was synthesized through the same steps as above, each step was confirmed by a Kaiser test after binding. The final p-nitrophenylsulfonylethoxycarbonyl group was not removed from the PNA oligomer for analysis. Deprotection of the MMT group and segmentation of the PNA oligomer from the resin were completed simultaneously by treatment with trifluoroacetic acid / m-cresol (4: 1) for 90 minutes. The PNA oligomer was precipitated by addition of diethyl ether and washed twice with diethyl ether. HPLC (260 nm) analysis under standard conditions yielded oligomers of 90% purity.

As described above, the PNA monomer according to the present invention introduces a p-nitrophenylsulfonylethoxycarbonyl group which is easily segmented in the weak base as a new main chain protecting group, and when the nucleobase portion is adenine, guanine or cytosine, 1 of the heterocycle Primary amino groups are protected by MMT groups that are easily deprotected from weak acids, making them easy to prepare and suitable for PNA oligomer synthesis. In particular, p-nitrophenylthioethoxycarbonyl group which is stable under basic conditions in the primary amino protecting group of the main chain in the hydrolysis reaction during the preparation process of the PNA monomer, and the target main chain protecting group p-nitrophenylsulfonyl by the oxidation reaction in the final step Obtaining a oxycarbonyl group solves the problem that some of the base-sensitive backbone protecting groups in the conventional PNA monomer production process must be removed in the hydrolysis step to reintroduce 20-30 mol% of protecting group reagent. Meanwhile, the p-nitrophenylsulfonylethoxycarbonyl group newly introduced in the present invention is more stable than the extreme sensitivity to the base of the Fmoc group, which is an existing protecting group, and is easier to handle, and in particular, it is more economical than the Fmoc group. This can be very useful.

Claims (4)

In the method for producing a peptide nucleic acid, 1) reacting 2-aminoethylglycine with p-nitrophenylthioethylsuccinimidyl carbonate to prepare 2-aminoethylglycine protected with p-nitrophenylthioethoxycarbonyl group; 2) preparing an alkyl ester hydrochloride thereof by reacting 2-aminoethylglycine protected by p-nitrophenylthioethoxycarbonyl group prepared in step 1 with cionyl chloride in an alcohol solvent; 3) reacting the nucleobase group with 4-methoxyphenyldiphenylmethylchloride to prepare a nucleobase group protected with 4-methoxyphenyldiphenylmethyl group; 4) 2-aminoethylglycinate hydrochloride protected with p-nitrophenylthioethoxycarbonyl group prepared in step 2 and the nucleobase group protected with 4-methoxyphenyldiphenylmethyl group prepared in step 3 above Reacted with -ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride to make the primary amino group of the main chain moiety represented by the following formula (2) protected by p-nitrophenylthioethoxycarbonyl group and Preparing an alkyl ester of a peptide nucleic acid monomer in which the primary amino group is protected with a 4-methoxyphenyldiphenylmethyl group; And 5) Peptide nucleic acid monomers in which the primary amino group of the main chain portion prepared in step 4 is protected with p-nitrophenylthioethoxycarbonyl group and the primary amino group of nucleobase portion is protected with 4-methoxyphenyldiphenylmethyl group. The alkyl ester is hydrolyzed using an alkali metal hydroxide, and then oxidized by hydrogen peroxide solution under a sodium molybdate catalyst to form p-nitrophenylthioethoxycarbonyl as a main chain protecting group and p-nitrophenylsulfonylethoxycarbonyl group. Preparing a peptide nucleic acid monomer represented by the following Chemical Formula 1; Method for producing a peptide nucleic acid monomer, characterized in that consisting of. Formula 1 In Chemical Formula 1; B represents adenine, guanine and cytosine, and R ₁ represents a 4-methoxyphenyldiphenylmethyl group. Formula 2 In Chemical Formula 2; B represents adenine, guanine and cytosine, R ₁ represents a 4-methoxyphenyldiphenylmethyl group, and R 'represents an alkyl group of C ₁ to C ₆ . The method of claim 1, wherein the alkali metal hydroxide of step 5 is lithium hydroxide or sodium hydroxide. An intermediate characterized by the following Formula 3 for preparing a peptide nucleic acid monomer represented by Formula 1. Formula 1 In Chemical Formula 1; B represents adenine, guanine and cytosine, and R ₁ represents a 4-methoxyphenyldiphenylmethyl group. Formula 3 In Chemical Formula 3; R 'represents an alkyl group of C ₁ ~ C _6. 4. The intermediate of claim 3 wherein R 'is a methyl or ethyl group.