OF OLIGOMERIC COMPOUNDS

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format.

The Sequence Listing is provided as a file entitled DVCM0043WOSEQ_ST25.txt created January 18, 2016, which is 4 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure is directed to the field of oligomer synthesis. In particular, improvements in the synthesis of oligomeric compounds comprising a terminally linked

monomethoxytrityl amino group are provided by modification of the final detritylation step. In certain embodiments, the final detritylation step is performed in solution at increased temperature and at a raised pH relative to standard methods. In certain embodiments, modification of the final detritylation step results in less depurination. In certain embodiments, the modification of the final detritylation step results in an improved yield. In certain embodiments, the modification of the final detritylation step results in a faster detritylation of the oligomeric compound compared to standard methods.

BACKGROUND OF THE INVENTION

Oligonucleotides have been used in various biological and biochemical applications.

Oligonucleotides have been used as primers and probes for the polymerase chain reaction (PCR), as antisense agents used in target validation, drug discovery and development, as ribozymes, as aptamers, and as general stimulators of the immune system. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, CA) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients. More recently, Kynamro® (Mipomersen sodium injectable; developed by Isis Pharmaceuticals Inc., Carlsbad, CA) has achieved marketing clearance (2013) from the U.S. Food and Drug Administration (FDA), and is currently a treatment of homozygous familial hypercholesterolemia (HoFH). The widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for their synthesis.

Synthesis of oligonucleotides is generally performed on solid support by the repeated coupling of phosphoramidite monomers until the predetermined length and sequence is achieved. The resulting full length oligonucleotide is then cleaved from the solid support and purified with a 5'-hydroxyl protecting group left on. The industry standard 5'-hydroxyl protecting group is the 4,4'- dimethoxytrityl (DMT) group. The phosphoramidite method is well known in the art (see for example: Beaucage and Caruthers (1981) Tetrahedron Letters 22: 1859-1862; McBride and

Caruthers (1983) Tetrahedron Letters 24:245-248; Sinha et al. (1984) Nucleic Acids Res. 12:4539- 4557 and Beaucage and Iyer (1992) Tetrahedron 48:2223-2311, each of which is incorporated herein by reference in its entirety).

Large scale synthesis of oligomeric compounds using the phosphoramidite approach is generally performed using solid phase chemistries wherein oligomeric compounds are assembled in an iterative process on a solid support. A first monomer subunit is coupled to a free hydroxyl group attached to a solid support via a series of chemical reactions. This series of chemical reactions is repeated in an iterative manner for each additional monomer subunit until an oligomeric compound having a predetermined length and base sequence is synthesized. After the oligomeric compound has been cleaved from the solid support the DMT-on oligomeric compound is purified by reverse phase liquid chromatography. When the 5'-terminal protecting group is a 4,4'-dimethoxytrityl (DMT) group the oligomeric compound is referred to as a DMT-on oligomeric compound. The 4,4'-dimethoxytrityl (DMT) group is normally left on to simplify the purification step.

Removal of 4,4'-dimethoxytrityl (DMT) protecting groups from terminal 5'-hydroxyl groups has been reported using warm conditions with mildly acidic buffers to try to limit depurination (see Salon et al, Nucleosides, Nucleotides and Nucleic Acids, 2011, 30, 271-279).

Removal of a monomethoxytrityl (MMT) group from a 5'-amino-modified oligonucleotide is performed using aqueous acid such as 20% glacial acetic acid in water (see MMT romoval, Glen Research, http://www.glenresearch.com/GlenReports/GR24-28.html).

SUMMARY OF THE INVENTION

Provided herein are methods of preparing oligomeric compounds wherein the standard detritylation step for removing the final 5'-trityl group is modified. Specifically, the final detritylation step is performed at reduced temperature and a lower pH relative to standard methods. DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods of preparing oligomeric compounds wherein the final detritylation step is performed at higher temperature and lower acidity (higher pH) compared to standard methods. The final detritylation step is performed for oligomeric compounds that comprise a terminal (5' or 3') linked monomethoxytrityl protected amino group as opposed to a terminal DMT protected hydroxyl group. These improved detritylation methods are particularly amenable to automated large scale solid phase synthesis of oligomeric compounds using the phosphoramidite approach. In certain embodiments, the detritylation methods disclosed herein provide oligomeric compounds having a lower percentage of depurination relative to oligomeric compounds prepared using standard methods. In certain embodiments, the final detritylation step is completed in less time using the detritylation methods provided herein compared to standard methods. In certain embodiments, the detritylation methods disclosed herein provide oligomeric compounds having increased purity relative to oligomeric compounds prepared using standard methods.

The synthesis of oligomeric compounds is generally performed using solid phase chemistries wherein oligomeric compounds are assembled in an iterative process. A first monomer subunit is coupled to a free hydroxyl group attached to a solid support via a series of chemical reactions. This series of chemical reactions is iteratively repeated for each additional monomer subunit until the desired oligomeric compound having a predetermined length and base sequence is synthesized.

The full length oligomeric compound still comprising a 5'-terminal protecting group is treated with reagents to deprotect the phosphorus groups and then treated with a strong base such as ammonium hydroxide to cleave the oligomeric compound from the solid support. Standard protocols provide a 4,4'-dimethoxytrityl (DMT) group and the oligomeric compound is referred to as a DMT-on oligomeric compound. The present methods provide a 5'-terminal (or 3')

monomethoxytrityl protected amino group that is linked to the oligomeric compound through a linker group such as an alkyl-phosphate group.

In certain embodiments, the MMT protected amino group is linked to the 5 '-terminus and is acid labile and base stable. In certain embodiments, this 5 -MMT group is intentionally left on (MMT-on) after the oligomeric compound has been synthesized as it serves as a chromatographic handle during reverse-phase HPLC purification. Following the purification process, the MMT removed from the oligonucleotide via the solution-phase detritylation reaction as shown

MMT-on oligomeric compound MMT-off oligomeric compound jyQ^j alcohol

Wherein only the 5 '-terminal monomer is shown and R' represents the remainder of the oligomeric compound and L is a linking group. A 2'-0-(CH ₂ )2-OCH ₃ substituted guanosine nucleoside is depicted as the monomer subunit at the 5 '-terminus for illustration only. Any monomer subunit having a terminally linked amino group protected by a monomethoxytrityl group is amenable to the illustrated deprotection reaction. Acetic acid is shown in the reaction sequence but other acids will work such as formic acid to provide essentially the same results.

An undesired side reaction that occurs during the final detritylation step is depurination as illustrated below.

apurinic site

Based on historical data, when all other reaction variables are held constant (solution composition, temperature, reaction pH) the rate of detritylation varies, based on the 5'-terminal nucleside, with the reaction rates taking on the order A>G>T>C. The rate of depurination also depends on the number of deoxyadenosine and deoxy guano sine nucleosides in the specific oligomeric compound being detritylated. It is desired to maximize the degree of completion of the detritylation reaction while minimizing the amount of depurination, but compounds that detritylate slowly and have several deoxypurines are at the greatest risk of yielding unacceptably high levels of depurination when using standard methods for detritylation.

The present examples illustrate that certain non-standard modifications, alone or in combination with the factors listed above, may also affect the rate and the amount of depurination of oligonucleotides when performing solution phase deprotection to remove the final trityl group. An intermediate oligonucleotide of ISIS-681257 was subjected to standard deprotection protocols and based on monitoring of the reaction progress and extrapolation of the data it was estimated that it would take 2050 minutes to go to completion. Such a long deprotection time would result in unacceptable levels of depurination.

ISIS-681257 intermediate oligonucleotide

After running a series of small scale deprotections of the ISIS-681257 intermediate oligonucleotide, a deprotection scheme was developed that provided the desired reaction time with an acceptable level of depurination. The small scale deprotection protocols were succesfully applied to a production scale run of 65.6 kg (Example 5).

It was found that modifying the pH and temperature during the final detritylation step such that the reaction is performed at about 35-40°C and at a pH of about 5 greatly decreased the rate of depurination and also increased the rate of detritylation for the ISIS-681257 intermediate oligonucleotide. In certain embodiments, these modified conditions provide a method for detritylating MMT-amino linked oligomeric compounds that are problematic and are not amenable to detritylation using standard methods. Detritylation of the ISIS-681257 intermediate

oligonucleotide using these modified conditions (pH 5.0, temperature 40°C) on a production scale provided acceptable product with 0.01% MMT-on and 0.20% total depurination.

The detritylation reactions have been performed at small and large scale with comparable results. For detritylation reactions on either a small scale (generally much smaller than about 100 mmol) or a large scale (from about 100 to about 900 mmol) the temperature of the solution throughout the detritylation and the quenching is maintained using a jacketed reaction

vessel. Production scale runs use a stainless steel jacketed tank controled at about 35-40°C.

In certain embodiments, the solution is first heated to at least about 35°C and the pH is adjusted to about 5.0. In certain embodiments, the temperature is maintained for the duration of the reaction. In certain embodiments, the pH is maintained for the duration of the reaction. In certain embodiments, after the reaction is complete the temperature of the solution is reduced to from bout 20 to about 22°C. In certain embodiments, after the solution is cooled the pH is adjusted to about 5.9 by addition of 10% v/v 2.0 M buffered sodium acetate solution (pH 7.2). While not wanting to be bound by theory it is believed that the choice of acid used during the final detritylation step is not limited to glacial acetic acid. It is also expected that the detritylation reaction could be performed using a stronger acid with no negative effect on the reaction rate.

Standard methods for detritylation generally use glacial acetic acid which is practical when targeting a pH of approximately 3.5. However, for lower pH values, larger quantities of this weak acid are required.

In certain embodiments, the present detritylation methods are performed using standard reagents as illustrated in the standard methods illustrated in the examples. In certain embodiments, the present detritylation methods are performed at a temperature of from about 30°C to about 50°C. In certain embodiments, the present detritylation methods are performed at a temperature of from about 35°C to about 45°C. In certain embodiments, the present detritylation methods are performed at a temperature of from about 35°C to about 40°C. In certain embodiments, the present detritylation methods are performed at about 35°C. In certain embodiments, the present detritylation methods are performed at about 40°C. In certain embodiments, the present detritylation methods are performed at a pH of from about 4.5 to about 5.5. In certain embodiments, the present detritylation methods are performed at a pH of about 5.0. In certain embodiments, the present detritylation methods are performed at a pH of about 5.0 and at a temperature of about 35°C to about 40°C. In certain embodiments, the present detritylation methods are performed at a pH of about 5.0 and at a temperature of about 35°C. In certain embodiments, the present detritylation methods are performed at a pH of about 5.0 and at a temperature of about 40°C. In certain embodiments, the selected temperature is maintained throughout the detritylation process. In certain embodiments, the pH is maintained at about 5.0 throughout the detritylation step.

The present detritylation methods are applicable to the preparation of oligomeric compounds comprising a wide range of monomer subunits such as nucleosides and modified nucleosides. In general, for the synthesis of oligomeric compounds each of the monomer subunits comprises a protected hydroxyl group and a phosphoramidite group. In certain embodiments, the hydroxyl protecting group is selected from substituted or unsubstituted trityl groups. In certain embodiments, the hydroxyl protecting group is 4,4'-dimethoxytrityl (DMT). In certain embodiments, the phosphoramidite group has the formula -P( R ₂ R3)(OR4), wherein R ₂ and R ₃ are each,

independently, Ci-C ₆ straight or branched alkyl, which includes but is not limited to, methyl, ethyl, n-propyl, 2-propyl, n-butyl, iso-butyl, and similar alkyl groups, and R ₄ is any group that is compatible with oligonucleotide synthesis that may be removed after synthesis is complete. Preferably, R4 is a substituted Ci-C ₆ alkyl including at least one heteroatom. Most preferably, R4 is -CH ₂ CH ₂ CN. A preferred phosphoramidite group is diisopropylcyanoethoxy phosphoramidite (-P(N(CH(CH ₃ ) ₂ ) ₂ )(0(CH ₂ ) ₂ CN)). In certain embodiments, the hydroxyl protecting group is 4,4'- dimethoxytrityl (DMT) and the phosphoramidite group is diisopropylcyanoethoxy phosphoramidite (-P(N(CH(CH ₃ ) ₂ ) ₂ )(0(CH ₂ ) ₂ CN)).

In certain embodiments, methods of synthesizing of oligomeric compounds are provided that utilize support medium. In certain embodiments, reactive groups on the support medium are first functionalized with Unylinker™ linking groups prior to addition of the first monomer subunit. A first monomer subunit is attached to a support medium with subsequent monomer subunits iteratively coupled to provide a desired oligomeric compound. The industry standard for large scale oligomeric compound synthesis uses solid support media in a reaction vessel. The growing oligomeric compound is reacted and washed with various reagents and solvents while attached to the solid support. In certain embodiments, support media can be selected having variable solubility in different solvents to allow the growing support bound oligomeric compound to be either in or out of solution at various points in the synthesis process as desired. In certain embodiments, soluble supports include soluble polymer supports that allow precipitating and dissolving the iteratively synthesized product at desired points in the synthesis (Gravert et al, Chem. Rev., 1997, 97, 489- 510).

The term "support media" is intended to include all forms of support, including those known to the art skilled for the synthesis of oligomeric compounds. Some representative support media that are amenable to the methods of the present invention include but are not limited to the following: crosslinked polystyrene (Primer Support 5G or NittoPhaseHL), controlled pore glass (CPG); oxalyl-controlled pore glass (see, e.g., Alul, et al, Nucleic Acids Research 1991, 19, 1527); silica-containing particles, such as porous glass beads and silica gel such as that formed by the reaction of trichloro-[3-(4-chloromethyl)phenyl]propylsilane and porous glass beads (see Parr and Grohmann, Angew. Chem. Internal. Ed. 1972, 11, 314, sold under the trademark "PORASIL E" by Waters Associates, Framingham, Mass., USA); the mono ester of 1,4-dihydroxymethylbenzene and silica (see Bayer and Jung, Tetrahedron Lett., 1970, 4503, sold under the trademark "BIOPAK" by Waters Associates); TENTAGEL (see, e.g., Wright, et al, Tetrahedron Letters 1993, 34, 3373); cross-linked styrene/divinylbenzene copolymer beaded matrix or POROS, a copolymer of polystyrene/divinylbenzene (available from Perceptive Biosystems); soluble support media, polyethylene glycol PEG's (see Bonora et al, Organic Process Research & Development, 2000, 4, 225-231).

As used herein the term "alkyl," refers to a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C ₁ -C ₁₂ alkyl) with from 1 to about 6 carbon atoms being more preferred. The term "lower alkyl" as used herein includes from 1 to about 6 carbon atoms. Alkyl groups as used herein may optionally include one or more further substituent groups.

As used herein the term "alkenyl," refers to a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, l-methyl-2- buten-l-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

As used herein the term "alkynyl," refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond.

Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

As used herein the term "aliphatic," refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups. As used herein the term "alicyclic" refers to a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.

As used herein the term "alkoxy," refers to a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n- butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

As used herein the term "aminoalkyl" refers to an amino substituted C1-C12 alkyl radical.

The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.

As used herein the terms "aryl" and "aromatic," refer to a mono- or poly cyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.

As used herein the terms "aralkyl" and "arylalkyl," refer to an aromatic group that is covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

As used herein the term "heterocyclic radical" refers to a radical mono-, or poly-cyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated or fully saturated, thereby including heteroaryl groups. Heterocyclic is also meant to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optionally contain no heteroatoms. A heterocyclic radical typically includes at least one atom selected from sulfur, nitrogen or oxygen. Examples of heterocyclic radicals include, [l,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as used herein may optionally include further substituent groups.

As used herein the terms "heteroaryl," and "heteroaromatic," refer to a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms.

Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen.

Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

As used herein the term "heteroarylalkyl," refers to a heteroaryl group as previously defined that further includes a covalently attached C1-C12 alkyl radical. The alkyl radical portion of the resulting heteroarylalkyl group is capable of forming a covalent bond with a parent molecule.

Examples include without limitation, pyridinylmethylene, pyrimidinylethylene,

napthyridinylpropylene and the like. Heteroarylalkyl groups as used herein may optionally include further substituent groups on one or both of the heteroaryl or alkyl portions.

As used herein the term "acyl," refers to a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula -C(0)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

As used herein the term "hydrocarbyl" includes radical groups that comprise C, O and H.

Included are straight, branched and cyclic groups having any degree of saturation. Such hydrocarbyl groups can include one or more additional heteroatoms selected from N and S and can be further mono or poly substituted with one or more substituent groups.

As used herein the term "mono or polycyclic ring system" is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic ring system can be further substituted with substituent groups such as for example phthalimide which has two =0 groups attached to one of the rings. Mono or polycyclic ring systems can be attached to parent molecules using various strategies such as directly through a ring atom, fused through multiple ring atoms, through a substituent group or through a bifunctional linking moiety.

As used herein the terms "halo" and "halogen," refer to an atom selected from fluorine, chlorine, bromine and iodine.

As used herein the term "oxo" refers to the group (=0).

As used herein the term "protecting group," refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.

Groups can be selectively incorporated into oligomeric compounds as provided herein as precursors. For example an amino group can be placed into a compound as provided herein as an azido group that can be chemically converted to the amino group at a desired point in the synthesis. Generally, groups are protected or present as precursors that will be inert to reactions that modify other areas of the parent molecule for conversion into their final groups at an appropriate time.

Further representative protecting or precursor groups are discussed in Agrawal et al , Protocols for Oligonucleotide Conjugates, Humana Press; New Jersey, 1994, 26, 1-72.

The term "orthogonally protected" refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany et al, J. Am. Chem. Soc, 1977, 99, 7363-

7365; Barany et al, J. Am. Chem. Soc , 1980, 102, 3084-3095). Orthogonal protection is widely used in for example automated oligonucleotide synthesis. A functional group is deblocked in the presence of one or more other protected functional groups which is not affected by the deblocking procedure. This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions. This allows for selective chemistry to arrive at a desired compound or oligomeric compound.

Examples of hydroxyl protecting groups include without limitation, acetyl, t-butyl, t- butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, l-(2-chloroethoxy)ethyl, p- chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2- acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMT), trimethoxytrityl, l(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Wherein more commonly used hydroxyl protecting groups include without limitation, benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyl- diphenylsilyl, benzoyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX).

Examples of amino protecting groups include without limitation, carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1 -methyl- l-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl.

Examples of thiol protecting groups include without limitation, triphenylmethyl (trityl), benzyl (Bn), and the like.

The compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, a or β, or as (D)- or (L)- such as for amino acids. Included herein are all such possible isomers, as well as their racemic and optically pure forms. Optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions, John Wiley & Sons, 1981. When the compounds described herein contain olefinic double bonds, other unsaturation, or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers or cis- and trans-isomers. Likewise, all tautomeric forms are also intended to be included. The configuration of any carbon- carbon double bond appearing herein is selected for convenience only and is not intended to limit a particular configuration unless the text so states.

The terms "substituent" and "substituent group," as used herein, are meant to include groups that are typically added to a parent compounds or to further substituted substituent groups to enhance one or more desired properties or provide other desired effects. Substituent groups can be protected or unprotected and can be added to one available site or many available sites on a parent compound. As an example if a benzene is substituted with a substituted alky it will not have any overlap with a benzene that is substituted with substituted hydroxyl. In such an example the alkyl portion of the substituted alkyl is covalently linked by one of its carbon atoms to one of the benzene carbon atoms. If the alky is Ci and it is substituted with a hydroxyl substituent group (substituted alkyl) then the resultant compound is benzyl alcohol (C6H ₅ CH ₂ OH). If the benzene were substituted with a substituted hydroxyl group and the hydroxyl was substituted with a Ci alkyl group then the resultant compound would be anisole (C6H ₅ OCH ₃ ).

Substituent groups amenable herein include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(0)R _aa ), carboxyl (-C(0)0-R _aa ), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-0-R _aa ), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(R _bb )(Rcc)), imino(= R _bb ), amido (-C(0)N(R _bb )(Rc _C ) or -N(R _bb )C(0)R _aa ), azido (-N ₃ ), nitro (-N0 ₂ ), cyano (-CN), carbamido (-OC(0)N(R _bb )(R _cc ) or -N(R _bb )C(0)OR _aa ), ureido (-N(R _bb )C(0)- N(R _bb )( cc)), thioureido (-N(R _bb )C(S)N(R _bb )(Rc _C )), guanidinyl (-N(R _bb )C(= R _bb )N(R _bb )(R _cc )), amidinyl (-C(= R _bb )N(R _bb )(Rc _C ) or -N(R _bb )C(= R _bb )(R _aa )), thiol (-SR _bb ), sulfinyl (-S(0)R _bb ), sulfonyl (-S(0) ₂ R _bb ) and sulfonamidyl (-S(0) ₂ N(R _bb )(R _cc ) or -N(R _bb )S(0) ₂ R _bb ). Wherein each R _aa , R _bb and R _cc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.

In this context, "recursive substituent" means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim. One of ordinary skill in the art of medicinal chemistry and organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by way of example and not limitation, physical properties such as molecular weight, solubility or logP, application properties such as activity against the intended target and practical properties such as ease of synthesis.

Recursive substituents are an intended aspect of the invention. One of ordinary skill in the art of medicinal and organic chemistry understands the versatility of such substituents. To the degree that recursive substituents are present in a claim of the invention, the total number will be determined as set forth above.

The terms "stable compound" and "stable structure" as used herein are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.

As used herein the term "nucleobase" generally refers to the nucleobase of a nucleoside or modified nucleoside. The term "heterocyclic base moiety" is broader than the term nucleobase in that it includes any heterocyclic base that can be attached to a sugar or sugar surrogate group to prepare a nucleoside or modified nucleoside. In one embodiment, a heterocyclic base moiety is any heterocyclic system that contains one or more atoms or groups of atoms capable of hydrogen bonding to a heterocyclic base of a nucleic acid. In certain embodiments, nucleobase refers to purines, modified purines, pyrimidines and modified pyrimidines. Such heterocyclic base moieties include but are not limited to naturally occurring nucleobases (adenine, guanine, thymine, cytosine and uracil) and protected forms of unmodified nucleobases (4-N-benzoylcytosine, 6-N- benzoyladenine and 2-N-isobutyrylguanine) as well as modified (5-methyl cytosine) or non- naturally occurring heterocyclic base moieties and synthetic mimetics thereof (such as for example phenoxazines). In certain embodiments, each heterocyclic base moiety is, independently, uracil, thymine, cytosine, 4-N-benzoylcytosine, 5 -methyl cytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine. In certain embodiments, each heterocyclic base moiety is, independently, uracil, thymine, cytosine, 5-methylcytosine, adenine, 6-N- benzoyladenine or guanine.

As used herein the term "sugar moiety" refers to naturally occurring sugars having a furanose ring system (ribose and 2'-deoxyribose), synthetic and/or non-naturally occurring sugars having a modified furanose ring system and sugar surrogates wherein the furanose ring has been replaced with a mono or polycyclic ring system such as for example a morpholino or hexitol ring system or a non-cyclic sugar surrogate such as that used in peptide nucleic acids. The sugar moiety of a monomer subunit provides the reactive groups that enable the linking of adjacent monomer subunits into an oligomeric compound. Illustrative examples of sugar moieties useful in the preparation of oligomeric compounds include without limitation, β-D-ribose, P-D-2'-deoxyribose, substituted sugars (such as 2', 5' and bis substituted sugars), 4'-S-sugars (such as 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2'-substituted ribose wherein the ring oxygen atom has been replaced with a sulfur atom), bicyclic modified sugars (such as the 2'-0-CH(CH ₃ )-4', 2'-0-CH ₂ -4' or 2'-0-(CH ₂ )2-4' bridged ribose derived bicyclic sugars) and sugar surrogates (such as for example when the ribose ring has been replaced with a morpholino, a hexitol ring system or an open non-cyclic system).

As used herein the term "sugar surrogate" refers to replacement of the nucleoside furanose ring with a non-furanose (or 4'-substituted furanose) group with another structure such as another ring system or open system. Such structures can be as simple as a six membered ring as opposed to the five membered furanose ring or can be more complicated such as a bicyclic or tricyclic ring system or a non-ring system such as that used in peptide nucleic acid. In certain embodiments, sugar surrogates include without limitation sugar surrogate groups such as morpholinos,

cyclohexenyls and cyclohexitols. In general the heterocyclic base is maintained even when the sugar moiety is a sugar surrogate so that the resulting monomer subunit will be able to hybridize.

As used herein the term "sugar substituent group" refers to a group that is covalently attached to a sugar moiety. In certain embodiments, examples of sugar substituent groups include without limitation halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, thio, substituted thio and azido. In certain embodiments the alkyl and alkoxy groups are Ci to C ₆ . In certain embodiments, the alkenyl and alkynyl groups are C ₂ to C ₆ . In certain embodiments, examples of sugar substituent groups include without limitation 2'-F, 2'-allyl, 2'-amino, 2'-azido, 2'-thio, 2'-0-allyl, 2'-OCF ₃ , 2'-O-Ci-Ci ₀ alkyl, 2'-OCH ₃ , 2'-0(CH ₂ ) _n CH ₃ , 2'-OCH ₂ CH ₃ , 2'-0-(CH ₂ ) ₂ CH ₃ , 2'-0-(CH ₂ ) ₂ -0-CH ₃ (MOE), 2'- 0[(CH ₂ ) _n O] _m CH ₃ , 2'-0(CH ₂ ) ₂ SCH ₃ , 2'-0-(CH ₂ ) ₃ -N(R _p )(R _q ), 2'-0(CH ₂ ) _n H ₂ , 2'-0-(CH ₂ ) ₂ -0- N(R _p )(Rq), 0(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ ] ₂ , 2'-0(CH ₂ ) _n O H ₂ , 2'-0-(CH ₂ ) ₂ -0-(CH ₂ ) ₂ -N(R _p )(R _q ), 2'-0- CH ₂ C(=0)-N(Rp)(R _q ), 2'-OCH ₂ C(=0)N(H)CH ₃ , 2'-0-CH ₂ C(=0)-N(H)-(CH ₂ ) ₂ -N(R _p )(R _q ) and 2'-0- CH ₂ -N(H)-C(= R _r )[N(Rp)(Rq)], wherein each R _p , Rq and R _r is, independently, H, substituted or unsubstituted Ci-Cio alkyl or a protecting group and where n and m are from 1 to about 10.

In certain embodiments, examples of sugar substituent groups include without limitation 2'- F, 2'-allyl, 2'-amino, 2'-azido, 2'-thio, 2'-0-allyl, 2'-OCF ₃ , 2'-O-Ci-Ci ₀ alkyl, 2'-0-CH ₃ , OCF ₃ , 2'-0- CH ₂ CH ₃ , 2'-0-(CH ₂ ) ₂ CH ₃ , 2'-0-(CH ₂ ) ₂ -0-CH ₃ (MOE), 2'-0(CH ₂ ) ₂ SCH ₃ , 2'-0-CH ₂ -CH=CH ₂ , 2'-0- (CH ₂ ) ₃ -N(R _m )(R„), 2'-0-(CH ₂ ) ₂ -0-N(R _m )(R„), 2'-0-(CH ₂ ) ₂ -0-(CH ₂ ) ₂ -N(R _m )(R„), 2'-0-CH ₂ C(=0)- N(R _m )(R„), 2'-0-CH ₂ C(=0)-N(H)-(CH ₂ ) ₂ -N(R _m )(R„) and 2'-0-CH ₂ -N(H)-C(= R _m )[N(R _m )(R„)] wherein each R _m and R _n is, independently, H, substituted or unsubstituted Ci-Cio alkyl or a protecting group. In certain embodiments, examples of 2,-sugar substituent groups include without limitation fluoro, -0-C¾, -0-CH ₂ CH ₃ , -0-(CH ₂ ) ₂ CH ₃ , -0-(CH ₂ ) ₂ -0-CH ₃ , -0-CH ₂ -CH=CH ₂ , -O- (CH ₂ ) ₃ -N(Ri)(R ₂ ), 0-(CH ₂ ) ₂ -0-N(Ri)(R ₂ ), -0-(CH ₂ ) ₂ -0-(CH ₂ ) ₂ -N(Ri)(R ₂ ), -0-CH ₂ C(=0)- N(Ri)(R ₂ ), -0-CH ₂ C(=0)-N(H)-(CH ₂ ) ₂ -N(Ri)(R ₂ ) and -0-CH ₂ -N(H)-C(= Ri)[N(Ri)(R ₂ )] wherein Ri and R ₂ are each independently, H or Ci-C ₂ alkyl. In certain embodiments, examples of sugar substituent groups include without limitation fluoro, -0-CH ₃ , -0-(CH ₂ ) ₂ -0-CH ₃ , -0-CH ₂ C(=0)- N(H)(CH ₃ ), -0-CH ₂ C(=0)-N(H)-(CH ₂ ) ₂ -N(CH ₃ ) ₂ and -0-CH ₂ -N(H)-C(=NCH ₃ )[N(CH ₃ ) ₂ ]. In certain embodiments, examples of sugar substituent groups include without limitation fluoro, -O- CH ₃ , -0-(CH ₂ ) ₂ -0-CH ₃ , -0-CH ₂ C(=0)-N(H)(CH ₃ ) and -0-CH ₂ C(=0)-N(H)-(CH ₂ ) ₂ -N(CH ₃ ) ₂ . Further examples of modified sugar moieties include without limitation bicyclic sugars (e.g. bicyclic nucleic acids or bicyclic nucleosides discussed below).

In certain embodiments, examples of "sugar substituent group" or more generally

"substituent group" include without limitation one or two 5'-sugar substituent groups independently selected from Ci-C ₆ alkyl, substituted Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₂ -C ₆ alkynyl and halogen. In certain embodiments, examples of sugar substituent groups include without limitation one or two 5'-sugar substituent groups independently selected from vinyl, 5'-methyl, 5 '-(^-methyl and 5'-(R)-methyl. In certain embodiments, examples of sugar substituent groups include without limitation one 5'-sugar substituent group selected from vinyl, 5 '-(^-methyl and 5'-(R)-methyl.

In certain embodiments, examples of sugar substituent groups include without limitation substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an oligomeric compound, and other substituents having similar properties. In certain embodiments, oligomeric compounds include modifed nucleosides comprising 2'-MOE substituent groups (Baker et al, J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution has been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'-0-methyl, 2'-0-propyl, and 2'-0-aminopropyl. Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, V ., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al, Chimia, 1996, 50, 168-176; Altmann et al, Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al, Nucleosides Nucleotides, 1997, 16, 917-926).

Sugar moieties can be substituted with more than one sugar substituent group including without limitation 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/101157, published on 8/21/08 for other disclosed 5', 2'-bis substituted nucleosides). Other combinations are also possible, including without limitation, replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) and 5'-substitution of a bicyclic nucleoside (see PCT International Application WO 2007/134181, published on 11/22/07 wherein a 4'-CH ₂ -0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group).

As used herein the term "monomer subunit" is meant to include all manner of monomers that are amenable to oligomer synthesis. In general a monomer subunit includes at least a sugar moiety having at least two reactive sites that can form linkages to further monomer subunits. Essentially all monomer subunits include a heterocyclic base moiety that is hybridizable to a complementary site on a nucleic acid target. Reactive sites on monomer subunits located on the termini of an oligomeric compound can be protected or unprotected (generally OH) or can form an attachment to a terminal group (conjugate or other group). Monomer subunits include, without limitation, nucleosides and modified nucleosides. In certain embodiments, monomer subunits include nucleosides such as β-D- ribonucleosides and P-D-2'-deoxyribnucleosides and modified nucleosides including but not limited to substituted nucleosides (such as 2', 5' and bis substituted nucleosides), 4'-S-modified nucleosides (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'-substituted ribonucleosides), bicyclic modified nucleosides (such as bicyclic nucleosides wherein the sugar moiety has a 2 -0- CHR _a -4' bridging group, wherein R _a is H, alkyl or substituted alkyl), other modified nucleosides and nucleosides having sugar surrogates. As used herein, the term "nucleoside" refers to a nucleobase- sugar combination. The two most common classes of such nucleobases are purines and pyrimidines. The term nucleoside includes β-D-ribonucleosides and P-D-2'-deoxyribonucleosides.

As used herein, the term "nucleotide" refers to a nucleoside further comprising a modified or unmodified phosphate internucleoside linking group or a non-phosphate internucleoside linking group. For nucleotides that include a pentofuranosyl sugar, the internucleoside linking group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. The phosphate and or a non-phosphate internucleoside linking groups are routinely used to covalently link adjacent nucleosides to one another to form a linear polymeric compound.

As used herein the term "modified nucleoside" refers to a nucleoside comprising a modified heterocyclic base and or a sugar moiety other than ribose and 2'-deoxyribose. In certain

embodiments, a modified nucleoside comprises a modified heterocyclic base moiety. In certain embodiments, a modified nucleoside comprises a sugar moiety other than ribose and 2'-deoxyribose. In certain embodiments, a modified nucleoside comprises a modified heterocyclic base moiety and a sugar moiety other than ribose and 2'-deoxyribose. The term "modified nucleoside" is intended to include all manner of modified nucleosides that can be incorporated into an oligomeric compound using standard oligomer synthesis protocols. Modified nucleosides include abasic nucleosides but in general a heterocyclic base moiety is included for hybridization to a complementary nucleic acid target.

In certain embodiments, modified nucleosides include a furanose ring system or a modified furanose ring system. Modified furanose ring systems include 4'-S analogs, one or more

substitutions at any position such as for example the 2', 3', 4' and 5' positions and addition of bridges for form additional rings such as a 2'-0-CH(CH ₃ )-4' bridge. Such modified nucleosides include without limitation, substituted nucleosides (such as 2', 5', and/or 4' substituted nucleosides) 4'-S- modified nucleosides, (such as 4'-S-ribonucleosides, 4'-S-2'-deoxyribonucleosides and 4'-S-2'- substituted ribonucleosides), bicyclic modified nucleosides (such as 2'-0-CH(CH ₃ )-4', 2'-0-CH ₂ -4' or 2'-0-(CH ₂ )2-4' bridged furanose analogs) and base modified nucleosides. The sugar can be modified with more than one of these modifications listed such as for example a bicyclic modified nucleoside further including a 5'-substitution or a 5' or 4' substituted nucleoside further including a 2' substituent. The term modified nucleoside also includes combinations of these modifications such as base and sugar modified nucleosides. These modifications are meant to be illustrative and not exhaustive as other modifications are known in the art and are also envisioned as possible modifications for the modified nucleosides described herein.

In certain embodiments, modified nucleosides comprise a sugar surrogate wherein the furanose ring has been replaced with a mono or polycyclic ring system or a non-cyclic sugar surrogate such as that used in peptide nucleic acids. Illustrative examples of sugar moieties for such modified nucleosides includes without limitation morpholino, hexitol, cyclohexenyl, 2.2.2 and 3.2.1 cyclohexose and open non-cyclic groups. In certain embodiments, modified nucleosides comprise a non-naturally occurring sugar moiety and a modified heterocyclic base moiety. Such modified nucleosides include without limitation modified nucleosides wherein the heterocyclic base moiety is replaced with a phenoxazine moiety (for example the 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2-one group, also referred to as a G-clamp which forms four hydrogen bonds when hybridized with a guanosine base) and further replacement of the sugar moiety with a sugar surrogate group such as for example a morpholino, a cyclohexenyl or a bicyclo[3.1.0]hexyl.

As used herein the term "bicyclic nucleoside" refers to a nucleoside comprising at least a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides having a furanosyl sugar that comprises a bridge between two of the non-geminal carbons atoms. In certain embodiments, bicyclic nucleosides have a bridge between the 4' and 2' carbon atoms.

Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to one of formulae: 4*-(CH ₂ )-0-2* (LNA); 4*-(CH ₂ )-S-2*; 4 ^, -(CH ₂ )2-0-2* (ENA); 4*-CH(CH ₃ )-0-2* and 4'-C- H(CH ₂ OCH ₃ )-0-2' (and analogs thereof see U. S. Patent 7,399,845, issued on July 15, 2008); 4'- C(CH ₃ )(CH ₃ )-0-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'-CH ₂ -N(OCH ₃ )-2' (and analogs thereof see published International Application WO2008/150729, published December 1 1, 2008); 4'-CH ₂ -0-N(CH ₃ )-2' (see U. S. Patent 7,96,345, issued on April 13, 2010,); 4'-CH ₂ -N(R)-0-2', wherein R is H, Ci-Ci ₂ alkyl, or a protecting group (see U. S. Patent 7,427,672, issued on September 23, 2008); 4'-CH ₂ -C(H)(CH ₃ )-2' (see Chattopadhyaya, et al, J. Org. Chem., 2009, 74, 1 18-134); and 4'-CH ₂ -CH ₂ -2' and 4'-CH ₂ -C- (=CH ₂ )-2' (and analogs thereof see published International Application WO 2008/154401, published on December 8, 2008). Further bicyclic nucleosides have been reported in published literature (see for example: Srivastava et al, J. Am. Chem. Soc, 2007, 129(26) 8362-8379; Frieden et al, Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al, Curr. Opinion Invens. Drugs, 2001, 2, 558- 561 ; Braasch et al, Chem. Biol, 2001, 8, 1-7; Orum et al, Curr. Opinion Mol. Then, 2001, 3, 239- 243; Wahlestedt et al, Proc. Natl. Acad. Sci. U. S. A, 2000, 97, 5633-5638; Singh et al, Chem. Commun, 1998, 4, 455-456; Koshkin et al, Tetrahedron, 1998, 54, 3607-3630; Kumar et al, Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al, J. Org. Chem., 1998, 63, 10035-10039; U. S. Patents Nos. : 7,741,457; 7,696,345; 7,547,684,;7,399,845; 7,053,207; 7,034, 133; 6,794,499; 6,770,748; 6,670,461 ; 6,525, 191 ; 6,268,490; U. S. Patent Publication Nos. : US2008-0039618; U.S. Patent Applications, Serial Nos. : 61/099,844; 61/097,787; 61/086,231 ; 61/056,564; 61/026,998; 61/026,995; 60/989,574; International applications WO2009/006478; WO2008/154401 ; WO2008/150729; WO 2007/134181; WO 2005/021570; WO 2004/106356; WO 94/14226). Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic nucleosides comprise a bridge between the 4' and the 2' carbon atoms of the pentofuranosyl sugar moiety including without limitation, bridges comprising 1 or from 1 to 4 linked groups (generally forming a 4 to 6 membered ring with the parent sugar moiety) independently selected from -[C(R _a )(R _b )] _n -, -C(R _a )=C(R _b )-, -C(R _a )=N-, -C(= R _a )-, -C(=0)- , -C(=S)-, -0-, -Si(R _a ) ₂ -, -S(=0)x-, and -N(R _a )-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R _a and R _b is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJ1J2, SJi, N ₃ , COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=0) ₂ -Ji), or sulfoxyl (S(=0)-Ji); and each Ji and is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2- C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is , -[C(R _a )(Rb)] _n - , -[C(R _a )(R _b )]n-0-, -C(R _a R _b )-N(R)-0- or -C(R _a R _b )-0-N(R)-. In certain embodiments, the bridge is 4'-CH ₂ -2', 4'-(CH ₂ )2-2', 4'-(CH ₂ ) ₃ -2', 4'-CH ₂ -0-2', 4'-(CH ₂ )2-0-2', 4'-CH ₂ -0-N(R)-2' and 4'-CH ₂ - N(R)-0-2'- wherein each R is, independently, H, a protecting group or C1-C12 alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-(CH ₂ )-0-2' bridge, may be in the a-L configuration or in the β-D configuration. Previously, a-L-methyleneoxy (4'-CH ₂ -0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids

Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4' to 2' bridge wherein such bridges include without limitation, a-L-4'-(CH ₂ )-0-2', p-D-4'-CH ₂ -0-2', 4'-(CH ₂ )2-0-2', 4'- CH ₂ -0-N(R)-2', 4'-CH ₂ -N(R)-0-2', 4'-CH(CH ₃ )-0-2', 4'-CH ₂ -S-2', 4'-CH ₂ -N(R)-2', 4'-CH ₂ -

CH(CH )-2', and 4'-(CH ₂ ) ₃ -2', wherein R is H, a protecting group or C1-C12 alkyl.

In certain embodiments, modified nucleosides include nucleosides having sugar surrogate groups that include without limitation, replacement of the ribosyl ring with a sugar surrogate such as a tetrahydropyranyl ring system (also referred to as hexitol) as illustrated below:

In certain embodiments, sugar surrogates are selected having the formula:

wherein:

Bx is a heterocyclic base moiety;

one of T ₃ and T is an internucleoside linking group attaching the tetrahydropyran nucleoside analog to the remainder of one of the 5' or 3' end of the oligomeric compound and the other of T and T ₄ is hydroxyl, a protected hydroxyl, a 5' or 3' terminal group or an internucleoside linking group attaching the tetrahydropyran nucleoside analog to the remainder of the other of the 5' or 3' end of the oligomeric compound;

qi, q ₂ , q , q ₄ , q ₅ , q ₆ and q ₇ are each independently, H, Ci-C ₆ alkyl, substituted Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl; and

one of Ri and R ₂ is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJiJ ₂ , SJi, N ₃ , OC(=X)Ji, OC(=X)NJiJ ₂ , and CN, wherein X is O, S or NJi and each Ji, J ₂ and J is, independently, H or Ci-C ₆ alkyl.

In certain embodiments, qi, q ₂ , q , q ₄ , q ₅ , q ₆ and q ₇ are each H. In certain embodiments, at least one of qi, q ₂ , q , q ₄ , q ₅ , q ₆ and q ₇ is other than H. In certain embodiments, at least one of qi, q ₂ , q , q ₄ , q ₅ , q ₆ and q ₇ is methyl. In certain embodiments, THP nucleosides are provided wherein one of Ri and R ₂ is F. In certain embodiments, Ri is fluoro and R ₂ is H; Ri is methoxy and R ₂ is H, and Ri is methoxyethoxy and R ₂ is H.

Such sugar surrogates can be referred to as a "modified tetrahydropyran nucleoside" or "modified THP nucleoside". Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), altritol nucleic acid (ANA), and mannitol nucleic acid (MNA) (see Leumann, C. J., Bioorg. & Med. Chem., 2002, 10, 841-854). In certain embodiments, oligomeric compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on April 10, 2010, Robeyns et al, J. Am. Chem. Soc, 2008, 130(6), 1979-1984; Horvath et al, Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al, J. Am. Chem. Soc, 2007, 129(30), 9340-9348; Gu et al.,, Nucleosides,

Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al, Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al, Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al, Tetrahedron, 2004, 60(9), 2111- 2123; Gu et al, Oligonucleotides, 2003, 13(6), 479-489; Wang et al, J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al, Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al, J. Org. Chem., 2001, 66, 8478-82; Wang et al, Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al, J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.

X

wherein independently for each of the at least one cyclohexenyl nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

one of T ₃ and T ₄ is an internucleoside linking group attaching the cyclohexenyl nucleoside to the remainder of one of the 5' or 3' end of the oligomeric compound and the other of T ₃ and T ₄ is hydroxyl, a protected hydroxyl, a 5' or 3' terminal group or an internucleoside linking group attaching the cyclohexenyl nucleoside to the remainder of the other of the 5' or 3' end of the oligomeric compound; and qi, q ₂ , q , q4, q ₅ , q ₆ , q7, q ₈ and qg are each, independently, H, Ci-C ₆ alkyl, substituted Ci-C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₂ -C ₆ alkynyl or other sugar substituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known in the art and are suitable as sugar surrogates that can be used to modify nucleosides for incorporation into oligomeric compounds as provided herein (see for example review article: Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to further enhance their activity.

Some representative U. S. patents that teach the preparation of such modified sugars include without limitation, U. S. : 4,981,957; 5, 1 18,800; 5,319,080; 5,359,044; 5,393,878; 5,446, 137;

5,466,786; 5,514,785; 5,519, 134; 5,567,81 1 ; 5,576,427; 5,591,722; 5,597,909; 5,610,300;

5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed June 2, 2005 and published as WO 2005/121371 on December 22, 2005 certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

As used herein the term "reactive phosphorus" is meant to include groups that are covalently linked to a monomer subunit that can be further attached to an oligomeric compound that are useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages. Such reactive phosphorus groups are known in the art and contain phosphorus atoms in P ^m or P ^v valence state including, but not limited to, phosphoramidite, H- phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. In certain embodiments, reactive phosphorus groups are selected from diisopropylcyanoethoxy

phosphoramidite (-0*-P[N[(CH(CH ₃ ) ₂ ] ₂ ]0(CH ₂ ) ₂ CN) and H-phosphonate (-0*-P(=0)(H)OH), wherein the O* is normally attached to the 3'-position of the Markush group of Formula I. A preferred synthetic solid phase synthesis utilizes phosphoramidites (P ⁱⁿ chemistry) as reactive phosphites. The intermediate phosphite compounds are subsequently oxidized to the phosphate or thiophosphate (P ^Y chemistry) using known methods to yield, phosphodiester or phosphorothioate internucleoside linkages. Chiral auxiliaries are known in the art (see for example: Wang et al, Tetrahedron Letters, 1997, 38(5), 705-708; Jin et al, J. Org. Chem, 1997, 63, 3647-3654; Wang et al., Tetrahedron Letters, 1997, 38(22), 3797-3800; and U. S. patent 6,867,294, issued March 15,

2005). Additional reactive phosphates and phosphites are disclosed in Tetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-231 1). As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

The term "oligonucleoside" refers to a sequence of nucleosides that are joined by

internucleoside linkages that do not have phosphorus atoms. Internucleoside linkages of this type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatom cycloalkyl, one or more short chain heteroatomic and one or more short chain heterocyclic. These internucleoside linkages include without limitation, siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl, sulfamate, methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide and others having mixed N, O, S and CH ₂ component parts.

As used herein, the term "oligomeric compound" refers to a contiguous sequence of linked monomer subunits. Each linked monomer subunit normally includes a heterocyclic base moiety but monomer subunits also include those without a heterocyclic base moiety such as abasic monomer subunits. In certain embodiments, at least some and generally most if not essentially all of the heterocyclic bases in an oligomeric compound are capable of hybridizing to a nucleic acid molecule, normally a preselected RNA target. The term "oligomeric compound" therefore includes oligonucleotides, oligonucleotide analogs and oligonucleosides. It also includes polymers having one or a plurality of nucleosides having sugar surrogate groups.

In certain embodiments, oligomeric compounds comprise a plurality of monomer subunits independently selected from naturally occurring nucleosides, non-naturally occurring nucleosides, modified nucleosides and nucleosides having sugar surrogate groups. In certain embodiments, oligomeric compounds are single stranded. In certain embodiments, oligomeric compounds are double stranded comprising a double-stranded duplex. In certain embodiments, oligomeric compounds comprise one or more conjugate groups and/or terminal groups.

As used herein the term "internucleoside linkage" or "internucleoside linking group" is meant to include all manner of internucleoside linking groups known in the art including but not limited to, phosphorus containing internucleoside linking groups such as phosphodiester and phosphorothioate, and non-phosphorus containing internucleoside linking groups such as formacetyl and methyleneimino. Internucleoside linkages also includes neutral non-ionic internucleoside linkages such as amide-3 (3'-CH ₂ -C(=0)-N(H)-5'), amide-4 (3'-CH ₂ -N(H)-C(=0)-5') and methylphosphonate wherein a phosphorus atom is not always present. In certain embodiments, each internucleoside linkage is, independently, a phosphorothioate or a phosphodiester internucleoside linkage. In certain embodiments, essentially each internucleoside linkage is a phosphodiester internucleoside linkage. In certain embodiments, essentially each internucleoside linkage is, a phosphorothioate internucleoside linkage.

In certain embodiments, oligomeric compounds as provided herein can be prepared having one or more internucleoside linkages containing modified e.g. non-naturally occurring

internucleoside linkages. The two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Modified internucleoside linkages having a phosphorus atom include without limitation, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phos- phoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionopho- sphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 -5' linkages, 2 -5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity can comprise a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus containing linkages include without limitation, U.S. : 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5, 177, 196;

5, 188,897; 5, 194,599; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321, 131; 5,399,676;

5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519, 126; 5,527,899; 5,536,821;

5,541,306; 5,550, 111; 5,563,253; 5,565,555; 5,571,799; 5,587,361; 5,625,050; 5,672,697 and

5,721,218, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

In certain embodiments, oligomeric compounds as provided herein can be prepared having one or more non-phosphorus containing internucleoside linkages. Such oligomeric compounds include without limitation, those that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH ₂ component parts.

Representative U. S. patents that teach the preparation of the above oligonucleosides include without limitation, U. S. : 5,034,506; 5, 166,315; 5, 185,444; 5,214, 134; 5,216, 141 ; 5,235,033;

5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;

5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;

5,633,360; 5,677,437; 5,677,439; 5,646,269 and 5,792,608, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

As used herein "neutral internucleoside linkage" is intended to include internucleoside linkages that are non-ionic. Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH ₂ -N(CH ₃ )-0-5'), amide-3 (3'-CH ₂ -C(=0)-N(H)- 5'), amide-4 (3'-CH ₂ -N(H)-C(=0)-5'), formacetal (3'-0-CH ₂ -0-5'), and thioformacetal (3'-S-CH ₂ -0- 5'). Further neutral internucleoside linkages include nonionic linkages comprising siloxane

(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH ₂ component parts.

In certain embodiments, oligomeric compounds as provided herein can be prepared having one or more optionally protected phosphorus containing internucleoside linkages. Representative protecting groups for phosphorus containing internucleoside linkages such as phosphodiester and phosphorothioate linkages include β-cyanoethyl, diphenylsilylethyl, δ-cyanobutenyl, cyano p-xylyl (CPX), N-methyl-N-trifluoroacetyl ethyl (META), acetoxy phenoxy ethyl (APE) and butene-4-yl groups. See for example U. S. Patents Nos. 4,725,677 and Re. 34,069 (β-cyanoethyl); Beaucage et al, Tetrahedron, 1993, 49(10), 1925-1963; Beaucage et al, Tetrahedron, 1993, 49(46), 10441- 10488; Beaucage et al, Tetrahedron, 1992, 48(12), 2223-231 1.

In certain embodiments, the steps for large scale synthesis of oligomeric compounds, other than coupling steps with bicyclic nucleosides of Formula I, are performed in accordance with published literature (see for example, Protocols for Oligonucleotides and Analogs, Agrawal, Ed., Humana Press, 1993, and/or RNA: Scaringe, Methods, 2001, 23, 206-217; Gait et al., Applications of Chemically synthesized RN A in RNA: Protein Interactions, Smith, Ed., 1998, 1-36; Gallo et al, Tetrahedron , 2001, 57, 5707-5713; Caruthers U.S. Patents Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5, 132,418; and Koster U.S. Patents Nos. 4,725,677 and Re. 34,069).

Commercially available equipment commonly used for the preparation of oligomeric compounds that utilize the solid support method is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. Suitable solid phase techniques, including automated synthesis techniques, are described in Oligonucleotides and Analogues, a Practical Approach, F. Eckstein, Ed., Oxford University Press, New York, 1991.

The synthesis of RNA and related analogs relative to the synthesis of DNA and related analogs has been increasing as efforts in RNA interference and micro RNA increase. The primary RNA synthesis strategies that are presently being used commercially include 5'-0-DMT-2'-0-t- butyldimethylsilyl (TBDMS), 5'-0-DMT-2'-0-[l(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP), 2'-0-[(triisopropylsilyl)oxy]methyl (2'-0-CH ₂ -0-Si(iPr) ₃ (TOM) and the 5'-0-silyl ether- 2'- ACE (5'-0-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2'-0-bis(2- acetoxyethoxy)methyl (ACE). A current list of some of the major companies currently offering RNA products include Pierce Nucleic Acid Technologies, Dharmacon Research Inc., Ameri Biotechnologies Inc., and Integrated DNA Technologies, Inc. One company, Princeton Separations, is marketing an RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. The primary groups being used for commercial RNA synthesis are:

TBDMS: 5'-0-DMT-2'-0-t-butyldimethylsilyl; TOM: 2'-0-[(triisopropylsilyl)oxy]methyl;

DOD/ACE: (5'-0-bis(trimethylsiloxy)cyclododecyloxysilyl ether-2'-0-bis(2-acetoxyethoxy)methyl; and FPMP: 5'-0-DMT-2'-0-[l(2-fluorophenyl)-4-ethoxypiperidin-4-yl]. In certain embodiments, each of the aforementioned RNA synthesis strategies can be used herein. In certain embodiments, the aforementioned RNA synthesis strategies can be performed together in a hybrid fashion e.g. using a 5 '-protecting group from one strategy with a 2'-0-protecting from another strategy.

All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

Those skilled in the art, having possession of the present disclosure will be able to prepare oligomeric compounds, comprising a contiguous sequence of linked monomer subunits, of essentially any viable length. While in certain embodiments, oligomeric compounds provided herein can be prepared as described, the following examples serve only to illustrate and are not intended to be limiting.

Example 1

Detritylation using standard methods

Industry standard methods for synthesizing oligomeric compounds are well known in the art. Generally, reactive sites on a solid support material are coupled to monomers or universal linkers that provide protected reactive sites. The process of deblocking reactive sites and coupling monomers is performed iteratively to eventually provide an oligomeric compound having a predetermined length and base sequence. The phosphorus groups are deprotected by treatment with a solution of triethylamine and the oligomeric compound is cleaved from the solid support by treatment with a solution of ammonium hydroxide. After cleavage the 5'-protected oligomeric compound is purified and optionally precipitated one or more times. When the 5'-protecting group is a trityl group the oligomeric compound is referred to as a DMT-on oligomeric compound. The most commonly used 5'-protecting group is the 4,4'-dimethoxytrityl (DMT) group.

Standard protocols for performing the final detritylation of an oligomeric compound include dissolution of the DMT-on oligomeric compound in water, adjustment of the temperature to 22°C and addition of acid to a pH of about 3.5. After the detritylation is complete the solution is quenched with base. In one such standard protocol (600 mmol support loading), the purified oligomeric compound is dissolved in purified water, the temperature is adjusted to 22°C and the pH is adjusted to 3.5 by addition of glacial acetic acid. After detritylation is complete the reaction is quenched by addition of sodium hydroxide and the oligomeric compound is precipitated by addition of ethanol (generally the ethanol is in a stirred container and the quenched solution is added to it). The solution is decanted or pumped away from the precipitate and the precipitate is washed with ethanol to provide the detritylated oligomeric compound. For doing repeat runs on the same scale for the same oligomeric compound the detritylation time would be essentially the same for each successive run.

Example 2

Detritylation using standard methods, large scale

DMT-on oligomeric compound is routinely prepared starting from an initial loading of the solid support of from about 200 to about 600 mmol. The DMT-on eluate (containing methanol and sodium acetate) from reverse phase HPLC purification is precipitated in ethanol to isolate the DMT- on oligomeric compound. After decanting the supernatant, the precipitated oligomeric compound is reconstituted with purified water to a targeted concentration. This typically yields a solution of 50 mg/g oligomeric compound, < 1% w/w sodium acetate, and <10% w/w organic solvent (ethanol and methanol). This DMT-on reconstituted oligomeric compound solution is typically detritylated by first adjusting the pH of the mixing oligomeric compound to pH 3.5 ± 0.2 with glacial acetic acid at 21-22 °C. The reaction is allowed to proceed until the detritylation reaction is complete (essentially all of the trityl groups removed < 0.2% DMT-on oligomeric compound relative to total oligomeric compound). The reaction time is generally based on laboratory pilot experiments or historical data. To stop the reaction, aqueous 10N sodium hydroxide is added to the solution to adjust the pH to 5.0- 6.0. The solution is then immediately precipitated in ethanol to isolate the detritylated oligomeric compound.

Example 3

Removal of MMT protecting groups from 5'-amino-modified oligomeric compounds using standard detritylation protocols

A particular 5'-GalNAc conjugated oligonucleotide (ISIS-681257) was prepared by reaction of the deprotected 5'-amino-modified oligonucleotide (intermediate oligonucleotide) with a reactive conjugate cluster. The MMT protected 5'-amino-modified oligonucleotide was prepared on an automated synthesizer using standard protocols. After cleavage, precipitation and purification the MMT protected 5'-amino-modified oligonucleotide was provided having Formula I:

I

Removal of the MMT group to provide the deprotected 5'-amino-modified oligonucleotide was performed following standard protocols as for a 5'-DMT protected hydroxyl group of an oligonucleotide as illustrated in Example 2. The deprotection step was monitored and proceded for an extended time without completion. Extrapolation of the data obtained indicated that removal of essentially all of the MMT protecting groups would take about 2050 minutes. Allowing the reaction to proceed for that much time would result in an unacceptable level of depurination.

SEQ ID NO. Composition (5' to 3') ISIS NO.

01/681257 GalNAc ₃ -7a-o'-TeGeo ^m Ceo eo ^m Ceo ^m CdGdTdTdGdGd TdGd ^m Cd TdTeoGeoTeT _e ^m Ce

Between adjacent nucleosides subscript "e" indicates that the preceding nucleoside comprises a 2'-0-(CH ₂ )2-OCH ₃ (MOE) substituent and all other nucleosides are P-D-2'-deoxyribo- nucleosides. A subscript "o" indicates that the internucleoside linkage between adjacent nucleosides is a phosphodiester internucleoside linkage and all other internucleoside linkages are

phosphorothioate internucleoside linkages and a subscript "ο'" indicates -0-P(=0)(OH)-. Capital letters indicate the nucleobase for each nucleoside and each " ^m C" indicates that this nucleoside comprises a 5-methyl cytosine heterocyclic base. The linked conjugate group GalNAc -7 _a - ₀ ' has the formula:

Example 4

Removal of MMT protecting groups from 5'-amino-modified oligomeric compounds using modified detritylation protocols (lab scale)

A small amount of protected 5'-amino-modified oligonucleotide (Formula I) was deprotected using modified protocols. The deprotection reaction was performed at pH 4.8 at 40°C (standard protocols: pH 3.5 ± 0.2 at 21-22 °C). The modified protocols increased the detritylation rate without a large increase in depurination rate. The reaction reached completion (<0.2% MMT-on) after 70 minutes of reaction and also resulted in an acceptable level (0.2%) of total depurination. Example 5

Removal of MMT protecting groups from 5'-amino-modified oligomeric compounds using modified detritylation protocols (production scale)

An aqueous solution of MMT protected ISIS-681257 intermediate having Formula I (1.128 kg, at a concentration of 24.5 mg/g) was placed in a jacketed stainless steel tank controlled to 40 °C. Once the temperature was at least 35 °C, glacial acetic acid (0.1 kg) was added to the mixing solution to achieve a reaction pH of 5.00, measured at the warm temperature. The reaction timer was started when the solution pH fell below 5.20. After approximately 5 hours of reaction, the reaction mixture was cooled to from 20 to 22 °C and the pH was raised to 5.92 by adding 10% v/v 2.0 M buffered sodium acetate solution (pH 7.2). The reaction was considered stopped at the completion of the sodium acetate addition (331 minutes). The material was then further processed following in house standard protocols. These reaction conditions produced acceptable material with 0.01% MMT-on and 0.20% total depurination.

Example 6

Removal of MMT protecting groups from 5'-amino-modified oligomeric compounds using modified detritylation protocols (multiple production scale runs)

Following the procedures illustrated in Example 5, multiple large scale production runs were preformed to provide the deprotected oligomeric compounds a summarized below.

Seq Id No./ Description

ISIS No.

01/681257i NH2-(CH2)6-o ^, -TeGeo ^m CeoTeo ^m Ceo ^m CGTTGGTG ^m CTTeoGeoTeT _e ^m Ce

02/696844i NH2-(CH2)6-o-AeTe ^m Ce ^m Ce ^m CeA ^m CG ^m C ^m C ^m C ^m CTGT ^m Ce ^m CeA _e Ge ^m Ce

03/721744i NH2-(CH2)6-o-TeGe ^m CeoAeoA _e GT ^m CT ^m CTTGG ^m CAeoAeoAe ^m CeAe 04/712408i H2-(CH ₂ )6-o _' -Ge ^m CeAeGe A _e GGTGAAG ^m CGAAeGeT _e Ge ^m Ce

05/703802i H2-(CH ₂ ) ₆ - _ol -GeGeoAeo ^m CeoAeoTTG ^m C ^m CAGTAATeo ^m CeoGe ^m C _e Ae

06/766720i H ₂ -(CH ₂ ) ₆ - _ol - ^m C _e ^m C _e A _eo ^m C _eo ^m C _e TTTGGGTGAAT _eo A _eo G _e ^m C _e A _e

07/757456i H ₂ -(CH ₂ ) ₆ - _ol - ^m Ce ^m Ae ^m CeAeAeA ^m CAAG ^m CTGGT ^m CeGeGeT _e Te

08/702843i H ₂ -(CH ₂ )6-o ^, - ^m CeTeoTeo eoAeoTT ^m C ^m CAAAGGG ^m CeoAeoG _e ^m CeTe

09/678354i H ₂ -(CH ₂ ) ₆ - _ol - AeGe ^m CeTeT _e ^m CTTGT ^m C ^m C AG ^m CT _e T _e T _e A _e T _e .

Between adjacent nucleosides subscript "e" indicates that the preceding nucleoside comprises a 2'-0-(CH ₂ ) ₂ -OCH ₃ (MOE) substituent and all other nucleosides are P-D-2'-deoxyribo- nucleosides. A subscript "o" indicates that the internucleoside linkage between adjacent nucleosides is a phosphodiester internucleoside linkage and all other internucleoside linkages are

phosphorothioate internucleoside linkages and a subscript "ο'" indicates -0-P(=0)(OH)-. Each " ^m C" indicates that this nucleoside comprises a 5-methyl cytosine heterocyclic base. The subscript i following the Seq Id Nos, indicates that the oligonucleotide is the aminohexyl intermediate and not the actual sequence listed which would include a conjugate group as illustrated above in Example 3.

Seq Id No./ Scale oligo (g) pH Temp Time pH

ISIS No. (lot No.) mmol rxn rxn rxn quench

01/681257i (086) 299 1128 5 40 331 5.92

02/696844i (090) 299 1213 4.5 40 145 5.3

03/721744i (096) 299 1386 4.5 40 150 5.48

04/712408i (097) 599 2721 4.5 40 153 5.34

05/70380¾ (098) 299 1269 4.5 40 145 5.36

06/766720i (102) 299 927 4.5 40 150 5.57

01/681257i (103) 900 3558 4.5 40 122 5.39

07/757456i 299 1132 4.5 40 189 5.43

08/702843i 449 1495 4.5 40 152 5.59

09/678354i 598 2613 4.5 40 108 5.39.

Seq Id No./ %MMT-on % Depurination

ISIS No. (lot No.) End Start End Difference

01/681257i (086) 0.01% 0.16% 0.20% 0.04%

02/696844i (090) 0.02% 0.32% 0.33% 0.01%

03/721744i (096) 0.02% 0.33% 0.35% 0.02%

04/712408i (097) 0.00% 1.1% 1.2% 0.10%

05/70380¾ (098) 0.02% 0.93% 0.83% -0.10%

06/766720i (102) 0.01% 0.41% 0.56% 0.15%

01/681257i (103) 0.01% 0.18% 0.23% 0.05%.

07/757456i 0.02% 0.59% 0.59% 0.00%

08/702843i 0.02% 0.44% 0.43% -0.01%

09/678354i 0.01% 0.30% 0.19% -0.11%.

change in depurination may be negative due to analytical variability.