Cap analogs and methods of use thereof

Abstract

Described herein are novel cap analogs and compositions, and methods of using the same. Also described herein is an RNA molecule comprising a 5'-cap, wherein the 5'-cap includes a cap analog as described herein. Methods of inducing a therapeutic effect in a subject are also described herein, the methods including a step of administering to the subject a cap analog or RNA molecule comprising the cap analog as described herein.

Description

CAP ANALOGS AND METHODS OF USE THEREOF CROSS-REFERENCE TO PRIORITY APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 530,592, filed August 3, 2023, and U.S. Provisional Application No.63 / 651,047, filed May 23, 2024, the contents of which are incorporated herein by reference in their entireties. TECHNICAL FIELD

[0002] The field of this invention relates to cap analogs and methods of using the same. Additionally, the invention relates to using the cap analogs for preparing 5’-capped RNAs, and the use of these analogs for inhibiting translation. BACKGROUND

[0003] Many types of cancer cells overexpress eIF4E, which can lead to increased expression of proteins that promote oncogenesis and metastasis (De Benedetti et al., (2004) Oncogene 23:3189-3199). Reducing eIF4E expression with antisense oligonucleotides or an eIF4E repressor can inhibit tumor growth and oncogenesis (Graff J. et al., (2007) J. Clin. Investigation 117:2638-2648; Herbert T., (2000) Curr. Biol.10:793-796).

[0004] Another way to inhibit eIF4E activity (inhibit translational activity of mRNAs) is to use synthetic cap analogs that are inhibitors of eIF4E. Several dinucleotide cap analogs have been shown to be inhibitors of eIF4E (Cai et al., (1999) Biochemistry 38:8538-8547; Kowalska et al., (2008) RNA 14:1119-1131; International Patent Publication WO2009 / 149253). BRIEF SUMMARY OF THE INVENTION

[0005] Described herein are cap analogs which may be inhibitors of eIF4E. These cap analogs can be used for preparing 5’-capped mRNAs, which are translational inhibitors. The advantage of trinucleotide cap analogs which are inhibitors of eIF4E over the dinucleotide cap analogs which are inhibitors of eIF4E, is that trinucleotide cap analogs are able to produce more 5’-capped mRNAs than dinucleotide cap analogs (via transcription). mRNAs capped with trinucleotide cap analogs can then be delivered to cells in order to inhibit or significantly reducetranslation.

[0006] In an aspect, provided herein is a compound of formula (I):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: N1is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, natural, modified, or unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside; each B1is independently a natural, modified, or unnatural nucleoside base; each X1is independently -O-, -CH2-, -CX2-, -CHX-, -N(R101)-, -BH-, or -S-; each Y1is independently O, S, or Se; each Z1is independently -OH, -SH, -BH3, substituted or unsubstituted -O-alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted -O-aryl;each R1is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR1A, -NR1AR1B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R5is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR5A, -NR5AR5B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R1and R5together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene; each R1A, R1B, R5A, and R5Bis independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1Aand R1Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R5Aand R5Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R101is hydrogen, oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H,-NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2,-OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; n is an integer from 0 to 3; m is an integer from 0 to 8; and X is -Cl, -Br, -I or –F.

[0007] In embodiments, provided herein is a compound of formula (IA):(IA), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0008] In embodiments, provided herein is a compound of formula (IB):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; R4is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR4A, -NR4AR4B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; each R2A, R3A, R4A, and R4B, is independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R4Aand R4Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

[0009] In embodiments, provided herein is a compound of formula (IC):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0010] In embodiments, provided herein is a compound of formula (ID):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0011] In embodiments, provided herein is a compound of formula (IE):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0012] In embodiments, provided herein is a compound of formula (IF):(IF), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0013] In embodiments, provided herein is a compound of formula (IG):(IG), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceuticallyacceptable salt, solvate, or hydrate thereof.

[0014] In embodiments, provided herein is a compound of formula (IH):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; and each R2Aand R3Ais independently hydrogen, -CX3, -CHX2, -CH2X,-C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^ ^NHNH2, ^ONH2,^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0015] In embodiments, N1is adenosine (A), cytidine (C), or uridine (U). In embodiments, N1is A or U. In embodiments, N1is A.

[0016] In embodiments, N1is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. In embodiments, N1is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted alkyl. In embodiments, N1is substituted alkyl or substituted heteroaryl. In embodiments, N1is substituted alkyl or substituted heteroaryl. In embodiments, N1i

[0017] In embodiments, each B1is independently A, m6A, G, C, or U. In embodiments, each B1is independently G. In embodiments, each B1is independently U.

[0018] In embodiments, each R1is independently hydrogen, halogen, or -OR1A. In embodiments, each R1is independently hydroxy, methoxy, ethoxy, propoxy, butoxy, or t-butoxy. In embodiments, each R1is independently methoxy.

[0019] In embodiments, R2is independently hydrogen or substituted or unsubstituted alkyl. In embodiments, R2is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, or hexyl. In embodiments, R2is independently hydrogen or methyl.

[0020] In embodiments, R3is independently hydrogen or substituted or unsubstituted alkyl. In embodiments, R3is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, or hexyl. In embodiments, R3is independently hydrogen ormethyl.

[0021] In embodiments, R4is hydrogen, halogen, or -NR4AR4B. In embodiments, R4is hydrogen, -F, -NHMe, -NH2, or -NMe2. In embodiments, R4is hydrogen or -NHMe. In embodiments, R4is hydrogen.

[0022] In embodiments, each R5is independently hydrogen. In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a locked nucleic acid (LNA).

[0023] In embodiments, each X1is independently -O-, -CH2-, -CF2-, or -S-. In embodiments, each X1is independently -O-.

[0024] In embodiments, each Y1is independently O or S. In embodiments, each Y1is independently O.

[0025] In embodiments, each Z1is independently -OH or -SH. In embodiments, each Z1is independently -OH.

[0026] In embodiments, m is 0 or 1. In embodiments, n is 1.

[0027] In an aspect, provided herein is a compound of the following structure:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0028] In an aspect, provided herein is a compound of the following structure:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0029] In an aspect, provided herein is a compound of the following structure: Ph-4-Bu-pppA2’OMepG:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0030] In an aspect, provided herein is a compound of the following structure: Thiamine-pppA2’OMepG:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof

[0031] In an aspect, provided herein is a compound of the following structure:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0032] In an aspect, provided herein is a compound of the following structure:ApppA2’OMepG:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0033] In embodiments, the pharmaceutically acceptable salt of any one of the compounds described herein is a sodium salt, a lithium salt, or a potassium salt. In embodiments, the pharmaceutically acceptable salt of any one of the compounds described herein is a sodium salt.

[0034] In an aspect, provided herein is an RNA molecule comprising a 5’-cap, wherein the 5’-cap comprises any one of the compounds described herein, including in embodiments. In embodiments, the RNA molecule is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule.

[0035] In an aspect, provided herein is a pharmaceutical composition comprising an RNA molecule comprising a 5’-cap, wherein the 5’-cap comprises any one of the compounds described herein, including in embodiments, and a pharmaceutically acceptable excipient.

[0036] In an aspect, provided herein is a pharmaceutical composition comprising any one of the compounds described herein, including in embodiments and a pharmaceutically acceptable excipient.

[0037] In an aspect, provided herein is an initiating capped oligonucleotide primer of formula Apppm6A2’OMepG. In an aspect, provided herein is an initiating capped oligonucleotideprimer of formula Ph-4-Bu-pppm6A2’OMepG. In an aspect, provided herein is an initiating capped oligonucleotide primer of formula Ph-4-Bu-pppA2’OMepG. In an aspect, provided herein is an initiating capped oligonucleotide primer of formula Thiamine-pppA2’OMepG. In an aspect, provided herein is an initiating capped oligonucleotide primer of formula Thiamine- pppm6A2’OMepG. In an aspect, provided herein is an initiating capped oligonucleotide primer of formula ApppA2’OMepG.

[0038] In an aspect, provided herein is an RNA molecule comprising any one of the initiating capped oligonucleotide primers, stereoisomer, or a pharmaceutically acceptable salt thereof, as described herein, including in embodiments. In embodiments, the pharmaceutically acceptable salt is a sodium salt, a lithium salt, or a potassium salt. In embodiments, the pharmaceutically acceptable salt is a sodium salt. In embodiments, the RNA molecule is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule.

[0039] In an aspect, provided herein is a pharmaceutical composition comprising an RNA molecule comprising any one of the initiating capped oligonucleotide primers, stereoisomer, or a pharmaceutically acceptable salt thereof, as described herein, including in embodiments, and a pharmaceutically acceptable excipient.

[0040] In an aspect, provided herein is a method of inducing a therapeutic effect in a subject, comprising administering to the subject any one of the RNA molecules described herein, including in embodiments. In an aspect, provided herein is a method of administering to a subject a therapeutic dose unit of an mRNA molecule comprising a 5’-cap, wherein the 5’-cap comprises any one of the compounds described herein, including in embodiments.

[0041] In an aspect, provided herein is a method of inhibiting eIF4E in a mammal comprising administering any one of the compounds, RNA molecules, or pharmaceutical compositions described herein, including in embodiments.

[0042] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (I), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: N1is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, natural, modified, or unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside; each B1is independently a natural, modified, or unnatural nucleoside base; each X1is independently -O-, -CH2-, -CX2-, -CHX-, -N(R101)-, -BH-, or -S-; each Y1is independently O, S, or Se; each Z1is independently -OH, -SH, -BH3, substituted or unsubstituted -O-alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted -O-aryl; each R1is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR1A, -NR1AR1B, -COOH, -CONH2,-NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R5is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR5A, -NR5AR5B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R1and R5together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene; each R1A, R1B, R5A, and R5Bis independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1Aand R1Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R5Aand R5Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R101is hydrogen, oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2,-OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; n is an integer from 0 to 3; m is an integer from 0 to 8; and X is -Cl, -Br, -I or –F; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0043] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IA), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template

[0044] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IB)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; R4is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR4A, -NR4AR4B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; each R2A, R3A, R4A, and R4B, is independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R4Aand R4Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0045] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IC)(IC); or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase underconditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template

[0046] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (ID)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template

[0047] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IE)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0048] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IF) (IF);or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0049] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IG)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0050] In an aspect, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IH)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; and each R2Aand R3Ais independently hydrogen, -CX3, -CHX2, -CH2X,-C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^ ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG.1 shows translation of EGFP encoding mRNA made with different cap analogs 4, 24, 48, and 72 hours post transfection in Expi293F cells. DETAILED DESCRIPTION OF THE INVENTION Definitions:

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, or other publication that is herein incorporated by reference, the definition set forth in this section prevails over the definition incorporated herein by reference.

[0053] As used herein, “a” or “an” means “at least one” or “one or more”. For example, reference to “a transcript” may include a plurality of transcripts.

[0054] “Or” is used in the inclusive sense, i.e., equivalent to “and / or”, unless the context clearly indicates otherwise.

[0055] The use of any and all examples or exemplary language (e.g., “such as”) provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0056] The terms “may,” “may be,” “can,” and “can be,” and related terms are intended to convey that the subject matter involved is optional (that is, the subject matter is present in some examples and is not present in other examples), not a reference to a capability of the subject matter or to a probability, unless the context clearly indicates otherwise.

[0057] The terms “optional” and “optionally” mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present as well as instances where it does not occur or is not present.

[0058] As used herein, the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value.

[0059] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0060] Ranges include the endpoints of the range. For example, “between 0 and 2” includes 0, 1, 2, and (unless the context requires otherwise) fractional values greater than 0 and less than 2.

[0061] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0062] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals. The alkyl may include a designated number of carbons (e.g., C1-C10means one to ten carbons). Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n- pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-). An alkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. An alkyl moietymay be fully saturated. An alkenyl may include more than one double bond and / or one or more triple bonds in addition to the one or more double bonds. An alkynyl may include more than one triple bond and / or one or more double bonds in addition to the one or more triple bonds.

[0063] The term “alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, - CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0064] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is an uncyclized chain. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, - CH2-S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N- OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. A heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P). The term “heteroalkenyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond. A heteroalkenyl may optionally include more than one double bond and / or one or more triple bonds in additional to the one or more double bonds. The term “heteroalkynyl,” by itself or in combination with another term, means, unless otherwisestated, a heteroalkyl including at least one triple bond. A heteroalkynyl may optionally include more than one triple bond and / or one or more double bonds in additional to the one or more triple bonds.

[0065] Similarly, the term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as - C(O)R', -C(O)NR', -NR'R'', -OR', -SR', and / or -SO2R'. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R'' or the like, it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R'' or the like.

[0066] The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0067] In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or a multicyclic cycloalkyl ring system. In embodiments, monocyclic ring systems are cyclichydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic. In embodiments, cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings. In embodiments, bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH2)w , where w is 1, 2, or 3). Representative examples of bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. In embodiments, fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring. In embodiments, cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia. In embodiments, the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia. In embodiments, multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring. In embodiments, multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independentlyselected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclic cycloalkyl groups include, but are not limited to tetradecahydrophenanthrenyl, perhydrophenothiazin-1-yl, and perhydrophenoxazin-1-yl.

[0068] In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl” is used in accordance with its plain ordinary meaning. In embodiments, a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system. In embodiments, monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond), but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In embodiments, bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings. In embodiments, bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CH2)w, where w is 1, 2, or 3). Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct 2 enyl. In embodiments, fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fused bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia. In embodiments, multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring. In embodiments, multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, abicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.

[0069] In embodiments, a heterocycloalkyl is a heterocyclyl. The term “heterocyclyl” as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle. The heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, P, and S where the ring is saturated or unsaturated, but not aromatic. The 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N, P, and S. The 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N, P, and S. The 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N, P, and S. The heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle. Representative examples of heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1- dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl. The heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system. Representative examples of bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl. In embodiments, heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fusedto a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia. Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. The multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring. In embodiments, multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclic heterocyclyl groups include, but are not limited to 10H-phenothiazin-10-yl, 9,10- dihydroacridin-9-yl, 9,10-dihydroacridin-10-yl, 10H-phenoxazin-10-yl, 10,11-dihydro-5H- dibenzo[b,f]azepin-5-yl, 1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H- benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.

[0070] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0071] The term “acyl” means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0072] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5- fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non- limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1- naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.

[0073] A fused ring heterocyloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fusedring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl- cycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.

[0074] Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom. The individual rings within spirocyclic rings may be identical or different. Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings. Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkyl rings). Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring. When referring to a spirocyclic ring system, substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.

[0075] The symbol “ ” or a dash (“-“) denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

[0076] The term “oxo,” as used herein, means an oxygen that is double bonded to a carbon atom.

[0077] The term “alkylsulfonyl,” as used herein, means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., “C1-C4 alkylsulfonyl”).

[0078] The term “alkylarylene” as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene group has the formula:.

[0079] An alkylarylene moiety may be substituted (e.g. with a substituent group) on thealkylene moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) with halogen, oxo, -N3, -CF3, - CCl3, -CBr3, -CI3, -CN, -CHO, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2CH3 -SO3H, , - OSO3H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, substituted or unsubstituted C1-C5alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl). In embodiments, the alkylarylene is unsubstituted.

[0080] Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,” “heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0081] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, - SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'- C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', - S(O)2NR'R'', -NRSO2R', ^NR'NR''R''', ^ONR'R'', ^NR'C(O)NR''NR'''R'''', -CN, -NO2, - NR'SO2R'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such radical. R, R', R'', R''', and R'''' each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R'', R''', and R'''' group when more than one of these groups is present. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).

[0082] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'- C(O)NR''R''', -NR''C(O)2R', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', - S(O)2NR'R'', -NRSO2R', ^NR'NR''R''', ^ONR'R'', ^NR'C(O)NR''NR'''R'''', -CN, -NO2, -R', -N3, - CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, -NR'SO2R'', -NR'C(O)R'', -NR'C(O)- OR'', -NR'OR'', in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R'', R''', and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R'', R''', and R'''' groups when more than one of these groups is present.

[0083] Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent). In such a case, the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings). When a substituent is attached to a ring, but not a specific atom (a floating substituent), and a subscript for the substituent is an integer greater than one, the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different. Where a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent), the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency. Where a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms. Where the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.

[0084] Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring- forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0085] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR')q-U-, wherein T and U are independently - NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, - S-, -S(O) -, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X'- (C''R''R''')d-, where s and d are independently integers of from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or - S(O)2NR'-. The substituents R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0086] As used herein, the terms “heteroatom” or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0087] A “substituent group,” as used herein, means a group selected from the following moieties:

[0088] (A) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,-OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10aryl, C10aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and

[0089] (B) alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10aryl, C10aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from:

[0090] (i) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,-OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and

[0091] (ii) alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 memberedheterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from:

[0092] (a) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8cycloalkyl, C3-C6cycloalkyl, or C5-C6cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and

[0093] (b) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8cycloalkyl, C3-C6cycloalkyl, or C5-C6cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from: oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, - SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3,-OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6cycloalkyl, or C5-C6cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 memberedheteroaryl).

[0094] A “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.

[0095] A “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 6 membered heteroaryl.

[0096] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

[0097] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C20alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.

[0098] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In some embodiments, the compound is a chemical species set forth herein.

[0099] In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., is an unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, and / or unsubstituted heteroarylene, respectively). In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene) is substituted (e.g., is a substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene, respectively).

[0100] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent group, wherein if the substituted moiety is substituted with a plurality of substituent groups, each substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of substituent groups, each substituent group is different.

[0101] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one size-limited substituent group, wherein if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of size-limitedsubstituent groups, each size-limited substituent group is different.

[0102] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one lower substituent group, wherein if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group is different.

[0103] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size- limited substituent group, and / or lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group is different.

[0104] Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those that are known in art to be too unstable to synthesize and / or isolate. The present disclosure is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.In certain embodiments, “optically active” and “enantiomerically active” refer to a collection of molecules, which has an enantiomeric excess of no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, no less than about 91%, no less than about 92%, no less than about 93%, no less than about 94%, no less than about 95%, no less than about 96%, no less than about 97%, no less than about 98%, no less than about 99%, no less than about 99.5%, or no less than about 99.8%. In certain embodiments, the compound comprises about 95% or more of one enantiomer and about 5% or less of the other enantiomer based on the total weight of the racemate in question.

[0105] As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

[0106] The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

[0107] It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure.

[0108] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[0109] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by13C- or14C-enriched carbon are within the scope of this disclosure.

[0110] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I), or carbon-14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the presentdisclosure.

[0111] It should be noted that throughout the application that alternatives are written in Markush groups, for example, each amino acid position that contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered separately, thereby comprising another embodiment, and the Markush group is not to be read as a single unit.

[0112] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0113] The term “solvate” refers to a complex or aggregate formed by one or more molecules of a solute, e.g., a compound provided herein, and one or more molecules of a solvent, which present in stoichiometric or non-stoichiometric amount. Suitable solvents include, but are not limited to, water, methanol, ethanol, n-propanol, isopropanol, and acetic acid. In certain embodiments, the solvent is pharmaceutically acceptable. In one embodiment, the complex or aggregate is in a crystalline form. In another embodiment, the complex or aggregate is in a noncrystalline form. Where the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, a hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate.

[0114] The term “salt thereof” means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0115] The term “pharmaceutically acceptable salt” means a salt which is acceptable for administration to a patient, such as a mammal, such as human (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and when the molecule contains a basic functionality, salts of organic or inorganic acids. The pharmaceutically acceptable salts of the compounds described herein are suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein. These salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like. Salts may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See S.M. Barge et al., J. Pharm. Sci. (1977) 66, 1; and Remington: The Science and Practice of Pharmacy, 23d Edition, Adejare et al. eds., Academic Press (2020); which are incorporated herein by reference in their entireties.)

[0116] As used herein, the term “cap analog” means a structural derivative of an RNA cap.

[0117] As used herein, the term “complement,” “complementary,” or “complementarity” refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are, generally, A and T (or A and U), and G and C. Complementarity, for example, between a capped oligonucleotide primer and a DNA template, may be “complete” or "total" where all of the nucleotide bases of two nucleic acid strands are matched according to recognized base pairing rules, it may be “partial” in which only some of the nucleotide bases of an initiating capped oligonucleotide primer and a DNA template are matched according to recognized base pairing rules, or it may be “absent” where none of the nucleotide bases of two nucleic acidstrands are matched according to recognized base pairing rules. Complementarity can also be “substantial complementarity” where the nucleotide bases of two nucleic acids are matched according to recognized base pairing rules, but include one or more mismatches (e.g., 1, 2, 3, 4) from total complementarity.

[0118] As used herein, a “deoxyribonuclease (DNase)” is an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA.

[0119] As used herein, the term “hybridize” or “specifically hybridize” refers to a process where initiating trinucleotide primer anneals to a DNA template under appropriately stringent conditions during a transcription reaction. Hybridizations to DNA are conducted with an initiating capped oligonucleotide primer which, in certain embodiments, is 3-10 nucleotides in length including the 5’-5’ inverted cap structure. Nucleic acid hybridization techniques are well known in the art (e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.(1989); Ausubel, F.M., et al., Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, N.J. (1994)).

[0120] As used herein, the term “impurities” refers to substances which differ from the chemical composition of the target material (e.g., mRNA transcripts). Impurities are also referred to as contaminants.

[0121] “Inorganic pyrophosphatase” refers to an enzyme that catalyzes the conversion of one ion of pyrophosphate to two phosphate ions, thus inhibiting aggregation and in some instances preventing interaction of pyrophosphate with magnesium ions during T7 transcription reactions.

[0122] As used herein, the term “internucleotide linkage” refers to the bond or bonds that connect two nucleosides of an oligonucleotide or nucleic acid and may be a natural phosphodiester linkage or modified linkage.

[0123] As used herein, the term “in vitro” refers to a process that takes place outside a living organism (e.g., a multi-cellular organism, such as a human or a non-human animal), for example, in a test tube, culture dish, or elsewhere outside a living organism.

[0124] As used herein, the term “in vivo” refers to events that occur within a living organism.

[0125] As used herein the term “in vivo assays” refer to methods used to detect and / or measure capacity of one or more of the compounds or molecules including the compounds (e.g.,mRNA molecules in, for example, a therapeutic dose) to increase or decrease a property relative to a control (e.g., biomarker levels). Optionally, in vivo assays as described herein can be used to determine a subject’s tolerability levels to a given compound or molecule. Exemplary measurements for assessing tolerability include one or more of body weight, organ weight, aspartate aminotransferase (AST) levels, alanine transaminase (ALT) levels, C-reactive protein (CRP) levels, procalcitonin (PCT) levels, interleukin-6 (IL-6) levels, erythrocyte sedimentation rate (ESR), serum amyloid A levels, and serum ferritin levels.

[0126] As used herein, “locked nucleic acid” (LNA) means a ribonucleotide having a bridge between the 2’O and 4’C methylene bicyclonucleotide monomers. An LNA moiety can have the following structure:.

[0127] As used herein, “messenger RNA transcript,” or “mRNA transcript,” is a transcript transcribed from a DNA template encoding a desired polypeptide. The mRNA transcript may contain coding and non-coding regions. For example, the DNA template can comprise an RNA polymerase promoter sequence, a 5’ UTR sequence, an open reading frame, and a 3’ UTR sequence. In some examples, the DNA template also comprises a nucleic acid sequence encoding a poly(A) tail.

[0128] As used herein, the term “nucleoside” refers to a nitrogenous base linked to a 5- carbon sugar (e.g., ribose or deoxyribose). The term includes all nucleosides, including all forms of nucleoside bases and furanoses. The term “natural nucleoside” refers to adenosine, guanosine, cytidine, uridine and thymidine.

[0129] According to Aduri et al (Aduri, R. et al., AMBER force field parameters for the naturally occurring modified nucleotides in RNA. Journal of Chemical Theory and Computation. 2006.3(4):1464-75) there are 107 naturally occurring nucleosides, including 1-methyladenosine, 2-methylthio-N6-hydroxynorvalyl carbamoyladenosine, 2-methyladenosine, 2-O- ribosylphosphate adenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6-glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6- threonylcarbamoyladenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,N6-hydroxynorvalylcarbamoyladenosine, 1,2-O-dimethyladenosine, N6,2-O-dimethyladenosine, 2-O-methyladenosine, N6,N6,O-2-trimethyladenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6- isopentenyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, 2-thiocytidine, 3- methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5- hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5-formyl-2-O-methylcytidine, 5,2-O-dimethylcytidine, 2-O-methylcytidine, N4,2-O-dimethylcytidine, N4,N4,2-O- trimethylcytidine, 1-methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2-O- ribosylphosphate guanosine, 7-methylguanosine, under modified hydroxywybutosine, 7- aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2,N2-dimethylguanosine, 4- demethylwyosine, epoxyqueuosine, hydroxywybutosine, isowyosine, N2,7,2-O- trimethylguanosine, N2,2-O-dimethylguanosine, 1,2-O-dimethylguanosine, 2-O- methylguanosine, N2,N2,2-O-trimethylguanosine, N2,N2,7-trimethylguanosine, peroxywybutosine, galactosyl-queuosine, mannosyl-queuosine, queuosine, archaeosine, wybutosine, methylwyosine, wyosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3- methyluridine, 4-thiouridine, 5-methyl-2-thiouridine, 5-methylaminomethyluridine, 5- carboxymethyluridine, 5-carboxymethylaminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinomethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5-methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5- (carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5- (isopentenylaminomethyl)-2-thiouridine, 3,2-O-dimethyluridine, 5-carboxymethylaminomethyl- 2-O-methyluridine, 5-carbamoylmethyl-2-O-methyluridine, 5-methoxycarbonylmethyl-2-O- methyluridine, 5-(isopentenylaminomethyl)-2-O-methyluridine, 5,2-O-dimethyluridine, 2-O- methyluridine, 2-thio-2-O-methyluridine, uridine 5-oxyacetic acid, 5- methoxycarbonylmethyluridine, uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5- aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl- 2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1-methylpseudouridine, 3- methylpseudouridine, 2-O-methylpseudouridine, inosine, 1-methylinosine, 1,2-O- dimethylinosine and 2-O-methylinosine. Each of these or the modified nucleobase thereof may be components of nucleic acids of the present invention.

[0130] All other nucleosides (not including the ones described above as natural nucleosides or modified natural nucleosides) are unnatural nucleosides.

[0131] As used herein, the term “nucleoside base” refers to a nitrogenous base. A “natural nucleoside base” includes purine and pyrimidine rings. Purine rings include, for example, adenine and guanine. Pyrimidine rings include, for example, cytosine, thymine, and uracil.

[0132] As used herein, the term “modified nucleoside base” describes natural modified nucleoside bases, including but is not limited to, for example, pseudouracil, 5-methylcytosine, N6-methyladenine, inosine, 5-hydroxymethylcytosine, 5-carboxylcytosine, N4-acetylcytosine, N4-methylcytosine, n1-methyladenine, N2, N2-dimethylguanine and the like. Also, see naturally occurring modified nucleosides above.

[0133] As used herein, the term “unnatural nucleoside base” refers to all nucleoside bases that are not natural (whether modified or not; see naturally occurring modified nucleosides above) including but is not limited to, for example, N1-methylpseudouracil, 7-deazaadenine, 2- aminoadenine, 5-methylisocytosine, 5-fluorouracil, 5-bromouracil, 5-iodouracil, 2-thiouracil, 2- methylthioadenine, 2-thio-5-methyluracil, 2-amino-6-methylthiopurine and the like. Natural nucleosides are described above,

[0134] As used herein, the terms “nucleoside analogs,” “modified nucleosides,” or “nucleoside derivatives” include synthetic nucleosides as described herein. Nucleoside derivatives also include nucleosides having modified base or / and sugar moieties, with or without protecting groups and include, for example, 2’-deoxy-2’-fluorouridine, 5-fluorouridine and the like. The compounds and methods provided herein include such base rings and synthetic analogs thereof, as well as unnatural heterocycle-substituted base sugars, and acyclic substituted base sugars. Other nucleoside derivatives that may be utilized with the present disclosure include, for example, LNA nucleosides, halogen-substituted purines (e.g., 6-fluoropurine), halogen- substituted pyrimidines, N6-ethyladenine, N4-(alkyl)-cytosines, 5-ethylcytosine, and the like (U.S. Patent No.6,762,298).

[0135] As used herein, the term “modified or unmodified G nucleoside” refers to a G nucleoside modified at any position or a natural (i.e. unmodified) G nucleoside. Some possible modification positions are shown with R, R1, R2, and R3(other modifications are also possible):can be any chemical group.

[0136] As used herein, the terms “nucleoside triphosphate,” “nucleoside 5’ triphosphate” or “NTP” refer to a nucleoside linked to three phosphate groups. The term encompasses natural NTPs (for example, adenosine triphosphate (ATP), uridine triphosphate (UTP), guanine triphosphate (GTP), and cytosine triphosphate (CTP)) as well as modified NTPs.

[0137] As used herein, the term “modified NTP” refers to a nucleoside 5’-triphosphate having a chemical moiety group bound at any position or substituted at any position, including the sugar, base, triphosphate chain, or any combination of these three locations. Optionally, the chemical moiety group may be a group of any nature compatible with the process of transcription. Examples of such NTPs include inosine triphosphate, dihydrouridine triphosphate, 2’-fluoro-2’-deoxycytidine triphosphate, pseudouridine triphosphate, N1-methylpseudouridine triphosphate, and 5-methyluridine triphosphate, and can be found, for example in “Nucleoside Triphosphates and Their Analogs: Chemistry, Biotechnology and Biological Applications,” Vaghefi, M., ed., Taylor and Francis, Boca Raton (2005).

[0138] As used herein, the term “modified oligonucleotide” or “modified trinucleotide” includes, for example, an oligonucleotide containing a modified nucleoside, a modified internucleotide linkage, or having any combination of modified nucleosides and internucleotide linkages. Non-limiting examples of internucleotide linkage modifications include, but are not limited to, phosphorothioate, phosphotriester and methylphosphonate derivatives (Stec, W.J., et al., Chem. Int. Ed. Engl., 33:709-722 (1994); Lebedev, A.V., et al., E., Perspect. Drug Discov. Des., 4:17-40 (1996); and Zon, et al., U.S. Patent Application No.20070281308). Other examples of internucleotide linkage modifications may be found in Waldner, et al., Bioorg. Med. Chem. Letters 6:2363-2366 (1996).

[0139] As used herein, “oligo dT purification” is an affinity chromatography method for purification of mRNA comprising or including a poly-A tail.

[0140] As used herein, “phosphorothioate linkage” refers to a linkage betweennucleosides in which the phosphorodiester linkage is modified by replacing one of the oxygen atoms, connected to a phosphorus atom, with a sulfur atom.

[0141] A “primary RNA” or “primary RNA transcript” means the RNA molecule that is newly synthesized by an RNA polymerase in vitro and which RNA molecule has a triphosphate on the 5′-carbon of its most 5′ nucleotide.

[0142] As used herein, the term “prematurely aborted RNA transcript” refers to incomplete products of an in vitro transcription reaction. Prematurely aborted RNA sequences may be any length that is less than the intended length of the desired transcriptional product.

[0143] The term “promoter” as used herein refers to a nucleotide sequence in a DNA template that directs and controls the initiation of transcription of a particular DNA sequence. Promoters are typically immediately adjacent to (or partially overlap with) the DNA sequence to be transcribed. Promoter sequences are typically located directly upstream or at the 5' end of the transcription initiation site. Nucleotide positions in the promoter are designated relative to the transcriptional start site, where transcription of DNA begins (position +1).

[0144] As used herein, the term “purified” or “purify” refers to separating a substance from at least some of the components (e.g., impurities or contaminants) with which it was associated when initially produced. For example, RNA transcripts are purified by removal of contaminating proteins or other undesired nucleic acid species (e.g., double-stranded RNA, DNA, and / or incomplete or aborted RNA transcripts). Purified substances (e.g., capped mRNA transcripts) can be separated from 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% of the other components with which they were initially associated.

[0145] As used herein, the term “RNase inhibitor” or “ribonuclease inhibitor” refers to a protein that inhibits RNAse activity for example, during an in vitro transcription reaction.

[0146] As used herein, the term “RNA polymerase” refers to an enzyme that synthesizes RNA using a DNA template. For in vitro transcription methods, single subunit phage RNA polymerases derived from T7, T3, SP6, K1-5, K1E, K1F or K11 bacteriophages, or variants thereof, are typically used. This family of polymerases has simple, minimal promoter sequences of about 17 nucleotides which require no accessory proteins and have minimal constraints of the initiating nucleotide sequence.

[0147] As used herein, “self-amplifying RNA,” or “saRNA,” is a linear, single-strandedRNA molecule that encodes the gene of interest. saRNA is a type of mRNA, but also includes non-structural proteins that encode a viral replicase. The viral replicase enables the RNA to self- replicate once delivered into the cell.

[0148] As used herein, the term “specific” when used in reference to an initiating trinucleotide primer sequence and its ability to hybridize to a DNA template is a sequence that has at least 50% sequence identity with a portion of the DNA template when the initiating trinucleotide primer and DNA strand are aligned. Higher levels of sequence identity include at least 66%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, and optionally 100% sequence identity.

[0149] As used herein, the term “substantially free” refers to a state in which relatively little or no amount of an undesired substance (e.g., prematurely aborted RNA sequences, DNA, and / or double-stranded RNA) is present in a sample. “Substantially free of impurities” means impurities are present at a level less than approximately 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (w / w) in a sample. For example, “substantially free of double-stranded RNA” means double-stranded RNA is present at a level less than approximately 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (w / w) in a sample.

[0150] As used herein, “tangential flow filtration (TFF)” is a type of filtration wherein the material to be filtered is passed tangentially across a filter rather than through it. In TFF, undesired permeate passes through the filter, while the desired retentate passes along the filter and is collected downstream. In TFF, the desired material is typically contained in the retentate, which is the opposite of what is encountered when performing traditional membrane or dead-end filtration.

[0151] As used herein, the term “transcription” refers to enzymatically making or synthesizing RNA that is complementary to a DNA template, thereby producing a number of RNA copies of a DNA sequence. The RNA molecule synthesized in a transcription reaction is an “RNA transcript,” “primary transcript,” or “transcript.” Transcription reactions involving the compositions and methods provided herein employ initiating capped oligonucleotide primers described herein. Transcription of a DNA template may be exponential, nonlinear or linear. A DNA template may be a double-stranded linear DNA, a partially double-stranded linear DNA, circular double-stranded DNA, DNA plasmid, PCR amplified product, or a modified nucleic acidtemplate that is compatible with RNA polymerase.

[0152] As used herein, the terms “universal base,” “degenerate base,” “universal base analog” and “degenerate base analog” include, for example, a nucleoside analog with an artificial base which is, in certain embodiments, recognizable by RNA polymerase as a substitute for one of the natural NTPs (e.g., ATP, UTP, CTP and GTP) or other specific NTP. Universal bases or degenerate bases are disclosed in Loakes, D., Nucleic Acids Res., 29:2437-2447 (2001); Crey- Desbiolles, C., et. al., Nucleic Acids Res., 33:1532–1543 (2005); Kincaid, K., et. al., Nucleic Acids Res., 33:2620-2628 (2005); Preparata, FP, Oliver, JS, J. Comput. Biol.753-765 (2004); and Hill, F., et. al., Proc Natl Acad. Sci. U S A, 95:4258-4263 (1998)).

[0153] As used herein, the term “unsubstituted” or “unmodified” in the context of the initiating capped oligonucleotide primer and NTPs refers to an initiating capped oligonucleotide primer and NTPs that have not been modified.

[0154] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0155] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0156] As used herein, the term “subject” or “patient” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0157] The term "immunization" or "vaccination" describes the process of treating a subject for therapeutic or prophylactic reasons.

[0158] The terms “effective amount,” “therapeutically effective amount” or “effective dose” or related terms may be used interchangeably and refer to an amount of the therapeutic agent that when administered to a subject, is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. Therapeutically effective amounts of the therapeutic agents provided herein, will vary depending upon the relative activity of the therapeutic agent, and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration, drugs used in combination or coincidental with the specific compound employed and the like, which can readily be determined by one of ordinary skill in the art. In one embodiment, a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques. In addition, as is known in the art, adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[0159] As used herein, “dosage form” means a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. A dosage forms can comprise inventive a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, in combination with a pharmaceutically acceptable excipient, such as a preservative, buffer, saline, or phosphate buffered saline. Dosage forms can be made using conventional pharmaceutical manufacturing and compounding techniques. Dosage forms can comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate,acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium deoxycholate), solution and / or cryo / lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). A dosage form formulated for injectable use can have a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, suspended in sterile saline solution for injection together with a preservative.

[0160] As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0161] The term “administering”, “administered” and grammatical variants refers to the physical introduction of a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include a topical, epidermalor mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0162] “Treating” is to be understood broadly and encompasses any beneficial effect, including, e.g., delaying, slowing, or arresting the worsening of symptoms associated with a viral disease or remedying such symptoms, at least in part. The term is intended to include the cure or elimination of the disease, disorder or condition. Those in need of treatment include those who already have the disease or disorder, as well as those who should prevent the disease or disorder. The patient to be treated is preferably a mammal, in particular a human being.

[0163] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0164] As used herein, the term “therapeutic agent” includes any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and / or physiologic effect by local and / or systemic action. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14thedition), the Physicians' Desk Reference (64thedition), and The Pharmacological Basis of Therapeutics (12thedition), and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. For example, the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; analgesics andanalgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) including both double- and single-stranded molecules, gene constructs, expression vectors, antisense molecules and the like), small molecules (e.g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas. The term "therapeutic agent" also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or pro- drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

[0165] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0166] “Analog,” or “analogue” is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar toanother compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.

[0167] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0168] The term a “sub-therapeutic amount” of an agent or therapy is an amount less than the effective amount for that agent or therapy as a single agent, but when combined with an effective or sub-therapeutic amount of another agent or therapy can produce a result desired by the physician, due to, for example, synergy in the resulting efficacious effects, or reduced side effects.

[0169] Combination therapy or “in combination with” refer to the use of more than one therapeutic agent to treat a particular disorder or condition. By “in combination with,” it is not intended to imply that the therapeutic agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of this disclosure. A therapeutic agent can be administered concurrently with, prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12weeks, or 16 weeks before), or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after), one or more other additional agents. The therapeutic agents in a combination therapy can also be administered on an alternating dosing schedule, with or without a resting period (e.g., no therapeutic agent is administered on certain days of the schedule). The administration of a therapeutic agent “in combination with” another therapeutic agent includes, but is not limited to, sequential administration and concomitant administration of the two agents. In general, each therapeutic agent is administered at a dose and / or on a time schedule determined for that particular agent.

[0170] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like. “Consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0171] Described herein are novel trinucleotide cap analogs and compositions, and methods of using the same. Also described herein is an RNA molecule comprising a 5’-cap, wherein the 5’-cap includes a trinucleotide cap analog as described herein. Methods of inducing a therapeutic effect in a subject are also described herein, the methods including a step of administering to the subject a trinucleotide cap analog or RNA molecule including the trinucleotide cap analog. Compounds

[0172] In an aspect, provided herein is a compound of formula (I):(I), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: N1is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, natural, modified, or unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside; each B1is independently a natural, modified, or unnatural nucleoside base; each X1is independently -O-, -CH2-, -CX2-, -CHX-, -N(R101)-, -BH-, or -S-; each Y1is independently O, S, or Se; each Z1is independently -OH, -SH, -BH3, substituted or unsubstituted -O-alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted -O-aryl; each R1is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR1A, -NR1AR1B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2,-OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R5is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR5A, -NR5AR5B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R1and R5together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene; each R1A, R1B, R5A, and R5Bis independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1Aand R1Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R5Aand R5Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R101is hydrogen, oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2,-OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;n is an integer from 0 to 3; m is an integer from 0 to 8; and X is -Cl, -Br, -I or –F.

[0173] In embodiments, each R1is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OR1A, -NR1AR1B, -NO2, -SH, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4 alkyl), substituted (e.g., substituted with at least one substituent group, size- limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6cycloalkyl), or substituted (e.g., substituted with at least one substituent group, size- limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10aryl, C10aryl, or phenyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

[0174] In embodiments, a substituted R1(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when a substituted R1is substituted, it is substituted with at least one substituent group. In embodiments, when a substituted R1is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a substituted R1is substituted, it is substituted with at least one lower substituent group.

[0175] In embodiments, each R1is independently hydrogen, halogen, or -OR1A. Inembodiments, each R1is independently hydrogen. In embodiments, each R1is independently halogen. In embodiments, each R1is independently -OR1A. In embodiments, each R1is independently chloro. In embodiments, each R1is independently bromo. In embodiments, each R1is independently iodo. In embodiments, each R1is independently fluoro.

[0176] In embodiments, each R1is independently hydroxy, methoxy, ethoxy, propoxy, butoxy, or t-butoxy. In embodiments, each R1is independently hydroxy. In embodiments, each R1is independently methoxy. In embodiments, each R1is independently ethoxy. In embodiments, each R1is independently propoxy. In embodiments, each R1is independently butoxy. In embodiments, each R1is independently t-butoxy.

[0177] In embodiments, R1Ais hydrogen or substituted or unsubstituted alkyl. In embodiments, R1Ais hydrogen. In embodiments, R1Ais substituted alkyl. In embodiments, R1Ais an unsubstituted alkyl.

[0178] In embodiments, R1Ais hydrogen, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).

[0179] In embodiments, a substituted R1A(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1Ais substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when a substituted R1Ais substituted, it is substituted with at least one substituent group. In embodiments, when a substituted R1Ais substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a substituted R1Ais substituted, it is substituted with at least one lower substituent group.

[0180] In embodiments, R1Ais hydrogen or substituted or unsubstituted alkyl. In embodiments, R1Ais hydrogen or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4alkyl). In embodiments, R1Ais hydrogen. In embodiments, R1Ais unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl). In embodiments, R1Ais substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl(e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).

[0181] In embodiments, R1Ais hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl. In embodiments, R1Ais hydrogen. In embodiments, R1Ais methyl. In embodiments, R1Ais ethyl. In embodiments, R1Ais propyl. In embodiments, R1Ais isopropyl. In embodiments, R1Ais butyl. In embodiments, R1Ais isobutyl. In embodiments, R1Ais t-butyl. In embodiments, R1Ais pentyl. In embodiments, R1Ais hexyl.

[0182] In embodiments, each R5is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OR5A, -NR5AR5B, -CONH2, -COOH, -NHC(O)OH, -NO2, -SH, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3- C8cycloalkyl, C3-C6cycloalkyl, or C5-C6cycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to6 membered heteroaryl).

[0183] In embodiments, a substituted R5(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R5is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when a substituted R5is substituted, it is substitutedwith at least one substituent group. In embodiments, when a substituted R5is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a substituted R5is substituted, it is substituted with at least one lower substituent group.

[0184] In embodiments, each R5is independently hydrogen.

[0185] In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene. In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a substituted or unsubstituted cycloalkylene. In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a substituted or unsubstituted heterocycloalkylene.

[0186] In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered heterocycloalkylene, 3 to 6 membered heterocycloalkylene, or 5 to 6 membered heterocycloalkylene). In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8 cycloalkylene, C3-C6 cycloalkylene, or C5-C6 cycloalkylene).

[0187] In embodiments, a substituted heterocycloalkylene formed by the joining of R1and R5within four atoms of said R1and the atoms to which they are connected, is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted heterocycloalkylene is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when heterocycloalkylene formed by the joining of R1and R5within four atoms of said R1and the atoms to which they are connected is substituted, it is substituted with at least one substituent group. In embodiments, when heterocycloalkyleneformed by the joining of R1and R5within four atoms of said R1and the atoms to which they are connected is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when heterocycloalkylene formed by the joining of R1and R5within four atoms of said R1and the atoms to which they are connected is substituted, it is substituted with at least onelower substituent group.

[0188] In embodiments, a substituted cycloalkylene formed by the joining of R1and R5within four atoms of said R1and the atoms to which they are connected is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted cycloalkylene is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when cycloalkylene formed by the joining of R1and R5within four atoms of said R1and the atoms to which they are connected is substituted, it is substituted with at least one substituent group. In embodiments, when cycloalkylene formed by the joining of R1and R5within four atoms of said R1and the atoms to which they are connected is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when cycloalkylene formed by the joining of R1and R5within four atoms of said R1and the atoms to which they are connected is substituted, it is substituted with at least one lower substituent group.

[0189] In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a substituted or unsubstituted 3 to 6 membered heterocycloalkylene. In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a substituted or unsubstituted 4 membered heterocycloalkylene.

[0190] In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a substituted 4 membered heterocycloalkylene. In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming an unsubstituted 4 membered heterocycloalkylene.

[0191] In embodiments, at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a locked nucleic acid (LNA).

[0192] In embodiments, R101is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10aryl, C10aryl, or phenyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R101is substituted with one or more substituent groups. In embodiments, R101is substituted with one or more size-limited substituent groups. In embodiments, R101is substituted with one or more lower substituent groups.

[0193] In embodiments, a substituted R101(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R101is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when R101is substituted, it is substituted with at least one substituent group. In embodiments, when R101is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R101is substituted, it is substitutedwith at least one lower substituent group.

[0194] In embodiments, X is -Cl, -Br, -I or –F. In embodiments, X is -Cl. In embodiments, X is -Br. In embodiments, X is -I. In embodiments, X is –F.

[0195] In embodiments, each R1A, R1B, R5A, and R5Bis independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl), substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8cycloalkyl, C3-C6cycloalkyl, or C5-C6cycloalkyl), substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or substituted (e.g., with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

[0196] In embodiments, a substituted R1A, R1B, R5A, or R5B(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1A, R1B, R5A, or R5Bis substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when R1A, R1B, R5A, or R5Bis substituted, it is substituted with at least one substituent group. In embodiments, when R1A, R1B, R5A, or R5Bis substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R1A, R1B, R5A, or R5Bis substituted, it is substitutedwith at least one lower substituent group.

[0197] In embodiments, each R1A, R1B, R5A, and R5Bis independently substituted with one or more substituent groups. In embodiments, each R1A, R1B, R5A, and R5Bis independently substituted with one or more size-limited substituent groups. In embodiments, each R1A, R1B, R5A, and R5Bis independently substituted with one or more lower substituent groups.

[0198] In embodiments, R5Aand R5Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), or substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, a substituted heterocycloalkyl or substituted heteroaryl formed by the joining of R5Aand R5Bsubstituents bonded to the same nitrogen atom is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted heterocycloalkyl or substituted heteroaryl is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size- limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when a heterocycloalkyl formed by the joining of R5Aand R5Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R5Aand R5Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R5Aand R5Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group. In embodiments, when a heteroaryl formed by the joining of R5Aand R5Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heteroaryl formed by the joining of R5Aand R5Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heteroaryl formed by the joining of R5Aand R5Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group.

[0199] In embodiments, R1Aand R1Bsubstituents bonded to the same nitrogen atom mayoptionally be joined to form a substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), or substituted (e.g., with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, a substituted heterocycloalkyl or substituted heteroaryl formed by the joining of R1Aand R1Bsubstituents bonded to the same nitrogen atom is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted heterocycloalkyl or substituted heteroaryl is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size- limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when a heterocycloalkyl formed by the joining of R1Aand R1Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R1Aand R1Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R1Aand R1Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group. In embodiments, when a heteroaryl formed by the joining of R1Aand R1Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heteroaryl formed by the joining of R1Aand R1Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heteroaryl formed by the joining of R1Aand R1Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group.

[0200] In embodiments, each B1is independently a natural, modified, or unnatural nucleoside base. In embodiments, each B1is independently a natural nucleoside base. In embodiments, each B1is independently a modified nucleoside base. In embodiments, each B1is independently an unnatural nucleoside base.

[0201] In embodiments, each B1is independently adenine (A), N6-methyladenine (m6A), guanine (G), cytosine (C), uracil (U), or thymine (T). In embodiments, each B1is independentlyA, m6A, G, C, or U. In embodiments, each B1is independently adenine (A). In embodiments, each B1is independently N6-methyladenine (m6A). In embodiments, each B1is independently guanine (G). In embodiments, each B1is independently cytosine (C). In embodiments, each B1is independently uracil (U). In embodiments, each B1is independently thymine (T).

[0202] In embodiments, each B1is independently 5-methylcytosine, pseudouracil, hypoxanthine, N1-methylpseudouracil, N6-methyladenine, N6-ethyladenine, 7-deazaadenine, N -(alkyl)-cytosines, or 5-ethylcytosine, and the like (U.S. Patent No.6,762,298).

[0203] In embodiments, B1includes, but is not limited to, pyrazolo[3,4-d]pyrimidines, 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8- bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7- deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3- deazaadenine, pyrazolo[3,4- d]pyrimidine, imidazo[l,5-a]l,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1 ,2,4-triazine, pyridazine; and 1 ,3,5 triazine.

[0204] In embodiments, each X1is independently -O-, -CH2-, -CX2-, -N(R101)-, or -S-. In embodiments, each X1is independently -O-, -CH2-, -CF2-, -NH-, or -S-. In embodiments, each X1is independently -O-, -CH2-, -CF2-, or -S-. In embodiments, each X1is independently -O-, - CH2-, or -NH-. In embodiments, each X1is independently -CH2- or -NH-.

[0205] In embodiments, each X1is independently -O-. In embodiments, each X1is independently -CH2-. In embodiments, each X1is independently -CX2-. In embodiments, each X1is independently -CF2-. In embodiments, each X1is independently -N(R101)-. In embodiments, each X1is independently -NH-. In embodiments, each X1is independently -BH-. In embodiments, each X1is independently -S-.

[0206] In embodiments, each Y1is independently O, S, or Se. In embodiments, each Y1is independently O or S. In embodiments, each Y1is independently O. In embodiments, each Y1is independently S. In embodiments, each Y1is independently Se.

[0207] In embodiments, each Z1is independently -OH, -SH, or substituted orunsubstituted -O-alkyl. In embodiments, each Z1is independently -OH, -SH, methoxy, ethoxy, propoxy, butoxy, or pentoxy. In embodiments, each Z1is independently -OH or -SH.

[0208] In embodiments, each Z1is independently -OH. In embodiments, each Z1is independently -SH. In embodiments, each Z1is independently methoxy. In embodiments, each Z1is independently ethoxy. In embodiments, each Z1is independently propoxy. In embodiments, each Z1is independently butoxy. In embodiments, each Z1is independently pentoxy.

[0209] In embodiments, n is an integer from 0 to 3. In embodiments, n is 1 or 2. In embodiments, n is 0. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3.

[0210] In embodiments, m is an integer from 0 to 8. In embodiments, m is 0 or 1. In embodiments, m is 0. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8.

[0211] In embodiments, N1is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, natural, modified, or unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside.

[0212] In embodiments, N1is a natural, modified, or unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside. In embodiments, N1is a natural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside. In embodiments, N1is a modified nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside. In embodiments, N1is an unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside.

[0213] In embodiments, N1is adenosine (A), cytidine (C), uridine (U), or thymidine. In embodiments, N1is adenosine (A), cytidine (C), or uridine (U). In embodiments, N1is adenosine (A) or uridine (U). In embodiments, N1is adenosine. In embodiments, N1is cytidine. In embodiments, N1is uridine. In embodiments, N1is thymidine.

[0214] In embodiments, N1includes, but is not limited to, 1-methyladenosine, 2- methylthio-N6-hydroxynorvalyl carbamoyladenosine, 2-methyladenosine, 2-O-ribosylphosphate adenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-acetyladenosine, N6- glycinylcarbamoyladenosine, N6-isopentenyladenosine, N6-methyladenosine, N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxynorvalylcarbamoyladenosine, 1,2-O-dimethyladenosine, N6,2-O-dimethyladenosine, 2-O-methyladenosine, N6,N6,O-2-trimethyladenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, 2-methylthio-N6-methyladenosine, 2-methylthio-N6- isopentenyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, 2-thiocytidine, 3- methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5- hydroxymethylcytidine, lysidine, N4-acetyl-2-O-methylcytidine, 5-formyl-2-O-methylcytidine, 5,2-O-dimethylcytidine, 2-O-methylcytidine, N4,2-O-dimethylcytidine, N4,N4,2-O- trimethylcytidine, epoxyqueuosine, hydroxywybutosine, isowyosine, peroxywybutosine, galactosyl-queuosine, mannosyl-queuosine, queuosine, archaeosine, wybutosine, methylwyosine, wyosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4-thiouridine, 5- methyl-2-thiouridine, 5-methylaminomethyluridine, 5-carboxymethyluridine, 5- carboxymethylaminomethyluridine, 5-hydroxyuridine, 5-methyluridine, 5-taurinomethyluridine, 5-carbamoylmethyluridine, 5-(carboxyhydroxymethyl)uridine methyl ester, dihydrouridine, 5- methyldihydrouridine, 5-methylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 3,2-O- dimethyluridine, 5-carboxymethylaminomethyl-2-O-methyluridine, 5-carbamoylmethyl-2-O- methyluridine, 5-methoxycarbonylmethyl-2-O-methyluridine, 5-(isopentenylaminomethyl)-2-O- methyluridine, 5,2-O-dimethyluridine, 2-O-methyluridine, 2-thio-2-O-methyluridine, uridine 5- oxyacetic acid, 5-methoxycarbonylmethyluridine, uridine 5-oxyacetic acid methyl ester, 5- methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5- methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl- 2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1- methylpseudouridine, 3-methylpseudouridine, 2-O-methylpseudouridine, inosine, 1- methylinosine, 1,2-O-dimethylinosine and 2-O-methylinosine.

[0215] In embodiments, N1includes, but is not limited to, LNA nucleosides, halogen- substituted purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines, N6-ethyladenine, N4- (alkyl)-cytosines, 5-ethylcytosine, and the like (U.S. Patent No.6,762,298).

[0216] In embodiments, N1is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl. Inembodiments, N1is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted alkyl. In embodiments, N1is substituted alkyl or substituted heteroaryl. [ e.

[0218] In embodiments, provided herein is a compound of formula (IA):(IA), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. N1, B1, R1, R5, X1, Y1, Z1, and n are each as defined herein, including in embodiments.

[0219] In embodiments, provided herein is a compound of formula (IB):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; R4is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR4A, -NR4AR4B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; each R2A, R3A, R4A, and R4B, is independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R4Aand R4Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl. N1, B1, R1, R5, X1, Y1, Z1, and n are each as defined herein, including in embodiments.

[0220] In embodiments, R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8cycloalkyl, C3-C6cycloalkyl, or C5-C6cycloalkyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

[0221] In embodiments, a substituted R2(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R2is independently substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when R2is substituted, it is substituted with at least one substituent group. In embodiments, when R2is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R2is substituted, it is substituted with at least one lower substituent group.

[0222] In embodiments, R2is independently hydrogen or substituted or unsubstituted alkyl. In embodiments, R2is independently hydrogen or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl). In embodiments, R2is independently hydrogen. In embodiments, R2is independently unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl). In embodiments, R2is independently substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4alkyl).

[0223] In embodiments, R2is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, or hexyl.

[0224] In embodiments, R2is independently hydrogen or methyl.

[0225] In embodiments, R2is independently hydrogen. In embodiments, R2is independently methyl. In embodiments, R2is independently ethyl. In embodiments, R2is independently propyl. In embodiments, R2is independently isopropyl. In embodiments, R2is independently butyl. In embodiments, R2is independently isobutyl. In embodiments, R2is independently t-butyl. In embodiments, R2is independently pentyl. In embodiments, R2is independently hexyl.

[0226] In embodiments, R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8cycloalkyl, C3-C6cycloalkyl, or C5-C6cycloalkyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

[0227] In embodiments, a substituted R3(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is independently substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R3is independently substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when R3is independently substituted, it is substituted with at least one substituent group. In embodiments, when R3is independently substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R3is independently substituted, it is substituted with at least onelower substituent group.

[0228] In embodiments, R3is independently hydrogen or substituted or unsubstituted alkyl. In embodiments, R3is independently hydrogen or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R3is independently hydrogen. In embodiments, R3is independently unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl). In embodiments, R3is independently substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4alkyl).

[0229] In embodiments, R3is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, or hexyl.

[0230] In embodiments, R3is independently hydrogen or methyl.

[0231] In embodiments, R3is independently hydrogen. In embodiments, R3is independently methyl. In embodiments, R3is independently ethyl. In embodiments, R3isindependently propyl. In embodiments, R3is independently isopropyl. In embodiments, R3is independently butyl. In embodiments, R3is independently isobutyl. In embodiments, R3is independently t-butyl. In embodiments, R3is independently pentyl. In embodiments, R3is independently hexyl.

[0232] In embodiments, R4is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CHCl2, -CHBr2, -CHF2, -CHI2, -CN, -OR4A, -NR4AR4B, -NO2, -SH, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8cycloalkyl, C3-C6cycloalkyl, or C5-C6cycloalkyl), or substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

[0233] In embodiments, a substituted R4(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R4is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when R4is substituted, it is substituted with at least one substituent group. In embodiments, when R4is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R4is substituted, it is substituted with atleast one lower substituent group.

[0234] In embodiments, R4is hydrogen, halogen, or -NR4AR4B. In embodiments, R4is hydrogen. In embodiments, R4is halogen. In embodiments, R4is -NR4AR4B. In embodiments, R4is -Cl. In embodiments, R4is -Br. In embodiments, R4is -I. In embodiments, R4is -F. In embodiments, R4is -NHMe. In embodiments, R4is -NH2. In embodiments, R4is -NMe2.

[0235] In embodiments, R4is hydrogen, -F, -NHMe, -NH2, or -NMe2. In embodiments, R4is hydrogen or -NHMe.

[0236] In embodiments, R4Ais hydrogen or substituted or unsubstituted alkyl. In embodiments, R4Ais hydrogen. In embodiments, R4Ais substituted alkyl. In embodiments, R4Ais an unsubstituted alkyl. In embodiments, R4Bis hydrogen or substituted or unsubstituted alkyl. In embodiments, R4Bis hydrogen. In embodiments, R4Bis substituted alkyl. In embodiments, R4Bis an unsubstituted alkyl.

[0237] In embodiments, R4Ais hydrogen, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R4Bis hydrogen, substituted (e.g., substituted with at least one substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl).

[0238] In embodiments, a substituted R4A(e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, and / or substituted heteroaryl) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R4Ais substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when R4Ais substituted, it is substituted with at least one substituent group. In embodiments, when R4Ais substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R4Ais substituted, it is substitutedwith at least one lower substituent group.

[0239] In embodiments, R4Ais hydrogen or substituted or unsubstituted alkyl. In embodiments, R4Ais hydrogen or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl). In embodiments, R4Ais hydrogen. In embodiments, R4Ais unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R4Ais substituted (e.g.with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R4Bis hydrogen or substituted or unsubstituted alkyl. In embodiments, R4Bis hydrogen or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R4Bis hydrogen. In embodiments, R4Bis unsubstituted alkyl (e.g., C1-C8alkyl, C1-C6alkyl, or C1-C4alkyl). In embodiments, R4Bis substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl).

[0240] In embodiments, R4Ais hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, or hexyl. In embodiments, R4Ais hydrogen. In embodiments, R4Ais methyl. In embodiments, R4Ais ethyl. In embodiments, R4Ais propyl. In embodiments, R4Ais isopropyl. In embodiments, R4Ais butyl. In embodiments, R4Ais isobutyl. In embodiments, R4Ais t-butyl. In embodiments, R4Ais pentyl. In embodiments, R4Ais hexyl. In embodiments, R4Bis hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, or hexyl. In embodiments, R4Bis hydrogen. In embodiments, R4Bis methyl. In embodiments, R4Bis ethyl. In embodiments, R4Bis propyl. In embodiments, R4Bis isopropyl. In embodiments, R4Bis butyl. In embodiments, R4Bis isobutyl. In embodiments, R4Bis t-butyl. In embodiments, R4Bis pentyl. In embodiments, R4Bis hexyl.

[0241] In embodiments, each R2A, R3A, R4A, and R4Bis independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), substituted (e.g. with asubstituent group, a size-limited substituent group or a lower substituent group) or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or substituted (e.g. with a substituent group, a size- limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).

[0242] In embodiments, each R2A, R3A, R4A, and R4Bis independently substituted with one or more substituent groups. In embodiments, each R2A, R3A, R4A, and R4Bis independently substituted with one or more size-limited substituent groups. In embodiments, each R2A, R3A, R4A, and R4Bis independently substituted with one or more lower substituent groups.

[0243] In embodiments, R4Aand R4Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), or substituted (e.g. with a substituent group, a size-limited substituent group or a lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, a substituted heterocycloalkyl or substituted heteroaryl formed by the joining of R4Aand R4Bsubstituents bonded to the same nitrogen atom is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted heterocycloalkyl or substituted heteroaryl is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and / or lower substituent group may optionally be different. In embodiments, when a heterocycloalkyl formed by the joining of R4Aand R4Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R4Aand R4Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when a heterocycloalkyl formed by the joining of R4Aand R4Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group. In embodiments, when a heteroaryl formed by the joining of R4Aand R4Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one substituent group. In embodiments, when a heteroaryl formed by the joining of R4Aand R4Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when a heteroaryl formed by the joining of R4Aand R4Bsubstituents bonded to the same nitrogen atom is substituted, it is substituted with at least one lower substituent group.

[0244] In embodiments, X is -Cl, -Br, -I or –F. In embodiments, X is -Cl. In embodiments, X is -Br. In embodiments, X is -I. In embodiments, X is –F.

[0245] In embodiments, provided herein is a compound of formula (IC):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. N1, B1, R2, R3, R4, R5, X1, Y1, Z1, and n are each as defined herein, including in embodiments.

[0246] In embodiments, provided herein is a compound of formula (ID):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. N1, B1, R4, R5, X1, Y1, Z1, and n are each as defined herein, including in embodiments.

[0247] In embodiments, provided herein is a compound of formula (IE):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. N1, B1, R4, R5, X1, Y1, Z1, and n are each as definedherein, including in embodiments.

[0248] In embodiments, provided herein is a compound of formula (IF):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. N1, R4, R5, X1, Y1, Z1, and n are each as defined herein, including in embodiments.

[0249] In embodiments, provided herein is a compound of formula (IG):(IG), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceuticallyacceptable salt, solvate, or hydrate thereof. N1, R4, R5, X1, Y1, Z1, and n are each as defined herein, including in embodiments.

[0250] In embodiments, provided herein is a compound of formula (IH):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; and each R2Aand R3Ais independently hydrogen, -CX3, -CHX2, -CH2X,-C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^ ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, 7-OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. N1, B1, R1, R5, X1, Y1, Z1, and n are each as defined herein, including in embodiments.

[0251] In an aspect, provided herein is a compound of the following structure:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0252] In an aspect, provided herein is a compound of the following structure:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0253] In an aspect, provided herein is a compound of the following structure:Ph-4-Bu-pppA2’OMepG:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0254] In an aspect, provided herein is a compound of the following structure: Thiamine-pppA2’OMepG:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0255] In an aspect, provided herein is a compound of the following structure:Thiamine-pppm6A2’OMepG:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0256] In an aspect, provided herein is a compound of the following structure: ApppA2’OMepG:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0257] As understood by those of skill in the art, the structures shown of the compounds described herein are representations of one form of the compound. Although such compounds may be drawn or described in protonated (free acid) form, in ionized (anionic) form, or ionized and in association with one or more cations (salt form), aqueous solutions of such compounds exist in equilibrium among such forms. For example, a phosphate linkage of a compounddescribed herein, in aqueous solution, exists in equilibrium among free acid, anion, and salt forms.

[0258] In embodiments, a compound of formula I can be depicted in protonated form (free acid):.

[0259] In embodiments, a compound of formula I can be depicted in ionized (anionic) form: .

[0260] In embodiments, a compound of formula I can be depicted in ionized and in association with one or more cations (salt) form:.

[0261] In embodiments, the sodium cations may be replaced by another suitable cation, e.g., lithium, potassium, or ammonium cations. In embodiments, where n and / or m are >1 the number of cations will be adjusted for neutrality.

[0262] As understood by those of skill in the art, the cation salt form may exist in which one, two, three, four, five or more cations are present (the number of cations depends on the number of Z1s present). Unless otherwise indicated, compounds described herein are intended to include all such forms. Moreover, certain compounds have several such linkages, each of which is in equilibrium. Thus, compounds in solution exist in an ensemble of forms at multiple positions all at equilibrium. Drawn structures necessarily depict a single form. Nevertheless, unless otherwise indicated, such drawings are likewise intended to include corresponding forms. Herein, a structure depicting the free acid of a compound followed by the term “or salts thereof” expressly includes all such forms that may be fully or partially protonated / de-protonated / in association with a cation.

[0263] In embodiments, the pharmaceutically acceptable salt is sodium salt, lithium salt, or potassium salt. In embodiments, the pharmaceutically acceptable salt is sodium salt. In embodiments, the pharmaceutically acceptable salt is lithium salt. In embodiments, thepharmaceutically acceptable salt is potassium salt.

[0264] In embodiments, provided herein is an initiating capped oligonucleotide primer selected from: Apppm6A2’OMepG, Ph-4-Bu-pppm6A2’OMepG, Ph-4-Bu-pppA2’OMepG, Thiamine- pppA2’OMepG, Thiamine-pppm6A2’OMepG, and ApppA2’OMepG.

[0265] In embodiments, provided herein is an initiating capped oligonucleotide primer of the following formula: Apppm6A2’OMepG.

[0266] In embodiments, provided herein is an initiating capped oligonucleotide primer of the following formula: Ph-4-Bu-pppm6A2’OMepG.

[0267] In embodiments, provided herein is an initiating capped oligonucleotide primer of the following formula: Ph-4-Bu-pppA2’OMepG.

[0268] In embodiments, provided herein is an initiating capped oligonucleotide primer of the following formula: Thiamine-pppA2’OMepG.

[0269] In embodiments, provided herein is an initiating capped oligonucleotide primer of the following formula: Thiamine-pppm6A2’OMepG.

[0270] In embodiments, provided herein is an initiating capped oligonucleotide primer of the following formula: ApppA2’OMepG. Methods of Making the Compounds and RNA molecules

[0271] The compounds described herein can be prepared in a variety of ways. The compounds can be synthesized using various synthetic methods. At least some of these methods are known in the art of synthetic organic chemistry. The compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions can vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0272] Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts, Greene’s Protective Groups in Organic Synthesis, 5th. Ed., Wiley & Sons, 2014, which is incorporated herein by reference in its entirety.

[0273] Product or intermediate formation can be monitored according to any suitablemethod known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high- performance liquid chromatography (HPLC) or thin layer chromatography.

[0274] The compounds described herein can be prepared via a two step process, including an activation step followed by a coupling step. During the activation step, a TEA salt of a desired (modified) diphosphate is dissolved in a solvent mixture (usually water / DMSO) and activated with an activating reagent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC.HCl salt), followed by addition of imidazole. After a period of time, an activated intermediate (imidazolide) is formed and prepared for use in a coupling step. The activated intermediate is then coupled with the desired (modified) dinucleotide.

[0275] Alternatively, a TEA salt of a diphosphate of the desired (modified) dinucleotide is dissolved in a solvent mixture (usually water / DMSO) and activated with an activating reagent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC.HCl salt), followed by addition of imidazole. After a period of time, an activated intermediate (imidazolide) is formed and prepared for use in a coupling step. The activated intermediate is then coupled with the TEA salt of the (modified) monophosphate.

[0276] Both general synthesis routes are depicted below in the Examples section as Scheme 1 and Scheme 2. Scheme 1 and Scheme 2 are provided for representative purposes only, and those of ordinary skill in the art will understand that the schemes can be applied, with modifications within the purview of those of skill in the art along with the disclosures in the Examples section, to any of the compounds described herein (i.e., compounds A-1 to A-7).

[0277] The synthetic methods described herein can be performed as a one-pot synthesis, such that all steps are performed in a single reactor with no isolation of intermediate products during the course of the synthetic method. Further description of these general synthetic methods can be found in International Patent Application No. PCT / US2023 / 061255 which is incorporated herein by reference in its entirety.

[0278] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (I), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: N1is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, natural, modified, or unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside; each B1is independently a natural, modified, or unnatural nucleoside base; each X1is independently -O-, -CH2-, -CX2-, -CHX-, -N(R101)-, -BH-, or -S-; each Y1is independently O, S, or Se; each Z1is independently -OH, -SH, -BH3, substituted or unsubstituted -O-alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted -O-aryl; each R1is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR1A, -NR1AR1B, -COOH, -CONH2,-NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R5is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR5A, -NR5AR5B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R1and R5together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene; each R1A, R1B, R5A, and R5Bis independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1Aand R1Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R5Aand R5Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R101is hydrogen, oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2,-OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; n is an integer from 0 to 3; m is an integer from 0 to 8; and X is -Cl, -Br, -I or –F; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0279] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: N1, B1, R1, R5, X1, Y1, Z1, and n are as defined herein, including in embodiments; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0280] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IB)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; R4is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR4A, -NR4AR4B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; each R2A, R3A, R4A, and R4B, is independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R4Aand R4Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; and N1, B1, R1, R5, X1, Y1, Z1, and n are as defined herein, including in embodiments; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0281] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IC), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof;wherein: N1, B1, R2, R3, R4, R5, X1, Y1, Z1, and n are as defined herein, including in embodiments; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0282] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein N1, B1, R4, R5, X1, Y1, Z1, and n are as defined herein, including in embodiments; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0283] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IE), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein N1, B1, R4, R5, X1, Y1, Z1, and n are as defined herein, including in embodiments; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0284] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IF)(IF), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein N1, R4, R5, X1, Y1, Z1, and n are as defined herein, including in embodiments; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template

[0285] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IG) (IG),or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein N1, R4, R5, X1, Y1, Z1, and n are as defined herein, including in embodiments; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

[0286] In embodiments, provided herein is a method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formulaor an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; and each R2Aand R3Ais independently hydrogen, -CX3, -CHX2, -CH2X,-C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^ ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and N1, B1, R1, R5, X1, Y1, Z1, and n are as defined herein, including in embodiments; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template Pharmaceutical Compositions, Formulations, and Methods of Using the Compounds

[0287] In embodiments, the compounds provided herein may be provided as a pharmaceutically acceptable salt (See, Berge et al., J. Pharm. Sci.1977, 66, 1-19; and “Handbook of Pharmaceutical Salts, Properties, and Use,” Stahl and Wermuth, Ed.; Wiley-VCH and VHCA, Zurich, 2002).

[0288] In embodiments, provided herein are pharmaceutical compositions including one or more of the compounds described herein and a pharmaceutically acceptable excipient. As used herein, the term pharmaceutically acceptable salt refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein. The term salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein. These salts can be prepared in situ during the isolation andpurification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See S.M. Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated herein by reference in its entirety, at least, for compositions taught therein.)

[0289] In embodiments, compounds described herein are in aqueous solution with sodium salt. In embodiments, compounds described herein are in aqueous solution with lithium salt. In embodiments, compounds described herein are in aqueous solution with potassium salt. In embodiments, compounds described herein are in aqueous solution with triethylammonium salt.

[0290] The compounds provided herein may also be provided as a prodrug, which is a functional derivative of the compound, for example, of formula I and is readily convertible into the parent compound in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have enhanced solubility in pharmaceutical compositions over the parent compound. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. See Harper, Progress in Drug Research 1962, 4, 221-294; Morozowich et al. in “Design of Biopharmaceutical Properties through Prodrugs and Analogs,” Roche Ed., APHA Acad. Pharm. Sci.1977; “Bioreversible Carriers in Drug in Drug Design, Theory and Application,” Roche Ed., APHA Acad. Pharm. Sci.1987; “Design of Prodrugs,” Bundgaard, Elsevier, 1985; Wang et al., Curr. Pharm. Design 1999, 5, 265-287; Pauletti et al., Adv. Drug. Delivery Rev.1997, 27, 235-256; Mizen et al., Pharm. Biotech.1998, 11, 345-365; Gaignault et al., Pract. Med. Chem.1996, 671-696; Asgharnejad in “Transport Processes in PharmaceuticalSystems,” Amidon et al., Ed., Marcell Dekker, 185-218, 2000; Balant et al., Eur. J. Drug Metab. Pharmacokinet.1990, 15, 143-53; Balimane and Sinko, Adv. Drug Delivery Rev.1999, 39, 183- 209; Browne, Clin. Neuropharmacol.1997, 20, 1-12; Bundgaard, Arch. Pharm. Chem.1979, 86, 1-39; Bundgaard, Controlled Drug Delivery 1987, 17, 179-96; Bundgaard, Adv. Drug Delivery Rev.1992, 8, 1-38; Fleisher et al., Adv. Drug Delivery Rev.1996, 19, 115-130; Fleisher et al., Methods Enzymol.1985, 112, 360-381; Farquhar et al., J. Pharm. Sci.1983, 72, 324-325; Freeman et al., J. Chem. Soc., Chem. Commun.1991, 875-877; Friis and Bundgaard, Eur. J. Pharm. Sci.1996, 4, 49-59; Gangwar et al., Des. Biopharm. Prop. Prodrugs Analogs, 1977, 409- 421; Nathwani and Wood, Drugs 1993, 45, 866-94; Sinhababu and Thakker, Adv. Drug Delivery Rev.1996, 19, 241-273; Stella et al., Drugs 1985, 29, 455-73; Tan et al., Adv. Drug Delivery Rev.1999, 39, 117-151; Taylor, Adv. Drug Delivery Rev.1996, 19, 131-148; Valentino and Borchardt, Drug Discovery Today 1997, 2, 148-155; Wiebe and Knaus, Adv. Drug Delivery Rev. 1999, 39, 63-80; and Waller et al., Br. J. Clin. Pharmac.1989, 28, 497-507.

[0291] Administration of the compounds and compositions described herein or pharmaceutically acceptable salts thereof can be carried out using therapeutically effective amounts of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein for periods of time effective to treat a disorder. The effective amount of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein may be determined by one of ordinary skill in the art.

[0292] Those of skill in the art will understand that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.

[0293] The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. Further, depending on the route of administration, one of skill in the art would know how to determine doses that result in a plasma concentration for a desired level of response in the cells,tissues and / or organs of a subject.

[0294] Any suitable formulation of the compounds described herein can be prepared. See generally, Remington's Pharmaceutical Sciences, (2000) Hoover, J. E. editor, 20 th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa., pages 780-857. A formulation is selected to be suitable for an appropriate route of administration. In embodiments, the compound of formula (I) is formulated for oral administration; in other embodiments, the compound is formulated for parenteral administration, such as injection or infusion.

[0295] Where contemplated compounds are administered in a pharmacological composition, it is contemplated that the compounds can be formulated in admixture with a pharmaceutically acceptable excipient and / or carrier. For example, contemplated compounds can be administered orally as neutral compounds or as pharmaceutically acceptable salts, or intravenously in a physiological saline solution. Conventional buffers such as phosphates, bicarbonates or citrates can be used for this purpose. Of course, one of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration. In particular, contemplated compounds may be modified to render them more soluble in water or other vehicle, which for example, may be easily accomplished with minor modifications (salt formulation, esterification, etc.) that are well within the ordinary skill in the art. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in a patient.

[0296] Depending on the intended mode of administration, the pharmaceutical composition can be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers / excipients or diluents. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.

[0297] To practice the method of the present invention, compounds having formula and pharmaceutical compositions thereof may be administered orally, parenterally, by inhalation, topically (including transdermally, buccally, and sublingually), rectally, nasally, vaginally, via an implanted reservoir, or other drug administration methods. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The compositions may be prepared by any method well known in the art of pharmacy.

[0298] Such methods include the step of bringing in association compounds of the invention or combinations thereof with any auxiliary agent. The auxiliary agent(s), also named accessory ingredient(s), include those conventional in the art, such as excipients (e.g., starch, lactose), fillers, binders (e.g., gelatin, cellulose, gum tragacanth), diluents, disintegrants (e.g., alginate, Primogel, and corn starch), lubricants (e.g., magnesium stearate, silicon dioxide), colorants, flavouring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint), anti-oxidants, wetting agents, or other material well known in the art for use in pharmaceutical formulations.

[0299] The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington: The Science and Practice of Pharmacy, 22d Edition, Loyd et al. eds., Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences (2012).

[0300] Examples of physiologically acceptable carriers include buffers, such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt- forming counterions, such as sodium; and / or nonionic surfactants, such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICSTM(BASF; Florham Park, NJ).

[0301] Compositions containing the compound described herein or derivatives thereofsuitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0302] These compositions may also contain adjuvants, such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like may also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0303] Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

[0304] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethyleneglycols, and the like.

[0305] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0306] Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

[0307] Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

[0308] Suspensions, in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

[0309] Routes of topical administration include nasal, bucal, mucosal, rectal, or vaginal applications. Compositions of the compounds described herein or derivatives thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers, such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.

[0310] Dosage forms for topical administration of the compounds described herein or derivatives thereof include ointments, lotions, creams, gels, pastes, suspensions, drops, powders,sprays, inhalants, and transdermal patches. One or more thickening agents, humectants, and stabilizing agents can be included in the formulations. Examples of such agents include, but are not limited to, polyethylene glycol, sorbitol, xanthan gum, petrolatum, beeswax, or mineral oil, lanolin, squalene, and the like. Methods for preparing transdermal patches are disclosed, e.g., in Brown, et al. (1988) Ann. Rev. Med.39:221-229 which is incorporated herein by reference. The compounds described herein or derivatives thereof are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required. Ophthalmic formulations, ointments, powders, and solutions are also contemplated as being within the scope of the compositions.

[0311] Optionally, the compounds described herein can be contained in a drug depot. A drug depot comprises a physical structure to facilitate implantation and retention in a desired site (e.g., a synovial joint, a disc space, a spinal canal, abdominal area, a tissue of the patient, etc.). The drug depot can provide an optimal concentration gradient of the compound at a distance of up to about 0.1 cm to about 5 cm from the implant site. A depot, as used herein, includes but is not limited to capsules, microspheres, microparticles, microcapsules, microfibers particles, nanospheres, nanoparticles, coating, matrices, wafers, pills, pellets, emulsions, liposomes, micelles, gels, antibody-compound conjugates, protein-compound conjugates, or other pharmaceutical delivery compositions. Suitable materials for the depot include pharmaceutically acceptable biodegradable materials that are preferably FDA approved or GRAS materials. These materials can be polymeric or non-polymeric, as well as synthetic or naturally occurring, or a combination thereof. The depot can optionally include a drug pump.

[0312] For transdermal administration, e.g. gels, patches or sprays can be contemplated. Compositions or formulations suitable for pulmonary administration e.g. by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurized aerosols, nebulisers or insufflators. A nasal aerosol or inhalation compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in, for example saline, employing suitable preservatives (for example, benzyl alcohol), absorption promoters to enhance bioavailability, and / or other solubilizing or dispersing agents known in the art.

[0313] The compositions may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of sterile liquid carrier, for example water, prior to use.

[0314] In addition, the compounds having formula (I)-formula (IH) or any of the exemplary compounds disclosed herein or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, may be administered alone or in combination with other therapeutic agents. Combination therapies according to the present invention comprise the administration of at least one exemplary compound of the present disclosure and at least one other therapeutic agent in a pharmaceutical composition. The at least one exemplary compound of the present disclosure and at least one other therapeutic agent(s) may be administered as a pharmaceutical composition separately or together. The amounts of the at least one exemplary compound of the present disclosure and the at least one other therapeutic agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.

[0315] In embodiments, provided herein are in vitro methods for synthesizing capped RNA transcripts, including capped messenger RNA (mRNA) transcripts. The methods described herein comprise (a) forming a reaction mixture comprising a compound as described herein (also referred to herein as a cap analog), a DNA template, and an RNA polymerase; and (b) incubating the reaction mixture under conditions that allow transcription of the DNA template to produce capped mRNA transcripts. In the methods described herein, the reaction mixture comprises NTPs, including ATP, CTP, GTP and UTP. One or more of the NTPs in the in vitro transcription reaction mixture can be a modified NTP. Exemplary nucleosides with modified bases include, but are not limited to, inosine, 7-deazaguanosine, 7-methylguanosine, dihyrouridine, 2'-O- methylguanosine, 2'-fluoro-2'-deoxycytidine, pseudouridine, N1-methylpseudouridine, 5- methyluridine. In embodiments, one or more uridines in the in vitro transcribed RNA are replaced by a modified nucleoside. In embodiments, the methods further comprise incubating the reaction mixture comprising the capped mRNA transcripts with a DNase I buffer including Ca2+and DNase I.

[0316] In embodiments, the methods further comprise subjecting the DNase treated reaction mixture to eliminate proteins from the in vitro transcription reaction. In embodiments, the methods further comprise subjecting the DNase treated reaction mixture to phosphatase treatment. In embodiments, the methods further comprise subjecting the DNase treated reactionmixture to one or more purification steps. The mRNA transcripts produced by the methods described herein can be purified using one or more purification techniques known to those of skill in the art. See, Baronti et al. “A guide to large-scale RNA sample preparation,” Anal. Bioanal. Chem.410(14): 3239-33252 (2018). For example, the mRNAs can be purified by liquid chromatography (e.g., HPLC, reversed-phase ion pairing HPLC (RP-IP-HPLC), anion- exchange chromatography, cation exchange chromatography, affinity chromatography, size- exclusion chromatography), precipitation, diafiltration, tangential flow filtration, oligo dT chromatography, silica membrane purification, or hydrophobic interaction chromatography, to name a few. The synthesized capped mRNA transcripts can be substantially free of impurities such as DNA, protein, double-stranded RNA and / or incomplete mRNA transcripts.

[0317] In embodiments, described herein are the resulting RNA molecules comprising a 5’-cap, wherein the 5’-cap comprises any one of the compounds described herein. In embodiments, the RNA molecule is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule. In embodiments, the RNA molecule is a messenger RNA (mRNA) molecule. In embodiments, the RNA molecule is a self-amplifying RNA (saRNA) molecule.

[0318] In embodiments, provided herein are pharmaceutical compositions comprising any one of the compounds described herein and a pharmaceutically acceptable excipient. In embodiments, provided herein are pharmaceutical compositions comprising an RNA molecule comprising any one of the compounds described herein and a pharmaceutically acceptable excipient.

[0319] In embodiments, provided herein are methods of inducing a therapeutic effect in a subject, the methods comprising administering to the subject an RNA molecule as described herein. In embodiments, the administered RNA may contain some amount of immunogenic double stranded RNA (dsRNA). The amount of dsRNA is calculated per each ug of administered RNA. In embodiments, provided herein are methods of administering to a subject a therapeutic dose unit of an mRNA molecule comprising a 5’-cap, wherein the 5’-cap comprises any one of the compounds described herein.

[0320] In embodiments, the mRNAs comprising the 5’-cap analogs described herein are minimally translated in vitro, as further detailed herein.

[0321] In embodiments, provided herein is a method of administering to a mammal a dose of an mRNA molecule comprising a 5’-cap as described herein. In embodiments, the 5’-cap comprises a compound selected from the group consisting of: ,Thiamine-pppA2’OMepG:and an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate, thereof.

[0322] In embodiments, provided herein is an RNA molecule comprising the initiating capped oligonucleotide primer, stereoisomer, or a pharmaceutically acceptable salt thereof, of any one of the compounds described herein. In embodiments, the pharmaceutically acceptable salt is sodium salt, lithium salt, or potassium salt. In embodiments, the pharmaceutically acceptable salt is sodium salt. In embodiments, the pharmaceutically acceptable salt is lithium salt. In embodiments, the pharmaceutically acceptable salt is potassium salt. In embodiments, the pharmaceutically acceptable salt is triethylamine salt.

[0323] In embodiments, the RNA molecule comprising the initiating capped oligonucleotide primer is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule. In embodiments, the RNA molecule comprising the initiating capped oligonucleotide primer is a messenger RNA (mRNA) molecule. In embodiments, the RNA molecule comprising the initiating capped oligonucleotide primer is a self-amplifying RNA (saRNA) molecule.

[0324] In embodiments, provided herein is a pharmaceutical composition comprising the RNA molecule comprising any one of the initiating capped oligonucleotide primer compounds, stereoisomer, or a pharmaceutically acceptable salt thereof, described herein and a pharmaceutically acceptable excipient. In embodiments, the pharmaceutically acceptable salt is sodium salt, lithium salt, or potassium salt. In embodiments, the pharmaceutically acceptable salt is sodium salt. In embodiments, the pharmaceutical composition comprising the RNA molecule comprising any one of the initiating capped oligonucleotide primer compounds, stereoisomer, or a pharmaceutically acceptable salt thereof, described herein and a pharmaceutically acceptable excipient, may be used in vaccines. In embodiments, the vaccines may be vaccines against infectious diseases or cancer. In embodiments, the pharmaceutical composition comprising the RNA molecule comprising any one of the initiating capped oligonucleotide primer compounds described herein and a pharmaceutically acceptable excipient, may be used in genetic vaccination, wherein an immune response is stimulated by introduction of a suitable mRNA, into a subject, which codes for an antigen or a fragment thereof. These vaccine strategies can require large quantities of capped RNA. The present methods facilitate such synthesis and subsequentpurification of capped RNA so as to make these vaccines commercially feasible. The present methods also describe strategies to increase the percentage of full-length capped RNA in a transcription reaction leading to a more homogenous product.

[0325] In embodiments, provided herein is a method of inducing a therapeutic effect in a subject, comprising administering to the subject an RNA molecule comprising any one of the initiating capped oligonucleotide primer compounds described herein.

[0326] In embodiments, provided herein is a method of administering to a subject a therapeutic dose unit of an mRNA molecule comprising a 5’-cap, wherein the 5’-cap comprises a compound of any one of the initiating capped oligonucleotide primer compounds described herein.

[0327] In an aspect, provided herein is a method of inhibiting eIF4E in a mammal comprising administering any one of the compounds, RNA molecules, or pharmaceutical compositions described herein, including in embodiments. Kits

[0328] In embodiments, provided herein are kits for performing transcription. A kit can include any of the compounds or compositions described herein. For example, a kit can include one or more compounds of Formula (I), Formula (IA), Formula (IB), Formula (IC), Formula (ID), Formula (IE), Formula (IF), Formula (IG), and / or Formula (IH). A kit can further include one or more additional reagents, such as reagents used for the synthesis of RNA. Optionally, a kit can contain a compound as described herein (also referred to herein as a cap analog), a container, and one or more reagents selected from one or more unmodified NTPs, one or more modified NTPs, an RNA polymerase, a reaction buffer, magnesium, and a DNA template. In some embodiments, a kit contains a compound as described herein (also referred to herein as a cap analog), a container, one or more NTPs, an RNA polymerase, a reaction buffer, and magnesium. A kit can additionally include directions for use of the kit (e.g., instructions for performing RNA synthesis), a means for administering transcribed mRNA (e.g., a syringe), and / or a carrier.Methods for Making Compounds as Described Herein

[0329] Compounds as described herein are readily prepared using methods illustrated in Scheme 1, Scheme 2, and the examples herein, in view of knowledge in the art.

[0330] As used herein, common organic chemistry abbreviations are defined as follows:

[0331] General methods for making trinucleotide cap analogs from a diphosphate and a dinucleotide. The methods below may be a one-pot synthesis.

[0332] Scheme 1.[

[0334] General Procedure for Scheme 1

[0335] To a stirred solution of diphosphate C-1 (triethylammonium (TEA) salt, 1.5 mol equivalent based on dinucleotide C-2) in a 10%water / DMSO (or 30%water / DMSO) solution at room temperature was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl salt; 2.4 mole equivalent based on dinucleotide C-2), followed by imidazole (4.5 mole equivalent based on dinucleotide C-2). The resulting mixture was allowed to stir at room temperature overnight (approximately 16-24 hours). The second day, magnesium chloride (3.15 M in water, 2.0 mole equivalent based on dinucleotide C-2) was added to the above solution, followed by the dinucleotide C-2 (TEA salt, 1 mole equivalent). The resulting solution was allowed to stir at room temperature overnight (approximately 16 to 24 hours). The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography (Q Sepharose Fast Flow (QFF) Resin, 20% acetonitrile in water as buffer A, 1.5 M trimethylamine acetate (TEAA) in 20% acetonitrile / water as buffer B, using a linear gradient from 25% to 45% buffer B for 4 column volumes (CV) and holding at 45% for 1.5 CV). The desired product was pooled and concentrated under vacuum, and the final product was precipitated as a sodium salt with sodium acetate and 95% absolute ethanol in water.

[0336] General Procedure for Scheme 2:

[0337] The procedure is the same as for Scheme 1, except it is the diphosphate of thedinucleotide (TEA salt) C-3 that is activated by imidazole followed by addition of the phosphate C-4 (TEA salt).

[0338] The syntheses of the following compounds followed the procedures described above and depicted in Scheme 1 or Scheme 2. EXAMPLES

[0339] The following examples are meant to be illustrative and can be used to further understand embodiments of the present disclosure and should not be construed as limiting the scope of the present teachings in any way.

[0340] The chemical reactions described in the Examples can be readily adapted to prepare a number of other compounds of the present disclosure, and alternative methods for preparing the compounds of this disclosure are deemed to be within the scope of this disclosure. For example, the synthesis of non-exemplified compounds according to the present disclosure can be successfully performed by modifications apparent to those skilled in the art, e.g., by utilizing other suitable reagents known in the art other than those described, or by making routing modifications of reaction conditions, reagents, and starting materials. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the present disclosure. Synthetic Examples Example S1: Synthesis of Compound A-1

[0341] To a stirred solution of compound 1 (AK Scientific Catalog #: E185; TEA salt, 300 mg, 0.61 mmol) in 10% water / DMSO (6.1 mL), at room temperature, was added EDC hydrochloride salt (197 mg, 1.03 mmol), followed by imidazole (166 mg, 2.44 mmol). The resulting mixture was allowed to stir at room temperature overnight. Upon completion of the reaction, 3.15M magnesium chloride (390 µL, 1.22 mmol) was added to the solution, followed by compound 2 (TEA salt, 680 mg, 0.73 mmol) (custom order Wuxi App Tec). The resulting solution was allowed to stir at room temperature overnight. The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography followed by reverse phase chromatography. Compound A-1 was pooled and concentrated under vacuum yielding the sodium salt of compound A-1. Compound A-1 was obtained as a white powder (191 mg, 26% yield).

[0342] 1H NMR (500 MHz, D2O): δ 8.18 (s, 1H), 8.15 (s, 1H), 8.02 (s, 1H), 7.93 (s,1H), 7.85 (s, 1H), 5.89-5.87 (m, 3H), 5.76 (d, J = 5.7 Hz, 1H), 4.88-4.85 (m, 1H), 4.73-4.71 (m, 2H), 4.53 (t, J = 5.3, 1H), 4.46-4.43 (m, 4H), 4.37-4.35 (m, 1H), 4.32-4.29 (m, 6H), 4.27-4.21 (m, 5H), 4.19-4.13 (m, 3H), 3.43 (s, 3H), 2.99 (s, 3H). 31P NMR (200 MHz, D2O): δ -0.40 (s, 1P), -10.75 (d, 1P), -10.89 (d, 1P), -22.25 (t, 1P). MS m / z =1150.7 [M+Na]. Example S2: Synthesis of Compound A-2

[0343] To a stirred solution of compound 2 (custom order Wuxi App Tec; TEA salt, 5.0 g, 6.94 mmol) in 20.0 ml of 10% H2O / DMSO, at room temperature, was added imidazole (2.20 g, 32.27 mmol), and EDC hydrochloride salt (2.59 g, 16.66 mmol). The resulting mixture was allowed to stir at room temperature for 22hrs.1M TBAP in DMF (85 ml, 85 mmol) was then added to the mixture and allowed to stir at room temperature for 18 hours. The crude reaction mixture was double purified by reverse phase chromatography followed by anion exchange chromatography to yield a solid (86% yield).

[0344] Compound 6 can be prepared from compound 5 according to methods describedby Kore et al. (2009) J. Am. Chem. Soc.131:6364-6365.

[0345] To a stirred solution of compound 4 (TEA salt, 500mg, 0.45mmol) in 10% water / DMSO (5mL), at room temperature, was added EDC hydrochloride salt (147mg, 0.77mmol), followed by imidazole (123mg, 1.8mmol). The resulting mixture was allowed to stir at room temperature overnight. To 2 mL of this solution 3.15M magnesium chloride (100µL, 0.55mmol) was added, followed by compound 6 (TEA salt, 110mg, 0.32mmol). The resulting solution was allowed to stir at room temperature overnight. The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography, followed by reverse phase chromatography. Compound A-2 was pooled and concentrated under vacuum. Compound A-2 was obtained as a white powder (67mg, 13% yield).

[0346] 1H NMR (500MHz, D2O) δ 8.39 (s, 1H), 8.05 (s, 1H), 7.91 (s, 1H), 7.09 (t, 2H, J=7.2 Hz), 7.03 (t, 1H, J=7.3 Hz), 6.88 (d, 1H, J=7.1 Hz), 6.01 (d, 1H, J=6 Hz), 5.81 (d, 1H, J=5.8 Hz), 4.95 (m, 1H), 4.51 (m, 1H), 4.47 (m, 2H), 4.45 (m, 1H), 4.21 (m, 4H), 3.84 (m, 2H), 3.41 (s, 3H), 3.02. (brs, 3H), 1.40 (m, 2H), 1.28 (m, 2H).31P NMR (200MHz, D2O) δ 1.29 (1P), -10.36 (1P), -10.91 (1P), -22.40 (1P).

[0347] MS m / z = 1012 (M-H). Example S3: Synthesis of Compound A-3

[0348] Compound 7 can be prepared from compound 6 according to methods described by Kore et al. (2009) J. Am. Chem. Soc.131:6364-6365).

[0349] To a stirred solution of compound 7 (TEA salt, 70mg, 0.18mmol) in 10% water / DMSO (5mL), at room temperature, was added EDC hydrochloride salt (74mg, 0.38mmol), followed by imidazole (61mg, 0.9mmol). The resulting mixture was allowed to stir at room temperature overnight. To 2 mL of this solution 3.15M magnesium chloride (143µL, 0.44mmol) was added, followed by compound 8 (TEA salt, 200mg, 0.22mmol) (custom order Wuxi App Tec). The resulting solution was allowed to stir at room temperature overnight. The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography, followed by reverse phase chromatography. Compound A-3 was then precipitated from sodium acetate and ethanol to give a white powder (70mg, 36% yield).

[0350] 1H NMR (500MHz, D2O) δ 8.46 (s, 1H), 8.08 (s, 1H), 7.93 (s, 1H), 7.12 (t, 2H, J=7.3 Hz), 7.04(t, 1H, J=7.3 Hz), 6.94 (d, 2H, J=7.2 Hz), 6.02 (d, 1H, J=6.35 Hz), 5.82, (d, 1H, J=6.35 Hz), 4.96 (m, 1H), 4.83 (t, 1H, J=11.25 Hz), 4.52 (m, 1H), 4.50 (m, 1H), 4.46 (m, 1H), 4.33 (m, 1H), 4.22-4.17 (m, 4H), 3.86 (m, 2H), 3.38 (s, 3H), 2.28 (t, 2H, J=8.15 Hz),1.43 (m, 2H), 1.34 (m, 2H). 31P NMR (200MHz, D2O) δ -.29 (1P), -10.35 (1P), -10.92 (1P), -22.40 (1P). MS m / z = 998 (M-H). Example S4: Synthesis of Compound A-4

[0351] To a stirred solution of compound 9 (Sigma Cat. # C8754; TEA salt, 250mg, 0.55mmol) in 10% water / DMSO (2.5mL), at room temperature, was added EDC hydrochloride salt (175mg, 0.9mmol), followed by imidazole (145mg, 2.2mmol). The resulting mixture was allowed to stir at room temperature overnight. Upon completion of the reaction, 3.15M magnesium chloride (175µL, 0.55mmol) was added to the solution, followed by compound 8 (TEA salt, 400mg, 0.46mmol) (custom order Wuxi App Tec). The resulting solution was allowed to stir at room temperature overnight. The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography, followed by reverse phase chromatography. Compound A-4 was pooled and concentrated under vacuum. Compound A-4 was obtained as a white powder (412mg, 65% yield).

[0352] 1H NMR (500 MHz, D2O): δ 8.45 (s, 1), 8.11 (s, 1), 7.94 (s, 1), 6.07 (d, 1H,J=6.2 Hz), 5.82 (d, 1H, J=6.0 Hz), 5.15 (q, 2H, j+6.0 Hz), 4.93 (m,1H), 4.48 (m, 3H), 4.32 (m, 1H), 4.19 (m, 4H), 4.12( q, 2H, J=5.8 Hz), 3.42 (s, 3H), 3.16 (t, 2H, J=5.5 Hz), 2.49 (s, 3H), 2.35 (s, 3H). 31P NMR (200 MHz, D2O): δ -.33 (1P), -11.14 (2P), -22.77 (1P). MS m / z =1111 [M-H]. Example S5: Synthesis of Compound A-5

[0353] To a stirred solution of compound 9 (TEA salt, 250mg, 0.55mmol) in 10% water / DMSO (2.5mL), at room temperature, was added EDC hydrochloride salt (175mg,0.9mmol), followed by imidazole (145mg, 2.2mmol). The resulting mixture was allowed to stir at room temperature overnight. Upon completion of the reaction, 3.15M magnesium chloride (175µL, 0.55mmol) was added to the solution, followed by compound 2 (TEA salt, 420mg, 0.46mmol). The resulting solution was allowed to stir at room temperature overnight. The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography, followed by reverse phase chromatography. Compound A-5 was pooled and concentrated under vacuum. Compound A-5 was obtained as a white powder (297mg, 45% yield).

[0354] 1H NMR (500 MHz, D2O): δ 8.41 (s, 1H), 8.12 (s, 1H), 6.10 (d, 1H, J=6.2, Hz), 5.82 (d, 1H, J=6.0 Hz), 5.09 (q, 2H, J=6.0 Hz), 4.93 (m, 1H), 4.75 (m, 1H), 4.48 (m 3H), 4.33 (m, 1H), 4.21-4.12 (m, 6H), 3.46 (s, 3H)3.18 (m, 1H), 3.01 (brs, 3H), 2.50 (s, 3H), 2.81 (s, 3H). 31P NMR (200 MHz, D2O): δ -.31 (1P), -11.13 (2P), -22.80(1P). MS m / z =1125 [M-H]. Example S6: Synthesis of Compound A-7

[0355] To a stirred solution of commercially available compound 1 (AK Scientific, Cat # E185; TEA Salt, 3.5g, 5.58mmol) in 10% water / DMSO (24mL), at room temperature, was added EDC hydrochloride salt (1.71g, 8.93mmol), followed by imidazole (1.17g, 17.3mmol). The resulting mixture was allowed to stir at room temperature overnight. Upon completion of the reaction, 3.15M magnesium chloride (2.3mL, 7.4mmol) was added to the solution, followed by compound 8 (TEA salt, 3.38g, 3.72mmol). The resulting solution was allowed to stir at room temperature overnight. The crude reaction mixture was then diluted with 10x water and purified by anion exchange chromatography. Compound A-7 was pooled and concentrated under vacuum and precipitated as a sodium salt with sodium acetate and 95% absolute ethanol in water. Compound A-7 was obtained as a white powder (2.3g, 56% yield).

[0356] 1H NMR (500 MHz, D2O): δ 8.27 (s, 1H), 8.24 (s, 1H), 8.04 (s, 1H), 7.98 (s, 1H), 7.89 (s, 1H), 5.91 (d, J=5.55Hz,1H), 5.87 (d, J=5.55Hz, 1H), 5.79 (d, J=5.91Hz, 1H), 4.89- 4.86 (m, 1H), 4.58 (t, J=5.37, 1H), 4.48-4.45 (m, 4H), 4.36-4.15 (m, 12H), 3.39 (s, 3H). 31P NMR (200 MHz, D2O): δ..42 (s, 1P), -10.84 (s, 1P), -22.34 (s, 1P) MS m / z = 1113.9 [M-2H] negative mode. Example S7: mRNA Synthesis

[0357] mRNAs including each of the Cap analogs, including compounds as described herein, were synthesized for further testing. See Table 1 for synthesized analogs.

[0358] Table 1. Synthesized mRNAsExample B1: Protein Expression in Cell-based Assays

[0359] Protein expression was measured by Structure Based Design in cell-based assays.

[0360] IVT template was produced by methods known in the art. The IVT template was used to prepare EGFP encoding mRNA capped with different cap analogs by in-vitro transcription. The mRNAs were purified using oligo dT prior to use in assays described below.

[0361] On the day of transfection, 500 µL of Expi293F cells in Expi293 expression medium were transferred to a well in a 24-well plate at concentration of 1.5 x 106 / mL (for each cap analog; each cap analog was tested in triplicate in three separate runs).

[0362] The cells were transfected with Lipofectamine™ MessengerMAX reagent following the manufacture’s instruction.

[0363] Each mRNA was diluted to 0.1 µg / µL and used in the next step.

[0364] In one tube, for each well to be transfected, 1 µL of mRNA (0.1 µg) was added to 25 µL Opti-MEM® I Reduced Serum.

[0365] In another tube, 0.75 µL of Lipofectamine™ MessengerMAX reagent was added to 25 µL Opti-MEM® I Reduced Serum, gently mixed, and then incubated at room temperature for 10 minutes.

[0366] The diluted mRNA was combined with the diluted Lipofectamine™ MessengerMAX. The mixture was mixed gently and incubated at room temperature for 10 minutes.

[0367] The mRNA- Lipofectamine™ MessengerMAX complexes were transferred to each well containing cells.

[0368] At 4, 24, 48 and 72 hours after the transfection, 50 µL of cells were transferred to a well in a 96-well plate for fluorescence quantification, and 50 µL of fresh Expi293 expression medium was added to each well in the 24-well plate containing cells.

[0369] The fluorescence intensity was measured using a Tecan Spark microplate reader at excitation 495 nm and emission 520 nm.

[0370] The mean fluorescent intensity for each mRNA was calculated and ratio of the mean intensity for each mRNA transfected cells vs non transfected cells was plotted on a graph.

[0371] Results:

[0372] FIG.1 shows relative fluorescence intensities (i.e., protein expression levels), 4, 24, 48, and 72 hours post transfection of mRNA capped with different cap analogs, in Expi293F cells. The “no mRNA” EGFP expression was normalized to 1, and all other EGFP expression values are displayed as a ratio to the “no mRNA’ control expression.

[0373] mRNA capped with N7-G(3’OMe)pppm6AmpG (M6) was used as a positivecontrol - .

[0374] “5’HO-AmpG” and “no mRNA” were used as negative controls.

[0375] Maximum fluorescence intensity was observed at 24 hours post transfection. Little change in fluorescence intensity was observed from 24 to 48 hours post transfection. About 33% decline in fluorescence intensity 72 hours post transfection was observed compared to 24 hours post transfection.

[0376] 24 hours post transfection mRNA capped with Cap N7-G(3’OMe)pppm6AmpG (M6) displayed the highest fluorescence intensity (i.e., highest protein expression). All other caps show very little protein expression.

[0377] The compounds and methods of the appended claims are not limited in scope by the specific compounds and methods described herein, which are intended as illustrations of a few aspects of the claims and any compounds and methods that are functionally equivalent are within the scope of this disclosure. Various modifications of the compounds and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, methods, and aspects of these compounds and methods are specifically described, other compounds and methods are intended to fall within the scope of the appended claims. Thus, a combination of steps, elements, components, or constituents can be explicitly mentioned herein; however, all other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

[0378] The detailed description set forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the presentinventive discovery. Such modifications are also intended to fall within the scope of the appended claims. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.

[0379] All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To the extent that any document incorporated by reference is inconsistent with the express content of this disclosure, the express content controls. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

Claims

WHAT IS CLAIMED IS:

1. A compound of formula (I):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: N1is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, natural, modified, or unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside; each B1is independently a natural, modified, or unnatural nucleoside base; each X1is independently -O-, -CH2-, -CX2-, -CHX-, -N(R101)-, -BH-, or -S-; each Y1is independently O, S, or Se; each Z1is independently -OH, -SH, -BH3, substituted or unsubstituted -O-alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted orunsubstituted -O-aryl; each R1is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR1A, -NR1AR1B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R5is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR5A, -NR5AR5B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R1and R5together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene; each R1A, R1B, R5A, and R5Bis independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1Aand R1Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R5Aand R5Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R101is hydrogen, oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H,-SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2,-OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; n is an integer from 0 to 3; m is an integer from 0 to 8; and X is -Cl, -Br, -I or –F.

2. The compound of claim 1, which is a compound of formula (IA):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; 3. The compound of claim 1, which is a compound of formula (IB):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; R4is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR4A, -NR4AR4B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; each R2A, R3A, R4A, and R4B, is independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R4Aand R4Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.

4. The compound of claim 1, which is a compound of formula (IC):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

5. The compound of claim 1, which is a compound of formula (ID):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

6. The compound of claim 1, which is a compound of formula (IE):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

7. The compound of claim 1, which is a compound of formula (IF):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

8. The compound of claim 1, which is a compound of formula (IG):, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

9. The compound of claim 1, which is a compound of formula (IH):or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; and each R2Aand R3Ais independently hydrogen, -CX3, -CHX2, -CH2X,-C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^ ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

10. The compound of any one of claims 1-9, wherein N1is A, C, or U.

11. The compound of claim 10, wherein N1is A or U.

12. The compound of claim 11, wherein N1is A.

13. The compound of any one of claims 1-9, wherein N1is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl.

14. The compound of claim 13, wherein N1is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted alkyl.

15. The compound of claim 14, wherein N1is substituted alkyl or substituted heteroaryl.1 wherein N1.

17. The compound of any one of claims 1-16, wherein each B1is independently A, m6A, G, C, or U.

18. The compound of any one of claims 3-6 or 9-17, wherein each B1is independently G.

19. The compound of any one of claims 3-6 or 9-17, wherein each B1is independently U.

20. The compound of any one of claims 1-3 or 9-19, wherein each R1is independently hydrogen, halogen, or -OR1A.

21. The compound of claim 20, wherein each R1is independently hydroxy, methoxy, ethoxy, propoxy, butoxy, or t-butoxy.

22. The compound of claim 21, wherein each R1is independently methoxy.

23. The compound of any one of claims 3, 4, or 9-22, wherein R2is independently hydrogen or substituted or unsubstituted alkyl.

24. The compound of claim 23, wherein R2is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, or hexyl.

25. The compound of claim 24, wherein R2is independently hydrogen or methyl.

26. The compound of any one of claims 3, 4, or 9-25, wherein R3is independently hydrogen or substituted or unsubstituted alkyl.

27. The compound of claim 26, wherein R3is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, or hexyl.

28. The compound of claim 27, wherein R3is independently hydrogen or methyl.

29. The compound of any one of claims 3-8 or 10-28, wherein R4is hydrogen, halogen, or -NR4AR4B.

30. The compound of claim 29, wherein R4is hydrogen, -F, -NHMe, -NH2, or -NMe2.

31. The compound of claim 30, wherein R4is hydrogen or -NHMe.

32. The compound of claim 31, wherein R4is hydrogen.

33. The compound of any one of claims 1-32, wherein each R5is independently hydrogen.

34. The compound of any one of claims 1-19 or 23-32, wherein at least one R1forms a ring together with the R5within four atoms of said R1and the atoms to which they are connected, thereby forming a locked nucleic acid (LNA).

35. The compound of any one of claims 1-34, wherein each X1is independently -O-, -CH2-, -CF2-, or -S-.

36. The compound of claim 35, wherein each X1is independently -O-.

37. The compound of any one of claims 1-36, wherein each Y1is independently O or S.

38. The compound of claim 37, wherein each Y1is independently O.

39. The compound of any one of claims 1-38, wherein each Z1is independently -OH or -SH.

40. The compound of claim 39, wherein each Z1is independently -OH.

41. The compound of any one of claims 1 or 10-40, wherein m is 0 or 1.

42. The compound of any one of claims 1-41, wherein n is 1.

43. A compound of the following structure:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

44. A compound of the following structure:or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. 4, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

46. A compound of the following structure:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

47. A compound of the following structure:or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

48. A compound of the following structure:, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

49. The compound of any one of claims 1-48, wherein the pharmaceutically acceptable salt is a sodium salt, a lithium salt, or a potassium salt.

50. The compound of claim 49, wherein the pharmaceutically acceptable salt is a sodium salt.

51. An RNA molecule comprising a 5’-cap, wherein the 5’-cap comprises a compound of any one of claims 1-50.

52. The RNA molecule of claim 51, wherein the RNA molecule is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule.

53. A pharmaceutical composition comprising a compound of any one of claims 1-50 and a pharmaceutically acceptable excipient.

54. A pharmaceutical composition comprising an RNA molecule of claim 51 and a pharmaceutically acceptable excipient.

55. An initiating capped oligonucleotide primer of the following formula: Apppm6A2’OMepG.

56. An initiating capped oligonucleotide primer of the following formula: Ph-4-Bu-pppm6A2’OMepG.

57. An initiating capped oligonucleotide primer of the following formula: Ph-4-Bu-pppA2’OMepG.

58. An initiating capped oligonucleotide primer of the following formula: Thiamine-pppA2’OMepG.

59. An initiating capped oligonucleotide primer of the following formula: Thiamine-pppm6A2’OMepG.

60. An initiating capped oligonucleotide primer of the following formula: ApppA2’OMepG.

61. An RNA molecule comprising the initiating capped oligonucleotide primer, stereoisomer, or a pharmaceutically acceptable salt thereof, of any one of claims 55-60.

62. The RNA molecule of claim 61, wherein the pharmaceutically acceptable salt is a sodium salt, a lithium salt, or a potassium salt.

63. The RNA molecule of claim 62, wherein the pharmaceutically acceptable salt is a sodium salt.

64. The RNA molecule of claim 63, wherein the RNA molecule is a messenger RNA (mRNA) molecule or a self-amplifying RNA (saRNA) molecule.

65. A pharmaceutical composition comprising an RNA molecule comprising the initiating capped oligonucleotide primer, stereoisomer, or a pharmaceutically acceptable salt thereof, of any one of claims 55-60, and a pharmaceutically acceptable excipient.

66. A method of inducing a therapeutic effect in a subject, comprising administering to the subject an RNA molecule according to claim 51 or 61 or a pharmaceutical composition according to claim 53 or 65.

67. A method of administering to a subject a therapeutic dose unit of an mRNA molecule comprising a 5’-cap, wherein the 5’-cap comprises a compound of any one of claims 1-50.

68. The RNA molecule of claim 51 or 61, for use in therapy.

69. The mRNA molecule comprising a 5’-cap, wherein the 5’-cap comprises a compound of any one of claims 1-50, for use in therapy.

70. A method of inhibiting eIF4E in a mammal comprising administering a compound of any one of claims 1-50, an RNA molecule of claim 51 or 61, or a pharmaceutical composition of claim 53 or 65.

71. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (I), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: N1is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, natural, modified, or unnatural nucleoside; with the proviso that N1is not a modified or unmodified G nucleoside; each B1is independently a natural, modified, or unnatural nucleoside base;each X1is independently -O-, -CH2-, -CX2-, -CHX-, -N(R101)-, -BH-, or -S-; each Y1is independently O, S, or Se; each Z1is independently -OH, -SH, -BH3, substituted or unsubstituted -O-alkyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted -O-aryl; each R1is independently hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR1A, -NR1AR1B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R5is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR5A, -NR5AR5B, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R1and R5together with the carbon atoms to which they are connected form a substituted or unsubstituted cycloalkylene or substituted or unsubstituted heterocycloalkylene; each R1A, R1B, R5A, and R5Bis independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1Aand R1Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl;R5Aand R5Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; R101is hydrogen, oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2,-OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; n is an integer from 0 to 3; m is an integer from 0 to 8; and X is -Cl, -Br, -I or –F; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

72. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IA), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase underconditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

73. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IB)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; R4is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, -CHCl2, -CHBr2, -CHF2, -CHI2, - CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OR4A, -NR4AR4B, -COOH, -CONH2,-NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, -N3, -SF5, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; each R2A, R3A, R4A, and R4B, is independently hydrogen, -CX3, -CHX2, -CH2X, -C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R4Aand R4Bsubstituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

74. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IC)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

75. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (ID)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

76. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IE), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

77. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IF) ,or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

78. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IG), or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

79. A method for preparing a fully templated RNA molecule comprising: introducing the initiating capped oligonucleotide primer of formula (IH)or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof; wherein: R2is independently hydrogen, –C(O)R2A, –C(O)OR2A, –OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R3is independently hydrogen, –C(O)R3A, –C(O)OR3A, –OR3A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or R2and R3together with the nitrogen atom to which they are connected form a substituted or unsubstituted heteroaryl or a substituted or unsubstituted heterocyclyl; and each R2Aand R3Ais independently hydrogen, -CX3, -CHX2, -CH2X,-C(O)OH, -C(O)NH2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, ^ ^NHNH2, ^ONH2, ^NHC=(O)NHNH2, ^NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)OH, -NHOH, -OCX3, -OCHX2, -OCH2X, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl; into a mixture comprising a polynucleotide template and an RNA polymerase under conditions conducive to transcription by the RNA polymerase of the polynucleotide template and incubating said mixture for a time sufficient to allow for transcription of said template.

80. The method of any one of claims 71-79, wherein B1, R1, R2, R3, R4, R5, N1, X1, Y1, Z1, m and n are as defined in any one of claims 1-42.

Images