WO2023039068A1

WO2023039068A1 - Compositions and methods for synthesis of peptide nucleic acid intermediates

Info

Publication number: WO2023039068A1
Application number: PCT/US2022/042912
Authority: WO
Inventors: James M. Coull; Brian D. Gildea
Original assignee: Neubase Therapeutics, Inc.
Priority date: 2021-09-08
Filing date: 2022-09-08
Publication date: 2023-03-16

Abstract

The present disclosure provides intermediates for the synthesis of peptide nucleic acid (PNA) backbones and monomers, such as cyclic intermediates, and methods of making the same.

Description

COMPOSITIONS AND METHODS FOR SYNTHESIS OF PEPTIDE NUCLEIC ACID INTERMEDIATES

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/241,800, filed September 8, 2021, which is entirely incorporated herein by reference.

BACKGROUND

[0002] The preparation of peptide nucleic acid intermediates typically entails multiple synthetic steps and can be associated with low yields and purity. As such, there is a need in the art for more efficient methods to synthesize peptide nucleic acid intermediates.

SUMMARY

[0003] The present disclosure provides intermediates for the synthesis of peptide nucleic acid (PNA) backbones and monomers, such as cyclic intermediates, and methods of making the same. PNAs are a class of nucleic acid mimics developed by Nielsen and co-workers in the early 1990s, and are distinguished from standard nucleic acids such as DNA and RNA in part by the chemical differences within the backbone structure (Nielsen et al (1991) Science 254: 1497). In contrast to the carbohydrate phosphodiester subunits found in DNA and RNA, each PNA monomer comprises a peptide-based backbone, such as a N-(2-aminoethyl) glycine unit, onto which a nucleobase may be tethered. PNAs are attractive for use as nucleic acid mimics for numerous reasons. For example, PNAs retain the sequence-specific binding of standard nucleic acids to a target sequence but exhibit increased stability to proteolytic degradation (Nielsen (1999) Acc. Chem. Res. 32:624)). PNAs may be employed for a number of promising uses, including diagnostic tools, biosensing, and molecular engineering (Sacui, et al. (2015) J. Am. Chem. Soc. 137:8603; Nolling, et al (2016) mBio 7:e00345).

[0004] The original PNAs designed by Nielsen et al were considerably less water soluble than standard nucleic acids; however, considerable effort in recent years has focused on engineering modifications to improve solubility. One promising approach has been to add a water soluble group along the PNA backbone, such as at the gamma position (Englund, et al. (2005) Org. Lett. 7:3465; Tedeschi, et al (2005) Tetrahedron Lett. 46:8395). This modification renders the PNA monomer chiral. Studies have shown that the right-handed stereoisomer has complementarity to DNA and/or RNA, and is capable of invading B-form DNA (Bahai, et al. (2012) ChemBioChem 13:56).

[0005] Current methods for preparing PNA backbones and PNA monomers is often associated with low yields and/or low enantioselectivity (Hseih et al, (2019) J. Org. Chem., 84:1276). For example, a common method to prepare these compounds makes use of the Mitsunobu reaction to convert an amino alcohol to a protected PNA backbone (e.g., a compound not containing a nucleobase moiety). Although this method preserves the chirality of the resulting product, the yield is often low and the required reactants can be costly and time-consuming to prepare. The synthetic methods disclosed herein feature a class of versatile cyclic compounds that may be efficiently employed to achieve a variety of useful PNA intermediates. These cyclic compounds are relatively inexpensive to prepare and can vastly improve the resulting yield of desired PNA intermediates, thus facilitating commercial scale up efforts.

[0006] In one aspect, the present disclosure provides a compound of Formula (IIA):

R⁶ R⁵ ^R o¹'=^N>s^>$ II-o o (IIA) or a salt thereof, wherein:

R¹ is 9-fluorenylmethyloxycarbonyl (Fmoc), 2-(4-nitropheylsulfonyl)ethoxycarbonyl (Nsc), 1,1 -di oxobenzo[b]thiophene-2-ylmethyloxy carbonyl (Bsmoc), 1,1 -di oxonaphthofl, 2- b]thiophene (Nsmoc), l-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl (ivDde), 2,7- di-tert-butylfluoren-9-ylmethoxy carbonyl (Fmoc*), 2-fluorofluoren-9-ylmethoxy carbonyl (Fmoc(2F)), 2-monoisooctylfluoren-9-ylmethoxy carbonyl (mio-Fmoc), 2,7-diisooctylfluoren-9- ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)-fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-t- butyl-9-fluorenylmethoxy carbonyl (Dtb-Fmoc), 2,7-bis(trimethylsilyl)- fluorenylmethoxycarbonyl (Bts-Fmoc), 9-(2,7-dibromo)fluorenylmethoxycarbonyl, 2- [phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms), ethanesulfonylethoxycarbonyl (Esc), 2-(4-sulfophenylsulfonyl)ethoxy carbonyl (Sps), or 1- cyano-2-methylpropan-2-ylcarbonyl (Cyoc), each of which is optionally substituted, or hydrogen; each of R⁵ and R⁶ are independently hydrogen, C3-C16 alkyl, cycloalkyl, C2-C16 alkenyl, C2-C16 alkynyl, Ce-Ci6 heteroalkyl, Ci-Ci6-haloalkyl, -OR^C, -CH2OR⁹, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkylene-heteroaryl, an optionally protected amino acid side chain,

, provided that one of R⁵ and R⁶ is not hydrogen, or both of R⁵ and R⁶ are not hydrogen; x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; and

R^c is hydrogen, C1-C12 alkyl, cycloalkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl, wherein each optionally protected amino acid side chain is independently selected from:

wherein each of Ill-a through III-z is independently and optionally protected.

[0007] In another aspect, the present disclosure provides a method of making a compound of Formula (IB):

salt thereof, wherein

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

R² is hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, C1-C16 heteroalkyl, C1-C16- haloalkyl, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkyl ene-heteroaryl; each of R³, R⁴, R⁵, and R⁶ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl, -N(R^A)(R^B), halo, -OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

, provided that each of R⁵ and R⁶ are not both hydrogen,

R⁷ is hydrogen or C1-C16 alkyl; each of R^A, R^B, and R^c is independently hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; n is an integer selected from 0, 1, 2, 3, or 4; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; wherein the method comprises use of an intermediate of Formula (IIB):

[0008] In another aspect, the present disclosure provides a method of making a compound of Formula (IB):

salt thereof, wherein

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

,

provided that each of R⁵ and R⁶ are not both hydrogen,

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; n is an integer selected from 0, 1, 2, 3, or 4; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; wherein the method comprises the following steps: (i) contacting a compound of Formula (IIB):

or a salt thereof, with an azide-containing compound to achieve the azide-containing intermediate of Formula (IVB):

(ii) contacting the azide-containing intermediate of Formula (IVB) with a reducing agent to achieve an intermediate of Formula (VIIB):

(iii) contacting the intermediate of Formula (VIIB) with an ester compound of Formula (VB):

to achieve a compound of Formula (VIIIB):

(iv) contacting the compound of Formula (VIIIB) with a nucleobase acetic acid to achieve the compound of Formula (IB).

[0009] In another aspect, the present invention provides pharmaceutical compositions comprising a compound of Formulas (I), (IB), (II), or (IIB), or a salt, solvate, hydrate, tautomer, stereoisomer, or isotopically labeled derivative thereof. The present invention may further comprise kits comprising a container with a compound of Formulas (I), (IB), (II), or (IIB), or a salt, solvate, hydrate, tautomer, stereoisomer, or isotopically labeled derivative thereof, or a pharmaceutical composition thereof.

[0010] The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 is an illustration of a generic peptide nucleic acid (PNA) subunit where B represents a nucleobase, and a, P, and y represent optionally substituted positions on the PNA backbone.

[0012] FIGS. 2A-2B are illustrations that depict a method to prepare peptide nucleic acid intermediates described herein. FIG. 2A depicts the conversion of amino alcohol (A), to a cyclic compound (B), and subsequent conversion to an acyclic compound (C) by nucleophilic addition. FIG. 2B depicts various routes to PNA monomers (I, G, and L) using methods and PNA intermediates described herein.

[0013] FIGS. 3A-3B are illustrations depicting an exemplary synthetic scheme for peptide nucleic acids intermediates using the Mitsunobu route. FIG. 3 A depicts the preparation of an intermediate used in the Mitsunobu route. FIG. 3B depicts the Mitsunobu route to prepare a PNA monomer.

[0014] FIG. 4 is an illustration depicting a scheme of exemplary routes to a chiral PNA monomer, e.g., using a method described herein.

[0015] FIG. 5 is an illustration of several common unprotected nucleobases (identified as “B” in FIG. 1) that can be linked to a PNA monomer.

[0016] FIG. 6 is an illustration of various exemplary nucleobases used in PNA synthesis.

[0017] FIGS. 7A-7B is an illustration of various side chains that can be linked to a PNA subunit, including the side chains of amino acids.

[0018] FIG. 8 is an illustration of several exemplary base-labile N-terminal amine protecting groups that can be used in an orthogonal protection scheme for the N-terminal amine group of PNA monomers or PNA Monomer Esters (e.g., as described herein) as contemplated by some embodiments of the present invention.

[0019] FIG. 9 an illustration of several exemplary acid-labile N-terminal amine protecting groups that can be used in an orthogonal protection scheme for the N-terminal amine group of PNA monomers or PNA Monomer Esters (e.g., as described herein) as contemplated by some embodiments of the present invention.

[0020] FIG. 10a is an illustration of several exemplary base-labile exocyclic amine protecting groups that can be used in an orthogonal protection scheme for the nucleobases of PNA monomers or PNA Monomer Esters (e.g., as described herein) as contemplated by some embodiments of the present invention.

[0021] FIG. 10b is an illustration of several exemplary acid-labile exocyclic amine protecting groups (or protecting group schemes such as Bis-Boc) that can be used in an orthogonal protection scheme for the nucleobases of PNA monomers or PNA Monomer Esters (e.g., as described herein) as contemplated by some embodiments of the present invention.

[0022] FIG. 10c is an illustration of several exemplary imide and lactam protecting groups that can be used in an orthogonal protection scheme for the nucleobases of PNA monomers or PNA Monomer Esters as contemplated by some embodiments of the present invention.

[0023] FIG Ila is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect amine containing side chain moieties such as those of formulas: Illi, Illj, Illk and Illm.

[0024] FIG 11b is an illustration of several exemplary base-labile protecting groups that can be used, inter alia, to protect amine containing side chain moieties such as those of formulas: Illi, Illj, Illk and Illm.

[0025] FIG. 12a is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect carboxylic acid containing side chain moieties such as those of formulas: lilt and IIIu.

[0026] FIG 12b is an illustration of several exemplary base-labile protecting groups that can be used, inter alia, to protect carboxylic acid containing side chain moieties such as those of formulas: lilt and IIIu.

[0027] FIG. 13 is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect amide containing side chain groups such as those of formulas: IIIv and IIIw.

[0028] FIG 14a is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect guanidinium containing side chain moieties such as those of formula: IIIx.

[0029] FIG 14b is an illustration of an exemplary base-labile protecting group that can be used, inter alia, to protect guanidinium containing side chain moieties such as those of formula: IIIx. [0030] FIG. 15a is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect thiol containing side chain moieties such as those of formula: Ilin.

[0031] FIG 15b is an illustration of several exemplary base-labile protecting groups that can be used, inter alia, to protect thiol containing side chain moieties such as those of formula: Ilin.

[0032] FIG. 16a is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect indole side chain moieties such as those of formula: Illy. [0033] FIG 16b is an illustration of an exemplary other protecting group that can be used, inter alia, to protect indole side chain moieties such as those of formula: Illy.

[0034] FIG. 17a is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect imidazole side chain moieties such as those of formula: IIIz.

[0035] FIG 17b is an illustration of several exemplary base-labile protecting groups that can be used, inter alia, to protect imidazole side chain moieties such as those of formula: IIIz.

[0036] FIG. 18a is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect hydroxyl containing moieties such as those of formulas: Illq and Illr. [0037] FIG 18b is an illustration of several exemplary other protecting groups that can be used, inter alia, to protect hydroxyl containing moieties such as those of formulas: Illq and Illr.

[0038] FIG. 19a is an illustration of several exemplary acid-labile protecting groups that can be used, inter alia, to protect phenolic containing moieties such as those of formula: Ills.

[0039] FIG 19b is an illustration of several exemplary other protecting groups that can be used, inter alia, to protect phenolic containing moieties such as those of formula: Ills.

[0040] FIG. 20a is an illustration of various examples of suitable nucleobases (in unprotected form) that can be used in some of the novel PNA Monomer Ester embodiments of the present invention.

[0041] FIG 20b is an illustration of various examples of suitable protected forms of the nucleobases illustrated in Fig. 18a that can be used in some of the novel PNA Monomer Ester embodiments of the present invention.

DETAILED DESCRIPTION

[0042] Described herein are compounds, compositions and methods for the synthesis of peptide nucleic acid (PNA) intermediates, such as PNA backbones and PNA monomers.

[0043] The compositions and synthetic methods disclosed herein feature a class of versatile cyclic compounds that may be efficiently employed to achieve a variety of useful PNA intermediates. These cyclic compounds are relatively inexpensive to prepare and can vastly improve the resulting yield of desired PNA intermediates, thus facilitating commercial scale up efforts.

Definitions

[0044] “Peptide nucleic acid,” “PNA,” or “PNA oligomer” as used herein, refer to a non-natural polymer composition comprising linked nucleobases capable of sequence specifically hybridizing to a target nucleic acid. A PNA oligomer is comprised of PNA subunits (e.g., a PNA monomer), each of which comprise a backbone moiety and, optionally, a nucleobase moiety that can form hydrogen bonds with the nucleobase of the target nucleic acid. Exemplary PNA oligomers are disclosed in or otherwise claimed in any of the following: U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 or 5,786,461; (each of the foregoing are herein incorporated herein by reference in its entirety). The term "peptide nucleic acid", "PNA", or “PNA oligomer” shall also apply to polymers comprising two or more PNA subunits of kind described in the following publications: Diderichsen et al., Tetrahedron Lett. 37:475-478 (1996); Fujii et al., Bioorg. Med. Chem. Lett. 7:637-627 (1997); Jordan et al., Bioorg. Med. Chem. Lett. 7:687-690 (1997); Krotz et al., Tetrahedron Lett. 36:6941-6944 (1995); Lagriffoul et al., Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1 :539-546; Lowe et al., J. Chem. Soc. Perkin Trans. 1, 1 :547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1 :555-560 (1997); Mitra R. and Ganesh, K.N. Chem Commun (Camb) 47:1198-1200 (2011); Petersen et al., Bioorg. Med. Chem. Lett. 6:793- 796 (1996); Diederichsen, U., Bioorg. Med. Chem. Lett., 8: 165-168 (1998); Cantin et al., Tetrahedron Lett., 38:4211-4214 (1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997);

Lagriffoule et al., Chem. Eur. J., 3:912-919 (1997); Mann A. et al, Methods Mol Biol. 1050: 1-12 (2014); Sugiyama T. and Kittaka, A. Molecules 18:287-310 (2012); and International Patent Publication No. W096/04000. Additional PNA oligomers include phosphono-PNA analogues (pPNAs) as described in van der Laan, A. C. et al., Tetrahedron Lett. 37:7857-7860 (1996); trans-4-hydroxy-L-proline nucleic acids (HypNAs) as described in Efimov et al., Nucleic Acids Res. 34(8):2247-2257 (2006); and (lS,2R/lR,2S)-cis-cyclopentyl PNAs (cpPNAs) as described in Govindaraju, T. et al., J. Org. Chem. 69(17):5725-34 (2004); each of the foregoing is herein incorporated herein by reference in its entirety.

[0045] An “abasic PNA monomer,” as used herein, refers to a single discrete heteroalkyl chain that comprises, for example, an aminoethylglycine moiety. An abasic PNA monomer may comprise a PNA backbone. An abasic PNA monomer, such as a PNA backbone, typically does not comprise a nucleobase moiety (e.g., a nucleobase acetic acid), and may exist as a free base or a salt thereof.

[0046] A “PNA monomer,” as used herein, refers to a single discrete building block for PNA synthesis. A PNA monomer comprises a backbone moiety and a nucleobase moiety. In an embodiment, a PNA monomer can form hydrogen bonds with a target nucleic acid. In an embodiment, a PNA monomer is responsible for recognition of a target nucleic acid. To form a PNA oligomer, a first PNA monomer is may be activated, for example, by exposure to an activating group (e.g., a carboxyl activating group such as PyBOP or HATU). The PNA monomer may then be coupled to a particular reactive moiety (e.g., a free amine terminus (i.e., N-terminus)) on second deprotected PNA monomer or a PNA oligomer to form a growing PNA oligomer chain. Exemplary PNA monomers include Fmoc/Bhoc PNA monomers, Fmoc/Boc PNA monomers, Boc/Z PNA monomers, Boc/Cbz PNA monomers, and others. Additional exemplary PNA monomers are included in WO 2012/138955; WO 2018/175927; Eriksson and Nielsen Quart. Rev. Biophys. 29(4):369-394 (1996); and Sugiyama et al., Bioorg Med Chem Lett 27(15):3337-3341 (2017); which are incorporated herein by reference in their entirety. The nucleobases within a PNA monomer may be naturally occurring or non-naturally occurring. In an embodiment, a nucleobase within a PNA monomer comprises any nucleobase found in nature, e.g., as a constituent of tRNA, mRNA, miRNA, and any other naturally occurring RNA or DNA. In an embodiment, a nucleobase within a PNA monomer comprises any nucleobase synthetically designed and incorporated into a nucleobase, nucleotide, synthetic nucleic acid, nucleic acid analog, nucleic acid mimic, e.g., incorporated by chemical, enzymatic, or other means. Exemplary nucleobases include adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2-thiopseudoisocytosine, 5-methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine (or 2,6-diaminopurine), 2-thiouracil, pseudoisouracil, 2-thiothymine, 2-thiocytosine, 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5-chlorocytosine, 5- bromocytosine, 5-iodocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7- deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 7-deaza-8 -aza guanine, 7- deaza-8-aza adenine, 5-propynyl uracil and 2-thio-5-propynyl, pyridazin-3(2H)-one (E), pyrimidin-2(lH)-one (P) and pyridin-2-amine (M), as well as tautomeric forms thereof.

Selected Chemical Definitions

[0047] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March ’s Advanced Organic Chemistry, 5^th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3^rd Edition, Cambridge University Press, Cambridge, 1987.

[0048] 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. [0049] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “Ci-Ce alkyl” is intended to encompass, Ci, C2, C3, C4, C5, Ce, C1-C6, C1-C₅, C1-C4, C1-C3, C1-C2, C2-C6, C₂-C₅, C2-C4, C2-C3, C3-C6, C₃-C₅, C3-C4, C4-C6, c₄- C5, and C5-C6 alkyl.

[0050] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention.

[0051] As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 18 carbon atoms (“Ci-Cis alkyl”). In some embodiments, an alkyl group has 1 to 16 carbon atoms (“C1-C16 alkyl”). In some embodiments, an alkyl group has 4 to 16 carbon atoms (“C4-C16 alkyl”). In some embodiments, an alkyl group has 6 to 16 carbon atoms (“Ce-Ci6 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-C12 alkyl”). In some embodiments, an alkyl group has 4 to 12 carbon atoms (“C4-C12 alkyl”). In some embodiments, an alkyl group has 6 to 12 carbon atoms (“C6-C12 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“Ci-Cs alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-C7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“Ci-Ce alkyl”). In some embodiments, an alkyl group has

1 to 5 carbon atoms (“C1-C5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-C4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-C3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-C2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6 alkyl”). Examples of C1-C24 alkyl groups include methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tert-amyl (C5), n-hexyl (Ce), octyl (Cs), nonyl (C9), decyl (C10), undecyl (Cu), dodecyl (or lauryl) (C12), tridecyl (C13), tetradecyl (or myristyl) (C14), pentadecyl (C15), hexadecyl (or cetyl) (Cie), heptadecyl (C17), or octadecyl (or stearyl) (Cis), and the like. Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0052] As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 18 carbon atoms (“C2-C18 alkenyl”). In some embodiments, an alkenyl group has 2 to 12 carbon atoms (“C2-C12 alkenyl”). In some embodiments, an alkenyl group has

2 to 8 carbon atoms (“C2-C8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-C7 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-C8 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2- Ce alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-C5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-C4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-C3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carboncarbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). The one or more carbon double bonds can have cis or trans (or E or Z) geometry. Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2- butenyl (C4), butadienyl (C4), and the like. Examples of C2-C24 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (Ce), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (Cs), octatrienyl (Cs), nonenyl (C9), nonadienyl (C9), decenyl (C10), decadienyl (C10), undecenyl (C11), undecadienyl (Cn), dodecenyl (C12), dodecadienyl (C12), tridecenyl (C13), tridecadienyl (C13), tetradecenyl (C14), tetradecadienyl (e.g., myristoleyl) (C14), pentadecenyl (C15), pentadecadienyl (C15), hexadecenyl (e.g., palmitoleyl) (Cie), hexadecadienyl (Cie), heptadecenyl (C17), heptadecadienyl (C17), octadecenyl (e.g., oleyl) (Cis), or octadecadienyl (e.g., linoleyl) (Cis), and the like. Each instance of an alkenyl group may be independently optionally substituted, /.< ., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkenyl group is unsubstituted C2-10 alkenyl.

[0053] As used herein, the term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 18 carbon atoms (“C2-C18 alkynyl”). In some embodiments, an alkynyl group has 2 to 12 carbon atoms (“C2-C12 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-C8 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-C6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-C5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-C4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-C3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carboncarbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1- butynyl (C4), 2-butynyl (C4), and the like. Each instance of an alkynyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkynyl group is unsubstituted C2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C2-6 alkynyl.

[0054] As used herein, the terms "heteroalkyl," “heteroalkenyl,” and “heteroalkynyl,” refer to a non-cyclic stable straight or branched alkyl, alkenyl, or alkynyl chains, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of 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) O, N, P, S, and Si may be placed at any position of the heteroalkyl, heteroalkenyl, or heteroalkynyl group. Exemplary heteroalkyl, heteroalkenyl, and heteroalkynyl groups include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH₂-CH₂-N(CH3)-CH3, -CH2-S-CH2-CH3, - CH₂-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, and -O-CH2-CH3. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0055] The terms "alkylene," “alkenylene,” “alkynylene,” or “heteroalkylene,” alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene. An alkylene, alkenylene, alkynylene, or heteroalkylene group may be described as, e.g., a Ci-Ce- membered alkylene, Ci-Ce-membered alkenylene, Ci-Ce-membered alkynylene, or Ci-Ce- membered heteroalkylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety. In the case of 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’- may represent both -C(O)2R’- and -R’C(O)2-. Each instance of an alkylene, alkenylene, alkynylene, or heteroalkylene group may be independently optionally substituted, z.e., unsubstituted (an “unsubstituted alkylene”) or substituted (a “substituted heteroalkylene) with one or more substituents.

[0056] As used herein, "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 it electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“Ce-Cu aryl”). In some embodiments, an aryl group has six ring carbon atoms (“Ce aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). An aryl group may be described as, e.g., a Ce-C 10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may be independently optionally substituted, /.< ., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted Ce-Cu aryl. In certain embodiments, the aryl group is a substituted Ce-Cu aryl.

[0057] As used herein, “cycloalkyl” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 7 ring carbon atoms (“C3-C7 cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 7 ring carbon atoms (“C5-C7 cycloalkyl”). A cycloalkyl group may be described as, e.g., a C4-C7-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (Ce), cyclohexenyl (Ce), cyclohexadienyl (Ce), and the like. Exemplary C3-C7 cycloalkyl groups include, without limitation, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), and cycloheptatrienyl (C7), bicyclo[2.1.1]hexanyl (Ce), bicyclo[3.1.1]heptanyl (C7), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated.

“Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may be independently optionally substituted, /.< ., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.

[0058] As used herein, the term “halo” refers to a fluorine, chlorine, bromine, or iodine radical (i.e., -F, -Cl, -Br, and -I, respectively).

[0059] As used herein, the term “heteroaryl,” refers to an aromatic heterocycle that comprises 1, 2, 3 or 4 heteroatoms selected, independently of the others, from nitrogen, sulfur and oxygen. As used herein, the term “heteroaryl” refers to a group that may be substituted or unsubstituted. A heteroaryl may be fused to one or two rings, such as a cycloalkyl, an aryl, or a heteroaryl ring. The point of attachment of a heteroaryl to a molecule may be on the heteroaryl, cycloalkyl, or aryl ring, and the heteroaryl group may be attached through carbon or a heteroatom. Examples of heteroaryl groups include imidazolyl, furyl, pyrrolyl, thienyl, thiazolyl, isoxazolyl, isothiazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, quinolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzisooxazolyl, benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl, pyrazolyl, triazolyl, oxazolyl, tetrazolyl, benzimidazolyl, benzoisothiazolyl, benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl, purinyl, pyrrolo[2,3]pyrimidyl, pyrazolo[3,4]pyrimidyl or benzo(b)thienyl, each of which can be optionally substituted. Each instance of a heteroaryl group may be independently optionally substituted, z.e., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.

[0060] As used herein, the term “heterocyclyl” refers to a radical of a heterocyclic ring system. Representative heterocyclyls include ring systems in which (i) every ring is non-aromatic and at least one ring comprises a heteroatom, e.g., tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl; (ii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is an aromatic carbon ring, e.g., 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl; and (iii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is aromatic and comprises a heteroatom, e.g., 3,4-dihydro-lH-pyrano[4,3-c]pyridine, and l,2,3,4-tetrahydro-2,6-naphthyridine. In certain embodiments, the heterocyclyl is a monocyclic or bicyclic ring, wherein each of said rings contains 3-7 ring atoms where 1, 2, 3, or 4 of said ring atoms are a heteroatom independently selected from N, O, and S. Each instance of a heterocyclyl group may be independently optionally substituted, /.< ., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents.

[0061] As used herein, the term “hydroxy” refers to the radical -OH.

[0062] As used herein, the terms “carbonyl” and “oxo” each refer to the radical -C=O.

[0063] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Exemplary substituents on a substitutable atom of an “optionally substituted” group (such as an atom within a alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocyclyl or heteroaryl) may include, for example, deuterium, halogen, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, -OH, -O-alkyl, - O-alkenyl, O-alkynyl, O-aryl, O-heteroaryl, O-cycloalkyl, O-heterocyclyl, -NO2, -CN, -N3, =0, =S, -SH, -S-alkyl, -S(O)-alkyl, -S(O)₂-alkyl, -C(O)OH, -C(O)alkyl, -C(O)O-alkyl, -C(0)NH2, - C(O)NH-alkyl, -C(O)N(alkyl)₂, -P(O)₂-alkyl, -P(O)-(alkyl)₂, -OP(O)alkyl, -OP(O)(O-(alkyl))₂, or -Si(alkyl)3. In an embodiment, an optionally substituted group may be itself optionally substituted. The symbol

as used herein refers to an attachment point to another moiety or functional group. For example, the -~^-may refer to an attachment point to a peptide nucleic acid backbone or the attachment point to another region or atom within a PNA intermediate. In some embodiments,

denotes an attachment point to a PNA monomer or PNA oligomer. [0064] As used herein, “hydrate” refers to a compound which is associated with water.

Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R xH₂0, wherein R is the compound and wherein x is a number greater than 0.

[0065] As used herein, a “pharmaceutically acceptable salt” refers to salts of the compounds that are prepared with acids or bases (e.g., relatively non-toxic acids or bases), depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of base addition salts (e.g., pharmaceutically acceptable base addition salts) include sodium, potassium, calcium, magnesium, ammonium, organic amino, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of acid addition salts (e.g., pharmaceutically acceptable acid addition salts) include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts may be prepared by methods known to those skilled in the art. Other carriers (e.g., pharmaceutically acceptable carriers) known to those of skill in the art are suitable for the present invention.

[0066] As used herein, “solvate” refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, dimethylsulfoxide, tetrahydrofuran, diethyl ether, and the like. The compounds described herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non- stoichiometric solvates.

[0067] The term “tautomer” as used herein refers to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of it electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest. [0068] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC), selective crystallization as chiral salts, or in the presence of chiral hosts, or from chiral solvents, as well as through enrichment using enzymes or chemical means including but not limited to dynamic kinetic resolution; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. [0069] As used herein, a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an “5” form of the compound is substantially free from the “A” form of the compound and is, thus, in enantiomeric excess of the “A” form. In some embodiments, ‘substantially free’, refers to: (i) an aliquot of an “A” form compound that contains less than 2% “S” form; or (ii) an aliquot of an “S” form compound that contains less than 2% “A” form. The term “enantiomerically pure” or “pure enantiomer” denotes that the compound comprises more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the single enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.

[0070] Compound described herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D or deuterium), and ³H (T or tritium); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Peptide Nucleic Acid Intermediates

Abasic Peptide Nucleic Acid Monomers

[0071] Described herein are methods of making peptide nucleic acid (PNA) intermediates, such as abasic PNA monomers. An abasic PNA monomer may also be referred to herein as a PNA backbone. PNA backbones encompass the heteroalkyl chain to which the nucleobase and other optional substituents are covalently bound (see, e.g., FIG. 1), and are convenient intermediates for synthesis of PNA monomers. A PNA backbone may, for example, comprise an aminoethylglycine moiety and an amine (e.g., a secondary amine) that may be further modified by conjugation to a nucleobase described herein. PNA backbones may further comprise protecting groups on both the N and C-termini, for example, an amine-protecting group and a carboxylate protecting group, which can be selectively added or removed as needed. Additionally, the PNA backbone may be obtained as a salt (e.g., a pharmaceutically acceptable salt), which can provide beneficial effects such as increased stability and ease of handling. PNA backbone salts can be obtained by a method described herein, e.g., by treating a PNA backbone with an acid (e.g., a stoichiometric or excess amount of acid). Exemplary PNA backbone salts include hydrochloride, hydrobromide, hydroiodide, acetate, trifluoroacetate, p-toluenesulfonate, methanesulfonate, or citrate salts. Other salts of PNA backbones have been described, e.g., in US Patent Publication No.: 2019/0055190, which is incorporated herein by reference in its entirety.

[0072] In an embodiment, R² is hydrogen, C1-C12 alkyl (e.g., t-butyl), C2-C12 alkenyl (e.g., allyl), or Ci-Ci2-haloalkyl (e.g., 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, or 2-iodoethyl). In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl.

[0073] A PNA backbone or salt thereof may be modified at any position along the backbone chain, for example, at the alpha, beta, or gamma positions (see, e.g., FIG. 1). For example, a PNA backbone may comprise a compound of Formula (VIII):

wherein R¹ is hydrogen, or an amine protecting group (e.g., an amine protecting group described herein; e.g., Fmoc); R² is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, or C1-C12 alkyl ene-heteroaryl; each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, - N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R⁷ is hydrogen or C1-C12 alkyl; and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[0074] In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen, Ci- C12 alkyl, C1-C12 heteroalkyl, or the side chain of an amino acid. In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen. In an embodiment, one of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is C1-C12 alkyl (e.g., methyl) or C1-C12 heteroalkyl, and the others of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² are each independently hydrogen. In an embodiment, one of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is methyl, and the others of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² are each independently hydrogen. In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen or a polyalkylene glycol (e.g., a polyethylene glycol (PEG), e.g., a PEG moiety comprising between 2 and 6 PEG units. In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen or the side-chain of an amino acid. The side-chain of an amino acid may be protected with a protecting group described herein.

[0075] In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen or has the structure of Formula (Illaa) or Formula (Illlab):

wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or Ci- C12 alkyl (e.g., t-butyl, methyl, or ethyl).

[0076] In an embodiment, one of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is

(Illaa)

In an embodiment, x is an integer selected from 0, 1, 2, 3,

4, 5, 6, 7, 8, 9, and 10. In an embodiment, x is an integer selected from 0, 1, 2, 3, and 4. In an embodiment, R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[0077] In an embodiment, R⁷ is hydrogen or C1-C12 alkyl (e.g., methyl). In an embodiment, R⁷ is hydrogen. In an embodiment, R⁷ is methyl.

[0078] In an embodiment, the compound of Formula (VIII) is a salt, e.g., a pharmaceutically acceptable salt. In an embodiment, the compound of Formula (VIII) is a chloride salt, a p- toluenesulfonate salt, an acetate salt, or a citrate salt. In an embodiment, the compound of Formula (VIII) is a chloride salt. In an embodiment, the compound of Formula (VIII) is a p- toluenesulfonate salt. In an embodiment, the compound of Formula (VIII) is an acetate salt. In an embodiment, the compound of Formula (VIII) is a citrate salt. In an embodiment, the compound of Formula (VIII) is a compound of Formula (Vlll-a) or Formula (Vlll-b):

(Vlll-b) wherein R¹ is an amine protecting group described herein (e.g., Fmoc); R² is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, or Ci-Ci2-haloalkyl; each of R³ or R⁵ is independently hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, -N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); and each of R^A, R^B, and R^c is independently hydrogen,

C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[0079] In an embodiment, R¹ is an amine protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R² is hydrogen, C1-C12 alkyl, or Ci-Ci2-haloalkyl. In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl. In an embodiment, R³ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R³ is hydrogen. In an embodiment, when R³ is not hydrogen, R³ is in either the (Reconfiguration or the (S)-configuration. In an embodiment, R³ is in the (Reconfiguration. In an embodiment, R³ is in the (S)-configuration. [0080] In an embodiment, R⁵ is hydrogen. In an embodiment, R⁵ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R⁵ is has the structure of Formula (Illaa) or Formula (Illlab):

[0081] In an embodiment,

(Illaa)

(Illbb). In an embodiment, x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In an embodiment, x is an integer selected from 0, 1, 2, 3, or 4. In an embodiment, R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[0082] In an embodiment, the compound of Formula (Vlll-a) of (Vlll-b) is a salt, e.g., a pharmaceutically acceptable salt. In an embodiment, the compound of Formula (Vlll-a) or (Vlll-b) is a chloride salt, a p-toluenesulfonate salt, an acetate salt, or a citrate salt. In an embodiment, the compound of Formula (Vlll-a) or (Vlll-b) is a chloride salt. In an embodiment, the compound of Formula (Vlll-a) or (Vlll-b) is a p-toluenesulfonate salt. In an embodiment, the compound of Formula (Vlll-a) or (Vlll-b) is an acetate salt. In an embodiment, the compound of Formula (Vlll-a) or (Vlll-b) is a citrate salt.

[0083] In an embodiment, the compound of Formula (VIII) is a compound of Formula (Vlll-b) or (VIII-c):

wherein R¹ is an amine protecting group described herein (e.g., Fmoc); R² is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, or Ci-Ci2-haloalkyl; R³ is hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, - N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R⁹ is hydrogen or C1-C12 alkyl; and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[0084] In an embodiment, R¹ is an amine protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R² is hydrogen, C1-C12 alkyl, or Ci-Ci2-haloalkyl. In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl. In an embodiment, R³ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R³ is hydrogen. In an embodiment, when R³ is not hydrogen, R³ is in either the (Reconfiguration or the (S)-configuration. In an embodiment, R³ is in the (Reconfiguration. In an embodiment, R³ is in the (S)-configuration. [0085] In an embodiment, R⁹ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁹ is hydrogen. In an embodiment, R⁹ is t-butyl. In an embodiment, R⁹ is methyl. In an embodiment, the compound of Formula (VIII-c) of (Vlll-d) is a salt, e.g., a pharmaceutically acceptable salt. In an embodiment, the compound of Formula (VIII-c) or (Vlll-d) is a chloride salt, a p-toluenesulfonate salt, an acetate salt, or a citrate salt. In an embodiment, the compound of Formula (VIII-c) or (Vlll-d) is a chloride salt. In an embodiment, the compound of Formula (VIII-c) or (Vlll-d) is a p-toluenesulfonate salt. In an embodiment, the compound of Formula (VIII-c) or (Vlll-d) is an acetate salt. In an embodiment, the compound of Formula (VIII-c) or (Vlll-d) is a citrate salt.

[0086] In an embodiment, the compound of Formula (VIII) is a compound of Formula (Vlll-e) or (Vlll-f):

wherein R¹ is an amine protecting group (e.g., an amine protecting group described herein; e.g., Fmoc); R² is hydrogen, C1-C12 alkyl, or Ci-Ci2-haloalkyl; R³ is hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R¹⁰ is hydrogen, C1-C12 alkyl, or Ci-Ci2-haloalkyl; and y is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0087] In an embodiment, R¹ is an amine protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R² is hydrogen or Ci-Ci2-haloalkyl. In an embodiment, R² is hydrogen. In an embodiment, R² is Ci-Ci2-haloalkyl. In an embodiment, R² is 2,2,2- tribromoethyl. In an embodiment, R² is 2-iodoethyl. In an embodiment, R³ is C1-C12 alkyl, Ci- C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R³ is hydrogen. In an embodiment, when R³ is not hydrogen, R³ is in either the (Reconfiguration or the (S)-configuration. In an embodiment, R³ is in the (R)-configuration. In an embodiment, R³ is in the (S)-configuration.

[0088] In an embodiment, R¹⁰ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R¹⁰ is hydrogen. In an embodiment, R¹⁰ is t-butyl. In an embodiment, R¹⁰ is methyl. In an embodiment, the compound of Formula (Vlll-e) or (Vlll-f) is a salt, e.g., a pharmaceutically acceptable salt. In an embodiment, the compound of Formula (Vlll-e) or (Vlll-f) is a chloride salt, a p-toluenesulfonate salt, an acetate salt, or a citrate salt. In an embodiment, the compound of Formula (Vlll-e) or (Vlll-f) is a chloride salt. In an embodiment, the compound of Formula (Vlll-e) or (Vlll-f) is a p-toluenesulfonate salt. In an embodiment, the compound of Formula (Vlll-e) or (Vlll-f) is an acetate salt. In an embodiment, the compound of Formula (Vlll-e) or (Vlll-f) is a citrate salt. In an embodiment, y is an integer selected from 1, 2, and 3. In an embodiment, y is an integer is 1.

[0089] In an embodiment, the compound of Formula (VIII) is a compound of Formula (Vlll-g) or (Vlll-h):

wherein R² is hydrogen, C1-C12 alkyl, or Ci-Cu-haloalkyl; R³ is hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R¹⁰ is hydrogen, C1-C12 alkyl, or Ci-Ci2-haloalkyl; and y is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0090] In an embodiment, R² is hydrogen or Ci-Ci2-haloalkyl. In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl. In an embodiment, R³ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R³ is hydrogen. In an embodiment, R¹⁰ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R¹⁰ is hydrogen. In an embodiment, R¹⁰ is t-butyl. In an embodiment, R¹⁰ is methyl.

[0091] In an embodiment, the compound of Formula (Vlll-g) or (Vlll-h) is a salt, e.g., a pharmaceutically acceptable salt. In an embodiment, the compound of Formula (Vlll-g) or (Vlll-h) is a chloride salt, a p-toluenesulfonate salt, an acetate salt, or a citrate salt. In an embodiment, the compound of Formula (Vlll-g) or (Vlll-h) is a chloride salt. In an embodiment, the compound of Formula (Vlll-g) or (Vlll-h) is a p-toluenesulfonate salt. In an embodiment, the compound of Formula (Vlll-g) or (Vlll-h) is an acetate salt. In an embodiment, the compound of Formula (Vlll-g) or (Vlll-g) is a citrate salt. In an embodiment, y is an integer selected from 1, 2, and 3. In an embodiment, y is 1.

[0092] Exemplary PNA backbones include:

thereof (e.g., a pharmaceutically acceptable salt).

[0093] In an embodiment, the PNA backbone comprises (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-N-(2-oxo-2-(2,2,2-tribromoethoxy)ethyl)propan-l -amine, (R)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-N-(2-(2- iodoethoxy)-2-oxoethyl)propan-l -amine, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-N-(2-oxo-2-(2,2,2-tribromoethoxy)ethyl)propan-l-amine, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(2-(2-iodoethoxy)-2-oxoethyl)-3-(2-(2- m ethoxy ethoxy)ethoxy)propan-l -amine, (S)-2-((((9H-fluoren-9-yl)m ethoxy )carbonyl)amino)-3- (2-(2-(tert-butoxy)ethoxy)ethoxy)-N-(2-(2-iodoethoxy)-2-oxoethyl)propan- 1 -amine, and (S)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-N-(2-oxo-2- (2,2,2-tribromoethoxy)ethyl)propan-l -amine.

Amine Protecting Groups

[0094] In an embodiment, R¹ is an amine-protecting group. The N-terminus of a PNA monomer generally comprises an appropriate amine protecting group. In standard PNA synthesis (as in peptide synthesis), this group protects the terminal amine (i.e. in PNA synthesis - the nitrogen in bold underline of the aminoethylglycine unit (-N-C-C-N-C-C(=O)-) from reaction during coupling of the PNA monomer to the growing polyamide (or to the support, as the case may be); wherein said coupling is effected by amide bond formation through reaction of a resin bound amine group with the carboxylic acid function of the PNA monomer.

[0095] As used herein, the abbreviation Ri is used to denote an N-terminal amine protecting group that can be acid-labile or that can be base-labile.

[0096] Non-limiting examples of suitable base-labile N-terminal amine protecting groups that can be used in PNA monomers according to embodiments of this invention include: Fmoc, Nsc, Bsmoc, Nsmoc, ivDde, Fmoc*, Fmoc(2F), mio-Fmoc, dio-Fmoc, TCP, Pms, Esc, Sps and Cyoc. These base-labile protecting groups are illustrated in Fig. 8.

[0097] Non-limiting examples of suitable acid-labile N-terminal amine protecting groups that can be used in PNA monomers according to embodiments of this invention include: Boc (or Boc), Trt, Ddz, Bpoc, Nps, Bhoc, Dmbhoc and Floc. These groups are illustrated in Fig. 9. [0098] Exemplary amine protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), 9-(2- fluoro)-fluorenylmethoxycarbonyl (Fmoc(2F), 9-(2-sulfo)-fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-t-butyl-9-fluorenylmethoxycarbonyl (Dtb-Fmoc), 2,7-di-t-butyl-9- fluorenylmethoxycarbonyl (FMoc*), 2,7-bis(trimethylsilyl)-fluorenylmethoxycarbonyl (Bts- FMoc), 9-(2,7-dibromo)fluorenylmethoxy carbonyl, 2-monoisooctyl-9- fluorenylmethoxycarbonyl (mio-FMoc), 2,7-diisooctyl-9-fluorenylmethoxycarbonyl (dio-Fmoc), Bsmoc, Bspoc, t-butyloxycarbonyl (Boc), carboxybenzyl (Cbz) and substituted carboxybenzyl, p-toluenesulfonyl (Ts), benzoyl (Bz), benzhydryloxy carbonyl (Bhoc), benzyl (Bn) and substituted benzyl, dibenzosuberyl (Sub), or trityl and substituted trityl derivatives. Additional protecting groups that may be used are described, e.g., in Greene et al., Protecting Groups in Organic Synthesis, Fourth Edition, Wiley, New York, 2011, and references cited therein, each of which are incorporated herein by reference in their entirety. In an embodiment, R¹ is 9- fluorenylmethyloxycarbonyl, t-butyloxycarbonyl, carboxybenzyl, p-toluenesulfonyl, benzoyl, or benzyl. In an embodiment, R¹ is 9-fluorenylmethyloxycarbonyl.

Peptide Nucleic Acid Monomers

[0099] Methods of making peptide nucleic acid intermediates, such as peptide nucleic acid monomers, are described herein. A peptide nucleic acid (PNA) monomer may comprise a nucleobase moiety, and a backbone moiety, e.g., an aminoethylglycine moiety. The nucleobase moiety may be a nucleobase that forms hydrogen bonds with another nucleobase, e.g., the nucleobase of a target nucleic acid. The nucleobases may be naturally occurring or non- naturally occurring. PNA monomers may be derived from a PNA backbone, e.g., a PNA backbone of Formula (VIII), by acylating the PNA backbone with a nucleobase carboxylic acid (e.g., a nucleobase acetic acid of Formula (XI)) using a method described herein. PNA monomers described herein may be used in methods to prepare peptide nucleic acid oligomers, e.g., tail-clamp PNA (tcPNA) oligomers.

[00100] In an embodiment, the PNA monomer is a compound of Formula (I):

or a salt thereof (e.g., a pharmaceutically acceptable salt), wherein B is a nucleobase (e.g., a nucleobase described herein); R¹ is hydrogen or an amine protecting group (e.g., an amine protecting group described herein; e.g., Fmoc); R² is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2- C12 alkynyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, or C1-C12 alkylene-heteroaryl; each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, -N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R⁷ is hydrogen or C1-C12 alkyl; each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl; and n is an integer selected from 0, 1, 2, 3, and 4.

[00101] In an embodiment, B is a nucleobase. In an embodiment, B is a naturally occurring nucleobase. In an embodiment, B is a non-naturally occurring nucleobase. In an embodiment, B is selected from adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2- thiopseudoisocytosine, 5-methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2,6-diaminopurine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-chlorouracil, 5-bromouracil, 5- iodouracil, 5-chlorocytosine, 5 -bromocytosine, 5-iodocytosine, 5-propynyluracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-azaadenine, 5-propynyluracil, 2-thio-5-propynyluracil, pyridazin-3(2H)-one, pyrimidin-2(lH)-one, and pyridin-2-amine, and tautomers thereof.

[00102] In an embodiment, R¹ is an amine protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R² is hydrogen, C1-C12 alkyl (e.g., t-butyl), C2-C12 alkenyl (e.g., allyl), or Ci-Ci2-haloalkyl (e.g., 2, 2, 2-tri chloroethyl, 2,2,2-tribromoethyl, or 2- iodoethyl). In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl.

[00103] In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen, Ci- C12 alkyl, C1-C12 heteroalkyl, or the side chain of an amino acid. In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen. In an embodiment, one of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is C1-C12 alkyl (e.g., methyl) or C1-C12 heteroalkyl, and the others of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² are each independently hydrogen. In an embodiment, one of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is methyl, and the others of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² are each independently hydrogen. In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen or a polyalkylene glycol (e.g., a polyethylene glycol (PEG), e.g., a PEG moiety comprising between 2 and 6 PEG units. In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen or the side-chain of an amino acid. The side-chain of an amino acid may be protected with a protecting group described herein.

[00104] In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen or has the structure of Formula (Illaa) or Formula (Illlab):

[00105] In an embodiment, one

a In an embodiment, x is an integer selected from 0, 1, 2, 3, 4, 5, 6,

7, 8, 9, and 10. In an embodiment, x is selected from 1, 2, 3, and 4. In an embodiment, R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[00106] In an embodiment, R⁷ is hydrogen or C1-C12 alkyl (e.g., methyl). In an embodiment, R⁷ is hydrogen. In an embodiment, R⁷ is methyl. In an embodiment, n is an integer selected from

0, 1, 2, 3, and 4. In an embodiment, n is 0. In an embodiment, n is 1. In an embodiment, n is 2.

In an embodiment, n is 3. In an embodiment, n is 4.

[00107] In an embodiment, the compound of Formula (I) is a compound of Formula (I-a) or (I- b):

or a salt thereof (e.g., a pharmaceutically acceptable salt), wherein B is a nucleobase; R¹ is hydrogen or an amine protecting group; R² is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, or Ci-Ci2-haloalkyl; each of R³ and R⁵ is independently hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, -N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[00108] In an embodiment, B is a nucleobase. In an embodiment, B is a naturally occurring nucleobase. In an embodiment, B is a non-naturally occurring nucleobase. In an embodiment, B is selected from adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2- thiopseudoisocytosine, 5-methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2,6-diaminopurine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-chlorouracil, 5-bromouracil, 5- iodouracil, 5-chlorocytosine, 5 -bromocytosine, 5-iodocytosine, 5-propynyluracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-azaadenine, 5-propynyluracil, 2-thio-5-propynyluracil, pyridazin-3(2H)-one, pyrimidin-2(lH)-one, and pyridin-2-amine, and tautomers thereof.

[00109] In an embodiment, R¹ is an amine protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R² is hydrogen, C1-C12 alkyl (e.g., t-butyl), C2-C12 alkenyl (e.g., allyl), or Ci-Ci2-haloalkyl (e.g., 2, 2, 2-tri chloroethyl, 2,2,2-tribromoethyl, or 2- iodoethyl). In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl.

[00110] In an embodiment, R³ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R³ is hydrogen. In an embodiment, when R³ is not hydrogen, R³ is in either the (R)-configuration or the (S)-configuration. In an embodiment, R³ is in the (R)-configuration. In an embodiment, R³ is in the (S)-configuration.

[00111] In an embodiment, R⁵ is hydrogen. In an embodiment, R⁵ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R⁵ is has the structure of Formula (Illaa) or Formula (Illlab):

8

[00112] In an embodiment,

(Illaa) or

(Illbb). In an embodiment, x is an integer selected from 0, 1, 2, 3, 4,

5, 6, 7, 8, 9, and 10. In an embodiment, x is an integer selected from 0, 1, 2, 3, or 4. In an embodiment, R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[00113] In an embodiment, the compound of Formula (I) is a compound of Formula (I-c) or (I- d):

or a salt thereof (e.g., a pharmaceutically acceptable salt), wherein B is a nucleobase (e.g., a nucleobase described herein); R¹ is an amine protecting group (e.g., an amine protecting group described herein; e.g., Fmoc); R² is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, or Ci-Ci2-haloalkyl; R³ is C1-C12 alkyl, C1-C12 heteroalkyl, -N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R⁹ is hydrogen or C1-C12 alkyl; and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[00114] In an embodiment, B is a nucleobase (e.g., a nucleobase described herein). In an embodiment, R¹ is an amine protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R² is hydrogen, C1-C12 alkyl, or Ci-Cu-haloalkyl. In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl. In an embodiment, R³ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R³ is hydrogen. In an embodiment, when R³ is not hydrogen, R³ is in either the (Reconfiguration or the (S)-configuration. In an embodiment, R³ is in the (R)- configuration. In an embodiment, R³ is in the (S)-configuration.

[00115] In an embodiment, R⁹ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁹ is hydrogen. In an embodiment, R⁹ is t-butyl. In an embodiment, R⁹ is methyl.

[00116] In an embodiment, the compound of Formula (I) is a compound of Formula (I-e) or (I- f):

or a salt thereof (e.g., a pharmaceutically acceptable salt), wherein B is a nucleobase; wherein R¹ is an amine protecting group; R² is hydrogen, C1-C12 alkyl, or Ci-Ci2-haloalkyl; R³ is hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R¹⁰ is hydrogen or C1-C12 alkyl; and y is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[00117] In an embodiment, B is a nucleobase described herein. In an embodiment, R¹ is an amine protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R² is hydrogen or Ci-Ci2-haloalkyl. In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl. In an embodiment, R³ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R³ is hydrogen. In an embodiment, when R³ is not hydrogen, R³ is in either the (R)- configuration or the (S)-configuration. In an embodiment, R³ is in the (R)-configuration. In an embodiment, R³ is in the (S)-configuration.

[00118] In an embodiment, R¹⁰ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R¹⁰ is hydrogen. In an embodiment, R¹⁰ is t-butyl. In an embodiment, R¹⁰ is methyl. In an embodiment, y is selected from 1, 2, and 3. In an embodiment, y is 1.

[00119] In an embodiment, the compound of Formula (I) is a compound of Formula (I-g) or (I- h):

or a salt thereof (e.g., a pharmaceutically acceptable salt), wherein B is a nucleobase (e.g., a nucleobase described herein), R² is hydrogen, C1-C12 alkyl, or Ci-Ci2-haloalkyl; R³ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R¹⁰ is hydrogen or C1-C12 alkyl; and y is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, and

[00120] In an embodiment, B is a nucleobase described herein. In an embodiment, R² is hydrogen or Ci-Cu-haloalkyl. In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl. In an embodiment, R³ is C1-C12 alkyl, C1-C12 heteroalkyl, or the optionally protected amino acid side chain. In an embodiment, R³ is hydrogen. In an embodiment, when R³ is not hydrogen, R³ is in either the (R)- configuration or the (S)-configuration. In an embodiment, R³ is in the (R)-configuration. In an embodiment, R³ is in the (S)-configuration.

[00121] In an embodiment, R¹⁰ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R¹⁰ is hydrogen. In an embodiment, R¹⁰ is t-butyl. In an embodiment, R¹⁰ is methyl. In an embodiment, y is selected from 1, 2, and 3. In an embodiment, y is 1.

[00122] Exemplary PNA monomers include the following compounds:

salt thereof (e.g., a pharmaceutically acceptable salt thereof).

[00123] Exemplary PNA monomers include 2,2,2-tribromoethyl (S)-N-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propyl)-N-(2-(5-m ethyl-2,4-di oxo-3, 4-dihydropyrimidin-l(2H)- yl)acetyl)glycinate, 2,2,2-tribromoethyl (R)-l l-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 2,2-dimethyl-13-(2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)acetyl)-3,6,9-trioxa-13- azapentadecan-15-oate, 2-iodoethyl (R)-l l-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-13- (2-(2,6-bis(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetyl)-2,2-dimethyl-3,6,9-trioxa-13- azapentadecan- 15 -oate, 2-iodoethyl (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12- (2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)acetyl)-2,5,8-trioxa-12-azatetradecan- 14-oate, 2-iodoethyl (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(4-((tert- butoxy carbonyl)amino)-2-oxopyrimidin-l(2H)-yl)acetyl)-2, 5, 8-trioxa-12-azatetradecan- 14-oate, 2-iodoethyl (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(6-((tert- butoxy carbonyl)amino)-9H-purin-9-yl)acetyl)-2, 5 , 8-trioxa- 12-azatetradecan- 14-oate, 2- iodoethyl (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(2-((tert- butoxycarbonyl)amino)-6-oxo-l,6-dihydro-9H-purin-9-yl)acetyl)-2,5,8-trioxa-12-azatetradecan- 14-oate, 2-iodoethyl (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(2-((tert- butoxycarbonyl)amino)-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)acetyl)-2,5,8- trioxa-12-azatetradecan- 14-oate, and 2-iodoethyl (S)-l l-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-13-(2-(2,6-bis(bis(tert-butoxycarbonyl)amino)-9H-purin-9- yl)acetyl)-2,2-dimethyl-3,6,9-trioxa-13-azapentadecan-15-oate, or a salt thereof (e.g., a pharmaceutically acceptable salt).

[00124] Additional PNA monomers include, for example, the following compounds:

or a salt thereof (e.g., a pharmaceutically acceptable salt).

[00125] Additional PNA monomers include (S)-N-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propyl)-N-(2-(5-m ethyl-2,4-di oxo-3, 4-dihy dropyrimidin- 1(2H)- yl)acetyl)glycine, (R)- 11 -((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl- 13 -(2-(5- methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)acetyl)-3,6,9-trioxa-13-azapentadecan-15-oic acid, (R)- 11 -((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 13-(2-(2,6-bis(bis(tert- butoxycarbonyl)amino)-9H-purin-9-yl)acetyl)-2,2-dimethyl-3,6,9-trioxa-13-azapentadecan-15- oic acid, (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(5-methyl-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oic acid, (R)-10-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(4-((tert-butoxycarbonyl)amino)-2-oxopyrimidin- l(2H)-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oic acid, (R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(6-((tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetyl)- 2, 5,8-trioxa- 12-azatetradecan- 14-oic acid, (R)- 10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(2-((tert-butoxycarbonyl)amino)-6-oxo-l,6-dihydro-9H- purin-9-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oic acid, and (R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(2-((tert-butoxycarbonyl)amino)-4-oxo-3,4-dihydro-7H- pyrrolo[2,3-d]pyrimidin-7-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oic acid, or a salt thereof (e.g., a pharmaceutically acceptable salt).

Cyclic Compounds

[00126] The present disclosure provides methods of preparing peptide nucleic intermediates, for example, through the use of a cyclic compound. A peptide nucleic acid intermediate, for example, a peptide nucleic acid backbone of Formula (VIII) or a peptide nucleic acid monomer of Formula (I), may prepared via a cyclic compound (e.g., a compound of Formula (II)).

[00127] The use of cyclic compounds in the synthesis of PNA intermediates provides an alternative to conventional methods of preparing peptide nucleic acid intermediates. An advantage of using the cyclic compound described herein is that it can provide PNA intermediates in a higher yield compared to conventional methods, such as a Mitsunobu-type reaction. A further advantage of using the cyclic compound is that it can provide peptide nucleic acid intermediates with higher purity (e.g., higher chemical purity and/or higher optical purity) and may assist in avoiding, for example, a difficult purification step. Additionally, the cyclic compounds can be facilely prepared using inexpensive reagents starting from commercially available and easily obtained N-protected beta-amino alcohols. Such compounds are typically obtained by reduction of N-protected amino acid esters or activated acids resulting in N- protected b-amino alcohols that are enantiomerically pure if so desired.

[00128] The cyclic compound may comprise a heterocycle. In an embodiment, the heterocycle comprises one or more of a nitrogen, sulfur, or oxygen atom. In an embodiment, a cyclic compound comprises a nitrogen, a sulfur, and an oxygen atom. In an embodiment, the cyclic compound comprises a sulfone moiety. In an embodiment, the cyclic compound comprises an oxathiazolidine ring. In an embodiment, a cyclic compound comprises an oxathiazolidine dioxide ring. In an embodiment, the cyclic intermediate is electrophilic. In an embodiment, the cyclic intermediate can be reacted with a nucleophile to prepare an acyclic product, using a method described herein. The cyclic compound may possess one or more substituents, each of which may be bound to a carbon or a heteroatom of the cyclic compound.

[00129] In an embodiment, a cyclic compound comprises a compound of Formula (II):

or a salt thereof (e.g., a pharmaceutically acceptable salt), wherein R¹ is hydrogen or a protecting group (e.g., a protecting group described herein; e.g., Fmoc); each of R⁵ and R⁶ are independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, Ci- Ci2-haloalkyl, N(R^A)(R^B), halo, -OR^C, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, Ci- C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, or C1-C12 alkylene-heteroaryl, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Ci- C12 heteroalkyl.

[00130] In an embodiment, R¹ is a protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, each of R⁵ and R⁶ is independently hydrogen, C1-C12 alkyl (e.g., methyl), or C1-C12 heteroalkyl (e.g., a polyethylene glycol). In an embodiment, each of R⁵ and R⁶ is independently hydrogen, methyl, or has the structure of Formula (Illaa) or Formula (Illab):

wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁵ is hydrogen, and R⁶ is methyl. In an embodiment, R⁵ is hydrogen, and R⁶ has a structure of Formula (Illaa) or Formula (Illab):

aa a

, or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁶ is hydrogen, and R⁵ is methyl. In an embodiment, R⁶ is hydrogen, and R⁵ has a structure of Formula (Illaa) or Formula (Illab):

wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, x is an integer of 0, 1, 2, 3, or 4. In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[00131] In an embodiment, the compound of Formula (II) is a compound of Formula (Il-a) or (Il-b):

or a salt thereof, wherein R¹ is hydrogen or a protecting group (e.g., a protecting group described herein; e.g., Fmoc); R⁵ is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, N(R^A)(R^B), halo, -OR^C, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, C1-C12 alkylene-heteroaryl, or the side chain of an optionally protected amino acid; and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[00132] In an embodiment, R¹ is a protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R⁵ is hydrogen, C1-C12 alkyl (e.g., methyl), or C1-C12 heteroalkyl (e.g., polyethylene glycol). In an embodiment, R⁵ is hydrogen. In an embodiment, R⁵ is methyl. In an embodiment, R⁵ has the structure of Formula (Illaa) or Formula (Illab):

, or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). . In an embodiment, x is an integer of 0, 1, 2, 3, or 4. In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl. or a salt thereof, wherein R¹ is hydrogen or a protecting group (e.g., Fmoc); and R⁵ is C1-C12 alkyl, C1-C12 heteroalkyl, C1-C12 haloalkyl, or the side chain of an optionally protected amino acid.

[00133] In some embodiments, the compound of Formula (II) is a compound of Formula (II-c) or (Il-d) :

or a salt thereof, wherein R¹ is hydrogen or a protecting group (e.g., Fmoc); R⁶ is hydrogen, Ci- C12 alkyl, C1-C12 heteroalkyl, C1-C12 haloalkyl, or the side chain of an optionally protected amino acid; and R⁹ is hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, or the side chain of an amino acid.

[00134] Exemplary cyclic compounds include:

salt thereof

(e.g., a pharmaceutically acceptable salt).

[00135] Exemplary oxathiazolidine dioxides include: (9H-fluoren-9-yl)methyl (S)-4-methyl- 1,2, 3 -oxathiazolidine-3 -carboxylate 2,2-dioxide, (9H-fluoren-9-yl)m ethyl (S)-4-((2-(2-(tert- butoxy)ethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide, (9H-fluoren-9- yl)methyl (S)-4-((2-(2-methoxyethoxy)ethoxy)methyl)- 1,2, 3 -oxathiazolidine-3 -carboxylate 2,2- dioxide, and (9H-fluoren-9-yl)methyl (R)-4-((2-(2-(tert-butoxy)ethoxy)ethoxy )m ethyl)- 1,2,3 - oxathiazolidine-3 -carboxylate 2,2-dioxide, or a salt thereof (e.g., a pharmaceutically acceptable salt).

[00136] Cyclic compounds disclosed herein may be obtained from a cyclic intermediate. For example, a cyclic compound may be prepared from a cyclic intermediate that has a different oxidation state (e.g., a lower oxidation state). In an embodiment, an intermediate with a lower oxidation state may be oxidized to provide a cyclic compound of Formula (II). A cyclic intermediate for the preparation of compound (II) includes, for example, a cyclic compound of Formula (II-I):

[00137] In an embodiment, a cyclic intermediate comprises a compound of Formula (II-I):

or a salt thereof, wherein R¹ is hydrogen or a protecting group (e.g., a protecting group described herein; e.g., Fmoc); each of R⁵ and R⁶ are independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, N(R^A)(R^B), halo, -OR^C, cycloalkyl, Ci- C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, C1-C12 alkylene-heteroaryl, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[00138] In an embodiment, R¹ is a protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, each of R⁵ and R⁶ is independently hydrogen, C1-C12 alkyl (e.g., methyl), or C1-C12 heteroalkyl (e.g., a polyethylene glycol). In an embodiment, each of R⁵ and R⁶ is independently hydrogen, methyl, or has the structure of Formula (Illaa) or Formula (Illab):

or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁵ is hydrogen, and R⁶ is methyl. In an embodiment, R⁵ is hydrogen, and R⁶ has a structure of Formula (Illaa) or Formula (Illab):

or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁶ is hydrogen, and R⁵ is methyl. In an embodiment, R⁶ is hydrogen, and R⁵ has a structure of a Formula (Illaa) or Formula (Illab):

wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, x is selected from 0, 1, 2, 3, and 4. In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[00139] In an embodiment, the compound of Formula (II-I) is a compound of Formula (Il-Ia) or (Il-Ib):

or a salt thereof, wherein R¹ is hydrogen or a protecting group (e.g., a protecting group described herein; e.g., Fmoc); R⁵ is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, N(R^A)(R^B), halo, -OR^C, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, C1-C12 alkylene-heteroaryl, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[00140] In an embodiment, R¹ is a protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R⁵ is hydrogen, C1-C12 alkyl (e.g., methyl), or C1-C12 heteroalkyl (e.g., polyethylene glycol). In an embodiment, R⁵ is hydrogen. In an embodiment, R⁵ is methyl. In an embodiment, R⁵ has the structure of Formula (Illaa) or Formula (Illab):

integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, x is selected from 0, 1, 2, 3, and 4. In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl. [00141] Exemplary cyclic intermediates include:

salt thereof (e.g., a pharmaceutically acceptable salt).

[00142] Exemplary cyclic intermediates include (9H-fluoren-9-yl)methyl (4S)-4-methyl-l,2,3- oxathiazolidine-3 -carboxylate 2-oxide, (9H-fluoren-9-yl)methyl (4S)-4-((2-(2-(tert- butoxy)ethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2-oxide, (9H-fluoren-9- yl)methyl (4S)-4-((2-(2-methoxyethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2- oxide, and (9H-fluoren-9-yl)methyl (4R)-4-((2-(2-(tert-butoxy)ethoxy)ethoxy)methyl)-l,2,3- oxathiazolidine-3 -carboxylate 2-oxide, or a salt thereof (e.g., a pharmaceutically acceptable salt).

Amino Alcohol Compounds

[00143] A cyclic intermediate (e.g., a compound of Formula (II)) used to prepare a peptide nucleic acid intermediate may itself be prepared from an amino alcohol. In general, amino alcohol compounds are inexpensive to produce or obtain commercially, and thus provide a great value when seeking to scale up the synthesis of PNA monomers. For example, an amino alcohol can be cyclized to afford the cyclic compound (e.g., a compound of Formula (II)) or a cyclic intermediate (e.g., a compound of Formula (II-I)) using a method described herein. In an embodiment, the amino alcohol comprises one or more protecting groups (e.g., an amine protecting group described herein). In some embodiments, the amino alcohol comprises an Fmoc protecting group on its terminal nitrogen. An amino alcohol may comprise one or more substituents alpha to the terminal nitrogen group (e.g., R⁵ or R⁶).

[00144] In some embodiments, the amino alcohol is a compound of Formula (XII):

or a salt thereof, wherein R¹ is hydrogen or a protecting group (e.g., a protecting group described herein; e.g., Fmoc); each of R⁵ and R⁶ are independently hydrogen, C1-C12 alkyl, or C1-C12 heteroalkyl, N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R⁷ is hydrogen, C1-C12 alkyl, or C1-C12 heteroalkyl; and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[00145] In an embodiment, R¹ is a protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, each of R⁵ and R⁶ is independently hydrogen, C1-C12 alkyl (e.g., methyl), or C1-C12 heteroalkyl (e.g., a polyethylene glycol). In an embodiment, each of R⁵ and R⁶ is independently hydrogen, methyl, or has the structure of Formula (Illaa) or Formula (Illab):

wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁵ is hydrogen, and R⁶ is methyl. In an embodiment, R⁵ is hydrogen, and R⁶ has the structure of Formula (Illaa) or Formula (Illab):

wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, x is selected from 0, 1, 2, 3, and 4. In an embodiment, R⁷ is hydrogen. In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[00146] Exemplary amino alcohols include:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof). [00147] Exemplary amino alcohols also include (9H-fluoren-9-yl)methyl (S)-(l-hydroxypropan- 2-yl)carbamate, (9H-fluoren-9-yl)methyl (R)-(l-(2-(2-(tert-butoxy)ethoxy)ethoxy)-3- hydroxypropan-2-yl)carbamate, (9H-fluoren-9-yl)methyl (R)-(l -hydroxy-3 -(2-(2- methoxyethoxy)ethoxy)propan-2-yl)carbamate, and (9H-fluoren-9-yl)methyl (S)-(l-(2-(2-(tert- butoxy)ethoxy)ethoxy)-3-hydroxypropan-2-yl)carbamate, or a salt thereof (e.g., a pharmaceutically acceptable salt thereof).

Azide-Containing Intermediates

[00148] A cyclic compound, e.g., a cyclic compound of Formula (II) may be converted to an acyclic compound such as an azide-containing intermediate. Azide containing intermediates are useful precursors in the preparation of peptide nucleic acid intermediates. In particular, azides provide convenient access to the corresponding amines, which can readily be alkylated to prepare peptide nucleic acid intermediates (e.g., an abasic peptide nucleic acid monomer), by methods described herein. A cyclic compound may be converted to an azide-containing intermediate using a method described herein. For example, a cyclic intermediate may be contacted with an azide source, leading to ring-opening and the formation of an acyclic azide product. Azide compounds described herein may further possess a terminal amino group. The amino group may be a free amino group, a salt of the amino group, or may be protected with a protecting group described herein (e.g., an Fmoc group). The azide-containing intermediate may further comprise one more substituents bonded to the carbon alpha to the terminal amino group (e.g., R⁵ or R⁶).

[00149] In an embodiment, an azide-containing intermediate comprises the structure of Formula (IV):

or a salt thereof, wherein R¹ is hydrogen or a protecting group (e.g., a protecting group described herein; e.g., Fmoc); each of R⁵ and R⁶ are independently hydrogen, C1-C12 alkyl, or C1-C12 heteroalkyl, N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); R⁷ is hydrogen or C1-C12 alkyl; and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl. [00150] In an embodiment, R¹ is a protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, each of R⁵ and R⁶ is independently hydrogen, C1-C12 alkyl (e.g., methyl), or C1-C12 heteroalkyl (e.g., a polyethylene glycol). In an embodiment, each of R⁵ and R⁶ is independently hydrogen, methyl, or has the structure of Formula (Illaa) or Formula (Illab):

or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁵ is hydrogen, and R⁶ is methyl. In an embodiment, R⁵ is hydrogen, and R⁶ has the structure of Formula (Illaa) or Formula (Illab):

wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, R⁶ is hydrogen, and R⁵ is methyl. In an embodiment, R⁶ is hydrogen, and R⁵ has a the structure of a Formula (Illaa) or Formula (Illab):

or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, x is selected from 0, 1, 2, 3, and 4. In an embodiment, R⁷ is hydrogen. In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[00151] Exemplary azide-containing intermediates include:

or a salt thereof (e.g., a pharmaceutically acceptable salt).

[00152] Exemplary azide-containing intermediates also include (9H-fluoren-9-yl)methyl (S)-(l- azidopropan-2-yl)carbamate, (9H-fluoren-9-yl)methyl (R)-(l-azido-3-(2-(2-(tert- butoxy)ethoxy)ethoxy)propan-2-yl)carbamate, (9H-fluoren-9-yl)methyl (R)-(l-azido-3-(2-(2- methoxyethoxy)ethoxy)propan-2-yl)carbamate, and (9H-fluoren-9-yl)methyl (S)-(l-azido-3-(2- (2-(tert-butoxy)ethoxy)ethoxy)propan-2-yl)carbamate, or a salt thereof (e.g., a pharmaceutically acceptable salt).

Diamine Compounds

[00153] Compounds containing a diamine moiety can be used to access peptide nucleic acid intermediates, e.g., a peptide nucleic acid backbone of Formula (VIII). For example, a diamine compound may be alkylated at one amine group, e.g., with an amino-acid derivative, to provide PNA backbones. Diamine compounds may comprise two terminal amino groups, wherein each can functionalized (e.g., with an alkyl group, a protecting group, or an amino-acid derivative, e.g., an amino acid ester). Diamine compounds may further comprise substituents alpha to one of the terminal amine groups. Additionally, diamine compounds may be obtained as a salt, which can facilitate isolation, handling, or storage of the diamine compound. The use of diamine compounds to produce PNA backbone compounds (and salts thereof) is described herein, and is also described in WO/2018/175927, the disclosure of which is incorporated herein by reference in its entirety.

[00154] Disclosed herein are methods to form such diamine compounds, as well as their use in the preparation of PNA backbones. For example, a diamine compound may be obtained by reducing an azide-containing intermediate (e.g., a compound of Formula (IV)). Further, diamine compounds may be modified through protecting group manipulation, for example, a compound of Formula (VII) may be converted to a compound of (IX) by selective removal and addition of protecting groups, using a method described herein.

[00155] In an embodiment, a diamine compound comprises a structure of Formula (VII) or Formula (IX):

or a salt thereof (e.g., a pharmaceutically acceptable salt), wherein each of R¹ and R¹³ is independently hydrogen or a protecting group (e.g., a protecting group described herein; e.g., Fmoc); each R⁵, R⁶, R¹¹, and R¹² are independently hydrogen, C1-C12 alkyl, or C1-C12 heteroalkyl, N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); each of R⁷ and R¹⁴ is hydrogen or C1-C12 alkyl; and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[00156] In an embodiment, each of R¹ and R¹³ is a protecting group described herein. In an embodiment, R¹ is Fmoc. In an embodiment, R¹³ is Fmoc. In an embodiment, each of R⁵, R⁶, R¹¹, and R¹² is independently hydrogen, C1-C12 alkyl (e.g., methyl), or C1-C12 heteroalkyl (e.g., a polyethylene glycol). In an embodiment, R⁵ is hydrogen. In an embodiment, R⁶ is hydrogen. In an embodiment, R¹¹ is hydrogen. In an embodiment, R¹² is hydrogen. In an embodiment, R⁵ is methyl. In an embodiment, R⁶ is methyl. In an embodiment, R¹¹ is methyl. In an embodiment, R¹² is methyl. In an embodiment, R⁵ is polyethylene glycol. In an embodiment, R⁶ is polyethylene glycol. In an embodiment, R¹¹ is polyethylene glycol. In an embodiment, R¹² is polyethylene glycol.

[00157] In an embodiment, each of R⁵, R⁶, R¹¹, and R¹² is independently hydrogen, methyl, or has the structure of Formula (Illaa) or Formula (Illab):

or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, each of R⁵ and R¹¹ is independently hydrogen, and each of R⁶ and R¹² is independently methyl. In an embodiment, each of R⁵ and R¹¹ is independently hydrogen, and each of R⁶ and R¹² independently has the structure of Formula (Illaa) or Formula

(Illab):

, wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, each of R⁶ and R¹² is independently is hydrogen, and each of R⁵ and R¹¹ is independently methyl. In an embodiment, each of R⁶ and R¹² is independently hydrogen, and each of R⁵ and R¹¹ independently has the structure of Formula (Illaa) or Formula (Illab):

, or a , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, x is an integer of 0, 1, 2, 3, or 4. In an embodiment, R⁷ is hydrogen. In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[00158] In an embodiment, the compound of Formula (VII) or (IX) is a salt (e.g., a salt described herein, e.g., a p-toluenesulfonate salt, an acetate salt, a chloride salt, or a citrate salt). In an embodiment, the compound of Formula (VII) or (IX) is a p-toluenesulfonate salt. In an embodiment, the compound of formula (VII) or (IX) is a chloride salt. In an embodiment, the compound of formula (VII) or (IX) is an acetate salt. In an embodiment, the compound of formula (VII) or (IX) is a citrate salt.

[00159] Exemplary diamine compounds include:

or a salt thereof (e.g., a chloride salt, a p-toluenesulfonate salt, an acetate salt, or a citrate salt). [00160] Exemplary diamine compounds also include (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propan-l -amine, (R)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)propan-l -amine, (R)-2-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-m ethoxy ethoxy)ethoxy)propan-l -amine, and (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)propan- 1 -amine, or a salt thereof (e.g., a chloride salt, a p-toluenesulfonate salt, an acetate salt, or a citrate salt).

Amino-Acid Derivatives and Nucleobase Intermediates

[00161] The preparation of peptide nucleic acid intermediates, e.g., a PNA backbone of Formula (VIII), may be achieved using an amino-acid derivative. For example, an amino-acid derivative of Formula (V) or Formula (VI):

or a salt thereof, wherein X is a leaving group (e.g., halo or sulfonyl); R² is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, or C1-C12 alkylene-heteroaryl; and each of R³ and R⁴ is independently hydrogen, Ci- C12 alkyl, C1-C12 heteroalkyl, -N(R^A)(R^B), halo, -OR^C, or the optionally protected amino acid side chain (see for example FIGS. 7A-7B); and each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl.

[00162] In an embodiment, X is halo or sulfonyl. In an embodiment, X is fluoro, chloro, bromo, iodo, p-toluenesulfonyl, triflouromethylsulfonyl or methanesulfonyl. In an embodiment, X is bromide. In an embodiment, R² is hydrogen, C1-C12 alkyl (e.g., t-butyl), C2-C12 alkenyl (e.g., allyl), or Ci-Ci2-haloalkyl (e.g., 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, or 2-iodoethyl). In an embodiment, R² is hydrogen. In an embodiment, R² is 2,2,2-tribromoethyl. In an embodiment, R² is 2-iodoethyl.

[00163] In an embodiment, each of R³ and R⁴ is independently hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, or the side chain of an amino acid. In an embodiment, each of R³ and R⁴ is independently hydrogen. In an embodiment, one of R³ and R⁴ is C1-C12 alkyl (e.g., methyl) or C1-C12 heteroalkyl, and the other of R³ and R⁴ is independently hydrogen. In an embodiment, one of R³ and R⁴ is methyl, and the other of R³ and R⁴ is independently hydrogen. In an embodiment, one of R³ and R⁴ is independently hydrogen or a polyalkylene glycol (e.g., a polyethylene glycol (PEG), e.g., a PEG moiety comprising between 2 and 6 PEG units. In an embodiment, each of R³ and R⁴ is independently hydrogen or the side-chain of an amino acid. The side-chain of an amino acid may be protected with a protecting group described herein. [00164] In an embodiment, each of R³, R⁴, R⁵, R⁶, R¹¹, and R¹² is independently hydrogen or has the structure of Formula (Illaa) or Formula (Illlab):

, (Illbb), wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or Ci- C12 alkyl (e.g., t-butyl, methyl, or ethyl). In an embodiment, x is an integer of 0, 1, 2, 3, or 4. In an embodiment, R⁸ is hydrogen. In an embodiment, R⁸ is t-butyl. In an embodiment, R⁸ is methyl.

[00165] The preparation of peptide nucleic acid intermediates, e.g., a PNA monomer of Formula (I), may be carried out by reacting a nucleobase carboxylic acid compound with an abasic PNA monomer (e.g., a PNA backbone of Formula (VIII)), using a method described herein.

Nucleobase carboxylic acids may comprise a short or long carboxylic acid chain (e.g., 1, 2, 3, 4, or 5 carbons in length), which can be selected to determine the distance between the nucleobase moiety and the backbone moiety in the PNA monomer. The nucleobase carboxylic acid may comprise a nucleobase acetic acid, having a carbon chain of 2 carbons. The nucleobase may further comprise protecting groups, e.g., a protecting group described herein. Exemplary nucleobases for use in a nucleobase carboxylic acid are shown in FIGS. 5 and 6.

[00166] A nucleobase carboxylic acid (e.g. nucleobase acetic acid) used in a method described herein may be a compound of Formula (XI):

or a salt thereof, wherein B is a nucleobase described herein, and n is an integer of 0, 1, 2, 3, 4, 5, or 6.

[00167] In an embodiment, B is selected from adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2-thiopseudoisocytosine, 5-methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine (or 2,6-diaminopurine), 2-thiouracil, 2-thiothymine, 2- thiocytosine, 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5-chlorocytosine, 5 -bromocytosine, 5- iodocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7- deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-aza adenine, 5-propynyl uracil and 2-thio-5-propynyl, pyridazin-3(2H)-one, pyrimidin-2(lH)-one, and pyridin-2-amine, and tautomeric forms thereof.

[00168] In an embodiment, n is an integer of 0, 1, 2, 3, or 4. In an embodiment, n is an integer of 1. In an embodiment, n is an integer of 2. In an embodiment, n is an integer of 3.

Amino Acid Side Chain Protecting Groups

[00169] As described in more detail herein, in some embodiments of this invention, PNA Monomer Ester compositions and PNA synthetic intermediates can comprise one or more optionally protected amino acid side chains. In some embodiments, these side chains are derived from (or have the chemical composition of) the side chains of naturally or non-naturally occurring amino acids.

[00170] For example and with reference to Fig. 7A and Fig. 7B, in some embodiments, the side chains can be compositions of formula: Illa (e.g., derived from alanine), Illb (e.g., derived from aminobutyric acid), IIIc (e.g., derived from valine), Illd (e.g., derived from leucine), Ille (e.g., derived from isoleucine), Illf (e.g., derived from norvaline), Illg (e.g., derived from phenylalanine) and/or Illh (e.g., derived from norleucine). These substituents are all alkanes and therefore generally considered unreactive under conditions used in PNA synthesis. Accordingly, they typically do not comprise any protecting group.

[00171] Again with reference to Fig. 7A, in some embodiments, the side chains can be compositions of formula: Illi (e.g., derived from 3 -aminoalanine), Illk (e.g., derived from 2,4- diaminobutanoic acid), Illj (e.g., derived from ornithine), and/or Illm (e.g., derived from lysine). These substituents all comprise an amine group. Consequently, the amine group of these substituents will typically comprise a protecting group. However, because this is a side chain protecting group generally remains intact during the entire synthesis of the PNA oligomer, this side chain protecting group can be orthogonal to the protecting group selected for the N-terminal amine or nitrogen atom in a synthetic intermediate that corresponds to the N-terminal amine in a PNA monomer product (i.e. denoted Ri). Thus, if Ri is base-labile, this side chain protecting group can be selected to be acid-labile or removed under conditions of neutral pH. A nonlimiting list of such acid-labile amine side chain protecting groups is illustrated in Fig. I la. These include, but are not limited to, Cl-Z, Boc, Bpoc, Bhoc, Dmbhoc, Nps, Floc, Ddz and Mmt.

[00172] Similarly, if Ri is acid-labile, this side chain protecting group can be selected to be base-labile or removed under conditions of neutral pH. A non-limiting list of such base-labile amine side chain protecting groups is illustrated in Fig. 1 lb. These include, but are not limited to, Fmoc, ivDde, Msc, tfa, Nsc, TCP, Bsmoc, Sps, Esc and Cyoc.

[00173] Again with reference to Fig. 7A, in some embodiments, the side chains can be compositions of formula: Ilin (e.g., derived from cysteine), IIIo (e.g., derived from S-methyl- cysteine), and/or IIIp (e.g., derived from methionine). These side chains all comprise a sulfur atom. While it is not essential that compounds of formula IIIo or IIIp comprise a protecting group (but they can optionally be protected), thiol containing compounds of formula Ilin typically comprise a protecting group. However, because this side chain protecting group generally remains intact during the entire synthesis of the PNA oligomer, this side chain protecting group can be orthogonal to the protecting group selected for the N-terminal amine or nitrogen atom in a synthetic intermediate that corresponds to the N-terminal amine in a PNA monomer product (i.e. Ri). Thus, if Ri is base-labile, this side chain protecting group can be selected to be acid-labile or removed under conditions of neutral pH. A non-limiting list of such acid-labile protecting groups suitable for thiol containing side chain moieties is illustrated in Fig. 15a. These include, but are not limited to, Meb, Mob, Trt, Mmt, Tmob, Xan, Bn, mBn, 1-Ada, Pmbr and *Bu.

[00174] Similarly, if Ri is acid-labile, this side chain protecting group can be selected to be base-labile or removed under conditions of neutral pH. A non-limiting list of such base-labile protecting groups suitable for thiol containing side chain moieties is illustrated in Fig. 15b. These include, but are not limited to, Fm, Dnpe and Fmoc.

[00175] Again with reference to Fig. 7A, in some embodiments, the side chains can be compositions of formula: Illq (e.g., derived from serine), Illr (e.g., derived from threonine), and/or Ills (e.g., derived from tyrosine). These side chains all comprise a -OH (hydroxyl or phenol) group. Compounds of formulas Illq, Illr and Ills will typically comprise a protecting group during PNA synthesis. However, because this is a side chain protecting group that generally remains intact during the entire synthesis of the PNA oligomer, this hydroxyl side chain protecting group can be orthogonal to the protecting group selected for the N-terminal amine or nitrogen atom in a synthetic intermediate that corresponds to the N-terminal amine in a PNA monomer product (i.e. Ri).

[00176] Thus, if Ri is base-labile, the side chain protecting group can be selected to be acid- labile or removed under conditions of neutral pH. A non-limiting list of such acid-labile protecting groups suitable for hydroxyl containing moieties is illustrated in Fig. 18a. These include, but are not limited to, Bn, Trt, cHx, TBDMS and ‘Bu. Because -OH of Tyrosine (Tyr) is phenolic, and there is a potentially broader group of protecting group available. A nonlimiting list of such acid-labile protecting groups for side chain moieties comprising a phenol is illustrated in Fig. 19a. These include, but are not limited to, Bn, ^lBu, BrBn, Deb, Z, BrZ, Pen, Boc, Trt, 2-Cl-Trt and TEGBn.

[00177] Similarly, if Ri is acid-labile, the side chain protecting group can be selected to be base- labile or removed under conditions of neutral pH. A non-limiting list of protecting groups for hydroxyl containing moieties that can be removed under conditions of neutral pH is illustrated in Fig. 18b. These include, but are not limited to, TBDPS, Dmnb and Poc. Because -OH of Tyrosine (Tyr) is phenolic, there is a potentially broader group of protecting group available. A non-limiting list of protecting groups for side chain moieties comprising a phenol that can be removed under conditions of neutral pH is illustrated in Fig. 19b. These include, but are not limited to, Al, oBN, Poc and Boc-Nmec.

[00178] With reference to Fig. 7B, in some embodiments, the side chains can be compositions of formula: lilt (e.g., derived from glutamic acid) and/or IIIu (e.g., derived from aspartic acid). These side chains all comprise a -COOH (carboxylic) group. Compounds of formulas lilt and IIIu will typically comprise a protecting group during PNA synthesis to thereby inhibit activation of the carboxylic acid group during the coupling reaction. However, because this is a side chain protecting group that generally remains intact during the entire synthesis of the PNA oligomer, this side chain protecting group can be orthogonal to the protecting group selected for the N-terminal amine (i.e. Ri).

[00179] Thus, if Ri is base-labile, the side chain protecting group can be selected to be acid- labile or removed under conditions of neutral pH. A non-limiting list of such acid-labile protecting groups suitable for use with carboxylic acid containing side chain moieties is illustrated in Fig. 12a. These include, but are not limited to, Bn, cHx, 'Bu, Mpe, Men, 2-Ph¹Pr and TEGBz.

[00180] Similarly, if Ri is acid-labile, the side chain protecting group can be selected to be base- labile or removed under conditions of neutral pH. A non-limiting list of such base-labile protecting groups suitable for use with carboxylic acid containing side chain moieties is illustrated in Fig. 12b. These include, but are not limited to, Fm and Dmab.

[00181] With reference to Fig. 7B, in some embodiments, the side chains can be compositions of formula: IIIv (e.g., derived from glutamine) and/or IIIw (e.g., derived from asparagine). These side chains all comprise a -C(=O)NH2 (amide) group. Compounds of formulas IIIv and IIIw do not necessarily require a protecting group during PNA synthesis but nevertheless, standard protecting groups used in peptide synthesis can be used. When used, this side chain protecting group can be orthogonal to the protecting group selected for the N-terminal amine (i.e. Ri).

[00182] Thus, if Ri is base-labile, the side chain protecting group can be selected to be acid- labile or removed under conditions of neutral pH. A non-limiting list of such acid-labile protecting groups for amide containing side chain moieties is illustrated in Fig. 13. These include, but are not limited to, Xan, Trt, Mtt, Mbh and Tmob. Similarly, if Ri is acid-labile, the side chain protecting group can be selected to be base-labile or removed under conditions of neutral pH.

[00183] With reference to Fig. 7B, in some embodiments, the side chains can be compositions of formula: IIIx (e.g., derived from arginine (Arg) - and containing a guanidinium moiety), IIIxl (e.g., derived from homoarginine - and containing a guanidinium moiety), Illy (e.g., derived from tryptophan (Trp) - and containing an indole moiety) and/or IIIz (e.g., derived from histidine (His) - and containing an imidazole moiety). Compounds of formulas IIIx, Illy and IIIz will typically comprise a protecting group during PNA synthesis. However, because this side chain protecting group generally remains intact during the entire synthesis of the PNA oligomer, this side chain protecting group can be orthogonal to the protecting group selected for the N-terminal amine (i.e. Ri)

[00184] Thus, if Ri is base-labile, the side chain protecting group can be selected to be acid- labile or removed under conditions of neutral pH. A non-limiting list of such acid-labile side chain protecting groups suitable for use with guanidinium containing side chain moieties is illustrated in Fig. 14a. These include, but are not limited to, Tos, Pmc, Pbf, Mts, Mtr, MIS, Sub, Suben, MeSub, Boc and NO2. A non-limiting list of such acid-labile side chain protecting groups suitable for use with indole containing side chain moieties is illustrated in Fig. 14a.

These include, but are not limited to, For, Boc, Hoc and Mts. A non-limiting list of such acid- labile side chain protecting groups suitable for use with imidazole containing side chain moieties is illustrated in Fig. 17a. These include, but are not limited to, Tos, Boc, Doc, Trt, Mmt, Mtt, Bom and Bum.

[00185] Similarly, if Ri is acid-labile, the side chain protecting group can be selected to be base- labile or removed under conditions of neutral pH. A non-limiting list of such base-labile side chain protecting groups suitable for use with guanidinium containing side chain moieties is illustrated in Fig. 14b. These include, but are not limited to, tfa. A non-limiting list of such side chain protecting groups removable under conditions of neutral pH suitable for use with indole containing side chain moieties is illustrated in Fig. 16b. These include, but are not limited to, Alloc. A non-limiting list of such base-labile side chain protecting groups suitable for use with imidazole containing side chain moieties is illustrated in Fig. 17b. These include, but are not limited to, Fmoc and Dmbz.

Nucleobase Protecting Groups

[00186] As in chemical DNA synthesis, certain of the functional groups of nucleobases (of the PNA monomers and growing PNA oligomers) are often protected during PNA synthesis. However, unprotected embodiments are also within the scope of the present disclosure. For this reason, the nucleobases can be said to ‘optionally comprise one or more protecting groups’, or are ‘optionally protected’.

[00187] For example, if the N-terminal amine protecting group (e.g., R¹) (which is typically removed at every synthetic cycle) is acid-labile, then any nucleobase protecting groups are generally selected to be base-labile or removed under conditions of neutral pH. In short, the protecting groups for the N-terminal amine and the protecting groups for the nucleobases can be orthogonal. For example, the exocyclic amine groups of nucleobases are typically protected during PNA synthesis so that no unwanted coupling of PNA monomers occurs by reaction with these amine groups. With reference to Fig. 10a, numerous base-labile protecting groups are illustrated and can be used to protect the exocyclic amine groups of PNA monomers, and synthetic intermediates thereto, that can be used in embodiments of this invention. These include (but are not limited to), formyl, acetyl, isobutyryl, methoxyacetyl, isopropoxyacetyl, Fmoc, Esc, Cyoc, Nsc, Clsc, Sps, Bsc, Bsmoc, Levulinyl, 3-methoxy-4-phenoxybenzoyl, benzoyl (and various other benzoyl derivatives) and phenoxyacetyl (and various other phenoxyacetyl derivatives). Other examples of nucleobase protecting groups can be found in Ref C-13.

[00188] Similarly, if the N-terminal amine protecting group (e.g., R¹) is base-labile, then any nucleobase protecting groups are generally selected to be acid-labile or removed under conditions of neutral pH. With reference to Fig. 10b, numerous acid-labile protecting groups are illustrated and can be used to protect the exocyclic amine groups of PNA monomers, and synthetic intermediates thereto, that are used embodiments of this invention. These include (but are not limited to), Boc (sometimes abbreviated Boc or t-Boc), Bis-Boc (which means two Boc groups linked to the same amine group - as illustrated in Fig. 10b), Bhoc, Dmbhoc, Floc, Bpoc, Ddz, Trt, Mtt, Mmt and 2-Cl-Trt.

[00189] Certain nucleobases, such as thymine and uracil often do not comprise a protecting group for PNA synthesis. However, the imide/lactam functional groups of pyrimidine nucleobases can be protected in some embodiments. Similarly, although the O-6 of the guanine is typically not protected, it can be protected in some embodiments. Some non-limiting examples of protecting groups that can be used in embodiments of this invention to protect the N3/O4 of a pyrimidine nucleobase (exemplary compounds 1001 or 1002 are illustrated) or the 06 of a purine nucleobase (exemplary compound 1000 is illustrated) can be found in Fig. 10c. [00190] In addition to those nucleobases illustrated in Figs. 5, 6, and 10c, Fig. 20a illustrates several common nucleobases herein identified as: A, D^, G, G*, C, 5^MC, T, T^2T, U, U^2T, Y, J and J^2T in unprotected form. Fig. 20b illustrates these nucleobases A, D^AP, G, G*, C, 5^MC, T, T^2T, U, U^2T, Y, J and J^2T as can be protected with an acid-labile protecting group for PNA synthesis (used for example where R¹ is selected to be base-labile).

[00191] A non-limiting list of nucleobases includes: adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2-thiopseudoisocytosine, 5-methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine (a.k.a. 2,6-diaminopurine), 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5-chlorocytosine, 5 -bromocytosine, 5 -iodocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7- deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-aza adenine, 5-propynyl uracil and 2-thio-5-propynyl uracil, including tautomeric forms of any of the foregoing.

Synthetic Routes

[00192] Disclosed herein are synthetic routes that may be used to prepare peptide nucleic acid intermediates, such as cyclic compounds and their cyclic intermediates, azide-containing intermediates, diamine compounds, peptide nucleic acid backbones, and peptide nucleic acid monomers.

Cyclic Compounds

[00193] A cyclic compound of Formula (II) may be prepared from an amino alcohol, e.g., an amino alcohol of Formula (XII). In an embodiment, an amino alcohol of Formula (XII) is converted to a compound of Formula (II). A compound of Formula (II) may be prepared via one of at least two methods as illustrated in Scheme 1. In a first method, the compound of Formula (II) is prepared through a cyclization step to form the cyclic intermediate of Formula (Il-aa) (as shown in Step A), followed by an oxidation step to yield the compound of Formula (II) (as shown in Step B. Alternatively, the compound of Formula (II) may be cyclized in one step, as shown in Scheme 1 via Step C.

Scheme 1. Direct or stepwise cyclization of amino alcohol of Formula (XII) (Steps A and C), and oxidation of cyclic compound of Formula (Il-aa) (Step C); R¹, R⁵, and R⁶ are as defined herein.

[00194] A compound of Formula (Il-aa) or Formula (II) may be formed through a cyclization reaction (e.g., a condensation reaction) as shown in Scheme 1. For example, a compound of Formula (Il-aa) may be prepared by incubating a compound of Formula (XII) with a cyclization agent and a base. Alternatively, a compound of Formula (II) may be prepared by incubating a compound of Formula (XII) with a cyclization agent and a base.

[00195] In an embodiment, the cyclization agent is a sulfur-containing compound. In an embodiment, the cyclization agent comprises a sulfoxide or a sulfone. In an embodiment, the sulfur-containing compound comprises one or more leaving groups. In an embodiment, the one or more leaving groups comprise halogen (e.g., chloride) or imidazole. In an embodiment, the cyclization agent comprises thionyl chloride, thionyl bromide, thionyl chloride fluoride, thionyl fluoride, l,r-sulfmyldiimidazole, lH-imidazole-4-sulfmyl chloride, sulfuryl chloride, 1,1’- sulfonyldiimidazole, lH-imidazole-4-sulfonyl chloride, sulfuryl chloride fluoride, or 1- (trifluoromethanesulfonyl)imidazole.

[00196] The base may be an organic base or an inorganic base. In an embodiment, the base is a nitrogen-containing compound. In an embodiment, base is a heterocyclic compound. In an embodiment, the heterocyclic compound is a nitrogen-containing ring or an oxygen-containing ring. In an embodiment, the heterocyclic compound is a four-membered ring, a five-membered ring, or a six-membered ring. In an embodiment, the base is a five-membered nitrogencontaining ring. In an embodiment, the base is a six-membered nitrogen-containing ring. In an embodiment, the base is an alkyl amine, an aryl amine, a heterocyclic amine, or a heteroaromatic amine. In an embodiment, the base comprises imidazole, 1 -methylimidazole, 4- methylimidazole, pyrazole, a triazole, pyridine, 4-dimethylaminopyridine, 2,6-lutidine, tri ethylamine, diisopropylethylamine, triisoproylamine, l,4-diazabicyclo[2.2.2]octane (DABCO), l,8-diazabicyclo(5.4.0)undec-7-ene (DBU), pyrrole, piperidine, or N- methylmorpholine. For other exemplary bases that may be used in the cyclization reaction, see, e.g., Journal of Organic Chemistry (2002), 67:5164, which is incorporated herein by reference in its entirety.

[00197] The amino alcohol of Formula (XII) may be cyclized to form a compound of Formula (Il-aa) as a mixture of diastereomers. In an embodiment, the cyclization of an amino alcohol of Formula (XII) comprising one or more stereocenters affords a mixture of one or more diastereomers. In an embodiment, an amino alcohol of Formula (XII) comprising a stereocenter with R configuration is cyclized to provide a diastereomeric compound of Formula (Il-aa) with R,R configuration. In an embodiment, an amino alcohol of Formula (XII) comprising a stereocenter with R configuration is cyclized to provide a diastereomeric compound of Formula (Il-aa) with R, S configuration. In an embodiment, an amino alcohol of Formula (XII) comprising a stereocenter with R configuration is cyclized to provide a mixture of diastereomers of Formula (Il-aa) with R, S and R, R configuration, respectively. In an embodiment, an amino alcohol of Formula (XII) comprising a stereocenter with S configuration is cyclized to provide a diastereomeric compound of Formula (Il-aa) with S,R configuration. In an embodiment, an amino alcohol of Formula (XII) comprising a stereocenter with S configuration is cyclized to provide a diastereomeric compound of Formula (Il-aa) with S,S configuration. In an embodiment, an amino alcohol of Formula (XII) comprising a stereocenter with S configuration is cyclized to provide a mixture of diastereomers of Formula (Il-aa) with S,R and S,S configuration, respectively.

[00198] As described above, a compound of Formula (II) may be achieved through oxidation of a compound of Formula (Il-a). For example, a compound of Formula (II) may be prepared through incubation of a compound of Formula (Il-aa) with an oxidizing agent, either alone or in the presence of a catalyst.

[00199] The oxidizing agent for the oxidation reaction may be any reagent capable of oxidizing other substances, e.g., adding one or more oxygens to a substance, or removing one or more electrons from a substance. In an embodiment, the oxidizing agent is an organic oxidant or an inorganic oxidant. In an embodiment, the oxidizing agent comprises a hypervalent iodine. In an embodiment, the inorganic oxidant comprises sodium periodate, potassium periodate, periodic acid, sodium peroxymonosulfate, potassium peroxymonosulfate (Oxone), sodium perborate, potassium perborate, sodium hypochlorite, potassium permanganate, pyridinium chlorochromate (PCC), chromic acid, hydrogen peroxide, oxygen, ozone, or combinations thereof. In an embodiment, the organic oxidant comprises Dess-Martin periodinane (DMP), 2-iodoxybenzoic acid (IBX), meta-chloroperoxybenzoic acid (mCPBA), or combinations thereof.

[00200] The catalyst used in the oxidation reaction may be an inorganic catalyst. In an embodiment, the catalyst comprises a transition metal. In an embodiment, the transition metal is ruthenium. In an embodiment, the catalyst comprises one or more ligands bound to the transition metal. In an embodiment, the ligand is a halogen or oxygen. In an embodiment, the catalyst is in the form of a hydrate. In an embodiment, the catalyst comprises ruthenium trichloride (e.g., ruthenium trichloride hydrate, ruthenium chloride trihydrate), ruthenium bromide (e.g., ruthenium bromide hydrate), ruthenium iodide (e.g., ruthenium iodide hydrate), ruthenium tetroxide, ruthenium nitrosyl nitrate, ruthenium acetylacetonate, or combinations thereof.

[00201] The reaction of an amino alcohol to a compound of Formula (II) may be carried out at a temperature of between about -100 to 50 °C (e.g., between about -80 to 40 °C, -50 to 30 °C, -15 to 15 °C, -10 to 10 °C, -5 to 15 °C, or 0 to 10 °C). In an embodiment, the reaction of an amino alcohol to a compound of Formula (II) is carried out at a temperature greater than -40 °C, -20 °C, -10 °C, -5 °C, 0 °C, 5 °C, 10 °C, 15 °C, 25 °C, or 30 °C. In an embodiment, the reaction of an amino alcohol to a compound of Formula (II) is carried out at a temperature lower than 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 0 °C, -5 °C, or -10 °C. In an embodiment, the reaction of an amino alcohol to a compound of Formula (II) is carried out at a temperature of 20 °C, 25 °C, or 30 °C. In an embodiment, the amino alcohol of Formula (XII) is cyclized at a temperature between about -100 to 50 °C (e.g., between about -90 to 40 °C, -80 to 30 °C, -78 to 25 °C, -90 to -20 °C, -85 to -60 °C, or -80 to -70 °C). In an embodiment, the amino alcohol of Formula (XII) is cyclized at a temperature greater than -100 °C, -90 °C, -80 °C, -78 °C, -50 °C, -20 °C, 0 °C, 10 °C, 25 °C, or 40 °C. In an embodiment, the amino alcohol of Formula (XII) is cyclized at a temperature lower than 50 °C, 30 °C, 10 °C, 0 °C, -10 °C, -30 °C, or -50 °C. In an embodiment, the amino alcohol of Formula (XII) is cyclized at a temperature of -78 °C, -20 °C, 0 °C, or at room temperature. In an embodiment, the compound of Formula (Il-aa) is oxidized at a temperature between about -50 to 50 °C (e.g., between about -40 to 40 °C, -30 to 30 °C, -15 to 15 °C, -10 to 10 °C, -5 to 15 °C, or 0 to 10 °C). In an embodiment, the compound of Formula (Il-aa) is oxidized at a temperature greater than -40 °C, -20 °C, -10 °C, -5 °C, 0 °C, 5 °C, 10 °C, 15 °C, 25 °C, or 30 °C. In an embodiment, the compound of Formula (Il-aa) is oxidized at a temperature lower than 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 0 °C, -5 °C, or -10 °C. In an embodiment, the compound of Formula (Il-aa) is oxidized at a temperature of 0 °C, 20 °C, 25 °C or 30 °C.

[00202] The reaction to form a compound of Formula (II) may be carried out in a solvent such as acetonitrile, methanol, ethanol, butanol, tetrahydrofuran, water, 1,4-di oxane, dimethylsulfoxide, dichloromethane, chloroform, toluene, diethyl ether, methyl tert-butyl ether, dimethylformamide, N-methylpyrrolidone, or mixtures thereof. In an embodiment, reaction to form a compound of Formula (II) is carried out in a solvent that comprises less than 10% water (e.g., less than 8%, 5%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%). In an embodiment, the reaction to form a compound of Formula (II) is carried out in a solvent that comprises less than 150 ppm of water (e.g., less than 120 ppm, less than 100 ppm, less than 80 ppm, less than 60 ppm, less than 40 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, less than 2 ppm, or less than 1 ppm). In an embodiment, the reaction to form a compound of Formula (II) is carried out in an anhydrous solvent. In an embodiment, the reaction to form a compound of Formula (II) is carried out in a biphasic solvent. In an embodiment, the amino alcohol of Formula (XII) is cyclized in a solvent comprising tetrahydrofuran (e.g., anhydrous tetrahydrofuran). In an embodiment, the amino alcohol of Formula (XII) is cyclized in a solvent comprising 1,4-dioxane (e.g., anhydrous 1,4-dioxane). In an embodiment, the compound of Formula (Il-aa) is oxidized in a solvent comprising dichloromethane, acetonitrile, methyl acetate, ethyl acetate, water, or mixtures thereof.

[00203] The reaction to form a compound of Formula (II) may be carried out at a pH of between about 2-13 (e.g., between about 3-10, 4-8, or 5-6). In an embodiment, the reaction to form a compound of Formula (II) is carried out at a pH greater than 2, 3, 4, 5, or 6. In an embodiment, the reaction to form a compound of Formula (II) is carried out at a pH of less than 9, 8, 7, 6, 5, or 4. In an embodiment, the compound of Formula (Il-aa) is oxidized at a pH of about 4, 5, or 6. [00204] In some embodiments, the cyclic compound of Formula (II) or the intermediate of Formula (Il-aa) has less than 80%, less than 60%, less than 40%, less than 20%, less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% of an impurity. The impurity may be residual starting materials, solvents, or by-products (e.g., a by-product from a side-reaction or from deprotection of a protecting group). In an embodiment, the compound of Formula (II) or the intermediate of Formula (Il-aa) has an impurity arising from the cyclization reaction comprising residual imidazole, residual, thionyl chloride, acyclic sulfite esters, or acyclic sulfonyl esters. In an embodiment, the compound of Formula (II) has an impurity arising from the oxidation reaction comprising residual ruthenium trichloride or sodium periodate.

[00205] The compound of Formulas (Il-aa) or (II) may be purified by any conventional purification technique. In an embodiment, the compound of Formula (Il-aa) or (II) is purified by chromatography, precipitation, crystallization, recrystallization, preparative HPLC, filtration, centrifugation, adsorption, distillation, evaporation, liquid-liquid extraction, lyophilization, trituration, or combinations thereof. In an embodiment, the cyclic compound of Formula (Il-aa) or (II) is purified by chromatography. In an embodiment, the cyclic compound of Formula (Il- aa) or (II) is purified by recrystallization. In an embodiment, the cyclic compound of Formula (Il-aa) or (II) is not purified.

[00206] In an embodiment, the compound of Formula (Il-aa) or (II), or a salt thereof, is obtained as a mixture of enantiomers. In an embodiment, the compound of Formula (Il-aa) or (II), or a salt thereof, has an enantiomeric excess greater than 60% (e.g., greater than 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9%). In an embodiment, the compound of Formula (Il-aa) or (II), or a salt thereof, has an enantiomeric excess lower than 99.99% (e.g., lower than 99.9%, 99.5%, 99%, 98%, 95%, or 90%). In an embodiment, the compound of Formula (Il-aa) or (II), or a salt thereof, is enantiopure.

Preparation of Azide-Containing Intermediates

[00207] An azide-containing intermediate of Formula (IV) may be prepared from a cyclic compound, e.g., a cyclic compound of Formula (II). In an embodiment, a cyclic compound of Formula (II) is converted to a compound of Formula (IV). Converting a compound of Formula (II) to a compound of Formula (IV) may involve the nucleophilic addition of an azide group, as shown in Scheme 2, via Step D.

Scheme 2. Conversion of a compound of Formula (II) to a compound of Formula (IV) (Step D); R¹, R⁵, R⁶, and R⁷ are as defined herein.

[00208] A compound of Formula (IV) may be formed through a nucleophilic addition reaction as shown in Scheme 2. For example, a compound of Formula (IV) may be prepared through incubation of a compound of Formula (II) with an azide source (e.g., an azide-containing reagent).

[00209] In some embodiments, the azide-containing reagent is an azide salt or an organic azide. In some embodiments, the azide-containing reagent comprises a substrate-bound azide (e.g., polymer-bound or silica-bound azide). In an embodiment, the azide-containing reagent comprises sodium azide, potassium azide, lithium azide, azidotrimethyltin, azidotributyltin, azidotrimethylsilane, trifluoromethanesulfonyl azide, imidazole- 1 -sulfonyl azide, p- toluenesulfonyl azide, benzenesulfonyl azide, 4-acetamidobenzenesulfonyl azide, diphenyl phosphoryl azide, 4-carboxybenzenesulfonazide, or combinations thereof.

[00210] In an embodiment, the nucleophilic addition reaction of a compound of Formula (II) to a compound of Formula (IV) is carried out at a temperature between about -20 °C to 100 °C (e.g., between about -10 to 90 °C, -5 to 80 °C, 0 to 70 °C, 10 to 60 °C, 20 to 60 °C, or 30 to 55 °C). In an embodiment, the nucleophilic addition reaction of a compound of Formula (II) to a compound of Formula (IV) is carried out at a temperature greater than -20 °C, -10 °C, 0 °C, 10 °C, 20 °C, 30 °C, 40 °C. In an embodiment, the nucleophilic addition reaction of a compound of Formula (II) to a compound of Formula (IV) is carried out at a temperature lower than 100 °C, 80 °C, 60 °C, 40 °C, 30 °C, 20 °C, 10 °C, or 0 °C. In an embodiment, the nucleophilic addition reaction of a compound of Formula (II) to a compound of Formula (IV) is carried out at a temperature of 45, 50 °C, or 55 °C. In an embodiment, the nucleophilic addition reaction of a compound of Formula (II) to a compound of Formula (IV) is carried out at room temperature. [00211] The nucleophilic addition reaction of a compound of Formula (II) to a compound of Formula (IV) may be carried out in a solvent such as methanol, ethanol, propanol (e.g., n- propanol or isopropanol), butanol, acetonitrile, dimethylsulfoxide, tetrahydrofuran, 1,4-di oxane, methyl tert-butyl ether, dimethyl formamide, toluene, diethyl ether, or dichloromethane. In an embodiment, the nucleophilic addition reaction of a compound of Formula (II) to a compound of Formula (IV) may be carried out in a solvent comprising methanol.

[00212] In some embodiments, the compound of Formula (IV) has less than 80%, less than 60%, less than 40%, less than 20%, less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% of an impurity. The impurity may be residual starting materials, solvents, or by-products (e.g., a by-product from a side reaction or from deprotection of a protecting group). In an embodiment, the impurity comprises residual azide-containing reagent or a by-product thereof.

[00213] The compound of Formulas (IV) may be purified by any conventional purification technique. In an embodiment, the compound of Formula (IV) is purified by chromatography, precipitation, crystallization, recrystallization, preparative HPLC, filtration, centrifugation, adsorption, distillation, evaporation, liquid-liquid extraction, lyophilization, trituration, or combinations thereof. In an embodiment, the compound of Formula (IV) is purified by chromatography. In an embodiment, the compound of Formula (IV) is purified by recrystallization. In an embodiment, the compound of Formula (IV) is not purified.

[00214] In an embodiment, the compound of Formula (IV), or a salt thereof, is obtained as a mixture of enantiomers. In an embodiment, the compound of Formula (IV), or a salt thereof, has an enantiomeric excess greater than 60% (e.g., greater than 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9%). In an embodiment, the compound of Formula (IV), or a salt thereof, has an enantiomeric excess lower than 99.99% (e.g., lower than 99.9%, 99.5%, 99%, 98%, 95%, or 90%). In an embodiment, the compound Formula (IV), or a salt thereof, is enantiopure.

Preparation of Diamine Compounds

[00215] An diamine compound of Formula (VII), or a salt thereof, may be prepared from an azide-containing intermediate, e.g., an azide-containing intermediate of Formula (IV). In an embodiment, a compound of Formula (VII) is prepared by reducing a compound of Formula (IV). The conversion of a compound of Formula (IV) to an compound of Formula (VII) may involve the reduction of an azide group to an amine group, as shown in Scheme 3 via Step E. In an embodiment, the compound of Formula (VII) is obtained as a salt.

Scheme 3. Reduction of an azide-containing intermediate of Formula (IV) to an intermediate of Formula (VII). R¹, R⁵, R⁶, and R⁷ are as described herein.

[00216] A compound of Formula (VII) may be formed through a hydrogenation reaction (e.g., a catalytic hydrogenation). For example, a compound of Formula (VII) may be prepared through incubation of a compound of Formula (IV) with a hydrogen source and a catalyst.

[00217] In an embodiment, the hydrogen source is a reagent that generates hydrogen in situ. In some embodiments, the hydrogen source comprises tri ethylsilane, phenylsilane, 1,4- cyclohexadiene, 1,3-cyclohexadiene, hydrazine, formic acid, ammonium formate, phosphinic acid, tributyltin hydride, sodium borohydride, or sodium hypophosphite.

[00218] In an embodiment, the hydrogen source is an exogenous hydrogen source (e.g., hydrogen gas). The hydrogen gas may be delivered to the reduction reaction by any suitable device, such as a hydrogen balloon or a hydrogen tank. The hydrogen gas may be delivered to the reduction reaction at any pressure, e.g., at a pressure suitable to reduce an azide group to an amine group. In some embodiments, the hydrogen gas is delivered to the reduction reaction at a pressure of between about 0.1 atm-200 atm (e.g., about 1 to 100 atm, about 1 to 50 atm, about 1 to 25 atm, about 1 to 10 atm, about 1 to 5 atm, about 1 to 3 atm). In an embodiment, the hydrogen gas is delivered to the reduction reaction at a pressure greater than 0.5 atm, 1 atm, 2 atm, 3 atm, 5 atm, 10 atm, 20 atm, 50 atm, or 100 atm. In an embodiment, the hydrogen gas is delivered to the reduction reaction at a pressure lower than 200 atm, 100 atm, 50 atm, 20 atm, 10 atm, 5 atm, or 3 atm. In an embodiment, the hydrogen gas is delivered to the reaction at atmospheric pressure.

[00219] The catalyst used in the reduction reaction may be a metal-containing catalyst. In an embodiment, the catalyst is a homogenous catalyst. In an embodiment, the catalyst is a heterogeneous catalyst. In an embodiment, the catalyst is in a reduced form. In an embodiment, the catalyst comprises a transition metal. In an embodiment, the transition metal is palladium, platinum, ruthenium, iridium, rhodium, nickel, or rhenium. In an embodiment, one or more ligands are bound to the transition metal to form a complex. In an embodiment, the transition metal is not ligand bound. In some embodiments, the catalyst comprises elemental palladium, palladium on carbon (e.g., palladium on charcoal), palladium hydroxide, palladium hydroxide on carbon, palladium acetate, palladium chloride, tetrakis(triphenylphosphine)palladium, Lindlar catalyst, Raney nickel, platinum dioxide, Crabtree’s catalyst, Wilkinson’s catalyst, or dichlorotri s(triphenylphosphine)ruthenium.

[00220] The conversion of a compound of Formula (IV) to a compound of Formula (VII), as shown in Scheme 3, may achieved without catalytic hydrogenation. For example, a compound of Formula (VII) may be prepared through incubation of a compound of Formula (IV) with a reducing agent. In an embodiment, a compound of Formula (VII) is prepared by incubating a compound of Formula (IV) with a reducing agent in the presence of a proton source.

[00221] The reducing agent may be an organic reducing agent or an inorganic reducing agent. In an embodiment, the reducing agent is a metal-containing reagent. In an embodiment, the reducing agent is in the form of granules, particles (e.g., nanoparticles), dispersion, powder, dust, solution, or a suspension. In an embodiment, the reducing agent has been activated (e.g., by treatment with acid). In an embodiment, the reducing agent comprises one or more elements selected from zinc, iron, indium, tin, copper, mercury, aluminium, lithium, sodium, boron, and silicon. In an embodiment, the reducing agent comprises elemental zinc, elemental iron, elemental copper, zinc amalgam, tin chloride, iron trichloride, indium trichloride, lithium aluminium hydride, diisobutylaluminium hydride, sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, boron trifluoride (e.g., boron trifluoride diethyletherate), borane tetrahydrofuran, diborane, borane-dimethyl sulfide, and triethylsilane. [00222] The proton source may be any reagent that can donate one or more protons to another substance. In an embodiment, the proton source is a solution (e.g., an aqueous solution). In an embodiment, the proton source is an acid, an alcohol, or a salt. In an embodiment, the proton source comprises ammonium chloride, acetic acid, trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, methanol, ethanol, propanol, butanol, or water.

[00223] The reduction of a compound of Formula (IV) to a compound of Formula (VI) may be carried out at a temperature of between about -20 to 120 °C (e.g., between about -10 to 100 °C, - 5 to 80 °C, 0 to 60 °C, 10 to 40 °C, or 15 to 30 °C). In an embodiment, the reduction of a compound of Formula (IV) is carried out at a temperature of greater than -10 °C, 0 °C, 10 °C, 20 °C, 25 °C, 30 °C, 40 °C, 50 °C, 60 °C, or 70 °C. In an embodiment, the reduction of a compound of Formula (IV) is carried out at a temperature of less than 100 °C, 70 °C, 60 °C, 50 °C, 40 °C, 30 °C, 20 °C, 10 °C. In some embodiments, the reduction of a compound of Formula (IV) is carried out at a temperature of 50 °C. In an embodiment, the reduction of a compound of Formula (IV) is carried out at room temperature.

[00224] The reduction of a compound of Formula (IV) to a compound of Formula (VI) may be carried out in a solvent such as ethanol, chloroform, methanol, propanol (e.g., n-propanol or isopropanol), butanol, water, cyclohexane, methyl cyclohexane, benzene, ethyl acetate, tetrahydrofuran, cyclohexane, diethyl ether, petroleum ether, dimethylformamide, toluene, acetone, acetonitrile, N-methylpyrrolidone, or mixtures thereof. In an embodiment, the reduction of a compound of Formula (IV) is carried out in a mixture of ethanol and chloroform. In an embodiment, reduction of a compound of Formula (IV) is carried out in a mixture of ethanol and water.

[00225] In some embodiments, the compound of Formula (VI) obtained from the reduction reaction has less than 80%, less than 60%, less than 40%, less than 20%, less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% of an impurity. The impurity may be residual starting materials, solvents, or byproducts. In an embodiment, the impurity comprises residual catalyst, residual hydrogen source, residual reducing agent, partially reduced side-products, or over-reduced side-products.

[00226] The compound of Formula (VI) formed in a reduction may be purified by any conventional purification technique. In an embodiment, the compound of Formula (VI) is purified by chromatography, precipitation, crystallization, recrystallization, preparative HPLC, filtration, centrifugation, adsorption, distillation, evaporation, liquid-liquid extraction, lyophilization, trituration, or combinations thereof. In an embodiment, the compound of Formula (VI) is purified by recrystallization. In an embodiment, the compound of Formula (VI) is purified by liquid-liquid extraction.

[00227] In an embodiment, the compound of Formula (VI), or a salt thereof, is obtained as a mixture of enantiomers. In an embodiment, the compound of Formula (VI), or a salt thereof, has an enantiomeric excess greater than 60% (e.g., greater than 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9%). In an embodiment, the compound of Formula (VI), or a salt thereof, has an enantiomeric excess lower than 99.99% (e.g., lower than 99.9%, 99.5%, 99%, 98%, 95%, or 90%). In an embodiment, the compound Formula (VI), or a salt thereof, is enantiopure.

Methods to Form PNA Backbones

[00228] A PNA backbone of Formula (VIII) may be prepared via one of at least three methods as illustrated in Scheme 4. The cyclic compound of Formula (II) is a common precursor for each of these methods, exemplifying its versatility in methods of preparing PNA intermediates. Each of the methods may afford a PNA backbone of Formula (VIII) comprising one or more functional groups at the alpha, beta, or gamma positions. In a first method, the PNA backbone of Formula (VIII-I) comprising functional groups at the gamma position (e.g., R⁵ or R⁶) can be prepared from the alkylation of a compound of Formula (VII) with a compound of Formula (V), via Step F. A second method, depicted as Step H in Scheme 4, involves alkylating the cyclic compound of Formula (II) directly with a compound of Formula (VI) to obtain the compound of Formula (VIII-II) comprising functional groups at the alpha position (e.g., R³ or R⁴) or the gamma position (R⁵ and R⁶). In a third method, a compound of Formula (IX) may be alkylated with a compound of Formula (V) to achieve the compound of Formula (VIII-III) comprising functional groups at the beta position (e.g., R¹¹ or R¹²) via step Step J.

Scheme 4. Exemplary methods for the synthesis of abasic PNA monomers of Formulas (VIII- I)-(VIII-III), from a cyclic compound of Formula (II); each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹¹, and R¹² are as defined herein.

[00229] The reaction to prepare a compound of Formula (VIII) may involve incubating a compound of Formula (V) with a compound of Formula (VII) or (IX) in the presence of a base (Steps F or J, Scheme 4). The compound of Formula (VII) or (IX) may in turn be derived from a compound of Formula (II) by a method described herein (see, e.g., Schemes 2, 3, and 5). In an embodiment, the compound of Formula (VIII-II) is prepared by incubating a compound of Formula (VI) with a compound of Formula (II) in the presence of a base. An abasic PNA monomer prepared by any one of the alkylation methods shown in Scheme 4 may be prepared as a salt (e.g., a tosyl salt or a hydrochloride salt), by further treating the abasic PNA monomer with an acid. For examine, an abasic PNA monomer of Formula (VIII-I)-(VIII-III) may be treated with an acid (e.g., p-toluenesulfonic acid) to obtain the compound as its salt.

[00230] In an embodiment, the base used in the alkylation reaction is an organic base or an inorganic base. In an embodiment, the base is a nitrogen-containing compound. In an embodiment, the base is an alkyl amine, an aryl amine, a heterocyclic amine, or a heteroaromatic amine. In an embodiment, the base used in the alkylation reaction comprises diisopropylethylamine, N-methylmorpholine, triethylamine, triisoproylamine, imidazole, pyrrole, piperidine, pyridine, 4-dimethylaminopyridine, 2,6-lutidine, 1,4- diazabicyclo[2.2.2]octane (DABCO), or l,8-diazabicyclo(5.4.0)undec-7-ene (DBU). In an embodiment, the base is N-methylmorpholine. In an embodiment, the base is dii sopropy 1 ethyl amine .

[00231] The compound of Formula (VIII) may comprise a diastereomer. In an embodiment, the compound of Formula (VIII) is isolated as a single diastereomer. In an embodiment, the compound of Formula (VIII) is isolated as a mixture of two or more diastereomers. In an embodiment, a compound of Formula (VII) or Formula (IX) comprising a stereocenter with R configuration is reacted with a compound of Formula (V) to provide a diastereomer with R, R or

R, S configuration. In an embodiment, a compound of Formula (VII) or Formula (IX) comprising a stereocenter with S configuration is reacted with a compound of Formula (V) to provide a diastereomer with S,R or S,S configuration. In an embodiment, a compound of Formula (VII) or Formula (IX) comprising a stereocenter is reacted with a compound of Formula (V) to provide a mixture comprising one or more diastereomers with R, R, R,S, S,R, or

S,S configuration. In an embodiment, a compound of Formula (II) comprising a stereocenter with R configuration is reacted with a compound of Formula (VI) to provide a diastereomer with R, R or R, S configuration. In an embodiment, a compound of Formula (II) comprising a stereocenter with S configuration is reacted with a compound of Formula (VI) to provide a diastereomer with S,R or S,S configuration. In an embodiment, a compound of Formula (II) comprising a stereocenter is reacted with a compound of Formula (VI) to provide a mixture comprising one or more diastereomers with R, R, R,S, S,R, or S,S configuration.

[00232] The abasic PNA monomer (e.g., a compound of Formula (VIII-I)-(VIII-III) may be isolated as a salt. The salt of the compound of Formula (VIII) may be achieved by treating the compound of Formula (VIII) with an acid. The treatment of the compound of Formula (VIII) with an acid to form a salt can be carried out during the alkylation reaction, prior to purification of the compound of Formula (VIII), or after the purification of the compound of Formula (VIII). In some embodiments, the compound of Formula (VIII) is more stable as a salt, as compared to the free base form. Acids that may be used to form the salt of Formula (VIII) include p- toluenesulfonic acid (p-TSA), hydrochloric acid, methanesulfonic acid, hydrobromic acid, hydroiodic acid, acetic acid, trifluoroacetic acid, citric acid, or an acid described in US Patent Publication No.: 2019/0055190 which is incorporated herein by reference in its entirety. In an embodiment, the acid used to form the salt of Formula (VIII) is p-toluenesulfonic acid (p-TSA). In an embodiment, the acid used to form the salt of Formula (VIII) is hydrochloric acid. In an embodiment, the compound of Formula (VIII) is prepared as a hydrochloride, hydrobromide, hydroiodide, acetate, trifluoroacetate, p-tolunesulfonate, methanesulfonate, or citrate. In some embodiments, the compound of Formula (VIII) is prepared as a p-toluenesulfonate.

[00233] The alkylation reaction to afford a compound of Formula (VIII) may be carried out at a temperature of between about -50 to 50 °C to (e.g., between about -40 to 40 °C, -30 to 30 °C, - 15 to 15 °C, -10 to 10 °C, -5 to 15 °C, or 0 to 10 °C). In an embodiment, the alkylation reaction is carried out at a temperature greater than -40 °C, -20 °C, -10 °C, -5 °C, 0 °C, 5 °C, 10 °C, 15 °C, 25 °C, or 30 °C. In an embodiment, the alkylation reaction is carried out at a temperature lower than 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 0 °C, -5 °C, or -10 °C. In an embodiment, the alkylation of a compound of Formula (VII) with a compound of Formula (V) is carried out at a temperature of 0 °C, 15 °C, or 25 °C. In an embodiment, the alkylation of a compound of Formula (VII) with a compound of Formula (V) is carried out at room temperature. In an embodiment, the alkylation of a compound of Formula (IX) with a compound of Formula (V) is carried out at a temperature of 0 °C, 15 °C, or 25 °C. In an embodiment, the alkylation of a compound of Formula (IX) with a compound of Formula (V) is carried out at room temperature. In an embodiment, the alkylation of a compound of Formula (II) with a compound of Formula (VI) is carried out at a temperature of 0 °C, 15 °C, or 25 °C. In an embodiment, the alkylation of a compound of Formula (II) with a compound of Formula (VI) is carried out at room temperature.

[00234] The alkylation reaction to form a compound of Formula (VIII) may be carried out in a solvent such as tetrahydrofuran, acetonitrile, methanol, ethanol, acetone, 1,4-di oxane, dimethylsulfoxide, dichloromethane, chloroform, toluene, diethyl ether, methyl tert-butyl ether, dimethylformamide, N-methylpyrrolidone, or mixtures thereof. In an embodiment, the alkylation reaction is carried out in a solvent that comprises less than 10% water (e.g., less than 8%, 5%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%). In an embodiment, the alkylation reaction is carried out in a solvent that comprises less than 150 ppm of water (e.g., less than 120 ppm, less than 100 ppm, less than 80 ppm, less than 60 ppm, less than 40 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, less than 2 ppm, or less than 1 ppm). In an embodiment, the reaction to form a compound of Formula (II) is carried out in an anhydrous solvent. In an embodiment, the alkylation of a compound of Formula (VII) with a compound of Formula (V) is carried out in tetrahydrofuran. In an embodiment, the alkylation of a compound of Formula (VII) with a compound of Formula (V) is carried out in acetonitrile. In an embodiment, the alkylation of a compound of Formula (IX) with a compound of Formula (V) is carried out in tetrahydrofuran. In an embodiment, the alkylation of a compound of Formula (IX) with a compound of Formula (V) is carried out in acetonitrile. In an embodiment, the alkylation of a compound of Formula (II) with a compound of Formula (VI) is carried out in tetrahydrofuran. In an embodiment, the alkylation of a compound of Formula (II) with a compound of Formula (VI) is carried out in acetonitrile.

[00235] The pH of the alkylation reaction may be adjusted in the course of the reaction, e.g., by the addition of an acid (e.g., acetic acid or hydrochloric acid). The pH of the alkylation reaction may also be adjusted using a buffer or base. In an embodiment, the pH of the alkylation reaction is adjusted with the addition of acetic acid. In an embodiment, the pH of the alkylation reaction is adjusted with the addition of hydrochloric acid. In an embodiment, the alkylation reaction is adjusted to a pH of between about 1-8 (e.g., between about 2-7, 2-4, or 4-7). In an embodiment, the pH of the alkylation reaction is adjusted to a pH greater than 1, 2, 3, 4, 5, or 6. In an embodiment, the pH of the alkylation reaction is adjusted to a pH lower than 8, 7, 6, 5, 4, or 3. In an embodiment, the pH of the alkylation reaction is adjusted to a pH of about 2, 3, 4, or 5. [00236] In an embodiment, the compound of Formula (VIII) has less than 80%, less than 60%, less than 40%, less than 20%, less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% of an impurity. The impurity may be residual starting materials, solvents, or by-products (e.g., a by-product from a sidereaction or from deprotection of a protecting group).

[00237] In an embodiment, the compound of Formula (III) or a salt thereof is purified by a conventional purification technique. In an embodiment, the compound of Formula (VIII) or a salt thereof is purified by chromatography, precipitation, crystallization, recrystallization, preparative HPLC, filtration, centrifugation, adsorption, distillation, evaporation, liquid-liquid extraction, lyophilization, trituration, or combinations thereof. In an embodiment, the compound of Formula (VIII) or a salt thereof is purified by chromatography. In an embodiment, the compound of Formula (VIII) or a salt thereof is purified by recrystallization. In an embodiment, the compound of Formula (VIII) or a salt thereof is not purified.

[00238] In an embodiment, the compound of Formula (III) or a salt thereof is obtained as a mixture of enantiomers. In an embodiment, the compound of Formula (III) or a salt thereof has an enantiomeric excess greater than 60% (e.g., greater than 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9%). In an embodiment, the compound of Formula (III) or a salt thereof has an enantiomeric excess lower than 99.99% (e.g., lower than 99.9%, 99.5%, 99%, 98%, 95%, or 90%). In an embodiment, the compound of Formula (III) or a salt thereof is enantiopure.

Preparation of the Diamine Compound of Formula (IX)

[00239] A compound of Formula (IX) may be prepared from a compound of Formula (VII), e.g., by protecting group manipulation. In an embodiment, the preparation of a compound of Formula (IX) is achieved using an orthogonal protecting group strategy, as shown in Scheme 5. In an embodiment, the compound of Formula (VII) comprising a first protecting group (e.g., R¹) is protected with a second protecting group, e.g., R¹³, as shown in Scheme 5, Step Gl. Then, the first protecting group, e.g., R¹, may be removed to afford the compound of Formula (IX), as shown in Scheme 5 via Step G2. In an embodiment, substituents R⁵ and R⁶ are the same as R¹¹ and R¹².

Scheme 5. Conversion of a compound of Formula (VII) to a compound of Formula (IX) via an intermediate of Formula (X); R¹, R⁵, R⁶, R⁷, R¹¹, R¹², R¹³, and R¹⁴ are as defined herein.

[00240] In an embodiment, the first and second protecting groups are orthogonal protecting groups. The protecting group may be any protecting group described herein (e.g., Fmoc, Boc, benzyl, or trityl). In an embodiment, the second protecting group, e.g., R¹³, is added to the compound of Formula (VII) by incubation with a protecting group reagent and a base. In an embodiment, the protecting group reagent comprises fluorenylmethyloxycarbonyl chloride, p- toluenesulfonyl chloride, or di-tert-butyl dicarbonate.

[00241] The base may be an organic base or an inorganic base. In an embodiment, the base is a nitrogen-containing compound. In an embodiment, the base is a hydroxide or a carbonate salt. In an embodiment, the base is an amine. In an embodiment, the base comprises triethylamine, diisopropylethylamine, triisopropylamine, piperidine, pyrrole, N-m ethylmorpholine, imidazole, pyridine, 4-dimethylaminopyridine, 2,6-lutidine, l,4-diazabicyclo[2.2.2]octane (DABCO), 1,8- diazabicyclo(5.4.0)undec-7-ene (DBU), sodium carbonate, potassium carbonate, cesium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide, or sodium hydride. [00242] In an embodiment, the first protecting group, e.g., R¹, is removed from the compound of Formula (X) after treatment with a deprotecting agent. In an embodiment, the deprotecting agent comprises an acid or a base. In an embodiment, the deprotecting agent comprises an amine. In an embodiment, the deprotecting agent comprises acetic acid, trifluoroacetic acid, hydrochloric acid, piperidine, piperazine, triethylamine, cyclohexylamine, ethanolamine, or diisopropylethylamine. In an embodiment, the deprotecting agent is hydrogen gas. In an embodiment, the deprotecting agent is a metal salt. Additional deprotecting agents are disclosed in Greene et al., Protecting Groups in Organic Synthesis, Fourth Edition, Wiley, New York, 2011, and references cited therein, each of which are incorporated herein by reference in their entirety.

[00243] The protection or deprotection steps may be carried out in at a temperature of between about -50 to 50 °C (e.g., between about -40 to 40 °C, -30 to 30 °C, -15 to 15 °C, -10 to 10 °C, -5 to 15 °C, or 0 to 10 °C). In an embodiment, the protection or deprotection steps are carried out at a temperature greater than -40 °C, -20 °C, -10 °C, -5 °C, 0 °C, 5 °C, 10 °C, 15 °C, 25 °C, or 30 °C. In an embodiment, the protection or deprotection steps are carried out at a temperature lower than 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 0 °C, -5 °C, or -10 °C. The protection or deprotection steps may be carried out in a solvent such as dichloromethane, acetonitrile, methanol, ethanol, tetrahydrofuran, water, 1,4-di oxane, dimethylsulfoxide, di chloromethane, chloroform, toluene, diethyl ether, methyl tert-butyl ether, dimethylformamide, N- methylpyrrolidone, or mixtures thereof.

[00244] The compound of Formula (IX) or Formula (X) may have less than 80%, less than 60%, less than 40%, less than 20%, less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% of an impurity. In some embodiments, the impurity is residual starting materials, solvents, or by-products of a protection or deprotection step. The compound of Formula (IX) or Formula (X) may be purified by any conventional purification technique. In an embodiment, the compound of Formula (IX) or (X) is purified by chromatography, precipitation, crystallization, recrystallization, preparative HPLC, filtration, centrifugation, adsorption, distillation, evaporation, liquid-liquid extraction, lyophilization, trituration, or combinations thereof.

[00245] In an embodiment, the compound of Formula (IX) or (X), or a salt thereof, is obtained as a mixture of enantiomers. In an embodiment, the compound of Formula (IX) or (X), or a salt thereof, has an enantiomeric excess greater than 60% (e.g., greater than 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9%). In an embodiment, the compound of Formula (IX) or (X), or a salt thereof, has an enantiomeric excess lower than 99.99% (e.g., lower than 99.9%, 99.5%, 99%, 98%, 95%, or 90%). In an embodiment, the compound Formula (IX) or (X), or a salt thereof, is enantiopure.

Preparation of Peptide Nucleic Monomers

[00246] A peptide nucleic acid (PNA) monomer of Formula (I) may be prepared from a PNA backbone, e.g., a PNA backbone of Formula (VIII), or a salt thereof. In an embodiment, a compound of Formula (VIII) is converted to a compound of Formula (I). A compound of Formula (I) may be prepared through an acylation step, as shown in Scheme 6 via Step K. A compound of Formula (VIII) may be acylated with a compound of Formula (XI). Following acylation, the compound of Formula (I) may be converted to a compound of Formula (I-I), e.g., by cleavage of an ester, via Step L.

Scheme 6. Acylation of a compound of Formula (VIII) with a compound of Formula (XI) to form a compound of Formula (I), and its conversion to a compound of Formula (I-I); each of R¹, R², R³, R⁴, R⁵, R⁶, R¹¹, R¹², B, and n are as defined herein.

[00247] A compound of Formula (I) may be formed through the acylation of a compound of Formula (VIII) with a compound of Formula (XI) as shown in Scheme 6, via Step K. For example, a compound of Formula (I) may be prepared by incubating a compound of Formula (VIII) and a compound of Formula (XI) in the presence of a coupling agent and a base. In an embodiment, the carboxylic acid group of the compound of Formula (XI) is activated with the coupling agent prior to contact with the compound of Formula (XIII).

[00248] In an embodiment, the coupling agent used in the acylation reaction is a peptide coupling agent. In an embodiment, the coupling agent comprises one or more carbocyclic rings, heterocyclic rings, or heteroaromatic rings. In an embodiment, the coupling agent comprises an acid halide. In an embodiment, the coupling agent is a carbodiimide, benzotri azole, phosphonium salt, uronium salt, a fluroformamidium salt, or an acid chloride. In an embodiment, the coupling agent comprises trimethylacetyl chloride, (1- [bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), N,N’ -di cyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), hydroxybenzotriazole (HOBt), l-hydroxy-7-azabenzotriazole (HOAt), 3- (diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one (DEPBT), benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrop), (2-(lH-benzotriazol-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(benzotriazol-l-yl)-N,N,N’,N’- tetramethyluronium tetrafluorob orate (TBTU), fluoro-N,N,N’,N’ -tetramethylformamidinium hexafluorophosphate (TFFH), or fluoro-N,N,N’,N’-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH).

[00249] The base used in the acylation reaction may be an organic base or an inorganic base. In an embodiment, the base is a nitrogen-containing compound. In an embodiment, the base is an amine. In an embodiment, the base comprises N-methylmorpholine, diisopropylethylamine, triethylamine, triisopropylamine, imidazole, pyridine, 4-dimethylaminopyridine, 2,6-lutidine, l,4-diazabicyclo[2.2.2]octane (DABCO), l,8-diazabicyclo(5.4.0)undec-7-ene (DBU), pyrrole, piperidine, or piperazine.

[00250] A compound of Formula (I) may in turn be converted to a compound of Formula (I-I), as shown in Scheme 6, Step L. In an embodiment, a compound of Formula (I-I) is prepared by the cleavage of an ester group. In an embodiment, the ester is cleaved with a saponification reaction. In an embodiment, the ester is cleaved in a reduction reaction. For example, a compound of Formula (I) may be subjected to cleavage of an ester group by incubating the compound with an ester cleavage agent and a buffer. In an embodiment, the ester group is selectively cleaved in the presence of other acyl-containing groups.

[00251] The ester cleavage agent may be a reducing agent, or a base (e.g., a hydroxide, e.g, lithium hydroxide, sodium hydroxide, or potassium hydroxide). In an embodiment, the reducing agent is a metal. The metal may be a single metal, or a combination of one or more metals. In an embodiment, the ester cleavage agent is a phosphine. The cleavage agent may be in the form of granules, particules (e.g., nanoparticles), powder, dust, suspension, or solution. In an embodiment, the reducing agent comprises zinc, copper, magnesium, lead, mischmetal, tri-n- butyl phosphine, (tris(2-carboxyethyl)phosphine), or any combination thereof. In an embodiment, the ester cleavage agent is a reducing agent described in US Patent Publication No.: 2018/0282375, the disclosure of which is incorporated herein by reference in its entirety. [00252] In an embodiment, the buffer comprises a mixture of an acid and one or more salts. The acid may be an organic acid or an inorganic acid. The components of the buffer may be added to the ester cleavage reaction as a solution or as a solid. In an embodiment, the buffer is an aqueous solution. The components of the buffer may be added to the cleavage reaction individually, or as a whole. In an embodiment, the acid comprises a carboxylic acid. In an embodiment, the one or more salts comprise a mixture of organic and inorganic salts. In an embodiment, the buffer comprises acetic acid, ethylenediaminetetraacetic acid (EDTA), EDTA zinc disodium hydrate, monopotassium phosphate (KH2PO4), or a buffer component described in US Patent Publication No.: 2018/0282375, the disclosure of which is incorporated herein by reference in its entirety.

[00253] The reaction to form PNA monomers of Formulas (I) and (I-I) may be carried out at a temperature of between about -50 to 50 °C (e.g., between about -20 to 40 °C, -10 to 30 °C, -5 to 25 °C, or 0 to 15 °C). In an embodiment, the reaction to form a compound of Formula (I) or Formula (I-I) is carried out at a temperature greater than -40 °C, -20 °C, -10 °C, -5 °C, 0 °C, 5 °C, 10 °C, 15 °C, 25 °C, or 30 °C. In an embodiment, the reaction to form a compound of Formula (I) or Formula (I-I) is carried out at a temperature lower than 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 0 °C, -5 °C, or -10 °C. In an embodiment, the acylation of a compound of Formula (VIII) to a compound of Formula (I) is carried out at a temperature of between about -50 to 50 °C (e.g., between about -20 to 40 °C, -10 to 30 °C, -5 to 25 °C, or 0 to 15 °C). In an embodiment, the acylation of a compound of Formula (VIII) to a compound of Formula (I) is carried out at a temperature greater than -40 °C, -20 °C, -10 °C, -5 °C, 0 °C, 5 °C, 10 °C, 15 °C, 25 °C, or 30 °C. In an embodiment, the acylation of a compound of Formula (VIII) to a compound of Formula (I) is carried out at a temperature lower than 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 0 °C, -5 °C, or -10 °C. In an embodiment, the acylation of a compound of Formula (VIII) to a compound of Formula (I) is carried out at a temperature of about 0 °C, 10 °C, 20 °C, or 25 °C. In an embodiment, the acylation of a compound of Formula (VIII) to a compound of Formula (I) is carried out at room temperature. In an embodiment, the ester cleavage of a compound of Formula (I) to a compound of Formula (I-I) is carried out at a temperature of between about -50 to 50 °C (e.g., between about -20 to 40 °C, -10 to 30 °C, -5 to 25 °C, or 0 to 15 °C). In an embodiment, the ester cleavage of a compound of Formula (I) to a compound of Formula (I-I) is carried out at a temperature greater than -40 °C, -20 °C, -10 °C, -5 °C, 0 °C, 5 °C, 10 °C, 15 °C, 25 °C, or 30 °C. In an embodiment, the ester cleavage of a compound of Formula (I) to a compound of Formula (I-I) is carried out at a temperature lower than 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 0 °C, -5 °C, or -10 °C. In an embodiment, the ester cleavage of a compound of Formula (I) to a compound of Formula (I-I) is carried out at a temperature of about -5 °C, 0 °C, 5 °C, or 10 °C. In an embodiment, the ester cleavage of a compound of Formula (I) to a compound of Formula (I-I) is carried out at room temperature.

[00254] The reaction to form a compound of Formula (I) or Formula (I-I) may be carried out in a solvent such as dimethyl formamide, acetonitrile, tetrahydrofuran, water, methanol, ethanol, 1,4-di oxane, dimethylsulfoxide, di chloromethane, chloroform, toluene, diethyl ether, methyl tert-butyl ether, N-methylpyrrolidone, or mixtures thereof. In an embodiment, acylation reaction to form a compound of Formula (I) is carried out in a solvent that comprises less than 10% water (e.g., less than 8%, 5%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%). In an embodiment, the acylation reaction is carried out in a solvent that comprises less than 150 ppm of water (e.g., less than 120 ppm, less than 100 ppm, less than 80 ppm, less than 60 ppm, less than 40 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, less than 2 ppm, or less than 1 ppm). In an embodiment, the acylation reaction to form a compound of Formula (I) is carried out in an anhydrous solvent. In an embodiment, the acylation reaction to form a compound of Formula (I) is carried out in acetonitrile or dimethylformamide. In an embodiment, the ester cleavage of Formula (I) to a compound of Formula (I-I) is carried out in a mixture of tetrahydrofuran and water.

[00255] In some embodiments, the compound of Formula (I) or Formula (I-I) has less than 80%, less than 60%, less than 40%, less than 20%, less than 10%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.75%, less than 0.5%, less than 0.25%, less than 0.2%, less than 0.1%, less than 0.05%, or less than 0.01% of an impurity. The impurity may be residual starting materials, solvents, or by-products. In an embodiment, the compound of Formula (I) has an impurity arising from the acylation reaction comprising residual coupling agent, a by-product of the coupling reagent, residual acyl chloride, or a coupling agent adduct. In an embodiment, the compound of Formula (I-I) has an impurity arising from the ester cleavage reaction comprising residual acid, residual salt, or a by-product of non-selective cleavage.

[00256] The compound of Formula (I) or (I-I) may be purified by any conventional purification technique. In an embodiment, the compound of Formula (I) or Formula (I-I) is purified by chromatography, precipitation, crystallization, recrystallization, preparative HPLC, filtration, centrifugation, adsorption, distillation, evaporation, liquid-liquid extraction, lyophilization, trituration, or combinations thereof. In an embodiment, the cyclic compound of Formula (I) or Formula (I-I) is purified by chromatography.

[00257] In an embodiment, the compound of Formula (I) or (I-I), or a salt thereof, is obtained as a mixture of enantiomers. In an embodiment, the compound of Formula (I) or (I-I), or a salt thereof, has an enantiomeric excess greater than 60% (e.g., greater than 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9%). In an embodiment, the compound of Formula (I) or (I-I), or a salt thereof, has an enantiomeric excess lower than 99.99% (e.g., lower than 99.9%, 99.5%, 99%, 98%, 95%, or 90%). In an embodiment, the compound of Formula (I-I) has an enantiomeric excess greater than 99.5%. In an embodiment, the compound of Formula (I-I) has an enantiomeric excess greater than 99.9%. In an embodiment, the compound of Formula (I) or (I-I), or a salt thereof, is enantiopure. NUMBERED EMBODIMENTS

[00258] 1. A compound of Formula (II):

or a salt thereof, wherein:

R¹ is hydrogen or a protecting group (e.g., Fmoc); each of R⁵ and R⁶ are independently C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, Ci- C12 heteroalkyl, Ci-Ci2-haloalkyl, -OR^C, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkyl ene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, or C1-C12 alkyleneheteroaryl, or the side chain of an optionally protected amino acid, provided that each of R⁵ and R⁶ are not both hydrogen; and

R^c is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl. [00259] 2. The compound of embodiment 1, wherein R¹ is 9-fluorenylmethyloxy carbonyl (Fmoc), t-butyloxy carbonyl (Boc), carboxybenzyl (Cbz), p-toluenesulfonyl (Ts), benzoyl (Bz), or benzyl (Bn).

[00260] 3. The compound of any one of embodiments 1-2, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc).

[00261] 4. The compound of any one of embodiments 1-3, wherein each of R⁵ and R⁶ is independently hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, or the side chain of an optionally protected amino acid.

[00262] 5. The compound of any one of embodiments 1-4, wherein each of R⁵ and R⁶ is

an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen, C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl), or C1-C12 haloalkyl.

[00263] 6. The compound of any one of embodiments 1-5, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently C1-C12 alkyl or C1-C12 heteroalkyl.

[00264] 7. The compound of any one of embodiments 1-6, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently

or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl).

[00265] 8. The compound of any one of embodiments 1-7, wherein the compound of Formula (II) is a compound of Formula (Il-a) or (Il-b):

or a salt thereof, wherein R¹ is hydrogen or a protecting group (e.g., Fmoc); R⁵ is hydrogen, Ci- C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, Ci-Ci2-haloalkyl, -OR^C, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, or C1-C12 alkylene-heteroaryl, or the side chain of an optionally protected amino acid; and R^c is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Ci-

C12 heteroalkyl.

[00266] 9. The compound of any one of embodiments 1-7, wherein the compound of

Formula (II) is a compound of Formula (II-c) or (Il-d):

[00267] 10. The compound of embodiment 9, wherein R⁶ is hydrogen.

[00268] 11. A method of making a compound of Formula (I):

or a salt thereof, wherein: B is a nucleobase;

R¹ is an amine protecting group (e.g., Fmoc);

R² is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 heteroalkyl, C1-C12- haloalkyl, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C12 alkylene-heterocyclyl, aryl, C1-C12 alkylene-aryl, heteroaryl, or C1-C12 alkyl ene-heteroaryl; each of R³, R⁴, R⁵, and R⁶ is independently hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, - N(R^A)(R^B), halo, -OR^C, or the optionally protected side chain of an amino acid, provided that each of R⁵ and R⁶ are not both hydrogen;

R⁷ is hydrogen or C1-C12 alkyl; each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl; and n is an integer selected from 0, 1, 2, 3, or 4; wherein the method comprises use of an intermediate of Formula (II):

or a pharmaceutically acceptable salt thereof to achieve the compound of Formula (I).

[00269] 12. The method of embodiment 11, wherein R¹ is 9-fluorenylmethyloxycarbonyl

(Fmoc), t-butyloxy carbonyl (Boc), carboxybenzyl (Cbz), p-toluenesulfonyl (Ts), benzoyl (Bz), or benzyl (Bn).

[00270] 13. The method of any one of embodiments 11-12, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc).

[00271] 14. The method of any one of embodiments 11-13, wherein R² is hydrogen, C1-C12 alkyl (e.g., t-butyl), or Ci-Ci2-haloalkyl (e.g., 2,2,2-tribromoethyl, 2-bromoethyl, 2,2,2- tri chloroethyl, or 2-iodoethyl).

[00272] 15. The method of any one of embodiments 11-14, wherein each of R³ and R⁴ is independently hydrogen or the optionally protected side chain of an amino acid.

[00273] 16. The method of any one of embodiments 11-15, wherein the side chain of an amino acid is selected from:

wherein each of Ill-a through III-z is independently and optionally protected.

[00274] 17. The method of any one of embodiments 11-16, wherein each of R³ and R⁴ is independently hydrogen. [00275] 18. The method of any one of embodiments 11-17, wherein each of R⁵ and R⁶ is independently hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, or the side chain of an amino acid that is optionally protected.

[00276] 19. The method of any one of embodiments 11-18, wherein each of R⁵ and R⁶ is independently hydrogen,

, or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl), provided that one of R⁵ and R⁶ is independently not hydrogen. [00277] 20. The method of any one of embodiments 11-19, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently C1-C12 alkyl or C1-C12 heteroalkyl.

[00278] 21. The method of any one of embodiments 11-20, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently

or

, wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen or C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl).

[00279] 22. The method of any one of embodiments 11-21, wherein R⁷ is hydrogen.

[00280] 23. The method of any one of embodiments 11-22, wherein B is a naturally occurring nucleobase or a non-naturally occurring nucleobase.

[00281] 24. The method of any one of embodiments 11-23, wherein B is selected from adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2-thiopseudoisocytosine, 5- methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2,6-diaminopurine, 2- thiouracil, 2-thiothymine, 2-thiocytosine, 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5- chlorocytosine, 5 -bromocytosine, 5-iodocytosine, 5-propynyluracil, 5-propynylcytosine, 6- azouracil, 6-azocytosine, 6-azothymine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-azaadenine, 5-propynyluracil, 2-thio-5-propynyluracil, pyridin-2-amine, 2- thiopseudoisocytosine, pyrimidin-2(lH)-one, and pyridazin-3(2H)-one.

[00282] 25. The method of any one of embodiments 11-24, wherein the compound of Formula (I) is a compound of Formula (I-a) or Formula (I-b):

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³ and B is as defined in embodiment 11, and R⁹ is hydrogen, C1-C12 alkyl, C1-C12 heteroalkyl, or the side chain of an amino acid.

[00283] 26. The method of any one of embodiments 11-25, wherein the compound of Formula (I) is a compound of Formula (I-c) or Formula (I-d):

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³ and B is as defined in embodiment 1; R¹⁰ is hydrogen, C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl), or C1-C12- haloalkyl; and y is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[00284] 27. The method of any one of embodiments 11-26, wherein the compound of Formula (I) is a compound of Formula (I-e) or Formula (I-f):

or a pharmaceutically acceptable salt thereof, wherein each of R¹, R², R³ and B is as defined in embodiment 1, and R¹⁰ is hydrogen, C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl), or C1-C12- haloalkyl.

[00285] 28. The method of any one of embodiments 11-27, wherein the method further comprises the reaction of the intermediate of Formula (II): R⁶ R⁵ ^R1'^N ₁ 7 o=s II-o 0 (II) with an azide-containing reagent to achieve the compound of Formula (I).

[00286] 29. The method of embodiment 28, wherein the azide-containing reagent is NaN

[00287] 30. The method of any one of embodiments 28-29, wherein each of R⁵ and R⁶ of

Formula (II) is hydrogen, C1-C12 alkyl (e.g., methyl), or C1-C12 heteroalkyl (e.g., a polyethylene glycol), provided that one of R⁵ and R⁶ of Formula (II) is independently not hydrogen.

[00288] 31. The method of any one of embodiments 28-30, wherein the intermediate of Formula (II) is a compound of Formula (Il-a) or Formula (Il-b):

wherein R¹ is defined as in embodiment 1, and R⁵ is C1-C12 alkyl (e.g., methyl), C1-C12 heteroalkyl (e.g., a polyethylene glycol), or an optionally protected amino acid side chain.

[00289] 32. The method of any one of embodiments 11-21, wherein R⁵ is hydrogen,

integer selected from 0, 1, 2,

3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen, C1-C12 alkyl (e.g., t-butyl, methyl, or ethyl), or Ci-

Ci2-haloalkyl.

[00290] 33. The method of any one of embodiments 28-32, wherein the reaction of the intermediate of Formula (II) and the azide-containing reagent followed by reduction yields an intermediate of Formula (IV):

or a salt thereof, wherein each of R¹, R⁵, R⁶, and R⁷ is as defined in embodiment 11.

[00291] 34. The method of any one of embodiments 11-33, wherein the method further comprises reaction of the intermediate of Formula (VII):

or a salt thereof with an ester compound of Formula (V):

wherein each of R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ is as defined in embodiment 11 and X is halo (e.g., chloro, bromo, or iodo), to achieve a compound of Formula (I).

[00292] 35. The method of embodiment 34, wherein each of R³ and R⁴ is independently hydrogen.

[00293] 36. The method of any one of embodiments 11-27, wherein the method further comprises reaction of the intermediate of Formula (II):

with a compound of Formula (VI):

wherein each of R¹, R², R³, R⁴, R⁵, and R⁶ is as defined in embodiment 11, to achieve a compound of Formula (I).

[00294] 37. A method of making a compound of Formula (I):

or a salt thereof, wherein:

B is a nucleobase; R¹ is an amine protecting group (e.g., Fmoc);

R⁷ is hydrogen or C1-C12 alkyl; each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl; and n is an integer selected from 0, 1, 2, 3, or 4; wherein the method comprises use of the intermediate of Formula (VII):

or a salt thereof to achieve the compound of Formula (I).

[00295] 38. A method of making a compound of Formula (I):

or a salt thereof, wherein:

B is a nucleobase;

R¹ is an amine protecting group (e.g., Fmoc);

R⁷ is hydrogen or C1-C12 alkyl; each of R^A, R^B, and R^c is independently hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, or C1-C12 heteroalkyl; and n is an integer selected from 0, 1, 2, 3, or 4; wherein the method comprises the following steps:

(i) contacting a compound of Formula (II):

or a salt thereof, with an azide-containing compound (e.g., NaNs) to achieve the azide- containing intermediate of Formula (IV):

(ii) contacting the azide-containing intermediate of Formula (IV) with a reducing agent (e.g., Pd/C or Zn) to achieve an intermediate of Formula (VII):

(iii) contacting the intermediate of Formula (VII) with an ester compound of Formula

(V):

to achieve a compound of Formula (VIII):

(iv) contacting the compound of Formula (VIII) with a nucleobase acetic acid to achieve a compound of Formula (I).

[00296] Bl. A compound of Formula (IIA):

or a salt thereof, wherein:

R¹ is 9-fluorenylmethyloxycarbonyl (Fmoc), 2-(4- nitropheylsulfonyl)ethoxy carbonyl (Nsc), 1, l-dioxobenzo[b]thiophene-2- ylmethyloxycarbonyl (Bsmoc), l,l-dioxonaphtho[l,2-b]thiophene (Nsmoc), l-(4,4- dimethyl-2, 6-dioxocy cl ohexylidene)-3 -methylbutyl (ivDde), 2,7-di-tert-butylfluoren-9- ylmethoxycarbonyl (Fmoc*), 2-fluorofluoren-9-ylmethoxycarbonyl (Fmoc(2F)), 2- monoisooctylfluoren-9-ylmethoxy carbonyl (mio-Fmoc), 2,7-diisooctylfluoren-9- ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)-fluorenylmethoxycarbonyl (Sulfmoc), 2,6- di-t-butyl-9-fluorenylmethoxy carbonyl (Dtb-Fmoc), 2,7-bis(trimethylsilyl)- fluorenylmethoxycarbonyl (Bts-Fmoc), 9-(2,7-dibromo)fluorenylmethoxycarbonyl, 2- [phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms), ethanesulfonylethoxycarbonyl (Esc), 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps), or l-cyano-2-methylpropan-2-ylcarbonyl (Cyoc), each of which is optionally substituted, or hydrogen; each of R⁵ and R⁶ are independently hydrogen, C3-C16 alkyl, cycloalkyl, C2-C16 alkenyl, C2-C16 alkynyl, Ce-Ci6 heteroalkyl, Ci-Ci6-haloalkyl, -OR^C, -CH2OR⁹, cycloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl, an optionally protected amino acid side chain,

provided that one of R⁵ and R⁶ is not hydrogen, or both of R⁵ and R⁶ are not hydrogen; x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; and R^c is hydrogen, C1-C12 alkyl, cycloalkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Ci-

C12 heteroalkyl, wherein each optionally protected amino acid side chain is independently selected from:

each of which is optionally protected.

[00297] B2. The compound of embodiment Bl, wherein each of R⁵ and R⁶ is independently hydrogen, C3-C16 alkyl, Ce-Ci6 heteroalkyl, or the optionally protected amino acid side chain, provided that one of R⁵ and R⁶ is not hydrogen, or both of R⁵ and R⁶ are not hydrogen.

[00298] B3. The compound of embodiment Bl, wherein each of R⁵ and R⁶ is independently

V°^(_O^OR⁸

1 1 111 aa ' X 111 l^d ah U ' / x hydrogen, , or ^{l l} , w ₁ herein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl, provided that one of R⁵ and R⁶ is not hydrogen, or both of R⁵ and R⁶ are not hydrogen.

[00299] B4. The compound of embodiment Bl, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently C3-C16 alkyl or Ce-Ci6 heteroalkyl.

[00300] B5. The compound of embodiment Bl, wherein one of R⁵ and R⁶ is independently

hydrogen and the other of R⁵ and R⁶ is independently or

, wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and

10 and R⁸ is hydrogen, cycloalkyl, or C1-C12 alkyl.

[00301] B6. The compound of embodiment Bl, wherein the compound of Formula (IIA) is a compound of Formula (IIA-a) or (IIA-b):

or a salt thereof, wherein

R¹ is 9-fluorenylmethyloxycarbonyl (Fmoc), 2-(4- nitropheylsulfonyl)ethoxy carbonyl (Nsc), 1, l-dioxobenzo[b]thiophene-2- ylmethyloxycarbonyl (Bsmoc), l,l-dioxonaphtho[l,2-b]thiophene (Nsmoc), l-(4,4- dimethyl-2, 6-dioxocy cl ohexylidene)-3 -methylbutyl (ivDde), 2,7-di-tert-butylfluoren-9- ylmethoxycarbonyl (Fmoc*), 2-fluorofluoren-9-ylmethoxycarbonyl (Fmoc(2F)), 2- monoisooctylfluoren-9-ylmethoxy carbonyl (mio-Fmoc), 2,7-diisooctylfluoren-9- ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)-fluorenylmethoxycarbonyl (Sulfmoc), 2,6- di-t-butyl-9-fluorenylmethoxy carbonyl (Dtb-Fmoc), 2,7-bis(trimethylsilyl)- fluorenylmethoxycarbonyl (Bts-Fmoc), 9-(2,7-dibromo)fluorenylmethoxycarbonyl, 2- [phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms), ethanesulfonylethoxycarbonyl (Esc), 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps), or l-cyano-2-methylpropan-2-ylcarbonyl (Cyoc), each of which is optionally substituted, or hydrogen;

R⁵ is C3-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, Ce-Ci6 heteroalkyl, C1-C16- haloalkyl, -OR^C, -CH2OR⁹, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl,

x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl; and

R^c is hydrogen, C1-C12 alkyl, cycloalkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Ci-

C12 heteroalkyl.

[00302] B7. The compound of any one of embodiments Bl, B2, and B6, wherein R⁵ is the optionally protected amino acid side chain.

[00303] B8. The compound of any one of embodiments Bl, B2, B6, and B7, wherein R⁵ is

, each of which is optionally protected.

[00304] B9. The compound of embodiment Bl, wherein the compound of Formula (IIA) is a compound of Formula (IIA-c) or (IIA-d):

or a salt thereof, wherein

R⁶ is hydrogen, C3-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, Ce-Ci6 heteroalkyl, Ci-Ci6-haloalkyl, -OR^C, cycloalkyl, Ci-C 12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkylene- heteroaryl, the optionally protected amino acid side chain,

, or

x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R^c is hydrogen, C1-C12 alkyl, cycloalkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Ci- C12 heteroalkyl; and

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or the side chain of an amino acid.

[00305] B10. The compound of embodiment B9, wherein R⁹ is methyl, ethyl, tert-butyl, cyclopentyl, or cyclohexyl.

[00306] Bl 1. The compound of any one of embodiments Bl -B10, wherein R⁶ is hydrogen. [00307] B12. The compound of any one of embodiments Bl-Bl 1, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc), 2, 7-di-tert-butylfluoren-9-ylmethoxy carbonyl (Fmoc*), 2- fluorofluoren-9-ylmethoxy carbonyl (Fmoc(2F)), 2-monoisooctylfluoren-9-ylmethoxy carbonyl (mio-Fmoc), 2,7-diisooctylfluoren-9-ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)- fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-t-butyl-9-fluorenylmethoxycarbonyl (Dtb-Fmoc), or 2,7-bis(trimethylsilyl)-fluorenylmethoxycarbonyl (Bts-Fmoc).

[00308] B13. The compound of any one of embodiments Bl-Bl 1, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc).

[00309] B14. A method of making a compound of Formula (IB):

or a salt thereof, wherein:

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

R² is hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkyl ene-heteroaryl; each of R³, R⁴, R⁵, and R⁶ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl, -N(R^A)(R^B), halo, -OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

provided that each of R⁵ and

R⁶ are not both hydrogen;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

[00310] B15. The method of embodiment Bl 4, wherein the method further comprises the reaction of the intermediate of Formula (IIB), or a salt thereof:

with an azide-containing reagent.

[00311] B16. The method of embodiment Bl 5, wherein the azide-containing reagent is NaN [00312] B 17. The method of embodiment B 15 or embodiment B 16, wherein the reaction of the intermediate of Formula (IIB) and the azide-containing reagent followed by reduction yields an intermediate of Formula (VIIB):

or a salt thereof, wherein

R¹ is an amine protecting group; each of R⁵ and R⁶ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl, - N(R^A)(R^B), halo, -OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

provided that one of both of

R⁵ and R⁶ are not hydrogen;

R⁷ is hydrogen or C1-C16 alkyl; each of R^A, R^B, and R^c is independently hydrogen, C1-C16 alkyl, C2-C16 alkenyl,

C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[00313] B18. The method of any one of embodiments B14-B17, wherein the method further comprises reaction of the intermediate of Formula (VIIB), or a salt thereof:

with an ester compound of Formula (VB), or a salt thereof:

to achieve a compound of Formula (VIIIB), or a salt thereof:

wherein

R¹ is an amine protecting group;

provided that each of R⁵ and

R⁶ are not both hydrogen;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and X is halo or tosyl.

[00314] B19. The method of embodiment Bl 4, wherein the method further comprises reaction of the intermediate of Formula (IIB), or a salt thereof:

with a compound of Formula (VIB), or a salt thereof:

to achieve a compound of Formula (VIIIB), or a salt thereof:

wherein

R¹ is an amine protecting group;

provided that each of R⁵ and

R⁶ are not both hydrogen; each of R^A, R^B, and R^c is independently hydrogen, C1-C16 alkyl, C2-C16 alkenyl,

C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

[00315] B20. The method of any one of embodiments B14-B19, wherein the intermediate of

Formula (IIB) is a compound of Formula (IIB-a) or Formula (IIB-b):

or a salt thereof, wherein

R¹ is an amine protecting group;

R⁵ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci- Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkylene-heteroaryl, -N(R^A)(R^B), halo, - OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

each of R^A, R^B, and R^c is independently hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

[00316] B21. The method of any one of embodiments B14-B20, wherein the compound of

Formula (IB) is a compound of Formula (IB-e) or Formula (IB-f):

or a salt thereof, wherein

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

R² is hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkylene-heteroaryl; each of R³ and R⁵ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkylene-heteroaryl, - N(R^A)(R^B), halo, -OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

[00317] B22. The method of any one of embodiments B14-B21, wherein the compound of

Formula (IB) is a compound of Formula (IB-a) or Formula (IB-b):

or a salt thereof, wherein

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

R² is hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkyl ene-heteroaryl;

R³ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci- Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkylene-heteroaryl, -N(R^A)(R^B), halo, - OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

[00318] B23. The method of any one of embodiments B14-B21, wherein the compound of Formula (IB) is a compound of Formula (IB-c) or Formula (IB-d):

or a salt thereof, wherein

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain;

R¹⁰ is hydrogen, C1-C16 alkyl, cycloalkyl, or Ci-Ci6-haloalkyl; x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and y is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[00319] B24. The method of any one of embodiments B14-B21, wherein the compound of

Formula (IB) is a compound of Formula (IB-e) or Formula (IB-f):

or a salt thereof, wherein

B is an optionally protected nucleobase;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

[00320] B25. The method of any one of embodiments B14-B19, wherein each of R³ and R⁴ is independently hydrogen or the optionally protected amino acid side chain.

[00321] B26. The method of any one of embodiments B14-B19, wherein each of R³ and R⁴ is independently hydrogen.

[00322] B27. The method of any one of embodiments B14-B19, B25, and B26, wherein each of R⁵ and R⁶ is independently hydrogen, C1-C16 alkyl, C1-C16 heteroalkyl, or the optionally protected amino acid side chain.

[00323] B28. The method of any one of embodiments B14-B19, B25, and B26, wherein each of R⁵ and R⁶ of Formula (Il-a) is hydrogen, Ci-C 12 alkyl, or C1-C12 heteroalkyl, provided that one of R⁵ and R⁶ of Formula (Il-a) is independently not hydrogen.

[00324] B29. The method of any one of embodiments B 14-B 19, B25, and B26, wherein each

of R⁵ and R⁶ is independently hydrogen, , or

, wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and

10; and R⁸ is hydrogen or C1-C16 alkyl, provided that one or both of R⁵ and R⁶ is not hydrogen. [00325] B30. The method of any one of embodiments B14-B19, B25, and B26, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently C1-C16 alkyl or C1-C16 heteroalkyl.

[00326] B31. The method of any one of embodiments B 14-B 19, B25, and B26, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently

wherein x is an integer selected from

0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen or C1-C16 alkyl.

[00327] B32. The method of any one of embodiments B14-B21, wherein R⁵ is the optionally protected amino acid side chain.

[00328] B33. The method of any one of embodiments B14-B21 and B32, wherein the

optionally protected amino acid side chain is , each of which is optionally protected.

[00329] B34. The method of any one of embodiments B14-B22, wherein R⁵ is CH2OR⁹.

[00330] B35. The method of any one of embodiments B14-B22 and B34, wherein R⁹ is methyl, ethyl, tert-butyl, cyclopentyl, or cyclohexyl.

[00331] B36. The method of any one of embodiments B14-B23 and B25-B35, wherein R¹ is 9- fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), p- toluenesulfonyl (Ts), benzoyl (Bz), or benzyl (Bn).

[00332] B37. The method of any one of embodiments B14-B23 and B25-B35, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc), 2-(4-nitropheylsulfonyl)ethoxy carbonyl (Nsc), 1,1- dioxobenzo[b]thiophene-2-ylmethyloxy carbonyl (Bsmoc), 1, 1 -di oxonaphthofl, 2-b]thiophene (Nsmoc), 1 -(4, 4-dimethyl-2,6-dioxocy cl ohexylidene)-3 -methylbutyl (ivDde), 2,7-di-tert- butylfluoren-9-ylmethoxycarbonyl (Fmoc*), 2-fluorofluoren-9-ylmethoxycarbonyl (Fmoc(2F)), 2-monoisooctylfluoren-9-ylmethoxy carbonyl (mio-Fmoc), 2,7-diisooctylfluoren-9- ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)-fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-t- butyl-9-fluorenylmethoxy carbonyl (Dtb-Fmoc), 2,7-bis(trimethylsilyl)- fluorenylmethoxycarbonyl (Bts-Fmoc), 9-(2,7-dibromo)fluorenylmethoxycarbonyl, 2- [phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms), ethanesulfonylethoxycarbonyl (Esc), 2-(4-sulfophenylsulfonyl)ethoxy carbonyl (Sps), or 1- cyano-2-methylpropan-2-ylcarbonyl (Cyoc), each of which is optionally substituted.

[00333] B38. The method of any one of embodiments B14-B23 and B25-B35, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc), 2, 7-di-tert-butylfluoren-9-ylmethoxy carbonyl (Fmoc*), 2- fluorofluoren-9-ylmethoxy carbonyl (Fmoc(2F)), 2-monoisooctylfluoren-9-ylmethoxy carbonyl (mio-Fmoc), 2,7-diisooctylfluoren-9-ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)- fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-t-butyl-9-fluorenylmethoxycarbonyl (Dtb-Fmoc), or 2,7-bis(trimethylsilyl)-fluorenylmethoxycarbonyl (Bts-Fmoc).

[00334] B39. The method of any one of embodiments B14-B23 and B25-B35, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc).

[00335] B40. The method of any one of embodiments B14-B25, wherein the optionally protected amino acid side chain is selected from:

each of which is optionally protected.

[00336] B41. The method of any one of embodiments B14-B40, wherein R² is hydrogen, Ci- Ci6 alkyl, or Ci-Ci6-haloalkyl.

[00337] B42. The method of any one of embodiments B14-B40, wherein R² is Ci-Cie- haloalkyl.

[00338] B43. The method of any one of embodiments B14-B42, wherein R² is 2,2,2- tribromoethyl, 2-bromoethyl, 2,2,2-trichloroethyl, or 2-iodoethyl.

[00339] B44. The method of any one of embodiments B14-B43, wherein B is a naturally occurring nucleobase or a non-naturally occurring nucleobase, each of which is optionally protected.

[00340] B45. The method of any one of embodiments B14-B44, wherein B is selected from adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2-thiopseudoisocytosine, 5- methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2,6-diaminopurine, 2- thiouracil, 2-thiothymine, 2-thiocytosine, 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5- chlorocytosine, 5 -bromocytosine, 5-iodocytosine, 5-propynyluracil, 5-propynylcytosine, 6- azouracil, 6-azocytosine, 6-azothymine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-azaadenine, 5-propynyluracil, 2-thio-5-propynyluracil, pyridin-2-amine, 2- thiopseudoisocytosine, pyrimidin-2(lH)-one, and pyridazin-3(2H)-one, each of which is optionally protected.

[00341] B46. The method of any one of embodiments B14-B45, wherein R⁷ is hydrogen.

[00342] B47. A method of making a compound of Formula (IB):

or a salt thereof, wherein:

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

provided that each of R⁵ and

R⁶ are not both hydrogen;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

or a salt thereof, with an azide-containing compound to achieve the azide- containing intermediate of Formula (IVB):

(iii) contacting the intermediate of Formula (VIIB) with an ester compound of

Formula (VB):

to achieve a compound of Formula (VIIIB):

(iv) contacting the compound of Formula (VIIIB) with a nucleobase acetic acid to achieve the compound of Formula (IB). EXAMPLES

[00343] In order that the disclosure described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the peptide nucleic acids, compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

[00344] The PNA intermediates (e.g., cyclic compounds, azide- or diamine compounds, PNA backbones, or PNA monomers) and compositions thereof provided herein can be prepared from readily available starting materials using modifications to the specific synthetic protocols set forth below in combination with what would be well known to those of skill in the art. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.

[00345] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in Greene et al., Protecting Groups in Organic Synthesis, Fourth Edition, Wiley, New York, 2011, and references cited therein, each of which are incorporated herein by reference in their entirety.

[00346] Exemplary PNA intermediates and compositions thereof may be prepared using any of the strategies described below.

Example 1: Synthesis of (9H-fluoren-9-yl)methyl (4S)-4-methyl-l,2,3-oxathiazolidine-3- carboxylate 2-oxide (2)

Ref: Adapted from J. Am. Chem. Soc. 2009, 131, 7917-7927

[00347] Imidazole (112.5 mmol, 7.695 g) was dissolved in 200 mL of dry tetrahydrofuran (THF) in an oven dried round bottom (RB) flask equipped with a large stir bar, addition funnel, and nitrogen inlet. The flask containing the mixture was purged with nitrogen and cooled to - 78°C using a dry ice/acetone bath. Thionyl chloride (25 mmol, 1.81 mL) was added dropwise with stirring whereupon a large amount of solid formed over 30 minutes. 10 mmol (2.976 g) of (9H-fluoren-9-yl)methyl (S)-(l-hydroxypropan-2-yl)carbamate (1) dissolved in 20 mL of dry THF was then added to the mixture, and the mixture was warmed to room temperature and stirred for 2 hours. The mixture was then concentrated to remove THF and the remaining white residue was partitioned between ethyl acetate (EtOAc) and deionized water. The water layer was discarded and the organic layer extracted once more with deionized water, twice with 5% (w/v) aqueous citric acid, once more with deionized water, then finally with saturated aqueous sodium chloride (brine). The EtOAc mixture was dried over granular anhydrous magnesium sulfate, then filtered and concentrated to a solid after concentration under vacuum. This solid was recrystallized from di-n-butyl ether to give 2.35 g (6.84 mmol; 68% yield) compound 2.

Example 2: Synthesis of (9H-fluoren-9-yl)methyl (S)-4-methyl-l,2,3-oxathiazolidine-3- carboxylate 2,2-dioxide (3)

Ref: Adapted from J. Am. Chem. Soc. 2009, 131, 7917-7927

[00348] (9H-fluoren-9-yl)methyl (4S)-4-methyl-l,2,3-oxathiazolidine-3-carboxylate 2-oxide (2) (2.34 g, 6.8 mmol) was dissolved in 40 mL of acetonitrile (ACN) and the resulting solution was cooled to 0°C, at which point crystals formed. Ruthenium trichloride (RuCh xJLO; 35 mg, 0.17 mmol) was added, followed by solid sodium periodate (NaIC ; 2.2 g, 10.3 mmol) and 30 mL of water. The mixture was warmed to RT and the slurry was stirred for 20 min. After 90 minutes, an additional 0.5 g (2.3 mmol) NaICU and 15 mg (0.072 mmol) RuCh xLbO were added. Dichloromethane (DCM) and deionized water were added to the mixture until two layers resulted. The DCM layer was removed and the water layer extracted with additional DCM. The initial DCM layer and DCM extracts were combined and then washed twice with deionized water and once with brine, then dried over granular anhydrous magnesium sulfate. This solution was filtered through Celite® and dried to afford 2.42 g (6.73 mmol; 99%) compound 3. This material was used without further purification.

Example 3: Synthesis of (9H-fluoren-9-yl)methyl (S)-(l-azidopropan-2-yl)carbamate (4)

Fmoc / NaN₃ I

N > - *- Fmoc._N ^N₃

^O=/S^o Methanol H

⁰ 3 4

[00349] (9H-fluoren-9-yl)methyl (S)-4-methyl-l,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (3) (2.17 g, 6 mmol) and 1.5 equivalents of NaNs (9 mmol, 0.6 g) were mixed in 25 mL of methanol and heated to 50 °C with rapid stirring. After 2 hours the mixture was concentrated to a thick oil. This residue was partitioned between 50 mL of EtOAc and 50 mL of water. The EtOAc layer was removed and the water layer extracted twice with 30 mL of EtOAc. The combined water layers were discarded. The combined EtOAc extracts were then mixed with dilute aqueous hydrochloric acid (HC1) with rapid stirring. Stirring was stopped and the aqueous layer that formed was then discarded. The remaining EtOAc layer was extracted once with 5% aqueous sodium bicarbonate and once with brine. The EtOAc layer was then dried over granular anhydrous magnesium sulfate and then filtered and concentrated to give a white solid. This material was recrystallized from a 1/1/3 (v/v/v) mixture of ethanol/ACN/water to provide a total of 1.5 g (4.7 mmol; 78%) of compound 4 as crystals.

Example 4: Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan-l- aminium chloride (5)

Ref: J. Org. Chem. 2007, 72(17), 6599-6601

[00350] (9H-fluoren-9-yl)methyl (S)-(l -azidopropan -2 -yl)carbamate (4) (1.4 g, 4.3 mmol) was combined under nitrogen with 170 mg of 10% Pd/C catalyst (unreduced form). Methanol (10 mL) and chloroform (2 mL) were then added, followed by the addition of triethylsilane (6.7 mL) dropwise with rapid stirring Vigorous evolution of hydrogen occurred throughout the silane addition then stopped shortly after addition was complete. The mixture was then filtered through Celite® and concentrated to a solid which was recrystallized from ACN/methanol to give a total of 0.832 g of compound 5 as solid (three crops, 2.5 mmol, 58%).

Example 5: Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(2-oxo-2- (2,2,2-tribromoethoxy)ethyl)propan-l-aminium 4-methylbenzenesulfonate (6)

O 100

Ref: WO/2018/175927, Example 17

[00351] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propan-l-aminium chloride (5)

(2.28 mmol, 0.80 g), 13 mL ethanol, and 16 mL of toluene were added to an oven dried 125 mL RB flask. The mixture was gently heated and once solids were dissolved the mixture was evaporated under reduced pressure, then co-evaporated twice with toluene (10 mL), and the residue was dried under vacuum for 1 hour. 2,2,2-Tribromoethyl 2-bromoacetate (100; 3.19 mmol, 1.28 g) and 15 mL of dry ACN were then added, and cooled in an ice bath for about 15 minutes under nitrogen. 6.15 mmol (1.06 mL) of N,N’-diisopropylethylamine (DIEA) was then added dropwise to the stirring solution and the reaction was allowed to proceed for 45 minutes, before warming to room temperature and stirring for an additional 30 minutes. IN HC1 (2-3 mL) was then added to lower the pH to 4-5, and the mixture was concentrated to about 1/3 of its volume by evaporation under reduced pressure. 50 mL of EtOAc was then added and the contents of the flask were transferred to a separatory funnel and the layers were separated. The EtOAc layer was washed with deionized water (25 mL), 2.5% (w/v) aqueous citric acid (3x 25 mL), deionized water (25 mL), ’A saturated Na2CO3 (2 x 25 mL), 5% (w/v) aqueous NaHCOs (25 mL), and then brine (25 mL). The EtOAc layer was then dried over granular anhydrous magnesium sulfate and filtered. p-Toluenesulfonic acid monohydrate (p-TSA) (1.83 mmol, 0.348 g) was then added to the filtrate and the solution was mixed until all of the p-TSA dissolved. The solution was evaporated to about 1/3 of its volume under reduced pressure and then placed in a refrigerator to crystallize. The product was then collected by vacuum filtration to yield 0.969 g (1.2 mmol; 54%) of 6 as crystals.

[00352] Note: 2,2,2-tribromoethyl 2-bromoacetate (100) was prepared as described in WO/2018/175927, Example 17, either in-house or by a commercial source.

Example 6: Synthesis of 2,2,2-tribromoethyl (S)-N-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propyl)-N-(2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin- l(2H)-yl)acetyl)glycinate (7)

[00353] To 1.32 mmol (0.243 g) of 2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)- yl)acetic acid (101) in a 100 mL RB flask was added about 8 mL of dry can, and the solution was then cooled in an ice bath under a nitrogen blanket for about 20 minutes. 1.43 mmol (0.176 mL) of trimethylacetyl chloride (TMAC) was then added followed by 5.5 mmol (0.604 mL) of N-methylmorpholine (NMM). When near complete mixed anhydride formation was confirmed by this TLC analysis,¹, 1.1 mmol (0.87 g) of (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-N-(2-oxo-2-(2,2,2-tribromoethoxy)ethyl)propan-l-aminium 4- methylbenzenesulfonate (6) was added to the stirring solution, and the mixture was allowed to stir for 1 hour while warming to room temperature. The solvent was then evaporated under reduced pressure partitioned with 25 mL of EtOAc and 20 mL of /i saturated KH2PO4. The mixture was transferred to a separatory funnel and the layers were separated. The EtOAc layer was then washed ’A saturated KH2PO4 (20 mL), 5% (w/v) aqueous NaHCOs (2x 20 mL), and brine (20 mL). The EtOAc layer was then dried over granular anhydrous magnesium sulfate, filtered and evaporated to yield 0.758 g (0.96 mmol; 88%) of crude 7. The crude 7 was then dissolved in a minimum of 1% MeOH in DCM and purified by injection on a 12g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 1-4% methanol in DCM gradient over 12 CV and then 4-6% MeOH over 3 CV after first holding at 1% methanol for 1.5 CV immediately after injection. Pooled fractions provided 0.486 g (0.62 mmol; 56%) of 7.

[00354] 'A TLC sample to monitor this reaction can be prepared as follows: add one drop of phenethylamine to about 1 mL of ACN; then add one drop of the reaction mixture to 100 pL of the phenethylamine containing solution; use 10% MeOH/DCM as the TLC eluent, and compound 101 as a reference.

Example 7: Synthesis of (S)-N-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propyl)-N- (2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)acetyl)glycine (8)

Zn dust, HOAc, KH₂PO₄, EDTA

Fmoc

[00355] To 0.618 mmol (0.486 g) of 2,2,2-tribromoethyl (S)-N-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propyl)-N-(2-(5-m ethyl-2,4-di oxo-3, 4-dihydropyrimidin-l(2H)- yl)acetyl)glycinate (7) in a RB flask was added 4 mL of THF. Once dissolved, 4 mL of TXE Buffer (prepared as described below) was added and this mixture was allowed to cool in a salt/ice bath for 30 minutes. Then acetic acid (1 mL), ice cold deionized water (1 mL), ice cold saturated KH2PO4 (1 mL), and 2.06 mmol (0.135 g) zinc dust were each added three times, in the same amount at 20 minute intervals. After 70 minutes from the first addition of zinc dust, the mixture was then filtered through a bed of Celite®, and the Celite® plug was washed with a mixture 4/1 (v/v) THF/deionized water (with 2-3 drops of added acetic acid). All filtrates were combined and evaporated under reduced pressure without heat to form a slurry. The ice-cold flask was removed from the evaporator and its contents were partitioned in 25 mL of EtOAc and 25 mL of Extraction Buffer (prepared as described below). The EtOAc layer was washed once with Extraction Buffer and once with brine. The EtOAc layer was then dried over granular anhydrous magnesium sulfate, filtered and evaporated to provide 0.308 g of crude 8. The crude 8 was then crystallized from ACN and the product collected by vacuum filtration to yield 0.215g (0.41 mmol; 66%) of 8 as crystals, with an enantiomeric excess (ee) of >99.9% as determined using the procedure described in Example 39, below.

[00356] Note(s): 1) TXE Buffer was prepared 1-3 days before being used in the reaction by adding 25 mmol (3.4 g) of KH2PO4, 12.5 mmol (4.99 g) ethylenediaminetetraacetic acid zinc disodium salt hydrate and 12.5 mmol ethylenediaminetetraacetic acid into a mixture of 100 mL of deionized water and 50 mL of glacial acetic acid. This mixture was allowed to stir overnight and then 50 mL of THF was added. After stirring an additional 30 minutes, the white solid was removed by filtration and the filtrate was used as TXE Buffer. 2) Extraction Buffer was prepared by dissolving 1.0 g of KH2PO4, and 0.5 g of KHSO4 (as potassium bisulfate) per 10 mL of water (prepared in proportion to the total volume of Extraction Buffer required for any particular reaction).

Example 8: Synthesis (9H-fluoren-9-yl)methyl (4S)-4-((2-(2-(tert- butoxy)ethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2-oxide (10)

[00357] In a three-necked RB flask equipped with a mechanical stirrer, addition funnel and N2 inlet was added 44.25 g (650 mmol) of imidazole, followed by 150 mL of anhydrous THF, and the mixture was stirred under N2 atmosphere until all imidazole had dissolved. The resulting solution was then cooled for 15 minutes in a salt/ice bath. Thionyl chloride (11.6 mL, 160 mmol) was then added dropwise over 5 minutes with stirring during which a solid formed. The mixture was then stirred a further 10 minutes following the addition, then a solution of (9H- fluoren-9-yl)methyl (R)-( 1 -(2-(2-(tert-butoxy)ethoxy)ethoxy)-3 -hydroxypropan-2-yl)carbamate (9) (22.86 g, 50 mmol) in 100 mL of anhydrous THF was added dropwise over 170 minutes. The mixture was stirred until consumption of compound 9, and then filtered and the solid obtained was washed with THF. The filtrate and washings were combined and concentrated, and the resulting oil was partitioned between in 200 mL of EtOAc and 80 mL of 0.5N aqueous HC1. The HC1 layer was discarded and the EtOAc layer was extracted once more with 0.5N aqueous HC1, once with saturated sodium bicarbonate, once with 5% (w/v) aqueous sodium bicarbonate, and once with brine. The EtOAc layer was then dried over granular anhydrous magnesium sulfate, filtered and concentrated to obtain 23g (45.7 mmol; 91%) of crude 10 as an oil. The oil was dissolved in about 20 mL of 25% (v/v) EtOAc in hexanes and purified by flash chromatography on silica gel using Teledyne ISCO CombiFlash Rf+ automated chromatography system and an ascending gradient of EtOAc in hexanes. Product fractions were pooled and concentrated to yield 15.81 g (31.4 mmol; 63% yield) of compound 10 as an oil.

[00358] Note: (9ELfluoren-9-yl)methyl (R)-(l-(2-(2-(tert-butoxy)ethoxy)ethoxy)-3- hydroxypropan-2-yl)carbamate (9) was obtained in high purity from a custom manufacturer.

Example 9: Synthesis of (9H-fluoren-9-yl)methyl (S)-4-((2-(2-(tert- butoxy)ethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (11)

[00359] Compound 10 obtained in Example 8 (15.81 g, 31.4 mmol) was dissolved in 60 mL of DCM and to this solution was added 60 mL of water. The bi-phasic mixture was stirred for 5 minutes then cooled in an ice bath for 15 minutes. NaICU (17.46 g, 81.6 mmol) was then added followed by RuCL xFLO (0.204 g, 0.98 mmol). The resulting black mixture was stirred for 15 minutes and then warmed to room temperature and stirred for two hours. An additional 0.02 g of RuChxEhO was then added and the mixture was stirred for an additional 3.75 hours, then filtered through Celite®. 150 mL DCM and 150 mL of brine were then added to the filtrate, and agitated, and the resulting emulsion was filtered. The filtrate was then separated, and the aqueous layer was further extracted with 100 mL of DCM. The combined DCM extracts were then extracted with 200 mL of brine, and then dried over granular anhydrous magnesium sulfate, filtered, and concentrated to afford an oil. The oil was then dissolved in minimal amount of 25% by volume EtOAc in hexanes and spun down in a centrifuge to remove residual catalyst. The supernatant was then carefully decanted and loaded onto a silica gel column and purified by flash chromatography using an isocratic elution of 25% EtOAc in hexanes. Product fractions were combined and concentrated to provide 8.16g (15.7 mmol, 50%) of 11 as an oil. Example 10: Synthesis of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2- (tert-butoxy)ethoxy)ethoxy)-N-(2-(2-iodoethoxy)-2-oxoethyl)propan-l-aminium 4- methylbenzenesulfonate (12a)

[00360] (9H-fluoren-9-yl)methyl (S)-4-((2-(2-(tert-butoxy)ethoxy)ethoxy)methyl)-l ,2,3- oxathiazolidine-3 -carboxylate 2,2-dioxide (11) (2.86 g, 5.5 mmol) and 2-(2-iodoethoxy)-2- oxoethan-l-aminium 2,2,2-trifluoroacetate (102) (5.66 g, 16.5 mmol) were combined in a flask. This mixture was co-evaporated with 50 mL of anhydrous THF to remove water, and concentrated to a solid, then purged with nitrogen. Anhydrous THF (33 mL) was then added and the mixture was stirred for 10 minutes at room temperature, then cooled in an ice bath for 10 minutes. 2.1 mL of anhydrous NMM was then added and the mixture was stirred for 5 minutes, and then warmed to room temperature. After 1.5 hours the flask was placed back in the ice bath. Aqueous 2N hydrochloric acid (approx. 7.5 mL) was then added dropwise until the pH of the mixture was about 3. The mixture was then partitioned between DCM and water and the water layer was discarded. The DCM layer was then extracted with 5% aqueous sodium bicarbonate, then brine. The DCM solution was then dried over granular anhydrous magnesium sulfate, and filtered. p-TSA (530 mg, 2.8 mmol) was then added to the filtrate to lower the pH of the solution to about 3. The DCM solution was then concentrated and re-dissolved in DCM, purified by silica gel flash chromatography using a Teledyne ISCO CombiFlash Rf+ automated chromatography system and an ascending gradient of methanol into DCM. Pooling of the product fractions and concentration gave 1.62g (1.9 mmol, 35%) of 12a as a foam.

[00361] Note: 2-(2-iodoethoxy)-2-oxoethan-l-aminium 2,2,2-trifluoroacetate (102) was prepared in house as described in WO/2018/175927, Example 3.

Example 11: Synthesis of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2- (tert-butoxy)ethoxy)ethoxy)-N-(2-oxo-2-(2,2,2-tribromoethoxy)ethyl)propan-l-aminium 4- methylbenzenesulfonate (12b)

[00362] (9H-fluoren-9-yl)methyl (S)-4-((2-(2-(tert-butoxy)ethoxy)ethoxy)methyl)-l ,2,3- oxathiazolidine-3 -carboxylate 2,2-dioxide (11) (1.91 mmol, 1.0g) was dissolved in 30 mL of dry THF in a flask fitted with stir bar and N2 inlet. The flask was cooled to 0°C and then 2.61g (5.8 mmol) of 2-oxo-2-(2,2,2-tribromoethoxy)ethan-l-aminium 2,2,2-trifluoroacetate (103) was added, followed by 0.737 mL (6.7 mmol) of NMM. The mixture was then stirred for one hour at room temperature until complete consumption of 11. Acetic acid was then added dropwise until the pH of the mixture was 5-6. The mixture was then concentrated and dissolved in 40 mL of EtOAc. 50 mL of IM monosodium phosphate (NaHzPCL) was then added and stirred rapidly for 1 hour. The EtOAc layer was then separated, and extracted sequentially with aqueous citric acid (pH 2), water, two amounts of 5% (w/v) aqueous sodium bicarbonate, and then brine. The EtOAc mixture was then dried over granular anhydrous magnesium sulfate, filtered and concentrated to about 30 mL. p-TSA (0.27g, 1.43 mmol) was then added, cooled, and filtered, and the filtrate was concentrated to an oil which was dissolved in 2% (v/v) methanol in DCM and purified by silica gel flash chromatography using a Teledyne ISCO CombiFlash Rf+ automated chromatography system and an ascending gradient of 2-10% methanol into DCM. Product fractions were pooled and concentrated to provide 0.95 g (1 mmol; 53%) of compound 12b

[00363] Note: 2-oxo-2-(2,2,2-tribromoethoxy)ethan-l-aminium 2,2,2-trifluoroacetate (103) was prepared in house as described in WO/2018/175927, Example 3.

Example 12: Synthesis of 2,2,2-tribromoethyl (R)-ll-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-2,2-dimethyl-13-(2-(5-methyl-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)acetyl)-3,6,9-trioxa-13-azapentadecan-15-oate (13)

[00364] 2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)acetic acid (101) (0.205, 1.1 mmol) was dissolved in 6 mL anhydrous ACN at 0°C under N2. 152 pL (1.37 mmol) of TMAC was then added followed by 0.5 mL (4.5 mmol) of anhydrous NMM. The mixture was stirred for about 20 min at 0°C. After the mixed anhydride had completely formed, 0.956 g (1 mmol) of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-N- (2-oxo-2-(2,2,2-tribromoethoxy)ethyl)propan-l-aminium 4-m ethylbenzenesulfonate (12b) dissolved in 4 mL of anhydrous ACN was added. After 10 min the mixture was warmed to room temperature and stirred for 30 min. The mixture was diluted with EtOAc and then extracted twice with water, twice with 5% (w/v) aqueous sodium bicarbonate, twice with 5% (w/v) aqueous citric acid, once with water, and then once with brine. The EtOAc layer was then dried over granular anhydrous magnesium sulfate, filtered and concentrated to provide a foam 0.70 g (0.74 mmol). This material was then dissolved in DCM and purified by silica gel flash chromatography using a Teledyne ISCO CombiFlash Rf+ automated chromatography system and an ascending gradient of 0-8% methanol into DCM over 8 CV. The product fractions were pooled and concentrated to provide 0.555 g (0.59 mmol; 59%) of compound 13 as a foam.

Example 13: Synthesis of (R)-ll-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2- dimethyl-13-(2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)acetyl)-3,6,9-trioxa-13- azapentadecan-15-oic acid (14)

[00365] 2,2,2-tribromoethyl (R)-l l-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2- dimethyl- 13 -(2-(5-methyl-2,4-dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)acetyl)-3 ,6,9-trioxa- 13- azapentadecan-15-oate (13) (0.516 g, 0.55 mmol) was dissolved in 5 mL of THF, then 5 mL of TXE Buffer (prepared as described above) was added. The biphasic mixture was then cooled to 0°C with vigorous stirring, and 0.82 mL of acetic acid, 0.82 mL deionized water, 0.82 mL saturated aqueous KH2PO4, and 119 mg (1.8 mmol) of zinc powder were added a total of three times in the same amounts at 20 minute intervals. After 1 hour the mixture was warmed to room temperature. The mixture was then filtered through Celite® and the filtrate and washings concentrated under reduced pressure without applying heat. The resulting slurry was then suspended in about 50 mL of EtOAc and the organic solution extracted three times with Extraction Buffer (~50 mL each time) and once with brine. The resulting solution was dried over granular anhydrous magnesium sulfate, filtered, and concentrated to obtained 0.370 g of crude 14, which was dissolved in 2% methanol/DCM (v/v) and purified by silica gel flash chromatography using an ascending gradient of 2-15% methanol in DCM over 13 CV. Product fractions were pooled and concentrated to give 0.200 g (0.3 mmol; 53%) of 14 as a foam, with an ee of 99.5% as determined using the procedure described in Example 38, below.

Example 14: Synthesis of 2-iodoethyl (R)-ll-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-13-(2-(2,6-bis(bis(tert-butoxycarbonyl)amino)-9H-purin-9- yl)acetyl)-2,2-dimethyl-3,6,9-trioxa-13-azapentadecan-15-oate (15)

[00366] 2.3 mmol (1.4 g) of 2-(2,6-bis(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetic acid (108), 1.92 mmol (1.62 g) of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2- (tert-butoxy)ethoxy)ethoxy)-N-(2-(2-iodoethoxy)-2-oxoethyl)propan- 1 -aminium 4- methylbenzenesulfonate (12a), 2.32 mmol (0.882 g) of l-[Bis(dimethylamino)methylene]-l/Z- l,2,3-triazolo[4,5-Z>]pyridinium 3-oxid hexafluorophosphate (HATU), and 7 mL of dry N,N’- dimethylformamide (DMF) were added to an oven dried RB flask. 4.6 mmol (0.795 mL) of DIEA was then added dropwise under nitrogen. After about 1 hour of stirring at room temperature, the solution was evaporated under reduced pressure, and the residue partitioned with 30 mL of EtOAc and 30 mL of deionized water, and separated. The EtOAc layer was then washed twice with ’A saturated KH2PO4, twice with 5% (w/v) aqueous NaHCOs and once with brine. The EtOAc layer was then dried over granular magnesium sulfate, filtered and concentrated to yield crude 15, which was then dissolved in a minimum of 1/1 EtOAc/hexanes and purified by injection on a 40g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 50-100% EtOAc in hexanes gradient over 10 CV with a hold at 100% EtOAc for 6 CV after first holding at 50% EtOAc for 1.0 CV immediately after injection. Pooled fractions provided 1.78 g (1.4 mmol; 73%) of 15 as a foam.

- I l l - Example 15: Synthesis of (R)-ll-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-13-(2-(2,6- bis(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetyl)-2,2-dimethyl-3,6,9-trioxa-13- azapentadecan-15-oic acid (16)

[00367] 2-iodoethyl (R)-l l-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-13-(2-(2,6- bis(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetyl)-2,2-dimethyl-3,6,9-trioxa-13- azapentadecan-15-oate (15) (1.78 g, 1.4 mmol) was dissolved in 10 mL of THF, and 10 mL of TXE Buffer (prepared as described above) was then added. The biphasic mixture was cooled to 0°C with vigorous stirring, and 1.5 mL of acetic acid, 1.5 mL water, 1.5 mL saturated aqueous KH2PO4, and 0.305 g (4.7 mmol) of zinc powder were added sequentially in the same amounts for a total of three times, at 20 minute intervals. Twenty minutes after the final addition the mixture was concentrated under reduced pressure without applying heat, and the resulting slurry was dissolved in about 50 mL of EtOAc, and the organic solution was extracted twice with Extraction Buffer (about 50 mL each time) and once with brine. The resulting solution was dried over granular anhydrous magnesium sulfate, filtered, and concentrated to provide 1.61g (1.46 mmol) of 16 as a foam. This material was dissolved in 2% methanol/DCM, (v/v), and purified by flash chromatography using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a gradient of 2-6% MeOH in DCM over 10 CV. Product fractions were pooled and concentrated to give a foam that was then re-dissolved in DCM and precipitated from a mixture of diethyl ether/hexanes. The precipitate was then collected by filtration and dried under vacuum to provide 1.0 g (0.9 mmol, 64%) of 16 as a powder.

Example 16: Synthesis of (9H-fluoren-9-yl)methyl (R)-(l-azido-3-(2-(2-(tert- butoxy)ethoxy)ethoxy)propan-2-yl)carbamate (17)

[00368] Sodium azide (0.41 g, 6.3 mmol) was dissolved in 16.5 mL of methanol, and then combined with a solution of 2.7g (5.6 mmol) of (9H-fluoren-9-yl)methyl (S)-4-((2-(2-(tert- butoxy)ethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (11) in 6.5 mL of methanol. The resulting mixture was stirred for 90 minutes, then partitioned between about 50 mL of EtOAc and 50 mL of brine. The EtOAc layer was separated and further washed with saturated aqueous KH2PO4, then dried over granular anhydrous magnesium sulfate, filtered, and concentrated to obtain 2.0 g (4.1 mmol, 74%) of crude 17 as an oil, which was used without further purification.

Example 17: Synthesis of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2- (tert-butoxy)ethoxy)ethoxy)propan-l-aminium chloride (18)

[00369] To (9H-fluoren-9-yl)methyl (R)-(l-azido-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)propan-2- yl)carbamate (17) (3.89 g, 8 mmol) was added ammonium chloride (1.0 g, 18.7 mmol), ethanol (22 mL) and deionized water (7 mL). After stirring briefly at room temperature, zinc powder (0.703 g, 10.8 mmol) was added, and the mixture was cooled in an ice bath. After one hour, the mixture was warmed to room temperature and 0.40 g (6.1 mmol) of zinc powder was added. After 2 hours additional zinc powder was added in two 0.2 g portions at 10 minute intervals. The mixture was then filtered through Celite®, and the Celite® was washed with methanol and the filtrate was concentrated. EtOAc was then added resulting in two layers, and the EtOAc layer was separated and washed with a dilute solution of aqueous disodium EDTA. The combined aqueous fractions were then extracted three times with small portions of EtOAc. Saturated aqueous sodium chloride solution was then added to the water/EDTA mixture which was again extracted with portions of EtOAc. The combined EtOAc extracts were then dried over granular anhydrous magnesium sulfate, filtered, and concentrated to provide 3.02 g (77% yield) of 18. Example 18: Synthesis of (9H-fluoren-9-yl)methyl (4S)-4-((2-(2- methoxyethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2-oxide (20)

[00370] To 330 mmol (22.46 g) of imidazole was added 200 mL of dry THF in an oven dried IL, three-neck round-bottom (RB) flask equipped with a mechanical stirrer. The solution was stirred under a nitrogen blanket while cooling in a salt/ice bath for about 20 minutes. 80 mmol (5.8 mL) of thionyl chloride was then while stirring briskly, and the resulting slurry was stirred for about 15 minutes. A solution of 25 mmol (10.39 g) of (9H-fluoren-9-yl)methyl (R)-(l- hydroxy-3-(2-(2-methoxyethoxy)ethoxy)propan-2-yl)carbamate (19) in about 75 mL of dry THF was then added to the mixture dropwise over about 120 minutes while keeping the RB flask in the salt/ice bath. About 10-15 minutes after the addition was complete, the mixture was filtered, and evaporated under reduced pressure to provide an oil, which was partitioned in 200 mL of EtOAc and about 80 mL of IN HC1. The layers were separated and the EtOAc layer was washed once with IN HC1, once with saturated aqueous NaHCCL, once with 5% (w/v) aqueous NaHCCL and once with brine. The EtOAc layer was then dried over granular magnesium sulfate, filtered and evaporated to yield 11.81g (25.6mmol; 102%) of 20 as an oil. This material could be used without further purification.

Notes: (9H-fluoren-9-yl)methyl (R)-(l -hydroxy-3 -(2-(2-methoxyethoxy)ethoxy)propan-2- yl)carbamate (19) was obtained in high purity from a custom manufacturer.

Example 19: Synthesis of (9H-fluoren-9-yl)methyl (S)-4-((2-(2- methoxyethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (21)

1 [00371] To 16 mmol (7.39 g) of (9H-fluoren-9-yl)methyl (4S)-4-((2-(2- m ethoxy ethoxy)ethoxy)methyl)- 1,2, 3 -oxathiazolidine-3 -carboxylate 2-oxide (20) in a 300 mL RB flask was added 35 mL of DCM and 35 mL of deionized water. The mixture was stirred to at room temperature until the solids dissolved and then the flask was cooled in ice bath for about 15 minutes. 41.6 mmol (8.90 g) of NaKL was then added followed by 0.5 mmol (103 mg) of RuCL XH2O, and the mixture was stirred for 5-10 minutes in the ice bath, then warmed to room temperature and stirred for 5 hours. 20 mg of RuCL xF O was then added, and after an additional 2 hours of stirring the mixture was filtered through Celite®. 100 mL of DCM and 100 mL of /i saturated NaCl were then added to the filtrate, and the DCM layer was separated. The aqueous layer was extracted once with 20 mL of DCM, and the combined DCM layers were then washed once with /i saturated NaCl and once with brine. The DCM layer was then dried over granular anhydrous magnesium sulfate, filtered and evaporated to provide crude 21 as an oil, which was dissolved in about 30 mL of 35/65 (v/v) EtOAc/hexanes, and then filtered to remove solids. The filtrate was then injected onto an 80g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running EtOAc/hexanes as the solvents. At injection, the system was running 35/65 (v/v) EtOAc/hexanes for 1.5 column volumes (CV) and then a gradient from 35% EtOAc to 55% EtOAc over 3 CV. The elution solvent was then held at 55% EtOAc until the product eluted. Fractions were pooled and evaporated to yield 5.48g (11.5 mmol, 72%) of 21 as an oil.

Note: LCMS analysis was performed using a Shimadzu LCMS 2020 running 0.025% formic acid in water (Buffer A) and 0.25% formic acid in ACN (Buffer B) through a 4.6mm x 150 mm, 5 pm Atlantis C18 reverse phase column. Analysis was based on the trace at 260nm.

Example 20: Synthesis of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(2-(2- iodoethoxy)-2-oxoethyl)-3-(2-(2-methoxyethoxy)ethoxy)propan-l-aminium 4- methylbenzenesulfonate (22)

[00372] To 11.5 mmol (5.48 g) of (9H-fhioren-9-yl)methyl (S)-4-((2-(2- methoxyethoxy)ethoxy)methyl)- 1,2, 3 -oxathiazolidine-3 -carboxylate 2,2-dioxide (21) in a 300 mL RB flask was added 34.5 mmol (11.83 g) of 2-(2-iodoethoxy)-2-oxoethan-l-aminium 2,2,2- trifluoroacetate (102) and about 50 mL of dry ACN. The flask was shaken to form a slurry and then the solvent was evaporated under reduced pressure to give a solid. The solid was then dissolved in about 50 mL of dry THF under nitrogen, and then cooled in an ice bath for about 20 minutes. 3 mL of NMM was then added dropwise to the mixture, and stirred for 10-15 minutes. An additional 0.64 mL of NMM was then added, and the mixture was warmed to room temperature. After 1.5 hours of stirring, 2.6 mL of acetic acid was added to lower the pH to 4-5. The solvent was then removed under reduced pressure, and the residue was partitioned between 100 mL EtOAc and 100 mL of IN HC1. This mixture was stirred for 2 hours at room temperature, and then the EtOAc layer was separated, and the aqueous layer was extracted once with EtOAc. The combined EtOAc layers were then washed once with 100 mL of IN HC1, once with a mixture of 50 mL saturated Na2CO3 and 50 mL of saturated NaHCOs, once with 5% (w/v) aqueous NaHCOs, and then once with brine. The EtOAc layer was dried over granular anhydrous magnesium sulfate and filtered. 10 mmol (1.9 g) of p-TSA was added to the filtrate, which was then evaporated to yield 8.97 g (11.2 mmol, 98%) of crude 22 as an oil. Crude 22 was dissolved in a minimum of DCM and purified by injection on a 80g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 0-6% methanol in DCM gradient over 15 C V with a hold at 6% methanol for 5 CV after first holding at 0% methanol for 1.5 CV immediately after injection. Pooled fractions provided 4.91 g (6.1 mmol; 53%) of 22 as an oil.

Example 21: Synthesis of 2-iodoethyl (R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)- yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oate (23)

[00373] To 3.9 mmol (0.718 g) of 2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)acetic acid (101) in a RB flask was added about 50 mL of dry ACN, and the mixture was stirred under nitrogen and cooled in an ice bath for about 10 minutes. 4.1 mmol (0.505 mL) of TMAC was then added, followed by 18 mmol (2.0 mL) of NMM, and the mixture was stirred for about 20 min at 0°C. When near complete mixed anhydride formation was confirmed, 3.4 mmol (2.7 g) of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(2-(2-iodoethoxy)-2-oxoethyl)-3-(2- (2-methoxyethoxy)ethoxy)propan-l-aminium 4-m ethylbenzenesulfonate (22) in about 20 mL of dry ACN was added by cannula. The mixture was then warmed to room temperature and stirred for 2 hours. The solvent was then evaporated under reduced pressure and the residue was partitioned with 35 mL of EtOAc and 35 mL of deionized water. The EtOAc layer was separated and washed twice with ’A saturated KH2PO4, twice with 5% (w/v) aqueous NaHCOs and once with brine. The EtOAc layer was then dried over granular magnesium sulfate, filtered and evaporated to yield 2.5 g (3.15 mmol; 93%) of crude 23, which was dissolved in a minimum of DCM and purified by injection on a 40g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 0-5% methanol in DCM gradient over 12 CV with a hold at 5% methanol for 5 CV after first holding at 0% methanol for 1.5 CV immediately after injection. Pooled fractions provided 1.98 g (2.5 mmol; 74%) of 23 as a foam.

Example 22: Synthesis of (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(5- methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14- oic acid (24)

[00374] To 2.5 mmol (1.98 g) of 2-iodoethyl (R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(5-methyl-2, 4-di oxo-3, 4-dihy dropyrimidin-1 (2H)-yl)acetyl)- 2,5,8-trioxa-12-azatetradecan-14-oate (23) in a RB flask was added 15 mL of THF. Once solids were dissolved, 15 mL of TXE Buffer (prepared as described above) was added, and the mixture was cooled in a salt/ice bath for 20-30 minutes. Acetic acid (2.5 mL), ice cold deionized water (2.5 mL), ice cold saturated KH2PO4 (2.5 mL), and 8.33 mmol (0.55 g) of zinc dust were then each added twice, in the same amounts at 20 minute intervals. 20 minutes after the second addition, further acetic acid (1.5 mL), ice cold deionized water (1.5 mL), ice cold saturated KH2PO4, and 8.33 mmol (0.55 g) of zinc dust were added. After 80 minutes from the first addition of zinc dust, the mixture filtered through a bed of Celite®, and the Celite® plug was washed with a mixture o f4/l (v/v) THF/deionized water (with 2-3 drops of added acetic acid). Combined filtrates were evaporated under reduced pressure without added heat, and the slurry was then partitioned in EtOAc and a mixture of about 1/1 (v/v) deionized water and Extraction Buffer (prepared as described above). The EtOAc layer was then separated and washed twice with 40 mL of Extraction Buffer and once with brine. The EtOAc layer was then dried over granular magnesium sulfate, filtered and evaporated to provide crude 24 as an oil, which was dissolved in a minimum amount of 2% MeOH in DCM and purified by injection on a 24g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 2-8% methanol in DCM gradient after first holding at 2% methanol for 1.5 CV immediately after injection. Pooled fractions provided 24 as a foam, which was dissolved in a minimum amount of DCM and precipitated from 80 mL hexanes and 20 mL of diethyl ether. The precipitate was collected by vacuum filtration to provide 1.43 g (2.2 mmol; 89%) of 24 as a solid. The identity and purity (>99.7%) of the product was confirmed with LCMS (at 260nm absorbance) and 'H-NMR. Minor impurities were residual hexane.

Example 23: Synthesis of 2-iodoethyl (10R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(4-((tert-butoxycarbonyl)amino)-2-oxo-3,4- dihydropyrimidin-l(2H)-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oate (25)

[00375] To 6.9 mmol (1.86 g) of 2-(4-((tert-butoxycarbonyl)amino)-2-oxopyrimidin-l(2H)- yl)acetic acid (104) was added 60-70 mL of dry ACN, and the solution was stirred under nitrogen and then cooled in an ice bath for about 20 minutes. 30 mmol (3.3 mL) of NMM was then added followed immediately by 7.4 mmol (0.911 mL) of TMAC. The mixture was stirred for about 20 min at 0°C. When near complete mixed anhydride formation was confirmed, 5.8 mmol (4.66 g) of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(2-(2-iodoethoxy)-2- oxoethyl)-3-(2-(2-methoxyethoxy)ethoxy)propan-l-aminium 4-methylbenzenesulfonate (22) in about 20-25 mL of dry ACN and was added to the mixture by cannula. After 30 minutes, the mixture was warmed to room temperature and stirred for 1.5 hours. The solvent was then evaporated under reduced pressure and the residue was partitioned with EtOAc and deionized water. The EtOAc layer was then separated and washed twice with ’A saturated KH2PO4, twice with 5% (w/v) aqueous NaHCOs and once with brine. The EtOAc layer was then dried over granular magnesium sulfate, filtered and evaporated to yield 4.66 g (5.3 mmol; 91%) of crude 25, which was dissolved in a minimum of DCM and purified by injection on a 80g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 0-5% methanol in DCM gradient over 15 CV with a hold at 5% methanol for 5 CV after first holding at 0% methanol for 1.5 CV immediately after injection. Pooled fractions provided 3.58 g (4.1 mmol; 70%) of 25 as a foam.

Example 24: Synthesis of (10R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(4- ((tert-butoxycarbonyl)amino)-2-oxo-3,4-dihydropyrimidin-l(2H)-yl)acetyl)-2,5,8-trioxa-12- azatetradecan- 14-oic acid (26)

[00376] To 4.1 mmol (3.58 g) of 2-iodoethyl (10R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(4-((tert-butoxycarbonyl)amino)-2-oxo-3,4- dihydropyrimidin-l(2H)-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oate (25) was added 20 mL of THF. Once solids were dissolved, 20 mL of TXE Buffer (prepared as described above) was added and the mixture was cooled in a salt/ice bath for 20-30 minutes. Acetic acid (6 mL), ice cold deionized water (6 mL), ice cold saturated KH2PO4 (6 mL), and 13.67 mmol (0.894 g) zinc dust were then added. After 20 minutes, acetic acid (4 mL), deionized water (4 mL), ice cold saturated KH2PO4 (4 mL), and 13.67 mmol (0.894 g) zinc dust were each added twice, in the same amount at 20 minute intervals. After 120 minutes from the first addition of zinc dust, the mixture was filtered through a bed of Celite®, and the Celite® plug was washed with a mixture 4/1 (v/v) THF/deionized water (with 2-3 drops of added acetic acid). Combined filtrates were evaporated under reduced pressure with no added heat, and the resulting slurry was partitioned with about 75 mL of EtOAc and a mixture of about 1/1 (v/v) deionized water and Extraction Buffer (prepared as described above). The EtOAc layer was then separated and washed thrice with Extraction Buffer and once with brine. The EtOAc layer was then dried over granular magnesium sulfate, filtered and evaporated to provide 2.89 g of crude 26 (4 mmol; 97%) as a foam, which was dissolved in a minimum of a solution of 2% MeOH in DCM and purified by injection on a 40g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 2-10% methanol in DCM gradient over 15 CV after first holding at 2% methanol for 1.5 CV immediately after injection. Pooled fractions provided 26 as a foam, which was dissolved in a minimum of DCM and precipitated from 110 mL hexanes and 15 mL of diethyl ether. The precipitate was collected by vacuum filtration to provide 2.22 g (3.1 mmol; 75%) of 26 as a solid, with 99.4% purity as confirmed by 'H-NMR and LCMS.

Example 25: Synthesis of 2-iodoethyl (R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(6-((tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetyl)- 2,5,8-trioxa-12-azatetradecan-14-oate (27)

[00377] To 3.6 mmol (1.06 g) of 2-(6-((tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetic acid (105) was added 25 mL of dry DMF, and after mixing the solution the solvent was evaporated under reduced pressure. 25 mL of dry DMF and 4 mmol (0.44 mL) of NMM were then added, and then the solvent was again evaporated under reduced pressure. 30 mL of dry DMF and 12 mmol (1.33 mL) of NMM were then added under nitrogen, and the mixture was cooled in an ice bath for about 20 minutes. 3.7 mmol (1.41 g) of HATU was added then added, and the mixture was warmed to room temperature. An additional 0.4 mmol of HATU was then added and the mixture was stirred for 20 minutes. 3 mmol (2.45 g) of (R)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-N-(2-(2-iodoethoxy)-2-oxoethyl)-3-(2-(2- methoxyethoxy)ethoxy)propan-l-aminium 4-m ethylbenzenesulfonate (22) in about 20-25 mL of dry ACN was then added to the mixture via a cannula, and the mixture was warmed to room temperature and stirred for 2 hours. The solvent was then evaporated under reduced pressure and the residue was partitioned with EtOAc and deionized water. Saturated NaHCCL and brine were added several times to separate the emulsion, and then the EtOAc layer was separated and dried over granular magnesium sulfate, filtered and evaporated to yield 2.35 g of crude 27 as an oil. Crude 27 was then dissolved in a minimum of DCM and purified by injection on a 40g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 0-4% methanol in DCM gradient over 12 CV with a hold at 4% methanol for 5 CV after first holding at 0% methanol for 1.5 CV immediately after injection. Pooled fractions provided 0.91 g (1.0 mmol; 34%) of 27. Example 26: Synthesis of (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(6- ((tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oic acid (28)

[00378] To 1 mmol (0.91 g) of 2-iodoethyl (R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(6-((tert-butoxycarbonyl)amino)-9H-purin-9-yl)acetyl)- 2,5,8-trioxa-12-azatetradecan-14-oate (27) was added 7 mL of THF. Once solids were dissolved, 7 mL of TXE Buffer (prepared as described above) was added and the mixture was cooled in a salt/ice bath for about 15 minutes. Acetic acid (1 mL), ice cold deionized water (1 mL), ice cold saturated KH2PO4 (1 mL), and 3.33 mmol (0.218 g) zinc dust were then added. After 20 minutes, further acetic acid (0.75 mL), deionized water (0.75 mL), and ice cold saturated KH2PO4 (0.75 mL), and 3.33 mmol (0.218 g) zinc dust were each added twice, in the same amount at 20 minute intervals. After 90 minutes from the first addition of zinc dust, the mixture was filtered through a bed of Celite®, and the Celite® plug was washed with a mixture of 4/1 (v/v) THF/deionized water (with 2-3 drops of added acetic acid). Combined filtrates were then evaporated under reduced pressure without heat, and the resulting slurry was partitioned with EtOAc and a mixture of about 1/1 (v/v) deionized water and Extraction Buffer (prepared as described above). The EtOAc layer was separated and washed thrice with Extraction Buffer and once with brine, then dried over granular magnesium sulfate, filtered and evaporated to provide 1.053 g of crude 28 as an oil. Crude 28 was then dissolved in a minimum of a solution of 2% MeOH in DCM and purified by injection on a 12g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 2-8% methanol in DCM gradient over 12 CV after first holding at 2% methanol for 1.5 CV immediately after injection. Pooled fractions provided 28 as a foam, that dissolved in a minimum of DCM and precipitated from 50 mL hexanes and 10 mL of diethyl ether. The precipitate was collected by vacuum filtration to provide 0.453 g (0.6 mmol; 60%) of 28 as a solid, with about 98.8% purity as confirmed by LCMS and ¹ H-NMR.

Example 27: Synthesis of 2-iodoethyl (R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(2-((tert-butoxycarbonyl)amino)-6-oxo-l,6-dihydro-9H- purin-9-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oate (29)

[00379] To 3.6 mmol (1.06 g) of 2-(2-((tert-butoxycarbonyl)amino)-6-oxo-l,6-dihydro-9H- purin-9-yl)acetic acid (106) was added 30 mL of dry DMF. The mixture was stirred and then the solvent was evaporated under reduced pressure. 30 mL of dry DMF and 4 mmol (0.44 mL) of NMM were added to the residue, which was again stirred and evaporated under reduced pressure. 30 mL of dry DMF, 12 mmol (1.33 mL) of NMM were added to the residue, followed by 3.7 mmol (1.41 g) of HATU, and the mixture was cooled in an ice bath. After 20 minutes, 3 mmol (2.43 g) of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(2-(2-iodoethoxy)-2- oxoethyl)-3-(2-(2-methoxyethoxy)ethoxy)propan-l-aminium 4-methylbenzenesulfonate (22) in about 25 mL of dry ACN was transferred to the mixture via a cannula. After 5 minutes the mixture was warmed to room temperature and stirred for 2 hours. The solvent was then evaporated under reduced pressure and the residue was partitioned with 50 mL of EtOAc and 25 mL of deionized water. The EtOAc layer was separated and then washed twice with ’A saturated KH2PO4, twice with 5% (w/v) aqueous NaHCOs and once with brine, then dried over granular magnesium sulfate, filtered and evaporated to yield 2.51 g (2.7 mmol; 91%) of crude 29 as a solid. Crude 29 was then dissolved in a minimum of DCM and purified by injection on a 40g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 0-5% methanol in DCM gradient over 12 CV after first holding at 0% methanol for 1.5 CV immediately after injection, and increasing to 6% MeOH/DCM. Pooled fractions provided 1.72 g (1.9 mmol; 63%) of 29 as a foam.

Example 28: Synthesis of (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(2- ((tert-butoxycarbonyl)amino)-6-oxo-l,6-dihydro-9H-purin-9-yl)acetyl)-2,5,8-trioxa-12- azatetradecan- 14-oic acid (30)

[00380] To 1.87 mmol (1.72 g) of 2-iodoethyl (R)-10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-12-(2-(2-((tert-butoxycarbonyl)amino)-6-oxo-l,6-dihydro-9H- purin-9-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oate (29) was added 10 mL of THF. Once solids were dissolved, 10 mL of TXE Buffer (prepared as described above) was added, and the mixture was cooled in a salt/ice bath for about 20 minutes. Acetic acid (2 mL), ice cold deionized water (2 mL), ice cold saturated KH2PO4 (2 mL), and 6.23 mmol (0.408 g) zinc dust were then added. After 20 minutes, further acetic acid (1.5 mL), deionized water (1.5 mL), ice cold saturated KH2PO4 (1.5 mL), and 6.23 mmol (0.408 g) zinc dust were added. After 20 minutes, acetic acid (1 mL), deionized water (1 mL), ice cold saturated KH2PO4 (1 mL), and 6.23 mmol (0.408 g) zinc dust were added. After 90 minutes from the first addition of zinc dust, the mixture was filtered through a bed of Celite®, and the Celite® plug was washed with a mixture 4/1 (v/v) THF/deionized water (with 2-3 drops of added acetic acid). Combined filtrates were evaporated under reduced pressure without adding heat, and the resulting slurry was partitioned with EtOAc and a mixture of about 1/1 (v/v) deionized water and Extraction Buffer (prepared as described above). The EtOAc layer was separated and washed twice with Extraction Buffer and twice with brine, then dried over granular anhydrous magnesium sulfate, filtered and evaporated to provide 1.5 g of crude 30 as a foam. Crude 30 was then dissolved in a minimum of a solution of 2% MeOH in DCM and purified by injection on a 24g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 2-6% methanol in DCM gradient over 12 CV with a hold at 6% MeOH/DCM for 6 CV, after first holding at 2% methanol for 1.5 CV immediately after injection. Pooled fractions provided 30 as a foam, which was dissolved in a minimum amount of DCM and precipitated from 60 mL hexanes and 15 mL of diethyl ether. The precipitate was collected by vacuum filtration to provide 0.933 g (1.2 mmol; 65%) of 30 as a solid.

Example 29: Synthesis of 2-iodoethyl (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 12-(2-(2-((tert-butoxycarbonyl)amino)-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7- yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oate (31)

[00381] 2-(2-((tert-butoxycarbonyl)amino)-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7- yl)acetic acid (107) (1.095 g, 3.5 mmol) was co-evaporated twice with dry ACN and then dissolved in 50 mL of dry ACN at 0°C under a N2 atmosphere. 0.85 mL (7.7 mmol) of NMM was then added to the stirred solution followed by 0.46 mL (3.8 mmol) of TMAC, and an additional 0.85 mL (7.7 mmol) of NMM. The mixture was stirred for about 30 min at 0°C, and then 2.47g (3.1 mmol) of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-(2-(2- iodoethoxy)-2-oxoethyl)-3-(2-(2-methoxyethoxy)ethoxy)propan-l-aminium 4- methylbenzenesulfonate (22) in 20 mL of dry ACN was added to the mixture by cannula. The mixture was stirred for 1 hour and 20 minutes, then warmed to room temperature. After 2 hours an additional 400 pL (3.6mmol) of NMM was added, and then the mixture was evaporated under reduced pressure, and partitioned between 100 mL of water and 100 mL of EtOAc. The EtOAc layer as separated and extracted twice with 100 mL of water, twice with 100 mL 5% (w/v) aqueous sodium bicarbonate solution, and once with 100 mL of brine, then dried over granular anhydrous magnesium sulfate, filtration, and concentrated by evaporation to yield compound 31 as a solid. Crude 31 was dissolved in DCM and purified by flash chromatography using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a gradient of 0% to 3% methanol in DCM. Product fractions were pooled and concentrated to give 1.01 g (1.1 mmol; 36%) of 31.

Example 30: Synthesis of (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(2- ((tert-butoxycarbonyl)amino)-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)acetyl)- 2,5,8-trioxa-12-azatetradecan-14-oic acid (32)

[00382] The 2-iodoethyl (R)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(2-((tert- butoxycarbonyl)amino)-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)acetyl)-2,5,8- trioxa-12-azatetradecan- 14-oate (31) (1.01 g, 1.1 mmol) was converted to (R)-10-((((9H- fluoren-9-yl)methoxy)carbonyl)amino)-12-(2-(2-((tert-butoxycarbonyl)amino)-4-oxo-3,4- dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)acetyl)-2,5,8-trioxa-12-azatetradecan-14-oic acid (32) using the zinc reduction procedure essentially as described in Example 28, adjusted appropriately for the difference in scale. Once complete, the mixture was filtered through Celite® and concentrated to a slurry which was then dissolved in 200 mL of DCM and extracted twice with 100 mL of Extraction Buffer. The organic layer was then dried over granular anhydrous magnesium sulfate, filtered, and concentrated by evaporation to provide crude 32 as an oil. Crude 32 was then dissolved in 2% methanol in DCM and purified by flash chromatography using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a gradient of 2% to 10% methanol in DCM. Product fractions were pooled and concentrated to give 0.517 g (0.68 mmol; 62%) of 32.

Example 31: (9H-fluoren-9-yl)methyl (4R)-4-((2-(2-(tert-butoxy)ethoxy)ethoxy)methyl)- l,2,3-oxathiazolidine-3-carboxylate 2-oxide (34)

[00383] 44.25 g (650 mmol) of imidazole and 400 mL of anhydrous THF were added to a threenecked RB flask equipped with a mechanical stirrer, addition funnel and N2 inlet, and the mixture was stirred under N2 atmosphere until all imidazole had dissolved. The solution was then cooled for 15 minutes in a salt/ice bath. Thionyl chloride (11.6 mL, 160 mmol) was then added dropwise over 10 minutes with stirring during which solid formed. The mixture was stirred a further 10 minutes following the addition, then a solution of (9H-fluoren-9-yl)methyl (S)-(l-(2-(2-(tert-butoxy)ethoxy)ethoxy)-3-hydroxypropan-2-yl)carbamate (33) (22.86 g, 50 mmol) in 150 mL of anhydrous THF was added dropwise over 160 minutes. The mixture was stirred for an additional 20 minutes and then filtered, and the solid obtained was washed with THF. The combined filtrate and washings were then concentrated and dissolved in 200 mL of EtOAc, which was extracted twice with 100 mL portions of 0.5N aqueous HC1, once with 100 mL of aqueous saturated sodium bicarbonate, once with 5% (w/v) aqueous sodium bicarbonate, and once with 100 mL of brine. The organic layer was then dried over granular anhydrous magnesium sulfate, filtered, and concentrated to obtain 24.06 g (47.8 mmol, 96%) of crude 34 as yellow oil. When repeated on a 15 g (32.8 mmol) scale of compound 33 in similar fashion, this procedure produced 16.7 g (33.1 mmol, 101%) of crude 34. When repeated again on 15 g (32.8 mmol) scale of compound 33 in similar fashion, this procedure produced 17.5 g (34.7 mmol, 106%) of crude 34. In all cases, the crude 34 was used in subsequent reactions without further purification.

[00384] Note: (9H-fluoren-9-yl)methyl (S)-(l-(2-(2-(tert-butoxy)ethoxy)ethoxy)-3- hydroxypropan-2-yl)carbamate (33) was obtained in high purity from a custom manufacturer. Example 32: Synthesis of (9H-fluoren-9-yl)methyl (R)-4-((2-(2-(tert- butoxy)ethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (35)

[00385] (9H-fluoren-9-yl)methyl (4R)-4-((2-(2-(tert-butoxy)ethoxy)ethoxy)m ethyl)- 1,2,3 - oxathiazolidine-3 -carboxylate 2-oxide (34) (24.06 g, 47.8 mmol) was dissolved in 100 mL of DCM, followed by addition of 100 mL of deionized water. The biphasic mixture was stirred for 5 minutes and then cooled in an ice bath for 15 minutes. NaICU (23.5 g, 110 mmol) was then added followed by RuChxEhO (0.31 g, 1.5 mmol), and the mixture was stirred for 15 minutes, then warmed to room temperature and stirred for two hours. An additional 0.03g of RuCh xFLO was then added and the mixture was stirred for an additional 2.5 hours, and then filtered through Celite® resulting in a bi-phasic filtrate. 200 mL of DCM and 200 mL of aqueous saturated sodium chloride were added to the filtrate, and the DCM layer was separated. The aqueous layer was extracted with an additional 100 mL of DCM, and the combined DCM layers were then extracted with 200 mL of brine. The DCM solution was then dried over granular anhydrous magnesium sulfate, filtered, and concentrated to obtain an oil that was purified by flash chromatography on silica gel using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running an isocratic elution of 25% EtOAc in hexanes. Product fractions were combined and concentrated to provide 19.6 g (37.7 mmol; 75.4% from Compound 33) of 35 as an oil. When repeated on 16.7 g scale of 34, substituting DCM with EtOAc , the oxidation was remarkably faster, and the procedure produced 9.7 g (18.7 mmol, 57% from Compound 33) of 35. The reaction was repeated again on 17.5 g scale of 34 using EtOAc as the solvent to produce 12.4 g (23.9 mmol; 72.8% from Compound 33) of 35.

Example 33: Synthesis of (9H-fluoren-9-yl)methyl (S)-(l-azido-3-(2-(2-(tert- butoxy)ethoxy)ethoxy)propan-2-yl)carbamate (36)

6 35 ^H 36

[00386] Sodium azide (1.07g, 16.5 mmol) was dissolved in 43.5 mL of methanol, and then combined with a solution of 7.15g (13.8 mmol) of (9H-fluoren-9-yl)methyl (R)-4-((2-(2-(tert- butoxy)ethoxy)ethoxy)methyl)-l,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (35) in 17 mL of methanol. The resulting mixture was stirred for 90 minutes, then partitioned between about 75 mL of EtOAc and 100 mL of brine. The EtOAc layer was separated and washed with saturated aqueous KH2PO4, then dried over granular anhydrous magnesium sulfate, filtered, and concentrated to obtain 5g (10.4 mmol, 75%) of 36 as clear oil. This reaction was repeated on 9.7g (18.7 mmol) scale of 35 to produce 8.8g (18.2 mmol, 98%) of 36. The reaction was repeated a third time on 12.4g (23.9 mmol) scale of 35 to produce 12.3g (25.5 mmol, 107%) of 36

Example 34: Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert- butoxy)ethoxy)ethoxy)propan-l-aminium 4-methylbenzenesulfonate (37)

[00387] Palladium carbon catalyst (100 mg, 10% Pd by weight, unreduced) was purged under nitrogen and cooled to 0°C in an ice bath. In a separate flask, (9H-fluoren-9-yl)m ethyl (S)-(l- azido-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)propan-2-yl)carbamate (36) (1.0g, 2.07 mmol) in 10 mL of methanol was mixed with 0.403 g of p-TSA, and the mixture was then added to the flask containing the palladium catalyst under N2. 3.31 mL (20.7 mmol) of tri ethylsilane was then added to the mixture dropwise over 5 minutes, during which cooling was maintained and hydrogen evolved. The mixture was stirred for an additional 10 minutes, then filtered through Celite®. The filtrate was then concentrated and dried under vacuum to afford 1.48 g (2.4 mmol, 114%) of solid (37) containing minor silane impurities. This reaction was repeated on 8.8 g (18.2 mmol) scale of 36 to produce 12.5 g (19.9 mmol, 109%) of 37. The reaction was repeated a third time on 13.3 g (27.6 mmol) scale of 36 to produce 17.66 g (28.1 mmol, 102%) of 37. Example 35: Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert- butoxy)ethoxy)ethoxy)-N-(2-oxo-2-(2,2,2-tribromoethoxy)ethyl)propan-l-aminium chloride (38)

[00388] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert- butoxy)ethoxy)ethoxy)propan-l-aminium 4-methylbenzenesulfonate (37) (17.56 g, 27.9 mmol) was purged under nitrogen, and then 2,2,2-tribromoethyl 2-bromoacetate (100) (15.8 g, 39.1 mmol) and 224 mL of anhydrous ACN were added. The mixture was stirred until all solids had dissolved then cooled in an ice bath. DIEA (13.14 mL, 75.4 mmol) was added to the chilled solution, and the mixture was then warmed to room temperature. After 40 minutes, 2N aqueous HC1 (~45 mL) was added to lower the pH to approx. 2. The mixture was then partitioned between EtOAc and deionized water, and the organic layer was separated and washed twice with 100 mL of 40 mM aqueous HC1, once with water, then once with brine, then dried over granular anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude solid (38). Crude 38 was dissolved in DCM and purified by flash chromatography on silica gel using Teledyne ISCO CombiFlash Rf+ automated chromatography system running a gradient from 0% to 15% methanol into DCM. Product fractions were pooled and concentrated to give 23g (28.2 mmol, 101%) of 38 as a foam, and as a mixture of tosyl and hydrochloride salts.

Example 36: Synthesis of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2- (tert-butoxy)ethoxy)ethoxy)-N-(2-(2-iodoethoxy)-2-oxoethyl)propan-l-aminium 4- methylbenzenesulfonate (39)

[00389] A mixture of (9H-fluoren-9-yl)methyl (S)-4-((2-(2-(tert-butoxy)ethoxy)ethoxy)methyl)- 1,2, 3 -oxathiazolidine-3 -carboxylate 2,2-dioxide (35) (19.6 g, 37.72 mmol) and 2-(2- iodoethoxy)-2-oxoethan-l-aminium 2,2,2-trifluoroacetate (102) (38.82 g, 113.16 mmol) were co-evaporated twice with 200 mL of anhydrous ACN. The resulting solid mixture was then purged under nitrogen, and 120 mL of anhydrous THF was added, and the mixture was stirred at room temperature. Once solids were dissolved the flask was cooled in an ice bath for 20 minutes, and then 11.9 mL of anhydrous NMM was added, and after five minutes the mixture was warmed to room temperature and stirred for 1.5 hours. The mixture was then cooled in the ice bath, and glacial acetic acid (~13 mL) was added dropwise to lower the pH to about 5. The mixture was then concentrated, and the resulting slurry was partitioned between 400 mL of EtOAc and 400 mL of water. The layers were separated and the water layer extracted with a further 250 mL of EtOAc. The combined EtOAc layers were then extracted twice with 250 mL of saturated aqueous monobasic potassium phosphate, twice with 250 ml of water, twice with 250 mL of saturated sodium bicarbonate, and with brine. The EtOAc solution was then dried over granular anhydrous magnesium sulfate, filtered, and concentrated to a gum that was then cooled to -20°C for about 3 days. The gum was then warmed to room temperature and dissolved in about 50 mL of DCM and purified (in two portions) by silica gel flash chromatography using a Teledyne ISCO CombiFlash Rf+ automated chromatography system and an ascending gradient of methanol into DCM. Fractions determined to contain the desired product (as its free base) were pooled, and p-TSA was added until the pH was about 2. Concentrating the solution gave 8.2 g (1.9 mmol, 26%) of 39 as a foam.

Note: Crude 39 is more stable after addition of p-TSA.

Example 37: Synthesis of 2-iodoethyl (S)-ll-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-13-(2-(2,6-bis(bis(tert-butoxycarbonyl)amino)-9H-purin-9- yl)acetyl)-2,2-dimethyl-3,6,9-trioxa-13-azapentadecan-15-oate (40)

[00390] To an oven dried 300 mL RB flask was added 5.4 mmol (3.28 g) of 2-(2,6-bis(bis(tert- butoxycarbonyl)amino)-9H-purin-9-yl)acetic acid (108) and 20 mL of dry DMF, and the mixture was stirred under nitrogen and cooled in an ice bath for about 15 minutes. 23.9 mmol (2.63 mL) of NMM was then added, followed immediately by 5.85 mmol (0.72 mL) of TMAC, and the mixture was stirred at 0°C for 20 minutes. An anhydrous solution of 4.5 mmol (3.78 g) of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-(tert-butoxy)ethoxy)ethoxy)-N- (2-(2-iodoethoxy)-2-oxoethyl)propan-l-aminium 4-methylbenzenesulfonate (39) in about 15 mL of dry ACN was then transferred to the mixture via a cannula, and after 15 minutes the mixture was warmed to room temperature and stirred for 2 hours. The solvent was then evaporated under reduced pressure and the residue was partitioned with 75 mL of EtOAc and 50 mL of /i saturated KH2PO4. The EtOAc layer was then separated and washed once with 50 mL of ’A saturated KH2PO4, twice with 50 mL of 5% (w/v) aqueous NaHCOs and once with 50 mL of brine, and then dried over granular magnesium sulfate, filtered and evaporated to yield 6.65 g of crude 40. Crude 40 was then dissolved in a minimum of DCM and purified by injection on a 80g ISCO Gold silica gel column and eluted using a Teledyne ISCO CombiFlash Rf+ automated chromatography system running a 0-4% methanol in DCM gradient over 12 CV with a hold at 4% methanol for 6 CV after first holding at 0% methanol for 1.5 CV immediately after injection. Pooled fractions provided 2.34 g (1.9 mmol; 41%) of 40 as a foam, which was further purified by chromatographic separation to obtain 2.14 g (1.7 mmol; 38%). 40 could be treated as described herein (e.g. Example 15) to thereby produce the comparable left-handed PNA monomer (i.e. (S)-l l-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-13-(2-(2,6-bis(bis(tert- butoxycarbonyl)-amino)-9H-purin-9-yl)acetyl)-2,2-dimethyl-3,6,9-trioxa-13-azapentadecan-15- oic acid).

Example 38: Synthesis of PNA Oligomers

/. PNA Synthesis Procedure:

[00391] All PNA oligomers were synthesized on an Intavis MultiPep RSi automated peptide synthesizer using Fmoc solid phase peptide synthesis protocol using rink amide TentaGel resin (Rapp polymer, R28023) as the solid support. The synthesis protocol comprised three synthetic steps (in addition to washing steps) wherein each of the steps was repeated for each new PNA monomer, linker, amino acid or other building block (e.g., synthon) until the polyamide was completely assembled. Specifically, a single synthetic cycle comprised: 1) deprotection of the N-terminal Fmoc group; 2) coupling of a new monomer, linker, amino acid or synthon to the growing polyamide; and 3) capping of the unreacted amino groups. Between each step in the cycle, the resin was washed extensively with DMF to remove unreacted reagents and other unwanted impurities and side products of the reaction.

2. Protocol for Small Scale Synthesis:

[00392] Approximately 45 mg (5.8 pmol) rink amide TentaGel resin was placed in the reaction column and treated with 800 pL DCM for 15 minutes. The swollen resin was then treated twice with 600 pL of 20% (v/v) piperidine/DMF for 5 min each to remove/deprotect the Fmoc group. After five washes with DMF, approximately 45 pmol of a PNA monomer, linker, amino acid (e.g., lysine) or other synthon (as applicable based on the sequence of the PNA oligomer to be prepared) was delivered to the resin from a solution comprising PNA monomer, linker, amino acid or synthon dissolved in dry DMF. A mixture of DIEA (approximately 56 pmol) dissolved in dry N-methyl pyrrolidone (NMP), and approximately 42 pmol of 1- [Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) in dry DMF were then added to the resin. 2,6-lutidine can be added to the DIEA solution in a ratio of approximately 1/1.5 DIEA/2.6-lutidine (v/v). After addition of the reagents the resin was agitated for 30 min, and the mixture was drained, followed by extensive washing with DMF. The capping step was then performed by treating the resin with 600 pL of capping solution (5% acetic anhydride and 6% lutidine in DMF (v/v)) while agitating the resin for 5 min. These three steps were repeated sequentially for each new PNA monomer, linker, amino acid or other building block until the PNA oligomer was completely assembled. In some cases, as a last step in the overall synthesis, the capping cycle was performed to thereby N-acetylate the terminal amine(s).

3. General Protocol for Cleavage and Deprotection of the PNA Oligomer:

[00393] Crude PNA oligomers were obtained by treating the dried resin with 1 mL of cleavage mixture containing trifluoracetic acid (TFA), m-cresol, water, and thioanisole (in a ratio of 95/2/2/1 : v/v/v/v) for 2 hours at room temperature. The sample was then filtered to remove the resin and the filtrate was subsequently treated with cold diethyl ether to cause precipitation of the PNA oligomer. After repeated suspension and pelleting of the PNA oligomer with cold diethyl ether, the crude PNA oligomer was dissolved in approximately 2 mL of a 1/1 (v/v) water/ ACN mixture. The crude PNA oligomer was then obtained for purification by lyophilization of this solution.

Example 39: Examination of the Optical Purity of Various PNA Monomers

[00394] The chiral purity of PNA monomers (See Table 1) was determined with HPLC by preparing a 6-mer oligomer of the sequence: Ac-L-Phe-X-gly-gly-gly-gly-NFE, wherein X is the PNA monomer to be examined for chiral purity. The L-enantiomer of phenylalanine (L-Phe) was used because it is relatively hydrophobic and can be obtained in near 100% optical purity. By substituting the chiral L-Phe molecule in the oligomer, a diastereomer is created by any chiral impurity (opposite enantiomer) of the X-PNA Monomer. A four residue C-terminal (gly)4 tail was used to add enough length to the oligomer to enable good separation of the diastereomeric oligomer products by conventional HPLC methods. The column used for analysis was a Waters Xbridge BEH C18 2.5um 4.6 x 100mm column, and the buffers used were: A Buffer: 0.1%TFA in water; and B Buffer: 0.1%TFA in ACN. The gradient was 0-2 min 5% B Buffer in A Buffer, then an ascending gradient of B Buffer into A Buffer from 5% to 35% from 2-15.1 min. The column was heated to 55 °C throughout the gradient. In our experience, the diastereomers of the 6-mer oligomers of this structure are well resolved by standard HPLC protocols using the separation conditions described above. By this test, chiral PNA monomers made by the methods of the Examples above and listed in Table 1 were found to have greater than 99% ee. There was little if any difference in optical purity of the PNA monomers prepared by the methodology described herein compared to PNA monomers prepared by the traditional Mitsunobu reaction (See Table 1 for comparative data) and Sahu et al, JOC, 2011, 76: 5614-5627.

Table 1. Comparison of Optical Purity of PNA monomers prepared by a Mitsunobu route or a method described herein.

Claims

We claim:

1. A compound of Formula (IIA):

R⁶ R⁵ ^R o¹'=^N>s^>S II-o o (IIA) or a salt thereof, wherein:

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R^c is hydrogen, C1-C12 alkyl, cycloalkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Ci- C12 heteroalkyl, wherein each optionally protected amino acid side chain is independently selected from:

each of which is optionally protected. The compound of claim 1, wherein each of R⁵ and R⁶ is independently hydrogen, C3-C16 alkyl, Ce-Ci6 heteroalkyl, or the optionally protected amino acid side chain, provided that one of R⁵ and R⁶ is not hydrogen, or both of R⁵ and R⁶ are not hydrogen. The compound of claim 1, wherein each of R⁵ and R⁶ is independently hydrogen, ,

, wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl, provided that one of R⁵ and R⁶ is not hydrogen, or both of R⁵ and R⁶ are not hydrogen. The compound of claim 1, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently C3-C16 alkyl or Ce-Ci6 heteroalkyl. The compound of claim 1, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently

, wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen, cycloalkyl, or C1-C12 alkyl. The compound of claim 1, wherein the compound of Formula (IIA) is a compound of

Formula (IIA-a) or (IIA-b):

or a salt thereof, wherein

x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl; and

R^c is hydrogen, C1-C12 alkyl, cycloalkyl, C2-C12 alkenyl, C2-C12 alkynyl, or Ci- C12 heteroalkyl. The compound of any one of claims 1, 2, and 6, wherein R⁵ is the optionally protected amino acid side chain.

The compound of any one of claims 1, 2, 6, and 7, wherein R⁵ is

, each of which is optionally protected. The compound of claim 1, wherein the compound of Formula (IIA) is a compound of Formula (IIA-c) or (IIA-d):

or a salt thereof, wherein

R⁶ is hydrogen, C3-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, Ce-Ci6 heteroalkyl, Ci-Ci6-haloalkyl, -OR^C, cycloalkyl, Ci-C 12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkyleneheteroaryl, the optionally protected amino acid side chain,

or

x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

The compound of claim 9, wherein R⁹ is methyl, ethyl, tert-butyl, cyclopentyl, or cyclohexyl. The compound of any one of claims 1-10, wherein R⁶ is hydrogen. The compound of any one of claims 1-11, wherein R¹ is 9-fluorenylmethyloxycarbonyl (Fmoc), 2,7-di-tert-butylfluoren-9-ylmethoxycarbonyl (Fmoc*), 2-fluorofluoren-9- ylmethoxycarbonyl (Fmoc(2F)), 2-monoisooctylfluoren-9-ylmethoxycarbonyl (mio- Fmoc), 2,7-diisooctylfluoren-9-ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)- fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-t-butyl-9-fluorenylmethoxycarbonyl (Dtb- Fmoc), or 2,7-bis(trimethylsilyl)-fluorenylmethoxycarbonyl (Bts-Fmoc). The compound of any one of claims 1-11, wherein R¹ is 9-fluorenylmethyloxycarbonyl (Fmoc). A method of making a compound of Formula (IB):

or a salt thereof, wherein:

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

provided that one of R⁵ and

R⁶ is not hydrogen, or both of R⁵ and R⁶ are not hydrogen;

R⁷ is hydrogen or C1-C16 alkyl;

- 139 - each of R^A, R^B, and R^c is independently hydrogen, Ci-Cie alkyl, C2-C16 alkenyl, C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

The method of claim 14, wherein the method further comprises the reaction of the intermediate of Formula (IIB), or a salt thereof:

with an azide-containing reagent. The method of claim 15, wherein the azide-containing reagent is NaN The method of claim 15 or claim 16, wherein the reaction of the intermediate of Formula (IIB) and the azide-containing reagent followed by reduction yields an intermediate of Formula (VIIB):

or a salt thereof, wherein

R¹ is an amine protecting group;

- 140 - each of R⁵ and R⁶ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl, - N(R^A)(R^B), halo, -OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

; provided that one of both of

R⁵ and R⁶ are not hydrogen;

C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The method of any one of claims 14-17, wherein the method further comprises reaction of the intermediate of Formula (VIIB), or a salt thereof:

with an ester compound of Formula (VB), or a salt thereof:

to achieve a compound of Formula (VIIIB), or a salt thereof:

wherein

R¹ is an amine protecting group; R² is hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkyl ene-heteroaryl; each of R³, R⁴, R⁵, and R⁶ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl, -N(R^A)(R^B), halo, -OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

R⁶ are not both hydrogen;

R⁷ is hydrogen or Ci-Cie alkyl; each of R^A, R^B, and R^c is independently hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and

X is halo or tosyl. The method of claim 14, wherein the method further comprises reaction of the intermediate of Formula (IIB), or a salt thereof:

with a compound of Formula (VIB), or a salt thereof:

to achieve a compound of Formula (VIIIB), or a salt thereof:

- 142 - wherein

R¹ is an amine protecting group;

provided that each of R⁵ and

C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The method of any one of claims 14-19, wherein the intermediate of Formula (IIB) is a compound of Formula (IIB-a) or Formula (IIB-b):

or a salt thereof, wherein

R¹ is an amine protecting group;

- 143 - each of R^A, R^B, and R^c is independently hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The method of any one of claims 14-20, wherein the compound of Formula (IB) is a compound of Formula (IB-e) or Formula (IB-f):

or a salt thereof, wherein

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

R² is hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkyl ene-heteroaryl; each of R³ and R⁵ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl, - N(R^A)(R^B), halo, -OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The method of any one of claims 14-21, wherein the compound of Formula (IB) is a compound of Formula (IB-a) or Formula (IB-b):

or a salt thereof, wherein

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

each of R^A, R^B, and R^c is independently hydrogen, C1-C16 alkyl, C2-C16 alkenyl,

C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The method of any one of claims 14-21, wherein the compound of Formula (IB) is a compound of Formula (IB-c) or Formula (IB-d):

or a salt thereof, wherein

B is an optionally protected nucleobase;

- 145 - R¹ is an amine protecting group;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R¹⁰ is hydrogen, C1-C16 alkyl, cycloalkyl, or Ci-Ci6-haloalkyl; x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and y is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. The method of any one of claims 14-21, wherein the compound of Formula (IB) is a compound of Formula (IB-e) or Formula (IB-f):

or a salt thereof, wherein

B is an optionally protected nucleobase;

R² is hydrogen, C1-C16 alkyl, C2-C16 alkenyl, C2-C16 alkynyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkylene-heteroaryl;

R³ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci- Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl,

- 146 - aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkylene-heteroaryl, -N(R^A)(R^B), halo, -

OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl;

R¹⁰ is hydrogen, C1-C16 alkyl, cycloalkyl, or Ci-Ci6-haloalkyl; x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and y is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. The method of any one of claims 14-19, wherein each of R³ and R⁴ is independently hydrogen or the optionally protected amino acid side chain. The method of any one of claims 14-19, wherein each of R³ and R⁴ is independently hydrogen. The method of any one of claims 14-19, 25, and 26, wherein each of R⁵ and R⁶ is independently hydrogen, C1-C16 alkyl, C1-C16 heteroalkyl, or the optionally protected amino acid side chain. The method of any one of claims 14-19, 25, and 26, wherein each of R⁵ and R⁶ of Formula (Il-a) is hydrogen, C1-C12 alkyl, or C1-C12 heteroalkyl, provided that one of R⁵ and R⁶ of Formula (Il-a) is independently not hydrogen. The method of any one of claims 14-19, 25, and 26, wherein each of R⁵ and R⁶ is independently hydrogen,

wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and R⁸ is hydrogen or C1-C16 alkyl, provided that one or both of R⁵ and R⁶ is not hydrogen. The method of any one of claims 14-19, 25, and 26, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently Ci-Cie alkyl or Ci- Ci6 heteroalkyl. The method of any one of claims 14-19, 25, and 26, wherein one of R⁵ and R⁶ is independently hydrogen and the other of R⁵ and R⁶ is independently

. or , wherein x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and R⁸ is hydrogen or C1-C16 alkyl. The method of any one of claims 14-21, wherein R⁵ is the optionally protected amino acid side chain. The method of any one of claims 14-21 and 32, wherein the optionally protected amino acid side chain i

each of which is optionally protected. The method of any one of claims 14-22, wherein R⁵ is CH2OR⁹. The method of any one of claims 14-22 and 34, wherein R⁹ is methyl, ethyl, tert-butyl, cyclopentyl, or cyclohexyl. The method of any one of claims 14-23 and 25-35, wherein R¹ is 9- fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), p- toluenesulfonyl (Ts), benzoyl (Bz), or benzyl (Bn). The method of any one of claims 14-23 and 25-35, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc), 2-(4-nitropheylsulfonyl)ethoxy carbonyl (Nsc), 1,1- dioxobenzo[b]thiophene-2-ylmethyloxy carbonyl (Bsmoc), 1, 1 -di oxonaphthofl, 2- b]thiophene (Nsmoc), l-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl (ivDde), 2,7-di-tert-butylfluoren-9-ylmethoxycarbonyl (Fmoc*), 2-fluorofluoren-9- ylmethoxycarbonyl (Fmoc(2F)), 2-monoisooctylfluoren-9-ylmethoxycarbonyl (mio- Fmoc), 2,7-diisooctylfluoren-9-ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)- fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-t-butyl-9-fluorenylmethoxycarbonyl (Dtb- Fmoc), 2,7-bis(trimethylsilyl)-fluorenylmethoxycarbonyl (Bts-Fmoc), 9-(2,7- dibromo)fluorenylmethoxy carbonyl, 2-[phenyl(methyl)sulfonio]ethyloxy carbonyl tetrafluoroborate (Pms), ethanesulfonylethoxycarbonyl (Esc), 2-(4- sulfophenylsulfonyl)ethoxy carbonyl (Sps), or l-cyano-2-methylpropan-2-ylcarbonyl (Cyoc), each of which is optionally substituted. The method of any one of claims 14-23 and 25-35, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc), 2, 7-di-tert-butylfluoren-9-ylmethoxy carbonyl (Fmoc*), 2-fluorofluoren-9-ylmethoxycarbonyl (Fmoc(2F)), 2-monoisooctylfluoren-9- ylmethoxycarbonyl (mio-Fmoc), 2,7-diisooctylfluoren-9-ylmethoxycarbonyl (dio-Fmoc), 9-(2-sulfo)-fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-t-butyl-9- fluorenylmethoxycarbonyl (Dtb-Fmoc), or 2,7-bis(trimethylsilyl)- fluorenylmethoxy carbonyl (Bts-Fmoc). The method of any one of claims 14-23 and 25-35, wherein R¹ is 9- fluorenylmethyloxy carbonyl (Fmoc). The method of any one of claims 14-25, wherein the optionally protected amino acid side chain is selected from:

- 149 -

and each of which is optionally protected.

41. The method of any one of claims 14-40, wherein R² is hydrogen, Ci-Cie alkyl, or C1-C16- haloalkyl.

42. The method of any one of claims 14-40, wherein R² is Ci-Ci6-haloalkyl.

43. The method of any one of claims 14-42, wherein R² is 2,2,2-tribromoethyl, 2- bromoethyl, 2,2,2-trichloroethyl, or 2-iodoethyl.

44. The method of any one of claims 14-43, wherein B is a naturally occurring nucleobase or a non-naturally occurring nucleobase, each of which is optionally protected.

45. The method of any one of claims 14-44, wherein B is selected from adenine, guanine, thymine, cytosine, uracil, pseudoisocytosine, 2-thiopseudoisocytosine, 5-methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2,6-diaminopurine, 2-thiouracil, 2- thiothymine, 2-thiocytosine, 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5- chlorocytosine, 5 -bromocytosine, 5-iodocytosine, 5-propynyluracil, 5-propynylcytosine,

6-azouracil, 6-azocytosine, 6-azothymine, 7-methylguanine, 7-methyladenine, 8- azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3- deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-azaadenine, 5-propynyluracil, 2-thio-5- propynyluracil, pyridin-2-amine, 2-thiopseudoisocytosine, pyrimidin-2(lH)-one, and pyridazin-3(2H)-one, each of which is optionally protected. The method of any one of claims 14-45, wherein R⁷ is hydrogen. A method of making a compound of Formula (IB):

or a salt thereof, wherein:

B is an optionally protected nucleobase;

R¹ is an amine protecting group;

R² is hydrogen, Ci-Cie alkyl, C2-C16 alkenyl, C2-C16 alkynyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, cycloalkyl, C1-C16 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene- heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, or C1-C16 alkyl ene-heteroaryl; each of R³, R⁴, R⁵, and R⁶ is independently hydrogen, C1-C16 alkyl, cycloalkyl, C1-C16 heteroalkyl, Ci-Ci6-haloalkyl, C1-C12 alkylene-cycloalkyl, heterocyclyl, C1-C16 alkylene-heterocyclyl, aryl, C1-C16 alkylene-aryl, heteroaryl, C1-C16 alkyl ene-heteroaryl, -N(R^A)(R^B), halo, -OR^C, -CH2OR⁹, an optionally protected amino acid side chain,

provided that each of R⁵ and

R⁶ are not both hydrogen;

C2-C16 alkynyl, or C1-C16 heteroalkyl;

R⁸ is hydrogen, C1-C12 alkyl, cycloalkyl, or C1-C12 haloalkyl; R⁹ is hydrogen, C1-C12 alkyl, cycloalkyl, C1-C12 haloalkyl, C1-C12 heteroalkyl, or an optionally protected amino acid side chain; n is an integer selected from 0, 1, 2, 3, or 4; and x is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; wherein the method comprises the following steps:

(i) contacting a compound of Formula (IIB):

(iii) contacting the intermediate of Formula (VIIB) with an ester compound of

Formula (VB): o x\ A ,R² °

R⁴ R³ (VB), to achieve a compound of Formula (VIIIB):

- 152 - (iv) contacting the compound of Formula (VIIIB) with a nucleobase acetic acid to achieve the compound of Formula (IB).

- 153 -