RU2791532C2 - Chiral reagents for production of homogenous oligomers - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to a method for the production of essentially diastereomeric pure phosphorodiamidate morpholino-oligomer containing chiral phosphoric bonds, which is used for the production of stereo-chemically pure oligonucleotide. The proposed method includes following stages: selection of essentially stereo-chemically pure phosphoramidochloride morpholino-monomers; and synthesis of essentially diastereomeric pure phosphorodiamidate morpholino-oligomer by stereo-specific combination of selected essentially stereo-chemically pure phosphoramidochloride morpholino-monomers, where the specified stereo-specific combination is carried out in an aprotic solvent in the presence of non-nucleophilic tertiary amine or aromatic base, at a temperature from 20°C to 50°C. At the same time, essentially stereo-chemically pure phosphoramidochloride morpholino-monomers are obtained by separation of a diastereomeric mixture of phosphoramidochloride morpholino-monomers into essentially stereo-chemically pure phosphoramidochloride morpholino-monomers. Essentially stereo-chemically pure, in this case, relates to enantiomers or diastereomers, which are in enantiomeric or diastereomeric excess, respectively, equal to or more than 87%, and essentially diastereomeric pure relates to diastereomers, which are in diastereomeric excess equal to or more than 87%.

EFFECT: obtainment of a method for the production of oligomers.

6 cl, 11 dwg, 2 tbl, 4 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under US Provisional Application No. 62/201510, filed Aug. 5, 2015. This application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Field of technology

[0003] Embodiments may relate to the preparation of substantially diastereomerically pure activated monomers that are morpholino-phosphoramidochloridate subunits. Embodiments may also refer to the use of substantially diastereomerically pure activated phosphorylated morpholine monomers to produce molecules by stereospecific coupling reactions.

[0004] State of the art

[0005] The synthesis of diastereomerically pure phosphorodiamidate oligonucleotides is significantly complicated by the presence of chiral phosphorus bonds. This contrasts, for example, with phosphodiester bonds, which do not have a chiral phosphorus. Examples are shown in Figure 1, which compares phosphodiester, phosphorothioate (which also includes chiral phosphorus), and phosphorodiamidate.

[0006] the Presence of chiral phosphorus presents significant difficulties in the synthesis, which involves the connection of a series of phosphorodiamide nucleotides. The absence of stereochemically pure reagents (matrices, subunits, structural units) that allow the stereospecific formation of phosphorodiamidate bonds leads to a reaction at the stereocenter, when it is impossible to control the phosphorus chirality of the resulting compound.

[0007] As shown graphically in Figure 2, the use of diastereomeric mixtures of nucleotides for a stereochemically uncontrolled coupling reaction to produce an oligonucleotide of any significant length and sequence produces a heterogeneous mixture of multiple diastereomers. The number of diastereomers is theoretically 2 ^(n-1) , where n is the number of nucleotides that join to form an oligonucleotide. As shown in Figure 2, even the simplest four-nucleotide oligonucleotide (tetranucleotide) can result in a mixture of eight individual diastereomers.

[0008] the Formation of a significant number of diastereomers may create a need for sensitive separation methods after synthesis. The output of the target product may adversely affect the use of raw materials due to the production of many diastereomers that are not target.

[0009] It would be useful to be able to select a particular diastereomer prior to synthesis and then synthesize the selected diastereomer in a stereochemically pure or substantially pure form.

SUMMARY OF THE INVENTION

[0010] Embodiments provide one or more of the stereochemically pure or substantially stereochemically pure compounds listed in Table 1. In additional embodiments, enantiomers of the compounds of Table 1 are also provided. Typically, the stereochemistry of these enantiomers differs from that of the compounds of Table 1 , by changing the stereochemistry of the morpholine ring.

[0011] TABLE 1

Compound formula number

20

21

22

23

24

25

26

27

28

29

thirty

31

[0012] R ₁ and R ₂ may be the same or different, and may be -H, optionally substituted C1-C3 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or with the nitrogen atom to which they are attached, they form an optionally substituted heterocycle which may be, for example, pyrrolidine, piperazine or morpholine.

[0013] Optionally substituted groups may be substituted with one or more of methyl, ethyl, halogen, nitro, methoxy, or cyano.

[0014] R ₃ may be trityl (Tr), which may be substituted trityl, including but not limited to, for example, MMTr (p-methoxyphenyldiphenylmethyl), optionally substituted benzyl, 4-methoxybenzyl (PMB, MPM), 3, 4-dimethoxybenzyl, diphenylmethyl (Dpm) or sulfonyl, which may be a cleavable sulfonyl. In some embodiments, the sulfonyl is 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl.

[0015] R ₄ , R ₅ , R ₆ may be -H, -C(O)R ₇ or -C(O)OR ₇ where R ₇ is alkyl (methyl, ethyl, isopropyl or other C1-C6 -alkyl), benzyl, 2,2,2-trichloroethyl or aryl (including but not limited to phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl). R ₉ may be optionally substituted alkyl, cyanoethyl, acyl, carbonate, carbamate, optionally substituted benzyl, 4-pivaloyloxybenzyl and silyl.

[0016] In additional embodiments, the morpholine nucleosides, in addition to those listed in Table 1, may be prepared in diastereomeric pure or substantially diastereomeric pure form.

[0017] In embodiments, methods can also be provided for separating diastereomeric mixtures of the compounds disclosed above into stereochemically pure or substantially stereochemically pure compounds. In additional embodiments, pharmaceutical compositions can be provided containing stereochemically pure or substantially stereochemically pure compounds as described herein. In other embodiments, pharmaceutical compositions can be provided containing pharmaceutically acceptable salts of stereochemically pure or substantially stereochemically pure compounds as described herein. The pharmaceutical compositions may be administered in an effective amount to patients in need of treatment. Pharmaceutical compositions may further include pharmaceutically acceptable carriers.

[0018] In some embodiments, the following group in each of the compounds of Table 1 may be substituted at positions a, b and e with one or two methyl groups, and may be substituted at positions c and d with one methyl group. In each case, the methyl group may be on either side of the plane of the morpholine ring. In other embodiments, an additional methylene, optionally substituted with one or more methyl groups, may be inserted adjacent to the nitrogen atom in the morpholine group to allow expansion to a seven-membered ring.

[0019] Embodiments of the invention further provide stereochemically pure oligonucleotides by stereospecific coupling of activated monomers. Additional embodiments relate to a substantially diastereomerically pure oligomer made by a stereospecific combination of activated monomers. Other embodiments relate to an essentially diastereomeric pure composition containing essentially diastereomeric pure compounds described herein.

DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows phosphodiester, phosphorothioate, and phosphorodiamidate oligonucleotide linkages.

[0021] In FIG. 2 shows the R- and S-phosphorus bonds in a phosphorodiamidate morpholino oligomer (PMO). In FIG. 2 also shows the increase in diastereomers that typically occurs when diastereomeric mixtures of phosphorodiamidate oligomer precursors are used to synthesize dinucleotides (2-mer), trinucleotides (3-mer), tetranucleotides (4-mer), and N-mer oligomers.

[0022] FIG. 3 shows the preparation of diastereomeric pure dinucleotides both with diastereomeric pure phosphoramidochloridates and with a diastereomeric mixture of phosphoramidochloridates.

[0023] FIG. 4A and FIG. 4B shows general diastereomeric mixtures of phosphoramidochloridates which may be useful in obtaining diastereomeric pure or substantially diastereomeric pure diastereomeric subunits.

[0024] FIG. 5 shows the preparation of diastereomerically pure (homogeneous) oligonucleotides using the diastereomerically pure phosphoramidochloridates described herein.

[0025] FIG. 6 shows a scheme for stereospecific synthesis of 16-mer PMO and stereoisomer differentiation by biophysical analysis.

[0026] FIG. 7 shows melting points for stereoisomers 1 and 2 from an example showing the stereospecific synthesis of 16-mer PMO and stereoisomer differentiation by biophysical analysis as described below.

[0027] In FIG. 8 shows ORTEP diagrams of crystalline compound 100 described below.

[0028] In FIG. 9A and FIG. 9B shows ORTEP diagrams of two fragments of compound 100 described below.

DETAILED DESCRIPTION

[0029] The inventors have found that the two diastereomers of the activated morpholine subunits can be separated by their physical properties, allowing the preparation of diastereomerically pure isomers. This allows the preparation of stereochemically pure or substantially stereochemically pure PMOs under controlled reaction conditions, which can then be used to selectively obtain oligonucleotides with the desired stereochemistry.

[0030] Embodiments may also relate to methods for separating diastereomeric mixtures of the compounds disclosed above into stereochemically pure or substantially stereochemically pure compounds. After separation, the pure diastereomers from the preceding diastereomeric mixture can be used to prepare diastereomeric pure compounds via stereospecific coupling reactions.

[0031] Diastereomerically pure compounds and substantially diastereomerically pure compounds prepared as described herein may be diastereomerically pure phosphorodiamidate oligonucleotides. These diastereomerically pure and substantially diastereomerically pure phosphorodiamidate oligonucleotides can have a wide range of applications. For example, they may be useful as medicines. They may be selected for properties that are potentially superior to those of heterogeneous (stereo-random) mixtures of phosphorodiamidate oligonucleotide diastereomers. For example, they may be selected for differences in reactivity, efficacy, stability, safety, and specificity. Diastereomerically pure and substantially diastereomerically pure oligomers may have physical, chemical and biological properties that differ from those of stereochemically heterogeneous mixtures of oligomers.

[0032] "Stereoisomers" refers to isomers that differ only in the arrangement of atoms in space.

[0033] "Diastereomers" refers to stereoisomers that are not mirror images of each other.

[0034] "Enantiomers" refers to stereoisomers that are non-superimposable mirror images of each other. Enantiomers include "enantiomerically pure isomers" that contain essentially a single enantiomer, such as more than either 90%, 92%, 95%, 98% or 99%, or 100% of a single enantiomer.

[0035] "Activated monomer" refers to 5'-O-phosphorylated morpholino subunits that have reactive phosphorus having leaving groups, including but not limited to chloride and halide leaving groups, that undergo nucleophilic displacement reactions, including but not limited to but not limited to amines and alcohols.

[0036] "R" and "S" as terms describing isomers are stereochemical configuration identifiers at asymmetrically substituted atoms, including, but not limited to: carbon, sulfur, phosphorus and nitrogen in the ammonium group. The designation of asymmetrically substituted atoms as "R" or "S" is accomplished by applying the Kahn-Ingold-Prelog precedence rules, as is well known to those skilled in the art and described in the International Union of Pure and Applied Chemistry (IUPAC) Rules for Organic Chemistry Nomenclature. Section E, Stereochemistry.

[0037] An enantiomer can be characterized by the direction in which it rotates the plane of plane polarized light, as is well known to those skilled in the art of chemistry. If it rotates the light clockwise (as seen by the observer towards whom the light is moving), then the enantiomer is denoted (+) and called dextrorotatory. Its mirror image will rotate plane polarized light counterclockwise, and will be referred to as (-) or left-handed. The direction of rotation of plane polarized light by an enantiomerically pure compound, called the sign of optical rotation, can be easily measured in a standard device known as a polarimeter.

[0038] "Racemic" refers to a mixture containing equal parts of the individual enantiomers.

[0039] "Non-racemic" refers to a mixture containing unequal portions of individual enantiomers. A non-racemic mixture may be enriched in the R or S configuration, including, but not limited to, about 50/50, about 60/40, and about 70/30 mixtures of R- relative to the S-enantiomer or S- relative to the R-enantiomer.

[0040] The terms "substantially stereochemically pure" and "substantial stereochemically pure" refer to enantiomers or diastereomers that are in an enantiomeric excess or diastereomeric excess, respectively, equal to or greater than 80%. In some embodiments, "substantially stereochemically pure" and "significantly stereochemically pure" refer to enantiomers or diastereomers that are in enantiomeric excess or diastereomeric excess, respectively, equal to or greater than 87%, equal to or greater than 90%, equal to or greater than 95%. , equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98%, or equal to or greater than 99%. "Essentially diastereomerically pure" refers to diastereomers that are in diastereomeric excess equal to or greater than 87%, equal to or greater than 90%, equal to or greater than 95%, equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98 % or equal to or greater than 99%.

[0041] The "enantiomeric excess" (EI) of an enantiomer is [(mole fraction of major enantiomer) minus (mole fraction of minor enantiomer)]×100. The diastereomeric excess (DE) of a diastereomer in a mixture of two diastereomers is determined similarly.

[0042] The term "pharmaceutically acceptable salt" as used herein refers to acid addition salts or base addition salts for the compounds herein. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not have an unduly deleterious or undesirable effect on the subject to whom it is administered and in the context in which it is administered. Pharmaceutically acceptable salts include, but are not limited to, metal complexes and salts of both inorganic and carboxylic acids. Pharmaceutically acceptable salts also include metal salts such as aluminium, calcium, iron, magnesium, manganese and complex salts. In addition, pharmaceutically acceptable salts include, but are not limited to, salts of acids such as acetic, aspartic, alkylsulfonic, arylsulfonic, axetyl, benzenesulfonic, benzoic, bicarboxylic, bisulfite, bitartaric, butyric, calcium edetate, camsylic, carboxylic, chlorobenzoic, citric , ethylenediaminetetraacetic, edisyl, estol, ezyl, ezyl, formic, fumaric, gluceptic, gluconic, glutamic, glycolic, glycolylarsanilic, hexamic, hexylresorcinol, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, malic, maleic , almond, methanesulfonic, methylnitric, methylsulphuric, mucinic, muconic, napsilic, nitric, oxalic, p-nitromethanesulfonic, pamic, pantothenic, phosphoric, monohydrophosphoric, dihydrophosphoric, phthalic, polygalacturonic, propionic, salicylic, stearic, succinic, sulfamic, sulfani lic, sulfonic, sulfuric, tannic, tartaric, theocline, toluenesulfonic, etc.

[0043] An "effective amount" of a combination of therapeutic agents (eg, Compound 1 and a CDK4/6 inhibitor) is an amount sufficient to provide an observable therapeutic effect compared to untreated HCC or IHCC in a subject or patient.

[0044] The active agents described herein may be combined with a pharmaceutically acceptable carrier to form pharmaceutical compositions thereof. The specific choice of carrier and composition will depend on the particular route of administration for which the composition is intended.

[0045] The term "pharmaceutically acceptable carrier" as used herein refers to a non-toxic carrier, adjuvant, or delivery vehicle that does not interfere with the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants, or delivery vehicles that may be used in the compositions of the present invention include, but are not limited to, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, disodium hydrogen phosphate, hydrogen phosphate potassium, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene glycol and lanolin.

[0046] The compositions of the present invention may be suitable for parenteral, oral, nebulizing, topical, rectal, nasal, buccal, vaginal or implanted reservoir administration, etc. In some embodiments of the invention, the composition contains ingredients that are of natural and non-natural origin. In some embodiments, the formulation or carrier may be provided in a sterile form. Non-limiting examples of a sterile carrier include endotoxin-free water or pyrogen-free water.

[0047] The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In specific embodiments, the compounds are administered intravenously, orally, subcutaneously, or by intramuscular injection. Sterile injectable forms of the compositions of the present invention may be in the form of an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as the solvent or suspending medium.

[0048] Embodiments of the present invention relate to the preparation of stereochemically pure isomers or substantially stereochemically pure isomers, followed by the use of pure isomers to stereospecifically prepare diastereomeric pure phosphorodiamidate morpholine oligomers (PMOs). The preparation can be made by separating a diastereomeric mixture of phosphoramidochloridate nucleotides. Separation can be performed, for example, using chromatography, such as high performance liquid chromatography or "HPLC". Separation can also be carried out by crystallization.

[0049] The separated monomers may be referred to as "active" monomers. "Active" means that the monomers include a phosphoramidochloridate group that is reactive with various nucleophiles, which include, but are not limited to: amines, alcohols/alkoxides, thiol/thiolate, alkyl lithium, and Grignard reagents.

[0050] I. Preparation of diastereomeric isomers

[0051] In one embodiment of the invention, stereochemically pure or substantially stereochemically pure activated monomers can be obtained by separation of a diastereomeric mixture of monomers. Separation can be carried out by methods that allow stereoisomers to be distinguished using physical properties. For example, the separation can be performed using chromatography or crystallization. Suitable types of chromatography, for example, include, but are not limited to, high performance liquid chromatography (HPLC), pseudo-moving bed chromatography, countercurrent chromatography, and other types of separation chromatography. For example, a diastereomeric mixture may be subjected to HPLC eluting a fast moving fraction and a slow moving fraction. Each of these fractions is a separate stereochemically pure or essentially stereochemically pure amount of monomer. As described below, these monomers can be used to obtain oligomers with the desired stereochemistry by stereospecific coupling using controlled reaction conditions.

[0052] The inventors have additionally found that after separation, stereochemically pure activated monomers have sufficient stability to be used in further chemical reactions. In addition, the inventors have come to the conclusion that stereochemically pure activated monomers can participate in stereospecific chemical reactions. Thus, as described in more detail below, these pure stereochemically activated monomers can be used in stereospecific coupling reactions to produce stereochemically pure products.

[0053] As indicated above, embodiments of the invention may relate to one or more stereochemically pure or essentially stereochemically pure compounds from Table 1, which can be obtained using different physical properties of stereoisomers. Other embodiments may also refer to the enantiomers of the compounds of Table 1. Typically, the stereochemistry of these enantiomers differs from that of the compounds of Table 1 by changing the stereochemistry of the morpholine ring.

[0054] TABLE 1

Compound formula number

20

21

22

23

24

25

26

27

28

29

thirty

31

[0055] Where R ₃ is optionally substituted triphenylmethyl (also referred to as "trityl"), optionally substituted benzyl, or sulfonyl. R ₄ , R ₅ and R ₆ may be -C(O)R ₇ or -C(O)OR ₈ where R ₇ is methyl, ethyl or phenyl and R ₈ is benzyl or 2,2,2 -trichloroethyl. R ₉ may be optionally substituted alkyl, cyanoethyl (for use as a protecting group see, for example, US Patent Application Publication No. US2013/0197220), acyl, sulfonyl, acetal/ketal, carbonate, carbamate, optionally substituted benzyl, 4 -pivaloyloxybenzyl and silyl.

[0056] In some embodiments, the optionally substituted benzyl is 4-methoxybenzyl (PMB, MPM). In some embodiments, the sulfonyl is a cleavable sulfonyl. In some embodiments, the sulfonyl is 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl, or 2,4-dinitrobenzenesulfonyl.

[0057] R ₁ and R ₂ may be the same or different, and may be -H, optionally substituted C1-C3 alkyl, optionally substituted phenyl, optionally substituted naphthyl, or with the nitrogen atom to which they are attached, they form an optionally substituted heterocycle which may be, for example, pyrrolidine, piperazine or morpholine.

[0058] Optionally substituted groups may be substituted with one or more of methyl, ethyl, halogen, nitro, or cyano.

[0059] R ₃ may be trityl (Tr), which may be substituted trityl, including but not limited to, for example, MMTr (p-methoxyphenyldiphenylmethyl), benzyl, 4-methoxybenzyl (PMB, MPM) and 3,4- dimethoxybenzyl, diphenylmethyl (Dpm).

[0060] R ₄ , R ₅ , R ₆ may be -H, -C(O)R ₇ or -C(O)OR ₇ where R ₇ is alkyl (methyl, ethyl, isopropyl, or other C1-C6 -alkyl) or aryl (including but not limited to phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl).

[0061] II. Stereo specific combination

[0062] In addition to determining that separation technology can be used to obtain essentially stereochemically pure amounts of activated monomer, the inventors have found that these activated monomers can, under certain reaction conditions, be used to perform stereospecific coupling to obtain stereochemically pure dinucleotides, stereochemically pure trinucleotides and longer stereochemically pure oligomers. Using the methods described herein, the chirality of the newly formed PMO bond can be specifically encoded by the chirality of the stereochemically pure active monomers used to form the oligomer.

[0063] Typical reaction conditions for stereospecific coupling include reaction in aprotic solvents. These solvents may, for example, include, but are not limited to, acetonitrile, tetrahydrofuran (THF), 1,3-dimethylimidazolidinone (DMI), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dimethylacetamide (DMAc), dichloromethane (DCM), 1,2-dichloroethane (DCE), chloroform, 1,4-dioxane, ethyl acetate, 2-methyltetrahydrofuran and isopropyl acetate. Coupling reactions can be carried out in the presence of non-nucleophilic bases, tertiary amines and aromatic bases. Suitable bases include, but are not limited to, diisopropylethylamine, triethylamine, 2,6-lutidine, trimethylpyridines (collidines), and N-ethylmorpholine. The reaction temperature may range from room temperature (about 20°C) to 50°C. In some cases, sonication may be used to dissolve the substrate(s).

[0064] In order to demonstrate the possibility of stereospecific coupling, essentially a variety of stereochemically pure PMO dinucleotides were obtained by stereospecific PMO coupling of slowly and rapidly eluting substantially stereochemically pure active monomers (phosphoramidochloridates). Table 2 below shows the HPLC retention profiles of these essentially stereochemically pure PMO dinucleotides. The table compares retention times for dinucleotides that were made from combinations of the 5' monomers listed on the left side of the table with both fast eluting and slower eluting isomers of the 3' monomers listed at the top of the table. The table demonstrates that a stereospecific combination of substantially stereochemically pure active monomers produces various diastereomerically pure dinucleotides having different physical properties.

TABLE 2:

Active monomers (3'end; electrophiles)

U C ^c A ^d G ^b T fast slow fast slow fast slow fast slow fast slow U ₁ U _{2 C1 C2} A _{1 A2 G1 G2 T1 T2} 5'-terminal monomers (nucleophiles)

U UU ₁ UU ₂ UC ₁ UC ₂ UA ₁ U.A. ₂ UG ₁ UG ₂ UT ₁ UT ₂ dinucleotide 9 < 27 9.1 > 6.5 12.6 > 10.9 12.5 < 18.8 8.4 < 27 Hold time (min) C ^a _{CU1 CU2} CC _{1 CC2} CA ₁ CA ₂ CG ₁ CG ₂ CT ₁ CT ₂ dinucleotide 5.6 < 6.6 6.7 > 5.7 8 > 7.4 10.4 < 13.3 5.6 < 6.1 Hold time (min) A ^a AU ₁ AU ₂ AC ₁ AC ₂ AA ₁ AA ₂ AG ₁ AG ₂ AT ₁ AT ₂ dinucleotide 3.5 < 3.6 3.6 = 3.6 4.2 > 4.1 7.6 < 8.5 3.5 < 3.6 Hold time (min) G ^b GU ₁ GU ₂ GC ₁ GC ₂ GA ₁ GA ₂ GG ₁ GG ₂ GT _{1 GT2} dinucleotide 7.6 < 8.1 7.2 = 7.2 9.7 > 8.9 14.2 > 11.3 7.4 < 7.5 Hold time (min) T TU ₁ TU ₂ TC _{1 TC2} TA ₁ TA ₂ TG ₁ TG ₂ TT ₁ TT ₂ dinucleotide 9.7 < 23.9 8.9 > 5.6 13.2 > 11.4 13.2 < 20.0 9 < 24 Hold time (min) ^a Unprotected nucleotide bases
^b The amino group of guanine is protected by an isobutyryl group
^c The amino group of cytosine is protected by: (1) an acetyl group when combined with U, C, A, and G, (2) a benzoyl group when combined with T
^d The amino group of adenine is protected by a benzoyl group

[0065] Analytical HPLC conditions for obtaining profiles of essentially stereochemically pure PMO dinucleotides from Table 2 are given below:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 30°C Speed 1.0 ml/ml mobile phase 10% n-heptane, 80% EtOAc and 10% MeOH-EtOH 1:1 with 0.1% diethylamine Gradient isocratic Run time 30 min Injection volume 1-2 µl (0.2 mg/ml, in dichloromethane) detection UV, 260 nm

[0066] EXAMPLES

[0067] III. Examples of diastereomeric separation of activated monomers

[0068] The following examples show the diastereomeric resolution of activated monomers, in accordance with some of the embodiments provided herein.

[0069] A. U-monomers

[0070] Analytical HPLC Conditions for Activated U Monomers:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase ethyl acetate Gradient isocratic Run time 15 minutes Injection volume 10 µl (5 mg/ml, in ethyl acetate) detection 260 nm retention time U ₁ 5 minutes U ₂ 7.5 min

[0071] Preparative HPLC conditions for activated U monomers:

[0072] Chiralpak IC, 21 x 250 mm, 5 µm; elution from the column at 11 ml/min with ethyl acetate at room temperature; detection at 260 nm.

[0073] [ ¹ H-NMR data for U ₁ ]

[0074] ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.18 (br, 1H), 7.45 (m, 6H), 7.15-7.32 (m, 10H), 6.12 (dd, 1H, J=2.0 and 9.6 Hz), 5.62 (d, 1H, J=8.0 Hz), 4.39 (m, 1H), 4.11 (m, 2H) , 3.39 (d, 1H, J=11 Hz), 3.15 (d, 1H, J=11 Hz), 2.65 (s, 3H), 2.62 (s, 3H), 1.49 (t, 1H, J=11 Hz), 1.39 (t, 1H, J=11 Hz).

[0075] [ ¹ H-NMR data for U ₂ ]

[0076] ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.07 (br, 1H), 7.44 (m, 6H), 7.14-7.34 (m, 10 H), 6, 12 (dd, 1H, J=2 and 9 Hz), 5.61 (d, 1H, 8.0 Hz), 4.39 (m, 1H), 4.08 (m, 2H), 3.39 ( d, 1H, J=12 Hz), 3.15 (d, 1H, J=12 Hz), 2.66 (s, 3H), 2.62 (s, 3H), 1.46 (t, 1H, J=11 Hz), 1.38 (t, 1H, J=11 Hz).

[0077] B. A-monomers

[0078] Analytical HPLC Conditions for Activated Monomers A:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase ethyl acetate Gradient isocratic Run time 15 minutes Injection volume 10 µl (5 mg/ml, in ethyl acetate) detection 260 nm retention time A ₁ 8.1 min A ₂ 11.7 min

[0079] Preparative HPLC Conditions for Activated Monomers A:

[0080] Chiralpak IC, 21 x 250 mm, 5 µm; elution from the column at 11 ml/min with 100% ethyl acetate at room temperature; UV detection at 260 nm.

[0081] [ ¹ H-NMR data for A ₁ ]

[0082] ¹ H-NMR (400 MHz, CDCl ₃ ) δ 9.01 (br, 1H), 8.79 (s, 1H), 8.00 (m, 3H), 7.58 (m, 1H ), 7.4-7.6 (m, 8H), 7.2-7.4 (m, 10H), 6.42 (d, 1H, J=8.4 Hz), 4.51 (m, 1H), 4.12 (m, 3H), 3.54 (d, 1H, J=12 Hz), 3.25 (d, 1H, J=12 Hz), 2.62 (s, 3H), 2 .59 (s, 3H), 1.81 (t, 1H, J=11 Hz), 1.62 (t, 1H, J=11 Hz).

[0083] [ ¹ H-NMR data for A ₂ ]

[0084] 1H-NMR (400 MHz, CDCl ₃ ) δ 9.04 (br, 1H), 8.79 (s, 1H), 8.00 (m, 3H), 7.56 (m, 1H) , 7.4-7.6 (m, 8H), 7.2-7.4 (m, 10H), 6.41 (d, 1H, J=8.4 Hz), 4.51 (m, 1H ), 4.12 (m, 3H), 3.54 (d, 1H, J=12 Hz), 3.25 (d, 1H, J=12 Hz), 2.64 (s, 3H), 2, 61 (s, 3H), 1.82 (t, 1H, J=11 Hz), 1.63 (t, 1H, J=11 Hz).

[0085] C. C-monomers

[0086] Analytical HPLC conditions for activated monomers C:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase 90% ethyl acetate, 10% n-heptane Gradient isocratic Run time 12 min Injection volume 10 µl (5 mg/ml, in dichloromethane) detection 260 nm retention time WITH ₁ 6.0 min WITH ₂ 6.2 min

[0087] Preparative HPLC conditions for activated monomers C:

[0088] The Chiralpak IC column was eluted at 15 ml/min with 75% ethyl acetate and 25% n-heptane. Room temperature and UV detection at 260 nm.

[0089] [ ¹ H-NMR data for C ₁ ]

[0090] ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.66 (d, 1H, J=7.8 Hz), 7:43 (m, 6H), 7:33 (d, 1H, J=7 .4 Hz), 7:15 to 7:32 (m, 9H), 6.18 (dd, 1H, J=2.2 and 9.2 Hz), 4.42 (m, 1H), 4.08 to 4.16 (m, 2H), 3:54 (d, 1H, J=11 Hz), 3.14 (d, 1H, J=12 Hz), 2.64 (s, 3H), 2.60 (s, 3H), 2.23 (s, 3H), 1.51 (t, 1H, J=11 Hz), 1.25 (m, 1H).

[0091] [ ¹ H-NMR data for C ₂ ]

[0092] ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.64 (d, 1H, J=7.8 Hz), 7:43 (m, 6H), 7:32 (d, 1H, J=7 .4 Hz), 7:15 to 7:32 (m, 9H), 6.19 (dd, 1H, J=2.1 and 9.2 Hz), 4.41 (m, 1H), 4.06 to 4.15 (m, 2H), 3:54 (d, 1H, J=11 Hz), 3.15 (d, 1H, J=12 Hz), 2.64 (s, 3H), 2.61 (s, 3H), 2.22 (s, 3H), 1:49 (t, 1H, J=11 Hz), 1.25 (m, 1H).

D. G-monomers (monoprotected guanine)

[0094] Analytical HPLC Conditions for Activated G Monomers:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase ethyl acetate Gradient isocratic Run time 30 min Injection volume 10 µl (5 mg/ml, in ethyl acetate) detection 260 nm retention time G ₁ 17.8 min G ₂ 22.3 min

[0095] Preparative HPLC Conditions for Activated G Monomers:

[0096] The Chiralpak IC column is eluted at 15 ml/min with 100% ethyl acetate. Room temperature and UV detection at 260 nm.

[0097] E. T-monomers

[0098] Analytical HPLC conditions for activated monomers T:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase ethyl acetate Gradient isocratic Run time 10 min Injection volume 10 µl (5 mg/ml, in ethyl acetate) detection 260 nm retention time T ₁ 4.5 min T ₂ 7.0 min

[0099] Preparative HPLC Conditions for Activated Monomers T:

Chiralpak IC, 50x500 mm, 20 µm. Column elution at 60 ml/min with ethyl acetate, room temperature, detection at 260 nm. Retention time 25 and 40 minutes.

[0100] [ ¹ H-NMR data for T ₁ ]

[0101] ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.4-7.5 (m, 5H), 7.26-7.33 (m, 6H), 7.16-7.22 (m, 3H), 7.04 (d, 1H, J=1 Hz), 6.12 (dd, 1H, J=2 and 10 Hz), 4.39 (m, 1H), 4.12 (m, 2H) , 3.37 (d, 1H, J=12 Hz), 3.15 (d, 1H, J=12 Hz), 2.66 (s, 3H), 2.63 (s, 3H), 1.83 (d, 1H, J=1 Hz), 1.49 (t, 1H, J=11 Hz), 1.41 (t, 1H, J=11 Hz).

[0102] [ ¹ H-NMR data for T ₂ ]

[0103] ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.4-7.5 (m, 6H), 7.24-7.35 (m, 6H), 7.14-7.22 (m , 3H), 7.03 (s, 1H), 6.12 (dd, 1H, J=2 and 10 Hz), 4.39 (m, 1H), 4.09 (m, 2H), 3.37 (d, 1H, J=11 Hz), 3.15 (d, 1H, J=11 Hz), 2.66 (s, 3H), 2.62 (s, 3H), 1.82 (s, 3H ), 1.48 (t, 1H, J=11 Hz), 1.40 (t, 1H, J=11 Hz).

[0104] F. C-monomers (NBz)

[0105] Analytical HPLC conditions for activated monomers C (NBz):

HPLC column Chiralpak IV, 4.6×150 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase 100% acetonitrile Gradient isocratic Run time 5 minutes Injection volume 10 µl (5 mg/ml, in acetonitrile) detection 260 nm retention time WITH ₁ 3.4 min WITH ₂ 4.5 min

[0106] Preparative HPLC conditions for activated monomers C (NBz):

A Chiralpak IB 20×250 mm column was eluted at 9 ml/min with 100% acetonitrile. Room temperature and UV detection at 260 nm. Retention times 13 and 16 minutes.

G. G-monomers (guanine with two protective groups)

[0108] Analytical HPLC conditions for activated monomers G (guanine with two protective groups):

HPLC column Chiralpak IA, 4.6×250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase 70% ethyl acetate/30% methylene chloride Gradient isocratic Run time 8 min Injection volume 10 µl (5 mg/ml, in ethyl acetate) detection 260 nm retention time G ₁ 3.9 min G ₂ 4.3 min

[0109] Preparative HPLC conditions for activated G monomers (guanine with two protecting groups):

[0110] Chiralpak IA 50×500 mm column, eluted at 60 ml/min with 100% ethyl acetate. Room temperature and UV detection at 260 nm. Retention time 20 and 24 minutes.

[0111] [ ¹ H-NMR data for G ₁ (guanine with two protective groups)]

[0112] ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.76 (s, 2H), 7.50 (d, 2H, J=9 Hz), 7.4-7.5 (m, 6H), 7.26-7.32(m, 6H), 7.16-7.22(m, 3H), 7.02(d, 2H, J=9Hz), 6.24(dd, 1H, J= 2 and 10 Hz), 5.61 (d, 1H, J=12 Hz), 5.56 (d, 1H, J=12 Hz), 4.48 (m, 1H), 4.1 (m, 2H ), 3.47 (d, 1H, J=11 Hz), 3.23 (d, 1H, J=12 Hz), 3.2 (m, 1H), 2.62 (s, 3H), 2, 59 (s, 3H), 1.75 (t, 1H, J=11 Hz), 1.57 (t, 1H, J=12 Hz), 1.33 (s, 9H), 1.33 (t, 6H, J=7 Hz).

[0113] [ ¹ H-NMR data for G ₂ (guanine with two protective groups)]

[0114] ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.78 (s, 1H), 7.77 (s, 1H), 7.50 (d, 2H, J=9 Hz), 7.4- 7.5(m, 6H), 7.26-7.33(m, 6H), 7.15-7.22(m, 3H), 7.02(d, 2H, J=9Hz), 6 .23 (dd, 1H, J=2 and 10 Hz), 5.61 (d, 1H, J=12 Hz), 5.56 (d, 1H, J=12 Hz), 4.47 (m, 1H ), 4.1 (m, 2H), 3.47 (d, 1H, J=11 Hz), 3.22 (d, 1H, J=12 Hz), 3.2 (m, 1H), 2, 64 (s, 3H), 2.60 (s, 3H), 1.75 (t, 1H, J=11 Hz), 1.58 (t, 1H, J=11 Hz), 1.33 (s, 9H), 1.33 (t, 6H, J=7 Hz).

[0115] IV. Examples of stereospecific PMO coupling with diastereomerically pure activated monomers.

[0116] The following examples describe the use of stereospecific coupling to obtain stereochemically homogeneous products.

[0117] A. Activated U-monomers (U ₁ & U ₂ )+U-morpholine-NH (1)

[0118] U ₁ (11 mg, 0.018 mmol, 1 eq., 99.0% CI (diastereomeric excess)) was dissolved in acetonitrile (0.11 ml) and mixed with diisopropylethylamine (8 μl, 0.05 mmol, 2. 5 eq.). U-morpholine-NH (14 mg, 0.030 mmol, 1.6 eq) was added and sonication was used to facilitate dissolution. After 0.5 h stirring, a small aliquot of the reaction mixture was diluted with CDCl ₃ and analyzed using ¹ H-NMR. The rest of the reaction mixture was diluted with acetonitrile (8 ml) for HPLC analysis and stored in a freezer. Stereospecific formation of compound 2 was confirmed by HPLC analysis (99.4% CI). The above protocol was also used for the U ₂ coupling reaction (95.6% CI) to produce compound 3 stereospecifically (96.0% CI).

[0119] Analytical HPLC conditions U/U-coupling:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase 80% methanol, 20% acetonitrile Gradient isocratic Run time 30 min Injection volume 10 µl (2 mg/ml, in acetonitrile) detection 260 nm retention time U ₁ 11.5 min U ₂ 21.5 min 2 12.0 min 3 22.1 min

Nucleophile activated U-monomer Product (UU dinucleotide) U-morpholine-NH ( 1 ) U ₁
(99.0% CI) → 2
(99.4% CI) U ₂
(95.6% CI) → 3
(96.0% CI)

[0120] [ ¹ H-NMR data for compound 2]

[0121] ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.6 (m, 4H), 7.2-7.5 (m, 20H), 7.1-7.2 (m, 3H), 6.15 (d, 1H, J=8.0 Hz), 5.73 (d, 1H, J=8.0 Hz), 5.66 (d, 1H, J=8.0 Hz), 5. 54 (d, 1H, J=8.0 Hz), 4.40 (m, 1H), 3.93 (m, 2H), 3.81 (m, 1H), 3.70 (m, 2H), 3.41 (m, 2H), 3.40 (m, 3H), 3.11 (d, 1H, J=12 Hz), 2.78 (m, 1H), 2.56 (s, 3H, Me ), 2.54 (s, 3H, NMe), 2.48 (m, 1H), 1.47 (t, 1H, J=11 Hz), 1.35 (t, 1H, J=11 Hz), 1.04 (s, 9H).

[0122] [ ¹ H-NMR data for compound 3]

[0123] ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.6 (m, 4H), 7.3-7.5 (m, 1 1H), 7.2-7.3 (m, 9H) , 7.1 (m, 3H), 6.12 (dd, 1H, J=2.0 and 9.6 Hz), 5.71 (d, 1H, J=8.4 Hz), 5.70 ( d, 1H, J=8.0 Hz), 5.47 (dd, 1H, J=2.0 and 10.4 Hz), 4.31 (m, 1H), 3.97 (m, 1H), 3.85 (m, 1H), 3.73 (m, 2H), 3.65 (m, 1H), 3.31 (m, 2H), 3.24 (m, 1H), 3.07 (d , 1H, J=12 Hz), 2.68 (m, 1H), 2.65 (s, 3H, NMe), 2.62 (s, 3H, NMe), 2.26 (m, 1H), 1 .45 (t, 1H, J=12 Hz), 1.29 (t, 1H, J=11 Hz), 1.04 (s, 9H).

[0124] B. Activated C-monomers (C ₁ & C ₂ )+C-morpholine-NH (4)

[0125] C ₁ (20 mg, 0.031 mmol, 1 eq., 93.5% CI) was dissolved/suspended in THF (0.40 ml) and mixed with diisopropylethylamine (12 μl, 0.069 mmol, 2.3 eq.) . Morpholino-cytosine (4; 16 mg, 0.035 mmol, 1.1 eq.) in THF (0.20 ml) was added. After stirring for 1.0-2.0 h, a small aliquot of the reaction mixture was diluted in acetonitrile and analyzed by LC/MS. An aliquot (30-50 μl) of the reaction mixture was diluted with dichloromethane (0.6 ml) for HPLC analysis. Stereospecific formation of compound 6 was confirmed by HPLC analysis (94.3% CI). The reaction mixture was loaded directly onto a silica gel column and eluted with a mobile phase gradient of 0-15% methanol in ethyl acetate. The above protocol was also used for C/C-coupling C ₂ (90.2% CI) to stereospecifically obtain compound 5 (90.0% CI).

[0126] Analytical HPLC conditions for C/C coupling:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase Solvent A n-heptane Solvent B 1:1 ethanol:methanol in 0.1% diethylamine (DEA) Gradient isocratic % A % IN 50 50 Run time 20 minutes Injection volume 10 µl (3 mg/ml, in dichloromethane) detection 260 nm retention time 4 5.4 min 5 10.5 min 6 12.9 min

Nucleophile activated C-monomer Product (CC dinucleotide) C-morpholine-NH ( 4 ) From ₁
(93.5% CI) → 6
(94.3% CI) From ₂
(90.2% CI) → 5
(90.0% CI)

[0127] [ ¹ H-NMR data for compound 5]

[0128] ¹ H-NMR (400 MHz, CDCl ₃ ) δ 10.9 (br, 1H), 7.69 (d, 1H, J=7.4 Hz), 7.62 (m, 5H), 7.35-7.44 (m, 13H), 7.21-7.35 (m, 6H), 7.15 (m, 4H), 6.14 (bd, 1H, J=7.8 Hz), 5.58 (dd, 1H, J=2.4 and 9.4 Hz), 5.53 (wide, 1H), 4.51 (dd, 1H, J=8.6 and 10 Hz) , 4.09 (m, 1H), 3.70-3.80 (m, 4H), 3.60 (dd, 1H, J=6.3 and 10 Hz), 3.56 (d, 1H, J =11 Hz), 3.28 (m, 1H), 2.96 (d, 1H, J=11 Hz), 2.69 (s, 3H, NMe), 2.67 (s, 3H, NMe), 2.65 (m, 1H), 2.25 (m, 1H), 2.07 (s, 3H), 1.31 (t, 1H, J=11 Hz), 1.13 (t, 1H, J =11 Hz), 1.04 (s, 9H).

[0129] [[ ¹ H-NMR data for compound 6]

[0130] ¹ H-NMR (400 MHz, CDCl ₃ ) δ 9.57 (br, 1H), 7.62-7.70 (m, 7H), 7.35-7.50 (m, 14H) , 7.23-7.35(m, 4H), 7.12(m, 4H), 6.31(m, 1H), 5.79(m, 1H), 5.70(m, 1H), 4.61 (m, 1H), 4.03 (m, 1H), 3.80-3.90 (m, 2H), 3.72 (m, 2H), 3.58 (m, 1H), 3 .48 (m, 1H), 3.09 (m, 1H), 2.75 (m, 1H), 2.58 (s, 3H, NMe), 2.55 (s, 3H, NMe), 2, 53 (m, 1H), 2.38 (m, 1H), 2.21 (s, 3H), 1.47 (t, 1H, J=10 Hz), 1.22 (t, 1H, J=10 Hz), 1.06 (s, 9H).

[0131] C. Activated A-monomers (A ₁ & A ₂ ) + U-morpholine-NH (1)

[0132] A ₁ (5.9 mg, 0.008 mmol) was resuspended in acetonitrile (118 μl). Diisopropylethylamine (5 μl, 0.03 mmol) was added followed by morpholino-uracil (1; 4.6 mg, 0.01 mmol). Sonication was used for 1 min and the resulting homogeneous mixture was stirred at room temperature. After stirring overnight, the mixture (thick white paste) was diluted with a mixture of acetonitrile (5.0 ml) and methanol (0.30 ml) to give a homogeneous clear solution. A small aliquot was directly analyzed by HPLC without further dilution.

[0133] A ₂ (F2; 5.0 mg, 0.007 mmol) was resuspended in acetonitrile (100 μl). Diisopropylethylamine (4 μl, 0.02 mmol) was added followed by morpholino-uracil (4.1 mg, 0.009 mmol). Sonication was used for 1 min and the resulting thick suspension was stirred at room temperature. After stirring overnight, acetonitrile (5.0 ml) was added and sonication was used to give a homogeneous clear solution. A small aliquot was directly analyzed by HPLC without further dilution.

[0134] Analytical HPLC U/A Coupling Conditions:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase Solvent A ethyl acetate Solvent B 1:1 ethanol:methanol in 0.1% diethylamine Gradient Isocratic: 98% Solvent A, 2% Solvent B Run time 30 min Injection volume 5 µl (1 mg/ml, in acetonitrile-methanol) detection 260 nm retention time 7 ( S isomer) 21.7 min 8 ( R isomer) 24.9 min

Nucleophile activated A-monomer Product (UA dinucleotide) U-morpholine-NH (1) A ₁
(97.8% CI) → 8
( R- isomer, 96.2% CI) A ₂
(98.4% CI) → 7
( S- isomer, 98.3% CI)

[0135] D. Activated G-monomers (G ₁ & G ₂ ) + U-morpholine-NH (1)

[0136] G ₁ (6.5 mg, 0.009 mmol, 1 eq., 99.9% CI) was dissolved/suspended in THF (0.13 ml) and mixed with diisopropylethylamine (3.6 μl, 0.02 mmol, 2.2 eq.). Morpholino-uracil (1; 4.7 mg, 0.010 mmol, 1.1 eq.) dissolved in THF (0.07 ml) was added. After stirring for 1.0-2.0 h, a small aliquot of the reaction mixture was diluted with acetonitrile and analyzed by LC/MS. An aliquot (100 μl) of the reaction mixture was diluted with dichloromethane (0.4 ml) for HPLC analysis. Stereospecific formation of compound 9 was confirmed by HPLC analysis (99.9% CI).

[0137] Analytical HPLC conditions for U/G coupling:

HPLC column Chiralpak IC, 4.6x250 mm, 5 µm Temperature 35°C Speed 1.0 ml/ml mobile phase Solvent A n-heptane Solvent B ethyl acetate Solvent C 1:1 ethanol:methanol in 0.1% diethylamine (DEA) Gradient isocratic % A % IN %WITH 55 40 5 Run time 30 min Injection volume 5 µl (2 mg/ml, in dichloromethane) detection 260 nm retention time 1 8.4 min 9 14.0 min 10 16.3 min

Nucleophile activated G-monomer Product (UG dinucleotide) U-morpholine-NH (1) G1 _{_}
(99.9% CI) → 9
(99.9% CI)

[0138] The substantially pure diastereomeric compounds described above can be used to prepare stereochemically pure oligonucleotides and other compounds. Examples of possible oligonucleotides are given, for example, in Summerton, J (1999). "Morpholino Antisense Oligomers: The Case for an RNase-H Independent Structural Type.". Biochimica et Biophysica Acta 1489 (1): 141-58; and in Summerton, J; Weller D. (1997). "Morpholino Antisense Oligomers: Design, Preparation and Properties". Antisense & Nucleic Acid Drug Development 7 (3): 187-95. Both of these documents are incorporated into the present description by reference.

[0139] V. Example of stereospecific synthesis of 16-membered PMOs and separation of stereoisomers by a biophysical method

[0140] This example describes a synthesis targeting a pair of stereopure 16-membered PMOs by stereospecific coupling using activated monomers. These PMOs have opposite stereochemical characteristics of their phosphorus bonds.

Target sequence:

(SEQ ID NO: 2)

Stereopure active monomers (structural units):

Target RMOs Stereopure active monomers used for the coupling reaction Stereoisomer 1 A ₂ , C ₂ , G ₁ and T ₁ Stereoisomer 2 A ₁ , C ₁ , G ₂ and T ₂

[0141] A scheme for the stereospecific synthesis of 16-membered PMOs and the separation of stereoisomers by a biophysical method is shown in FIG. 6. 16-membered PMO stereoisomers 1 and 2 were manually prepared by solid phase synthesis on aminomethyl polystyrene disulfide resin (load ~300 µmol/g, see US Patent Application Publication No. 20090131624A1, which is incorporated herein by reference) at a scale of 50 mg ( resin initial weight).

[0142] Stock solutions for solid phase synthesis:

Detritylation 4-cyanopyridine trifluoroacetate (CYTFA), 2% (w/v), and 0.9% ethanol (v/v) in 20% trifluoroethanol in DCM (v/v). Neutralization 5% diisopropylethylamine (v/v) in 25% isopropanol in DCM (v/v). Combination Freshly prepared 55 mM solution in NEM-DMI* for each of the stereopure active monomers (A ₂ , C ₂ , G ₁ and T ₁ for stereoisomer 1; A ₁ , C ₁ , G ₂ and T ₂ for stereoisomer 2)
*0.11 M N-ethylmorpholine in 1,3-dimethylimidazolidinone (DMI)

[0143] Operating cycle for each PMO combination:

Stage Volume (ml) Time (min) DCM 1-2 2-5 Detritylation 1-2 5 Detritylation 1-2 5 Detritylation 1-2 5 Detritylation 1-2 5 Detritylation 1-2 5 DCM 1-2 2-5 Neutralization 1-2 2-5 Neutralization 1-2 2-5 Neutralization 1-2 2-5 Neutralization 1-2 2-5 DCM 1-2 2-5 DCM 1-2 2-5 DCM 1-2 2-5 Combination 1 >180* DCM 1-2 2-5 Neutralization 1-2 2-5 Neutralization 1-2 2-5 DCM 1-2 2-5 DCM 1-2 2-5 DCM 1-2 2-5 DCM 1-2 2-5

*40°C for 3 hours or room temperature for 12 hours.

[0144] Removal from resin and removal of protection:

[0145] To the resin-bound 16-membered oligonucleotide (after detritylation) was added 1:3 (v/v) of 28% aqueous ammonia in ethanol (˜5 ml). The mixture was sealed and heated at 45° C. for 20 hours. After cooling to room temperature, the mixture was filtered and washed with methanol. The filtrate was concentrated and diafiltered against 15 mM triethylammonium (TEAA) buffer, pH 7.0. The device used was an Amicon stirred cell (50 ml) with a 1 kDa Ultracel UF membrane. Samples were diafiltered by dilution/concentration until the concentration of the original solvent was reduced to 1% of the original concentration (approximately 5 cycles) and then purified using preparative reverse phase HPLC.

[0146] Method for purifying RMO by reverse phase preparative HPLC:

HPLC column Column XBridge Prep C8 OBD, 9×150 mm, 5 µm Column temperature Room temperature Speed 30.0 ml/min Gradient Time (min) %A %B Elementary 85 15 18 80 20 20 0 100 mobile phase Solvent A: 15mM triethylammonium acetate (TEAA) buffer, pH7+10% MeOH
Solvent B: acetonitrile + 10% MeOH Dilution solution 15 mM TEAA buffer Run time 20 minutes detection UV, 260 nm retention time Stereoisomer 1 13.98 min Stereoisomer 2 14.03 min

[0147] LC/MS method for assessing the quality of the RMO:

HPLC column Waters BEH C18 Oligo 2.1×50 mm, 130 Angstrom 1.7 µm Column temperature 45°C Speed 0.3 ml/min Gradient Time (min) %A %B Elementary 95 5 2 95 5 20 50 50 24 50 50 24.1 95 5 thirty 95 5 mobile phase Solvent A: 50 mM ammonium acetate
Solvent B: acetonitrile/methanol 1/1 v/v with 50 mM ammonium acetate retention time 30 min Sample volume 25 µl, diluent: water or 10 mM triethylamine acetate detection UV, 260 nm MS/ionization mode Synapt G2/electrospray, positive mode Cone tension/extraction 30V/4V/2.8KV Pace. Source/ Collision Energy/MS Function/Analysis 100°C / 4 eV (low energy) 40-70 eV (high energy) / MS ^E mode / deconvolution and isotope correction using Waters MSe Viewer retention time Stereoisomer 1 10.83 min Stereoisomer 2 10.86 min

[0148] Materials and conditions for measuring the melting point (Tm)

Complementary RNA (16-mer) 5'-UUCCUUGAUGUUGGAG-3' (SEQ ID NO. 1) (IDT Integrated DNA Technologies) dilution buffer 10 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, pH 7.0 (supplied with phosphoric acid) Melting point measuring device UV-Visible Spectrophotometer - Shimadzu 2700 Equipped with Shimadzu S-1700 Temperature Module Vacuum Centrifuge Concentrator Labconco Centrivap Concentrator Model 7810015 Stock solutions 8 micromolar each sample and complementary RNA in 250 µl of buffer were prepared using dilution buffer from samples concentrated and dried by vacuum centrifugation Procedure Each sample was then mixed with an equal volume of 8 micromolar complementary RNA.
The mixtures were then heated to 95°C and then cooled to 25°C for annealing before measuring Tm.
Tm analysis (UV 260 nm) was performed from 25° C. to 105° C. at 0.5° C./min (temperature returned to baseline after each run) and then repeated under the same conditions.
An analytical program (Shimadzu) was used to calculate Tm using an "averaging function".

[0149] The results of thermal melting of complexes of stereochemically different PMO with complementary RNA:

Purity by LC/MS (% area) Tm (°C) Stereoisomer 1 97.6 62.8 Stereoisomer 2 94.2 57.2

[0150] Melting points for stereoisomers 1 and 2 are shown in FIG. 7. Based on the different melting points, it can be concluded that certain amounts of essentially pure stereoisomers have been obtained.

[0151] VI. An example of stereospecific synthesis and absolute stereochemical determination of a stereopure PMO dinucleotide compound 100 (5'-ΤΑ ₂ -3')

[0152] The late eluting active monomer A ( A ₂ ; 200 mg, 0.277 mmol, 1 eq.) was dissolved in a mixture of acetonitrile (2.0 ml) and DIPEA (0.12 ml, 0.69 mmol, 2.5 eq. ). Then T-morpholine-NH ( 1 ; 146 mg, 0.305 mmol, 1.1 eq) was added and the resulting suspension was sonicated for several minutes until a clear solution was obtained. The reaction mixture was stirred at room temperature overnight. After completion of the reaction according to LC/MS, the mixture was concentrated and column chromatographed (in 3% methanol in DCM, Biotage SnapUltra 10 g SiO ₂ ). Pure fractions containing product were combined and concentrated in vacuo to give fully protected stereopure 5'-TA-3' dinucleotide 2 as a white solid (240 mg, 0.206 mmol, 74% yield).

[0153] To fully protected dinucleotide 2 (500 mg, 0.429 mmol) in a 25 ml flask was added 2,2,2-trifluoroethanol (TFE, 4.0 ml) and acetic acid (1.0 ml) at room temperature. The resulting mixture was stirred at room temperature and the progress of the reaction was monitored by LC/MS. After 30 minutes, the reaction mixture was quenched with saturated aqueous NaHCO ₃ and DCM. After separation of the two layers, the aqueous layer was re-extracted. All organic layers were combined, washed with half saturated mother liquor, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give the crude product as a white foam. The crude product was purified by column chromatography (in 20% MeOH in acetone on a 25 g SiO ₂ Biotage Snap Ultra cartridge) to give the partially protected dinucleotide 3 as a glassy solid (300 mg, 0.325 mmol, 76% yield).

[0154] Partially protected dinucleotide 3 (250 mg, 0.271 mmol) was dissolved in a mixture of methanol (12.5 ml) and THF (12.5 ml) and treated with 1 M NaOH (10.8 ml) at room temperature. After stirring at room temperature for 22 hours (progress monitored by LC/MS), the mixture was neutralized with 1 M HCl (10.8 ml) to bring the pH to 8, and then concentrated in vacuo to dryness. The residue was dissolved in water (5 ml) and washed with EtOAc (5 ml). The aqueous layer was concentrated to dryness in vacuo to give the crude product as a white solid (480 mg). The crude product was purified by size exclusion chromatography (on Sephadex® LH-20 in methanol/water 4:1) to give compound 100 (fully deprotected dinucleotide) as a white solid (137 mg, 0.236 mmol, 87 yield %).

[0155] A drop of an aqueous solution of compound 100 (200 mg/ml) was sealed in a well of pure water for one day to grow single crystals. X-ray diffraction analysis of the single crystal confirmed the absolute configuration of the phosphorus bond as S. This x-ray structure is shown as an ORTEP diagram in FIG. 8. ORTEP diagrams of individual fragments are shown in FIG. 9A and FIG. 9B. X-ray diffraction data were obtained as described below.

[0156] Acquisition of X-ray diffraction data

[0157] A single crystal of compound 100 (C ₂₂ H ₃₃ N ₁₀ O ₇ P) was mounted on glass fiber. All measurements were carried out on a diffractometer using monochromatic radiation (Cu-Kα, graphite monochromator).

[0158] The cell parameters and orientation matrix for the data were derived from least squares refinement using 36473 set angles of well-centered reflections in the range 7.75<2θ<147.10°, and corresponded to a C-centered monoclinic cell with dimensions:

a=33.3523(2)

β=96,8075(6)°b=13.80020(11)

β=96.8075(6)°

c=14.19956(10)

³ V=6489.53(8)

³

[0159] For Z=4 and FW=580.54, the calculated density is 0.594 g/cm ³ . Based on the reflection conditions:

hkl: h+k=2n

packing, statistical analysis of the intensity distribution and successful solution and refinement of the structure, the space group was defined as:

С2(#5)

[0160] The data were acquired at 23±1°C using the ω-2θ scan method up to a maximum 2θ value of 147.7°. Omega scans of several intense reflections taken prior to data collection had an average FWHM of 0.00° with a reflection angle of 6.0°. Scans (0.00+0.00 tan θ)° were taken at a rate of 0.0°/min (in ω).

[0161] Reducing the amount of data

[0162] 50,795 reflections were collected, of which 12,008 were unique (R _int = 0.0453). The data was collected and processed using CrysAlisPro (Rigaku Oxford Diffraction). (CrysAlisPro: Data Collection and Processing Software, Rigaku Corporation (2015) Tokyo 196-8666, Japan.). No decay correction was applied.

[0163] The linear absorption coefficient, μ, for Cu-Κα radiation is 6.011 cm ^-1 . An empirical absorbance correction was applied, giving factors ranging from 0.341 to 1.000. The data were corrected for Lorentz and polarization effects.

[0164] Decision and structure refinement

[0165] The structure was solved by direct methods ( SHELXT Version 2014/5 : Sheldrick, GM (2014). Acta Cryst . A70, C1437) and extended using Fourier methods. Non-hydrogen atoms were refined anisotropically. The hydrogen atoms were refined using the rider model. The final round of full-matrix least squares refinement (using the minimized least squares function: (SHELXL Version 2014/7); Σ w (F _o 2-F _s 2) ² , where w=least squares weight) on F ² was based on 12008 observed reflections and 849 variable parameters and converged (the largest parameter shift was 0.00 from its estimated standard deviation) with unweighted and weighted goodness of fit factors:

R1=Σ||Fo|-|Fc||/Σ|Fo|=0.0522

wR2=[Σ(w(Fo ² -Fc ² ) ² )/Σw(Fo ² ) ² ] ^1/2 =0.1632

[0166] The degree of agreement was 1.45. The degree of agreement is defined as follows: [Σ w (Fo2-Fc2)2/(No-Nv)] ^1/2 where: N _O =number of observations and Nv=number of variables.

[0167] Specific gravity was used. The maximum and minimum peaks on the final difference Fourier map corresponded to 1.79 and -0.69 e ^- /Å ³ , respectively. The final Fleck parameter was 0.029(7), indicating that the structure is a twin of the inversion. (Parsons, S. and Flack, H. (2004), Acta Cryst . A60, s61; Flack, HD and Bernardinelli (2000), J. Appl. Cryst. 33, 114-1148).

[0168] The scattering factors for neutral atoms were taken from the International Tables for Crystallography (IT), Volume C, Table 6.1.1.4. (International Tables for Crystallography, Vol. C (1992). Ed. AJC Wilson, Kluwer Academic Publishers, Dordrecht, Netherlands, Table 6.1.1.4, pp. 572). Anomalous dispersion effects have been included in Fcalc (Ibers, JA & Hamilton, WC; Acta Crystallogr., 17, 781 (1964)); values for Δf' and Δf" were taken from Creagh and McAuley. (Creagh, DC & McAuley, WJ; "International Tables for Crystallography", Vol C, (AJC Wilson, ed.), Kluwer Academic Publishers, Boston, Table 4.2.6.8, pages 219-222 (1992) Values for mass attenuation coefficients were taken from Creagh and Hubbell (Creagh, DC & Hubbell, JH.; "International Tables for Crystallography", Vol C, (AJC Wilson, ed. ), Kluwer Academic Publishers, Boston, Table 4.2.4.3, pages 200-206 (1992)). 4.2 : Crystal Structure Analysis Package, Rigaku Corporation (2000-2015) Tokyo 196-8666, Japan SHELXL Version 2014/7 : Sheldrick, GM (2008) Acta Cryst A64, 112-122).

[0169] Crystalline data, intensity measurements, and structure resolution and refinement are as follows:

A. Crystal Data

Empirical formula C22H33N10O7P Molecular weight 580.5 Crystal color and shape Missing Crystal dimensions Not described Crystal system Monoclinic Lattice type C-centered The number of reflections used for the cell Cell definition (2θ range) 36473 (7.7-147.1°) Omega Peak Width at half height 0.00° Lattice parameters

a=33.3523(2)

b=13.80020(11)

c=14.19956(10)

β=96.8075(6)° V=6489.53(8)

³ space group С2 (#5) Z-value 4 D _calc. 0.594 g/ ^cm3 _F000 1224.00 μ(CuKα) 6.011 cm ^-1

B. Intensity measurements

Diffractometer Radiation

)CuKα (λ=1.54187

) graphite monochromatizer Reflection angle 2.8° Detector aperture 2.0-2.5mm horizontal 2 mm vertical Distance from crystal to detector 21 mm Temperature 23.0°C Scan type ω-2θ Scan speed 0.00°/min (in ω) (up to 0 scans) Scan Width (0.00+0.00 tan θ)° 2θ _max. 147.7° Number of measured reflections Total: 50795 Unique: 12008 ( _Rint =0.0453) Parson coefficients (Fleck x-parameter): 4813 Corrections Lorentz polarization Absorption (transmission factors: 0.341-1.000)

C. Decision and structure refinement

Structure decision Direct Methods (SHELXT Version 2014/5) Clarification Full-matrix least squares on F ² Minimized Function Σw(F _o 2-F _s 2) ² Least squares weight w=1/[σ ² (Fo ² )+(0.1000⋅P) ² +0.0000⋅P] where P=(Max(Fo ² ,0)+2Fc ² )/3 2θ _max cutoff 147.7° Anomalous dispersion All non-hydrogen atoms Number of observations (all reflections) 12008 Number of variables 849 Reflection coefficient/parameter 14.14 Residual errors: R1 (I>2.00σ(I)) 0.0522 Residual errors: R (all reflections) 0.0534 Residuals: wR2 (all reflections) 0.1632 Agreement score 1,450 Fleck parameter (Parson coefficients=4813) 0.029(7) Maximum shift/error in the last cycle 0.001 Maximum peak in final diffraction map

³ 1.79 e ^- /

³ Minimum peak in final diffraction map -0.69 e ^- /

³

[0170] [ ¹ H-NMR data for compound 100]

[0171] ¹ H-NMR (400 MHz, D ₂ O) δ 8.25 (s, 1H), 8.15 (s, 1H), 7.40 (s, 1H), 5.85 (d, 1H ), 5.45 (d, 1H), 4.25 (m, 2H), 4.05 (m, 1H), 3.85 (m, 1H), 3.6 (m, 2H), 3.4 (m, 4H), 2.90 (m, 4H), 2.60 (d, 6H), 1.8 (s, 3H).

[0172] All documents referred to in this application are incorporated herein by reference. If there is any discrepancy between the included document and this document, then this document shall prevail.

Claims (25)

1. A method for producing a substantially diastereomerically pure phosphorodiamidate morpholino oligomer containing chiral phosphorus bonds, comprising: selection of substantially stereochemically pure phosphoramidochloridate morpholino monomers; And synthesizing a substantially diastereomerically pure phosphorodiamidate morpholino oligomer by stereospecific coupling of selected substantially stereochemically pure phosphoramidochloridate morpholino monomers, wherein said stereospecific coupling is carried out in an aprotic solvent in the presence of a non-nucleophilic tertiary amine or aromatic base at a temperature of 20°C to 50°C; wherein substantially stereochemically pure phosphoramidochloridate morpholino monomers are prepared by separating a diastereomeric mixture of phosphoramidochloridate morpholino monomers into substantially stereochemically pure phosphoramidochloridate morpholino monomers, and where essentially stereochemically pure refers to enantiomers or diastereomers that are in an enantiomeric or diastereomeric excess, respectively, equal to or greater than 87%, and substantially diastereomerically pure refers to diastereomers that are in a diastereomeric excess equal to or greater than 87%. 2. The method according to claim 1, where the separation of the diastereomeric mixture occurs using at least one method from the group consisting of chromatography and crystallization. 3. The method of claim 2, wherein the chromatography is selected from the group consisting of high performance liquid chromatography (HPLC), pseudo-moving bed chromatography, and countercurrent chromatography. 4. The method according to any one of claims 1 to 3, wherein the substantially stereochemically pure phosphoramidochloridate morpholino monomers are selected from the group consisting of a compound of formula 20, a compound of formula 21, a compound of formula 22, a compound of formula 23, a compound of formula 24, a compound of formula 25 , compounds of formula 26, compounds of formula 27, compounds of formula 28, compounds of formula 29, compounds of formula 30 and compounds of formula 31, Compound formula number

20

21

22

23

24

25

26

27

28

29

thirty

31 where R ₁ and R ₂ may be the same or different and selected from the group consisting of -H, C ₁ -C ₃ -alkyl, phenyl, naphthyl, or with the nitrogen atom to which they are attached, they form a substituted heterocycle; where R ₃ is selected from the group consisting of trityl (Tr), substituted trityl, benzyl, 4-methoxybenzyl (PMB, MPM), 3,4-dimethoxybenzyl, diphenylmethyl (Dpm) and sulfonyl; R ₄ , R ₅ and R ₆ are selected from the group consisting of -H, -C(O)R ₇ and -C(O)OR ₇ where R ₇ is C ₁ -C ₆ -alkyl, benzyl, 2, 2,2-trichloroethyl and an aryl group selected from phenyl, 4-methoxyphenyl, 4-bromophenyl and 4-nitrophenyl; R ₉ is selected from the group consisting of alkyl, cyanoethyl, acyl, carbonate, carbamate, benzyl, 4-pivaloyloxybenzyl and silyl, and their enantiomers. 5. The method of claim 4 wherein the sulfonyl is a cleavable sulfonyl selected from the group consisting of 2-nitrobenzenesulfonyl, 4-nitrobenzenesulfonyl and 2,4-dinitrobenzenesulfonyl. 6. A substantially diastereomerically pure phosphorodiamidate morpholino oligomer containing chiral phosphorus bonds obtained by the method of any one of claims 1 to 5 to obtain a stereochemically pure oligonucleotide, wherein said oligonucleotide is selected from the group consisting of:

;

;

;

;

;

;

;

.

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