Synthesis of backbone-modified morpholino oligonucleotides and chimeras using phosphoramidite chemistry

Abstract

A method for making phosphorodiamidite monomers is provided. [Solution] The method includes step a: contacting 5'-O-dimethoxytritylribo-base with sodium periodate and ammonium diborate; step b: reducing the hydroxyl group on the product obtained in step a with sodium cyanoborohydride to produce a morpholino monomer; and step c: phosphitylating the morpholino monomer with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphordiamidite and 4,5-dicyanoimidazole (DCI) in dichloromethane to produce a phosphorodiamidite monomer.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Patent Application No. 62 / 513,000, filed May 31, 2017. No. 89 and U.S. Provisional Patent Application No. 62 / 397,27, filed September 20, 2016 No. 7, both of which are incorporated herein by reference. is part of.

[0002] The present disclosure relates to methods for synthesizing morpholino oligonucleotides and derivatives thereof. [Background technology]

[0003] Morpholino oligonucleotides are known for their high affinity for DNA and RNA, Resistance to various nucleases, in vivo stability, and low toxicity make it an excellent candidate for antisense oligonucleotides. These phosphorodiamidate morpholines show promise for use as therapeutic oligonucleotides. PMO (compound 3, Figure 1) is a nonionic internucleotide linkage. These N,N-dimethylamino groups have morpholino groups that replace the deoxyribose in DNA. Tyraminophosphoradiamidate morpholino oligonucleotides inhibit RNA splicing It inhibits gene expression by interfering with transcription and preventing translation (Non-patent Document 1). , and is resistant to cellular nucleases (Non-patent Documents 2 and 3), and has a higher It has RNA binding affinity (Non-Patent Document 4). The development of treatments for PMO has been promoted by, for example, Duchess Treatment of Marburg muscular dystrophy (DMD) and the hemorrhagic filovirus Marburg It is being tested in clinical trials for the prevention of viral infections (Patent Document 1 and Patent Document 2). 2) Furthermore, PMOs have applications in nanotechnology (Non-Patent Document 5) and surface hysteresis. Hybridization (Non-Patent Document 6) has been discussed.

[0004] Unfortunately, these promising applications for PMOs are hindered by the lack of efficient synthetic methodologies. The standard method for chemically producing DNA and RNA on an automated synthesizer is very limited. In contrast, PMOs are currently synthesized in a 5' to 3' orientation on polystyrene resin. In the first step, 5'-hydroxyl-N-trityl-morpholino nucleoside and N, Condensation with N-dimethylaminodichlorophosphoramidate gives N,N-dimethylaminodichlorophosphoramidate. Coupling in the presence of a base produces a pyrrolophosphoramidate synthon. Further detritylation with acid leads to a dimer attached to the lipid. This approach has several recognized advantages. For example, the condensation yield is low (the recovery yield of dithymidine morpholino is 45%). Furthermore, the 5'-chloro-phosphoramidate monomer is unstable, and coupling of two morpholino monomers in the presence of a large amount of base is difficult. Furthermore, this approach is not readily available in many laboratories. Special procedures, techniques and materials (e.g., swellable polystyrene resins, It requires a specific reaction vessel and the pyridinium salt of trifluoroacetic acid as a detritylation agent. It is essential.

[0005] Recently, a new method using H-phosphonate chemistry for the synthesis of polythymidine PMOs has been developed. However, this approach still presents challenges. For example, The phosphorus atom is electrophilic and lacks a lone pair of electrons, making it ideal for most P(III) compounds. It is much more resistant to oxidation under ambient conditions than H-phosphonate morpholines. The coupling yield for the formation of the lino dimer is low (77%). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. 2016 / 0040162 [Patent Document 2] U.S. Patent Application Publication No. 2015 / 0038462 [Non-patent literature]

[0007] [Non-Patent Document 1] Summerton, J. Biochim. Biophys. Acta 1999, 1489, 141-158 [Non-patent document 2] Hudziak, et al. Antisense Nucleic Acid Drug Dev. 1996, 6, 267-272 [Non-patent document 3] Arora, et al. J. Pharm. Sci. 2002, 91, 1009-18. [Non-patent document 4] Summerton, JE Morpholinos and Related Antisense Biomolecules (Janson, CG, and During, MJ, Eds.), 2003, Kluwer / Plenum Publishers. [Non-Patent Document 5] Paul, et al., Chem. Commun., 2013, 49, 11278-80 [Non-patent document 6] Tercero, et al., J. Am. Chem. Soc., 2009, 131:4953 [Non-Patent Document 7] Bhadra, et al., Tetrahedron Letters 2015, 56: 4565-68 Summary of the Invention [Problem to be solved by the invention]

[0008] Each of the foregoing disadvantages is overcome by the method of the present disclosure. Further, the method of the present disclosure comprises: Other advantages are achieved which are discussed more fully below. [Means for solving the problem]

[0009] The present disclosure combines phosphorodiamidate morpholinos (PMOs) and PMO-DNA chimeras. These methods provide novel methods for producing phosphodiaminodiaminodiphosphate nucleotides. Oligomers containing intermolecular linkages are phosphoramiditized in high yields on an automated DNA synthesizer. The process is very robust because it can be made using morpholino phosphatase chemistry. Starting with the incorporation of sulfamidites into DNA, boronation, and subsequent various Various PMOs and PMO-DNA chimeras were synthesized by oxidation with iodine in the presence of amines. The above method is general and allows for the synthesis of a large number of PMO linkages. Unlike procedures using chlorophosphoramidate synthons, the more reactive Phosphorodiamidite synthons can be used to synthesize these analogs in high yield. To assemble these analogs, dicyanoimidazole-mediated coupling was used. Selective activation of the diisopropylamino moiety of the morpholinophosphordiamidite is used. can be.

[0010] Advantageously, the previously developed N,N-dimethylamino PMO synthon is Unlike chemistry designed to synthesize these analogs in a directional manner, morpholino phosphorylation Both the amidite and 2'-deoxynucleoside synthons are oligonucleotides in the 3' to 5' direction. Since PMO and PMO-DNA chimeras were designed for the synthesis of DNA fragments, Furthermore, this new approach allows for the synthesis of iodine-containing amines with suitable amines. Borane phosphoramidates that can be used to generate diverse PMO chimeras by oxidation of the corresponding boranes. This is achieved by using internucleotide bonds, such as PMO- LNA, PMO-RNA, PMO-DNA phosphorothioate / phosphorodithioate, and PMO-DNA phosphonoacetate / phosphonoformate chimeras were used in these methods. It can be made using

[0011] These PMOs are described for their potential use in various biological and biochemical applications. -DNA chimeras have three potential advantages over some of their analogues: For example, aminomorpholino phosphorodiamidate derivatives can be used to bind unmodified DNA / RNPs. A or standard N,N-dimethylamino PMO analogs. It was found that it forms a more stable duplex with RNA (one duplex with RNA (Approximately 1.75 times more stable than the N,N-dimethyl analogue per modification). PMO-DNA chimeras are active with RNase H1, which is consistent with fully substituted PM Compared to standard N,N-dimethylamino PMO analogs, which are inactive against RNase H1, These cap / gap N,N-dimethylamino PMO chimeras are promising candidates for R By activating Nase H1, various biochemicals useful for various applications in biology can be provide analogs with the same biological properties (e.g., aminoamidate derivatives have the ability to inhibit duplex formation) (compared to unmodified duplexes with complementary RNA) The increased stabilization of these PMO-DNA chimeras allows for the formation of cap / gamma-like domains. tAP analogs can use shorter single-stranded antisense oligonucleotides Therefore, off-target effects are reduced. Furthermore, these PMO-DNA chimeras are generally Cells can be easily transfected using commonly known transfection reagents. Microinjection and hybridization of PMO with DNA and delivery by ethoxylated polyethyleneimine, or peptide or dendritic molecule transporters. Eliminate the challenges associated with delivering PMOs via methods such as conjugation to one of the transporters .

[0012] Therefore, the present disclosure provides an efficient method for synthesizing PMO derivatives with minimal by-product formation. These synthetic methods provide efficient and cost-effective methods for synthesizing PMOs. The method is based on the previously reported method by the present inventors to prepare borane phosphonate DN in the presence of iodine and an amine. By incorporating the stereoselective conversion of A to phosphoramidate DNA (Paul, S., Roy, S ., Monfregola, L., Shang, S., Shoemaker, R., Caruthers, MH, J. Am. Chem. Soc. 2015, 137:3253-64), which provides three notable advantages of the disclosed synthetic method: 1) These methods are similar to standard DNA / RNA synthesis methods using phosphoramidite chemistry. PMO synthesis is orthogonal and compatible with conventional DNA synthesis. A synthesis can be performed in the synthesis apparatus. 2) N,N-dimethylaminophosphoramidate containing internucleotide bonds other than morpholino For the first time, we have been able to synthesize a PMO with 3) These synthesis methods allow the synthesis of PMO-DNA chimeras. These chimeras are , because it is anionic, it is water-soluble and hybridizes to RNA, Activates ase H.

[0013] This Summary is intended to represent the complete extent and scope of the invention. Furthermore, nothing in this specification to the "disclosure" or aspects thereof shall be construed as including, but not limited to, the following: Reference to a particular embodiment of the present disclosure is understood to refer to a particular embodiment, and not necessarily to all embodiments. The disclosure is not to be construed as being limited to the specific description. and the detailed description are set forth in various levels of detail, and no limitation on the scope of the disclosure is intended. No inclusion or exclusion of elements, components, etc. in the summary is intended to convey the present disclosure. These and other features will become more apparent from the detailed description, particularly when considered in conjunction with the drawings. become. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the chemical structures of DNA (1), RNA (2), N,N-dimethylaminophosphoramidate morpholino (3), and borane phosphoramidate morpholino (4) oligonucleotides. [Figure 2]

[0023] Figure 1 is a synthetic scheme depicting the current approach to the synthesis of N,N-dimethylamino phosphorodiamidate morpholino oligonucleotides. In morpholino numbering, the primary hydroxyl group must be 6', but for purposes of this disclosure, this hydroxyl group is shown as 5' to relate to conventional nucleic acid chemistry. [Figure 3]

[0023] Figure 1 is a synthetic scheme illustrating the synthesis of a BIBS-protected morpholinophosphordiamidite synthon. Reaction conditions: (i) lutidine (4.0 equiv.) and 1,4-dioxane; (ii) 1.0 M NH3 / MeOH; (iii) DMT-Cl (1.2 equiv.), pyridine; and (iv) NaIO4 (1.1 equiv.), (NH4)2B4O7 (1.1 equiv.), MeOH; (v) NaCNBH3 (2.0 equiv.), AcOH (2.0 equiv.), MeOH; (vi) P(OCH2CH2CN)(NiPr2)2 (1.2 equiv.), 4,5-dicyaminomimidazole (0.5 equiv.), and CHCl2. [Figure 4] Figure 1 shows a solid-phase synthesis scheme for phosphorodiamidate morpholino oligonucleotides (PMOs) using BIBS-protected phosphoramidites. Compound F is a PMO containing only N,N-dimethylamino-phosphorodiamidate internucleotide linkages, where "x" is the selected number of N,N-diethylamino-PMO nucleotide units. Compound A is a thymidine ribonucleoside, although this ribonucleoside may be replaced by any of four 2'-deoxyribonucleosides or morpholino nucleosides. In examples where cytosine, adenine, and guanine bases are used, the bases must be protected with bis(tertbutyl)isopropylsilyl (BIBS) groups. [Figure 5] FIG. 1 shows substitutions at the C-4 and C-6 positions of cytosine caused by the use of dimethylamino in an oxidative amination reaction performed after removal of the silyl protecting group. [Figure 6] FIG. 1 shows the synthesis of N,N-dimethylamino PMO trimer (left panel) and the LC profile from LCMS analysis of the entire reaction mixture (right panel). [Figure 7]Figures 7A-7F show visualization of ODN27 transfection by fluorescence microscopy. ODN27 (100 nM) in serum-reduced medium (Opti-MEM) was transfected into HeLa cells using Dharmafect 1 and then incubated at 37°C for 18 hours. The cells were then washed with PBS and fixed using a buffered formalin solution. The nuclei of the cells were counterstained with DAPI (blue). Figures 7A and 7D show images of fluorescein localization of ODN27. Figures 7B and 7E show images of DAPI localization of the nuclei in the same cells. Figure 7C is an image overlay of the images from Figures 7A and 7B. Figure 7F is a phase-contrast image of the cells shown in Figure 7D. DETAILED DESCRIPTION OF THE INVENTION

[0015] Unless otherwise indicated, the following terms have the following meanings:

[0016] The term "oligonucleotide analog" refers to oligonucleotides with modified backbone structures, e.g., oligonucleotides that are not naturally occurring. backbones other than the standard phosphodiester bond found in nucleotides and polynucleotides , and optionally a modified sugar moiety, e.g., other than a ribose or deoxyribose moiety. Analogs refer to oligonucleotides having a morpholino moiety of the formula: maintains bases capable of hydrogen bonding by Watson-Crick base pairing with the thiol base, Here, the analog backbone is a structural analog of the oligonucleotide analog molecule and a standard polynucleotide ( For example, the hydrogen bonds between bases in a sequence-specific manner (e.g., single-stranded RNA or single-stranded DNA) The bases are presented to allow binding. Preferred analogs are substantially uncharged. It has a phosphorus-containing skeleton.

[0017] The "nucleobase" is not particularly limited as long as it can be used in the synthesis of nucleic acids, and examples thereof include a cytosyl group. pyrimidine bases such as uracil and thyminyl groups, and purine bases such as adenyl and guanyl groups An "optionally protected nucleobase" includes, for example, an adenyl group, a guanyl group, or a silyl group. It means a nucleic acid base having an amino group which can be protected by a tosyl group, and the amino group is morpholine. The nitrogen atom of the morpholine ring of a nucleotide is protected by a protecting group that can be maintained under the deprotection conditions. Preferred are nucleobases that are

[0018] The "amino protecting group" is not particularly limited, and examples thereof include Greene's PROTECTIVE GROUPS I N Organic Synthesis, 4th edition, Wiley-Interscience, 2006, etc. Specific examples of the "amino protecting group" include a pivaloyl group and a pivaloyloxymethyl group. group, trifluoroacetyl group, phenoxyacetyl group, 4-isopropylphenoxyacetyl group ethyl group, 4-tert-butylphenoxyacetyl group, acetyl group, benzoyl group, iso Butyryl group, dimethylformamidinyl group, 9-fluorenylmethyloxycarbonyl group Among them, phenoxyacetyl group, 4-isopropylphenoxy Acetyl group, acetyl group, benzoyl group, isobutyryl group and dimethylformamidinyl Furthermore, the carbonyl group of the nucleobase may be protected, for example by a phenol group. 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol , 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol phenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol , pentafluorophenol, 4-pivaloyloxybenzyl alcohol, 4-nitrophenyl Phenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl ) Ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethyl Carbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride Chloride, 1-pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride, difluoromethane The compound can be protected by reacting it with phenylcarbamoyl chloride or the like.

[0019] In some cases, a carbonyl protecting group may not be specifically introduced. In addition to the group, any one to three substituents (e.g., , a halogen atom, an alkyl group, an aralkyl group, an alkoxy group, an acyl group, an alkoxyalkoxy group alkyl group, hydroxy group, amino group, monoalkylamino, dialkylamino, carboxy modified nucleobases (e.g., substituted nucleobases with cyano, nitro, etc.); , 8-bromoadenylic group, 8-bromoguanyl group, 5-bromocytosyl group, 5-iodosyl group Tosyl group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, 5 -methylcytosyl group, 8-oxoguanyl group, hypoxanthinyl group, etc.) are also referred to as "nucleic acid salts" The term "group" is included in the above.

[0020] The substantially uncharged phosphorus-containing backbone in the oligonucleotide analogues is The majority of the bonds, e.g., 60% to 100%, are uncharged at physiological pH and have one linker. The analogues contain 5 to 40 subunits, typically about 8 to 10 subunits. The analogue contains 25, preferably about 12 to 25, monomeric subunits. It may have exact sequence complementarity to the sequence, or may have an approximate complementarity, as defined below. It may have complementary properties.

[0021] A "monomer" or "subunit" of an oligonucleotide analog is one nucleic acid of the analog. refers to a nucleotide (or nucleotide analog) unit. The term refers to the attached subunits. It refers to a nucleotide unit with or without inter-linkages, but also refers to a "charged subunit" In such cases, the charge is typically at the intersubunit bond (e.g., phosphate or phosphorothioate). oate bond).

[0022] "Morpholino oligonucleotide analogs" refer to morpholino subunits of the type shown in FIG. An oligonucleotide analogue is composed of a unit structure, wherein the structure is composed of 1 atom to The morpholino nitrogen of one subunit is linked together by a phosphorus-containing bond three atoms long. The "B" portion connects the base to the 5' exocyclic carbon of the adjacent subunit, forming a base-specific hydrogen bond. a purine or pyrimidine base available for binding to a base in a polynucleotide by The base pairing moiety of a purine or pyrimidine is typically adenine, cytosine, or thiazolinone. The amino acids are cysteine, guanine, uracil, or thymine. The properties of the composite are described in U.S. Patent Nos. 5,698,685, 5,217,866, and 5,1 No. 42,047, No. 5,034,506, No. 5,166,315, No. 5,52 Nos. 1,063 and 5,506,337, all of which are incorporated herein by reference. The present application is hereby incorporated by reference in its entirety.

[0023] The subunits and bonds shown in Figure 4 are used for the six atom repeating unit backbone, where The six atoms are the morpholino nitrogen, the attached phosphorus atom, and the carbon atom connecting the phosphorus atom to the 5' exocyclic carbon. the atom (usually oxygen), the 5' exocyclic carbon, and the next two carbon atoms of the morpholino ring) In these structures, the atom connecting the 5' exocyclic morpholino carbon to the phosphorus group is sulfur, nitrogen, The X moiety pendant from the phosphorus may be carbon or oxygen. Any stable group that does not interfere with the function of the alkyl group, including fluoro, alkyl, alkoxy, thioalkoxy, and It includes alkylaminos containing cyclic amines, all of which do not interfere with base-specific binding. Alkyl, alkoxy and thioalkoxy can be substituted in various ways unless otherwise specified. may contain 1 to 6 carbon atoms. Alkylamino refers to a lower alkyl (C1-C6) substituent. It is preferable that the cyclic amine optionally contains 1 to 2 atoms selected from oxygen, nitrogen and sulfur. Preferably, Y is a 5- or 7-membered nitrogen heterocycle containing an additional heteroatom. is oxygen.

[0024] Preferred morpholino oligomers are phosphorodiamidate-linked morpholino oligomers. and are referred to herein as PMOs. Such oligomers are shown in FIG. morpholino subunit structures, wherein X=NH2, NHR, or NR2 (where , R is lower alkyl, preferably methyl), Y=O, and Z=O, and B is a salt A polymer that is effective in binding to bases in polynucleotides by group-specific hydrogen bonding. In FIG. 4, X is a base pairing moiety of a methyl group or a pyrimidine group. Y=NH or NR (where R is lower alkyl), for example methoxy or ethoxy; ), structures with alternative phosphorodiamidate linkages where Y=O are also preferred.

[0025] In particular, with respect to an alkyl group, an alkoxy group, a thioalkyl group, or an alkylamino group, The term "is" refers to, for example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino, oxo (keto), nitro, cyano, etc. Substituents containing heteroatoms or various acids such as carboxylic acids, sulfonic acids, phosphonic acids, etc. or ester. The term "substituted" also refers to the replacement of a hydrogen atom on a carbon by Hydrogen atoms on heteroatoms (amino) by alkyl, carbonyl, or other carbon-containing groups It can also refer to the substitution of an alkyl group (e.g., aryl hydrogen).

[0026] Sequences are "complementary" to one another if hybridization occurs in an antiparallel configuration A double-stranded polynucleotide is described as "complementary" to another polynucleotide. The targeting sequence can be "approximately" or "substantially" the target sequence. Even if they have complementarity, they can still function for the purposes of the present invention. The tide analog preferably has at most one mistake in 10 nucleotides with the target sequence. Preferably, there is at most one mismatch in 20 matches. The antisense oligomers shown comprise the exemplary targeting sequences shown herein and It has at least 90% sequence identity, preferably at least 95% sequence identity. Thus, oligomers produced by the methods of the present disclosure are applicable to antisense therapy. These compounds are particularly useful as therapeutic antisense molecules when administered to treat various disease states. be.

[0027] Substantially above 45°C, preferably at least 50°C, typically 60°C to 80°C or When the oligomer hybridizes to the target under physiological conditions, the Tm is higher than Oligonucleotide analogs "specifically hybridize" to target polynucleotides Such hybridization is preferably performed under stringent hybridization conditions. At a given ionic strength and pH, the Tm corresponds to the 50% target sequence affinity. is the temperature at which a given polynucleotide will hybridize to a complementary polynucleotide. The amplification is as effective as the antisense amplification of the target sequence, as it is with exact complementarity. This can occur through "approximate" or "substantial" complementarity of the oligomers.

[0028] A "nuclease-resistant" oligomeric molecule (oligomer) is one whose backbone is made of a non-hybridized In its purified or hybridized form, it is resistant to common extracellular and intracellular nucleases in the body. The term "oligomer" refers to an oligomer that is substantially resistant to nuclease cleavage by nucleases. - little or no activity under normal nuclease conditions in the body to which the oligomer is exposed No nuclease cleavage is shown.

[0029] A "heteroduplex" is a duplex formed between an oligonucleotide analog and the complementary portion of a target RNA. "Nuclease-resistant heteroduplex" refers to a heteroduplex that is a double-stranded RNA / Intracellular and extracellular enzymes such as RNase H, which can cleave RNA or RNA / DNA complexes The phase is substantially resistant to in vivo degradation by extracellular nucleases. refers to the heteroduplex formed by binding of an antisense oligomer to its complementary target. vinegar.

[0030] The oligonucleotide analogs of the present disclosure are capable of specifically binding to target RNA sequences in cells. The base specificity of such binding is sequence specific. For example, The nucleotides are capable of specifically binding to single-stranded polynucleotides that are complementary in sequence. .

[0031] As noted above, the antisense oligomer is directed to the viral genome, preferably the 5' and a base sequence directed to a targeting moiety of either -CS or 3'-CS. The oligomers, when administered to infected host cells, e.g., to infected animal subjects, This requirement is due to the fact that the oligomer If the compound (a) has the ability to be actively taken up by mammalian cells, and (b Once incorporated, it forms a duplex with the target ssRNA at a Tm greater than about 50°C. It is fulfilled when

[0032] As described below, the ability to be taken up by cells depends on whether the oligomeric backbone is substantially The oligomeric structure must be free of charge and preferably has an active crossing mechanism across the cell membrane. or facilitated transport. The ability to form a stable duplex with RNA is determined by the factors mentioned above, antisense oligonucleotides for the target. The length and degree of complementarity of the oligomer, the ratio of G:C to A:T base matches, and any The degree of mismatch depends on the oligomer backbone as well as the position of the mismatched base. The ability of oligomers to resist cellular nucleases is crucial for survival and delivery of oligomers to the cytoplasm of cells. This promotes final delivery.

[0033] The following method converts any given substantially uncharged backbone into a molecule that meets these requirements. It is useful for testing the ability of

[0034] Active or facilitated uptake by cells Antisense oligomers penetrate host cell membranes when administered in free (uncomplexed) form. by facilitated or active transport over the It can be taken up by the host cell by the endocytic mechanism.

[0035] In instances where the agent is administered in free form, the antisense compound should be substantially uncharged. This means that the majority of the intersubunit bonds are uncharged at physiological pH. Experiments conducted in support of this technology have shown that the detection of small net charges, e.g., 15-mer One to two charges on a ~20-mer oligomer is, in fact, essentially uncharged bone. This shows that the cellular uptake of certain oligomers with a charge can be enhanced. , the oligomer itself, e.g., may be carried in the backbone bonds, or may have terminal charged group appendages ( The number of charged bonds may be Preferably, there is no more than one charged bond for every four uncharged bonds. Not more than 1 band per 10 uncharged bonds or not more than 1 band per 20 uncharged bonds More preferably, the oligomer is fully charged. do not have.

[0036] Oligomers can also be negatively and positively charged, as long as the opposite charges are present in approximately equal numbers. The oligomer may contain more than 3 to 5 bonds of either charge. Preferably, the oligomer does not contain a run of consecutive subunits. represents a given number of anionic linkages, e.g., phosphorothioate or N3'→P5' phospho A comparable number of phosphoramidate bonds, such as N,N-diethylenediamine phosphoramidate The net charge may be neutral or at most 100 per oligomer. A net charge of 1-2 is preferred.

[0037] In addition to being substantially or completely uncharged, antisense agents have the ability to cross cell membranes and interact with membranes that facilitate or actively transport oligomers; are substrates (i.e., membrane protein(s)) for the ATP transporter system This feature is preferably achieved by the following: It can be identified by one of several tests.

[0038] The first test places selected charged oligomers of oligomeric compounds on the cell surface. Ability to be replaced or displaced by selected charged oligomers on the cell surface Binding at cell surface receptors is assessed by measuring the force. Approximately 10 nM to 300 with a given amount of test oligomer, which is typically fluorescently labeled, at a final oligomer concentration of 100 nM. Immediately after, for example, 10 to 30 minutes (for the test oligomer), the cells are incubated with the Before significant internalization can occur), gradually increasing concentrations of the displacement compound are added. If the test compound is able to bind to the cell surface receptor, the displacing compound is The displacing compound is observed at a concentration 10 times or less than the test compound concentration. If the test compound is shown to cause 50% displacement, it is considered a displacing compound. They are thought to bind at recognition sites for the same cellular transport system.

[0039] The second test involves determining whether the test compound binds a labeled reporter, such as a fluorescent reporter, to the cell. Cellular transport is measured by examining the ability of the drug to transport ATP. Incubate cells in the presence of labeled test compound added at a final concentration of 1 for 30 min. After a 20-minute incubation, intracellular labeling is assessed, e.g., by microscopy. The presence of significant intracellular labeling indicates that the test compound is involved in facilitated or active transport. Therefore, it is evidence of transportation.

[0040] Antisense compounds may also be administered in a complexed form, where the complexing agent is typically Typically, the polymer, e.g., has a charge opposite to any net charge on the antisense compound. , cationic lipids, polypeptides or non-biological cationic polymers. The present invention also includes bilayer complexes between cationic oligonucleotides and cationic lipids or other polymeric components. There are many well-known methods for forming complexes, such as the cationic lipid DOTM. A(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-tolumethylan ammonium chloride) and the neutral phospholipid DOPE (dioleylphosphatidylethanolamine) Lipofectin™, a liposomal composition containing methylamine, is widely used. After administration, the complex typically undergoes endosomal encapsulation, involving particle encapsulation in endosomal bodies. It is taken up by cells through the endocytic mechanism.

[0041] Alternatively, the antisense compound may be linked to the 5' or 3' end of the antisense oligomer. The peptide may be administered in the form of a conjugate with an arginine-rich peptide. is typically 8 to 16 amino acids, including arginine, phenylalanine, and Consists of a mixture of cysteine ​​and other amino acids. Peptide conjugate oligomers Exposure of cells to the RNA results in enhanced cellular uptake and delivery to the RNA target.

[0042] Alternatively, the labeled compound may be administered to the animal, and the animals may be assayed several hours after the oligomer is administered. A simple method is to assay a body fluid sample taken from a subject for the presence of heteroduplexes with a target RNA. Extensive in vivo testing is required to establish the essential properties of an oligomer with any given backbone. It can be recognized.

[0043] Substantial resistance to RNase H Two general mechanisms to explain the inhibition of expression by antisense oligonucleotides. First, the formation of a chromosome between the oligonucleotide and the viral RNA has been proposed. The teloduplex acts as a substrate for RNase H, resulting in cleavage of the target RNA. Oligonucleotides that belong or are proposed to belong to this class include phosphatidylcholinesterase inhibitors, Phosphothioates, phosphotriesters and phosphodiesters (unmodified "natural" Such compounds include oligomers in an oligomer:RNA duplex structure. It exposes the viral RNA to hydrolysis by RNase H, rendering it nonfunctional.

[0044] "steric blockers," or "RNase H inactive" or "RNase H inactive" The second class of oligonucleotide analogs, termed "RNase H resistant," are resistant to RNase H. It has not been observed to act as a substrate for target RNAs, and it has not been shown to mediate nucleocytoplasmic transport or spiracle transport. It is thought to act by sterically blocking isolating, translation, or replication. The classes are methylphosphonates, morpholino oligonucleotides, and peptide nucleic acids (PNAs). ), certain 2'-O-allyl or 2'-O-alkyl modified oligonucleotides, and N3 '→N5' phosphoramidate.

[0045] The test oligomer is allowed to form an RNA:oligomer duplex with the test compound, and then by incubating the duplex with RNase H under standard assay conditions. Exposure to RNase H can be used to assay for RNase H resistance. The presence or absence of an intact duplex can then be monitored by gel electrophoresis or mass spectrometry. Cut.

[0046] In vivo uptake The rapid test is to determine whether a given antisense oligomer type has the necessary properties mentioned above, i.e. High Tm, ability to be actively taken up by host cells, and resistance to RNase H This method exists to ensure that a properly designed amplifier provides adequate resistance. The compound stably binds to the complementary portion of the target RNA when administered to a mammalian subject. The heteroduplexes then appear in urine (or other body fluids). The details of this method are based on the discovery that non-invasive methods for detecting target RNA are U.S. patent application Ser. No. 09 / 099,599, entitled "A Non-Invasive Method for Detecting Target RNA" No. 736,920, the disclosure of which is incorporated herein by reference. Briefly, a test scaffold containing a target sequence against a known RNA is used. The experimental oligomer is injected into an animal, e.g., a mammalian subject. , directed against any intracellular RNA, including host RNA or the RNA of an infectious virus A few hours after administration (typically 8 to 72 hours), the antisense RNA The urine is assayed for the presence of heteroduplexes. If heteroduplexes are detected, the bone marrow The oligonucleotides are suitable for use in the antisense oligomers of the invention.

[0047] The oligomers, when appropriate for mammalian subjects, may be used to facilitate subsequent analysis. For example, the assay may be labelled with a fluorescent or radioactive tag. They may be in solid phase or fluid format. Generally, solid phase assays are performed by first Binding the double-stranded analyte to a solid support, such as a particle or polymer, or a test strip substrate. In a fluid phase assay, the presence / amount of the analyte is detected by The sample is typically pretreated to remove interfering sample components. If present, the presence of the heteroduplex is confirmed by detecting the labeled tag. For the compound, the heteroduplex can be identified by immunoassay in the case of a solid phase format. or in solution or suspension format by mass spectrometry or other known methods. It can be detected by

[0048] Exemplary Oligomeric Backbones Examples of non-ionic linkages that can be used in oligonucleotide analogs are shown in Figure 4. wherein B is a polynucleotide preferably selected from adenine, cytosine, guanine and uracil. Purine available to bind to the base in a nucleotide by base-specific hydrogen bonding or pyrimidine base pairing moiety. A preferred backbone structure is carbonate (R=O). and carbamate (R=NH2) bonds; alkylphosphonate bonds and phosphotriester bonds ester bonds (R=alkyl or -O-alkyl); amide bonds; sulfone bonds and sulfo bonds amide bond (R1, R2 = CH2); and thioformacetyl bond (2E). The latter is related to phosphorothioate antisense compounds, which have enhanced duplex and It has been reported to have triplex stability. Compounds containing benzophenone have also been reported.

[0049] Preferred oligomer structures have base-pairing moieties joined by uncharged bonds. The antisense oligomers employ morpholino-based subunits. Oligonucleotides are described, for example, in commonly owned U.S. Pat. Nos. 5,698,685, 5,202,413, and 5,326,722. No. 17,866, No. 5,142,047, No. 5,034,506, No. 5,16 Nos. 6,315, 5,185,444, 5,521,063, and 5,5 No. 06,337, all of which are expressly incorporated herein by reference. To form a part.

[0050] An important property of morpholino-based subunits is their stable, uncharged backbone bonds. the ability to be linked in oligomeric form by ligation; the polymers formed are linked to target R It hybridizes with complementary target nucleic acids containing NA, as well as with short oligomers of 10 to 14 bases. nucleotide bases (e.g., adenine, cytosine, guanine or the ability to support uracil; the ability of oligomers to be actively transported into mammalian cells; and and the ability of oligomer:RNA heteroduplexes to resist RNase degradation.

[0051] The backbone structures for the antisense oligonucleotides of the present disclosure are uncharged phosphorus-containing The morpholino subunit types shown in Figure 4 are linked by heterosubunit bonds. FIG. 4 shows the phosphorus-containing linkages forming the five atom repeat unit backbone, where The phosphorino rings are linked by one-atom phosphoamide bonds. Figure 4 shows the six-atom repeating unit backbone. In this structure, the atom connecting the 5' morpholino carbon to the phosphorus group is It may be sulfur, nitrogen, carbon, or preferably oxygen. X moiety from phosphorus The pendant of the moiety may be fluorine, alkyl or substituted alkyl, alkoxy or substituted alkyl. Alkoxy, thioalkyl or substituted thioalkyl, or morpholine or piperidine The nitrogen may be unsubstituted, monosubstituted or disubstituted, including cyclic structures such as alkyl, The alkoxy and thioalkoxy preferably contain 1 to 6 carbon atoms. The moiety is sulfur or oxygen, preferably oxygen.

[0052] As mentioned above, the substantially uncharged oligomers are advantageously present in a limited number For example, up to about 1 band per every 5 uncharged bonds, more preferably every 10 There is a maximum of about one charged bond per uncharged bond. Alternative linkages, such as charged phosphoramidates or phosphorothioates, can also be used in oligomers. can be incorporated into

[0053] Another aspect of the present disclosure is a 5'-unprotected-2'-deoxy Starting with a ribonucleoside, this 5'-unprotected-2'-deoxyribonucleoside is Phosphoramidite in anhydrous acetonitrile containing 4,5-dicyaminimidazole (DCI) Diester synthons (i.e., monomers) are reacted to form phosphoramidite diester nucleic acids. and forming a dimer having an internucleotide linkage. The dimer is then subjected to boronation, sulfurization, or oxidation to produce a dimer analog. The dimer is then chemically activated by either These steps (capping and detritylation) result in the formation of a phosphoramidite. The oligomer is formed by adding a monomer to the growing oligomer. elongating the dimer (growing stepwise from dimer to trimer, tetramer, pentamer, hexamer, etc.) This process is repeated as desired to obtain a morpholino phosphoramidate oligomer. is contacted with iodine and dimethylamine in tetrahydrofuran to give morpholinoborane. The sulphoamidate is converted to N,N-dimethylamino PMO. The oligomer is then hydrated. from the polystyrene support by contacting it with a solution containing ammonium oxide and ethylenediamine. The addition of repeated monomers to these oligomers is generally This may be done in a commercially available DNA synthesizer, allowing synthesis in a cost-effective manner. Efficiency and speed can be greatly enhanced.

[0054] Exemplary monomers for use in the synthetic methods of the present disclosure include the following: [ka] (In the formula, B or B * was a silyl protecting group, or an acid labile protecting group, or a base labile protecting group. It is okay to X = cyanoethyl or its derivatives, alkyl, thioalkyl, thiocarbonyl, carboxylate, acetate, or formate derivatives, R=dimethoxytrityl, trimethoxytrityl, or any silyl-based protecting group can be, R1 and R2 are independently isopropyl, C 2~20 Straight or branched alkyl chain or a 5- to 7-membered aliphatic ring) Examples include luamidite.

[0055] Each publication or patent cited herein is hereby incorporated by reference in its entirety. It forms part of a book.

[0056] The disclosure generally described herein is merely illustrative of certain aspects of embodiments of the disclosure. This will be more readily understood by reference to the following examples, which are included for purposes of illustration. The techniques and methods of the present invention satisfy the claims and deviate from the scope of the claimed disclosure. It will be appreciated by those skilled in the art from the teachings above and the examples below that the present invention can be used without the need for As such, they are not intended to limit the disclosure. [Example]

[0057] Example 1 Synthesis of N-di-tertbutylisobutyl-protected morpholinophosphoramidite To generate borane phosphoramidate morpholino bonds, all four bases are required. A phosphoradiamidite synthon was developed (see the synthesis scheme in Figure 3). Protocol (Zhang, et al., Tetrahedron Letters 2008, 49, 3570, Pattanayak, et al ., Nucleosides, Nucleotides and Nucleic Acids, 2012, 31, 763-782) The synthesis of the radiamidite thymidine morpholino monomer synthon was carried out. Thymidine was dissolved in dimethoxytrityl chloride (DMT-Cl) in anhydrous pyridine under atmospheric pressure. This is treated with 5'O-DMTr-thymidine (7) to give 5'O-DMTr-thymidine (7), which is then reacted with sodium periodate. Treatment with sodium followed by ammonium diborate gave 2',3'-dihydroxy-morpho Cyanoborohydride was used under mildly acidic conditions to generate cyano-thymidine (not shown). Reduction of the 2' and 3' hydroxyl groups was carried out using sodium (without further purification). Then, under an argon atmosphere, dichloromethane was added to generate the thymine morpholino monomer (8). 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidin in acetone Phosphitylation of compound (8) with methylaminoimidazole and 4,5-diaminoimidazole (DCI) The basic unit thymidine synthon (9) was prepared in 86% yield. Compound (9) does not require base protection.

[0058] We also prepared 5'-dimethoxytrityl-morpholinomethylidin according to a similar protocol. Thymidine-3'-O-methyl-N,N-diisopropylphosphoradiamidite was synthesized. However, solid-phase coupling with cyanoethyl phosphoramidites was more efficient than with the O-methyl counterparts. was also found to be efficient.

[0059] The synthesis of the corresponding monomers of cytosine, adenine, and guanine was carried out using various previously reported methods. Protocol (Roy, S., Olesiak, M., Shang, S., Caruthers, M. H., J. Am. Chem. Soc. 2013, 135:6234-41) to first convert the nucleoside amino group to bis(tert-butyl) ) isobutylsilyl (BIBS) protection.

[0060] 5',3',2'-tri-O-acetylcytidine (10 in) in the presence of 2,6-lutidine ) with BIBS-OTf (Tf = triflate) under argon atmosphere for 60 After stirring at 5°C for 2 hours, 11 was obtained in 76% yield. However, the reaction The synthesis of 16 and 21 was achieved in 25%–31% (16) and 74% (21) yields, respectively. A much longer reaction time (3 days) was required. As shown in the synthesis scheme in Figure 3, These protected (silylated) ribonucleosides (11, 16, 21) were first reacted with ammonium hydroxide. to remove the acetyl protecting group to give the 5'-dimethoxy-trityl compound (12, 17, 22), which were then converted to morpholino derivatives (13, 18, and 23). This mixture was used to generate phosphorodiamidite synthons 14, 19 and 24.

[0061] General Procedure for the Synthesis of Morpholino Nucleosides The 5'-dimethoxytrityl protected nucleoside was dissolved in methanol, and then 1.2 equivalents of Sodium periodate and ammonium diborate tetrahydrate (1.2 equivalents) were added. The mixture was stirred at room temperature for 3 hours, after which TLC showed complete consumption of the starting material. The material was filtered through a pad of celite and the activated powder 4A° molecular weight was After adding 2.0 equivalents of cyanoborohydride to each sieve (0.4 g / mmol), Sodium hydroxide and glacial acetic acid were added, and the reaction mixture was stirred for 4 to 5 hours. The intermediate diol was completely reduced. The reaction mixture was filtered through a pad of Celite. The product was dissolved in chloroform and washed with saturated NaHCO3 and brine. The organic phase was collected, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The product was purified by flash chromatography on a silica gel column. The silica gel slurry was mixed with the starting eluent containing an additional 5% triethylamine. After pouring the slurry, two column volumes of triethylamine-free The column was washed with the starting solvent mixture: 8, 13, and 18 were purified with 19:1 chloroform:methanol. The column was eluted using a gradient of chloroform to alcohol. Compound 23 was purified using a gradient from 7:3 to 7:3 ethyl acetate:hexane. All yields given are for 5'-dimethoxytrityl-N-BIBS protected nucleosides. The results are shown for two steps starting from

[0062] 5'-Dimethoxytritylmorpholinothymidine (8): Yield: 53%. 1 H NMR(C DCl3,400MHz)δ:7.46~7.44(2H,m), 7.35~7.20( 7H,m), 6.85~6.81(4H,m), 5.78~5.75(1H,dd), 4 .02~3.98(1H,m), 3.79(6H,s), 3.28~3.25(1H,m ), 3.16~3.12(1H,dd), 3.08~3.04(2H,m), 2.43( 3H,s). 13 C NMR(CDCl3)δ:164.22, 158.49, 150. 45, 144.72, 135.90, 135.78, 135.32, 130.06, 13 0.03, 128.12, 127.81, 126.83, 113.10, 113.09, 110.82, 86.02, 80.90, 64.43, 55.20, 45.93, 11. 84. ESI-MS(m / z):561.2314(M+H) + .

[0063] N 2 -Di(tert-butyl)isobutylsilyl-5'-dimethoxytritylmorpholino Cytidine (13): Yield: 64%. 1 H NMR (CDCl3, 400 MHz) δ: 7. 59~7.58(1H,d), 7.53~7.51(2H,m), 7.41~7.25( 7H,m), 6.91~6.87(4H,m), 5.88~5.87(1H,d), 5. 79~5.76(1H,dd), 4.70(1H,bs), 4.03~4.00(1H, m), 3.82(6H,s), 3.29~3.24(2H,m), 3.14~3.10( 1H,m), 3.04~3.00(1H,d), 2.67~2.62(1H,m), 2. 52~2.46(1H,m), 2.15~2.08(1H,m), 1.18~1.17( 19H,m), 1.07~1.03(8H,m). 13 C NMR(CDCl3) δ:1 68.25, 158.63, 154.57, 145.06, 140.50, 136.02 , 135.95, 130.06, 130.02, 128.11, 127.88, 126. 73, 113.06, 96.3588.95, 81.76, 77.94, 64.70, 5 5.17, 50.01, 47.32, 46.30, 28.68, 26.24, 26.14 , 24.82, 21.02, 20.72, 20.67, 11.65. ESI-MS(m / z):727.4047(M+H) + .

[0064] N 2 -Di(tert-butyl)isobutylsilyl-5'-dimethoxytritylmorpholino Adenosine (18): Yield: 61%. 1 H NMR (CDCl3, 400 MHz) δ:8 .38(1H,s), 8.01(1H,s), 7.51~7.48(2H,m), 7.3 9~7.24(7H,m), 6.88~6.85(4H,m), 5.90~5.86(1 H,dd), 4.11~4.08(1H,m), 3.82(6H,s), 3.33~3. 26(2H,m), 3.15~3.10(2H,m), 2.79~2.73(1H,m) , 2.19~2.12(1H,m), 1.22~1.02(27H,m). 13 CNM R(CDCl3)δ:158.64, 158.11, 152.55, 148.92, 14 4.97, 144.96, 137.76, 135.90, 130.04, 128.06, 127.80, 126.74, 121.60, 121.52, 117.79, 117.7 1, 113.04, 86.06, 80.44, 76.89, 64.39, 60.14, 5 9.88, 50.16, 49.93, 48.49, 47.36, 47.12, 46.08 , 46.00, 43.93, 43.83, 43.71, 26.82, 26.28, 26. 19, 24.80, 24.36, 24.27, 24.18, 21.04, 20.81, 2 0.79, 20.35, 20.07. ESI-MS(m / z):751.4238(M+ H) + .

[0065] N 2 -O 6 -bis[di(tert-butyl)isobutylsilyl-5'-dimethoxytrimethylsilyl] Morpholinoguanosine (23): Yield: 47%. 1 H NMR (CDCl3, 400 M Hz)δ:7.80(1H,s), 7.51~7.49(2H,m), 7.39~7.3 2(6H,m), 7.28~7.25(1H,m), 6.89~6.87(4H,m), 5.74~5.71(1H,m), 4.05(1H,bs), 3.82(6H,s), 3 .31~3.26(1H,m), 3.23~3.15(2H,m), 3.13~3.07 (1H,m), 2.76~2.70(1H,m), 2.26~2.19(1H,m), 2 .15~2.09(1H,m), 1.93(1H,bs), 1.21~1.18(36H ,m), 1.09~1.07(2H,m), 1.03-0.99(16H,m). 13 C NMR(CDCl3)δ:160.69, 159.39, 158.62, 154.10 , 145.01, 135.56, 135.85, 130.02, 128.06, 127. 79, 126.73, 116.84, 113.06, 85.99, 80.73, 77.4 1, 64.42, 60.23, 49.83, 47.78, 26.31, 24.89, 24 .72, 21.94, 21.40, 20.73. ESI-MS(m / z):965.59 44(M+H) + .

[0066] Referring again to Figure 3, synthesis of appropriately protected synthons (9, 14, 19 or 24) The procedure for is as follows:

[0067] The 5'-O-DMT-N-BIBS protected morpholino nucleosides (8, 1 3, 18 or 23) were dried under vacuum overnight. After dissolving them in anhydrous CH₂Cl₂, 1.2 equivalents of 2-cyanoethyl-N,N,N',N'-tetraisopropyl Phosphorodiamidite was added. 0.5 equivalents of 4,5-dicyanoimidazole was added. After that, the reaction was allowed to proceed at room temperature under an argon atmosphere for 5 hours with stirring. At this time, TLC showed complete conversion of the starting material. The reaction mixture was diluted with CH2Cl2 and The organic layer was washed with 5% NaHCO3 solution and then with brine. The organic layer was dried over Na2SO4. The product was purified by silica gel column chromatography. A mixture of the starting eluent containing an additional 5% triethylamine was used to elute the silica gel slide. After pouring the slurry, two column volumes of starting solution without triethylamine were added. The column was washed with the solvent mixture. A gradient of 3:7 ethyl acetate:hexane to 1:1 ethyl acetate:hexane was used to refine the Made.

[0068] 5'-O-Dimethoxytritylmorpholinothymine-3'-O-cyanoethyl-N,N-di Isopropyl phosphorodiamidite (9): Yield: 86%. 31 P NMR (CDCl 2) δ: 127.21, 126.08. 1 H NMR (CD2Cl2, 400 MHz) δ :9.51(1H,bs), 7.50~7.47(2H,m), 7.38~7.25(8 H,m), 6.89~6.86(4H,m), 5.78~5.75(0.5H,dd), 5.65~5.62(0.5H,dd), 4.08~4.02(1H,m), 3.99~ 3.86(3H,m), 3.82(6H,s), 3.65~3.52(2H,m), 3. 48~3.34(1H,m), 3.31~3.27(1H,m), 3.13~3.09( 1H,m), 2.75~2.68(2H,m), 2.53~2.47(2H,m), 1. 96(3H,m), 1.25~1.18(12H,m). 13 C NMR (CD2Cl2 )δ:164.02, 163.96, 158.66, 150.13, 144.95, 13 5.92, 135.90, 135.79, 135.68, 135.52, 129.99, 128.07, 127.77, 126.77, 117.89, 117.74, 113.0 5, 110.48, 110.39, 86.05, 80.54, 80.49, 80.20, 77.36, 77.21, 64.37, 60.09, 59.84, 55.20, 49.0 5, 48.83, 47.58, 47.06, 46.83, 45.87, 45.78, 43 .91, 43.74, 24.36, 24.29, 24.22, 24.20, 20.77, 20.33, 20.66, 12.29. ESI-MS(m / z):727.4047(M +H) + .

[0069] N 2 -Di(tert-butyl)isobutylsilyl-5'-dimethoxytrityl-morpholino Nocitosine-3'-O-cyanoethyl-N,N-diisopropylphosphordiamidite ( 14): Yield: 83%.31 P NMR(CD2Cl2)δ:126.32、124.8 8。 1 H NMR(CD2Cl2,400MHz)δ:7.58~7.55(1H,m) 、7.50~7.48(2H,m)、7.38~7.31(6H,m)、7.28~7. 24(1H,m)、6.88~6.85(4H,m)、5.79~5.76(1.5H, m)、5.63~5.59(0.5H,m)、4.54(1H,bs)、4.04~3. 87(3H,m)、3.82(6H,s)、3.74~3.71(0.5H,m)、3. 65~3.45(3H,m)、3.30~3.24(1.5H,m)、3.14~3.1 0(1H,m)、2.80~2.75(1H,m)、2.71~2.68(1H,m)、 2.55~2.46(1H,m)、2.38~2.31(1H,m)、2.11~2.0 7(1H,m)、1.78(1H,bs)、1.25~1.15(31H,m)、1.0 4~1.02(8H,m)。 13 C NMR(CD2Cl2)δ:168.14、158 .61、154.46、144.98、140.62、140.42、135.98、1 35.88、130.03、128.07、127.75、126.71、117.96 、117.75、113.02、96.20、96.09、85.97、81.68、8 1.34、81.23、77.05、77.00、64.50、60.21、60.09 、59.85、55.18、49.63、49.41、47.98、47.13、46. 18、45.91、45.83、43.96、43.84、43.77、28.58、2 6.21、26.07、24.78、24.38、24.26、24.17、24.12 , 20.98, 20.76, 20.71, 20.62, 20.31, 20.23. ESI -MS(m / z):927.5329(M+H) + .

[0070] N 2 -Di(tert-butyl)isobutylsilyl-5'-dimethoxytritylmorpholino Adenine-3'-O-cyanoethyl-N,N-diisopropylphosphordiamidite (1 9): Yield: 79%. 31 P NMR(CD2Cl2)δ:127.96, 125.36 . 1 H NMR(CD2Cl2,400MHz):8.38~8.37(1H,d), 8 .01~8.00(1H,d), 7.51~7.48(2H,m), 7.38~7.31 (6H,m), 7.28~7.24(1H,m), 6.88~6.86(4H,m), 5 .93~5.90(0.5H,m), 5.82~5.79(0.5H,m), 5.39( 1H,bs), 4.14~4.10(0.5H,m), 4.06~4.02(0.5H, m), 4.0~3.90(2H,m), 3.82(6H,s), 3.71~3.63(1 H,m), 3.60~3.52(2.5H,m), 3.42~3.37(0.5H,m) , 3.34~3.30(1H,m), 3.17~3.13(1H,m), 3.02~2. 91(1H,m), 2.78~2.75(1H,m), 2.72~2.68(1H,m) , 2.66~2.59(1H,m), 1.26~1.18(31H,m), 1.10~1 .08(8H,m). 13 C NMR(CD2Cl2)δ:158.64, 158.11 , 152.55, 148.92, 144.97, 137.76, 135.92, 135. 80, 130.02, 128.06, 127.77, 126.74, 121.60, 11 7.79, 117.71, 113.04, 86.50, 80.50, 80.44, 80. 34, 80.22, 76.94, 76.81, 64.39, 60.14, 59.91, 5 5.18, 50.16, 49.93, 48.49, 48.44, 47.35, 47.12 , 46.08, 46.00, 43.95, 43.83, 43.71, 28.62, 26. 28, 26.19, 24.80, 24.35, 24.29, 24.18, 21.04, 2 0.79, 20.36, 20.27. ESI-MS(m / z):951.5437(M+ H) + .

[0071] N 2 ,O 6 -Bis[di(tert-butyl)isobutylsilyl]-5'-dimethoxytrimethylsilyl methyl-morpholinoguanine-3'-O-cyanoethyl-N,N-diisopropylphosphoran Diamidite (24): Yield: 82%. 31 P NMR(CD2Cl2) δ: 127.6 2, 126.99. 1 H NMR(CD2Cl2,400MHz):7.79~7.76 (1H,d), 7.49~7.45(2H,m), 7.37~7.29(6H,m), 7 .27~7.23(1H,m), 6.87~6.84(4H,m), 5.76~5.64 (1H,dd), 4.55(1H,s), 4.05~3.88(3H,m), 3.82( 6H,s), 3.67~3.54(3H,m), 3.49~3.45(1H,m), 3. 32~3.23(1H,m), 3.19~2.90(2H,m), 2.71~2.68( 2H,m), 2.61~2.54(1H,m), 2.23~2.16(1H,m), 2. 13~2.06(1H,m), 1.27~1.15(49H,m), 1.06-0.97 (27H,m). 13 C NMR(CD2Cl2)δ:160.67, 159.38, 1 58.61, 154.33, 154.06, 144.96, 136.47, 135.90 , 135.79, 130.00, 128.02, 127.76, 126.69, 117. 58, 116.77, 113.03, 85.99, 80.69, 80.58, 79.55 , 79.49, 76.95, 76.45, 76.36, 64.22, 60.20, 60. 12, 59.88, 59.86, 55.16, 49.71, 49.47, 47.77, 4 7.68, 47.46, 47.26, 45.96, 45.89, 43.99, 43.88 , 43.77, 43.65, 28.76, 28.07, 26.39, 26.31, 26. 27, 24.87, 24.68, 24.44, 24.37, 24.27, 24.19, 2 1.90, 21.37, 20.83, 20.76, 20.75, 20.69, 20.35 , 20.32, 20.27, 20.24. ESI-MS(m / z):1165.7203 (M+H) + .

[0072] For the synthesis of PMO-DNA chimeras, 5'-DMT-2'-deoxyribonucleoside -3'-phosphoramidite synthons 26, 27 and 28 were synthesized according to literature protocols (Roy, et al. ., J. Am. Chem. Soc. 2013, 135, 6234-6241). Compound 25 was commercially available. was obtained from a readily available supplier (Glen Research).

[0073] General method for the synthesis of 26, 27, and 28 5'-Dimethoxytrityl-N-BIBS protected 2'-deoxynucleosides were Anhydrous dichloromethane and 2-cyanoethyl-N-methylpropional were added to a round-bottom flask containing 2-cyanoethyl-N-methylpropional. ,N,N',N'-tetraisopropylphosphorodiamidite (1.2 equivalents) was added via syringe 1.0 equivalent of tetrazole (obtained from Glen Research in 0.1% CO₂ in CH₃CN) was added via To this solution was added dropwise with stirring over 30 minutes. The reaction was stirred at room temperature for 2-3 hours. The reaction mixture was stirred for 1 hour, at which time TLC showed the complete disappearance of the starting material. The mixture was diluted in ethanol and extracted twice with saturated NaHCO3 solution. The organic layer was dried over Na2SO4. The product was purified by flash chromatography on a silica column. The starting eluent mixture containing an additional 5% triethylamine was used to purify the silica gel. A gel slurry was prepared. After pouring the slurry, two column volumes containing no triethylamine were The column was washed with a mixture of starting solvents: 26 and 27 in 3:7 ethyl acetate:hexane. 28 was purified using a gradient from 1:1 ethyl acetate:hexane to 7:3 hexamethyl. Purification was carried out using a mixture of ethanol and dimethyl ether.

[0074] N 2 -Di(tert-butyl)isobutylsilyl-5'-dimethoxytrityl-2'-de Oxycytidine 3'-O-cyanoethyl-N,N-diisopropylphosphoramidite ( 26). Yield: 87%. 31 P NMR(CD2Cl2): 148.67, 148.43 . 1 H NMR(CD2Cl2,400MHz)δ:7.88~7.80(1H,dd) , 7.49~7.45(2H,m), 7.37~7.27(7H,m), 6.90~6. 86(4H,m), 6.35~6.29(1H,m), 5.53~5.51(1H,m) , 4.71~4.62(1H,m), 4.49(1H,bs), 4.20~4.16(1 H,m), 3.89~3.77(7H,m), 3.72~3.58(3H,m), 3.5 1~3.35(2H,m), 2.68~2.58(2H,m), 2.52~2.49(1 H,m), 2.30~2.22(1H,m), 2.11~2.04(1H,m), 1.2 9~1.25(1H,m), 1.23~1.20(9H,m), 1.14~1.10(2 2H,m), 1.02-0.97(9H,m). 13 C NMR(CD2Cl2)δ:1 58.71, 155.07, 144.68, 140.67, 135.58, 130.13 , 128.15, 127.89, 126.90, 117.75, 117.60, 113. 12, 96.12, 96.04, 86.60, 85.82, 85.77, 85.16, 8 5.11, 84.98, 84.92, 62.91, 62.61, 58.64, 58.26 , 43.30, 43.14, 40.75, 40.53, 28.59, 26.10, 24. 79, 24.33, 24.26, 20.66, 20.41, 20.18. ESI-MS( m / z): 928.5201 (M+H) + .

[0075] N 2 -Di(tert-butyl)isobutylsilyl-5'-dimethoxytrityl-2'-de Oxyadenosine 3'-O-cyanoethyl-N,N-diisopropylphosphoramidite (27). Yield: 81%. 31 P NMR(CD2Cl2): 148.51, 148.4 9. 1H NMR(CD2Cl2,400MHz)δ:8.30(1H,s)、7.98 ~7.96(1H,d)、7.47~7.44(2H,m)、7.37~7.34(4H ,m)、7.31~7.23(3H,m)、6.87~6.83(4H,m)、6.46 ~6.43(1H,m)、4.86~4.78(1H,m)、4.34~4.28(1H ,m)、3.92~3.82(7H,m)、3.78~3.62(3H,m)、3.47 ~3.32(2H,m)、3.02~2.94(1H,m)、2.70~2.57(2H ,m)、2.55~2.52(1H,m)、2.18~2.11(1H,m)、1.25 ~1.16(30H,m)、1.10~1.08(3H,m)、1.03~1.00(7 H,m)。 13 C NMR(CD2Cl2)δ:158.64、158.09、152. 37、149.19、144.86、138.72、135.86、135.67、13 0.11、129.99、128.09、127.78、126.74、122.27、 117.76、117.64、113.05、86.31、85.66、85.45、8 4.27、74.14、73.97、73.54、73.37、63.67、63.53 、58.54、58.26、43.30、43.18、39.03、38.93、28. 62、26.25、26.21、24.80、24.42、24.38、24.31、2 1.02、20.79、20.44、20.37、20.29、20.22。ESI-M S(m / z):952.5279(M+H) + 、974.5130(M+Na) + 。

[0076] N 2-Di(tert-butyl)isobutylsilyl-5'-dimethoxytrityl-2'-de Oxyguanosine 3'-O-cyanoethyl-N,N-diisopropylphosphoramidite (28). Yield: 79%. 31 P NMR(CD2Cl2): 149.03, 148.5 2. 1 H NMR(CD2Cl2,400MHz)δ:7.83~7.77(1H,d) , 7.51~7.48(2H,m), 7.39~7.24(7H,m), 6.90~6. 86(4H,m), 6.41~6.38(1H,t), 4.65~4.60(1H,m) , 4.54(1H,bs), 4.30~4.28(1H,m), 3.86~3.76(7 H,m), 3.73~3.61(3H,m), 3.43~3.33(2H,m), 2.8 5~2.45(4H,m), 2.25~2.17(1H,m), 2.12~2.05(1 H,m), 1.23~1.15(48H,m), 1.06-0.97(18H,m). 1 3 C NMR(CD2Cl2)δ:160.64, 159.27, 158.67, 154 .21, 144.77, 136.28, 135.71, 135.60, 130.08, 1 28.11, 127.87, 126.80, 117.56, 117.14, 113.13 , 86.39, 85.40, 85.09, 83.79, 83.63, 63.78, 63. 48, 58.37, 58.16, 43.34, 43.18, 40.88, 40.55, 2 8.74, 28.09, 26.30, 26.17, 24.89, 24.69, 24.52 , 24.45, 24.36, 24.29, 21.90, 21.37, 20.77, 20. 73, 20.71, 20.62, 20.40, 20.33, 20.22, 20.15. E SI-MS(m / z):1188.6903(M+Na) + .

[0077] Example 2 Morpholino oligonucleotides using BIBS-protected morpholino phosphoramidites Synthesis of Once the synthon described in Example 1 was available, the next goal was to develop a borane phospho To generate morpholino derivatives of thiamidates and convert these compounds to the corresponding PMOs, The goal was to optimize the solid-phase synthesis cycle. The synthesis cycle is outlined in Figure 4 and Table 1. Prior to synthesis, 5′-deoxyribothymidine was attached to a polystyrene support. The '-DMT group was removed by cleavage in 10% trimethylphosphite borane (TMPB) in chloroform. The residue was removed with 0.5% trifluoroacetic acid in HCl.

[0078] In anhydrous acetonitrile containing 4,5-dicyanoimidazole (DCI), 5' Unprotected 2'-deoxyribonucleosides (Figure 4, Compound A) were synthesized using synthons 9, 14, and 1 9 or 24 (Figure 4, "1. Condensation") to give a phosphoramidite diene A dimer with a steric internucleotide linkage was generated (Figure 4, compound B). The waiting time was 300 seconds. Following detritylation (Figure 4, "4. Deprotection"), this cycle Repeated rounds of cloning produced a product ready for further conversion to PMO.

[0079] [Table 1]

[0080] After synthesis, the support was washed with acetonitrile to remove the cyanoethyl groups from the internucleotide bonds. To remove the ions, the mixture was treated with a 1:1 mixture of triethylamine and acetonitrile for 600 seconds ( (See Figure 4, "5. Removal of cyanoethyl ester"), and wash with acetonitrile and dichloromethane several times. The residual triethylamine was removed by filtration and dried. The polystyrene support was then removed from the column. The solution was removed and placed in a 1.5 mL screw-cap glass reaction vial.

[0081] To convert morpholinoborane phosphoramidates to N,N-dimethylamino PMOs , morpholinoborane phosphoramidate, 0.05 M iodine, and 2.0 M dimethylamine The mixture was treated with a tetrahydrofuran solution of iodine overnight (Figure 4, "6. Iodine / dimethylamine"). The resin was washed repeatedly with acetonitrile, and then the oligonucleotide was added to tetrabutylammonium chloride. The BIBS protecting group was removed by treatment with a 1.0M THF solution of tetraethylammonium fluoride (TBAF). (Figure 4, "7. Silyl deprotection"). (Et3NHF adds a phosphorodiamidate bond.) This reagent could not be used to remove these silyl groups due to hydrolysis. Ta).

[0082] When the silyl protecting group was removed and then oxidative amination was performed, the N-4 of cytosine was Amine substitution was observed (Figure 5). As shown in reaction steps 6 and 7 of Figure 4, This problem was overcome by performing an oxidative amination reaction prior to desilylation.

[0083] Once the silyl groups are removed, the oligonucleotides are cleaved using 30% ammonium hydroxide. The cord was cut from the support (Figure 4, "8. Cutting from the support") and centrifuged with a 0.2 μm centrifuge filter. The polystyrene resin was removed using a filter, and the solution was then washed with Illustra NAP-25 colorants. The fluoride salts were removed by passing the sample through an Amicon™ Ultra-15 3K centrifuge. Using a separate filter device (to remove shorter failed sequences), The oligonucleotides were then used for further characterization and other studies. Used for.

[0084] The synthesis was carried out on an ABI 394 synthesizer. All syntheses were carried out via succinate linkage. a small amount of 5'-DMT2'-deoxythymidine tethered to a polystyrene solid support. The synthesis of morpholino oligonucleotides was carried out on a 0.2 micromolar scale using Therefore, the coupling time was increased to 300 seconds to achieve a standard 2.0 μmol synthesis size. After the detritylation step, the cyclohexyl methyl ester was washed with methanol. Dite (9, 14, 19 or 24; 0.1M) was dissolved in anhydrous CH3CN. Detritylation was performed using a 0.5% solution of TFA in anhydrous CHCl3 containing PB. Before use, the solution for boronation (0.05M BH3-THF complex in THF) For activation (4,5-dicyanoimidazole) and capping, The reagents were purchased from Glen Research. A step-by-step description of the synthesis cycle is given in Table 1. Deprotection was carried out in two steps: 1) the borane-phosphoramidate monolayer attached to the solid support; The fluorophore oligonucleotides were first prepared by dissolving them in a 1:1 solution of triethylamine in acetonitrile. After 10 minutes of treatment, the mixture was washed extensively with acetonitrile. The resin was dried by filtration and transferred to a glass vial. A solution of 2.0 M of dimethylamine was added to the glass vial. The same amount of iodine was dissolved in ammonia (2.0 M) in isopropanol, and this solution was The glass vials were then placed on a mechanical shaker for 24 hours. For the synthesis of diethylamine and morpholinodiamidate derivatives, 2.0 M THF of each amine was used. solution was used.

[0085] The vial containing the resin was centrifuged at 4000 rpm and the supernatant was removed with a pipette. After this, wash the resin 4-5 times with 2 mL aliquots of acetonitrile and shake vigorously. The mixture was centrifuged at 4000 rpm for 5 minutes and the supernatant was removed. For the morpholine, the ammonia solution in isopropanol was removed under vacuum. The lino oligonucleotides were incubated overnight in tetrabutylammonium fluoride (tetrabutylammonium fluoride). The product was then desilylated by treatment with 1.0 mL of 1.0 M THF solution. To remove the residue, the resin was treated with 1 mL of 37% ammonium hydroxide solution for 1 hour and centrifuged at 0.2 μm. A centrifuge filter was used to remove the polystyrene resin beads.

[0086] Bring the total volume of each solution up to 2.5 mL by adding 0.5 mL of Millipore water. The PMO was diluted with 1000 kJ / ml and then loaded onto an Illustra™ NAP™-25 column (GE Healthcare The columns were first equilibrated with 25.0 mL of water. After the water had completely entered the gel bed by gravity flow, 2.5 mL of sample was loaded onto the column and M The purified sample was eluted using 3.5 mL of AMICON (trademark) water. ) Using an Ultra-4 3K instrument, oligonucleotides with more than 10 nucleotides The oxidase was subjected to the second step of purification. The accumulated solution after Nap column purification (total volume 3.5 mL) was loaded into an Amicon device and centrifuged at 4000 x g for 30 minutes. The solute was similarly washed twice with 3.0 mL of Millipore water. were collected and used in various experiments.

[0087] Uridine morpholinophosphorylation with 5-(ethylthio)-1H-tetrazole (ETT) Luamidite (B * = uracil) to the siRNA sequence Incorporation and cleavage of the resulting morpholino phosphoramidite internucleotides by aqueous iodine Subsequent conversion to the corresponding phosphoramidate morpholino linkage has been reported (Te Trahedron Letters 2008, 49:3570-73, Bioorg. Med. Chem., 2009, 17:2441-46). However, we found that compounds 9, 14, 19, and 24 exhibited ETT (pK a =4. 3) activated both morpholine and N,N-diisopropylamine. (Figure 3). As a result, activation of morpholine followed by boronation and iodine / dimethylamine acid The 5'-N,N-diisopropylamino-phosphoro A capped PMO oligonucleotide bearing a diamidate (Figure 4, Compound C) was also used. Therefore, very low yields of the final PMO product were obtained according to their literature protocol. To further explore this issue, several less acidic active compounds were tested. Chemical substances (tetrazole, saccharin-1-methylimidazole, 4,5-dicyanoimidazole azole) was tested to determine the pK a =11.1) However, morpholine (pK a ODNs that reacted only minimally with α- and β-blockers (=8.3) were identified. Activators screened in the synthesis of PMOs with sequences corresponding to 1 (Table 2) Among the substances, 4,5-dicyanoimidazole (pK a =5.2, 0.12M, 300 seconds The highest yield (67%) was achieved with low levels of by-products. The desired PMO was generated (Supplementary Information, Figure S2). The phosphoramidite diester (Figure 4, compound B) is unstable in the capping solution. Therefore, prior to the capping step, a phosphorus(IV) morpholino compound (Figure 4, compound After boronation, the support was washed with acetonitrile. Cap failed sequences using acetic anhydride, 10% TMBP and 0.5% TFA. Detritylation was carried out using chloroform solution (compound D was obtained by these acidic detritylation reactions). (It was stable under various conditions.)

[0088] Using this synthetic procedure outlined in Figure 4, a trimer containing only thymine nucleoside bases was prepared. The compound was synthesized and N,N-dimethylamino PMO derivative was prepared by iodine / N,N-dimethylamine. The compound was converted to a conductor, removed from the support with ammonia, and analyzed by LCMS. The four main peaks shown in the LC profile (Figure 6, right panel) correspond to the four products. It has the mass spectrum expected for the diastereomers (calculated mass 902.3, experimental mass 902.3). The combined area of ​​all peaks from the crude reaction mixture was calculated using these diastatic By comparing the peak area with that of the dimer, the yield of compound 29 was calculated to be 94%. In similar experiments in which dimers were made, the first coupling yields were higher than other reported procedures. The results showed a significantly better result of 96% (Bioorg. Med. Chem. Lett. 2012, 22:1445-47, Tetrahedron Letters, 2015, 56;4565-68).

[0089] [Table 2]

[0090] This approach was used to synthesize oligothymidins (Table 2, ODNs 1 and 2) and all four bases. N,N-dimethylamino PMOs (Table 2, ODN3-8) were synthesized and analyzed by LCMS. PMOs with lengths of 12 to 16 nucleosides were characterized by their effectiveness in RNA expression. are effective inhibitors (either via interference with splicing or interference with mRNA translation). Since it was established that the 16-metabolite has all four bases, we next The synthesis of r (Table 2, ODN8) was carried out. The LCMS chromatogram of the crude reaction mixture was , yield (isolated 10 A 260 Both the mass spectrometry of this ODN were satisfactory. It was shown that it was something.

[0091] Example 3 Synthesis of PMO-DNA chimeras For the synthesis of PMO-DNA chimeras, 4,5-dicyaminomidazole (0.12 M and and 300 seconds coupling time) and ETT (0.25M and 180 seconds coupling time) between the morpholino phosphoramidite synthon (9, 14, 19 or 24) and the 5' -Dimethoxytrityl-2'-deoxyribonucleoside-3'-phosphoramidite ( 25, 26, 27 or 28), respectively. After condensation, morpholinophosphoric acid was The amidite diester was converted to a phosphorus(IV) borane bond and 0 Phosphate the phosphite triester using standard oxidation with 0.02M iodine This procedure was repeated until a PMO-DNA chimera of the desired sequence / length was produced. These synthetic steps were repeated. Table 1 outlines these synthetic steps.

[0092] PMO-DNA chimeras are novel to the scientific community and useful for various research projects. Since it could be demonstrated that PMO-DNA chimeras could be synthesized, several PMO-DNA chimeras were synthesized (Table 2, O These chimeras were first synthesized by cleaving four N,N-dimethylamino PMO bonds. These 21-mers were prepared as a series of 21-mer oligothymidine fragments containing the 21-mer. and a morpholinodiamidate bond is located adjacent to either the 5' or 3' end. , or placed close to the middle of the 21mer and every third position throughout the oligomer ( Table 2; ODNs 9, 10, 11, 12, and 13). The substitution of borane with diethylamine during activation proceeded efficiently, and LC of the crude reaction mixture MS analysis revealed that the expected phosphorodiamidate bond was formed in nearly quantitative yield. These promising results demonstrate that the DNA fragments containing all four nucleobases in various locations The synthesis of PMO-DNA chimeras containing multiple PMO linkages (Table 2; ODN14, 15 and 16) LCMS analysis of the reaction mixture and 31 P NMR confirmed the expected PM O-DNA chimeras were synthesized with an average yield of 10-20 A260 units (from 0.2 μM synthesis cycles). These experiments also demonstrated that dimethylamine was used to obtain the desired product in high yield. The treatment does not result in measurable cleavage of the succinate bond and loss of product during synthesis. This demonstrated that:

[0093] Example 4 Solid-phase synthesis of PMOs with amino, N-methylamino, and morpholino bonds The borane phosphonate bond is cleaved by iodine against displacement by a number of nucleophiles. It is known that N,N-dimethylamino-phosphorodiamine can be activated. We tested this new synthetic route by synthesizing PMO analogs containing amidate bonds. In addition to this, we have used other methods to generate several new PMO-DNA derivatives. We decided to investigate whether it would be possible to use amines such as

[0094] First, four borane phosphoramidate morpholino bonds were attached near the center of this oligomer. A 21-mer oligothymidine having the following structure was synthesized. were treated with N-methylamine, ammonia, and morpholine under iodine oxidation conditions. The sample was then purified using reversed-phase column chromatography. Mixed sequences containing all four bases and amino-phosphorodiamidate internucleotide linkages PMO-DNA chimeras were synthesized, and the position of the diamidate linkage was determined for these chimeras. The sequences and LCMs of these PMOs were The mass spectrometry results by S are listed in Table 3. The yields were The results were comparable to those obtained by

[0095] [Table 3]

[0096] Example 5 Melting temperature of PMO-DNA chimeras To evaluate the target binding ability of the modified PMO-DNA chimeras, amino, methylamino, and mono 2'-Deoxymorpholino and Dimethylamino-morpholino Internucleotide Linkages Duplex hybridization studies were performed using ribozyme. The total duplex concentration was 1 0.0 μM, buffer (1.0 M NaCl, 10 mM sodium phosphate, pH 7.1) PMO11, 17, 18, and 19 were mixed at a 1:1 ratio with 21-nucleotide 2'-deoxyribonucleic acid (2'-deoxyribonucleic acid) The samples were denatured at 96°C and mixed with ribo- or riboadenosine-oligonucleotides. The sample was then cooled to 5°C. The sample was then heated at a rate of 1°C / min. 260 Time was recorded The melting temperatures were taken as the temperatures of half dissociation and were obtained from the first derivative plots (Table 4).

[0097] Melting temperature studies of RNA heteroduplexes of PMO11, 17, 18, and 19 showed that the phosphorylation The introduction of amidate internucleotide bonds increases the PMO:RN duplex size compared to the unmodified duplex. The trend of increasing stabilization was amino > N-methylamino > N,N-dimethylamino>morpholino. PMO-DNA chimera was 2'-deoxy When duplexing with riboadenosine 21mer, the modifications, except for the morpholino analogue, Unclear DNA-DNA duplex T m A similar trend was observed when compared with

[0098] [Table 4]

[0099] Example 6 RNase H1 activity of chimeric PMO-RNA heteroduplexes N,N-dimethylamino PMO-DNA chimeras stimulate RNase H1 activity. The test system consisted of 5'-O-fluorescein-labeled RNA and complementary It was composed of N,N-dimethylamino PMO chimeras.

[0100] Synthesis of 5'-fluorescein PMO-DNA chimeras. Borane phosphoramidate morphotransferase After completion of DMT-ON synthesis of the rhino chimera, ODNs were synthesized using a standard DNA synthesis cycle. and conjugated with 5'-Amino-Modifier C6-TFA (Glen Research). After iodine oxidation of the exocyclic amine and desilylation, the product was purified using reversed-phase HPLC. Oligonucleotides were purified (Buffer A: triethylammonium bicarbonate, 0.0 5M, Buffer B: acetonitrile; 0% → 100% B for 50 min; 55°C; flow rate 4. The purified ODN was dissolved in 1 mL of 1:1 NH4OH:CH3NH2 solution. The reaction mixture was dissolved and heated at 65°C for 3 hours to remove the trifluoroacetamide group. Dry and place in a buffer containing 20 mM sodium phosphate and 0.15 M NaCl (200 The concentration was measured by dissolving the solution in 100µL of 5-(2-chloro-2-methyl-2-propanol) at pH 8.0. 6-)Carboxy-fluorescein succinimidyl ester (Thermo Fisher Scientific ic) was dissolved in DMSO and added to the ODN solution. After stirring the reaction mixture at room temperature for 1 hour, The reaction mixture was diluted with 300 μL of water and stirred at 4° C. for 3 hours. Excess NHS-fluorescein was purified using a USTRA™ NAP™-5 column. The analyzed PMOs contained an N,N-dimethylamino PMO bond at either end. and analogs containing a cap / gap sequence with 3-7 phosphodiester bonds in the center The control was a complementary oligothymidine 14-mer (ODN24 to ODN26, Table 5). 2'-O-methylated DNA and 2'-O-methylated RNA (which activate RNase H1 activity, respectively) These cap / gap oligonucleotide analogs were Both of these were found to activate RNA hydrolysis.

[0101] Hydrolysis of RNA heteroduplexes by E. coli RNase H1. RNase H1 (Promega) experiments were performed as previously described (J. Am. Chem. Soc., 2003, 125:940-50) was used. 50 mM Tris-HCl (pH 8.0), 20 mM KCl, 9 mM MgCl2, 1 mM β-mercaptoethanol and 250 μg / m The above reaction was carried out using the assay buffer for bovine serum albumin. Oligodeoxynucleotide or modified oligodeoxynucleotide (200 pmol) and 5 '-O-Fluorescein-labeled complementary oligoribonucleotides in assay buffer (35 Following the addition of E. coli RNase H1 (3 units), the cells were incubated at 25°C for 12 h. The reaction was carried out over a period of 1 hour. The same volume of 80% formamide gel containing a tracking dye was added. Dilute the reaction mixture with loading buffer and analyze by polyacrylamide gel electrophoresis. (20%, 19:1 crosslinked, 7M urea). All reactions were performed in triplicate. olecular Dynamics Typhoon Phosphorimager The developed gel was analyzed using

[0102] [Table 5]

[0103] Example 7 Cellular uptake Delivering uncharged PMOs into cells using lipid-based transfection reagents Therefore, the commonly used siRNA transfection reagent, Dh We examined the cellular uptake of these PMO-DNA chimeras in the presence of Armafect 1. PMO-DNA chimera was synthesized (ODN27, 5'-FL-T * G * T * A * a p a p c p c p a p t p g p a p t p g p t p g p c p t p G * C * T * A * t, these abbreviations See Table 2 for a description of the nucleotides), where the internal normal nucleotides are It is flanked at the - and 3'-ends by N,N-dimethylamino PMO nucleotides. ODN27 also contains a fluorescein dye (FL) linked by a 6-carbon linker. In the presence of Dharmafect 1, live and fixed HeLa cells were used. Cells were transfected with ODN27 (100 nM concentration) (Figure 7), and 20 After 18 hours of incubation, the cells were imaged by fluorescence microscopy.

[0104] Lipid transfection observed by microscope image. ODN27 stock solution was diluted to 200 μl. The ODN was diluted with MicroL's OptiMEM to a final concentration of 0.1 μM. In a Diaper tube, add 5.0 microliters of DharmaFECT 1 to 200 ml of Dilute 200 microL of ODN27 solution with 200 microL of Dham Mix the aFECT 1 solution, equilibrate for 20 minutes, and add 600 microliters of OptiMEM DME containing 10% FBS and penicillin streptomycin (penstrep) was added. 0.3 × 10 cells on glass coverslips in 6-well plates in M ​​medium 6 cells / well After 24 hours, the medium was removed and the cells were transfected at 80% confluency. Before washing, cells were washed twice (2.0 mL D-PBS / wash). The cells were then removed and 1.0 mL of the transfection mixture was added to each well. The cells were then incubated at 37°C for 18 hours and washed twice (2.0 mL D- PBS / wash). Cells were covered with 1.0 mL of 10% neutral buffered formalin for 15 min. The formalin solution was removed, and the cells were covered with 3.0 mL of DPBS for 10 minutes at room temperature. Remove the burr glass from the well and mount it in Floromount with DAPI as mounting agent. Mounted upside down on a cover slide using -G and Hamamatsu C4742 An inverted microscope (Oly) equipped with a -95 CCD and a CoolSNAP ES digital camera Observations were made using a mpusIX 81).

[0105] An increase in the fluorescent signal was observed when cells were incubated with 100 nM ODN27. The transfection was dose-dependent, as the fluorescence was mainly distributed in the nucleus. Although the structure appears to be distributed, the common punctuated structure seen in many analogues is There is also evidence of a cytoplasmic distribution without cytoplasmic markers.

[0106] The above-described embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the form or forms disclosed herein. Variations and modifications commensurate with the teachings herein, and the skill or knowledge of the relevant art, are within the scope of the present invention. The specific embodiments described in the examples provided herein are intended to be illustrative and not restrictive of the principles of the present invention. It is intended to further describe the best mode known, and it is understood that those skilled in the art will be familiar with such and other embodiments. The invention can be utilized with various modifications required for a particular application or use of the invention. The appended claims are intended to be as broad as permitted by the prior art. It is intended that the terms "conventional" and "conventional" be construed to encompass alternative embodiments.

[0107] This is to comply with the formal requirements of jurisdictions that do not recognize such multiple dependencies. It was only made to the extent that the accompanying claims were drafted without multiple dependencies. All possible combinations of features that can be implied by giving It should be noted that the present invention is not limited to the above and should be considered as part of the present invention.

Claims

[Claim 1] The following formula: [[ID=0=0]]【Chemical 1】 (where, B is a nucleobase selected from adenine, guanine, and cytosine, said nucleobase protected with a silyl protecting group, said silyl protecting group being 【Chemistry 2】 and R １ and R ２ are each independently C ２～２０ is a straight or branched chain alkyl; R ３ is cyanoethyl, R ４ is dimethoxytrityl) A method for preparing a phosphorodiamidite monomer represented by the formula: a. contacting 5'-O-dimethoxytrityl ribo-base with sodium periodate and ammonium diborate; wherein said base is a protected nucleobase corresponding to B; b. reducing the hydroxyl group on the product obtained in step a with sodium cyanoborohydride to form a morpholino monomer; c. phosphitylating the morpholino monomer with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite and 4,5-dicyanoimidazole (DCI) in dichloromethane to form a phosphorodiamidite monomer; A method comprising:

Images