Methods and reagents for the synthesis of nucleoside analogues, and uses thereof

Abstract

The present invention relates to methods for the synthesis of nucleoside analogues. More specifically, the present invention provides methods for the synthesis of 6-disubstituted nucleoside analogues of Formula (I).

Description

10102-040W01METHODS AND REAGENTS FOR THE SYNTHESIS OF NUCLEOSIDE ANALOGUES,AND USES THEREOFFIELD

[0001] The present invention relates to methods for the synthesis of nucleoside analogues. More specifically, the present invention relates to methods for the synthesis of 6-disubstituted nucleosides.BACKGROUND

[0002] Nucleosides play key roles in diverse cellular processes ranging from cell signalling to metabolism (1 ). Nucleosides are composed of a nucleobase - canonically composed of adenine, guanine, cytosine, thymine and uracil, and a sugar moiety, typically ribose of 2’-deoxyribose. Nucleosides can be further modified with a 5’-phosphate or phosphate-like group, and RNA oligomers include nucleotides linked via phosphate or phosphate-like linkages from 5’ to 3’. Nucleosides can be modified in several ways, including modifications to the ribose moiety, modifications to the base moiety or modifications to the phosphate moiety, leading to compounds referred to as “nucleoside analogues” (NAs).

[0003] NAs have a long and rich history in the field of medicinal chemistry and as tool compounds in chemical biology. The naturally occurring nucleosides are a unique and valuable starting point for drug design due to their involvement in numerous biological processes. Synthetic NAs have been designed to mimic their natural counterparts (2-18). Single NAs have been primarily used as treatments for parasitic, bacterial and fungal infections as well as potent and effective anticancer drugs. In addition to this “small molecule” modality, NAs can be incorporated into oligomeric structures that can modulate gene expression, thus bypassing the complexities associated with protein inhibition. For example, NAs can be incorporated into oligonucleotide sequences to enhance nuclease resistance, binding affinity or to reduce toxicity. Such oligomeric structures can include short interfering RNA (siRNA), microRNA (miRNA), inhibitory antisense oligonucleotides (ASOs), small activating RNA (saRNA) and messenger RNA (mRNA) that may reduce the expression of one or more specific gene products and may therefore find use in therapeutic, diagnostic, and research applications.

[0004] NAs have been used in the treatment of cancer (2, 6) and represent the largest class of small molecule antivirals (3, 4). Mechanistically, NAs can operate as toxic antimetabolites that interfere with nucleic acid synthesis (4). Alternatively, following in vivo phosphorylation, the resulting nucleotide analogues can inhibit enzymes involved in cancer10102-040W01cell growth or viral replication (e.g., DNA / RNA polymerases, ribonucleotide reductases or nucleoside phosphorylases) (2, 4). NAs have also demonstrated promise as epigenetic modulators, and both decitabine and azacitidine inhibit DNA methyltransferase and have been approved for cancer therapy (4).

[0005] While several decades of organic and medicinal chemistry have yielded numerous valuable nucleoside analogues, the synthesis of further nucleoside analogues presents some challenges. Nucleoside analogues are often synthesized from naturally occurring carbohydrates, which limits patterns of substitution and furanose stereochemistry e.g., 19-29). The addition of nucleobases to activated ribose derivatives often fails or proceeds with poor diastereoselectivity with C2’ or C4’ modified nucleosides and efficient strategies for producing C4’ modified nucleosides, including thionucleosides are limited.

[0006] Synthesis of deprotected methyl locked nucleic acid (LNA) involved 12 steps from a chiral starting material (31). Furthermore, LNAs may be toxic and oligonucleotides containing locked nucleic acid may improve potency but cause significant hepatotoxicity in animals. In addition to toxicity, the pharmacokinetic properties of many oligonucleotide therapeutics are limited, as are options for extrahepatic delivery.

[0007] Bicyclic nucleosides or “BNAs” are NAs in which the furanose portion of the nucleoside includes a bridge connecting two atoms on the furanose ring thereby forming a bicyclic ring system (32-34; WO 2009 / 006478 A2).

[0008] Synthesis of nucleosides and nucleoside analogues have been described in (30; WO 2021 / 191830).

[0009] Attachment of a lipid(35), aromatic(36) or charged(37) moiety to an oligonucleotide can improve biodistribution, efficacy, cell permeability or toxicity.SUMMARY

[0010] The present invention relates to 6-disubstituted nucleosides, and methods of synthesis and use thereof.

[0011] In one aspect, there is provided a compound of Formula (I) or a salt thereof:10102-040W01(I)whereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group;one of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4 can be O, S or NX;X and Y can each independently be H, halogen, optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C1-C6alkoxy, OJ1, SJ1, SOJ1, SO2J1, NJ1J2, N3, CN, C(=O)OJ1, C(=O)NJ1J2, C(=O)J1, O-C(=O)NJ1J2, N(H)C(=NH)NJ1J2, N(H)C(=O)NJ1J2, N=O-J1or N(H)C(=S)NJ1J2;or X and Y together can be =C(q3)(q4), where q3 and q4 can each, independently, be H, halogen, (=O), or optionally substituted C1-C6alkyl;Z and Q can each independently be CJ1J2, NJ1J2, or O;wherein J1and J2can each independently be H, optionally substituted C1-C6alkyl, optionally substituted C1-C6acyl, optionally substituted C2-C6alkenyl, C2-C6alkynyl, optionally substituted C1-C6aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.10102-040W01

[0012] In some embodiments, there is provided a compound of Formula (II) or a salt thereof:IIwhereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R5can be halogen, optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C1-C6alkoxy, OJ1, SJ1, SOJ1, SO2J1, NJ1J2, N3, CN, C(=O)OJ1, C(=O)NJ1J2, C(=O)J1, O-C(=O)NJ1J2,N(H)C(=NH)NJIJ2, N(H)C(=O)NJIJ2, N=O-J1 or N(H)C(=S)NJIJ2;X and Y can each independently be H, halogen, optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C1-C6alkoxy, OJ1, SJ1, SOJ1, SO2J1, NJ1J2, N3, CN, C(=O)OJ1, C(=O)NJ1J2, C(=O)J1, O-C(=O)NJ1J2, N(H)C(=NH)NJ1J2, N(H)C(=O)NJ1J2, N=O-J1or N(H)C(=S)NJ1J2;or X and Y together can be =C(q3)(q4), wherein q3 and q4 can each independently be H, halogen, (=O), or optionally substituted C1-C6alkyl;10102-040W01Z can be CJ1J2, NJ1J2, or O; andJ1and J2can each independently be H, optionally substituted C1-C6alkyl, optionally substituted C1-C6acyl, optionally substituted C2-C6alkenyl, C2-C6alkynyl, optionally substituted C1-C6aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.

[0013] In some embodiments, there is provided a compound of Formula (III) or a salt thereof:IIIwhereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;X and Y can each independently be H, halogen, optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, optionally substituted C1-C6alkoxy, OJ1, SJ1, SOJ1, SO2J1, NJ1J2, N3, CN, C(=O)OJ1, C(=O)NJ1J2, C(=O)J1, O-C(=O)NJ1J2, N(H)C(=NH)NJ1J2, N(H)C(=O)NJ1J2, N=O-J1or N(H)C(=S)NJ1J2;10102-040W01or X and Y together can be =C(q3)(q4), wherein q3 and q4 can each independently be H, halogen, (=O), or optionally substituted C1-C6alkyl; and

[0014] J1and J2can each independently be H, optionally substituted C1-C6alkyl, optionally substituted C1-C6acyl, optionally substituted C2-C6alkenyl, C2-C6alkynyl, optionally substituted C1-C6aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.

[0015] In some embodiments, there is provided a compound of Formula (IV) or a salt thereof:whereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2- oxo-l,2-dihydropyrimidine-l-yl group;J1and J2can each independently be:10102-040W0110102-040W01Ji and J2 together can be:where R9 can be:; and10102-040W01

[0017] In some embodiments, there is provided a compound of Formula (V) or a salt thereof:OR2R4whereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;X and Y can each independently be:10102-040W01where R12can be:10102-040W01or N; and

[0018] * can be “R” or “S” configuration10102-040W01

[0019] In some embodiments, there is provided a compound of Formula (VI) or a salt thereof:J-IN- OR2R4R5whereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;J1and R5can each independently be:10102-040W01where R12can be:10102-040W01where R9 can be:N'\; and

[0020] * can be “R” or “S” configuration.

[0021] In some embodiments, there is provided a compound of Formula (VII) or a salt thereof:whereR1 and R2can each independently be H, a hydroxyl protecting group, a terrminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;10102-040W01Rs can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l, 2-dihydropyrimidine-l-yl group;R15 can be:10102-040W01where R12can be:10102-040W01where R9 can be:N'\ andcan be “R” or “S” configuration.

[0022] In some embodiments, the compound can be one or more of compound B, Ba, Bb, Be, Bd, Bx, Bxa, Bxb, Bxc, Bxd, C, Ca, Cb, Cc, Cd, D, Da, Db, De, Dd, E, Ea, Eb, Ec, Ed, F, Fa, Fb, Fc, Fd, G, Ga, Gb, Gc, or Gd.

[0023] In some embodiments, the compound can be one or more of the compound listed in Table 1.

[0024] In an alternative aspect, there is provided a nucleic acid molecule including a compound as described herein. The nucleic acid molecule may be an oligomer or a polymer.

[0025] In an alternative aspect, there is provided a method of synthesizing a compound as described herein, by providing an alkene of the structure (A):where10102-040W01R1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 can be O, S or NX; andperforming a heteroatom cycloaddition reaction in the presence of an isocyanate to yield a compound as described herein; orperforming a cycloaddition on the alkene to yield a dichloroketone; reducing the dichloroketone to a ketone; performing a reductive alkylation on the ketone; orperforming a cycloaddition with a keteniminium salt on the alkene followed by hydrolysis to yield a ketone, and performing a reductive alkylation, a 1,2 addition, a condensation, or a reductive amination on the ketone to yield a compound as described herein.

[0026] In an alternative embodiment, there is provided a method of synthesizing a compound as described herein, by providing a compound (C)OR2R4whereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; or10102-040W01one of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 can be O, S or NX;and performing a reductive alkylation to yield the compound as described herein.

[0027] In an alternative embodiment, there is provided a method of synthesizing a compound as described herein, by providing a compound (C)OR2R4whereRi and R2are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of Ri or R2is an internucleoside linking group and the other of Ri or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orRi is a hydroxyl protecting group and R2is a reactive phosphorous group;R3is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 is O, S or NX;10102-040W01ii) performing a 1,2 nucleophile addition to yield the compound (D)D,whereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4 can be O, S or NX;X can be:10102-040W0110102-040W01; and10102-040W01* can be “R” or “S” configuration;and alkylating the compound (D) to yield the compound as described herein.

[0028] In an alternative embodiment, there is provided a method of synthesizing a compound as described herein, by providing a compound (A):whereRi and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 can be O, S or NX;performing a heteroatom cycloaddition reaction in the presence of an isocyanate to yield the compound (E)10102-040W01, andwhereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4 can be O, S or NX; and* can be “R” or “S” configuration; andalkylating or acylating the compound (E), to yield the compound as described herein.

[0029] In an alternative embodiment, there is provided a method of synthesizing a compound as described herein, by providing a compound (C)OR2R410102-040W01whereR1and R2can each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 can be O, S or NX; andperforming a condensation reaction on the compound (C), in the presence of a hydroxylamine or performing a Wittig reaction on the compound (C), to yield the compound as described herein.

[0030] In some embodiments, there is provided a method of preparing an oligomer or a polymer, the method comprising providing a compound as described herein, and reacting the compound with one or more nucleotides or nucleic acid analogues under suitable synthetic conditions to yield an oligomer or a polymer.

[0031] This summary of the invention does not necessarily describe all features of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a graph showing relative MALAT1 expression levels in different tissues from mice dosed with CB050 and CB054 at 5 mg / kg and 15 mg / kg. Values are an average of 5 distinct replicates for their respective tissue and dose regime and are reported with their standard error of the mean (SEM). PBS = phosphate buffered saline.10102-040W01DETAILED DESCRIPTION

[0033] The present disclosure provides, in part, 6-disubstituted nucleosides and methods and uses thereof.

[0034] In one aspect, the present disclosure provides a compound according to Formula I:Formula Iwhere Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4 can be O, S or NX;X and Y can each, independently, be H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-Ce alkenyl, optionally substituted C2-Ce alkynyl, optionally substituted Ci-Ce alkoxy, OJ1, SJ1, SOJ1, SO2Ji, NJIJ2, N3, CN, C(=O)OJi, C(=O)NJIJ2, C(=O)Ji, O-C(=O)NJIJ2, N(H)C(=NH)NJIJ2, N(H)C(=O)NJIJ2, N=O-J1 or N(H)C(=S)NJIJ2;or X and Y together can be =C(q3)(q4), where q3 and q4 can each, independently, be H, halogen, (=O), or optionally substituted C1-C6alkyl;Z and Q can each independently be CJ1J2, NJ1J2, or O;10102-040W01where J1and J2can each, independently, be H, optionally substituted C1-C6alkyl, optionally substituted C1-C6acyl, optionally substituted C2-C6alkenyl, C2-C6alkynyl, optionally substituted C1-C6aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.

[0035] In an alternative aspect, the present disclosure provides a compound according to Formula II:Formula IIwhere R1and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R5can be halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted Ci-Ce alkoxy, OJ1, SJ1, SOJ1, SO2Ji, NJ1J2, N3, CN, C(=O)OJI, C(=O)NJIJ2, C(=O)JI, O-C(=O)NJIJ2,N(H)C(=NH)NJIJ2, N(H)C(=O)NJIJ2, N=O-J1 or N(H)C(=S)NJIJ2;X and Y can each, independently, be H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted Ci-Ce alkoxy, OJ1, SJ1, SOJ1, SO2Ji, NJ1J2, N3, CN, C(=O)OJi, C(=O)NJIJ2, C(=O)Ji, O-C(=O)NJIJ2, N(H)C(=NH)NJIJ2, N(H)C(=O)NJIJ2, N=O-J1 or N(H)C(=S)NJIJ2;10102-040W01or X and Y together can be =C(q3)(q4), where q3 and q4 can each, independently, be H, halogen, (=O), or optionally substituted C1-C6alkyl;Z can be CJ1J2, NJ1J2, or O;where J1and J2can each, independently, be H, optionally substituted C1-C6alkyl, optionally substituted C1-C6acyl, optionally substituted C2-C6alkenyl, C2-C6alkynyl, optionally substituted C1-C6aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.

[0036] In an alternative aspect, the present disclosure provides a compound according to Formula III:Formula IIIwhere R1 and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;X and Y can each, independently, be H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-Ce alkenyl, optionally substituted C2-Ce alkynyl, optionally substituted Ci-Ce alkoxy, OJ1, SJ1, SOJ1, S02Ji, NJIJ2, N3, CN, C(=O)OJi, C(=O)NJIJ2, C(=O)Ji, O-C(=O)NJIJ2, N(H)C(=NH)NJIJ2, N(H)C(=O)NJIJ2, N=O-J1 or N(H)C(=S)NJIJ2;10102-040W01or X and Y together can be =C(q3)(q4), where q3 and q4 can each, independently, be H, halogen, (=O), or optionally substituted C1-C6alkyl;where J1and J2can each, independently, be H, optionally substituted C1-C6alkyl, optionally substituted C1-C6acyl, optionally substituted C2-C6alkenyl, C2-C6alkynyl, optionally substituted C1-C6aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.

[0037] In an alternative aspect, the present disclosure provides a compound according to Formula IV:Formula IVwhere R1and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2- oxo-l,2-dihydropyrimidine-l-yl group;10102-040W01J1and J2can each independently be:10102-040W01; andcan be “R” or “S” configuration.10102-040W01

[0038] In an alternative aspect, the present disclosure provides a compound according to Formula V:Formula Vwhere Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;X and Y can each independently be:10102-040W0110102-040W01; andcan be “R” or “S” configuration.10102-040W01

[0039] In an alternative aspect, the present disclosure provides a compound according to Formula VI:Formula VIwhere Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;J1and R5can each independently be:o10102-040W01where R12can be:10102-040W01where R9 can be:nandcan be “R” or “S” configuration.

[0040] In an alternative aspect, the present disclosure provides a compound according to Formula VII:Formula VIIwhere R1 and R2can both be H, a hydroxyl protecting group, a terrminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3 can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;10102-040W01R15can be:Q3 ^4 O or ^|r|zv where Ji can be:q3 and q4 can each independently be:10102-040W01where R12can be:10102-040W01* can be “R” or “S” configuration.

[0041] In some embodiments, the present disclosure provides the following compounds, as set forth in Table 1.Table 1: List of Compounds10102-040W017b-U10102-040W01NHBz8a-C NHBzF8b-CNHBz10102-040W0110102-040W01NHBz 0 NHBzft fti ft (S X JHHO- N O^ HO— N^O HO— N^OV-O-TI ^^iQT—13b-mC 13b-C13b-UA° ofti ft (S < xHO—. N" X)\-0~J VZz ^HO^oH^T— 0^NHF\-NH AftFft A14a-mC 14a-C14a-UA° 0fti ftHO— N^O (S\-0~J yo~^ \-O-7lHo^IHO^4H<^^— 0Ho^i ^F\ ^NH \ ftft A14b-mC 14b-C14b-UAr < 1 Jft ft ftft \-O-J(iPr)2N J>— / V b'Afto,p-? A-o0\Z°\ V-* ANZ 15-C0 NHBzft(S ft J5— (\H^ HO— N^O HO—0yo-J j ^HO^4H(2^o16-C16-Uftftft ftft V-o-J'r^V^o r °0c°10102-040W01NHBz o NHBz / Y° YS AXH(S 1 J N" > HO—HO— I HO— N^O N O18 HOXZJH(^F F F F F F18-mC 18-C18-U(S XAP^ XX(r°r-9V~o r° r° / ~° Sr y0A 15 X TO— N^O> T-0 yy? yyF F21 -U AlX (^swX / A ( <NI5 c y ° r° v X ^9^0r Y y r° r° y °

[0042] In alternative aspects, the present disclosure provides methods of synthesizing a compound, for example a ketone, as described herein, by for example:10102-040W01providing an alkene, as described herein, and performing a 2+2 cycloaddition using a dichloroketene generated from the action of Zn-Cu on trichloroacetyl chloride or the action of base on dichloroacetyl chloride to yield a dichloroketone as described herein;reducing the dichloroketone, as described herein, to a ketone as described herein via a suitable method, for example, by using zinc and a proton source such as acetic acid or ammonium chloride; orproviding an alkene, as described herein, and performing a cycloaddition with a keteniminium salt, followed by hydrolysis to yield a ketone.

[0043] In some embodiments, present disclosure provides methods of performing:a reductive alkylation on the ketone as described herein to yield an amine as described herein;performing a 1,2 addition of a nucleophile on the ketone as described herein to yield an alcohol as described herein, which can be further elaborated via alkylation or acylation;performing a 2+2 heterocyclic cycloaddition on the alkene as described herein with for example chlorosulfonyl isocyanate (CSI) to yield a B-lactam which can be further elaborated via alkylation or acylation;performing condensation reactions or Wittig reactions with an appropriate hydroxylamine to form oxime ethers as described herein, or alkenes as described herein.

[0044] Accordingly, in some embodiments, there is provided a method of synthesizing a compound B, Ba, Bb, Be or Bd, the method comprising:i) providing a compound A, Aa, Ab, Ac, or Ad, as appropriate; andii) performing a cycloaddition reaction in the presence of a chloroketene to yield the compound B, Ba, Bb, Be or Bd, as described herein.

[0045] In some embodiments, there is provided a method of synthesizing a compound C, Ca, Cb, Cc or Cd, the method comprising:i) providing a compound B, Ba, Bb, Be or Bd, as appropriate; and

[0046] ii) reducing the compound B, Ba, Bb, Be or Bd in the presence of zinc and a proton source to yield the compound C, Ca, Cb, Cc or Cd.10102-040W01

[0047] The proton source may be without limitation acetic acid, ammonium chloride, etc.

[0048] Accordingly, in some embodiments, the present disclosure provides a method of synthesizing a compound as described herein, by providing a compound A:Awhere Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Rs can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 can be O, S or NX,and performing a cycloaddition reaction on the compound A as, for example, described herein in Scheme 1, where in compounds B and C:Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;10102-040W01Rs can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l, 2-dihydropyrimidine-l-yl group; andR4 can be O, S or NX.Scheme 1

[0049] In some embodiments, the present disclosure provides a method of synthesizing a compound as described herein, by providing a compound Aa:where R1 and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group; andRe can be O, NH or a benzoyl protected amine (NBz),10102-040W01and performing a cycloaddition reaction on Aa as, for example, described herein in Scheme 2a where, in Ba and Ca:R1 and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orRi can be a hydroxyl protecting group and R2 can be a reactive phosphorous group; andRe can be O, NH or a benzoyl protected amine (NBz).Aa Ba CaScheme 2a

[0050] Similarly, in alternative embodiments, the present disclosure provides a method of synthesizing a compound as described herein, as shown in Schemes 2b-2d.

[0051] In the compounds of Schemes 2b-2d,R1 and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; or10102-040W01Ri can be a hydroxyl protecting group and R2 can be a reactive phosphorous group; andRe, if present, can be O, NH or a benzoyl protected amine (NBz),R8, if present, can be H or an amino protecting group,R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.Ad Bd CdScheme 2d10102-040W01

[0052] In some embodiments, there is provided a method of synthesizing a compound C, Ca, Cb, Cc, or Cd, the method comprising:i) providing a compound A, Aa, Ab, Ac, or Ad, as appropriate; andii) performing a cycloaddition with a keteniminium salt to yield a compound Bx, Bxa, Bxb, Bxc, or Bxd followed by hydrolysis to yield the compound C, Ca, Cb, Cc, or Cd, as shown in Schemes 3 or 4a-d.

[0053] In Schemes 3 or 4a-d,Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group; R3, if present, can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4, if present, can be O, S or NX,Re, if present, can be O, NH or a benzoyl protected amine (NBz),R8, if present, can be H or an amino protecting group,R10 if present, can be H or an amino protecting group, andR11, if present, can be can be H or an amino protecting group.A Bx CScheme 310102-040W01Scheme 4aAd Bxd Cd Scheme 4d10102-040W01

[0054] By performing cycloaddition reactions on alkene compounds A, Aa, Ab, Ac, or Ad, as described herein, a spirocyclobutanone C, Ca, Cb, Cc, or Cd can be synthesized in a scale that allows the synthesis of further analogues via condensations, 1,2 additions or reductive aminations or a beta-lactam structure can be obtained via a heteroatom cycloaddition that can be further elaborated, for example as described herein.

[0055] Accordingly, in some embodiments, there is provided a method of synthesizing a compound of Formula IV, IVa, IVb, IVc or IVd, the method comprising:i) providing a compound C, Ca, Cb, Cc, or Cd, as appropriate, andii) performing reductive alkylation on the compound C, Ca, Cb, Cc, or Cd to yield the compound according to Formula IV, IVa, IVb, IVc or IVd, as shown in Schemes 5 or 6a-d.

[0056] In Schemes 5 or 6a-d,Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Rs, if present, can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4, if present, can be O, S or NX;Re, if present, can be O, NH or a benzoyl protected amine (NBz);R8, if present, can be H or an amino protecting group,R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group,Ji and J2, if present, can each independently be:10102-040W0110102-040W01where R9 can be:or; and, if present, can be “R” or “S” configuration.10102-040W014iC Formula IV Scheme 5Ca Formula IVa Scheme 6aJ2Cb Formula IVb Scheme 6b10102-040W01Cc Formula IVcScheme 6cCd Formula IVdScheme 6d

[0057] In some embodiments, there is provided a method of synthesizing a compound D, Da, Db, Dc or Dd, the method comprising:i) providing a compound C, Ca, Cb, Cc, or Cd, as appropriate; andii) performing a 1,2 nucleophile addition to yield the compound D, Da, Db, Dc or Dd, as shown in Schemes 7 or 8a-d.

[0058] In some embodiments, there is provided a method of synthesizing a compound of Formula V, Va, Vb, Vc, or Vd, the method comprising:i) providing a compound D, Da, Db, Dc or Dd, as appropriate; andii) alkylating the compound D, Da, Db, Dc or Dd to yield the compound of Formula V, Va, Vb, Vc, or Vd as shown in Schemes 7 or 8a-d.

[0059] In Schemes 7 or 8a-d,10102-040W01Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3, if present, can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4, if present, can be O, S or NX;Re, if present, can be O, NH or a benzoyl protected amine (NBz);R8, if present, can be H or an amino protecting group,R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group,X and Y, if present, can each independently be:10102-040W01 where R12can be:10102-040W01; and10102-040W01can, if present, be “R” or “S” configuration.Formula Va Scheme 8aScheme 8bCc Dc Formula Vc Scheme 8c10102-040W01HO Cd Dd Formula Vd Scheme 8d

[0060] In some embodiments, there is provided a method of synthesizing a compound E, Ea, Eb, Ec or Ed, the method comprising:i) providing a compound A, Aa, Ab, Ac, or Ad, as appropriate; andii) performing a heteroatom cycloaddition reaction in the presence of an isocyanate to yield the compound E, Ea, Eb, Ec or Ed, as shown in Schemes 9 or 10a-d.

[0061] The isocyanate may include without limitation chlorosulfonyl isocyanate (CSI).

[0062] In some embodiments, there is provided a method of synthesizing a compound of Formula VI, Via, Vlb, Vic, or Vid, the method comprising:i) providing a compound E, Ea, Eb, Ec or Ed, as appropriate; andii) alkylating or acylating the compound E, Ea, Eb, Ec or Ed to yield the compound of Formula VI, Via, Vlb, Vic, or Vid, as shown in Schemes 9 or 10a-d.

[0063] The alkylating agent may be without limitation an alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, arylalkyl, heteroarylalkyl, acyl, aminoalkyl, alkylthio or a protected version thereof with a covalently bonded leaving group (such as bromide, iodide, chloride, tosylate, mesylate, tritiate, etc.) to for example allow alkylation of a nucleophile. Suitable alkylating agents are described herein or known in the art.

[0064] The acylating agent may be without limitation an alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, arylalkyl, heteroarylalkyl, acyl, aminoalkyl, alkylthio or a protected version thereof with an activated carboxylic acid group to for example allow reaction with a nucleophile. Suitable acylating agents are described herein or known in the art.

[0065] In Schemes 9 or 10a-d,10102-040W01Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R3, if present, can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4, if present, can be O, S or NX;Re, if present, can be O, NH or a benzoyl protected amine (NBz);R8, if present, can be H or an amino protecting group,R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group,Ji and R5, if present, can each independently be:Owhere R12can be:10102-040W01NH10102-040W01where R9 can be:and, if present, can be “R” or “S” configuration.1) J1-LG 2) base, R5-LG E Formula VI Scheme 91) J1-LG 2) base, R5-LGFormula Via Scheme 10a10102-040W01Ab Eb Formula Vlb Scheme 10bAc Ec Formula Vic Scheme 10cAd Ed Formula Vid Scheme 10d

[0066] In some embodiments, there is provided a method of synthesizing a compound F, Fa, Fb, Fc or Fd, the method comprising:i) providing a compound C, Ca, Cb, Cc, or Cd, as appropriate; andii) performing a condensation reaction on the compound C, Ca, Cb, Cc, or Cd in the presence of a hydroxylamine to yield the compound F, Fa, Fb, Fc or Fd, as shown in Schemes 11 or 13a-d,10102-040W01

[0067] The hydroxylamine may be, without limitation, an alkyl, aryl, alkylaryl, acyl, amino alkoxy, heteroaryl heteroalkylaryl, alkenyl, alkynyl hydroxylamines or protected versions thereof, for example as described herein.

[0068] In some embodiments, there is provided a method of synthesizing a compound G, Ga, Gb, Gc, or Gd, the method comprising:i) providing a compound C, Ca, Cb, Cc, or Cd, as appropriate; andii) performing a Wittig reaction on the compound C, Ca, Cb, Cc, or Cd, to yield the compound G, Ga, Gb, Gc, or Gd, as shown in Schemes 12 or 14a-d.

[0069] In any one of Schemes 11, 12, 13a-d or14a-d,Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Rs, if present, can be an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4, if present, can be O, S or NX;Re, if present, can be O, NH or a benzoyl protected amine (NBz);R8, if present, can be H or an amino protecting group,R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group,Ji, if present, can be:10102-040W01Rl2q3 and q4, if present, can each independently be:orwhere R12can be:10102-040W01where R9 can be:10102-040W01⊕ PPh3Scheme 11 Scheme 12Cb Fb Cb Gb Scheme 13b Scheme 14b10102-040W01Cc Fc Cc Scheme 13c Gc Scheme 14cCd Cd Gd Scheme 13d Scheme 14d

[0070] In some embodiments, compounds as described herein can be prepared inaccordance with Scheme 15 as follows:Scheme 15whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or aninternucleoside linking group; orone of Ri or R2can be an internucleoside linking group and the other of Ri or R2canbe H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2can be H and the other of Ri or R2can be a hydroxyl protectinggroup; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz),10102-040W01R8, if present, can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0071] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 16 as follows:NR8NR,[F], solventOR20F— F OR20FScheme 16whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz), andRs, if present, can be H or an amino protecting group.

[0072] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 17 as follows:10102-040W01[F], solventScheme 17whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz),R8, if present, can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0073] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 18 as follows:NR NR0R200R20Scheme 18where10102-040W01Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz), andRs, if present, can be H or an amino protecting group.

[0074] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 19 as follows:Scheme 19whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz),10102-040W01R8, if present, can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0075] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 20 as follows:Scheme 20whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz),R8, if present, can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0076] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 21 as follows:10102-040W01Scheme 21whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz),R8, if present, can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0077] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 22 as follows:Scheme 2210102-040W01whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz),Rs, if present, can be H or an amino protecting group and*, if present, can be “R” or “S” configuration.

[0078] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 23 as follows:Scheme 23whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; or10102-040W01Ri can be a hydroxyl protecting group and R2 can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz),Rs, if present, can be H or an amino protecting group and*, if present, can be “R” or “S” configuration.

[0079] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 24 as follows:Scheme 24whereR1 and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;Re, if present, can be O, NH or a benzoyl protected amine (NBz),R8, if present, can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0080] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 25 as follows:10102-040W01Scheme 25whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0081] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 26 as follows:Scheme 26where10102-040W01Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R10if present, can be H or an amino protecting group, andR11, if present, can be can be H or an amino protecting group.

[0082] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 27 as follows:Scheme 27whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and10102-040W01*, if present, can be “R” or “S” configuration.

[0083] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 28 as follows:Scheme 28whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R if present, can be H or an amino protecting group,Rn, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0084] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 29 as follows:10102-040W01Scheme 29whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0085] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 30 as follows:Scheme 30where10102-040W01Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0086] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 31 as follows:Scheme 31whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; or10102-040W01Ri can be a hydroxyl protecting group and R2 can be a reactive phosphorous group;R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0087] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 32 as follows:HF2C NH2Scheme 32whereR1 and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0088] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 33 as follows:10102-040W01Scheme 33whereR1 and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0089] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 34 as follows:MeO X N‘Scheme 34whereR1 and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; or10102-040W01one of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0090] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 35 as follows:oScheme 35whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R8, if present, can be H or an amino protecting group,R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and, if present, can be “R” or “S” configuration.10102-040W01

[0091] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 36 as follows:[F], solventScheme 36whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R8, if present, can be H or an amino protecting group,R if present, can be H or an amino protecting group, andRn, if present, can be can be H or an amino protecting group.

[0092] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 37 as follows:Scheme 37where10102-040W01Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R8, if present, can be H or an amino protecting group,R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0093] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 38 as follows:Scheme 38whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;10102-040W01R8, if present, can be H or an amino protecting group,R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0094] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 39 as follows:Scheme 39whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R8, if present, can be H or an amino protecting group,R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0095] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 40 as follows:10102-040W01Scheme 40whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R8, if present, can be H or an amino protecting group,R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0096] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 41 as follows:10102-040W01[H]Scheme 41whereR₁ and R₂ can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of Ri or R2can be an internucleoside linking group and the other of Ri or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orRi can be a hydroxyl protecting group and R2 can be a reactive phosphorous group;R8, if present, can be H or an amino protecting group,R10 if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0097] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 42 as follows:10102-040W01oScheme 42whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R8, if present, can be H or an amino protecting group,R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0098] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 43 as follows:Scheme 43where10102-040W01Ri and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;R8, if present, can be H or an amino protecting group,R10if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group, and*, if present, can be “R” or “S” configuration.

[0099] In some embodiments, compounds as described herein can be prepared in accordance with Scheme 44 as follows:Scheme 44whereRi and R2can both be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2can be an internucleoside linking group and the other of R1or R2can be H, a terminal group, a linking group, or a conjugate group; orone of R1or R2can be H and the other of R1or R2can be a hydroxyl protecting group; orR1can be a hydroxyl protecting group and R2can be a reactive phosphorous group;10102-040W01R8, if present, can be H or an amino protecting group,R if present, can be H or an amino protecting group,R11, if present, can be can be H or an amino protecting group and*, if present, can be “R” or “S” configuration.

[0100] The alkylating agent may be without limitation an alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, arylalkyl, heteroarylalkyl, acyl, aminoalkyl, alkylthio or a protected version thereof with a covalently bonded leaving group (such as bromide, iodide, chloride, tosylate, mesylate, triflate, etc.) to for example allow alkylation of a nucleophile. Suitable alkylating agents are described herein or known in the art.

[0101] The acylating agent may be without limitation an alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, arylalkyl, heteroarylalkyl, acyl, aminoalkyl, alkylthio or a protected version thereof with an activated carboxylic acid group to for example allow reaction with a nucleophile. Suitable acylating agents are described herein or known in the art.

[0102] In some embodiments, a synthetic solid phase synthesis that utilizes phosphoramidites (P(lll) chemistry) as reactive phosphites may be used. The intermediate phosphite compounds are subsequently oxidized to the P(V) state using known methods to yield, in certain embodiments, phosphodiester or phosphorothioate internucleotide linkages.

[0103] The compounds in accordance with the present disclosure, for example, any one or more of Formula I, II, III, IVa, IVb, IVc, IVd, V, Va, Vb, Vc, Vd, VI, Via, Vlb, Vic, Vidor VII;Compound B, Ba, Bb, Be, Bd, Bx, Bxa, Bxb, Bxc, Bxd, C, Ca, Cb, Cc, Cd, D, Da, Db, De, Dd, E, Ea, Eb, Ec, Ed, F, Fa, Fb, Fc, Fd, G, Ga, Gb, Gc, or Gd; or as listed in Table 1, or as shown in the Schemes and Examples herein, may form a nucleic acid molecule, such as an oligomer or a polymer, with each other or with naturally occurring nucleotides or other, known NAs, when one of Ri or R2 is an internucleoside linking group and the other of R1 or R2 is a terminal group, a linking group, or a conjugate group. It is to be understood that a protecting group, active phosphorus group, or similar group in a compound, as disclosed herein, may be modified as necessary to allow for a suitable use, such as incorporation into an oligomer or a polymer, using standard procedures. For example, the protecting group, active phosphorus group, or similar group can be removed or used in oligonucleotide coupling techniques using standard procedures, as known in the art.

[0104] In some embodiments, an oligonucleotide in accordance with the present disclosure may be:10102-040W01CB050CB05410102-040W01

[0105] Accordingly, in an alternative aspect, there is provided a method of using a compound as described herein in the preparation of a nucleic acid molecule, such as a polymer, for example, vaccine mRNA, or an oligomer, for example, an antisense oligomer, by: combining at least one compound, as disclosed herein with one or more nucleotides or NAs, under suitable synthetic conditions.

[0106] In some embodiments, the compound includes an internucleoside linking group. In some embodiments, the compound includes a lipid group, an aromatic group or a charged moiety or a combination thereof. In alternative embodiments, the suitable synthetic conditions may include conditions suitable for reaction of an internucleoside linking group to form an oligomer or a polymer Suitable synthetic conditions for the preparation of nucleic acid molecules are known in the art.

[0107] A “nucleoside” is a glycosylamine having a nitrogenous base or “nucleobase” or “NB” and a sugar ring e.g., ribose or deoxyribose) containing oxygen, in which the anomeric carbon is linked through a glycosidic bond to the N9 of a purine e.g., adenine or guanine) or the N1 of a pyrimidine e.g., cytosine, thymine, or uracil). Nucleosides include both L- and D-nucleoside isomers. Examples of naturally occurring nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine.

[0108] Nucleoside analogues (NAs) are compounds that are structurally similar to naturally occurring nucleosides. NAs may include, without limitation, compounds with modifications or “functionalizations” at positions CT, C2', C3', C4' and / or C5' of the sugar ring, relative to a naturally occurring nucleoside. In some embodiments, NAs may exist as a free triol or may be phosphorylated at C3' and / or C5'. In some embodiments, NAs may include, without limitation, compounds with a saturated or unsaturated carbocyclic ring. In some embodiments, NAs may include nitrogen or sulphur in the sugar ring, for example as a replacement for the naturally occurring oxygen, and / or may include N-R groups, where R may be without limitation alkyl, allyl, alkynyl or benzyl. NAs may include “locked nucleic acids” or “LNAs” which are RNA derivatives in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. In some embodiments, the nucleoside analogues disclosed herein may be modified to function as a phosphoramidate or phosphonamidate compound, e.g., a “ProTide,” which includes a 5'-nucleoside monophosphate in which the two hydroxyl groups are masked with an amino acid ester and an aryloxy component which can be enzymatically metabolized to deliver free 5'-monophosphate, which is further transformed to the active 5'-triphosphate form of the nucleoside analogue, once delivered to a cell. The nucleobase of NAs (“NB”) may be any aryl or heteroaryl attached from the C1 position to a carbon or nitrogen atom. NBs may also10102-040W01be modified, for example, may be 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, 5,5,5-trifluoromethylthymine, 5-fluorouracil, 2-thiouracil, 4-methylbenzimidazole, hypoxanthine, 7-deazaguanine, 7-deazaadenine, indole, imidazole, triazole, pyrrole, pyrazole, etc.

[0109] Nucleosides and NAs may form nucleic acid molecules. The terms “nucleic acid” or “nucleic acid molecule” encompass both RNA (plus and minus strands) and DNA, including cDNA and synthetic (e.g., chemically synthesized) DNA. The nucleic acid molecule may be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By “RNA” is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. One example of a modified RNA included within this term is phosphorothioate RNA. By “DNA” is meant a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides. By “cDNA” is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Thus a “cDNA clone” means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector. By “complementary” is meant that two nucleic acids, e.g., DNA or RNA, contain a sufficient number of nucleotides which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acids. Thus, adenine in one strand of DNA or RNA pairs with thymine in an opposing complementary DNA strand or with uracil in an opposing complementary RNA strand. It will be understood that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. A nucleic acid molecule is “complementary” to another nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule.

[0110] A nucleic acid molecule may be an oligomeric compound (“oligomer”) or a polymeric compound (“polymer”) which, as used herein, include repeating units of nucleosides and NAs linked through their 5'- and 3'- terminal groups. It is to be understood that an oligomer or a polymer: may include naturally occurring nucleosides and NAs; may be DNA and / or RNA; and / or may be single stranded or double stranded. It is also to be understood that an oligomer or a polymer, as described herein, is not limited by length and can be of any suitable size, for example, depending on use. It is further to be understood that a compound in accordance with the present disclosure may be incorporated into an oligomer or a polymer, as described herein, irrespective of the length of the oligomer or a polymer.10102-040W01

[0111] An “oligomer,” as used herein, refers to a molecule having a relatively small number of repeating units, for example, 2 to 49, or for example, 8 to 30, or any integer in between. Oligomers can include DNA or RNA or analogs thereof and can include without limitation short interfering RNA (siRNA), microRNA (miRNA), inhibitory antisense oligonucleotides (ASOs), small activating RNA (saRNA), DNA-RNA hybrids, etc. Oligomers can be single stranded or double stranded.

[0112] A “polymer,” as used herein, refers to a molecule having a relatively large number of repeating units, for example, 50 to 9000, or any integer in between, or more than 9000. Polymers can include DNA or RNA or analogs thereof and can include without limitation messenger RNA (mRNA), cDNA, DNA-RNA hybrids, etc. Polymers can be single stranded or double stranded.

[0113] An “internucleoside linking group” is a phosphate-containing group that allows the linkage of multiple nucleotides to form nucleic acid molecules, such as oligomers and polymers. An internucleoside linking group may be an amide group, a phosphoryl guanidine group, a phosphoryl amidate group, such as a mesyl phosphoramidate group, a phosphodiester or a phosphorothioate. In some embodiments, an internucleoside linking group may be a phosphorothioate.

[0114] A “linking group,” as used herein, is a group that can attach two or more groups, such as an oligomer or polymer and a chemical functional group or a conjugate group. In certain embodiments, a linking group is a group that can attach two or more groups, such as an oligomer and a chemical functional group or a conjugate group. A linking group can be a bifunctional linking group including a hydrocarbyl moiety having two functional groups. In such a bifunctional linking group, one of the functional groups is selected to bind to an oligomer or polymer and the other is selected to bind to another any selected group such as a chemical functional group or a conjugate group or another oligomer or polymer, which may be the same or may be different. In certain embodiments, multivalent oligomers and / or polymers may be connected by a linking group. In alternative embodiments, multivalent oligomers may be connected by a linking group. In certain embodiments, the linking group may include repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in bifunctional linking groups include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like. Some nonlimiting examples of bifunctional linkingmoieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC) and 6-10102-040W01aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C₁-C₁₀ alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0115] The term "terminal group," as used herein is meant a group that can be attached to one or both of the termini (3'- and / or 5'-) of an oligomeric or a polymeric nucleic acid molecule. A terminal group includes without limitation, a conjugate group (for example, that is positioned at one or both termini), a linking group (for example, that is positioned at one or both termini), a phosphate moiety that is non-reactive ( / .e., not a reactive phosphate group as described herein) and non-internucleoside phosphate ( / .e., not capable of linking nucleosides), a 3’- hydroxyl, a 5’- hydroxyl, or a protecting group.

[0116] Terminal groups are non-labile chemical moieties that can be used for various purposes such as enabling the tracking and / or isolation of the oligomeric compound (a fluorescent label or other reporter group), or attenuating or halting the reactivity of the 3’ or 5’ terminus of the oligomer. A terminal group can include without limitation biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, dyes, etc.

[0117] A “5'-terminal group,” as used herein, is a hydroxyl group linked to the 5’ carbon (C5') of the sugar ring of a nucleoside or NA.

[0118] A “3'-terminal group” is a hydroxyl group linked to the 3’ carbon (C3') of the sugar ring of a nucleoside or NA.

[0119] A “conjugate group,” as used herein, refers to a group that is covalently attached to an oligomeric compound and modify one or more properties of the oligomeric compound including but not limited to pharmakodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance properties. A conjugate group may be linked directly or via an optional linking moiety or linking group to the oligomeric compound. Conjugate groups may include, without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, etc.

[0120] A “protecting group” as used herein, means is a labile chemical moiety i.e., a reversibly formed derivative of an existing functional group in a molecule that is temporarily attached to decrease reactivity such that the protected functional group does not react under10102-040W01synthetic conditions to which the molecule is subjected in one or more subsequent steps. A protecting group may be used selectively and / or orthogonally to protect sites during reactions at other reactive sites and may then be removed to leave the unprotected group as is or be available for further reactions. A protecting group includes, without limitation, hydroxyl groups, amino groups, thiol groups, benzyl (Bn) groups, benzoyl (Bz) groups, t-butyl carbamate (Boc) groups, benzyl carbamate (CBZ) groups, p-methoxybenzyl (PMB) groups, substituted silanes (TMS, TBS, TBDMS, TIPS), acetyl (Ac) groups, benzyloxymethyl (BOM) groups, methyloxymethyl (MOM) groups, tetrahydropyranyl (THP) groups, trityl (Tr) groups, dimethoxytrityl (DMT) groups, pivaloyl (Piv) groups, t-butyl ether (tBu) groups, tosyl (Ts) groups, allyl (Al) groups, benzyl esters, t-butyl esters or dimethyl acetonide groups.Protecting groups are as described herein or as known in the art. Protecting groups as known in the art are described generally in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999. Also included are those protecting groups adapted for nucleoside and nucleotide chemistry as described in Chapter 2, Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06 / 2012. It is to be understood that a person skilled in the art will readily be able to determine a suitable protecting group for a particular synthesis.

[0121] Exemplary amino protecting groups include without limitation methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2- trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1 -methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1, 1 -dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1 -methyl-1 -(4-biphenylyl)ethyl carbamate (Bpoc), 1 -(3, 5-di-t-butylphenyl)-1 -methylethyl carbamate (t-Bumeoc), 2-(2'-and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N, N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1 -isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4- dimethylthiophenyl carbamate (Bmpc), 2-10102-040W01phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N'-p-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclo hexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycar bonylvinyl carbamate, o-(N, N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N, N-dimethylcarboxamido) propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2- pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1 -methylcyclobutyl carbamate, 1 -methylcyclohexyl carbamate, 1-methyl-1 -cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1 -methyl -1 -phenylethyl carbamate, 1 -methyl -1 -(4-pyridyl) ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethyl ammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloro acetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino) acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1, 1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-trimethylsilyl)ethoxy]methylamine (SEM), N-3- acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3- pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5- dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fem), N-2-picolylamino N'-oxide, N-1, 1 -dimethylthiomethyleneamine, N-benzylideneamine, N-p-10102-040W01methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesi-tyl]methyleneamine, N-(N', N' dimethylaminomethylene) amine, N, N'-isopropylidenediamine, N-p- nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5- dimethyl -3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl (pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxy benzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimeth ylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracene-sulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzene sulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, or phenacylsulfonamide.

[0122] A “hydroxyl protecting group,” includes, without limitation, a suitable ether, for example, a phenylmethyl or trimethylsilyl ether; a suitable ester, or a suitable acetal. A hydroxyl protecting group includes, without limitation, acetonide, silyl protecting group, alkyl protecting group or aryl protecting group (including cyclic or acyclic), such as a silyl ether for example, tert-butyl(dimethyl)s / 7y / (TBS), triisopropylsilyl (TIPS), trimethylsilyl (TMS), tertbutyldiphenylsilyl (TBDPS), triethylsilyl (TES), 2-(trimethylsilyl)ethoxymethyl ether (SEM), etc.; acetate (Ac), pivalate (Piv) or other ester or carbonate protecting group etc.; benzyl, allyl, methoxymethyl or p-methoxybenzyl (PMB) or other ether protecting group, etc.; tetrahydropyranyl (THP) ether; methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphe noxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxym ethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl) ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1 -methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetra hydrothiopyranyl S, S-dioxide, 1-[(2-chloro-4-10102-040W01methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2, 3, 3a, 4, 5, 6, 7, 7a octahydro-7, 8, 8-trimethyl-4,7-methanobenzofuran-2-yl, 1 -ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1 -methoxyethyl, 1-methyl-1-benzyloxyethyl, 1 -methyl -1 -ben zyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl) diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris (benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1, 1 -bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S, S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkylmethyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-di methoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzene sulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthio methoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkyl N, N, N', N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and10102-040W01tosylate (Ts). For protecting 1,2- or 1,3- diols, the protecting groups include methylene acetal, ethylidene acetal, 1 -t-butylethylidene ketal, 1 -phenylethyl idene ketal, (4-methoxyphenyl) ethylidene acetal, 2,2,2- trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, ben zylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, Ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1 -ethoxyethylidine orthoester, 1,2-dimethoxyethylidene orthoester, a-methoxybenzylidene orthoester, 1-(N, N-dimethylamino)ethylidenederivative, a-(N, N'-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, or phenyl boronate.

[0123] Halogens include bromine, chlorine, fluorine, iodine, etc. “Halo” refers to halogen groups such as bromo, chloro, fluoro, iodo, etc.

[0124] A “reactive phosphorus group,” as used herein, is meant a group that permits formation of an internucleoside linking group. A reactive phosphorus group includes unstable phosphorus atoms, for example in P(lll) or P(V) valence state, as known in the art. A reactive phosphorus group includes, without limitation, an orthogonal protecting group, orthophosphate, phosphoramidite, H-phosphonate, phosphate triesters, or phosphorus containing chiral auxiliaries, or any other reactive phosphorus group as known in the art. For example, reactive phosphates and phosphites are disclosed in Beaucage and Iyer (38), Huang et al. (39).

[0125] “Alkyl” or “lower alkyl” refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing no unsaturation and including, for example, from one to twelve carbon atoms, or any value in between, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, and which is attached to the rest of the molecule by a single bond. In some embodiments, alkyl may refer to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing no unsaturation and including from one to six carbon atoms, or any value in between, such as 1, 2, 3, 4, 5, or 6 carbon atoms, and which is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, the alkyl group may be optionally substituted by one or more substituents as described herein. Unless stated otherwise specifically herein, it is understood that the substitution can occur on any carbon of the alkyl group.10102-040W01

[0126] “Alkenyl” or “lower alkenyl” as used herein refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing at least one double bond and including, for example, from two to twelve carbon atoms, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, and which is attached to the rest of the molecule by a single bond. In alternative embodiments, the alkenyl group may contain from two to eight carbon atoms, such as 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In alternative embodiments, the alkenyl group may contain from three to six carbon atoms, such as 3, 4, 5, or 6 carbon atoms. Unless stated otherwise specifically in the specification, the alkenyl group may be optionally substituted by one or more substituents as described herein. Unless stated otherwise specifically herein, it is understood that the substitution can occur on any carbon of the alkenyl group.

[0127] “Alkynyl” or “lower alkynyl” as used herein refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing at least one triple bond and including, for example, from two to twelve carbon atoms, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, and which is attached to the rest of the molecule by a single bond. In alternative embodiments, the alkynyl group may contain from two to eight carbon atoms, such as 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In alternative embodiments, the alkynyl group may contain from three to six carbon atoms, such as 3, 4, 5, or 6 carbon atoms. Unless stated otherwise specifically in the specification, the alkynyl group may be optionally substituted by one or more substituents as described herein.

[0128] “Alkoxy” as used herein, 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 e.g., a group of the formula -ORa, where Ramay be alkyl, alkenyl, alkynyl, etc. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Unless stated otherwise specifically herein, the term “alkoxy” is meant to include alkoxy groups optionally substituted by one or more substituents as described herein.

[0129] By “aryl” is meant a monocyclic or bicyclic aromatic ring containing only carbon atoms, including for example, 5-14 members, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 members. Examples of aryl groups include phenyl, biphenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-benzodioxanyl, and the like. Unless stated otherwise specifically herein, the term “aryl” is meant to include aryl groups optionally substituted by one or more substituents as described herein.10102-040W01

[0130] By “heteroaryl” or "heteroaromatic” is meant a radical including a mono- or polycyclic aromatic ring, ring system or fused ring system where at least one of the rings is aromatic and includes one or more heteroatom, for example sulfur, nitrogen or oxygen, and is unsaturated, partially saturated or fully saturated, thus including heteroaryl group.Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain at least one heteroatom and the other ring(s) can contain one or more heteroatoms or optionally contain no heteroatoms. Accordingly, a heteroaryl group includes a single or fused aromatic ring group containing one or more heteroatoms in the ring, for example sulfur, nitrogen or oxygen, and including for example, 5-14 members, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 members. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Examples of heteroaryl groups include, without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolyl, oxazolyl, isooxazolyl, isothiazolyl, thiadiazolyl e.g., 1,3,4-thiadiazolyl, oxadiazolyl e.g., 1,2,3-oxadiazolyl, triazolyl e.g., 1,2,3-triazolyl or 1,2,4-triazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, tetrazolyl, pyridazinyl, 2,6-dichloropyrimidinyl, 1,3,5-triazinyl, benzothiazolyl, indolizinyl, indolyl, isoindolyl, benzofuranyl, benzothiophenyl, 1 H-indazolyl, purinyl, 4H-quinolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, 1,8-naphthyridinyl, pteridinyl, uracilyl, thyminyl, deazadeninyl, phthalimideyl, adeninyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, [1,3]dioxolane, and the like. Unless stated otherwise specifically herein, the term “heteroaryl” is meant to include heteroaryl groups optionally substituted by one or more substituents as described herein.

[0131] “Arylalkyl " or "aralkyl," as used herein, refers to a radical formed between an C1-C6 alkyl group and an aryl group wherein the alkyl group is used to attach the aralkyl group to a parent molecule. Examples include, but are not limited to, 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. Accordingly, unless stated otherwise specifically herein, the term “arylalkyl” or "aralkyl” is meant to include “arylalkyl” or "aralkyl” groups optionally substituted by one or more substituents as described herein.

[0132] By "heteroarylalkyl," is meant a heteroaryl group as indicated herein and having an alky radical that can attach the heteroarylalkyl group to a parent molecule. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl, and the like.10102-040W01Heteroarylalkyl groups as used herein may optionally include further substituent groups on one or both of the heteroaryl or alkyl portions.

[0133] By “acyl,” as used herein, refers to a radical formed by removal of a hydroxyl group from an organic acid and is meant a group having the structure C(O) Rb, where Rb may be aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Unless stated otherwise specifically herein, the term “acyl” is meant to include acyl groups optionally substituted by one or more substituents as described herein.

[0134] By “aminoalkyl,” as used herein, refers to an amino substituted alkyl radical. This term is meant to include C1-C6 alkyl groups having an amino substituent at any position and where the alkyl group attaches the aminoalkyl group to the parent molecule. The alkyl and / or amino portions of the aminoalkyl group can be further optionally substituted by one or more substituents as described herein.

[0135] By “alkylthio,” as used herein, refers to a thio substituted alkyl radical. This term is meant to include C1-C6 alkyl groups having an thiol substituent at any position and where the alkyl group attaches the alkylthio group to the parent molecule. The alkyl and / or thiol portions of the alkylthio group can be further optionally substituted by one or more substituents as described herein.

[0136] “Optional” or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs one or more times and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl group may or may not be substituted and that the description includes both substituted alkyl groups and alkyl groups having no substitution, and that the alkyl groups may be substituted one or more times.Similarly, “optionally substituted alkenyl” means that the alkenyl group may or may not be substituted and that the description includes both substituted alkenyl groups and alkenyl groups having no substitution, and that the alkenyl groups may be substituted one or more times; “optionally substituted alkynyl” means that the alkynyl group may or may not be substituted and that the description includes both substituted alkynyl groups and alkynyl groups having no substitution, and that the alkynyl groups may be substituted one or more times; and “optionally substituted alkoxy” means that the alkoxy group may or may not be substituted and that the description includes both substituted alkoxy groups and alkoxy groups having no substitution, and that the alkoxy groups may be substituted one or more times.10102-040W01

[0137] Accordingly, an “optionally substituted” purine-9-yl group or 2-oxo-l,2-dihydropyrimidine-l-yl group, may or may not be substituted and therefore includes both a substituted purine-9-yl group or a substituted 2-oxo-l,2-dihydropyrimidine-l-yl group and a purine-9-yl group or 2-oxo-l,2-dihydropyrimidine-l-yl group having no substitution. A purine-9-yl group or 2-oxo-l,2-dihydropyrimidine-l-yl group may be substituted with one or more of the following substituents: a hydroxyl group, a hydroxyl group protected by a protective group for nucleic acid synthesis, O, NH, a Ci to Ce linear alkyl group, a Ci to Ce linear alkoxy group, a mercapto group, a mercapto group protected by a protective group for nucleic acid synthesis, a Ci to Ce linear alkylthio groups, an amino group, a Ci to Ce linear alkylamino group, an amino group protected by a protective group for nucleic acid synthesis, a benzoyl protected amine (NBz), for example, methyl cytidine, or a halogen atom.

[0138] Similarly, a Ci-Ce alkyl, C2-C6 alkenyl, C2-Cealkynyl, Ci-Ce acyl, Ci-Ce aminoalkyl, Ci-Ce alkoxy, alkylaryl, or aryl group may be substituted with one or more of the following substituents: halogen, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, OJ1, SJ1, NJ1J2, N3, CN, C(=O)OJ1, C(=O)NJIJ2, C(=O)JI, O-C(=O)NJIJ2, N(H)C(=O)NJIJ2, N(H)C(=NH)NJIJ2, or N(H)C(=S)NJIJ2, or C(=N-OJi).

[0139] An optional substituent may be a suitable substituent. As used herein, the term "suitable substituent" means a chemically and pharmaceutically acceptable group, i.e., a moiety that does not significantly interfere with the preparation of or negate the efficacy of the compounds as described herein. Such suitable substituents may be routinely chosen by those skilled in the art. Suitable substituents may be selected from the group consisting of halo, Ci-Ce alkyl, C2-C6 alkenyl, Ci-Ce haloalkyl, Ci-Ce alkoxy, Ci-Ce haloalkoxy, C2-C6 alkynyl, C3-C6 cycloalkenyl, (C3-C6 cycloalkyl)Ci-C6 alkyl, (C3-C6 cycloalkyl)C2-C6 alkenyl, (C3-Ce cycloalkyl)Ci-C6 alkoxy, C3-C6 heterocycloalkyl, (C3-C6 heterocycloalkyl) Ci-Ce alkyl, (C3-Ce heterocycloalkyl)C2-C6 alkenyl, (C3-C6 heterocycloalkyl)Ci-C6 alkoxy, hydroxy, carboxy, oxo, sulfanyl, Ci-Ce alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl, heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, Ci-Ce alkylamino, di-(Ci-Ce alkyl)amino, carbamoyl, (Ci-Ce alkyl)carbonyl, (Ci-Ce alkoxy)carbonyl, (Ci-Ce alkyl)aminocarbonyl, di-( Ci-Ce alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (Ci-Ce alkyl)sulfonyl, and arylsulfonyl.

[0140] In some embodiments, an optionally substituted Ci-Ce alkyl group may include, without limitation: monovalent saturated aliphatic hydrocarbon radical having the indicated number of carbon atoms. The radical may be a linear or branched chain and, where specified, optionally substituted with one to three suitable substituents as defined above. Illustrative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-10102-040W01butyl, n-pentyl, n- hexyl, isopropyl, isobutyl, isopentyl, amyl, sec-butyl, tert-butyl, tertpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl and the like. In some embodiments, alkyl groups include without limitation methyl, ethyl, n-propyl and isopropyl. In some embodiments, suitable substituents include without limitation halo, methoxy, ethoxy, cyano, nitro and amino. In some embodiments, an optionally substituted Ci-Ce alkyl group may include, without limitation:

[0141] In some embodiments, an optionally substituted Ci-Ce alkenyl group may include, without limitation: a monovalent aliphatic hydrocarbon radical having the indicated number of carbon atoms and at least one carbon-carbon double bond. The radical may be a linear or branched chain, in the E or Z form, and where specified, optionally substituted with one to three suitable substituents as defined above. Illustrative examples of alkenyl groups include, but are not limited to, vinyl, 1 -propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2- butenyl, isobutenyl, 2-methyl-1 -propenyl, 1 -pentenyl, 2-pentenyl, 4-methyl-2-pentenyl, 1,3-pentadienyl, 2,4-pentadienyl, 1,3-butadienyl and the like. In some embodiments, alkenyl groups include without limitation vinyl, 1 -propenyl and 2-propenyl. In some embodiments, suitable substituents include without limitation halo, methoxy, ethoxy, cyano, nitro and amino.

[0142] In some embodiments, an optionally substituted Ci-C6alkynyl group may include, without limitation: a monovalent aliphatic hydrocarbon radical having the indicated number of carbon atoms and at least one carbon-carbon triple bond. The radical may be a linear or branched chain and, where specified, optionally substituted with one to three suitable substituents as defined above. Illustrative examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1 -butynyl, 2-butynyl, 1 -pentynyl, 2- pentynyl, 3-methyl-1 -pentynyl, 3-pentynyl, 1 -hexynyl, 2-hexynyl, 3-hexynyl and the like. In some embodiments, alkynyl groups include without limitation ethynyl, 1-propynyl and 2-propynyl. In some embodiments, suitable substituents include without limitation halo, methoxy, ethoxy, cyano, nitro and amino.

[0143] In some embodiments, an optionally substituted Ci-Ce acyl group may include, without limitation an aliphatic radical of the form alkyl-C(O)-, where alkyl is as described10102-040W01herein. In some embodiments, an optionally substituted Ci-Ce acyl group may include, without limitation:

[0144] In some embodiments, an optionally substituted Ci-Ce aminoalkyl group may include, without limitation an aliphatic radical of the form alkyl-N-, where alkyl is as described herein. In some embodiments, an optionally substituted Ci-Ce aminoalkyl group may include, without limitation:

[0145] In some embodiments, an optionally substituted Ci-Ce alkoxy group may include, without limitation an aliphatic radical of the form alkyl-O-, where alkyl is as described herein.10102-040W01Illustrative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, neopentoxy, tertiary pentoxy, hexoxy, isohexoxy, heptoxy, octoxy and the like. In some embodiments, alkoxy groups include methoxy or ethoxy. In some embodiments, an optionally substituted Ci-Ce alkoxy group may include, without limitation:

[0146] In some embodiments, an optionally substituted alkylaryl group may include, without limitation an alkyl radical of one to six carbons as described herein substituted with an aryl group as described herein. In some embodiments, an optionally substituted alkylaryl group may include, without limitation:

[0147] In some embodiments, an optionally substituted aryl group may include, without limitation a monovalent aromatic hydrocarbon radical having five to ten carbon atoms forming a carbocyclic ring and, where specified, optionally substituted with one to three suitable substituents as defined above. Illustrative examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and the like. In some embodiments, an optionally substituted aryl group may be phenyl or naphthyl, optionally mono- or disubstituted by identical or different suitable substituents selected from halo, cyano, Ci-Ce alkyl, C3-C6 cycloalkyl, difluoromethyl, trifluoromethyl, C1-C3 alkoxy, difluoromethoxy or trifluoromethoxy.

[0148] In some embodiments, a compound as disclosed herein may be useful in modulating gene expression pathways, including those relying on mechanisms of action such10102-040W01as RNaseH, siRNA and dsRNA enzymes, as well as other antisense mechanisms based on target degradation or target occupancy.

[0149] In some embodiments, a compound as disclosed herein may be useful in preparing an antisense compound i.e., a single stranded oligonucleotide having a sequence that is complementary to a target single stranded DNA molecule or to a target RNA molecule, such as mRNA, viral RNA, or other RNA species, and that inhibits the function of the target molecule after sequence-specific binding.

[0150] In some embodiments, a compound as disclosed herein may be useful in preparing a vaccine.

[0151] In some embodiments, a compound as disclosed herein may be useful as a tool for drug design.

[0152] In some embodiments, the compounds disclosed herein may be used as small molecule therapeutics or as monomers in oligonucleotide therapeutics.

[0153] In some embodiments, the methods disclosed herein may be useful in the preparation of diversity libraries. For example, larger collections of tricyclic nucleic acids e.g., focused screening libraries) can be generated using the methods described herein.

[0154] As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. For example, “a compound” refers to one or more of such compounds. Throughout this application, it is contemplated that the term “compound” or “compounds” refers to the compounds discussed herein and includes precursors and derivatives of the compounds. The compounds of the present invention may contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers.Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and it is intended that all of the possible optical isomers and diastereomers in mixtures and as pure or partially purified compounds are included within the ambit of this invention, unless specifically indicated otherwise. An asterisk (*) in a Formula or compound described herein denotes “R” or “S” configuration. Any formulas, structures or names of compounds described in this specification that do not specify a particular stereochemistry are meant to encompass any and all existing isomers as described above and mixtures thereof in any proportion. When stereochemistry is specified, the invention is meant to encompass that particular isomer in pure form or as part of a mixture with other isomers in any10102-040W01proportion. Single enantiomers, i.e., optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemates. Resolution of the racemates can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent; chromatography, using, for example a chiral HPLC column; or derivatizing the racemic mixture with a resolving reagent to generate diastereomers, separating the diastereomers via chromatography, and removing the resolving agent to generate the original compound in enantiomerically enriched form. These procedures can be repeated, if desired, to increase the enantiomeric purity of a compound. When the compounds described herein contain olefmic double bonds or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include the cis, trans, Z- and E- configurations. Likewise, all tautomeric forms are also intended to be included.

[0155] The starting materials can be obtained from commercial sources, prepared from commercially available organic compounds, prepared using known synthetic methods.

[0156] The present invention will be further illustrated in the following examples.

[0157] Examples:

[0158] General Procedure A. Reductive amination of cyclobutanone 1

[0159] To a rt, stirred solution of cyclobutanone 1 (1 eq., 0.14 mmol) in THF (0.14 M) was added in sequence the appropriate amine (2.2 eq.), AcOH (2.0 eq.), NaBH(OAc)3(3.0 eq). The resultant suspension was then stirred overnight for 16-22 h. After this time, the reaction mixture was quenched by addition of saturated aqueous NaHCO3(1.5 mL). The aqueous layer was extracted with EtOAc (4x 1.5 mL), dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude products were purified on SiC>2 via flash column chromatography. Appropriate fractions were pooled, and solvent was removed in vacuo to furnish the pure products as a mixture of diastereomers, which were used immediately in the subsequent step.

[0160] General Procedure B. Acetonide deprotection of substituted cyclobutane analogues

[0161] To a rt, stirred solution of the appropriate cyclobutane analogue (1 eq., mixture of diastereomers) in MeCN and H2O (8:3 v / v), was added neat TFA (1.2 eq.) and the reaction mixture was allowed to stir for 1-3 h. After this time, starting material was consumed as monitored by TLC analysis and the reaction mixture was then evaporated to dryness and the10102-040W01residue was co-evaporated from MeOH (3x 7 mL). The products were isolated in high to quantitative yields as diastereomeric mixtures that were typically unresolvable by conventional SiC>2 chromatography. A portion of each mixture was purified via HPLC prior to acquiring analytical characterization data on each diastereomer. Thus, the yields refer to post-workup mixtures of diastereomers. However, in cases where the entire batch of material was purified via HPLC, a post-HPLC yield is also included.

[0162] Example 1Zn-Cu, THF rt2

[0163] Synthesis of 2:

[0164] Alkene 1 (30) (495 mg, 1.54 mmol) was dissolved in 30 mL dry THF, and zinc-copper couple (706 mg, 10.78 mmol, 7 eq) was added. Trichloroacetylchloride (344 uL, 3.08 mmol, 2 eq) was then added slowly so that the solution temperature did not exceed 50 oC. After ~1 h TLC analysis indicated that the starting material was consumed. The reaction mixture was filtered through celite and washed with EtOAc. Satd. NaHCO3 was then added and the biphasic mixture was stirred vigorously, resulting in a precipitate. The biphasic mixture was filtered through celite and washed with EtOAc. The mixture was transferred to a separatory funnel and the layers separated. The aqueous layer was saturated with NaCI and extracted a second time with EtOAc. The combined organic layers were dried over Na2SO4 and concentrated to yield 844 mg of an orange solid that was used without further purification.

[0165] Synthesis of 3:

[0166] Crude 2 (1.54 mmol assumed) was dissolved in 25 mL dry THF. Zn powder (1.00g, 15.4 mmol, 10 eq) and HOAc (1.32 mL, 23.1 mmol, 15 eq) were added, and the resulting mixture was heated to 50 oC. After ~6-8h, the starting material was consumed as evidenced by TLC analysis. The reaction mixture was then filtered through celite and washed with EtOAc. The organic layer was washed with satd. NaHCO3, dried over Na2SO410102-040W01and concentrated to yield crude 3, which was purified by column chromatography to yield ketone 3 (390 mg, 1.07 mmol, 70 % over 2 steps) as a light yellow solid.

[0167] Data for 3:1H NMR (400 MHz CDCl3) 58.99 (s, 1H), 7.26 (d, J = 1.3 Hz, 1H), 5.5 (s, 1 H), 4.82 (s, 1 H), 4.47 (d, J = 11.8 Hz, 1 H), 4.18 (d, J = 11.8 Hz, 1 H), 4.04 (m, 1 H), 3.93 (s, 1 H), 3.54 (m, 1 H), 3.30 (m, 2H), 1.99 (d, J = 1.3 Hz, 3H), 1.59 (s, 3H), 1.47 (s, 3H). ms (ESI, +ve mode): calcd: 364.2, found 365.2.

[0168] Synthesis of 4:

[0169] Ketone 3 (29.0 mg, 0.067 mmol) was dissolved in MeOH and the solution was cooled to -78oC. NaBH4 (3 mg, 0.081 mmol, 1.2 eq) was added, and the solution was stirred at -78 oC for 45 min. At this point, the reaction was quenched cold with satd. NH4CI. EtOAc was added and the layers were partitioned. The organic layer was dried over Na2SO4, concentrated and purified by column chromatography to yield 4 (13.1 mg, 53 %) as a 2:1 mixture of compounds.major diastereomer minor diastereomer

[0170] Synthesis of 4a and 4b:

[0171] To a cold (0 degrees C), stirred solution of cyclobutanone 3 (410 mg, 1 Eq, 1.13 mmol) in MeOH (11.3 mL) was added NaBH4 (85.1 mg, 2 Eq, 2.25 mmol) and the reaction was allowed to proceed for 20 min at 0 °C. After this time, the reaction mixture was10102-040W01quenched with sat. aq. NH4CI (10 mL). After effervescence had subsided (~10 min) the biphasic mixture was transferred to a separatory funnel and the aqueous layer was extracted with EtOAc (3x 30 mL). The combined organic layers were dried (Na2SO4), filtered, and solvent was removed in vacuo. The resultant crude residue was triturated with EtOAc (~3x 10 mL) and the extracts were passed through a short SiO2 plug eluting with EtOAc.Evaporation of solvent furnished pure cyclobutanols 4a / b (365.0 mg, 89%) as a 3:1 mixture of diastereomers. This diastereomeric mixture was typically used as is for the subsequent steps but could also be resolved via HPLC on an Agilent Prep 100A C18, 50 x 100 mm, 5 pm; isocratic 20% MeCN in H2O; flow rate = 80 mL / min, A = 254 nm; retention time = 4.28 min for minor diastereomer 4b; retention time = 4.96 min for major diastereomer 4a.

[0172] Data for 4a (Major Diastereomer):1H NMR (600 MHz, CDCl3) 59.13 (s, 1H), 7.23 (q, = 1.2 Hz, 1 H), 5.40 (s, 1 H), 4.67 (s, 1 H), 4.36 (d, = 11.5 Hz, 1 H), 4.12 - 4.08 (m, 1 H), 4.08 -4.02 (m, 1H), 3.78 (s, 1H), 3.40 (ddd, J = 13.1, 8.8, 3.7 Hz, 1H), 2.93 (ddd, J = 12.6, 7.3, 5.0 Hz, 1 H), 2.43 (s, 1 H), 2.33 - 2.22 (m, 2H), 1.95 (d, = 1.2 Hz, 3H), 1.53 (s, 3H), 1.46 (s, 3H). HRMS (ESI+) Anal. Calcd. For C17H23N2O7+[M+H]+367.1500, found 367.1506.

[0173] Data for 4b (Minor Diastereomer):1H NMR (600 MHz, CDCl3) 58.73 (s, 1H), 7.24 (q, = 1.4 Hz, 1 H), 5.41 (s, 1 H), 4.65 (s, 1 H), 4.59 (p, J = 7.0 Hz, 1 H), 4.39 - 4.30 (m, 2H), 3.74 (s, 1H), 2.68 (ddd, J = 13.7, 7.0, 2.1 Hz, 1H), 2.54 (dt, J = 13.4, 6.5 Hz, 1H), 2.47 (dt, J = 12.8, 6.4 Hz, 1H), 2.33 (ddd, J = 13.1, 6.8, 2.1 Hz, 1H), 2.06 (s, 1H), 1.95 (d, J = 1.2 Hz, 3H), 1.53 (s, 3H), 1.43 (s, 3H). HRMS (ESI+) Anal. Calcd. For C17H23N2O7+[M+H]+367.1500, found 367.1502.

[0174] Synthesis of 5:

[0175] Under N2, ketone 3 (1.53 g, 4.20 mmol) was slurried in 25 mL diethylene glycol. KOH (4.7 g, 84 mmol, 20 eq) was added, followed by H2NNH2-H2O (4.07 mL, 84 mmol, 20 eq). The resulting slurry was then heated to 130 oC with rapid stirring for 1h. The reaction mixture was removed from the heat, allowed to cool and poured into 100 mL H2O. This aqueous solution was saturated with NH4CI(s), then filtered. The resulting clear aqueous10102-040W01solution was extracted with 3 x CH2CI2, and the pooled organic layers were washed with 3 x H2O. The organic layer was dried over Na2SO4, and concentrated to dryness. The crude material was purified by column chromatography (3% MeOH in CH2CI2) to yield cyclobutane 5 (568 mg, 1.62 mmol, 39 %) as a white solid.

[0176] 1H NMR (400 MHz CDCl3) δ 9.08 (s, 1H), 7.25 (d, J = 1.25 Hz, 1H), 5.41 (s, 1H), 4.66 (s, 1 H), 4.40 (d, J = 11.7 Hz, 1 H), 4.30 (d, J = 11.7 Hz, 1 H), 3.77 (s, 1 H), 2.94 (m, 1 H), 2.51 (m, 1 H), 2.24 (m, 2H), 2.00 (m, 1 H), 1.97 (d, J = 1.25 Hz, 3H), 1.55 (s, 3H), 1.47 (s, 3H).

[0177] Alternate Synthesis of 5:4a 4b major diastereomer minor diastereomer

[0178] To a cold (0 °C), stirred solution of diastereomeric cyclobutanols 4a / b (203.0 mg, 1 Eq, 0.5541 mmol) in dry CH2CI2 (5 mL) was added pyridine (131.5 mg, 134 pL, 3 Eq, 1.662 mmol) followed by dropwise addition of Tf20 via syringe (234.5 mg, 140 pL, 1.500 Eq, 0.8311 mmol) over approximately 1 min. The reaction was allowed to stir at 0 °C for 25 min. After this time starting material was consumed as monitored by TLC analysis (70% EtOAc / hexanes). Next, reagent grade DMF (5 mL) was added followed by portion wise addition of NaBH4 (209.6 mg, 10 Eq, 5.541 mmol) to the cold reaction mixture over 5 minutes [caution! hydrogen gas vigorously evolved]. During addition of NaBH4the reaction mixture turns from orange to pale yellow. Once the evolution of gas had subsided (~5 min.), the reaction mixture was warmed to rt and allowed to stir for 45 min. After this time, the intermediate triflate was consumed as monitored by TLC analysis the reaction mixture was re-cooled to 0 °C and quenched by slow addition of saturated NH4CI (10 mL) [caution! hydrogen gas vigorously evolved]. After evolution of hydrogen gas had ceased (~20 min) reaction mixture was poured into a separatory funnel and diluted with 10 mL H2O and 20 mL EtOAc. The organic layer was separated, and aqueous layer was extracted with EtOAc (4x 20 mL). Combined organic extracts were dried (Na2SC>4), decanted, and solvent was evaporated in vacuo. The resultant residue was evaporated from toluene (3x 15 mL) to remove most of the remaining DMF. The crude product was purified on SiO2via flash column chromatography (30% to 75% EtOAc / CH2Cl2). Appropriate fractions were pooled,10102-040W01and solvent was removed in vacuo to give 138 mg of an off-white semi-solid. Co-evaporation of the crude residue from toluene, followed by diethyl ether furnished cyclobutane 5 (128.2 mg, 66%) as a yellowish solid.

[0179] Data for 5:1H NMR (500 MHz, CDCl3) δ 8.15 (s, 1H), 7.25 (q, J = 1.3 Hz, 1H), 5.39 (s, 1 H), 4.64 (s, 1 H), 4.38 (d, = 11.3 Hz, 1 H), 4.29 (dd, = 11.3, 0.9 Hz, 1 H), 3.76 (s, 1 H), 2.93 (dddt, J = 15.8, 6.6, 4.3, 2.1 Hz, 1 H), 2.55 - 2.47 (m, 1 H), 2.29 - 2.17 (m, 2H), 2.04 - 1.93 (m, 4H), 1.65 (dtt, = 11.3, 9.2, 6.6 Hz, 1 H), 1.54 (s, 3H), 1.45 (s, 3H). HRMS (ESI+) Anal. Calcd. For C17H23N2O6+[M+H]+351.1551, found 351.1552.

[0180] Synthesis of 6a and 6b:1. BOMCI, tBuOK, THF, rt 2. NaBH4, MeOH, 0°C 3. NaH, MOE-I, DMF, rt 4. TFA, toluene, A °'^xx'O'X6b major diasteromer minor diasteromer

[0181] To a rt, stirred solution of cyclobutanone 3 (65 mg, 1 Eq, 0.18 mmol) in anhydrous DMF (1.5 mL) was added KO'Bu (20 mg, 0.18 mL, 1 molar, 1 Eq, 0.18 mmol) via syringe and the solution was allowed to stir for 5 min at rt. After this time, BOMCI (28 mg, 25 pL, 1 Eq, 0.18 mmol) was added via microsyringe and the solution was allowed to stir at rt for 23 h.After this time, starting material was consumed as monitored by TLC analysis. The reaction mixture was quenched by addition of sat. aq. NH4CI (2 mL) and diluted with H2O (2 mL) and EtOAc (5 mL). The organic layer was separated, and the aqueous layer was extracted with EtOAc (3x 5 mL). The combined organic layers were dried (Na2SO4), filtered, and solvent was removed in vacuo to give the crude product as a brown oil. The crude product was was co-evaporated with toluene (3x 10 mL) to remove DMF, and purified on SiO2via flash column chromatography (10% to 40% EtOAc / hexanes). Appropriate fractions were pooled, and solvent was removed in vacuo to give intermediate BOM protected cyclobutanone analogue (32 mg), which was used immediately in the subsequent step. To a cold (0 °C), stirred solution of crude BOM protected cyclobutanone analogue from the previous step (32 mg, 1.0 Eq, 66 pmol) was added NaBH4(5.0 mg, 2.0 Eq, 0.13 mmol) and the reaction mixture was stirred at the same temperature for 1 h 15 min. After this time, the reaction mixture was quenched by slow addition of sat. aq. NH4CI (2 mL). Once all effervescence had ceased (~15 min) the mixture was transferred to a separatory funnel and the aqueous layer was extracted10102-040W01with EtOAc (3x 5 mL). The combined organic layers were dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude product was purified on SiO2via flash column chromatography (75% EtOAc / hexanes). Appropriate fractions were pooled, and solvent was removed in vacuo to give intermediate cyclobutanols as a 2:1 mixture of diastereomers, which was used immediately in the subsequent step. To a rt, stirred solution of the BOM protected cyclobutanol analogues from the previous step (33 mg, 1.0 Eq, 68 pmol) in a mixture of dry THE (0.8 mL) and DMF (0.2 mL) was added NaH (11.2 mg, 60% Wt, 4.1 Eq, 280 pmol) in one portion. The reaction mixture was stirred for 5 min to ensure complete deprotonation. After this time, MOE-I (38 mg, 21 pL, 3 Eq, 0.20 mmol) was added via microsyringe and the reaction mixture was allowed to proceed at rt. After 2 h 40 min, additional NaH (10.0 mg, 60% Wt, 3.7 Eq, 250 pmol) and MOE-I (22 mg, 12 pL, 1.7 Eq, 0.12 mmol) were added and the reaction. After a further 1 h 45 min at rt, the reaction mixture was cooled to 0 °C and carefully quenched with sat. aq. NH4CI (~1 mL). After evolution of hydrogen gas had ceased, the aqueous layer was extracted with EtOAc (10x 1 mL). The combined organic layers were dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude residue was co-evaporated from toluene (1x 20mL) to remove DMF and the product was purified on SiO2via flash column chromatography (55% EtOAc / hexanes).Appropriate fractions were pooled, and solvent was removed in vacuo to give diastereomeric methoxyethyl cyclobutane analogues as a colorless oil (21 mg), which was used immediately in the subsequent step. A rt, stirred solution of the methoxyethyl cyclobutane diastereomeric analogues from the previous step (21 mg, 1 Eq, 39 pmol) were dissolved in a mixture of toluene (0.2 mL) and TFA (1 mL) and the reaction vessel was submerged in a pre-heated oil bath (60 °C). After 1 h, the reaction mixture was cooled to rt, diluted with CH2CI2 (10 mL), and the solvent was removed in vacuo. The crude product was purified via HPLC on an Agilent Prep 100A C18, 50 x 100 mm, 5 pm; isocratic 15.5% MeCN in H2O + 0.1% TFA (v / v) in both mobile phases; flow rate = 80 mL / min, A = 254 nm; retention time = 21.04 min for minor diastereomer; retention time = 22.52 min for major diastereomer. Appropriate fractions were pooled, and solvent was freeze dried in vacuo to give minor diastereomer 6b (0.6 mg, 0.9%, 4 steps) and major diastereomer 6a (2.0 mg, 2.9%, 4 steps) as white solids.

[0182] Data for 6a (Major Diastereomer):1H NMR (601 MHz, MeOD) δ 7.80 (q, J = 1.2 Hz, 1 H), 5.36 (s, 1H), 4.25 (s, 1H), 4.16 (s, 1 H), 4.00 (d, J = 12.6 Hz, 1 H), 3.92 (d, J = 12.6 Hz, 1 H), 3.61 (p, J = 6.9 Hz, 1 H), 3.53 - 3.47 (m, 4H), 3.36 (s, 3H), 2.92 - 2.80 (m, 2H), 2.17 (ddd, J = 20.9, 13.2, 7.0 Hz, 2H), 1.88 (d, = 1.2 Hz, 3H). HRMS (ESI+) Anal. Calcd. For C17H25N2O8+[M+H]+385.1605, found 385.1615.10102-040W01

[0183] Data for 6b (Minor Diastereomer):1H NMR (601 MHz, MeOD) δ 7.83 (q, J = 1.2 Hz, 1 H), 5.37 (s, 1H), 4.27 (s, 1H), 4.15 (s, 1 H), 4.15 - 4.07 (m, 2H), 4.01 (d, J = 13.0 Hz, 1 H), 3.53 (t, J = 4.2 Hz, 2H), 3.50 - 3.45 (m, 2H), 3.37 (s, 3H), 2.47 - 2.37 (m, 3H), 2.33 -2.27 (m, 1 H), 1.88 (d, = 1.2 Hz, 3H). HRMS (ESI+) Anal. Calcd. For C17H25N2O8+[M+H]+385.1605, found 385.1610.

[0184] Synthesis of 7a and 7b1 NaH, THF, Mel, 0°C to rt 2. TFA, H2O, MeCN, rt MeO 7bminor diasteromer (25%, 2 steps)

[0185] To a rt, stirred solution of cyclobutanol 4a / b as a 3:1 mixture of diastereomers (43.3 mg, 1 Eq, 118 pmol) in dry THF (1 mL) was added NaH (10.0 mg, 60% Wt, 2.12 Eq, 250 pmol) and the deprotonation was allowed to proceed at rt for 5 min. After this time, CH3I (31.6 mg, 14.0 pL, 1.89 Eq, 223 pmol) was added via microsyringe and the reaction was placed in a pre-heated oil bath (50 °C) and allowed to proceed at this temperature for 22 h. After this time, the reaction mixture was cooled to rt and quenched by slow addition of sat. aq. NH4CI (2 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (4x 1 mL). The combined organic layers were dried (Na2SO4), filtered and solvent was removed in vacuo. The crude product was purified on SiO2via flash column chromatography (20 to 80% EtOAc / hexanes). Appropriate fractions were pooled, and solvent was removed in vacuo to give a 3:1 diasteromeric mixture of the acetonide protected methoxy cyclobutanes analogues as a white solid (22.5 mg), which was used immediately in the subsequent step. To a rt, stirred solution of of the acetonide protected methoxy cyclobutane analogues from the previous step (22.5 mg, 1 Eq, 59.1 pmol) in MeCN (0.4 mL) and H2O (0.8 mL) was added TFA (8.09 mg, 5.47 pL, 1.2 Eq, 71.0 pmol) and the reaction mixture was stirred at rt for 1.5 h. After this time, starting material was consumed as monitored by TLC analysis and the reaction mixture was diluted with MeOH (10 mL) and evaporated to dryness in vacuo. The residue was co-evaporated from additional MeOH (2x 10 mL) to remove water. The crude residue was purified via HPLC on an Agilent Prep 100A C18, 50 x 100 mm, 5 pm; isocratic 7% MeCN in H2O + 0.1% TFA (v / v) in both mobile phases; flow rate = 100 mL / min, A = 254 nm; retention time = 19.27 min for minor diastereomer; retention time = 20.11 min for major diastereomer. Appropriate fractions were10102-040W01pooled, and solvent was freeze dried in vacuo to give minor diastereomer 7b (2.6 mg, 6%, 2 steps) and major diastereomer 7a (7.6 mg, 19%, 2 steps) as white solids.

[0186] Data for 7a (Major Diastereomer):1H NMR (601 MHz, MeOD) 57.80 (q, J = 1.1 Hz, 1H), 5.36 (d, J = 0.6 Hz, 1H), 4.25 (s, 1H), 4.17 (s, 1H), 4.01 (d, J = 12.6 Hz, 1H), 3.93 (d, = 12.6 Hz, 1 H), 3.50 (p, J = 6.9 Hz, 1 H), 3.22 (s, 3H), 2.91 - 2.85 (m, 1 H), 2.82 (dtd, J = 13.3, 6.0, 5.4, 1.1 Hz, 1H), 2.18-2.08 (m, 2H), 1.88 (d, J = 1.2 Hz, 3H). HRMS (ESI+) Anal. Calcd. For C15H21N2O7+[M+H]+341.1343, found 341.1360.

[0187] Data for 7b (Minor Diastereomer):1H NMR (601 MHz, MeOD) 57.82 (t, = 1.2 Hz, 1H), 5.37 (s, 1H), 4.27 (s, 1H), 4.15 (s, 1H), 4.07-3.96 (m, 3H), 3.22 (s, 3H), 2.46-2.36 (m, 3H), 2.26 (ddd, J = 14.1, 4.9, 2.5 Hz, 1H), 1.88 (d, J = 1.3 Hz, 3H). HRMS (ESI+) Anal.Calcd. For C15H21N2O7+[M+H]+341.1343, found 341.1355.

[0188] Synthesis of 8a and 8b1. Tf2O, pyridine, THF, 0 °C then TBAF 2. TFA, H2O, MeCN, rt4a 4b major diasteromer minor diasteromer (48%, 2 steps)

[0189] To a cold (0 °C), stirred solution of diastereomeric (3:1) cyclobutanols 4a / b (44 mg, 1 Eq, 0.12 mmol) in dry CH2CI2(0.5 mL) was added pyridine (19 mg, 19 pL, 2 Eq, 0.24 mmol) and Tf2O (41 mg, 24 pL, 1.2 Eq, 0.14 mmol). The reaction mixture was allowed to stir while warming to rt. After 10 min, starting material was consumed as monitored by TLC analysis. A solution of TBAF (0.22 g, 0.84 mL, 1 molar, 7 Eq, 0.84 mmol) in THF was added via syringe and the reaction mixture was allowed to stir at rt for a further 2 h. After this time, the reaction mixture was quenched by addition of sat. aq. NaHCOs (0.5 mL) followed by sat. aq. NH4CI (2 mL) and water (~2 mL). The biphasic mixture was then transferred to a separatory funnel and the aqueous layer was extracted with EtOAc (5x 5mL). The combined organic layers were dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude product was purified on SiO2via flash column chromatography (10 to 60% EtOAc / hexanes). Appropriate fractions were pooled, and solvent was removed in vacuo to give a diastereomeric mixture of fluorocyclobutanes (24 mg) as a white solid, which was used immediately in the subsequent step. To a rt, stirred suspension of fluorocyclobutanes from the previous step (24 mg, 1 Eq, 65 pmol) in H2O (0.8 mL) and MeCN (0.4 mL) was added10102-040W01TFA (8.9 mg, 6.0 pL, 1.2 Eq, 78 pmol) via microsyringe. The reaction was allowed to proceed at rt for 2 h and gradually became homogeneous. After this time, the solvent was removed in vacuo and the crude residue was evaporated from MeOH (5x 5 mL) to give the product (19 mg, 48%, 2 steps). The diastereomers were separated via HPLC on an Agilent Prep 100A C18, 50 x 100 mm, 5 pm; 10.5% MeCN in H2O + 0.1% TFA (v / v) in both mobile phases; flow rate = 100 mL / min, A = 254 nm; retention time = 8.59 min for minor diastereomer; retention time = 9.51 min for major diastereomer. Appropriate fractions were pooled, and solvent was freeze dried in vacuo to give minor diastereomer 8b (3.6 mg, 9%, 2 steps) and major diastereomer 8a (9.6 mg, 24%, 2 steps) as white solids.

[0190] Data for 8a (Major Diastereomer):1H NMR (500 MHz, MeOD) 57.83 (d, = 1.3 Hz, 1H), 5.38 (s, 1H), 5.17 (dddd, J = 56.8, 10.9, 6.5, 4.5 Hz, 1H), 4.29 (s, 1H), 4.18 (s, 1H), 4.10 -4.02 (m, 2H), 2.72 - 2.61 (m, 1H), 2.61 - 2.46 (m, 3H), 1.88 (d, J = 1.2 Hz, 3H).19F NMR (471 MHz, MeOD) 5 -177.59. HRMS (ESI+) Anal. Calcd. For C14H18FN2O6+[M+H]+329.1143, found 329.1156.

[0191] Data for 8b (Minor Diastereomer):1H NMR (500 MHz, MeOD) 57.76 (d, = 1.4 Hz, 1H), 5.37 (s, 1H), 4.71 (dp, J = 56.4, 6.4 Hz, 1H), 4.28 (s, 1H), 4.14 (s, 1H), 4.00 (d, J = 12.7 Hz, 1H), 3.91 (d, J = 12.7 Hz, 1 H), 3.07 - 2.89 (m, 2H), 2.52 - 2.32 (m, 2H), 1.88 (d, J = 1.2 Hz, 3H).19F NMR (471 MHz, MeOD) 5 -170.84. HRMS (ESI+) Anal. Calcd. For C14H18FN2O6+[M+H]+329.1143, found 329.1158.

[0192] Synthesis of 9:DAST, CH2CI2rt

[0193] Ketone 3 (31.1 mg, 0.085 mmol) was dissolved in 1 mL CH2CI2. DAST (77 uL, 0.58 mmol, 8 eq) was added, and the resulting solution was stirred overnight at rt. The reaction was quenched with the addition of satd. NaHCO3 and extracted with EtOAc. The organic layer was dried over Na2SO4, concentrated and purified via column chromatography to yield 5 (15 mg, 46 % yield).10102-040W01

[0194] 1H NMR (400 MHz CDCI3) 68.44 (s, 1H), 7.24 (d, J = 1.1 Hz, 1H), 5.43 (s, 1H), 4.75 (s, 1 H), 4.41 (d, J = 11.3 Hz, 1 H), 4.27 (d, J = 11.3 Hz, 1 H), 3.85 (s, 1 H), 3.65 (m, 1 H), 3.09 (m, 1 H), 2.85 (m, 2H), 1.99 (d, J = 1.1 Hz, 3H), 1.58 (s, 3H), 1.48 (s, 3H);19F NMR (CDCI3) 5 -91.5 (d, J = 200 Hz), -96.2 (d, J = 200 Hz.

[0195] Alternate synthesis of 9:

[0196] To a solution of cyclobutanone 3 (840 mg, 2.3 mmol, 1.0 equiv.) in dry DCM (c = 0.1 M, 23.5 mL) in a 50-mL round-bottom flask equipped with a magnetic stir bar and N2inlet, a solution of DAST (2.1 mL, 16.1 mmol, 7.0 equiv.) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, then allowed to warm to room temperature and stirred for an additional 14 h. Upon completion (monitored by TLC), the reaction was quenched by the addition of triethylamine (Et3N) (7 mL, 20 equiv.) and stirred for 5 min. The mixture was then poured into a saturated aqueous solution of NaHCO3at 0 °C. After CO2evolution ceased, additional NaHCO3was added until gas evolution was complete. Subsequently, trimethylsilyl methoxide (TMS-OMe, 15 equiv., to quench F ) was added to the mixture and stirred for 5 min. The aqueous phase was extracted with DCM (3 x 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford the crude product. The resultant crude product was purified by silica gel column chromatography using a gradient of EtOAc / hexanes (30% — > 50% — > 80%). Appropriate fractions were combined and concentrated under reduced pressure to afford the product, which contained residual Et3N. Residual base was removed by co-evaporation with EtOAc (3 x 5 mL). The product was then dissolved in minimal Et2O and concentrated under reduced pressure to give 9 as a white foam (742 mg, 83%).

[0197] Data for 9:1H NMR (400 MHz, CD30D) 57.55 (d, = 1.3 Hz, 1 H), 5.43 (s, 1 H), 4.64 (d, = 11.4 Hz, 2H), 4.20 (dd, = 11.6, 0.9 Hz, 1 H), 3.96 (s, 1 H), 3.66 - 3.53 (m, 1 H), 3.19 -3.08 (m, 1 H), 2.80 - 2.66 (m, 2H), 1.92 (d, J = 1.3 Hz, 3H), 1.58 (s, 3H), 1.40 (s, 3H).HRMS (ESI+) calcd for [C17H21F2N2O6]: 387.1368 m / z found: 387.1370 m / z.10102-040W01

[0198] Synthesis of 10a and 10b1. NaBH(OAc)3, THF, Et2NH2, rt 2. TFA, H2O, MeCN, rt10b3 major diasteromer minor diasteromer

[0199] A suspension of cyclobutanone 3 (50.0 mg, 1 Eq, 137 pmol), Et2NH (22.1 mg, 31.2 pL, 2.2 Eq, 302 pmol), AcOH (16.5 mg, 15.7 pL, 2.00 Eq, 274 pmol), and NaBH(OAc)3(87.3 mg, 3.00 Eq, 412 pmol) in THF (1 mL) was stirred at rt for 16 h. Workup and purification on SiC>2 via flash column chromatography (wet loaded in eluent, 5% to 10% MeOH / CFfeCh) furnished acetonide protected diethylamino cyclobutane analogue (34 mg) as a 5:1 mixture of diastereomers. A solution of this acetonide protected diethylamino cyclobutane analogue (34 mg, 1 eq, 81 pmol) and TFA (11 mg, 7.5 pL, 1.2 Eq, 97 pmol) in a mixture of H2O (1 mL) and MeCN (0.1 mL) was allowed to stir for 1 h. Workup furnished 10a / b as a mixture of diastereomers (40 mg, 59%, 2 steps). The entire batch of material was purified via HPLC on an Agilent Prep 100A C18, 50 x 100 mm, 5 pm; 2 to 12% MeCN in H2O + 0.1% TFA (v / v) in both mobile phases over 35 min; flow rate = 25 mL / min, A = 254 nm; retention time = 28.80 min for minor diastereomer; retention time = 30.70 min for major diastereomer. Appropriate fractions were pooled, and solvent was freeze dried in vacuo to give minor diastereomer 10b (2.1 mg, 3%, 2 steps) and major diastereomer 10a (9.7 mg, 14%, 2 steps) as the corresponding TFA salts.

[0200] Data for 10a (Major Diastereomer):1H NMR (600 MHz, CD3CN) 5 10.62 (s, 1 H), 9.16 (s, 1H), 7.59 (d, J = 1.4 Hz, 1H), 5.30 (s, 1H), 4.21 (s, 1H), 4.16 (s, 1H), 4.06 (d, J = 12.9 Hz, 1H), 3.94 (d, J = 12.8 Hz, 1H), 3.26 (h, J = 8.2 Hz, 1H), 3.12 (dp, J = 14.3, 7.3 Hz, 2H), 2.99 (dq, J = 13.5, 6.7 Hz, 2H), 2.90 - 2.82 (m, 2H), 2.64 (dd, J = 13.5, 8.7 Hz, 1 H), 2.59 (dd, J = 14.4, 8.8 Hz, 1H), 1.82 (d, J = 1.2 Hz, 3H), 1.20 (td, J = 7.3, 5.1 Hz, 6H).19F NMR (471 MHz, CD3CN) 5 -76.67. HRMS (ESI+) Anal. Calcd. For C18H28N3O6+[M+H]+382.1973, found 382.1983.

[0201] Data for 10b (Minor Diastereomer):1H NMR (600 MHz, CD3CN) 5 10.78 (s, 1H), 9.08 (s, 1 H), 7.53 (q, J = 1.2 Hz, 1 H), 5.36 (s, 1 H), 4.26 (s, 1 H), 4.07 - 4.02 (m, 2H), 3.98 (d, J = 13.3 Hz, 1H), 3.89 (q, J = 8.1 Hz, 1H), 3.18-3.06 (m, 2H), 3.05- 2.90 (m, 3H), 2.79 (ddd, J = 12.1, 8.6, 2.6 Hz, 1H), 2.48-2.39 (m, 2H), 1.82 (d, J = 1.2 Hz, 3H), 1.20 (q, J =10102-040W016.3, 5.8 Hz, 6H).19F NMR (471 MHz, CD3CN) 5 -76.75. HRMS (ESI+) Anal. Calcd. For C18H28N3O6+[M+H]+382.1973, found 382.1979.

[0202] Synthesis of 11 a and 11b11a 11bmajor diasteromer minor diasteromer

[0203] A suspension of cyclobutanone 3 (50.0 mg, 1 Eq, 137 pmol), morpholine (26.3 mg, 26.0 pL, 2.2 Eq, 302 pmol), in dry THF(1 mL) was added in sequence AcOH (16.5 mg, 15.7 pL, 2.0 Eq, 274 pmol) and NaBH(OAc)3(87.3 mg, 3.0 Eq, 412 pmol) and the reaction mixture was stirred at rt for 21.5 h. Workup and purification on SiO2via flash column chromatography (wet loaded in eluent, 5% to 10% MeOH / CH2CI2) furnished intermediate acetonide protected morpholino cyclobutane analogue (43.5 mg) as a 7:1 mixture of diastereomers. A solution of this acetonide protected morpholino cyclobutane analogue (43.5 mg, 1 Eq, 99.9 pmol) and TFA (13.7 mg, 9.23 pL, 1.2 Eq, 120 pmol) in a mixture of MeCN (0.8 mL) and H2O (0.3 mL) was allowed to stir for 2 h. Workup furnished 11a / b as a mixture of diastereomers (51 mg, 73%, 2 steps). A small portion of the material was purified via HPLC on an Agilent Prep 100A C18, 50 x 100 mm, 5 pm; 2 to 7% MeCN in H2O + 0.1% TFA (v / v) in both mobile phases over 40 min; flow rate = 25 mL / min, A = 254 nm; retention time = 31.28 min for minor diastereomer; retention time = 32.13 min for major diastereomer. Appropriate fractions were pooled, and solvent was freeze dried in vacuo to give minor diastereomer 11 b (1.5 mg) and major diastereomer 11 a (10.0 mg) as the corresponding TFA salts.

[0204] Data for 11a (Major Diastereomer):1H NMR (601 MHz, MeOD) 57.72 (d, J= 1.5 Hz, 1 H), 5.40 (s, 1H), 4.32 (s, 1H), 4.17 (s, 1 H), 4.12 (d, J = 12.7 Hz, 1 H), 4.06 (d, J = 12.8 Hz, 1 H), 4.02 (very broad s, 2H), 3.75 (very broad s, 2H), 3.43 (p, J = 8.0 Hz, 1 H), 3.39 (very broad s, 2H), 3.10 - 2.99 (m, 2H), 2.96 (very broad s, 2H), 2.59 - 2.39 (m, 2H), 1.89 (d, J = 1.3 Hz, 3H).13C NMR (151 MHz, MeOD) 5 166.40, 151.87, 136.67, 110.89, 89.40, 87.86, 81.84, 80.10, 72.93, 64.96, 57.65, 53.54, 50.66, 37.69, 35.53, 12.61.19F NMR (471 MHz, MeOD) 5 -76.77. HRMS (ESI+) Anal. Calcd. For C18H26N3O7+[M+H]+396.1765, found396.1775.10102-040W01

[0205] Data for 11b (Minor Diastereomer):1H NMR (601 MHz, Acetone-d6) 59.96 (s, 1H), 7.76 (d, J = 1.5 Hz, 1H), 5.39 (d, J = 0.7 Hz, 1H), 4.31 (s, 1H), 4.25 (s, 1H), 4.12 (d, J = 13.2 Hz, 1 H), 4.01 (d, = 13.2 Hz, 1 H), 3.89 (s, 4H), 2.86 (very broad s, ~9H), 2.41 (ddt, J = 26.4, 12.9, 5.8 Hz, 2H), 1.81 (d, = 1.2 Hz, 3H). Several protons overlap with the broad water signal at 2.86 ppm.19F NMR (471 MHz, Acetone-d6) 5 -75.25. HRMS (ESI+) Anal.Calcd. 396.1765 For C18H26N3O7+[M+H]+, found 396.1773.

[0206] Synthesis of 12a and 12b12a 12b major diasteromer minor diasteromer

[0207] A suspension of cyclobutanone 3 (50.0 mg, 1 Eq, 137 pmol), N, O-dimethylhydroxylamine hydrochloride (26.3 mg, 26.0 pL, 2.2 Eq, 302 pmol), in dry THF(1 mL) was added in sequence AcOH (16.5 mg, 15.7 pL, 2.0 Eq, 274 pmol) and NaBH(OAc)3(87.3 mg, 3.0 Eq, 412 pmol) and the reaction mixture was stirred at rt for 21 h. Workup and purification on SiC>2 via flash column chromatography (wet loaded in eluent, 20% to 60% EtOAc / hexanes) furnished intermediate acetonide protected N,0-dimethylhydroxyl amino cyclobutane analogue (40.1 mg) as a 6:1 mixture of diastereomers. A solution of this acetonide protected N, O-dimethylhydroxyl amino cyclobutane (40.1 mg, 1 Eq, 97.9 pmol) and TFA (13.4 mg, 9.1 pL, 1.2 Eq, 118 pmol) in a mixture of MeCN (0.8 mL) and H2O (0.3 mL) was allowed to stir for 3 h. The crude material was co-evaporated from MeOH (2x 10 mL) and the residue was treated with sat. aq. NaHCO3(1 mL) and allowed to stir 5 min. before being extracted with 1:1 CH2Cl2 / MeOH (10x 1 mL). The organic layer was dried was evaporated to dryness and the crude product was purified via flash column chromatography. Appropriate fractions were pooled, and solvent was removed in vacuo to give minor diastereomer 12b (2.5 mg, 5%, 2 steps) and major diastereomer 12a (8.5 mg, 17%, 2 steps) as white solids.

[0208] Data for 12a (Major Diastereomer):1H NMR (600 MHz, MeOD) 57.82 (q, J = 1.2 Hz, 1H), 5.38 -5.34 (m, 1H), 4.26 -4.24 (m, 1H), 4.18 (s, 1H), 4.05 (d, J = 12.6 Hz, 1H), 3.99 (d, = 12.6 Hz, 1 H), 3.53 (s, 3H), 2.76 (p, J = 7.8 Hz, 1 H), 2.70 - 2.64 (m, 1 H), 2.64 -10102-040W012.59 (m, 1 H), 2.44 (s, 3H), 2.27 (very broad s, 2H), 1.88 (d, J = 1.2 Hz, 3H). HRMS (ESI+) Anal. Calcd. For C16H24N3O7+[M+H]+370.1609, found 370.1622.

[0209] Data for 12b (Minor Diastereomer):1H NMR (600 MHz, MeOD) 57.80 (q, J= 1.1 Hz, 1 H), 5.38 (s, 1H), 4.28 (s, 1H), 4.15 (d, J = 13.1 Hz, 1 H), 4.12 (s, 1 H), 4.02 (d, J = 13.2 Hz, 1 H), 3.54 (s, 3H), 3.27 (p, J = 7.2 Hz, 1 H), 2.46 (s, 3H), 2.42 (very broad s, 2H), 2.34 -2.25 (m, 2H), 1.88 (d, = 1.2 Hz, 3H). HRMS (ESI+) Anal. Calcd. For C16H24N3O7+[M+H]+370.1621, found 370.1622.

[0210] Synthesis of 13a and 13b1. NaBH(OAc)3, THF, pyrrolidine, rt 2. TFA, H2O, MeCN, rt3major diasteromer minor diasteromer

[0211] Following general procedure A, a suspension of cyclobutanone 3 (50.0 mg, 1 Eq, 137 pmol), pyrrolidine (21.5 mg, 24.8 pL, 2.2 Eq, 302 pmol), in dry THF(1 mL) was added in sequence AcOH (16.5 mg, 15.7 pL, 2.0 Eq, 274 pmol) and NaBH(OAc)3(87.3 mg, 3.0 Eq, 412 pmol) and the reaction mixture was stirred at rt for 18 h. Workup and purification on SiO2via flash column chromatography (wet loaded in eluent, 5% to 15% MeOH / CH2CI2) furnished intermediate acetonide protected pyrrolidino cyclobutane analogue (39 mg) as a 5:1 mixture of diastereomers. A solution of this acetonide protected pyrrolidino cyclobutane analogue (39 mg, 1 Eq, 93 pmol) and TFA (13.0 mg, 8.6 pL, 1.2 Eq, 110 pmol) in a mixture of MeCN (0.3 mL) and H2O (0.8 mL) was allowed to stir for 2 h. Workup furnished 13a / b as a mixture of diastereomers (40.1 mg, 68%, 2 steps). A small portion of the material was purified via HPLC on an Agilent Prep 100A C18, 50 x 100 mm, 5 pm; 2 to 10% MeCN in H2O + 0.1% TFA (v / v) in both mobile phases over 17 min; flow rate = 80 mL / min, A = 254 nm; retention time = 15.06 min for minor diastereomer; retention time = 15.55 min for major diastereomer. Appropriate fractions were pooled, and solvent was freeze dried in vacuo to give minor diastereomer 13b (3.6 mg) and major diastereomer 13a (9.1 mg) as white solids.

[0212] Data for 13a (Major Diastereomer):1H NMR (601 MHz, MeOD) 57.72 (d, J= 1.3 Hz, 1 H), 5.39 (s, 1H), 4.32 (s, 1H), 4.16 (s, 1 H), 4.12 (d, J = 12.8 Hz, 1 H), 4.06 (d, J = 12.8 Hz, 1 H), 3.64 - 3.53 (m, 2H), 3.49 (p, J = 7.9 Hz, 1 H), 3.09 - 3.04 (m, 1 H), 3.01 (dddd, J =10102-040W0112.7, 7.3, 4.9, 2.5 Hz, 3H), 2.50 -2.41 (m, 2H), 2.21 - 2.10 (m, 2H), 2.08- 1.97 (m, 2H), 1.89 (d, J = 1.3 Hz, 3H).19F NMR (565 MHz, MeOD) 5 -76.87. HRMS (ESI+) Anal. Calcd. For C18H26N3O6+[M+H]+380.1816, found 380.1829.

[0213] Data for 13b (Minor Diastereomer):1H NMR (601 MHz, MeOD) 57.67 (q, J= 1.0 Hz, 1H), 5.44 (s, 1H), 4.36 (s, 1H), 4.15 (d, J = 0.8 Hz, 2H), 4.08 (s, 1H), 3.96 (p, J = 7.8 Hz, 1H), 3.58 (s, 2H), 3.04 (s, 2H), 2.81 (dd, J = 13.3, 7.6 Hz, 1H), 2.62 (dd, J = 7.9, 2.4 Hz, 2H), 2.60 - 2.55 (m, 1 H), 2.16 (s, 2H), 2.04 (s, 2H), 1.89 (s, 3H).19F NMR (565 MHz, MeOD) 5 -76.91. HRMS (ESI+) Anal. Calcd. For C18H26N3O6+[M+H]+380.1816, found 380.1826.

[0214] Synthesis of 14a and 14b1. NaBH(OAc)3, THF, difluoroethylamine, rt 2. TFA, H2O, MeCN, rt14a 14bmajor diasteromer minor diasteromer

[0215] A suspension of cyclobutanone 3 (50.0 mg, 1 Eq, 137 pmol), 2,2-difluoroethylamine (24.5 mg, 2.2 Eq, 302 pmol), in dry THF(1 mL) was added in sequence AcOH (16.5 mg, 15.7 pL, 2.0 Eq, 274 pmol) and NaBH(OAc)3(87.3 mg, 3.0 Eq, 412 pmol) and the reaction mixture was stirred at rt for 18 h. Workup and purification on SiO2via flash column chromatography (wet loaded in eluent, 100% EtOAc) furnished intermediate acetonide protected 2,2-difluoroethylamino cyclobutane analogue (55 mg) as a 4:1 mixture of diastereomers. A solution of this acetonide protected 2,2-difluoroethylamino cyclobutane analogue (55 mg, 1 Eq, 130 pmol) and TFA (18 mg, 12 pL, 1.2 Eq, 150 pmol) in a mixture of MeCN (0.3 mL) and H2O (0.8 mL) was allowed to stir for 2 h at rt. Workup furnished 14a / b as a mixture of diastereomers (53.5 mg, 93%, 2 steps). A small portion of the material was purified via HPLC on an Agilent Prep 100A C18, 50 x 100 mm, 5 pm; isocratic 3% MeCN in H2O + 0.1% TFA (v / v) in both mobile phases; flow rate = 80 mL / min, A = 254 nm; retention time = 19.53 min for minor diastereomer; retention time = 20.73 min for major diastereomer. Appropriate fractions were pooled, and solvent was freeze dried in vacuo to give minor diastereomer 14b (3.2 mg) and major diastereomer 14a (13.0 mg) as white solids.

[0216] Data for 14a (Major Diastereomer):1H NMR (601 MHz, MeOD) 57.71 (q, J= 1.2 Hz, 1H), 6.28 (ft, = 53.7, 3.0 Hz, 1 H), 5.39 (d, J = 0.6 Hz, 1H), 4.32 (s, 1H), 4.15 (s, 1H),10102-040W014.13 (d, J = 12.8 Hz, 1H), 4.06 (d, J = 12.7 Hz, 1H), 3.54 (p, J = 7.9 Hz, 1H), 3.47 (tdd, J = 15.7, 3.0, 1.8 Hz, 2H), 3.07 (dddd, J = 13.1, 7.9, 5.1, 1.1 Hz, 1H), 3.01 (dddd, J = 12.8, 7.8, 5.1, 1.2 Hz, 1H), 2.49 -2.42 (m, 2H), 1.89 (d, J = 1.2 Hz, 3H).19F NMR (565 MHz, MeOD) 5 -76.93, -124.44. HRMS (ESI+) Anal. Calcd. For C16H22F2N3O6+[M+H]+390.1471, found 390.1485.

[0217] Data for 14b (Minor Diastereomer)1H NMR (601 MHz, MeOD) 57.62 (q, J= 1.2 Hz, 1 H), 6.27 (tt, J = 53.6, 2.8 Hz, 1 H), 5.42 (s, 1 H), 4.35 (s, 1 H), 4.21 (d, J = 2.8 Hz, 2H), 4.05 (s, 1 H), 3.94 (ddd, J = 14.8, 8.2, 6.6 Hz, 1 H), 3.46 (tt, J = 16.0, 3.0 Hz, 2H), 2.87 - 2.79 (m, 1 H), 2.72 - 2.58 (m, 3H), 1.89 (d, = 1.2 Hz, 3H).19F NMR (565 MHz, MeOD) 5 -76.96, -124.66, -124.67. HRMS (ESI+) Anal. Calcd. For C16H22F2N3O6+[M+H]+390.1471, found 390.1480.

[0218] Example 2

[0219] Thymine and 5-methyl cytosine LNA cyclobutane phosphoramidite compounds were prepared in accordance with Scheme 45, as follows.10102-040W011. 1,2,4-triazole, POCI3, Et3N MeCN, 0°Cto it 2. NH4OH, H20, dioxane, rt 3. BzCI, pyridine, CH2CI2, rt 4. TFA, H2O, MeCN, rt1. TFA, MeCN, H2O, rt 2. DMTrCI, pyridine, rt 3. (N(iPr)2)2P-OCH2CH2CN,X 'N MeCN, rt N-N 26%, 3 steps 1. DMTrCI, pyridine 2. (N(iPr)2)2P-OCH2CH2CN MeCN, rt 29%, 6 stepsMALAT 1 targeting oligonucleotidesfrom 15 and 17 LEGENDScheme 45

[0220] Synthesis of 1510102-040W01

[0221] To a rt, stirred solution cyclobutane 5 (330 mg, 1 Eq, 942 pmol) in acetonitrile (5 mL) and water (5 mL) was added TFA (15 mg, 10 pL, 0.14 Eq, 0.13 mmol) and the reaction mixture was allowed to stir at rt. After 1 h, additional TFA (15 mg, 10 pL, 0.14 Eq, 0.13 mmol) was to the reaction mixture via microsyringe. After a further 1 h starting material was consumed as monitored by TLC analysis. At this point, the reaction mixture was evaporated to dryness and co-evaporated in sequence with MeCN (3 x 20 mL), toluene (1 x 20 mL), Et20 (1 x 20 mL) to give the crude diol (-308 mg) as a white solid, which was used immediately in the subsequent step. To a rt, stirred solution of the crude diol (-308 mg, 1 Eq, -942 pmol) in dry pyridine (9.4 mL) was added DMTrCI (638 mg, 2 Eq, 1.88 mmol) and the reaction mixture was allowed to stir for 23 h at rt. After this time, solvent was removed in vacuo and the crude residue was purified on SiC>2 via flash column chromatography (2% to 5% EtOH / CFfeCh). Appropriate fractions were pooled, and solvent was removed in vacuo to give DMTr protected analogue (297 mg) as a white solid, which was used immediately in the subsequent step. To a cold (0 °C), stirred solution of DMTr protected analogue from the previous step (297 mg, 1 Eq, 0.485 mmol)and ETT (94.8 mg, 1.50 Eq, 0.728 mmol) in dry MeCN (2.4 mL) and CH2CI2 (2.4 mL) was added 2-Cyanoethyl N, N, N', / -tetraisopropylphosphorodiamidite (219 mg, 231 pL, 1.50 Eq, 0.727 mmol) via syringe and the reaction was warmed to rt. After 2 h 15 min substantial precipitate had formed and the reaction mixture was filtered through a cotton plug, and the precipitate was washed with EtOAc (2x 0.5 mL). The filtrate was concentrated to dryness in vacuo, and the resultant residue was purified on SiC>2 via flash column chromatography (10 to 70% EtOAc / hexanes + 1% v / v Et3N). Appropriate fractions were pooled, and solvent was removed in vacuo. The resultant solid was dissolved in minimal toluene (-2 mL) and hexanes (-10-15 mL) was added with rapid stirring (-10-15 mL) and a white precipitate rapidly crashes out. The precipitate was collected on a filtering frit with suction, and the filtrate was concentrated in vacuo and reprecipitated a second time. The two precipitation crops were combined to furnish phosphoramidite 15 (196 mg, 26%, 3 steps) as a 3:1 mixture of P(lll) diastereomers and as a white solid.

[0222] Data for 15:1H NMR (600 MHz, C6D6) 58.87 (s, 0.25H), 8.72 (s, 0.75H), 7.79 -7.67 (m, 3H), 7.57 (t, J = 8.7 Hz, 1 H), 7.53 (t, J = 9.1 Hz, 3H), 7.27 - 7.20 (m, 2H), 7.08 (td, 7.3, 1.4 Hz, 1 H), 6.87 - 6.77 (m, 4H), 5.59 (s, 0.25H), 5.58 (s, 0.75H), 4.94 (s, 0.75H), 4.92 (s, 0.25H), 4.69 - 4.62 (m, 1 H), 3.89 (d, = 10.6 Hz, 1 H), 3.82 - 3.75 (m, 1 H), 3.42 - 3.34 (m, 2H), 3.34-3.23 (m, 6H), 3.13 -3.02 (m, 1.5H), 3.02 -2.95 (m, 0.5H), 2.60 -2.52 (m, 1H), 2.38-2.29 (m, 1H), 2.29 -2.15 (m, 2H), 1.76 (d, J = 1.2 Hz, 0.75H), 1.70 (d, J = 1.2 Hz, 2.25H), 1.68 - 1.42 (m, 4H), 1.06 - 0.89 (m, 12H).31P NMR (162 MHz, C6D6) 5 148.96, 148.85. HRMS (ESI+) Anal. Calcd. C44H54N4O9P+For [M+H]+813.3623, found 813.3648.10102-040W01

[0223] Synthesis of 16

[0224] To a cold (0 °C), stirred suspension of 1 H-1,2,4-triazole (1.09 g, 10 Eq, 15.8 mmol) in dry MeCN (20 mL) was added POCh (485 mg, 0.295 mL, 2.00 Eq, 3.17 mmol) via syringe and the reaction mixture was stirred for 5 min at the same temperature. After this time, EtsN (1.60 g, 2.20 mL, 10 Eq, 15.8 mmol) was added and the reaction mixture was stirred at the same temperature for a further 5 min before adding cyclobutane 5 (554 mg, 1 Eq, 1.58 mmol) as a solution in EtsN (1.12 g, 1.54 mL, 7 Eq, 11.1 mmol) and MeCN (5 mL + 3 mL rinse). After a further 5 min at 0 °C, the reaction mixture was warmed to rt and stirred. After a further 35 min at rt, starting material was consumed as monitored by TLC analysis. The reaction mixture was quenched in sequence with 20 mL sat. NaHCOs and 20 mL H2O and the biphasic mixture was transferred to a separatory funnel. The aqueous layer was extracted with EtOAc (4x 50 mL) and the combined organic extracts were dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude product was purified on SiO2via flash column chromatography (30 to 75% EtOAc / hexanes). Appropriate fractions were pooled and solvent was removed in vacuo to give intermediate triazole (620 mg) as a white foam, which was used immediately in the subsequent step. To a rt, stirred solution of the triazole from the previous step (620 mg, 1 Eq, 1.54 mmol) in 1,4-dioxane (10 mL) was added aqueous NH4OH (812 mg, 1.54 mL, 15 molar, 15 Eq, 23.2 mmol). The reaction mixture was allowed to stir at rt 2 h. After this time, the solvent was concentrated to dryness in vacuo and the residue was co-evaporated from MeCN (5x 20 mL) to remove water. The resultant solid was dissolved CH2CI2(20 mL), dried (Na2SC>4), and filtered. The filtrate was concentrated to dryness in vacuo to and the residue was co-evaporated from a mixture of CH2CI2(5 mL) and Et2O (20 mL) to give the intermediate amine as fluffy white foam (650 mg) which was contaminated with 1 H-1,2,4-triazole, which was used immediately in the subsequent step. To a cold (0 °C) stirred solution of amine intermediate from the previous step (650 mg crude, 1 Eq, -1.54 mmol) in anhydrous pyridine (10 mL) was added BzCI (0.65 g, 0.54 mL, 3.0 Eq, 4.6 mmol) in one portion via syringe. The reaction mixture was then allowed to warm to rt and stirred at ambient temperature for 2.5 h. After this time, the reaction mixture was cooled (0 °C) and quenched by addition of sat. aq. NaHCOs (30 mL). The biphasic mixture was transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (3x 50 mL). The combined organic layers were dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude product was purified on SiO2via flash column chromatography (5 to 25% EtOAc / hexanes). Appropriate fractions were pooled, and solvent was removed in vacuo to give the benzoylated amine intermediate (417 mg) as a white powder, which was used immediately in the subsequent step. To a rt, stirred solution of benzoylated amine from the previous step (418 mg, 1 Eq, 0.922 mmol) in MeCN (13 mL) and H2O (2 mL) was added TEA10102-040W01(49.2 mg, 33.0 pL, 0.468 Eq, 431 pmol) and the reaction was allowed to proceed for 2 h 40 min. After this time, starting material was consumed as monitored by TLC analysis, and solvent was removed in vacuo. The resultant residue was co-evaporated from MeCN (3x 10 mL) to remove water. The crude product was purified on SiC>2 via flash column chromatography (3% MeOH / CFfeCh). Appropriate fractions were pooled, and solvent was removed in vacuo to give diol 16 (233 mg) as a white powder, which was used immediately in the subsequent step.

[0225] Synthesis of 17

[0226] To a rt, stirred solution of diol 16 from the previous step (233 mg, 1 Eq, 564 pmol) in anhydrous pyridine (7.5 mL) was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (217 mg, 1.14 Eq, 640 pmol) in one portion, and the reaction mixture was allowed to proceed at rt for 3.5 h. After this time, the reaction mixed was quenched with 10 mL sat. NaHCOs at 0 °C and the biphasic mixture was transferred to a separatory funnel. The aqueous layer was extracted with EtOAc (3x 30 mL) and the combined organic layers were dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude product was purified on SiC>2 via flash column chromatography (35 to 55% EtOAc / hexanes + 1% v / v EtsN). Appropriate fractions were pooled, and solvent was removed in vacuo to give intermediate DMTr protected analogue (332 mg) as a white solid, which was used immediately in the subsequent step. To a cold (0 °C) stirred solution of DMTr protected analogue from the previous step (332 mg, 1 Eq, 0.464 mmol) and ETT (96.6 mg, 1.60 Eq, 0.742 mmol) in MeCN (6 mL) was added 2-Cyanoethyl / V, / V, / V, / V-tetraisopropylphosphorodiamidite (210 mg, 0.221 mL, 1.50 Eq, 696 pmol) and the reaction was warmed to rt. After 2 h, a significant amount of white precipitate had formed. At this point the reaction mixture was filtered through a cotton plug and the precipitate was rinsed with minimal EtOAc (2x 2 mL). The filtrate was concentrated to dryness in vacuo and the crude product was purified on SiO2via flash column chromatography (20 to 65% Et2O / hexanes). Appropriate fractions were pooled, and solvent was removed in vacuo to give phosphoramidite 17 as 3:1 mixture of P(lll) diastereomers and as a white solid (356 mg, 29%, 6 steps).

[0227] Data for 17:1H NMR (500 MHz, C6D6) 58.75 - 8.63 (m, 2H), 7.94 (s, 0.25H), 7.90 (s, 0.75H), 7.75 (d, J = 7.8 Hz, 0.5H), 7.72 (d, J = 7.8 Hz, 1.5H), 7.62 – 7.50 (m, 4H), 7.33 – 7.17 (m, 6H), 7.14 - 7.10 (m, 1 H), 6.94 - 6.72 (m, 4H), 5.60 (s, 1 H), 5.00 (s, 0.75H), 4.96 (s, 0.25H), 4.72 (d, J = 6.9 Hz, 0.25H), 4.69 (d, J = 7.9 Hz, 0.75H), 3.92 (d, J = 10.5 Hz, 1H), 3.81 (d, J = 10.5 Hz, 1H), 3.34 (t, J = 5.5 Hz, 8H), 3.22 - 3.14 (m, 0.5H), 3.03 (qt, J = 10.7, 5.5 Hz, 1.5H), 2.65 -2.48 (m, 1H), 2.35 (q, J = 10.7 Hz, 1H), 2.30 -2.14 (m, 2H), 1.93 (s,10102-040W010.75H), 1.86 (s, 2.25H), 1.60 - 1.32 (m, 3H), 1.25- 1.14 (m, 1H), 1.05- 0.91 (m, 12H).31P NMR (162 MHz, C6D6) 5149.04. HRMS (ESI+) Anal. Calcd. C44H54N4O9P+For [M+H]+916.4045, found 916.4073.

[0228] Example 3

[0229] Thymine and 5-methyl cytosine LNA difluorocyclobutane phosphoramidite compounds were prepared in accordance with Scheme 46 as follows.DMTrCI, pyridine, rt 83%18 9 1. 1,2,4-triazole, POCI3, Et3N MeCN, 0°Cto rt 2. NH4OH, H2O, dioxane, rt 3. BzCI, pyridine, CH2CI2, rt 63%, 3 steps!NHBz(N(IPr)2)2P-OCH2CH2CN MeCN, rt 63%MALAT1 targeting oligonucleotides from 20 and 23 LEGEND CB054 = 5' mCTAgttcactgaaTGmC 3' bold caps = LNA, underlined = difluorocyclobutyl modified LNAlowercase = DNA, all linkages are stereorandom PS mC = 5-methyl cytosineScheme 4610102-040W01

[0230] Synthesis of 18

[0231] A sample of compound 9 (210 mg, 0.5435 mmol) was dissolved in a mixture of trifluoroacetic acid (TFA) and water (c = 0.1 M, 5.2 mL: 0.28 mL) at room temperature and stirred for 15 min. The solvent was then removed under reduced pressure. The residue was dissolved in MeOH and concentrated under reduced pressure to azeotrope off residual TFA. This process was repeated three times to ensure complete removal of TFA. The product was obtained as a white semisolid (180 mg, 95%).

[0232] Data for 18:1H NMR (500 MHz, CD3OD) 57.82 (d, J = 1.3 Hz, 1H), 5.42 (d, J = 0.6 Hz, 1H), 4.34 (s, 1H), 4.23 (s, 1H), 4.01 (q, J = 12.8 Hz, 2H), 3.19 -3.06 (m, 1H), 3.05-2.95 (m, 1 H), 2.85 - 2.67 (m, 2H), 1.88 (d, = 1.3 Hz, 3H). HRMS (ESI+) calcd for [C14H17F2N2O6]: 347.1055 m / z found: 347.1056 m / z.

[0233] Synthesis of 19

[0234] To a stirred solution of compound 18 (178 mg, 0.5140 mmol, 1.0 equiv.) in pyridine (pre-dried over 3 A MS, 0.5 M, 1.0 mL) at room temperature, 4,4'(chloro(phenyl)methylene)bis(methoxybenzene) (DMTrCI) (191.6 mg, 0.5654 mmol, 1.1 equiv.) was added. The reaction mixture was stirred for 20 h at room temperature and monitored by TLC. Upon completion, the reaction was quenched with methanol and stirred for an additional 5-10 min. The solvent was evaporated under ambient air. To prepare for column chromatography, Et3N and silica gel were added to form a slurry. Before elution, the silica gel column was neutralized using 1% Et3N in 100 mL of a 1:4 mixture of acetone and hexane (acetone / hexane). Elution was performed using a gradient of acetone / hexane (30% — > 50% — > 80%). compound 19 was obtained as a white semisolid (277 mg, 83%). (TLC Note: For accurate monitoring and column fraction analysis, TLC plates should be pre-dipped in the eluent system (50% acetone / hexane + 1% v / v Et3N) before development in the same solvent system.)

[0235] Data for 19:1H NMR (500 MHz, CD3OD) 57.74 (d, J = 1.4 Hz, 1H), 7.52 -7.47 (m, 2H), 7.40 - 7.35 (m, 4H), 7.35 - 7.29 (m, 2H), 7.28 - 7.22 (m, 1 H), 6.91 - 6.86 (m, 4H), 5.42 (s, 1H), 4.40 (d, J = 19.4 Hz, 2H), 3.78 (d, J= 1.6 Hz, 6H), 3.70 (d, J = 11.1 Hz, 1H), 3.56 (d, J = 11.1 Hz, 1H), 3.08-3.00 (m, 1H), 2.82 -2.60 (m, 3H), 1.54 (d, J = 1.2 Hz, 3H).HRMS (ESI+) calcd for [C35H35F2N2O8]: 649.2361 m / z found: 649.2366 m / z.

[0236] Synthesis of 2010102-040W01

[0237] To a cold (0 °C), stirred suspension of compound 19 (195 mg, 1 Eq, 0.300 mmol) and DCI (44.6 mg, 1.26 Eq, 0.378 mmol) in dry CH2CI2 (2 mL) was added 2-Cyanoethyl / V, / V, / V, / V-tetraisopropylphosphorodiamidite (114 mg, 0.12 mL, 1.26 Eq, 0.378 mmol) via syringe and the reaction was warmed to rt. After 1 h 10 min additional DCI (11.8 mg, 0.33 Eq, 0.10 mmol) and 2-Cyanoethyl / V, / V, / V, / V-tetraisopropylphosphorodiamidite (30 mg, 31.5 pL, 0.33 Eq, 0.10 mmol) were added to the reaction mixture. After a further 50 min (2 h total), starting material was consumed as monitored by TLC analysis. The filtrate was concentrated to dryness in vacuo, and the resultant residue was purified on SiC>2 via flash column chromatography (40 to 70% EtOAc / hexanes + 1% v / v Et3N). Appropriate fractions were pooled, and solvent was removed in vacuo. The resultant solid was dissolved in minimal toluene (~1.5 mL) and hexanes (-7-8 mL) was added with rapid stirring and a white precipitate rapidly crashes out. The precipitate was collected on a filtering frit with suction. The precipitate was subjected to an identical second reprecipitation to furnish phosphoramidite 20 (195 mg, 77%) as a 3:1 mixture of P(lll) diastereomers and as a white solid.

[0238] Data for 20:1H NMR (600 MHz, C6D6) 59.80 (s, 1 H), 7.73 - 7.65 (m, 3H), 7.57 -7.53 (m, 1 H), 7.53 - 7.49 (m, 3H), 7.27 - 7.21 (m, 2H), 7.11 - 7.07 (m, 1 H), 6.86 - 6.79 (m, 4H), 5.49 (s, 1 H), 5.03 (s, 0.75H), 4.98 (s, 0.25H), 4.73 (d, J = 7.1 Hz, 0.25H), 4.70 (d, J = 7.6 Hz, 0.75H), 3.90 - 3.84 (m, 1 H), 3.81 - 3.76 (m, 1 H), 3.36 - 3.28 (m, 8H), 3.16 - 2.93 (m, 3H), 2.88- 2.70 (m, 3H), 1.76 (d, = 1.3 Hz, 0.75H), 1.72 (d, J = 1.1 Hz, 2.25H), 1.71 -1.58 (m, 2H), 1.02 (d, J = 6.7 Hz, 4.5H), 0.95 (d, J = 6.9 Hz, 1,5H), 0.93 (d, J = 6.8 Hz, 4.5H), 0.85 (d, J = 6.7 Hz, 1.5H).19F NMR (471 MHz, C6D6) 5 -88.14 (d, J = 200.3 Hz), -88.40 (d, J = 203.1 Hz), -97.28 (d, J = 200.3 Hz), -97.46 (d, = 203.1 Hz).31P NMR (162 MHz, C6D6) 5 149.70, 149.15. HRMS (ESI+) Anal. Calcd. C44H52F2N4O9P+For [M+H]+849.3434, found 849.3462.

[0239] Synthesis of 21

[0240] A cold (0 °C), stirred suspension of 1 A7-1,2,4-triazole (2.66 g, 38.6 mmol, 50.0 equiv.) in dry MeCN (18.6 mL) was treated with POCI3(456 mg, 720 pL, 4.93 mmol, 1.0 equiv.) via syringe and stirred for 5 min at 0 °C. Et3N (4.43 mL, 31.8 mmol) was then added, and the mixture was stirred for an additional 5 min at the same temperature. Separately, compound 9 (301.0 mg, 0.775 mmol, 1.0 equiv.) was suspended in Et3N (1.05 mL, 7.54 mmol.) and MeCN (4.75 mL) and sonicated to aid dispersion. The vial was rinsed with MeCN (0.9 mL), and the rinse was added to the reaction mixture. The combined mixture was stirred for 5 min at 0 °C, then allowed to warm to room temperature and stirred for an additional 35 min. The reaction progress was monitored by TLC until complete consumption of the starting10102-040W01material. The reaction was quenched with sat. aq. NaHCO3(18.6 mL) and H2O (18.6 mL). The mixture was transferred to a separatory funnel, and the organic layer was separated. The aqueous layer was extracted with EtOAc (4 x 47 mL). (Note: Triazole derivatives are sensitive to silica; column chromatography should be avoided). The resultant crude product was dissolved in 1,4-dioxane (10 mL) and stirred at room temperature. Ammonium hydroxide (15 M, 775 pL, 11.63 mmol, 15.0 equiv.) was added, and the reaction mixture was stirred for 6 h at ambient temperature. TLC analysis confirmed complete consumption of the starting material. The mixture was evaporated to dryness under reduced pressure, and the residue was co-evaporated with MeCN (5 x 20 mL) to remove residual water. The solid was dissolved in CH2CI2(20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using a gradient of acetone / hexanes (30% — > 50 — > 75%). Appropriate fractions were combined and concentrated under reduced pressure to afford the amine as a white fluffy solid (270 mg, 90%), which was used immediately in the subsequent step. To a cold (0 °C), stirred solution of the amine (270 mg, 0.6487 mmol, 1.0 equiv.) in anhydrous pyridine (0.1 M, 7 mL) was added benzoyl chloride (BzCI, 113 pL, 0.973 mmol, 1.5 equiv.) in one portion via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 8 h. The mixture was cooled to 0 °C and quenched by the addition of sat. aq. NaHCO3(30 mL). The biphasic mixture was transferred to a separatory funnel, and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using a gradient of acetone / hexanes (15% — > 30% — > 60%). Appropriate fractions were pooled, and solvent was removed in vacuo to give the product, which contained residual pyridine. The residual pyridine was removed via coevaporation from EtOAc (3x 5 mL). The product was dissolved in minimal Et2O, and the solvent was removed in vacuo to give benzoyl protected amine 21 as a white foam (240 mg, 63% over 3 steps from thymine substrate).

[0241] Data for 21:1H NMR (500 MHz, CD3OD) 58.33 - 8.28 (m, 2H), 7.56 - 7.52 (m, 1 H), 7.45 (dd, J = 8.4, 7.0 Hz, 2H), 7.37 (d, = 1.4 Hz, 1 H), 5.45 (s, 1 H), 4.77 (s, 1 H), 4.40 (d, J = 11.6 Hz, 1H), 4.27 (dd, J = 11.6, 0.9 Hz, 1H), 3.83 (s, 1H), 3.67-3.59 (m, 1H), 3.13 -3.04 (m, 1H), 2.95 -2.77 (m, 2H), 2.16 (d, J = 1.2 Hz, 3H), 1.56 (s, 3H), 1.46 (s, 3H).

[0242] Synthesis of 22

[0243] A sample of compound 21 (90 mg, 0.184 mmol, 1.0 equiv.) was dissolved in a mixture of TFA and water (0.1 M, 1.75 mL: 92 pL) at room temperature and stirred for 15 min. The solvent was then removed under reduced pressure. The residue was dissolved in10102-040W01MeOH and concentrated under reduced pressure to azeotrope off residual TFA. This process was repeated three times to ensure complete removal of TFA, and the crude was carried forward for the next reaction. To a stirred solution of the crude mixture (1.0 equiv.) in pyridine (pre-dried over 3 A MS, 0.4 M, 0.4 mL) at room temperature, DMTrCI (94 mg, 0.276 mmol, 1.5 equiv.) was added. The reaction mixture was stirred for 20 h at room temperature and monitored by TLC. Upon completion, the reaction was quenched with MeOH and stirred for an additional 5-10 min. The solvent was evaporated under ambient air. To prepare for column chromatography, Et3N and silica gel were added to form a slurry. Before elution, the silica gel column was neutralized using 1% Et3N in 100 mL of a 1:4 mixture of acetone and hexane (acetone / hexane). Elution was performed using a gradient of acetone / hexane (30% — > 50% — > 80%). The product 22 was obtained as an off-white fluffy solid (75 mg, 54%).

[0244] Data for 22:1H NMR (500 MHz, Acetone-d6) 58.34 - 8.25 (m, 2H), 7.95 (d, J = 1.2 Hz, 1 H), 7.60 - 7.54 (m, 3H), 7.50 - 7.43 (m, 6H), 7.38 (dd, J = 8.5, 7.0 Hz, 2H), 7.32 -7.27 (m, 1 H), 6.98 - 6.91 (m, 4H), 5.54 (s, 1 H), 4.62 (s, 1 H), 4.53 (s, 1 H), 3.85 (d, = 11.2 Hz, 1H), 3.81 (s, 6H), 3.62 (d, J= 11.1 Hz, 1 H), 3.24 - 3.15(m, 1 H), 2.92 - 2.75 (m, 6H), 2.74 - 2.60 (m, 1H), 1.85 (d, J = 1.1 Hz, 3H) HRMS (ESI+) calcd for [C42H40F2N3O8]: 752.2783 m / z found: 752.2812 m / z.

[0245] Synthesis of 23

[0246] To a cold (0 °C) stirred solution of compound 22 from the previous step (152 mg, 1 eq, 0.210 mmol) and DCI (37.2 mg, 1.5 Eq, 0.315 mmol) in CH2CI2 (1.5 mL) was added 2-Cyanoethyl / V, / V, / V, / V-tetraisopropylphosphorodiamidite (94.9 mg, 0.10 mL, 1.5 Eq, 0.315 mmol) and the reaction was warmed to rt. After 2 h, starting material was consumed as monitored by TLC analysis. The reaction mixture was concentrated dryness in vacuo and the crude product was purified on SiC>2 via flash column chromatography (10 to 30% EtOAc / hexanes + 1% v / v EtsN). Appropriate fractions were pooled, and solvent was removed in vacuo. The resultant solid was dissolved in minimal toluene (~1 mL) and hexanes (~7-8 mL) was added with rapid stirring and a white precipitate rapidly crashes out. The precipitate was collected on a filtering frit with suction. The precipitate was subjected to an identical second reprecipitation to furnish phosphoramidite 23 (121 mg, 63%) as a 7:3 mixture of P(lll) diastereomers and as a white solid.

[0247] Data for 23:1H NMR (600 MHz, C6D6) 58.76 - 8.65 (m, 2H), 7.86 (s, 0.3H), 7.81 (s, 0.7H), 7.75 - 7.71 (m, 0.6H), 7.71 - 7.66 (m, 1,4H), 7.57 (dd, J = 8.9, 7.3 Hz, 1.2H), 7.54 (t, J = 8.3 Hz, 2.8H), 7.31 - 7.20 (m, 6H), 7.14-7.11 (m, 1H), 6.90 -6.82 (m, 4H), 5.48 (s, 0.7H), 5.47 (s, 0.3H), 5.05 (s, 0.7H), 5.00 (s, 0.3H), 4.77 (d, J = 6.8 Hz, 0.3H), 4.72 (d, J =10102-040W017.5 Hz, 0.7H), 3.93 - 3.86 (m, 1 H), 3.83 - 3.76 (m, 1 H), 3.36 - 3.32 (m, 6H), 3.31 - 3.24 (m, 2H), 3.14 - 2.89 (m, 3H), 2.86 - 2.72 (m, 3H), 1.92 (s, 0.9H), 1.87 (s, 2.1 H), 1.71 - 1.64 (m, 0.6H), 1.56 - 1.46 (m, 1.4H), 0.98 (d, J = 6.8 Hz, 4H), 0.90 (d, J = 6.8 Hz, 6H), 0.83 (d, J = 6.8 Hz, 2H).19F NMR (471 MHz, C6D6) 5 -88.03 (d, J = 203.1 Hz), -88.32 (d, J = 203.1 Hz), -97.51 (d, = 202.8 Hz), -97.74 (d, = 200.3 Hz).31P NMR (162 MHz, C6D6) 5 149.53, 148.92. HRMS (ESI+) Anal. Calcd. For C51H57F2N5O9P+[M+H]+952.3856, found 952.3889.

[0248] Synthesis of 251. Zn / Cu, THFCI3C2. Zn, AcOH, THF50%, 2 steps

[0249] To a cold (0 °C), stirred suspension of exocyclic alkene 24 (42.0 mg, 1 Eq, 0.136 mmol) and zinc copper couple (62.3 mg, 7 Eq, 0.954 mmol) in anhydrous THF (1.80 mL) was added TCACI (119 mg, 73 pL, 4.8 Eq, 0.654 mmol) dropwise via syringe pump over 15 minutes. The reaction mixture was then filtered through a short pad of celite into 10 mL of saturated aqueous NaHCOs. The aqueous layer was extracted with EtOAc (3x 10 mL) and the combined organic extracts were dried (MgSC ), filtered, and solvent was removed in vacuo to yield the crude dichloroketone.

[0250] To a rt, stirred suspension of crude dichloroketone (-57 mg, -0.136 mmol, 1 Eq) and zinc dust (89.1 mg, 10 Eq, 1.36 mmol) was added glacial AcOH (123 mg, 0.117 mL, 15 Eq, 2.04 mmol) and the reaction vessel was heated to 55 °C in an oil bath. After 3.5 hours the reaction was complete and was cooled to 0 °C, followed by quenching with EtsN (0.400 mL, 2.86 mmol, 21 Eq.) and the reaction mixture filtered through a celite pad into 10 mL of saturated aqueous NaHCOs. The aqueous layer was extracted with EtOAc (3x 10 mL). The combined organic layers were dried (MgSO4), filtered, and solvent was removed in vacuo. The crude ketone was purified by flash column chromatography (loaded in CH2CI2 and eluted with 40% to70% to 80% to 90% EtOAc / hexanes). Appropriate fractions were pooled, and solvent was removed in vacuo to give cyclobutanone 25 (24.0 mg, 50% over two steps) as a white solid.

[0251] Data for 25:1H NMR (500 MHz, Acetone) 5 10.12 (s, 1H), 7.88 (d, J = 8.1 Hz, 1 H), 5.58 (d, J = 8.6 Hz, 1 H), 5.56 (s, 1 H), 4.70 (d, = 11.7 Hz, 1 H), 4.70 (s, 1 H), 4.40 (d, = 11.5 Hz, 1 H), 4.13 (s, 1H), 3.98 (ddd, J = 18.7, 6.1, 2.3 Hz, 1 H), 3.67 (ddd, J = 17.9, 6.1, 2.0 Hz,10102-040W011 H), 3.12 (ddd, J = 18.1, 7.4, 2.3 Hz, 1 H), 3.05 (ddd, J = 18.4, 7.4, 2.0 Hz, 1H), 1.60 (s, 3H), 1.38 (s, 3H). HRMS (ESI+): Anal. Calcd. for C16H19N2O7+[M+H+] 351.1187, found 351.1198.

[0252] Synthesis of 26a and 26b

[0253] To a cold (0 °C), stirred solution of cyclobutanone 25 (18.6 mg, 0.0531 mmol, 1 Eq) in MeOH (0.53 mL) was added sodium borohydride (4.0 mg, 0.11 mmol, 2 Eq). The mixture was stirred for 20 minutes before quenching with 10 mL of saturated aqueous ammonium chloride. The aqueous layer was extracted with EtOAc (3x 10 mL) and the combined organic layers were dried (MgSC ), filtered, and solvent was removed in vacuo. The crude material was run through a short plug of silica with EtOAc and concentrated to yield the cyclobutanol (12.6 mg, 64%) as a white solid and mixture of diastereomers (7:3).

[0254] Data for 26a+b (7:3 mixture of diastereomers):1H NMR (500 MHz, CDCh) 5 9.19 (s, 1 H), 7.52 - 7.44 (m, 1 H), 5.86 - 5.78 (m, 1H), 5.47 (s, 0.3H), 5.45 (s, 0.7H), 4.67 (s, 1 H), 4.39 -4.34 (m, 1H), 4.17 -4.10 (m, 2H), 3.76 (s, 0.7H), 3.73 (s, 0.3H), 3.47 - 3.37 (m, 0.7H), 3.01 - 2.91 (m, 0.7H), 2.74 - 2.64 (m, 1 H), 2.56 (dt, J = 13.3, 6.5 Hz, 0.3H), 2.50 (dt, J = 12.8, 6.4 Hz, 0.3H), 2.40 - 2.24 (m, 2H), 1.55 (s, 2.1H), 1.54 (s, 0.9H), 1.49 (s, 2.1H), 1.45 (s, 0.9H). LRMS (ESI+): Anal. Calcd. for C16H24N3O7+[M+NH4+] 370.2, found 370.2.

[0255] Synthesis of 27Tf2O, pyridine, CH2CI2, 0 °C then DMF, NaBH4, 0 °C to rt 54%26ab

[0256] To a cold (0 °C), stirred solution of diastereomeric cyclobutanols 26ab (12.6 mg, 1 Eq, 35.8 pmol) and pyridine (8.49 mg, 8.68 pL, 3.0 Eq, 107 pmol) in dry CH2CI2 (0.25 mL) was added Tf2O (15.1 mg, 9.06 pL, 1.5 Eq, 53.6 pmol). The reaction mixture was stirred at 0 °C for 15 min. After this time, DMF (0.25 mL) and sodium borohydride (13.5 mg, 10 Eq, 35810102-040W01pmol) were added in sequence and the reaction mixture was warmed to rt. After stirring for 1 h at rt, the reaction mixture was cooled to 0 °C in an ice-bath and carefully quenched by dropwise addition of sat. aq. NH4CI (~0.5 mL) over 2-3 min. Subsequently EtOAc (0.5 mL) was added and the reaction mixture was allowed to stir at rt for 15 min until all effervescence had ceased. The EtOAc layer was then separated, and the aqueous layer was extracted with further EtOAc (4x 1 mL). The combined organic extracts were dried (Na2SO4), filtered, and solvent was removed in vacuo. The crude product was purified on SiO2via flash column chromatography (20 to 40% acetone / hexanes). Appropriate fractions were pooled, and solvent was removed in vacuo to give 27 (6.5 mg, 54%) as a film on the sides of the flask.

[0257] Data for 27:1H NMR (500 MHz, Acetone) 510.07 (br s, 1H), 7.86 (d, J = 8.2 Hz, 1H), 5.59 (d, J = 8.1 Hz, 1H), 5.43 (s, 1H), 4.64 (d, J = 11.3 Hz, 1H), 4.56 (s, 1H), 4.35 (dd, J = 11.3, 0.9 Hz, 1 H), 3.99 (s, 1 H), 3.02 - 2.95 (m, 1 H), 2.64 - 2.56 (m, 1 H), 2.21 - 2.11 (m, 2H), 1.94 (dtt, = 11.0, 9.8, 6.6 Hz, 1 H), 1.75- 1.63 (m, 1 H), 1.59 (s, 3H), 1.39 (s, 3H).HRMS (ESI+) Anal. Calcd. for C16H21N2O6+[M+H]+337.1394, found 337.1404.

[0258] Example 4

[0259] Synthesis of CB050 and CB054

[0260] Oligonucleotides CB050 and CB054 were synthesized using DMT phosphoroamidites 15 / 17 (CB050) and 20 / 23 (CB054) on an ABI 394 synthesizer using standard phosphoroamidite methods (Table 2). Following global deprotection (28% aqueous NH4OH solution, 24h, rt), the oligos were purified by RP-HPLC.

[0261] Data for CB050 (TOF MS ES) Anal. Calcd. For C176H218N57O86P15S155453.4, found 5455.1.

[0262] Data for CB054 (TOF MS ES') Anal. Calcd. For C176H210F8N57O86P15S155597.4, found 5599.1.Table 2. MALAT1 Targeting Oligonucleotides tested in vivoSEQ ID NO: jO^ Oligonucleotide sequence 6 CB050 5' mC*T*A*g*t*t*c*a*c*t*g*a*a*T*G*mC 3'7 CB054 5' mC*T*A*g*t*t*c*a*c*t*g*a*a*T*G*mC 3'Legend: bold caps = LNA, bold underlined caps for CB050 = 15 (T), 17 (me), for CB054 = 20 (T) and 23 (mC), lowercase = DNA, * = stereorandom PS linkage, mC = 5-methyl cytosine, T = thymine, A = adenine, G = guanine.10102-040W01CB050CB05410102-040W01

[0263] Equimolar amounts of surrogate Malatl RNA 5’ GCAUUCAGUGAACUAG 3’ (SEQ ID NO: 1) and test oligonucleotide were combined in 1 x PBS (pH 7.2) to obtain a final concentration of 1 mM of each strand (3 ml). Duplex samples were then annealed by heating at 90°C, followed by slow cooling to 4°C and storage at 4°C. UV absorbance at 254 nm was recorded at intervals of 30 s as the temperature was raised from 15°C to 95°C at a rate of +0.5°C per min, using a Cary Series UV-Vis spectrophotometer (Agilent Technologies).Absorbance was plotted against the temperature and the Tm values were calculated by taking the first derivative of each curve (Table 3).Table 3. Synthesized MALAT1 Targeting Oligonucleotides with Melting temperatures (Tm). Oligonucleotide Oligonucleotide sequence Tm(°C). ID >CB050 5' mC*T*A*a*t*t*c*a*c*t*g*a*a*T*G*mC 3' | 57.133ONS001 5' mC*T*A*g*t*t*c*a*c*t*g*a*a*T*G*mC 3' 54.347Legend: bold caps - LNA. bold underlined caps - 15 (T) and 17 (mC). lowercase - DNA. * -stereorandom PS linkage, mC = 5-methyl cytosine, T = thymine, A = adenine, G = guanine.

[0264] The test oligonucleotides (6 to 7.5 pL / g) were administered to the tail vein of 6-week old C57BL / 6 mice (5 mice / group) to the indicated final concentration. After 72 hours, the mice were sacrificed and the tissues of interest were collected. RNA extraction (Maxwell simplyRNA for tissue) was performed, followed by the reverse transcription of the extracted RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The mRNA levels of MALAT1 were measured by qPCR (One Step or QuantStudio 6 Pro; SYBR Green PCR Master Mix [Applied Biosystems]). MALAT1 levels were normalizedagainst RPL10 to obtain relative RNA levels using the AACt method, with the following primer sequences for qPCR: (q-Malat1 F: TGG GTT AGA GAA GGC GTG TAC TG (SEQ ID NO: 2); q-Malat1 R: TCA GCG GCA ACT GGG AAA (SEQ ID NO: 3); q-Rpl10 F: TCA TGT CCA TCC GAA CCA AG (SEQ ID NO: 4); q-Rpl10 R: GCA TTA AAC TTG GTG AAG CCC (SEQ ID NO: 5)). The results are shown in Figure 1.

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[0266] All citations are hereby incorporated by reference.10102-040W01

[0267] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. Therefore, although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to,” and the word “comprises” has a corresponding meaning. It is to be however understood that, where the words “comprising” or “comprises,” or a variation having the same root, are used herein, variation or modification to “consisting” or “consists,” which excludes any element, step, or ingredient not specified, or to “consisting essentially of” or “consists essentially of,” which limits to the specified materials or recited steps together with those that do not materially affect the basic and novel characteristics of the claimed invention, is also contemplated. The elements of the present invention as described may be indicated specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications are incorporated herein by reference as if each individual publication was specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples.

Claims

10102-040W01WHAT IS CLAIMED IS:

1. A compound of Formula (I) or a salt thereof:(I)whereinRi and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group;one of R1 or R2is an internucleoside linking group and the other of R1 or R2is H, a terminal group, a linking group, or a conjugate group; orone of R1 or R2is H and the other of R1 or R2is a hydroxyl protecting group; orR1 is a hydroxyl protecting group and R2 is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4 is O, S or NX;X and Y are each independently H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted Ci-Ce alkoxy, OJ1, SJ1, SOJ1, SO2Ji, NJ1J2, N3, CN, C(=O)OJi, C(=O)NJIJ2, C(=O)Ji, O-C(=O)NJIJ2, N(H)C(=NH)NJIJ2, N(H)C(=O)NJIJ2, N=O-J1 or N(H)C(=S)NJIJ2;or X and Y together are =C(q3)(q4), where q3 and q4 are each independently H, halogen, (=0), or optionally substituted Ci-Ce alkyl;Z and Q are each independently CJ1 J2, NJ1 J2, or O;wherein Ji and J2 are each independently H, optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce acyl, optionally substituted C2-C6 alkenyl, C2-C6 alkynyl,10102-040W01optionally substituted Ci-Ce aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.

2. The compound of claim 1 wherein the compound has the chemical structure of Formula (II):IIwhereinRi and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R5is halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted Ci-Ce alkoxy, OJ1, SJ1, SOJ1, SO2Ji, NJ1J2, N3, CN, C(=O)OJI, C(=O)NJIJ2, C(=O)JI, O-C(=O)NJIJ2, N(H)C(=NH)NJIJ2,N(H)C(=O)NJIJ2, N=O-J1 or N(H)C(=S)NJIJ2;X and Y are each independently H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted Ci-Ce alkoxy, OJ1, SJ1, SOJ1, SO2Ji, NJ1J2, N3, CN, C(=O)OJi, C(=O)NJIJ2, C(=O)Ji, O-C(=O)NJIJ2, N(H)C(=NH)NJIJ2, N(H)C(=O)NJIJ2, N=O-J1 or N(H)C(=S)NJIJ2;10102-040W01or X and Y together are =C(q3)(q4), wherein q3 and q4 are each independently H, halogen, (=0), or optionally substituted Ci-Ce alkyl;Z is CJi J2, NJ1 J2, or O; andJi and J2 each independently H, optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce acyl, optionally substituted C2-C6 alkenyl, C2-C6 alkynyl, optionally substituted Ci-Ce aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.

3. The compound of claim 1 wherein the compound has the chemical structure of Formula (III):IIIwhereinR1 and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1 or R2is an internucleoside linking group and the other of R1 or R2 is H, a terminal group, a linking group, or a conjugate group; orone of R1 or R2is H and the other of R1 or R2is a hydroxyl protecting group; orR1 is a hydroxyl protecting group and R2 is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;X and Y are each independently H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted Ci-Ce alkoxy, OJ1, SJ1, SOJ1, S02Ji, NJ1J2, N3, CN, C(=O)OJi, C(=O)NJIJ2, C(=O)Ji, O-C(=O)NJIJ2, N(H)C(=NH)NJIJ2, N(H)C(=O)NJIJ2, N=0-J1 or N(H)C(=S)NJIJ2;10102-040W01or X and Y together are =C(q3)(q4), wherein q3 and q4 are each independently H, halogen, (=0), or optionally substituted Ci-Ce alkyl; andJi and J2 are each independently H, optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce acyl, optionally substituted C2-C6 alkenyl, C2-C6 alkynyl, optionally substituted Ci-Ce aminoalkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted alkylaryl, or a protecting group.

4. The compound of claim 1 wherein the compound has the chemical structure of Formula (IV):J2IVwhereinR1 and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1 or R2is an internucleoside linking group and the other of R1 or R2is H, a terminal group, a linking group, or a conjugate group; orone of R1 or R2is H and the other of R1 or R2is a hydroxyl protecting group; orR1 is a hydroxyl protecting group and R2 is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2- dihydropyrimidine-l-yl group;Ji and J2 are each independently:10102-040W0110102-040W01; and10102-040W015. The compound of claim 1 wherein the compound has the chemical structure of Formula (V):whereinRi and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;X and Y are each independently:O10102-040W01wherein R12 is:10102-040W01; and10102-040W016. The compound of claim 1 wherein the compound has the chemical structure of Formula (VI):whereinRi and R2 are each independently be H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;Ji and Rs are each independently:o10102-040W01wherein R12 is:10102-040W01; and* is “R” or “S” configuration.

7. The compound of claim 1 wherein the compound has the chemical structure of Formula (VII):10102-040W01whereinRi and R2 are each independently H, a hydroxyl protecting group, a terrminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R15 is:q3 q4N-O-J1 orjxjjrvwherein Ji is:10102-040W01q3 and q4 are each independently:owherein R12 is:10102-040W01wherein R9 is:10102-040W01N'\ and* is “R” or “S” configuration.

8. The compound of claim 1 wherein the compound is compound B, Ba, Bb, Be, Bd, Bx, Bxa, Bxb, Bxc, Bxd, C, Ca, Cb, Cc, Cd, D, Da, Db, De, Dd, E, Ea, Eb, Ec, Ed, F, Fa, Fb, Fc, Fd, G, Ga, Gb, Gc, or Gd.

9. The compound of claim 1 wherein the compound is as listed in Table 1.

10. A nucleic acid molecule comprising a compound of any one of claims 1 to 9.

11. The nucleic acid molecule of claim 10, wherein the nucleic acid molecule is an oligomer or a polymer.

12. A method of synthesizing a compound of claim 1, the method comprising:i) providing an alkene of the structure (A):wherein10102-040W01Ri and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 is O, S or NX;ii) performing a heteroatom cycloaddition reaction in the presence of an isocyanate to yield a compound according to claim 1; oriii) performing a cycloaddition on the alkene to yield a dichloroketone;iv) reducing the dichloroketone to a ketone;v) performing a reductive alkylation on the ketone; orvi) performing a cycloaddition with a keteniminium salt on the alkene followed by hydrolysis to yield a ketone, andvi) performing a reductive alkylation, a 1,2 addition, a condensation, or a reductive amination on the ketone to yield a compound according to claim 1.

13. A method of synthesizing a compound of claim 4, the method comprising:i) providing a compound (C)OR2R4whereinRI and R2are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; or10102-040W01one of Ri or R2 is an internucleoside linking group and the other of R1 or R2is H, a terminal group, a linking group, or a conjugate group; orone of R1 or R2is H and the other of R1 or R2is a hydroxyl protecting group; orR1 is a hydroxyl protecting group and R2is a reactive phosphorous group;R3is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4is O, S or NX; andii) performing a reductive alkylation to yield the compound according to claim 4.

14. A method of synthesizing a compound of claim 5, the method comprising:i) providing a compound (C)OR2R4whereinR1 and R2are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1 or R2is an internucleoside linking group and the other of R1 or R2is H, a terminal group, a linking group, or a conjugate group; orone of R1 or R2is H and the other of R1 or R2is a hydroxyl protecting group; orR1 is a hydroxyl protecting group and R2is a reactive phosphorous group;R3is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 is O, S or NX;10102-040W01ii) performing a 1,2 nucleophile addition to yield the compound (D)D,whereinRi and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4 is O, S or NX;X is:10102-040W0110102-040W01; and10102-040W01* is “R” or “S” configuration; andiii) alkylating the compound (D) to yield the compound of claim 5.

15. A method of synthesizing a compound of claim 6, the method comprising:i) providing a compound (A):whereinRi and R2are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4 is O, S or NX;ii) performing a heteroatom cycloaddition reaction in the presence of an isocyanate to yield the compound (E)10102-040W01, andwhereinRi and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of Ri or R2is H and the other of Ri or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3 is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group;R4is O, S or NX; and* is “R” or “S” configuration; andiii) alkylating or acylating the compound (E), to yield the compound of claim 6.

16. A method of synthesizing a compound of claim 7, the method comprising:i) providing a compound (C)10102-040W01whereinRi and R2 are each independently H, a hydroxyl protecting group, a terminal group, or an internucleoside linking group; orone of R1or R2is an internucleoside linking group and the other of R1or R2is H, a terminal group, a linking group, or a conjugate group; orone of R1or R2is H and the other of R1or R2is a hydroxyl protecting group; orR1is a hydroxyl protecting group and R2is a reactive phosphorous group;R3is an optionally substituted purine-9-yl group or an optionally substituted 2-oxo-l,2-dihydropyrimidine-l-yl group; andR4is O, S or NX; andii) performing a condensation reaction on the compound (C), in the presence of a hydroxylamine or performing a Wittig reaction on the compound (C), to yield the compound of claim 7.

17. A method of preparing an oligomer or a polymer, the method comprising;i) providing a compound according to any one of claims 1 to 9, andii) reacting the compound with one or more nucleotides or nucleic acid analogues under suitable synthetic conditions to yield an oligomer or a polymer.

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