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

Abstract

To provide amine substituted morpholino oligonucleotides, other than the classical N,N-dimethylamino PMO analogue, and methods of efficiently synthesizing these oligonucleotides with high yield.SOLUTION: Morpholino oligonucleotides having thiophosphoramidate, phosphoramidate, phosphoramidate diester, and alkyl phosphoramidate linkers. Chimeras containing unmodified DNA / RNA and other analogs of DNA / RNA can be prepared. These oligonucleotides form duplexes with complementary DNA or RNA that are more stable than natural DNA or DNA / RNA complexes, are active with RNAse H1, and may be transfected into cells using standard lipid reagents. These analogues are therefore useful for numerous applications.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 513,089, filed May 31, 2017, and U.S. Provisional Patent Application No. 62 / 397,277, filed September 20, 2016, both of which are incorporated herein by reference.

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

[0003] Morpholino oligonucleotides are promising for use as antisense oligonucleotide therapeutics due to their high affinity for DNA and RNA, resistance to various nucleases, in vivo stability, and low toxicity. Phosphorodiamidate morpholino oligonucleotides (PMO; compound 3, FIG. 1) have nonionic internucleotide linkages and morpholinos that replace deoxyribose in DNA. These N,N-dimethylamino phosphorodiamidate morpholino oligonucleotides inhibit gene expression by interfering with RNA splicing and preventing translation (Non-Patent Document 1), are resistant to cellular nucleases (Non-Patent Document 2, Non-Patent Document 3), and have higher RNA binding affinity than DNA (Non-Patent Document 4). Therapeutic development of PMOs is ongoing in clinical trials, for example, for the treatment of Duchenne muscular dystrophy (DMD) and for the prevention of infection with the hemorrhagic Filoviridae Marburg virus (Patent Document 1 and Patent Document 2). Furthermore, PMOs have been considered for applications in nanotechnology (Non-Patent Document 5) and surface hybridization (Non-Patent Document 6).

[0004] Unfortunately, these promising applications for PMOs are severely limited by the lack of efficient synthetic methodologies. In contrast to the standard method of chemically producing DNA and RNA on automated synthesizers, PMOs are currently synthesized in the 5' to 3' direction on polystyrene resins. As a first step, condensation of a 5'-hydroxyl-N-trityl-morpholino nucleoside with N,N-dimethylaminodichlorophosphoramidate produces the N,N-dimethylaminochlorophosphoramidate synthon. Coupling in the presence of base results in a dimer attached to the resin. Further detritylation with acid produces a dimer that can be extended by repeating the cycle. This approach has several recognized challenges. For example, the condensation yield is low (recovery yield of dithymidine morpholino was 45%) and requires long reaction times. Furthermore, the 5'-chloro-phosphoramidate monomer is unstable, and it is difficult to couple two morpholino monomers in the presence of large amounts of base (Non-Patent Document 7). Furthermore, this approach requires specialized procedures, techniques and materials (e.g., swellable polystyrene resins, specialized reaction vessels, and pyridinium salts of trifluoroacetic acid as detritylation agents) that are not readily available in many laboratories.

[0005] Recently, a new method was developed using H-phosphonate chemistry for the synthesis of polythymidine PMOs. Challenges exist with this approach as well. For example, the phosphorus atom in H-phosphonates is electrophilic and lacks a lone pair of electrons, making it much more resistant to oxidation under ambient conditions than most P(III) compounds. Furthermore, the coupling yield for the formation of H-phosphonate morpholino dimers is low (77%). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2016 / 0040162 [Patent Document 2] US 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, which further achieves other advantages that are discussed more fully below. [Means for solving the problem]

[0009] The present disclosure provides new methods for synthesizing phosphorodiamidate morpholinos (PMOs) and PMO-DNA chimeras. These methods are very robust since oligomers containing phosphophosphorodiamidate internucleotide linkages can be made using phosphoramidite chemistry in high yields on an automated DNA synthesizer. The process begins with the incorporation of morpholino phosphoramidites into DNA, followed by boronation and subsequent oxidation with iodine in the presence of various amines to form a diverse set of PMOs and PMO-DNA chimeras. The method is general and results in the synthesis of a large number of PMO linkages. Unlike procedures using chlorophosphoramidate synthons, the more reactive phosphorodiamidite synthons of the present disclosure can be used to synthesize these analogs in high yields. To assemble these analogs, selective activation of the diisopropylamino moiety of morpholino phosphorodiamidites via dicyanoimidazole during coupling is used.

[0010] Advantageously, PMO and PMO-DNA chimeras can be synthesized on a DNA synthesizer because both the morpholino phosphoramidite and 2'-deoxynucleoside synthons were designed for the synthesis of oligonucleotides in the 3' to 5' direction, unlike the previously developed chemistry where the N,N-dimethylamino PMO synthons were designed to synthesize these analogs in the 5' to 3' direction. Furthermore, this new approach relies on the use of borane phosphoramidate internucleotide linkages that can be used to generate a variety of PMO chimeras by oxidation of iodine with appropriate amines. For example, PMO-LNA, PMO-RNA, PMO-DNA phosphorothioate / phosphorodithioate, and PMO-DNA phosphonoacetate / phosphonoformate chimeras, among others, can be made using these methods.

[0011] Regarding their potential use in various biological and biochemical applications, these PMO-DNA chimeras exhibit three potential advantages over some other analogues: For example, the aminomorpholino phosphorodiamidate derivatives bind to complementary DNA more easily than either unmodified DNA / RNA or standard N,N-dimethylamino PMO analogues. or RNA (approximately 1.75-fold more stable per modification than the N,N-dimethyl analog in duplex formation with RNA). Furthermore, these PMO-DNA chimeras are active with RNase H1. This is encouraging compared to standard N,N-dimethylamino PMO analogs, where fully substituted PMOs are inactive with RNase H1. By activating RNase H1, these cap / gap N,N-dimethylamino PMO chimeras provide analogs with different biochemical properties that are useful for various applications in biology (e.g., aminoamidate derivatives have enhanced stability for duplex formation). Due to the increased stabilization of these PMO-DNA chimeras (relative to unmodified duplexes) with complementary RNA, this type of cap / gap analogs allows the use of shorter single-stranded antisense oligonucleotides, thus reducing off-target effects. Furthermore, these PMO-DNA chimeras can be easily transfected into cells using commonly known transfection reagents, eliminating the challenges associated with delivery of PMOs by techniques such as microinjection, hybridization of PMOs with DNA, and delivery via ethoxylated polyethyleneimine, or conjugation to either peptide or dendritic molecular transporters.

[0012] Thus, the present disclosure provides PMO synthesis methodologies that provide an efficient and cost-effective way to synthesize PMO derivatives with minimal by-product formation. These synthetic methods incorporate our previously reported stereoselective conversion of borane phosphonate DNA to phosphoramidate DNA in the presence of iodine and amines (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 orthogonal to and compatible with standard DNA / RNA synthesis methods that use phosphoramidite chemistry, so PMO synthesis can be performed on a standard DNA synthesizer; 2) For the first time, we were able to synthesize PMOs with internucleotide linkages other than N,N-dimethylaminophosphoramidate morpholino, 3) These synthetic methods allow the synthesis of PMO-DNA chimeras that are anionic, and therefore water-soluble, and that activate RNase H upon hybridization to RNA.

[0013] This summary is not intended, nor should it be construed, to be representative of the complete extent and scope of the present invention. Moreover, references herein to "the disclosure" or aspects thereof are understood to mean certain embodiments of the disclosure, and are not necessarily to be construed as limiting all embodiments to the particular description. The disclosure is described in various levels of detail in this summary, as well as in the accompanying drawings and detailed description, and no limitation on the scope of the disclosure is expressed by the inclusion or exclusion of elements, components, etc. in this summary. Additional aspects of the disclosure will become more apparent from the detailed description, particularly when taken in conjunction with the drawings. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the chemical structures of DNA (1), RNA (2), N,N-dimethylamino phosphoramidate morpholino (3), and borane phosphoramidate morpholino (4) oligonucleotides. [Diagram 2] FIG. 1 is a synthetic scheme illustrating 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 the purposes of this disclosure, this hydroxyl group is shown as 5' to relate to conventional nucleic acid chemistry. [Diagram 3] FIG. 1 is a synthetic scheme illustrating the synthesis of a BIBS-protected morpholino phosphorodiamidite 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-dicyaminomidazole (0.5 equiv.), and CH2Cl2. [Figure 4] FIG. 1 shows a synthetic scheme of solid-phase synthesis for phosphorodiamidate morpholino oligonucleotides (PMOs) using BIBS-protected phosphoramidites. Compound F is a PMO with only N,N-dimethylamino-phosphorodiamidate internucleotide linkages, where "x" is the selected number of N,N-diethylamino-PMO nucleotide units, and compound A is a thymidine ribonucleoside, but this ribonucleoside may be replaced by any of the four 2'-deoxyribonucleosides or morpholino nucleosides. In examples where cytosine, adenine, and guanine bases are used, the bases must be protected by bis(tertbutyl)isopropylsilyl (BIBS) groups. [Diagram 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. 13: Synthesis of N,N-dimethylamino PMO trimer (left panel) and LC profile from LCMS analysis of the total 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, followed by incubation at 37°C for 18 hours. 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 are images of fluorescein localization of ODN27. Figures 7B and 7E are images of DAPI localization of the nucleus 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 PREFERRED EMBODIMENTS

[0015] Unless otherwise stated, the following terms have the following meanings.

[0016] The term "oligonucleotide analog" refers to an oligonucleotide having a modified backbone structure, e.g., a backbone other than the standard phosphodiester linkage found in natural oligonucleotides and polynucleotides, and, optionally, modified sugar moieties, e.g., morpholino moieties other than ribose or deoxyribose moieties. The analog maintains bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents bases that allow such hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Preferred analogs are those that have a substantially uncharged phosphorus-containing backbone.

[0017] The "nucleobase" is not particularly limited as long as it can be used in the synthesis of a nucleic acid, and includes, for example, pyrimidine bases such as cytosyl, uracil, and thyminyl groups, and purine bases such as adenyl and guanyl groups. The "optionally protected nucleobase" means, for example, a nucleobase having an amino group that can be protected in an adenyl, guanyl, or cytosyl group, and is preferably a nucleobase in which the amino group is protected by a protecting group that can be maintained under the deprotection conditions of the nitrogen atom of the morpholine ring of a morpholinonucleotide.

[0018] The "amino protecting group" is not particularly limited, and examples thereof include protecting groups described in Greene's PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th edition, Wiley-Interscience, 2006, etc. Specific examples of the "amino protecting group" include a pivaloyl group, a pivaloyloxymethyl group, a trifluoroacetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, an acetyl group, a benzoyl group, an isobutyryl group, a dimethylformamidinyl group, a 9-fluorenylmethyloxycarbonyl group, etc. Among these, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, an acetyl group, a benzoyl group, an isobutyryl group, and a dimethylformamidinyl group are preferred. Furthermore, the carbonyl group of the nucleic acid base may be protected, for example, by reacting with phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride, diphenylcarbamoyl chloride, or the like.

[0019] In some cases, carbonyl protecting group may not be introduced. In addition to the above-mentioned groups, modified nucleic acid bases (e.g., 8-bromoadenylic group, 8-bromoguanyl group, 5-bromocytosyl group, 5-iodocytosyl group, 5-bromouracil group, 5-iodouracil group, 5-fluorouracil group, 5-methylcytosyl group, 8-oxoguanyl group, hypoxanthinyl group, etc.) are also included in "nucleobase", which are nucleic acid bases substituted with any one to three substituents (e.g., halogen atom, alkyl group, aralkyl group, alkoxy group, acyl group, alkoxyalkyl group, hydroxyl group, amino group, monoalkylamino, dialkylamino, carboxy, cyano, nitro, etc.) at any position (may be multiple).

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

[0021] A "monomer" or "subunit" of an oligonucleotide analog refers to one nucleotide (or nucleotide analog) unit of the analog. The term refers to a nucleotide unit with or without an attached intersubunit linkage, although when referring to a "charged subunit," the charge is typically in the intersubunit linkage (e.g., a phosphate or phosphorothioate linkage).

[0022] A "morpholino oligonucleotide analog" is an oligonucleotide analog comprised of morpholino subunit structures of the form shown in FIG. 4, where the structures are linked together by phosphorus-containing linkages of one to three atoms in length connecting the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and the "B" moiety is a purine or pyrimidine base pairing moiety effective to bind to a base in a polynucleotide by base-specific hydrogen bonding. The purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, or thymine. The synthesis, structure, and linkage characteristics of morpholino oligomers are described in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,113, and 5,217,866. Nos. 42,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, all of which are incorporated herein by reference.

[0023] The subunits and bonds shown in FIG. 4 are used for a 6-atom repeat unit backbone (where the 6 atoms include the morpholino nitrogen, the connected phosphorus atom, the atom connecting the phosphorus atom to the 5' exocyclic carbon (usually oxygen), the 5' exocyclic carbon, and the two carbon atoms of the next morpholino ring). In these structures, the atom connecting the 5' exocyclic morpholino carbon to the phosphorus group may be sulfur, nitrogen, carbon, or oxygen. The X moiety pendant from the phosphorus is any stable group that does not interfere with base-specific hydrogen bonding, including fluoro, alkyl, alkoxy, thioalkoxy, and alkylamino, including cyclic amine, all of which may be variously substituted so long as base-specific bonding is not disrupted. The alkyl, alkoxy, and thioalkoxy may contain 1-10 carbon atoms. The alkylamino preferably refers to lower alkyl (C1-C6) substitutions, and the cyclic amine is preferably a 5- or 7-membered nitrogen heterocycle, optionally containing 1-2 additional heteroatoms selected from oxygen, nitrogen, and sulfur. Y is sulfur or oxygen.

[0024] A preferred morpholino oligomer is a phosphorodiamidate-linked morpholino oligomer, herein referred to as PMO. Such oligomers are composed of morpholino subunit structures as shown in Figure 4, where X=NH2, NHR or NR2 (where R is lower alkyl, preferably methyl), Y=O, and Z=O, and B is a purine or pyrimidine base pairing moiety that is effective for binding to a base in a polynucleotide by base-specific hydrogen bonding. Also preferred are structures with alternative phosphorodiamidate linkages, where X=lower alkoxy, such as methoxy or ethoxy, Y=NH or NR (where R is lower alkyl), and Y=O in Figure 4.

[0025] The term "substituted," particularly with respect to an alkyl, alkoxy, thioalkyl, or alkylamino group, refers to the replacement of a hydrogen atom on a carbon with a heteroatom-containing substituent, such as, for example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino, oxo(keto), nitro, cyano, or various acids or esters, such as carboxylic acids, sulfonic acids, phosphonic acids, etc. The term "substituted" can also refer to the replacement of a hydrogen atom on a heteroatom (such as an amine hydrogen) with an alkyl, carbonyl, or other carbon-containing group.

[0026] Sequences are described as "complementary" to each other when hybridization occurs in an antiparallel configuration. A double-stranded polynucleotide can be "complementary" to another polynucleotide. A targeting sequence can have "approximate" or "substantial" complementarity to a target sequence and still function for the purposes of the present invention. The oligonucleotide analogs of the present disclosure preferably have at most one mismatch with the target sequence in 10 nucleotides, preferably at most one mismatch in 20 nucleotides. Alternatively, the antisense oligomers of the present disclosure have at least 90% sequence homology, preferably at least 95% sequence homology, with the exemplary targeting sequences shown herein. Thus, the oligomers produced by the methods of the present disclosure are particularly useful as therapeutic antisense molecules when administered to treat disease conditions amenable to antisense therapy.

[0027] An oligonucleotide analogue "specifically hybridizes" to a target polynucleotide if the oligomer hybridizes to the target under physiological conditions with a Tm substantially greater than 45° C., preferably at least 50° C., typically 60° C. to 80° C. or higher. Such hybridization is preferably referred to as stringent hybridization. At a given ionic strength and pH, the Tm is the temperature at which 50% of the target sequence hybridizes to a complementary polynucleotide. Again, such hybridization can occur with "approximate" or "substantial" complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity.

[0028] A "nuclease-resistant" oligomeric molecule (oligomer) refers to one whose backbone, in either unhybridized or hybridized form, is substantially resistant to nuclease cleavage by common extracellular and intracellular nucleases in the body, i.e., the oligomer exhibits little or no nuclease cleavage under typical nuclease conditions in the body to which the oligomer is exposed.

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

[0030] The oligonucleotide analogue of the present disclosure preferably specifically binds to target RNA sequence in cells.The base specificity of such binding is sequence specific.For example, single-stranded polynucleotide can specifically bind to the single-stranded polynucleotide that has complementary sequence.

[0031] As described above, antisense oligomers have a base sequence directed to the targeting portion of the viral genome, preferably either 5'-CS or 3'-CS.Furthermore, when the oligomer is administered to an infected host cell, for example, when administered to an infected animal subject, it can effectively target the infectious virus.This requirement is met when the oligomeric compound (a) has the ability to be actively taken up by mammalian cells, and (b) once taken up, forms a duplex with the target ssRNA with a Tm higher than about 50°C.

[0032] As described below, the ability to be taken up by cells requires that the oligomer backbone is substantially uncharged, and preferably requires that the oligomer structure is recognized as a substrate for active or facilitated transport across the cell membrane. The ability of an oligomer to form a stable duplex with a target RNA also depends on the oligomer backbone, as well as the factors mentioned above, the length and degree of complementarity of the antisense oligomer with respect to the target, the ratio of G:C to A:T base matches, and the position of any mismatched bases. The ability of an antisense oligomer to resist cellular nucleases promotes survival and eventual delivery of the oligomer to the cytoplasm of a cell.

[0033] The following method is useful for testing any given substantially uncharged scaffold for its ability to meet these requirements.

[0034] Active or facilitated uptake by cells Antisense oligomers may be taken up by host cells by facilitated or active transport across the host cell membrane when administered in free (unconjugated) form, or by endocytic mechanisms when administered in a complexed form.

[0035] In instances where the agent is administered in free form, the antisense compound should be substantially uncharged, meaning that the majority of its intersubunit bonds are uncharged at physiological pH. Experiments conducted in support of this technology indicate that a small number of net charges, e.g., 1-2 charges for 15-mer to 20-mer oligomers, can indeed enhance cellular uptake of certain oligomers with a substantially uncharged backbone. The charges may be carried on the oligomer itself, e.g., on the backbone linkages, or may be terminal charged-group appendages. The number of charged linkages may be determined by the number of charged linkages. Preferably, there is no more than 1 charged bond per 4 uncharged bonds. More preferably, the number is no more than 1 charged bond per 10 uncharged bonds, or no more than 1 charged bond per 20 uncharged bonds. In one embodiment, the oligomer is completely uncharged.

[0036] Also, oligomers may contain both negatively and positively charged backbone linkages, so long as the opposite charges are present in approximately equal numbers. It is preferred that oligomers not contain a run of more than 3-5 consecutive subunits of either charge. For example, oligomers may have a given number of anionic linkages, e.g., phosphorothioate or N3'→P5' phosphoramidate linkages, and a comparable number of cationic linkages, such as N,N-diethylenediamine phosphoramidate, etc. The net charge is preferably neutral or at most 1-2 net charges per oligomer.

[0037] In addition to being substantially or completely uncharged, the antisense agents are preferably substrates for a membrane transporter system (i.e., a membrane protein(s)) that can facilitate or actively transport the oligomer across the cell membrane. This characteristic can be identified by one of several tests for oligomer interaction or cellular uptake, as follows:

[0038] The first test evaluates binding at cell surface receptors by examining the ability of an oligomeric compound to displace or be displaced by a selected charged oligomer on the cell surface. Cells are incubated with a given amount of test oligomer, typically fluorescently labeled, at a final oligomer concentration of about 10 nM to 300 nM. Shortly thereafter, for example after 10 to 30 minutes (before significant internalization of the test oligomer can occur), a displacing compound is added in gradually increasing concentrations. If the test compound is able to bind to the cell surface receptor, it is observed that the displacing compound displaces the test compound. If the displacing compound is shown to result in 50% displacement at a concentration 10 times or less than the test compound concentration, the test compound is considered to bind at the same recognition site for the cellular transport system as the displacing compound.

[0039] The second test measures cellular transport by examining the ability of the test compound to transport a labeled reporter, e.g., a fluorescent reporter, into the cells. Cells are incubated in the presence of labeled test compound added at a final concentration of about 10 nM to 300 nM. After 30 to 120 minutes of incubation, the cells are examined, e.g., by microscopy, for intracellular labeling. The presence of significant intracellular labeling is evidence that the test compound is transported by facilitated or active transport.

[0040] Antisense compounds may also be administered in complexed form, where the complexing agent is typically a polymer, e.g., a cationic lipid, polypeptide, or nonbiological cationic polymer, that has an opposite charge to any net charge on the antisense compound. Methods for forming complexes, including bilayer complexes between anionic oligonucleotides and cationic lipids or other polymeric components, are well known. For example, Lipofectin™, a liposome composition containing the cationic lipid DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and the neutral phospholipid DOPE (dioleylphosphatidylethanolamine), has been widely used. After administration, the complexes are typically taken up by cells through an endocytic mechanism, involving particle encapsulation in endosomal bodies.

[0041] Antisense compounds may also be administered in conjugated form with arginine-rich peptides linked to the 5' or 3' end of the antisense oligomer. The peptides are typically 8-16 amino acids long and consist of a mixture of arginine and other amino acids, including phenylalanine and cysteine. Exposure of cells to the peptide-conjugated oligomer results in enhanced cellular uptake and delivery to the RNA target.

[0042] Alternatively, the requisite properties of an oligomer with any given backbone can be confirmed by a simple in vivo test in which a labeled compound is administered to an animal and a sample of body fluid taken from the animal several hours after the oligomer is administered is assayed for the presence of heteroduplexes with the target RNA.

[0043] Substantial resistance to RNase H Two general mechanisms have been proposed to explain the inhibition of expression by antisense oligonucleotides. First, the heteroduplex formed between the oligonucleotide and the viral RNA 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 phosphorothioates, phosphotriesters and phosphodiesters (unmodified "natural" oligonucleotides). Such compounds expose the viral RNA in the oligomer:RNA duplex structure to hydrolysis by RNase H, resulting in loss of function.

[0044] "Steric blockers," or "RNase H inactive" or "RNase A second class of oligonucleotide analogs, termed "RNase H-resistant," have not been observed to act as substrates for RNase H and are believed to act by sterically blocking nucleocytoplasmic transport, splicing, translation, or replication of the target RNA. This class includes methylphosphonates, morpholino oligonucleotides, peptide nucleic acids (PNAs), certain 2'-O-allyl or 2'-O-alkyl modified oligonucleotides, and N3'→N5' phosphoramidates.

[0045] A test oligomer can be assayed for its RNase H resistance by forming an RNA:oligomer duplex with a test compound and then incubating the duplex under standard assay conditions with RNase H. After exposure to RNase H, the presence or absence of an intact duplex can be monitored by gel electrophoresis or mass spectrometry.

[0046] In vivo uptake Rapid tests exist to confirm that a given antisense oligomer type provides the necessary properties mentioned above, namely high Tm, ability to be actively taken up by host cells, and substantial resistance to RNase H. The method is based on the discovery that properly designed antisense compounds, when administered to a mammalian subject, will form stable heteroduplexes with complementary portions of the target RNA, which then appear in the urine (or other body fluids). Details of the method are presented in U.S. Patent Application No. 09 / 736,920, entitled "Non-Invasive Method for Detecting Target RNA" (Non-Invasive Method), the disclosure of which is incorporated herein by reference. Briefly, a test oligomer containing the backbone to be evaluated, with a base sequence targeted against a known RNA, is injected into an animal, e.g., a mammalian subject. The antisense oligomer may be directed against any intracellular RNA, including host RNA or the RNA of an infectious virus. Several hours after administration (typically 8-72 hours), the antisense-RNA is expressed in a 50-μg / mL IgG antibody. The urine is assayed for the presence of heteroduplexes. If a heteroduplex is detected, the backbone is suitable for use in an antisense oligomer of the invention.

[0047] The oligomers may be labeled, for example with a fluorescent or radioactive tag, to facilitate subsequent analysis, if appropriate for the mammalian subject. The assay may be in any suitable solid-phase or fluid format. In general, solid-phase assays involve first binding the heteroduplex analyte to a solid-phase support, such as a particle or polymer or test strip substrate, and detecting the presence / amount of bound heteroduplex. In fluid-phase assays, the analyte sample is typically pretreated to remove interfering sample components. If the oligomer is labeled, the presence of the heteroduplex is confirmed by detecting the label tag. For unlabeled compounds, the heteroduplex may be detected by immunoassay in the case of solid-phase format, or by mass spectrometry or other known methods in the case of solution or suspension format.

[0048] Exemplary Oligomeric Backbones Examples of non-ionic linkages that can be used in oligonucleotide analogs are shown in Figure 4. In these figures, B represents a purine or pyrimidine base pairing moiety that is effective to bind by base-specific hydrogen bonding to bases in a polynucleotide, preferably selected from adenine, cytosine, guanine and uracil. Suitable backbone structures include carbonate (R=O) and carbamate (R=NH2) linkages; alkylphosphonate and phosphotriester linkages (R=alkyl or -O-alkyl); amide linkages; sulfone and sulfonamide linkages (R1, R2=CH2); and thioformacetyl linkages (2E). The latter have been reported to have enhanced duplex and triplex stability with respect to phosphorothioate antisense compounds. 3'-methylene-N-methylhydroxyamino compounds have also been reported.

[0049] Preferred oligomer structures employ morpholino-based subunits with base-pairing moieties linked by uncharged bonds.Morpholino oligonucleotides, including antisense oligomers, are described in detail, for example, in co-owned U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and 5,506,337, all of which are expressly incorporated herein by reference.

[0050] Important properties of morpholino-based subunits include their ability to be linked in oligomeric form by stable, uncharged backbone linkages; their ability to support nucleotide bases (e.g., adenine, cytosine, guanine, or uracil) such that the polymers formed can hybridize to complementary base target nucleic acids, including high Tm target RNAs, as well as short oligomers of 10-14 bases; the ability of the oligomers to be actively transported into mammalian cells; and the ability of oligomer:RNA heteroduplexes to resist RNase degradation.

[0051] Backbone structures for the antisense oligonucleotides of the present disclosure include the morpholino subunit type shown in Figure 4, which is linked by an uncharged phosphorus-containing subunit bond. Figure 4 shows phosphorus-containing bonds forming a 5-atom repeat unit backbone, in which the morpholino rings are linked by a 1-atom phosphoamide bond. Figure 4 shows bonds resulting in a 6-atom repeat unit backbone. In this structure, the atom linking the 5' morpholino carbon to the phosphorus group can be sulfur, nitrogen, carbon, or preferably oxygen. The X moiety pendant from the phosphorus can be fluorine, alkyl or substituted alkyl, alkoxy or substituted alkoxy, thioalkyl or substituted thioalkyl, or unsubstituted, mono- or di-substituted nitrogen, including cyclic structures such as morpholine or piperidine. Alkyl, Alkoxy and thioalkoxy preferably contain from 1 to 6 carbon atoms. The Y moiety is sulfur or oxygen, preferably oxygen.

[0052] As mentioned above, a substantially uncharged oligomer advantageously contains a limited number of charged linkages, e.g., up to about 1 per every 5 uncharged linkages, more preferably up to about 1 per every 10 uncharged linkages. Thus, some charged linkages, e.g., charged phosphoramidates or phosphorothioates, may also be incorporated into the oligomer.

[0053] Another aspect of the disclosure provides a method for making morpholino-containing oligonucleotide analogs, starting with a 5'-unprotected-2'-deoxyribonucleoside linked to a polystyrene support, and reacting the 5'-unprotected-2'-deoxyribonucleoside with a phosphoramidite synthon (i.e., monomer) in anhydrous acetonitrile containing 4,5-dicyaminimidazole (DCI) to produce a dimer having a phosphoramidite diester internucleotide linkage. The dimer is then chemically activated by either boronation, sulfurization, or oxidation of the dimer. The dimer is then capped and detritylated. These steps (adding phosphoramidite synthons via capping and detritylation steps) are repeated as desired to extend the oligomer by adding monomers to the growing oligomer (growing stepwise from dimers to trimers, tetramers, pentamers, hexamers, etc.). The morpholino phosphoramidate oligomers are then contacted with iodine and dimethylamine in tetrahydrofuran to convert the morpholino borane phosphoramidates to N,N-dimethylamino PMOs. The oligomers are then contacted with a solution containing ammonium hydroxide and ethylenediamine to remove the oligomers from the polystyrene support. The repeated addition of monomers to these oligomers may be carried out on a common commercial DNA synthesizer, which can greatly enhance the efficiency and speed of synthesis in a cost-effective manner.

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

[0055] Each publication or patent cited herein is hereby incorporated by reference in its entirety.

[0056] The disclosure generally described herein is merely illustrative of certain aspects of embodiments of the present disclosure. This will be more readily understood by reference to the following examples, which are included for purposes of illustration and are not intended to limit the present disclosure, as one of ordinary skill in the art will recognize from the teachings above and the following examples that other techniques and methods may be used that satisfy the claims and do not depart from the scope of the claimed disclosure. EXAMPLES

[0057] Example 1 Synthesis of N-di-tertbutylisobutyl-protected morpholinophosphoramidite To generate the borane phosphoramidate morpholino linkage, phosphoradiamidite synthons were developed for all four required bases (see synthesis scheme in Figure 3). The synthesis of phosphoradiamidite thymidine morpholino monomer synthons was carried out according to literature protocols (Zhang, et al., Tetrahedron Letters 2008, 49, 3570; Pattanayak, et al., Nucleosides, Nucleotides and Nucleic Acids, 2012, 31, 763-782). First, thymidine was treated with dimethoxytrityl chloride (DMT-Cl) in anhydrous pyridine under argon atmosphere to give 5'O-DMTr-thymidine (7), which was then treated with sodium periodate followed by ammonium diborate to give 2',3'-dihydroxyl-morpholino-thymidine (not shown). Reduction of the 2' and 3' hydroxyl groups was performed using sodium cyanoborohydride under mildly acidic conditions (without further purification) to generate the thymine morpholino monomer (8). The building block thymidine synthon (9) was then prepared in 86% yield by phosphitylation of compound (8) with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite and 4,5-dicyaminimidazole (DCI) in dichloromethane under an argon atmosphere. The thymidine synthon (9) does not require base protection.

[0058] We also synthesized 5'-dimethoxytrityl-morpholinothymidine-3'-O-methyl-N,N-diisopropylphosphoradiamidite following a similar protocol, but found that solid-phase coupling with the cyanoethyl phosphorradiamidite was more efficient than the O-methyl counterpart.

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

[0060] Reaction of 5',3',2'-tri-O-acetylcytidine (10) with BIBS-OTf (Tf = triflate) in the presence of 2,6-lutidine afforded 11 in 76% yield after 2 h stirring at 60 °C under argon atmosphere. However, synthesis of 16 and 21 from 15 and 20 required much longer reaction times (3 days) with yields of 25%-31% (16) and 74% (21), respectively. As represented in the synthetic scheme in Figure 3, these protected (silylated) ribonucleosides (11, 16, 21) were first treated with ammonium hydroxide to remove the acetyl protecting group and were converted to the 5'-dimethoxy-trityl compounds (12, 17, 22) and then to the morpholino derivatives (13, 18 and 23). These compounds were 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, followed by the addition of 1.2 equivalents of sodium periodate and ammonium diborate tetrahydrate (1.2 equivalents). The mixture was stirred at room temperature for 3 hours, at which time TLC indicated complete consumption of the starting material. The reaction mixture was filtered through a pad of celite and added to activated powdered 4A° molecular sieves (0.4 g / mmol), followed by addition of 2.0 equivalents each of cyanoborohydride. Sodium chloride and glacial acetic acid were added. The reaction mixture was then stirred for 4-5 hours, at which time the intermediate diol was completely reduced. The reaction mixture was filtered through a pad of Celite and evaporated to dryness. 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. In all cases, a silica gel slurry was made with a mixture of starting eluents containing an additional 5% triethylamine. After pouring the slurry, the column was washed with two column volumes of the starting solvent mixture without triethylamine. 8, 13, and 18 were eluted using a gradient of chloroform up to 19:1 chloroform:methanol. Compound 23 was purified using a gradient from 1:1 ethyl acetate:hexane to 7:3 ethyl acetate:hexane. All yields listed in the following columns represent those obtained for two steps starting with 5'-dimethoxytrityl-N-BIBS protected nucleosides.

[0062] 5'-Dimethoxytritylmorpholinothymidine (8): Yield: 53%. 1 H NMR(CDCl3,400MHz)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, 130.03, 1 28.12, 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'-dimethoxytritylmorpholinocytidine (13): Yield: 64%. 1 H NMR(CDCl3,400MHz)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:168.25, 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, 55.17, 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'-dimethoxytritylmorpholinoadenosine (18): Yield: 61%. 1 H NMR(CDCl3,400MHz)δ:8.38(1H,s), 8.01(1H,s), 7.51~7.48(2H,m), 7.39~7.24(7H,m), 6.88~6.85(4H,m), 5.90~5.86(1H,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). 13C NMR(CDCl3)δ:158.64, 158.11, 152.55, 148.92, 144.97, 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, 59.88, 50.16, 49.93, 48.49, 47.36, 47.12, 46.08, 46.00, 4 3.93, 43.83, 43.71, 26.82, 26.28, 26.19, 24.80, 24.36, 24.27, 24.18, 21.04, 20.81, 20.79, 20.35, 20.07. ESI-MS(m / z):751.4238(M+H) + .

[0065] N 2 -O 6 -Bis[di(tert-butyl)isobutylsilyl-5'-dimethoxytritylmorpholinoguanosine (23): Yield: 47%. 1 H NMR(CDCl3,400MHz)δ:7.80(1H,s), 7.51~7.49(2H,m), 7.39~7.32(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, 1 16.84, 113.06, 85.99, 80.73, 77.41, 64.42, 60.23, 49.83, 47.78, 26.31, 24.89, 24.72, 21.94, 21.40, 20.73. ESI-MS(m / z):965.5944(M+H) + .

[0066] Referring again to FIG. 3, the procedure for the synthesis of an appropriately protected synthon (9, 14, 19 or 24) is as follows.

[0067] The 5'-O-DMT-N-BIBS protected morpholino nucleosides (8, 13, 18 or 23) described above were dried under vacuum overnight. They were dissolved in anhydrous CHCl, and then 1.2 equivalents of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite was added under argon. After adding 0.5 equivalents of 4,5-dicyanoimidazole, the reaction was allowed to proceed with stirring at room temperature under an argon atmosphere for 5 hours, at which time TLC showed complete conversion of the starting material. The reaction mixture was diluted with CHCl and washed first with 5% NaHCO solution and then with brine. The organic layer was dried over NaSO, filtered and evaporated to dryness. The product was purified by silica gel column chromatography. A silica gel slurry was made using a mixture of the starting eluents containing an additional 5% triethylamine. After pouring the slurry, the column was washed with 2 column volumes of the starting solvent mixture without triethylamine. The phosphorodiamidite compounds 9, 14, 19 and 24 were purified using a gradient of 3:7 ethyl acetate:hexane to 1:1 ethyl acetate:hexane.

[0068] 5'-O-Dimethoxytritylmorpholinothymine-3'-O-cyanoethyl-N,N-diisopropylphosphordiamidite (9): Yield: 86%. 31 P NMR(CD2Cl2)δ:127.21, 126.08. 1H NMR (CD2Cl2,400MHz)δ:9.51(1H,bs), 7.50~7.47(2H,m), 7.38~7.25(8H,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), 3.99~3.8 6(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, 135.92, 135.90, 135.79, 135.68, 135.52, 1 29.99, 128.07, 127.77, 126.77, 117.89, 117.74, 113.05, 110.48, 110.39, 86.05, 80.54, 8 0.49, 80.20, 77.36, 77.21, 64.37, 60.09, 59.84, 55.20, 49.05, 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-morpholinocytosine-3'-O-cyanoethyl-N,N-diisopropylphosphordiamidite (14): Yield: 83%. 31 P NMR(CD2Cl2)δ:126.32, 124.88. 1H 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), 6.88~6.8 5(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), 3.14~3.10(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), 2.11~2.07(1H,m), 1.78(1H,bs), 1.25~1.15(31H,m), 1.04~1.02(8H,m). 13 C NMR(CD2Cl2)δ:168.14, 158.61, 154.46, 144.98, 140.62, 140.42, 135.98, 135.88, 130.03, 128.07, 12 7.75, 126.71, 117.96, 117.75, 113.02, 96.20, 96.09, 85.97, 81.68, 81.34, 81.23, 77.05, 77.00, 64.5 0, 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, 26.21, 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'-dimethoxytritylmorpholinoadenine-3'-O-cyanoethyl-N,N-diisopropylphosphordiamidite (19): Yield: 79%. 31 P NMR(CD2Cl2)δ:127.96, 125.36. 1H 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(1H,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(1 H,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, 117.79, 117.71, 113.04, 86.50, 80.50, 80.44, 80. 34, 80.22, 76.94, 76.81, 64.39, 60.14, 59.91, 55.18, 50.16, 49.93, 48.49, 48.44, 47.35, 47.12, 46.08, 4 6.00, 43.95, 43.83, 43.71, 28.62, 26.28, 26.19, 24.80, 24.35, 24.29, 24.18, 21.04, 20.79, 20.36, 20.27. ESI-MS(m / z):951.5437(M+H) + .

[0071] N 2 ,O 6 -Bis[di(tert-butyl)isobutylsilyl]-5'-dimethoxytrityl-morpholinoguanine-3'-O-cyanoethyl-N,N-diisopropylphosphoradiamidite (24): Yield: 82%. 31 P NMR(CD2Cl2)δ:127.62, 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), 2.6 1~2.54(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, 158.61, 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, 47.68, 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, 21.90, 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 prepared according to literature protocols (Roy, et al., J. Am. Chem. Soc. 2013, 135, 6234-6241). Compound 25 was commercially available. The IgG1A1 and IgG2A1 peptides were obtained from a commercially available supplier (Glen Research).

[0073] General method for the synthesis of 26, 27, and 28 The 5'-dimethoxytrityl-N-BIBS protected 2'-deoxynucleoside was added to an argon-purged round-bottom flask. Anhydrous dichloromethane and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.2 eq.) were added via syringe. 1.0 eq. of tetrazole (0.01 ml in CH3CN obtained from Glen Research) was added via syringe. 0.4M) was added dropwise to this solution with stirring over 30 min. The reaction was stirred at room temperature for 2-3 h, at which time TLC showed complete disappearance of starting material. The reaction mixture was diluted in dichloromethane and extracted twice with saturated NaHCO3 solution. The organic layer was dried over Na2SO4, filtered and evaporated to dryness. The product was purified by flash chromatography on a silica column. A silica gel slurry was made with a mixture of starting eluents containing an additional 5% triethylamine. After pouring the slurry, the column was washed with 2 column volumes of starting solvent mixture without triethylamine. 26 and 27 were purified using a gradient from 3:7 ethyl acetate:hexane to 1:1 ethyl acetate:hexane. 28 was purified using a 7:3 hexane:dimethyl ether mixture.

[0074] N 2 -Di(tert-butyl)isobutylsilyl-5'-dimethoxytrityl-2'-deoxycytidine 3'-O-cyanoethyl-N,N-diisopropylphosphoramidite (26). Yield: 87%. 31 P NMR (CD2Cl2): 148.67, 148.43. 1H 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(1H,m), 3.89~3.7 7(7H,m), 3.72~3.58(3H,m), 3.51~3.35(2H,m), 2.68~2.58(2H,m), 2.52~2.49(1H,m), 2.30~2.22(1H ,m), 2.11~2.04(1H,m), 1.29~1.25(1H,m), 1.23~1.20(9H,m), 1.14~1.10(22H,m), 1.02-0.97(9H,m). 13 C NMR(CD2Cl2)δ:158.71, 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, 85.11, 84.98, 84.92, 62.91, 62.61, 58.64, 58.26, 43.30, 43.1 4, 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'-deoxyadenosine 3'-O-cyanoethyl-N,N-diisopropylphosphoramidite (27). Yield: 81%. 31 P NMR (CD2Cl2): 148.51, 148.49. 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(7H,m). 13 C NMR(CD2Cl2)δ:158.64, 158.09, 152.37, 149.19, 144.86, 138.72, 135.86, 135.67, 130.11, 129.99, 128.09, 127.78, 126.74, 122.27, 117.76, 117.64, 113.05, 86.31, 85.66, 85.45, 84. 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, 2 8.62, 26.25, 26.21, 24.80, 24.42, 24.38, 24.31, 21.02, 20.79, 20.44, 20.37, 20.29, 20.22. ESI-MS(m / z):952.5279(M+H) + , 974.5130(M+Na) + .

[0076] N 2 -Di(tert-butyl)isobutylsilyl-5'-dimethoxytrityl-2'-deoxyguanosine 3'-O-cyanoethyl-N,N-diisopropylphosphoramidite (28). Yield: 79%. 31 P NMR (CD2Cl2): 149.03, 148.52. 1H 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(7H,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(1H,m), 1.23~1.15(48H,m), 1.06-0.97(18H,m). 13 C NMR(CD2Cl2)δ:160.64, 159.27, 158.67, 154.21, 144.77, 136.28, 135.71, 135.60, 130.08, 128 .11, 127.87, 126.80, 117.56, 117.14, 113.13, 86.39, 85.40, 85.09, 83.79, 83.63, 63.78, 63.4 8, 58.37, 58.16, 43.34, 43.18, 40.88, 40.55, 28.74, 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. ESI-MS(m / z):1188.6903(M+Na) + .

[0077] Example 2 Synthesis of morpholino oligonucleotides using BIBS-protected morpholino phosphoramidites. Once the synthons described in Example 1 were available, the next goal was to optimize the solid-phase synthesis cycle to generate borane phosphoramidate morpholino derivatives and convert these compounds to the corresponding PMOs. The synthesis cycle is outlined in Figure 4 and Table 1. Prior to synthesis, the 5'-DMT group on 2'-deoxyribothymidine linked to a polystyrene support was removed with 0.5% trifluoroacetic acid in chloroform containing 10% trimethylphosphiteborane (TMPB).

[0078] 5'-Unprotected-2'-deoxyribonucleosides (Figure 4, Compound A) were reacted with one of the synthons 9, 14, 19 or 24 (Figure 4, "1. Condensation") in anhydrous acetonitrile containing 4,5-dicyanoimidazole (DCI) to generate dimers with phosphoramidite diester internucleotide linkages (Figure 4, Compound B). The coupling wait time was 300 s. Following borylation, capping (Figure 4, "2. Boronation" and 3. Capping") and detritylation (Figure 4, "4. Deprotection"), repetition of this cycle generated products ready for further conversion to PMOs.

[0079] [Table 1]

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

[0081] To convert the morpholino borane phosphoramidate to the N,N-dimethylamino PMO, the morpholino borane phosphoramidate was treated overnight with a solution of 0.05 M iodine and 2.0 M dimethylamine in tetrahydrofuran (Figure 4, "6. Iodine / Dimethylamine"). The resin was washed repeatedly with acetonitrile, and the oligonucleotide was then treated with a solution of 1.0 M THF of tetrabutylammonium fluoride (TBAF) to remove the BIBS protecting groups (Figure 4, "7. Silyl Deprotection"). (Et3NHF hydrolyzed the phosphorodiamidate bond, so this reagent could not be used to remove these silyl groups.)

[0082] When oxidative amination was performed after removal of the silyl protecting group, amine substitution at N-4 of cytosine was observed (Figure 5). This challenge was overcome by performing the oxidative amination reaction prior to desilylation, as depicted in reaction steps 6 and 7 in Figure 4.

[0083] Once the silyl groups were removed, the oligonucleotides were cleaved from the support using 30% ammonium hydroxide (Figure 4, "8. Cleavage from Support"), the polystyrene resin was removed using a 0.2 μm centrifugal filter, and the solution was passed through an Illustra NAP-25 column to remove the fluoride salt. An additional purification step was performed with each PMO using an Amicon™ Ultra-15 3K centrifugal filter device (to remove shorter failed sequences). The oligonucleotides were then used for further characterization and other studies.

[0084] The synthesis was carried out on an ABI 394 synthesizer. All syntheses were carried out on a 0.2 micromolar scale using 5'-DMT2'-deoxythymidine tethered to a small amount of polystyrene solid support via a succinate bond. For the synthesis of morpholino oligonucleotides, a standard 2.0 micromolar synthesis cycle was used with an increased coupling time of 300 seconds. The detritylation step was followed by washing with methanol. All phosphorodiamidites (9, 14, 19 or 24; 0.1 M) were dissolved in anhydrous CH3CN. Detritylation was carried out using a 0.5% solution of TFA in anhydrous CHCl3 containing 10% TMPB. Solutions for boronation (0.05 M BH3-THF complex in THF) were made fresh prior to use. Reagents for activation (4,5-dicyanoimidazole) and capping were purchased from Glen Research. A step-by-step description of the synthesis cycle is given in Table 1. Deprotection was performed in two steps: the borane-phosphoramidate morpholino oligonucleotides linked to the solid support were first treated with a 1:1 solution of triethylamine in acetonitrile for 10 min, followed by extensive washing with acetonitrile. The resin was then dried using a stream of argon and transferred to a glass vial. A solution of iodine (0.05 M) and dimethylamine (2.0 M) in THF was added to the glass vial. For the reaction with ammonia, the same amount of iodine was dissolved in an isopropanol solution of ammonia (2.0 M) and this solution was added to the resin. The glass vial was then placed on a mechanical shaker for 24 hours. For the synthesis of methylamine and morpholinodiamidate derivatives, a 2.0 M THF solution of each amine was used.

[0085] The vial containing the resin was centrifuged at 4000 rpm and the supernatant was removed with a pipette. The resin was then washed 4-5 times with 2 mL aliquots of acetonitrile and shaken vigorously. The resin was centrifuged at 4000 rpm for 5 minutes and the supernatant was removed. For amino-modified morpholino derivatives, the ammonia solution in isopropanol was removed under vacuum. These morpholino oligonucleotides were desilylated by overnight treatment with fluoride (1.0 mL of 1.0 M THF solution of tetrabutylammonium fluoride). The resin was then treated with 1 mL of 37% aqueous ammonium hydroxide for 1 hour to remove the product from the resin, and the polystyrene resin beads were removed using a 0.2 μm centrifugal filter.

[0086] The total volume of each solution was diluted to 2.5 mL by adding 0.5 mL of Milipore water, and the PMOs were desalted using Illustra™ NAP™-25 columns (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 3.5 mL of Millipore water was used to elute the purified sample. The second step of purification was carried out for oligonucleotides with more than 10 nucleotides using an AMICON™ Ultra-4 3K device. The accumulated solution after Nap column purification (total volume 3.5 mL) was loaded onto the Amicon device and centrifuged at 4000×g for 30 minutes. The concentrated solute was washed twice with 3.0 mL of Millipore water in the same manner. The oligonucleotides were collected and used for various experiments.

[0087] Uridine morpholino phosphoramidite (B * = uracil) into siRNA sequences and subsequent conversion of the resulting morpholino phosphoramidite internucleotide linkages to the corresponding phosphoramidate morpholino linkages with aqueous iodine has been reported (Tetrahedron Letters 2008, 49:3570-73; Bioorg. Med. Chem., 2009, 17:2441-46). However, we have not found any significant difference in the ETT (pK a We found that a 5'-N,N-diisopropylamino-phosphorodiamidate (pK = 4.3) activated both morpholine and N,N-diisopropylamine (Figure 3). As a result, very low yields of the final PMO product were observed following their literature protocol, as activation of morpholine followed by boronation and iodine / dimethylamine oxidation resulted in a capped PMO oligonucleotide bearing a 5'-N,N-diisopropylamino-phosphorodiamidate (Figure 4, compound C) that could not be further extended (Figure 4). To further explore this issue, several less acidic activators (tetrazole, saccharin-1-methylimidazole, 4,5-dicyanoimidazole) were tested to obtain a 5'-N,N-diisopropylamine (pK = 4.3) activation with N,N-diisopropylamine (pK = 4.3). a =11.1), but morpholine (pK aAmong the activators screened in the synthesis of a PMO with a sequence corresponding to ODN1 (Table 2), 4,5-dicyanoimidazole (pK a = 5.2, 0.12 M, with a coupling time of 300 s) produced the highest yield (67%) of the desired PMO with low levels of by-products (Supporting Information, Figure S2). Because the morpholino phosphoramidite diester synthesized after the coupling step (Figure 4, compound B) was unstable in the capping solution, boronation was performed to generate the phosphorus(IV) morpholino compound (Figure 4, compound D) prior to the capping step. After boronation, the support was washed with acetonitrile, failed sequences were capped using acetic anhydride, and detritylation was performed using 10% TMBP and 0.5% TFA in chloroform (compound D was stable to these acidic detritylation conditions).

[0088] Using this synthetic procedure outlined in Figure 4, a trimer with only thymine nucleoside bases was synthesized, converted to the N,N-dimethylamino PMO derivative with iodine / N,N-dimethylamine, removed from the support with ammonia, and analyzed by LCMS. The four main peaks shown in the LC profile (Figure 6, right panel) have the expected mass spectra for the four diastereomers of the product (calculated mass 902.3, observed 901.3). The combined areas of all peaks from the crude reaction mixture were used to calculate the mass spectra for these diastereomers. By comparing the peak area to the dimer, compound 29 was calculated to be in 94% yield. In a similar experiment in which the dimer was made, the first coupling yield was found to be 96%, which is much better than other reported procedures (Bioorg. Med. Chem. Lett. 2012, 22:1445-47, Tetrahedron Letters, 2015, 56;4565-68).

[0089] [Table 2]

[0090] Using this approach, oligothymidines (Table 2, ODN1 and 2) and N,N-dimethylamino PMOs bearing all four bases (Table 2, ODN3-8) were synthesized and characterized by LCMS. Since it had been established that PMOs 12-16 nucleosides in length are effective inhibitors of RNA expression (either via interfering with splicing or interfering with mRNA translation), we next pursued the synthesis of a 16-mer bearing all four bases (Table 2, ODN8). LCMS chromatograms of the crude reaction mixtures showed high yields (10 isolated A). 260 Both the NMR spectroscopy (NMR spectroscopy) and mass spectrometry of this ODN showed to be satisfactory.

[0091] Example 3 Synthesis of PMO-DNA chimeras For the synthesis of PMO-DNA chimeras, 4,5-dicyaminimidazole (0.12 M and 300 s coupling time) and ETT (0.25 M and 180 s coupling time) were used for the morpholino phosphoramidite synthon (9, 14, 19 or 24) and 5'-dimethoxytrityl-2'-deoxyribonucleoside-3'-phosphoramidite (25, 26, 27 or 28), respectively. After condensation, the morpholino phosphoramidite diester was converted to a phosphorus(IV) borane bond, and the phosphite triester was converted to a phosphate triester using standard oxidation with 0.02 M iodine in THF / water / pyridine. These synthetic steps were repeated until the PMO-DNA chimera of the desired sequence / length was produced. Table 1 outlines these synthetic steps.

[0092] Since PMO-DNA chimeras are novel to the scientific community and could prove useful for various research projects, we synthesized several PMO-DNA chimeras (Table 2, ODNs 9-16). First, these chimeras were synthesized by combining four N,N-dimethylamino PMO bonds. In these 21-mers, the morpholinodiamidate linkage was placed adjacent to either the 5' or 3' end, or near the middle of the 21-mer, and at every third position throughout the oligomer (Table 2; ODNs 9, 10, 11, 12, and 13). For these chimeras, replacement of borane with diethylamine upon activation with iodine proceeded efficiently, and analysis by LCMS of the crude reaction mixtures revealed that the expected phosphorodiamidate linkages were formed in near quantitative yield. These encouraging results led to the synthesis of PMO-DNA chimeras containing all four nucleobases in various locations and multiple PMO linkages (Table 2; ODNs 14, 15, and 16). LCMS analysis of the reaction mixtures and the 31 P NMR demonstrated that the predicted PMO-DNA chimeras were obtained in high yield with an average yield of 10-20 A260 units (from a synthesis cycle of 0.2 μM). These experiments also demonstrated that treatment with dimethylamine did not result in measurable cleavage of the succinate bond and loss of product during synthesis.

[0093] Example 4 Solid-phase synthesis of PMOs with amino, N-methylamino and morpholino bonds It is known that the borane phosphonate bond can be activated by iodine toward displacement by a number of nucleophiles. Therefore, in addition to testing this new synthetic route by synthesizing PMO analogs with N,N-dimethylamino-phosphorodiamidate bonds, we decided to examine whether other amines could be used to generate some new PMO-DNA derivatives.

[0094] First, an oligothymidine 21-mer was synthesized with four borane phosphoramidate morpholino linkages near the center of the oligomer. The support containing this oligonucleotide was divided into three samples that were treated with N-methylamine, ammonia, and morpholine under iodine oxidation conditions and then purified using reverse-phase column chromatography. In addition, mixed-sequence PMO-DNA chimeras were synthesized with all four bases and amino-phosphorodiamidate internucleotide linkages, where the positions of the diamidate linkages were placed at the 5', 3', and 5' / 3' ends of these chimeras. The sequences of these PMOs and their mass analyses by LCMS are listed in Table 3. The yields were comparable to those obtained for the N,N-dimethylamino PMO chimeras.

[0095] [Table 3]

[0096] Example 5 Melting temperature of PMO-DNA chimeras To evaluate the target binding ability of the modified PMO-DNA chimeras, duplex hybridization studies were performed with 2'-deoxyoligothymidines bearing amino, methylamino, morpholino and dimethylamino-morpholino internucleotide linkages. PMO11, 17, 18 and 19 were mixed with 21 nucleotide long 2'-deoxyribo- or riboadenosine-oligonucleotides at 1:1 ratio in buffer (1.0 M NaCl, 10 mM sodium phosphate, pH 7.1) at 0.0 μM. The samples were denatured at 96°C and cooled to 15°C. The samples were then heated at a rate of 1°C / min and cooled to 15°C. 260 The melting temperature was taken as the temperature of half dissociation and was obtained from the first derivative plot (Table 4).

[0097] Melting temperature studies of RNA heteroduplexes of PMOs 11, 17, 18, and 19 showed that the introduction of phosphorodiamidate internucleotide linkages stabilized the PMO:RNA duplexes compared to the unmodified duplexes. The trend in increasing stabilization was amino > N-methylamino > N,N-dimethylamino > morpholino. When PMO-DNA chimeras were duplexed with a 2'-deoxyriboadenosine 21mer, the T values ​​of the unmodified DNA·DNA duplexes were significantly higher than those of the unmodified DNA·DNA duplexes, except for the morpholino analogue. m A similar trend was observed when comparing

[0098] [Table 4]

[0099] Example 6 RNase H1 activity of chimeric PMO-RNA heteroduplexes N,N-dimethylamino PMO-DNA chimeras were tested for their ability to stimulate RNase H1 activity. The test system consisted of 5'-O-fluorescein-labeled RNA and the complementary N,N-dimethylamino PMO chimera.

[0100] Synthesis of 5'-fluorescein PMO-DNA chimeras. After completion of the DMT-ON synthesis of the borane phosphoramidate morpholino chimera, the ODN was conjugated with 5'-Amino-Modifier C6-TFA (Glen research) using a standard DNA synthesis cycle. After iodine oxidation and desilylation of the exocyclic amine, the oligonucleotides were purified using reversed-phase HPLC (Buffer A: triethylammonium bicarbonate, 0.05 M, Buffer B: acetonitrile; 0% to 100% B for 50 min; 55° C.; flow rate 4.0 mL / min). The purified ODN was dissolved in 1 mL of 1:1 NH4OH:CH3NH2 and heated at 65° C. for 3 h to remove the trifluoroacetamide group. The reaction mixture was dried and dissolved in 200 microL of buffer containing 20 mM sodium phosphate and 0.15 M NaCl (pH 8.0) and the concentration was measured. A 20-fold molar excess of 5-(and 6-)carboxy-fluorescein succinimidyl ester (Thermo Fisher Scientific) was dissolved in DMSO and added to the ODN solution. The reaction mixture was stirred at room temperature for 1 h and then at 4° C. for 3 h. The reaction mixture was diluted with 300 microliters of water and excess NHS-fluorescein was removed using an ILLUSTRA™ NAP™-5 column. The PMOs analyzed were oligothymidine 14-mers (ODN24-ODN26, Table 5) containing a cap / gap sequence with an N,N-dimethylamino PMO linkage at either end and 3-7 phosphodiester linkages in the center of the analog. Controls included complementary DNA and 2'-O-methyl RNA (which activate RNase H1 activity, respectively). Both of these cap / gap oligonucleotide analogs were found to activate RNA hydrolysis.

[0101] Hydrolysis of RNA heteroduplexes by E. coli RNase H1. E. coli RNase H1 (Promega) experiments were performed using conditions previously described (J. Am. Chem. Soc., 2003, 125:940-50). 50 mM Tris-HCl (pH 8.0), 20 mM The reactions were carried out using an assay buffer of KCl, 9 mM MgCl2, 1 mM β-mercaptoethanol and 250 μg / mL bovine serum albumin. Oligodeoxynucleotides or modified oligodeoxynucleotides (200 pmol) and 5'-O-fluorescein-labeled complementary oligoribonucleotides were added to the assay buffer (35 μL). Following the addition of E. coli RNase H1 (3 units), the reactions were carried out for 12 hours at 25°C. The reaction mixtures were diluted with an equal volume of 80% formamide gel loading buffer containing tracking dye and analyzed by polyacrylamide gel electrophoresis (20%, 19:1 cross-linked, 7M urea). All reactions were carried out in triplicate. Developed gels were analyzed using a Molecular Dynamics Typhoon Phosphorimager.

[0102] [Table 5]

[0103] Example 7 Cellular uptake Because it is not possible to deliver uncharged PMOs to cells using lipid-based transfection reagents, we examined the cellular uptake of these PMO-DNA chimeras in the presence of Dharmafect 1, a commonly used siRNA transfection reagent. PMO-DNA chimeras were 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, see Table 2 for explanation of these abbreviations), where an internal normal nucleotide is flanked by N,N-dimethylamino PMO nucleotides at the 5'- and 3'-ends. ODN27 also contained a fluorescein dye (FL) tethered by a 6-carbon linker. HeLa cells were transfected with ODN27 (100 nM concentration) in the presence of Dharmafect 1 using live and fixed cells (Figure 7) and imaged by fluorescence microscopy after 20 and 18 hours of incubation, respectively.

[0104] Lipid transfection observed by microscopy images. ODN27 stock solution was diluted in 200 microL of OptiMEM to a final concentration of 0.1 microM ODN. In a separate Eppendorf tube, 5.0 microL of DharmaFECT 1 was diluted in 200 microL of OptiMEM. 200 microL of ODN27 and DhamaFECT 1 solutions were mixed and equilibrated for 20 minutes, and 600 microL of OptiMEM was added. 0.3×10 cells were transfected onto glass coverslips in a 6-well plate in DMEM medium with 10% FBS and penstrep. 6 HeLa cells were plated at 1000 cells / well. After 24 hours, the medium was removed and transfection was performed at 80% confluency. Before transfection, cells were washed twice (2.0 mL D-PBS / wash). D-PBS was removed from HeLa cells and 1.0 mL of the transfection mixture was added to each well. Cells were then incubated at 37°C for 18 h and washed twice (2.0 mL D-PBS / wash). Cells were covered with 1.0 mL 10% neutral buffered formalin for 15 min. The formalin solution was removed and cells were covered with 3.0 mL DPBS for 10 min at room temperature. Coverslips were removed from wells and mounted upside down on cover slides using Floromount-G with DAPI as mounting agent and observed using an inverted microscope (OlympusIX 81) equipped with a Hamamatsu C4742-95 CCD and a CoolSNAP ES digital camera.

[0105] Transfection was dose-dependent, as an increase in the fluorescent signal was observed when cells were incubated with 100 nM ODN27. The fluorescence appears to be distributed primarily in the nucleus, although there is also evidence of cytoplasmic distribution without the common punctuated structures seen in many analogues.

[0106] The above examples of the present invention are presented for purposes of illustration and description. Moreover, these examples are not intended to limit the present invention to the forms disclosed herein. As a result, variations and modifications in accordance with the teachings of the present specification, as well as 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 further illustrate the best modes known for carrying out the present invention, and it is intended that those skilled in the art will be able to utilize the present invention in such or other embodiments with various modifications as required for the particular application or use of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.

[0107] This has been done only to the extent that the appended claims are drafted without multiple dependencies in order to comply with formal requirements in competent patent offices that do not recognize such multiple dependencies. It should be noted that all possible combinations of features that may be implied by providing multiple dependent claims are expressly contemplated and are made part of the present invention.

Claims

1. The following formula: 【Chemistry 1】 (In the formula, B is a nucleobase selected from adenosine, guanosine, uracil, thymine, cytosine, and inosine, or said nucleobase protected with a silyl protecting group or an acid-labile protecting group or a base-labile protecting group; Y is oxygen or sulfur; X is oxygen, sulfur, BH ３ or N,N-dimethylamino, and at least one X in the oligomer is N,N-dimethylamino.

1. A method for forming an oligomer comprising a monomeric subunit represented by a. Providing a 5'-unprotected-2'-deoxyribonucleoside linked to a polystyrene / CPG support; b. reacting the 5'-unprotected-2'-deoxyribonucleoside with a nucleoside phosphorodiamidite in anhydrous acetonitrile containing 4,5-dicyanoimidazole (DCI), tetrazole, 5-(ethylthio)-1H-tetrazole, or other activating agent to form an adduct; c. chemically activating the adduct, comprising at least one of boronizing, sulfurizing, or oxidizing the adduct; d. capping any unreacted nucleosides of step b; e. Detritylation of the adduct; f. Repeating steps b to e to extend the adduct to form an oligomer; g. Removing the cyanoethyl groups from the oligomer; h. when X is N,N-dimethylamino, contacting the oligomer with iodine and dimethylamine in tetrahydrofuran to convert BH 3 to an N,N-dimethylamino group; i. removing the oligomers from the support by contacting the oligomers with a solution comprising ammonium hydroxide and ethylenediamine; Including, when X is N,N-dimethylamino, step c comprises boronation of said adduct; As at least one nucleoside phosphorodiamidite in step b and / or repeated step b, a nucleoside phosphorodiamidite having the following formula: 【Chemistry 2】 (In the formula, B is the same as above; R is trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl; R １ and R ２ are each independently ２～２０ It is a straight or branched chain alkyl. The method of claim 1, wherein a nucleoside phosphorodiamidite of step b is used,

2. 2. The method of claim 1, further comprising the additional step of contacting the oligomer with a solution of tetrabutylammonium fluoride (TBAF) in THF to remove the BIBS protecting group from the oligomer if the oligomer has a BIBS protecting group prior to step i.

3. 2. The method of claim 1, wherein said iterative synthesis step f is carried out on a commercially available DNA synthesizer.

4. 2. The method of claim 1, wherein step c comprises sulfurizing the adduct to form a thiophosphoramidate internucleotide linkage.

5. The nucleobase protecting group is 【Chemistry 3】 The method of claim 1 , wherein the compound is selected from the group consisting of

6. 10. The method of claim 1, further comprising forming a duplex between the oligomer and a complementary DNA or RNA which is more stable than a natural DNA or DNA / RNA complex.

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