JP2005027569A - New dna conjugate and antisense agent with the same as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new DNA conjugate producible by a simple method using an inexpensive raw material, exhibiting excellent antisense properties, and giving a double-stranded hybrid in between the conjugate and a complementary DNA in a stable condition. <P>SOLUTION: The new DNA conjugate is represented by the general formula( wherein, A<SP>1</SP>is an alkylene group or an alkylene group interrupted by an oxygen atom; A<SP>2</SP>is an alkylene group; R is a residue of a peptide, saccharide or functional amine; and n is 0 or 1 ). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel DNA conjugate having antisense activity.

  Oligonucleotide and peptide complexes have the potential to be used as antisense agents for gene expression, and the peptide facilitates an increase in the intercellular concentration of active oligonucleotides, so many Attempts have been made.

  For example, a conjugate of DNA and peptide siphon has been obtained by introducing a thiol group at the 5′-end of a synthetic oligonucleotide (see Non-Patent Document 1). In addition, functional organic compounds such as amino groups, carboxylic acid groups, hydroxyl groups, phenol groups, etc. present in protein molecules are utilized via bifunctional linkers such as dialkylating reagents, dimaleimides, dialdehydes, etc. A combination of angiotensin I, insulin, prazikinin, topramycin, and other anticancer substances is also known, and the use of aromatic dithioisocyanate as a linker has also been proposed (see Non-Patent Document 2).

  However, these oligonucleotide and peptide conjugates are expensive because of the high cost of raw materials required for their production, complicated production processes, low yields, and insufficient antisense properties. However, it was not always satisfactory.

“Bioconjugate Chemistry”, 1994, Vol. 5, p. 373-378 “Science”, 1964, vol. 144, p. 1344

  The present invention is a novel DNA that can be produced by a simple method using an inexpensive raw material, exhibits excellent antisense characteristics, and stably provides a double-stranded hybrid with a complementary DNA. The purpose is to provide a conjugate.

  As a result of intensive research to develop a novel DNA conjugate, the present inventors have used solid-phase fragment condensation method to convert ω-aminoalkyl phosphoroate and aliphatic diisocyanate or carbonyl to DNA having a terminal hydroxyl group. By condensing peptides, sugars or functional amines with diimidazole as a linker-forming agent, DNA conjugates exhibiting excellent antisense properties and forming stable double-stranded hybrids with complementary DNA Based on this finding, the present inventors have found that it can be obtained in a yield.

（式中のＡ¹はアルキレン基又は酸素原子で中断されたアルキレン基、Ａ²はアルキレン基、Ｒはペブチド、糖又は機能性アミンの残基、ｎは０又は１である）
で表わされるＤＮＡコンジュゲート、及びそれを有効成分としてなるアンチセンス剤を提供するものである。 That is, the present invention has the general formula

(In the formula, A ¹ is an alkylene group or an alkylene group interrupted by an oxygen atom, A ² is an alkylene group, R is a residue of a peptide, sugar or functional amine, and n is 0 or 1)
And an antisense agent comprising the same as an active ingredient.

  The DNA in the general formula (I) is not particularly limited, and may be used among DNA derived from various animal cells, DNA derived from various bacteria, or a DNA segment obtained by chopping them with an enzyme. Although it can be arbitrarily selected and used according to the purpose, in the present invention, it is necessary to use an amino group introduced by reacting a 5'-terminal hydroxyl group with an aminating agent.

Next, as the alkylene group of the A ¹ bond in the general formula (I), a polymethylene group having 2 to 10 carbon atoms, preferably 4 to 8 carbon atoms such as an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, etc. Can be mentioned. The alkylene group may be one having a carbon chain interrupted by an oxygen atom, such as a 2-oxapropylene group, a 3-oxapentylene group, or a 4-oxaheptylene group.
Further, the alkylene group having an A ² bond in the general formula (I) is preferably a polymethylene group having 4 to 12 carbon atoms or a branched alkylene group.

  The peptides introduced into the DNA via a linker include peptides obtained by degradation of various proteins, for example, nuclear export signal peptides, antigen-derived nuclear localization signal peptides, synthesized amphiphilic properties. Examples of the α-helix and β-sheet peptide include sucrose and galactosamine as saccharides, and examples of functional amines include spermine and lipofectamine.

（式中のＲ¹及びＲ²は保護基、Ａ¹は前記と同じ意味をもつ）
で表わされる亜リン酸エステルを反応させ、５´末端を化学修飾し、一般式

（式中のＲ¹、Ｒ²及びＡ¹は前記と同じ意味をもつ）
で表わされるＤＮＡ誘導体を形成させたのち、酸化して、一般式

（式中のＲ¹、Ｒ²及びＡ¹は前記と同じ意味をもつ）
で表わされるＤＮＡ誘導体とし、次いで一般式
ＯＣＮ−Ａ²−ＮＣＯ （Ｖ）
（Ａ²は前記と同じ意味をもつ）
で表わされる脂肪族ジイソシアネートを反応させることにより、一般式

（式中のＲ¹、Ａ¹及びＡ²は前記と同じ意味をもつ）
で表わされる化合物を製造したのち、これにペプチド、糖又は機能性アミンを反応させ、最後にアルカリ例えばアンモニア水で処理して固相担体からの切りはなし、及び保護基の脱離を行うことにより製造することができる（以下合成法１という）。 Among the DNA conjugates of the present invention, those of general formula (I) where n = 1 are in accordance with the solid phase fragment condensation method, such as solid phase carriers such as porous glass, controlled porous glass (CPG), polyethylene glycol-polystyrene. In the presence of a catalyst, DNA such as an oligonucleotide having a hydroxyl group at the 5 ′ end is condensed.

(Wherein R ¹ and R ² are protecting groups, and A ¹ has the same meaning as above)
A phosphite ester represented by the following formula:

(Wherein R ¹ , R ² and A ¹ have the same meaning as above)
After the formation of a DNA derivative represented by

(Wherein R ¹ , R ² and A ¹ have the same meaning as above)
And then the general formula OCN-A ² -NCO (V)
(A ² has the same meaning as above)
Is reacted with an aliphatic diisocyanate represented by the general formula

(Wherein R ¹ , A ¹ and A ² have the same meaning as described above)
By reacting this with a peptide, sugar or functional amine, and finally treating with an alkali such as aqueous ammonia to cut off the solid phase carrier and removing the protecting group. Can be produced (hereinafter referred to as Synthesis Method 1).

で表わされるカルボニルジイミダゾールを用いて、一般式

（式中のＲ¹、Ａ¹は前記と同じ意味をもつ）
で表わされる化合物を製造したのち、これにペプチド、糖又は機能性アミンを反応させ、さらにアルカリ処理することにより製造することができる（以下合成法２という）。
これらの反応は、適当な溶媒、例えばアセトニトリル、ジメチルホルムアミドなどを用いて行われる。 On the other hand, in the general formula (I), those in which n = 0 are substituted with the aliphatic diisocyanate represented by the general formula (V).

Using the carbonyldiimidazole represented by the general formula

(Wherein R ¹ and A ¹ have the same meaning as described above)
Can be produced by reacting it with a peptide, sugar or functional amine and further subjecting it to an alkali treatment (hereinafter referred to as synthesis method 2).
These reactions are performed using a suitable solvent such as acetonitrile, dimethylformamide and the like.

Next, aqueous ammonia is added to the reaction product thus obtained to excise the DNA conjugate from the solid support, and optionally remove the protecting group of the peptide present.
As the protecting group represented by R ¹ and R ² , for example, a ω-cyanoalkyl group such as 2-cyanoethyl group or a triphenylmethyl derivative residue such as 4-methoxyphenyldiphenylmethyl group is used.

Formula DNA-PO ₃ obtained in this manner _{CH 2 CH 2 OCH 2 CH 2} NH-R
As examples of the DNA conjugate of the present invention represented by the formula (I), CI to CXI can be mentioned in which DNA and R have the structures shown in Table 1 below.

In general formula _{DNA-PO 3 CH 2 CH 2} OCH 2 CH 2 NHCONH (CH 2) 6 NHCONH-R
As examples of the DNA conjugate of the present invention represented by the formula, CXII to CXVI in which DNA and R have the structures shown in Table 2 below can be mentioned.

  According to the present invention, a DNA conjugate suitable as an antisense agent, which has excellent antisense characteristics and can form a stable double-stranded hybrid with complementary DNA, is produced by a simple reaction operation. be able to.

  Next, the present invention will be described in more detail with reference to examples.

  First, using an automatic DNA synthesizer (product name “PS250” manufactured by Kurachem, product name “PS250”), the 5′-end of the controlled porous glass (product name “500 mm support”, hereinafter abbreviated as CPG) is used. After supporting an oligonucleotide NI (ttttttctctctctct-3 ′) having a hydroxyl group protected with a dimethoxytrityl group, it was treated with a 3% by mass acetonitrile solution of trichloroacetic acid to remove the protecting group, and a commercially available amino group was removed from the free hydroxyl group. The amination reagent N-methoxytrityl-2- (2-aminoethyloxy) ethyl phosphoramidite was aminated by reaction with a dimethylformamide solution containing 1H tetrazole at room temperature for 30 minutes.

  Next, after capping the unreacted 5'-hydroxyl group with acetic anhydride, the phosphorus atom portion was oxidized with an ioacetic acid solution to form a phosphate ester. The intermediate compound thus obtained was reacted with a 3% by mass acetonitrile solution of trichloroacetic acid to remove the methoxytrityl group, which is a protecting group for the terminal amino group, and then hexamethylene diisocyanate (HMI) or carbonyldiimidazole ( CDI) and dimethylformamide containing diisopropylethylamine were reacted at room temperature for 2 to 12 hours, and then the peptides or galactosamines shown in Table 3 were reacted in dimethylformamide containing diisopropylethylamine for 24 hours at room temperature.

Next, the reaction product thus obtained is treated in concentrated aqueous ammonia at 55 ° C. for 4 hours to excise from CPG and remove the protecting group bonded to the oligonucleotide and peptide. The DNA conjugate was obtained as a crude product.
This crude product was purified using a reverse phase liquid chromatograph, and the resulting compound was analyzed by liquid chromatography and laser-excited time-of-flight mass spectrometry (MALDI-TOF MS). The results are shown in Table 3.

  Peptides shown in Table 3 using oligonucleotide NII (5′-cagttaggggttag-3 ′) or oligonucleotide SI (5′-cagttaggggttag-3 ′) instead of oligonucleotide NI (ttttttctctctctct-3 ′) in Example 1 Or it couple | bonded with the amine like Example 1, and the DNA conjugate samples 7-15 were manufactured. The results are shown in Table 4.

  Instead of the oligonucleotide NI (ttttttctctctctct-3 ') in Example 1, the oligonucleotide RI [5'-r- (agagagagaaaaaaa) was used, and the peptide or sugar used in Example 1 was used in the same manner as in Example 1. The DNA conjugate samples 16 to 21 shown in Table 5 were produced by the reaction.

DNA conjugate sample Nos. Obtained in Examples 1 and 3 1-6 and no. 16 to 21 were tested for their stability with complementary DNA as follows.
Using a UV detector (manufactured by JASCO Corporation, product name “V-560 UV / Vis spectrometer”), each sample was 1 μM in a buffer solution (pH 7.0) composed of 100 mM NaCl, 50 mM Tris-HCl, 20 mM MgCl _2. After dissolving at a concentration and adding complementary strand DNA thereto and heating at 92 ° C. for 5 minutes, the double strand was once melted and then slowly cooled to 4 ° C. to recombine the double strand.
Next, this was heated from 5 ° C. to 80 ° C. at a rate of 0.5 ° C./min, the change in absorbance at 260 nm was measured with a UV detector, and the temperature at the 50% absorbance rise point was calculated as the melting temperature. The results are shown in Table 6.
Note that + Mg in the table indicates that MgCl ₂ is present in the measurement solution, and −Mg indicates that MgCl ₂ is not present.

  From this table, the DNA conjugate molecule of the present invention can form a hybrid duplex with complementary DNA, and the melting temperature of the hybrid with complementary DNA is a cationic amino acid. Sample No. conjugated with a peptide having a high content of Lys or Arg. 1, no. 4, no. 5, no. 16 and the sample No. conjugated with sugar. Rising at 21 and stabilizing double strands, especially those conjugated with synthetic β-sheet peptides, significantly stabilized hybrid duplexes, conjugated peptides rich in hydrophobic amino acids Sample No. 3, no. No. 18 shows no increase in the stability of the hybrid duplex between complementary DNA, but it does not become unstable.

The resistance test of the DNA conjugate of the present invention against the DNA-degrading enzyme DNase 1 was performed as follows.
Sample No. obtained in Example 1 1 was adjusted to a concentration of 1 μM with 0.1 M NaCl, and 5 ml of 160 Kunitz units of DNase 1 (manufactured by Sigma) was added thereto, and maintained at 37 ° C., and its degradation rate was measured over time. The degradation rate in minutes was 20%.
Moreover, the degradation rate in natural DNA performed for comparison was 100%.
Note that 1 Kunitz is a unit for increasing the absorbance of the UV spectrum at 260 nm by 0.001 per minute for DNA in 1 ml at 25 ° C. and pH 5.0.

Sample No. obtained in Example 1 2 and no. For the 4 DNA conjugates, a degradation test for ribonuclease H was performed as follows.
Adjust the fluorescent label at the 5 'end of the RNA complementary to the sample to a concentration of 10 μM to form a hybrid duplex, then add 2.5 units of ribonuclease H (Sigma-Aldrich) and reaction buffer The degradation rate of RNA in the solution was measured over time by 20% polyacrylamide gel electrophoresis. As a result, sample no. 2 and no. It was found that 4 showed almost the same activity as natural DNA.

Sample No. obtained in Example 1 2 and no. The stability in serum of the 6 DNA conjugates was measured as follows.
Each sample was dissolved in 120 μl of ultrapure water at a concentration of 1 nM, 20 μl of 10% non-immobilized fetal calf serum was added, and the degradation state of DNA was monitored over time using RP-HPLC. The reaction was stopped at each time by adding the sample to a reaction solution (pH 8.4) composed of 0.1 M Tris-HCl, 0.09 M boric acid and 7 M urea and freezing with liquid nitrogen.
The monitoring was performed by measuring changes at 260 nm using RP-HPLC (manufactured by Hewlett-Packard, series 1100) and ODS column (5 × 125 × 4 mm). As a result, the natural DNA was degraded by 95% or more after 180 minutes. 2 is 35%, sample no. 6 was found to remain at 28% degradation.

For the DNA conjugates obtained in Examples 2 and 3, the human telomerase inhibitory activity in the cell extract was measured as follows. This human telomerase is an enzyme that is specifically expressed in cancer cells and causes immortalization of the cells.
Using a solution extracted from leukemia cell-derived Jurkat with a lysis solution, the effect of inhibiting the activity by the DNA conjugate was expressed as the concentration required for 50% inhibition (IC ₅₀ ). The results are shown in Table 7.

  As can be seen from this table, the DNA conjugate of the present invention exhibits stronger human telomerase inhibitory activity than natural DNA.

  Since the DNA conjugate of the present invention has excellent antisense properties and can form a stable hybrid duplex with complementary DNA, it can be used as an antisense agent.

Claims (2)

General formula

(In the formula, A ¹ is an alkylene group or an alkylene group interrupted by an oxygen atom, A ² is an alkylene group, R is a residue of a peptide, sugar or functional amine, and n is 0 or 1)
A DNA conjugate represented by An antisense agent comprising the DNA conjugate according to claim 1 as an active ingredient.