JP2011004682A - Oligonucleotide derivative and oligonucleotide structure using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an oligonucleotide derivative having a small load on a living body such as toxicity to cells, excellent permeability to cell membrane and improved nuclease resistance, and an oligonucleotide structure using the same.SOLUTION: The oligonucleotide derivative contains a hydroxy group at the 3'-terminal of oligonucleotide, modified with a substituent represented by formula (1) (wherein Ris a 1-10C alkylene group which may be a straight chain or branched chain; and Ris a protective group for preventing reaction between hydrogen or an amino group and an amidite reagent).

Description

  The present invention relates to an oligonucleotide derivative and an oligonucleotide construct using the oligonucleotide derivative.

  In recent years, various oligonucleotides such as DNA and RNA have been used for therapeutic and diagnostic purposes. For example, in the diagnostic use, a DNA chip and a DNA microarray can be mentioned. In the therapeutic use, in addition to the introduction of a treatment-related gene, expression suppression by knockdown of a disease-related gene can be mentioned. Attempts have also been made to use aptamers that are nucleic acid molecules or peptides that specifically bind to specific molecules as therapeutic agents.

  Nucleic acid technology that has received particular attention includes a knockdown method for specific genes using RNA interference (RNAi). RNAi refers to a phenomenon in which the action of a gene whose sequence is homologous to it is suppressed by the action of double-stranded RNA (dsRNA). In the suppression of gene expression by RNAi, dsRNA is recognized by Dicer, a member of the RNase III family, and cleaved into 21-23 mer siRNAs (short interfering RNAs). This is because mRNA that is taken up and subsequently homologous to the taken-in siRNA is cleaved at the center and degraded.

  However, exogenous DNA and RNA in vivo are exposed to various nucleases, and in particular, RNA is easily degraded by nucleases. There was a problem that it was difficult to maintain.

  In order to solve these problems, it has been studied to improve nuclease resistance by chemically modifying RNA (Non-Patent Documents 1 to 3).

  For example, various chemical modifications have been attempted for siRNA as shown in FIG. 5 (Non-patent Document 4). In addition, the present inventor converted the phosphodiester bond portion of the 3 ′ end dangling end of siRNA into a carbamate bond or a urea bond, thereby neutralizing the negative charge of the bond portion and allowing the permeability of the cell nucleus to the membrane. It has succeeded in enhancing the nuclease resistance and silencing activity of siRNA by improving the above (Non-patent Document 5). The present inventor has also succeeded in enhancing nuclease resistance by introducing a unit having a benzene skeleton in place of a nucleoside into a nucleic acid oligomer such as RNA, and further, an amidite reagent for introducing a benzene skeleton Has been successfully modified into a CPG resin and nucleotide modification using the same (Patent Document 1).

WO2007 / 094135

L. Beigelman., J.A McSwiggen. , K. G. Draper et al., J Biolchem 270, 25702-25708 (1995) 27 S.P.Zinnen K. Domenico., M. Wilson et al., RNA 8,214-228 (2002) S.Agrawaland E.R.KandimaIla., Curr.CanGer Drug Targets., 1,197-209 (2001) H. Hoshi, FEBS Letters 521, 197-199 (2002) Y. Ueno, T. Naito, K. Kawada, A. Shibata, Hye-Sook Kim Y. Wataya, Y. Kidade, Biochem Biophys Res Commun330, 1168-1175 (2005) Z.Paroo, D.R.Corey, Trends Biotechnol., 8, 390-394 (2004) H. de Martimprey, C. Vauthier, C. Malvy, P. Couvreur, Eur. J. Pharm. Biopharm., 71, 490-504 (2009)

  The technique of introducing a unit having a benzene skeleton into a nucleic acid oligomer such as RNA described in Patent Document 1 described above improves nuclease resistance and silencing activity by chemically modifying the 3 ′ end dangling end of siRNA. It has been confirmed that However, chemically modified siRNA is inferior in cell membrane passage compared to unmodified siRNA, and it is considered difficult to supply chemically modified siRNA into cells. Moreover, the chemical modification group to the oligonucleotide is artificial, and no particular consideration was given to biocompatibility such as toxicity to cells.

  The present invention has been made in view of the above-described conventional circumstances, and has a low burden on a living body such as toxicity to cells, good permeability to a cell membrane, and excellent nuclease resistance and oligonucleotide derivatives and oligonucleotides It is an object to provide an oligonucleotide construct using a derivative.

The present inventor considered modifying a functional group having a cationic property into an oligonucleotide in order to improve the ability of the chemically modified oligonucleotide to pass through a cell membrane. This is due to the following reason. That is, when siRNA is administered to a living body from a vein, it disappears from the blood in a short time due to uptake by the reticuloendothelial system, excretion from the kidney, and degradation by nuclease in the blood. This is probably because siRNA is a relatively small water-soluble compound having a molecular weight of about 15,000 and is negatively charged, so that it is quickly filtered from the glomeruli after administration in blood. In fact, administration of siRNA was radiolabeled with I ¹²³ in mouse body, as a result of investigating, blood levels of siRNA decreased the boundary of about 5 minutes after the administration, distribution increased correspondingly to the kidneys and bladder Has been reported (Non-patent Document 6). In addition, by forming a complex of siRNA with a cationic substance or neutral substance, electrostatic repulsion with an anionic substance present in the blood or in the cell is alleviated, nuclease resistance, and blood retention It has been reported that the intracellular introduction rate is greatly improved (Non-patent Document 7).

For these reasons, the present inventor considered modifying a functional group having a cationic property into an oligonucleotide. Further, in consideration of biocompatibility such as toxicity to cells, glucosamine, which is a kind of natural amino sugar, was chemically modified to oligonucleotide. Glucosamine is one of amino sugars in which the hydroxyl group at the 2-position of glucose is substituted with an amino group, and exhibits cationic properties by ionization of the amino group (see the following chemical structural formula).

  Glucosamine is an amino sugar that forms chitin contained in crab and shrimp shells in large quantities. Chitin has biodegradability, and N-acetylglucosamine produced in the degradation process is important for the human body. It exists in many glycoproteins that are essential constituents. For this reason, glucosamine is considered to have low toxicity and high biocompatibility compared to other cationic substances.

Further, as a result of intensive studies on chemically modifying this glucosamine to an oligonucleotide, the present invention has been completed.
That is, in the oligonucleotide derivative of the present invention, the hydroxyl group at the 3 ′ end of the oligonucleotide is a substituent represented by the following formula (1) (wherein R ¹ may have a linear or branched carbon number of 1 to 10 represents an alkylene group, and R ² is modified with hydrogen or an amino group which represents a protective group for preventing reaction with an amidite reagent.

  The oligonucleotide derivative of the present invention has NH-based cationicity and cell membrane permeability due to binding of the glucosamine derivative substituent represented by the formula (1) to the hydroxyl group at the 3 ′ end of the oligonucleotide. It becomes good. In addition, since the glucosamine derivative substituent is less toxic than other cationic substances and has high biocompatibility, the burden on the living body such as toxicity to cells is reduced. Furthermore, according to the test results of the present inventor, the oligonucleotide derivative of the present invention has good nuclease resistance, and the effect is longer even when the oligonucleotide derivative of the present invention is placed in a cell. Can last for hours. For this reason, for example, by binding the substituent of the above formula (1) to the 3 ′ dangling end of siRNA, the gene expression can be suppressed by RNAi and the action can be continued for a longer time.

The oligonucleotide derivative of the present invention has a glucosamine (or a derivative in which a protective group for preventing the amino group of glucosamine reacting with an amidite reagent) bonded to the hydroxyl group at the 3 ′ end of the oligonucleotide via a substituent R ^1. Are connected. For this reason, the substituent R is compared to the case where glucosamine (or a derivative in which a protective group for preventing the amino group of glucosamine from reacting with an amidite reagent) is directly bonded to the hydroxyl group at the 3 ′ end of the oligonucleotide. ¹ is easy to synthesize order to reduce the steric hindrance of the hexose ring portion and amidite reagent glucosamine (or derivative in which a protecting group is bound to prevent reacting with the amidite reagent to the amino group of glucosamine).

R ² in the oligonucleotide derivative of the present invention can be a functional group represented by the following formula (2).

According to the test results of the present inventors, when R ² is a substituent having the above structure, a higher gene expression suppression effect is exhibited than when R ² is hydrogen.

  In the oligonucleotide derivative of the present invention, the oligonucleotide portion may have a partial sequence of a predetermined gene mRNA or a complementary sequence thereof. The chain length of the oligonucleotide may be 10 or more and 35 or less. Furthermore, the oligonucleotide may be an oligoribonucleotide.

The oligonucleotide construct of the present invention is an oligonucleotide construct for regulating gene expression, characterized by having any of the above oligonucleotide derivatives. This oligonucleotide construct can be an oligonucleotide construct selected from single and double stranded DNA, single and double stranded RNA, DNA / RNA chimeras and DNA / RNA hybrids, and its function. From the aspect, it can be selected from antigene, antisense, aptamer, siRNA, miRNA, shRNA and lipozyme. Furthermore, the substituent represented by the following formula (1) in the dangling end portion (where R ¹ represents a linear or branched alkylene group having 1 to 10 carbon atoms, and R ² represents Which represents a protecting group for preventing hydrogen or amino groups from reacting with the amidite reagent.

  Moreover, it is siRNA, Comprising: The oligonucleotide derivative can contain the substituent represented by the said Formula (1) in 3 'terminal dangling end part.

  Furthermore, according to the present invention, an oligonucleotide construct for gene diagnosis having any of the above-described oligonucleotide derivatives can be obtained. Furthermore, the construct can be a probe or primer.

(Oligonucleotide derivatives)
The oligonucleotide derivative of the present invention has a unit represented by the above formula (1) at the 3 ′ end of the oligonucleotide derivative. Due to the presence of this unit, excellent nuclease resistance can be obtained. Such a unit can be introduced by addition to an oligonucleotide having a known sequence or an unknown sequence, and thus can be an oligonucleotide derivative of the present invention. In addition, the oligonucleotide here is an oligonucleotide that may be modified.

  Moreover, when constructing an antigene, an antisense, an aptamer, a miRNA and a ribozyme using the oligonucleotide derivative of the present invention, a unit represented by the formula (1) or a unit may be provided. When the probe is immobilized on a solid phase carrier, the unit represented by the formula (1) can be provided on the free end side. Furthermore, the primer may be appropriately provided with a unit represented by the formula (1) as necessary.

  In the present specification, the term “oligonucleotide” generally means a polymer having a plurality of monomer units, each of which is a monomer that constitutes an oligonucleotide or a polynucleotide. The oligonucleotide means deoxyribonucleotide and / or ribonucleotide as a monomer unit. In general, a polymer having deoxyribonucleotides as monomer units as nucleotides is referred to as DNA, and a polymer having ribonucleotides as monomer units is referred to as RNA, but the oligonucleotide derivative of the present invention is generally used for DNA and RNA, These oligomer unit oligomers are included. Oligonucleotides also include RNA / DNA chimeras. The oligonucleotides that may be modified include not only oligonucleotides comprising only natural bases such as guanine, cytosine, thymine, adenine, uracil or methylcytosine, which are purines and pyrimidines, but also various portions of the oligonucleotide. That is, oligonucleotides having one or more nucleotides with some chemical modification in the base, sugar moiety and phosphate ester moiety.

  The oligonucleotide derivative of the present invention can have a sense strand of DNA of a predetermined gene, an antisense strand thereof, a partial sequence of mRNA, or a complementary sequence thereof. By having such complementarity, it is possible to hybridize with various target nucleic acids, thereby expressing the intended function of the oligonucleotide derivative. In the oligonucleotide derivative of the present invention, the length of the oligonucleotide is not particularly limited and can be a length according to the use. However, considering the ease of oligonucleotide synthesis and the expected effect, it is 10 or more. It is preferable to be 35 or less. In the case of antisense, it can be about 10 or more and 30 or less. In the case of siRNA, the total chain length of A and B is preferably 15 or more and 35 or less, more preferably 30 or less. . In the case of a primer, it is preferably 10 or more and 30 or less, and in the case of a probe, it is preferably 10 or more and 30 or less.

  When the oligonucleotide derivative of the present invention is used for, for example, siRNA, shRNA, antisense, ribozyme, and aptamer, the monomer unit can be an oligoribonucleotide that may be modified.

(Oligonucleotide construct)
The oligonucleotide construct of the present invention has the oligonucleotide derivative of the present invention. Depending on the type of the oligonucleotide derivative in the oligonucleotide construct, the construct can be in the form of single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, DNA / RNA chimera, DNA / RNA hybrid, etc. Each or a combination can be used. As already described, since the oligonucleotide portion constituting the oligonucleotide derivative contains a modified oligonucleotide, the oligonucleotide construct may also contain a modified form of the oligonucleotide.

  Since the oligonucleotide construct of the present invention has improved nuclease resistance, it can be used for gene expression regulation or for various uses for research and diagnosis. Examples of gene expression regulation applications include antigene, antisense, aptamer, siRNA, miRNA, shRNA and ribozyme. In particular, by introducing the unit represented by the formula (1) into dT at the 3 ′ end overhang site in siRNA and shRNA, both nuclease resistance and silencing activity can be improved.

  Diagnostic or research applications include probes and primers. Probes are oligonucleotides that, by design or selection, have a sequence that is specifically defined for a target nucleic acid and that are obtained so that they hybridize under a given stringency. Nuclease resistance is improved by using this oligonucleotide derivative as a probe, so the effect of nuclease mixed in the sample containing the target nucleic acid is suppressed or avoided, and even if the degree of nuclease removal is low or nuclease removal treatment Sample preparation without omission becomes possible. As a result, genetic diagnosis and testing can be performed easily. Such hybridization between the probe and the target can be performed by immobilizing the probe on an appropriate glass substrate, a plastic substrate, or a solid phase carrier such as beads. The present invention also includes a solid phase carrier on which a probe containing the present oligonucleotide derivative is immobilized.

(Method for producing oligonucleotide derivative)
In order to obtain the oligonucleotide derivative of the present invention, an amidite method is used, using an immobilized glucosamine derivative immobilized on a solid phase carrier via a linking group while protecting the hydroxyl group and amino group of glucosamine with a protecting group. Methods for preparing oligonucleotide derivatives by various nucleic acid synthesis methods can be used. The hydroxyl protecting group of glucosamine is not particularly limited, and various conventionally known hydroxyl protecting groups can be used. Specific examples of such a protecting group include a benzyl group, an acetyl group, and a benzoyl group, and a particularly preferred protecting group is a benzyl group. Specific examples of the amino-protecting group include a phthalic acid group and a benzoyl group, with a phthalic acid group being particularly preferred.

(Use of oligonucleotide derivatives)
The oligonucleotide derivative of the present invention can be used as a gene expression inhibitor by being constructed so as to function as siRNA or antisense. Further, the oligonucleotide derivative of the present invention can be used as an active ingredient of a pharmaceutical composition for prevention / treatment of diseases in humans and non-human animals. For example, for a disease associated with gene expression, the oligonucleotide derivative of the present invention constructed as a gene expression inhibitor is effective for the prevention and treatment of such diseases.

  Furthermore, the oligonucleotide derivative of the present invention can be used as a test reagent or a diagnostic reagent such as a probe or a primer by constructing it so as to exhibit its hybridization function. Furthermore, what hold | maintained these oligonucleotide constructs to solid supports, such as a chip | tip and a bead, can be utilized as a test | inspection apparatus, a diagnostic apparatus, or these one part. Furthermore, such test reagents and diagnostic agents can be used as test or diagnostic kits in combination with other reagents, diagnostic agents or devices.

  The oligonucleotide derivative of the present invention can also be used for a gene expression suppression method using the gene expression suppression action of an oligonucleotide construct containing the oligonucleotide derivative of the present invention. Furthermore, it can also be used in a gene detection method using the hybridization function of the oligonucleotide construct of the present invention.

In this example, the measuring instrument, chromatography carrier and reagent shown in Table 1 were used.

In this specification, the following abbreviations may be used for the reagents used in this example.
AcOEt: ethyl acetate
Ac ₂ O: Acetic anhydride
APS: ammonium peroxodisulfate
BF ₃・ Et ₂ O: Boron trifluoride ・ diethyl ether complex
BF ₃・ Me ₂ O: Boron trifluoride ・ dimethylethyl ether complex
BnOEtOH: 2-Benzyloxyethanol
BzCl: benzyl chloride
CPG: Controlled-pore glass
DMAP: N, N-dimethyl-4-aminopyridine
DMF: N, N-dimethylformamide
DMTrCl: 4,4'-dimethoxytrityl chloride, 95%
EDTA: ethylenediaminetetraacetic acid
HPLC: High performance liquid chromatography
MALDI-TOF: matrix assisted laser desorption ionization-time of flight
MeONa: Sodium methoxide
mRNA: Messenger RNA
MS: Mass Spectrometry
NIS: N-Iodosuccimide
NPhth: phthalimide group
PAGE: Polyacrylamide gel electrophoresis
Pd (OH) ₂ / C: Palladium hydroxide / carbon (Pd20%) (approx. 50% water wet product).
TBAF: Tetrabutylammonium fluoride 1.0M in THF
TBE: Trisborate EDTA
TBDMSCl: tert-butyldimethylchlorosilane
TEA: Trimethylamine
TEAA: Triethylammonium acetate
TEMED: N, N, N´, N´-tetramethyl-ethylenediamine
TfOH: trifluoromethanesulfonic acid
Tris: Tris (hydroxymethyl) aminomethane
WSC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

  In Examples, an oligonucleotide derivative in which glucosamine was introduced at the 3 ′ end of siRNA was prepared by a solid phase synthesis method using CPG resin. The details are shown below.

<Introduction of a linker to the hydroxyl group at the 1-position of glucosamine>
First, a linker was introduced into the hydroxyl group at the 1-position of glucosamine according to the synthesis route shown in Chemical Formula 5 below. Here, the 2-position amino group was protected by a phthaloyl group, and the 3,4-positions were protected by a benzoyl group.

Details of the synthesis method are shown below.
Synthesis of 3,4,6-Tri-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl acetate (4)
Methanol (200 ml) was added to 10 g (46.4 mmol) of D-Glucosamine hydrochloride (3), sodium methoxide (10.7 g, 55.7 mmol) was further added, and the mixture was stirred at room temperature for 2 hours. Thereafter, phthalic anhydride (13.8 g, 92.8 mmol) was further added, and the mixture was stirred at room temperature for 4 hours. Then, after the solvent was distilled off under reduced pressure, acetic anhydride (100 ml, 1.06 mol) and pyridine (150 ml) were added and stirred for 48 hours. The reaction mixture was diluted with methanol (200 ml), the solvent was distilled off under reduced pressure, and the mixture was partitioned with 2N hydrochloric acid and chloroform, and pyridine was transferred to the aqueous layer. The organic layer was dehydrated by adding anhydrous sodium sulfate. The solvent was distilled off while reducing the pressure using a water pump, and the residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1: 1). The solvent was distilled off under reduced pressure, and then recrystallized with a mixed solvent of ethyl acetate and hexane to obtain a white powder of compound (4) (14.2 g, 64%).

Synthesis of 2-Benzyloxyethyl 3,4,6-tri-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (5) Compound (4) 2 g (4.19) obtained as described above mmol) was dissolved in 20 ml of acetonitrile, BnOEtOH (5.95 ml, 41.9 mmol) and BF ₃ .Me ₂ O (1.17 ml, 12.6 mmol) were added, and the mixture was heated to reflux with stirring for 5 hours. The reaction solution was neutralized with triethylamine, and the solvent was distilled off under reduced pressure to obtain compound (5).

Synthesis of 2-Benzyloxyethyl 2-deoxy-2-N-phthalimido-β-D-glucopyranoside (6) After dissolving the compound (5) thus obtained in 30 ml of methanol and adding sodium methoxide solution to pH 12 The mixture was stirred at 0 ° C. for 3 hours. The reaction mixture was neutralized with acetic acid and the solvent was distilled off under reduced pressure. The oily substance thus obtained was purified by silica gel column chromatography (CHCl ₃ → CHCl ₃ : MeOH = 15: 1 → 8: 1) to obtain compound (6) 1.03 g, yield 55%) as white crystals. It was. The ¹ H NMR spectrum of this product is shown below.
¹ H NMR (DMSO-d ₆ ): (400 MHz)
δ 7.88-6.93 (m, 9H, Ph, Phth), 5.38 (d, 1H, J _{3, OH-3} = 4.9Hz, OH-3), 5.17 (d, 1H, J _{4, OH-4} = 5.6Hz , OH-4), 5.07 (d, 1H, J _1,2 = 8.8Hz, H-1)

<Synthesis of protecting group-introduced glucosamine derivatives>
According to the synthetic route shown in the following chemical formula 6, starting from the compound (6) obtained as described above, a glucosamine derivative (11) having a protective group introduced at the 1,3,4-position hydroxyl group and the 2-position amino group ) Was synthesized.

Details of the synthesis method are shown below.
2-Benzyloxyethyl6-O-tert-butyldimethylsilyl-2-deoxy-2-N-phthalimido-β-D-lucopyranoside (7) compound (6) 400 mg (0.902 mmol) in anhydrous pyridine 9 ml, TBDMSCl246 mg, (1.63 mmol, 1.8 equivalents) was added and stirred at room temperature for 5 hours. Then, after quenching by adding an appropriate amount of methanol, vacuum distillation with addition of toluene was performed twice to remove pyridine. The oil layer thus obtained was purified by silica gel column chromatography (CHCl ₃ : MeOH = 18: 1 → 15: 1), whereby 492 mg (97% yield) of the compound (7) was obtained as a viscous transparent oily substance. As obtained.

2-Benzyloxyethyl 3,4-di-O-benzoyl-6-O-tert-butyldimethylsilyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (8) synthetic compound (7) 380 mg (0.681 mmol) Anhydrous dichloromethane was added to the mixture, and 0.5 ml (6.18 mmol) of anhydrous pyridine and 0.4 ml (3.44 mmol) of benzyl chloride were added under ice cooling, followed by stirring at room temperature for 6 hours. Then, after quenching by adding an appropriate amount of methanol, the solvent was distilled off under reduced pressure. The obtained oil was purified by silica gel column chromatography (AcOEt: Hexane = 1: 10 → 1: 3 → 1: 1). As a result, 495 mg (yield 95%) of the compound (8) was found to be viscous and transparent. Obtained as an oil. The ¹ H NMR data of the compound (8) is shown below.
¹ H NMR (DMSO-d ₆ ): (400 MHz)
δ 7.88-6.97 (m, 19H, Ph, Phth, PhCO), 6.06 (t, 1H, J _3,4 = 9.516, H-3), 5.57 (d, 1H, J _1,2 = 8.54, H-1 ), 5.52 (t, 1H, H-4), 4.30 (t, 1H, H-2), 4.12-4.09 (m, 1H, H-5)

2-Hydroxyethyl 3,4-di-O-benzoyl-6-O-tert-butyldimethylsilyl-2-deoxy-2-N-phthalimido-
Compound (8) 506 mg (0.661 mmol) was weighed into a synthesis flask of β-D-glucopyranoside (9) , the inside of the system was made an argon atmosphere, Pd (OH) ₂ / C 120 mg was added, and then EtOH / 20 ml of a mixed solvent of THF = 3: 1 was added and stirred. The solution was filled with hydrogen gas in a flask while removing argon using a suction device, and stirred at room temperature for 20 minutes. Subsequently, Pd (OH) ₂ / C was removed by Celite filtration, and then purified by silica gel column chromatography (CHCl ₃ : MeOH = 30: 1 → 25: 1) to obtain 425 mg of compound (9) (yield 95%) was obtained as a viscous clear oil.

Synthesis flask of 2- (4,4´-Dimethoxytrityl) oxyethyl 3,4-di-O-benzoyl-6-O-tert-butyldimethylsilyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (10) Then, 251 mg (0.371 mmol) of the compound (9) was weighed, 7 ml of anhydrous pyridine was added, and 161 ml (0.475 mmol) of DMTrCl was further added, followed by stirring at room temperature for 6 hours. The reaction solution was azeotroped with Toluene (twice) and then purified by silica gel column chromatography (AcOEt: Hexane = 1:10 → 1: 3 → 1: 1). As a result, 318 mg of compound (10) (88% yield) Was obtained as a viscous yellow oil. ¹ H NMR data of this product is shown below.
¹ H NMR (DMSO-d ₆ ): (400 MHz)
δ 8.01-7.07 (m, 27H), 6.11 (t, 1H, J _3,4 = 9.6, H-3), 5.66 (d, 1H, J _1,2 = 8.8, H-1), 5.61 (t, 1H, J _4,5 = 10, H-4), 4.44 (t, 1H, H-2), 4.15 (m, 1H, H-5), 4.08 (m, 1H, H-6)

Synthesis of 2- (4,4′-Dimethoxytrityl) oxyethyl-3,4-di-O-benzoyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (11) 0.487 mmol) was weighed and dissolved in 25 ml of anhydrous THF, 0.625 ml (0.625 mmol) of TBAF was added, and the mixture was stirred at 0 ° C. for 3 hours. Then, after quenching with methanol, the solvent was distilled off under reduced pressure, and purification by silica gel column chromatography (AcOEt: Hexane = 1: 10 → 1: 3 → 1: 1) revealed that 145 mg of compound (11) was Obtained as a viscous clear yellow oil.

<Modification to CPG carrier>
The compound (11) obtained as described above was modified to a CPG carrier using succinic acid as a linker (see the synthesis route of the following chemical formula 7). Details are shown below.

2- (4,4´-Dimethoxytrityl) oxyethyl-3,4-di-O-benzoyl-6- (succinyl-hydroxymethyl) -2-deoxy-
In a synthesis flask of 2-N-phthalimido-β-D-glucopyranoside (12), 213 mg (0.247 mmol) of compound (11) sufficiently dried under vacuum was weighed and dissolved in 4 ml of anhydrous pyridine, then DMAP0. 8 mg (6.55 × 10 ⁻³ mmol) and 90 mg (0.899 mmol) of succinic anhydride were added and stirred at room temperature for 72 hours. The reaction solution thus obtained was subjected to liquid separation treatment in the order of water, an aqueous NaHCO ₃ solution, and an aqueous NaCl solution, and the organic layer was extracted with diethyl ether and then dried over anhydrous sodium sulfate overnight. Then, the solvent was distilled off under reduced pressure, and the residue was further azeotroped with Toluene to obtain Compound (12).

2- (4,4´-Dimethoxytrityl) oxyethyl-3,4-di-O-benzoyl-6- (CPG-longchain-alkylamino-succinyl-hydroxymethyl) -2-deoxy-2-N-phthalimido-β-D- In a synthetic flask of glucopyranoside (13), weighed out compound (12) (0.247 mmol, CPG: 4e.q.) that had been thoroughly dried under vacuum, and dissolved in 6.2 ml of anhydrous DMF (0.01 M for CPG). did. CPG522mg (1e.q.) was added and lightly shaken to adjust to the solution, then WSC 48mg (0.247mmol, 4e.q.) was quickly added and shaken at room temperature for 72 hours. The treated CPG was transferred to a glass filter, washed 3 times with pyridine, and after sufficient drying, 15 mL of 0.1MDMAP solution (pyridine: Ac ₂ O = 9: 1) was added, and further at room temperature under Ar atmosphere. Shake overnight. Then, the CPG was transferred to a glass filter and thoroughly washed with pyridine, ethanol and acetonitrile in this order, and then dried in a vacuum overnight to obtain a modified CPG (13). The amount of the modifying group supported was measured by the following method. That is, 6 mg of dried modified CPG (13) was transferred to a glass filter, washed with a solution of HClO ₄ : EtOH = 3: 2, and the absorbance of the washed liquid was measured at UV 498 nm (DMTr group absorption wavelength). It was calculated by substituting into the following equation. As a result, it was 28.4 mmol / g.

<Synthesis of glucosamine-linked oligonucleotide>
Using CPG (13) modified as described above, an oligonucleotide having a glucosamine derivative introduced at the 3 ′ end was synthesized. The nucleotide sequences of the synthesized oligonucleotides are two types, 5′-r (CUUCUUCGUCGAGACCAUGtt) -3 ′ and 3′-r (ttGAAGAAGCAGCUCUGGUAC) -5 ′ having a complementary sequence thereof. For comparison, two kinds of oligonucleotides having the same base sequence were also synthesized. The details are shown below.

Oligonucleotides were synthesized on a 1 μmol scale according to the phosphoramidite method.
That is, normal phosphoramidite 0.1M in MeCN was prepared using an automatic DNA synthesizer (Applied Biosystem Model 3400) and synthesized according to the RNA protocol. The synthesis was completed with the DMTr group removed at the 5 ′ end of the oligonucleotide, and the CPG resin was dried through argon gas.

After that, transfer the oligonucleotide-bound CPG to an Eppendorf tube, add 1.2 ml of NH ₄ OH: EtOH = 3: 1 (v / v) aqueous solution, shake for 12 hours at room temperature, cut out from CPG and deprotect Went. Then, after transferring the supernatant to an Eppendorf tube, the CPG was further washed with 1.2 ml of H ₂ O: EtOH = 3: 1 (v / v) aqueous solution, and the washing solution was transferred to an Eppendorf tube and dried overnight under reduced pressure. Solidified. Then, 1 ml of TBAF (1M in THF) was added to the residue and shaken at room temperature for 15 hours to deprotect the benzoyl group and phthaloyl group. Thereafter, the reaction solution was diluted by adding 30 ml of TEAA buffer (0.1 M), passed through C-18 reverse phase column chromatography (Sep-Pak), and adsorbed on the column. Incidentally, 0.1 N TEAA buffer is a plus triethylamine 277.6 ml acetic 114.38 ml to 1 L with H ₂ O, prepared by the pH 7.0 with acetic acid, was prepared by diluting it 20-fold .

The column was washed with sterilized water to remove the salt, eluted with 3 ml of 50% MeCN in H ₂ O, and dried under reduced pressure. 200 μl of loading solution (90% formamide in 1 × TBE) was added to the residue, and the target oligonucleotide was isolated by 20% PAGE. 20% PAGE was dissolved by adding 45 ml of 40% acrylamide (19: 1) solution, 37.8 g of urea and 9 ml of 10 × TBE buffer, and H ₂ O was added to make 90 ml. Finally, 62 mg of APS was added and dissolved, and then 45 μl of TEMED was added and shaken to prepare. In addition, the electrophoresis gel was prepared by pouring 20% PAGE between two glass plates fixed with a 1.5 mm spacer in between, and allowing to stand until solidified. In addition, 1 × TBE buffer was used as the electrophoresis buffer. After the electrophoresis, the target oligonucleotide band was cut out, 0.1 M TEAA buffer and 0.1 M EDTA aqueous solution (15 ml) were added, and the mixture was shaken overnight. The filtrate was purified by C-18 reverse phase column chromatography (Sep-Pak) and evaporated to dryness under reduced pressure. The oligonucleotide thus obtained was dissolved in H ₂ O (1 ml), and the absorbance at 260 nm of the diluted solution was measured to determine the yield. The calculation method is as follows.

That is, the absorbance at a wavelength of 260 nm (Abs ₂₆₀ ) was diluted so that it was within the effective range of the absorptiometer, and Abs ₂₆₀ was measured at room temperature using a quartz cell for absorbance measurement with an optical path length (1) of 1 cm. The following formula was used to calculate the OD ₂₆₀ value. Here, V represents the total amount of the solution.
OD ₂₆₀ (Mε ^-1 · mL ^-1 · cm ^-1 ) = Abs ₂₆₀ (Mε ^-1 ) · V ^-1 (mL) · l ^-1 (cm)
Further, the following equation was used to calculate the molar extinction coefficient ε ₂₆₀ of the single-stranded oligonucleotide represented by N ₁ pN ₂ pN ₃ p... N _n-1 pN _n .
ε = 2 {ε (N ₁ pN ₂ ) + ε (N ₂ pN ₃ ) + ・ ・ ・ + ε (N _n-1 pN _n )}-{ε (N ₂ ) + ε (N ₃ ) + ・ ・・ + Ε (N _n-1 )}
Here, ε (N _n ) represents ε ₂₆₀ of a certain nucleic acid N _n , and ε (N _n-1 pN _n ) represents ε ₂₆₀ of a certain nucleic acid dimer N _n-1 pN _n .
The ε value of the glucosamine-linked oligonucleotide was assumed to be the same as that of the natural oligonucleotide.
Further, the following formula was used to calculate the concentration C (mol / l).
C = Abs ₂₆₀・ ε ₂₆₀ ⁻¹・ l ^-1

  30 pmol of the purified oligonucleotide was evaporated to dryness, dissolved in 3 μl of sterilized water, mixed well with 3 μl of the matrix solution, and then the sample was applied to the plate. The matrix solution was prepared by dissolving 4.85 mg of 3-hydroxypicolinic acid (3-HPA) and 0.8 mg of di-Ammonium hydrogen citrate in 50 μl of 50% MeCN in Milli Q. After the sample was dried, the structure was confirmed by MALDI-TOF / MS. As a result, four types of oligonucleotide derivatives (SSG, SSGN, ASSG and ASSGN) shown in Table 2 below were identified. In addition, Y which is a glucosamine derivative substituent is a phthalimide protecting group remaining without being removed.

<Verification of RNAi>
SiRNA was prepared for the glucosamine derivative-binding oligonucleotide and unmodified oligonucleotide obtained as described above, and the RNAi expression was verified.
Preparation of siRNA and measurement of Tm Using the four types of oligonucleotides prepared as described above and an unmodified oligonucleotide, annealing was performed in the combinations shown in Table 3 to prepare siRNA.

And 50% dissociation temperature _Tm value was measured and the presence or absence of formation of double stranded RNA was confirmed. That is, the concentration of each strand in the 50% melting temperature (T _m ) measurement of the oligonucleotide was adjusted to 3 μM, and the measurement buffer (10 mM NaH ₂ PO ₄ -Na ₂ HPO ₄ , 100 mM NaCl (pH 7.0)) Dissolved in 400 μl, heated at 95 ° C. for 5 minutes, annealed, and allowed to stand until it reached room temperature. 180 μl of the sample was placed in a dedicated cell, heated from 20 ° C. to 95 ° C., and the change in absorbance was measured to determine the 50% melting temperature (T _m ). At this time, the _Tm value was measured using the midline method.

As a result, as shown in FIG. 1, the curves drawn by the T _m values were all sigmoid curves, and the T _m values were almost the same as the unmodified natural siRNA. This suggests that the synthesized siRNA retains thermal stability and forms a stable duplex as in the natural type.

RNAi activity evaluation RNAi activity evaluation was performed by dual-luciferase reporter assay using HeLa cells which are human breast cancer cells. This method is a method for evaluating the inhibition of protein expression of Renilla luciferase by siRNA from the ratio of each luminescence using a vector expressing Firefly luciferase and Renilla luciferase (see FIG. 2). .

Samples were prepared using the seven types of siRNA annealed as described above. Details of the procedure are shown below.
HeLa cells were adjusted to 4000 cell / ml, and 100 μl was added to each well of a 96 well plate and cultured for 24 hours. Each strand of the synthesized siRNA was dissolved in TE buffer (100 mM NaCl) and annealed. Each siRNA volume, each medium (OPTI-MEM) volume, 0.1 μg / μL psi-CHECK (vector with Firefly and Renilla sequences) 1 μl, transfast (transfection reagent) 1.5 μl to a total volume of 175 μl After mixing, 35 μl was added to each well of a 96-well plate from which the medium had been aspirated. After 1 hour, 100 μl of the medium was added and cultured for 24 hours. After 24 hours, the medium was aspirated and stored frozen. At the time of measurement, after thawing, 24 μl of Dual glo substrate (Firefly substrate) was added and allowed to stand for 10 minutes, and then 23 μl of the sample was transferred to a 96 well plate for luminescence measurement, and Firefly luciferase was measured. Thereafter, 23 μl of Stop and glo substrate was added and allowed to stand for 10 minutes, and then Renilla luciferase was measured. Renilla luciferase values were divided by Firefly luciferase values and compared using% of control. In addition, Luminescenser JNR was used for luciferase measurement.

  As a result, as shown in FIG. 3, although a slight decrease was observed, it was found that all siRNAs did not lose the protein inhibitory effect. In addition, it was found that siRNA introduced with phthalimide-type glucosamine protected at the 2-position with a phthaloyl group had a higher inhibitory effect than those introduced with glucosamine at the 3 ′ end. This is thought to be due to the hydrophobic pocket that is said to recognize the 3 ′ end present in the PAZ domain in RISC. In addition, when either the sense strand or the antisense strand was modified with glucosamine, the siRNA activity was slightly decreased, but when both 3 ′ ends were modified, the activity was increased. Furthermore, siRNA in which phthalimide-type glucosamine was introduced at both 3 ′ ends resulted in a higher protein inhibitory effect than unmodified natural siRNA.

<3 'exonuclease resistance of glucosamine-linked oligonucleotide>
Fluorescent substituents are modified at the 5 ′ end of natural oligonucleotide 5′-r (CUUCUUCGUCGAGACCAUGtt) -3 ′ and glucosamine-linked oligonucleotide 5′-r (CUUCUUCGUCGAGACCAUGttX) -3 ′ synthesized as described above. Then, the 3 ′ exonuclease resistance was examined. The fluorescent substituent was fluorescein, and a phosphoramidite form of fluorescein was introduced into the 5 'end by a phosphoramidite method using a DNA / RNA automatic synthesizer.

The nuclease resistance of 5′flu-r (CUUCUUCGUCGAGACCAUGtt) -3 ′ and 5′-flu-r (CUUCUUCGUCGAGACCAUGttX) -3 ′ synthesized in this way was examined.
Specifically, 25 μl of SVPD (snake venom phosphodiesterase) (100 μmol / mL) and 475 μL of buffer solution (250 mM Tris-HCl, 50 mM MgCl ₂ (pH 8.0)) were added to prepare a total volume of 500 μL. . Add 300 pmol of the synthesized oligonucleotide to an Eppendorf tube to dryness, add 100 μL of the prepared SVPD (5.0 × 10 ^-3 unit / mL), incubate at 37 ° C, 1 min, 5 min, 10 min, 30 5 μL of the reaction solution was added to 15 μL of a loading solution (9M urea XC BPB) that had been dispensed into separate Eppendorf tubes every min, 1 h, 3 h, and used as a reaction solution for each hour. The 0 minute sample was assumed to have no enzyme added.
After separating them by 20% PAGE, the nuclease resistance was tested by analyzing the fluorescence intensity of fluorescein with a fluorescence scanner.

As a result, as shown in FIG. 4, in the natural 5′-flu-r (CUUCUUCGUCGAGACCAUGtt) -3 ′, the original band almost disappeared after about 5 minutes, whereas the 5′-flu-r ( In CUUCUUCGUCGAGACCAUGttX) -3 ′, the original band remained even after about one hour, indicating that it has excellent 3 ′ exonuclease resistance.
Further, as described above, siRNA synthesized from glucosamine-linked oligonucleotide exhibits a protein inhibitory effect equivalent to or higher than that of siRNA synthesized from natural oligonucleotide (FIG. 3). From these results, it was revealed that siRNA synthesized from glucosamine-linked oligonucleotide can exert a protein suppression effect over a longer period of time than siRNA synthesized from natural oligonucleotide.

  From the above results, the same applies when glucosamine-linked oligonucleotides are used for single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, DNA / RNA chimeras, DNA / RNA hybrids, etc. It became clear to have nuclease resistance.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

It is a graph which shows the relationship between the temperature and the light absorbency of siRNA synthesize | combined from the glucosamine derivative coupling | bonding oligonucleotide and the unmodified oligonucleotide. It is a conceptual diagram showing the evaluation method of RNAi activity. It is a graph of RNAi activity of siRNA synthesize | combined from the glucosamine derivative coupling | bonding oligonucleotide and unmodified oligonucleotide. It is a drawing substitute photograph which shows the result of electrophoresis for every predetermined time in a nuclease tolerance test. It is a figure which shows the example of the chemical modification of siRNA.

  INDUSTRIAL APPLICABILITY The present invention can provide a useful means in the medical field using nucleic acid oligomers such as RNA drug discovery that is expected to be developed into personalized medicine.

Claims (13)

A substituent whose hydroxyl group at the 3 ′ end of the oligonucleotide is represented by the following formula (1) (wherein R ¹ represents a linear or branched alkylene group having 1 to 10 carbon atoms, and R ² represents An oligonucleotide derivative characterized by being modified with a hydrogen or amino group (which represents a protecting group for preventing reaction with an amidite reagent).

The oligonucleotide derivative according to claim 1, wherein R ² is a functional group represented by the following formula (2).

The oligonucleotide derivative according to claim 1 or 2, wherein R ¹ is an ethylene group.   The oligonucleotide derivative according to any one of claims 1 to 3, wherein the oligonucleotide has a partial sequence of a predetermined gene mRNA or a complementary sequence thereof.   The oligonucleotide derivative according to any one of claims 1 to 4, wherein the oligonucleotide has a chain length of 10 or more and 35 or less.   The oligonucleotide derivative according to any one of claims 1 to 5, wherein the oligonucleotide is an oligoribonucleotide. An oligonucleotide construct for regulating gene expression comprising:
A construct comprising the oligonucleotide derivative according to any one of claims 1 to 6.   The construct according to claim 7, which is an oligonucleotide construct for regulating gene expression selected from single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, DNA / RNA chimera and DNA / RNA hybrid.   9. The construct according to claim 7 or 8, selected from antigene, antisense, aptamer, siRNA, miRNA, shRNA and lipozyme. A substituent represented by the following formula (1) in the dangling end portion (where R ¹ represents a linear or branched alkylene group having 1 to 10 carbon atoms, R ² represents hydrogen or amino 10. A construct according to any one of claims 7 to 9 having a protecting group for preventing the group from reacting with an amidite reagent.

siRNA, a substituent represented by the following formula (1) at the dangling end portion at the 3 ′ end (where R ¹ represents a linear or branched alkylene group having 1 to 10 carbon atoms) , R ² represents a protecting group for preventing hydrogen or an amino group from reacting with an amidite reagent).

  An oligonucleotide construct for gene diagnosis, comprising the oligonucleotide derivative according to any one of claims 1 to 7.   The construct according to claim 12, which is a probe or a primer.

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